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Hereditary Disease
Foundation
Huntington's Disease: New Progress and
New Directions
January 10 and 11, 1998
Santa Monica, California
Prepared by Christopher Nichols
HEREDITARY DISEASE FOUNDATION WORKSHOP
"Huntington's Disease: New Progress and New Directions"
January 10 and 11, 1998
Santa Monica, California
Participants
Norman Arnheim
Director, Molecular Biology Program
Hedco Professor of Biological Sciences
Department of Biological Sciences
University of Southern California
University Park
Los Angeles, California 90089-0371
Gillian Bates
Senior Lecturer
Division of Medical and Molecular Genetics
United Medical and Dental Schools
Guy's and St. Thomas's Hospital
8th Floor Guy's Tower
London SE1 9RT, United Kingdom
M. Flint Beal
Chairman of Neurology
Department of Neurology and Neuroscience
Room A568
The New York Hospital-Cornell Medical Center
525 East 68th Street
New York, New York 10021
Marie-Franáoise Chesselet
Charles H. Markham Professor of Neurology
Department of Neurology
Reed Neurological Center, Room B114
University of California Los Angeles
School of Medicine
710 Westwood Plaza
Los Angeles, California 90095-1769
Don Cleveland
Professor of Medicine and Neuroscience
Ludwig Institute for Cancer Research
CMM-East 3080
University of California San Diego
9500 Gilman Drive
La Jolla, California 92093-0660
Robert Darnell
Assistant Professor, Associate Physician
Head, Laboratory of Molecular Neurooncology
The Rockefeller University
1230 York Avenue
New York, New York 10021
Stephen Davies
Reader in Neuroscience
Department of Anatomy and Developmental
Biology
University College London
Gower Street
London WC1E 6BT, United Kingdom
Stephen B. Dunnett
Reader in Neurobiology and
Director of Scientific Programmes
MRC Cambridge Centre for Brain Repair
University of Cambridge
University Forvie Site, Robinson Way
Cambridge CB2 2PY, United Kingdom
David Eisenberg
Director,
UCLA-DOE Laboratory of Structural Biology
Molecular Biology Institute
University of California Los Angeles
Box 951570
Los Angeles, California 90095-1570
Hans Lehrach
Professor
Max Planck Institut fur Molekulare Genetik
Ihnestrasse 73
Berlin - Dahlem
Berlin D14195, Germany
Greg E. Lemke
Professor and Director
Molecular Neurobiology Laboratory
The Salk Institute for Biological Studies
10010 North Torrey Pines Road
La Jolla, California 91037
John C. Mazziotta
Pierson-Lovelace Investigator
Director, UCLA Brain Mapping
Division of Brain Mapping
Department of Neurology
University of California Los Angeles
School of Medicine
710 Westwood Boulevard
Los Angeles, California 90024-1769
Edward R.B. McCabe
Professor and Executive Chair
Department of Pediatrics
Physician-in-Chief, UCLA Children's Hospital
Room 22-412 MDCC
University of California Los Angeles
10833 LeConte Avenue
Los Angeles, California 90095-1752
Christopher Nichols
Graduate Student
Brain Research Institute
University of California Los Angeles
73-369 Center for the Health Sciences
Los Angeles, California 90095-1761
Ethan Signer
Executive Director, Cure HD Initiative
Hereditary Disease Foundation
1427 7th Street, Suite 2
Santa Monica, California 90401
Professor of Biology Emeritus
Massachusetts Institute of Technology
Room 68-150
Cambridge, Massachusetts 02139
Larry W. Swanson
Milo Don and Lucille Appleman
Professor of Biological Sciences
Director, Neuroscience Graduate Program
Department of Biological Sciences
Neurobiology Section
University of Southern California
Hedco Neuroscience Building MC 2520
Los Angeles, California 90089-2520
Danilo Tagle
Investigator
Head - Molecular Neurogenetics Section
Genetics and Molecular Biology Branch
National Human Genome Research Institute
National Institutes of Health
Buiding 49, Room 3A14
49 Convent Drive MSC 4442
Bethesda, MD 20892-4442
Allan Tobin
Scientific Director
Hereditary Disease Foundation
1427 7th Street, Suite 2
Santa Monica, California 90401
Eleanor Leslie Endowed Chair in Neuroscience
Director, Brain Research Institute
University of California Los Angeles
73-369 Center for the Health Sciences
Los Angeles, California 90095-1761
Nancy Wexler
President
Hereditary Disease Foundation
1427 7th Street, Suite 2
Santa Monica, California 90401
Higgins Professor of Neuropsychology
Departments of Neurology and Psychiatry
College of Physicians and Surgeons
Columbia University
722 West 168th Street, Box 58
New York, New York 10032
Ai Yamamoto
Predoctoral Graduate Student
Center for Neurobiology and Behavior
College of Physicians and Surgeons
Columbia University
722 West 168th Street
New York, New York 10032
Anne B. Young
Julieanne Dorn Professor of Neurology
Harvard Medical School
Chief, Neurology Service
Neurology Service, VBK 915
Massachusetts General Hospital
32 Fruit Street
Boston, Massachusetts 02114
Parkinson's Disease versus Huntington's
Motor symptoms of both Huntington's disease (HD) and Parkinson's disease
(PD) result from the loss of function within the basal ganglia. In both PD and HD,
people have difficulty with fine motor coordination and with the initiation of
movement. The classical explanation for the differences between HD and PD posits
two interconnected neural pathways, one suppressive and the other facilitative.
According to this model, in PD the suppressive pathway is over-active relative
to the facilitative, leading to akinesia and bradykinesia. Conversely, the standard
model posits that in HD the suppressive pathway is underactive and the executive
movement pathway dominates, giving rise to hyperkinesia.
In addition, people with HD report difficulties with concentration, e. g. in
reading, and they have difficulty in maintaining attention. Nancy Wexler has also
noted obsessive compulsive symptoms in people with HD. This behavior can take
the form of lists, rituals, and other obsessive-compulsive behavior.
Several studies have addressed the relationship of pathology to behavior.
Most conclude that the caudate is the first site of pathology where investigators
have found gliosis and cell loss in the tail of the caudate. This gliosis may underlie
the earliest symptoms of HD. Recent MRI studies suggest, however, that the onset
of symptoms occurs only after the putamen is affected.
Cognitive defects are common in HD, but the origin of these defects is
unknown. Discrete lesions of the basal ganglia in baboons result in cognitive
deficits; Stephen Dunnett suggested that neuropsychology might provide clues to
the pathology. To understand cognitive deficits in mice, however, will be a
formidable challenge. Still, cognitive defects may precede movement disorders, and
it will be important to devise sophisticated tests to detect early changes in behavior.
Huntington's Disease is a CAG Repeat Disease
The hallmark of HD, SCA1, SCA2, MJD, and similar diseases are expansions
of DNA CAG repeats that encode polyglutamine stretches in different proteins.
Proteins with expanded polyglutamine repeats may form insoluble aggregates in
vitro as well as intracellular inclusions in the cytoplasm and/or nucleus. Hans
Lehrach, Erich Wanker, Gillian Bates, Stephen Davies and their collaborators have
proposed that polyglutamine aggregates cause cell death. The number of CAG
repeats is inversely correlated with age of disease onset, with, in general, longer
repeats leading to earlier onset than shorter repeats.
The correlation only accounts for approximately 50% of the variability in age
of onset. Marie-Franáoise Chesselet mentioned a family from Crete with
comparatively late age of onset, and Hans Lehrach suggested that analysis of
people who lie off the curve of age-of-onset versus repeat length might reveal
modifier genes.
Two major areas of HD research are the molecular disease basis of cellular
pathology and the development of animal models. Huntingtin (htt), the protein
product of the gene affected in Huntington's disease, may itself cause HD or it may
act in concert with other factors. Trophic factors and mitochondria, for example, may
play important roles. The researchers at the workshop addressed the role of
expanded repeats, proteolytic processing of huntingtin, nuclear transport,
intracellular inclusions, protein-protein interactions, cell death, and cellular
dysfunction.
Most adult-onset HD patients have axonal inclusions ("dystrophic neurites"),
but not nuclear inclusions. On the other hand, brains from people who have died
from HD do not always contain inclusions.
Huda Zoghbi discussed SCA1, a CAG repeat disease, in which many of the
Purkinje cells that die do not have inclusions of the corresponding protein, ataxin-1.
When inclusions are present, both in humans and in a mouse model, they number
only one per cell. In contrast, 85% of COS cells that transiently express full length
ataxin-1 have 2-3 nuclear inclusions each, and these aggregates are not
ubiquitinated. These COS cells can survive for many weeks despite the aggregates.
Huda Zoghbi also noted that overexpression of wild-type ataxin-1 in COS cells
results in multiple, tiny inclusions in 1-2% of the cells.
Marie-Franáoise Chesselet raised the question of age-related factors playing
a role. In the formation and distribution of inclusion bodies. She described SCA6
(Ca2+ channel), a disease in which relatively short repeat lengths cause inclusions,
and she suggested that cells might, with age, lose the ability to degrade nuclear
inclusions.
Allan Tobin compared the pathology of sickle cell anemia to that of HD. If a
known gene defect could be detected at the cellular level long before symptoms,
clinicians could screen young people with a family history of HD for subtle cellular
defects preceding the onset of symptoms, such as changes in membrane fluidity,
glutamate toxicity, and DNA repair. Murine models, cell lines, and transient
expression assays will allow researchers to address these questions.
Murine HD Models
Huntingtin is essential in embryonic development. In htt knockouts, embryos
die at day E7-8, but it is not yet known whether htt is required in the adult. Nancy
Wexler noted that this question is being studied by Scott Zeitlin (Columbia
University), using the Cre-Lox system to excise an htt gene after birth.
The laboratories of Gillian Bates, Danilo Tagle, and Rene Hen have made
transgenic murine models of Huntington's disease with different numbers of
expanded repeats, promoters, expression, incorporation sites, and gene copies.
Transgenic Mice from Gillian Bates' Lab
Line
Copy #
Repeat Length
Protein
Onset of Phenotype*
R6/1
1
115
Yes
5 mo.
R6/2
1
150
Yes
2 mo.
R6/5
4
(3)130, (1)155
Yes
9 mo.
* Bates' group could not perform age tests because of UK animal regulations.
The R6/1 line was kept for 1 year and the R6/5 line for 2 years.
Gillian Bates' lines express a ~2 Kb huntingtin gene fragment encoding htt
exon 1 and a small part of intron 1. The group has analyzed the behavior and the
brain pathology of each transgenic line. All the lines display some movement
disorders. The three lines shown above have neuronal pathology, although a fourth,
R6/4, does not.
All of the transgenic mice (except the R6/4 line) have a hopping gait, and
they lose their balance when grooming or reaching out of the cage. They are less
active than the wild type controls, and they shake, shudder, and twitch while
displaying persistent stereotypical grooming movements. They are less robust and
all eventually start losing weight without alterations in respiration rates. They exhibit
tremors, although no seizure activity has been detected by EEG. Age of onset is
correlated with the level of mRNA expression as assessed by Northern blotting, but
not with protein levels.
Stephen Dunnett, working with Gillian Bates' mice, noted that rodents can
mimic some aspects of human neuropathology. Although transgenic mice may not
display exactly the same symptoms as humans, behaviors such as impulsiveness
may be useful. The difficulty lies in unraveling the hodge-podge of symptoms
exhibited by transgenic mice. Still, the mice exhibit clear behavioral abnormalities
long before cell death is evident. Stephen Dunnett stressed using detailed analyses
of movement and dexterity as two ways to detect disease. Once researchers can
profile the disorder in rodents, they may be able to develop and standardize
screening tests for humans. Dunnett's group will soon have results from cognitive
assessments such as open field tests and T-mazes.
Stephen Davies has found that the nuclear inclusions always appear before
behavioral symptoms. Most sensitive visualization is with an antibody to the htt N-
terminus, and anti-ubiquitin staining only appears a week later. In transmission EM,
the inclusions can be seen without antibody staining. He finds inclusions most often
in neurons, but he also sees them (rarely) in oligodendrocytes, astrocytes, and
microglia. At late times he also finds inclusions in dystrophic neurites (myelinated
and unmyelinated axons, dendrites and nerve terminals), but neither in the
cytoplasm nor, he thinks, outside cells. Changes occur in the nuclear membranes
of striatal neurons, except for striatal interneurons that contain NADPH diaphorase,
neuropeptide Y, NO, choline acetyltransferase, calretinin, and parvalbumin. Davies
observed no indentations in the nuclear membrane initially, but after the inclusions
appear, the nuclear pores become rounded, with finger-like protrusions, and the
number of pores increases. Freeze-fracture EM showed increases in the number
of individual nuclear pores and clusters. He did not find inclusions in cells that did
not express high levels of huntingtin. Nor was there evidence of huntingtin-
associated proteins (HAP-1, Hip-1, Hip-2, or GAPDH) within the inclusions. Nuclear
blebbing, cellular disintegration, or other signs of apoptosis were absent.
Immunohistochemical analysis, with both an antibody raised to the N-
terminus of huntingtin and another to ubiquitin showed fibrous aggregates in the
nucleus before the onset of symptoms in both the R6/1 and R6/2 lines. TEM also
showed such fibrous aggregates.
Stephen Davies observed degeneration in the caudate and in the deep
cortex (adjacent to the corpus callosum). The cells show no nuclear blebbing or
disintegration. Their cytoplasm is condensed, the cells are smaller, and the
distinction between nucleus and cytoplasm is lost. The cell membranes are ruffled,
the Golgi apparatus is disrupted, and the mitochondria have degenerated. He also
found spongiform degeneration in the cortex and caudate, with membrane-bounded
vacuoles in neuropil, in cytoplasm, and especially in the nucleus.
Davies does not find any evidence of cell death in the brain until after 17
weeks and he thinks the cells with the largest nuclear inclusions are dying the most
rapidly. R6/2 mice have inclusions in glial cells at 17 weeks which are rare overall
and take a long time to form, but not in activated glia. Rare inclusions are also
present in myelinated and non-myelinated axons, in terminals with synaptic vesicles,
and in dendrites, and these inclusions appear late in development. The R6/2 mice
show no inclusions or signs of necrosis in somatic tissues.
In R6/1 mice, abnormal behavior occurs earlier in homozygotes than in
heterozygotes. Older animals have nuclear inclusions in glia but these are very rare
(10 cells among 100 mice). These glial inclusions do not occur in activated glia.
In the R6/2 line, behavioral alterations are evident by about 2 months, with
some changes as early as 6 weeks. By 12-13 weeks, the mice are severely
affected, with some sudden deaths, possibly due to seizures. Nuclear inclusions are
present in 90% of the cells in the caudate but are not limited to the striatum or
cortex. Visualized by immunohistochemistry with antibodies to the N-terminus of
huntingtin the inclusions begin to appear at 3 weeks in the cortex and 4 weeks in
the striatum. By 9 weeks, nuclear inclusions are present in Purkinje cells, with some
inclusions also in their axons. Similar axonal inclusions are also present in motor
neurons.
R6/5 homozygotes show nuclear inclusions in glia in older animals (as in
R6/1), but the heterozygotes have no degeneration and no nuclear inclusions until
22 months. A few compound heterozygotes between lines have been made, and
have a more severe phenotype than simple heterozygotes.
No phenotype is evident in the R6/4 line at 22 months.
Gillian Bates noted a delayed onset in the R6/2 line with aggregates at 3.5
weeks, and she suggested a possible involvement of age-dependent factors in
mice. Between 3.5 and 12 weeks cells do not die, yet the mice show severe
behavioral abnormalities.
Neurotransmitter Receptor Changes in Gillian Bates' Mice
The fact that the death of the R6/2 transgenic mice occurs before cell death
suggests that the causes of behavioral abnormalities can be cell dysfunction rather
than cell death.
A hallmark of sick cells is low neurotransmitter activity. Gillian Bates
proposed that neurons lose part of their capacity for their neurochemical repertoire
in response to subtle effects of the aggregates. To address this question, Anne
Young's group has been investigating the levels of neurotransmitters and their
receptors in the R6/2 at 12 weeks.
The receptors they studied included: NMDA, AMPA, GABAA, GABAB,
muscarinic, cholinergic, dopamine D1 and D2, and glutamate all of which
contribute to cell signaling in the basal ganglia. They found no changes in NMDA,
AMPA or GABAA receptors and little change in GABAB receptors. However,
dopamine D1 and D2 receptors were decreased by 60% and adenosine A2A
receptors were reduced more than 60% at 8 weeks of age. Anne Young and her
colleagues also found a decrease in metabotropic glutamate receptors and a
decrease in mRNA levels for metabotropic type II receptors in deep cortical
projections into the striatum.
Stephen Davies also found clear changes in dopamine receptors in the R6/2
line both at the mRNA level (by in situ hybridization) and at the protein level (by
immunoblotting). Both protein and mRNA levels were down 20-30% at 2 weeks, with
D1 receptors reduced more than D2. By 8 weeks both protein and mRNA levels
were severely depressed. Since he noted inclusions in the striatum at 3.5 weeks
and in the cortex at 4.5 weeks, the dopamine receptor decreases precede the
appearance of any visible inclusions.
Marie-Franáoise Chesselet commented that both neuronal inputs and outputs
are decreased in the R6/2 mice. She might have expected a more conventional
compensatory increase in receptors in response to decreased signal. Perhaps the
transgene has subtle effects on specific components of a neurotransmitter system,
such as dopamine decarboxylase. Such effects, in turn, could work to alter the
circuitry. If, for example, the glutamate metabotropic type II receptors are strikingly
changed in the cortex, it would be important to clarify events preceding the
development of altered behavior. Allan Tobin noted that events that start in the
nucleus can affect other factors and snowball into a more global phenomenon.
Transgenic Mice from Danilo Tagle's Lab
Transgene
CAG Repeat #
Lines
Htt present?
CAG16
16
5 (A-E)
Yes
CAG48
48
3 (A-C)
Yes
CAG89
89
3 (A-C)
Yes
Danilo Tagle has produced 11 transgenic lines with a full length huntingtin
cDNA driven by a CMV promoter, including 5 lines with 16 repeats (CAG16), 3 with
48 repeats (CAG48), and 3 with 89 repeats (CAG89). Copy number ranges from 1
to 6. Expression of huntingtin was confirmed via Western blotting with an antibody
against the C-terminus of htt. The line with the highest expression was 48B, with
five fold expression above endogenous huntingtin. Expression is evident in liver,
heart, spleen, lung, gonads, and brain, with the highest expression levels found in
brain and spleen, and sometimes gonads.
Behavioral Alterations in Danilo Tagle's Mice
Time (mo.)
Phenotype
CAG16
CAG48
CAG89
2
Feet-Clasping
none
100%
100%
4
Hyperactivity
none
40%
40%
10
Hypoactivity
none
100%
100%
11-12
Premature Death
none
100%
100%
CAG48 and CAG89 heterozygous transgenic mice, but not CAG16, exhibit
feet clasping at two months of age. At 4 months, about 40% of the mice exhibit
hyperactivity, running in unidirectional circles for as long as 6-8 hours. The mice
also swim in circles, with tighter circles in larger enclosures. Other abnormal
behavior includes backflips and excessive grooming. No inner ear abnormalities
were found. There was no difference in average age of onset or duration between
the CAG48 and CAG89 lines.
One CAG48 line, 48C, was anomalous with the mice carrying this transgene,
showing only feet clasping with no twisting or contortions.
Tagle used a vertical pole test to test muscle strength and coordination in his
mice. Wild type control mice hung onto the pole for 30 seconds, while transgenic
mice fell off after about 10-15 seconds. This hyperactive behavior differed from
behavior induced with striatal lesions but were similar to the effects of cocaine and
amphetamines.
All the transgenic mice entered a hypokinetic stage at about 9-10 months for
1 month to 6 weeks following an abrupt transition. In this hypoactive stage, the mice
sat and groomed but were resistant to stimulation. The mice died at 11-12 months.
Tagle mentioned that researchers have documented a similar two-stage behavior
with 3-NP lesions.
Homozygous transgenic mice also exhibited feet clasping, with onset at the
same time as heterozygotes, and they showed hyperactivity at about 2 months.
There was no significant weight difference between homozygotes and
heterozygotes.
There was no obvious cause of death. The mice did not die from starvation
as they had post-mortem ingesta, nor did they have any evident brain pathology at
2 months. However at the hypokinetic stage, Danilo Tagle found profound HNE
staining, and degeneration of the striatum, cortex, thalamus, and hippocampus. A
few nuclear inclusions were detected with an anti-ubiquitin antibody. Tagle also
found condensed nuclei and neurodegeneration, and TUNEL staining with
chromatin fragmentation.
Transgenic Mice from Rene Hen's Lab (presented by Ai Yamamoto)
Rene Hen's transgenic mice have a Tet-Off regulated exon 1 construct with
94 repeats driven by a CaMKII promoter (from Eric Kandel's lab). The construct is
bidirectional (biTetO), with tTA expressed in one direction and exon 1 with 94
repeats and LacZ with a nuclear localization signal (a control for promoter activity)
in the other. Expression was strongest in the forebrain including expression in the
hippocampus (CA1), striatum, and cortex, as judged from inspection of brain
sections with a -galactosidase assay.
Two litters of transgenic mice have been studied to date. In the first litter, the
pups were mobile but failed to nurse, resulting in a 70% mortality. The remaining
30% died within 21 days of birth and displayed a 50-60% decrease in body weight.
The animals were rigid, could not right themselves, and were diagnosed as a
"seizure death" by the veterinarian. In the second litter, based on the previous
result, doxycycline (2 mg/ml) was added to the mother's water for one week
beginning at E12 to E14 to repress exon 1 transcription, and this resulted in a 100%
survival rate; doxycycline was then discontinued at birth.
Pups treated with doxycycline in utero exhibited clasping of hind limbs at 4-6
weeks, with fisting, unilateral hind limb abductions, and little fore paw movement
when dangled. The clasping developed from grabbing and release of hind limbs at
2 months to full time clasping by 10 weeks. Animals maintained on doxycycline for
2, 3, and 14 weeks postnatally showed no motor phenotype.
Aggregates: Cause or Effect?
The 3 alternative hypotheses concerning the possible role of htt-containing
aggregates in HD: (a) homomeric aggregates of htt alone cause disease directly,
(b) heteromeric interactions of htt and unknown factors cause disease directly, (c)
aggregates do not contribute to the disease caused by htt.
Stephen Davies noted that Marian DiFiglia (Massachusetts General Hospital)
has seen aggregates in dystrophic neurites. She observed an important difference
between the juvenile and adult forms of the illness. In the juvenile form, aggregates
are predominantly nuclear while in the adult form they are characteristically in
dystrophic neurites.
Immunohistochemistry with an antibody against the C-terminus showed wild-
type huntingtin is diffuse in the cytoplasm. Thus, huntingtin may be proteolized to
a form, denoted htt*, that leads to the disease.
In vivo, the aggregates stain with anti-ubiquitin antibodies. The presence of
ubiquitin suggests the involvement of the main proteolizing element in the cell, the
26S proteasome. Perhaps the polyglutamine stretches in huntingtin are not properly
degraded by the proteosome, possibly leading to the accumulation of htt*, which
then aggregates. This aggregate might be similar to Alzheimer's disease, where
cells may lose the ability to clear protein early in the disease process. Marie-
Franáoise Chesselet proposed that age-related factors may disrupt the
proteasome's ability to process huntingtin.
If huntingtin needs to be partially digested to become pathological, the
proteolysis sites must be identified. One way might be to isolate the aggregates and
determine the molecular weight by mass spectrometry. Another way might be to
"walk" down the protein with a battery of antibodies, in order to identify which
epitopes remain in the aggregates. Allan Tobin suggested starting at low resolution,
e.g. with chymotrypsin which cuts after aromatics, and move up to more specific
proteases in order to locate a domain of local folding. This is akin to the
characterization of nucleosomes, where investigators repeated serial digests with
increasing specificity to identify protease sites and local domains.
Hans Lehrach and collaborators have shown that glutamine polypeptides
synthesized in vitro form aggregates similar to those found in vivo. This in vitro
aggregation suggests the aggregates are composed of htt alone and do not include
huntingtin-associated proteins. Exon 1 fused to GST (glutathione S-transferase)
expressed in E. coli does not aggregate. However, following enzymatic cleavage of
the GST tag, exon 1 polypeptides with long repeats aggregate into a -pleated sheet
in vivo and in vitro, while exon 1 polypeptides with short repeats yield no
aggregates.
Hans Lehrach's group have developed a high throughput system to screen
compounds that may prevent aggregation: they soak a filter in the fusion protein,
apply droplets of the test compound, spray on proteolytic enzyme to cleave the
fusion, and screen the filter for the absence of precipitated aggregate. Compounds
tested thus far have not prevented aggregation. Hans Lehrach proposed that
aggregates induce apoptosis, but the relationship between aggregates and
apoptosis has yet to be defined experimentally.
Allan Tobin commented that a simple interaction between polyglutamine
repeats could form aggregates and subsequently disease: nothing else may be
necessary. On the other hand, although htt is expressed ubiquitously, the disease
is restricted to the brain and primarily to GABAergic striatal neurons, suggesting
heterologous interactions of htt with as yet unknown factors. This paradox raises
two questions: what triggers HD in the brain? and conversely, what prevents
pathology in the rest of the body? Potential interactions might include transcription
factors, axonal transporters, and proteins required for mitochondrial function.
Many transcription factors contain polyalanine and polyglutamine stretches
and act via protein-protein interactions. In Kennedy's disease, the polyglutamine
containing protein is the androgen receptor, a transcription factor. Huntingtin,
however, has no DNA-binding domain, but it might decrease or augment the activity
of other transcription factors. It might be informative to examine mRNA profiles from
htt-expressing cells, perhaps by differential display, to compare patterns of gene
expression before and after htt expression.
Lehrach reported a comparison of mRNA (by hybridization screening) and
protein (by 2-D gels) from transgenic mice versus wild type mice. The transgenics
studied , however, where not inbred, complicating the analysis. The mRNA showed
changes that have not yet been defined, and the protein analysis may have showed
changes but was less certain.
Alternatively, huntingtin might cause defects in axonal transport. Such
defects could be addressed by studying transport of labeled products in mice and/or
cells. Don Cleveland pointed out that axonal transport requires approximately 30
microtubule associated proteins, and although subtle changes are difficult to
measure, crude experiments could be done in mice. Stephen Davies noted that
immuno-EM with a C-terminal antibody showed no difference in distribution of
mouse htt in transgenics versus wild-type lines. These data suggest that axonal
transport was normal, but quantification was poor.
A third hypothesis maintains that the expanded repeats affect the
mitochondria, whether directly or through the nuclear inclusions. Flint Beal reported
metabolic abnormalities at 6-8 weeks in Bates' R6/2 mice by magnetic resonance
imagery (MRI) studies. He noted that the inclusions appeared too quickly to detect
any abnormalities that might occur earlier, and suggested that the later onset lines
(like R6/1) might be better subjects in which to look for changes that precede the
inclusions.
Flint Beal went on to cite studies of others in which transgenic mice
expressed abnormal amounts of neurofilaments in the cytoplasm of motor neurons.
The result of this neurofilament overexpression was an increase of up to four times
their normal cell size, but the cells nevertheless survived. Even though the
overabundance of neurofilament was not pathological, it does not rule out subtle
changes in cell- or compartment-specific sensitivity to the abundance of htt*. In
addition, inclusions in axons or dendrites might send signals to the nucleus which
lead to pathology.
Marie-Franáoise Chesselet noted that in Parkinson's disease, Lewy bodies
found in cytoplasm and neurites contain only alpha -synuclein and no other protein,
and are ubiquitinated. Stephen Davies mentioned that Kurt Frischbeck's group has
found aggregates staining with 1C2 antibody in a person with Neuronal Inclusion
Disease.
Finally, Anne Young pointed out a problem with the hypothesis that
aggregates cause disease. In both people with HD and mouse models, neurons
appear still to function for many weeks or years even with aggregates present.
Aggregates and Nuclear Export/Import
In both people with HD and in current mouse models, aggregates are present
in neurite processes and in the nucleus. But normally huntingtin is cytoplasmic and
may be targeted to dendrites and axon terminals. Stephen Davies noted that EM
data suggests that in Gillian Bates' transgenic mice, faulty nuclear export may lead
to the deposition of aggregates.
These data suggest several questions: (a) are the proteins small enough
simply to diffuse into the nucleus?; (b) do aggregated proteins fold into a de facto
nuclear targeting signal?; (c) are proteins imported into the nucleus via a complex
("karyoferrins or importins")?; (d) are proteolytic events required?; (e) do the
proteins aggregate first in the cytoplasm before transport to the nucleus?; and (f)
do the proteins accumulate in the nucleus and then aggregate?
These questions gave rise to five suggestions for experiments to test whether
or not polyglutamine proteins enter isolated nuclei in vitro: (a) Bob Darnell
suggested looking at nuclear transport in a heterokaryon made from an HD nucleus
and wild-type cytoplasm; (b) Norman Arnheim suggested looking at nuclear import
in vitro by treating isolated nuclei with digitonin to wash out import factors, and then
adding the factors back one by one; (c) Bob Darnell suggested using an inducible
system to perform a pulse-chase study; such a study could follow nuclear entry and
exit; (d) Ethan Signer suggested using a nuclear export tag on the protein; (e)
Stephen Davies suggested introducing a non-nuclear protein marked with CAG's
and tracking its import and export.
These studies could start to identify factors, conditions, and stoichiometry
between inclusions and HD proteins. Eventually, such experiments could define the
seeding event of aggregation.
The In Vitro Approach: Cell Lines
The use of inducible cell culture systems could answer how aggregates form,
how fast, and whether aggregation can be reversed. Distinct neuronal and non-
neuronal cell lines could be engineered to express the HD gene. If overexpression
is itself lethal, an inducible construct might be useful to study differences in the
effects of wild-type and mutant htt with expanded polyglutamine stretches.
Workshop participants discussed 3 approaches to making inducible cell lines:
(a) immortalization with a temperature-sensitive oncogene, allowing cell proliferation
at the lower temperature and differentiation at the higher temperature; (b) induction
by an ecdysone derivative developed by Ron Evans (Salk Institute) and made by
Signal Pharmaceuticals; and (c) induction with the commercial Tet-On and Tet-Off
vectors. One consideration in choosing among these alternatives is the time it takes
for the system to be activated or deactivated: the steroid system, for example, works
within minutes whereas the tetracycline systems can take hours.
Beal and Young noted that Marian DiFiglia has stable transfectants in a
clonal human striatal line (from Allan Heller) that carry either a full length or exon 1-
3 constructs with a FLAG tag, driven by the NSE promoter. Expression is low, and
the protein can not be visualized by immunohistochemistry although its presence
has been confirmed via Western blots. These cells die, but their survival is
prolonged by treatment with a caspase-3 inhibitor. In a transient assay, the htt
fragment encoded by the CAG16 construct remained in the cytoplasm whereas the
htt fragment encoded by CAG48 and CAG89 were found in the nucleus after 4-6
days. It is not known whether these htt fragments enter the processes of the
neuronal cell lines.
Lowering protein levels in cell lines via gene regulation might eventually be
feasible. Bates noted that David Rubensztein (Cambridge, UK) is investigating
huntingtin promoter structure. High through-put screening for compounds that affect
gene expression or bio-informatic sequence analysis might identify factors that bind
to the promoter.
Therapeutic Approaches
Allan Tobin pointed out that other diseases of protein aggregates have been
successfully treated without intervention in the aggregation process. In the case of
sickle cell anemia, for example, the mutant subunits of hemoglobin can be partially
replaced by the stimulated synthesis of the subunits, thereby preventing Hb's
precipitation.
Hans Lehrach and Don Cleveland noted that blocking protein-protein
interactions may be risky and difficult, but that HD is very different from sickle cell
anemia. Whereas sickle cell anemia cannot be blocked by a single inhibitor,
polyglutamine aggregation may be simple. An aggregation inhibitor might therefore
be a viable therapeutic option.
Ethan Signer noted that chemicals passing though the blood brain barrier
(BBB) are generally small, non-polar, and slightly lipophilic. If an aggregation
inhibitor could be obtained, it could subsequently be modified to cross the BBB. For
a chemical with a long half-life in the bloodstream, even slight crossing of the BBB
at each pass could accumulate in the brain to therapeutic drug levels. Many drugs
on the market cross the BBB with low efficiency but are nevertheless effective.
Hans Lehrach argued for in vitro and mouse experiments to identify
compounds that block aggregation, with the caveat that there may be some subtle
changes. Even partial inhibition could significantly delay the onset of disease. High
through-put screening to identify drug leads could be followed by testing in cell-
based systems and then in transgenic mice.
Closing Remarks and Priorities
Norman Arnheim:
Support the Lehrach hypothesis with drug screening and a focus on the
fundamental biological properties.
Gillian Bates:
Several models are necessary: in vivo, in vitro, and cell lines. Other models
such as Drosophila melanogaster and C. elegans can be utilized. Alternative types
of therapeutics and other high-through-put screens can attack proteases and
promoters. We do not require the molecular mechanism to drive clinical research.
Flint Beal:
Transgenic mice are important. It is also important to test Lehrach's
aggregate hypothesis, first in mice and then in humans. Investigators should isolate
the inclusions and determine where they are and what they do. They also should
define proteolytic cleavage sites through mass spectrometry. Overall, the huntingtin
protein is a good target for therapeutics possibly tied into cell death, necrosis or
apoptosis, and the role of inclusions. Further targets might be upstream or
downstream in the disease pathway.
Marie-Franáoise Chesselet:
Study people who have a late onset of HD. The focus should be on what is
changing with age that is critical for the development of the disease, such as (a)
protease activation or expression, (b) conditions of aggregation (pH, etc.), and (c)
possible age-related decrease in the ability to dispose of the aggregates.
Don Cleveland:
He has an optimistic outlook on HD research. The aggregate hypothesis
must be tested because it can be tested. But still, what else is in the aggregates?
Can they be blocked in vitro? We should examine humans to confirm the presence
of nuclear aggregates. Then, use cell culture to confirm the formation of nuclear
aggregates in vitro. It is of the utmost importance to avoid unilateral approaches and
maintain open mindedness in the scientific community. The HDF should invest in
priorities but without the exclusion of others.
Robert Darnell:
It remains unproved whether the aggregates are a result of the disease or a
cause. If the aggregates are at the end of the disease pathway, then prioritize from
the last step and work backward. Scientists should examine the biochemical
pathways up and downstream of aggregate deposition. It is preferable to seek
therapeutics that can block early steps before formation of the aggregates.
Cellular localization of huntingtin should be determined in both people with
HD and unaffected individuals.
Changes in transcription are important because there is a direct relationship
between polyglutamine stretches and transcription factors. It would be useful to
compare/contrast transgenic knock-in mice and knock-out mice to find modifying
elements. It is important to find a way to standardize the behavioral aspects of the
transgenic mice.
Stephen Davies:
Conduct studies in both mice and humans, investigating neurite versus
nuclear aggregates. Search the inclusions for other proteins besides the mutant
huntingtin. Murine models with the phenotypes and histopathology are good for
screening drugs for humans. Aggregation is important; find some inhibitors and start
testing.
Stephen Dunnett:
Examine aggregates and nuclear transport because we can. Examine
protein-protein interactions, patterns of expression, systems involved, and
distribution of the htt* protein. Explore the details and patterns of behavioral defects.
In vitro studies are not systems specific but can be combined with systems
information if they are run in parallel. Use this combination to illuminate the circuits
and systems involved.
Greg Lemke:
Lehrach's hypothesis can be tested and that is good. Delivery, therapeutics
follow Lehrach's work. Expand the use of inducible expression systems in cell lines.
Good questions can be asked with regulatable cell lines. We must keep in mind that
science is punctuated and not continuous, and there are waves of research as data
is collected and models are developed. The ultimate goal is a pill and therapy for
HD.
Hans Lehrach:
Drug Screening is worthwhile and more biochemical information is
necessary. Modifier genes in man and mouse can be identified and genetically
tested for structural analysis and prediction. Drugs to impede aggregation should
be obtainable via high-through put methods. Focus on protein-protein interaction
research. Utilize yeast expression systems. Once an assay is found, drugs that treat
HD can be found. Phenotypic consequences may be species specific with a
common mechanism due to the pathways. Target the steps between the mutation
and the disease. Mice and humans are similar enough for models to be useful. The
HDF should focus on modifier genes, inhibitors of aggregates and inclusions.
Edward McCabe:
Hypothesis drives science, whereas pharmacology is often empirical.
Rational Drug Design is not the only way, and we should explore other methods
such as study of cell lines to develop drugs on an empirical basis.
Ethan Signer:
There are two areas of progress to focus on in the next 6 months to 1 year;
(a) Protein Structure, (b) Cell Biology. Work with inducible cell lines is key.
Ai Yamamoto:
Investigators should examine upstream and downstream stretches of the HD
gene.
Anne Young:
Bring information from transgenic animals to humans. Examine human brains
to define pathways involved in symptoms. Specific therapies might be targeted to
different points in disease with a variety of approaches to minimize side effects.
Limitation is now personnel and time. We must also study brain circuitry and getting
the drugs to the brain.
Allan Tobin:
HD research is now in an optimistic, open-minded, and dynamic era. A cure
or prophyleaxis has now become a feasible goal. Cooperation of current
researchers and encouragement of new people to join the research are essential. |
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