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Workshop Reports
Huntington's Disease: New Progress
The Brain Center, August 9-10, 1998
Summary Report
Prepared by Shawn D. Handran
Summary of Presentations
Over forty scientists delivered brief (10 minute) presentations on the
latest research progress toward the understanding and treatment of Huntington's
disease. This report summarizes these presentations with the goal of providing
a lucid and cohesive account of the current state of HD research. Hopefully, this
summary will help each individual investigator in their own particular area of
expertise.
This report is divided into the following categories:
a. Pathology in human cases of HD and other CAG repeat disorders
b. Transgenic rodent models of HD
c. Behavioral studies in transgenic HD mice
d. Transgenic mouse models of other CAG repeat disorders
e. Invertebrate models of HD and other CAG repeat disorders
f. In vitro models of HD and huntingtin aggregate formation
g. In vitro models of other CAG repeat disorders
h. Cellular mechanisms of huntingtin and/or degenerative processes
i. Potential screening assays and therapeutic treatments for HD
Pathology in human cases of HD and other CAG repeat disorders. Dr.
Hersch presented huntingtin immunohistochemistry labelling by EM48 antibody
in grade 1 and 2 HD patients. Interestingly, EM48 staining in early grade HD
cortex revealed extensive neuropil aggregation with very few instances of
nuclear inclusions. In addition, there was a paucity of aggregate staining of any
sort in the striatum. Later grade HD cases had increasing proportion of
aggregates in the nuclei of HD cortex and striatal neurons, but still had an equal
or higher proportion of neuropil aggregates. By electron microscopy, neuropil
aggregates appeared to be located in dendrites. Light microscopy revealed a
predominance of neuropil aggregates in the apical dendrites of cortical pyramidal
neurons. The aggregates (either nuclear or neuropil) appeared to be randomly
distributed throughout different neuronal classes of the striatum, did not
correspond to patch and matrix compartments and were not enriched in the
dorsal/medial portions of the striatum (which degenerates earlier in HD).
Interestingly, the calbindin-positive neurons (the most sensitive population in HD)
had relatively few aggregates, but non-sensitive neurons, such as diaphorase
and cholinergic interneurons had a higher incidence of nuclear inclusions (e.g.,
50% for diaphorase-positive neurons).
Dr. Davies reported on the structural features of aggregates in HD brain,
which included three types (fibrous, granular, mixed fibrous/granular). In
contrast to the human disease where all three types are observed, HD exon 1
transgenic mice show predominately the mixed fibrous/granular type. Dr. Davies
also shared another interesting finding in the pathology of HD during a
discussion period. His group observed anterior cingulate cortex degeneration in
the HD exon 1 mouse model of HD, and retrospectively confirmed that human
HD anterior cingulate cortex did indeed degenerate early in the disease and went
previously unreported. If verified in additional HD cases, this would be the
second example of an HD animal model leading to a new discovery about the
human disease. The first case was when neuronal intranuclear inclusions were
discovered in these same mice.
Dr. Sharp's group investigated nuclear inclusions in HD brain using anti-
peptide antibodies generated against various epitopes spanning between 1-700
amino acids of the N-terminus of huntingtin. The group also conducted similar
experiments in cell lines transfected with huntingtin cDNA. Their results suggest
that huntingtin is cleaved somewhere between amino acid 55 and 513 (and
possibly before residue153).
Dr. Macdonald's group presented evidence that normal and mutant
huntingtin from human brain extracts were both able to bind Huntingtin Yeast
Partners (HYP) A,B,and C; these are WW domain-containing proteins that were
identified in yeast two-hybrid screening. HYP A (the mouse homolog of FBP11,
a splicesome protein) and HYP C (a novel protein of unknown function) were
localized to both cytoplasm and nuclei of neurons in control and HD brain by
immunostaining. Transfection of HYP B in COS cells revealed nuclear
localization.
Dr. Djian's group reported that huntingtin from affected brain regions was
predominately found in high molecular weight aggregates, whereas extracts from
unaffected regions were monomeric. In vitro studies suggest that the high-
molecular weight form of huntingtin is due to transglutaminase cross-linking and
protein insolubility.
Dr. Ross reported their group has observed increased amounts of
neuronal intranuclear inclusions in HD patients with higher number of CAG
repeats, and also that DRPLA brain autopsy specimens, like HD, have nuclear
inclusions in affected regions.
Dr. Auburger presented pathological examination of brains from patients
with Sca-2 disease. A family group of 9548 members was studied; 1143 people
have died of Sca-2, 1400 are affected and 1000 have been genotyped. Sca-2 is
a multisystem neurodegenerative disease that affects Purkinje cells, brain stem,
substantia nigra, striatum and cortex. The normal protein, ataxin-2 is
cytoplasmic, but the expanded protein is primarily localized to the nucleus,
although aggregates or inclusions have not been observed. Interestingly,
ubiquitin staining also revealed intranuclear presence in some Purkinje neurons,
but also in the dentate nucleus, which is spared in Sca-2.
Dr. Paulson's group investigated MJD/Sca3 in human patients. MJD is
caused by a glutamine expansion in in the C-terminus of the ataxin-3 protein,
which is located in both the cytoplasm and nucleus. The brain stem neurons are
primarily affected and exhibit excessive amounts of inclusions in brain autopsy.
Some inclusions are immunopositive for HSP-40 and HSP-70 epitopes.
Transgenic rodent models of HD. There were more presentations on
transgenic models of HD than on any other subject. The following summary is
arranged according to the principle investigator of the laboratory that generated
the mice or conducted the experiments.
Dr. Tagle's group generated five mouse lines containing the full length
huntingtin with 16, 48 or 89 CAG repeats, under the CMV promoter. The 48
CAG repeat line with high expression, as well as all of the 89 CAG repeat mice
exhibited a progressive phenotype which included onset of clasping, circling
behavior, and finally culminating in a hypokinetic state, at which point, brain
pathology was observed (neuronal loss, gliosis, TUNEL-positive staining in the
striatum, and some instances of nuclear and peri-nuclear aggregates in
neurons). Striatal loss of medium spiny neurons approached 20%. Transgenic
mice with expanded repeats were also heavier than wild-type littermates.
Dr. Ross reported on transgenic mice expressing amino acids 1-171 of
huntingtin with 18,44 and 82 CAG repeats, under the prion protein promoter. All
repeat lengths of N171 show cleavage product. A line expressing 44 CAG
repeats was normal up to 2 years, and approximately 10% of the transgene
protein was degraded and detectable at 44 kD. Certain lines expressing 82 CAG
repeats exhibited an obvious phenotype, which included lower weight than wild-
type mice, abnormal gait and tremors, clasping, hypokinesis and lack of
grooming. Nuclear inclusions were observed in cortex, hippocampus, amygdala,
striatum, and cerebellum of end-stage mice. Interesting, nearly all cerebellar
granular neurons had nuclear inclusions. Thalamus, brain stem and subthalamic
nucleus were devoid of nuclear inclusions, but the latter had large, swollen axon
terminals (also prevalent in the amygdala).
Dr. Zeitlin's group generated mice with a conditional knockout of the
endogenous HD gene using Cre-loxP recombination. Two lines were
established, one under the CAMK-II-CreR1 promoter with a nuclear localization
signal (NLS), and the second line was inducible using the HSP70-CreR1
promoter (there was background expression in this line in the absense of heat-
shock). Conditional knockout mice exhibited clasping by weening age, are
smaller (80% of WT), and interestingly, are viciously attacked by the WT
littermates.
Dr. Hen's group generated repressible transgenic expression of HD exon
1with 94 CAG repeats (interrupted by an Arg at position 42) under the Tet-
On/Tet-Off and CAMK-II promoters. The transgene included lacZ reporter gene
and NLS. If dams were not treated with doxicycline, the transgenic pups died at
P1, whereas doxicycline treatment from E14-21 resulted in viable animals. After
doxicycline cessation, a progressive phenotype was observed including clasping
and tremor by 14 months. Transgene expression was high in cortex and
striatum, but absent in cerebellum. Immunostaining for huntingtin revealed
diffuse nuclear staining and punctate labelling through the cell. Ventricles were
enlarged and striatal area was decreased 27%. Some gliosis was observed, as
well as loss of D1 dopamine receptor expression in the dorsal striatum. Ubiquitin
immunolabeling was tentatively reported to be localized in axons.
Dr. Hayden's group established mouse lines expressing the full length HD
gene including introns , exons and endogeneous promoter (~600 kb) using a
yeast artificial chromosome (YAC). The HD gene had 18, 46 or 72 CAG repeats,
and the YAC is expressed in cortex, cerebellum and testis, but these mice lacked
an observable phenotype up to 22 months. The YAC construct rescues the
phenotype of embryonic lethality in HD null mice. Long-term potentiation (LTP)
in the Schaeffer collatoral fibers of the hippocampus were deficient at 6 months
and absent at 10 months in mice with expanded repeats. Foot shock
conditioning was absent in mice with 46 CAG repeats. One mouse with 4-5 YAC
copies expressing 72 CAG repeats developed ataxia and had neuronal
intranuclear inclusions that appeared to be within degenerating neurons.
Interestingly, in a YAC transgenic line expressing 72 CAG repeats but with lower
copy number, there was no observable phenotype, but some neurodegeneration
was observed.
Dr. Davies presented patholgical observations in the HD exon 1 mouse.
He noted that axonal and dendritic aggregates are observed in mice with ~150
CAG repeats, but only very late in the life of the mouse (in constrast to the
human disease, where neuropil aggregates appear to precede nuclear
inclusions). He has also observed glial inclusions very late in the mouse, but has
not observed glia inclusion in any human HD brains. The nuclear inclusions in
these mice were immunopositive for SH3/GL3 epitopes (but not in humans), and
negative for epitopes of HAP1, HIP1 and GAPDH. Interestingly, proteasome
components (notably, the 20S core protein) as well as additional proteasome
components (ATPase of 19S, P31-19S, P28 ý subunit of IIS) were also present
in nuclear inclusions.
Dr. Reiner's group characterized huntingtin immunoreactivity in different
classes of striatal neurons of rats. Huntingtin expression in three classes of
interneurons (which are largely unaffected in human HD) were measured: CHAT-
positive cholinergic neurons all expressed high levels of huntingtin; 20% of
parvalbumin-containing neurons had high expression, but 80% had low to
moderate levels of huntingtin; and somatostatin neurons had no detectable
huntingtin. Approximately 60% of medium spiny neurons in both the matrix and
patch compartments were immunopositive for huntingtin. Layer V cortical
neurons (which project extensively to the striatum) have high expression levels of
huntingtin protein. Single-cell reverse transcriptase-PCR confirmed that 99% of
cholinergic interneurons contained huntingtin mRNA, while 65% of Substance
P/enkephalin medium spiny neurons expressed huntingtin. Further
immunostaining was conducted in R6/2 HD exon 1 mice. At 12 weeks, 80% of
calbindin-positive medium spiny striatal neurons have nuclear inclusions;
likewise 80% of parvalbumin and 50% of cholinergic interneurons had nuclear
inclusions, suggesting that there is poor correlation between inclusions and
subsequent neuronal vulnerability. Nuclear inclusions were not observed in the
huntingtin-deficient somatostatin neurons.
Dr. Goldowitz reported that even though R6/2 mice have a 25% decrease
in body weight, he observed a 25% increase in abdominal wall fat, suggesting
that lipolysis may be detrimentally affected by the HD exon 1 transgene. In
collaboration with Dr. Reiner, he is generating two types of chimeric mice: (1) WT
and HD exon 1 (R6/2) chimeras; and (2) R6/2 and HD knockout chimeras. The
R6/2-WT chimeras survived past the 3 month period typical for R6/2 mice, but
also exhibit edsimilar alterations in neurotransmitter systems (including
substance P, enkephalin, calbindin and parvalbumin). Twenty-five chimeric mice
have been generated from R6/2 and HD knockout-derived cells and are
presently under investigation.
Dr. Cha presented work of his group characterizing extensive analyses of
striatal neurotransmitter changes measured by receptor autoradiography and
mRNA in situ hybridizaiton in three lines of HD exon 1 mice (R6/2, R6/5 and
R6/1). R6/2 mice develop neurotransmitter decreases by 8 weeks, including
decreases in AMPA, group I and II mGluR (the latter of which are on the
corticostriatal synaptic terminal and regulate glutamate release onto the medium
spiny striatal neurons), D1 and D2 dopamine receptors. Notably, by 12 weeks,
enkephalin levels were decreased and adenosine A2a receptors were decreased
90%. mRNA levels for adenosine, D1 dopamine and mGluR receptors were
detectably lower as early as 4 weeks, well before the onset of symptoms. D1,
D2, and A2a receptor levels were diminished in the R6/5 and R6/1 lines as well.
Dr. Levine's group assayed cell swelling and slice electrophysiology in WT
and transgenic mice expressing 71, 94 or 115-150 CAG repeats. Cell swelling
during NMDA exposure was differentially higher in HD transgenic brain slices
from mice with 150 CAG repeats and severity of swelling was dose-dependent
(NMDA concentration). Brain slices from mice expressing 94 CAG repeats were
also more susceptable to NMDA, but the response in slices from mice with 71
CAG repeats was not different from controls. In brain slices from mice with 150
CAG repeats, a difference was observed only in mice over 60 d, and with higher
doses of NMDA (above 25 M). Slice electrophysiology revealed enormous
spontaneous inward currents of >100 pA, observed in 40-50% of transgenic
mice, but was never seen in control brain slices. This finding suggests that
glutamate release onto striatal neurons is abnormally high in these mice.
Dr. Greengard's group investigated dopaminergic signaling pathways, in
particular, DARPP-32, a phosphoprotein enriched in striatum, substantia nigra
and nucleus accumbens neurons (also in kidney and brown fat cells as well).
DARPP-32 integrates dopamine and glutamate receptor signalling in medium
spiny striatal neurons. In DARPP-32 knockout mice, GluR1 receptor
phosphorylation is markedly decreased, dopamine-induced calcium channel
conductances are reduced, and the mice are less sensitive to cocaine-induced
hyperactivity. They also found that HD exon 1 mice had substantially decreased
levels of DARPP-32, decreased D1 receptor expression, decreased GluR1
receptor subunits and lower cAMP levels. In acutely dissociated neostriatal
neurons, calcium conductances were attenuated and GABA conductances
increased.
Dr. Sharp's group investigated cleavage products of huntingtin in
transgenic mice expressing amino acids 1-171 of the N-terminus of huntingtin
with 18 or 44 CAG repeats. They observed fragments of 7500 and 9800 daltons,
which might correspond to fragments spanning amino acids 45-66 and 59-87,
respectively. There was no nuclei staining with anti-peptide antibodies raised
against amino acid residues 139-145 (approximate), suggesting that cleavage
occurs prior to residue 139. Antibodies against residues 1-17 and 55-68 did
label nuclei, suggesting that the approximate cleavage occurs between amino
acid residues 97-139. Huntingtin contain dibasic residues (consensus:
KKxxxxxKK) between residues 55-150, which may serve as a substrate for ICE-
like proteases.
Behavioral studies in transgenic HD mice. In addition to the aforementioned
reports of behavioral abnormalities observed inseveral different HD mouse
models, two investigators presented their results of extensive behavioral testing
in HD exon 1 mice. Dr. Bolivar's group measured habituation in WT and HD
transgenic mice (B6CBA strain) in response to placement in an open field. Pre-
symptomatic HD transgenic female mice did not habituate to the open field,
whereas pre-symptomatic HD transgenic male mice had reduced exploratory
activity, but appeared to habituate after several trials. The reduced activity and
habituation was more pronounced in symptomatic male HD mice. In contrast,
control male mice did not habituate.
Dr Dunnett presented data from a collaborative study between his group and the
group of Jenny Morton in Cambridge, involving extensive motor behavior testing
results in the R6/2 line (HD exon 1) of trangenic mice, which included a battery of
tests, such as open field activity, balance in crossing elevated bridges of varying
difficulty, ability to stay on a rotating rod (rotorod), gait measured by labelling
front and hind paws with different colors of ink, swimming in a water tank, and
startle response to a loud and abrupt noise. Body weight was also monitored.
The group measured mouse performance in each task beyond 14 weeks of age.
Transgenic mice expressing 141-157 CAG repeats developed progressive motor
deficits, the earliest at 5-5.5 weeks in the most difficult tasks (raised bridges,
rotorod and swimming), followed by abnormal gait (8 weeks), abnormal
responses in acoustic startle reflex (8.5-10 weeks), body weight loss (10-13
weeks) and open field exploratory deficits (12 weeks). The investigators also
noted unusual posture in affected mice.
Both groups of investigators commented on the applicability and utility of
complex behavioral testing in future therapeutic screening, as certain behavioral
deficits are detected prior to other symptoms such as weight loss or formation of
neuronal intranuclear inclusions.
Transgenic mouse models of other CAG repeat disorders. Several groups
generated transgenic models of neurodegenerative disease other than HD that
are caused by trinucleotide repeat expansion. In addition, some investigators
produced animals with transgenes that express a pathologic-length
polyglutamine stretch either alone or in the context of a protein that does not
normally contain a polyglutamine stretch.
Dr. Detloff's group generated transgenic mice expressing 70 or 146 CAG
repeats within the Hprt gene (which does not normally contain a polyglutamine
stretch). They observed an abnormal phenotype in mice express Q146,
including decreased open cage activity and lower rotorod performance, with
eventual ataxia and lethargy. All mice died by 53 weeks, apparently of of seizure
related symptoms. Neuronal intranuclar inclusions were observed in brain
neurons, and they observed a weight gain increase of 10-15 g in affected mice.
Homo- and hemi-zygous mice with 2 copies of the transgene die earlier than
heterozygotes and exhibit a progressive ataxic phenotype, but no obvious
neuronal death, although there may be axonal tract degeneration.
Dr. Gage's laboratory has established an interesting rat model of CAG
repeat disease by adeno-associated viral infection 97 CAG repeats and Green
Fluorescent Protein (GFP). Viral constructs are stereotatically-delivered into the
striatum of adult mice and GFP expression was observed as early as 72 h post-
infection. Rats were sacrificed at 5, 12 or 35 days and tissue sections were
observed for GFP expression and TUNEL labeling. Expression was observed in
cortex, striatum and hippocampus, and neurons were almost exclusively labeled
(rarely, oligodendrocytes were infected, but expression in other glial cells was
never observed). By 5 days, cytoplasmic and nuclear aggregates were
apparent, and by 12 days, neuronal nuclear inclusions were widespread with a
high degree of TUNEL positive labelling. By 35 d, there were nuclear inclusions,
very little TUNEL staining, and low amounts of GFP-expressing neurons. In
addition, there was no evidence of tissue necrosis. In contrast, control animals
infected with normal number of CAG repeats had cytoplasmic localization and
expression persisted for several months.
Dr. Ross' group has generated transgenic mice expressing mutant
atrophin-1 (DRPLA disease). These mice develop ataxia, trembling, seizures,
exhibit clasping and cease grooming behavior. All transgenic mice died by 25
weeks. Nuclear inclusions as well as diffuse cytoplasmic staining was observed
in brain, and Western blot analsysis revealed cleavage products of atrophin-1 in
cerbellum, cortex and hippocampus.
Invertebrate models of HD and other CAG repeat disorders. Several groups
generated D. melanogaster or C. elegans models of trinucleotide repeat
disorders.
Dr. Bonini established a Drosophila model of MJD by expressing the C-
terminal fragment or full length protein with 27 or 78 glutamines. Flies
expressing the truncated mutant protein in the eye using the glass promoter
have pigmentation loss and eye degeneration during development, and cells
contain nuclear inclusions. The full length mutant MJD protein causes mild
pigmentation loss, and an "adult" onset of degeneration. The eyes-absent
protein (which contains interrupted segments of polyglutamines) and HSP-70 are
recruited into nuclear aggregates. Their group has identified 12 potential
suppressor mutations through screening.
Dr. Marsh's group has developed a Drosophila model of polyglutamine
disease by expressing 22 or 108 glutamines, either alone or in the disheveled
protein, under a variety of promoters including glass, ELAV, sevenless and
dpp/blink. In addition, the group generated contructs containing the reverse
sequence for glutamine (CTG) which encoded either 22 or 108 leucines. They
found that flies expressing Q22 under any promoter had no abnormal phenotype.
Flies expressing Q108 under the ELAV promoter resulted in 80% lethality, and
100% lethality under the sevenless promoter. The flies expressing Q108 under
the glass promoter had an eye defect, and there was no abnormal phenotype in
flies with the dpp/blink promoter. When the polyglutamine segments were
placed in the disheveled protein (which normally has a ~27 residue glutamine
stretch in flies, but only 2Q in frog, mouse and human homologs), there was no
phenotype in any transgenic flies with the ELAV promoter, but a mild phenotype
was observed in transgenic flies with the sevenless promoter with either Q27 or
no (deleted) glutamine stretch, but Q108 was lethal. In rescue experiments,
adding glutamines was worse than removing the endogenous polyglutamine
stretch; that is, expansion of the glutamine stretch was more detrimental than
deletion.
Dr. Zipursky's group developed transgenic Drosophila expressing
huntingtin (amino acid residues 1-171) with 2, 75 or 150 glutamines under the
glass promoter. They observed eye degeneration in flies with Q150 by 10 days
after eclosion, and even later and milder degeneration in flies with Q75. They
observed paracrystalline arrays in cells by electron microscopy. In addition, p35
was unable to rescue the HD-mediated effect, in constrast to other known eye
mutations, and also in contrast to the partial rescue observed in transgenic flies
expressing truncated mutant MJD protein.
Dr. Hart's group developed a C. elegans model of HD by expressing
truncated huntingtin (amino acid residues 1-171) with 2, 23, 95 or 150
glutamines, under the OSM-10 promoter (expressed in a neuronal subpopulation
of ASH sensory neurons). They observed an age -and polyglutamine-
dependant dye-filling defect in ASH neurons (which have a sensory process in
contact with the environment, which contains the lipophilic tracer DiD), and was
more severe in sensitized worms (those expressing contstructs with GFP
reporter gene). The dye-filling defect was completely abolished in worms with
the CED3 knockout background. They also observed cytoplasmic inclusions in
worms expressing Q150, but never saw nuclear inclusions. In a behavioral
assay (retraction from nose touch), worms with Q95 or Q150 were respectively
less responsive to perturbation (64% and 41%, respectively), approaching the
level of unresponsiveness seen in ASH laser ablation experiments (35%). Dye
filling defects, along with these results suggest that the mutant protein causes a
defect in the sensory process of ASH neurons, without obligate neuronal death
required for a phenotype. Supressor mutations can be screened using the nose-
touch assay.
Dr. Chalfie's group also generated a C. elegans model of HD by
expressing varying polyglutamate stretches (19 or 84 glutamines) flanked on
either side by 17 amino acids of the huntingtin sequence, with mec3 and GFP
proteins on the N- and C-terminus , respectively. The promoter was mec3,
which is expressed in 10 neurons, 6 of which are sensory touch-responsive
neurons. They observed obvious cytoplasmic and perinuclear aggregates in
worms expressing Q84, and worms developed a defect in tail-touch response
over time, but no neuronal death was seen.
In vitro models of HD and huntingtin aggregate formation. This section
summarizes results of experiments conducted in cell culture models to further
elucidate the cell biology of huntingtin, as well as aggregate formation.
Dr. Cattaneo investigated huntingtin toxicity in immortalized embyronic
striatal neurons (ST14a). These cells were transformed by SV40 infection, retain
the ability to differentiate into neurons and glia, and the neurons express
DARPP-32 as well as GABA, dopamine and glutamate receptors. She
transfected varying size huntingtin fragments (full length, nucleotides 1-1955, 1-
580, 1-436) with 15, 82 and 128 CAG repeats. Shorter huntingtin fragments
were more toxic than full length at 33 C, and likewise at 39 C, except that longer
huntingtin fragments were protective (i.e., cells transfected with longer fragments
of huntingtin, or full length huntingtin had less toxicity than non-transfected WT
cells).
Dr. Gusella's group presented evidence that huntingtin isolated from HD
brain contained an N-terminal portion of huntingtin (179-595 residues), which
forms aggregates in vitro at a threshold similar to number of repeats that cause
the human disease (41 CAG repeats). In addition, these aggregates contain
TATA-binding protein, a predominant transcription factor.
Dr. Housman's group investigated aggregate formation in mammalian cell
lines transfected with CAG/CAA triplet repeat lengths of 25-300. Their
constructs contained 17 amino acids of the N-terminus of huntingtin, and either a
c-myc or EGFP tag. Expression was exclusively localized to the cytoplasm, and
aggregate formation occured with CAG repeat lengths between 100-300. Only
addition of a nuclear localization signal was sufficient to induce entry of the
protein into the nucleus. In addition, protein with 104 glutamines was able to
recruit protein with 25 glutamines. Extraction of aggregates led to insoluble
material that was highly resistant to detergents.
Dr. Hayden's group established neuronal cultures from embryonic stem
cells that were transfected with full length huntingtin cDNA with 15 or 138 CAG
repeats (under the endogenenous HD promoter). Cytoplasmic aggregates were
observed in neuronal lines expressing 138 polyglutamines, but not in neurons
expressing 15 polyglutamines.
In addition, Dr. Hayden's group further characterized the possible
interaction sites between huntingtin and the interactors HIP1, HIP2, HAP1 and
GAPDH. They report that all four interactors bind near huntingtin amino acid
residues 427-548, and therefore may compete with one another, or may form
multimeric complexes. In addition, they report that GAPDH binding to huntingtin
is a relatively weak interaction.
Dr. Hayden's group also investigated cell toxicity in relation to aggregate
formation in 293 cells transfected with huntingtin constructs. The found
increasing amounts of aggregates, more nuclear inclusions and higher levels of
toxicity in cells expressing shorter fragments of mutant huntingtin. To further
characterize the cause of toxicity, they transfected cells with the huntingtin
fragment (amino acid residues 1-1955) with 128 repeats (which is normally
targeted to the cytoplasm), with either a nuclear localization signal (NLS) or a
nuclear export sequence (NES). Toxicity was assayed by MTT. All cells (100%)
transfected with the NLS had nuclear inclusions, whereas a mutation of the NLS
resulted in 100% of cells expressing cytoplasmic huntingtin aggregates. The
toxicity in either case was identical, suggesting that aggregates and not their
location was the determining factor for toxicity. In cells expressing the NES
constructs (these huntingtin fragments were amino acid residues 1-171 with 128
repeats, which is normally targeted to the nucleus), there was a significant
decrease in the number of cells expressing nuclear aggregates. The toxicity was
identical to cells expressing a construct that lacked NES, again indicating that
toxicity was not dependent on the site of aggregate formation but rather that the
amount of aggregates (in any compartment) resulted in higher toxicity.
Dr. Ross' group also investigated the effect of aggregates on cell toxicity
by transfecting neuroblastoma cells with truncated N-terminal huntingtin
fragments (1-171, 1-63, 1-43 amino acid residues) with either a NLS or NES.
Toxicity was assayed by counting the number of viable cells expressing the GFP
reporter protein. They observed increased toxicity (less cells with GFP staining)
in cells expressing constructs with NLS, and decreased toxicity (more cells with
GFP staining) in cells expressing contructs with NES, indicating that nuclear
presence of huntingtin increased toxicity.
Dr Greenberg's laboratory investigated mutant huntingin toxicity in primary
striatal neurons transfected with a huntingtin fragment (amino acid residues 1-
480) with 17 or 68 CAG repeats and the lacZ reporter gene. Striatal neurons
expressing Q68-huntingtin was more toxic in enkephalin-positive neurons,
whereas there was no difference in toxicity between Q17- and Q68-huntingtin in
enkephalin-negative neurons. Expression levels of either construct were similar.
Neuronal intranuclear inclusions were observed in neurons expressing Q68-
huntington. Addition of CNTF or BDNF protected neurons from death, and
apoptosis was blocked either by Bcl-XL expression or addition of DEVD caspase
inhibitor. In cells transfected with shorter huntingtin fragments (amino acid
residues 1-171) that also included a nuclear export signal (NES), there were no
more nuclear inclusions and Q68-huntingtin protein was no longer toxic. Finally,
addition of BDNF or CTNF (which are protective) resulted in higher incidence of
nuclear inclusions, but were also observed in non-affected neurons.
Transfection of a shorter huntingtin fragment (amino acid residues 1-171) also
resulted in higher incidence of nuclear inclusions but there was no difference in
toxicity compared to the longer huntingtin fragment. Therefore, nuclear presence
of mutant huntingtin is required for apoptosis, the formation of nuclear inclusions
precedes apoptosis, but nuclear inclusions do not appear to cause apoptosis,
and may even serve a protective role in striatal neurons.
Dr. Carpo investigated the effect of transglutaminase on aggregate
formation. She observed a dose-dependent increase in aggregate formation of
huntingtin (amino acids 1-310 with 23 or 41 glutamines) in this presence of
increasing concentrations of transglutaminase. Aggregates were not
birefringent, and aggregate formation was inhibited by transglutaminase
inhibitors. She also reported that transglutaminase is a cytoplasmic protein that
is enriched in the cortex and striatum.
Dr. Kahlem also reported on transglutaminase activity on involucrin, Sca1,
Sca3 and DRPLA protein. He reported that transglutaminase catalyzed the
formation of insoluble involucrin protein containing 2 or more glutamine residues.
Transglutaminase was able to saturate 50% of soluble Sca1 protein and 90% of
DRPLA protein, suggesting that these proteins are excellent substrates for
transglutamine-mediated aggregate formation. Dr. Messer's group generated
immortalized fibroblast lines from the HD exon 1 mice. They compared control
and HD exon 1 immortalized fibroblast response to a variety of potential toxic
agents and found a difference only in proteasome inhibitors, which were more
toxic to the HD exon 1-derived fibroblasts. They also observed reduced levels of
HSP70 in mutant fibroblasts.
Dr. Li's group investigated PC12 cells transfected with huntingtin (amino
acid residues 1-67) with 20 or 150 glutamines. Using the EM48 antibody, no
staining was detected in WT untransfected cells, but cytoplasmic staining was
observed in cells transfected with Q20-huntingtin, and nuclear staining was
observed in cells transfected with Q150-huntingtin. Observation of the latter
cells by electron microscopy revealed that the nuclear membrane appeared
normal. When PC12 cells with Q150-huntingtin were grown at 4 C, EM48
staining revealed much higher amounts of huntingtin in the cytoplasm,
suggesting that nuclear localization is an energy-dependent process. Also,
PC12 cells transfected with Q150-huntingtin were unable to differentiate upon
addition of NGF, and neurite formation was severly hindered. In differentiated
cells with Q20-huntingtin, huntingtin diffused into the neurite processes.
Differential display assay showed that the Q20-huntingtin cells had one increase
and one decrease in band intensity compared to Q150 cells, suggesting that
alterations at least two messages were evident.
Dr. DiFiglia's group examined mutant huntingtin expression in
immortalized murine striatal neurons. They transfected huntingtin full length
cDNA or 1-3221 (amino acid residues), with 18, 46 and 100 glutamine repeats,
which also included the Flag epitope. Cells transfected with 46 or 100 CAG
repeats developed nuclear and cytoplasmic inclusions, in addition to diffuse
cytoplasmic, vacuole and perinuclear staining observed in cells expressing Q18-
huntingtin. Inclusions were seen in cells expressing the full length construct.
Apoptotic hallmarks in neuronal morphology (by electron microscopy) were
observed in cells expressing pathogenic length of glutamines. Contructs
containing a C-terminal GFP tag were also localized to inclusions. Aggregate
staining remained on the surface of the dish even after the obvious demise of the
neuron. There was dose-dependant toxicity with repeat length. Treatment of
ZVAD-FMK increased survival of neurons but had no effect on the amount of
aggregates observed. Caspase-3 inhibitors (ZVAD and IETD) did not affect
survival. Western analysis revealed a number of huntingtin fragments at 140,
100, 90 and 80 kDa, and in cells treated with DEVD, the intensity of the 90 kDa
band was depleted.
Dr. Thompson's group investigated the effect of inducible huntingtin
expression in PC12 cells. The Ecdysone inducible gene expression system was
employed, and mutant huntingtin expression resulted in perinuclear and
intranuclear inclusions in PC12 cells. In this system, PC12 cells were able to
differentiate and extend neuritic processes.
Dr. McMurray's group investigated full length huntingtin with 19 or 82 CAG
repeats transfected into DRG neurons. They found that full length huntingtin had
to be cleaved in order to enter the nucleus, and that aggregate formation was
necessary for programmed cell death to occur. However, addition of a nuclear
export signal to the huntingtin constructs prevented aggregate formation but did
not affect neuronal death. They also isolated huntingtin from affected (cortex and
striatum) and unaffected regions (cerebellum) of HD brain, and found only large
complexes, whereas both high and low-molecular weight forms of huntingtin
were seen in control brains. Even the high molecular weight complexes were
soluble. Huntingtin appears to be involved in cytoskeleton interactions (tubulin,
kinesin and dynactin in particular), and mutant huntingtin may exert a detrimental
effect on cytoskeletal trafficking.
In vitro models of other CAG repeat disorders. Several investigators also
studied cell culture models of other trinucleotide repeat disorders.
Dr. Gage's laboratory expressed proteins in cell lines that contained 13 or
97 (with an argenine interruption) polyglutamine repeats fused to GFP, with or
without a nuclear localization signal. Cells expressing 97 repeats had
cytoplasmic inclusions; nuclear inclusions were observed in cell expressing
contructs that contained the nuclear localization signal. In co-transfection
experiments, aggregates caused by Q97 polyglutamines recruited both Q13-GFP
proteins as well as Q97 proteins that lacked GFP. In inducible expression
systems, Q97 was toxic after successive passages, approximately 30% of 293
cells had cytoplasmic aggregates which were also unbiquitin immunopositive.
Aggregates were also observed with Q97 in Ecdysone-inducible B3 cells.
Dr. Paulson's group investigated ataxin-3 (MJD/Sca3 protein) in cultured
293 cells. In cells transfected with the ataxin-3 cDNA, they observed that ataxin-
3 was located strictly within the cytoplasm (in contrast to localization of ataxin-3
in humans, which is observed in both cytoplasmic and nuclear compartments).
Addition of a NLS was required for ataxin-3 presence in the nucleus. The full
length protein with the NLS and either normal or expanded glutamine did not
result in any nuclear aggregates. However, the truncated protein with a normal
number of glutamines resulted in the formation of small aggregates,. Cells
expressing the truncated protein with expanded glutamines had many nuclear
inclusions. In this latter cell line, the investigators then transfected ataxin-1 and
observed recruitment of ataxin-1 into the ataxin-3 nuclear inclusions.
Interestingly, if the polyglutamine region of ataxin-3 was deleted, recruitment of
ataxin-1 still occurred, indicating that other regions of ataxin-3 are necessary for
recruitment of ataxin-1. Finally, truncated ataxin-3 with expanded glutamines
was able to recruit a short protein consisting of 19 glutamines fused with GFP.
They also investigated TATA-binding protein (TBP) and while they found very
little recruitment into ataxin-3 aggregates in cells, they did find TBP in a small
percentage of NII in MJD patient brain. In COS cells, aggregates form in every
cell and HSP-70 expression is much higher than in cell transfected with proteins
containing the normal number of glutamine repeats. Ataxin-3 aggregates recruit
the Q19-GFP protein as well as a portion of the HSP-70 pool; HSP-70 doesn't
appear to prevent aggregate formation, however HDJ2 (a component of the
HSP40 complex) did appear to inhibit aggregate formation. They next
investigated the 20S subunit of the proteasome, and found that it was recruited
into the ataxin-3 inclusions as well. The cells also have increased staining for
ubiquitin. In a cell line transfected with an MJD protein construct (with 48Q) that
has very little aggregate formation (4% of cells have nuclear inclusions),
proteasome inhibition with lactacystin caused 50% of cells to develop
aggregates.
Dr. Merry's group investigated SBMA disease, which is caused by a
glutamine expansion in the androgen receptor. They transfected cell lines with
constructs containing 16, 65, 97, 106 and 112 glutamines in a truncated form of
the androgen receptor. COS and MN1 cells expressing the higher repeats (65
and above) had increased toxicity, aggregate formation and abnormal proteolytic
processing. Aggregates remain in the stacking gel in Western analysis, and
antibody mapping experiments suggest that the androgen receptor protein is
cleaved, perhaps near or within the polyglutamine region. Treatment of cells
with proteasome inhibitors caused increased amounts of androgen receptor
aggregates and monomers. However, treatment with inhibitors after aggregate
formation did not have any effect. Both HSP-70 and HSP-40 proteins are
recruited into aggregates.
Dr. Burke's group investigated interactions between polyglutamine
proteins and cytoskeletal components in TR1 neuroblastoma cells.
Polyglutamine stretches of 19, 56 or 80 residues were fused with GFP.
Transfected cells with expanded polyglutamine (Q56 or Q80) developed large
cytoplasmic inclusions that were immunopositive for phosphorylated and non-
phosphorylated neurofilament protein. Neurofilament staining was also enriched
in the region immediately surrounding aggregates, but such a pattern was not
observed with antibodies to actin and tubulin, and these proteins were not
associated with aggregates.
Dr. Yuan's group investigated the role of caspase-8 in apoptotic cell death
of cells transfected with truncated ataxin-1 with 35 or 79 glutamine residues and
the GFP reporter. Only truncated ataxin-1 protein with 79 glutamines was toxic
in cultured cells, with ~70% programmed cell death observed in HeLa cells.
They found that programmed cell death was inhibited by CrmA, Bcl-2 and a
dominant-negative form of FADD (FADDN), but not by Bcl-X. FADDN was
recruited into aggregates and found in the insoluble aggregate fraction.
Caspase-8 also was recruited into aggregates, and CrmA, Bcl-2, but not Bcl-X
were able to prevent recruitment. A GFP- tagged caspase -8 mutant was not
recruited into Q79 aggregates. Finally, HD patient brain extracts have a 46 kD
band immunoreactive for caspase-8, suggesting that caspase-8 may play a role
in the pathophysiology of polyglutamine expansion disorders.
Cellular mechanisms of huntingtin and/or other degenerative processes.
This section summarizes the work presented on investigating cellular
biochemistry potentially involved in HD pathophysiology.
Dr. Chen investigated the electrophysiological properties of NMDA
receptors in transfected 293 cells expressing full length huntingtin protein with
138 glutamines. He observed higher current densities following NMDA receptor
activitation in cells expressing NR1/NR2B subunits, whereas the current density
was unaffected in cells co-expressing the NR1/NR2A subunits. The NR2A
protein was insignificantly decreased by Western analysis, but the NR2B was
unaffected. Likewise, surface expression studies suggest that NR2A was slightly
diminished (not significant), and the NR2B was unaffected. There were also no
differences in the open channel probability time nor in single channel
conductances.
Dr. Schwarcz investigated kynurenic acid (KYNA), an endogenous brain
metabolite that is markedly reduced in grade 3-4 HD. KYNA is an NMDA
receptor antagonist, produced by glia, and released during synaptic activity.
Dopamine agonists such as d-amphetamine acutely decrease KYNA levels and
may contribute to glutamate receptor excitotoxicity, which might partially explain
selective vulnerability of the neostriatum in HD. In rats, pre-treatment with d-
amphetamine increased the lesion size after intrastriatal injection of NMDA.
Agents that increase KYNA levels attenutated d-amphetamine-mediated
rotational behavior. Because of extensive dopaminergic input to the striatum,
HD neurodegeneration may result when KYNA levels are persistently decreased.
Because of the acute decrease in KYNA levels following dopamine receptor
activation, striatal neurodegeneration might be exacerbated in HD patients, in
particular, those treated with dopamine agonists.
Dr. Sherman investigated c-Jun N-terminal kinase (JNK) involvement in
programmed cell death. Dr. Sherman's hypothesis is that damaged proteins
(that might be present in HD) inhibit JNK phosphatase, which in turn causes JNK
activation and induction of apoptosis. They found that HSP70 expression was
able to suppress JNK activation and programmed cell death.
Dr. Segovia investigated tyrosine hydroxlase (TH) expression under the
GFAP (glia-specific) promoter in C6 rat glioma cells and transgenic mice. In the
former case, lacZ reporter gene expression increased upon in vitro
differentiation. In the latter, TH expression was found to be responsive to gliosis,
making the GFAP promoter of particular interest in future HD models, as gliosis
is a hallmark of the disease.
Potential screening assays and therapeutic treatments for HD. Three
investigators discussed possible screening assays or future therapeutic
interventions for the treatment of HD.
Dr. Waldo presented a novel approach to study protein mis-folding. GFP
is fused to the C-terminus of a protein in question, and if the tertiary structure of
the protein is correctly processed and folded, GFP is likewise correctly folded. If
a mutation results in protein mis-folding (which may occur with mutant
huntingtin), a screening assay in E. coli can be performed to identify mutations
that rescue the mis-folding phenotype. Dr. Waldo presented evidence that a
single mutation of glutamine to arginine was sufficient to bring an insoluble mis-
folded protein into solution, and raises the possibility that expanded
polyglutamine in HD may lead to mis-folding or insolubility of huntingtin. If so, it
may be feasible to use this assay to screen for agents that inhibit aggregation or
enhance solubility of mutant huntingtin.
Dr. Thomford's group is designing an assay to prevent aggregate
formation using "decoy" peptides. Peptides will be screened in GST-fusion
proteins of huntingtin (amino acid residues 1-171) with 13, 35, 57 and 97
glutamines.
Dr. Yen discussed an intriguing and novel therapeutic approach to the
treatment of HD. He proposed to identify potential regions of huntingtin mRNA
that could be cleaved by a DNA enzyme. The DNA enzyme cleaves single-
stranded RNA and consists of a catalytic core (5'-RGGCTAGCTACAACGAR-3')
and short flanking sequences that bind to specific huntingtin sequence. He
identified 15 putative regions of huntingtin mRNA that may exist in single-
stranded form (by computer analysis), and of these, he observed two regions
that the DNA enzymes were able to cleave. Together, the two DNA enzymes
cleaved huntingtin mRNA at multiple sites, and reduced mature protein
expression in transfected cells by 50%. Neither DNA enzyme targeted the
polyglutamine region, and therefore would not be specific to the mutant
transcript, but further screening may identify a site that preferentially cleaves
mutant huntingtin mRNA, or further experiments may generate an enzyme that is
able to cleave double-stranded mRNA, in which case, a specific tertiary structure
associated with the mutant transcript might be identified. Even if a selective
DNA enzyme were not identified, this therapeutic approach might still be of
therapeutic use by reducing both normal and mutant huntingtin expression in
HD carriersto a level that is sufficient to significantly delay onset by decreasing
the amount of mutant huntingtin protein expression without detrimentally
affecting the level of normal transcripts.
Finally, Dr. Signer provided investigators with two important criteria for
successful high-throughput therapeutic screening: (1) a robust and (2) positive
signal.
Concluding Remarks. As evidenced in this report, the presentations largely fell
into two categories of study: transgenic mouse models of HD (and other CAG
repeat disorders); and in vitro cell biology and toxicity of aggregates in cultured
cells transfected with mutant huntingtin.
Regarding transgenic models, pathogenic-length glutamine stretches in
any protein context appeared to cause a progressive abnormal movement
phenotype in many different transgenic lines (but certainly not all). Impairment of
certain motor tasks (i.e., especially the more difficult tests, such as rotarod,
swimming, or bridge crossing) tended to precede any obvious neuropathology.
Other investigators have transgenic mice with little or no motor abnormalities and
no brain pathology. One striking feature of all transgenic mouse lines with an
obvious phenotype is that the number of glutamine repeats necessary to cause
the disturbance is markedly higher than what is necessary to cause disease in
humans. The propensity to form intraneuronal aggregates appears to be higher
in such mice as well. With these two caveats in mind, a question that continually
lingered during the meeting was whether aggregates were really the causative
agent in the patholophysiology of HD. Clearly, anyone can consider tens to
thousands of reasons that may explain the difference between mice and men,
but the root of the question is whether such transgenic models are: (1) useful in
understanding the pathophysiological cause of HD (and other CAG repeat
disorders) and (2) whether these mice are appropriate models of the human
disease so that potential therapeutic treatments for HD can be developed and
tested. Otherwise, why is there such a great push to generate transgenic mice
with the largest possible number of CAG repeats? It can't possibly be to study
the normal function of huntingtin.
One question is: does it matter (for purposes of developing a treatment for
HD) that these mice may not only be a model of HD, but a generalized model of
CAG repeat disorders? Every CAG repeat disease affects specific
subpopulations of neurons, whereas most transgenic mouse lines exhibit little or
no specificity or neurodegeneration. Mice and men may vary in key components
of certain biochemical pathway (s) that are present in humans. It is possible that
inappropriate expression or overexpression of the mutant protein may lead to
pathological processes in mice that are not a part of the pathophysiology of the
respective human diseases. The finding that several different mouse lines have
abnormal weight gain or body fat distribution (just the opposite is observed in
humans) suggests that the mechanism of pathology in these mice is different
from humans. It is difficult to test cell-specific strategies easily using a general
CAG model, but this argues for developing both general and specific models.
However, there are two important reasons to believe that the answer is
"no". First, the pathophysiology of CAG repeat disorders is likely to share a
common mechanism. Certainly, it was evident at the meeting that our
understanding of HD is accelerated by the investigation of other CAG repeat
disorders. In addition, testing and developing a treatment in a "generalized"
mouse model of polyglutamine disease may lead to a therapeutic strategy that
treats all groups of patients. Second, the transgenic mice will be an invaluable
tool for testing therapeutic treatments. For example, heterozygous transgenic
mice with a normal and expanded HD allele are absolutely required to test
whether a putative RNA-cleaving DNA enzymes specific for the expanded HD
mRNA is able to reduce the amount of mutant HD mRNA without dramatically
changing the normal HD mRNA levels.
Regarding the results presented on the in vitro pathology and toxicity of
aggregate formation, it is much more difficult to effectively summarize.
Presentations on this topic included just about every possibility: that aggregates
were sufficient for toxicity regardless of cellular distribution; that only nuclear
aggregates were sufficient for toxicity; or that aggregates were not sufficient for
toxicity but mutant huntingtin had to enter the nucleus for toxicity. It is premature
to further speculate on the in vitro findings presented at this meeting, and the
possibility of exchanging constructs, cell lines and reagents among investigators
was encouraged to address and help resolve these discrepancies.
The same questions that were raised above also apply here. But in light
of additional investigations in human patient material (i.e., the paucity of
huntingtin aggregates in the striatum of early grade HD), as well as in studies of
the neuronal distribution of huntingtin in human and rodent striatum (i.e., that
aggregates are prevalent in unaffected neuronal populations), raised some doubt
that the prevention of aggregate formation is the cure for HD. However, extracts
of mutant huntingtin obtained from autopsy specimens did not only form
aggregates at the expected threshold for the disease (around 40 glutamines) but
formed more aggregates as the size of the glutamine stretch increased. These
investigator's findings, also presented at this meeting, certainly warrant further
study. In vitro models are currently the best available means of investigating
aggregate formation and toxicity, although toxicity is not necessarily the best
outcome to measure since it is clear from some mouse models that neurologic
disease can develop without substantial cell loss.
In conclusion, HD research is so rapidly advancing that many participants
felt overwhelmed by the amount of data presented. As slow as we often think
research progresses (and certainly HD families are anxious, often desperate for
a treatment), it seems now nearly impossible to keep pace. To the credit of the
scientists that participated, there was relatively selfless sharing of data, and this
atmosphere makes the inability to keep abreast of HD progress tolerable! But
more importantly, this type of scientific openness, when responsibly, respectfully
and wisely utilized, will certainly accelerate the development of a treatment for
HD and other CAG repeat disorders.
Acknowledgments. I thank Dr. Debra Chao for providing notes she had taken
at this meeting; for without these, this report would not have been possible. I
also thank Dr. Anne Young for proof-reading and editing an earlier version of this
report and Dr. Diane Merry for essential edits. |
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