Karen N. McFarland

Consensus paper: Pathological mechanisms underlying neurodegeneration in spinocerebellar ataxias

Intensive scientific research devoted in the recent years to understand the molecular mechanisms or neurodegeneration in spinocerebellar ataxias (SCAs) are identifying new pathways and targets providing new insights and a better understanding of the molecular pathogenesis in these diseases. In this consensus manuscript, the authors discuss their current views on the identified molecular processes causing or modulating the neurodegenerative phenotype in spinocerebellar ataxias with the common opinion of translating the new knowledge acquired into candidate targets for therapy. The following topics are discussed: transcription dysregulation, protein aggregation, autophagy, ion channels, the role of mitochondria, RNA toxicity, modulators of neurodegeneration and current therapeutic approaches. Overall point of consensus includes the common vision of neurodegeneration in SCAs as a multifactorial, progressive and reversible process, at least in early stages. Specific points of consensus include the role of the dysregulation of protein folding, transcription, bioenergetics, calcium handling and eventual cell death with apoptotic features of neurons during SCA disease progression. Unresolved questions include how the dysregulation of these pathways triggers the onset of symptoms and mediates disease progression since this understanding may allow effective treatments of SCAs within the window of reversibility to prevent early neuronal damage. Common opinions also include the need for clinical detection of early neuronal dysfunction, for more basic research to decipher the early neurodegenerative process in SCAs in order to give rise to new concepts for treatment strategies and for the translation of the results to preclinical studies and, thereafter, in clinical practice.

Genome-Wide Histone Acetylation Is Altered in a Transgenic Mouse Model of Huntington's Disease

In Huntingtons disease (HD; MIM ID #143100), a fatal neurodegenerative disorder, transcriptional dysregulation is a key pathogenic feature. Histone modifications are altered in multiple cellular and animal models of HD suggesting a potential mechanism for the observed changes in transcriptional levels. In particular, previous work has suggested an important link between decreased histone acetylation, particularly acetylated histone H3 (AcH3; H3K9K14ac), and downregulated gene expression. However, the question remains whether changes in histone modifications correlate with transcriptional abnormalities across the entire transcriptome. Using chromatin immunoprecipitation paired with microarray hybridization (ChIP-chip), we interrogated AcH3-gene interactions genome-wide in striata of 12-week old wild-type (WT) and transgenic (TG) R6/2 mice, an HD mouse model, and correlated these interactions with gene expression levels. At the level of the individual gene, we found decreases in the number of sites occupied by AcH3 in the TG striatum. In addition, the total number of genes bound by AcH3 was decreased. Surprisingly, the loss of AcH3 binding sites occurred within the coding regions of the genes rather than at the promoter region. We also found that the presence of AcH3 at any location within a gene strongly correlated with the presence of its transcript in both WT and TG striatum. In the TG striatum, treatment with histone deacetylase (HDAC) inhibitors increased global AcH3 levels with concomitant increases in transcript levels; however, AcH3 binding at select gene loci increased only slightly. This study demonstrates that histone H3 acetylation at lysine residues 9 and 14 and active gene expression are intimately tied in the rodent brain, and that this fundamental relationship remains unchanged in an HD mouse model despite genome-wide decreases in histone H3 acetylation.

Transgenic mice with SCA10 pentanucleotide repeats show motor phenotype and susceptibility to seizure: A toxic RNA gain-of-function model

Spinocerebellar ataxia type 10 (SCA10) is an autosomal dominant neurodegenerative disorder manifested by ataxia and seizure. SCA10 is caused by a large expansion of an intronic ATTCT pentanucleotide repeat in the ATXN10 gene. We have recently postulated a toxic RNA‐mediated gain of function in the pathogenesis of spinal cerebellar ataxia type 10 (SCA10). The spliced intron‐9 RNA containing the expanded AUUCU repeat aggregates in SCA10 cells and sequesters hnRNP K. hnRNP K sequestration triggers the translocation of protein kinase Cδ (PKCδ) to mitochondria, leading to activation of caspase‐3 and apoptosis. To confirm the toxic RNA‐mediated gain of function, we generated a new transgenic mouse model in which the expanded pentanucleotide repeats are constructed in the 3′‐untranslated region (3′UTR) to ensure transcription without translation of the repeat. We constructed an artificial transgene containing the SCA10 (ATTCT)500 track within the 3′UTR of the LacZ gene driven by the rat prion promoter (PrP) and used this to generate a new transgenic mouse model for SCA10. We then examined these mice for neurological phenotypes and histopathological, molecular, and cellular changes. The transgenic mice showed irregular gait and increased seizure susceptibility at the age of 6 months, resembling the clinical phenotype of SCA10. The cerebral cortex, hippocampus, and pontine nuclei showed neuronal loss. The brains of these animals also showed molecular and cellular changes similar to those previously found in an SCA10 cell model. Expression of the expanded SCA10 AUUCU repeat within the 3′UTR of a gene results in neuronal loss with associated gait abnormalities and increased seizure susceptibility phenotypes, which resemble those seen in SCA10 patients. Moreover, these results bolster the idea that the SCA10 disease mechanism is mediated by a toxic RNA gain‐of‐function mutation of the expanded AUUCU repeat.

MeCP2: a novel Huntingtin interactor

Transcriptional dysregulation has been proposed to play a major role in the pathology of Huntingtons disease (HD). However, the mechanisms that cause selective downregulation of target genes remain unknown. Previous studies have shown that mutant huntingtin (Htt) protein interacts with a number of transcription factors thereby altering transcription. Here we report that Htt directly interacts with methyl-CpG binding protein 2 (MeCP2) in mouse and cellular models of HD using complimentary biochemical and Fluorescent Lifetime Imaging to measure Förster Resonance Energy Transfer approaches. Htt-MeCP2 interactions are enhanced in the presence of the expanded polyglutamine (polyQ) tract and are stronger in the nucleus compared with the cytoplasm. Furthermore, we find increased binding of MeCP2 to the promoter of brain-derived neurotrophic factor (BDNF), a gene that is downregulated in HD, in the presence of mutant Htt. Finally, decreasing MeCP2 levels in mutant Htt-expressing cells using siRNA increases BDNF levels, suggesting that MeCP2 downregulates BDNF expression in HD. Taken together, these findings suggest that aberrant interactions between Htt and MeCP2 contribute to transcriptional dysregulation in HD.

ADAR2-dependent GluA2 editing regulates cocaine seeking

Activation of AMPA receptors (AMPARs) in the nucleus accumbens is necessary for the reinstatement of cocaine-seeking behavior, an animal model of drug craving and relapse. AMPARs are tetrameric protein complexes that consist of GluA1–4 subunits, of which GluA2 imparts calcium permeability. Adenosine deaminase acting on RNA 2 (ADAR2) is a nuclear enzyme that is essential for editing GluA2 pre-mRNA at Q/R site 607. Unedited GluA2(Q) subunits form calcium-permeable AMPARs (CP-AMPARs), whereas edited GluA2(R) subunits form calcium-impermeable channels (CI-AMPARs). Emerging evidence suggests that the reinstatement of cocaine seeking is associated with increased synaptic expression of CP-AMPARs in the nucleus accumbens. However, the role of GluA2 Q/R site editing and ADAR2 in cocaine seeking is unclear. In the present study, we investigated the effects of forced cocaine abstinence on GluA2 Q/R site editing and ADAR2 expression in the nucleus accumbens. Our results demonstrate that 7 days of cocaine abstinence is associated with decreased GluA2 Q/R site editing and reduced ADAR2 expression in the accumbens shell, but not core, of cocaine-experienced rats compared with yoked saline controls. To examine the functional significance of ADAR2 and GluA2 Q/R site editing in cocaine seeking, we used viral-mediated gene delivery to overexpress ADAR2b in the accumbens shell. Increased ADAR2b expression in the shell attenuated cocaine priming-induced reinstatement of drug seeking and was associated with increased GluA2 Q/R site editing and surface expression of GluA2-containing AMPARs. Taken together, these findings support the novel hypothesis that an increased contribution of accumbens shell CP-AMPARs containing unedited GluA2(Q) promotes cocaine seeking. Therefore, CP-AMPARs containing unedited GluA2(Q) represent a novel target for cocaine addiction pharmacotherapies.

Repeat interruptions in spinocerebellar ataxia type 10 expansions are strongly associated with epileptic seizures

Spinocerebellar ataxia type 10 (SCA10), an autosomal dominant neurodegenerative disorder, is the result of a non-coding, pentanucleotide repeat expansion within intron 9 of the Ataxin 10 gene. SCA10 patients present with pure cerebellar ataxia; yet, some families also have a high incidence of epilepsy. SCA10 expansions containing penta- and heptanucleotide interruption motifs, termed “ATCCT interruptions,” experience large contractions during germline transmission, particularly in paternal lineages. At the same time, these alleles confer an earlier age at onset which contradicts traditional rules of genetic anticipation in repeat expansions. Previously, ATCCT interruptions have been associated with a higher prevalence of epileptic seizures in one Mexican-American SCA10 family. In a large cohort of SCA10 families, we analyzed whether ATCCT interruptions confer a greater risk for developing seizures in these families. Notably, we find that the presence of repeat interruptions within the SCA10 expansion confers a 6.3-fold increase in the risk of an SCA10 patient developing epilepsy (6.2-fold when considering patients of Mexican ancestry only) and a 13.7-fold increase in having a positive family history of epilepsy (10.5-fold when considering patients of Mexican ancestry only). We conclude that the presence of repeat interruptions in SCA10 repeat expansion indicates a significant risk for the epilepsy phenotype and should be considered during genetic counseling.

Paradoxical effects of repeat interruptions on spinocerebellar ataxia type 10 expansions and repeat instability

Spinocerebellar ataxia type 10 (SCA10) is an autosomal dominant neurodegenerative disorder caused by a noncoding ATTCT pentanucleotide expansion. An inverse correlation between SCA10 expansion size and age at onset has been reported, and genetic anticipation has been documented. Interruptions in the ATTCT expansion are known to occur within the expansion. In order to determine the effect of repeat interruptions in SCA10 expansions, we designed a PCR assay to easily identify ATCCT repeat interruptions in the 5′-end of the expansion. We screened a cohort of 31 SCA10 families of Mexican, Brazilian and Argentinean ancestry to identify those with ATCCT repeat interruptions within their SCA10 expansions. We then studied the effects of ATCCT interruptions on intergenerational repeat instability, anticipation and age at onset. We find that the SCA10 expansion size is larger in SCA10 patients with an interrupted allele, but there is no difference in the age at onset compared with those expansions without detectable interruptions. An inverse correlation between the expansion size and the age at onset was found only with SCA10 alleles without interruptions. Interrupted expansion alleles show anticipation but are accompanied by a paradoxical contraction in intergenerational repeat size. In conclusion, we find that SCA10 expansions with ATCCT interruptions dramatically differ from SCA10 expansions without detectable ATCCT interruptions in repeat-size-instability dynamics and pathogenicity.

SMRT sequencing of long tandem nucleotide repeats in SCA10 reveals unique insight of repeat expansion structure

A large, non-coding ATTCT repeat expansion causes the neurodegenerative disorder, spinocerebellar ataxia type 10 (SCA10). In a subset of SCA10 patients, interruption motifs are present at the 5’ end of the expansion and strongly correlate with epileptic seizures. Thus, interruption motifs are a predictor of the epileptic phenotype and are hypothesized to act as a phenotypic modifier in SCA10. Yet, the exact internal sequence structure of SCA10 expansions remains unknown due to limitations in current technologies for sequencing across long extended tracts of tandem nucleotide repeats. We used the third generation sequencing technology, Single Molecule Real Time (SMRT) sequencing, to obtain full-length contiguous expansion sequences, ranging from 2.5 to 4.4 kb in length, from three SCA10 patients with different clinical presentations. We obtained sequence spanning the entire length of the expansion and identified the structure of known and novel interruption motifs within the SCA10 expansion. The exact interruption patterns in expanded SCA10 alleles will allow us to further investigate the potential contributions of these interrupting sequences to the pathogenic modification leading to the epilepsy phenotype in SCA10. Our results also demonstrate that SMRT sequencing is useful for deciphering long tandem repeats that pose as “gaps” in the human genome sequence.

Molecular biology of Huntington's disease.

It has been more than 17 years since the causative mutation for Huntingtons disease was discovered as the expansion of the triplet repeat in the N-terminal portion of the Huntingtin (HTT) gene. In the intervening time, researchers have discovered a great deal about Huntingtins involvement in a number of cellular processes. However, the role of Huntingtin in the key pathogenic mechanism leading to neurodegeneration in the disease process has yet to be discovered. Here, we review the body of knowledge that has been uncovered since gene discovery and include discussions of the HTT gene, CAG triplet repeat expansion, HTT expression, protein features, posttranslational modifications, and many of its known protein functions and interactions. We also highlight potential pathogenic mechanisms that have come to light in recent years.

Expansion of the Spinocerebellar Ataxia Type 10 (SCA10) Repeat in a Patient with Sioux Native American Ancestry

Spinocerebellar ataxia type 10 (SCA10), an autosomal dominant cerebellar ataxia, is caused by the expansion of the non-coding ATTCT pentanucleotide repeat in the ATAXIN 10 gene. To date, all cases of SCA10 are restricted to patients with ancestral ties to Latin American countries. Here, we report on a SCA10 patient with Sioux Native American ancestry and no reported Hispanic or Latino heritage. Neurological exam findings revealed impaired gait with mild, age-consistent cerebellar atrophy and no evidence of epileptic seizures. The age at onset for this patient, at 83 years of age, is the latest documented for SCA10 patients and is suggestive of a reduced penetrance allele in his family. Southern blot analysis showed an SCA10 expanded allele of 1400 repeats. Established SNPs surrounding the SCA10 locus showed a disease haplotype consistent with the previously described “SCA10 haplotype”. This case suggests that the SCA10 expansion represents an early mutation event that possibly occurred during the initial peopling of the Americas.