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Workshop Reports
Hereditary Disease
Foundation
Nuclear Inclusions and the Pathogenesis of
Polyglutamine Diseases
December 13 and 14, 1997
Playa Del Rey, California
Prepared by Jang-Ho Cha
HEREDITARY DISEASE FOUNDATION WORKSHOP
"Nuclear Inclusions and the Pathogenesis of Polyglutamine Diseases"
December 13 and 14, 1997
Playa del Rey, CA
Participants
Barbara Brodsky
Professor, Department of Biochemistry
University of Medicine and Dentistry of New Jersey
Robert Wood Johnson Medical School
Piscataway, New Jersey 08854
Jang-Ho Cha
Instructor in Neurology, Department of Neurology
Neurological Research, WRN408
Massachusetts General Hospital
Fruit Street
Boston, Massachusetts 02114
Bruce Chesebro
Laboratory Chief
Rocky Mountain Laboratories
903 South 4th Street
Hamilton, Montana 59840
Robert Hughes
Research Scientist
Department of Genetics, Box 357360
University of Washington
J-205 Health Sciences Building
Seattle, Washington 98195-7360
Vernon Ingram
John & Dorothy Wilson Professor of Biochemistry
Director, Experimental Study Group
Housemaster, Ashdown House
Department of Biology
Massachusetts Institute of Technology
Room 56-601
Cambridge, Massachusetts 02139-4307
Jeffery Kelly
Lita Annenberg Hazen Professor of Chemistry
The Scripps Research Institute
10550 North Torrey Pines Road
Mail Stop MB12
La Jolla, California 92037
Diane Merry
Research Assistant
Professor of Neurology and Genetics
Department of Neurology
University of Pennsylvania
415 Curie Boulevard
Philadelphia, Pennsylvania 19104
Cameron Mura
Graduate Student
Department of Chemistry and Biochemistry
University of California Los Angeles
Box 951570
Los Angeles, California 90095-1570
Henry Paulson
Assistant Professor, Neurology
Department of Neurology
University of Iowa Hospitals
Iowa City, Iowa 52242
Terry Reisine
Scientific Director
Davis Biomedical Association
2121 Avenue of the Stars
Suite 2800
Los Angeles, California 90067
Christopher Ross
Professor of Psychiatry and Neuroscience
Laboratory of Molecular Neurobiology
Ross Building 618
Johns Hopkins University
720 Rutland Avenue
Baltimore, Maryland 21205-2196
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
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
Martin Vey
Postdoctoral Fellow
Department of Neurology HSE-781
University of California San Francisco
School of Medicine
San Francisco, California 94143-0518
Jonathan Weissman
Assistant Professor, Cellular & Molecular
Pharmacology and Biochemistry & Biophysics
University of California San Francisco
513 Parnassus Avenue
Post Office Box 0450
San Francisco, California 94143Ronald Wetzel
Professor of Medicine
Department of Medicine
University of Tennessee Medical Center
1924 Alcoa Highway
Knoxville, Tennessee 37920
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 Overview
The recent observation of neuronal intranuclear inclusions (NII) in the brains
of Huntington's disease (HD) patients and transgenic HD mouse models has
provided a novel perspective on the pathogenesis of this and other so-called
polyglutamine diseases (Davies et al., 1997; DiFiglia et al., 1997; Ross, 1997). This
workshop brought together scientists of various disciplines to discuss the implication
of NII and their possible role in causing disease.
Introduction
The meeting opened with introduction of a 25 year old young woman with HD
and her husband. She discussed how her HD had been diagnosed and the impact
that this diagnosis has had on her life. Her husband described the difficulties which
they have had as her disease symptoms progressed. Although she was diagnosed
just earlier in the year, she has already had to give up many of her favorite activities
such as skiing and many other sports, and is no longer able to continue in her
profession of modeling and architecture. Both she and her husband cited their
religious faith as a source of strength, as well as their optimism in the progress of
medical research.
For many of the researchers present, it was the first face-to-face encounter
with someone with Huntington disease. Several researchers remarked how
inspiring it was to actually meet someone with HD, bringing home to them the reality
of the disease, rather than just an abstract concept. All concurred that keeping in
mind the family's perspectives is a true motivating force in any disease-based
research. The ensuing discussion centered around the question of whether it was
fair to families to raise their hopes by offering the prospect of a "cure" for HD. Allan
Tobin pointed out that in the case of HD, a small change in the rate of progression
of disease could functionally amount to a cure. A similar example might be
Alzheimer disease, in which effective treatments appear likely, while ultimate cures
may be impossible. Another point is that most medical successes are examples of
effective treatments rather than pure "cures." While a "cure" for HD in the sense of
total eradication of the disease might not be immediately feasible, all agreed that
treatments might lie within the realm of the possible and should be aggressively
sought. Nancy Wexler pointed out that in 1968, the use of the neuroleptic Haldol
was standard treatment for people with HD, while now Haldol is not used nearly as
much, and antidepressants are widely and effectively used.
Transgenic Mice and Neuronal Intranuclear Inclusions
Chris Ross initiated the next portion of the workshop by showing two
videotapes of transgenic mice.
The first video showed transgenic mice into which were inserted the N-
terminal portion of truncated HD gene containing an expanded CAG (82 repeats)
region. Working with David Borchelt and Gabrielle Schilling, Dr. Ross has created
three lines of mice each containing 82 repeats. All three mouse lines develop
neurologic symptoms, but with different ages of onset, ranging from 2 to 6 months
of age. Depending on the line, these mice have a life span of 5 to 10 months
(normal life span for a mouse is greater than 1 year). Symptoms include difficulty
walking, tremor, and uncoordinated movements. When control mice walk, they
demonstrate a fluid flexible movements, shifting weight easily, striding rapidly.
When picked up by the tail, control mice attempt to escape. Transgenic mice
appear less well groomed and unsteady on their feet. When picked up by the tail,
they are especially uncoordinated and unable to escape. Ross' mice demonstrate
clasping of their back paws, a trait also reported in Gill Bates' transgenic HD mice.
As the Ross mice age, they become more slow, stiff and lethargic. At end stage,
they are very hypomotile.
Following the video presentation, Chris Ross described the approach they
have taken in making their transgenic mice. For the HD mouse, they have used a
cDNA construct which conatins the N-terminal 160 residues, including 82 glutamine
repeats (N-160, Q-82). Their numbering system is based on the original HD gene
paper in which the reported sequence has 23 CAG repeats (Huntington's Disease
Collaborative Research Group, 1993). The transgene is under the control of the
prion promoter, which is highly expressed in brain, with lower expression levels in
heart and other tissues. They have made three different mouse lines. The mouse
lines express 20 to 50 copies of the transgene, but overall transgenic protein
expression levels are lower than the levels of endogenous mouse huntingtin.
Gill Bates' group was the first to make a transgenic mouse model. This model
also expresses a truncated exon 1 of huntingtin, but (i) the construct is a genomic
as opposed to a cDNA construct, (ii) expression is under the control of the human
HD promoter, and (iii) the repeat number is higher (120 to 150 glutamines)
(Mangiarini et al., 1996). Ross' group found in analyzing expression of their
transgene that, although RNA levels are relatively high, protein expression is
inexplicably low. They have been able to immunostain with the AP 194 antibody (an
antibody which recognizes the N-terminus of huntingtin made by Allan Sharp at
John Hopkins University) and by the 1C2 antibody; developed by Yvon Trottier in
Jean-Louis Mandel's laboratory, this antibody recognizes polyglutamine regions with
expanded repeats.
The second video showed transgenic mice expressing a full length
dentatorubropallidoluysian atrophy (DRPLA) gene, again, with an expanded CAG
region (61 repeats). These mice demonstrate a constant tremor, uncoordinated
movements, and rapid involuntary forepaw movements which are reminiscent of
chorea. Their gait is abnormal, resembling a hopping movement as opposed to the
more fluid striding of normal mice. In addition, these mice have seizures, as
evidenced in the video.
Ross then went on to comment on the pathbreaking discovery of inclusions
which Steve Davies has made while analyzing the Bates mice (Davies et al., 1997).
Ross' group have made these observations as well in its transgenic mice. In certain
neurons of both the Bates and Ross transgenic mice, spherical intranuclear
inclusions are seen. These inclusions are roughly the size of the nucleolus, and
they immunostain with antibodies directed against the N-terminus and against
ubiquitin. Davies has recently shown that formation of inclusions precedes the
onset of symptoms in the Bates mice. Following the description of NII in mice,
Marian DiFiglia confirmed the presence of these so-called neuronal intranuclear
inclusions (NII) in human HD brain, and made the first observation that inclusions
are also found in dystrophic neurites. Mark Becher at Hopkins has confirmed the
presence of inclusions in dystrophic neurites in human HD brain. Both Bates' and
Ross' groups have subsequently found inclusions in dystrophic neurites in the
brains of their transgenic mice.
Ross then summarized some of the recent results of Michael Hayden, who
has found that huntingtin can be cleaved by caspase-3 (formerly known as apopain,
also known as cpp-32 and yama) (Goldberg et al., 1996). Caspase-3 recognizes
the amino acid motif DXXD. Since the caspases are known to participate in certain
aspects of programmed cell death, the question of whether HD is an apoptotic
disease was raised. The role of caspases in CAG repeat diseases remains unclear.
Jang-Ho Cha described his work analyzing neurotransmitter receptors in the
brains of the Bates mice. Although the transgenic mice brains were originally
described as appearing morphologically normal, brain weights were decreased
compared to controls. Using receptor binding assays, Cha found that there were
selective decreases in receptor binding in the brains of these mice (receptor and
binding levels in transgenic mice as a percentage of control mice: NMDA 100%;
AMPA 85%; kainate 70%, group I metabotropic (G-protein linked) 100%, group II
metabotropic 65%, GABA 100%, muscarinic acetylcholine 65%, D1 dopamine 33%,
D2 dopamine 33%). Receptor binding changes occurred not only in striatum but
also in cortex. Decreases in corresponding receptor proteins were confirmed by
immunoassay. In situ hybridization demonstrated decreases in corresponding
mRNA levels (e.g. for the metabotropic glutamate receptors mGluR2 and mGluR3,
mRNA levels were decreased to 50% of control whereas mGluR5 mRNA levels
were unchanged). D1 and D2 dopamine receptor mRNA were changed as early as
4 weeks of age, preceding receptor binding changes and the development of
symptoms. One correlate of NII, then, could be altered transcription of
neurotransmitter receptor messages.
Other transgenic animals
Ethan Signer then reviewed other HD transgenic animals which have been
developed. Danilo Tagle at NIH has made two different transgenic mice, one using
full length cDNA and the other a truncated cDNA. Both of Tagle's mice develop
symptoms, although the mice expressing the truncated version develop symptoms
more quickly than the mice expressing the full length gene. Diane Merry pointed
out that the CMV promoter used by Tagle results in very high level of protein
expression.
Signer then went on to describe Rick Myers' (Stanford University) "knock-in"
mouse. This mouse has the expanded CAG repeats inserted into the mouse's
endogenous huntingtin gene which to this point show a slightly abnormal
phenotype, demonstrating increased aggressiveness and some mild hippocampal
cellular abnormalities. Wexler then related that Efstratiadis & Scott Zeitlin's knock-
ins were as yet asymptomatic at one year of age. Wexler then went on to explain
that efforts to make inducible transgenics are underway.
Helices and Sheets
Barbara Brodsky reviewed the properties of glutamine (Gln or Q) residues
in proteins. Gln's can exist in ý-helical formations, -sheets, and have also been
reported to be found in random coil. Brodsky also noted that Gln's are often found
in the neighborhood of polyproline stretches, as is the case with huntingtin. Gln's
can thus exist in numerous conformations, but they will not exist on their own. They
are most likely to exist in -sheets or binding to something else. PolyGln's are
highly insoluble, so even with wildtype huntingtin proteins (with normal numbers of
CAG repeats), something is likely acting to keep them soluble.
Jeffery Kelly told us that -sheets are 6 to 8 residues per strand, and that
Gln is the residue that favors sheet formation most. Kelly went on to speculate that
the Xray crystallographic structure of the mutated huntingtin would be instructive.
An abnormally large polyglutamine moiety could drag the whole protein into a
sheet configuration, thereby destabilizing the protein. He further noted that Gln-
containing peptides can have very many different properties.
Cameron Mura (from David Eisenberg's lab) told us about his experiments
in making polyGln proteins expressed as fusion proteins linked to glutathione-s-
transferase (GST). Thus far, he has been successful in obtaining crystallization
leads from fusion proteins containing 0, 19, and 35 Gln's. However, he has not
been able to crystallize GST fusion proteins containing either 62 or 81 Gln's,
because these proteins abnormally aggregate. The resultant aggregates are
difficult to work with and will not even enter a stacking gel, indicating their high
molecular weight. The aggregates are resistant to dissolution by 8M guanidinium,
boiling, DTT, or urea. These results illustrate the dilemma of working with an
aggregatable protein: if one is too successful in producing an aggregatable protein,
an insoluble, hence unanalyzable, dense aggregate will result. Kelly hypothesized
that an incremental approach would be needed, for example, starting with a simpler
protein, e.g. prion protein.
Lessons from Prions
Bruce Chesebro noted that the structure of prion proteins has now been
solved in two different laboratories. However, the final configuration of prion is half
structured and half unstructured. Several caveats were offered. Kelly noted that
glycosylation has not been taken into account as far as the final prion structure, and
Martin Vey noted that the recombinant protein has not yet been shown to be
infectious. Nevertheless, prion protein was considered to be a good model of a
disease-related protein which can assume abnormal configurations. Chesebro
noted that the repeat regions in prion protein have both Gln and glycine (Gly).
Chesebro and Vey then briefly reviewed the biology of prion protein. The
structure has been partially solved. NMR evidence indicates that the N-terminal
portion of the prion protein (amino acid residues 1-100) is amorphous. In any case,
this portion of the prion protein has yet to be successfully crystallized. The structure
of prion protein has been solved starting from amino acid 122. Residues 1-23
constitute the signal peptide of the 254 residue protein. Residues 23-231 constitute
the mature protein whose C-terminal is lipidated, and contains two N-linked sugars
near the COOH terminal. The first structure that was solved was of the 121-231
fragment, and the 89-231 structure can be folded into the scrapie isoform. The
whole protein assumes the following conformation:
N-terminal (unstructured) -- sheet -- ý helix -- ý helix -- ý helix
Within the "amorphous" region of the prion protein lie five octapeptide
repeats. Mutations within these repeats can lead to inherited prion diseases, raising
a possible similarity to HD, which is a disease of repeated protein structures.
Chesebro then related an important experiment by Aguzzi in which normal brain
tissue was transplanted into the brains of mice lacking PrP (prion protein). PrP null
mice are resistant to infection by scrapie. However, PrP null mice which had
received grafts of normal brain were infected with scrapie, and the affected areas
extended beyond the areas of the graft. Thus, infection with an infectious particle
depended on some crucial interaction with the normal protein. Paulson then
observed that "pathologic" markers may not themselves be pathogenic. That is,
aggregation of huntingtin may not be the pathologic agent, but the neuronal
intranuclear inclusions (NII) may only be an abnormal marker.
Fibrils & Aggregates
How does one study the formation of abnormal protein structures? Prion
protein research has taught us that sheets are potentially pathogenic protein
formations. The propensity to form sheet correlates with (i) protease resistance
and (ii) ability to be pelleted by centrifugation. These pelleted aggregates bind
Congo Red, and electron microscopy reveals them to be fibrils. However, both
electron microscopy and Congo Red staining are not definitive proof of sheet
structure. The actual mechanism by which Congo Red stains sheet structures is
unknown. Paulson noted that the neuronal intranculear inclusions which he has
observed in Machado-Joseph disease are negative for Congo Red and thioflavin S
staining, but that does not guarantee that these inclusions have no sheet. Vernon
Ingram noted that different batches of commercial Alzheimer amyloid (A ) have
different amounts of aggregate (as opposed to monomer), and Ronald Wetzel
noted that these differences may result in part from different amounts of
endogenous aggregate. In short, aggregation is a nucleation-dependent event
which itself can be nucleated.
Protein-protein interactions
The yeast two hybrid system has been used to search for proteins which may
interact with huntingtin. Robert Hughes noted that the ability to find interacting
proteins in the yeast 2 hybrid system depends on the proteins' capacity to exist in
a soluble state. Extremely aggregatable proteins may be missed using this
methodology.
Transthyretin
Jeffery Kelly reviewed recent advances in transthyretin, a protein that can
assume abnormal configurations leading to disease. Transthyretin is a serum
protein which is affected in one form of systemic amyloidosis. Its normal function
is to bind retinol and thyroxine. Transthyretin exists as a tetramer, two coassembled
dimers. Thyroxine binds at the interface between the two dimers. Under acidic
conditions (pH 5.5) the protein undergoes a conformational change into an amyloid-
forming configuration. As a functional definition, "amyloid" refers to a protein
substance which (i) binds Congo Red and (ii) exhibits birefringence. The structure
of amyloid is cross- helices. Colin Blake has found the structure of -helix to be
composed of overlapping strands oriented at 15¯ per turn, ascending at a rate of
24 strands per cycle, that is, a twisted sheet which goes on and on. Transthyretin
coassembles into 4 protofilaments. These protofilaments in turn coalesce into the
amyloid fibril.
Amyloid aggregates have only been found extracellularly. Amyloid deposition
is potentially a reversible process, since patients with systemic amyloidosis who
receive liver transplants clear large amount of amyloid. Transgenic mice have been
created expressing 90 copies of the transthyretin gene. They develop abnormal
deposits of amyloid at 17 months of age.
Amyloidosis resulting from transthyretin occurs when transthyretin switches
from the normally folded form into the disease-associated amyloid form. Kelly
explained a recent experiment published in Science in which analogs of thyroxine
were used to bind to transthyretin, specifically targeted to the active site. These
analogues stabilized the normally folded form of transthyretin. Kelly went on to note
that fibril formation is the thermodynamic minimum of all proteins, if they can
achieve that thermodynamic state. But most proteins are destined not to get there.
Proteolysis
Terry Reisine reviewed the role of proteolysis. NII in the nuclei of cells in HD
brains are described as containing fragments of huntingtin, suggesting that some
processing has occurred. What is the possible role of proteolysis? What are the
enzymes involved? Proteolysis is a highly regulated event. For example, peptides
necessarily undergo proteolysis for their normal function. As an example, the opiate
precursor molecule POMC requires 3 to 5 proteases to achieve biological activity.
These enzymes may be differentially distributed throughout the brain, for example,
the anterior pituitary produces predominantly -endorphin, and the intermediate lobe
produces primarily -MSH.
Where might cleavage sites exist in huntingtin? The recent report from
DiFiglia et al. (DiFiglia et al., 1997) describes a truncated 42kD portion of huntingtin
detectable in nuclear subcellular fractions. Several discussants speculated that
determining sites of cleavage would be desirable, as this could provide a major clue.
All agreed that elucidating huntington's normal proteolytic processing would be
important. Similarly, looking for enzymes which could preferentially cleave mutant
protein would be fruitful.
Lessons from Androgen Receptor
Diane Merry noted that the function of one of the CAG repeat disease
proteins is known. Androgen receptor is the protein affected in spinobulbar
muscular atrophy (SBMA). Proteolysis does not contribute to processing, but in
COS cells, they have seen repeat length-dependent differences in localization. She
has also seen aggregates in her cell culture models. Using antibodies directed
against the N-terminal region, she and her colleagues have observed the formation
of aggregates. The high molecular weight aggregates are recognized by C-terminal
antibodies, suggesting that recruitment of normal full length molecules is occurring.
Also the 1C2 antibody fails to recognize the high molecular weight aggregates.
What is the nature of the high molecular weight aggregates? Are these fragments
which are specifically cleaved at a certain point or are the aggregates a
conglomeration of fragments of various lengths?
Kelly theorized that one could use stronger solvents to dissociate the
aggregates. Fluorinated alcohols are traditionally used, although recent data
suggest that chlorinated alcohols (e.g. dichloro-butanol or 2-Cl-butanol) may be
more effective in dissociating aggregates. Chlorinated alcohols are rumored to
much more effective than, for instance, guanidine. Merry noted that she has
observed similar aggregates in a cell-free system (GST fusion proteins), so if
aggregation does involve covalent linking, it is not through enzymatic mechanisms.
Proteasomes and Chaperones
Jonathan Weissman reviewed proteasomes. The cell has two proteolytic
pathways: the lysosomal system for degrading extracellular proteins and the
proteasome for degrading intracellular proteins. The proteasome is composed of
a quadruple ring structure with an antechamber and a catalytic ring. Ubiquitinated
proteins are targeted to the proteasome where they are degraded by the 26S moiety
of the proteasome. The 26S activity contains within it a deubiquitinating enzyme.
Conceivably, if a polyGln-containing protein was not cut, the uncleaved portions of
protein could inhibit the proteasome. Inhibition of proteasome function in yeast
leads to cell cycle arrest. An hypothesis would be that proteasomes are inhibited
by larger uncleaved pieces of polyGln containing-proteins and these fragments
would accumulate in post-mitotic cells, since they would not be diluted by mitosis.
One possible experimental strategy to approaching the role of the proteasome
would be to construct an exon 1 fragment of huntingtin which lacks Lys, which would
preclude its ubiquitination. Weissman also noted that NF- B processing by
proteasomes is a process which is sequence-independent. Finally, lactacystin is a
proteasome inhibitor (Fenteany et al., 1995), although it is somewhat difficult to
obtain.
Polyglutamine by itself is undeniably a highly aggregatable moiety. Many
proteins have the potential to form aggregates, and yet do not. Chaperones are
proteins which can stabilize non-aggregatable conformations of potentially
aggregating proteins. Weissman reviewed an experiment by Charnoff and Lindquist
in which they examined the overexpression of HSP 104 on the behavior of Sup35.
Sup35 is a yeast protein with a prion domain which can exist in either a soluble or
aggregate form. Once aggregated, Sup35 is passed on from generation to
generation of yeast and is stably transmitted. HSP 104 is the major heat shock
protein expressed by yeast in response to heat exposure. HSP 104 has homology
to clpA, which itself possesses similar activity to the 26S proteasome. HSP 104
also has ATPase activity. When HSP 104 is overexpressed in yeast with the
aggregated form of Sup35, HSP 104 can reverse the aggregation of Sup35.
Interestingly, HSP 104 knockout yeast are unable to propagate Sup35 aggregates.
Thus, HSP 104 seems to have two potential roles with regard to aggregates: (i) HSP
104 can reverse aggregation of Sup35 and (ii) the presence of HSP 104 may be in
some way necessary to allow aggregation to occur in the first place. Two
hypotheses have been put forth to explain the curious result with the HSP 104
knockouts. Lindquist has argued that HSP 104 binding to Sup35 is required for
assembly of monomers into aggregates, but that binding of too much HSP 104 ties
up the monomer and prevents aggregation. Ter-Avanesyan has theorized that HSP
104 is required to break up aggregates so they are distributed to both mother and
daughter upon cell division. Without HSP 104 the aggregates are not broken up and
are distributed to only one of the two resulting cells, whereupon the cell that did not
receive them has a selective advantage and overgrows the population.
HSP 104 possesses other interesting properties in that it can break apart
aggregates of many types of proteins, not just of Sup35. HSP 104 has the ability
to disrupt strong noncovalent bonds. In mammalian cells, HSP 70 might be
expected to serve some of the roles which are served by HSP 104 in yeast.
Inclusions and Recruitment
Paulson noted that inclusions are found in a number of neurologic diseases.
Marinesco bodies have been described in normal aging but have some
resemblance to the nuclear bodies in SCA3. Lewy bodies are cytoplasmic
structures which are immunopositive for ubiquitin and ý-synuclein. Paulson
described the process of recruitment, citing examples drawn from spinocerebellar
ataxia 3 (SCA3, also known Machado-Joseph disease). SCA3 is the most common
inherited ataxia, in which the brainstem and substantia nigra undergo degeneration.
Whereas huntingtin is a large protein of 350kD, the protein involved in SCA3,
ataxin-3 is only 42kD. In distinction to huntingtin, in which the polyGln stretch is
located closer to the N-terminus, in ataxin-3 the polyGln is located closer to the C-
terminus. The shortest number of repeats known to cause SCA3 disease is 61.
Paulson and his group have made constructs of either full length ataxin-3 or
truncated forms of ataxin-3 containing the C-terminal domain with the glutamine
repeat. For both full length and truncated ataxin-3, the repeat length is either
normal (n = 27) or expanded (n = 78). If cells are transfected with (i) a full length
construct containing an expanded repeat or (ii) a truncated construct with an
unexpanded CAG repeat, immunostaining for ataxin-3 reveals a diffuse cytoplasmic
pattern. However, if cells are transfected instead with a truncated ataxin-3 with an
expanded CAG repeat region, inclusions are seen. Some small intranuclear
inclusions are seen, but there are also large perinuclear inclusions. In coexpression
experiments, truncated expanded molecules form insoluble aggregates which can
recruit full length proteins with expanded CAG repeats. Recruitment seems to be
complete, in that immunofluorescence staining reveals that all of the full length
molecules are conglomerated into the inclusions, and seem to require a glutamine
domain in the recruited protein. Paulson noted that he did not know the
stoichiometry of the truncated:recruited protein interaction.
Another example of recruitment has been described by Michael Hayden. In
his transfected cells, truncated copies of huntingtin containing an expanded CAG
repeat are able to form inclusions in which are also found copies of full length
huntingtin proteins.
Final Thoughts & Suggested Experiments
Brodsky suggested making transgenic mice with the remainder of the
huntingtin molecule (i.e. without the N-terminus). Does this affect the protein?
Study the nature of polyGln: what is the nature of aggregated polyGln? Then, try
to dissolve it using chlorinated alcohols. Make an aggregate affinity column and see
what binds to it.
Cha proposed stopping aggregation and seeing if pathology would be
arrested. Also, would the proteasome really be inhibited by huntingtin with
expanded repeat?
Chesebro suggested using FT-IR or CD to get structural information, but
also to look for other compounds, e.g. glycosaminoglycans (GAG's). Another
suggestion was to create tetraparental mice such that animals would grow up
expressing mixtures of HD exon 1 (truncated) and wild type huntingtin to addresss
the issue of recruitment.
Hughes proposed making cell based models to monitor aggregation, which
can then be used to screen compounds and gene libraries. Also, understanding cell
specificity would be key to understanding the pathogenesis.
Ingram wondered if there was a threshold phenomenon which differentiated
normal and pathological length huntingtin. He would screen compounds which
interfere with (i) the normal role of huntingtin and (ii) stabilize conformation due to
expanded length. Next, was there a difference in long Gln aggregates and shorter
exon 1 with peptide motifs, that is, what is the abnormal function of the aggregated
structure?
Merry wondered about the high resolution structure of huntingtin. She would
then design compounds to block aggregation and test the hypothesis that
aggregation is pathologic. She would also use high throughout screening in vitro to
find compounds which could prevent aggregates. Also what roles do chaperones
play in processing and aggregation?
Mura proposed to: 1) look at structure of polyGln and Fab structure of 1C2.
2) What does "aggregate" really mean? One should create Cys mutants to
distinguish conformations. 3) Use mass spectrometry to see if protein is covalently
bound in the aggregates. 4) Do genome searches for Gln motifs Paulson
proposed finding the 42kD fragment, which has been observed in HD brain by
DiFiglia et al., and seeing if it is really in NII and dystrophic neurites. If so,
determine the exact cleavage site, and determine the protease involved.
Reisine suggested screening for drug compounds which can bind to the
polyGln region, and also drugs which inhibit the catabolism of huntingtin.
Signer suggested looking for the mRNA species which are first affected
following transfection with huntingtin. He also noted that we need more basic
structural information regarding huntingtin.
Tobin concluded that it would be nice to know what the protein does.
Certain crystallographic efforts are underway: Perutz, using Merry's construct for
androgen receptor; Mura looking at huntingtin; and Pat Law (UPenn) looking at
ataxin3. Maybe sickle cell anemia is a more appropriate model for HD than
metabolic diseases, since it is a protein-protein interaction disease.
Vey stated that using inducible knockouts, one should explore the role of
huntingtin in adult life. Is it necessary for normal functioning? One could then use
antisense or ribozyme strategies to knockout the function of huntingtin. One could
also make a construct linking the huntingtin promoter to GFP and then screen for
compounds which could turn off huntingtin expression. He would also express
GFP-polyGln in cell lines to screen compound libraries, using the FRET double
fluorescence system or by using inducible promoters.
Weissman thought purified aggregates should be subjected to mass
spectrometry in order to discern the components of the aggregates.
Wetzel stated that recruitment of proteins was tantalizing. Therefore
analyzing the components of the inclusions was key. He proposed to test
recruitment in vitro: is it possible to recruit into these inclusions?
References
Davies SW, Turmaine M, Cozens BA, DiFiglia M, Sharp AH, Ross CA,
Scherzinger E, Wanker EE, Mangiarini L, and Bates GP (1997). Formation of
neuronal intranuclear inclusions underlies the neurological dysfunction in mice
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