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Workshop Reports
Hereditary Disease
Foundation
What's Killing Neurons in Huntington's
Disease?
November 8 and 9, 1997
Playa Del Rey, California
Prepared by Shawn Handran
What's Killing Neurons in Huntington's Disease?
November 8 and 9, 1997
Playa Del Rey, California
Participants
M. Flint Beal
Massachusetts General Hospital
Joseph S. Beckman
The University of Alabama at Birmingham
John Caviness
Mayo Clinic Scottsdale
Marie-Franáoise Chesselet
University of California Los Angelesr>
Robert E. Davis
MitoKor
Laura L. Dugan
Washington University
Michael A. Forte
Oregon Health Sciences University
J. Timothy Greenamyre
Emory University
Shawn Handran
Washington University
Maurico S. Montal
University of California San DiegoAnne N. Murphy
George Washington University Medical Center
David Nicholls
Dundee University
Diane M. Papzaian
University of California Los Angeles
Serge Przedborski
Columbia Universityr>
Ian J. Reynolds
University of Pittsburgh
Ellen V. Rothenberg
California Institute of Technology
Ethan R. Signer
Massachusetts Institute of Technology
Russ Swerdlow
University of Virginia
Allan J. Tobin
University of California Los Angeles
Nancy S. Wexler
Columbia University
What's Killing Neurons in Huntington's Disease
November 8 and 9, 1997
Playa Del Rey, California
A. Introduction to Huntington's Disease
Huntington's Disease (HD) is presently an untreatable
neurodegenerative disorder caused by an expanded trinucleotide repeat in a
gene of unknown function (Huntington's Disease Collaborative Research
Group 1993). The normal gene product, huntingtin, contains a stretch of 25-
30 polyglutamine (polyQ) residues near the N-terminus; an HD phenotype
results when greater than 35 polyQ repeats are present. The typical clinical
presentation for HD is progressive involuntary choreoform movements,
abnormal ocular saccades, disturbed balance and coordination, personality
changes, depression, dementia, and paradoxical weight loss; advancing to a
vegetative state and eventual death 15-20 years after onset (Albin et al.,
1989; Sharp & Ross, 1996). Onset is usually in mid-life, often after
reproductive years, but earlier onset is associated with the inheritance of a
greater number of CAG repeats on the HD allele (Stine et al., 1993).
Pathologically, HD is characterized by marked hypertrophy of the striatum
with moderate to severe cortical degeneration (Albin et al., 1989), which
accounts for the symptoms typical for most HD patients.
HD belongs to a family of neurodegenerative diseases including
Kennedy's disease, spinocerebellar ataxia type 1 (SCA1), Machado-
Joseph's disease (SCA3/MJD), spinocerebellar type 6, dentatorubral pallidal
luysian atrophy, which are dominant gain-of-function mutations caused by
polyQ expansions above 35 glutamine residues present in distinct gene
products (Khan et al., 1996; La Spada et al., 1991; Riess et al., 1997; Ross
et al., 1993). Thus, proteins containing polyQ repeats appear to play an
important role in the cellular biochemistry of neurons, with the polyQ region
likely being a protein structural motif whose normal function is critically
dependent on the length of the polyQ stretch.
Recently, several investigators have observed neuronal intranuclear
inclusions (NII), immunoreactive for huntingtin and ubiquitin epitopes in HD
autopsy specimens (DiFiglia et al., 1997) and in a transgenic mouse lineage
expressing exon 1 of huntingtin with 115-156 CAG repeats (Davies et al.,
1997). Similar findings have been reported in transgenic mice expressing
the human SCA3/MJD gene (Paulson et al., 1997).
The function of the huntingtin protein is presently unknown, but
studies indicate the normal protein is located in the cytoplasm and
association with vesicle proteins (DiFiglia et al., 1995). There appears to be
no direct co-localization of huntingtin with mitochondria; a primary candidate
underlying the cellular pathophysiology of HD, based on reports of defective
electron transport activity in post-mortem HD brain (Brennan et al., 1985;
Browne et al., 1997; Parker et al., 1990), and extensive animal studies that
model HD pathology in rodents or non-human primates by exposure to
excitotoxins or mitochondrial inhibitors (Beal et al., 1986; Beal et al., 1991;
Beal et al., 1993b; Beal et al., 1993a; Brouillet et al., 1993; Brouillet et al.,
1995; Coyle, Schwarcz, 1976; Matthews et al., 1997; McGeer, McGeer,
1976). Support for mitochondrial impairment is also strengthened by in vivo
MR imaging in humans that show increased lactate in HD cortex (Jenkins et
al., 1993; Koroshetz et al., 1997), suggesting greater reliance on anaerobic
metabolism for ATP production in HD neurons.
The purpose of the Mary Jennifer Selznick Workshop was to discuss
the connection, if any, between the polyQ expansion in the huntingtin protein
and mitochondrial dysfunction, and how the HD mutation could cause such
metabolic defects.
B. Roundtable Discussion
1. Summary of evidence for mitochondrial impairment in HD. As stated
above, there is substantial experimental evidence for an impairment of
mitochondrial function or energy metabolism in the etiology of HD.
Biochemical studies in HD brain autopsy specimens have revealed
decreased enzyme activity in components of the electron transport system
(Brennan et al., 1985; Browne et al., 1997; Parker et al., 1990). Rodents or
non-human primates treated with specific inhibitors of energy metabolism
develop selective striatal lesions that show remarkable similarity to striatal
losses in HD (Beal et al., 1991; Beal et al., 1993a; Beal et al., 1993b;
Brouillet et al., 1993; Brouillet et al., 1994; Brouillet et al., 1995; Greene et
al., 1993; Henshaw et al., 1994; Matthews et al., 1997; Messam et al., 1995;
Palfi et al., 1996; Schulz et al., 1994; Storey et al., 1992). Lesions are
markedly attenuated by glutamate receptor antagonists (Messam et al.,
1995; Greene, Greenamyre, 1995).
Finally, in vivo MR imaging in HD patients show increased lactate
production in occipital cortex and striatum (Jenkins et al., 1993).
2. Summary of research on huntingtin function. The gene product of
huntingtin is a high molecular weight (249 kD) protein with unknown function.
Embryonic expression of huntingtin is required for gastrulation, as
inactivation of both huntingtin alleles is lethal at embryonic day 7 or 8 (Duyao
et al., 1995; White et al., 1997; Zeitlin et al., 1995). It is presently unknown if
huntingtin expression is required in the adult. Normal huntingtin protein is
localized to the cytoplasm and vesicular organelles (DiFiglia et al., 1995).
The participants discussed the recent findings that neuronal
intranuclear inclusions were observed in HD brain autopsy , in neurons of
transgenic mice expressing exon 1 of huntingtin containing 150 CAG repeats
(Davies et al., 1997; DiFiglia et al., 1997), as well as in a transgenic mouse
expressing the MJD protein, which is the cause of another CAG expansion
disorder (Paulson et al., 1997). NII were ubiquinated, suggesting that
neurons were unable to complete a degradation pathway (Davies et al.,
1997; DiFiglia et al., 1997; Kalchman et al., 1996). Studies in the Bates
transgenic mice showed that ubiquitin-positive staining of NII occurred
several weeks after the formation of huntingtin-positive NII, suggesting that
unbiquitination was a later event (Davies et al., 1997), perhaps an attempt by
the neuron to eliminate the inclusion. In vitro studies indicate that protein
aggregates form when a critical number of polyQ expansions have been
exceeded, around 40 repeats (Scherzinger et al., 1997), suggesting that
proteins containing polyQ expansions beyond a certain length result in
conformational changes that lead to aggregation and insolubility. It is
presently unclear whether NII are composed of the entire HD protein or a
fragment, but initial studies by immunostaining and sub-cellular fractionation
suggest that NII may contain only the N-terminal portion of huntingtin
(DiFiglia et al., 1997). If this is indeed the case, it is unclear which cellular
compartment that the cleavage takes place, and how huntingtin becomes
localized to the nucleus. One participant noted that mutant huntingtin binds
to the glycolytic enzyme GAPDH (Burke et al., 1996), which is putatively
transported into the nucleus during an apoptotic program. Likewise,
processing of mutant huntingtin may be influenced by the effect of the
expansion on caspase activity, as recently indicated by Goldberg and
colleagues (1996).
These findings suggest that inappropriate induction of an apoptotic
program may be the initiating cause of huntingtin pathophysiology, but no
consensus was reached on this issue, or on the issue of the pathological
relevance of NII. Some participants felt that the NII were not etiological, and
were a consequence of some other pathological process, while others felt
that the formation of insoluble aggregates as well as the presence of NII in
the nucleus was critically involved in the pathogenesis of HD.
The possibility that the presence of huntingtin aggregations in the nucleus
could alter gene expression or transcription was also considered. A recent
report by Cha and colleagues (1997) showing specific changes in striatal
glutamate receptor expression in the Bates transgenic mice indicates the
possibility that mutant huntingtin may indeed influence the expression of
nuclear-encoded proteins.
3. Paradigm shift: Does long-term neuronal dysfunction (but not
neuronal death) underlie the clinical symptoms of chronic
neurodegenerative disease? Perhaps the most interesting topic
considered at the workshop was the possibility that neurons in HD and other
neurological disorders (including other CAG expansion disorders, PD or
schizophrenia) might actually persist for months or years in a dysfunctional
state, and that neuronal death occurs very late (or not at all) in disease
progression. The dysfunctional neuron in HD may undergo atrophy, severe
reduction in metabolic activity, loss of neurotransmitter expression (and
therefore loss the normal functioning in the neostriatal circuitry), and yet
persists in a marginally- or non-functional state that resembles neuronal
death. Evidence in support of such a theory comes from several lines.
First, autopsy specimens from patients that had confirmed clinical
symptoms of HD can have marked brain atrophy with little or no obvious
striatal or cortical pathology (Vonsattel et al., 1985). Likewise, transgenic
mice expressing exon 1 of the huntingtin gene containing 150 CAG repeats
have marked reduction in overall brain weight, prior to exhibiting motor
dysfunction or obvious neurodegeneration (Davies et al., 1997).
Second, in animal models of PD, where substantia nigra
dopaminergic neurons are selectively lesioned with the complex I toxin
MPTP, substantial recovery of tyrosine hydroxylase histochemistry was
observed after treatment with glial-derived neurotrophic factor (Tomac et al.,
1995); suggesting that the neurons affected by the toxin were not killed, but
rather existed in a chronic state of minimal metabolic activity. A similar
situation appears to occur in PD, as autopsy brain reveals a marked loss of
pigmentation in neurons of the substantial nigra, and may represent
dysfunctional neurons that are no longer expressing enzymes of dopamine
neurotransmission. The observation that some PD patients recover motor
activity with L-DOPA treatment further supports the hypothesis that the
circuitry of the nigrostriatal neurons may actually be intact, rather than
irreversibly degenerated. Schizophrenia is another disorder involving
monoamine neurotransmitter systems, which might result from
neurotransmitter depletion and persistent dysfunction in select cortical
circuitry; and may even be caused by an expanded nucleotide repeat
mutation (Brown et al., 1986; Ross et al., 1993; Selemon et al., 1995; Sirugo
et al., 1997; Thibaut et al., 1995).
Finally, neurons cultured from transgenic mice with deletion of the Bax
protein are unable to complete an apoptotic program upon growth factor
deprivation (Deckwerth et al., 1996, and are characterized by neuronal
atrophy and decreased metabolism but can persist in such a state for
several weeks, with recovery to a normal phenotype upon re-addition of
nerve growth factor. A similar result was observed in an analogous
experimental setting, where cultured SCG neurons deprived of NGF and
treated with the caspase inhibitor BAF, were indefinitely prevented from
undergoing neuronal apoptosis, and resulted in atrophied but viable neurons
with decreased metabolism (Deshmukh et al., 1996). Electrophysiological
studies of the "NGF-deprived, BAF-saved" SCG neurons show marked
reduction of nicotinic responses, broadened action potentials, slower re-
polarization kinetics and disrupted cytosolic calcium handling (Werth et al.,
1997). Re-addition of NGF resulted in rapid recovery (1-2 days) to normal
morphology and physiological responses (Deshmukh et al., 1996; Werth et
al., 1997).
A similar state may occur in HD as the result of an incomplete
apoptotic program; recent studies have shown increased cleavage of mutant
huntingtin by the caspase CPP32 (apopain, caspase-3) (Goldberg et al.,
1996) and another study has implicated caspase cleavage of huntingtin in
the etiology of NII (DiFiglia et al., 1997). Either mouse model system (NGF-
deprivation in Bax-deficient or BAF-saved neurons) may provide a valuable
experimental approach to understanding the pathophysiology of CAG
expansion disorders. More importantly, if vulnerable neurons in HD are
actually alive but chronically dysfunctional, delivery of the appropriate growth
factor to these neurons may prove to be an effective treatment. Similar
studies animal models of neurodegenerative disease (either using
excitotoxins or mitochondrial toxins) have shown marginal neuronal
protection by growth factor treatment (Emerich et al., 1997; Tomac et al.,
1995); however, since these models use insults that are presumably much
more acutely neurotoxic than that introduced by a polyQ expansion protein, it
is reasonable to suggest that growth factor treatment may be more protective
in HD or other CAG expansion disorders that in current in vivo models.
4. Experimental research of immediate importance.
a. Experiments addressing mitochondrial impairment in HD.
There was little disagreement that a priority in HD research is to determine
whether mutant huntingtin protein directly or indirectly causes mitochondrial
dysfunction or if such observations are secondary to the true (and yet to be
determined) pathophysiology. Experiments were discussed that would
directly address this issue in experiments already in progress, or using
techniques and assays that could be easily adapted to the study of HD cell
or animal models. The following includes a brief description of the
experiments discussed at the workshop.
i. In vivo MRI experiments in pre-symptomatic and symptomatic HD
patients. If the HD mutations results in a state of progressive and chronic
neuronal dysfunction that occurs without or prior to large scale degeneration,
one would predict that energy impairment would precede development of the
clinical phenotype. This hypothesis could be directly tested in pre-
symptomatic HD patients using established methodologies already described
(Jenkins et al., 1993; Martin et al., 1995).
ii. Testing the mitochondrial impairment hypothesis in transgenic
models of HD. Another experiment that would directly test the effect of
polyQ expansions on mitochondrial function was proposed by Professors
Murphy and Nicolls. The hypothesis to be tested is that any detrimental
effect of polyQ expansions on mitochondrial function would be directly
observable as a defect in O2 consumption in isolated mitochondria.
Mitochondria are prepared from synaptosomal fraction of acutely dissected
neostriatum of the Bates HD transgenic mouse. Professor Nicolls suggested
that striata from 12 mice would be sufficient to perform a single experiment.
Professor Chesselet observed that a synaptosomal preparation from the
striatum would include very little striatal mitochondria (the striatum contains
predominately cortical and thalamic terminals), and that obtaining striatal
mitochondria from mouse globus pallidus would be too little yield. However,
these experiments would still prove useful, as cortical terminals from striatal
synaptosome preparations would presumably be affected as well, since the
cortex is also severely affected in HD. No specific plans were made for the
performance of these experiments.
iii. Mitochondrial impairment arising from the nucleus. Another set of
experiments was proposed that would address the development of
mitochondrial impairment in HD. The hypothesis to be tested is that the
nuclear expression of the huntingtin mutation will cause normal mitochondria
to gradually develop respiratory chain defects, as observed in HD brain
autopsy specimens (Brennan et al., 1985; Browne et al., 1997; Parker et al.,
1990. The experiment requires the re-population of normal mitochondria into
a cell expressing the huntingtin mutation, which is achieved by the "rho 0"
hybrid cell technique. Simply, fibroblast or lymphoblast cells obtained from
juvenile HD patients (which generally have >80 CAG repeats in the
huntingtin gene) are depleted of mtDNA by treatment with ethidium bromide
or dideoxycytidine, thus generating a cell with the mutant HD gene in the
nucleus but are lacking functional mitochondria. Normal mitochondrial from
control platelets are reintroduced by fusion with the rho 0 cells, and
biochemical assays for mitochondrial enzyme activity or vital dye assays
(such as measuring mitochondrial membrane potential or cytosolic calcium
handling) are easily performed to determine if mutant huntingtin causes the
development of mitochondrial defects. This methodology is established in
Professor Swerdlow's laboratory, and HD tissues are available from
Professor Greenamyre. Professors Greenamyre, Murphy and Swerdlow
agreed to collaborate on these experiment which received overwhelming
approval from the workshop attendees.
iv. Mitochondrial impairment arising from the cytoplasm. Another
interesting possibility is that the presence of mutant huntingtin in the
cytoplasm was sufficient for development of cellular pathology. Professor
Murphy proposed that abnormal cleavage of mutant huntingtin, which may
be caused by caspase activity, results in cytosolic peptide fragments that can
directly gate the mitochondrial permeability transition, a putative pore that
opens only under pathological conditions; see review by Zoratti and Szabo
(1995). An assay for mitochondrial swelling, which follows PTP opening,
could be performed in the previously mentioned rho 0 cells, and Professor
Murphy agreed to perform the experiments, if possible.
v. Mitochondrial transport as the site of huntingtin pathology.
Participants discussed the possibility that abnormal interactions between
huntingtin and cytoskeletal components, perhaps through the interacting
proteins HAP-1 or HIP-2, may lead to mitochondrial impairment. Preliminary
evidence for HAP-1 involvement in such a mechanism has recently been
presented (Li et al., 1997. Since neurons have extensive processes that
require high volume trafficking of mitochondria; slight impairment of, or
interference with cytoskeletal components by polyQ expansions may be
sufficient to cause energy failure in cortical and striatal projection neurons.
Glia and other non-neuronal tissues are thus not overly affected by the
huntingtin mutation. Selective vulnerability is not explained by such a
mechanism, but cannot be ruled out as a participatory process in HD
pathophysiology. No experiments were proposed by the participants.
vi. Mitochondrial involvement in apoptotic programs in the
pathophysiology of huntingtin. The workshop participants discussed the
possible role of mitochondria in the direct activation of apoptotic cascades,
based on recent evidence showing cytochrome c released from mitochondria
directly activates caspases (Liu et al., 1996. Although specific experiments
were not proposed, many participants felt that it was worthwhile to determine
whether the putative caspase cleavage of huntingtin was a cause or
consequence of HD pathophysiology. Several investigators are further
characterizing the possible role of apoptosis in HD (Chuang & Ishitani, 1996;
Dragunow et al., 1995; Goldberg et al., 1996; Portera-Cailliau et al., 1995;
Thomas et al., 1995). One hypothesis was suggested at the workshop, that
impaired retrograde transport of growth factors by striatal neurons (through
cytoskeletal abnormalities mentioned above) may result in mild growth-factor
deprivation, leading to chronic dysfunction. Professor Forte emphasized the
importance of model genetic systems (e.g., C. elegans, S. cerevisiae,
Zebrafish) or cell-free experimental systems (Ellerby et al., 1997; Liu et al.,
1996), in the elucidation of these pathways.
b. Experiments investigating the structure and function of
normal and mutant huntingtin protein. A second area that workshop
participates felt was of immediate important was further investigation of
neuronal intranuclear inclusions. No specific experiments were proposed,
perhaps because many participants felt such approaches were out of their
field of expertise. But several questions were raised that the participants felt
were of great importance.
i. Is the proteosome unable to degrade proteins with polyQ
expansions above a critical repeat number? The participants discussed the
possibility that mutant huntingtin accumulate in neurons because of the
inability of the proteosome to process a protein containing a polyQ repeat.
In such a scenario, the chronic presence of mutant huntingtin in neuron may
adopt new and potentially pathogenic functions. Professor Montal
suggested that in vitro biochemical experiments could be devised that test
the hypothesis that proteosomes are unable to process proteins with polyQ
expansion in the disease-causing range.
ii. What immediate changes in gene expression, if any, result from the
presence of NII? Professor Signer suggested that messenger profiling
assays could be performed in the Bates transgenic mice.
5. Potential therapeutic agents for treatment of HD and CAG expansion
disorders. As mentioned above, several potentially successful therapeutic
treatments were suggested during the course of the workshop. A brief
summary follows, however, as several attendees noted, such treatments are
based on current knowledge and assumptions about HD pathophysiology,
and as such, are subject to abandonment pending the outcome of future
investigations.
Agents that improve bioenergetics. If HD results from insufficient
bioenergetics, then treating patients with agents that improve mitochondrial
function should slow the progression of the disease. An investigation by
Koroshetz and colleagues (1997) revealed that coenzyme Q treatment in HD
patients resulted in decrease cortical lactate production; suggesting that
such agents may indeed partially ameliorate defective energy metabolism in
HD. Professor Davis suggested that creatine may prove the most promising
bioenergetic-improving agent and worth pursuing in human clinical trials.
Many participants believed that such avenues of treatment will
probably not cure HD, but would rather shift the age-of-onset, or in the worst
case scenario, not prove effective at all (as indicated by clinical trials of
coenzyme Q in mitochondrial encephalomyopathies; Matthews et al., 1993).
Agents that prevent mitochondrial damage. Several participants
discussed the possible therapeutic potential of inhibitors of the permeability
transition pore (PTP), and ideal target, since the opening of the PTP appears
to happen only under pathological conditions. However, the present
understanding of the biochemical components and pharmacology of the PTP
is limited, and will require much more investigation before a suitable and
selective agent will be available.
Agents that rescue dysfunctional neurons. All workshop participants were
excited about the possibility that neurons in HD may be persistently
dysfunctional rather than irreversibly lost (e.g., dead). Such a scenario could
allow for the regeneration of dysfunctional neurons through the therapeutic
administration of a particular growth factor (such as CNTF or GDNF). Many
participants felt that a clinical trial testing the efficacy of growth factor
treatment in ameliorating HD degeneration would be worthwhile. Effective
treatment may require prolonged exposure to growth factors in HD patients
prior to the onset of clinical symptoms; necessitating the availability of pre-
symptomatic, gene-positive individuals for such a trial.
Agents that prevent the biochemical recognition of polyQ expansions, or
prevent huntingtin aggregation. The ideal treatment, as proposed by
Professor Signer, would be one that prevents the neuron from recognizing
the presence of the expanded polyQ repeat. Such an approach may be
possible by the in vivo production of a single-chain antibody directed at the
polyQ expansion (as evidenced by the finding that expanded polyQ repeats
result in a different epitope that the non-expanded wild-type protein; Trottier
et al., 1995). Such treatments may have to await the development of genetic
therapies such as viral gene delivery, and may be many years in the future.
If huntingtin expression is not required in the adult, anti-sense therapy may
be another possible therapeutic avenue to pursue.
Another treatment was suggested by Professor Davis, who stated that
agents such as Congo Red, which inhibit aggregation and precipitation of
amyloids, might be effective in preventing the formation of huntingtin
aggregates. If effective, Congo Red could conceivably reduce the amount
"pathological" huntingtin present in neurons, thereby delaying the age-of-
onset.
Inhibiting death-promoting apoptotic pathways might also prove
effective treatment for neurodegenerative polyQ expansion diseases. The
most apparent targets based on current knowledge include caspase
inhibitors, or agents that prevent mitochondrial involvement in apoptosis,
(see discussion on PTP, above).
Combinatorial or synergistic treatments. Finally, a combination of
treatments that ameliorate energy depletion, inhibit death-promoting
apoptotic enzymes, and reduce the expression or recognition of polyQ
expansions may prove synergistically effective in the treatment of HD. Many
participants felt that such an approach would most likely be successful in the
short term, until more advanced (and presently undeveloped) treatments
such as gene therapy are available and proven safe and effective.
C. Concluding Remarks
The participants of the Mary Jennifer Selznick workshop were
overwhelmingly positive about the success of the meeting. The goal of the
HDF in organizing this workshop was to generate relevant and immediately
testable hypotheses regarding the pathophysiology of HD and potential
therapeutic avenues worth exploring. Such a goal was achieved, and two
specific experiments were designed to test the hypothesis that the HD
mutation causes mitochondrial dysfunction and are being pursued by
Professors Greenamyre, Murphy and Swerdlow. The first was to determine
if the introduction of normal mitochondria into cells expressing the HD
mutation results in the development of observable mitochondrial injury. The
second was to isolate synaptosomal mitochondria from the striatum of the
Bates transgenic mouse and assay electron transport respiratory enzyme
function, in order to determine if the polyQ expansion results in enzyme
defects similar to those observed in HD brain autopsy specimens.
One of the most exciting aspects of this meeting was the possibility
that trinucleotide repeat disorders are caused by the persistent dysfunction
of neuronal activity, rather than the progressive degeneration of neurons
over time. If true, the meeting was inappropriately named ("What's killing
neurons in Huntington's Disease?"), and in the words of the workshop
coordinator Professor Tobin, what was discussed represents a "paradigm
shift" in our thinking of chronic neurodegenerative disease. But the most
exciting aspect of this hypothesis is the possibility of restoring neuronal
function in patients suffering from HD.
References Cited
Albin RL, Young AB, Penney JB (1989) The functional anatomy of basal
ganglia disorders. Trends.Neurosci. 12:366-375.
Beal MF, Brouillet E, Jenkins B, Henshaw R, Rosen B, Hyman BT (1993a)
Age-dependent striatal excitotoxic lesions produced by the
endogenous mitochondrial inhibitor malonate. J.Neurochem.
61:1147-1150.
Beal MF, Brouillet E, Jenkins BG, Ferrante RJ, Kowall NW, Miller JM, Storey
E, Srivastava R, Rosen BR, Hyman BT (1993b) Neurochemical and
histologic characterization of striatal excitotoxic lesions produced by
the mitochondrial toxin 3-nitropropionic acid. J.Neurosci. 13:4181-
4192.
Beal MF, Kowall NW, Ellison DW, Mazurek MF, Swartz KJ, Martin JB (1986)
Replication of the neurochemical characteristics of Huntington's
disease by quinolinic acid. Nature 321:168-171.
Beal MF, Swartz KJ, Hyman BT, Storey E, Finn SF, Koroshetz W (1991)
Aminooxyacetic acid results in excitotoxin lesions by a novel indirect
mechanism. J.Neurochem. 57:1068-1073.
Brennan WAJ, Bird ED, Aprille JR (1985) Regional mitochondrial respiratory
activity in Huntington's disease brain. J.Neurochem. 44:1948-1950.
Brouillet E, Hantraye P, Ferrante RJ, Dolan R, Leroy-Willig A, Kowall NW,
Beal MF (1995) Chronic mitochondrial energy impairment produces
selective striatal degeneration and abnormal choreiform movements
in primates. Proc.Natl.Acad.Sci.U.S.A. 92:7105-7109.
Brouillet E, Hyman BT, Jenkins BG, Henshaw DR, Schulz JB, Sodhi P,
Rosen BR, Beal MF (1994) Systemic or local administration of azide
produces striatal lesions by an energy impairment-induced excitotoxic
mechanism. Exp.Neurol. 129:175-182.
Brouillet E, Jenkins BG, Hyman BT, Ferrante RJ, Kowall NW, Srivastava R,
Roy DS, Rosen BR, Beal MF (1993) Age-dependent vulnerability of
the striatum to the mitochondrial toxin 3-nitropropionic acid.
J.Neurochem. 60:356-359.
Brown R, Colter N, Corsellis JA, Crow TJ, Frith CD, Jagoe R, Johnstone EC,
Marsh L (1986) Postmortem evidence of structural brain changes in
schizophrenia. Differences in brain weight, temporal horn area, and
parahippocampal gyrus compared with affective disorder.
Arch.Gen.Psychiatry 43:36-42.
Browne SE, Bowling AC, MacGarvey U, Baik MJ, Berger SC, Muqit MM, Bird
ED, Beal MF (1997) Oxidative damage and metabolic dysfunction in
Huntington's disease: selective vulnerability of the basal ganglia.
Ann.Neurol. 41:646-653.
Burke JR, Enghild JJ, Martin ME, Jou YS, Myers RM, Roses AD, Vance JM,
Strittmatter WJ (1996) Huntingtin and DRPLA proteins selectively
interact with the enzyme GAPDH. Nat.Med. 2:347-350.
Cha JH, Huffard BH, Kerner JA, Mangiarini L, Davies SW, Bates GP, Young
AB (1997) Reduced metabotropic glutamate receptor expression in
Huntington's Disease transgenic mouse brain. Soc.Neurosci.Abstr.
23:861(Abstract)
Chuang DM, Ishitani R (1996) A role for GAPDH in apoptosis and
neurodegeneration [letter; comment]. Nat.Med. 2:609-610.
Coyle JT, Schwarcz R (1976) Lesion of striatal neurones with kainic acid
provides a model for Huntington's chorea. Nature 263:244-246.
Davies SW, Turmaine M, Cozens BA, DiFiglia M, Sharp AH, Ross CA,
Scherzinger E, Wanker EE, Mangiarini L, Bates GP (1997) Formation
of neuronal intranuclear inclusions underlies the neurological
dysfunction in mice transgenic for the HD mutation. Cell 90:537-548.
Deckwerth TL, Elliott JL, Knudson CM, Johnson EM, Jr., Snider WD,
Korsmeyer SJ (1996) BAX is required for neuronal death after trophic
factor deprivation and during development. Neuron 17:401-411.
Deshmukh M, Vasilakos J, Deckwerth TL, Lampe PA, Shivers BD, Johnson
EMJ (1996) Genetic and metabolic status of NGF-deprived
sympathetic neurons saved by an inhibitor of ICE family proteases.
J.Cell Biol. 135:1341-1354.
DiFiglia M, Sapp E, Chase K, Schwarz C, Meloni A, Young C, Martin E,
Vonsattel JP, Carraway R, Reeves SA (1995) Huntingtin is a
cytoplasmic protein associated with vesicles in human and rat brain
neurons. Neuron 14:1075-1081.
DiFiglia M, Sapp E, Chase KO, Davies SW, Bates GP, Vonsattel JP, Aronin
N (1997) Aggregation of huntingtin in neuronal intranuclear inclusions
and dystrophic neurites in brain. Science 277:1990-1993.
Dragunow M, Faull RL, Lawlor P, Beilharz EJ, Singleton K, Walker EB, Mee
E (1995) In situ evidence for DNA fragmentation in Huntington's
disease striatum and Alzheimer's disease temporal lobes.
Neuroreport. 6:1053-1057.
Duyao MP, Auerbach AB, Ryan A, Persichetti F, Barnes GT, McNeil SM, Ge
P, Vonsattel JP, Gusella JF, Joyner AL (1995) Inactivation of the
mouse Huntington's disease gene homolog Hdh. Science 269:407-
410.
Ellerby HM, Martin SJ, Ellerby LM, Naiem SS, Rabizadeh S, Salvesen GS,
Casiano CA, Cashman NR, Green DR, Bredesen DE (1997)
Establishment of a cell-free system of neuronal apoptosis:
comparison of premitochondrial, mitochondrial, and postmitochondrial
phases. J.Neurosci. 17:6165-6178.
Emerich DF, Winn SR, Hantraye PM, Peschanski M, Chen EY, Chu Y,
McDermott P, Baetge EE, Kordower JH (1997) Protective effect of
encapsulated cells producing neurotrophic factor CNTF in a monkey
model of Huntington's disease. Nature 386:395-399.
Goldberg YP, Nicholson DW, Rasper DM, Kalchman MA, Koide HB, Graham
RK, Bromm M, Kazemi-Esfarjani P, Thornberry NA, Vaillancourt JP,
Hayden MR (1996) Cleavage of huntingtin by apopain, a proapoptotic
cysteine protease, is modulated by the polyglutamine tract.
Nat.Genet. 13:442-449.
Greene JG, Greenamyre JT (1995) Exacerbation of NMDA, AMPA, and L-
glutamate excitotoxicity by the succinate dehydrogenase inhibitor
malonate. J.Neurochem. 64:2332-2338.
Greene JG, Porter RH, Eller RV, Greenamyre JT (1993) Inhibition of
succinate dehydrogenase by malonic acid produces an "excitotoxic"
lesion in rat striatum. J.Neurochem. 61:1151-1154.
Henshaw R, Jenkins BG, Schulz JB, Ferrante RJ, Kowall NW, Rosen BR,
Beal MF (1994) Malonate produces striatal lesions by indirect NMDA
receptor activation. Brain Res. 647:161-166.
Huntington's Disease Collaborative Research Group (1993) A novel gene
containing a trinucleotide repeat that is expanded and unstable on
Huntington's disease chromosomes. The Huntington's Disease
Collaborative Research Group. Cell 72:971-983.
Jenkins BG, Koroshetz WJ, Beal MF, Rosen BR (1993) Evidence for
impairment of energy metabolism in vivo in Huntington's disease
using localized 1H NMR spectroscopy. Neurology 43:2689-2695.
Kalchman MA, Graham RK, Xia G, Koide HB, Hodgson JG, Graham KC,
Goldberg YP, Gietz RD, Pickart CM, Hayden MR (1996) Huntingtin is
ubiquitinated and interacts with a specific ubiquitin- conjugating
enzyme. J.Biol.Chem. 271:19385-19394.
Khan FA, Margolis RL, Loev SL, Sharp AH, Li SH, Ross CA (1996) cDNA
cloning and characterization of an atrophin-1 (DRPLA disease gene)-
related protein. Neurobiol.Dis. 3:121-128.
Koroshetz WJ, Jenkins BG, Rosen BR, Beal MF (1997) Energy metabolism
defects in Huntington's disease and effects of coenzyme Q10.
Ann.Neurol. 41:160-165.
La Spada A, Wilson EM, Lubahn DB, Harding AE, Fischbeck KH (1991)
Androgen receptor gene mutations in X-linked spinal and bulbar
muscular atrophy. Nature 352:77-79.
Li SH, Gutekunst CA, Hersch SM, Li XJ (1997) Huntingtin associated protein
(HAP1) implicated in neuronal vesicle transport. Soc.Neurosci.Abstr.
1910(Abstract)
Liu X, Kim CN, Yang J, Jemmerson R, Wang X (1996) Induction of apoptotic
program in cell-free extracts: requirement for dATP and cytochrome c.
Cell 86:147-157.
Martin WR, Hoskinson M, Kremer B, Maguire C, McEwan A (1995)
Functional caudate imaging in symptomatic Huntington's disease:
positron emission tomography versus single-photon emission
computed tomography. J.Neuroimaging. 5:227-232.
Matthews PM, Ford B, Dandurand RJ, Eidelman DH, O'Connor D, Sherwin
A, Karpati G, Andermann F, Arnold DL (1993) Coenzyme Q10 with
multiple vitamins is generally ineffective in treatment of mitochondrial
disease. Neurology 43:884-890.
Matthews RT, Ferrante RJ, Jenkins BG, Browne SE, Goetz K, Berger S,
Chen IY, Beal MF (1997) Iodoacetate produces striatal excitotoxic
lesions. J.Neurochem. 69:285-289.
McGeer EG, McGeer PL (1976) Duplication of biochemical changes of
Huntington's chorea by intrastriatal injections of glutamic and kainic
acids. Nature 263:517-519.
Messam CA, Greene JG, Greenamyre JT, Robinson MB (1995) Intrastriatal
injections of the succinate dehydrogenase inhibitor, malonate, cause
a rise in extracellular amino acids that is blocked by MK-801. Brain
Res. 684:221-224.
Palfi S, Ferrante RJ, Brouillet E, Beal MF, Dolan R, Guyot MC, Peschanski
M, Hantraye P (1996) Chronic 3-nitropropionic acid treatment in
baboons replicates the cognitive and motor deficits of Huntington's
disease. J.Neurosci. 16:3019-3025.
Parker WDJ, Boyson SJ, Luder AS, Parks JK (1990) Evidence for a defect in
NADH: ubiquinone oxidoreductase (complex I) in Huntington's
disease. Neurology 40:1231-1234.
Paulson HL, Perez MK, Trottier Y, Trojanowski JQ, Subramony SH, Das SS,
Vig P, Mandel JL, Fischbeck KH, Pittman RN (1997) Intranuclear
inclusions of expanded polyglutamine protein in spinocerebellar ataxia
type 3. Neuron 19:333-344.
Portera-Cailliau C, Hedreen JC, Price DL, Koliatsos VE (1995) Evidence for
apoptotic cell death in Huntington disease and excitotoxic animal
models. J.Neurosci. 15:3775-3787.
Riess O, Schols L, Bottger H, Nolte D, Vieira-Saecker AM, Schimming C,
Kreuz F, Macek MJ, Krebsova A, Macek MS, Klockgether T, Zuhlke
C, Laccone FA (1997) SCA6 is caused by moderate CAG expansion
in the alpha1A-voltage- dependent calcium channel gene.
Hum.Mol.Genet. 6:1289-1293.
Ross CA, McInnis MG, Margolis RL, Li SH (1993) Genes with triplet repeats:
candidate mediators of neuropsychiatric disorders. Trends.Neurosci.
16:254-260.
Scherzinger E, Lurz R, Turmaine M, Mangiarini L, Hollenbach B, Hasenbank
R, Bates GP, Davies SW, Lehrach H, Wanker EE (1997) Huntingtin-
encoded polyglutamine expansions form amyloid-like protein
aggregates in vitro and in vivo. Cell 90:549-558.
Schulz JB, Henshaw DR, Jenkins BG, Ferrante RJ, Kowall NW, Rosen BR,
Beal MF (1994) 3-Acetylpyridine produces age-dependent excitotoxic
lesions in rat striatum. J.Cereb.Blood Flow Metab. 14:1024-1029.
Selemon LD, Rajkowska G, Goldman-Rakic PS (1995) Abnormally high
neuronal density in the schizophrenic cortex. A morphometric analysis
of prefrontal area 9 and occipital area 17. Arch.Gen.Psychiatry
52:805-818.
Sharp AH, Ross CA (1996) Neurobiology of Huntington's disease.
Neurobiol.Dis. 3:3-15.
Sirugo G, Pakstis AJ, Kidd KK, Matthysse S, Levy DL, Holzman PS, Parnas
J, McInnis M, Breschel T, Ross CA (1997) Detection of a large
CTG/CAG trinucleotide repeat expansion in a Danish schizophrenia
kindred. Am.J.Med.Genet. 74:546-548.
Stine OC, Pleasant N, Franz ML, Abbott MH, Folstein SE, Ross CA (1993)
Correlation between the onset age of Huntington's disease and length
of the trinucleotide repeat in IT-15. Hum.Mol.Genet. 2:1547-1549.
Storey E, Hyman BT, Jenkins B, Brouillet E, Miller JM, Rosen BR, Beal MF
(1992) 1-Methyl-4-phenylpyridinium produces excitotoxic lesions in rat
striatum as a result of impairment of oxidative metabolism.
J.Neurochem. 58:1975-1978.
Thibaut F, Martinez M, Petit M, Jay M, Campion D (1995) Further evidence
for anticipation in schizophrenia. Psychiatry Res. 59:25-33.
Thomas LB, Gates DJ, Richfield EK, O'Brien TF, Schweitzer JB, Steindler
DA (1995) DNA end labeling (TUNEL) in Huntington's disease and
other neuropathological conditions. Exp.Neurol. 133:265-272.
Tomac A, Lindqvist E, Lin LF, Ogren SO, Young D, Hoffer BJ, Olson L
(1995) Protection and repair of the nigrostriatal dopaminergic system
by GDNF in vivo. Nature 373:335-339.
Trottier Y, Lutz Y, Stevanin G, Imbert G, Devys D, Cancel G, Saudou F,
Weber C, David G, Tora L (1995) Polyglutamine expansion as a
pathological epitope in Huntington's disease and four dominant
cerebellar ataxias. Nature 378:403-406.
Vonsattel JP, Myers RH, Stevens TJ, Ferrante RJ, Bird ED, Richardson EPJ
(1985) Neuropathological classification of Huntington's disease.
J.Neuropathol.Exp.Neurol. 44:559-577.
Werth JL, Cocabo J, Deshmukh M, Johnson EM, Rothman SM (1997)
Physiologic properties of SCG neurons prevented from undergoing
programmed cell death. Soc.Neurosci.Abstr. 23:2225(Abstract)
White JK, Auerbach W, Duyao MP, Vonsattel JP, Gusella JF, Joyner AL,
MacDonald ME (1997) Huntingtin is required for neurogenesis and is
not impaired by the Huntington's disease CAG expansion. Nat.Genet.
17:404-410.
Zeitlin S, Liu JP, Chapman DL, Papaioannou VE, Efstratiadis A (1995)
Increased apoptosis and early embryonic lethality in mice nullizygous
for the Huntington's disease gene homologue. Nat.Genet. 11:155-
163.
Zoratti M, Szabo I (1995) The mitochondrial permeability transition.
Biochim.Biophys.Acta 1241:139-176.
Appendix I: Report Summaries from Meeting Attendees
(as compiled and outlined by the workshop recorder)
Swerdlow
A. There is mitochondrial dysfunction is HD
1. How does htt affect mito function?
a. Htt not in mito
b. Htt may disrupt transport of nuc-encoded p into mito
i. not specific to HD, applicable to other trinucl.
disorders
ii. perhaps by affecting import of mtDNA/RNA
polymerases which contain short polyQ repeats - thus
leading to mtDNA depletion, energy failure
c. Htt may disrupt transport of mito along axons
2. How does mito dysfunction lead to neuronal death?
a. Energy impairment, oxidative stress, perturbed Ca2+
homeostasis, and lower apoptotic threshold conspire to impair
normal neuronal physiology
b. Beal hypothesis - glutamate excitotoxocity under energy
impairment
c. Pharmacological targets: improve bioenergetics, reduce
oxidative stress, block glutamate excitotoxicity, prevent PTP,
start therapy in pre-symptomatic patients, but these will only
delay onset, won't cure HD
d. Molecular gene therapy - fix production of mutant htt
B. NII- important to pursue, but until further evidence, NII should be
considered markers of cell sickness, and not distract from elucidation of real
pathophysiology of HD
Caviness
A. Solving HD will resolve 4 issues
1. mito dysfunction
a. Complex II/III decreases in HD
i. Does htt disrupt transcription of these complexes in
nucleus?
ii. Do htt peptide fragments disrupt mito function in
cytoplasm?
(Excitotoxicity and ROS then contribute to
death/dysfunction)
2. Morphology and distribution of mutant htt in nucleus and
cytoplasm
a. Normal htt in vesicles, cytoplasm
b. Mutant htt - aggregates in cytoplasm [?] and nucleus, or both
i. nucleus - interference with DNA expression
ii. cytoplasm - interference with mito function or
cytoskeleton
cleavage - necessary for pathological effect?
iii. Interacting proteins required for pathology?
3. selective death/dysfunction of neurons in HD
a. Protein aggregates in susceptible neurons
b. Excitotoxic models resemble pathology of HD
c. Connection unclear but specificity still an important clue
4. chronic progressive character of HD
a. If neuronal death is rapid, then trigger of events leading to
death must occur seemingly haphazard to various neurons over
many years
i. May be observed as calcium dysregulation or mito
swelling
b. If death is slow, damage would occur slowly
i. Apoptosis? Evidence in HD is weak
B. Successful treatment
1. Need to know molecular events better
2. Final answer will satisfactorily address these 4 issues
3. Experiments should be directed at bringing together htt
accumulation and mito dysfunction
4. Type of treatment [currently] based on inexact information
a. Energy metabolism treatment in clinical trials, not clear how
[effective]
b. Growth factor treatment to keep sick neurons functioning -
hasn't worked in other diseases [?]
c. Multiple treatments - may be synergistic
Greenamyre
Little disagreement that mito dysfunction exists in HD
What about contradictory views?
Most important task - how is htt related to mito function
1. Murphy's suggestion that peptide fragments might uncouple or
interfere with protein import are testable
2. HAP1/htt might interfere with mitochondrial/subunit transport or
turnover
3. Collaboration between Greenamyre, Murphy, Swerdlow - see if htt
nucleus causes disruption of normal mitochondria (reverse rho 0
cell line: take HD fibro/lymphoblast depleted of mtDNA, fuse with
normal platelets and assay mito function)
a. But what is appropriate control?
Forte
Reductionist approach - model genetic systems
1. How do cells handle large polyQs?
a. Is processing or folding affected by larger than [35] repeats?
b. Or do expansions interfere with transcription or axonal
transport
c. Three issues:
i. Do polyQ larger than 50 adopt novel conformations?
a. Use high energy NMR methodology
ii. Toxic effect associated with insolubility of polyQ? Or
ameliorated by co-treatment with agent that can mitigate
insol.
iii. What cellular machinery recognize and deal with
polyQ repeats?
a. Simple systems yeast, drosophila, worms
cannot be underestimated, relevant process are
likely conserved, thus identifiable by genetic
strategies
2. How are mito implicated in HD?
a. HD nuclei/normal mito cybrids - most easily developed
i. If defect - focus on cause and effect, progression
ii. Reservation: what does absence of mito defect in
fibroblasts mean?
b. Striatal neurons may undergo incomplete apoptosis
i. If so, apoptotic trigger must be related to issue #1
above
3. Why are striatal neurons sensitive to polyQ expression
a. HD is specific failure of striatal neurons to deal with polyQ
repeats
What are some key differences from other neurons?
i. Handling of HD protein -> slow cascade -> mito
dysfunction -> disease
a. Need good model systems - current mouse
models a good start, but need better. Is there a
simpler system? E.g., Zebrafish?
Montal
Two main areas to explore
1. Non-invasive NMR imaging to identify mito defic in HD patients
2. Structural studies on normal and mutant htt protein in conjunction
with studies on:
a. mechanism of protein processing
b. accumulation of misfolded proteins
Reynolds
Transgenic mice a good resource, but are they actual models of HD?
Primary cultures from HD transgenics would be valuable
Two issues regarding the role of mito in brain injury
1. Chronic impair of metabolic capacity contributes to neuron dys?
2. Mitochondria the final common pathway for neuronal death
processes?
Need more evidence for mito impair in humans to help illuminate therapeutic
targets
Safety margin of vulnerable neurons proposed by Nicholls
1. Vulnerable neurons are closer to the point where they can no
longer respond to a challenge with increased metabolism
2. Lactate in HD visual ctx following photic stimulation suggests that
neurons are consistent with this hypothesis
3. How to test?
a. Monitor DY or NADH levels in relevant neuronal population
(HD transgenic mice, engineered cell lines)
b. Use this method to determine rate limiting step
c. Increase margin with agents such as CoQ?
d. rho 0 cell line might be very useful
Intervention
Maybe more useful to study pathways involved in cell death
1. Work of Nicholls, Reynolds - Ca entry by mitochondria crucial for
excitotox
2. Bredesen - mitochondrial involvement in apoptosis
3. But is mitochondrial failure the consequence of calcium loading or
PTP?
4. Bioenergic failure - cause or consequence of [neurodegeneration]
5. Mito ROS the critical death signal OR cyt c (Bredesen)
6. Excitotoxic related mechanism seems likely in HD
7. Manipulation of mito function a possible therapeutic approach
a. block mito calcium uptake
b. ROS scavengers/antioxidants specific for mt SOD
production
c. block PTP
i. Agents blocking PTP are ideal since they don't affect
normal functioning (such as NMDA antagonists)
Beal
Topics reviewed:
1. HD is not a primary defect of mito function (rho 0 have no HD mito
defect)
a. Reverse experiment rho 0 HD cells repopulated with normal mito
worth doing!
2. Caspase in the development of NII
a. see if caspase inhibitors block the formation of NII in [system]
expressing the full length huntingtin protein
3. HD patients in metabolic chamber
a. Caloric expedenture correlated with chorea, may not support
energy defect in HD
i. Unknown if this method could detect metabolic defect in patients
with ME
ii. Unknown if chorea is more energy consuming than normal
movement
iii. Results don't rule out metabolic defect in HD
4. Transgenic models of HD
a. Perhaps best method to prove (disprove) if HD results from mito
dys
b. [Beal's] results indicate increased glucose utilization in striatum of
MacDonald mouse [knock-in of repeats to mouse htt gene]
c. MRI data suggests TCA defect in these mice (both need
replication)
d. Testing in other mice would be useful (NMR or post-mortem
biochem.)
5. Complex II/III defects in human post-mortem tissue
a. If true, would suggest a defect of the nuc-encoded SDH
Przedborksi
Bates transgenic mice develops neurological disorder associated with brain
weight loss
Normal htt - no aggregatates
Aggregates - secondary?
Complex II/III defect in human HD - secondary?
Dysfunction before death [undead hypothesis]
1. neurons shrink
2. loss of expression of phenotypic markers
3. possible down-regulation of oxidation metabolism
4. mutant htt causes incomplete death program (do not push cells to
die immediately)
5. trophic factors may rescue from hibernation
a. test in Tg mice
6. Is this process [undead] found in PD or ALS, etc.
7. what processes cause neuronal shrinkage, etc
Davis
1. What is the normal function of the huntingtin (HTT) protein?
a. Critical to understand normal htt function
b. Could HD be a loss of normal htt function, not polyQ expansion?
i. Answer by generating Tg mouse w/ inducible promoter
(TET), e.g., no induction of mutant htt until after development
ii. But HD may also be developmental abnormalities, like
schizophrenia
2. Is the nuclear inclusion (NT) of HTT fragments important in disease
pathogenesis.
a. Are NII epiphenomenon, or do they interact with gene regulators
i. Study gene transcription in Bates mice with SAGE,
differential display, etc
ii. Positive results can be rapidly tested in HD autopsy brain
3. Is the HTT fragmentation pattern important in this disease?
a. Is Htt with or without expansions a substrate for caspases?
b. Do cleavage products affect mitochondrial dysfunction (test in cell-
free systems)
4. What are near-term drug targets for this disease?
a. Agents that improve mitochondrial function
i. Creatine - best agent to start with
ii. Can HDF provide incentive for clinical trials and drug
development through HD, AD and PD centers?
b. Preventing aggregate formation with inhibitors
i. Congo red inhibition of conversion of beta-pleated sheet
protein to aggregates- may work in AD, possibly HD
a. Test in vitro [Wanker's system]
ii. Agents that prevent protein-protein interactions can be tested
in Bates mouse
c. Caspase inhibitors (if involved in HD pathology, see point 3)
i. Many pharmaceutical companies are already working on
these agents
ii. HDF should establish relationship with these companies
Rothenberg
Two views of HD discussed - nuclear aggregates and mitochondrial
dysfunction
Four key points:
1. NII are unbiquinated - targeted for breakdown, but not achieved
a. Huntingtin is mis-folded or mis-located
b. Pathology from aberrant proteolysis/degradation?
2. Is HD caused by the death of neurons or chronic dysfunctional
state?
a. Non-neuronal cells with polyQ repeats may be eliminated
through apoptosis at the first sign of dysfunction
b. But neurons might accumulate sub-lethal damage while
attempting to stay alive; the huntingtin mutation does not
necessarily have to be an efficient trigger of a caspase-related
pathway in order to participate in the death of vulnerable
neurons
3. Does huntingtin affect nuclear gene expression in striatal neurons?
a. Is gene expression altered by polyQ expansions?
b. Do vulnerable neurons have distinct changes that result in
undead state?
4. Model for "undead" neuron in growth factor deprived Bax knockout
mice
Murphy
1. Most obvious hypothesis for neuron dysfunction in HD- accumulation of
repeat in nuclear, affecting transcription or other nuclear functions
2. Another hypothesis - neuronal dysfunction arises from the attempt to
proteolyze or dispense mutant protein
a. ubiquintin-labeling supports such a hypothesis
b. Proteolytic fragments may cause PTP opening (similar to
mastoparan)
c. Fragments may compete with import of mito targeted protein
3. Experiments
a. Do repeats induce mitochondrial injury?
i. In HD trangenic mice test:
a. substrate-dependent rate of respiration
b. ATP coupling to respiration
c. mito membrane potential
d. ion transport
e. PTP sensitivity
ii. Do cells transfected w/ repeat exhibit these defects?
i. Do isolated mito exposed to repeat protein exhibit these
defects?
ii. Does the cytosol of cells transfected with repeat cause
aggregates or caspase activation in cell-free systems?
b. Are mito swollen by EM, in HD autopsy brain?
Appendix II: Notes by Shawn Handran
(square brackets indicate uncertain or inferred items)
DAY 1
Tobin - PolyQ expansion effect on mito??
Beal - Mito defect in HD is secondary
Complex II/III activity decreased in post-mortem HD brain
GAPDH translocation to nuc during apoptosis
Mutant htt binds GAPDH
Experiment - knock out GAPDH by antisense, see if block htt
translocation to nucleus?
Beckman - ko GAPDH and all translocation will be affected
Przedborski - Complex II/III def. - less protein?
Davis - AD has COX deficiency but protein levels are not reduced
Forte - covalent modification affects enzyme activity?
Davis - Cybrids
neuroblastoma rho 0 + platelet (mito)
AD transmits COX catalytic impairment
PD transmits Complex I impairment
HD - no impairment transferred, therefore II/III defect must be
nDNA
Greenamyre - How is amount of enzyme measured
Davis - complexes are rigidly associated - can pull out by IP, then denaturing
gel
Forte - What component of mito is de novo and what is based on a
preexisting template?
Greenamyre - What regulates mito turnover?
Tobin - can you repopulate mtDNA?
Davis - nuclear fusion protein - acts as primer for mtDNA replication
Handran - redox state of mito a signal to nucleus for transcription of mito
proteins?
Tobin - What is a redox protein and how does it work?
Beckman- response to oxidation (H2O2, thiol reagents)
Proteins very sensitive to ox state:
Fe/S
Zinc finger
SOX gene of E coli (an SOD) [?]
FGF acidic
in brain 1 NGF per 2500 FGF molecules
released during ox stress
re-enters, relocated to nucleus
Pappazian - Post-synaptic Density molecule PD2 (a protein dimer)
Tobin - What is ox stress?
Nicolls - Mito production of superoxide [?]
Rothenberg - ox signals for T cell differentiation - not assoc with pathology
Dugan - ox set points (rather than "stress") - oxygen tension
Tobin - ROS a second messenger?
Beckman - Redox state - not a fan of this idea - different compartments
have diff states
Hydroxyl radical not that toxic - it's too reactive - can't be a signaling
molecule, need a lot to cause death
Tobin - How much?
Beckman - 10 nm reaction - reacts w/ anything, 10/mole/sec
Nicholls - Fenton rxt is not important?
Beckman - no
Dugan - but there still is a reactive electron
Rothenberg - What is causal link between CAG & mito impairment?
Beckman - NO production (Beal's work) - HD stimulates ox stress?
Greenamyre - HAP1 assoc w/ cytoskel trafficking
HAP1 assoc w/ mito not in [?]
Degenerating mito [?]
Interfer w/ mito turnover?
Signer - use cybrids plus nuclear compliment to tease apart signaling
Davis - hasn't been done in HD
Swerdlow - nuclear protein affect mito function
PolyQ - natures' velcro
mt polymerase and mtRNA- have polyQ repeat (15)
polyQ might interfer w/ HAP1
Brennan/Bird showed COX defect in HD [?]
Beckman - no peak means COX c is reduced and the others are oxid.
Tobin - ox/red III/IV?
Beal - in post-mortem tissue, complexes are either fully ox or red
Davis - mito encoding[?] defect
Beal - or import problem
Nicolls - What is the normal function of huntingtin?
Wexler - homozygous results - dominant gain of function
Signer - Is htt required in adults?
Not known
MacDonald - intermed levels of htt [?]
huntingtin required perinatally, may not be same as embryonic
requirement
Forte - 2 types of gain-of-function mutation
1. new function (neomorphic)
2. dysregulation of normal function
Tobin - neomorphic [?]
BREAK
Murphy - ubiquitin results - what is conseq of proteolytic fragments?
Fragments (amphiphilic peptides) - compete w/ PTP?
Greenamyre - protein import studies
HD homozygous - see if it affects protein import?
Murphy - signal peptide sequence for COX may affect PTP
amphiphilic peptides - induce mito swelling
Ataxin-3 - localized/assoc. with mito
Reynolds - caspase makes fragments then affects mito?
How do you treat/target this?
Nicholls - explains glutamate excitotx and mito inhibitors
Do mito initiate or try to protect [excitotoxicity]?
Oligo + ETS inhib. - glutamate is no longer excitotoxic (NMDA rec
mediated)
calcium accumulation
Reynolds - cytosolic calcium load not important - but mito Ca is the key
calbindin - knockout greatly protects against ischemia (UCLA)
Nicholls - chelators are not buffers - slows the rise, doesn't prevent rise
buffers - lowers amount of calcium (e.g., mitochondria)
Murphy - Ca and apoptosis induction
Tobin - somebody explain caspases and PTP
Montal - PTP
molecular component unknown, primarily known by pharm
macromolecular pore - blocked by CyA
Rothenberg - FK506 - effect on calcineurin (like CyA)
Reynolds - CsA + Glut toxicity
pharmacol of PTP is hopeless
Montal - connection with apoptosis
Why bcl-2 has same effect as CyA? What do they have in common?
Rothenberg - Why are neurons impaired for so long?
Montal - Follow up - synaptic terminals have lots of mito
Dugan - Wong-Riley studied transport of mito components in dendrites
Davis - mito have to made near nucleus and transported
Forte - Neurons are trying to die, but can't?
Davis [?]
Wexler [?]
Signer - little cell death [observed], even though they are [presumably] dead
[?]
Chesselet - microtubules - long projections, neurons are mostly affected
Beal - mito transport (kinesins)
Greenamyre - HAP1
Beal [?]
Dugan - nuclear indentations described in paper - seen also in growth factor
deprivation, add growth factors back?
Chesselet - mito participate in growth factor
Beckman - PTP connection by thiol group
Nicholls - isolated mitochondria - not physiological
No Mg, adenine nucleotides
much different behavior in cytoplasm
Greenamyre - Lemasters studies [PTP]
Forte - doesn't agree [with Lemasters studies?]
Nicholls - negative staining [seen by Lemasters group] could be quenching
effect
Reynolds - PTP in hepatocytes - relation to neurons?
Nicolls - PTP - cyA - 10 min protection is all
Murphy - CyA is toxic not protective [in her hands]
Montal - Bcl-2 makes ion channels?
[more PTP discussion]
Handran - cycle involving htt, caspases, aggregates affecting physiol.
Greenamyre - JC1 measurements in HD fibroblasts
subtle defect in mito calcium buffering
Tobin - is this consistent with Anne's hypothesis (amphiphilic peptide
fragments) - are compromising mito
Murphy [?]
Caviness - GAPDH binding - what causes mito comprimise - specific or non-
specific?
Greenamyre - protein import impaired by depolarization of DY
Dugan - SOD knockout - II activ decreased, aconitase decreased, III
decreased
a mito radical problem?
Murphy - most sensitive model of ROS stress [?]
Nicholls - ionomycin - efflux ca - uncouples mito
Comment on Greenamyre's results
repeated exposure, ion causes ATP [energy] crisis
or perhaps control fibroblasts have higher ATP levels
Pappazian - Inclusions are not an epiphenomenon
Adult vs juv
Changes in nuclear membrane
Signer - inclusions may not be cause but effect
Nicholls - NII - neurons are not dead yet
Chesselet [?]
Forte [?]
Rothenberg- cell type specific accumulation
Przedborski - how much [inclusions?] are necessary?
Chesselet - expanded repeat prevents degradation, fragments might then be
the cause of HD?
Signer - MJD/ataxin-3
The smaller the fragment of polyQ, easier entry into nucleus
Chesselet - inclusion in non-neuronal cells?
Signer - yes (he thinks)
[Handran - no NII in glia, according to DiFiglia paper]
Tobin - Kennedy's disease protein is a nuclear protein w/ NII
Chesselet - high levels - dividing cells can dilute by division
Pappazian- concentration dependent?
Non-neuronal cells can dilute by division
Transport specificity then becomes very important
Signer - TATA binding protein (TBP) - Binds polyQ?
Tobin - [Are polyQ proteins] zippers (homodimers) or velcro (heterodimers)?
Pleotrophic mutation - binds to many polyQ containing proteins?
Little difference between adult and juv HD
Beal - probably the same processes occur in adult
Signer - Back to Anne's theory
Is the nucleus necessary? Maybe it's all mito?
Let's assume cleavage - signal to nucleus or mito?
Full length htt excluded from nuc? [DiFiglia] results were by Western -
evidence for cleavage not clear, but possible
BREAK
Montal - normal function of huntingtin not known
Tobin - only in KD is the protein known [androgen receptor]
Some minor defects in sexual function, secondary to
neurodegeneration
Signer - Ab selective for expanded repeats - this Ab was raised against
TATA binding protein (TBP)
New conformation is bad - expect TBP to be likewise affected?
Chesselet - are transcription factors made in large quantities?
Can neurons handle larger quantities betters?
Forte - expansions affects nuclear proteins?
Signer - not necessary at gene expression level
Beal - htt protein levels correspond well in [vulnerable neurons]
Greenamyre - in striatum
Beal - in cortex as well, layer 5/6, disease has to do with distribution of
protein
Murphy - argues for degredation defect, a proteolytic issue
Rothenberg [?]
Interview with HD patient Anette [sp]
Father 39 yrs - hit by car - chorea - change in behavior
in retrospect - probably already had disease before accident
Grandfather died in world war
Grandmother very late onset AD, no HD
Living in states for 4 years
Noticed shakiness in fingers 1.5 - 3 yrs ago
Repeated knee injury
Realized she made extra steps and balance problems w/ chorea
Diagnosed April 8, 1997 at UCLA
Had episodes of uncontrolled behavior - brief, did not understand episode
afterwards
Has had an unusual gate as long as husband has known her (3 years)
[stopped taking notes]
BREAK
Caviness - metabolic chamber measuring caloric activity in humans
1. Energy expenditure during sleep
2. Energy cost during arousal
3. thermic affect of food
4. cost of muscle activity
5. (also records movement)
Measured in full HD, presymptomatic and mild HD
Results
1. no difference between control & HD in #1 and #2
2. energy expend during movement (in day) was different
between control and HD
3. controls - 1500-2000 cal/day
4. chorea correlates w/ energy expediture
5. presymptomatic not different that control (n=4)
Nicholls - how many cal are consumed by chorea alone? Can we rule out
leaky mito?
Murphy - uncoupled mito [?]
Nicholls - higher state IV (O2 consumption) is indicative of uncoupled state
Handran - energetics of non-neuronal tissues
Beal - mito abnormalities in other tissues?
Tobin - caloric intake?
Murphy - 16X caloric intake of normals [?]
Nicholls - increased lactate [potential causes for observation]
1. shunt into lactate
2. protein leak
3. ATP utilization is defective
Rothenberg - lymphoblast experiments?
Murphy - easy experiment - measure O2 consumption in Tg mouse brain
Beal - neurons use glycolysis to repolarize
Shown in normal people
HD patients have rebound decrease [in ?]
Phosph MR altered in muscle
13C glucose - detectable by NMR
Peak in brain - [reveals] glutamate, GABA, C4 carbons
Tg mice - show block in TCA
Beckman - Increase in glutamate? Aconitase might be affected
Signer - is it useful to check brain in Bates mouse?
Murphy - problem is mito preps are whole brain homogenate
Nicholls - problem solved by synaptosome preparation
Signer - how many mice are needed to check striatum
Nicholls - 12 - experiments done with microchamber
Murphy -monitor redox state by measuring pyr nucleotide
Reynolds - can be done in str cultures with microscopy
Chesselet - must do globus pallidus [to get striatal synapses]
Tobin [?]
Beckman - 3NPA in HD Tg mice?
Greenamyre [?]
Chesselet - Dopamine + glutamate [effect], DA in ischemia
Greenamyre - unilateral depletion of DA protects against 3NPA
Beal - DA enhances NMDA rec toxicity
Tobin - Is there an experiment where abnormal mitochondria do or do not
contribute to HD?
Murphy - repopulate with HD nucleus
Greenamyre - HD fibroblast (homozygous) rho 0, repopulate with normal
platelet mito
Davis - use DDC, causes rapid depletion of mito
Greenamyre/Swerdlow/Murphy - agree to do experiments
Beckman - another experiment - overexpress mutant fragment
Murphy - will test this as well
Tobin - What about amphiphilic peptides - what concentration?
Murphy [?]
Montal - [possible link to] hyperthryoidism - no known neuronal damage
Przedborski - not chronic
Beal - isolated organ is affected
Davis - thyroxin - no neuronal receptor
Greenamyre/Beal/Chesselet - hyperthyroidism has chrorea and psychosis
Chesselet - an uncoupling [protein]?
Tobin - So you don't need NII or cell death [to develop HD]?
Davis - yes, need cell death
Davis - in AD, neurons don't die
Tobin - [Is] Dysfunction or death [the cause of HD]?
Chesselet - neurons stay alive in brain for many years in a state of dys in HD
Beal - Orr showed ataxia appears before cerebellar neuronal death
Greenamyre - 6-OHDA can turn off DA neurons but they are not dead
Signer - Is it worth pursuing apoptotic inhibitors?
Nicholls - What is the definition of a dysfunctional cell?
Chesselet - neurons don't make neurotransmitter but are still alive
Tobin - Is this meeting a paradigm shift?
Signer [?]
DAY 2
Tobin - Laura, talk about Bax
Dugan - explains Bax knockout in SCG neurons [prevents neuronal death]
NGD deprived BAF (caspase inhibitor) saved neurons similar to Bax
KO mice
BAF aborts an apoptotic program
Handran - describes results by Werth/Cocabo/Rothman [electrophysiology of
BAF saved neurons]
Altered action potential - broadened, with delayed repolarization - K
current?
Decreased nicotinic response
Rapid recovery to normal in 24-48 hrs
Dugan - GT1-7 and trkA [?]
ROS response to NGF withdrawal was blocked by ETS and AA
inhibitors
Beal - is there ROS after caspase activity? Suggests that ROS after casp is
death signal
ZVAD after caspase - 9 hr protection
ZVAD + MK - 12 hr protection
ROS scavenger - 9-12 hr protection
[test in] ICE dominant negative mice [?]
Nicholls - in isolated mito - state IV, highly polarized, lots of ROS
but in intact cells - mito depolization results in ROS production
Note on ethidium and DHR123- fluorescence is dep on DY,
hyrdoethidium is better probe
Murphy - Anebo and Chase - only conclusive evidence that depolarization or
uncoupled state results in ROS production?
Dugan - uncoupling is leak
Nicholls - what is causing ROS?
Tobin - Estavation - not murder or suicide
Rothenberg - "undead" neurons might prevent other neurons from
functioning properly
Tobin - Is it better to be dead or defunct?
Przedborski - no possibility of saving dead cells
Tobin - Kordower is trying CNTF in HD patients
Greenamyre - how do ETS inhibitors block ROS?
Dugan - non-depolarizing conc of ETS inhibitors prevents mito ROS
formation
Tobin - GABA synthesis is shunted from succinate
Chesselet - what experimental differences are there between mice and rats?
Dugan [?]
Rothenberg [?]
Reynolds - guinea pig models closer to human - quinolinate production after
trauma
Chesselet - Bcl/Bax?
Reynolds - Bcl-2 and calcium mobilization
Murphy - Bcl-2 protein is targeted to mito, overexpression of bcl-2 prevents
Ca redistribution
Montal - dimerization of bcl family members
Bak peptides - bind to Bcl-XL, becomes amphiphilic helix
Does htt affect this by preventing binding?
Tobin - does wild type htt bind bax?
Montal - Fesik structural studies on alpha helix of proapoptotic molecule
which binds to pocket in anti-apoptotic molecule [thus preventing apoptosis]
Perhaps htt interacts with binding site
Expanded htt precipitation affected by this interaction?
Test by assaying binding of Bak or Bcl with different repeat
sizes
Tobin - testable hypothesis - how/what?
Forte - Yeast assay system?
Rothenberg - Maybe the reverse occurs - expanded protein can't
bind/conform to site
Chesselet/Greenamyre - [loss of function] by htt - interfers with apoptosis
Montal - Studies in C. elegans?
Signer - 3 groups are working on this right now
Tobin [?]
BREAK
Tobin - What is HD - cell death or cell undeath (Rothenberg hypothesis)
Rothenberg - rescue undead with growth factors
Chesselet - PD - has similar "undead" - lesion GP and patients get better
Striatal growth factors
In vitro - many (CNTF, BDNF, NT, etc)
In vivo - ?? maybe NT-3, others
Rothenberg - HD a failure to transport growth factors?
Beal [?]
Tobin - BDNF in rats - stem cells of striatum (Subventricular Zone
development)
Beal/Beckman - FGF effects - protect against stroke & 3NP/malonate
Greenamyre - effect of growth factors on mito?
Murphy/Dugan - emerging story
Swerdlow - growth factors rescue the calcium handling defect in AD cybrids
Greenamyre - Fred Fay's group - CaM kinase and mitochondrial calcium [?]
Beckman - tRNA [cytosolic] - disrupt polyQ by mischarged tRNAs?
Forte - is death of neuron related to insolubility of htt?
Montal - What is special about polyQ or polyAsn?
Murphy - [is there/need an] inducible system
Beckman [?]
Tobin - What about the proteosome?
Beckman - a good inhibitor [?]
Rothenberg - ubiquitin feeds proteins into the proteosome
Beckman - proteosome is 26S size protein complex that cleans up proteins
Montal - Robert Hubert has extensively studied proteosome protein structure
Tobin - Tie back to protein fragment effect (Murphy's hypothesis)
Beal - need to do glucose and O2 utilization
We saw a difference in glc util in Marcies [MacDonald] knock in mice
Pappazian - Drosphila "Zest" - DNA binding protein
Montal - htt processing?
Davis - cleanup of lipofuscin/amyloids - no clinical improvement
Beckman - protease activity starts disease?
Dugan - TGF [?]
Rothenberg - TGF - a stop signal?
Beal - induction of TGF by injury/stroke
Murphy [?]
Dugan [?]
Chesselet - induction of protease
BREAK
Reynolds - Excitotoxicity overview
What about quinolinic acid models?
Chesselet - Striatum has mechanism for quin toxicity [?]
Dugan - Change in receptor numbers?
Davis - specific protein in AD [not known yet]
Tobin - Excitotoxicity cascade targets?
Nicholls - respiratory controls
cells have 5X capacity to pump ions (above what's necessary)
TCA enzymes modulated by Calcium
backup of glycolysis - Pasteur effect (glycolysis)
inhibit respiration - erode the safety net
glutamate safety margin - eroded in 30 - 60 min (in vitro)
"Erosion of safety margin"
in vitro - 30 min
stroke - 1-2 days
HD - 20 years
[What is] safety margin
Stress -> lose function -> recover -> etc
Murphy/Reynolds - apoptotic cascades
Nicholls - Outer mitochondrial membrane [?]
Montal [?]
Rothenberg - What's a "brown-out" [bioenergetic brown-out]
Nicholls - energy demands following glutamate [receptor activation]
Glutamate leads to calcium [influx]
extrusion or
mitochondrial [buffering] - which competes with ATP production
Przedborski - acute mitochondrial catastrophy?
Davis - [30-40X activity of ETS] - add stress - then mito "dies"
Tobin - what would argue against the brown out theory in HD?
Nicolls - PET studies, but might miss [a mito defect]
Montal - NMR imaging - [observe] safety margin?
Beckman - Nitric Oxide link [to safety margin]
MnSOD [in mito] - nitrotyr [indicator of ROS mtDNA damage?]
NOS+ neurons in striatum
Tobin - Head trauma precipitating a neurological disease [e.g., PD]?
Rothenberg - CAG might chelate Zinc, pull it away from transcription factors
What experiments could be done to test?
What treatment could be performed?
BREAK
Summary statement by attendees:
Pappazian - schematic summarizing the meeting
Experiments should focus on nucleus - use cytosolic parameters as a
"readout" measurement
Przedborski - Shrinking and dysfunction of neurons [Rothenberg hypothesis]
[What would be effect of] bcl2 overexpress and KO [on htt]
Dugan - trophic factor injection in mice
Nicholls - the undead
BAX KO - a model of the "undead" neuron [BAF saved neurons]
Therapy [to remedy] reduced safety margin
e.g., CoQ, others
Montal - two approaches
Investigate safety margin by NMR imaging in patients
Protein structural studies [of huntingtin] - crystallography
Murphy - does the expansion affect mito?
Develop an inducible transfection system/model
Test cytosolic fractions on isolated mito
Therapy - prevent/reduce mito swelling?
Handran - glutamate receptor activation causes cortical and str degeneration
in HD, how does htt affect/induce an inappropriate excitotox/apoptotic
cascade
Forte - Trinucleotide repeat
"constipation" of the proteosome?
How do cells deal with polyQ expansion?
How do striatal cells deal with polyQ expansion?
Rothenberg - 2 areas to focus
Nucleus
Rescue [of undead neurons]
Conformation studies [of htt] by FRET methodology
Caviness - bridge gap between NII cause and effect
Signer - Is there a direct effect [of htt] on mito (that doesn't go through the
nucleus), e.g., Anne Murphy's hypothesis of Amphiphilic fragments?
Amphiphilic peptide experiements
Messenger Profiling - what is the 1st change in message [in the
nucleus]
Intervention - "utterly bewildering" at this time
Best idea - make cell blind to the presence of polyQ expansion
In vivo production of single chain Ab that binds to expanded
repeat
Chesselet - microtubules - axonal transport [disrupted by htt] - worth
exploring
Swerdlow - mutation on chr 4 - ends at the mitochondria?
Other CAG repeat disorders
[What is the connection between] PolyQ and mito
Experiment - htt nucleus effect on normal mito (cybrid expts)
Parallel "undead" disorder - schizophrenia?? (no neuronal loss, but
may be an undead disorder)
Prevention - ameliorate/block/protect mitochondrial function
Reynolds - safety margin worth further investigation
END OF NOTES |
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