Download Dysfunction of Wild-Type Huntingtin in Huntington

Dysfunction of Wild-Type Huntingtin in
Huntington disease
Elena Cattaneo
Department of Pharmacological Sciences and Center for Excellence in Neurodegenerative Diseases, University of Milano, 20133 Milano, Italy
Huntingtin is the protein involved in Huntington disease (HD), an inherited neurodegenerative disease. Research activities have focused on the abnormal functions of mutant huntingtin. However,
recent results indicate that wild-type huntingtin has important activities in brain neurons, suggesting that loss of these activities may play a role in HD.
untingtin is a 348-kDa cytoplasmic protein that is widely
expressed in the body (15). Interest in this protein stems
from the demonstration that an aberrantly expanded repetition
of a CAG nucleotide triplet into the huntingtin gene causes
Huntington disease (HD), a neurodegenerative disease that
hits at a mean age of 35 years. HD is greatly disabling and
progressive, leading to death within 1520 years (7).
Despite the wide pattern of expression of wild-type and
mutant huntingtin, selective degeneration of the mediumsized spiny neurons of the striatum and, at later stages of the
disease, of the neurons of the cerebral cortex represents the
typical neuropathological hallmark of the disease. Due to this
peculiar pattern of neurodegeneration, HD is characterized
primarily by motor dysfunctions (“chorea,” which in Greek
means “dance”) and cognitive and behavioral disturbances.
These neuropathological and clinical manifestations are evident when the number of CAG repetitions in the huntingtin
gene exceeds the limit of 35 that are present in the normal
population (15). Repeats above 36 define an HD allele. CAG
translates into glutamine (Gln), and the resulting mutant huntingtin protein is therefore characterized by an expanded polyGln stretch. There is an inverse relationship between CAG
repeat number and age of disease onset; the greater the number of CAG repeats, the earlier the age of onset, with cases of
juvenile onset of HD characterized by expansions of >60
repeats (15). Age of disease onset is, however, not only influenced by the number of CAG repeats but also by other modulating factors. For example, variations in the kainate-specific
glutamate receptor GluR6 genotype have been shown to
account for some of the fluctuations in age of onset of HD not
accounted for by CAG repeat length (14).
The gained toxic activity of the mutant huntingtin protein
The current hypothesis in HD is that the disease arises from
a gained toxic activity of the mutant huntingtin protein. The
first evidence against a simple loss of normal huntingtin function in HD was derived from the demonstration that deletion
of a large portion of chromosome 4, which includes the IT15
locus, does not cause HD (1). Yet most patients with this deletion do not survive long enough to develop HD. Additional
evidence obtained in animal models indicates that expression
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of mutant huntingtin in the background of a mouse having two
normal copies of the wild-type huntingtin gene does cause
neurological dysfunction and aggregate formation (13, 15).
Although these observations support the notion that the
mutant protein, or the CAG per se, initiates the disease, they
do not completely rule out the possibility of a loss of normal
huntingtin activity in HD (1).
Since the cloning of the HD gene in 1993 (7), most of the
efforts toward understanding the pathogenic mechanisms
were driven by the gain-of-function hypothesis and focused
on the analyses of the mutant protein. A striking feature,
shared by mutant huntingtin and other proteins involved in
other CAG repeat diseases, is their tendency to form insoluble
aggregates either through “polar zippers” or by transglutaminase-mediated events (15). Such aggregates, composed of
NH2-terminal mutant huntingtin, accumulate in striatal and
cortical neurons of HD patients both in the nucleus and in
dystrophic neurites (15). Aggregates of mutant huntingtin are
also common to many transgenic mouse models of HD,
although description of their nuclear localization in striatal
neurons varies greatly depending on the mouse model (15).
The role of these aggregates in the disease is still debated.
Indeed, cell death seems to occur in mice and cultured cells
in the absence of visible aggregates, and conversely the presence of aggregates does not always cause cell death (17).
Therefore toxicity of mutant huntingtin is not conclusively
shown to be mediated by its aggregation, and a deeper understanding of what mutant huntingtin inclusions do inside the
cells remains crucial.
One important set of data indicates that mutant huntingtin
impairs gene transcription, either by intranuclear aggregate
formation and/or by sequestration of important transcription
factors (2). Various authors demonstrated that transcription
factors such as CBP [cAMP-response element binding protein
(CREB) binding protein], TATA binding protein, and Sin3A can
be recruited into the intranuclear aggregates, thus reinforcing
the hypothesis of a role for transcriptional dysregulation in HD
(15). Cha and other colleagues (9, 10, 16, 19) were the first to
suggest this possibility and initiate more through investigations
aimed at examining the early changes in gene expression in
mice and cells by gene chip analyses. This large body of evidence indicates that the presence of mutant huntingtin is associated with a reduced transcription of several important genes.
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FIGURE 1. Huntingtin, the key player. Some of the activities of huntingtin and its interactions with key cellular elements involved in apoptotic cascades are summarized. Htt, huntingtin; MLK2, mixed-lineage kinase 2; JNK, c-Jun NH2-terminal kinase; C9, caspase-9; C8, caspase-8; C7, caspase-7; C6, caspase-6; pro-C9,
procaspase-9; Cyt.C, cytochrome c; BDNF, brain-derived neurotrophic factor. Modified from Ref. 15 with permission.
In addition, it was shown that the poly-Gln-containing
domain of huntingtin directly binds to two distinct proteins,
CBP and p300/CBP-associated factor (P/CAF) and inhibits
their acetyltransferase activity, anticipating the possibility of a
reduced histone acetylation as demonstrated in vitro (18).
Importantly, this reduction could be reversed by administration of inhibitors of histone deacetylase (18). Sequestration of
CBP by elongated poly-Gln tracts leading to altered gene transcription and cell toxicity is also invoked in another beautiful
study by Nucifora (10).
Additional observations in favor of the gain-of-function
hypothesis indicate that huntingtin is a substrate for caspases.
Huntingtin cleavage by caspases occurs in a poly-Gln-dependent manner, i.e., the longer the poly-Gln in huntingtin, the
more efficient is the cleavage, which then generates NH2-terminal fragments of mutant huntingtin endowed with cell toxicity (6, 15). An alternative hypothesis is suggested by a recent
study demonstrating that mutant huntingtin from human brain,
transgenic animals, and cells is more resistant to proteolysis
than normal huntingtin (4). This study proposes that inhibition
of mutant huntingtin proteolysis leads to aggregation and toxicity through sequestration of important targets, including normal huntingtin (4).
Most of these data indicate that mutant huntingtin toxicity
in HD occurs via the formation of NH2-terminal fragments
that aggregate. However, it is unclear how these events lead to
striatal vulnerability. One study has compared the patterns of
inclusion formation in HD transgenic mice expressing only
exon 1-containing expanded repeats with those in knock-in
mice expressing full-length mutant huntingtin. Although the
former develop inclusions in most brain regions, the latter
develop inclusions only in striatum (8). Although inclusion
formation is not a direct measure of cell dysfunction or death,
these data seem to indicate that tissue-specific proteolysis of
mutant huntingtin could determine which cell population may
be affected. Yet these data indicating a preferential cleavage of
mutant huntingtin are in contrast with data by Dyer (4) showing preferential cleavage of wild-type huntingtin.
Physiology of wild-type huntingtin
In a research context mostly focused on the analyses of
mutant huntingtin toxicity, interest in the function of the normal protein has been very scant. However, new investigations,
some of which were performed in our laboratory, indicate that
wild-type huntingtin has important roles in striatal neurons,
implying that loss of normal huntingtin activity may occur in
the disease, leading to striatal vulnerability (20).
Distribution of wild-type huntingtin has been analyzed by
various groups that demonstrated its presence in all brain neurons. These studies also showed the lack of any correlation
between huntingtin expression and neuronal vulnerability
(15). More recently, variable expression of huntingtin in brain
regions was reported. In particular, one study showed low
expression of wild-type huntingtin in the striatal neurons. In
contrast, cortical neurons were found to be rich in huntingtin
protein and mRNA (15). Huntingtin is mostly cytoplasmic,
although some reports indicate its presence in the nucleus
(15). Of note, whereas the human huntingtin gene carries a
normal polymorphic CAG stretch ranging from 9 to 35
repeats, in all investigated species so far the number of CAG
found was equal or inferior to the normal range of human IT15
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FIGURE 2. Wild-type huntingtin increases the transcription of the BDNF gene
(left). BDNF is produced in cortex and anterogradely transported to the striatum via the corticostriatal afferents. Striatal neurons require cortical BDNF for
their survival. In Huntington disease (right), loss of wild-type huntingtin results
in a decreased production of cortical BDNF, leading to increased vulnerability of the striatal neurons.
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ing in inhibition of the apoptotic cascade (1, 12, 15) (Fig. 1).
Alternatively, the antiapoptotic effect of wild-type huntingtin
may occur via sequestration of proapoptotic molecules like
Hip1-Hippi, resulting in cell survival (5). Subsequent studies
demonstrated that wild-type huntingtin also protects against
apoptosis in the testis of mice expressing mutant huntingtin
and that overexpression of wild-type huntingtin significantly
reduces the cellular toxicity of mutant huntingtin in various
cell types (1, 13, 15).
These data suggest that one of the roles of ubiquitously
expressed wild-type huntingtin may include a protective
mechanism from various apoptotic stimuli. Huntingtin also
affects neuronal stability because its deletion in mice causes
degeneration of axon fibers (3). Various evidence has converged to indicate that wild-type huntingtin may be involved
in cellular vesicle trafficking, endocytosis, and synaptic function. Several proteins interacting with huntingtin are associated with membrane vesicles and interact with cytoskeletal
proteins (15). Huntingtin also codistributes with intracellular
and plasma membranes containing clathrins, and it appears to
be associated with clathrin through huntingtin-interacting proteins (HIP-1) (15). Together these data argue in favor of the
possibility that loss of wild-type huntingtin function may occur
in HD, therefore contributing to neuronal death.
Despite this large body of evidence, a mechanism explaining the selective striatal vulnerability in HD is still elusive. It is
worth mentioning that HD is one of eight inherited neurological diseases caused by the presence of a CAG expansion in
genes encoding for different proteins of known [like for the
androgen receptor involved in spinobulbar muscular atrophy
(SBMA)] or unknown function and characterized by a different pattern of neurodegeneration. In SBMA, for example, the
expanded CAG is found in the gene encoding for the androgen receptor and the degeneration specifically hits the motor
neurons. It is therefore possible that although it is the CAG per
se that evokes toxicity it is the context of the protein in which
the poly-Gln expansion is present that defines which neurons
will undergo cell death. In other words, we hypothesized that
wild-type huntingtin could have important physiological functions for the striatal neurons that were lost in HD. Interestingly,
we found that both full-length wild-type huntingtin and its
548-amino acid terminal (N548) were equally protective for
cells, whereas a shorter NH2 terminal (of 63 amino acids) had
no effect. This suggested that huntingtin may be organized into
several functional domains (12).
In search of striatal-specific functions of wild-type huntingtin that, when lost, could account for the selective neurodegeneration observed in the disease, we recently reported
that wild-type huntingtin upregulates the transcription of
brain-derived neurotrophic factor (BDNF) (20), a prosurvival
factor produced by cortical neurons and necessary for the survival of the striatal neurons in the brain. Cortical BDNF is
delivered to striatum via the corticostriatal afferents, known to
release glutamate and to control the striatal output. Our study
demonstrated that this beneficial activity of wild-type huntingtin is lost when huntingtin is mutated, resulting in
decreased production of cortical BDNF and leading to insufficient trophic support to the striatal neurons, which then die
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alleles. A longer uninterrupted CAG stretch was found in the
pig and consists of 21 CAG, whereas rat and mouse genes
have eight and seven repeats, respectively, the pufferfish has
only four repeats, and Drosophila does not have any (1, 11). It
is possible that increased CAG length during evolution has
endowed the normal protein with specific functions.
One of the first pieces of evidence about the function of
wild-type huntingtin was discovered in 1995, with the recognition that embryos of huntingtin homozygous knockout mice
die during gestation (1). This evidence indicated that wild-type
huntingtin is required for embryonic development. Of note, in
one constitutive knockout mouse, much higher apoptotic cell
death was observed in the embryonic ectoderm of null
mutants with respect to normal (1). Huntingtin is also important in adulthood since heterozygous knockout mice displayed increased motor activity and significant neuronal loss
in the subthalamic nucleus (1). One recently engineered
mouse has allowed more rigorous testing of the role of wildtype huntingtin in mature neurons by conditional inactivation.
It was found that depletion of endogenous wild-type huntingtin in postmitotic neurons results in neurological deficits
and neurodegeneration, indicating a function for the wild type
in the brain (3). Studies by our group (12) have provided the
first direct evidence for a role of huntingtin in the survival of
central nervous system cells. Central nervous system cells that
were engineered to express wild-type huntingtin were resistant to the lethal effect of stresses such as serum deprivation,
exposure to mitochondrial toxins, or transfection of proapoptotic Bcl-2 family proteins. In particular, we found that wildtype huntingtin acts as an antiapoptotic protein (12). We also
reported that the antiapoptotic effect of wild-type huntingtin
takes place upstream of caspase-3 activation. Specifically, in
our experimental conditions wild-type huntingtin inhibited
the conversion of procaspase-9 into active caspase-9, result-
(Fig. 2). We also reported that the ability of wild-type huntingtin to modulate BDNF gene transcription depends on a
specific activity of the BDNF promoter (20).
In conclusion, analyses of the physiology of wild-type huntingtin has disclosed unexpected information about its activity
and biology, also anticipating that strategies aimed at restoring
wild-type huntingtin functions may represent one more strategy to interfere with the pathogenic mechanisms underlying
HD.
The work conducted in our laboratory is supported by the Huntington’s
Disease Society of America, the Hereditary Disease Foundation, Telethon
(#840 Italy), and Ministero dell’Istruzione e della Ricerca Scientifica (Miur,
MM06278849-005).
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