Sci. Aging Knowl. Environ., 17 September 2003
Vol. 2003, Issue 37, p. pe26
[DOI: 10.1126/sageke.2003.37.pe26]


How Does the Huntington's Disease Mutation Damage Cells?

David C. Rubinsztein

The author is in the Department of Medical Genetics, Cambridge Institute for Medical Research, Wellcome/MRC Building, Addenbrooke's Hospital, Cambridge, CB2 2XY, UK. E-mail: dcr1000{at};2003/37/pe26

Key Words: polyglutamine expansion • huntingtin • neurodegeneration • Huntington's disease

Huntington's disease (HD) is a devastating autosomal dominant neurodegenerative condition that manifests with abnormal movements (including chorea, an involuntary, irregular, randomly distributed, and abrupt type of movement), cognitive deterioration, and psychiatric abnormalities [reviewed in (1)]. The disease typically presents between the ages of 35 and 50 years but can strike at any age. HD is caused by the expansion of a CAG trinucleotide repeat stretch (to >35 repeats) in the coding sequence of the HD gene. This mutation is translated into an abnormally long polyglutamine (polyQ) tract in the N-terminal region of the huntingtin protein, and the age at disease onset correlates inversely with the number of repeats. CAG/polyglutamine mutations cause eight other neurodegenerative diseases that show the same type of repeat-length/age-at-onset relationships. All of the known diseases caused by this class of mutation are characterized pathologically by intraneuronal aggregates (also known as inclusions) that contain the mutant protein and other cellular proteins. HD aggregates are seen in both the nucleus and the neuronal processes. The physiological effects of these aggregates have been the subject of ongoing debate, and a recent paper in Nature Genetics adds another important piece to this complex puzzle (2).

How does the polyglutamine mutation cause HD? Mutations can cause disease by loss-of-function, dominant negative, or gain-of-function mechanisms. Loss-of-function mutations cause phenotypes similar to those that would be caused by mRNA null mutations for the allele. Dominant negative mutations arise when the mutant allele interferes with the function of the remaining wild-type allele, causing a >50% loss of function. This can occur when the gene product forms active complexes with itself (and possibly with other proteins). In this scenario, a dominant mutation in the coding sequence that allows the synthesis of significant concentrations of mutant protein will produce a phenotype that is more severe but qualitatively similar to that produced by an mRNA null mutation. This situation is exemplified in the brittle bone disease osteogenesis imperfecta, which is caused by mutations in the COLA1 gene. Type I collagen is made up of two alpha 1 chains (which are encoded by COLA1) and one alpha 2 chain. Point mutations in one of the alpha 1 chains disrupt 75% of the collagen complexes, whereas mRNA null mutations result in the loss of only 50% of the complexes. However, if a dominant coding mutation causes a phenotype that is qualitatively different from that produced by a null mutation, or if such a mutation causes a more severe phenotype than does the complete absence of both allele products [as seen with the polyalanine expansion mutation in the HOXD13 gene associated with synpolydactyly (3)], then one can infer that the mutant allele is interfering with the functions of other gene products and thus constitutes a gain-of-function mutation.

HD is unlikely to be caused by a straight forward loss-of-function. In humans, hemizygous loss of one of the two wild-type huntingtin alleles has been observed as a result of either a terminal deletion of one copy of chromosome 4 (which includes the HD gene) in patients with Wolf-Hirschhorn syndrome (4), or of a balanced translocation with a break point between exons 40 and 41, physically disrupting one of the HD gene copies in one female (5). In these cases, hemizygous inactivation of one of the two huntingtin genes does not cause an HD phenotype. In addition, mice that have only one functioning HD gene do not show features of the disease (6-9). Thus, 50% loss of huntingtin function is not sufficient to cause HD.

A polyQ expansion in the androgen receptor gene causes spinobulbar muscular dystrophy (SBMA). This is an X-linked disease, so that only the mutant allele is expressed in affected male individuals. Mutations that inactivate the androgen receptor, including genomic deletions, do not result in the neurological features of SBMA but cause the testicular feminization of androgen insensitivity syndrome [reviewed in (10)]. This suggests that the polyQ mutation in this gene causes neurological disease by a gain-of-function. However, patients with SBMA do show mild feminization; thus, it is possible that some loss of function may occur in other polyQ diseases that could enhance the gain-of-function toxicity of these mutations, even if the loss of function is neither necessary nor sufficient to cause disease.

A gain-of-function mechanism is supported by a mouse model in which a 146 CAG repeat sequence was inserted into the hypoxanthine phosphoribosyltransferase (HPRT) gene, which is not involved in any of the known CAG repeat disorders (11). While previous work had shown that inactivation of the HPRT gene in mice does not cause deleterious effects, these mutant mice produced a polyglutamine-expanded form of the HPRT protein and developed a late-onset neurological phenotype that progressed to premature death. These data indicate that the primary effect of the HD mutation is a toxic gain of function. This hypothesis is consistent with the HD-like phenotypes seen in a number of different HD transgenic mouse models that express either the entire mutant HD gene or mutated N-terminal fragments of the gene, in addition to the endogenous murine huntingtin orthologs [reviewed in (1)].

The recent paper from the Cattaneo lab raises the possibility that HD may not be caused only by gain-of-function effects. This study complements an earlier one from the same group. These researchers reported that brain-derived neurotrophic factor (BDNF) concentrations are lower in cells overexpressing a mutant HD gene product (in addition to endogenous wild-type huntingtin), as compared to wild-type cells, but that overexpression of a wild-type huntingtin transgene resulted in elevated concentrations of BDNF (12). Their more recent paper provides part of the molecular explanation for their initial observation. They showed that wild-type huntingtin (a largely cytosolic protein) binds to and sequesters in the cytoplasm a negatively acting transcription factor called neuron restrictive silencer factor (NRSF) (2). This sequestration prevents NRSF from entering the nucleus, where it would bind to NRSF-binding sites (NRS elements) in certain gene promoters (such as the BDNF gene) and repress their transcription. Mutant huntingtin overexpression results in a shift of NRSF localization to the nucleus, as compared to untransfected wild-type cells; however, the mechanism for this is not clear. They also confirmed the prediction that wild-type huntingtin overexpression in transgenic mice induces the expression of genes normally inhibited by NRSF, while transgenic mice overexpressing mutant huntingtin show a reduction in the expression of these genes in comparison to wild-type type littermates. In this situation, the wild-type protein interferes with NRSF function by sequestering it in the cytoplasm. In contrast, the mutant protein somehow allows more NRSF to exist in the nucleus and repress transcription than in wild-type nontransfected cells. These findings suggest that mutant huntingtin blocks the function of the endogenous wild-type huntingtin: a dominant negative model (Fig. 1).

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Fig. 1. Schematic representation of the different effects on a model pathway that would be observed if one overexpresses either a wild-type protein that acts positively on the pathway or mutant proteins with dominant negative or loss-of-function mutations.

Additional evidence for dominant negative effects in HD may be found in the study of huntingtin interacting protein-1 (HIP-1) (13). HIP-1 also binds to HIPPI (HIP-1 protein interactor), and this heterodimer can recruit procaspase-8 into a HIP-1/HIPPI/procaspase 8 complex and launch apoptosis by activating caspase 8. Gervais et al. (13) observed that overexpression of wild-type huntingtin (in addition to the cell's endogenous wild-type huntingtin) reduces the availability of free HIP-1, as compared to wild-type cells, and suppresses apoptosis by making less HIP-1 available to form complexes with HIPPI and procaspase 8. Overexpression of mutant huntingtin, however, increases the availability of free HIP-1, as compared to wild-type cells, which would predict the formation of more proapoptotic HIP-1/HIPPI/procaspase 8 complexes (13). This observation creates a possible paradox. Previous work from Hayden and colleagues showed that mutant huntingtin does bind to HIP-1, although the increased polyglutamine length in mutant huntingtin reduces its binding as compared to wild-type huntingtin (14). Thus, one would expect overexpression of mutant huntingtin to show a partial loss of function as compared to wild-type huntingtin. Mutant huntingtin would be expected to reduce the availability of free HIP-1, as compared to wild-type untransfected cells, but have relatively more HIP-1 available as compared to cells overexpressing the wild-type constructs; that is, unless mutant huntingtin is acting in a dominant negative fashion (Fig. 1).

In addition to raising the possibility that dominant negative processes may contribute to HD (on top of gain-of-function toxicity), the study of Cattaneo et al. (2) also highlights a new transcriptional pathway that may be impaired in HD. This pathway is independent from other transcriptional pathways shown to be affected in HD. In previous studies, perturbations were observed in the expression of genes regulated by CBP, CREB, Sp1, and certain other transcription factors and cofactors [reviewed in (15)]. A number of other abnormalities have been reported in HD, including impaired proteasome function (16), abnormal calcium signaling (17), caspase activation (18), and excitotoxicity (19). A crucial question raised by these new findings is whether the polyglutamine mutation causes disease by influencing one key pathway early in the disease process, and most of the diverse changes observed in HD are secondary consequences of effects on this pathway; or whether the HD mutation can induce many parallel, but not necessarily temporally synchronous, pathway lesions (Fig. 2). Although the answer to this question is unclear at present, I believe that the latter model deserves serious consideration. Many loss-of-function mutations show phenotypes that can be attributed to the reduction of a number of distinct molecular functions of the relevant gene, because many genes have multiple cellular roles. Similarly, one would expect that many gain-of-function mutations would have multiple physiological consequences.

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Fig. 2. Schematic representation of the two extreme models of HD pathogenesis. In the top model, the polyglutamine mutation causes disease by influencing one key pathway early in the disease process, and most of the diverse observed changes are secondary consequences of this effect. Pictured in the lower schematic is a scenario where the HD mutation induces many parallel, but not necessary temporally synchronous, pathway lesions.

In HD, the mutation is likely to exert most, if not all, of its effects at the protein level. However, we cannot discount effects at the DNA or mRNA levels; for instance, the expanded CUG repeats in mRNA encoding the myotonic dystrophy gene (DMPK) bind to and alter the activities of CUG-binding proteins, perturbing alternative splicing of other transcripts. Such hypothetical effects may contribute to pathology even if they are not sufficiently deleterious to cause disease by themselves. Because the polyQ expansion makes the huntingtin protein sticky and also changes the conformation of the mutant protein at sites adjacent to the repeats (20), one may expect multiple proteins to interact abnormally with mutant huntingtin. These interactions could occur at the level of both the soluble protein and the aggregated/aggregating protein. A proportion of these abnormal interactions will result in large enough changes in the concentrations and activities of the binding partners to influences their functions on a cellular level.

Mutant huntingtin fragments, which result from cleavage of the full-length protein, can interact and coaggregate with wild-type huntingtin fragments (21, 22) and possibly also with the full-length, wild-type huntingtin protein. Therefore, such interactions may provide a mechanism for a dominant negative process, whereas interactions with other proteins would result in a gain of function. Another possibility that could account for dominant negative effects is that that the polyQ mutation may induce enhanced proteolytic cleavage of huntingtin (both the wild-type and mutant gene products). Aggregates could also contribute to pathology by acting as space-occupying lesions; for instance, by blocking transport in neuronal processes. These mechanisms can account for a model for polyQ pathogenesis that invokes multiple parallel pathway disturbances. Thus, many of the pathways described as playing a role in HD models to date may impact on the disease independently. It is possible that different combinations of pathways are induced by the polyQ mutation in different cell types. If the HD mutation does induce multiple parallel pathways, then therapies that aim to alleviate single pathways perturbed in such diseases may be much less effective than combination therapies that attack multiple independent pathways or strategies that selectively remove the mutant mRNA/protein (23).

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Citation: D. C. Rubinsztein, How Does the Huntington's Disease Mutation Damage Cells? Sci. SAGE KE 2003 (37), pe26 (2003).

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