Expression levels of DNA replication and repair genes predict regional somatic repeat instability in the brain but are not altered by polyglutamine disease protein expression or age.

Expansion of CAG/CTG trinucleotide repeats causes numerous inherited neurological disorders, including Huntington's disease (HD), several spinocerebellar ataxias and myotonic dystrophy type 1. Expanded repeats are genetically unstable with a propensity to further expand when transmitted from parents to offspring. For many alleles with expanded repeats, extensive somatic mosaicism has been documented. For CAG repeat diseases, dramatic instability has been documented in the striatum, with larger expansions noted with advancing age. In contrast, only modest instability occurs in the cerebellum. Using microarray expression analysis, we sought to identify the genetic basis of these regional instability differences by comparing gene expression in the striatum and cerebellum of aged wild-type C57BL/6J mice. We identified eight candidate genes enriched in cerebellum, and validated four--Pcna, Rpa1, Msh6 and Fen1--along with a highly associated interactor, Lig1. We also explored whether expression levels of mismatch repair (MMR) proteins are altered in a line of HD transgenic mice, R6/2, that is known to show pronounced regional repeat instability. Compared with wild-type littermates, MMR expression levels were not significantly altered in R6/2 mice regardless of age. Interestingly, expression levels of these candidates were significantly increased in the cerebellum of control and HD human samples in comparison to striatum. Together, our data suggest that elevated expression levels of DNA replication and repair proteins in cerebellum may act as a safeguard against repeat instability, and may account for the dramatically reduced somatic instability present in this brain region, compared with the marked instability observed in the striatum.

Sporadic ALS has compartment-specific aberrant exon splicing and altered cell-matrix adhesion biology.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by progressive weakness from loss of motor neurons. The fundamental pathogenic mechanisms are unknown and recent evidence is implicating a significant role for abnormal exon splicing and RNA processing. Using new comprehensive genomic technologies, we studied exon splicing directly in 12 sporadic ALS and 10 control lumbar spinal cords acquired by a rapid autopsy system that processed nervous systems specifically for genomic studies. ALS patients had rostral onset and caudally advancing disease and abundant residual motor neurons in this region. We created two RNA pools, one from motor neurons collected by laser capture microdissection and one from the surrounding anterior horns. From each, we isolated RNA, amplified mRNA, profiled whole-genome exon splicing, and applied advanced bioinformatics. We employed rigorous quality control measures at all steps and validated findings by qPCR. In the motor neuron enriched mRNA pool, we found two distinct cohorts of mRNA signals, most of which were up-regulated: 148 differentially expressed genes (P

Thermoregulatory and metabolic defects in Huntington's disease transgenic mice implicate PGC-1alpha in Huntington's disease neurodegeneration.

Huntington's disease (HD) is a fatal, dominantly inherited disorder caused by polyglutamine repeat expansion in the huntingtin (htt) gene. Here, we observe that HD mice develop hypothermia associated with impaired activation of brown adipose tissue (BAT). Although sympathetic stimulation of PPARgamma coactivator 1alpha (PGC-1alpha) was intact in BAT of HD mice, uncoupling protein 1 (UCP-1) induction was blunted. In cultured cells, expression of mutant htt suppressed UCP-1 promoter activity; this was reversed by PGC-1alpha expression. HD mice showed reduced food intake and increased energy expenditure, with dysfunctional BAT mitochondria. PGC-1alpha is a known regulator of mitochondrial function; here, we document reduced expression of PGC-1alpha target genes in HD patient and mouse striatum. Mitochondria of HD mouse brain show reduced oxygen consumption rates. Finally, HD striatal neurons expressing exogenous PGC-1alpha were resistant to 3-nitropropionic acid treatment. Altered PGC-1alpha function may thus link transcription dysregulation and mitochondrial dysfunction in HD.

CTCF cis-regulates trinucleotide repeat instability in an epigenetic manner: a novel basis for mutational hot spot determination.

At least 25 inherited disorders in humans result from microsatellite repeat expansion. Dramatic variation in repeat instability occurs at different disease loci and between different tissues; however, cis-elements and trans-factors regulating the instability process remain undefined. Genomic fragments from the human spinocerebellar ataxia type 7 (SCA7) locus, containing a highly unstable CAG tract, were previously introduced into mice to localize cis-acting "instability elements," and revealed that genomic context is required for repeat instability. The critical instability-inducing region contained binding sites for CTCF -- a regulatory factor implicated in genomic imprinting, chromatin remodeling, and DNA conformation change. To evaluate the role of CTCF in repeat instability, we derived transgenic mice carrying SCA7 genomic fragments with CTCF binding-site mutations. We found that CTCF binding-site mutation promotes triplet repeat instability both in the germ line and in somatic tissues, and that CpG methylation of CTCF binding sites can further destabilize triplet repeat expansions. As CTCF binding sites are associated with a number of highly unstable repeat loci, our findings suggest a novel basis for demarcation and regulation of mutational hot spots and implicate CTCF in the modulation of genetic repeat instability.

Bergmann glia expression of polyglutamine-expanded ataxin-7 produces neurodegeneration by impairing glutamate transport.

Non-neuronal cells may be pivotal in neurodegenerative disease, but the mechanistic basis of this effect remains ill-defined. In the polyglutamine disease spinocerebellar ataxia type 7 (SCA7), Purkinje cells undergo non-cell-autonomous degeneration in transgenic mice. We considered the possibility that glial dysfunction leads to Purkinje cell degeneration, and generated mice that express ataxin-7 in Bergmann glia of the cerebellum with the Gfa2 promoter. Bergmann glia-specific expression of mutant ataxin-7 was sufficient to produce ataxia and neurodegeneration. Expression of the Bergmann glia-specific glutamate transporter GLAST was reduced in Gfa2-SCA7 mice and was associated with impaired glutamate transport in cultured Bergmann glia, cerebellar slices and cerebellar synaptosomes. Ultrastructural analysis of Purkinje cells revealed findings of dark cell degeneration consistent with excitotoxic injury. Our studies indicate that impairment of glutamate transport secondary to glial dysfunction contributes to SCA7 neurodegeneration, and suggest a similar role for glial dysfunction in other polyglutamine diseases and SCAs.

Androgen receptor YAC transgenic mice recapitulate SBMA motor neuronopathy and implicate VEGF164 in the motor neuron degeneration.

X-linked spinal and bulbar muscular atrophy (SBMA) is an inherited neuromuscular disorder characterized by lower motor neuron degeneration. SBMA is caused by polyglutamine repeat expansions in the androgen receptor (AR). To determine the basis of AR polyglutamine neurotoxicity, we introduced human AR yeast artificial chromosomes carrying either 20 or 100 CAGs into mouse embryonic stem cells. The AR100 transgenic mice developed a late-onset, gradually progressive neuromuscular phenotype accompanied by motor neuron degeneration, indicating striking recapitulation of the human disease. We then tested the hypothesis that polyglutamine-expanded AR interferes with CREB binding protein (CBP)-mediated transcription of vascular endothelial growth factor (VEGF) and observed altered CBP-AR binding and VEGF reduction in AR100 mice. We found that mutant AR-induced death of motor neuron-like cells could be rescued by VEGF. Our results suggest that SBMA motor neuronopathy involves altered expression of VEGF, consistent with a role for VEGF as a neurotrophic/survival factor in motor neuron disease.

Polyglutamine-expanded ataxin-7 promotes non-cell-autonomous purkinje cell degeneration and displays proteolytic cleavage in ataxic transgenic mice.

Spinocerebellar ataxia (SCA) type 7 is an inherited neurodegenerative disorder caused by expansion of a polyglutamine tract within the ataxin-7 protein. To determine the molecular basis of polyglutamine neurotoxicity in this and other related disorders, we produced SCA7 transgenic mice that express ataxin-7 with 24 or 92 glutamines in all neurons of the CNS, except for Purkinje cells. Transgenic mice expressing ataxin-7 with 92 glutamines (92Q) developed a dramatic neurological phenotype presenting as a gait ataxia and culminating in premature death. Despite the absence of expression of polyglutamine-expanded ataxin-7 in Purkinje cells, we documented severe Purkinje cell degeneration in 92Q SCA7 transgenic mice. We also detected an N-terminal truncation fragment of ataxin-7 in transgenic mice and in SCA7 patient material with both anti-ataxin-7 and anti-polyglutamine specific antibodies. The appearance of truncated ataxin-7 in nuclear aggregates correlates with the onset of a disease phenotype in the SCA7 mice, suggesting that nuclear localization and proteolytic cleavage may be important features of SCA7 pathogenesis. The non-cell-autonomous nature of the Purkinje cell degeneration in our SCA7 mouse model indicates that polyglutamine-induced dysfunction in adjacent or connecting cell types contributes to the neurodegeneration.

A SCA7 CAG/CTG repeat expansion is stable in Drosophila melanogaster despite modulation of genomic context and gene dosage.

CAG and CTG repeat expansions are the cause of at least a dozen inherited neurological disorders. In these so-called "dynamic mutation" diseases, the expanded repeats display dramatic genetic instability, changing in size when transmitted through the germline and within somatic tissues. As the molecular basis of the repeat instability process remains poorly understood, modeling of repeat instability in model organisms has provided some insights into potentially involved factors, implicating especially replication and repair pathways. Studies in mice have also shown that the genomic context of the repeat sequence is required for CAG/CTG repeat instability in the case of spinocerebellar ataxia type 7 (SCA7), one of the most unstable of all CAG/CTG repeat disease loci. While most studies of repeat instability have taken a candidate gene approach, unbiased screens for factors involved in trinucleotide repeat instability have been lacking. We therefore attempted to use Drosophila melanogaster to model expanded CAG repeat instability by creating transgenic flies carrying trinucleotide repeat expansions, deriving flies with SCA7 CAG90 repeats in cDNA and genomic context. We found that SCA7 CAG90 repeats are stable in Drosophila, regardless of context. To screen for genes whose reduced function might destabilize expanded CAG repeat tracts in Drosophila, we crossed the SCA7 CAG90 repeat flies with various deficiency stocks, including lines lacking genes encoding the orthologues of flap endonuclease-1, PCNA, and MutS. In all cases, perfect repeat stability was preserved, suggesting that Drosophila may not be a suitable system for determining the molecular basis of SCA7 CAG repeat instability.

CTCF regulates ataxin-7 expression through promotion of a convergently transcribed, antisense noncoding RNA.

Spinocerebellar ataxia type 7 (SCA7) is a neurodegenerative disorder caused by CAG/polyglutamine repeat expansions in the ataxin-7 gene. Ataxin-7 is a component of two different transcription coactivator complexes, and recent work indicates that disease protein normal function is altered in polyglutamine neurodegeneration. Given this, we studied how ataxin-7 gene expression is regulated. The ataxin-7 repeat and translation start site are flanked by binding sites for CTCF, a highly conserved multifunctional transcription regulator. When we analyzed this region, we discovered an adjacent alternative promoter and a convergently transcribed antisense noncoding RNA, SCAANT1. To understand how CTCF regulates ataxin-7 gene expression, we introduced ataxin-7 mini-genes into mice, and found that CTCF is required for SCAANT1 expression. Loss of SCAANT1 derepressed ataxin-7 sense transcription in a cis-dependent fashion and was accompanied by chromatin remodeling. Discovery of this pathway underscores the importance of altered epigenetic regulation for disease pathology at repeat loci exhibiting bidirectional transcription.

Activation of the extrinsic caspase pathway in cultured cortical neurons requires p53-mediated down-regulation of the X-linked inhibitor of apoptosis protein to induce apoptosis.

Cultured cortical neurons exposed to the Human Immunodeficiency Virus gp120 coat protein undergo apoptosis involving activation of both caspase-8 and caspase-9. Additionally, gp120-mediated neuronal apoptosis requires the pro-apoptotic transcription factor p53. As caspase-8-induced apoptosis does not typically require p53, we examined the possibility of a novel role for p53 in caspase-8 activation initiated by gp120. We observed that gp120 treatment of cultured cortical neurons induced caspase-8 activity and Bid cleavage independently of p53, but induction of caspase-3 enzymatic activity required p53 expression. These findings suggested the possibility that p53 down-regulates a caspase-3 inhibitor. We observed high-level expression of the caspase-3/9 inhibitor X-linked inhibitor of apoptosis protein (XIAP) in cultured cortical neurons. Adenoviral expression of p53 or induction of endogenous p53 by camptothecin treatment reduced XIAP protein in neurons. Infection with a p53 expressing adenovirus increased expression of the mRNA for Omi/HtrA2, a protease that cleaves and inactivates XIAP. These findings suggest that p53 regulates neuronal apoptosis, in part, by suppressing the anti-apoptotic protein XIAP via transcriptional activation of Omi/HtrA2.

Genomic context drives SCA7 CAG repeat instability, while expressed SCA7 cDNAs are intergenerationally and somatically stable in transgenic mice.

Spinocerebellar ataxia type 7 (SCA7) is an autosomal dominant cerebellar ataxia caused by a CAG repeat expansion in the ataxin-7 gene. In humans, SCA7 is characterized by marked anticipation due to intergenerational repeat instability with a bias toward expansion, and is thus regarded as the most unstable of the polyglutamine diseases. To study the molecular basis of CAG/CTG repeat instability and its pathological significance, we generated lines of transgenic mice carrying either a SCA7 cDNA construct or a 13.5 kb SCA7 genomic fragment with 92 CAG repeats. While the cDNA transgenic mice showed little intergenerational repeat instability, the genomic fragment transgenic mice displayed marked intergenerational instability with an obvious expansion bias. We then went on to generate additional lines of genomic fragment transgenic mice, and observed that deletion of the 3' genomic region significantly stabilized intergenerational transmission of the SCA7 CAG92 repeat. These results suggest that cis-information present on the genomic fragment is driving the instability process. As the SCA7 genomic fragment contains a large number of replication-associated motifs, the presence of such sequence elements may make the SCA7 CAG repeat region more susceptible to instability. Small-pool and standard PCR analysis of tissues from genomic fragment mice revealed large repeat expansions in their brains and livers, but no such changes were found in any tissues from cDNA transgenic mice that have been shown to undergo neurodegeneration. As large somatic repeat expansions are absent from the brains of SCA7 cDNA mice, our results indicate that neurodegeneration can occur without marked somatic mosaicism, at least in these mice.