Mouse Huntington's disease homolog mRNA levels: variation and allele effects

Huntington's disease homolog (Hdh) mRNA levels in mice with different Hdh alleles were measured. Brain Hdh mRNA levels varied up to threefold in genetically identical wild-type mice, indicating nongenetic factors influence Hdh expression. Striatal Hdh mRNA levels from an allele with a repeat expanded to 150 CAGs were diminished compared with wild-type and showed variation that might contribute to phenotypic variability in the Hdh(CAG)150 knock-in mouse model. To determine whether Hdh mRNA levels are tightly regulated, we assessed these levels in mice heterozygous for a deletion of the Hdh promoter. The loss of one allele reduced Hdh mRNA levels in most tissues, suggesting mechanisms to maintain Hdh mRNA levels are not in effect and should not impede therapies designed to destroy mutant huntingtin mRNA. Finally, we found a correlation between tissue mRNA levels and the susceptibility of the Hdh locus to Cre-mediated deletion. The two tissues with the highest levels of Hdh mRNA, testes and brain, were the only tissues susceptible to Cre-mediated recombination between loxP sites at Hdh locus. In contrast, the same Cre-expressing line caused recombination in every tissue for loxP sites at another genomic location. The pattern of Cre susceptibility at Hdh suggests a correlation between chromatin accessibility and high levels of Hdh expression in testes and brain.     

Mismatch repair gene Msh2 modifies the timing of early disease in Hdh(Q111) striatum

Somatic instability of expanded HD CAG repeats that encode the polyglutamine tract in mutant huntingtin has been implicated in the striatal selectivity of Huntington's disease (HD) pathology. Here in Hdh(Q111) mice, we have tested whether a genetic background deficient in Msh2, expected to eliminate the unstable behavior of the 109 CAG array inserted into the murine HD gene, would alter the timing or striatal specificity of a dominant disease phenotype that predicts late-onset neurodegeneration. Our analyses of Hdh(Q111/+):Msh2(+/+) and Hdh(Q111/+): Msh2(-/-) progeny revealed that, while inherited instability involved Msh2-dependent and -independent mechanisms, lack of Msh2 was sufficient to abrogate progressive HD CAG repeat expansion in striatum. The absence of Msh2 also eliminated striatal mutant huntingtin with somatically expanded glutamine tracts and caused an approximately 5 month delay in nuclear mutant protein accumulation, but did not alter the striatal specificity of this early phenotype. Thus, somatic HD CAG instability appears to be a consequence of a striatal-selective disease process that accelerates the timing of an early disease phenotype, via expansion of the glutamine tract in mutant huntingtin. Therefore Msh2, as a striking modifier of early disease onset in a precise genetic HD mouse model, provides a novel target for the development of pharmacological agents that aim to slow pathogenesis in man.     

A Huntington's disease CAG expansion at the murine Hdh locus is unstable and associated with behavioural abnormalities in mice

Huntington's disease (HD) is a dominant disorder characterized by premature and progressive neurodegeneration. In order to generate an accurate model of the disease, we introduced an HD-like mutation (an extended stretch of 72-80 CAG repeats) into the endogenous mouse Hdh gene. Analysis of the mutation in vivo reveals significant levels of germline instability, with expansions, contractions and sex-of-origin effects in evidence. Mice expressing full-length mutant protein display abnormal social behaviour in the absence of acute neurodegeneration. Given that psychiatric changes, including irritability and aggression, are common findings in HD patients, our data are consistent with the hypothesis that some clinical features of HD may be caused by pathological processes that precede gross neuronal cell death. This implies that effective treatment of HD may require an understanding and amelioration of these dysfunctional processes, rather than simply preventing the premature death of neurons in the brain. These mice should facilitate the investigation of the molecular mechanisms that underpin the pathway from genotype to phenotype in HD.     

N-methyl-D-aspartate (NMDA) receptor function and excitotoxicity in Huntington's disease

Many lines of evidence support a role for neuronal damage arising as a result of excessive activation of glutamate receptors by excitatory amino acids in the pathogenesis of Huntington disease. The N-methyl-d-aspartate subclass of ionotropic glutamate receptors (NMDARs) is more selective and effective than the other subclasses in mediating this damage. As well, neurons expressing high levels of NMDARs are lost early from the striatum of individuals affected with Huntington's disease (HD), and injection of NMDAR agonists into the striatum of rodents or non-human primates recapitulates the pattern of neuronal damage observed in HD. Altered NMDAR function has been reported in corticostriatal synapses in one mouse model of HD, and NMDAR-mediated current and/or toxicity have been found to be potentiated in striatal neurons from several HD mouse models as well as heterologous cells expressing the mutant huntingtin protein. Changes in NMDAR activity have been correlated with altered calcium homeostasis, mitochondrial membrane depolarization and caspase activation. NMDAR stimulation is also closely linked to mitochondrial activity, as treatment with mitochondrial toxins has been demonstrated to produce striatal damage that can be reversed by the addition of NMDAR antagonists. Recent efforts have focused on the elucidation of molecular pathways linking huntingtin to NMDARs, as well as the mechanisms which underlie the enhancement of NMDAR activity by mutant huntingtin. Here, we review the literature to date and recent findings concerning the role of NMDARs in HD pathogenesis.     

Phonemic fluency deficits in asymptomatic gene carriers for Huntington's disease

The aim of the present study was to investigate verbal fluency in preclinical Huntington's disease (HD). Phonemic and semantic fluency and the rate of word production over time were assessed for 29 asymptomatic gene carriers and 34 noncarriers of HD. The relationship between fluency tasks and other cognitive domains was investigated. Compared to noncarriers, carriers produced fewer words and produced them more slowly in the phonemic fluency task but not in the semantic fluency task. When the carrier group was divided on the basis of Predicted-Years-To-Onset (PYTO), only carriers with <12 PYTO performed worse than noncarriers on both fluency tasks. Correlational analyses revealed that phonemic fluency was associated with cognitive speed and working memory, while semantic fluency was linked with crystallized abilities. The difference between carriers and noncarriers in phonemic fluency and a difference between the two carrier groups (<12 PYTO and >or=12 PYTO) in semantic fluency, but not in phonemic fluency, suggest that frontostriatal deficits may precede temporal involvement in preclinical HD.     

New DNA markers in the Huntington's disease gene candidate region

The search for the Huntington's disease (HD) gene has prompted construction of a complete long-range restriction map of a 2.5-Mb candidate region, distal to the DNA markerD4S10. To facilitate the procurement of cloned DNA from this candidate region, we have augmented the existing regional mapping panel of somatic cell hybrids with hybrid HHW1071 containing a t(4p16;12) chromosome from a patient with Wolf-Hirschhorn syndrome. This translocation maps betweenD4S180 andD4S127, subdividing theHD candidate region and setting a proximal limit to the Wolf-Hirschhorn syndrome region. Using the expanded mapping panel, we have regionally assigned 14 independently cloned cosmids, five proximal to the t(4;12) breakpoint in the same region asD4S10 and nine distal to the breakpoint. By a combination of overlap with previously mapped cosmids and pulsed-field gel analysis, each of these cosmids has been positioned on the long-range restriction map of 4p16.3, increasing the clone coverage of the candidate region to approximately 40%. Single-copy probes from mapped cosmids were used to identify eight new DNA polymorphisms spanning theHD candidate region. These new DNA markers should prove valuable for analysis of recombination and linkage disequilibrium inHD, as well as for preclinical diagnosis of the disorder.     

Early cognitive deficits in Swedish gene carriers of Huntington's disease

The primary focus of this study was to examine whether there is early neuropsychological impairment in presymptomatic Huntington's disease (HD). A broad neuropsychological assessment battery was administered to 24 asymptomatic gene carriers (HD+) and 31 noncarriers (HD-). The gene carriers revealed inferior cognitive functioning as compared with the noncarriers in memory and executive functions. When the gene carriers were assigned to 2 groups based on predicted years to onset (with 15 and over being HD+ late and under 15 being HD+ near), the HD+ near group performed significantly worse than the HD+ late group in all domains but ability to shift conceptually and visuospatial memory. Results suggest that early cognitive deficits are detectable prior to motor symptoms, first in memory functions and then in executive functions and perceptual motor speed.     

Huntington's disease.

The most recent findings in the elucidation of the molecular pathology of Huntington's disease are reviewed. Particular interest has been paid to the role of huntingtin and its associated proteins in excitotoxicity mediated via NMDA and kainate receptors.     

Regional and cellular gene expression changes in human Huntington's disease brain

Huntington's disease (HD) pathology is well understood at a histological level but a comprehensive molecular analysis of the effect of the disease in the human brain has not previously been available. To elucidate the molecular phenotype of HD on a genome-wide scale, we compared mRNA profiles from 44 human HD brains with those from 36 unaffected controls using microarray analysis. Four brain regions were analyzed: caudate nucleus, cerebellum, prefrontal association cortex [Brodmann's area 9 (BA9)] and motor cortex [Brodmann's area 4 (BA4)]. The greatest number and magnitude of differentially expressed mRNAs were detected in the caudate nucleus, followed by motor cortex, then cerebellum. Thus, the molecular phenotype of HD generally parallels established neuropathology. Surprisingly, no mRNA changes were detected in prefrontal association cortex, thereby revealing subtleties of pathology not previously disclosed by histological methods. To establish that the observed changes were not simply the result of cell loss, we examined mRNA levels in laser-capture microdissected neurons from Grade 1 HD caudate compared to control. These analyses confirmed changes in expression seen in tissue homogenates; we thus conclude that mRNA changes are not attributable to cell loss alone. These data from bona fide HD brains comprise an important reference for hypotheses related to HD and other neurodegenerative diseases.     

Immunoreactive somatostatin-28(1-12) is increased in Huntington's disease

Somatostatin-28(1-12) concentrations were measured in Huntington's disease (HD) postmortem tissue using a specific radioimmunoassay. Concentrations of immunoreactive somatostatin-28(1-12) were significantly increased in the caudate and putamen but were unchanged in cortical areas A9 and A17. Since somatostatin-28(1-12) terminates with the amino acids Arg-Glu-OH, we examined whether this dodecapeptide compound might exert a neurotoxic effect. Injections of somatostatin-28(1-12) into rat striatum showed no evidence of histologic damage.     

Expression of the Huntington's disease (IT15) protein product in HD patients

Huntington's disease (HD) is an inherited, neurodegenerativedisorder caused by expansion of a CAG repeat in the IT15 gene,leading to an expanded glutamine repeat in the HD protein. Themechanism by which the expanded repeat causes expression ofthe disease is not known, though there do not appear to be changesin the mRNA levels. We have conducted quantitative Western blotanalyses of HD patients and controls. Expression of the IT15protein is essentially equal in control and HD frontal cortex.In caudate from HD patients, IT15 protein is decreased in parallelwith the decrease in a neuronal marker, suggesting that lossof IT15 protein is secondary to neuronal loss. In order to determineexpression of the two alleles of the IT15 protein we used Westernblots of 4% polyacrylamide gels. Both alleles of the IT15 proteinwere expressed at similar levels in HD lymphoblastoid cell linesand HD post-mortem hippocampus and cerebellum (regions relativelyspared in HD), indicating that even very long CAG repeats canbe translated into polyglutamine. In contrast, in cerebral cortexand caudate (regions severely affected in HD), in the longerexpanded repeat cases the expanded allele of the IT15 proteinwas present at a significantly lower level (compared with thenormal length allele), often with a smear of more slowly migratingreactivity above it. These data suggest the possibility of alteredstructure, abnormal processing or abnormality of protein—proteininteractions involving the IT15 protein with the expanded glutaminerepeat.