49
focl4S
Biotechnol. 21,394~96
71 Gold, H. M., Glenn, J. K. and Alice, M. (1988) in Methods of Enzy-
mology, Vol. 16IB, Lignin, Pectin and Chitin (Wood, W. A. and
Kellog, S. T., eds), pp. 74-78, Academic Press
72 de Jong, E., de Vries, F. P., Field, J. A., van der Zwan, 1L. P. and
Alzheimer's disease- from cause
to cure?
The tools of molecular biology have only been applied to the study of the brain
since the early 1980s, but it is already apparent that this approach holds promise
for identifying strategies and drugs that are able to modify the activity and function
of particular neural pathways. One of the greatest prizes - both for society and
commercially-will be the discovery of a safe and effective drug to treat Alzheimer's
disease.
The incidence o f Alzheimer's disease (AD) is high
compared with that o f other neurodegenerative dis-
eases, such as Huntington's disease (HD) and motor
neurone disease (MND). Throughout the world,
-10% of people in their 70s and 30% o f people in their
80s suffer from AD. In the U K alone, this amounts to
-500 000 people - nearly as high as the combined
incidence of all forms o f cancer. As the proportion of
elderly people in the populafon rises, the incidence of
AD will rise, and could be doubled by the year 2050.
A further incentive for finding effective prevent-
ative, or therapeutic treatments for AD is the distress-
ing nature o f the disease for both the affected indi-
vidual and their relatives since, as Brian Anderton
(Institute o f Psychiatry, London, UK) explains;
"There is a relentless erosion of a whole range of men-
tal facilifes, including memory, and everyday skills like
making tea or tying shoelaces. It progresses to a loss of
social behaviour and the disappearance of personality".
The relentless decline o f affected individuals occurs
over five to 15 years following the initial onset of the
disease.
Identifying therapeutic targets
Attempts to find potential targets for therapeutic
intervention need to focus on the primary defect that
is responsible for initiating the neural degeneration.
Alzheimer's disease is characterized by widespread
neuronal dysfunction in the brain (Fig. 1); the appear-
ance ofproteinaceous fibrils intraneuronally; and the
progressive deposition of amyloid, both intra-
G. Vines is afieelance sdence writer.
© 1993, ElsevierScience Publishers Ltd (UK)
de Bont, J. A. M. (1992) Mycol. Res. 96, 1098 1104
73 deJong, E., Field, J. A., Dings, J. A. F. M., Wijnberg, J. B. P. A. and
de Bont, J. A. M. (1992) FEBS Lett. 305, 220-224
74 Kelley, R. L., Ramasamy, K. and 1<eddy, A. (1986) Arch. Microbiol.
144, 254-257
Gail Vines
neuronally and, as amyloid plaques, extracellularly and
in blood vessels. There are two characteristic and diag-
nostic degenerative changes detectable in brain biop-
sies o f individuals who have died of AD. One of these
is the presence of'senile plaques', aggregates o f cellu-
lar debris with a core composed olin-pleated sheets of
amyloid proteins, scattered among the dying nerve
cells (P,.ef. 1; Fig. 2). The second is the presence of
neurofibrillary tangles within dying nerve cells. These
tangles result from the accumulation of cross-linked
fibrils termed paired-helical filaments. While the exact
composition o f these fibrils remains controversial, one
component has been identified as a modified form of
the protein t a u 2. In normal nerve ceils, tau forms part
o f the cytoskeleton, a complex network found
throughout the cytoplasm of eukaryotic ceils, which
is involved in coordinating the movement of
organelles and molecules within the cell. This trans-
port system fails if the cytoskeleton is disrupted by the
formation o f paired-helical filaments. The tau protein
appears to play a key role in regulating cytoskeletal
assembly - a function that depends, in part, on its
phosphorylation state. There is some evidence that tau
is hyperphosphorylated in sufferers o f AD. The dis-
ease is also characterized by deficits in the brain of
neurotransmitters, enzymes involved in their
metabolism and neurotransmitter receptors 3.
What, therefore, is the primary defect? In the past
year or so, there has been a growing consensus that
amyloid plaques are the cause, rather than the conse-
quence, of neuronal death, with the first step in the
developing pathology being the accumulation o f
[3-amyloid protein ([3-AP) in neurons. If this is true,
TIBTECHFEBRUARY1993 (VOL 11)
 5O
feature
Figure 1
Positron emission tomography (PET) scan of brain from a 75-year old patient with
Alzheimer's disease, four to five years after onset of the disease, The scan of the
localization of a radioactive tracer reveals variations in metabolic activity within the
brain - degeneration of neural tissue results in decreased metabolic activity. Gamma
rays emitted during annihilation of positrons from the tracer are recorded and colour-
coded according to intensity. This scan indicates that brain activity is confined to the
upper and lower regions of the image; dark blue/black areas represent virtually no
activity. (PET scan image from Science Photo Library, London, UK.)
blocking the pathways which lead to the synthesis or
deposition of [3-AP might cure the disease, or at least
prevent further deterioration.
~-amyioid protein
Since deposition of[3-AP in the core region of senile
plaques is a key feature of AD, much research is
directed at understanding the metabolic pathways
leading to production of this protein. [3-amyloid is a
small polypeptide fragment, of approximately 40
amino acids which is generated by proteolytic cleav-
age of a much larger, membrane spanning protein, the
[3-amyloid precursor protein ([3-APP) 4.
Inherited Alzheimer' s disease
To date, the best evidence for the involvement of
[3-AP in neuronal death comes from studies of human
genetics. In 1987, several groups mapped the gene
encoding [3-APP to chromosome 21. This was of par-
ticular interest since people with Down's syndrome
(caused by aberrant inheritance of an extra copy of
chromosome 21), usually develop [3-AP deposits and
exhibit neuronal degeneration and dementia resem-
bling that of AD, between the ages of 20 and 30. The
TIBTECHFEBRUARY1993 (VOL11)
additional copy of the [3-APP gene carried on the
extra chromosome of people with Down's syndrome
may result in the production of higher levels of
[3-APP. However, studies of other forms of hereditary
(familial) AD (FAD), which strikes people in their 40s
and 50s (and is less common than late-onset AD),
failed to reveal either defects or duplications in the
[3-APP gene, or linkage of AD with defects in chromo-
some 21. It would appear, therefore, that although
deposition of [3-AP is the common metabohc end-
result, AD is a genetically heterogeneous disease.
However, a breakthrough came in 1991 when, in
the search for the genes responsible for FAD, John
Hardy and colleagues (St Mary's Hospital Medical
School, London, UK) discovered a link between a
mutation in the [3-APP gene and AD in a few famil-
ies in which the mutation is inherited as an autosomal
dominant. The 'Hardy' mutation results in substi-
tution of isoleucine for valine at codon 717 in the
[3-APP protein s. This, at least, was demonstration
of a specific cause of AD at the molecular level, and;
"... that aberrations in the amyloid precursor protein
are sufficient, if not necessary" for AD. Eight variant
sequences of [3-APP have now been reported 6. It is
likely that mutations in other genes also result in AD.
Indeed, Gerard Schellenberg (University of Washing-
ton, Seattle, WA, USA), has recently linked a defect
on chromosome 14 to an inherited form of early-onset
AD. As yet, the gene responsible has not been ident-
ified, but it is suspected to be related to [3-APP syn-
thesis and processing7.
Amyloid processing and cellular pathology
Despite these advances in identifying genetic defects,
progress in correlating such defects with changes in
cellular metabolism, and understanding the role of
amyloid proteins in the pathology of the disease has
been slow - elucidating the metabolic pathways lead-
ing to the abnormal deposition of [3-AP is proving
more difficult than expected. Intact [3-APP does not
appear to damage cells but, as yet, a role in normal
cellular function has not been ascribed to it. [3-APP is
embedded in the nerve-cell membrane, with approxi-
mately one-third of the [3-amyloid fragment buried in
the membrane, and the remainder projecting outside
the cell. Sam Sisodia and colleagues (Johns Hopkins
Medical School, Baltimore, MD, USA) s and shortly
after, Fred Esch and co-workers (Athena Neuro-
sciences, South San Francisco, CA, USA) 9 showed
that in normal cells ~3-APP is cleaved proteolytically
within the [3-amyloid fragment region, resulting in the
extracellular release of the N-terminal portion of the
molecule (Fig. 3). This 'secretase' reaction, which
might be controlled by a specific [3-APP protease,
could not, therefore, be responsible for the ac-
cumulation of [3-AP within the cell. Alternative
patterns of proteolytic cleavage, which release the
intact [3-AP fragment by cleaving [3-APP at two sites,
on each side of the ~-AP sequence, are probably
involved in AD. However, the exact mechanism by
which [3-APP is cleaved is unclear, since in the intact
 51

feature
[3-APP molecule embedded in the membrane,
approximately two-thirds of the [3-amyloid fragment
is on the cell exterior, but one-third is buried within
the membrane and is thus inaccessible to proteases.
The identity o f the proteolytic enzymes involved in
either 'normal' or 'abnormal' [3-APP-processing path-
ways are still unknown. In 1992, however, Steve
Younkin and colleagues (Case Western Reserve Uni-
versity School o f Medicine, Cleveland, O H , USA)
discovered a proteolytic pathway that yields intact
[3-amyloid fragments that accumulate in the lyso-
some¢ °. A route that involves internalization of
[3-APP from the cell surface and its targeting to the
lysosomes, where the [3-AP is generated, was recently
reported to exist in cultured human cells [Dennis
Selkoe (1992)_/. Cell. Biochem. Suppl. 16E, p. 198].
The author hypothesized that, during aging, an
alternative, but normal pathway for ~-APP proteolysis
results in the release ofamyloidogenic fragments con-
taining [3-AP. In Down's syndrome, this alternative
path is overutilized due to the higher [3-APP gene
dosage, whereas in FAD, mutations in the [3-APP
gene, or elsewhere on chromosome 21, lead either to
structurally abnormal [3-APP, or disregulation of
[3-APP synthesis, either of which could lead to
increased processing through the alternative pathway.
At present, much research centres on the search for
the mechanism by which the balance between the two
degradation pathways is shifted in favour o f the lyso-
somal [3-AP-producing route.
A structural mutation in [3-APP (such as the 'Hardy'
mutation) may alter the way in which [3-APP is pro-
cessed. However, such mutations probably account for
only a small proportion o f AD cases. Defects in other
genes, perhaps involved in the secretion pathway, or
in the lysosomal processing pathway, may also cause
the disease. A further question that remains to be
answered is h o w [3-AP leaves the lysosomes. One
possibility is that it is secreted; another is that the
accumulation o f [3-AP might lead to cell damage, or
even death, thus resulting in the release of lysosomal
contents.
In an attempt to identify some of these genes, Hardy
is continuing the search for AD-linked mutations in
the rare families with the inherited form of the disease.
Approximately a dozen families have been identified
so far, and from his studies Hardy concludes; "There
is now no disagreement - a mutation in the [3-APP
gene is one cause of Alzheimer's". However, he esti-
mates that this defect probably only accounts for, at
most, 20% o f the cases of AD. H e continues; "We
need to find the causes of the other 80%, and find
mutations in other genes, and create animal models. It
might take a very long time".
Hardy and his team are looking for candidate genes
among those involved in [3-APP metabolism. In ad- Figure 2
dition, many biotechnology and pharmaceutical com- Light micrograph of human brain tissue from an Alzheimer's patient, showing
panies are now focusing on [3-APP processing. For (a) neurofibrillary tangles, formed of thickened filaments in the cytoplasm of neurons
instance, researchers at Cephalon, Inc. (Westchester, and (b) senile plaques, consisting of tangled masses of filaments and granules
PA, USA), and at Pfizer Central Research (Groton, surrounding a core of 13-amyloid. (Micrographs courtesy of C. Harrington, Cambridge
CT, USA), are working to identify [3-APP proteases. Brain Bank, Cambridge, UK.)

TIBTECH FEBRUARY1993 (VOL 11)

 52

feature
I NH2
ebrospinal fluid1~14, suggesting that cleavage ofl3-APP
NH2 to release [3-AP also occurs in the membranes of nor-
Alternative mal cells. Alzheimer's disease-related amyloid depo-
processing 'Secretase' sition may therefore involve changes in a pathway that
route(s) pathway normally produces extracellular [3-AP. But the chal-
lenge - to understand what controls the rate of [3-AP
production and removal, and why it is abnormally
•APP cleaved transformed into insoluble fibres in AD - remains.
o yield intact
I]-AP ....~ -AP P cleaved Animal models
fragment >:,!~;.:,]~ region ]-AP region One investigative approach is to develop an animal
model of human AD. Transgenic mice engineered to
express [3-APP in their brains could be expected to
develop neuropathologies and behavioural symptoms
similar to those of the human disease. This would not
only confirm the role of ~-AP in disease pathogen-
esis, but also provide an animal model in which to test
drugs for their efficacy against AD. By March 1992,
three such transgenic-mouse models had been
reported, of which two showed apparent [3-AP
deposits, and a third developed neurofibrillary tangles
and neurodegeneration 1s<7. However, two of these
reports were subsequently retracted: one for technical
-- COOH --- COOH reasons (the 'changes' appeared in nontransgenic ani-
mals of the same mouse strain); the second report is
I~-APP
under investigation by N I H as a possible case of fraud
(the photomicrograph showing brain tissue with
Figure 3
plaques and tangles may be from a human sufferer of
Possible processing pathways of beta-amyloid precursor protein (13-APP). 13-APP
embedded in the cell membrane is normally cleaved within the 6-amyloid (13-AP)region, AD, not from a transgenic mouse). The remaining
and the N-terminal portion is then secreted: intact 13-APcould not therefore be pro- transgenic-mouse model, produced by Barbara
duced by this 'secretase' pathway. An alternative processing reaction, thought to Cordell and colleagues (California Biotechnology
occur in the lysosomes, yields intact 13-AP,and may lead to 13-APdeposition. Inc., Mountain View, CA, USA) shows only diffuse
[3-AP deposition and has yet to prove its worth TM.
Despite the early disappointments, further attempts
to produce a transgenic model are being made. Ken-
Robert Siman of Cephalon argues that the multi- neth Kosik (Harvard Medical School, MA, USA) sees
catalytic protease should be considered as a candidate the preparation oftransgenic animals as "probably the
for aberrant processing of 13-APP in AD. highest priority of most laboratories researching
The researchers at Cephalon are also trying to study Alzheimer's disease around the world". Companies
aberrant [3-APP processing by overexpressing [3-APP such as Upjohn (Kalamazoo, MI, USA) are also in the
and site-specific mutants of [3-APP in cell culture. transgenic race. Hardy says he is going to start making
Barry Greenberg and colleagues (Upjohn, Kalama- mice as well. "There are lots more experiments to do
zoo, MI, USA) are attempting to identify the compo- before I would regard it as futile," he says. "Each trans-
nents that aggregate to form the senile plaques, and to genic animal is a single experiment, testing a certain
understand how molecules such as fibronectin and genetic construct in a particular strain of mice. And
other extracellular matrix proteins bind together. there are an infinite number of constructs."
Meanwhile, American Cynanamid Company (Pearl Another approach towards an animal model for AD
River, NY, USA) is looking at the presence of hep- is being explored by Sarah-Jane Richards (Adden-
arin-binding growth factors in the amyloid plaques. brooke's Hospital, Cambridge, UK). In the mouse,
The normal physiological functions of [3-APP, chromosome 16 has many genes in common with
however, remain unknown. Current hypotheses (John human chromosome 21, and researchers have created
Hardy) include protease inhibition; a role in cell ad- mice with three copies of chromosome 16, in an
hesion; and the regulation of cell growth. In addition, attempt to partially simulate human trisomy 21
researchers at Athena Neurosciences (South San Fran- (Down's syndrome). Unfortunately, mice with tri-
cisco, CA, USA) have found that secreted [3-APP somy 16 usually die in utero. Richards has therefore
stimulates fibroblasts to release interleukin 6 (IL-6) and transplanted brain tissue from the trisomy 16 fetuses
so conclude that [3-APP may be involved in the brain's into other, genetically normal mice. As the grafts from
inflammatory response 11. the hippocampal region of the fetal brain of the tri-
Recently, three teams have established indepen- somy 16 mice grew, they began to show signs of
dently that soluble 13-AP is produced by healthy cells Alzheimer-like pathology, including the production
in tissue culture, and in human and animal cer- of amyloid plaques and paired-helical filaments con-

TIBTECH FEBRUARY1993 (VOL 11)

 53
feature
raining tau protein. However, this model is still at an
early stage of development, and it is too soon to say
whether the observed pathological changes are specifi-
cally related to the expression of genes from chromo-
some 16. Nevertheless, this technique may facilitate a
systematic appraisal of the pathological events associ-
ated with AD.
At the Karolinska Institute (Stockholm, Sweden),
researchers attempting to develop an animal model
have transplanted neural tissue from aborted human
fetuses with trisomy 21. The human tissue was intro-
duced into the cortex of nude rats, which are
immunodeficient and thus do not reject transplants.
Immunohistochemistry using anti-[3-APP antibodies
revealed distinct aggregates of[3-APP in, and close to,
the site of the transplants. " O u r human-rat model pro-
vides a new tool to investigate the progression of
Alzheimer's disease-like neuropathological processes,"
say the researchers.
As an alternative approach, nerve-cell cultures may
also eventually prove useful, although, so far, it has
proven difficult to obtain sufficient numbers of pri-
mary neuronal cells, and to introduce genes into them.
N e w transplant techniques using neuronal cell lines
immortalized by infection with a retrovirus bearing an
oncogene may ultimately prove to be powerful
tools19,20.
Despite the current lack of a 'perfect' animal model,
there are several plausible theories as to how [3-AP
causes damage. [3-amyloid itself may be toxic to
neurons, or may render them more sensitive to dam-
age by excitatory amino acids, oxygen or glucose
deprivation. The protein is known to disrupt the
cellular Ca 2+ ion balance leading to an increase in Ca 2+
concentration within nerve cells. This, in turn, could
lead to the formation o f neurofibrillary tangles, since
raised intracellular Ca 2+ levels induce the hyperphos-
phorylation oftau. T o explore this possibility, Athena
Neurosciences Inc. and Eli Lilly (Indianapolis, IN,
USA) are both supporting research on the role of
[3-APP and [3-AP in the regulation of Ca 2+ levels
within cells in tissue culture. Since neurofibrillary
tangles are another possible target for therapeutic
intervention, both SmithKline Beecham (Welwyn,
Hefts., UK) and ICI Pharmaceuticals (Wilmington,
DE, USA) are backing research into identifying the
kinase that phosphorylates tau in the paired-helical
filaments.
Neurotoxicity of ~-AP
Factors affecting the site at which neuronal death
starts and its subsequent spread also remain unknown.
Bruce Yankner and colleagues (Children's Hospital,
Boston, MA, USA) explain the selective vulnerability
of certain regions of the brain as due to a neurotoxic
interaction between [3-AP and nerve growth factor
(NGF). Hardy, however, suggests that the brain
damage spreads as a result of a cascade of events after
initial deposition o f [3-AP in the limbic system. The
neurotoxicity o f [3-AP, Hardy speculates, leads to "an
upregulation of proteins [including [3-APP] that
respond to stress, either via heat shock, or interleukin-
mediated responses in surrounding neurons". This
stress response causes an increase in amyloid depo-
sition. As [3-APP is axonally transported, it would
therefore lead to increased [3-APP turnover at the ter-
minals of those neurons being damaged, and thus to
initiation o f the same process in the neurons with
which they make contact. Thus, the disease propagates
along neuronal pathways. If this idea is correct, then
drugs that antagonize the stress response should slow
the disease process.
Gareth Roberts (St Mary's Hospital Medical School,
London, UK) supports this view. He sees [3-APP as
part o f the acute-phase response of the brain to any
type of trauma 2~, with any factor causing stress to
neurons leading to an increase in [3-APP production.
"This explains why Alzheimer's is so common - there
must be a whole range of causes - but the common
event in the end is stress to neurons in the brain."
Physical trauma to the brain is the cause o f [3-APP
mismetabolism - the 'punch-drunk' syndrome of
boxers produces dementia and brain lesions that are
very similar to those o f AD. Aluminium poisoning
may be another cause of mismetabolism. Dialysis
patients with encephalopathy caused by large amounts
of aluminium in the brain also have enhanced [3-APP
levels in their neurons. In addition within hours of
suffering cerebral ischaemia, brain cells upregulate
[3-APP production.
The response of neurons to trauma is extremely
rapid, and is characterized by the production ofl3-APP
and the upregulation of other neuron-specific proteins
within hours. In most people, the trauma may lead to
only a brief disruption of [3-APP metabolism. H o w -
ever, John Hardy feels that it is possible that in some
individuals the entire pathological cascade leading to
AD would be initiated.
Unfortunately, the regulation ofl3-APP production
during the acute-phase response remains poorly
understood. Roberts examinedi the brains o f people
who had died within days, or months of head injury,
and found [3-AP deposition in 30% of t h e m - an
intriguing finding given that only 20 to 30% of the
elderly develop AD. "The mechanisms that regulate
it [[3 -AP deposition], and why it becomes deleterious
and out-of-control in some people - that's the funda-
mental question: if we knew the answer, we'd under-
stand why some people get Alzheimer's and most
don't," says Roberts.
Roberts also draws parallels with arthritis, in which
inflammation races out-of-control. If this comparison
is valid, some o f the anti-inflammatory drugs that help
arthritis patients might halt the progress of AD.
Roberts and Nancy Rothwell ( University o f Manch=
ester, Manchester, UK) suspect that an interleukin 1
(IL-1) receptor antagonist might downregulate the
acute-phase response and so suppress the expression of
[3-APP. According to Roberts, Synergen (Boulder,
CO, USA) is ready to take its recombinant IL-1 recep-
tor antagonist to clinical trials involving people with
AD.
TIBTECH FEBRUARY1993 (VOL ] t)
 54
feature
Retrieving lost memory
In 1992, Bruce Yankner and colleagues at Harvard
claimed that rats whose brains had been injected with
[3-AP developed neurodegeneration, but were pro-
tected if treated with another naturally occurring brain
peptide, substance P, which has a similar amino acid
sequence to a region of ~-AP. Yankner suggested that
substance P may prevent [3-AP aggregation. How-
ever, other groups have been unable to repeat the
work and thus contest both findings22.
Alternative treatments that might slow the decline
of cognitive abilities, such as learning and memory in
AD patients have, over the past few years, focused on
'cognition enhancers', i.e. drugs that increase the lev-
els of the neurotransmitter acetylcholine in the brain,
either directly, or indirectly by inhibiting its break-
down by interfering with the enzyme cholinesterase23.
Most of the major pharmaceutical companies, includ-
ing Merck Sharp and Dohme ( West Point, PA, USA),
Du Pont Merck (Wilmington, DE, USA) and
Hoechst Neuroscience (Somerville, NJ, USA) are
testing potential cognition enhancers - mostly cholin-
ergic drugs. Merck Sharp and Dohme, for instance,
has been testing eptastigxnine, a congener ofphysostig-
mine which produces long-lasting inhibition ofacetyl-
cholinesterase. Hoechst has developed velnacrine
maleate, an acetylcholinesterase inhibitor, and Du
Pont Merck is developing a class of compounds
(linopiridines) which enhance potassium-stimulated
release of a variety of neurotransmitters, including
acetylcholine.
Merck Sharp and Dohme (Harlow, Essex, UK) are
studying agonists that are selective for different sub-
types ofmuscarinic receptor, to assess their impact on
memory and learning in rats. Advanced Immunothera-
peutics (Irvine, CA, USA) claims that a derivative of
the purine hypoxanthine has a memory restorative
effect in aged animals afflicted with mild, or moder-
ate memory deficits. Cognitive Drug Research Ltd
( Reading, UK) has claimed that giving healthy young
people anti-muscarinic drags, such as scopolamine can
produce a 'human model' of AD by temporarily
impairing learning and memory. However, Hans
Fibiger (University of British Columbia, Vancouver,
Canada) indicates that recent research has cast doubt
on the adequacy of the scopolamine syndrome as a
model for the memory defects of AD 24. Fibiger also
doubts that the cognitive decline in patients with AD
is due solely to the loss of cholinergic cells in parts of
the brain. He argues that, in general, traditional phar-
macological replacement strategies are unlikely to suc-
ceed in AD because so many neurochemically distinct
systems degenerate. The neurons lost in AD do not
seem to share any particular transmitter - deficits have
been found in cholinergic, noradrenergic, serotoner-
gic and dopaminergic systems, as well as in the amino
acid and peptide neurotransmitters. In addition,
Fibiger says structures, such as the hippocampus and
cortex, which are among the presumed post-synaptic
targets for cholinergic drugs, are themselves damaged
in AD.
TIBTECHFEBRUARY1993 (VOL11)
Many argue, however, that cholinergic-based thera-
pies may be helpful, at least in the early stages of the
disease. David Bowen (Institute of Neurology, Lon-
don, UK), for instance, remains convinced that even
if researchers eventually discover how to block the
deposition of [3-AP, drugs that affect neurotrans-
mission will "still be needed in most patients to rescue
functional activities that are already lost". He thinks
more attention should be paid to glutamatergic pyr-
amidal cells in the neocortex, and has proposed that the
antibiotic and glutamate partial agonist, D-cycloserine,
should be tested in clinical trials. He argues that a new
class of drug, the selective 5-hydroxytryptamine 1A
(5-HT1A) receptor antagonist, might also help 2s~v.
Researchers concerned with the fundamental bio-
chemistry of the disease remain sceptical of this
approach, since memory enhancers do not address the
underlying pathology of the disease. However, cog-
nition enhancers are currently undergoing clinical
trials, and this field of research is likely to remain
active, if only because any promising products could
find alternative markets in the treatment of other
neurological disorders.
Alzheimer's diagnostics
Although the desirability of diagnosis without the
possibility of treatment is controversial, efforts are
being made to develop tests for AD. Abbott Labora-
tory (Abbott Park, IL, USA) is attempting to devise a
biochemical test for a marker protein in cerebral spinal
fluid (CSF). Tests based on 'Mzheimer's disease associ-
ated protein' and 'neural thread protein' (NTP)
"indicate that we are on the right road to developing
a biochemical test for Alzheimer's," the company
claims. Its NTP-based test is an automated micropar-
ticle enzyme immunoassay. Gene-based diagnostic
tests might also one day prove to be a commercial
proposition. Already, the discovery of the first gene
implicated in AD has led to ethical dilemmas for
researchers, who, despite the absence of any cure, can
now offer presymptomatic diagnoses to members of
those rare families at risk of developing FAD.
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Selection of defined cell types by

flow-cytometric cell sorting
Hans J. Tanke and Maarten van der Keur

Cell sorting by flow cytometry usually involves preliminary staining with fluorescent
dyes or reagents (antibodies or probes) that interact specifically with cellular
constituents. Passage through a focused beam of light enables cells to be sorted
on the basis of their light-scattering or fluorescence characteristics. Integration with
other techniques and refinement of labelling specificity is enabling high-speed
sorting to be developed for an expanding range of both analytical and preparative
applications -including isolation of specific cells for PCR amplification, establishing
high-expressing cell clones and chromosome sorting.

In flow cytometry, cells or cellular components in sus-
pension are passed t h r o u g h a sensing region, thus gen-
erating optical or electrical signals w h i c h m a y sub-
sequently be analysed. T h e cells to be analysed are
usually stained with fluorescent dyes, t h o u g h valuable
information can be derived f r o m unstained cells by
measurement o f their light-scattering properties or
electrical resistance. Light-scatter measurements pro-
vide information about cell size and internal structure,
whereas the electrical resistance o f cells in suspension
is related to cell v o l u m e (also referred to as Coulter
volume) 1.
Flow cytometer design
T h e design o f early flow cytometers bore a strong
resemblance to that o f microscopes, since epi-illumi-
nation and high numerical aperture oil-immersion
lenses to focus the illumination were used. All flow
cytometers currently available commercially have
essentially the same configuration (Fig. 1; P,,efs 2-4)
and use similar technology for sample presentation,
sample illumination and data capture. T h e differences
H . d . Tanke and M. van der Keur are at the Department of Cyto-
chemistry and Cytometry, University of Leiden, Wassenaarseweg 72,
2333 A L Leiden, The Netherlands.
lie predominantly in the n u m b e r and p o w e r o f light
sources and the capability for data analysis. Cells in sus-
pension are injected into a laminar fluid stream (a
process termed 'hydrodynamic focusing') s, and thus
are made to flow single-file through a focused beam
o f light from a high intensity source - usually a laser
or high-pressure arc lamp. Intersection o f the cells
with the laser beam generates two types o f emitted
light - scattered and fluorescent. In most flow cyto-
meters, cells are subjected to the laser beam while c o n -
tained within a quartz chamber - this eliminates light
scatter resulting from the stream o f liquid, and
improves the resolution o f signals from the cells. Care-
fully orientated photodetectors collect a fraction o f the
emitted light and generate electrical signals that are
proportional to the optical signals. Light scatter is
measured at low angles (ranging from 2 ° to 19°), and
at 90 ° to the incident light; both light scatter and
fluorescence at one or m o r e wavelengths are detected.
T h e electrical signals generated by the photomultiplier
tubes at the detectors are amplified, accumulated, and
analysed, and presented as single- or multi-parameter
frequency distributions (dot-plots). Data from differ-
ent measurements (e.g. scatter and fluorescence) can
thus be stored in matrices (multi-parameter analysis)
enabling correlating measurements from individual
events to be made.

© 1993,ElsevierSciencePublishersLtd(UK) TIBTECHFEBRUARY
1993(VOL11)