Showing papers by "Marcy E. MacDonald published in 1997"

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

Abstract: 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 lineaar 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.

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

Abstract: 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.

Heterogeneous Topographic and Cellular Distribution of Huntingtin Expression in the Normal Human Neostriatum

Abstract: 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.

Reduced Penetrance of the Huntington's Disease Mutation

Abstract: 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.

Frataxin gene of Friedreich's ataxia is targeted to mitochondria

Abstract: 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.

The Genetic Defect Causing Huntington’s Disease: Repeated in Other Contexts?

Abstract: Huntington's disease (HD) is a dominantly inherited, untreatable neurodegenerative disorder involving progressive chorea, psychiatric changes, and intellectual decline (1). The most characteristic feature of HD is its peculiar movement disorder which begins subtly and progresses to exaggerated dance-like motions that consume the entire body. HD occurs equally in both sexes and is found in all races, but most frequently (-1 in 10,000) in people of Western European descent (2). Although symptoms may begin at any age, they are usually first manifested between the ages of 30 and 55 and they progressively worsen until death 12-18 years later. The clinical progression of HD is paralleled by neuronal degeneration in the brain. The hallmark of HD is the loss of medium spiny GABAergic projection neurons in a gradient progressing along posteroanterior, dorsoventral, and mediolateral axes of the caudate nucleus (3). Prior to cell death, signs of neuronal dysfunction are evident in recurved dendritic endings and changes in spine density, shape, and size (4). The disorder eventually destroys the architecture of the caudate nucleus and the adjacent putamen, although extensive cell loss also occurs in other regions of the basal ganglia and in the deep layers of the cerebral cortex (5,6). Overall brain weight may be reduced by 25% or more. Although the proximate cause of the neuronal dysfunction and death is not yet known, it is ultimately due to the presence of a mutant gene

High resolution characterization of an interstitial deletion of less than 1.9 Mb at 4p16.3 associated with Wolf-Hirschhorn syndrome.

Abstract: Wolf-Hirschhorn syndrome (WHS) caused by 4p16.3 deletions comprises growth and mental retardation, distinct facial appearance and seizures. This study characterized a subtle interstitial deletion of 4p16.3 in a girl with mild retardation and possessing facial traits characteristic of WHS. The patient had generalized seizures in conjunction with fever at 3 and 5 years of age. Fluorescence in situ hybridization (FISH) with a series of markers in the 4p16.3 region showed that the interstitial deletion in this patient was between the probes D4S96 and D4S182, enabling the size of the deletion to be estimated as less than 1.9 Mb. This is the smallest interstitial deletion of 4p16.3 which has been reported. The patient contributes to a refinement of the phenotypic map of the WHS region in 4p16.3. The critical region for the characteristic facial changes of WHS, failure to thrive and developmental delay is now localized to a region of less than 700 kb. The mental retardation of this patient was mild suggesting that small interstitial deletion may have less severe phenotypic consequences.

Modulation of macrophage function for defence of the lung against Pseudomonas aeruginosa.

Abstract: Pseudomonas aeruginosa is a common respiratory tract pathogen in certain groups of compromised hosts, most notably those with cystic fibrosis. The pathogenicity of P. aeruginosa may depend in part upon its capacity to resist normal phagocytic cell clearance. We have recently shown that phagocytosis of P. aeruginosa by macrophages is a unique two-step process; binding is glucose-independent but ingestion occurs only in the presence of D-glucose or D-mannose. P. aeruginosa is the only particle we have found which is ingested by macrophages in a glucose-dependent manner. Since glucose is present in only negligible quantities in the endobronchial space, P. aeruginosa may be pathogenic by virtue of its capacity to exploit the opportunity presented in the lower airway to resist normal nonspecific phagocytic defences. The purpose of the studies reported here is to better understand the glucose-dependent phagocytosis of P. aeruginosa and to design novel therapies to facilitate phagocytic cell clearance of it from the lower respiratory tract. We have shown that phagocytosis of unopsonized P. aeruginosa depends upon facilitated transport of glucose into macrophages via the GLUT1 isoform. After transport into the macrophage, the glucose must be metabolized to trigger phagocytosis of P. aeruginosa; pretreatment with 2-deoxyglucose or 5-thioglucose abrogates glucose-dependent ingestion. We have recently demonstrated that pulmonary alveolar macrophages (as opposed to all other macrophage phenotypes studied) lack the capacity to transport glucose and to phagocytose unopsonized P. aeruginosa; however, after the cells have been cultured in vitro for 48 hours, they are able to perform both functions. Whereas most macrophages (such as peritoneal cells) primarily depend upon glycolysis for metabolic energy, pulmonary alveolar macrophages reside in a high oxygen tension environment and appear to utilize oxidative phosphorylation. Treatment of freshly explanted pulmonary alveolar macrophages with sodium azide (to poison oxidative respiration) dramatically enhances both glucose transport and glucose-dependent phagocytosis of P. aeruginosa. We are currently investigating the compromised phagocytic function of pulmonary alveolar macrophages and the mechanism by which azide enhances glucose transport and phagocytosis of P. aeruginosa. Although physiological measurements have indicated that glucose is removed from the endobronchial space by an active transport process of the lung epithelium, the types of glucose transporters that are expressed in the lung are as yet unknown. Using RT-PCR, we have amplified a product from human and murine lung RNA which has a high degree of homology with members of the sodium-dependent glucose transporter (SGLT) family. The ultimate goal of these studies is to design novel agents for enhancing the phagocytic function of pulmonary alveolar macrophages. Delivery of simple glucose by aerosol would not be effective because (i) it would be exported by sodium-dependent active transport and (ii) pulmonary alveolar macrophages lack the capacity to transport glucose. Various approaches for targeting glucose to alveolar macrophages by receptor-mediated endocytosis are under investigation.

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

Abstract: We have applied exon amplification, GRAIL2 exon prediction and EST database searching to a 2 Mb segment of chromosome 4p 16.3. 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 toDrosophila 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.

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

Abstract: 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.