Guangbin Xia

Muscleblind-like 2-Mediated Alternative Splicing in the Developing Brain and Dysregulation in Myotonic Dystrophy

The RNA-mediated disease model for myotonic dystrophy (DM) proposes that microsatellite C(C)TG expansions express toxic RNAs that disrupt splicing regulation by altering MBNL1 and CELF1 activities. While this model explains DM manifestations in muscle, less is known about the effects of C(C)UG expression on the brain. Here, we report that Mbnl2 knockout mice develop several DM-associated central nervous system (CNS) features including abnormal REM sleep propensity and deficits in spatial memory. Mbnl2 is prominently expressed in the hippocampus and Mbnl2 knockouts show a decrease in NMDA receptor (NMDAR) synaptic transmission and impaired hippocampal synaptic plasticity. While Mbnl2 loss did not significantly alter target transcript levels in the hippocampus, misregulated splicing of hundreds of exons was detected using splicing microarrays, RNA-seq, and HITS-CLIP. Importantly, the majority of the Mbnl2-regulated exons examined were similarly misregulated in DM. We propose that major pathological features of the DM brain result from disruption of the MBNL2-mediated developmental splicing program.

Generation of human induced pluripotent stem cells to model spinocerebellar ataxia type 2 in vitro

Spinocerebellar ataxia type 2 (SCA2) is caused by triple nucleotide repeat (CAG) expansion in the coding region of the ATAXN2 gene on chromosome 12, which produces an elongated, toxic polyglutamine tract, leading to Purkinje cell loss. There is currently no effective therapy. One of the main obstacles that hampers therapeutic development is lack of an ideal disease model. In this study, we have generated and characterized SCA2-induced pluripotent stem (iPS) cell lines as an in vitro cell model. Dermal fibroblasts (FBs) were harvested from primary cultures of skin explants obtained from a SCA2 subject and a healthy subject. For reprogramming, hOct4, hSox2, hKlf4, and hc-Myc were transduced to passage-3 FBs by retroviral infection. Both SCA2 iPS and control iPS cells were successfully generated and showed typical stem cell growth patterns with normal karyotype. All iPS cell lines expressed stem cell markers and differentiated in vitro into cells from three embryonic germ layers. Upon in vitro neural differentiation, SCA2 iPS cells showed abnormality in neural rosette formation but successfully differentiated into neural stem cells (NSCs) and subsequent neural cells. SCA2 and normal FBs showed a comparable level of ataxin-2 expression; whereas SCA2 NSCs showed less ataxin-2 expression than normal NSCs and SCA2 FBs. Within the neural lineage, neurons had the most abundant expression of ataxin-2. Time-lapsed neural growth assay indicated terminally differentiated SCA2 neural cells were short-lived compared with control neural cells. The expanded CAG repeats of SCA2 were stable throughout reprogramming and neural differentiation. In conclusion, we have established the first disease-specific human SCA2 iPS cell line. These mutant iPS cells have the potential for neural differentiation. These differentiated neural cells harboring mutations are invaluable for the study of SCA2 pathogenesis and therapeutic drug development.

Generation of Neural Cells from DM1 Induced Pluripotent Stem Cells As Cellular Model for the Study of Central Nervous System Neuropathogenesis

Dystrophia myotonica type 1 (DM1) is an autosomal dominant multisystem disorder. The pathogenesis of central nervous system (CNS) involvement is poorly understood. Disease-specific induced pluripotent stem cell (iPSC) lines would provide an alternative model. In this study, we generated two DM1 lines and a normal iPSC line from dermal fibroblasts by retroviral transduction of Yamanakas four factors (hOct4, hSox2, hKlf4, and hc-Myc). Both DM1 and control iPSC clones showed typical human embryonic stem cell (hESC) growth patterns with a high nuclear-to-cytoplasm ratio. The iPSC colonies maintained the same growth pattern through subsequent passages. All iPSC lines expressed stem cell markers and differentiated into cells derived from three embryonic germ layers. All iPSC lines underwent normal neural differentiation. Intranuclear RNA foci, a hallmark of DM1, were detected in DM1 iPSCs, neural stem cells (NSCs), and terminally differentiated neurons and astrocytes. In conclusion, we have successfully established disease-specific human DM1 iPSC lines, NSCs, and neuronal lineages with pathognomonic intranuclear RNA foci, which offer an unlimited cell resource for CNS mechanistic studies and a translational platform for therapeutic development.

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.

RAN Translation Regulated by Muscleblind Proteins in Myotonic Dystrophy Type 2

Several microsatellite-expansion diseases are characterized by the accumulation of RNA foci and RAN proteins, raising the possibility of a mechanistic connection. We explored this question using myotonic dystrophy type 2, a multisystemic disease thought to be primarily caused by RNA gain-of-function effects. We demonstrate that the DM2 CCTG⋅CAGG expansion expresses sense and antisense tetrapeptide poly-(LPAC) and poly-(QAGR) RAN proteins, respectively. In DM2 autopsy brains, LPAC is found in neurons, astrocytes, and glia in gray matter, and antisense QAGR proteins accumulate within white matter. LPAC and QAGR proteins are toxic to cells independent of RNA gain of function. RNA foci and nuclear sequestration of CCUG transcripts by MBNL1 is inversely correlated with LPAC expression. These data suggest a model that involves nuclear retention of expansion RNAs by RNA-binding proteins (RBPs) and an acute phase in which expansion RNAs exceed RBP sequestration capacity, are exported to the cytoplasm, and undergo RAN translation. VIDEO ABSTRACT.

Depression and clinical progression in spinocerebellar ataxias

BACKGROUND Depression is a common comorbidity in spinocerebellar ataxias (SCAs) but its association with ataxia progression is not well understood. OBJECTIVES To study the prevalence and influence of depressive symptoms in SCAs. METHODS We studied 300 participants with SCA 1, 2, 3 and 6 from the Clinical Research Consortium for Spinocerebellar Ataxias (CRC-SCA) and repeatedly measured depressive symptoms by the 9-item Patient Health Questionnaire (PHQ-9) along with other clinical features including ataxia, functional status, and quality of life every 6 months for 2 years. We employed regression models to study the effects of depressive symptoms on clinical progression indexed by Scale for Assessment and Rating of Ataxia (SARA), Unified Huntingtons Disease Rating Scale Part IV (UHDRS-IV) and EQ5D after adjusting for age, sex and pathological CAG repeats. RESULTS Comorbid depression is common in SCAs (26%). Although the baseline prevalence of depression was similar among different SCA types, suicidal ideation was more frequently reported in SCA3 (65%). Depressive symptoms were associated with SARA scores but did not significantly progress over time within 2 years or deteriorate by increased numbers of pathological CAG repeats. The effects of depression on ataxia progression varied across different SCA types. Nevertheless, depression had consistently negative and significant impact on functional status and quality of life in all SCAs, even after accounting for ataxia progression. CONCLUSIONS Depressive symptoms are not simply the consequence of motor disability in SCAs. Comorbid depression per se contributes to different health outcomes and deserves more attention when caring patients with SCAs.

A knockin mouse model of spinocerebellar ataxia type 3 exhibits prominent aggregate pathology and aberrant splicing of the disease gene transcript

Polyglutamine diseases, including spinocerebellar ataxia type 3 (SCA3), are caused by CAG repeat expansions that encode abnormally long glutamine repeats in the respective disease proteins. While the mechanisms underlying neurodegeneration remain uncertain, evidence supports a proteotoxic role for the mutant protein dictated in part by the specific genetic and protein context. To further define pathogenic mechanisms in SCA3, we generated a mouse model in which a CAG expansion of 82 repeats was inserted into the murine locus by homologous recombination. SCA3 knockin mice exhibit region-specific aggregate pathology marked by intranuclear accumulation of the mutant Atxn3 protein, abundant nuclear inclusions and, in select brain regions, extranuclear aggregates localized to neuritic processes. Knockin mice also display altered splicing of the disease gene, promoting expression of an alternative isoform in which the intron immediately downstream of the CAG repeat is retained. In an independent mouse model expressing the full human ATXN3 disease gene, expression of this alternatively spliced transcript is also enhanced. These results, together with recent findings in other polyglutamine diseases, suggest that CAG repeat expansions can promote aberrant splicing to produce potentially more aggregate-prone isoforms of the disease proteins. This report of a SCA3 knockin mouse expands the repertoire of existing models of SCA3, and underscores the potential contribution of alternative splicing to disease pathogenesis in SCA3 and other polyglutamine disorders.

Dynamic changes of nuclear RNA foci in proliferating DM1 cells

Abstract Nuclear RNA foci are molecular hallmarks of myotonic dystrophy type 1 (DM1). However, no designated study has investigated their formation and changes in proliferating cells. Proliferating cells, as stem cells, consist of an important cellular pool in the human body. The revelation of foci changes in these cells might shed light on the effects of the mutation on these specific cells and tissues. In this study, we used human DM1 iPS-cell-derived neural stem cells (NSCs) as cellular models to investigate the formation and dynamic changes of RNA foci in proliferating cells. Human DM1 NSCs derived from human DM1 iPS cells were cultured under proliferation conditions and nonproliferation conditions following mitomycin C treatment. The dynamic changes of foci during the cell cycle were investigated by fluorescence in situ hybridization. We found RNA foci formed and dissociated during the cell cycle. Nuclear RNA foci were most prominent in number and size just prior to entering mitosis (early prophase). During mitosis, most foci disappeared. After entering interphase, RNA foci accumulated again in the nuclei. After stopping cell dividing by treatment of mitomycin C, the number of nuclear RNA foci increased significantly. In summary, DM1 NSC nuclear RNA foci undergo dynamic changes during cell cycle, and mitosis is a mechanism to decrease foci load in the nuclei, which may explain why dividing cells are less affected by the mutation. The dynamic changes need to be considered when using foci as a marker to monitor the effects of therapeutic drugs.

Impeding Transcription of Expanded Microsatellite Repeats by Deactivated Cas9

Transcription of expanded microsatellite repeats is associated with multiple human diseases, including myotonic dystrophy, Fuchs endothelial corneal dystrophy, and C9orf72-ALS/FTD. Reducing production of RNA and proteins arising from these expanded loci holds therapeutic benefit. Here, we tested the hypothesis that deactivated Cas9 enzyme impedes transcription across expanded microsatellites. We observed a repeat length-, PAM-, and strand-dependent reduction of repeat-containing RNAs upon targeting dCas9 directly to repeat sequences; targeting the non-template strand was more effective. Aberrant splicing patterns were rescued in DM1 cells, and production of RAN peptides characteristic of DM1, DM2, and C9orf72-ALS/FTD cells was drastically decreased. Systemic delivery of dCas9/gRNA by adeno-associated virus led to reductions in pathological RNA foci, rescue of chloride channel 1 protein expression, and decreased myotonia. These observations suggest that transcription of microsatellite repeat-containing RNAs is more sensitive to perturbation than transcription of other RNAs, indicating potentially viable strategies for therapeutic intervention.

Coenzyme Q10 and spinocerebellar ataxias

The aim of this study was to investigate the association between drug exposure and disease severity in SCA types 1, 2, 3 and 6. The Clinical Research Consortium for Spinocerebellar Ataxias (CRC‐SCA) enrolled 319 participants with SCA1, 2, 3, and 6 from 12 medical centers in the United States and repeatedly measured clinical severity by the Scale for Assessment and Rating of Ataxia (SARA), the Unified Huntingtons Disease Rating Scale part IV (UHDRS‐IV), and the 9‐item Patient Health Questionnaire during July 2009 to May 2012. We employed generalized estimating equations in regression models to study the longitudinal effects of coenzyme Q10 (CoQ10), statin, and vitamin E on clinical severity of ataxia after adjusting for age, sex, and pathological CAG repeat number. Cross‐sectionally, exposure to CoQ10 was associated with lower SARA and higher UHDRS‐IV scores in SCA1 and 3. No association was found between statins, vitamin E, and clinical outcome. Longitudinally, CoQ10, statins, and vitamin E did not change the rates of clinical deterioration indexed by SARA and UHDRS‐IV scores within 2 years. CoQ10 is associated with better clinical outcome in SCA1 and 3. These drug exposures did not appear to influence clinical progression within 2 years. Further studies are warranted to confirm the association.