February 2010 Issue 9 alzheimers.org.

uk/research

Research e-journal
Alzheimers Society scientific journal with lay versions of every article

Brain donation for dementia research

Brains for Dementia Research: Developing a gold standard in brain donation Professor Paul Francis Research into the cholinergic system: From brain bank, to bench, to bedside Professor Elaine Perry Biomarkers for Alzheimers disease - spinal taps, brain scans, blood tests and the critical role of brain donation Professor Simon Lovestone
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Editorial
Professor Clive Ballard, Academic Editor
Alzheimers Society, in partnership with the Alzheimers Research Trust, has invested substantially in establishing Brains for Dementia Research (BDR). BDR is an initiative to standardise procedures across a number of major UK brain banks with two major goals. Firstly, to ensure that donated tissue is available to the national and international research community, thus supporting world-class research, and secondly, to increase the number of people donating their brains to dementia research following their death and improve the support available to those individuals and their families. Research based on donated tissue was of course what led to the discovery of Alzheimers disease in the first place and also underpinned the development of the licensed drugs for the symptomatic treatment of Alzheimers disease that we now have available. In the current edition, articles by Professor Paul Francis and Professor Elaine Perry review further progress in these areas, looking at developments in our understanding of the pathology, biochemistry and molecular biology of Alzheimers disease and other dementias based upon autopsy research. Autopsy studies have been invaluable in enabling us to verify procedures for the clinical diagnosis of Alzheimers disease and will play a key role in identifying and validating biomarkers to improve diagnostic accuracy and to begin to diagnose Alzheimers disease before the onset of clinical symptoms. The emergence of new technologies such as proteomics and transcriptomics enable us to look at the alteration of proteins and gene expression in Alzheimers disease and other dementias, creating exciting opportunities for novel cutting-edge research. The implications for discovering better biomarkers and some of the research at the forefront of new technologies is reviewed in this edition by Professor Simon Lovestone. The creation of BDR has been one of the most important research contributions of Alzheimers Society. The reviews in this volume of the e-journal highlight the achievements and the potential of autopsy research to lead the way to a brighter future for people with Alzheimers disease and other dementias.

Each article in this e-journal is accompanied by a lay version which summarises the scientific versions without any technical language or need for any previous scientific knowledge. The lay versions are contributed by science writer Caroline Bradley, to whom we are very grateful for her expertise and hard work. Quick-read summaries are also included to provide the main points of each article.

Brains for Dementia Research: Developing a gold standard in brain donation

Professor Paul Francis, Dr Gillian Hayes* & Dr Richard Hudspith
Brains for Dementia Research, Wolfson Centre for Age Related Diseases, Kings College London, St Thomas Street, London SE1 1UL *Corresponding author email: Gillian.hayes@kcl.ac.uk The importance of brain donation
For over a century, from Alois Alzheimers initial work to the present day, examination of postmortem brain tissue has been associated with all the major milestones in dementia research. For example, although Alzheimer described plaques, it was not until the 1980s that the protein deposited in these structures, amyloid-beta, was identified. Treatments aimed at removing amyloid-beta have been validated in part by examining post-mortem brains from people receiving vaccination therapy. However, there is still much to learn and understand. We now know from post-mortem studies that there are a number of pathological patterns associated with dementia, and the term dementia describes a collection of cognitive impairments, of which Alzheimers disease is one. It is also recognised that for every 100 people diagnosed with dementia during life, 20 will not have pathology of dementia upon post-mortem. There is clearly a need for more accurate clinical diagnosis of the dementias so the search is on for biomarkers of the disease, but again, validation is dependent on post-mortem analysis, and on the continuing donation of brain tissue from people with and without dementia. Post-mortem brains have also been vital in the quest for new treatments for Alzheimers disease. The rationale for cholinesterase inhibitors such as Aricept was based on studies of human brain demonstrating reductions in the transmitter acetylcholine in Alzheimers disease in relation to cognitive decline. As with any group of allied conditions, it is unlikely that one drug will be effective across the whole disease. Treatments targeting specific, troublesome symptoms need to be developed. This highlights one critical research area observing symptoms during life and correlating them with post-mortem biochemical analysis. Current treatments have limited efficacy but are being given to a broad spectrum of memory impairments. The lesson from medical treatments elsewhere, such as cancer chemotherapy, is that in order to be effective treatments need to be specifically targeted. However, the development of better and more effective treatments is unlikely without increased availability of brain tissue for study.

Brains for Dementia Research is a brain banking initiative joint funded by Alzheimers Society and the Alzheimers Research Trust

Brains for Dementia Research

Brains for Dementia Research (BDR) is an initiative funded by the Alzheimers Research Trust and Alzheimers Society to address the shortage of brain tissue available for research through prospective recruitment. BDR has a network of five centres which recruit potential brain donors and then conduct assessments of memory, thinking, behaviour and daily living skills every one or two years until death, when the brain donation can be received. In this way, the highest quality of tissue is collected, with standardised clinical information, to enable the best research, essential to finding new generation treatments for this devastating and debilitating group of conditions. Tissues from individuals without a diagnosis of dementia are particularly in short supply. Some major brain tissue banks receive only one or two suitable donations per year. However, without normal control brains, establishing what is abnormal and what is normal is challenging. BDR registers people both with and without a diagnosis of memory impairment. BDR has developed a gold standard in brain donation procedure in order to provide researchers with fully categorised tissue with, where possible, a full medical history. The standardised donation and processing protocol means that donated tissue from BDR centres is uniformly treated, thus removing possible inconsistencies that arise from tissue that

has been treated in different ways. All BDR tissue is available to researchers on request when they register with the online database.

Raising the question of brain donation

Those in clinical practice are understandably concerned about approaching patients about brain donation. In the clinician-patient relationship it is very difficult to find the right time to raise the issue. However, if it is to be raised, arguably it is not ethical to consciously leave the topic until the memory-impaired individual cannot give consent for themselves. Our experience has been that people are willing to consider brain donation, and that large numbers of people without a diagnosis of memory impairment are also very open to it. Indeed at the BDR coordinating centre 70 per cent of our enquirers are non-memory impaired. What reasons are there for this surprising proportion? Many spouses or partners have an intimate understanding of how terrible dementia can be and what few treatment options are available. As a result they are very willing to participate in BDR as it gives them something they can do to fight this dreadful condition. Others feel it is something they can do together with the person affected. A number of potential donors have lost parents or other family members to the condition, and readily understand that brain donation, whilst it may not benefit their own generation, is a generous gift that could help fight the condition for their children. A number of control participants have been on the organ donor register for many years but are at an age where that is no longer applicable or they have a condition which prevents organ donation, yet are very keen to help others after their death. Overall, BDR has had a very positive response to discussing brain donation with those with and without a diagnosis of dementia.

is reasonably possible, to minimise tissue deterioration. Once the death certificate has been issued the brain bank can liaise with the family and their chosen funeral director, arrange for the body of the donor to be transported to a local mortuary for the post-mortem, and then returned to the funeral directors. Brain donation does not interfere with family funeral arrangements and does not prevent open casket viewing (donation of the spinal cord does involve an incision on either the front or back of the donors body, but this is not usually visible).

Clinical assessment of donors

The annual assessment of potential donors will be made by trained research nurses and old age psychiatrists visiting the participant at their place of residence, attending hospital, or by telephone contact The initial assessment is in the form of face-to-face interview, and takes about 90 minutes. Diagnostic information will include a structured baseline history , demographics and family history, and clinical dementia subtype diagnosis by operational criteria. The minimal clinical assessment criteria for individuals with a clinical diagnosis of dementia will include the following scales: Mini Mental State Examination (or if appropriate, Telephone Interview for Cognitive Status), Neuropsychiatric Inventory, Bristol Activities of Daily Living Scale, Geriatric Depression Scale and Clinical Dementia Rating. The Cornell Depression Scale may be used to assess mood. Normal aged controls will be similarly assessed, except that the Global Depression Scale will be used in place of the Cornell Depression Scale, and assessment will take place every other year. Some normal control participants may develop memory problems as indicated by the assessments. This is discussed with the participant and they can be advised appropriately. Development of dementia during the project does not affect participation.

Answers to practical questions surrounding brain donation

What happens when someone who has given consent for brain donation during life, dies? It is important that the brain bank is contacted as soon as possible they can all be contacted out of hours. The families have the contact information, and we ask that those involved in the end of life care for the person are also informed. Once informed, the brain bank staff can help the family and end of life carers/clinicians through the donation process. The brain banks are very experienced at dealing with bereaved families and the procedures following donor death. However, there is a need to proceed as quickly as

How can you help, and how can we support you?

Health and care professionals are key figures in helping us to recruit people for brain donation. This can be a difficult subject to broach but it has been our experience that there is much less reluctance to think about end of life events when it is nearer, both for those with and without memory impairment. BDR has developed information resources for use by health and care professionals to facilitate these discussions and provide key information for prospective donors. Our leaflets, web content and posters provide information about participating in the project and

about brain donation. Donor cards can be carried by participants, and made available to carers and family members so that those responsible for informing the brain bank of a death have the necessary contact information. Participants and their families/carers can also contact the research nurses with any queries they have. These materials have been prepared in consultation with lay people, including people with dementia and carers so the information is sensitive, clear and straightforward. We would encourage anyone working with people with dementia to contact us for materials or to visit our website to find out more.

Quick-read summary Brains for Dementia Research

Being able to examine brains after death is vital for improving understanding of what causes dementia and how best to treat it Brains for Dementia Research promotes brain donation and supports the development of brain tissue banks in England and Wales. Brain donation can be a difficult subject to broach, but BDR reports a high level of positive responses. Donations are needed from individuals who do not have dementia as well as people with the condition. Researchers can register for access to the online database and apply to receive tissue. All donated tissue is subject to a gold standard treatment, ensuring high quality tissue for research.

How can scientists access donated tissue?

Dementia researchers can register and access the tissue database online from the BDR website. Full details of clinical assessments and associated medical histories are available, as are details of the tissue preparation procedure.

Contacting BDR
Dr Gillian Hayes BDR Coordinating Centre, Wolfson Centre for Age Related Diseases, Kings College London, St Thomas Street, London SE1 1UL W www.brainsfordementiaresearch.org.uk T 020 7848 8377 F 020 7848 6515 E:bdr.office@kcl.ac.uk

Lay summary

Brains for Dementia Research: Developing a gold standard in brain donation

Our brains are complex and difficult to study. Being able to examine brains after death is a well established method of developing understanding of the underlying causes of dementia. Dementia is a term used to describe various different brain disorders that involve a loss of brain function and lead to symptoms such as the gradual loss of memory, communication skills and the ability to think and reason clearly. Post-mortem studies of brains have shown that there are different patterns to the type of damage in the brain that causes dementia.

The difficult subject of brain donation

Brain donation can be a difficult subject for clinicians to bring up with patients. From an ethical point of view it is important to address the issue at a time when someone with memory impairment is able to give informed consent. 70 per cent of the enquiries received by the BDR coordinating centre are from people without memory impairment on behalf of their partner or relation. Many are from the partners or family members of people with dementia who feel that donating their brain is something they can do to improve the treatments available and reduce the impact that dementia will have on other peoples lives in the future. In general BDR has experienced a positive response to the possibility of brain donation from individuals with and without dementia. Health and care professionals are vital for recruiting potential brain donors. BDR has developed a range of sensitive and straightforward information resources to assist them in this process.

Much to learn and understand

Matching information about how a persons brain functioned in life with what it looks like after death helps researchers to understand what type of damage in the brain is linked with particular symptoms of dementia. Being able to do this will, over time, help researchers to develop better, more targeted treatments. These are desperately needed, as the current situation is that treatments that have limited effects are given to a broad range of patients. Studying brain tissue helps researchers to understand: changes in the brain that cause different types of dementia how particular symptoms in life match with brain changes seen after death the effects that new anti-dementia drugs have on the brain how to diagnose dementia more accurately before death.

The practicalities
The brain bank needs to be contacted as soon as possible after someone who has consented to be a donor dies. The brain bank staff can then help the family or carers through the donation process. It is important to proceed as quickly as is reasonably possible to minimise tissue deterioration. Once the death certificate has been issued, the brain bank can liaise with the funeral director to organise removal of the brain and spinal cord. Donation does not interfere with funeral arrangements.

Brains for Dementia Research

Brains for Dementia Research (BDR) is a partnership between the Alzheimers Research Trust and Alzheimers Society to promote brain donation and develop a network of brain tissue banks in England and Wales for dementia research. BDR has developed a gold standard for brain donation procedures as well as the protocols to follow to provide researchers with material that has been classified and stored in the best ways possible. BDR collects and stores brain tissue from people over 65 who had no sign of dementia as well as people who had memory problems or dementia. Tissues from individuals without a diagnosis of dementia are generally in short supply. These donations of normal control brains are vital to establish what is and what is not normal in the ageing brain.

Gathering valuable information

BDR asks potential donors to go through a short assessment session every year for people with dementia and every two for people who do not have the condition. The assessment is a chance for BDR medical staff to capture information on each persons health, background, day-to-day life and any symptoms they might have. Linking these regular snapshots to the donated brain tissue after death provides the very best resource for researchers working to understand dementia. Further information about the BDR tissue database, including details of how researchers can register to use it are available at www.brainsfordementiaresearch.org.uk

Research into the cholinergic system in dementia: From brain bank, to bench, to bedside
Professor Elaine Perry
Institute for Ageing and Health, Newcastle University, Campus for Ageing and Vitality, Newcastle upon Tyne, NE4 5PL Correspondence: e.k.perry@newcastle.ac.uk The history of brain bank research in dementia
There have been milestones in the development of cholinergic therapy for people with dementia that, in retrospect, are both fascinating and surprising. Going back in history to the early 1970s there were just a few small private collections of formalin-fixed autopsy brain tissue for research in the UK (beyond those samples taken for diagnosis). Almost no one believed anything useful, like an active enzyme or receptor could be measured in frozen tissue, and research on Alzheimers disease (AD) was embryonic. Most researchers had never heard of the disease and thought it was something of little significance to do with general senility. By a curious coincidence three research groups almost simultaneously decided to store unfixed tissue as frozen material for biochemical studies, and to include measures of the cholinergic system amongst other transmitter signalling and related activities. Each independently found the same consistent abnormality in cerebral cortical tissue from individuals affected by the plaque and tangle hallmarks of Alzheimers disease (AD) loss of choline acetyltransferase (the enzyme that makes the transmitter acetylcholine). The groups published within a year of each other [1, 2, 3]. This was a striking development in brain pathology in dementia, a field with very few previous publications other than the historical papers published earlier in the century and the seminal clinico-pathological papers from a group in Newcastle which linked plaques and tangles to mental impairment for the first time [4, 5]. These neurochemical findings raised the prospect of a rational therapy for Alzheimer-type dementia for the first time since acetylcholine was already associated with memory function. The identification of a dopamine deficit in Parkinsons disease through frozen autopsy tissue led to effective therapy with the drug Levodopa [6]. This inspired many to pursue a similar objective in the AD area. At the same time the idea caught on that carefully stored autopsy brain tissue might retain activities which could provide a useful index of functional pathologies. Throughout the 1980s groups in Europe and the United States set up collections of frozen brain tissue from people with AD, other types of dementia and age-matched healthy controls. International meetings were called to reach agreement about how to optimally section (based on brain maps to identify standard, Brodmann cortical areas), freeze and fix tissue, and how best to store brain tissue for neurochemical analysis. This was the beginning of the idea that the private collections set up by local individuals could instead become brain banks open to use by any bona fide investigator. Research into AD-related dementias consequently took off with exponential numbers of papers appearing on topics from biochemical/ neurochemical to molecular/genotypically related activities. For the cholinergic system this included new enzymes, transporters and receptors variously affected in AD, linking such indices to pathological features and clinical measures before death [7]. Research linked neuronal loss from the nucleus of Meynert which projects cholinergic fibres to the cortex, particularly to the hippocampus [8]. Meanwhile, further studies observed that cholinergic deficits are not confined to AD and are even more extensive in Lewy body types of dementia (dementia with Lewy bodies or Parkinsons disease dementia) [9, 10]. This last discovery should perhaps have alerted those hoping for the kind of dramatic awakenings that were seen in people with Parkinsons disease treated with Levodopa. The fact that cognitive impairment is generally more severe in AD than the Lewy body dementia type, despite the extent of cholinergic deficits being the reverse, signalled that other non-cholinergic pathology was contributing to cognitive dysfunction in AD.

Analysing the efficacy of cholinergic therapies

The 1990s saw the first publications on the effects of cholinergic therapy in people with AD. Disappointingly, transmitter precursor treatment with choline or lecithin (analogous to Levodopa in Parkinsons) did not work. However, as more trials on cholinesterase inhibitors were undertaken (starting with tacrine), ranging from small open to large controlled trials, significant effects on

cognition, behaviour and quality of life were apparent, and continue to be so up to this time [11]. Comparison of tissue from Alzheimer and Lewy body dementias have helped substantiate the clinical impression that more clinically relevant responses to cholinesterase inhibitors are seen in people with Lewy body dementia [12]. Debates on funding, regulation and the development of other drugs such as memantine are modern history still in the making. While brain bank material undoubtedly underpinned the development of cholinergic therapy for AD, the hope that it might also help identify the characteristics of AD responders versus non-responders has not yet been realised and progress in this area has been limited. Using MRI however, Kanetaka et al. reported that responders had significantly greater atrophy of the substantia innominata [13]. There is accumulating autopsy tissue evidence that groups treated with cholinesterase inhibitors show reductions in pathology such as amyloid plaques [14], consistent with various reports of fewer abnormalities in treated compared to untreated patients subjected to neuroimaging. Some studies have even suggested that this treatment even leads to neurogenesis, the generation of neural stem cells [15]. This correlates with a wealth of data on animal models that these or other cholinergic drugs counter disease-related effects [16] and promote potentially regenerative mechanisms such as neurogenesis [17]. The latest paper on this topic demonstrates that a muscarinic agonist induces restoration of hippocampal neurogenesis and cognitive improvement in a model of cholinergic denervation [18].

being applied post-mortem and before brain dissection to establish links between disease changes earlier in the disease and at end stages when most material is collected for banking. Specific cholinergic markers that can be used in SPECT or PET imaging such as acetylcholinesterase have revealed that cholinergic deficits can be detected early in the disease using this enzyme as a marker [19]. These findings have not yet been incorporated into clinical practice, contrasting with dopamine transporter imaging to identify Lewy body types of dementia. Incidentally, beta CIT imaging was originally investigated as a result of differential activities of the dopamine transporter identified from autopsy brain tissue [20].

A tool for the future

As with cholinergic imaging, the search for other biomarkers such as cerebrospinal fluid (CSF) cholinesterases has yielded positive data that has not yet been applied clinically. These approaches may reach the clinic if more effective symptomatic or preventative cholinergic drugs are developed. Both CSF and blood samples collected during life are now often included in brain bank stores. This indicates a need to modify nomenclature, for example in Newcastle the Brain Bank name changed to Tissue Resource. Blood samples obviously also provide the opportunity for genetic analysis, although in many instances this can be performed using brain tissue samples. Genotyping of cholinergic molecular markers such as acetyl and butyryl cholinesterases or cholinergic receptors has generated some interesting but not yet definitive data on how genetic variants impact on disease manifestation and response to treatment. A key question concerning cholinergic pathology in AD continues to be whether this hallmark of the disease is a later stage development, so accounting for the modest therapeutic drug response, or whether it is a core feature that should be targeted by more effective cholinergic therapy. As indicated above, some imaging data suggests early pathology in the disease although collections of brain tissue across all the disease stages have indicated changes occurring only on reaching the early Braak 1 or 2 stages. These are necessarily cross-sectional studies and there is still a need to address this question in follow-up studies for example analysing biomarkers in presymptomatic familial AD families. New in vivo imaging evidence has established a link between degeneration of specific cholinergic compartments of the basal forebrain (which projects to the hippocampus and cerebral cortex) and cognitive-related deficits in subjects with MCI at high risk of developing AD [21].

Combining brain bank research with complimentary techniques

With respect to disease modelling, the early brain bank findings on the cholinergic system in AD led to a variety of models of cholinergic dysfunction, including surgical or toxin-induced lesions of the basal forebrain nucleus. These findings were in turn used to monitor the potentially therapeutic effects of cholinergic drugs. Much research today uses brain bank material in a reverse sequence often to establish if the human brain is affected by the same features as seen in animal models generated using manipulation of disease implicated genes. The cholinergic era in dementia research has been further enhanced by developing techniques that are not directly linked to brain banking although they have impacted on banking procedures. Neuroimaging, particularly techniques for structural imaging, has led to similar procedures

While the key presynaptic marker on the cortex, choline acetyltransferase, does not decrease until stage 2 [22] we have just obtained evidence that it is reduced at the earliest stage 1 in the brain areas where neurogenesis occurs [15]. Also, in Lewy body dementias this marker is affected at the earliest disease stages in the cerebral cortex. This debate is highly relevant to what is almost certainly the most important goal in dementia research developing a preventative strategy. Even if, as most assume, AD is so complex and variable in pathological contributions and cascade that no unitary mechanism or global therapy exists, ways of preventing degeneration of the cholinergic system per se would clearly be highly relevant. Here, brain bank tissue could again play a key role, answering key questions such as: In subgroups of normal elderly or people with mild cognitive impairment, what are the biological factors (modulators of plasticity, neurogenesis, apoptosis for example) and other factors (psychological, social, environmental) associated with the highest cholinergic activity? Do people with high amyloid burden but normal cognition have normal cholinergic systems? Moving beyond the realms of dementia there are an increasing number of other diseases or conditions of the central nervous system for which cholinergic therapy has been considered with some promising trial data, albeit often not extensive or necessarily placebo-controlled. These include: attention deficit disorder, autism and other developmental disorders, myalgic encephalopathy (ME), multiple sclerosis (MS), post-operative delirium, schizophrenia and others. Few of these applications have arisen as a result of brain tissue studies, exceptions being autism, with a marked nicotinic and muscarinic receptor loss [23], and schizophrenia with muscarinic receptor loss [24]. Brain bank material which exists for some of these diseases therefore holds great potential for identifying potential cholinergic pathologies.

Quick-read summary The cholinergic system

In the 1970s researchers, using brain bank tissue, discovered that the brains of people with Alzheimers disease all had low levels of a particular enzyme that was part of the cholinergic system, which helps to regulate memory and learning. The 1980s saw a boom in the development of brain banks as well as the level of research into Alzheimers disease and other forms of dementia. In the 1990s, results of the first cholinergic therapies were disappointing, but later trials of cholinesterase inhibitors revealed significant effects on cognition, behaviour and quality of life. Research into the role that the cholinergic system plays in dementia and other conditions is continuing and brain bank tissue continues to be a vital tool for researchers.

A personal note From Professor Elaine Perry

In the late 1970s my Director of Studies advised me to move my researches on from the cholinergic system (youve done that now). I did not take his advice and no doubt missed other opportunities. But from medical research into Alzheimers and other disorders of the brain, through explorations of medicinal plants as novel therapies to more ethereal subjects such as consciousness and shamanism, this simple molecule in all its system complexities has provided an immensely satisfying and absorbing career in research. None of this would have taken place without the establishment of a brain bank in Newcastle almost 40 years ago. And a final surprise is that even after all those years new papers are still appearing on the cholinergic system in dementia it has not all been done yet! 9

References
[1] Davies, P., Maloney, A.J. (1976). Selective loss of central cholinergic neurons in Alzheimer's disease. Lancet, 25;2(8000), p.1403.
[2] Smith, C.B., Bowen, D.M. (1976). Soluble proteins in normal and diseased human brain. J Neurochem, 27(6), pp.1521-8.

[3] Perry, E.K. et al. (1977). Neurotransmitter enzyme abnormalities in senile dementia. Choline acetyltransferase and glutamic acid decarboxylase activities in necropsy brain tissue. J Neurol Sci, 34(2), pp.247-65.
[4] Roth, M., Tomlinson, B.E., Blessed, G. (1966). Correlation between scores for dementia and counts of 'senile plaques' in cerebral grey matter of elderly subjects. Nature, 1;209(5018), pp.10910. [5] Roth, M., Tomlinson, B.E., Blessed, G. (1967). The relationship between quantitative measures of dementia and of degenerative changes in the cerebral grey matter of elderly subjects. Proc R Soc Med, 60(3), pp.254-60. [6] Bernheimer, H., Birkmayer, W., Hornykiewicz, O. (1966). [Homovanillic acid in the cerebrospinal fluid: studies in Parkinson's syndrome and other diseases of the CNS]. Wien Klin Wochenschr, 10;78 (23), pp.417-9. [7] Perry, E.K. et al. (1978). Correlation of cholinergic abnormalities with senile plaques and mental test scores in senile dementia. Br Med J, 25;2(6150), pp.1457-9. [8] Whitehouse, P.J. (1987). Clinical and neurochemical consequences of neuronal loss in the nucleus basalis of Meynert in Parkinson's disease and Alzheimer's disease. Adv Neurol, 45, pp.393-7. [9] Perry, E.K. et al. (1990). Cholinergic and dopaminergic activities in senile dementia of Lewy body type. Alzheimer Dis Assoc Disord, 4(2), pp.8795. [10] Ruberg, M., Ploska, A., Javoy-Agid, F., Agid, Y. (1982). Muscarinic binding and choline acetyltransferase activity in Parkinsonian subjects with reference to dementia. Brain Res, 232(1), pp.129-39. [11] Pepeu, G., Giovannini, M.G. (2009). Cholinesterase inhibitors and beyond. Curr Alzheimer Res, 6(2), pp.86-96. [12] Liberini, P., Valerio, A., Memo, M., Spano, P.F. (1996) Lewy-body dementia and responsiveness to cholinesterase inhibitors: a paradigm for heterogeneity of Alzheimer's disease? Trends Pharmacol Sci, 17(4), pp.155-60. [13] Kanetaka, H. et al. (2008) Prediction of response to donepezil in Alzheimer's disease: combined MRI analysis of the substantia innominata and SPECT measurement of cerebral perfusion. Nucl Med Commun, 29(6), pp.568-73.

[14] Ballard, C.G. (2002) Advances in the treatment of Alzheimer's disease: benefits of dual cholinesterase inhibition. Eur Neurol, 47(1), pp.6470. [15] Perry, E.K., Ballard, C., McKeith, L., Johnson, M. (In prep). Cholinergic therapy associated with increased progenitor activity in lewy body types of dementia. [16] Muoz-Torrero, D. (2008). Acetylcholinesterase inhibitors as diseasemodifying therapies for Alzheimer's disease. Curr Med Chem, 15(24), pp.2433-55. [17] Kotani, S., Yamauchi, T., Teramoto, T., Ogura, H. (2008). Donepezil, an acetylcholinesterase inhibitor, enhances adult hippocampal neurogenesis. Chem Biol Interact, 175(1-3), pp.227-30. [18] Van Kampen, J.M., Eckman, C.B. (2009). Agonist-induced restoration of hippocampal neurogenesis and cognitive improvement in a model of cholinergic denervation. Neuropharmacology, Epub in Press. [19] Herholz, K. (2008). Acetylcholine esterase activity in mild cognitive impairment and Alzheimer's disease. Eur J Nucl Med Mol Imaging, 35 Suppl 1:S25-9. [20] Piggott, M.A. et al. (1999). Striatal dopaminergic markers in dementia with Lewy bodies, Alzheimer's and Parkinson's diseases: rostrocaudal distribution. Brain, 122 ( Pt 8), pp.1449-68. [21] Grothe, M. et al. (2009). Reduction of Basal Forebrain Cholinergic System Parallels Cognitive Impairment in Patients at High Risk of Developing Alzheimer's Disease. Cereb Cortex, Epub ahead of print. [22] Pedersen, W.A., Guo, Q., Hartman, B.K., Mattson, M.P. (1997). Nerve growth factorindependent reduction in choline acetyltransferase activity in PC12 cells expressing mutant presenilin-1. J Biol Chem, 5;272(36), pp.22397-400. [23] Perry, E.K. et al. (2001). Cholinergic activity in autism: abnormalities in the cerebral cortex and basal forebrain. Am J Psychiatry, 158(7), pp.105866. [24] Dean, B., Bymaster, F.P., Scarr, E. (2003). Muscarinic receptors in schizophrenia. Curr Mol Med 3(5), pp.419-26.

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Lay summary

Research into the cholinergic system in dementia: From brain bank, to bench, to bedside
In the 1970s there were only a few, small collections of preserved brain tissue available for research in the UK. Researchers generally had little interest in using such tissue to explore the underlying causes of dementia. However, by curious coincidence three research groups decided to have a go. Each group independently found the same thing: that the brains of people with Alzheimers disease all had low levels of a particular enzyme, called choline acetyltransferase, that is responsible for making the neurotransmitter acetylcholine. The system of brain cells that uses acetylcholine is known as the cholinergic system and its role is to help regulate memory and learning. These findings suddenly raised the prospect of a possible treatment for Alzheimers disease. Discovering that people with Parkinsons disease had low levels of dopamine in the brain had paved the way to an effective treatment (with Levodopa in 1968) and there was real hope that these findings would lead to a similar development for Alzheimers. cholinesterase inhibitors for people with different types of dementia, although what makes some people with Alzheimers disease more likely to respond to treatment than others remains unclear. There is evidence that the treatments can reduce physical signs of dementia and might even help to generate new cells. Evidence from animal models supports these ideas..

Brain tissue plus

Observing the damaged cholinergic system in human brains led to the development of animal models that enabled researchers to test the effects of cholinergic drugs. Researchers continue to crosscheck what they see in genetic animal models of the condition with human tissue held in brain banks. Neuroimagaing has provided brain tissue researchers with a new tool. Structural imaging can provide valuable data post-mortem, before dissection. Also, scans that measure levels of the enzyme acetylcholinesterase show that it falls below normal levels at an early stage of the disease. It is possible to measure levels of cholinergic chemicals in the cerebrospinal fluid (CSF) of people with dementia. This has yet to be put to clinical use, but it is important for brain banks to collect CSF and blood to widen the scope of future research. Although genetic analysis can be carried out on brain tissue samples, blood samples provide a valuable alternative.

Brain bank boom

In the 1980s support grew for the idea that carefully stored brain tissue could provide valuable evidence about the causes and development of dementia. Groups in Europe and the US established collections of brain tissue from people with all types of dementia and age-matched healthy controls. Researchers met to agree international rules for preparing and storing the tissue to ensure its usefulness. There was also a huge increase in levels of dementia research, which really did lift the lid on what was going on inside the brain when the condition developed. For the cholinergic system this included the discovery of new enzymes and other chemicals involved in the communication between cells as well as improved understanding of how they systems cells became damaged. Research also revealed that people who developed Lewy body dementias had more damaged cholinergic systems than people with Alzheimers disease.

Sooner rather than later?

A key question is whether problems with the cholinergic system develop at an earlier or later stage of dementia. Some imaging data suggests earlier, but brain bank tissue collected at all stages of the disease suggests changes tend to happen later. Researchers continue to debate this argument and seek new evidence. If degeneration of the cholinergic system is found to be a fundamental cause of dementia it would need to be targeted by better treatments than those currently available. In any case, the ability to prevent degeneration of the cholinergic system would be an important advance.

Analysing cholinergic therapies

First results of the effects of cholinergic therapy were published in the 1990s. Initial work on transmitter precursor treatment proved disappointing, but later trials of cholinesterase inhibitors revealed that they could have significant effects on cognition, behaviour and quality of life. Brain bank tissue can provide information on the physical effects of treatment with

A wider view
There is some evidence that cholinergic therapies could help in a range of other conditions, including autism, attention deficit disorder, multiple sclerosis (MS) and myalgic encephalopathy (ME). Brain bank material that exists for these conditions needs to be explored to reveal whether the cholinergic system does play a role.

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Biomarkers for Alzheimers disease spinal taps, brain scans, blood tests and the critical role of brain donation
Professor Simon Lovestone
Professor of Old Age Psychiatry, NIHR Biomedical Research Centre for Mental Health, King's College London, Institute of Psychiatry, De Crespigny Park, London SE5 8AF
Corresponding author email: simon.lovestone@kcl.ac.uk

Suppose you have memory problems and go to your doctor what happens next? At best, and the best is not always achieved, then an assessment of memory and other cognitive function is made and, in some cases, a referral is made to a memory clinic where there may be more memory tests and perhaps a brain scan. If the outcomes of these tests are not severe enough to warrant a clear-cut diagnosis then, usually, the cognitive tests are repeated after a year to see if they have got worse. This is unsatisfactory as the wait must seem interminable to patients and their relatives. For professionals too, not being able to make an early diagnosis is frustrating. When treatments for Alzheimers disease (AD) go beyond symptomatic treatment to therapies for the illness itself, this wait will be unacceptable since it is in this early phase, before dementia is established, that the drugs are most likely to be effective. This, then, is one of the most important drivers for research into biomarkers of AD. A biomarker is a biological signal that can be detected for diagnosis, ideally very early and before doctors are currently able to diagnose the condition. Biomarkers are also useful as the basis of tests for measuring how a disease is progressing. This latter use of biomarkers would be especially useful in research to find new treatments for dementia. Currently clinical trials rely almost entirely on memory tests which are sometimes less reliable than we would like as measures of disease. A biomarker that reflected the disease progression in the brain would be immensely useful as a measure against which to judge new treatments.

and tau is the protein that forms tangles. Interestingly in the spinal fluid amyloid levels are reduced in Alzheimers disease and tau levels increase. It is thought that this is because less amyloid reaches the spinal fluid as it gets bound up in the plaques, and more tau reaches the fluid because the nerve cells are dying and releasing their tau protein. Whatever the explanation, the finding has been very widely reproduced and a test has been produced that clinicians can use to measure amyloid and tau in spinal fluid. This test looks very promising as an early biomarker of dementia. It is now being incorporated into clinical trials of new drugs to prevent amyloid build-up and tangle formation and some evidence is beginning to suggest that it is proving to be a useful marker in these drug trials. This is all hugely encouraging and an international collaborative group is now working to ensure that the test is used in the same way, and gives the same results, in laboratories across the world. However, collection of spinal fluid has disadvantages due to patient discomfort and some minor side-effects. An alternative approach is brain scans. These are used today mostly to exclude other brain disease that might cause memory problems. However, new scans and techniques for analysing data have been introduced. The new analytical techniques use computer algorithms to measure shrinkage in the brain in different regions. This is a hugely complex task and in the past could only be done manually. It is now possible to do it in an automated fashion and such analysis, pioneered by Professor Nick Fox and others in London, is now being used to measure the effects of novel treatments in clinical trials. At the Maudsley Hospital in London we have just begun to use these automated analyses of brain scans in our routine clinical work. Other approaches to biomarkers are using imaging technologies to directly measure the amount of amyloid plaques in the brain in living patients. This is known as amyloid-PET (positron emission tomography) and involves the injection into the bloodstream of a chemical that is radioactive for a very short time and seeks out and binds to amyloid deposits. Other kinds of PET imaging have revolutionised the measurement of

Spinal taps and brain scans

Today, we do not have any biomarkers widely accepted either as diagnostic markers or as progression markers. But there is a huge effort underway to discover markers either in spinal fluid, using brain scans or in blood. Spinal fluid is perhaps the best place to start and nearly ten years ago researchers found that two proteins amyloid and tau are changed in the spinal fluid of people with Alzheimers. Amyloid is the protein that forms plaques in the brain in people with AD

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some cancers and are routinely used in diagnosis and in evaluating the success of treatments. It is very exciting seeing this technology applied to the brain in Alzheimers disease. However, the limitation is that PET scanners are few and far between and the short-lived radioactive isotopes currently used are so short-lived that the chemists need to make them no more than half an hour or so before they are used. Research is underway to find other, slightly longer-lived probes that would make amyloid-PET imaging more widely available.

problems go on to get Alzheimers in the next year, based on a blood test and a brain scan.

The future of biomarker research

These findings are very encouraging indeed. They suggest that in the future the person worried about their memory who goes to the doctor will not face the prospect of a years wait to see if it gets worse before a diagnosis is made, but will instead be able to have a blood test, a spinal fluid test or a brain scan (or perhaps all three) which will help the doctor come to a much faster diagnosis very early in the course of the illness. However, we are not quite there yet and more research needs to be done. Specifically, we need larger, longer term studies involving more people donating blood and spinal fluid samples. We need to discover ever better and more accurate biomarkers and in particular we need to work harder to convert scientific discoveries into useable tests. This has been something of a gap in research until recently but large funding agencies, including the Medical Research Council and the National Institute of Health research in the UK, have recognised this and have put more effort and funding into this translation gap. Not just funding but volunteers are essential for this research and we all have reason to be grateful to the many who have contributed to our cohort studies over the years and agree, year after year, to give up their time and come and have another assessment and another blood test. There is one particular kind of cohort study that is hugely valuable in the search for biomarkers and that is the so-called prospective study where volunteers are assessed over time in life and then donate their brain after death. Through the Brains for Dementia Research programme we have been able to show that one of our biomarkers (lets call it biomarker A) is specifically associated with plaques in the brain and that the amount of this protein in blood in life matches the amount of the protein in the brain after death. In another study we find a different protein in the blood (biomarker B), although clearly associated with plaques in the brain, does not show the same blood-protein matching. These are rare and special studies. They allow us not only to find biomarkers but to begin the difficult process of asking why they are biomarkers. We are able to conclude that biomarker A is acting in the brain but biomarker B is acting in the blood. As both affect amyloid we think biomarker A stops plaques from forming but biomarker B draws amyloid out of the brain and into blood where it can do no harm. Armed with this knowledge we can begin to look for other proteins that do the same and then even plan

A blood biomarker for Alzheimers

Our own research has focused on trying to find a biomarker for Alzheimers disease in blood. When we started this work, nearly ten years ago, it seemed unlikely that there would be such a marker; after all, the brain is separated from the blood by a nearly impenetrable membrane. Despite this, because most biomarkers in medicine are blood-based and because getting blood samples analysed is so routine in healthcare, we persevered and were funded to do so by both Alzheimers Society and the Alzheimers Research Trust. We employed proteomics to look for protein differences in the blood of people with Alzheimers and elderly controls. Somewhat to our surprise we did find some differences and went on to confirm these in quite a large group of people. We published this finding and then an Alzheimers Society fellow, Dr Madhav Thambisetty, showed that the changes we see in the blood in proteins called complement factor H (CFH) and alpha-2macroglobulin (A2M) matched changes in the function of the brain in people not only with Alzheimers but also those with pre-Alzheimer states such as mild cognitive impairment. This is very encouraging as it suggests that these proteins act as very early markers of Alzheimers disease pathology. Encouraged by this finding we have concentrated all our efforts in the last year or two on finding more very early markers of AD pathology. We have found one protein that is consistently increased in blood very early in the disease process and is increased especially in people who go on to decline quickly. We have been able to study mice with Alzheimers pathology and find this protein is increased in the blood here also and, in collaboration with Madhav, who is now working at the National Institutes of Ageing in the US, we have shown our protein is increased in people with large amounts of amyloid as shown by PET. This takes us ever closer to a blood-based biomarker for Alzheimers. Our preliminary findings suggest we can be more than 90 per cent accurate in predicting which patients with mild memory

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studies designed to find new treatments based on this knowledge. There are very few studies anywhere in the world that are able to combine approaches in this way. By establishing cohorts of people assessed in research in life and then donating their brains to research after death, Brains for Dementia Research looks well set to make a huge contribution to the search for biomarkers for better and earlier diagnosis as well as contributing to our understanding, and hopefully one day, to treatment of Alzheimers disease.

Quick-read summary Biomarkers of Alzheimers disease

A biomarker is an indicator that can reveal whether someone has a disease or how far a disease has progressed. There is an urgent need to develop accurate tests that can diagnose Alzheimers disease and other forms of dementia at an early stage. Researchers are currently validating a test that can detect Alzheimers disease by measuring changes in levels of proteins in cerebrospinal fluid. Brain scans and blood tests offer other possible routes to a reliable test and good progress is being made. Biomarker research is greatly assisted by large-scale cohort studies and is particularly enhanced by access to comprehensive data on peoples health in life complemented by access to their brain tissue after death.

References
Huang, H.C., Jiang, Z.F. (2009). Accumulated amyloid-beta peptide and hyperphosphorylated tau protein: relationship and links in Alzheimer's disease. J Alzheimers Dis, 16(1), pp.15-27. Mattsson, N., Blennow, K., Zetterberg, H. (2009). CSF biomarkers: pinpointing Alzheimer pathogenesis. Ann N Y Acad Sci, 1180, pp.28-35. Barnes, J., Ourselin, S., Fox, N.C. (2009). Clinical application of measurement of hippocampal atrophy in degenerative dementias. Hippocampus, 19(6), pp.510-6. Lovestone, S. et al. (2007). Proteomics of Alzheimer's disease: understanding mechanisms and seeking biomarkers. Expert Rev Proteomics, 4 (2), pp.227-38. Lovestone, S., Francis, P., Strandgaard, K. (2007) Biomarkers for disease modification trials--the innovative medicines initiative and AddNeuroMed. J Nutr Health Aging, Jul-Aug;11(4), pp.359-61. Thambisetty, M. et al. (2008). Proteome-based identification of plasma proteins associated with hippocampal metabolism in early Alzheimer's disease. J Neurol, 255(11), pp.1712-20. Hye, A. et al. (2006). Proteome-based plasma biomarkers for Alzheimer's disease. Brain, 129(11), pp.3042-50.

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Lay summary

Biomarkers for Alzheimers disease spinal taps, blood tests and the critical role of brain donation
Patients, medics and researchers are all crying out for a clear and measurable way of identifying the early stages of Alzheimers disease. The technical term for something that enables you to know whether or not you have a disease or are likely to develop one is a biomarker. A biomarker can also tell us how far a disease has progressed. At the moment, the lack of a clear-cut test can make the process of diagnosis long, worrying and wearisome for everyone involved. However, such a test will be vital once treatments become available that can tackle Alzheimers disease at the very earliest stages, even before symptoms start to show. Finding a biomarker that can reveal whether the disease is progressing would also be of huge benefit to medical researchers, enabling them to accurately measure the difference that possible new treatments make to the condition, rather than having to rely on sometimes unreliable memory tests. PET scans can measure the level of amyloid deposits in the brain by tracking the movement of radioactive chemicals that seek out and stick to the deposits. This technology is routinely used in the diagnosis and measurement of some cancers. Unfortunately PET scanners are not widely available so there are limits on this technique.

Blood biomarkers
Researchers at the Institute of Psychiatry have identified blood protein differences between people with and without Alzheimers disease. Changes in two specific blood proteins have been matched with changes in the function of the brain in people with Alzheimers, but also those with states which can sometimes lead to dementia such as mild cognitive impairment. This suggests that these proteins act as very early markers of Alzheimers disease. Further research has identified one protein that is consistently increased in blood very early in the disease process and is increased especially in people who go on to decline quickly. Early results suggest that combining analysis of a blood test and a brain scan can be more than 90 per cent accurate in predicting which patients with mild memory problems go on to develop Alzheimers within the following year.

Biomarks, get set, go!

The race to discover reliable markers in spinal fluid, blood or by using brain scans is now on. So far spinal fluid has proved to be the most successful area. Almost ten years ago researchers found that levels of two proteins were different in the spinal fluid of people with Alzheimers disease. Amyloid protein levels went down, whilst the tau protein went up. A test to measure these changes is currently being used in clinical trials of new drugs for Alzheimers disease. Initial results are promising and researchers are working together to establish an international protocol to ensure the test is used consistently. Unfortunately, testing spinal fluid can be uncomfortable for patients and carries the risk of sideeffects. It also needs to be carried out by a doctor in a hospital so isnt suitable for a primary care service.

Testing times
Although progress is being made in developing accurate and reliable biomarkers, there is a clear need for more research lab discoveries to be pursued and converted into useable tests. New funding is being made available to fill this translation gap. Large-scale prospective cohort studies are also needed. These involve the collection of information about a group of people with the same condition over time. When people offer to donate their brain after death it adds a valuable dimension to a cohort study. Matching data from life with details obtained from the brain after death enriches the evidence that investigators can explore. For example, the Institute of Psychiatry researchers used information and brain tissue samples from Brains for Dementia Research to make definitive links between levels of specific proteins in the blood in life and levels of amyloid plaques in the brain after death. The data and samples made available to researchers by Brains for Dementia Research will make a substantial contribution to the search for biomarkers for better and

Another route
Brain scans offer another possible route to an effective biomarker. They can detect changes in the brain such as the loss of cells from a particular region. Professor Nick Fox is part of a team at University College London that has pioneered the idea of using a pattern-spotting computer programme to diagnose Alzheimers disease. The programme can identify the typical pattern of damage to the brain caused by Alzheimers disease that shows up on MRI scans. This technique is now being used to measure the effects of new treatments in clinical trials.

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Call for Proposals

Scientists, clinicians and healthcare professionals can now apply for our research grants into the cause, cure, care and prevention of all types of dementia. Project grants In collaboration with the Bupa Foundation

Contacts
Academic Editor / Director of Research Professor Clive Ballard Head of Research Dr Susanne Sorensen T 020 7423 3600 Commissioning Editor / Research Communications Officer Dr Anne Corbett T 020 7423 3609 E anne.corbett@alzheimers.org.uk Scientific Liaison Officer Dr James Pickett T 020 7423 3607 E james.pickett@alzheimers.org.uk Executive Administrative Officer Brett Kerrigan T 020 7423 3603 E brett.kerrigan@alzheimers.org.uk General enquiries research@alzheimers.org.uk Research funding enquiries grantenquiries@alzheimers.org.uk Website alzheimers.org.uk/research

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research@alzheimers.org.uk

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