Full-length Mutant Huntingtin Research Articles

Huntingtin Expression Stimulates Endosomal–Lysosomal Activity, Endosome Tubulation, and Autophagy

An expansion of polyglutamines in the N terminus of huntingtin causes Huntington's disease (HD) and results in the accrual of mutant protein in the nucleus and cytoplasm of affected neurons. How mutant huntingtin causes neurons to die is unclear, but some recent observations suggest that an autophagic process may occur. We showed previously that huntingtin markedly accumulates in endosomal-lysosomal organelles of affected HD neurons and, when exogenously expressed in clonal striatal neurons, huntingtin appears in cytoplasmic vacuoles causing cells to shrink. Here we show that the huntingtin-enriched cytoplasmic vacuoles formed in vitro internalized the lysosomal enzyme cathepsin D in proportion to the polyglutamine-length in huntingtin. Huntingtin-labeled vacuoles displayed the ultrastructural features of early and late autophagosomes (autolysosomes), had little or no overlap with ubiquitin, proteasome, and heat shock protein 70/heat shock cognate 70 immunoreactivities, and altered the arrangement of Golgi membranes, mitochondria, and nuclear membranes. Neurons with excess cytoplasmic huntingtin also exhibited increased tubulation of endosomal membranes. Exogenously expressed human full-length wild-type and mutant huntingtin codistributed with endogenous mouse huntingtin in soluble and membrane fractions, whereas human N-terminal huntingtin products were found only in membrane fractions that contained lysosomal organelles. We speculate that mutant huntingtin accumulation in HD activates the endosomal-lysosomal system, which contributes to huntingtin proteolysis and to an autophagic process of cell death.

Huntingtin's WW domain partners in Huntington's disease post-mortem brain fulfill genetic criteria for direct involvement in Huntington's disease pathogenesis.

An elongated glutamine tract in mutant huntingtin initiates Huntington's disease (HD) pathogenesis via a novel structural property that displays neuronal selectivity, glutamine progressivity and dominance over the normal protein based on genetic criteria. As this mechanism is likely to involve a deleterious protein interaction, we have assessed the major class of huntingtin interactors comprising three WW domain proteins. These are revealed to be related spliceosome proteins (HYPA/FBP-11 and HYPC) and a transcription factor (HYPB) that implicate huntingtin in mRNA biogenesis. In HD post-mortem brain, specific antibody reagents detect each partner in HD target neurons, in association with disease-related N-terminal morphologic deposits but not with filter trapped insoluble-aggregate. Glutathione S:-transferase partner 'pull-down' assays reveal soluble, aberrantly migrating, forms of full-length mutant huntingtin specific to HD target tissue. Importantly, these novel mutant species exhibit exaggerated WW domain binding that abrogates partner association with other huntingtin isoforms. Thus, each WW domain partner's association with huntingtin fulfills HD genetic criteria, supporting a direct role in pathogenesis. Our findings indicate that modification of mutant huntingtin in target neurons may promote an abnormal interaction with one, or all, of huntingtin's WW domain partners, perhaps altering ribonucleoprotein function with toxic consequences.

Early kynurenergic impairment in Huntington's Disease and in a transgenic animal model

Several neuroactive metabolites of the kynurenine pathway of tryptophan degradation have been speculatively linked to the pathophysiology of Huntington's Disease (HD). Here we demonstrate that the levels of two of these metabolites, the free radical generator 3-hydroxykynurenine (3HK) and the neuroprotectant kynurenate (KYNA), are increased in the neostriatum of stage 1 HD patients and in the brain of mice transgenic for full-length mutant huntingtin. In both cases, the elevation in 3HK was far more pronounced, resulting in significant increases in the 3HK/KYNA ratios. These data suggest that abnormal kynurenine pathway metabolism may play a role during the early phases of the neurodegenerative process in HD.

Modelling Huntington’s disease

Huntington’s disease (HD) is one of a group of inherited neurodegenerative disorders caused by a polyglutamine (CAG)-repeat expansion. The pathology of these disorders is associated with the presence of neuronal intranuclear inclusions, which contain the polyglutamine- repeat-containing protein that is specific to the disease, along with ubiquitin. Sathasivam et al.1xFormation of polyglutamine inclusions in non-CNS tissue. Sathasivam, K. et al. Hum. Mol. Genet. 1999; 8: 813–822Crossref | PubMedSee all References1 report the formation of nuclear inclusions (NIs) in non-neuronal tissue in previously described transgenic mice that express exon 1 of the human HD gene that contains a highly expanded CAG repeat. In this model, NIs were found in several tissues, including cardiac and skeletal muscle, liver, kidney, adrenal gland and pancreas. They report a correlation between the presence of NIs and a progressive decrease in organ size. It is not known whether similar non-neuronal NIs occur in human HD patients, and as such the relevance of this aspect of the model is unclear. Despite this, the presence of NIs in tissues such as skeletal muscle provides a good system to study NIs and to investigate ways to prevent their formation.Although the association of NIs with HD is well documented, their role in the pathology of the disease remains unproven, and an unknown property of the expanded polyglutamine repeat that is independent of NI formation might underlie certain aspects of the disease. A new mouse model, described by Shelbourne et al.2xA Huntington’s disease CAG expansion at the murine Hdh locus is unstable and associated with behavioural abnormalities in mice. Shelbourne, P.F. et al. Hum. Mol. Genet. 1999; 8: 763–774Crossref | PubMedSee all References2, provides a valuable tool to study this possibility. This mouse has a stretch of 72–80 CAG repeats inserted into the endogenous Hdh gene. This ‘knockin’ mutation is similar to the human HD mutation as the mice express full-length mutant huntingtin in a wild-type genomic context. From a genetic viewpoint, this model appears to closely mirror the human HD mutation. As in humans, the expanded CAG repeat is unstable and a parent-of-origin effect on the repeat length is observed, with expansions tending to be associated with paternal transmission and contractions being associated with maternal transmission. The brains of the mutant mice are histologically normal with no evidence of NI formation. However, a reduction in brain size is seen in the mutant strains. The mutant mice also displayed chronic aggressive behaviour; human HD patients often exhibit symptoms such as irritability and aggression and ∼50% of patients present with psychiatric symptoms. These mice might therefore provide a model for the psychiatric disorder in HD.The knockin model was used to study the possible basis of cognitive impairment in HD (Ref. 3xImpaired synaptic plasticity in mice carrying the Huntington’s disease mutation. Usdin, M.T. et al. Hum. Mol. Genet. 1999; 8: 839–846Crossref | PubMedSee all ReferencesRef. 3). Long-term potentiation (LTP) is the sustained increase in synaptic strength following a high-frequency conditioning stimulus. LTP provides a model for a mechanism that underlies some forms of learning and memory. LTP was significantly reduced in the stratum radiatum of area CA1 of hippocampal slices from the mutant mice. Post-tetanic potentiation and paired-pulse facilitation were also shown to be impaired, suggesting that the presynaptic terminals might be less able to sustain neurotransmitter release with repetitive stimulation. This was tested by monitoring the opening of N-methyl-d-aspartate (NMDA) channels by glutamate release. Glutamatergic synapses in area CA1 were shown to transmit normally in response to single stimuli but were impaired in their ability to release neurotransmitter at moderate levels of stimulation. The authors propose that the polyglutamine expansion might interfere with neurotransmitter release at the presynaptic terminals. This hypothesis is consistent with previous reports of wild-type HD protein being associated with microtubules and synaptic vesicles.This new mouse model provides evidence that some of the symptoms of HD might be independent of NI formation and might precede neurodegeneration. The two mouse models provide insights into distinct aspects of HD; taken together they should complement each other to facilitate research into the molecular basis and treatment of this disease.

A YAC mouse model for Huntington's disease with full-length mutant huntingtin, cytoplasmic toxicity, and selective striatal neurodegeneration.

We have produced yeast artificial chromosome (YAC) transgenic mice expressing normal (YAC18) and mutant (YAC46 and YAC72) huntingtin (htt) in a developmental and tissue-specific manner identical to that observed in Huntington's disease (HD). YAC46 and YAC72 mice show early electrophysiological abnormalities, indicating cytoplasmic dysfunction prior to observed nuclear inclusions or neurodegeneration. By 12 months of age, YAC72 mice have a selective degeneration of medium spiny neurons in the lateral striatum associated with the translocation of N-terminal htt fragments to the nucleus. Neurodegeneration can be present in the absence of macro- or microaggregates, clearly showing that aggregates are not essential to initiation of neuronal death. These mice demonstrate that initial neuronal cytoplasmic toxicity is followed by cleavage of htt, nuclear translocation of htt N-terminal fragments, and selective neurodegeneration.

Mutant Huntingtin Expression in Clonal Striatal Cells: Dissociation of Inclusion Formation and Neuronal Survival by Caspase Inhibition

Neuronal intranuclear inclusions are found in the brains of patients with Huntington's disease and form from the polyglutamine-expanded N-terminal region of mutant huntingtin. To explore the properties of inclusions and their involvement in cell death, mouse clonal striatal cells were transiently transfected with truncated and full-length human wild-type and mutant huntingtin cDNAs. Both normal and mutant proteins localized in the cytoplasm, and infrequently, in dispersed and perinuclear vacuoles. Only mutant huntingtin formed nuclear and cytoplasmic inclusions, which increased with polyglutamine expansion and with time after transfection. Nuclear inclusions contained primarily cleaved N-terminal products, whereas cytoplasmic inclusions contained cleaved and larger intact proteins. Cells with wild-type or mutant protein had distinct apoptotic features (membrane blebbing, shrinkage, cellular fragmentation), but those with mutant huntingtin generated the most cell fragments (apoptotic bodies). The caspase inhibitor Z-VAD-FMK significantly increased cell survival but did not diminish nuclear and cytoplasmic inclusions. In contrast, Z-DEVD-FMK significantly reduced nuclear and cytoplasmic inclusions but did not increase survival. A series of N-terminal products was formed from truncated normal and mutant proteins and from full-length mutant huntingtin but not from full-length wild-type huntingtin. One prominent N-terminal product was blocked by Z-VAD-FMK. In summary, the formation of inclusions in clonal striatal cells corresponds to that seen in the HD brain and is separable from events that regulate cell death. N-terminal cleavage may be linked to mutant huntingtin's role in cell death.

Protein Fate in Neurodegenerative Proteinopathies: Polyglutamine Diseases Join the (Mis)Fold

Protein misfolding is now recognized to be a central feature of neurodegenerative diseases. Alzheimer disease (AD), Parkinson disease (PD), amyotrophic lateral sclerosis (ALS), prion diseases, and the polyglutamine (polygln) diseases all represent proteinopathies—diseases in which a particular protein or set of proteins misfolds and aggregates. In the past decade, mutations have been identified in an increasing number of genes underlying specific forms of neurodegeneration—for example, genes encoding amyloid precursor protein (APP) and presenilins 1 and 2 in AD, parkin and alpha-synuclein in PD, Cu/Zn superoxide dismutase in ALS, tau in frontotemporal dementia, prion protein in Creutzfeldt-Jacob and Gerstmann-Straussler diseases, and at least eight different proteins in the polygln diseases (Hardy and GwinnHardy 1998; Price et al. 1998). Identification of these mutations was a first step in understanding the disease process; next, it will be necessary to explain how specific changes in disease proteins may alter the neuron’s physiology and to further question how neuronal responses to the mutant protein influence the disease process. This review examines the fate of mutant protein in neurons and the potential role of posttranslational events in pathogenesis, with particular focus on the polygln diseases. Posttranslational events have already been implicated