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M E MacDonald

Publications and source records attributed to M E MacDonald.

At least 37 records · Page 2Linked to original sources
PubMed1999

Polyglutamine-mediated dysfunction and apoptotic death of a Caenorhabditis elegans sensory neuron.

The effect of expressing human huntingtin fragments containing polyglutamine (polyQ) tracts of varying lengths was assessed in Caenorhabditis elegans ASH sensory neurons in young and old animals. Expression of a huntingtin fragment containing a polyQ tract of 150 residues (Htn-Q150) led to progressive ASH neurodegeneration but did not cause cell death. Progressive cell death and enhanced neurodegeneration were observed in ASH neurons that coexpressed Htn-Q150 and a subthreshold dose of a toxic OSM-10::green fluorescent protein (OSM-10::GFP) fusion protein. Htn-Q150 huntingtin protein fragments formed protein aggregates in ASH neurons, and the number of ASH neurons containing aggregates increased as animals aged. ASH neuronal cell death required ced-3 caspase function, indicating that the observed cell death is apoptotic. Of interest, ced-3 played a critical role in Htn-Q150-mediated neurodegeneration but not in OSM10::GFP-mediated ASH neurodegeneration. ced-3 function was important but not essential for the formation of protein aggregates. Finally, behavioral assays indicated that ASH neurons, coexpressing Htn-Q150 and OSM10::GFP, were functionally impaired at 3 days before the detection of neurodegeneration, cell death, and protein aggregates.

Age Factors↗
PubMed1999

Mutant huntingtin forms in vivo complexes with distinct context-dependent conformations of the polyglutamine segment.

Huntington'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.

Animals↗
PubMed1999

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Animals↗
PubMed1999

Length-dependent gametic CAG repeat instability in the Huntington's disease knock-in mouse.

The CAG repeats in the human Huntington's disease (HD) gene exhibit striking length-dependent intergenerational instability, typically small size increases or decreases of one to a few CAGs, but little variation in somatic tissues. In a subset of male transmissions, larger size increases occur to produce extreme HD alleles that display somatic instability and cause juvenile onset of the disorder. Initial efforts to reproduce these features in a mouse model transgenic for HD exon 1 with 48 CAG repeats revealed only mild intergenerational instability ( approximately 2% of meioses). A similar pattern was obtained when this repeat was inserted into exon 1 of the mouse Hdh gene. However, lengthening the repeats in Hdh to 90 and 109 units produced a graded increase in the mutation frequency to >70%, with instability being more evident in female transmissions. No large jumps in CAG length were detected in either male or female transmissions. Instead, size changes were modest increases and decreases, with expansions typically emanating from males and contractions from females. Limited CAG variation in the somatic tissues gave way to marked mosaicism in liver and striatum for the longest repeats in older mice. These results indicate that gametogenesis is the primary source of inherited instability in the Hdh knock-in mouse, as it is in man, but that the underlying repeat length-dependent mechanism, which may or may not be related in the two species, operates at higher CAG numbers. Moreover, the large CAG repeat increases seen in a subset of male HD transmissions are not reproduced in the mouse, suggesting that these arise by a different fundamental mechanism than the small size fluctuations that are frequent during gametogenesis in both species.

Age Factors↗
PubMed1999

Analysis of germline mutation spectra at the Huntington's disease locus supports a mitotic mutation mechanism.

Trinucleotide repeat disease alleles can undergo 'dynamic' mutations in which repeat number may change when a gene is transmitted from parent to offspring. By typing >3500 sperm, we determined the size distribution of Huntington's disease (HD) germline mutations produced by 26 individuals from the Venezuelan cohort with CAG/CTG repeat numbers ranging from 37 to 62. Both the mutation frequency and mean change in allele size increased with increasing somatic repeat number. The mutation frequencies averaged 82% and, for individuals with at least 50 repeats, 98%. The extraordinarily high mutation frequency levels are most consistent with a mutation process that occurs throughout germline mitotic divisions, rather than resulting from a single meiotic event. In several cases, the mean change in repeat number differed significantly among individuals with similar somatic allele sizes. This individual variation could not be attributed to age in a simple way or to ' cis ' sequences, suggesting the influence of genetic background or other factors. A familial effect is suggested in one family where both the father and son gave highly unusual spectra compared with other individuals matched for age and repeat number. A statistical model based on incomplete processing of Okazaki fragments during DNA replication was found to provide an excellent fit to the data but variation in parameter values among individuals suggests that the molecular mechanism might be more complex.

Adolescent↗
PubMed1998

Polyglutamine-expanded human huntingtin transgenes induce degeneration of Drosophila photoreceptor neurons.

Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder. Disease alleles contain a trinucleotide repeat expansion of variable length, which encodes polyglutamine tracts near the amino terminus of the HD protein, huntingtin. Polyglutamine-expanded huntingtin, but not normal huntingtin, forms nuclear inclusions. We describe a Drosophila model for HD. Amino-terminal fragments of human huntingtin containing tracts of 2, 75, and 120 glutamine residues were expressed in photoreceptor neurons in the compound eye. As in human neurons, polyglutamine-expanded huntingtin induced neuronal degeneration. The age of onset and severity of neuronal degeneration correlated with repeat length, and nuclear localization of huntingtin presaged neuronal degeneration. In contrast to other cell death paradigms in Drosophila, coexpression of the viral antiapoptotic protein, P35, did not rescue the cell death phenotype induced by polyglutamine-expanded huntingtin.

Animals↗
PubMed1998

Huntingtin: a single bait hooks many species.

Cloning of the Huntington's disease gene uncovered huntingtin, which is remarkable for its lack of similarity with known proteins despite its large size, approximately 350 kDa. Subsequent experiments established that huntingtin has an as yet unknown function, crucial for embryonic development and neurogenesis. Recent protein trapping to identify huntingtin interactors now reveals that many different prey fall victim to huntingtin bait.

Animals↗
PubMed1998

Amyloid formation by mutant huntingtin: threshold, progressivity and recruitment of normal polyglutamine proteins.

Huntington'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.

Adult↗
PubMed1998

Huntingtin interacts with a family of WW domain proteins.

The hallmark neuropathology of Huntington's disease (HD) is due to elongation of a polyglutamine segment in huntingtin, a novel approximately 350 kDa protein of unknown function. We used a yeast two-hybrid interactor screen to identify proteins whose association with huntingtin might be altered in the pathogenic process. Surprisingly, no interactors were found with internal and C-terminal segments of huntingtin. In contrast, huntingtin's N-terminus detected 13 distinct proteins, seven novel and six reported previously. Among these, we identified a major interactor class, comprising three distinct WW domain proteins, HYPA, HYPB and HYPC, that bind normal and mutant huntingtin in extracts of HD lymphoblastoid cells. This interaction is mediated by huntingtin's proline-rich region and is enhanced by lengthening the adjacent glutamine tract. Although HYPB and HYPC are novel, HYPA is human FBP-11, a protein implicated in spliceosome function. The emergence of this class of proteins as huntingtin partners argues that a WW domain-mediated process, such as non-receptor signaling, protein degradation or pre-mRNA splicing, may participate in HD pathogenesis.

Amino Acid Sequence↗
PubMed1997

Heterogeneous topographic and cellular distribution of huntingtin expression in the normal human neostriatum.

A 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.

Aged↗
PubMed1997

CAG repeat number governs the development rate of pathology in Huntington's disease.

We compared the number of CAG repeats, the age at death, and the severity of neuropathology in 89 Huntington's disease brains. We found a linear correlation between the CAG repeat number and the quotient of the degree of atrophy in the striatum (the brain region most severely affected in Huntington's disease) divided by age at death, with an intercept at 35.5 repeats. The largest CAG repeat length, therefore, at which no pathology is expected to develop is 35.5. These results imply that striatal damage in Huntington's disease is almost entirely a linear function of the length of the polyglutamine stretch beyond 35.5 glutamines multiplied by the age of the patient. Thus, it is predicted that the pathological process develops linearly from birth. Analysis of other measures of striatal function could test this hypothesis and might determine when treatment for CAG repeat diseases should start.

Adult↗
PubMed1997

Frataxin gene of Friedreich's ataxia is targeted to mitochondria.

Friedreich's ataxia is caused by a triplet repeat expansion in intron 1, a noncoding region of the frataxin gene (X25). We have generated a chimeric gene composed of the frataxin gene fused with the green fluorescent protein (GFP) gene as a reporter. Transfection of the fusion construct into living COS cells revealed that the frataxin-GFP construct localizes to organelles that double-label with 8-(4'-chloromethyl) phenyl-2,3,5,6,11,12,14,15-octahydro-1H,4H,10H-13H-diquinolizin o-8H-xanthylium chloride (CMXRos), a novel mitochondrial dye. Thus, frataxin appears to be a nuclear-encoded mitochondrial protein.

Animals↗
PubMed1997

Exon trapping and sequence-based methods of gene finding in transcript mapping of human 4p16.3.

We have applied exon amplification, GRAIL2 exon prediction and EST database searching to a 2 Mb segment of chromosome 4p16.3. Experimental and computational methods of identifying exons were comparable in efficiency and apparent false positive rate, but were complementary in gene identification, revealing distinct overlapping sets of expressed sequences. EST searching was most powerful when we considered only those ESTs that show evidence of splicing relative to the genomic sequence. The combination of the three gene finding methods produced a transcription map of 30 loci in this segment of 4p16.3 that includes known human genes, homologs of loci identified in rodents and several anonymous transcripts, including a putative novel DNA polymerase and a gene related to Drosophila ash1. While most of the genes in the region have been found, our data suggest that even with the entire DNA sequence available, complete saturation of the transcript map will require additional, focused experimental effort.

Chromosome Mapping↗
PubMed1997

Assignment of the human gene encoding eukaryotic initiation factor 4E (EIF4E) to the region q21-25 on chromosome 4.

We recently cloned genomic sequences containing the promoter region for the messenger RNA cap binding protein (eIF4E). As the rate-limiting step in translation, eukaryotic initiation factor 4E is important in cellular growth control. Using oligonucleotide primers specific for the promoter region in polymerase chain reactions (PCR), we amplified the human gene in a chromosome 4-specific human/rodent somatic cell panel. This panel mapped single copy genomic sequences for eIF4E in the region 4q21 to 4q25.

Animals↗
PubMed1997

Huntingtin is required for neurogenesis and is not impaired by the Huntington's disease CAG expansion.

Huntington's disease (HD) is an autosomal-dominant neurodegenerative disorder caused by a CAG repeat expansion that lengthens a glutamine segment in the novel huntingtin protein. To elucidate the molecular basis of HD, we extended the polyglutamine tract of the mouse homologue, Hdh, by targetted introduction of an expanded human HD CAG repeat, creating mutant HdhneoQ50 and HdhQ50 alleles that express reduced and wild-type levels of altered huntingtin, respectively. Mice homozygous for reduced levels displayed characteristic aberrant brain development and perinatal lethality, indicating a critical function for Hdh in neurogenesis. However, mice with normal levels of mutant huntingtin did not display these abnormalities, indicating that the expanded CAG repeat does not eliminate or detectably impair huntingtin's neurogenic function. Thus, the HD defect in man does not mimic complete or partial Hdh inactivation and appears to cause neurodegenerative disease by a gain-of-function mechanism.

Alleles↗
PubMed1997

Reduced penetrance of the Huntington's disease mutation.

Controversy persists concerning the significance of Huntington disease (HD) alleles in the 36-39 repeat range. Although some clinically affected persons have been documented with repeats in this range, elderly unaffected individuals have also been reported. We examined 10 paternal transmissions of HD alleles of 37-39 repeats in collateral branches of families with de novo HD. All 10 descendants, including many who are elderly, are without symptoms of HD. Forty percent of the transmissions were unstable, although none varied by more than one repeat. The observation that individuals with alleles of 37-39 repeats may survive unaffected beyond common life expectancy supports the presence of reduced penetrance for HD among some persons with repeat sizes which overlap the clinical range. Non-penetrance may be increased in the collateral branches of de novo mutation families when compared to penetrance estimates from patient series. There was no CAA-->CAG mutation for the penultimate glutamine in either a de novo expanded 42 repeat allele or the corresponding non-penetrant 38 repeat allele in a family with fresh mutation to HD.

Adult↗