Expression analysis of the ataxin-1 protein in tissues from normal and spinocerebellar ataxia type 1 individuals.

Spinocerebellar ataxia type 1 (SCA1) is an autosomal dominant neurodegenerative disorder caused by expansion of a CAG trinucleotide repeat which codes for glutamine in the protein ataxin-1. We have investigated the effect of this expansion on ataxin-1 by immunoblot analysis. The wild-type protein is detected in both normal and affected individuals; however, a mutant protein which varies in its migration properties according to the size of the CAG repeat is detected in cultured cells and tissues from SCA1 individuals. The protein has a nuclear localization in all normal and SCA1 brain regions examined but a cytoplasmic localization of ataxin-1 was also observed in cerebellar Purkinje cells. Our data show that in SCA1, the expanded alleles are faithfully translated into proteins of apparently normal stability and distribution.

Evidence for a mechanism predisposing to intergenerational CAG repeat instability in spinocerebellar ataxia type I.

Spinocerebellar ataxia type I (SCAI) is an autosomal dominant neurodegenerative disease caused by the expansion of a CAG trinucleotide repeat on chromosome 6p. Normal alleles range from 19-36 repeats while SCA1 alleles contain 43-81 repeats. We now show that in 63% of paternal transmissions, an increase in repeat number is observed, whereas 69% of maternal transmissions showed no change or a decrease in repeat number. Sequence analysis of the repeat from 126 chromosomes reveals an interrupted repeat configuration in 98% of the unexpanded alleles but a contiguous repeat (CAG)n configuration in 30 expanded alleles from seven SCA1 families. This indicates that the repeat instability in SCA1 is more complex than a simple variation in repeat number and that the loss of an interruption predisposes the SCA1 (CAG)n to expansion.

Gametic and somatic tissue-specific heterogeneity of the expanded SCA1 CAG repeat in spinocerebellar ataxia type 1.

Spinocerebellar ataxia type 1 is associated with expansion of an unstable CAG repeat within the SCA1 gene. Male gametic heterogeneity of the expanded repeat is demonstrated using single sperm and low-copy genome analysis. Low-copy genome analysis of peripheral blood also reveals somatic heterogeneity of the expanded SCA1 allele, thus establishing mitotic instability at this locus. Comparative analysis of a large normal allele and a small affected allele suggests a role of midstream CAT interspersions in stabilizing long (CAG)n stretches. Within the brain, tissue-specific mosaicism of the expanded allele is also observed. The differences in SCA1 allele heterogeneity between sperm and blood and within the brain parallels the findings in Huntington disease, suggesting that both disorders share a common mechanism for tissue-specific instability.

Rett syndrome is caused by mutations in X-linked MECP2, encoding methyl-CpG-binding protein 2.

Rett syndrome (RTT, MIM 312750) is a progressive neurodevelopmental disorder and one of the most common causes of mental retardation in females, with an incidence of 1 in 10,000-15,000 (ref. 2). Patients with classic RTT appear to develop normally until 6-18 months of age, then gradually lose speech and purposeful hand use, and develop microcephaly, seizures, autism, ataxia, intermittent hyperventilation and stereotypic hand movements. After initial regression, the condition stabilizes and patients usually survive into adulthood. As RTT occurs almost exclusively in females, it has been proposed that RTT is caused by an X-linked dominant mutation with lethality in hemizygous males. Previous exclusion mapping studies using RTT families mapped the locus to Xq28 (refs 6,9,10,11). Using a systematic gene screening approach, we have identified mutations in the gene (MECP2 ) encoding X-linked methyl-CpG-binding protein 2 (MeCP2) as the cause of some cases of RTT. MeCP2 selectively binds CpG dinucleotides in the mammalian genome and mediates transcriptional repression through interaction with histone deacetylase and the corepressor SIN3A (refs 12,13). In 5 of 21 sporadic patients, we found 3 de novo missense mutations in the region encoding the highly conserved methyl-binding domain (MBD) as well as a de novo frameshift and a de novo nonsense mutation, both of which disrupt the transcription repression domain (TRD). In two affected half-sisters of a RTT family, we found segregation of an additional missense mutation not detected in their obligate carrier mother. This suggests that the mother is a germline mosaic for this mutation. Our study reports the first disease-causing mutations in RTT and points to abnormal epigenetic regulation as the mechanism underlying the pathogenesis of RTT.

Chaperone suppression of aggregation and altered subcellular proteasome localization imply protein misfolding in SCA1.

Spinocerebellar ataxia type 1 (SCA1) is an autosomal dominant neurodegenerative disorder caused by expansion of a polyglutamine tract in ataxin-1. In affected neurons of SCA1 patients and transgenic mice, mutant ataxin-1 accumulates in a single, ubiquitin-positive nuclear inclusion. In this study, we show that these inclusions stain positively for the 20S proteasome and the molecular chaperone HDJ-2/HSDJ. Similarly, HeLa cells transfected with mutant ataxin-1 develop nuclear aggregates which colocalize with the 20S proteasome and endogenous HDJ-2/HSDJ. Overexpression of wild-type HDJ-2/HSDJ in HeLa cells decreases the frequency of ataxin-1 aggregation. These data suggest that protein misfolding is responsible for the nuclear aggregates seen in SCA1, and that overexpression of a DnaJ chaperone promotes the recognition of a misfolded polyglutamine repeat protein, allowing its refolding and/or ubiquitin-dependent degradation.

Expansion of an unstable trinucleotide CAG repeat in spinocerebellar ataxia type 1.

Spinocerebellar ataxia type 1 (SCA1) is an autosomal dominant disorder characterized by neurodegeneration of the cerebellum, spinal cord and brainstem. A 1.2-Megabase stretch of DNA from the short arm of chromosome 6 containing the SCA1 locus was isolated in a yeast artificial chromosome contig and subcloned into cosmids. A highly polymorphic CAG repeat was identified in this region and was found to be unstable and expanded in individuals with SCA1. There is a direct correlation between the size of the (CAG)n repeat expansion and the age-of-onset of SCA1, with larger alleles occurring in juvenile cases. We also show that the repeat is present in a 10 kilobase mRNA transcript. SCA1 is therefore the fifth genetic disorder to display a mutational mechanism involving an unstable trinucleotide repeat.

A high resolution deletion map of human chromosome Xp22.

We have developed a 32-interval deletion panel for human chromosome Xp22 spanning about 30 megabases of genomic DNA. DNA samples from 50 patients with chromosomal rearrangements involving Xp22 were tested with 60 markers using a polymerase chain reaction strategy. The ensuing deletion map allowed us to confirm and refine the order of previously isolated and newly developed markers. Our mapping panel will provide the framework for mapping new sequences, for orienting chromosome walks in the region and for projects aimed at isolating genes responsible for diseases mapping to Xp22.

Identification and characterization of the gene causing type 1 spinocerebellar ataxia.

Spinocerebellar ataxia type 1 (SCA1) is a neurodegenerative disorder caused by expansion of a CAG trinucleotide repeat. In this study, we describe the identification and characterization of the gene harbouring this repeat. The SCA1 transcript is 10,660 bases and is transcribed from both the wild type and SCA1 alleles. The CAG repeat, coding for a polyglutamine tract, lies within the coding region. The gene spans 450 kb of genomic DNA and is organized in nine exons. The first seven fall in the 5' untranslated region and the last two contain the coding region, and a 7,277 basepairs 3' untranslated region. The first four non-coding exons undergo alternative splicing in several tissues. These features suggest that the transcriptional and translational regulation of ataxin-1, the SCA1 encoded protein, may be complex.

Autosomal dominant cerebellar ataxia (SCA6) associated with small polyglutamine expansions in the alpha 1A-voltage-dependent calcium channel.

A polymorphic CAG repeat was identified in the human alpha 1A voltage-dependent calcium channel subunit. To test the hypothesis that expansion of this CAG repeat could be the cause of an inherited progressive ataxia, we genotyped a large number of unrelated controls and ataxia patients. Eight unrelated patients with late onset ataxia had alleles with larger repeat numbers (21-27) compared to the number of repeats (4-16) in 475 non-ataxia individuals. Analysis of the repeat length in families of the affected individuals revealed that the expansion segregated with the phenotype in every patient. We identified six isoforms of the human alpha 1A calcium channel subunit. The CAG repeat is within the open reading frame and is predicted to encode glutamine in three of the isoforms. We conclude that a small polyglutamine expansion in the human alpha 1A calcium channel is most likely the cause of a newly classified autosomal dominant spinocerebellar ataxia, SCA6.

Retinal degeneration characterizes a spinocerebellar ataxia mapping to chromosome 3p.

A heterogeneous group of neurological disorders known as the spinocerebellar ataxias (SCA) are characterized by degeneration of the cerebellum, spinal cord and brainstem. We describe linkage analysis in four unusual SCA families revealing a distinct disease locus on chromosome 3p14-21.1. The disease in these families is distinguished from other forms of SCA by concomitant retinal degeneration. Initial visual problems leading to blindness, disabling ataxia and anticipation are seen in all kindreds. The anticipation in these families suggests a dynamic mutation at this locus. Eventual molecular characterization of this disease may provide valuable insights into the processes of both neural and retinal degeneration.

Large expansion of the ATTCT pentanucleotide repeat in spinocerebellar ataxia type 10.

Spinocerebellar ataxia type 10 (SCA10; MIM 603516; refs 1,2) is an autosomal dominant disorder characterized by cerebellar ataxia and seizures. The gene SCA10 maps to a 3.8-cM interval on human chromosome 22q13-qter (refs 1,2). Because several other SCA subtypes show trinucleotide repeat expansions, we examined microsatellites in this region. We found an expansion of a pentanucleotide (ATTCT) repeat in intron 9 of SCA10 in all patients in five Mexican SCA10 families. There was an inverse correlation between the expansion size, up to 22.5 kb larger than the normal allele, and the age of onset (r2=0.34, P=0.018). Analysis of 562 chromosomes from unaffected individuals of various ethnic origins (including 242 chromosomes from Mexican persons) showed a range of 10 to 22 ATTCT repeats with no evidence of expansions. Our data indicate that the new SCA10 intronic ATTCT pentanucleotide repeat in SCA10 patients is unstable and represents the largest microsatellite expansion found so far in the human genome.

Requirement of Math1 for secretory cell lineage commitment in the mouse intestine.

The mouse small intestinal epithelium consists of four principal cell types deriving from one multipotent stem cell: enterocytes, goblet, enteroendocrine, and Paneth cells. Previous studies showed that Math1, a basic helix-loop-helix (bHLH) transcription factor, is expressed in the gut. We find that loss of Math1 leads to depletion of goblet, enteroendocrine, and Paneth cells without affecting enterocytes. Colocalization of Math1 with Ki-67 in some proliferating cells suggests that secretory cells (goblet, enteroendocrine, and Paneth cells) arise from a common progenitor that expresses Math1, whereas absorptive cells (enterocytes) arise from a progenitor that is Math1-independent. The continuous rapid renewal of these cells makes the intestinal epithelium a model system for the study of stem cell regeneration and lineage commitment.

Math1: an essential gene for the generation of inner ear hair cells.

The mammalian inner ear contains the cochlea and vestibular organs, which are responsible for hearing and balance, respectively. The epithelia of these sensory organs contain hair cells that function as mechanoreceptors to transduce sound and head motion. The molecular mechanisms underlying hair cell development and differentiation are poorly understood. Math1, a mouse homolog of the Drosophila proneural gene atonal, is expressed in inner ear sensory epithelia. Embryonic Math1-null mice failed to generate cochlear and vestibular hair cells. This gene is thus required for the genesis of hair cells.

atonal regulates neurite arborization but does not act as a proneural gene in the Drosophila brain.

Drosophila atonal (ato) is the proneural gene of the chordotonal organs (CHOs) in the peripheral nervous system (PNS) and the larval and adult photoreceptor organs. Here, we show that ato is expressed at multiple stages during the development of a lineage of central brain neurons that innervate the optic lobes and are required for eclosion. A novel fate mapping approach shows that ato is expressed in the embryonic precursors of these neurons and that its expression is reactivated in third instar larvae (L3). In contrast to its function in the PNS, ato does not act as a proneural gene in the embryonic brain. Instead, ato performs a novel function, regulating arborization during larval and pupal development by interacting with Notch.

Proprioceptor pathway development is dependent on Math1.

The proprioceptive system provides continuous positional information on the limbs and body to the thalamus, cortex, pontine nucleus, and cerebellum. We showed previously that the basic helix-loop-helix transcription factor Math1 is essential for the development of certain components of the proprioceptive pathway, including inner-ear hair cells, cerebellar granule neurons, and the pontine nuclei. Here, we demonstrate that Math1 null embryos lack the D1 interneurons and that these interneurons give rise to a subset of proprioceptor interneurons and the spinocerebellar and cuneocerebellar tracts. We also identify three downstream genes of Math1 (Lh2A, Lh2B, and Barhl1) and establish that Math1 governs the development of multiple components of the proprioceptive pathway.

Mutation of the E6-AP ubiquitin ligase reduces nuclear inclusion frequency while accelerating polyglutamine-induced pathology in SCA1 mice.

Mutant ataxin-1, the expanded polyglutamine protein causing spinocerebellar ataxia type 1 (SCA1), aggregates in ubiquitin-positive nuclear inclusions (NI) that alter proteasome distribution in affected SCA1 patient neurons. Here, we observed that ataxin-1 is degraded by the ubiquitin-proteasome pathway. While ataxin-1 [2Q] and mutant ataxin-1 [92Q] are polyubiquitinated equally well in vitro, the mutant form is three times more resistant to degradation. Inhibiting proteasomal degradation promotes ataxin-1 aggregation in transfected cells. And in mice, Purkinje cells that express mutant ataxin-1 but not a ubiquitin-protein ligase have significantly fewer NIs. Nonetheless, the Purkinje cell pathology is markedly worse than that of SCA1 mice. Taken together, NIs are not necessary to induce neurodegeneration, but impaired proteasomal degradation of mutant ataxin-1 may contribute to SCA1 pathogenesis.

Methyl-CpG-binding protein 2 mutations in Rett syndrome.

The X-linked methyl-CpG-binding protein 2 gene (MECP2) encodes a protein that links DNA methylation to transcriptional repression mediated by histone deacetylases. Mutations in MECP2 have been found in 76% of classic Rett syndrome patients. Favourable nonrandom X chromosome inactivation ameliorates the phenotype.

Polyglutamine expansion down-regulates specific neuronal genes before pathologic changes in SCA1.

The expansion of an unstable CAG repeat causes spinocerebellar ataxia type 1 (SCA1) and several other neurodegenerative diseases. How polyglutamine expansions render the resulting proteins toxic to neurons, however, remains elusive. Hypothesizing that long polyglutamine tracts alter gene expression, we found certain neuronal genes involved in signal transduction and calcium homeostasis sequentially downregulated in SCA1 mice. These genes were abundant in Purkinje cells, the primary site of SCA1 pathogenesis; moreover, their downregulation was mediated by expanded ataxin-1 and occurred before detectable pathology. Similar downregulation occurred in SCA1 human tissues. Altered gene expression may be the earliest mediator of polyglutamine toxicity.

Polyglutamine diseases: protein cleavage and aggregation.

Neuronal aggregates of the disease-causing protein, often in the nucleus of affected cells, are a pathological hallmark of the neurodegenerative diseases known as polyglutamine disorders. It was suggested that these nuclear aggregates are the cause of these disorders. However, recent evidence suggests that the aggregates, in fact, are not the pathogenic basis and, instead, may play a role in sequestration of the pathogenic protein.