Zeron, M. M., O. Hansson, et al. (2002). "Increased sensitivity to
N-methyl-D-aspartate receptor-mediated excitotoxicity in a mouse model of
Huntington's disease." Neuron33(6): 849-60.
Previous work suggests N-methyl-D-aspartate receptor (NMDAR) activation may be
involved in degeneration of medium-sized spiny striatal neurons in
Huntington's disease (HD). Here we show that these neurons are more vulnerable
to NMDAR-mediated death in a YAC transgenic FVB/N mouse model of HD expressing
full-length mutant huntingtin, compared with wild-type FVB/N mice. Excitotoxic
death of these neurons was increased after intrastriatal injection of
quinolinate in vivo, and after NMDA but not AMPA exposure in culture. NMDA-induced
cell death was abolished by an NR2B subtype-specific antagonist. In contrast,
NMDAR-mediated death of cerebellar granule neurons was not enhanced,
consistent with cell-type and NMDAR subtype specificity. Moreover, increased
NMDA-evoked current amplitude and caspase-3 activity were observed in
transgenic striatal neurons. Our data support a role for NR2B-subtype NMDAR
activation as a trigger for selective neuronal degeneration in HD.
Yu, Z. X., S. H. Li, et al. (2002). "Huntingtin inclusions do not deplete
polyglutamine-containing transcription factors in HD mice." Hum Mol Genet11(8): 905-14.
A pathological hallmark of polyglutamine diseases is the presence of
inclusions or aggregates of the expanded polyglutamine protein. Polyglutamine
inclusions are present in the neuronal nucleus in a number of inherited
neurodegenerative disorders, including Huntington disease (HD). Recent studies
suggest that polyglutamine inclusions may sequester polyglutamine-containing
transcription factors and deplete their concentration in the nucleus, leading
to altered gene expression. To test this hypothesis, we examined the
expression and localization of the polyglutamine-containing or glutamine-rich
transcription factors TBP, CBP and Sp1 in HD mouse models. All three
transcription factors were diffusely distributed in the nucleus, despite the
presence of abundant intranuclear inclusions. There were no differences in the
nuclear staining of these transcription factors between HD and wild-type mouse
brains. Although some CBP staining appeared as dots in the selective brain
regions (e.g. hypothalamus and amygdala), double labeling showed that most CBP
was not co-localized with huntingtin nuclear inclusions. Electron microscopy
confirmed that CBP was diffusely distributed in the nucleus. Western blots
showed that these transcription factors were not trapped in huntingtin
inclusions. In the striatum of HD mice, which suffers a significant reduction
in the expression of a number of genes, mutant huntingtin was present in both
an aggregated and a diffuse form. These findings suggest that altered gene
expression may result from the interactions of soluble mutant huntingtin with
nuclear transcription factors, rather than from the depletion of transcription
factors by nuclear inclusions.
Wyttenbach, A., O. Sauvageot, et al. (2002). "Heat shock protein 27 prevents
cellular polyglutamine toxicity and suppresses the increase of reactive oxygen
species caused by huntingtin." Hum Mol Genet11(9): 1137-1151.
Neuronal loss and intraneuronal protein aggregates are characteristics of
Huntington's disease (HD), which is one of 10 known neurodegenerative
disorders caused by an expanded polyglutamine [poly(Q)] tract in the disease
protein. N-terminal fragments of mutant huntingtin produce intracellular
aggregates and cause toxicity. Several studies have shown that chaperones
suppress poly(Q) aggregation and toxicity/cell death, but the mechanisms by
which they prevent poly(Q)-mediated cell death remain unclear. In the present
study, we identified heat shock protein 27 (HSP27) as a suppressor of poly(Q)
mediated cell death, using a cellular model of HD. In contrast to HSP40/70
chaperones, we showed that HSP27 suppressed poly(Q) death without suppressing
poly(Q) aggregation. We tested the hypotheses that HSP27 may reduce poly(Q)-mediated
cell death either by binding cytochrome c and inhibiting the mitochondrial
death pathway or by protecting against reactive oxygen species (ROS). While
poly(Q)-induced cell death was reduced by inhibiting cytochrome c (cyt c)
release from mitochondria, protection by HSP27 was regulated by its
phosphorylation status and was independent of its ability to bind to cyt c.
However, we observed that mutant huntingtin caused increased levels of ROS in
neuronal and non-neuronal cells. ROS contributed to cell death because both
N-acetyl-L-cysteine and glutathione in its reduced form suppressed poly(Q)-mediated
cell death. HSP27 decreased ROS in cells expressing mutant huntingtin,
suggesting that this chaperone protects cells against oxidative stress. We
propose that a poly(Q) mutation can induce ROS that directly contribute to
cell death and that HSP27 is an antagonist of this process.
Wheeler, V. C., C. A. Gutekunst, et al. (2002). "Early phenotypes that presage
late-onset neurodegenerative disease allow testing of modifiers in Hdh CAG
knock-in mice." Hum Mol Genet11(6): 633-40.
In Huntington's disease (HD), CAG repeats extend a glutamine tract in
huntingtin to initiate the dominant loss of striatal neurons and chorea.
Neuropathological changes include the formation of insoluble mutant N-terminal
fragment, as nuclear/neuropil inclusions and filter-trap amyloid, which may
either participate in the disease process or be a degradative by-product. In
young Hdh knock-in mice, CAGs that expand the glutamine tract in mouse
huntingtin to childhood-onset HD lengths lead to nuclear accumulation of
full-length mutant huntingtin and later accumulation of insoluble fragment.
Here we report late-onset neurodegeneration and gait deficits in older
Hdh(Q111) knock-in mice, demonstrating that the nuclear phenotypes comprise
early stages in a disease process that conforms to genetic and pathologic
criteria determined in HD patients. Furthermore, using the early
nuclear-accumulation phenotypes as surrogate markers, we show in genetic
experiments that the disease process, initiated by full-length mutant protein,
is hastened by co-expression of mutant fragment; therefore, accrual of
insoluble-product in already compromised neurons may exacerbate pathogenesis.
In contrast, timing of early disease events was not altered by normal
huntingtin or by mutant caspase-1, two proteins shown to reduce inclusions and
glutamine toxicity in other HD models. Thus, potential HD therapies in man
might be directed at different levels: preventing the disease-initiating
mechanism or slowing the subsequent progression of pathogenesis.
Song, C., G. Perides, et al. (2002). "Expression of full-length polyglutamine-expanded
Huntingtin disrupts growth factor receptor signaling in rat pheochromocytoma
(PC12) cells." J Biol Chem277(8): 6703-7.
We reported previously that normal Huntingtin is associated with epidermal
growth factor receptor (EGF) signaling complex (Liu, Y. F., Deth, C. R., and
Devys, D. (1997) J. Biol. Chem. 272, 8121-8124). To investigate the potential
role of normal and polyglutamine-expanded Huntingtin in the regulation of
growth factor receptor-mediated cellular signaling and biological function, we
stably transfected full-length Huntingtin containing 16, 48, or 89
polyglutamine repeats into PC12 cells where cellular signaling mechanisms,
mediated by nerve growth factor (NGF) or EGF receptors, are well
characterized. Expression of polyglutamine-expanded Huntingtin, but not normal
Huntingtin, leads to a dramatic morphological change. In clones carrying the
mutated Huntingtin, both NGF and EGF receptor-mediated activation of mitogen-activated
protein kinase, c-Jun N-terminal kinase, and Akt are significantly attenuated,
and NGF receptor-mediated neurite outgrowth is blocked. Co-immunoprecipitation
studies show that the associations of NGF or EGF receptors with growth factor
receptor-binding protein 2 (Grb2) and phosphoinositide 3-kinase are
significantly inhibited. NGF-induced tyrosine phosphorylation of NGF receptors
(TrkA) is also consistently suppressed. Our data demonstrate that
polyglutamine-expanded Huntingtin disrupts cellular signaling mediated by both
EGF and NGF receptors in PC12 cells. It is known that Huntington's disease
patients exhibit an extremely low incidence of a variety of cancers and are
deficient in glucose metabolism. Thus, our results may reflect an important
molecular mechanism for the pathogenesis of the disease.
Rubinsztein, D. C. (2002). "Lessons from animal models of Huntington's
disease." Trends Genet18(4): 202-9.
Huntington's disease (HD) is an autosomal-dominant neurodegenerative disorder
caused by a CAG trinucleotide repeat expansion in the HD gene. The expanded
repeats are translated into an abnormally long polyglutamine tract close to
the N-terminus of the HD gene product, huntingtin. Studies in mouse models and
human suggest that the mutation is associated with a deleterious gain of
function. There is now a wide range of mouse models for HD, providing
important insights into processes associated with disease pathogenesis. These
models have been complemented by studies in Drosophila and Caenorhabditis
elegans that have allowed the identification of possible modifier loci through
suppressor screens.
Peters, P. J., K. Ning, et al. (2002). "Arfaptin 2 regulates the aggregation
of mutant huntingtin protein." Nat Cell Biol4(3): 240-5.
Huntington's disease (HD) is an inherited neurodegenerative disorder. Here we
demonstrate that expression of arfaptin 2/POR1 (partner of Rac1) in cultured
cells induces the formation of pericentriolar and nuclear aggregates, which
morphologically resemble mutant huntingtin aggregates characteristic of HD.
Endogenous arfaptin 2 localizes to aggregates induced by expression of an
abnormal amino-terminal fragment of huntingtin that contains polyglutamine (polyQ)
expansions. A dominant inhibitory mutant of arfaptin 2 inhibits aggregation of
mutant huntingtin, but not in the presence of proteasome inhibitors. Using
cell-free biochemical assays, we show that arfaptin 2 inhibits proteasome
activity. Finally, we show that expression of arfaptin 2 is increased at sites
of neurodegeneration and the protein localizes to huntingtin aggregates in HD
transgenic mouse brains. Our data suggest that arfaptin 2 is involved in
regulating huntingtin protein aggregation, possibly by impairing proteasome
function.
Perutz, M. F., B. J. Pope, et al. (2002). "Aggregation of proteins with
expanded glutamine and alanine repeats of the glutamine-rich and asparagine-rich
domains of Sup35 and of the amyloid beta-peptide of amyloid plaques." Proc
Natl Acad Sci U S A99(8): 5596-600.
The exon-1 peptide of huntingtin has 51 Gln repeats and produces the symptoms
of Huntington's disease in transgenic mice. Aggregation of the yeast Sup35
protein into prions has been attributed to its glutamine-rich and asparagine-rich
domain. Here, we show that poly-L-asparagine forms polar zippers similar to
those of poly-L-glutamine. In solution at acid pH, the glutamine-rich and
asparagine-rich 18-residue Sup35 peptide, rendered soluble by the addition of
two aspartates at the amino end and two lysines at the carboxyl end, gives a
beta-sheet CD spectrum; it aggregates at neutral pH. A poly-alanine peptide
D(2)A(10)K(2) gives an alpha-helical CD spectrum at all pHs and does not
aggregate; a peptide with the sequence of the C-terminal helix of the
alpha-chain of human hemoglobin, preceded by two aspartates and followed by
two lysines, exhibits a random coil spectrum and does not aggregate either.
Alignment of several beta-strands with the sequence of the 42-residue
Alzheimer's amyloid beta-peptide shows that they can be linked together by a
network of salt bridges. We also asked why single amino acid replacements can
so destabilize the native structures of proteins that they unfold and form
amyloids. The difference in free energy of a protein molecule between its
native, fully ordered structure and an amorphous mixture of randomly coiled
chains is only of the order of 10 kcal/mol. Theory shows that destabilization
of the native structure by no more than 2 kcal/mol can increase the
probability of nucleation of disordered aggregates from which amyloids could
grow 130,000-fold.
Muchowski, P. J., K. Ning, et al. (2002). "Requirement of an intact
microtubule cytoskeleton for aggregation and inclusion body formation by a
mutant huntingtin fragment." Proc Natl Acad Sci U S A99(2):
727-32.
Huntington's disease is caused by the expansion of CAG repeats coding for a
polyglutamine tract in the huntingtin protein. The major pathological feature
found in Huntington's disease neurons is the presence of detergent-insoluble
ubiquitinated inclusion bodies composed of the huntingtin protein. However,
the mechanisms that underlie inclusion body formation, and the precise
relationship between inclusion bodies and events that initiate toxicity,
remain unclear. Here, we analyzed the effects of drugs or genetic mutations
that disrupt the microtubule cytoskeleton in a Saccharomyces cerevisiae model
of the aggregation of an amino-terminal polyglutamine-containing fragment of
huntingtin exon 1 (HtEx1). Treatment of yeast with drugs that disrupt
microtubules resulted in less than 2% of the detergent-insoluble HtEx1
observed in mock-treated cells and prevented the formation of large
juxtanuclear inclusion bodies. Disruption of microtubules also unmasked a
potent glutamine length-dependent toxicity of HtEx1 under conditions where
HtEx1 exists in an entirely detergent-soluble nonaggregated form. Results from
the yeast model paralleled those from neuronal pheochromocytoma cells, where
disruption of microtubules eliminated the formation of juxtanuclear and
intranuclear inclusion bodies by HtEx1. Our results suggest that active
transport along microtubules may be required for inclusion body formation by
HtEx1 and that inclusion body formation may have evolved as a cellular
mechanism to promote the sequestration or clearance of soluble species of
HtEx1 that are otherwise toxic to cells.
Menalled, L. B. and M. F. Chesselet (2002). "Mouse models of Huntington's
disease." Trends Pharmacol Sci23(1): 32-9.
Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder.
In 1993 the mutation that causes HD was identified as an unstable expansion of
CAG repeats in the IT15 gene. Since then one of the most important advances in
HD research has been the generation of various mouse models that enable the
exploration of early pathological, molecular and cellular abnormalities
produced by the mutation. In addition, these models have made it possible to
test different pharmacological approaches to delay the onset or slow the
progression of HD. In this article, insights gained from mouse models towards
the understanding of HD and the design of new therapeutic strategies are
discussed.
Mattson, M. P. (2002). "Accomplices to neuronal death." Nature415(6870):
377-9.
MacGibbon, G. A., L. C. Hamilton, et al. (2002). "Immediate-early gene
response to methamphetamine, haloperidol, and quinolinic acid is not impaired
in Huntington's disease transgenic mice." J Neurosci Res67(3):
372-8.
Striatal neurons in symptomatic Huntington's disease (HD) transgenic mice are
resistant to a variety of toxic insults, including quinolinic acid (QA),
kainic acid and 3-nitropropionic acid. The basis for this resistance is
currently unknown. To investigate the possibility that the immediate-early
gene (IEG) response is defective in symptomatic HD mice leading to a lack of
response to these compounds, we examined the expression of c-Fos and Krox 24
after administration of the indirect dopamine agonist methamphetamine, the
dopamine D(2) receptor antagonist haloperidol and the neurotoxin QA in 5- and
10-week-old R6/2 transgenic HD and wild-type mice. Unlike wild-type and
pre-symptomatic R6/2 transgenic HD mice, 10-week-old symptomatic HD mice were
resistant to methamphetamine-induced gliosis and QA lesion. There was,
however, no difference in the number or distribution of c-Fos-immunoreactive
nuclei 2 hr after single injections of methamphetamine or haloperidol among 5-
and 10-week-old wild-type mice and 5- and 10-week-old R6/2 HD mice. Similarly,
despite their resistance to QA-induced lesioning and lower basal levels of
krox-24 mRNA, the symptomatic R6/2 mice had equivalent increases in the amount
of c-fos and krox-24 mRNA compared to wild-type and pre-symptomatic R6/2 HD
mice as determined by in situ hybridization and densitometry 2 hr after QA
administration. These data demonstrate that the c-Fos and Krox 24 IEG response
to dopamine agonists, dopamine antagonists and neurotoxic insult is functional
in symptomatic R6/2 HD mice. Resistance to toxic insult in R6/2 mice may be
conferred by interactions of mutant huntingtin with proteins or
transcriptional processes further along the toxic cascade.
MacGibbon, G. A., L. C. Hamilton, et al. (2002). "Immediate-early gene
response to methamphetamine, haloperidol, and quinolinic acid is not impaired
in Huntington's disease transgenic mice." J Neurosci Res67(3):
372-378.
Striatal neurons in symptomatic Huntington's disease (HD) transgenic mice are
resistant to a variety of toxic insults, including quinolinic acid (QA),
kainic acid and 3-nitropropionic acid. The basis for this resistance is
currently unknown. To investigate the possibility that the immediate-early
gene (IEG) response is defective in symptomatic HD mice leading to a lack of
response to these compounds, we examined the expression of c-Fos and Krox 24
after administration of the indirect dopamine agonist methamphetamine, the
dopamine D(2) receptor antagonist haloperidol and the neurotoxin QA in 5- and
10-week-old R6/2 transgenic HD and wild-type mice. Unlike wild-type and
pre-symptomatic R6/2 transgenic HD mice, 10-week-old symptomatic HD mice were
resistant to methamphetamine-induced gliosis and QA lesion. There was,
however, no difference in the number or distribution of c-Fos-immunoreactive
nuclei 2 hr after single injections of methamphetamine or haloperidol among 5-
and 10-week-old wild-type mice and 5- and 10-week-old R6/2 HD mice. Similarly,
despite their resistance to QA-induced lesioning and lower basal levels of
krox-24 mRNA, the symptomatic R6/2 mice had equivalent increases in the amount
of c-fos and krox-24 mRNA compared to wild-type and pre-symptomatic R6/2 HD
mice as determined by in situ hybridization and densitometry 2 hr after QA
administration. These data demonstrate that the c-Fos and Krox 24 IEG response
to dopamine agonists, dopamine antagonists and neurotoxic insult is functional
in symptomatic R6/2 HD mice. Resistance to toxic insult in R6/2 mice may be
conferred by interactions of mutant huntingtin with proteins or
transcriptional processes further along the toxic cascade. Copyright 2002
Wiley-Liss, Inc.
Li, S. H., A. L. Cheng, et al. (2002). "Interaction of Huntington disease
protein with transcriptional activator Sp1." Mol Cell Biol22(5):
1277-87.
Polyglutamine expansion causes Huntington disease (HD) and at least seven
other neurodegenerative diseases. In HD, N-terminal fragments of huntingtin
with an expanded glutamine tract are able to aggregate and accumulate in the
nucleus. Although intranuclear huntingtin affects the expression of numerous
genes, the mechanism of this nuclear effect is unknown. Here we report that
huntingtin interacts with Sp1, a transcription factor that binds to GC-rich
elements in certain promoters and activates transcription of the corresponding
genes. In vitro binding and immunoprecipitation assays show that polyglutamine
expansion enhances the interaction of N-terminal huntingtin with Sp1. In HD
transgenic mice (R6/2) that express N-terminal-mutant huntingtin, Sp1 binds to
the soluble form of mutant huntingtin but not to aggregated huntingtin. Mutant
huntingtin inhibits the binding of nuclear Sp1 to the promoter of nerve growth
factor receptor and suppresses its transcriptional activity in cultured cells.
Overexpression of Sp1 reduces the cellular toxicity and neuritic extension
defects caused by intranuclear mutant huntingtin. These findings suggest that
the soluble form of mutant huntingtin in the nucleus may cause cellular
dysfunction by binding to Sp1 and thus reducing the expression of
Sp1-regulated genes.
Lesort, M., W. Chun, et al. (2002). "Does tissue transglutaminase play a role
in Huntington's disease?" Neurochem Int40(1): 37-52.
Tissue transglutaminase (tTG) likely plays a role in numerous processes in the
nervous system. tTG posttranslationally modifies proteins by transamidation of
specific polypeptide bound glutamines (Glns). This reaction results in the
incorporation of polyamines into substrate proteins or the formation of
protein crosslinks, modifications that likely have significant effects on
neural function. Huntington's disease is a genetic disorder caused by an
expansion of the polyglutamine domain in the huntingtin protein. Because a
polypeptide bound Gln is the determining factor for a tTG substrate, and
mutant huntingtin aggregates have been found in Huntington's disease brain, it
has been hypothesized that tTG may contribute to the pathogenesis of
Huntington's disease. In vitro, polyglutamine constructs and huntingtin are
substrates of tTG. Further, the levels of tTG and TG activity are elevated in
Huntington's disease brain and immunohistochemical studies have demonstrated
that there is an increase in tTG reactivity in affected neurons in
Huntington's disease. These findings suggest that tTG may play a role in
Huntington's disease. However in situ, neither wild type nor mutant huntingtin
is modified by tTG. Further, immunocytochemical analysis revealed that tTG is
totally excluded from the huntingtin aggregates, and modulation of the
expression level of tTG had no effect on the frequency of the aggregates in
the cells. Therefore, tTG is not required for the formation of huntingtin
aggregates, and likely does not play a role in this process in Huntington's
disease brain. However, tTG interacts with truncated huntingtin, and
selectively polyaminates proteins that are associated with mutant truncated
huntingtin. Given the fact that the levels of polyamines in cells is in the
millimolar range and the crosslinking and polyaminating reactions catalyzed by
tTG are competing reactions, intracellularly polyamination is likely to be the
predominant reaction. Polyamination of proteins is likely to effect their
function, and therefore it can be hypothesized that tTG may play a role in the
pathogenesis of Huntington's disease by modifying specific proteins and
altering their function and/or localization. Further research is required to
define the specific role of tTG in Huntington's disease.
Lastres-Becker, I., F. Berrendero, et al. (2002). "Loss of mRNA levels,
binding and activation of GTP-binding proteins for cannabinoid CB(1) receptors
in the basal ganglia of a transgenic model of Huntington's disease." Brain
Res929(2): 236-42.
Data obtained from the basal ganglia of postmortem Huntington's disease (HD)
brains have revealed that the level of cannabinoid CB(1) receptors in striatal
efferent neurons decreases in parallel to the dysfunction and subsequent
degeneration of these neurons. These findings, and others from rat models of
HD generated by lesions with mitochondrial toxins, suggest that the loss of
CB(1) receptors may be involved in the pathogenesis of the disease. To explore
further the changes in the endocannabinoid system, as well as the potential of
endocannabinoid-related compounds, we examined the status of CB(1) receptors
in the HD94 transgenic mouse model of HD. These mice express huntingtin exon 1
with a polyglutamine tract of 94 repeats in a tissue-specific and conditional
manner using the tet regulatable system. They develop many features of HD,
such as striatal atrophy, intraneuronal aggregates and progressive dystonia.
In these animals, we analyzed mRNA levels for the CB(1) receptor, in addition
to the number of specific binding sites and the activation of GTP-binding
proteins by CB(1) receptor agonists. mRNA transcripts of the CB(1) receptor
were significantly decreased in the caudate-putamen of HD transgenic mice
compared to age-matched littermate controls. The decrease concurred with a
marked reduction in receptor density in both the caudate-putamen and its
projection areas such as the globus pallidus, entopeduncular nucleus and
substantia nigra pars reticulata. Furthermore, the efficacy of CB(1) receptor
activation was reduced in the globus pallidus, as determined by
agonist-induced [35S]GTPgammaS binding, and tended towards a decrease in the
substantia nigra. None of these changes was seen in the cerebral cortex and
hippocampus, despite high levels of expression of the mutant protein in these
regions. The decrease in CB(1) receptor levels was accompanied by a decrease
in the proenkephalin-mRNA levels but not in substance P-mRNA levels. Taken
together, these results suggest that the loss of CB(1) receptor might be
preferential to the enkephalinergic CB(1) receptor-containing striatopallidal
neurons, and further implicate the CB(1) receptor to the subsequent HD
symptomatology and neuropathology.
Klug, A. (2002). "Structural biology and biochemistry. Retrospective: Max
Perutz (1914-2002)." Science295(5564): 2382-3.
Khoshnan, A., J. Ko, et al. (2002). "Effects of intracellular expression of
anti-huntingtin antibodies of various specificities on mutant huntingtin
aggregation and toxicity." Proc Natl Acad Sci U S A99(2):
1002-7.
We have generated eight mAbs (MW1-8) that bind the epitopes polyglutamine (polyQ),
polyproline (polyP), or the C terminus of exon 1 in huntingtin (htt) protein.
In the brains of Huntington's disease (HD) mouse models, the anti-polyQ mAbs
bind to various cytoplasmic compartments, whereas the anti-polyP and anti-C
terminus mAbs bind nuclear inclusions containing htt. To use these mAbs as
intracellular perturbation agents, we have cloned and expressed the
antigen-binding domains of three of the mAbs as single-chain variable region
fragment Abs (scFvs). In 293 cells cotransfected with htt exon 1 containing an
expanded polyQ domain, MW1, MW2, and MW7 scFvs colocalize with htt exon 1.
Moreover, these scFvs coimmunoprecipitate with htt exon 1 in cell extracts. In
perturbation experiments, MW7 scFv, recognizing the polyP domains of htt,
significantly inhibits aggregation as well as the cell death induced by mutant
htt protein. In contrast, MW1 and MW2 scFvs, recognizing the polyQ stretch,
stimulate htt aggregation and apoptosis. Therefore, these anti-htt scFvs can
be used to investigate the role of the polyP and polyQ domains in HD
pathogenesis, and antibody binding to the polyP domain has potential
therapeutic value in HD.
Kegel, K. B., A. R. Meloni, et al. (2002). "Huntingtin is present in the
nucleus, interacts with the transcriptional corepressor C-terminal binding
protein, and represses transcription." J Biol Chem277(9):
7466-76.
Huntingtin is a protein of unknown function that contains a polyglutamine
tract, which is expanded in patients with Huntington's disease (HD). We
investigated the localization and a potential function for huntingtin in the
nucleus. In human fibroblasts from normal and HD patients, huntingtin
localized diffusely in the nucleus and in subnuclear compartments identified
as speckles, promyelocytic leukemia protein bodies, and nucleoli. Huntingtin-positive
nuclear bodies redistributed after treatment with sodium butyrate. By Western
blot, purified nuclei had low levels of full-length huntingtin compared with
the cytoplasm but contained high levels of N- and C-terminal huntingtin
fragments, which tightly bound the nuclear matrix. Full-length huntingtin co-immunoprecipitated
with the transcriptional corepressor C-terminal binding protein, and
polyglutamine expansion in huntingtin reduced this interaction. Full-length
wild-type and mutant huntingtin repressed transcription when targeted to DNA.
Truncated N-terminal mutant huntingtin repressed transcription, whereas the
corresponding wild-type fragment did not repress transcription. We speculate
that wild-type huntingtin may function in the nucleus in the assembly of
nuclear matrix-bound protein complexes involved with transcriptional
repression and RNA processing. Proteolysis of mutant huntingtin may alter
nuclear functions by disrupting protein complexes and inappropriately
repressing transcription in HD.
Kazantsev, A., H. A. Walker, et al. (2002). "A bivalent Huntingtin binding
peptide suppresses polyglutamine aggregation and pathogenesis in Drosophila."
Nat Genet.
Huntington disease is caused by the expansion of a polyglutamine repeat in the
Huntingtin protein (Htt) that leads to degeneration of neurons in the central
nervous system and the appearance of visible aggregates within neurons. We
have developed and tested suppressor polypeptides that bind mutant Htt and
interfere with the process of aggregation in cell culture. In a Drosophila
model, the most potent suppressor inhibits both adult lethality and
photoreceptor neuron degeneration. The appearance of aggregates in
photoreceptor neurons correlates strongly with the occurrence of pathology,
and expression of suppressor polypeptides delays and limits the appearance of
aggregates and protects photoreceptor neurons. These results suggest that
targeting the protein interactions leading to aggregate formation may be
beneficial for the design and development of therapeutic agents for Huntington
disease.
Kazantsev, A., H. A. Walker, et al. (2002). "A bivalent Huntingtin binding
peptide suppresses polyglutamine aggregation and pathogenesis in Drosophila."
Nat Genet30(4): 367-76.
Huntington disease is caused by the expansion of a polyglutamine repeat in the
Huntingtin protein (Htt) that leads to degeneration of neurons in the central
nervous system and the appearance of visible aggregates within neurons. We
have developed and tested suppressor polypeptides that bind mutant Htt and
interfere with the process of aggregation in cell culture. In a Drosophila
model, the most potent suppressor inhibits both adult lethality and
photoreceptor neuron degeneration. The appearance of aggregates in
photoreceptor neurons correlates strongly with the occurrence of pathology,
and expression of suppressor polypeptides delays and limits the appearance of
aggregates and protects photoreceptor neurons. These results suggest that
targeting the protein interactions leading to aggregate formation may be
beneficial for the design and development of therapeutic agents for Huntington
disease.
Karpuj, M. V., M. W. Becher, et al. (2002). "Evidence for a role for
transglutaminase in Huntington's disease and the potential therapeutic
implications." Neurochem Int40(1): 31-6.
Transglutaminase (TGase) activity is increased in affected regions of brains
from patients with Huntington's disease (HD). TGase activity is particularly
elevated in the nucleus compared with the cytoplasm from these brains. Gamma-glutaminyl-lysyl
cross-links have been detected in nuclear inclusions in HD brain, indicating
that TGase may play a prominent role in the aggregation of huntingtin (htt).
Attempts to ameliorate experimental disease, via inhibition of TGase in
transgenic models of HD in mice, are under investigation.
Karpuj, M. V., M. W. Becher, et al. (2002). "Prolonged survival and decreased
abnormal movements in transgenic model of Huntington disease, with
administration of the transglutaminase inhibitor cystamine." Nat Med
8(2): 143-9.
An expanded polyglutamine domain in huntingtin underlies the pathogenic events
in Huntington disease (HD), characterized by chorea, dementia and severe
weight loss, culminating in death. Transglutaminase (TGase) may be critical in
the pathogenesis, via cross-linking huntingtin. Administration of the TGase
competitive inhibitor, cystamine, to transgenic mice expressing exon 1 of
huntingtin containing an expanded polyglutamine repeat, altered the course of
their HD-like disease. Cystamine given intraperitoneally entered brain where
it inhibited TGase activity. When treatment began after the appearance of
abnormal movements, cystamine extended survival, reduced associated tremor and
abnormal movements and ameliorated weight loss. Treatment did not influence
the appearance or frequency of neuronal nuclear inclusions. Unexpectedly,
cystamine treatment increased transcription of one of the two genes shown to
be neuroprotective for polyglutamine toxicity in Drosophila, dnaj (also known
as HDJ1 and Hsp40 in humans and mice, respectively). Inhibition of TGase
provides a new treatment strategy for HD and other polyglutamine diseases.
Hoffner, G., P. Kahlem, et al. (2002). "Perinuclear localization of huntingtin
as a consequence of its binding to microtubules through an interaction with
beta-tubulin: relevance to Huntington's disease." J Cell Sci115(Pt
5): 941-8.
Huntington's disease results from an expansion of a series of glutamine
repeats in the protein huntingtin. We have discovered from immunopurification
studies that huntingtin combines specifically with the beta subunit of tubulin.
This binding explains why huntingtin can be shown on assembled microtubules by
electron microscopy. Immunostaining shows that most of the huntingtin in the
cytoplasm is associated with microtubules. Huntingtin is particularly abundant
in the perinuclear region, where it is also associated with microtubules and
in the centrosomal region, where it co-localizes with gamma-tubulin. In
Huntington's disease, inclusions are often nuclear or perinuclear. Since the
perinuclear concentration of huntingtin does not depend on the number of its
glutamine repeats, we propose that inclusions are found in perinuclear and
intranuclear locations because the beta-tubulin binding property of huntingtin
brings it to the perinuclear region, from which it readily gains access to the
nucleus. The mutational glutamine expansion then promotes insolubility and
results in an inclusion.
Gervais, F. G., R. Singaraja, et al. (2002). "Recruitment and activation of
caspase-8 by the Huntingtin-interacting protein Hip-1 and a novel partner
Hippi." Nat Cell Biol4(2): 95-105.
In Huntington disease, polyglutamine expansion of the protein huntingtin (Htt)
leads to selective neurodegenerative loss of medium spiny neurons throughout
the striatum by an unknown apoptotic mechanism. Binding of Hip-1, a protein
normally associated with Htt, is reduced by polyglutamine expansion. Free
Hip-1 binds to a hitherto unknown polypeptide, Hippi (Hip-1 protein interactor),
which has partial sequence homology to Hip-1 and similar tissue and
subcellular distribution. The availability of free Hip-1 is modulated by
polyglutamine length within Htt, with disease-associated polyglutamine
expansion favouring the formation of pro-apoptotic Hippi-Hip-1 heterodimers.
This heterodimer can recruit procaspase-8 into a complex of Hippi, Hip-1 and
procaspase-8, and launch apoptosis through components of the 'extrinsic'
cell-death pathway. We propose that Htt polyglutamine expansion liberates
Hip-1 so that it can form a caspase-8 recruitment complex with Hippi. This
novel non-receptor-mediated pathway for activating caspase-8 might contribute
to neuronal death in Huntington disease.
Ferrante, R. J., O. A. Andreassen, et al. (2002). "Therapeutic effects of
coenzyme Q10 and remacemide in transgenic mouse models of Huntington's
disease." J Neurosci22(5): 1592-9.
There is substantial evidence that bioenergetic defects and excitotoxicity may
play a role in the pathogenesis of Huntington's disease (HD). Potential
therapeutic strategies for neurodegenerative diseases in which there is
reduced energy metabolism and NMDA-mediated excitotoxicity are the
administration of the mitochondrial cofactor coenzyme Q10 and the NMDA
antagonist remacemide. We found that oral administration of either coenzyme
Q10 or remacemide significantly extended survival and delayed the development
of motor deficits, weight loss, cerebral atrophy, and neuronal intranuclear
inclusions in the R6/2 transgenic mouse model of HD. The combined treatment,
using coenzyme Q10 and remacemide together, was more efficacious than either
compound alone, resulting in an approximately 32 and 17% increase in survival
in the R6/2 and N171-82Q mice, respectively. Magnetic resonance imaging showed
that combined treatment significantly attenuated ventricular enlargement in
vivo. These studies further implicate defective energy metabolism and
excitotoxicity in the R6/2 and N171-82Q transgenic mouse models of HD and are
of interest in comparison with the outcome of a recent clinical trial
examining coenzyme Q10 and remacemide in HD patients.
Ellerby, L. M. (2002). "Hunting for excitement: NMDA receptors in Huntington's
disease." Neuron33(6): 841-2.
Excitotoxic cell death stimulated by quinolinic acid injection into the
striatum has a long history of "mimicking" many aspects of motor, behavioral,
and neurochemical changes observed in Huntington's disease patients. In this
issue of Neuron, provide insight into the role of NMDA receptors in the
cell-specific excitotoxic death observed in Huntington's disease (HD) using a
HD mouse model expressing full-length mutant huntingtin (htt).
Dunah, A. W., H. Jeong, et al. (2002). "Sp1 and TAFII130 Transcriptional
Activity Disrupted in Early Huntington's Disease." Science.
Huntington's Disease (HD) is an inherited neurodegenerative disease caused by
expansion of a polyglutamine tract in the huntingtin protein. Transcriptional
dysregulation has been implicated in HD pathogenesis. Here we report that
huntingtin interacts with the transcriptional activator Sp1 and coactivator
TAFII130. Co-expression of Sp1 and TAFII130 in cultured striatal cells from
wild type and HD transgenic mice reverses the transcriptional inhibition of
the dopamine D2 receptor gene caused by mutant huntingtin, as well as protects
neurons from huntingtin-induced cellular toxicity. Furthermore, soluble mutant
huntingtin inhibits Sp1 binding to DNA in postmortem brain tissues of both
presymptomatic and affected HD patients. Understanding these early molecular
events in HD may provide an opportunity to interfere with the effects of
mutant huntingtin before development of disease symptoms.
de Almeida, L. P., C. A. Ross, et al. (2002). "Lentiviral-mediated delivery of
mutant huntingtin in the striatum of rats induces a selective neuropathology
modulated by polyglutamine repeat size, huntingtin expression levels, and
protein length." J Neurosci22(9): 3473-83.
A new strategy based on lentiviral-mediated delivery of mutant huntingtin (htt)
was used to create a genetic model of Huntington's disease (HD) in rats and to
assess the relative contribution of polyglutamine (CAG) repeat size, htt
expression levels, and protein length on the onset and specificity of the
pathology. Lentiviral vectors coding for the first 171, 853, and 1520 amino
acids of wild-type (19 CAG) or mutant htt (44, 66, and 82 CAG) driven by
either the phosphoglycerate kinase 1 (PGK) or the cytomegalovirus (CMV)
promoters were injected in rat striatum. A progressive pathology characterized
by sequential appearance of ubiquitinated htt aggregates, loss of dopamine-
and cAMP-regulated phosphoprotein of 32 kDa staining, and cell death was
observed over 6 months with mutant htt. Earlier onset and more severe
pathology occurred with shorter fragments, longer CAG repeats, and higher
expression levels. Interestingly, the aggregates were predominantly located in
the nucleus of PGK-htt171-injected rats, whereas they were present in both the
nucleus and processes of CMV-htt171-injected animals expressing lower
transgene levels. Finally, a selective sparing of interneurons was observed in
animals injected with vectors expressing mutant htt. These data demonstrate
that lentiviral-mediated expression of mutant htt provides a robust in vivo
genetic model for selective neural degeneration that will facilitate future
studies on the pathogenesis of cell death and experimental therapeutics for
HD.
Cooper, A. J., T. M. Jeitner, et al. (2002). "Cross linking of polyglutamine
domains catalyzed by tissue transglutaminase is greatly favored with
pathological-length repeats: does transglutaminase activity play a role in (CAG)(n)/Q(n)-expansion
diseases?" Neurochem Int40(1): 53-67.
Protein aggregates are a hallmark of Huntington's disease (HD) and other
inherited neurodegenerative diseases caused by an elongated (CAG)(n) repeat in
the genome and to a corresponding increase in the size of the Q(n) domain in
the expressed protein. When the protein associated with HD (huntingtin)
contains <35 Q repeats disease does not occur. However, an n>/=40 leads to
disease. Some investigators have proposed that aggregates in the nuclei of
affected cells are toxic, but other workers have suggested that the aggregates
may be neutral or even protective. Whether or not they are toxic, an
understanding of the processes whereby the aggregates develop may shed light
on the neuropathological processes involved in the (CAG)(n)/Q(n)-expansion
disorders. Q(n) domains have a tendency to non-covalently self align as 'polar
zippers' rendering them less soluble, but evidence that such polar zippers
occur in the aggregates in intact HD brain has so far been limited. The human
brain contains at least three Ca(2+)-dependent enzymes (transglutaminases,
TGases) that catalyze protein cross-linking reactions, namely TGase 1, TGase 2
(tissue transglutaminase, tTGase) and TGase 3. Q(n) aggregates have been found
by several groups to be excellent substrates of tTGase. Moreover, the activity
toward the Q(n) domains increases greatly as n is increased to 40 or beyond.
tTGase mRNA and total TGase activity are elevated in HD brain. Moreover, some
evidence suggests that Ca(2+) homeostasis is disrupted in HD brain. We propose
that the combination of increased huntingtin (or huntingtin fragment
containing the Q(n) domain) in the nucleus, increased the ability of the Q(n)
domains to act as substrate, increased Ca(2+) levels and increased inherent
TGase activity all contribute to increased cross-linking of proteins in HD
brain. At first the proteasome machinery can recognize and degrade the
cross-linked proteins, but over time the proteasome machinery may be
overwhelmed and protein aggregates will accumulate.
Clifford, J. J., J. Drago, et al. (2002). "Essential fatty acids given from
conception prevent topographies of motor deficit in a transgenic model of
Huntington's disease." Neuroscience109(1): 81-8.
Transgenic R6/1 mice incorporate a human genomic fragment containing promoter
elements exon 1 and a portion of intron 2 of the Huntingtin gene responsible
for Huntington's disease. They develop late-onset neurological deficits in a
manner similar to the motor abnormalities of the disorder. As essential fatty
acids are phospholipid components of cell membranes which may influence cell
death and movement disorder phenotype, R6/1 and normal mice were randomised to
receive a mixture of essential fatty acids or placebo on alternate days
throughout life. Over mid-adulthood, topographical assessment of behaviour
revealed R6/1 transgenics to evidence progressive shortening of stride length,
with progressive reductions in locomotion, elements of rearing, sniffing,
sifting and chewing, and an increase in grooming. These deficits were either
not evident or materially diminished in R6/1 transgenics receiving essential
fatty acids. R6/1 transgenics also showed reductions in body weight and in
brain dopamine D(1)-like and D(2)-like quantitative receptor autoradiography
which were unaltered by essential fatty acids.These findings indicate that
early and sustained treatment with essential fatty acids are able to protect
against motor deficits in R6/1 transgenic mice expressing exon 1 and a portion
of intron 2 of the Huntingtin gene, and suggest that essential fatty acids may
have therapeutic potential in Huntington's disease.
Chun, W., M. Lesort, et al. (2002). "Mutant huntingtin aggregates do not
sensitize cells to apoptotic stressors." FEBS Lett515(1-3):
61-5.
It has been postulated that neuronal inclusions composed of mutant huntingtin
may play a causative role in the pathogenesis of Huntington's disease. To
study the putative role of aggregates in modulating apoptotic vulnerability,
SH-SY5Y cell lines stably expressing truncated huntingtin with 18 (wild-type)
(N63-18Q) or 82 (mutant) (N63-82Q) glutamine repeats were established.
Aggregates were observed in approximately 13% of the N63-82Q cells; no
aggregates were observed in the N63-18Q cells. In response to apoptotic
stimuli such as staurosporine or hyperosmotic stress, caspase-3 activity was
significantly greater in the N63-82Q cells compared to the N63-18Q cells.
However, double immunostaining for huntingtin and active caspase-3 revealed
that the presence of aggregates did not correlate with the presence of active
caspase-3, indicating that aggregates do not contribute to the increase in
apoptosis in the N63-82Q cells.
Chuang, J. Z., H. Zhou, et al. (2002). "Characterization of a brain-enriched
chaperone, MRJ, that inhibits Huntingtin aggregation and toxicity
independently." J Biol Chem.
Molecular chaperones are involved in a wide range of cellular events, such as
protein folding and oligomeric protein complex assembly. DnaK- and DnaJ-like
proteins are the two major classes of molecular chaperones in mammals. Recent
studies have shown that DnaJ-like family proteins can inhibit polyglutamine
aggregation, a hallmark of many neurodegenerative diseases including
Huntington disease. While most DnaJ-like proteins studied are ubiquitously
expressed, some have restricted expression, so it is possible that some
specific chaperones may affect polyglutamine aggregation in specific neurons.
In this report, we describe the isolation of a DnaJ-like protein MRJ and the
characterization of its chaperone activity. Tissue distribution studies showed
that MRJ is highly enriched in the central nervous system. In an in vitro cell
model of Huntington disease (HD), overexpressed MRJ effectively suppressed
polyglutamine-dependent protein aggregation, caspase activity, and cellular
toxicity. Collectively, these results suggest that MRJ has a relevant
functional role in neurons.
