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Hereditary Disease
Foundation
Why Do Polyglutamine Proteins Enter and
Leave the Nucleus?
June 28 and 29, 1998
New York, New York
Prepared by Ai Yamamoto
Hereditary Disease Foundation
Why Do Polyglutamine Expansions Enter and Leave the Nucleus?
June 28 and 29, 1998
New York, New York
Participants
Stephen Adam
Assistant Professor
Department of Cell and Molecular Biology
Northwestern University
MD W129
303 East Chicago Avenue
Chicago, Illinois 60611
GÅnter Blobel
John D. Rockefeller, Jr. Professor
Investigator, HHMI
Laboratory of Cell Biology
Howard Hughes Medical Institute
Rockefeller University
1230 York Avenue
New York, New York 10021
Marian DiFiglia
Associate Professor HMS, MGH
Department of Neurology
Massachusetts General Hospital
Neuroscience Center
149 13th Street, 6th Floor
Charlestown, Massachusetts 02129
Paul Ferrigno
Postdoctoral Fellow
Department of Biological Chemistry and
Molecular Pharmacology
Harvard Medical School
Dana Farber Cancer Institute
Boston, Massachusetts 02115
Kenneth H. Fischbeck
Chief, Neurogenetic Branch, NINDS
Neurogenetics Branch
NINDS, NIH
10/3B14, 10 Center Drive
MSC 1250
Bethesda, Maryland 20892-1250
Michael E. Greenberg
Professor of Neurology and Neurobiology
Department of Neuroscience
Children's Hospital
Harvard University
Enders 260
300 Longwood Avenue
Boston, Massachusetts 02115
John Hanover
Chief, LCBB, NIDDK, NIH
Laboratory of Cell Biochemistry and Biology
NIDDKD
National Institutes of Health
Building 8, Room 402
8 Center Drive, MSC 0850
Bethesda, Maryland 20892-0850
Cynthia McMurray
Associate Professor
Department of Pharmacology
Mayo Clinic and Foundation
Rochester, Minnesota 55905
Mary Shannon Moore
Assistant Professor
Department of Cell Biology
Baylor College of Medicine
One Baylor Plaza
Houston, Texas 77030
Sara Nakielny
Postdoctoral Fellow
Howard Hughes Medical Institute
University of Pennsylvania School of Medicine
328 Clinical Research Building
415 Curie Boulevard
Philadelphia, Pennsylvania 19104-6148
Michael Rout
Assistant Professor, Head of Laboratory
Rockefeller University
1230 York Avenue
New York, New York 10021
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
Robert H. Singer
Professor and Director, Institute for Molecular
Medicine
Department of Anatomy and Structural Biology
Department of Cell Biology
Albert Einstein College of Medicine
Golding Building, Room 601
Bronx, New York 10461
Carsten Strubing
Postdoctoral Fellow
Department of Neurobiology
Harvard Medical School
Children's Hospital of Boston
Room 1309 Enders Building, P.O. Box EN-306
320 Longwood Avenue
Boston, Massachusetts 02115
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
Overview
Huntington's Disease (HD) is an autosomal dominant, almost fully penetrant,
neurodegenerative disorder characterized by movement dysfunction, cognitive decline
and psychological disturbances (1,2). On a genetic level, patients afflicted with HD all
share an expanded trinucleotide repeat mutation in a gene of unknown function (3).
Studies over the past two years have demonstrated that this mutation is sufficient to
cause aggregation of the protein as well as a striking change in its intracellular
localization from exclusively cytosolic to nuclear (4,5,6,7). In both human tissue and
transgenic animal models of this and other trinucleotide expansion diseases,
intranuclear inclusions have thus been characterized. The question remains, are the
inclusion bodies the cause of the pathology associated with HD? The workshop
sponsored by the Hereditary Disease Foundation entitled, "Why Do Polyglutamine
Expansions Enter and Leave the Nucleus?" held at the Cornell Club in New York
(June 28-29, 1998) concentrated on this question and two others: Are the expanded
polyaminoacids being actively or passively transported into the nucleus? In addition, is
there a means by which we can target the nuclear transport such that a therapeutic
strategy can be formulated?
Introduction
We were fortunate to have an extraordinary couple, Liz and Bob Regan, come to
speak to the participants to open the meeting. Liz was diagnosed with HD 10 years
ago, at the age of 31, the only one of her 5 siblings to be diagnosed. Liz's father, from
whom she inherited the HD gene, had represented a pharmaceutical firm as a
salesman. He was one of the first people in the newly created Terrence Cardinal
Cooke Health Center's HD Unit, where he recently passed away from the disease. Liz
worked as a schoolteacher before her diagnosis. She and Bob have two children of
their own. The participants were extraordinarily moved by the wisdom, vivacity and
courage of this indomitable couple. They gained a first hand knowledge of the disease
and its family dynamic.
HD belongs to a family of triplet repeat expansion diseases including spinocerebellar
ataxia diseases (SCA) 1, 2, 3 (Machado Joseph's Disease), 6 and 7; dentatorubral
pallidolysian atrophy (DRPLA); and spinobulbar muscular atrophy (SBMA). The
pathology of these diseases were described by Kenneth Fischbeck (National
Institute of Health) and summarized in Table 1 (For review see 8,9). These diseases
are all dominant gain-of-function diseases caused by polyQ expansions in distinct
gene products. It is likely that the mechanism underlying the pathogenesis of these
diseases are in common with HD.
The mutation involved in HD was characterized in 1993 as an unstable
polyglutamine (polyQ) expansion found in exon 1 of the 67 exon-containing gene, IT15
(3). As explained by Marian DiFiglia (Massachusetts General Hospital,
Massachusetts), the normal polyQ expansion ranges from 6 to about 34 units, while
the mutation is about 37 polyQs and above. As it is an autosomal dominant disease,
only one mutated allele is necessary for the disease phenotype to occur. The age of
onset of HD has been correlated with the length of the expansion such that the longer
the expansion, the earlier onset (10). While the majority of adult-onset disorders have
a polyQ range of 40 to 55 CAG units, the more severe juvenile onset disorder has a
range of 70 units or more.
The function of the 350 kDa protein encoded by IT15, huntingtin, is unknown.
Sucrose gradient analysis of the protein reveals that it is associated to the cytosolic
and membrane bound fractions of brain tissue (11) and the clathrin-coated vesicle
(trans-Golgi) fraction in fibroblasts (Marian DiFiglia). Studies show that huntingtin
associates with proteins involved in vesicle membranes and microtubules (12),
implying that it may be involved in vesicle trafficking. An important role during
embryogenesis has been suggested by the creation of IT15 null mutants by several
groups (13,14,15). Animals that do not express this gene die early in development.
Huntingtin is ubiquitously expressed, showing no particular enrichment in the brain or
caudate nucleus. On a cellular level, in the adult brain, it has an exclusively
cytoplasmic expression and is found in the cell body, in axon terminals and in
dendrites in all brain regions (16,17). Nuclear localization of wildtype huntingtin is seen
in some cultured cells.
The elongation of the polyQ expansion results in an altered intracellular
localization of the huntingtin protein. The exclusively cytoplasmic distribution changes
to include an intranuclear accumulation. Further, intranuclear and cytoplasmic
aggregations of the mutant proteins have been identified. The incidence of
intranuclear aggregate formation is also correlated to the length of the polyQ. In
juvenile onset disorder cases, 38 to 52% of the cortical neurons examined were
positive, while adult-onset cortical neurons demonstrated only 3 to 6% (Marian
DiFiglia).
Aggregate Composition: Is huntingtin truncated?
The composition of the aggregates is still unclear. The difficulty of isolating and
dissociating the highly insoluble "rocks" has greatly complicated the issue.
Ultrastructural analysis reveals that they are a heterogeneous mixture of filaments and
fibrils (18). Studies using antibodies that recognize different portions of the huntingtin
protein demonstrate that only the N-terminal portion of the protein proximal to the
polyQ is present in the aggregates (Marian DiFiglia). Antibodies closer to the C-
terminal end of the protein do not recognize the aggregates, though there presence or
absence is not yet clear. Putative caspase-3 cleavage sites have also been identified
along the huntingtin protein, further indicating the possible involvement of a truncated
huntingtin (19).
Transgenic animals demonstrate that a truncated form of the protein that
contains an expanded polyQ region is sufficient to form aggregates. One such
transgenic, the BatesR6/2 mice, express exon 1 of the human HD gene with a polyQ
of 115 to 152 units, under the control of the HD gene promoter (20). In neurons of
these animals, nuclear inclusions similar to those in humans have been found, albeit
in more widespread regions. The incidence of cytoplasmic inclusions is, however,
minimal. These mice exhibit a progressive phenotype including a gradual irregular
gait, a decrease in body weight, a resting tremor, stereotypic grooming and seizures.
Curiously, unlike human HD, neuronal loss and reactive gliosis in the striatum are
absent.
Studies using clonal striatal cell lines indicate that the HD protein involved in
cytoplasmic and nuclear aggregates differ. Cells were transfected with a partial or full
length cDNA of huntingtin with an N-terminal FLAG tag and C-terminal GFP tag
(Marian DiFiglia). Most nuclear inclusions were FLAG positive while very few were
GFP positive. This is in contrast to the cytoplasmic aggregates that were positive for
both. Mary Shannon Moore (Baylor College, Texas) suggested that perhaps
cleavage precedes nuclear import. This was supported by Dr. DiFiglia's finding that
caspase-3 inhibition decreased the number of neurons with intranuclear aggregates.
Nuclear Aggregates, Nuclear Localization and Cell Death: Cell Culture Studies.
The toxicity of nuclear aggregates on neurons was addressed in three cell culture
studies by Marian DiFiglia (Massachusetts General Hospital), Michael Greenberg
(Harvard University, Massachusetts) and Cynthia McMurray (Mayo Clinic,
Minnesota). The results are summarized in Table 2. Although different constructs and
repeat lengths were used, both full length and truncated constructions carrying the
mutated polyQ expansion lengths formed aggregates in the cytoplasm and/or nucleus.
Several other interesting observations were also disclosed. The previously described
clonal striatal cell line study conducted by Dr. DiFiglia demonstrated that one caspase
inhibitor decreased by half the number of nuclear aggregates. Despite this decrease,
however, the survival of the neurons was not affected. The use of another caspase
inhibitor, on the other hand, had no effect on the number of aggregates formed; yet,
the rate of survival of the neurons was increased.
Michael Greenberg described experiments conducted on primary cultures of
rat striatum and hippocampus. In 20% of the striatal neurons, intranuclear aggregates
were seen. Only some glia demonstrated perinuclear aggregates. The number of
intranuclear aggregates peaked 6 days post-transfection. Interestingly, cell death
occurred at day 9, when the number of aggregates began to decrease. Moreover, the
mutant construct was toxic only for the met-enkephalin-positive striatal neurons, the
neurons that preferentially die in HD. The other striatal neurons as well as
hippocampal neurons were not affected. This nuclear aggregate formation and cell
death was reversed when the construct contained a nuclear export sequence (NES).
Further, the use of leptomycin B, an inhibitor of nuclear export, reversed this finding,
and led to the formation of intranuclear aggregates despite the NES. In this study, the
nuclear localization of the mutant construct appears to be important for cell death.
However, this cell death may not be caused by the aggregates themselves, but the
inability of the cells to further sequester the fragments into the inert aggregate.
These findings however, differed from the findings presented by Cynthia
McMurray. Using primary culture of dorsal root ganglion (DRG) cells and studies from
dividing cells, a truncated fragment with a polyQ expansion of 82 units with a GFP
marker was expressed. Aggregation was monitored using time-lapse video allowing
the living cells to be viewed in situ. Despite the use of a truncated mutant fragment,
intranuclear inclusions were not seen. Instead, perinuclear and diffuse cytoplasmic
staining was observed. A full length fragment was also studied in a neuroblastoma cell
line. 12-hours after transfection, diffuse cytoplasmic GFP was detected, but none in
the nucleus. After 48-hours nuclear inclusions were detected, however at this time the
cells already were compromised and dying. The cell death observed in all cases was
largely apoptotic. Another point that was unlike Dr. Greenberg's study was that the
addition of a NES had no effect on cell survival. Dr. McMurray concluded that nuclear
inclusions are not required for cell death, although inclusions forming in the nucleus
are undoubtedly not good for the cell.
The contrasts in the studies are difficult to interpret since the experimental designs of
each were distinct on many points. For example, the cell types used in each studied
differed. The study that did not see nuclear inclusions used DRG cells. It is therefore
difficult to interpret whether this is a cell dependent phenomenon or not. Kenneth
Fischbeck mentioned that in his SBMA model, in which the polyQ is found on the
androgen receptor, non-neuronal cells can have nuclear inclusions. The most striking
contrast between the latter two studies was the differential effect on cell survival of the
NES. Stephen Adam (Northwestern Medical University, Illinois) cautioned that the
NES data should be interpreted with care. He explained that a forced interaction with
the nuclear transport machinery could have a chaperone effect on the fragment,
increasing the solubility of the fragment and/or allowing more efficient degradation by
proteasomes. Another caution was stated by John Hanover (National Institute of
Health) in regards to GFP, whose over-expression may itself be contributing to the
aggregation phenotype observed. Michael Rout (Rockefeller University, New York)
summarized the general consensus by stating that it is crucial to resolve the apparent
discrepancies between these two latter studies to determine if nuclear HD is
associated with disease pathology. Ethan Signer (MIT, Massachusetts) suggested
that Drs. Greenberg and McMurray can conduct an exchange of constructs and/or
cell systems to see if the respective results can be repeated. GÅnter Blobel
(Rockefeller University, New York) suggested that the Drosophila system designed
by Nancy Bonini and colleagues might resolve this discrepancy.
The common thread in all three studies appeared to be the separation between
nuclear inclusion formation and cell death. Dr. DiFiglia's study showed that despite a
decrease in nuclear aggregates after caspase-3 inhibition, cell survival was not
changed. Dr. Greenberg's work showed that cell death occurred when the number of
aggregates was decreasing and not at the maximum level. Paul Ferrigno (Harvard
University, Massachusetts) ventured that perhaps if the aggregates are going away
before cell death, that it might imply that early stage patients have more inclusions
than at end stage. Finally, Dr. McMurray demonstrated that nuclear localization may
not be required at all. Instead, she proposes that cytoplasmic aggregation may be the
key, and that cell death is likely due to progressive sequestration of cellular proteins,
thus causing progressive damage to normal cell function. Stephen Adam suggested
that transglutaminases might be one of these sequestered proteins. If so, then the
intracellular localization of the aggregates might not be critical.
Passive vs. Active Transport into the Nucleus.
Dr. McMurray also presented a study determining the exclusion limits of different cell
types ranging from cardiac myocytes, which allow up to 30 kDa proteins to passively
diffuse, to the DRG, which allow up to 70 kDa. Mary Shannon Moore added that on
average, 45 kDa is the size exclusion limit for a nucleus. Stephen Adam noted that
due to the small size and insolubility of the polyQ fragment, the appearance of the
nuclear aggregates is likely due to diffusion of the protein fragment into the nucleus
and spontaneous aggregation. Based on the presented data, he felt that there was no
evidence for direct transport of the mutant protein into the nucleus.
Michael Rout indicated that by using a simple well-characterized system such
as yeast (Saccharomyces) it might be possible to distinguish between the pathology
and cell biology of the disease. He explained further that it would require the
construction of stable cell lines which can inducibly express mutant or normal HD, or
polyglutamine tracts fused to a variety of control or reporter proteins. Should it be
shown that nuclear transport is required, then a yeast system would be one of the
fastest ways of determining the method of transport. Another system that might be
used is the digitonin system (Stephen Adam). This in vitro system consists of a
permeabilized plasma membrane while the nuclear membrane is maintained intact.
Through addition of cystolic factors, an energy source and a reporter construct with a
nuclear localization sequence (NLS), to track nuclear accumulation, one could
determine if htt is actively transported. GÅnter Blobel cautioned that for this system to
work, the huntingtin must be in the soluble form and not in the aggregate form.
Stephen Adam added that a negative result would be meaningless in this study,
especially if the transport is neuron specific due to a neuronal a-importin.
Parting Remarks
As best summarized by GÅnter Blobel, it is necessary to answer the question, "Does
the fragment have a nuclear lifestyle." To answer this question may help determine not
only the underlying pathogenic mechanism of HD, but to also aid in therapy-design.
Several studies were proposed by many of the panel members to address the
differences seen upon introduction of a NES. The four particular experiments
proposed included; the determination of the effect of a NES on the full length protein
(Greenberg); the creation of transgenic animals that express a mutated form of exon
1 together with a NES (Rout, McMurray); the creation of inducible stable cell lines
(DiFiglia); and the use the Drosophila system to study the effect of the NES
(Fischbeck).
Other studies proposed, parting remarks and comments can be summarized as
follows:
Stephen Adam: It is clear that polyQ accumulation in neurons lead to cell damage or
death. It appears unlikely that the non-polyQ region plays a major role in the disease,
although it cannot be excluded that aggregation or co-aggregation with the polyQ can
exacerbate things. The experiments that were proposed to investigate the effects of
the polyQ fragment on transport, using yeast as a model system, could be very
promising if yeast show a phenotype upon expression of the fragment. Since HD is a
gain-of-function disease, the function gained may be only indirectly related to
huntingtin or the polyQ fragment. Competitive inhibition of ubiquitination, proteasome
or transglutaminase pathways via accumulation of the polyQ could causes another
regulatory pathway to be up-regulated leading to cellular damage.
GÅnter Blobel: The polyQ is the key to the pathology as it is a common mutation in
several diseases. One must have a broad consideration of cell physiology and pursue
studies looking at microtubules, dynein and anterograde and retrograde transport.
Moreover, it is important to pursue the question, how does the cell deal with
aggregates and inclusions?
In addition, it would be interesting to see if microglia can degrade polyQ
inclusions. Studies have been conducted in cultured neurons that demonstrate that
co-cultivation with microglia can induce cell death. It appears that the microglia
secrete a toxin known as phenolic toxin. Further, aggregates in a few cells may be
attacked by microglia. This attack may produce bystander effects. Co-cultivation
experiments with microglia and cells expressing the aggregates could be done.
Marian DiFiglia: We will be pursuing more biochemical studies on the human brain
and in HD transgenic mice. Also, it is important to determine if there is an effect due to
the level of protein expression. Does a higher level of expression lead to a more
severe phenotype? Or is there a correlation at all? Finally, we will begin to define the
signal transduction pathway(s) that may be responsible for cell death.
Paul Ferrigno: As someone who did not study HD before this meeting, I would like to
say that this has been an inspiring and fascinating meeting. I would like to first outline
the questions that are of interests to me, and what I believe can be done in yeast. The
questions are as follows: Are there any RNA in the nuclear inclusions? Are there
ubiquitin-like proteins rather than ubiquitin in the inclusions? And finally is the ubiquitin
at any times removed from the inclusions. The experiments I would like to do in yeast
is to first determine the localization and toxicity of full length huntingtin, using yeast in
the stationary phase. Next, using a purified dynactin complex that we already have, I
will determine the relationship between HAP-1 dynactin and huntingtin. Third, using an
aptamer screen, we can screen for drugs as well as the possible interactors of wild
type huntingtin. Finally, we can perform a screen on human library for proteins that
make yeast sensitive to the polyQ.
Kenneth Fischbeck: We are currently trying to determine if and how the flanking
sequences of the polyQ and the protein determine specificity. We will be using the
Drosophila system.
Michael Greenberg: We will first try to resolve the discrepancies with Cynthia
McMurray, and continue with work in our system to determine the effect of the NES on
full-length. Also, is it clear that the HD phenotype is due to the protein rather than the
RNA? And if anything is known about possible transcription factor activity of polyQ
proteins.
John Hanover: The mouse model discussion largely confirmed the human findings
but pointed out a number of differences. Since the knockout of huntingtin in mouse in
embryonic lethal, I feel an important control is to knock in a form lacking the amino-
terminus to see if one can rescue survival. This could have implications for gene
therapy. Cell models were then discussed. The key question of whether huntingtin
needs to enter the nucleus was difficult to resolve. The use of a very sensitive two-
hybrid approach, such as in the Notch transcription factor experiments, may identify
cellular factors that interact with exon 1 constructs. Finally, it is likely that huntingtin
enters the nucleus as a fragment. This suggests that a processing event must occur.
This processing event is an obvious drug target and it should be defined.
Cynthia McMurray: There appears to be little evidence to suggest that polyQ proteins
are actively transported in the nucleus. Evidence from several sources indicates that
HD-derived proteins move into the nucleus by passive diffusion. Experiments we
intend to do include resolving the export experiment with Michael Greenberg. Second,
determine the regional pathology of HD, using promoter swapping. Third, investigate
the components of the aggregates by isolating the high molecular weight fractions
containing huntingtin protein then repeat this as a function of grade. Fourth, identify
the cleavage sites along the huntingtin protein then try to block proteolysis with
expressed competitive inhibitors of the target sites. For possible cures, it can be
determined whether huntingtin is required during adulthood. If not, then gene targeting
constructs that are designed to silence the gene can be designed. Moreover, excision
of the expanded region by small molecules can lead to a cure for the family, while
specific targeting and neutralization of the huntingtin protein can lead to a cure for the
patient.
Mary Shannon Moore: It is not clear whether there is a role for nuclear trafficking. If
the protein is available, it can be determined if the digitonin system can be used as a
means to study the protein/protein fragment. In terms of therapy, it may be important
to determine whether the protein is required in the adult. If not, it leads to a possibility
for gene therapy.
Michael Rout: The polyQ may be the important factor. The HPRT transgenic mouse
should be studied in greater depth to lead to a better understanding. Further to study
the "nuclear lifestyle" of the protein, yeast may be an efficient manner in which the
studies can be conducted. Finally, changes in gene expression due to the aggregates
should be examined by using technology including the DNA chip.
Ethan Signer: Other polyaminoacids aside from polyQ should be explored. Is the
phenomenon that we see specific to the glutamine? Another question that should be
addressed is what molecule in the cell is the first to recognize and bind to polyQ.
Robert Singer: The inclusions that are seen might be a red herring. The inclusions
might be a secondary process that does not address what the polyQ is doing. Instead,
forget the huntingtin protein/fragment and focus on the polyQ. Is this acting as a
transcription factor? Finally, if the protein is not necessary in adults, then ribozymes
may be designed to degrade the mRNA.
Carsten Strubing (Harvard University, Massachusetts): This workshop was an
inspiring experience. I think there was a consensus between the participants that the
form of the workshop, i.e. an informal presentation of data and free exchange of
ideas, provided the basis for the interesting and fruitful discussions. Clear
experimental approaches (in vitro import assay, microinjection of huntingtin, in vivo
import assay in yeast) hopefully will reveal the mechanism of nuclear huntingtin import
in the near future. It was pointed out the cellular pathophysiology of HD is not well
understood. Previous data suggests that mitochondrial function and metabotropic
glutamate receptor expression are compromised in HD. Both mitochondria and
mGluRs are implicated in intracellular Ca2+ signaling. Therefore, fluorometric Ca2+ -
measurements as well as electrophysiological studies on isolated neurons and brain
slices from transgenic HD models could help better understand the cellular processes
underlying the HD phenotype.
TABLE 1: Triplet repeat expansion disorders.
Disease Affected Region Protein Normal Location Nuclear inclusion?
HD Cortex htt cytoplasm yes
Striatum
SBMA DRG androgen cytoplasm/ yes
Motor neurons receptor nucleus
SCA 1 Cerebellum nucleus yes
Brain stem
SCA 2 Cerebellum cytoplasm yes
Brain stem
SCA 3 Cerebellum nucleus yes
Brain stem
SCA 6 Cerebellum Ca2+ nucleus yes
Brain stem channel
SCA 7 Cerebellum yes
Brain stem
DRPLA Brain stem nucleus yes
Basal Ganglia
TABLE 2: Presented cell culture studies
Marian DiFiglia
Michael Greenberg
Cynthia McMurray
Cell
type(s)
Clonal striatal line
Rat striatal primary
culture
DRG
Neuroblastoma
Full length
Construct
FLAG-tagged
None
GFP-Full length
(Neuroblastoma study)
Localization
and
inclusions
Nuclear & cytoplasmic.
Few cells at Day 1. All
cells at Day 6.
N/A
Nuclear inclusions (occur
late well cell already
compromised.)
Truncated
Construct
3 kb w/ N-terminal
FLAG tag, and C-
terminal GFP tag.
480 residues with
polyQ= 17 or 68
171 residues with
polyQ= 17 or 68
Truncated within
polypropylene region. N-
terminal GFP
PolyQ= 19 or 82
(DRG study)
Localization
and
inclusions
Nuclear and
cytoplasmic inclusions
480-Q17 cytoplasmic, non
aggregate; -Q68 nuclear
inclusions in 20% of cells.
171-Q68 nuclear
inclusions in 80% cells.
Maximum no. of
inclusions at day 6.
Q19: small degree of
death
Q82: Inclusions and
diffuse staining in
cytoplasm and
perinuclear. No nuclear
aggregates
Inclusions
vs.
Cell Death
The use of different
caspase inhibitors
dissociated the
formation of
aggregates from cell
survival.
Cell death in met-enk
positive cells only. Death
occurred on day 9.
Addition of NES- no
inclusions, no death.
Protein in cytoplasm.
Addition of NES had no
effect on survival
Notes
WT huntingtin stops
cell proliferation.
Mutant approx. 20%
keep proliferating.
Living cells were
monitored in vivo.
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