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STUDENT HANDBOOK
2013 - 2014
Master of Research Strands: Biology of Cancer Cellular & Molecular Physiology Drug Safety Medical Sciences Molecular and Clinical Gastroenterology Molecular and Clinical Pharmacology Nanomedicine and Biostatistics Neuroscience Stem Cells, Tissues and Disease Womens, Childrens and Perinatal Health
Contents
Section 1 Introduction Page 3
Page 8
Page 14
Section 4 Assessment
Page 23
Page 28
Page 54
Page 77
Page 100
Page 102
Section 10 Appendix
Page 103
Section 1 Introduction
1.1 A welcome to new students
I am pleased to welcome you as a new student into the Institute of Translational Medicine and hope that you will find your time studying on the MRes in Biomedical Sciences and Translational Medicine programme an enriching experience. The course combines hands-on laboratory research work with lectures and tutorials to give you direct knowledge and experience of cutting-edge biomedical and clinical research. You will also receive training in transferable skills, which will broaden your existing skill set and help prepare you for your subsequent careers. Feedback from previous MRes students indicates that you will need to work hard, but that the course is both enjoyable and rewarding. This handbook contains essential information about all aspects of the MRes programme, so it is important that you read and understand it. Additional information and announcements about the programme will also be issued during the course, either electronically by email or through VITAL (the Universitys online teaching resource), or via posters on the MRes notice boards. It is therefore very important that you check your e-mail, relevant VITAL pages and the notice board every day, as there may be important messages relating to the course or changes in schedule, etc. One of the first things you will want to know is who to contact for help, information and advice. The MRes programme is run by a team consisting of the Programme Director, the Programme Administrators and the Strand Convenors. They are happy to help you if you are having any difficulties, whether academic, administrative or personal in nature, including disability-related issues. Any problems should initially be discussed with your Strand Convenor, who will either deal with the issue directly or will refer the matter to the Programme Director or Administrator as appropriate. Prof Andrea Varro is the Institute Director of Postgraduate Research of the Institute of Translational Medicine and she will be happy to discuss any unresolved problems with you provided that the appropriate line of communication within the MRes team has been followed and exhausted. Contact details for each member of the team can be found in Section 2.1 of this handbook, along with a brief description of their area of responsibility, to guide you to the most suitable person to deal with your question. I hope that you find the MRes in Biomedical Sciences and Translational Medicine both useful and enjoyable. Good luck with your studies.
Programme Director: Professor Alan Morgan Email: amorgan@liv.ac.uk Telephone: 0151 794 5333
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MORNING
AFTERNOON
12:00 13:00 Campus and Building Tour 13:00 14:00 Lunch break 13.45 14.30 Overseas students Paperwork completion - bring passport, visa, original certificates/transcript LOCATION: PC Centre, Sherrington Building 14.30 15.30 Overview of MRes in Biomedical Sciences talk by Professor Alan Morgan LOCATION: Physiology Seminar Room 15.30 16.30 Strand Inductions 16.30 ITM Postgrad Society talk and welcome drinks LOCATION: Physiology Seminar Room
12.00 13.00 Q&A session with Professor Alan Morgan LOCATION: Physiology Seminar Room 13.00 13.30 Lunch break 13.30 14.30 UK/EU students, surname A to H, bring passport, original certificates/transcript LOCATION: PC Centre, Sherrington Building
MORNING
AFTERNOON
12.30 13.00 Q&A session with Professor Alan Morgan LOCATION: Wolfson Suite, Harold Cohen Library 13.00 13.30 Lunch break 13.30 14.30 UK/EU students, surname K to Z, bring passport, original certificates/transcript LOCATION: PC Centre, Sherrington Building
16.30 17.00 Meeting with Professor Alan Morgan and the ITM PGR Team LOCATION: TBC
LATER EVENTS:
Some induction activities are scheduled to take place after the first 2 weeks. Details of these are given below: Event: University Safety Seminar Date: to be confirmed Time: Location: Event: Prospective Licensee Training Course Examination Date: to be confirmed Time: Location: Event: University Radiation Protection (BASIC) Date: to be confirmed Time: Location: Event: English Language Classes for International Students Date: Classes will run throughout the academic year on a Friday starting 4th October Time: 09:00 10:00 Location: Brodie Tower Event: Demonstrator Training Workshop Date: 10th October 2013 Time: 1.30pm top 4.30pm Location: to be confirmed Event: University Radiation Protection (LASER) Date: to be confirmed Time: Location: Event: Biostatistics Workshop Date: 6th & 13th November Time: 2.00pm to 5.00pm Location: Physiology Seminar Room Event: IP and Commercialization Workshop Date: July 2014 Time: To Be Confirmed Location: To be confirmed 6
email: Barclayj@liverpool.ac.uk
email: Dom@liverpool.ac.uk
email: amorgan@liverpool.ac.uk
Molecular and Clinical Gastroenterology: Dr. John Jenkins Tel: 0151 794 6828 Molecular and Clinical Pharmacology: Dr. Jean Sathish Tel: 0151 794 5477 Nanomedicine and Biostatistics: Prof Andrew Owen Tel: 0151 794 8211 Dr. Marta van der Hoek Tel: 0151 794 5083 Neuroscience: Dr. Graeme Sills
email: jrj1@liverpool.ac.uk
email: sathish@liverpool.ac.uk
email: g.sills@liverpool.ac.uk
Stem Cells, Tissues and Disease: Prof David Edgar Tel: 0151 794 5493 Womens, Childrens and Perinatal Health: Dr. Dharani Hapangama Tel: 0151 795 9559
email: dhedgar@liverpool.ac.uk
email: dharani@liverpool.ac.uk
Programme Director Prof Alan Morgan Responsible for the overall organisation of the MRes programme, he will help with general academic and administrative problems relating to the MRes course. He will also help with any specific issues that are not able to be resolved by strand convenors. Alans office, room G.37, is situated in Red Block on the ground floor of the main Physiology Building on Crown Street (Building 313 on the campus map). He can be contacted by email, amorgan@liv.ac.uk, or by telephone on (0151) 794 5333. Issues that cannot be resolved by the Programme Director will be referred to the Institute Director of Postgraduate Studies (Prof Andrea Varro). Programme Administrator Rachel Flynn Responsible for the general administration of the MRes programme, she will help with non-academic problems related to the course, including attendance issues, deadlines, schedule alterations, etc. Rachel can be found in the Postgraduate Office on the ground floor of the Sherrington Building on Ashton Street She can be contacted by e-mail at rflynn@liv.ac.uk or by telephone on (0151) 794 5455. Institute Senior Postgraduate Administrator Lisa Crimmins Responsible for recruitment and registration onto the MRes programme, including supervisor registration, she will help with problems related to these areas. She will also assist with wider University level postgraduate issues, such as financial arrangements, visa problems, etc. Lisa can be found in the Postgraduate Office on the ground floor of the Sherrington Building on Ashton Street. She can be contacted by e-mail at crimmins@liverpool.ac.uk or by telephone on (0151) 794 5465 Postgraduate Students Team The team is based in Room LG43, Sherrington Building, they will help you with general administrative issue and non-academic related problems, the team consists of: Lisa Crimmins (0151 794 5465), Rachel Flynn (0151 794 5455), Michelle Jackson (0151 794 8293), Jack Carter-Hallam (0151 794 8032).
2.3 Safety
Institute of Translational Medicine has a dedicated Safety Officer. All students must attend a safety talk given by Geoff Williams in the first week and will receive written guidelines from him on safety within the Institute. You are also required to attend University training sessions dealing with general safety and radiation protection, as detailed in the induction timetable in Section 1.2. Additional safety information will be given by Departmental safety officers within the Departments in which you conduct your Research Projects. Risk assessment forms must be completed for your Research Projects and copies of these provided to the appropriate safety officers in your department. 9
Working with human subjects and/or human material Supervisors have a responsibility to ensure that all work involving human subjects is covered by appropriate Ethics Comittee Permission. They should also ensure that students conducting research projects involving human subjects and/or material understand the permission given for their work, and in writing their dissertation, they make a clear statement of the Ethics Comittee Approval for the work. Working with animals Supervisors have a responsibility to ensure that the appropriate Home Office Authority (both personal and project licence) are in place before working with experimental animal is started. They should also ensure that students conducting research projects involving experimental animals understand the permission given for their work, and in writing their dissertation, they make a clear statement of the Home Office Approval for the work. You need to read carefully and obey all the instructions regarding safety that have been given to you before commencing experimental work in the laboratory. Normal working hours are 9.00-5.30 Monday Friday. Work outside these hours, including weekends, is only permitted if either your supervisor or a suitably qualified person approved by your supervisor is present.
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events for international students information about UK life support for international students with children workshops and presentations guidance notes and publications, including a monthly newsletter covering the latest issues affecting international students.
Welcome event The IST hosts a Welcome event in September, which all new international MRes students should attend. The event is designed to introduce you to the city and the University and to also give you the opportunity to meet all the other new international students. Contacts For more information contact: Tel: +44 (0)151 794 4716 Email: ist@liv.ac.uk Web: www.liv.ac.uk/studentsupport/ist/
inform academic departments about your support requirements arrange appropriate support in using the libraries and other academic support services organise study assistants find financial support for services.
With consent, and when appropriate, the team can liaise on a continual basis with your academic department and prepare documentation to confirm your support arrangements.
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support requirements how to apply for Disabled Students Allowance and other sources of funding who to contact for support and advice.
You can find further information and details of who to contact regarding disability issues from the following website: http://www.liv.ac.uk/studentsupport/disability/index.htm The Institute contact for disability matter is Professor Andrea Varro. regarding disability matters can be made to Lisa Crimmins. Informal enquiries
government student loans and grants previous study and how it will affect your funding entitlement welfare benefits tax credits debt counselling and advice.
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Aims The degree program aims to provide students with a first class training in research at Masters level, in Biomedical Sciences and Translational Medicine.
Objectives To conduct independent pieces of research and provide Masters level research training via three 12-week research projects in an interdisciplinary research environment. To demonstrate a critical awareness of a range of modern techniques and to enable students to increase their knowledge of current research in Biomedical Sciences to Masters level. To provide the acquisition and training of transferable skills and knowledge, appropriate to postgraduate research students.
Learning outcomes The three objectives above directly map on to the three separately assessed components of the programme: A) Research Projects; B) Techniques and Frontiers in Biomedical sciences; C) Transferable Skills. The learning outcomes of each of these components are detailed below:
A)
Research Projects
Students will be enabled to develop the skills required for: Data gathering and interpretation in an area of biomedical research. Planning and managing research and achieving goals. The presentation and discussion of scientific data, both verbally and in writing. The acquisition of a detailed knowledge of the experimental foundation of a specific area of biomedical research. Working in a group to achieve a common objective. Communication in science. Accessing information, including the use of electronic systems, and PC technology.
B)
Students will be enabled to: Develop a critical understanding of the experimental methods that underpin modern ideas in biomedical sciences. 14
Appreciate current experimental limitations and likely technical developments Develop an understanding of the concepts fundamental to modern ideas in biomedical sciences. Relate emerging and future developments to existing knowledge. Develop the ability to access, collate and discuss in writing the subject literature.
C)
Transferable skills
Students will be enabled to: Develop the skills required to access information, including the use of electronic systems, and computer technology. Develop the skills required for communication in science, to both specialists and nonspecialists Be appraised of the skills required, and mechanisms for, transferring basic science into a business setting Develop the ability to access, collate and discuss in writing technical information e.g. for reports, publications. Develop the skills of group working and leadership to achieve objectives Improve their management skills, in terms of personal time management and research management.
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strand convenors to enhance awareness of research that is particularly relevant to individual MRes strands. Finally, strand-specific activities are an important part of the Techniques and Frontiers module, as they facilitate awareness of the science associated with particular research strands. Students will be assessed on this module via one short review based on a Techniques lecture, one short review based on a Frontiers lecture, and a referees report based on a Journal Club.
C) Transferable Skills Training in this module is on-going throughout the year and is delivered by staff involved with the program and the University via central provisions. It includes training in research techniques and the development of personal and professional transferable skills. Topics include research philosophy, principles and ethics, managing research progress, data analysis and presentation, health and safety, scientific and technical writing, patent law, exploitation of research, team work skills, time and resource management, communication skills, self-assessment skills and leadership skills. In addition, there is a weekly English conversation session run by The English Language Unit to improve communication skills for students with English as a second language. Important and innovative parts of the transferable skills module include the IP and Commercialization and Writing a PhD Studentship workshops and debates for public understanding of science (science & society). At the end of the module you will need to prepare a Portfolio of Assessment commentary as part of the transferable skills training. Finally, you will attend a Demonstrator Training session as part of the transferable skills training. Further information on the 3 assessed components of the module is given below (detailed information can be found later in this handbook): The IP and Commercialization Workshop will raise awareness of the issues associated with, and necessary for, successful commercialisation of academic research. Intellectual property is a key component for business success. You will learn about the types and importance of intellectual property and how to search for patent information on the Web. The routes available for creating value from basic research will be described. There will be a comparison of the licensing and spin-out routes, together with detail of the business planning process and the role of the technology transfer office. You will be working as a group to prepare a written business plan for potential commercialisation of research. In addition, you will be required to present your idea as a pitch to a panel of judges in a similar way to the popular television programme Dragons Den. The Writing a PhD Studentship workshop will enable students to create a coherent and feasible research proposal for a scientific project to continue studying for a PhD, and to present this in a form suitable for external scrutiny. The Portfolio of Assessment Commentary will enable students to evaluate and reflect on the aims and objectives of the individual programme components, as well as their own achievements. Students will be required to assemble a professional-looking portfolio that documents their progression through the programme in a form suitable for external scrutiny. Students will be assessed on the business plan and presentation given in the IP and Commercialisation component, on the PhD Studentship application, and on the Portfolio of Assessment Commentary.
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Research Project 1 (30 September 6 December) Oral/poster presentations: 10 January 2014 Project report deadline: 13 January 2014
Research Project 2 (13 January 21 March) Oral/poster presentations: 11 April 2014 Project report deadline: 14 April 2014
Research Project 3 (14 April 20 June) Oral/poster presentations: 4 July 2014 Project report deadline: 7 July 2014
Writing a PhD Studentship Workshop 8 July 2013 12.00-2.00pm Application deadline 8 Aug 2014 Submission of portfolio by 15 August 2014 English Conversation Classes Fridays 9.00 -10.00am
Please note: Students need to be resident in Liverpool until 15 August 2014 except for the timetabled holidays indicated below. Permission to be absent from University outside of these holidays can only be granted by the Programme Director following a written request. Timetabled holidays: 15 December 2013 5 January 2014 29 March 2014 6 April 2014 16 August 2014 7 September 2014 (Inclusive) Students MUST attend a viva with the MRes external examiner in order to be awarded an MRes degree. The specific date for your viva will be set nearer the time but you must be available in Liverpool from 8 - 12 September 2014, when vivas will be held. English Conversation Classes are compulsory for students with English as a second language.
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TBA
Location Physiology Seminar Room Venue to be confirmed later Venue to be confirmed later
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Semester 2
Time: 2.00pm 5.00pm Wednesdays Venue: Physiology Seminar Room (plus Physiology Meeting Room for Journal Clubs) Date/time
15 January 2pm 3pm 4pm 22 January 2pm 3pm 4pm 29 January 2pm 3pm 4pm 05 February 2pm 3pm 4pm 12 February 2pm 3pm 4pm 19 February 2pm 3pm 4pm 26 February 2pm 3pm 4pm 05 March 2pm 3pm 4pm 12 March 2pm 3pm 4pm 19 March 2pm 3pm 4pm Dr. Lake/Prof. Wray Prof. Alan Morgan Prof. Alan Morgan Journal club or SSA Debate 3 (therapeutic use of cannabis) Review/feedback session Dr. Sathish/Prof. Probert Dr. Sarah Lake Prof. Susan Wray Journal club or SSA Lecture Lecture External speakers External speakers External speakers Careers workshop Careers workshop Careers workshop Prof. Edgar/Prof. Varro Dr. Jean Sathish Prof. Chris Probert Journal club or SSA Lecture Medical applications of GCMS Dr. Williams/Dr. Sills Prof. David Edgar Prof. Andrea Varro Journal club or SSA Stem cells and induced pluripotent cells Biomarkers, cancer microenvironment and upper GI carcinogenesis Prof. Alan Morgan Dr. Dominic Williams Dr Graeme Sills Journal club demonstration (all strands) Lecture Lecture Prof. Mike Clague Prof. Bob Burgoyne Prof. Alan Morgan Journal club demonstration (all strands) Journal club demonstration (all strands) Molecular mechanisms of ageing Prof. Alan Morgan Prof. Mike Clague Prof. Bob Burgoyne Introduction to Frontiers lectures and journal clubs Lecture Calcium sensor proteins in the regulation of neuronal function Dr. Hannah McCue Dr. Chris Goldring Prof. Alan Morgan Production and use of recombinant proteins Fine-tuning the expression of proteins in cell and animal models Science skills (publishing in journals) Dr. Lakis Liloglou Prof. John Quinn Prof. Alan Morgan Epigenetic techniques Nurturing Nature: How Polymorphic Variation Helps Shape The Individual Science skills (organisation/record keeping)
Presenter
Session
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Semester 3
Time: 2.00pm 5.00pm Wednesdays Venue: Physiology Seminar Room (plus Physiology Meeting Room for Journal Clubs) Date/time
16 April 2pm 3pm 4pm 23 April 2pm 3pm 4pm 30 April 2pm 3pm 4pm 07 May 2pm 3pm 4pm 14 May 2pm 3pm 4pm 21 May 2pm 3pm 4pm 28 May 2pm 3pm 4pm 04 June 2pm 3pm 4pm 11 June 2pm 3pm 4pm 18 June 2pm 3pm 4pm Prof. Pritchard/Prof. Weeks Prof. Alan Morgan Prof. Alan Morgan Journal club or SSA Debate 5 (topic chosen by students) Review/feedback session Dr. Criddle/Dr. Antoine Prof. Mark Pritchard Prof. Andrew Weeks Journal club or SSA Why do some people develop cancers of the GI tract? Lecture Dr. Slupsky/Dr. Campbell Dr. David Criddle Dr. Dan Antoine Journal club or SSA Mitochondrial dysfunction in acute pancreatitis Lecture Dr. Plagge/Prof. Owen Dr. Joe Slupsky Dr. Barry Campbell Journal club or SSA Pathogenetic role of PKCbetaII in chronic lymphocytic leukaemia Lecture Dr. Jenkins/Prof. Marson Dr. Toni Plagge Prof. Andrew Owen Journal club or SSA Lecture Lecture Dr. Alfirevic/Prof. Boyd Dr. John Jenkins Prof. Tony Marson Journal club or SSA Proteomic Approaches: Colon Cancer Biomarkers Lecture Prof. Urbe/Dr. Green Dr. Ana Alfirevic Prof Mark Boyd Journal club or SSA Pharmacogenetics Lecture Prof. Khoo/Dr. Murray Prof. Sylvie Urbe Dr. Tim Green Journal club or SSA Lecture Obtaining and using structural information Dr. Coulson/Dr. Hapangama Prof. Saye Khoo Dr Patricia Murray Journal club or SSA Lecture Lecture Prof. Alan Morgan Dr. Judy Coulson Dr. Dharani Hapangama Debate 4 (topic chosen by students) Transcription in cancer: regulating the regulators Lecture
Presenter
Session
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Section 4 Assessment
4.1 Overview of Assessment
The accreditation for a Research Masters Degree is regulated by The University of Liverpool Ordinances and Regulations. The award of MRes requires that a minimum of 180 credits be obtained. In order to be eligible for the award of an MRes, candidates must achieve a minimum mark of 50% in each of the 3 modules (Research projects, Techniques and Frontiers in Biomedical Sciences, Transferable Skills). However, where the average of the total marks in all modules is 50% or above, a mark in the range 40-49% may be deemed compensatable. Candidates who fail to satisfy the examiners in a module assessment shall be permitted to re-present the failed work on one further occasion only, at a time specified by the examiners.
4.1.1 Late submission of work Please note that the standard University penalty for late submission of written work applies; 5% of the total marks available for the assessment will be deducted from the final mark for each day after the submission date up to maximum of seven days. Work received after seven days will receive a mark of zero.
4.1.2 Marking of submitted work We aim to assess all written work within three working weeks after submission.
4.1.3 Mitigating Circumstances When awarding degrees, the Board of Examiners will take into consideration any mitigating circumstances that may have adversely affected a candidates performance providing these have been notified in writing to the Programme Director. Where illness is involved a medical certificate should be supplied. Rules and regulations can be downloaded from the University website: http://www.liv.ac.uk/tqsd/pol_strat_cop/cop_assess/appendix_M_cop_assess.pdf Please note that the appropriate application form needs to be filled out to be eligible for consideration. A copy of the mitigating circumstances form can be found at the back of this handbook. Documentation must be supplied within a reasonable timeframe, forms submitted long after the event will not be considered. The form and supporting evidence must be submitted as soon as possible (normally within five days) after the events under consideration occur, and no later than one week before the meeting of the Board of Examiners at which the results of the assessments concerned will be considered. If you are unable to submit the form within the normal five days please contact the MRes Programme Administrator.
4.1.4. External Examiner The External Examiner(s) will oversee course assessment procedures and assess annually the quality and relevance of the subjects taught. The External Examiner(s) will conduct a viva voce examination on the research elements of all candidates. Attendance at the viva is a prerequisite for obtaining the MRes degree.
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4.1.5 Award of Distinction A distinction will be awarded to MRes candidates who obtain an overall mark of 70% or over AND achieve marks of 70% or over in at least 2 out of the 3 assignments in each of the three assessed components of the course. In other words, a distinction will be awarded if the following conditions are met: Average mark of 70% or over for the 3 Research Project reports AND 70% or over in at least 2 out of these 3 assignments Average mark of 70% or over for the 3 Techniques & Frontiers in Biomedical Sciences assignments AND 70% or over in at least 2 out of these 3 assignments Average mark of 70% or over for the 3 Transferable Skills assignments AND 70% or over in at least 2 out of these 3 assignments
For example, a student who obtained the following marks would not be awarded for a distinction despite obtaining 70% overall, because they had two marks of less than 70% in the Techniques/Frontiers component:
Transferable Skills
3 Review 1 Review 2 Referee Portfolio Studentship Business 100% 100% 100% 100% 100% 100% 100% Total Mark % 71 65 66 80 80 72 70 70
In addition, the award of Distinction requires a minimum of 70% recorded attendance at the compulsory sessions in the Techniques/Frontiers and Transferable Skills modules.
4.1.6 Progression to PhD Progression to PhD of MRes graduates is subject to obtaining 65% or above in the Research Projects and is also subject to the discretion of the supervisor.
External examiners perform a significant role at the final Examiners Board meeting. The examiner might comment at this meeting on each students performance and on the course in general. All external examiner write an annual report on the course and on the assessment process.
There will be several Strand external examiners representing research expertise in the main strands of the MRes degree programme. The vivas will take place in September (8-12 September 2014) and attendance at the viva is a prerequisite for obtaining the degree.
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between a student and another person in the preparation and production of work which is presented as the students own. Coercive collusion would be considered a serious breach of academic integrity.
Copying Copying occurs when a student consciously presents as their own work material copied directly from a fellow student or other person without their knowledge. It includes the passing off of anothers intellectual property, not in the public domain, as ones own. It differs from collusion in that the originator of the copied work is not aware of or party to the copying. Copying of work from published sources would be dealt with as plagiarism. Submission of commissioned or procured coursework The dishonest practice occurs when a student presents as their own work coursework assessment tasks (or parts thereof) which have been intentionally procured (by financial or other inducement means) for this purpose. The definition includes the practice of requesting another party to prepare all or part of a course assignment (with or without payment) on the students behalf. Fabrication Throughout this policy the term fabrication is used to cover one or more of the following: Embellishment or Falsification of Data occurs when a proportion of the total data is altered, enhanced or exaggerated in order to emphasise data which has been obtained by legitimate means Fabrication of Data occurs when a student creates and presents an extensive amount or significant piece of data in order to conceal a paucity of legitimate data; or wholly fabricates a set of data in the absence of legitimate data. Plagiarism Plagiarism occurs when a student misrepresents, as his/her own work, the work, written or otherwise, of any other person (including another student) or of any institution. Examples of forms of plagiarism include: the verbatim (word for word) copying of anothers work without appropriate and correctly presented acknowledgement and citation of the source; the close paraphrasing of anothers work by simply changing a few words or altering the order of presentation, without appropriate and correctly presented acknowledgement and citation of the source; failure to reference appropriately or to adequately identify the source of material used; unacknowledged quotation of phrases from anothers work; the deliberate and detailed presentation of anothers concept as ones own.
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Laboratory Books Laboratory books are the property of Liverpool University and are to be handed to the supervisor at the end of the project placement along with a Completion of project work A Completion Form must be filled out and signed by your project supervisor(s) at the conclusion of the research projects, to confirm that all material has been safely accounted for and that any useful data has been passed on to your supervisor.
should be cross-referenced to the relevant figure or table. Is it not necessary to reproduce the same material in tables and figures. This section must not take the form of a diary of your experimental observations in the laboratory, nor need every single experimental observation be recorded. Instead, you must take responsibility for collating the data, and whatever statistical analysis are appropriate, and presenting your findings in a way that makes it possible for the reader to understand your major conclusions. Each figure should have an explanatory legend that enables the reader to understand how the experiment was performed. Figures and tables should be inserted into the main body of the text as close as possible to the relevant section. 5. Discussion. This section should focus on the interpretation of your results, and set them in the context of current knowledge in the field. It should not be necessary to repeat your description of the experimental data, but you will want to summarise your main findings and explain how they are meaningful. 6. References. Again, this should follow the the Biochemical Journal style. It is strongly recommended that you use reference organising software (such as EndNote) to construct your references, which will ensure that you use the correct Biochemical Journal style. References in the text should be cited as a number in the order in which they appear. The reference list should be correspondingly numbered, and references listed in order of their citation in the text. 7. Footnotes. Abbreviations and acronyms used in the text must be defined immediately after the first use of the abbreviation. In addition, a complete list of all abbreviations used should also be cited in a single Footnote section. The abbreviations of some important biochemical compounds, e.g. ATP, NADH, DNA, and amino acids in proteins, need not be defined.
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Mark 1 - Project report, 40% Mark 2 - Continual Assessment, 10% Mark 3 - Oral/poster Presentation, 10% Total = 60%
The average of the marks for the three Research Projects will contribute 60% of the total MRes mark (marks for Project 1 + 2 + 3 divided by 3).
Details of the assessment criteria used and the assessment forms that will be used by markers are given on the on the pages that follow.
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90% - 80%
80% - 70%
Pass Level
54% - 50%
Fail Level
49% - 45%
44% - 40%
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90% - 80%
80% - 70%
Pass Level
69% - 65%
64% - 60%
44% - 40%
39% - 35%
34% - 10% 0%
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Scientific content Excellent understanding of the topic and interpretation of the science. Answers to questions were rational and confident. Good understanding of the topic and interpretation of the science. Answered most questions well. Covered the basic concepts fairly well. Provided some interpretation of the science. Problems with questions. answering some (more complex)
Mark
60 69%
50 59%
Very limited understanding of the topic. Little or no interpretation of the science Had difficultly answering even basic questions. Unsatisfactory
40 49%
< 40%
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Presentation, grammar & readability Presented in a logical and very easy to follow format. Excellent use of language with no or very few grammatical/spelling errors. Excellent quality figures and tables, with clear and informative legends. Presented in a clear, easy to follow style with good use of English. Few grammatical or spelling errors. Figures, tables and associated legends well presented. Some issues with style and format that make it somewhat difficult to follow in parts. Several grammatical and/or spelling errors. Figures, tables and legends reasonable, but clarity and/or content could be improved. Significant issues with style and format. Difficult to read and follow. Numerous problems with grammar and spelling. Figures, tables and/or legends are not well presented, or are absent. Unsatisfactory
Guide >70%
Mark %
60 69%
50 59%
40 49%
< 40%
Scientific content Background: Excellent introduction to topic. Very clear project aims. Results: Data presented extremely clearly and logically. Interpretation faultless. Verbal Presentation: Explained the project impecably. Answers to questions were rational and confident. Background: Good introduction of the topic and project aims. Data: Very clear presentation of data. Few problems of interpretation. Verbal Presentation: Explained the project competantly. Answered most questions well. Background: Introduced basic concepts and aims. Data: Data reasonably well presentated, but gaps and/or errors in interpretation. Verbal Presentation: Explained the project fairly well. Problems with answering more complex questions. Background: Poor description of background and aims. Data: Significant issues with both presentation and analysis of the data. Verbal Presentation: Explanation of project poor; had difficultly answering even basic questions. Unsatisfactory
Mark %
60 69%
50 59%
40 49%
< 40%
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5.6 Front sheet for all Research Project Report submissions (example)
Supervisor: [Insert name] Internal Assessor: [Insert name] Strand: [Insert name of your strand]
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vivo with the PD protein, -synuclein, for preventing neurodegeneration (6). The profound neurodegeneration and lethality in CSP mutant flies and mice can be ameliorated by transgenic overexpression of normal -synuclein (6-8) and exacerbated in -synuclein knock-out mice. Similarly, it was also suggested that CSP binds to mutant huntingtin (Htt), but not to normal Htt. The sequestration and subsequent depletion of CSP by expanded polyQ stretches eliminates the robust inhibition of N-type Ca2+ channels promoted by CSP (9). This would be a prominent mechanism contributing to accelerated neurodegeneration due to lack of CSP availability for neuroprotection. However, the mechanistic insight into why CSP absence results in neurodegeneration and how this may be alleviated by -synuclein remain largely obscure. In theory, one way to characterise the evolutionarily conserved pathway through which CSP functions and gain insight into how CSP may be of physiological or pathological importance for preventing neurodegeneration is to identify genetic modifiers, which are genes that modulate the manifestations of neurodegenerative disease-induced primary mutations (3). Genetic modifiers are classified as suppressors or enhancers by conferring either neuroprotection or enhancement of neurodegeneration. Their isolations and analysis of the underlying pathophysiology could lead to the identification of proteins whose expression has the potential to modulate CSP activity and in particular protect from neurodegeneration in CSPs absence, and elucidate the molecular mechanisms and genetic susceptibility of multiple NDs. The employment of a plethora of powerful model organisms with shorter generation times such as C. elegans, S. cerevisiae and D. melanogaster, has not only expedited screening of potential genetic modifiers of the late-onset cellular and behaviour phenotypes, but also facilitated high-throughput testing of hypotheses to illuminate a prospective cellular cause of protein-misfolding diseases like HD, PD, Amyotrophic Lateral Sclerosis (ALS) and AD or neuroprotective mechanisms against underlying functional aspects of neurodegeneration (1,10).
Screening can be performed by molecular, genetic and chemical manipulations of gene function, i.e. using mutagenesis (deletion libraries, transposon based insertion), transgenic overexpression of exogenous human misfolding disease-related proteins, or RNA interference (RNAi)-mediated knockdown to determine the loss- or gain-of-function phenotypes (11). Previous genome-wide screens have used C. elegans to develop multiple tissue-specific transgenic models manifesting pathological phenotypes that faithfully recapitulate many salient cellular and molecular pathologies of complex neurodegenerative disease processes based on muscular or neuronal expression of aggregation-prone proteins such as mutant tau, superoxide dismutase (SOD1) and -synuclein proteins, polyQ constructs, Htt fragment and toxic amyloid beta 1-42 (A1-42), and identified modifiers and cellular processes of -synuclein inclusion formation (12), -synuclein and Htt misfolding-induced toxicity (13), tau-induced pathology (14), presenilin function (15), polyQ (16) and mutant SOD1 aggregation (17). However, whole genome RNAi screening of genetic modifiers of neurodegeneration or protein aggregation in worms lacks efficiency in identifying positive hits (12,14,16). An alternative way to identify potential modifier genes in a specific pathway is to perform a targeted screen (a candidate approach/reverse genetic, hypothesis-driven approach) based on hypothesis generated by screens in lower model organisms or existing knowledge of disease mechanisms and pathways (10,18). In view of this, we adopted a targeted gene approach to mine and integrate multiple heterogeneous data accumulated in previous RNAi knockdown and mutagenesis studies in model organisms, and selected a small number of worm genetic disease modifiers which fit both adult neuronal expression and non-RNAi lethality criteria. Comparative analysis of diverse genetic modifier screens indicates that the pathological processes underlying multiple neurodegenetive diseases are largely distinct. Upon closer look, common classes of modifier genes from our candidate list that act on pathology of most disease proteins are those involved in genetic regulation of protein
41
homeostasis, stress responsiveness and cellular ageing. Furthermore, we subjected six transgenic lines containing either pan-neuronal or discrete neuronal promoters to confocal microscopy and three-dimensional (3D) reconstructions and verified the accuracy of the reported GFP localisations in targeted neuronal types, as revealed by the location of the fluorescent GFP reporter. EXPERIMENTAL PROCEDURES Data Mining Published literature was manually curated to compile a collection of experimentally delineated genetic modifiers of protein aggregation, misfolding and neurodegeneration in C. elegans, S. cerevisiae and D. melanogaster (Table 1). PDF files containing full lists of modifiers in the online supplemental materials were converted and imported into Microsoft Excel (version 2007; Microsoft, Redmond, WA) by PDF2XL Software (Cogniview). Individual worm orthologues of yeast and fly modifier genes were identified by consulting the Princeton Protein Orthology Database (P-POD) [http://ortholog.princeton.edu/findorthofamily.ht ml], Saccharomyces Genome Database (SGD) [http://www.yeastgenome.org] and FlyBase [http://flybase.org/]. To extract genetic modifiers with known expression in adult neurons that also non-RNAi lethal, the list of modifiers was further refined by conducting search queries in bioinformatic interfaces such as WormBase [WormBase Web site, available at www.wormbase.org, release version WS224, April, 2011], Biomart [http://www.biomart.org/], and most predominantly GExplore 1.1 [http://genome.sfu.ca/gexplore/]. First, a list of all adult neuronal genes within the genome of C. elegans was obtained by searching within GExplore for genes belonging to the expression pattern neuron, nerve, nervous system and life stage adult. The candidate list was then checked using available RNAi phenotypic data provided by GExplore and Wormbase to specifically exclude modifiers for which existing RNAi experimental data indicate to have elicited severe deleterious phenotypes due to the
potential for non-specific effects from knocking down well known important housekeeping genes. All C. elegans genes were filtered for the RNAi phenotype lethal under GExplore Phenotype search field. For data analyses, all annotations returned were imported into Microsoft Excel and were processed manually for subsequent categorisations. Each genetic modifier was then cross-compared with these GExplore data using the Microsoft Excel VLOOKUP function to identify those which fit our exact criteria. As an additional test of the validity of the data, all the non-RNAi lethal genetic modifiers with adult neuronal expression were searched within GExplore Combined Search Interface. Note: Preselected modifier gene sets, data downloaded from GExplore 1.1 and all data analyses performed are not appended. Please contact the author for any additional supporting data. Nematode culture C. elegans provided by the Caenorhabditis Genetics Center (CGC) were grown and maintained on seeded nematode growth medium (NGM) agar plates at 20C. Six transgenic lines used for confocal microscopy were kindly provided by Dr. James R. Johnson: N2;Ex[Prab-3::EGFP], N2;Ex[Pdat-1::GFP] and RM2754;Ex[Pgcy-8::GFP] carry the GFP reporter alone without an experimental modify transgene; PS3551;Ex[Punc-17::hsf-1 + Punc-17::GFP], PS3551;Ex[Pglr-1::hsf-1 + Pglr-1::GFP] and PS3551;Ex[Posm-6::hsf-1 + Posm-6::GFP] carry both the GFP reporter as well as the full length hsf-1 rescuing construct. Fluorescence Confocal Microscopy and 3D Image and Animation Processing Before the procedure, young adult worms, four days posthatch from individual GFP expressing lines were cleansed of adhering bacteria by transferring to an unseeded NGM plate, and were allowed to crawl for 5 minutes. Microscopy of living animals was performed by mounting the worms on cover glass (22X40 mm, VWR International, PA) in a 5l drop of PEG/glycerol. PEG/glycerol mounting medium is a blend of equal percentage (20:20) of PEG 20000 and glycerol in PBS which immobilises the nematode (personal communication). Slides were rapidly sealed with a clean glass coverslip (13 mm, VWR
42
Reference
Nollen (16) Wang (17) van Ham (12) Kraemer (14) Hamamichi (20) Kuwahara (21) Bates (22) Parker (13) Cohen (23) Kraemer (24) Guthrie (25) Yamanaka (26) Faber (27) Willingham (28) Yeger-Lotem (29) Giorgini (30) Cooper (31) Gitler (32) Blard (33) Kaltenbach (34) Branco (35) Shulman (36) Bilen (37) Fernandez-Funez (38) Kazemi-Esfarjani (39) Auluck (40)
Model Organism
C .elegans C .elegans C .elegans C .elegans C .elegans C .elegans C .elegans C .elegans C .elegans C .elegans C .elegans C .elegans C .elegans S. cerevisiae S. cerevisiae S. cerevisiae S. cerevisiae S. cerevisiae D. melanogaster D. melanogaster D. melanogaster D. melanogaster D. melanogaster D. melanogaster D. melanogaster D. melanogaster Total
(12,13,16,17,19-40)
TABLE 1: Candidate modifier genes gleaned from literature. For each published study, the model organism used, the number of experimentally verified genetic modifiers and the number of genetic modifiers found in C. elegans are indicated. We identified 887 genetic modifiers in total, of which 656 C. elegans modifiers were collected. Note these numbers also encompass genes that have appeared multiple times in various screens. Additional orthologues of a modifier gene (*). Number of modifiers if taking into account of the multiple C. elegans orthologues found for that particular gene (**).
International, PA). All fluorescence images were acquired on a Leica TCS-SP-MP AOBS laser scanning confocal microscope (Leica Microsystems, Heidelberg, Germany) using a 20X Plan Apo dry objective for imaging the entire worm, and a LEICA DM IRB E laserscanning confocal microscope (Leica Microsystems AG, Wetzlar, Germany) with a 1.3 numerical aperture 63X Plan Apo oilimmersion objective for high resolution imaging of the head region of worms. Fluorochromes were excited using an argon laser source at 488 nm. For high quality 3D reconstruction, optimised sections of Z stacks were scanned with a line average of 4-8. 3D data stacks of confocal images and animations are shown as maximum-intensity projections generated by Leica Confocal Software, and were exported as TIFF and AVI files respectively.
RESULTS A smaller than expected bioinformatically prioritised target gene set was obtained We first compiled a candidate list of hitherto experimentally verified genetic disease modifiers in C. elegans by assessing available literature. To identify additional conserved modifier genes and to further extend our target gene set, worm orthologues of modifiers that were uncovered via genome-wide screens for effectors of neurodegeneration in S. cerevisiae and in D. melanogaster were also incorporated (Table 1). As a further stricture in the analysis, we selected for modifier genes based on their respective expression pattern and RNAi phenotypes [obtained from (41,42)] to retrieve only those with known expression in adult neurons that are also non-RNAi lethal. We specifically excluded modifiers that have been
43
Total Genetic Modifiers Availability in RNAi library Overlap with Sieburth Screen
RNAi Lethal
Non-RNAi Lethal
618
331
287
580
130
314
266
44
108
33
51
57
TABLE 2: Refinement of candidate modifier genes. The table summarises the findings following selection of the target modifier gene set based on their respective expression pattern and RNAi phenotypes which are available from GExplore and WormBase (WS224). Our stringent selection criteria preferentially include modifier genes with known expression in adult neurons and non-lethal phenotypes arising from existing RNAi experiments. The majority of the RNAi clones for our modifiers are available in both Vidal and Ahringer RNAi feeding libraries. To gain increased confidence in our candidate gene set, we also compared our modifier set to a published list of 2072 C. elegans genes reported by Sieburth et al. which includes genes that influence synaptic function. 108 overlapping genes were identified to be present in both gene lists. Note that the total number of modifier genes in Table 2 is less than the number represented in Table 1, because the complete list of candidate genes collected in Table 1 also include ones that have appeared in multiple screens and were subsequently discounted so that the data displayed in Table 2 contains only the unique worm genetic modifiers.
experimentally proven as essential for worm survival and development due to the potential for non-specific, off-target effects and false positive results (20). As shown in both Table 2 and Figure.1, a total number of 618 unique worm genetic modifiers were collected. Within this dataset, 21% (132/618) are adult neuronal; 46% (287/618) are non-RNAi lethal. Applying both criteria together, only 8% (47/618) of all unique modifier genes we extracted correspond to genes that are currently known to be both adult neuronal and non-RNAi lethal, which are listed in Table 3. 91% of the modifiers within this specific set have clear human orthologues and function in a variety of basal cellular processes. 94% of all modifier genes collected are also represented in the two available RNAi feeding libraries. Given that CSP has presynaptic localisation and defective presynaptic transmission has recently been implicated as an early mechanism preceding neurodegeneration (43), we subsequently compared our candidate modifier set with a set of 2072 candidate genes previously screened for genes involved in the C. elegans neuromuscular junction (44), with the aim of providing confidence in the shortlist of testable targets generated and enriching for modifier
FIGURE 1: Venn Diagram analysis of genetic modifier set. Analysed data in adjacent Table 2 were used to generate the Venn diagram and are represented as colour-coded circles. A total of 618 unique worm modifiers were collected. Of these, 132 (21%) are adult neuronal genes (green). After removing modifiers which existing RNAi experimental data indicate as RNAi lethal positives (red), 287 (46%) non-RNAi lethal modifiers (light blue) were subjected to further analysis and we have an end list of 47 genes (8%) that are both adult neuronal and non-RNAi lethal (yellow intersecting region). Comparison with C. elegans genes reported by Sieburth et al. (44) are also shown.
genes known to influence presynaptic function when targeting the respective genetic modifier in the future by genetic and/or chemical means. Of the 2072 candidate genes that were subjected to
44
Gene name F08H9.4 T09B4.10 F55B12.3 Y61A9LA.8 T05H10.1 T26A5.5 K09A9.6 F47G6.1 T04D1.3 Y22F5A.3 EEED8.9 T25F10.2 M04C9.5 K11E8.1 K07A9.2 B0334.8 W08G11.4 R11A8.4 R13H8.1 H21P03.1 C04G6.3 F02E9.4 F39C12.2 K05B2.3 R05D3.7 F47G4.7 C06E7.1 F44E7.2 Y56A3A.13 C30H7.2 K08F4.7 F35E8.8 C50C3.9 R05G6.7 ZK256.1 T23G5.5 C28C12.7 M88.5 K11H3.1 T05A12.2 W05E10.4 C04B4.2 R74.3 R13A1.8 F52C9.8 F08C6.3 Y55F3AM.14
CGC Name chn-1 sel-10 sut-2 dyb-1 unc-57 ric-4 pink-1 dbl-1 dyf-5 unc-43 cmk-1 age-1 pptr-1 sir-2.1 daf-16 mbf-1 pld-1 sin-3 add-1 ifa-4 unc-116 smd-1 sams-3 nft-1 gst-4 gst-38 unc-36 pmr-1 dat-1 spp-10 gpdh-2 tre-2 tre-3 xbp-1 glb-23 pqe-1 tag-197 -
Description heat shock protein (HSP) of the HSP16 class mammalian carboxyl-terminus of Hsc70 interacting protein F-box and WD-repeat-containing protein of CDC4/CUL1 family of E2-E3 ubiquitin ligases nuclear polyadenylated RNA binding protein putative ubiquitin-specific protease F-box protein JEMMA aspartyl beta-hydroxylase ZZ type zinc nger endophilin A, required for synaptic vesicle endocytosis intracellular protein transport, neurotransmitter secretion BRPK/PTEN-induced protein kinase a member of the transforming growth factor beta (TGFbeta) superfamily a putative MAP kinase type II calcium/calmodulin-dependent protein kinase Ca2+/calmodulin-dependent protein kinase I functions in an insulin-like signalling (ILS) pathway Serine/threonine protein phosphatase 2A NAD-dependent deacetylase sirtuin-1 sole C. elegans forkhead box O (FOXO) homologue transcription factor MBF1 phospholipase D1 SIN3 family of histone deacetylase subunits cytoskeletal protein alpha-adducin a nonessential intermediate filament protein kinesin-1 heavy chain ortholog S-adenosylmethionine decarboxylase S-adenosylmethionine synthetase p-nitrophenyl phosphatase carbon-nitrogen hydrolase human thioredoxin domain-containing protein 4 precursor a putative glutathione-requiring prostaglandin D synthase unclassified alpha2/delta subunit of a voltage-gated calcium channel porin/voltage-dependent anion-selective channel protein a Golgi P-type ATPase Ca2+/Mn2+-pump a plasma membrane dopamine transporter sphingolipid metabolism/lysosomal IGF-II mRNA-binding protein IMP glycerol 3-phosphate dehydrogenases putative trehalases of unknown function putative trehalases of unknown function unclassified unclassified unclassified unclassified unclassified unclassified
RNAi library Vidal Ahringer Vidal Not available Ahringer Vidal Ahringer Vidal Vidal Vidal Ahringer Vidal Ahringer Vidal Vidal Ahringer Not available Ahringer Vidal Vidal Vidal Vidal Vidal Vidal Ahringer Vidal Vidal Vidal Vidal Vidal Vidal Vidal Vidal Vidal Not available Vidal Vidal Vidal Vidal Vidal Vidal Vidal Vidal Vidal Vidal Vidal Vidal
worm, fly STUB1 CHIP worm, yeast yeast fly fly fly worm worm fly worm worm yeast yeast yeast fly fly worm, fly worm yeast fly fly fly fly fly worm yeast yeast yeast worm fly fly fly fly yeast worm worm fly fly yeast yeast worm worm worm worm yeast fly FBXW7 ZC3H14 USP47 FBXL19 ASPHD2 DTNA SH3GL2 SNAP-25 PINK1 BMP4 MAK/ICK CaMKII CaMK1 PI3K PP2A SIRT1 FOXO4 EDF1 PLD1 SIN3 ADD1 IFB-1 KIF5 AMD1 MAT1A PDXP FHIT ERP44 GSTA3 GSTA4 CACNA2D3 VDAC2 ATP2C1 SLC6A2/A3 PSAP IGF2BP2 GPD1L TREH TREH None None None REXO1 VPS52 None
Wang (17), YegerLotem (29) Guthrie (25) Bilen (37) Bilen (37) Kaltenbach (34)
Signal transduction
Hamamichi (20) Wang (17) Yeger-Lotem (29) Willingham (28) Willingham (28) Branco (35) Shulman (36)
Transcription cofactor
van Ham (12), Branco (35) Cohen (23) Giorgini (30) Bilen (37) Branco (35) Kaltenbach (34) Kaltenbach (34) Kaltenbach (34)
ECM/Cytoskeletal organisation
Enzymes
Kraemer (14) Willingham (28) Willingham (28) Willingham (28) Wang (17) Branco (35) Branco (35)
Redox/oxidative stress
Voltage-gated ion channels Transport ATPase Dopamine metabolism Lipid metabolism RNA metabolism Carbohydrate metabolism Trehalose biosynthesis Unclassified
Kaltenbach (34) Kaltenbach (34) Yeger-Lotem (29) Wang (17) van Ham (12) Bilen (37) Kaltenbach (34) Yeger-Lotem (29) Yeger-Lotem (29) Kraemer (14) Kraemer (14) Kraemer (14) Faber (27) Willingham (28) Kaltenbach (34)
TABLE 3: Set of 47 modifier genes with known adult neuronal expression and non-RNAi lethality. Manual analysis of modifier genes compiled identified a list of 47 genes out of the 618 unique genetic modifiers for further dissection. For each genetic screen, assigned functional category, C. elegans gene name, CGC approved name, available description, model organism used and human orthologue are shown. RNAi feeding library coverage of our modifier genes is also indicated. Three modifiers that are common to multile screens are highlighted in red, while the nine modifiers that overlapped with genes reported by Sieburth et al. are highlighted in orange. Four modifier genes are nematode specific and six genes remain uncharacterised. UPS: Ubiquitin-proteasome system, ECM: Extracellular matrix. 45
-synuclein
Sc Dm Ce
Tau
Sc Dm
SOD
Ce
classes of genes that are specifically mentioned in original literature classes of genes mentioned, but found only 1 or 2 genes in that respective class either no genes in that specific class were found or mentioned in the original publication Worm screens that utilised neuronal expression system to drive exogenous neurodegenerative proteins
TABLE 4: Functional profiling of genetic modifiers identified in diverse screens. Comparative analysis of worm modifiers of polyQ, tau, SOD and -synuclein aggregation, yeast modifiers of misfolded -synuclein and Htt toxicity and fly modifiers of misfolded tau and polyQ toxicity from diverse screens reveals that the modifier genes identified in most screens function in a wide variety of biological processes. The molecular mechanisms mediating neuronal toxicity in tauopathies, synucleopathy and polyQ diseases appear to be largely distinct due to the lack of overlap exists between the underlying causative pathways. A larger proportion of the neurodegeneration studies using C. elegans have focused more on overexpression of either wild-type or mutant forms of -synuclein and polyQ constructs in comparison with other aggregation prone disease proteins. The functional class of vesicle trafficking appears specifically enriched in synuclein screens, whereas protein degradation and transcriptional regulation are more enriched in polyQ screens. Screen numbers correspond to the following publications: 1Nollen et al., 2004 (16); 2Faber et al., 2002 (27); 3Bates et al., 2006 (22); 4Willingham et al., 2003 (28); 5Giorgini et al., 2005 (30); 6Branco et al., 2008 (35); 7Bilen et al., 2007 (37); 8Kaltenbach et al., 2007 (34); 9Fernandez-Funez et al., 2000 (38); 10Kazemi-Esfarjani et al., 2000 (39); 11van Ham et al., 2008 (12); 12Kuwahara et al., 2008 (21); 13Hamamichi et al., 2007 (20); 14Willingham et al., 2003 (28); 15Cooper et al., 2006 (31); 16Yeger-Lotem et al., 2009 (29); 17Gitler et al., 2009 (32); 18Auluck et al., 2002 (40); 19Kraemer et al., 2006 (14); 20Kraemer et al., 2007 (24); 21Guthrie et al., 2009 (25); 22Blard et al., 2007 (33); 23Shulman et al., 2003 (36); and24Wang et al., 2009 (17).
Overlapping classes of genes Heat shock transcription factor Molecular chaperones Ubiquitin-proteasome system (UPS) Transcriptional cofactor Kinases and phospatases Nucleoporin Transport ATPase SNARE protein GTPase activator protein Putative cargo transport protein ERV29 Acyl-CoA oxidase Degree of overlap and models used 3 X worm 4 X worm, 11 X fly 1 X worm, 1 X yeast, 5 X fly 3 X worm,11 X fly 2 X worm, 2 X fly 2 X fly 2 X worm, 3 X yeast, 1 X fly 2 X yeast 2 X yeast 1 X worm, 2 X yeast 1 X yeast, 1 X fly Disease proteins tau, SOD and A1-42 -synuclein, polyQ and tau -synuclein, polyQ and tau -synuclein, polyQ and tau tau and SOD polyQ ans tau -synuclein and polyQ -synuclein -synuclein -synuclein -synuclein and polyQ
TABLE 5: Classes of modifier genes common to multiple disease proteins in small model organisms. This table is a complete listing, restricted to our 618 unique modifier genes that have been identified in multiple screens of the associated animal models. The common genes shared among different screens function in a variety of cellular processes, including protein folding (hsf-1, hsp-1, dnj-13), UPS (chn-1, ubc-8) and transcriptional cofactor (sir-2.1) Overlapping genes are categorised according to their roles; the degree of overlap between specific models of neurodegeneration and protein misfolding, the overall times the modifiers appeared in screens of a particular model, 46 and the pathology of disease proteins they act on are also illustrated.
RNAi-mediated knockdown in Sieburth et al. (44), 185 positive genes were identified in their subsequent multi-tiered drug screens for assessing C. elegans exocytosis. In comparing these genes to our list of 618 modifiers, we observed limited overlap, since only 108 genes were common to both candidate gene sets (Table 2, Fig. 1). Among these overlapping modifier genes, only 21 modifiers overlapped with Sieburth et al. positive hits. Molecular mechanisms mediating neurodegenerative processes in tauopathies, synucleopathies and polyQ diseases are largely distinct The majority of the experimentally delineated modifiers identified fall into distinct functional categories. In order to discern whether their cellular mechanisms underlying the pathology are common to several or all disease proteins, or whether some specialise in mediating pathology of particular disease proteins, models from diverse modifier screens expressing different aggregation-prone proteins using either RNAi or biochemical approaches were compared (Table 4). Ideally, we are looking for modifier genes that may have a widespread rather than disease-specific role in neurodegeneration. However, there appears to be little overlap between the modifier genes pulled out from most worm, yeast and fly screens, thereby highlighting that the molecular steps preceding the pathology of various disease protein are superficially very different. Functional classes of modifier genes such as vesicle trafficking are found exclusively for synuclein pathologies in all of yeast as well as C. elegans -synuclein screens, whereas those associated with RNA metabolism, protein folding and degradation, and transcriptional regulation appear to influence the pathology of various disease proteins, but are more specifically enriched in polyQ aggregation and toxicity screens (Table 4), hence accentuating the importance of these evolutionarily conserved molecular processes as integral pathways affected by respective disease proteins. Positive genes that are consistently shared between various datasets are involved in cellular protein quality control and ageing To further ascertain if specific modifiers within a common class of genes act on the pathology of different
or same disease proteins, we focused on the overlapping genes within our candidate modifier gene set and identified 25 modifiers that are consistently shared between multiple screens (Table 5). In support of the above functional class comparison, modifier genes involved in protein folding, degradation and cellular ageing appear to be potent regulators of multiple neurodegenerative disorders. Heat shock transcription factor (hsf-1), a critical evolutionarily conserved principal regulator of chaperone gene expression was found in three C .elegans screens for modifiers of tau, SOD and A1-42, indicating that this may be a modifier common to these misfolding disease proteins. Stress responsive and neuroprotective molecular chaperones, such as hsp-1 and dnj-13 and UPS components i.e. chn-1 and ubc-8, appear to specialise in mediating degradation of synuclein, tau and polyQ disease proteins; while activation and depletion of genetic regulator of lifespan sir-2.1 have been shown to either pharmacologically or genetically ameliorate the polyQ-induced toxicity, and accelerate agedependent aggregation of -synuclein respectively. Selected C. elegans neuronal promoters correctly represent their endogenous gene expression Modifier genes we selected could be examined in a subsequent RNAi screen to identify the positive candidate hits. To further explore possible effects of RNAi silencing and overexpression of positive genes on dnj-14-/-induced age-dependent neurodegeneration, appropriate C. elegans neuronal promoters would need to be selected in order to incorporate targeted transgene expression of a positive gene and gain insights into its role in neuronal subtype survival, vulnerability and function. Direct visual screens for neurodegeneration have become feasible as neuronal loss can be reliably assayed in live worms by monitoring the morphological and degenerative changes of all or a discrete subset of neurons labelled with green fluorescent protein (GFP) reporter (45,46). However, it remains possible that some of the localisation patterns reported do not accurately reflect the localisation of the corresponding endogenously expressed proteins (3,44). Here, we subjected 6 transgenic lines containing either
47
C
*
Prab-3::EGFP
Pdat-1::GFP
D B
Posm-6::GFP Punc-17::GFP
E
FIGURE 2: Localisation of GFP expression in transgenic C. elegans is Pglr-1::GFP confirmed by fluorescent reporter constructs in extrachromosomal arrays. Fluorescence microscopy of whole body (A,B;C-F: left panels) or magnified 3D confocal reconstruction of neuronal panarchitecture in the head region (C-F: right F panels). A, The pan-neuronal localisation of enhanced GFP (EGFP) expression is consistent with the expected rab-3 promoter driven expression pattern in all worm neurons. B, Punc-17::GFP labels most P ::GFP gcy-8 regions of the nervous system, predominantly cholinergic motor neuronal processes. C, The dat-1 promoter labels all eight dopamine (DA) neurons: six anterior neurons in the head and two posterior deirid neurons (PDEs, thick arrow). 3D confocal reconstruction of the magnified head region (right), detailing the six anterior-most DA neurons: four cephalic CEP neurons (asterisks) project dendrites (thin arrows) to the tip of the nose and two anterior deirid ADE neurons (arrow head) extend ciliated processes posteriorly. The CEP dendritic processes of another worm are also visible. D, Posm-6::GFP selectively labels all 60 ciliated sensory neurons. E, The glr-1 promoter drives expression of the GFP in the ventral cord interneurons, motorneurons and nerve ring. F, gcy-8::GFP localises exclusively to one thermo-sensory AFD pair (dashed arrow). 3D confocal reconstruction (right) shows that the AFD dendrites extend anteriorly to the tip of the nose. The sensory endings of both neurons are positioned at the tip of the dendrites (double asterisks). All animals imaged are young adults, four days post-hatch. They include transgenic N2 and RM2754 lines overexpressing GFP reporter alone (A, C and F) or together with HSF-1 in the targeted neurons of PS3551 strain, a hsf-1 mutant (B,D,E). All 3D confocal reconstructions shown are maximal projections of z-stacks. Anterior to the left (C,E,F: right panesl); Anterior to the right (D, right panel). Scale bars: A,B;C-F: left panels, 100 m; C-F: right panels, 10 m. Representative images of at least 5 worms from each transgenic line are shown. 48
neuronal or discrete neuronal promoters to confocal microscopy and 3D reconstructions and verified the accuracy of their GFP localisations. We observed in N2;Ex[Prab-3::EGFP] transgenic line, that the rab-3 reporter for synaptic vesicle does indeed drive the expected pan-neuronal enhanced GFP (EGFP) expression in the nerve ring, axons of ventral and dorsal nerve cords, neuronal cell bodies, dendrites of sensory neurons as well as outgrowth of motor neuron commissures that connect ventral and dorsal cord (Fig. 2A). The unc-17 promoter is active in most regions of the nervous system, including nerve ring, dorsal and ventral nerve cord and pharyngeal nervous system. GFP expression pattern observed correspond exactly to the expected localisation of unc-17 (Fig. 2B). C. elegans haemarphrodites have precisely eight dopaminergic (DA) neurons and their anatomic locations can be readily visualised with an endogenous dopaminespecifc transporter promoter (Pdat-1) which drive transgene expression exclusively in all DA neurons (Fig. 2C). Expression of osm-6 gene is limited to 60 ciliated sensory neurons, where it has a role in transducing chemo- and mechanosensory stimuli. GFP is easily detected in the complex neuronal process bundles that extend to the tip of the nose of the worm (Fig. 2D). glr-1 encodes an AMPA-type ionotropic glutamate receptor subunit. Expression of GFP is limited to four of the five pairs of command interneurons of the motor circuit, as well as several classes of motor neurons, ventral nerve cord and nerve ring (Fig. 2E). gcy-8 encodes a receptor-type guanylyl cyclase and is expressed exclusively to a pair of AFD thermosensory neurons that regulate thermotaxis (Fig. 2F). DISCUSSION The candidate gene strategy described herein is in keeping with the recent preselected screens (20,21) which unlike previous genome-wide screens, pooled genes that were preselected based on their established role in neuronal function or for their predicted role in neurodegeneration (47). We have mined and refined genetic modifiers of three well-studied model organisms to produce a tractable list of 47 C. elegans candidates practical for detailed future investigation by genetic and/or chemical means with low false-negative rate, that could contribute to the identification of proteins whose expression has the potential to modulate CSP activity and in particular protect from neurodegeneration in CSPs absence. For several reasons, we believe that our target gene set is likely to be an underestimate of the true conservation among all disease modifier genes. Firstly, the present approach differs from previous genome-wide screening for genetic modifiers. Although this focused strategy may enhance our ability to identify functionally relevant effectors or enrich for certain groups or categories of genes, the selection of genetic modifiers in our study was based on prior knowledge and will therefore consist of only biased, preselected subsets which are likely to miss many important genes, gene families, or novel pathways, that may be potential effectors of protein misfolding and neuroprotection in C. elegans, which would otherwise be identified by genome-wide screening (12,20). Secondly, before the discovery of neuronal RNAi hypersensitive strains such as eri-1, rrf-3, lin-35, eri-1;lin-15B, and sid-1 transgenic TU3311 strains, RNAi resistance in WT C. elegans postmitotic neurons was a stumbling block for interrogating neuronal gene function with confidence. In order to overcome the potential problems caused by the neuronal intractability of this species, many research groups performed genetic modifier RNAi screens by overexpressing human proteins in non-neuronal cells i.e. body wall muscles and intestine cells in order to rapidly pre-screen potential modifiers (12,15,20,48,49). Muscle cells may be large and easy to score, and certain models may also express certain postsynaptic receptors that receive synaptic signals from neurons, but given that processes such as threshold for pathology relevant to neurons are likely very different to muscle cells, expression in muscle is not a suitable model. This could not only lead to identification of regulators not expressed in neurons (i.e. muscle-specific), but certain neuronal tissue-specific genes that affect neurodegeneration and the actual site where the misfolded proteins in question cause disease, may have also been missed (11,47,50). Thirdly, within WormBase, most of the modifier genes are annotated with more than one RNAi phenotype. Amongst all the RNAi lethal genes we excluded, some are definitive essential genes with 100% lethality reported in all prior studies, whereas others may be false-negative
49
or low-confidence lethal that were automatically eliminated from our set because of a single RNAi experiment. The strong penetrance reported by one RNAi assay may not genuinely reflect the phenotypic outcome and should be interpreted cautiously (51). RNAi in C. elegans is known to yield varied efficacy on all tissues due to inherent variability in the level of target mRNA knock-down for different genes and tissues, resulting in different phenotypes in RNAi screens. As such, some potential hits may have been overlooked because the genes may not be knocked down far enough to result in a phenotype, leading to insufficient or incorrect annotation of RNAi phenotypes (11,12,16,50). With regard to the specificity and confidence of this subset, there is not a strong consensus between the candidates we collected and the Sieburth et al. (44) data, suggesting that our target genes lack enrichment for synaptic function. Most genes pulled-out by Sieburth et al. that are expressed in neurons are also expressed outside the nervous system, which could explain why our modifier might not be enriched among the positives of their screen. In general, a large proportion of the modifiers that have been robustly demonstrated to alter the toxicity and aggregation phenotypes screened in animal models have diverse cellular functions that act either directly (components of protein folding and ubiquitin-proteasome system) or indirectly (signal transduction pathway components, cytoskeletal proteins and transcription factors) in cellular protein quality control and different ageing pathways. Recent genetic screens have further expanded the range of modifiers to include additional classes of modifiers, some are clearly novel and are at present not well explained i.e. RNAi metabolism (12,16,50,52), but possess potentially broad significance to neurodegeneration. It was striking to see that the pathology and functional deficits of each of the NDs can be induced or influenced by alterations of a multitude of disease-protein-specific molecular processes. Components of the vesicular-trafficking machinery specifically modify synuclein toxicity and aggregation, but are rarely found as modifiers of polyQ and tau toxicity and aggregation. This indicates that the -synuclein misfolding and toxicity may be more sensitive to perturbations in this specific biological process than the underlying common denominator of polyQ diseases, the cellular protein quality control system. However, this does not wean the magnitude of protein folding and degradation in pathological processes associated with -synuclein toxicity and accumulation since several of them such as Hsp70 are known to alter -synuclein toxicity in a fly -synuclein overexpression model (40). It is possible that vesicle trafficking may be more important as an early stage effect related to the initiation of -synuclein pathology than the typical protein aggregation modifiers (12). As suggested previously, the technical differences between the screens could also confound making direct comparisons between studies. Considering the many variables in each of the genetic modifier screens conducted, including different model organisms and transgenic constructs, different modes of gene targeting and scoring criteria for different phenotypes, insufficient congruence even between screens that have a similar scope is not entirely surprising. Furthermore, genetic screens to date have only implicated a limited number of tau, SOD and A1-42 toxicity modifier genes, in comparison with those identified for -synuclein and polyQ. Given that components of ubiquitin-degradation systems were found to co-localise with aggregated proteins and various molecular chaperone in the brain of patients (47), and integration of stress response and ageing has recently been demonstrated to facilitate agerelated failures in proteostasis (53), it is not surprising to find that hsf-1, hsp-1 and sir-2.1 play pivotal roles in ameliorating the onset, development and progression of protein misfolding, aggregation and toxicity associated with expression of different disease proteins in models of multiple NDs (53). Despite its apparent neuronal simplicity, C. elegans has different neuronal classes, including motor neurons, sensory neurons, and interneurons (3). Certain neurodegenerative disorders such as HD have been reported to confer differential vulnerability of specific neuronal populations which lead to distinct clinical phenotypes (54). This can therefore be reproduced in C. elegans as neurons manifesting neurodegeneration are characterised by distinctive loss of neuron soma and breakdown of neurites (45). Screens have used panneuronal promoters (1,17,19) as well as neuronal specific promoters (21,55,56) to direct expression of aggregation-prone proteins such as mutant SOD1, tau, -synuclein, polyQ
50
constructs, and Htt fragment in targeted C. elegans neurons to mimic the selective neuronal vulnerability and pathology observed in higher organisms. In particular, dat-1 promoter has been well established as a valid means of probing -synuclein-age-dependent neurodegeneration in multiple studies (45,46,57,58). The six neuronal promoters we selected direct GFP expression exclusively in targeted neurons, and are therefore feasible for future fluorescence imaging of transgenic worms to gain insights into the roles of positive genetic modifiers in neuronal subtype vulnerability and CSP KO-induced neurodegeneration. Taken together, modifier genes identified in disparate models are clearly pertinent to many aspects of neurological phenotype expression, though most have a disease-specific role in certain disease proteins rather than a general role affecting every disease protein. The small number of candidates isolated in this study which modulate phenotypic consequences, directly or indirectly, provides a basis for more detailed molecular analyses. A hypothesisdriven RNAi screen would be performed and to maximise neuronal RNAi efficacy, we would create a dnj-14; rrf-3 double mutant by crossing, with the latter strain being recently reported as a mutant most sensitised to neuronal RNAi (59). This would reveal conserved genes and pathways that are important for CSP loss of function defects or alternatively independent factors that regulate neurodegeneration. With either outcome, they are vital for future development of neuroprotective therapeutic strategies. Acknowledgments....
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(c) A final concluding statement may take the form of an overview, summary or outline of prospects for future work (or all three). Short reviews should be 2,500 250 words. This word limit covers all text sections of the report, including legends to figures/tables, except for the References section (i.e., the list of citations at the end does not count toward the 2,500 words). Marks will be deducted proportionally for not adhering this limit (for example, 3000 words = 20% over the limit = 20% deducted; 2000 words = 20% below the limit = 20% deducted;), down to a minimum cap set at 50% for the assignment. Short reviews should include up to 30 citations to peer-reviewed papers. It will often be the case that many more papers have been consulted; the process of selecting the most appropriate literature citations is therefore a matter of judgement and this will be reflected in the quality of the final product. Submitting your Short Reviews: Three copies of the short review should be submitted in printed form (i.e. produced on a word-processor) - Submit one copy to the lecturer giving the topic - Submit two copies to the ITM Postgraduate Student Team office, Room LG43, Sherrington Building, by 10.00am on the submission date. - Submit an electronic copy of the final version (including any Figures) via Turnitin. Please attach a front cover sheet to your report stating your name, the title of your review, your student number, the name of the lecturer giving the topic, the title of the report, and the word count. A template cover sheet is supplied at the end of this Section for this purpose. Short review 1 Choose a technique that was discussed in the lecture course Techniques in Biomedical Sciences in semester 1. This should not be connected to the main methods used in your research projects, and should not be a lecture given by your strand convenor. It is essential that you contact the lecturer of your chosen Techniques lecture well in advance of the deadline to check that they are available to assess your review and to get advice on the content of the planned short review. Once the chosen lecturer has agreed to mark your short review, you should immediately contact your strand convenor to inform them of the arrangement. Prepare a short review style article discussing the principles on which the technique is based, any practical problems in applying the technique, and the importance of the technique in modern Biomedical Sciences. Short review 2 Choose a topic covered in Frontiers in Biomedical Sciences in semesters 2 and 3. This should not be connected to the main methods used in your research projects, and should not be a lecture given by your strand convenor. It is essential that you contact the lecturer of your chosen Techniques lecture well in advance of the deadline to check that they are available to assess your review and to get advice on the content of the planned short review. Once the chosen lecturer has agreed to mark your short review, you should immediately contact your strand convenor to inform them of the arrangement. Prepare a short review style article discussing the importance of the subject, its topicality, and why or how recent progress has been made. Journal Club The idea behind a journal club is to provide insights into how to critically analyse papers and allow an appreciation of recent advances. Our journal club will be organised as follows: one strand will be allocated to each paper. The paper will be chosen by the presenter of the preceding lecture. An electronic copy will be made available (or a reference to the paper given) at the lecture. The paper will be chosen to illustrate some important technical aspects or novel biological findings, or to highlight problems with the data/interpretations in the paper. Excessively long papers should preferably be avoided. All participating students will be expected to read the paper and prepare a brief summary, as a series of bullet points, of the major issues. These will be helpful for the discussions of the paper and should be emailed to the chairperson in advance of each journal club. 55
Referees report A paper that was discussed at a Journal Club should provide the basis for this submission. Your strand convenor will inform you which journal club is to be chosen for this nearer the time. After the Journal Club, you should produce a written account in the style of an extended referees report. Guidance on how to construct referees reports will be given by the Programme Director in the Science Skills session on Scientific Publishing and also in the introductory lecture to the Frontiers module, and these can be used to provide the basis for the written account. Your referees report should be 1000 100 words and the word count should be indicated. Marks will be deducted proportionally for not adhering to this limit (for example, 1200 words = 20% over the limit = 20% deducted; 800 words = 20% below the limit = 20% deducted), down to a minimum cap set at 50% for the assignment. Submitting your Referees Report: Three copies of the referees report should be submitted in printed form (i.e. produced on a word-processor) - Submit one copy to the lecturer giving the journal club - Submit two copies to the ITM Postgraduate Student Team office, Room LG43, Sherrington Building, by 10.00am on the submission date. - Submit an electronic copy of the final version (including any Figures) via Turnitin. A copy of the refereed paper should be attached to each copy of the referees report submitted. Please attach a front cover sheet to your report stating your name, your student number, the name of the lecturer giving the topic, the title of the report, and the word count. A template cover sheet is supplied at the end of this Section for this purpose.
6.2 Assessment
Each short review will contribute 8%, and the referees report 4% of the total marks. Therefore, the Techniques and Frontiers module contributes 20% of the total marks available for the MRes. Both short reviews and referees reports will be double marked by the person who gave the lecture or journal club and a 2nd internal assessor (usually the strand convenor). The mark awarded will be the average of these two marks. Please note that the standard university penalty for late submission of written work applies (see earlier, under overview of assessment). Details of the assessment criteria used and the assessment forms that will be used by markers are given on the on the pages that follow, as are examples of a short review and a referees report.
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90% - 80%
80% - 70%
Pass Level
59% - 55%
54% - 50%
Fail Level
49% - 45%
44% - 40%
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6.2.3
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Distinction Level
100% - 90%
90% - 80%
80% - 70%
Pass Level
59% - 55%
54% - 50%
Fail Level
49% - 45%
44% - 40%
Outstanding. No better result conceivable at Masters level. Extensive evidence of critical thinking and cogent scientific argument. Evidence of reading around the subsect of the paper reviewed Excellent. Essentially correct and complete, with evidence of critical thinking and excellent use of the paper reviewed. Logical structure, well written and presented, displaying a cogent scientific argument. Very Good. Content essentially without any major flaws, very well explained with clear evidence of a high level of scientific competence, and mature, critical scientific judgement. Good. Well explained, showing good evidence of critical scientific judgement. Quite Good. A well explained review, with good understanding and some evidence of critical scientific judgement. Fairly Good. A generally sound review with a good or quite good level of understanding, evidence of sound scientific competence and judgement. Adequate. Showing some progress but with some deficiencies in one or more aspects of understanding of the paper and scientific judgement. A poor report with an overall superficial approach. Essentially an incomplete report with major omissions in several areas and evidence of a poor understanding. A poor report with an overall superficial approach and more errors, omissions and evidence of a deficiency of effort and poor understanding. A marked deficiency in content of understanding the paper and the task set. Even more marked deficiencies in content of understanding and application. A complete absence of relevant work presented.
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6.3 Front sheet for referees report and short review submissions (example)
Lecturer (who gave the Techniques/Frontiers lecture): [Insert name] Strand: [Insert name of your strand]
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Galectin-3 is a member of a family of animal lectins that bind -galactosides. It is involved in a vast range of biological processes and its expression has been related to cancer progression and metastasis. Galectin-3 exerts different functions depending on its cellular localisation and binding partners. It has been implicated in regulation of cell growth, proliferation, cellular transformation, immune reactions and spreading of cancer metastases, to name a few. This review summarises the diversity of its functions with particular emphasis on its role in cancer pathogenesis and metastasis. INTRODUCTION Lectins are carbohydrate-binding proteins that serve as a link between glycan recognition and cellular responses (56). One of lectin families comprises galectins multifunctional animal proteins that share highly conserved carbohydrate recognition domains (CRDs), which bind to -galactoside (6). To date, 15 members of this family have been isolated and classified into subgroups depending on their structure and the number of CRDs (63). Prototypical galectins (galectin-1, -2, -5, -7, -10, -11, -13, -14 and -15) contain one CRD, whereas the tandem-repeat galectins (galectin-4, -6, -8, -9 and 12) have two CRDs separated by a linker region in a single polypetide chain. Galectin-3, in turn, is the exclusive member of the chimera-type subgroup and it contains a CRD connected to an extended non-lectin N-terminal domain. Galectin-3 is probably the best-studied animal lectin and since its discovery in 1982 (22, 14) the diversity of its functions in physiological and pathological processes seems to expand continuously. It is involved in growth and apoptosis regulation, proliferation, cell-cell adhesion, angiogenesis and regulation of immune response, just to mention a few. Similarly to some other galectins, galectin-3 is ubiquitously expressed in mammalian tissues and is often deregulated in human cancers (63). The plethora of tumours in which it is overexpressed, makes it a promising therapeutic target. Moreover, galectin-3 is now often used as a prognostic marker for certain cancers. Thorough understanding of the cellular mechanisms that involve galectins-3 is pivotal to delineate the complexity of transformation and tumour metastasis, as well as the many physiological processes, in which this particular lectin is implicated. This review will present a comprehensive summary of the multitude of tasks performed by galectin-3 and will focus on its role in cancer development and progression. GENERAL CHARACTERISTICS Structural properties Galectin-3 was first characterised as a 32kDa antigen called Mac-2 expressed on the surface of murine macrophages (22). It was also previously known as CBP-35, HL-29, RL-29, IgEBP, hL-31 and LBP, as a result of its isolation from different species (14). Its structure remains highly conserved across animal kingdom and is unique in the whole galectin family (13). It comprises a CRD and an unusual flexible N-terminal domain (ND) (Fig.1) of about 110-130 amino acids, depending on species of origin. The ND consists of 7-14 repeats of a 62
FIG. 1. A schematic representation of monomeric (left) and pentameric (right) structures of galectin3. The carbohydrate-binding domain consists of about 130 amino acid residues, the proline-, glycine- and tyrosine-rich repeats of N-terminal domain contain about 100 residues and the small Nterminal domain of galectin-3 contains about 30 residues. Adapted from (6). nine amino acid sequence: Pro-Gly-Ala-Tyr-Pro-Gly and 3 extra amino acids (8, 14). It is also referred to (as) a collagen-like ND, due to its homology to collagen 1 (II) chain (49). The ND is essential for multimerisation of galectin-3 and is sensitive to proteolysis by MMP-2 and MMP-9 matrix metalloproteinases (37, 45). Moreover, it is indispensable for the activity of galectin-3 (54) and it has been demonstrated that it takes part in carbohydrate-binding via its Tyr102 residue (5). The first 12 amino acids of the ND, referred to as small ND, are necessary for galectin-3 secretion as well as its nuclear localization (20, 40) and the highly conserved Ser6 is responsible for the anti-apoptotic activity (65), which will be discussed later. The C-terminal CRD of galectin-3 contains approximately 130 amino acids that form a globular structure comprising five- and six-stranded -sheets arranged in a -sandwich (54), similar to galectin-1 and -2. The CRD constitutes the galectin activity, as it creates the entire carbohydrate binding site (25, 46, 54). It contains a NWGR (Asp-Trp-Gly-Arg) motif, which is essential for binding galactosides (2, 13) as well as for homodimerisation (61). This motif is also present in the B-cell lymphoma 2 (Bcl2) family members of apoptosis regulators and it has been confirmed to be responsible for the anti-apoptotic activity of galectin-3 (62). Both lactose (Lac) and N-acetyllactosamine (LacNAc) are specifically recognised by the CRD of galectin-3, which has a 5-times higher binding affinity for the latter (3, 53, 54). What is more, the ND of galectin-3 seems to contribute to its increased affinity for larger oligosaccharides (extended Lac or LacNAc), as compared to other galectins (21). It has been demonstrated that binding of carbohydrate ligands results in a conformational change of galectin-3 (3) and it can be modulated by phosphorylation of Ser6 (39). Furthermore, the multiple interacting partners of galectin-3 also encompass many intracellular proteins that are recognised in a carbohydrate-independent manner (15, 41, 48, 57, 58). Multimerisation and cellular localisation As mentioned before, oligomerisation of galectin-3 is accredited to both the CRD and the ND (37, 61), but the formation of pentamers (Fig.1) occurs only via the NDs in the presence of multivalent carbohydrate ligands (1). In solution, however, galectin-3 has been shown to primarily occur as a monomer (42). Extracellularly, galectin-3 can be found in the extracellular matrix (ECM) and in biological fluids (14) and its multimers form lattice-like structures by cross-linking glycoconjugates on the cell surface (1, 9). This brings about cell-cell associations or bridging of cells to extracellular matrix proteins and, hence, results in triggering of downstream signalling (35, 44, 63), as discussed later. Galectin-3 does not contain endoplasmic reticulum (ER) translocation signal sequence, and, 63
therefore, it is not secreted via the classical ER/Golgi pathway, but by the not fully understood process of ectocytosis, which is dependent on the ND (27, 40). Intracellularly, galectin-3 is present in both the cytoplasm and nucleus, depending on the cell proliferation status and a range of factors, such as cell type and the stage of tumour progression (14). For some cancer types, there is a general shift from nucleus to the cytoplasm, where galectin-3 can interact with a multitude of its binding partners (4, 52). The translocation from the nucleus involves the nuclear export signal (NES) contained within the CRD (60), whereas the nuclear import might be attributed to the ND (20), but there is some evidence for the implication of the CRD in this process (19). Physiological functions The different cellular localisations of galectin-3 imply its vast range of functions in normal and pathological conditions. Extracellular galectin-3 has been shown to be involved in cell adhesion via glycan-dependent interactions with integrins: 11 and subunit of M1 (14). Besides, it mediates cell adhesion by associating with glycosylated cell surface and ECM proteins, such as collagen IV, elastin, fibronectin and laminin. In this way, extracellular galectin-3 plays important role in autocrine and paracrine signalling. Galectin-3 has a particular function in immune response. For example, it stimulates production of superoxide and induces production of interleukin-1 by monocytes, resulting in increased inflammation (29, 34). Among its other effects, galectin-3 enhances phagocytosis of neutrophils (16) and oxidative response of immune cells (17). Besides, extracellular activity of galectin-3 is involved in morphogenesis of endothelial cells and angiogenesis (43). At high concentration, its function is also associated with chemoattraction and an increased Ca2+ influx in monocytes, whereas at low concentration galectin-3 promotes chemokinesis (14). It also participates in endocytosis of modified low density lipoproteins and advanced glycation end products. Functions of galectin-3 inside the cell are better described than for other galectins (63). In the cytoplasm, galectin-3 binds to Bcl-2 and inhibits cell apoptosis (62). What is more, it has also been demonstrated that it interacts with a death receptor CD95 (APO-1/Fas), as well as with nucling and Alix/AIP1 proteins involved in apoptosis regulation (14). Moreover, cytosolic galectin-3 exhibits important effects on cell survival, differentiation and proliferation. To substantiate these multiple functions, it has been shown that galectin-3 interacts with activated GTP-bound K-Ras (15, 55) and it affects Akt signaling (33, 47). On the other hand, nuclear galectin-3, like galectin-1, regulates splicing of pre-mRNA and takes part in spliceosome assembly (14, 63). Its interaction with pre-mRNA may occur indirectly via complexes including its nuclear binding partner Gemin4. In nucleus, galectin-3 has also been shown to regulate gene transcription by enhancing transcription factor association with Spi1 and CRE elements in gene promoter sequences, e.g. to induce cyclin D1 expression (14). Also, intranuclear galectin-3 seems to regulate Wnt pathway signalling. Certainly, this myriad of functions exhibited by galectin-3 is a result of juxtaposition of its unique characteristics: the atypical ND, the diverse cellular localisations and wide tissue distribution (14, 21). Galectin-3 has been found widely expressed in human tissues, including immune cells, epithelial cells of many organs as well as respiratory and gastrointestinal tracts and in some sensory neurons (14, 26). However, during early stages of human embryogenesis, galectin-3 shows a more specific expression pattern, mainly in the epithelia, kidney, chondrocytes and the liver (14). Yet, galectin-3 knock-out mice remain viable and display no obvious abnormalities (13), which indicates that galectin-3 functions may be redundant. Conversely, galectin-3 overexpression is a predominant feature of many cancers and, therefore, the insights into its functions in physiological processes are of great importance in elucidating its role in these pathological states (14, 63). ROLE IN TUMOUR DEVELOPMENT Galectin-3 overepression has been shown to be associated with tumourigenesis and increased metastatic potential of transformed cells (14, 63). Its role in cancer pathogenesis encompasses 64
regulation of a diverse range of cancer-related processes: transformation, cell survival, proliferation, inhibition of apoptosis and spreading of metastasis (Fig.2, Fig.3). In majority of tumours galectin-3 expression is enhanced and its cellular distribution is altered (14). Its intracellular localisation is mainly cytoplasmic and is related to its anti-apoptotic activity (62). In turn, extracellular galectin-3 is associated with dissemination of metastatic cells (63). The following sections will compile the various functions of galectin-3 in different stages and aspects of cancer development and metastasis, with particular emphasis on its binding partners.
FIG. 2. A simplified representation of the many roles of galectin-3 in cancer pathogenesis for further details refer to text.
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FIG. 3. The diversity of galectin-3 functions in cancer progression and metastasis only few main processes in which galectin-3 is involved could be depicted this figure. Galectin-3 has a crucial role in cellular transformation, inhibition of apoptosis and possibly in cell cycle regulation. During metastasis, galectin-3 is engaged in several distinct processes, which include cell-cell and cell-matrix adhesions and angiogenesis. Adapted from (35). Initiation of transformation Substantial evidence is accumulating regarding the role of galectin-3 in neoplastic transformation (15, 23, 35, 66). In highly malignant human breast carcinoma cells, suppression of galectin-3 resulted 66
in reversion of the altered cell morphology as well as in loss of anchorage- and serum-independent growth (23). Moreover, it led to inhibition of tumour growth in immunologically suppressed mice. Likewise, inhibition of galectin-3 by antisense cDNA in human thyroid papillary carcinoma cell line led to suppression of anchorage-independent growth (66). What is more, transfection of galectin-3 cDNA into normal thyroid follicular cells resulted in altered gene expression, loss of contact inhibition and serum-independent growth (59). These results suggest that the maintenance of the transformed phenotype in malignant breast and thyroid cells is dependent on high levels of galectin3 expression. The molecular mechanisms behind galectin-3 function in transformation initiation may involve interactions with oncogenic K-Ras4B (15). Galectin-3 selectively promotes Ras-dependent phosphatidylinositol 3-kinase (PI3-K) and Raf-1 activation and attenuates ERK that results in altered transcriptional outcomes. Hence, galectin-3 might be necessary for Ras anchorage to the plasma membrane and for Ras-dependent cell transformation. The tumourigenic potential of galectin-3 may also involve interactions with -catenin that subsequently result in enhanced expression of cyclin D and c-MYC (58, 63). Nuclear localisation of galectin-3 may also imply its role in regulation of gene transcription and, indeed, it has been shown to interact with transcription factors involved in activation of cyclin D1 expression and possibly expression of cyclin A, E, p27/KIP1 and p21/CIP1 (14). In this way galectin-3 might be involved in regulation of the cell cycle. Also, it has been demonstrated that galectin-3 bind to the thyroidspecific transcriptiona factor 1 (TTF-1) in human papillary thyroid cancer cells, and, hence contributes to cell proliferation (48). Inhibition of apoptosis Regulation of apoptosis is probably the best studied function of galectin-3 related to cancer progression (35). Galectin-3 bears high sequence homology to anti-apoptotic Bcl-2 (2, 62), to which it binds in vitro. Point mutations within the homologous NWGR motif lead to the attenuation of the anti-apoptotic activity of galectin-3. Following apoptotic stimuli, overexpressed galectin-3 has been shown to translocate to the mitochondria, where it protects the cell from the production of reactive oxygen species, blocks the alteration of mitochondrial membrane potential and inhibits cytochrome c release (38, 67). Interestingly, cytoplasmic galectin-3 interactcs with synexin in human breast epithelial cells (67). This calcium-dependent and phospholipid-binding protein is responsible for the translocation of galectin-3 to the mitochondria and is essential for its anti-apoptotic activity. Furthermore, phosphorylation of galectin-3 at serine6 by casein kinase I is indispensable for its export from the nucleus and activation of the mitogen activated protein kinase (MAPK) pathway, and, hence, it is essential for anti-apoptotic activity of galectin-3 (35). Besides, galectin-3 is capable of preventing anoikis a type of apoptosis induced by loss of cell-ECM interactions of human breast carcinoma cells, possibly due to mediating cell cycle arrest (31). On the other hand, inhibition of apoptosis in human bladder carcinoma cells by galectin-3 seems to be mediated via Akt signalling (47). Conversely, in human prostate cancer cell line galectin-3 plays a dual role in apoptosis regulation (11). In the cytosol it exhibits anti-apoptotic activity, whereas in the nucleus it promotes apoptosis. Moreover, extracellular galectin-3 also exerts a pro-apoptotic effect, presumably by binding to cell surface glycoproteins (35). Taken together, the effects of galectin-3 on the regulation of apoptosis are complex and depend on its cellular localisation. In oncogenesis, however, its role is predominatly anti-apoptotic and it might be regulated by upstream p53 pathway (63). ROLE IN METASTASIS Neoplastic metastases are the result of increased tumour cell motility, alteration of cell adhesion, angiogenesis and immune response evasion (35). There is substantial evidence that galectin-3 is implicated in all of these pathological processes; thus, understanding of its function is imperative in elucidating the mechanisms of tumour invasiveness and dissemination through biological fluids. Yet, overexpression of galectin-3 in some cancer types appears to be a potential diagnostic marker for their malignancy. For instance, high levels of galectin-3 expression could be predictive for high 67
invasiveness of a neuroendocrine tumour pheochromocytoma (51), epithelial ovarian carcinoma (31), primary cutaneous melanoma (10), thyroid cancer (12), colorectactal cancer (7) and pituitary tumours (50), to name a few. Henceforth, the importance of galectin-3 in cancer prognosis seems to be invaluable and its role in different aspects of metastasis will be discussed below. Angiogenesis Formation of new blood vessels from pre-existing vessels angiogenesis is considered to be a crucial process in tumour invasiveness (35). New blood vessels supply oxygen and the essential nutrients to tumour cells and serve as a link between primary and secondary tumour sites. The role of galectin-3 in angiogenesis has been extensively documented and it appears that it might be connected to induction of endothelial cells morphogenesis and their increased motility (43). In fact, in vivo mice studies revealed that exogenous galectin-3 induces formation of new capillaries surrounding the transplanted human breast tumour (43). The mechanism by which galectin-3 exhibits this angiogenic activity may involve formation of complexes containing 31 integrin and a transmembrane proteoglycan NG2 (18). Also, it may be a result of galectin-3dependent clustering of v integrin and activation of focal adhesion kinase that leads to modulation of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF)mediated angiogenesis (36). Finally, galectin-3 could mediated vascularisation via binding to aminopeptidase N/CD13 - the endothelial cell surface enzyme involved in early steps of angiogenesis (64). Cell adhesion Loss of cell-cell and cell-matrix adhesion is linked to initiation of tumour metastases (35). As a result, cancer cells detach from the primary tumour site and start migration to secondary sites. Indeed, galectin-3, as well as galectins -1 and -8, inhibits cancer cell-ECM proteins adhesion possibly via disruption of normal interactions that consequently leads to escape of cancer cells from their primary tumour site (44). On the contrary, galectin-3 can promote cell adhesion thanks to its multivalent properties (35). In this way, galectin-3 could augment homotypic adhesion of tumour cell and could enhance tumour-endothelial cells adhesion at secondary tumour sites, therefore, promoting metastasis. For example, highly invasive human breast carcinoma cells show enhanced galectin-3-dependent adhesion to endothelial cells (30). This process may involve galectin-3 binding to Mgat5-modified Nglycans on the tumour cell surface and triggering 51 integrin translocation to fibrillar adhesions (32). Some potential galectin-3 cell adhesion ligands in human colon carcinoma include laminin and lysosome-associated membrane glycoproteins. Also, cross-linking and preventing of endocytosis of epidermal growth factor receptor (EGFR) or transforming growth factor- receptor (TGFR) by galectin-3 cell-surface lattices may affect cell growth and receptor density and, hence, could result in increased invasiveness of tumour cells (7). Tumour cell spreading Circulating galectin-3 has been detected at a 5-fold higher concentration in the bloodstream of patients with colorectal cancer as compared to healthy individuals (28). Increased levels of galectin-3 in blood circulation could be prognostic for metastatic potential of colorectal tumours (7). In the bloodstream, galectin-3 has been shown to bind to the oncofoetal Thomsen-Friedenreich (galactose1,3N-acetylgalactosamine, TF) antigen present on MUC1 a glycosylated transmembrane mucin highly expressed in many cancers (68). This interaction triggers polarisation and redistribution of MUC1 on the cell surface and subsequent exposure of smaller cell adhesion molecules. As a result, there is increased cancer cell adhesion to vascular endothelial cells and increased homotypic interactions between transformed cells. The latter effect leads to formation of cancer cell aggregates (emboli) that survive longer in blood circulation and, hence, allow tumour cell spreading to sites distant from the primary tumour. Furthermore, overexpressed galectin-3 in human breast carcinoma cells contributes to the remodelling of the cytoskeletal microfilaments, which are involved in cell spreading (35). Finally, 68
because galectin-3 binds to and stimulates expression of integrins, which take part in cell migration, it may also regulate tumour invasion by activating integrins. Importantly, galectin-3 can suppress immune response and consequently may aid tumour cells in evasion of the immune surveillance (35). Unfortunately, due to space restrictions, it is beyond the scope of this review to describe the complexity of immune reactions regulated by galectin-3. In short, galectin-3 is responsible for T-cell apoptosis and cross-linking of T-cell receptors (TCRs) that results in TCR-dependent signal inhibition. Also, it is capable of reducing IL5 production and inhibiting B lymphocyte differentiation (35). CONCLUSION AND PERSPECTIVES Galectin-3 is a multi-talented lectin that mediates a myriad of physiological processes. Its implication in cancer pathogenesis is obvious and is associated with a multitude of protein-protein and proteinglycan interactions. Galectin-3 is a promising therapeutic target, as its inhibition by the CRD-specific ligands has been shown to be effective in reducing lung and liver metastases in melanoma and sarcoma mouse models, respectively (7). However, future studies are necessary for developing clinically significant therapeutics. Clearly, galectin-3 is a potential diagnostic and prognostic biomarker in several cancers. However, care should be taken in interpretation of the results, as they may not always reflect the correct outcome of tumour progression/diagnosis (14). Moreover, many of the aforementioned interactions of galectin-3 with its binding partners/ligands have been investigated only in vitro and there is no data regarding binding affinities of its intracellular ligands (7); therefore, it is important not to over-interpret galectin-3 function at physiological levels. Much remains to be learned about the role of galectin-3 in cancer progression and metastasis, as well as in non-pathological processes. The mechanistic detail of its action at different cellular locations might be crucial in understanding tumourigenesis and developing better anticancer therapies. Additionally, galectin-3 has been shown to be involved in other disease conditions, such as diabetes, atherosclerosis and inflammation (63), and, hence, it may also be a promising therapeutic target for these pathological states. REFERENCES 1. Ahmad N, Gabius HJ, Andre S, Kaltner H, Sabesan S, Roy R, Liu B, Macaluso F, Brewer CF. Galectin-3 precipitates as a pentamer with synthetic multivalent carbohydrates and forms heterogeneous cross-linked complexes. J. Biol. Chem. 279: 1084110847, 2004. 2. Akahani S, Nangia-Makker P, Inohara H, Kim HR & Raz A. Galectin-3: a novel antiapoptotic molecule with a functional BH1 (NWGR) domain of Bcl-2 family. Cancer Res. 57: 52725276, 1997. 3. Agrwal N, Sun Q, Wang SY, Wang JL. Carbohydrate-binding protein 35. I. Properties of the recombinant polypeptide and the individuality of the domains. J. Biol. Chem. 268: 14932 14939, 1993. 4. Andre S, Kojima S, Yamazaki N, Fink C, Kaltner H, Kayser K, Gabius HJ. Galectin-1 and -3 and their ligands in tumour biology. Non-uniform properties in cell surface presentation and modulation of adhesion to matrix glycoproteins for various tumor cell lines, in biodistribution of free and liposome-bound galectins and in their expression by breast and colorectal carcinomas with/without metastatic propensity. J. Cencer Res. Clin Oncol 125: 461-474, 1999. 5. Barboni EA, Bawumia S, Henrick K, Hughes RC. Molecular modeling and mutagenesis studies of the N-terminal domains of galectin-3: evidence for participation with the Cterminal carbohydrate recognition domain in oligosaccharide binding. Glycobiology 10: 12011208, 2000. 6. Barondes SH, Castronovo V, Cooper DN, Cummings RD, Drickamer K, Feizi T, Gitt MA, Hirabayashi J, Hughes C, Kasai K, Leffler H, Liu F-T, Lotan R, Mercurio AM, Monsigny M, 69
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Pillai S, Poirer F, Raz A, Rigby PWJ, Rini JM, Wang JL. Galectins: a family of animal betagalactoside-binding lectins. Cell 76: 597598, 1994. Barrow H, Rhodes JM, Yu L-G. The role of galectins in colorectal cancer progression. Int J Cancer 129: 1-8, 2011. Birdsall B, Feeney J, Burdett IDJ, Bawumia S, Barboni EAM and Hughes RC. NMR solution studies of hamster galectin-3 and electron microscopic visualization of surface-adsorbed complexes: evidence for interactions between the N- and C-terminal domains. Biochemistry 40: 48594866, 2001. Brewer CF, Miceli MC & Baum LG. Clusters, bundles, arrays and lattices: novel mechanisms for lectin-saccharide-mediated cellular interactions. Curr. Opin. Struct. Biol. 12: 616623, 2002. Buljan M, Situm M, Tomas D, Miloevi M, Krulin B. Prognostic value of galectin-3 in primary cutaneous melanoma. J Eur Acad Dermatol Venereol. 2010 (ahead of print) Califice S, Castronovo V, Bracke M & van Den Brule F. Dual activities of galectin-3 in human prostate cancer: tumor suppression of nuclear galectin-3 vs tumor promotion of cytoplasmic galectin-3. Oncogene 23: 75277536, 2004. Cheng S, Serra S, Mercado M, Ezzat S, Asa SL. A high-throughput proteomic approach provides distinct signatures for thyroid cancer behavior. Clin Cancer Res. 17(8): 2385-2394, 2011. Colnot C, Fowlis D, Ripoche MA, Bouchaert I, and Poirier F. Embryonic implantation in galectin 1/galectin 3 double mutant mice. Dev. Dyn. 211: 306313, 1998. Dumic J, Dabelic S, Flgel M. Galectin-3: an open-ended story. Biochim Biophys Acta. 1760(4): 616-635, 2006. Elad-Sfadia G, Haklai R, Ballan E. & Kloog Y. Galectin-3 augments K-Ras activation and triggers a Ras signal that attenuates ERK but not phosphoinositide 3-kinase activity. J. Biol. Chem. 279: 3492234930, 2004. Fernandez GC, Ilarregui JM, Rubel CJ, Toscano MA, Gomez SA, Beigier Bompadre M, Isturiz MA, Rabinovich GA, Palermo MS. Galectin-3 and soluble fibrinogen act in concert to modulate neutrophil activation and survival: involvement of alternative MAPK pathways. Glycobiology 15: 519527, 2005. Feuk-Lagerstedt E, Jordan ET, Leffler H, Dahlgren C, Karlsson A. Identification of CD66a and CD66b as the major galectin-3 receptor candidates in human neutrophils. J. Immunol. 163: 55925598, 1999. Fukushi J, Makagiansar IT, Stallcup WB. NG2 proteoglycan promotes endothelial cell motility and angiogenesis via engagement of galectin-3 and alpha3beta1 integrin. Mol. Biol. Cell 15: 35803590, 2004. Gaudin JC, Mehul B, Hughes RC. Nuclear localisation of wild type and mutant galectin-3 in transfected cells. Biol. Cell 92: 4958, 2000. Gong HC, Honjo Y, Nangia-Makker P, Hogan V, Mazurak N, Bresalier RS, Raz A. The NH2 terminus of galectin-3 governs cellular compartmentalization and functions in cancer cells. Cancer Res. 59: 62396245, 1999. Hirabayashi J, Hashidate T, Arata Y, Nishi N, Nakamura T, Hirashima M, Urashima T, Oka T, Futai M, Muller WEG, Yagi F and Kasai K. Oligosaccharide specificity of galectins: a search by frontal affinity chromatography. Biochim. Biophys. Acta 1572: 232254, 2002. Ho MK, Springer TA. Mac-2, a novel 32,000 Mr mouse macrophage subpopulation-specific antigen defined by monoclonal antibodies, J. Immunol. 128: 12211228, 1982. Honjo Y, Nangia-Makker P, Inohara H & Raz A. Downregulation of galectin-3 suppresses tumorigenicity of human breast carcinoma cells. Clin. Cancer Res. 7: 661668, 2001. Houzelstein D, Goncalves IR, Fadden AJ, Sidhu SS, Cooper DN, Drickamer K, Leffler H, Poirier F. Phylogenetic analysis of the vertebrate galectin family. Mol. Biol. Evol. 21: 1177 1187, 2004. Hsu DK, Zuberi RI, Liu F-T. Biochemical and biophysical characterization of human recombinant IgE-binding protein, an S-type animal lectin. J. Biol. Chem. 267: 1416714174, 1992. 70
26. Hughes RC. The galectin family of mammalian carbohydrate-binding molecules. Biochem. Soc. Transact. 25: 11941198, 1997. 27. Hughes R.C. Secretion of the galectin family of mammalian carbohydrate-binding proteins. Biochim. Biophys. Acta 1473: 172185, 1999. 28. Iurisci I, Tinari N, Natoli C, Angelucci D, Cianchetti E, Iacobelli S. Concentrations of galectin3 in the sera of normal controls and cancer patients. Clin Cancer Res 6: 1389-1393, 2000. 29. Jeng KC, Frigeri LG, Liu FT. An endogenous lectin, galectin-3 (epsilon BP/Mac-2), potentiates IL-1 production by human monocytes. Immunol. Lett. 42: 113116, 1994. 30. Khaldoyanidi SK, Glinsky VV, Sikora L, Glinskii AB, Mossine VV, Quinn TP, Glinsky GV, Sriramarao P. MDA-MB-435 human breast carcinoma cell homo- and heterotypic adhesion under flow conditions is mediated in part by Thomsen-Friedenreich antigengalectin-3 interactions. J. Biol. Chem. 278: 41274134, 2003. 31. Kim MK, Sung CO, Do IG, Jeon HK, Song TJ, Park HS, Lee YY, Kim BG, Lee JW, Bae DS. Overexpression of Galectin-3 and its clinical significance in ovarian carcinoma. Int J Clin Oncol. 2011 (ahead of print) 32. Lagana A, Goetz JG, Cheung P, Raz A, Dennis JW, Nabi IR. Galectin binding to Mgat5modified N-glycans regulates fibronectin matrix remodeling in tumor cells. Mol Cell Biol. 26(8): 3181-3193, 2006. 33. Lee YJ, Song YK, Song JJ, Siervo-Sassi RR, Kim HR, Li L, Spitz DR, Lokshin A, Kim JH. Reconstitution of galectin-3 alters glutathione content and potentiates TRAIL-induced cytotoxicity by dephosphorylation of Akt. Exp. Cell Res. 288: 2134, 2003. 34. Liu FT, Hsu DK, Zuberi RI, Kuwabara I, Chi EY, Henderson WR Jr. Expression and function of galectin-3, a beta-galactoside-binding lectin, in human monocytes and macrophages. Am. J. Pathol. 147: 10161028, 1995. 35. Liu FT, Rabinovich GA. Galectins as modulators of tumour progression. Nat Rev Cancer. 5(1): 29-41, 2005. 36. Markowska AI, Liu FT, Panjwani N. Galectin-3 is an important mediator of VEGF- and bFGFmediated angiogenic response. J Exp Med. 207(9): 1981-1993, 2010. 37. Massa SM, Cooper DN, Leffler H, Barondes SH. L-29, an endogenous lectin, binds to glycoconjugate ligands with positive cooperativity, Biochemistry 32 260267, 1993. 38. Matarrese P, Tinari N, Semeraro ML, Natoli C, Iacobelli S, Malorni W. Galectin-3 overexpression protects from cell damage and death by influencing mitochondrial homeostasis. FEBS Lett. 473: 311315, 2000. 39. Mazurek N, Conklin J, Byrd JC, Raz A, Bresalier RS. Phosphorylation of the beta-galactosidebinding protein galectin-3 modulates binding to its ligands. J. Biol. Chem. 275: 3631136315, 2000. 40. Menon RP, Hughes RC. Determinants in the N-terminal domains of galectin-3 for secretion by a novel pathway circumventing the endoplasmic reticulumGolgi complex. Eur. J. Biochem. 264: 569576, 1999. 41. Menon RP, Strom M, Hughes RC. Interaction of a novel cysteine and histidine-rich cytoplasmic protein with galectin-3 in a carbohydrate-independent manner. FEBS Lett. 470: 227231, 2000. 42. Morris S, Ahmad N, Andr S, Kaltner H, Gabius HJ, Brenowitz M, Brewer F. Quaternary solution structures of galectins-1, -3, and -7. Glycobiology 14(3): 293-300, 2004. 43. Nangia-Makker P, Honjo Y, Sarvis R, Akahani S, Hogan V, Pienta KJ, Raz A. Galectin-3 induces endothelial cell morphogenesis and angiogenesis. Am. J. Pathol. 156: 899909, 2000. 44. Ochieng J, Furtak V & Lukyanov P. Extracellular functions of galectin-3. Glycoconj. J. 19: 527535 (2004). 45. Ochieng J, Green B, Evans S, James O, Warfield P. Modulation of the biological functions of galectin-3 by matrix metalloproteinases. Biochim. Biophys. Acta 1379: 97106, 1998. 46. Ochieng J, Platt D, Tait L, Hogan V, Raz T, Carmi P, Raz A. Structurefunction relationship of a recombinant human galactosidebinding protein. Biochemistry 32: 44554460, 1993. 71
47. Oka N, Nakahara S, Takenaka Y, Fukumori T, Hogan V, Kanayama HO, Yanagawa T, Raz A. Galectin-3 inhibits tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis by activating Akt in human bladder carcinoma cells. Cancer Res. 65: 75467553, 2005. 48. Paron I, Scaloni A, Pines A, Bachi A, Liu F-T, Puppin C, Pandolfi M, Ledda L, Di Loreto C, Damante G, Tell G. Nuclear localization of Galectin-3 in transformed thyroid cells: a role in transcriptional regulation. Biochem. Biophys. Res. Commun. 302: 545553, 2003. 49. Raz A, Pazerini G, Carmi P. Identification of the metastasis-associated, galactoside-binding lectin as a chimeric gene product with homology to an IgE-binding protein. Cancer Res. 49: 34893493, 1989. 50. Righi A, Jin L, Zhang S, Stilling G, Scheithauer BW, Kovacs K, Lloyd RV. Identification and consequences of galectin-3 expression in pituitary tumors. Mol Cell Endocrinol. 326(1-2): 814, 2010. 51. Saffar H, Sanii S, Heshmat R, Haghpanah V, Larijani B, Rajabiani A, Azimi S and Tavangar SM. Expression of galectin-3, nm-23, and cyclooxygenase-2 could potentially discriminate between benign and malignant pheochromocytoma. Am. J. Clin. Pathol. 135 (3): 454-460, 2011. 52. Sanjuan X, Fernandez PL, Castells A, Castronovo V, van den Brule F, Liu FT, Cardesa A, Campo E. Differential expression of galectin-3 and galectin-1 in colorectal cancer progression. Gastroenterology 113: 1906-1915, 1997. 53. Sato S, Hughes RC. Binding specificity of a baby hamster kidney lectin for H type I and II chains, polylactosamine glycans, and appropriately glycosylated forms of laminin and fibronectin. J. Biol. Chem. 267: 69836990, 1992. 54. Seetharaman J, Kanigsberg A, Slaaby R, Leffler H, Barondes SH, Rini JM. X-ray crystal structure of the human galectin-3 carbohydrate recognition domain at 2.1-A resolution. J. Biol. Chem. 273: 1304713052, 1998. 55. Shalom-Feuerstein R, Cooks T, Raz A, Kloog Y. Galectin-3 regulates a molecular switch from N-Ras to K-Ras usage in human breast carcinoma cells. Cancer Res. 65: 72927300, 2005. 56. Sharon N. Lectins: carbohydrate-specific reagents and biological recognition molecules. J Biol Chem. 282(5):2753-2764, 2007. 57. Shimura T, Takenaka Y, Fukumori T, Tsutsumi S, Okada K, Hogan V, Kikuchi A, Kuwano H, Raz A. Implication of galectin-3 in Wnt signaling. Cancer Res. 65: 35353537, 2005. 58. Shimura T, Takenaka Y, Tsutsumi S, Hogan V, Kikuchi A, Raz A. Galectin-3, a novel binding partner of beta-catenin. Cancer Res. 64: 63636367, 2004. 59. Takenaka Y, Inohara H, Yoshii T, Oshima K, Nakahara S, Akahani S, Honjo Y, Yamamoto Y, Raz A, Kubo T. Malignant transformation of thyroid follicular cells by galectin-3. Cancer Lett. 195: 111119, 2003. 60. Tsay YG, Lin NY, Voss PG, Patterson RJ, Wang JL. Export of galectin-3 from nuclei of digitonin-permeabilized mouse 3T3 fibroblasts. Exp.Cell Res. 252: 250261, 1999. 61. Yang R-Y, Hill PN, Hsu DK, Liu F-T. Role of the carboxylterminal lectin domain in selfassociation of galectin-3. Biochemistry 37: 40864092, 1998. 62. Yang R-Y, Hsu DK & Liu F-T. Expression of galectin-3 modulates T-cell growth and apoptosis. PNAS 93: 67376742, 1996. 63. Yang R-Y, Rabinovich GA, Liu F-T. Galectins: structure, function and therapeutic potential. Expert Rev Mol Med. 10: e17, 2008. 64. Yang E, Shim JS, Woo HJ, Kim KW, Kwon HJ. Aminopeptidase N/CD13 induces angiogenesis through interaction with a pro-angiogenic protein, galectin-3. Biochem Biophys Res Commun. 363(2): 336-341, 2007. 65. Yoshii T, Fukumori T, Honjo Y, Inohara H, Kim HR, Raz A. Galectin-3 phosphorylation is required for its anti-apoptotic function and cell cycle arrest. J. Biol. Chem. 277: 68526857, 2002. 66. Yoshii T, Inohara H, Takenaka Y, Honjo Y, Akahani S, Nomura T, Raz A, Kubo T. Galectin-3 maintains the transformed phenotype of thyroid papillary carcinoma cells. Int. J. Oncol. 18: 787792, 2001. 72
67. Yu F, Finley RL Jr, Raz A & Kim HR. Galectin-3 translocates to the perinuclear membranes and inhibits cytochrome c release from the mitochondria. A role for synexin in galectin-3 translocation. J. Biol. Chem. 277: 1581915827, 2002. 68. Yu LG, Andrews N, Zhao Q, McKean D, Williams JF, Connor LJ, Gerasimenko OV, Hilkens J, Hirabayashi J, Kasai K, Rhodes JM. Galectin-3 interaction with Thomsen-Friedenreich disaccharide on cancer-associated MUC1 causes increased cancer cell endothelial adhesion. J Biol Chem. 282(1): 773-781, 2007.
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General comments:
The article demonstrates that deubiquitylase HAUSP plays essential role in the maintenance of neuronal progenitor cells. The various methods used by the authors provide solid arguments to claim that HAUSP is a critical factor in positive regulation of REST and indeed it acts directly on REST as its specific deubiquitylase to counteract -TrCP-dependent ubiquitylation. Certainly, these results give important insights into the regulation of neuronal lineage commitment and, hence, could significantly aid in elucidating the causes of pathological states, such as neurodegenerative diseases and brain cancers. Also, the authors are aware of the potential use of deubiquitylase HAUSP as a therapeutic target and as an invaluable tool in regenerative medicine. The article is well-written and sounds convincing; however, the data presented does not fully explain or validate some of the conclusions 74
drawn by the authors. Therefore, to substantiate their final conclusions, I recommend the following changes before submission of the paper:
Major points:
1) Figure 2 immunoblotting experiments use CoREST as a negative control to show that HAUSP knockdown specifically reduces REST protein levels. Although CoREST is a primary co-factor of REST, the authors do not show whether HAUSP knockdown affects the levels of other factors in the repressor complex with REST, such as HDAC-1 and -2 (Abrajano , J.J. et al.). It would be good to confirm whether these protein levels remain unchanged to validate whether HAUSP specifically affects REST. 2) Figure 4 similarly to Figure 2, CoREST is used as a negative control for rtPCR to check whether mRNA levels of REST are affected by HAUSP knockdown. The authors should also investigate mRNA levels of other REST co-factors and possibly other transcription factors involved in NPC differentiation. 3) Figure S4d the authors argue that forced expression of flag-HAUSP (...) increased REST protein levels. This is not convincing as immunoblotting does not actually show any significant increase in protein levels. 4) Figure 5e in vitro deubiquitylation assay utilises proteins that were overexpressed in 293T cells and subsequently purified. This means that there is a potential risk of co-purifying other DUBs and/or other factors or scaffold proteins that could affect REST polyubiquitination status. The result might be particularly dubious if WT-HAUSP, but not Mt-HAUSP was contaminated, but this could be checked by mass spectrometry or protein sequencing. 5) Figure 7 the authors claim to identify a consensus HAUSP binding sequence in REST, but fail to show a direct loss of interaction between HAUSP and mutant REST. Although decreased deubiquitination of REST(S313A) is demonstrated, immunoprecipitation of HAUSP and immunoblotting with mutant REST would be crucial to validate the authors conclusions. 6) Figure 8 the concept of direct modulation of REST protein levels and, hence, lineage commitment and self-renewal, by -TrCP and HAUSP as presented in the Ying-Yang model is very simplified. The authors fail to discuss other potential factors involved in this regulation, only with a brief mention of TRF2. Certainly, REST is a critical factor in regulating NPC differentiation, but other factors are also implicated. For example, RAinduced differentiation results in activation of LIF (Asano, H. et al). Finally, authors do not show how -TrCP and HAUSP antagonise each other at the molecular level and whether modulation of REST protein levels is phosphorylation-dependent, as -TrCP requires a phosphodegron for ubiquitination (Westbrook, T.F. et al.).
Minor points
1) Figure 1 the authors show protein levels of HAUSP, -TrCP, REST and TUJ1 by immunoblotting during NPC differentiation and the results are confirmed by immunostaining of all proteins, but -TrCP. It would be nice to include -TrCP in this experiment and also to be consistent with the cell line used, as the authors sometimes utilise 15167 and sometimes ENStemA NPCs.
2) Figure 3e immunoblot shows protein levels of ectopically expressed Flag-REST after HAUSP knockdown; it would be good to see also total REST levels. 75
3) Figure 7b immunoblot shows incomplete disruption of deubiquitylation of REST(S313A) (lane 4) as compared to control not overexpressing HAUSP (lane 2) this is not discussed in text. 4) Figure S6: a REST is almost completely degraded in differentiated cells (lane 2), although a proteasomal inhibitor is used. b REST ubiquitination in cells overexpressing HAUSP is only slightly reduced (lane 1) as compared to control (lane 2). These results are not explained. 5) Most of the immunoprecipitation experiments do not include input REST levels (Figure 5c, e, f, Figure 6d, Figure 7b, c, d, Figure S4c and Figure S6), so it is hard to assess the relevancy of the results of deubiquitination assays. 6) Figure 1, 3 and 6 apart from TUJ1, it would be nice to use antibodies against other neuronal markers to substantiate the results.
References
1. Abrajano JJ, Qureshi IA, Gokhan S, Zheng D, Bergman A, Mehler MF. REST and CoREST modulate neuronal subtype specification, maturation and maintenance. PLoS One. (2009) 4(12):e7936. 2. Asano H, Aonuma M, Sanosaka T, Kohyama J, Namihira M, Nakashima K. Astrocyte differentiation of neural precursor cells is enhanced by retinoic acid through a change in epigenetic modification. Stem Cells. (2009) 27(11):2744-52. 3. Westbrook TF, Hu G, Ang XL, Mulligan P, Pavlova NN, Liang A, Leng Y, Maehr R, Shi Y, Harper JW, Elledge SJ. SCF-TRCP controls oncogenic transformation and neural differentiation through REST degradation. Nature (2008) 452, 370-374.
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7.2
The Writing a PhD Studentship workshop will enable students to create a coherent and feasible research proposal for a scientific project to continue studying for a PhD, and to present this in a form suitable for external scrutiny. Research project supervisors should be also be consulted for advice on preparing the studentship application during the write-up period. An overview of the material covered in this workshop is given below. Aims: To improve your understanding of the concepts of hypothesis testing and experimental planning. To practice the skills required for producing a scientific proposal in writing.
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Learning outcomes: On completion of this assignment, you will have improved your ability to: - formulate testable hypotheses - explain the rationale and design of experiments to test specific hypotheses - write a scientific proposal in a structured way. How is this relevant to me? In your later career you may need to think analytically about new experiments, and may need to explain your thinking in writing. Almost all scientific activity that occurs in the outside world (and almost all new scientific activity in Universities) is based on written proposals that justify the work to be done. In some cases, your future salary (or that of your colleagues) may even depend on the ability to write a convincing proposal describing what you intend to do.
Examples of types of proposal Deciding what experiments are worth doing, how to design them to produce useful data, and then how to explain these processes to other people, is central to all modern scientific activity. The same generic approach is used at many different levels, including Ph.D. projects proposal you will need write as part of your transferable skill task, and proposals for grants that provide the salary for a post-doctoral researcher or a research fellow. The same general principles also apply to the formulation of proposals for large projects in industry designed to produce new products (eg drug discovery and development), and for big international research projects that might involve the collaboration of many different research groups. The elements of a scientific proposal The general questions that provide the basis for any scientific proposal are: (a) What is already known about the specific topic ? This component often takes the form of a review of previous studies in the area. (b) What are the gaps in present knowledge ? A critical analysis of previous work in the field may identify unanswered questions, or there may be conflicting data that need to be explained and reconciled. Developments in one field, including new types of technology, may suggest new applications in another field, or new ways of looking at specific questions in other fields. (c) What specific hypothesis will provide the basis for the proposed experiments ? The concept of hypothesis-testing lies at the foundation of much of modern science. A section below deals with this in more detail. (d) What experimental plan will be followed ? For a proposal to be convincing, it needs to be clear what types of experiment are to be done and how; what problems are anticipated, and what strategies are available to overcome these problems ? There are often several different types of experiment that can be done to address a specific question, so it is useful to ask what is the rationale for a particular approach. Can multiple lines of evidence be brought to a support a particular conclusion ? If so what are they ? (e) What outcomes are anticipated ? Any problems that are anticipated with data interpretation should be identified. Virtually all experimental work costs money: will the financial backer be able to use the outcomes of the experiment, will there by value for money ?
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Hypothesis testing The definition of a hypothesis that most scientists would recognise goes something like this: a supposition made as the basis for experiment without assumption of its truth. A typical example in physiology might take the form substance X plays a role in regulating process Y. Several obvious lines of experiment to test the hypothesis might include measuring the activity of Y after removing substance X, after over-stimulation by X, and after blocking the effects of X. It may be possible to do these experiments both in vivo (for example in experimental animals) and in vitro (for example in cell lines). Depending on the outcomes, the data would be said to be either consistent with the hypothesis (in which case one might then ask about the relevant mechanisms), or they are inconsistent in which case the hypothesis should be rejected (and an alternative one developed). There are some major scientific projects that are not strongly based on hypothesis. An example would be the sequencing of the human genome which has sometimes been called hypothesis-generating activity. In general, however, most modern science is based on the formulation of specific testable hypotheses. The generation of hypotheses is one of the most important parts of any scientific plan. Good hypotheses are sometimes also described as useful. In turn, useful hypotheses generally possess most if not all of the following: they take account of existing knowledge and indicate ways in which this knowledge could be extended (if correct), and they provide the basis for designing specific experiments (ie tests of the hypothesis) that should yield clear-cut answers one way or the other. They may also provide a way of unifying disparate sets of observations. Writing a proposal The proposal you are asked to complete in this assignment is for a PhD studentship and should have the following sections: Summary, Background, Hypothesis, Specific aims & objectives, Experimental plan, Outcomes, & Sources of information (ie references etc). In addition, you are required to write a short summary of the proposal for both scientific and lay audiences. Each section should address a distinct set of points. Summary: this should provide a brief overview of the entire proposal. The lay summary should avoid the use of technical terms and jargon that would not be familar to the average lay person. Background: existing knowledge should be reviewed (and sources referenced at the end of the proposal). If there are pilot data, or preliminary experimental findings available (see below), these can be described at the end of this section. The objective in writing this section should be the identification of worthwhile new experiments. You would like to reader to recognise youre your proposed experiments are exactly the right ones to do next. Hypothesis: The workshop will provide help in formulating hypotheses and in distinguishing between useful and inappropriate hypotheses. A specific hypothesis can be quite brief (one or two sentences) but may represent a lot of hard thinking. Aims and objectives. These can be quite briefly expressed. In effect they should translate the hypothesis into specific questions that your intended experiments will answer. Experimental plan: at least in part this section may be written in the future tense since it should describe what you intend to do. It might help you to think in terms of three subsections; (1) The first part of the plan should outline the general strategy to be followed, and may explain why other strategies are rejected. If you have pilot data, or other reasons for supposing that a particular strategy will be successful, this should be explained. (2) You may already have an idea of the specific experimental protocols that you propose to use: if so, explain this but consider also, what will happen if things dont go exactly according to plan? 79
What priorities will govern your decisions in this case. <HINT: dont put all your experimental eggs in one basket>. If the experimental methods are not yet worked out in detail, explain how you will go about optimising the methods to yield useful data. What criteria will be applied to evaluating any data from optimisation experiments. Depending on the specific experiments, you may need to define the number of observations required to yield useful conclusions; in addition, you should already consider what statistical methods will be applied to the analysis of the data. (3) For many complex projects it is useful to provide a time-line, or milestones; these are generally regarded as useful tools for project management. Ask yourself, should some projected tasks be completed before others, or by specific times within the project as a whole? If so, how is this incorporated into the experimental plan? In the real world, time and money are always limited: what resources will you need to achieve your objectives in an efficient and cost-effective way. Outcomes: Briefly summarise how, if everything works well, you will have achieved your objectives (and how a financial backer will have obtained value for money). Justification for support requested: Briefly describe how the funds requested will be spent, breaking down consumables funds to give an idea of how much individual parts of the experimental plan will cost. References: cite any sources of information quoted in the application. The application typically should include up to 25 citations to peer-reviewed papers. Not more than 2,000 words should be used to describe the research proposal (excluding the scientific and lay summaries, and excluding references). Each section should be written in good English (not note form or bullet points). Make sure you also choose an informative title. A template for you to enter the application on will be provided for the application nearer the time and an example PhD Studentship application is given in section 7.3.
Your portfolio will be viewed by the main External Examiner and therefore should look professional and presentable. 80
7.3.2 Portfolio Self-Assessment (Commentary) Students need to assess: 1. 2. 3. The Research Projects. The Techniques and Frontiers in Biomedical Sciences component. The Transferable Skills component.
Profile for a portfolio self-assessment: 1. 2. 3. 4. 5. Describe the work to be assessed. Give the aim of the assignment. Describe your learning objectives. Critically evaluate your achievements. What are the learning outcomes?
It is essential to use the same template for each item of work using the same headings (description, aims, learning objectives, achievements, learning outcomes; ~ one paragraph for each). A template for this purpose will be provided by the Programme Director.
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Distinction Level
100% - 90%
90% - 80%
80% - 70%
Outstanding. No better result conceivable at Masters level. Extensive evidence of critical thinking and cogent scientific argument. Evidence of reading around the topic. Excellent experimental design. Excellent. Essentially correct and complete, with evidence of critical thinking and excellent use of the literature. Logical structure, well written and presented, displaying a cogent scientific argument. Very good experimental design. Very Good. Content essentially without any major flaws, very well explained with clear evidence of a high level of scientific competence. Good experimental design. Good. Well explained, showing good evidence of critical scientific judgement. Good experimental design. Quite Good. A well explained application, with good understanding and some evidence of critical scientific judgment. Satisfactory experimental design. Fairly Good. A generally sound review with a good or quite good level of understanding, evidence of sound scientific competence and judgment. Adequate. Showing some progress but with some deficiencies in one or more aspects of understanding of the paper and scientific judgment. A poor application with an overall superficial approach. Essentially an incomplete application with major omissions in several areas and evidence of a poor understanding. A poor application with an overall superficial approach and more errors, omissions and evidence of a deficiency of effort and poor understanding. A marked deficiency in content of understanding of the work proposed and the task set. Even more marked deficiencies in content of understanding and application. A complete absence of relevant work presented.
Pass Level
59% - 55%
54% - 50%
Fail Level
49% - 45%
44% - 40%
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2.
3.
SUMMARY OF RESEARCH a) For scientifically qualified assessors (no more than 200 words)
In spite of exciting progress, the molecular determinants involved in the aetiology and susceptibility of various chronic age-related neurodegenerative disorders (NDs) such as Alzheimers disease (AD), Parkinsons disease (PD), Huntingtons disease (HD), and amyotrophic lateral sclerosis (ALS), remain mechanistically undefined. Numerous genetic and pharmacological screens have been conducted in Caenorhabditis elegans disease models to unravel candidate genes and /or specific cellular pathways pertinent to multiple neurodegenerative cascades and successfully identified and validated many neuroprotective factors. The overall aim of this project is to elucidate critical cellular mediators of neuropathology and neuron vulnerability by exploiting the experimental attributes of C. elegans and integrating genetic and pharmacological evaluation of a set of experimentallydelineated genetic modifiers. Multiple transgenic, non-transgenic and toxicant C. elegans ND models will be generated and optimised by examining their pathological phenotypes. The bioinformatically prioritised modifiers will then be characterised and validated by phenocopy using a reverse genetic approach by applying RNA interference (RNAi). In addition, a chemical reverse genetic screen will be performed to illuminate neuroprotective compounds molecular targets, mechanism of action and affected molecular pathways. For lay readers (no more than 200 words)
The biological underpinnings of a diverse spectrum of progressive neurological disorders have remained elusive. Large-scale screens have been performed in model organisms to isolate genes that either rescue or exacerbate the manifestations of neurodegenerative disorders (NDs), which in turn yield valuable insights into mediators of disease within cells and reveal targets for potential therapies. This project will utilise microscopic nematode roundworm, Caenorhabditis elegans to elucidate the incompletely understood disease mechanisms and genetic susceptibility of multiple NDs. Worms are used because of their 84
facile genetics and strikingly similar neuronal mechanisms to the humans. Worms will be manipulated to simulate the cardinal features of several NDs by inserting human disease proteins, removing or depleting worm version of the disease proteins and administering toxic compounds, and the animals will be observed for any effects. A collection of previously identified C. elegans genes already known to influence the severity of dysfunction will be suppressed in a subsequent screen to discern how they work. In addition, a set of drugs known to alleviate some disease symptoms will also be applied to reveal their mechanism of action. Ultimately, the project aims to expand our knowledge of the mechanisms of NDs.
4.
RESEARCH QUESTION a) What is your research question/hypothesis (no more than 100 words)
neurodegeneration and neuroprotection? 3. Through what mechanisms do therapeutic candidates mediate their neuroprotective effects? 4. 5. Do modifier genes have a widespread or disease-specific role in neurodegeneration? Are pathogenic mechanism shared between several or all NDs, irrespective of the specific disease? 6. Which types of neurons are most vulnerable to alterations of modifier genes?
Neurodegenerative diseases (NDs) pose a tremendous economical and societal burden to our rapidly ageing populations. Though toxic functions of specific disease proteins have been ascribed following the identification of many predisposition genes, the lack of mechanistic insights into why and how these genes contribute or influence the aetiology and progression of the NDs has severe repercussions for treatment of these crippling diseases since effective therapeutic agents with disease-modifying or preventive properties remain acutely deficient and development of new drugs continues to be a challenging job. Definitive insights into the complex mechanistic connections between specific cellular pathways and specific misfolded protein pathologies are likely to explain the conundrums and facilitate the development of new disease biomarkers and promising disease-modifying neuroprotective therapies for timely intervention.
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5.
DETAILS OF RESEARCH PROJECT: Summarise under the following headings:a) b) c) d) e) f) Aims and objectives of the project Work which has led up to the project Detailed experimental plan of investigation Outcomes Contingency plans Timetable and milestones
NO MORE THAN 2,000 WORDS SHOULD BE USED TO DESCRIBE THE RESEARCH PROJECT (PLEASE INCLUDE A WORD COUNT)
a)
This project aims to use a combined application of appropriate genetic and toxicant Caenorhabditis elegans (C. elegans) neurodegenerative diseases (NDs) models to unravel the incompletely understood key molecular pathways, disease mechanisms and genetic susceptibility of multiple NDs. The main focus of the investigation is the genetic and pharmacological characterisation of modifier genes involved in neuropathology and neuron vulnerability, which contribute toward the identification of putative novel targets and therapeutic leads.
b)
Essentially, the majority of neurodegenerative disorders (NDs) are characterised by the mechanistic unifying theme of aggregation, accumulation and deposition of prominent intraor extracellular toxic proteinaceous inclusions in a particular population of neurons (1) where subsequent necrotic or apoptotic neurodegeneration results in irreversible neuronal loss, synaptic impairments and eventual neuronal death. The use of Caenorhabditis elegans (C. elegans) as a primary platform to probe the poorly defined disease mechanisms of several major NDs and a more rapid means towards novel gene and drug discovery has escalated over the last decade, since worms are amenable to high-throughput genetic, genomic, proteomic, and drug screening approaches. Indeed, a multitude of informative, tissuespecific C. elegans ND models have been developed manifesting abnormal behavioural or pathological phenotypes that partially recapitulate the salient cellular, molecular and pathological aspects of complex ND processes (Table 1), to explore the dynamics of aggregate formation, cellular malfunctions, genetic and environmental susceptibility factors and putative neuroprotective genes and compounds against disease-protein toxicity (2). As shown in Figure 1, these models can be subjected to medium or high-throughput genomewide forward and reverse genetic screens to identify genetic modifiers, which are essential physiologic determinants of aggregation and toxicity and yield new potential therapeutic targets or models for the given disease, as well as low-throughput chemical screens that 86
directly explore effective lead compounds that rescue the phenotype to identify pharmacological treatments that block neurodegeneration. Targets identified in several disease models (Table 1) using RNAi screening and microarray analysis have already advanced to the stage of drug validation for efficacy in mammalian system (3-5). Substantial evidence implicates that while genetic regulators of protein homeostasis, stress responsiveness and ageing, including molecular chaperones and components of the ubiquitin-degradation systems, are common to most disease proteins, the onset,
development and progression of each of the NDs can be induced or influenced by alterations of a multitude of disease-protein-specific molecular processes (6). All C. elegans and yeast -synuclein screens identified components of the vesicular-trafficking machinery to specifically modify -synuclein toxicity and aggregation, but are rarely found as modifiers of polyglutamine (polyQ) and tau toxicity and aggregation, whereas those associated with RNA metabolism, protein folding and degradation, and transcriptional regulation appear to influence the pathology of various disease proteins, but are specifically enriched in polyQ aggregation and toxicity screens including those that have since been validated in other model organisms and mammals. It also remains unclear which populations of neurons are most susceptible to the loss of modifier genes when combined with ectopic-overexpression of aggregation-prone proteins. Though genome-wide screening in C. elegans may facilitate the identification of all possible effectors of protein misfolding and neuroprotection, this approach is limited by its inefficiency in detecting positive hits (2,7,8), due to inclusion of large gene sets that are not necessarily unique which in turn result in some redundancy, as well as important house-keeping genes that give rise to non-specific, off-target effects and false positive results. Few screens have recently used a more focused strategy - a candidate approach/hypothesis-driven approach to reveal potential modifier genes in a specific pathway based on hypothesis generated by screens in lower model organisms or existing knowledge of disease mechanisms and pathways (9,10). Preliminary work in this laboratory adopted a candidate approach to mine the literature and compile a collection of experimentally delineated genetic modifiers of protein aggregation, misfolding and toxicity in C. elegans. Using online databases WormBase
(www.wormbase.org) and GExplore (http://genome.sfu.ca/gexplore/), modifier genes were further selected for to retrieve only those with known expression in adult neurons that are also non-RNAi lethal (Table 2) which form the basis for a hypothesis-driven RNAi screen and subsequent in-depth analyses and mechanistic dissection. Our recent work also used fluorescence confocal imaging of transgenic C. elegans lines to confirm the promoter specificity of six neuronal promoters (Fig. 2) which are applicable as means of probing the role of positive genetic modifiers in the survival, vulnerability and function of specific sets of neurons and their subsequent validations. 87
TABLE 1. Summary of all available C. elegans neurodegeneration models. Worms are subjected to molecular, genetic and chemical manipulations to initiate the neurodegenerative cascade. Corresponding pathological phenotypes and identified drug compounds that confer neuroprotection are indicated. Models highlighted in beige are most suitable for this project. Human diseases: AD, Alzheimers disease; HD, Huntingtons disease; PD, Parkinsons disease; ALS, Amyotrophic lateral sclerosis; SMA, Spinal muscular atrophy
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Neurodegenerative diseases
AD
Transgene expression
Phenotype
Drug
References
Ginkgo biloba extract EGb 761 and Ginkgolide A (12) Soya isoflavone glycitein (35) Reserpine (48) Thioflavin T; curcumin (21)
Link et al (15)
Molecular manipulation Molecular manipulation Molecular manipulation Molecular manipulation Molecular manipulation Molecular manipulation Molecular manipulation Molecular manipulation Molecular manipulation Molecular manipulation Molecular manipulation Molecular manipulation Molecular manipulation Molecular manipulation Molecular manipulation Molecular manipulation Molecular manipulation
AD AD AD AD HD HD HD HD HD HD HD HD PD PD PD PD PD
Psnb-1::A1-42 - inducible pan-neuronal Paex-3::tau pan-neuronal Pmec-7::tau mechanosensory neurons Prgef-1:: PHP tau- pan-neuronal Pmyo-3::polyQGFP inducible muscle Punc-54::polyQGFP - constitutive muscle Punc-54::DRPLAP::GFP- constitutive muscle Pmec-3::polyQGFP - mechanosensory neurons Posm-10::polyQGFP - chemosensory neurons Prgef-1::polyQGFP - pan-neuronal pqe-1(rt13); Htn-Q150(rtIs11) pha-1(e2123); Htn-Q150(rtIs11) Paex-3::-syn WT and A53T - pan-neuronal Psnb-1::-syn WT - pan-neuronal Punc-119::-syn A53T- pan-neuronal Punc-51 ::-syn WT,A30P and A53T - pan-neuronal Pdat-1 ::-syn WT, A30P, A53T, A56P and A76P DA neurons
Paralysis Unc, tau phosphorylation and aggregation, neurodegeneration Mec, tau phosphorylation, neurodegeneration Unc, decreased lifespan, defective neuronal development Paralysis, age-dependent polyQ aggregation Motility defect Mec, axon morphology abnormalities polyQ aggregation, ASH neuron degeneration, nose-touch defective Unc, motility defects, paralysis Accelerated neurodegeneration ASH neuronal death Unc, DA dendritic abnormalities, reduced motor movement Mitochondrial stress Mitochondrial stress Motor/developmental defects, endocytosis defects DA neurodegeneration, -syn accumulation in DA neurons, reduced DA levels, altered DA neuronal activity Trichostatin (38) Acetaminophen (39) Bromocriptine, Quinpirole (3) Resveratrol Lithium chloride; mithramycin (23) -
Wu et al (12) Kraemer et al(16) Miyasaka et al (17) Brandt et al (18) Caldwell et al (41) Wang et al (19) Saytal et al (20) Yamanaka et al (36) Parker et al (37) Faber et al (42) Brignull et al (22) Voisine et al (23) Voisine et al (23) Lakso et al (24) Springer et al (25) Ved et al (26) Ved et al (26) Kuwahara et al (27) Lakso et al (24) Settivari et al (32) Cao et al (31) Kuwahara et al (27) Karpinar et al (40) Lakso et al (24) Kuwahara et al (27) Hamamichi et al (10) Van Ham et al (2) Saha et al (28) Kuwahara et al (27)
Molecular manipulation Molecular manipulation Molecular manipulation Molecular manipulation Molecular manipulation Molecular manipulation Molecular manipulation Molecular manipulation Mutant/RNAi Mutant/RNAi Mutant/RNAi Mutant/RNAi Mutant/RNAi
PD PD PD PD PD
Pacr-2::-syn WT and A53T motor neurons Pmec-7::-syn WT and A53T - mechanosensory neurons Punc-54 ::-syn::GFP - constitutive muscle Psnb-1::LRRK2 WT, R1441C, G2019S panneuronal eri-1 (mg366);Pdat-1 ::-syn WT, eri-1 (mg366);Pdat-1 ::-syn A53T eri-1 (mg366);Pdat-1 ::-syn A30P pdr-1(lg103); -syn A53T
Reduced motor movement Impaired touch response -syn misfolding and accumulation Mitochondrial stress, reduced DA levels, DA degeneration Unc, growth retardation (Gro)
PD
Highly penetrant, temperature sensitive lethality, early larval arrest phenotype Paraquat hypersensitivity Locomotory defects, intra-neuronal aggregation, abnormal processes and synaptic function Egg laying defective (Egl) Accelerated neurodegeneration ASH neuronal death Mitochondrial stress, ER stress sensitive Mitochondrial stress, ER stress sensitive, decreased life span Oxidative stress sensitive, neurite outgrowth defects, mitochondrial cristae defects -syn misfolding Mitochondrial stress Neuromuscular function deficits DA neurodegeneration Oxidative stress, mitochondrial stress, enhanced DA neurodegeneration, reduced DA levels DA neurodegeneration, motility defects, lethality Oxidative stress Mitochondrial stress, reduced viability
Kuwahara et al (27)
ALS ALS AD HD HD PD PD
Pmyo-3::SOD-1 hsp-16::SOD-1 - body wall muscle or inducible Psnb-1::SOD-1 WT, G85R, H46R/H48Q YFP pan-neuronal sel-12 or hop-1 pqe-1(rt13) pha-1(e2123) lrk-1 (km17), (km41), (tm1898) and (RNAi) pdr-1 (lg103), (XY1046, Parkin KO3) and (RNAi)
Oeda et al (43) Wang et al (44) Lakowski et al (45) Voisine et al (23) Voisine et al (23) Saha et al (28) Samann et al (29) Springer et al (25) Ved et al (26) Samann et al (29) Gitler et al (46) Ved et al (26) Briese et al (47) Nass et al (30) Cao et al (31) Settivari et al (32) Braungart et al. (33) Samann et al (29) Ved et al (26) Saha et al (28) Caldwell et al (34)
Mutant/RNAi Mutant/RNAi Mutant/RNAi Mutant/RNAi Chemical treatment Chemical treatment Chemical treatment Chemical treatment Chemical treatment
PD PD PD SMA PD PD PD PD PD
pink-1 (tm1779) catp-6 (RNAi) djr-1.1 (RNAi) smn-1 (ok355) Pdat-1::GFP subjected to 6-hydroxydopamine (6OHDA) N2 , Pdat-1::GFP, Pdat-1::-syn subjected to Manganese (Mn2+) N2, Pcat-2::GFP subjected to MPTP and MPP+ pink-1 (tm1779) subjected to Paraquat pdr-1 (XY1046), Psnb-1::-syn WT, Punc-119::-syn A53T, N2, lrk-1 (km17), Psnb-1::LRRK2 WT, R1441C, G2019S subjected to Rotenone Pdat-1::GFP subjected to Streptomyces venezuelae secondary metabolite
Chemical treatment
PD
DA neurodegeneration
89
Pharmacological screen
suppressor enhancer
suppressor enhancer
EMS
Screening goal drug sensitivity drug resistance Determine optimal drug concentrations Distribution of compounds
gene expression changes miRNA microarray Quantitative real-time PCR Target analysis
Mutagenesis
Phenotype analysis Positive hits identified Confirmation of RNAi clones Gene mapping genetic complementation and sequencing of mutant alleles Hits prioritisation Confirmation of drug efficacy Exclude drug effects on growth/development and aging pathway
Hits validation
Secondary analyses in multiple worm models Pharmacological intervention in worms if successful Mammalian assays and identification of human orthologues
Drug Validation
FIGURE 1. Schematic diagram of the various screening routes for the identification of genetic or 90 pharmacological targets of a C. elegans disease model
CGC Name
chn-1 sel-10 sut-2 dyb-1 unc-57 ric-4 pink-1 dbl-1 dyf-5 unc-43 cmk-1 age-1 pptr-1 sir-2.1 daf-16 mbf-1 pld-1 sin-3 add-1 ifa-4 unc-116 smd-1 sams-3 nft-1 gst-4 gst-38 unc-36 pmr-1 dat-1 spp-10 gpdh-2 tre-2 tre-3 xbp-1 glb-23 pqe-1 tag-197 -
Description
heat shock protein (HSP) of the HSP16 class mammalian carboxyl-terminus of Hsc70 interacting protein F-box and WD-repeat-containing protein of CDC4/CUL-1 family of E2-E3 ubiquitin ligases Nuclear polyadenylated RNA binding protein Putative ubiquitin-specific protease F-box protein JEMMA Aspartyl beta-hydroxylase ZZ type zinc nger endophilin A, required for synaptic vesicle endocytosis Intracellular protein transport, neurotransmitter secretion BRPK/PTEN-induced protein kinase a member of the transforming growth factor beta (TGFbeta) superfamily a putative MAP kinase type II calcium/calmodulin-dependent protein kinase Ca2+/calmodulin-dependent protein kinase I functions in an insulin-like signalling (ILS) pathway Serine/threonine protein phosphatase 2A NAD-dependent deacetylase sirtuin-1 sole C. elegans forkhead box O (FOXO) homologue Transcription factor MBF1 Phospholipase D1 SIN3 family of histone deacetylase subunits cytoskeletal protein alpha-adducin a nonessential intermediate filament protein kinesin-1 heavy chain ortholog S-Adenosylmethionine decarboxylase S-adenosylmethionine synthetase p-Nitrophenyl phosphatase Carbon-nitrogen hydrolase human thioredoxin domain-containing protein 4 precursor a putative glutathione-requiring prostaglandin D synthase unclassified alpha2/delta subunit of a voltage-gated calcium channel Porin/voltage-dependent anion-selective channel protein a Golgi P-type ATPase Ca2+/Mn2+-pump a plasma membrane dopamine transporter sphingolipid metabolism/lysosomal IGF-II mRNA-binding protein IMP glycerol 3-phosphate dehydrogenases putative trehalases of unknown function putative trehalases of unknown function unclassified unclassified unclassified unclassified unclassified unclassified
Human orthologues
HSPB6 STUB1 CHIP FBXW7 ZC3H14 USP47 FBXL19 ASPHD2 DTNA SH3GL2 SNAP-25 PINK1 BMP4 MAK/ICK CaMKII CaMK1 PI3K PP2A SIRT1 FOXO4 EDF1 PLD1 SIN3 ADD1 IFB-1 KIF5 AMD1 MAT1A PDXP FHIT ERP44 GSTA3 GSTA4 CACNA2D3 VDAC2 ATP2C1 SLC6A2/A3 PSAP IGF2BP2 GPD1L TREH TREH None None None REXO1 VPS52 None
RNAi library
Vidal Ahringer Vidal Not available Ahringer Vidal Ahringer Vidal Vidal Vidal Ahringer Vidal Ahringer Vidal Vidal Ahringer Not available Ahringer Vidal Vidal Vidal Vidal Vidal Vidal Ahringer Vidal Vidal Vidal Vidal Vidal Vidal Vidal Vidal Vidal Not available Vidal Vidal Vidal Vidal Vidal Vidal Vidal Vidal Vidal Vidal Vidal Vidal
Transcription cofactor
Enzymes
Unclassified
TABLE 2: Set of 47 modifier genes with known adult neuronal expression and non-RNAi lethality. Assigned functional category, C. elegans gene name, CGC approved name, available description and human orthologue are shown. RNAi feeding library coverage of the modifier genes is also indicated. Four modifier genes are nematode specific and six genes remain uncharacterised. UPS: ubiquitin-proteasome system, ECM: extracellular matrix.
91
E
*
Prab-3::EGFP Pdat-1::GFP
Posm-6::GFP Punc-17::GFP
FIGURE 2: Promoter specificity of transgenic C. elegans is confirmed by Pglr-1::GFP fluorescent reporter constructs in extrachromosomal arrays. Fluorescence microscopy of whole body (A,B;C-F left panels) or magnified 3D confocal reconstruction of neuronal architecture in the head region (C-F right panels). A, The pan-neuronal localisation of enhanced GFP (EGFP) expression is consistent with the expected rab-3 promoter driven expression pattern in all worm neurons. B, Punc-17::GFP labels most regions of the nervous system, predominantly cholinergic Pgcy-8::GFP motor neuronal processes. C, The dat-1 promoter labels all 8 dopaminergic (DA) neurons: six anterior neurons in the head and two posterior deirid neurons (PDEs, thick arrow). 3D confocal reconstruction of the magnified head region (right), detailing the six anterior-most DA neurons: four cephalic CEP neurons (asterisks) project dendrites (thin arrows) to the tip of the nose and two anterior deirid ADE neurons (arrow head) extend ciliated processes posteriorly. The CEP dendritic processes of another worm are also visible. D, Posm-6::GFP selectively labels all 60 ciliated sensory neurons. E, The glr-1 promoter drives expression of the GFP in the ventral cord interneurons, motorneurons and nerve ring. F, gcy-8::GFP localises exclusively to one thermo-sensory AFD pair (dashed arrow). 3D confocal reconstruction (right) shows that the AFD dendrites extend anteriorly to the tip of the nose. The sensory endings of both neurons are positioned at the tip of the dendrites (double asterisks). All animals imaged are young adults, four days post-hatch. They include transgenic N2 and RM2754 lines overexpressing GFP reporter alone (A, C and F) or together with HSF-1 in the targeted neurons of PS3551 strain, a hsf-1 mutant (B,D,E). All 3D confocal reconstructions shown are maximal projections of z-stacks. Anterior to the left (C,E,F right panels); Anterior to the right (D, right panel). Scale bars: A,B;C-F: left panels, 100 m; C-F right panels, 10 m. Representative images of at least 5 worms from each transgenic line are shown.
92
c)
Since the availability of a reliable disease model that recreates the molecular characteristics and associated major pathological hallmarks of neurodegenerative disorders (NDs), with a pronounced, measurable phenotype is the prerequisite for targets screening (5,11), this project will commence by generating and characterising multiple transgenic, nontransgenic and pharmacological disease models (Table 1, highlighted in beige) to choose the optimal models. Mutant models will be requested from the C. elegans mutant collection at the Caenorhabditis elegans Genetic Center, while standard transformation technique of microinjection will be used to generate transgenic models overexpressing a variety of wildtype and toxic gain-of-function aggregation-prone proteins under different promoters. Neuronal expression models will be favoured over muscle expression strains. The transgene will be chromosomally integrated by subsequent treatment with long-wave UV using a UV cross-linker. Immunohistochemistry, single-worm PCR and Western blotting will be performed to verify the expression of transgenes in transgenic lines. Two Parkinsons disease (PD) pharmacological models will also be generated by exposing transgenic backgrounds to Parkinsonism-inducing neurotoxins which will be purchased from Sigma (St Louis, MO). Among the behavioural assays that have been well established in C. elegans to assess neuronal toxicity-evoked phenotypic abnormalities (including thrashing/swimming, body bend, paralysis, pharyngeal pumping, and egg-laying) (12), this project will focus on locomotion and aldicarb (paralysis) assays to characterise and optimise disease models in comparison with wild-types (WT). These assays examine motor impairments and defective exocytosis rate respectively, and can be applied generically to majority of the models. The food-sensing assay which is exclusive to PD transgenic, mutant and compound intoxicated models with enhanced dopaminergic (DA) neuron degeneration will be performed to determine aberrant basal slowing behavioural phenotype since C. elegans food-sensing behaviour is mediated exclusively through an intact DA neural circuitry. Typically 20-30 agesynchronised worms per strain will be evaluated at all developmental stages and statistical significance will be determined by a t-test for single strains and one-way ANOVA for multiple strains. Disease models with prominent phenotypic deviations from control strains will be selected. Next a selective RNAi genetic modifier screen will be carried out in which bioinformatically prioritised modifiers retrieved from disparate datasets of previous genome-wide analyses (Table 2) will be specifically knocked down in the chosen disease models to characterise their function and the pathways through which they act. This laboratory has the published RNAi feeding libraries, which together cover 95% of expressed worm genes. To circumvent RNAi resistance in WT C. elegans postmitotic neurons and to maximise RNAi efficacy, RNAi hypersensitive rrf-3 (mg373) strain which has recently been reported as a mutant most sensitised to neuronal RNAi (13) will be injected with transgene constructs of the chosen 93
models together with a green fluorescent protein (GFP) reporter, while models that were generated by mutagenesis will be crossed onto the rrf-3 background to construct the new double transgenic models. Synchronised larvae will be grown to young adults on agar plates containing the dsRNA-expressing bacterial culture that will target a specific C. elegans modifer gene (and one empty vector control per experiment), and then transferred to fresh RNAi plates containing FUdR (to prevent progeny growth) every five days. The initial round of screening will be carried out based on examining locomotion at adult day 0, 2, 5, 7 and 9. These days have been selected to enable detection of either amelioration or exacerbation of the model-specific phenotypes. Knock-down rate of the target gene will be confirmed by reverse transcriptase polymerase chain reaction (RT-PCR) and Western blot analysis. After initial screening, positive hits will be re-tested three times to verify the initial findings and determine reproducibility. A neurodegeneration assay which has more direct implications for NDs will also be performed using the six validated pan-neuronal and discrete neuronal promoters (Fig. 2) to directly assess age-dependent neuronal loss during the lifetime of the worms, and identify particular classes of neurons that are most vulnerable to the loss of modifier genes when combined with disease protein overexpression. However, this project aims to use methods for culturing C. elegans neurons to facilitate accurate quantitative evaluation of cumulative neurodegenerative changes in distinct C. elegans neuronal classes (14), as opposed to conventional neurodegeneration assays in whole nematode studies which have many considerable challenges i.e. living-worm microscopy, following neuronal loss in the same worm consecutively that would require repetitive worm recoveries etc. Following dissociation from early embryos, cultured embryonic C. elegans cells are plated on culture dish surfaces covered with peanut lectin where they adhere and subsequently differentiate into neurons. Rate of degeneration will be determined by following GFP-labeled neurons over time which can undergo further manipulation to provide a means for discerning cellular mechanisms associated with ND (14). These molecular regulators will be validated further by overexpressing the proteins in WT and additional worm models before entry into secondary analyses. It is conceivable that neuroprotective compounds mitigate the behavioural deficits and delay degenerative phenotype through unexplored pathways parallel, upstream or downstream of targets. Therefore, this project will aim to further identify the therapeutic targets and pathways involved in neuroprotective compounds efficacy, and illuminate their mechanism of action. A collection of twelve chemicals (Table1) i.e. antioxidants, kinase inhibitors,
dopamine D2 receptor agonists and amyloid-binding compounds (Sigma) that have been previously identified to confer neuroprotection will be tested in a chemical reverse genetics screen. Synchronised larvae of disease models expressing GFP in the targeted neurons will be incubated with and without solubilised drugs for three days in 24-well plates. Each well will contain a bacteria culture from the RNAi library expressing dsRNA to a C. elegans modifier 94
gene (12 wells per bacteria), followed by addition of individual or a combination of compounds per well. The presence or absence of GFP fluorescence will be evaluated in each well to determine neuronal survival prior to replating on solid media for further behaviour assays for confirmation. Wells that contain a weak or absent fluorescent signal would indicate that the modifier is involved in suppression of the compounds neuroprotective effects. Significance will be determined using a Fishers exact test which is appropriate for small sample sizes.
d)
Outcomes
These investigations will help to reveal conserved molecular and therapeutic mechanisms which contribute not only to the molecular understanding of human NDs but also eventually to the development of effective therapeutic preventions and interventions for those who are at genetic risk or are affected clinically.
e)
Contingency plans
Project progression may be temporarily slowed by some unexpected events, or potential difficulties in the operations. In such a case, efforts will be made to tackle the bottleneck as detailed below:
Difficulties to integrate transgene in the germ-line of the animals. The difficulty with transgene expression in the C. elegans germ-line resides in uncharacterised repression mechanisms of this organism and has long been a problem. In the event that no satisfactory transgene expression can be obtained by conventional microinjection, double transgenic models will be created by genetic crossing. Alternatively, this problem can also be circumvented by the new Mos1 techniques developed by Jorgensen Lab.
RNAi knock-down rate cannot be confirmed by PCR GFP reporter will be used as an alternate to confirm reduced gene expression. Strains with GFP-labelled neurons will have gradual loss of fluorescence if RNAi have successfully suppressed gene expressions. Technical challenges of RNAi-based phenotypic analysis arise from the inherent limitations of RNA (i.e., neuronal intractability, inherent variability in the level of target mRNA knock-down for different genes, possibility of off-target effects). If this proves too difficult, alternative loss of function experiments can be validated with a forward genetic screen using knockout mutants.
95
Generation of transgenic strains Screen and select Generate RNAi sensitive double transgenic models Identification of positive hits Validation of positive hits Obtain Screen and examine
Milestone 3. Suppressor chemical screens Potential difficuties and contingency plans Genetic crossing Forward genetic screens
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26.
27. 28.
47. 48.
Ved, R., Saha, S., Westlund, B., Perier, C., Burnam, L., Sluder, A., Hoener, M., Rodrigues, C. M., Alfonso, A., Steer, C., Liu, L., Przedborski, S., and Wolozin, B. (2005) J Biol Chem 280, 4265542668 Kuwahara, T., Koyama, A., Gengyo-Ando, K., Masuda, M., Kowa, H., Tsunoda, M., Mitani, S., and Iwatsubo, T. (2006) J Biol Chem 281, 334-340 Saha, S., Guillily, M. D., Ferree, A., Lanceta, J., Chan, D., Ghosh, J., Hsu, C. H., Segal, L., Raghavan, K., Matsumoto, K., Hisamoto, N., Kuwahara, T., Iwatsubo, T., Moore, L., Goldstein, L., Cookson, M., and Wolozin, B. (2009) J Neurosci 29, 9210-9218 Smann, J., Hegermann, J., von Gromoff, E., Eimer, S., Baumeister, R., and Schmidt, E. (2009) J Biol Chem 284, 16482-16491 Nass, R., Miller, D. M., and Blakely, R. D. (2001) Parkinsonism & Related Disorders 7, 185-191 Cao, S., Gelwix, C. C., Caldwell, K. A., and Caldwell, G. A. (2005) J Neurosci 25, 3801-3812 Settivari, R., LeVora, J., and Nass, R. (2009) J. Biol. Chem., M109.051409 Braungart, E., Gerlach, M., Riederer, P., Baumeister, R., and Hoener, M. C. (2004) Neurodegener Dis 1, 175-183 Caldwell, K. A., Tucci, M. L., Armagost, J., Hodges, T. W., Chen, J., Memon, S. B., Blalock, J. E., DeLeon, S. M., Findlay, R. H., Ruan, Q., Webber, P. J., Standaert, D. G., Olson, J. B., and Caldwell, G. A. (2009) PLoS One 4, e7227 Gutierrez-Zepeda, A., Santell, R., Wu, Z., Brown, M., Wu, Y., Khan, I., Link, C. D., Zhao, B., and Luo, Y. (2005) BMC Neurosci 6, 54 Yamanaka, K., Okubo, Y., Suzaki, T., and Ogura, T. (2004) J Struct Biol 146, 242-250 Parker, J. A., Arango, M., Abderrahmane, S., Lambert, E., Tourette, C., Catoire, H., and Neri, C. (2005) Nat Genet 37, 349-350 Bates, E. A., Victor, M., Jones, A. K., Shi, Y., and Hart, A. C. (2006) J Neurosci 26, 2830-2838 Locke, C. J., Fox, S. A., Caldwell, G. A., and Caldwell, K. A. (2008) Neurosci Lett 439, 129-133 Karpinar, D. P., Balija, M. B., Kugler, S., Opazo, F., Rezaei-Ghaleh, N., Wender, N., Kim, H. Y., Taschenberger, G., Falkenburger, B. H., Heise, H., Kumar, A., Riedel, D., Fichtner, L., Voigt, A., Braus, G. H., Giller, K., Becker, S., Herzig, A., Baldus, M., Jackle, H., Eimer, S., Schulz, J. B., Griesinger, C., and Zweckstetter, M. (2009) EMBO J 28, 3256-3268 Caldwell, G. A., Cao, S., Sexton, E. G., Gelwix, C. C., Bevel, J. P., and Caldwell, K. A. (2003) Hum Mol Genet 12, 307-319 Faber, P. W., Alter, J. R., MacDonald, M. E., and Hart, A. C. (1999) Proc Natl Acad Sci U S A 96, 179-184 Oeda, T., Shimohama, S., Kitagawa, N., Kohno, R., Imura, T., Shibasaki, H., and Ishii, N. (2001) Hum Mol Genet 10, 2013-2023 Wang, J., Farr, G. W., Hall, D. H., Li, F., Furtak, K., Dreier, L., and Horwich, A. L. (2009) PLoS Genet 5, e1000350 Lakowski, B., Eimer, S., Gbel, C., Bttcher, A., Wagler, B., and Baumeister, R. (2003) Development 130, 2117-2128 Gitler, A. D., Bevis, B. J., Shorter, J., Strathearn, K. E., Hamamichi, S., Su, L. J., Caldwell, K. A., Caldwell, G. A., Rochet, J. C., McCaffery, J. M., Barlowe, C., and Lindquist, S. (2008) Proc Natl Acad Sci U S A 105, 145-150 Briese, M., Esmaeili, B., Fraboulet, S., Burt, E. C., Christodoulou, S., Towers, P. R., Davies, K. E., and Sattelle, D. B. (2009) Hum Mol Genet 18, 97-104 Arya, U., Dwivedi, H., and Subramaniam, J. R. (2009) Exp Gerontol 44, 462-466
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6.
SUMMARY OF FINANCIAL SUPPORT REQUESTED Specify the funds required for staff, animals, consumables and travel in the Table below.
YEAR 2 13590
YEAR 3 13590
ANIMALS CONSUMABLES
10,000
10,000
10,000
TRAVEL TOTAL
1000 24590
1000 24590
1000 24590
7.
JUSTIFICATION OF FINANCIAL SUPPORT REQUESTED In this section, justify the funds requested for animals, consumables and travel (no more than 200 words).
The applicant will be working full time and participating in the experimental design, application and analysis. She has gained experience working with C. elegans in our
laboratory during three 10-week Masters-level rotation projects. The requested funding will cover the purchase for project-specific consumables such as routine chemicals and labware, the molecular biology techniques outlined above e.g. microinjections, primary culture of embryonic neurons, as well as obtaining knockout mutants. The collection of drug compounds required for chemical screening costs around 5000 if to be purchased from Sigma, therefore contributes to a significant portion of the total costs. In addition, there are also charges relating to use of communal facilities (i.e. confocal microscope). Finally, funds requested will cover travel expenses to attend and present research findings at relevant conferences.
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Attendance Monitoring
Punctual attendance at all scheduled activities in the MRes course is compulsory and will be monitored carefully. Note that a documented attendance record of at least 70% of all scheduled sessions is a pre-requisite for the award of Distinction. An attendance record of at least 50% is required to pass the MRes degree. Experience has shown us that students who are absent from scheduled activities are often having either academic or personal problems. We therefore monitor attendance closely to identify problems as early as possible. The University also has a responsibility to report international students with poor attendance to the UK Border Agency (UKBA) and this could lead to visa problems. Students will be asked to scan their student ID card at each scheduled activity to register their attendance. It is therefore important that students always carry their ID cards with them. If students forget their card, there is a temporary paper register available for signing. Students must scan their card or sign the register BEFORE the lecture commences. Students who arrive after the lecture has begun will not be able to scan/sign in and will be marked as absent. To avoid this, we request that students arrive 10 minutes before the scheduled start time (e.g., at 1.50 pm for lectures starting at 2.00 pm). Daily laboratory attendance from 9am is required for your research projects. Supervisors are required to monitor this and to report any unauthorised absence to the Programme Administrator and the Institute Senior Postgraduate Administrator. If any scheduled activity or lab work is missed for good reason (e.g. illness, family circumstances, medical appointment) students should complete an absence form no later than 5 days after the last day of absence (see below). Their attendance records will then be amended accordingly. Any student who displays poor attendance will be contacted by their strand convenor and required to explain their absence. If the situation is not resolved, the issue will subsequently be dealt with by the Programme Director, and the Institute Director of Postgraduate Studies.
Absence Reporting
If for any reason you need to be absent (e.g. other meetings, courses, illness, etc) you should inform your supervisor and strand convenor as soon as possible, at the latest by 9.30am on the day that you will be away from the lab, by telephoning or emailing them. You must provide information on when and why you will be absent, and ask him/her to make arrangements for any ongoing experiments that you cannot complete that day. You should also email the Programme Administrator to formally report your absence. If your absence is for 5 consecutive days or less, then you should use the Self-Certificates as detailed below (see items 1 and 2 below). Absences of longer than 5 consecutive days will require a Medical Certificate authorised by a medical practitioner (see item 3 below). Each of these three forms can be found in the Appendix at the back of this handbook. 100
1.
Self-Certificate of Absence caused by ILLNESS This can be used for a period of 5 consecutive days and does not require the signature of a medical practitioner. It should be submitted no later than 5 days after the last day of absence. a. b. c. d. Thoroughly complete the Self-Certificate of Absence caused by ILLNESS form. Obtain your supervisors signature. Obtain your strand convenors authorisation. Submit the form to the Programme Administrator.
2.
Self-Certificate of Absence caused by OTHER REASONS (i.e. attending meetings, hospital appointments, interviews). The form should be submitted no later than 5 days after the last day of absence. a. b. c. d. Thoroughly complete the Self-Certificate of Absence caused by OTHER REASONS form. Obtain your supervisors signature. Obtain your strand convenors authorisation. Submit the form to the Programme Administrator.
3.
Absence due to Illness VERIFIED BY A MEDICAL CERTIFICATE This form and accompanying medical certificate should be submitted no later than 5 days after the last day of absence. a. b. c. d. Thoroughly complete the Absence due to Illness VERIFIED BY A MEDICAL PRACTICTIONER form. Obtain your supervisors signature. Obtain your strand convenors authorisation. Submit the form and medical certificate to the Programme Administrator.
Mitigating Circumstances
If you feel that your performance has been adversely affected by circumstances beyond your control, such as illness etc, and wish this to be taken into account, you must fill out the appropriate form in this handbook. This form must be submitted to the Programme Administrator as soon as possible after the event and should be accompanied by all relevant documentation.
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Terms of Reference (i) (ii) (iii) All aspects of curriculum delivery; Resources provided for teaching and learning; Matters of general relevance and interest to the course.
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Section 10 APPENDIX
The following pages contain useful additional forms and information. A signed copy of the plagiarism form should be attached to all submitted written assignments (Research Project reports, Short Reviews, Referees Report, PhD studentship application). A copy of the appropriate absence reporting form, signed by you, your supervisor and your strand convenor, should be submitted to the Programme Secretary to formally report any absences. The mitigating circumstances form should be copied, completed and submitted to the Programme Administrator if you feel that your performance has been adversely affected by circumstances beyond your control such as illness etc and wish this to be taken into account. This form must be submitted as soon as possible after the event and should be accompanied by relevant documentation. The campus map shows the location of the various University buildings on campus.
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DECLARATION ON PLAGIARISM AND COLLUSION NAME.STUDENT NUMBER.... MODULE TITLE/CODE TITLE OF WORK. This form should be completed by the student and appended to any piece of work that is submitted for summative assessment. Section 8.1 of the Universitys Code of Practice on Assessment provides the following definition of plagiarism: Plagiarism occurs when a student misrepresents, as his/her own work, the work, written or otherwise, of any other person (including another student) or of any institution. Examples of forms of plagiarism include: the verbatim (word for word) copying of anothers work without appropriate and correctly presented acknowledgement; the close paraphrasing of anothers work by simply changing a few words or altering the order of presentation, without appropriate and correctly presented acknowledgement; unacknowledged quotation of phrases from anothers work; the deliberate and detailed presentation of anothers concept as ones own. Anothers work covers all material, including, for example, written work, diagrams, designs, charts, musical compositions and pictures, from all sources, including, for example, the internet, journals, textbooks and essays. You must also avoid self-plagiarism, i.e. re-using text, data or images that you have previously submitted for assessment. In other words, you are expressly forbidden from copying any such material from one of your written assignments to another. Section 8.1 of the Universitys Code of Practice on Assessment provides the following definition of collusion: Collusion occurs when, unless with official approval (e.g. in the case of group projects), two or more students consciously collaborate in the preparation and production of work which is ultimately submitted by each in an identical, or substantially similar, form and/or is represented by each to be the product of his or her individual efforts. Collusion also occurs where there is unauthorised co-operation between a student and another person in the preparation and production of work which is presented as the students own. Further information on plagiarism and collusion can be found in departmental/programme handbooks. Students found to have committed plagiarism or to have colluded in the production of work for assessment are liable to receive a mark of zero for the assessment concerned. Subsequent offences will attract more severe penalties, including possible termination of studies. STUDENT DECLARATION I confirm that I have read and understood the above definitions of plagiarism, self-plagiarism and collusion. I confirm that I have not committed plagiarism when completing the attached piece of work, nor have I colluded with any other student in the preparation and production of this work.
SIGNATUREDATE..
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ASSESSMENT(S) AFFECTED (TICK AS APPROPRIATE): my absence prevented my attendance at, or affected my submission of, the following assessed work.
Research Project Report submission Research Project Oral or Poster presentation Short Review submission Referees Report Submission IP & Comm. Workshop Business Plan PhD Studentship Application submission Final Portfolio submission ACTIVITY
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STUDENT: I certify that I was absent from the University through illness during the period:
Date from: My attendance resumed on: The reason for illness was:
Student Signature:
Date:
SUPERVISORS SIGNATURE:
Supervisors Name: Signature:
FOR OFFICE USE ONLY: Absence: [ ] Number of self-certificates submitted: 1[ ] 2[ ] 3[ ] 4[ ] more than 4 [ ]
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1 day [ ]
2 days [ ]
3 days [ ]
4 days [ ]
5 days [ ]
ASSESSMENT(S) AFFECTED (TICK AS APPROPRIATE): my absence prevented my attendance at, or affected my submission of, the following assessed work.
Research Project Report submission Research Project Oral or Poster presentation Short Review submission Referees Report Submission IP & Comm. Workshop Business Plan PhD Studentship Application submission Final Portfolio submission ACTIVITY
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STUDENT: I certify that I was absent from the University during the period:
Date from: My attendance resumed on: The reason(s) for my absence were:
SUPERVISORS SIGNATURE:
Supervisors Name: Signature:
FOR OFFICE USE ONLY: Absence: [ ] Number of self-certificates submitted: 1[ ] 2[ ] 3[ ] 4[ ] more than 4 [ ]
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1 day [ ]
2 days [ ]
3 days [ ]
4 days [ ]
5 days [ ]
ASSESSMENT(S) AFFECTED (TICK AS APPROPRIATE): my absence prevented my attendance at, or affected my submission of, the following assessed work.
Research Project Report submission Research Project Oral or Poster presentation Short Review submission Referees Report Submission IP & Comm. Workshop Business Plan PhD Studentship Application submission Final Portfolio submission ACTIVITY
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STUDENT: I certify that I was absent from the University through illness during the period:
Date from: My attendance resumed on: Student Signature: Date:
SUPERVISORS SIGNATURE:
Supervisors Name: Signature:
FOR OFFICE USE ONLY: Absence: [ ] Number submitted: 1[ ] 2[ ] 3[ ] 4[ ] more than 4 [ ] 1 day [ ] 2 days [ ] 3 days [ ] 4 days [ ] 5 days [ ] more than 5 days
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Modules affected by mitigating circumstances Module Code Module Name Assessment missed/affected
Details of mitigating circumstances Please provide a description of the mitigating circumstances that may have affected your performance in the above modules, including the time period over which these circumstances occurred. Please state what aspect(s) of the assessment you feel have been affected.
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Supporting documentation Please list all the documentation provided in support of your claim. The documentation should be stapled to this form. Medical claims should be supported by a medical note, other claims should be supported by appropriate documentation (for example, police reports, insurance reports).
Student declaration I confirm that all the information contained in this statement is accurate and complete to the best of my knowledge. I consent to the information being used by the Mitigating Circumstances Committee, and understand that the information will be treated in the strictest confidence. Signature of student:. Date:
FOR USE BY THE CHAIR OF THE MITIGATING CIRCUMSTANCES COMMITTEE ONLY I recommend that the following action be taken in respect of this claim:
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