The clinical and genetic spectrum of spinocerebellar ataxia 14.

Spinocerebellar ataxia 14 (SCA14) is associated with missense mutations in the protein kinase C gamma gene (PRKCG), rather than a nucleotide repeat expansion. In this large-scale study of PRKCG in patients with ataxia, two new missense mutations, an in-frame deletion, and a possible splice site mutation were found and can now be added to the four previously described missense mutations. The genotype/phenotype correlations in these families are described.

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.

Mutations in the gene encoding methyl-CpG-binding protein 2 cause Rett syndrome.

Rett syndrome is an X-linked dominant neurodevelopmental disorder primarily affecting girls. About 80% of classic Rett syndrome is caused by mutations in the gene for methyl-CpG-binding protein (MeCP2) in Xq28. MeCP2 links DNA methylation to transcriptional repression, and MECP2 mutations likely cause partial or complete loss of function of the protein, leading to inappropriate transcription of downstream genes at critical times in brain development. More severe and milder variant forms can all be caused by similar mutations. Most classic Rett syndrome patients have random X-chromosome inactivation (XCI), but skewed patterns are present in a few. All asymptomatic or mildly mentally delayed female carriers studied to date have non-random XCI patterns, suggesting that this attenuates the deleterious effects of the MECP2 mutations in these women. The finding of non-random XCI patterns in some patients with very early truncations is consistent with this observation and supports that many mutations could cause partial and not complete loss of function. Our observation that the mutant mRNA is stable in three patients with truncating mutations supports this possibility. Further studies will have to be performed to better understand the functional consequences of MECP2 mutations in RTT.

Reduction of Purkinje cell pathology in SCA1 transgenic mice by p53 deletion.

The expansion of a polyglutamine tract in the ataxin-1 protein beyond a critical threshold causes spinocerebellar ataxia type 1 (SCA1). To investigate the mechanism of neuronal degeneration in SCA1, we analyzed the phenotype of an SCA1 transgenic mouse model in the absence of p53, an important regulator of cell death. p53 deficiency did not affect the early features of SCA1 mice such as impaired motor coordination and ataxin-1 nuclear inclusion formation but caused a notable reduction in later pathological features, including Purkinje cell heterotopia, dendritic thinning, and molecular layer shrinkage. To determine if this protective effect was mediated by an anti-apoptotic property of p53 deficiency, we looked for apoptosis in SCA1 mice but failed to detect any evidence of it even in the presence of p53. We propose that p53 acts after the initial pathogenic events in SCA1 to promote the progression of neuronal degeneration in SCA1 mice, but this activity may be unrelated to apoptosis.

Increased expression of alpha 1A Ca2+ channel currents arising from expanded trinucleotide repeats in spinocerebellar ataxia type 6.

The expansion of polyglutamine tracts encoded by CAG trinucleotide repeats is a common mutational mechanism in inherited neurodegenerative diseases. Spinocerebellar ataxia type 6 (SCA6), an autosomal dominant, progressive disease, arises from trinucleotide repeat expansions present in the coding region of CACNA1A (chromosome 19p13). This gene encodes alpha(1A), the principal subunit of P/Q-type Ca(2+) channels, which are abundant in the CNS, particularly in cerebellar Purkinje and granule neurons. We assayed ion channel function by introduction of human alpha(1A) cDNAs in human embryonic kidney 293 cells that stably coexpressed beta(1) and alpha(2)delta subunits. Immunocytochemical analysis showed a rise in intracellular and surface expression of alpha(1A) protein when CAG repeat lengths reached or exceeded the pathogenic range for SCA6. This gain at the protein level was not a consequence of changes in RNA stability, as indicated by Northern blot analysis. The electrophysiological behavior of alpha(1A) subunits containing expanded (EXP) numbers of CAG repeats (23, 27, and 72) was compared against that of wild-type subunits (WT) (4 and 11 repeats) using standard whole-cell patch-clamp recording conditions. The EXP alpha(1A) subunits yielded functional ion channels that supported inward Ca(2+) channel currents, with a sharp increase in P/Q Ca(2+) channel current density relative to WT. Our results showed that Ca(2+) channels from SCA6 patients display near-normal biophysical properties but increased current density attributable to elevated protein expression at the cell surface.

Genomic organization of Tropomodulins 2 and 4 and unusual intergenic and intraexonic splicing of YL-1 and Tropomodulin 4.

BACKGROUND: The tropomodulins (TMODs) are a family of proteins that cap the pointed ends of actin filaments. Four TMODs have been identified in humans, with orthologs in mice. Mutations in actin or actin-binding proteins have been found to cause several human diseases, ranging from hypertrophic cardiomyopathy to immunodeficiencies such as Wiskott-Aldrich syndrome. We had previously mapped Tropomodulin 2 (TMOD2) to the genomic region containing the gene for amyotrophic lateral sclerosis 5 (ALS5). We determined the genomic structure of Tmod2 in order to better analyze patient DNA for mutations; we also determined the genomic structure of Tropomodulin 4 (TMOD4). RESULTS: In this study, we determined the genomic structure of TMOD2 and TMOD4 and found the organization of both genes to be similar. Sequence analysis of TMOD2 revealed no mutations or polymorphisms in ALS5 patients or controls. Interestingly, we discovered that another gene, YL-1, intergenically splices into TMOD4. YL-1 encodes six exons, the last of which is 291 bp from a 5' untranslated exon of TMOD4. We used 5' RACE and RT-PCR from TMOD4 to identify several intergenic RACE products. YL-1 was also found to undergo unconventional splicing using non-canonical splice sites within exons (intraexonic splicing) to produce several alternative transcripts. CONCLUSIONS: The genomic structure of TMOD2 and TMOD4 have been delineated. This should facilitate future mutational analysis of these genes. In addition, intergenic splicing at TMOD4/YL-1 was discovered, demonstrating yet another level of complexity of gene organization and regulation.

SCA1 molecular genetics: a history of a 13 year collaboration against glutamines.

Spinocerebellar ataxia type 1 (SCA1) is a relatively rare autosomal-dominant neurological disorder. SCA1 has the intriguing feature that the disease-causing mutation is the expansion of an unstable trinucleotide repeat, specifically a CAG repeat that encodes the amino acid glutamine in ataxin-1. During the past 10 years, substantial progress has been made towards understanding the pathogenic mechanism in this disease. The nucleus has been identified as the subcellular site where the mutant protein acts to cause disease. Evidence indicates that expansion of the glutamine tract alters the folding properties of ataxin-1. Finally, several cellular pathways have been identified which are able to impinge on the SCA1 disease process. The characterization of these pathways and their role in SCA1 will guide research over the next several years.

Altered trafficking of membrane proteins in purkinje cells of SCA1 transgenic mice.

Spinocerebellar ataxia type 1 (SCA1) is a neurodegenerative disease caused by the expression of mutant ataxin-1 that contains an expanded polyglutamine tract. Overexpression of mutant ataxin-1 in Purkinje cells of transgenic mice results in a progressive ataxia and Purkinje cell pathology that are very similar to those seen in SCA1 patients. Two prominent aspects of pathology in the SCA1 mice are the presence of cytoplasmic vacuoles and dendritic atrophy. We found that the vacuoles in Purkinje cells seem to originate as large invaginations of the outer cell membrane. The cytoplasmic vacuoles contained proteins from the somatodendritic membrane, including mGluR1, GluRDelta1/Delta2, GluR2/3, and protein kinase C (PKC) gamma. Further examination of PKCgamma revealed that its sequestration into cytoplasmic vacuoles was accompanied by concurrent loss of PKCgamma localization at the Purkinje cell dendritic membrane and decreased detection of PKCgamma by Western blot analysis. In addition, the vacuoles were immunoreactive for components of the ubiquitin/proteasome degradative pathway. These findings present a link between vacuole formation and loss of dendrites in Purkinje cells of SCA1 mice and indicate that altered somatodendritic membrane trafficking and loss of proteins including PKCgamma, are a part of the neuronal dysfunction in SCA1 transgenic mice.

Over-expression of inducible HSP70 chaperone suppresses neuropathology and improves motor function in SCA1 mice.

Many neurodegenerative diseases are caused by gain-of-function mechanisms in which the disease-causing protein is altered, becomes toxic to the cell, and aggregates. Among these 'proteinopathies' are Alzheimer's and Parkinson's disease, prion disorders and polyglutamine diseases. Members of this latter group, also known as triplet repeat diseases, are caused by the expansion of unstable CAG repeats coding for glutamine within the respective proteins. Spinocerebellar ataxia type 1 (SCA1) is one such disease, characterized by loss of motor coordination due to the degeneration of cerebellar Purkinje cells and brain stem neurons. In SCA1 and several other polyglutamine diseases, the expanded protein aggregates into nuclear inclusions (NIs). Because these NIs accumulate molecular chaperones, ubiquitin and proteasomal subunits--all components of the cellular protein re-folding and degradation machinery--we hypothesized that protein misfolding and impaired protein clearance might underlie the pathogenesis of polyglutamine diseases. Over-expressing specific chaperones reduces protein aggregation in transfected cells and suppresses neurodegeneration in invertebrate animal models of polyglutamine disorders. To determine whether enhancing chaperone activity could mitigate the phenotype in a mammalian model, we crossbred SCA1 mice with mice over-expressing a molecular chaperone (inducible HSP70 or iHSP70). We found that high levels of HSP70 did indeed afford protection against neurodegeneration.

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.

Calcium dynamics and electrophysiological properties of cerebellar Purkinje cells in SCA1 transgenic mice.

Cerebellar Purkinje cells (PCs) from spinocerebellar ataxia type 1 (SCA1) transgenic mice develop dendritic and somatic atrophy with age. Inositol 1,4,5-trisphosphate receptor type 1 and the sarco/endoplasmic reticulum Ca(2+) ATPase pump, which regulate [Ca(2+)](i), are expressed at lower levels in these cells compared with the levels in cells from wild-type (WT) mice. To examine PCs in SCA1 mice, we used whole-cell patch clamp recording combined with fluorometric [Ca(2+)](i) and [Na(+)](i) measurements in cerebellar slices. PCs in SCA1 mice had Na(+) spikes, Ca(2+) spikes, climbing fiber (CF) electrical responses, parallel fiber (PF) electrical responses, and metabotropic glutamate receptor (mGluR)-mediated, PF-evoked Ca(2+) release from intracellular stores that were qualitatively similar to those recorded from WT mice. Under our experimental conditions, it was easier to evoke the mGluR-mediated secondary [Ca(2+)](i) increase in SCA1 PCs. The membrane resistance of SCA1 PCs was 3.3 times higher than that of WT cells, which correlated with the 1.7 times smaller cell body size. Most SCA1 PCs (but not WT) had a delayed onset (about 50--200 ms) to Na(+) spike firing induced by current injection. This delay was increased by hyperpolarizing prepulses and was eliminated by 4-aminopyridine, which suggests that this delay was due to enhancement of the A-like K(+) conductance in the SCA1 PCs. In response to CF stimulation, most PCs in mutant and WT mice had rapid, widespread [Ca(2+)](i) changes that recovered in <200 ms. Some SCA1 PCs showed a slow, localized, secondary Ca(2+) transient following the initial CF Ca(2+) transient, which may reflect release of Ca(2+) from intracellular stores. Thus, with these exceptions, the basic physiological properties of mutant PCs are similar to those of WT neurons, even with dramatic alteration of their morphology and downregulation of Ca(2+) handling molecules.

Molecular genetics of Rett syndrome and clinical spectrum of MECP2 mutations.

Rett syndrome, a neurodevelopmental disorder that is a leading cause of mental retardation in females, is caused by mutations in the X-linked gene encoding methyl-CpG-binding protein 2 (MeCP2). MECP2 mutations have subsequently been identified in patients with a variety of clinical syndromes ranging from mild learning disability in females to severe mental retardation, seizures, ataxia, and sometimes neonatal encephalopathy in males. In classic Rett syndrome, genotype-phenotype correlation studies suggest that X chromosome inactivation patterns have a more prominent effect on clinical severity than the type of mutation. When the full range of phenotypes associated with MECP2 mutations is considered, however, the mutation type strongly affects disease severity. MeCP2 is a transcriptional repressor that binds to methylated CpG dinucleotides throughout the genome, and mutations in Rett syndrome patients are thought to result in at least a partial loss of function. Abnormal gene expression may thus underlie the phenotype. Discovering which genes are misregulated in the absence of functional MeCP2 is crucial for understanding the pathogenesis of this disorder and related syndromes.

The spinocerebellar ataxia type 1 protein, ataxin-1, has RNA-binding activity that is inversely affected by the length of its polyglutamine tract.

Spinocerebellar ataxia type 1 (SCA1) is an autosomal dominant neurodegenerative disease caused by the expansion of a polyglutamine tract within the SCA1 product, ataxin-1. Previously, using transgenic mice, it was demonstrated that in order for a mutant allele of ataxin-1 to cause disease it must be transported to the nucleus of the neuron. Using an in vitro RNA-binding assay, we demonstrate that ataxin-1 does bind RNA and that this binding diminishes as the length of its polyglutamine tract increases. These observations suggest that ataxin-1 plays a role in RNA metabolism and that the expansion of the polyglutamine tract may alter this function.

Identification of genes that modify ataxin-1-induced neurodegeneration.

A growing number of human neurodegenerative diseases result from the expansion of a glutamine repeat in the protein that causes the disease. Spinocerebellar ataxia type 1 (SCA1) is one such disease-caused by expansion of a polyglutamine tract in the protein ataxin-1. To elucidate the genetic pathways and molecular mechanisms underlying neuronal degeneration in this group of diseases, we have created a model system for SCA1 by expressing the full-length human SCA1 gene in Drosophila. Here we show that high levels of wild-type ataxin-1 can cause degenerative phenotypes similar to those caused by the expanded protein. We conducted genetic screens to identify genes that modify SCA1-induced neurodegeneration. Several modifiers highlight the role of protein folding and protein clearance in the development of SCA1. Furthermore, new mechanisms of polyglutamine pathogenesis were revealed by the discovery of modifiers that are involved in RNA processing, transcriptional regulation and cellular detoxification. These findings may be relevant to the treatment of polyglutamine diseases and, perhaps, to other neurodegenerative diseases, such as Alzheimer's and Parkinson's disease.

Neurobiology of disease.

Advances in technology and basic sciences this past decade have transformed neurobiological research. Practitioners looking prospectively in 1990 could hardly have hoped for the diagnostics and rational therapeutics that have become part of regular practice today. Here, we discuss three areas that have had great impact: genetics, cell death, and stem cell/gene therapy research.

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.