1,721,040 research outputs found
Mutant huntingtin forms in vivo complexes with distinct context-dependent conformations of the polyglutamine segment
No full textHuntington's disease (HD) is caused by an expanded glutamine tract, which confers
a novel aggregation-promoting property on the 350-kDa huntingtin protein. Using
specific antibodies, we have probed the structure of the polyglutamine segment in
mutant huntingtin complexes formed in cell culture from either truncated or
full-length protein. Complexes formed by a mutant amino terminal fragment most
frequently entail a change in conformation that eliminates reactivity with the
polyglutamine-specific mAb 1F8, coincident with production of insoluble
aggregate. By contrast, complexes formed by the full-length mutant protein remain
soluble and are invariably 1F8-reactive, indicating a soluble polyglutamine
conformation. Therefore, aggregates in HD may form by different biochemical
mechanisms that invoke different possibilities for the pathogenic process. If
pathogenesis is triggered by a truncated fragment, it probably involves the
formation of an insoluble aggregate. However, the observation of soluble
complexes in which an HD-specific pathogenic conformation of the glutamine tract
remains accessible suggests that pathogenesis could also be triggered at the
level of full-length huntingtin by abnormal aggregation with normal or abnormal
protein partners
Amyloid formation by mutant huntingtin: threshold, progressivity and recruitment of normal polyglutamine proteins
No full textHuntington's disease (HD) is caused by an expanded CAG trinucleotide repeat
encoding a tract of consecutive glutamines near the amino terminus of huntingtin,
a large protein of unknown function. It has been proposed that the expanded
polyglutamine stretch confers a new property on huntingtin and thereby causes
cell and region-specific neurodegeneration. Genotype-phenotype correlations
predict that this novel property appears above a threshold length (approximately
38 glutamines), becomes progressively more evident with increasing polyglutamine
length, is completely dominant over normal huntingtin and is not appreciably
worsened by a double genetic dose in HD homozygotes. Recently, an amino terminal
fragment of mutant huntingtin has been found to form self-initiated fibrillar
aggregates in vitro. We have tested the capacity for aggregation to assess
whether this property matches the criteria expected for a fundamental role in HD
pathogenesis. We find that that in vitro aggregation displays a threshold and
progressivity for polyglutamine length remarkably similar to the HD disease
process. Moreover, the mutant huntingtin amino terminus is capable of recruiting
into aggregates normal glutamine tract proteins, such as the amino terminal
segments of both normal huntingtin and of TATA-binding protein (TBP). Our
examination of in vivo aggregates from HD post-mortem brains indicates that they
contain an amino terminal segment of huntingtin of between 179 and 595 residues.
They also contain non-huntingtin protein, as evidenced by immunostaining for TBP.
Interestingly, like the in vitro aggregates, aggregates from HD brain display
Congo red staining with green birefringence characteristic of amyloid. Our data
support the view that the expanded polyglutamine segment confers on huntingtin a
new property that plays a determining role in HD pathogenesis and could be a
target for treatment. Moreover, the new property might have its toxic
consequences by interaction with one or more normal polyglutamine-containing
proteins essential for the survival of target neurons
Huntingtin immunoreactivity in the rat neostriatum: differential accumulation in projection and interneurons
No full textHuntington's disease is caused by a mutation of the gene encoding the protein
huntingtin. Features of the human disease, characterized by selective loss of
neurons from the neostriatum, can be replicated in rodents by administration of
excitotoxins. In both affected individuals and the rodent model, there is massive
loss of striatal projection neurons with selective sparing of interneurons.
Furthermore, in the human disease the earliest evidence of striatal injury is
found in striosomal regions of the striatum. The mRNA encoding huntingtin is
known to be expressed by neurons throughout the brain, a distribution which does
not account for the selective patterns of neuronal death which are observed.
Using fluorescence immunocytochemistry and confocal microscopy with an antibody
to huntingtin, we have observed that in rats a subset of striatal projection
neurons contains dense accumulations of huntingtin immunoreactivity (HT-ir),
while most neurons in the striatum contain much smaller amounts. The intensely
stained neurons are concentrated within the striatal striosomes, as defined by
calbindin-D28K staining. In the matrix regions, relatively few neurons contain
dense accumulations of HT-ir, and these cells always lack perikaryal staining for
calbindin-D28K. Striatal interneurons, identified by the presence of
immunoreactivity for choline acetyltransferase, parvalbumin, calretinin, or
neuronal nitric oxide synthase, exhibit little or no HT-ir. The paucity of HT-ir
in striatal interneurons, as well as the prominence of staining in a subset of
striosomal neurons, mirrors the selective vulnerability of these different types
of cells in early stages of human Huntington's disease and in rodent excitotoxic
models of the disorder. Our observations suggest that mechanisms which modulate
the accumulation of huntingtin may play a central role in the neuronal
degeneration of Huntington's disease
Specific progressive cAMP reduction implicates energy deficit in presymptomatic Huntington's disease knock-in mice
No full textDefects in gene transcription and mitochondrial function have been implicated in
the dominant disease process that leads to the loss of striatal neurons in
Huntington's disease (HD). Here we have used precise genetic HD mouse and
striatal cell models to investigate the hypothesis that decreased cAMP responsive
element (CRE)-mediated gene transcription may reflect impaired energy metabolism.
We found that reduced CRE-signaling in Hdh(Q111) striatum, monitored by brain
derived neurotrophic factor and phospho-CRE binding protein (CREB), predated
inclusion formation. Furthermore, cAMP levels in Hdh(Q111) striatum declined from
an early age (10 weeks), and cAMP was significantly decreased in HD postmortem
brain and lymphoblastoid cells, attesting to a chronic deficit in man. Reduced
CRE-signaling in cultured STHdh(Q111) striatal cells was associated with
cytosolic CREB binding protein that mirrored diminished cAMP synthesis. Moreover,
mutant cells exhibited mitochondrial respiratory chain impairment, evidenced by
decreased ATP and ATP/ADP ratio, impaired MTT conversion and heightened
sensitivity to 3-nitropropionic acid. Thus, our findings strongly suggest that
impaired ATP synthesis and diminished cAMP levels amplify the early HD disease
cascade by decreasing CRE-regulated gene transcription and altering energy
dependent processes essential to neuronal cell survival
Heterogeneous topographic and cellular distribution of huntingtin expression in the normal human neostriatum
No full textA striking heterogeneous distribution of topographic and cellular huntingtin
immunoreactivity was observed within the human neostriatum using three distinct
huntingtin antibodies. Patchy areas of low huntingtin immunoreactivity were
present in both the caudate nucleus and putamen, surrounded by an intervening
area of greater immunoreactivity. Comparison of huntingtin immunoreactivity with
contiguous serial sections stained for enkephalin and calbindin D28k
immunoreactivities showed that the topographic heterogeneity of huntingtin
immunostaining corresponded to the patch (striosome) and matrix compartments
within the striatum. Huntingtin immunoreactivity was confined primarily to
neurons and neuropil within the matrix compartment, whereas little or no neuronal
or neuropil huntingtin immunostaining was observed within the patch compartment.
There was marked variability in the intensity of huntingtin immunolabel among
medium-sized striatal neurons, whereas a majority of large striatal neurons were
only faintly positive or without any immunoreactivity. Combined techniques for
NADPH-diaphorase enzyme histochemistry and huntingtin immunocytochemistry, as
well as double immunofluorescence for either nitric oxide synthase or calbindin
D28k in comparison with huntingtin expression, revealed a striking correspondence
between calbindin D28k and huntingtin immunoreactivities, with little or no
colocalization between NADPH-diaphorase or nitric oxide synthase neurons and
huntingtin expression. These observations suggest that the selective
vulnerability of spiny striatal neurons and the matrix compartment observed in
Huntington's disease is associated with higher levels of huntingtin expression,
whereas the relative resistance of large and medium-sized aspiny neurons and the
patch compartments to degeneration is associated with low levels of huntingtin
expression
The mouse Huntington’s disease gene homologue (Hdh)
No full textThe incurable neurodegenerative disorder, Huntington's disease (HD), is caused by
an expanded, unstable CAG repeat encoding a stretch of polyglutamine in a 4p16.3
gene (HD) of unknown function. Near the CAG repeat is a polyproline-encoding CCG
repeat that shows more limited allelic variation. The mouse homologue, Hdh, has
been mapped to chromosome 5, in a region devoid of mutations causing any
comparable phenotype. We have isolated overlapping cDNAs from the Hdh gene and
compared their sequences with the human transcript. The consensus mouse coding
sequence is 86% identical to the human at the DNA level and 91% identical at the
protein level. Despite the overall high level of conservation, Hdh possesses an
imperfect CAG repeat encoding only seven consecutive glutamines, compared to the
13-36 residues that are normal in man. Although no evidence for polymorphic
variation of the CAG repeat was seen, a nearby CCG repeat differed in length by
one unit between several strains of laboratory mouse and Mus spretus. The absence
of a long CAG repeat in the mouse is consistent with the lack of a spontaneous
mouse model of HD. The information presented concerning the sequence of the mouse
gene should facilitate attempts to create such a model
Differential expression of normal and mutant Huntington’s disease gene alleles
No full textHuntingtin expression was examined by Western blot and immunoprecipitation
studies of lymphoblastoid cell lines from Huntington's disease (HD) homozygotes,
heterozygotes, and a phenotypically normal individual with a t(4p16.3;12p13.3)
breakpoint in the HD gene. The latter produced a reduced level of normal
huntingtin without evidence of an altered protein, indicating that simple loss of
huntingtin activity does not cause HD. In juvenile onset HD heterozygotes, NH2-
and COOH-terminal antisera revealed reduced relative expression from the mutant
allele. Pulse-chase studies indicated that huntingtin is a stable protein whose
differential allelic expression is not due to destabilization of the mutant
isoform. No stable breakdown products specific to mutant huntingtin were detected
in either HD homozygotes or heterozygotes. These data are consistent with HD
involving either a gain of function or a dominant negative loss of function that
operates within severe constraints and suggest that in either case the pathogenic
process is usually saturated by the amount of abnormal huntingtin produced from a
single mutant allele
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