Co-expression networks in generation of induced pluripotent stem cells.

We developed an adenoviral vector, in which Yamanaka's four reprogramming factors (RFs) were controlled by individual CMV promoters in a single cassette (Ad-SOcMK). This permitted coordinated expression of RFs (SOX2, OCT3/4, c-MYC and KLF4) in a cell for a transient period of time, synchronizing the reprogramming process with the majority of transduced cells assuming induced pluripotent stem cell (iPSC)-like characteristics as early as three days post-transduction. These reprogrammed cells resembled human embryonic stem cells (ESCs) with regard to morphology, biomarker expression, and could be differentiated into cells of the germ layers in vitro and in vivo. These iPSC-like cells, however, failed to expand into larger iPSC colonies. The short and synchronized reprogramming process allowed us to study global transcription changes within short time intervals. Weighted gene co-expression network analysis (WGCNA) identified sixteen large gene co-expression modules, each including members of gene ontology categories involved in cell differentiation and development. In particular, the brown module contained a significant number of ESC marker genes, whereas the turquoise module contained cell-cycle-related genes that were downregulated in contrast to upregulation in human ESCs. Strong coordinated expression of all four RFs via adenoviral transduction may constrain stochastic processes and lead to silencing of genes important for cellular proliferation

Muscleblind-like 3 deficit results in a spectrum of age-associated pathologies observed in myotonic dystrophy.

Myotonic dystrophy type I (DM1) exhibits distinctive disease specific phenotypes and the accelerated onset of a spectrum of age-associated pathologies. In DM1, dominant effects of expanded CUG repeats result in part from the inactivation of the muscleblind-like (MBNL) proteins. To test the role of MBNL3, we deleted Mbnl3 exon 2 (Mbnl3(ΔE2)) in mice and examined the onset of age-associated diseases over 4 to 13 months of age. Accelerated onset of glucose intolerance with elevated insulin levels, cardiac systole deficits, left ventricle hypertrophy, a predictor of a later onset of heart failure and the development of subcapsular and cortical cataracts is observed in Mbnl3(ΔE2) mice. Retention of embryonic splice isoforms in adult organs, a prominent defect in DM1, is not observed in multiple RNAs including the Insulin Receptor (Insr), Cardiac Troponin T (Tnnt2), Lim Domain Binding 3 (Ldb3) RNAs in Mbnl3(ΔE2) mice. Although rare DM1-like splice errors underlying the observed phenotypes cannot be excluded, our data in conjunction with the reported absence of alternative splice errors in embryonic muscles of a similar Mbnl3(ΔE2) mouse by RNA-seq studies, suggest that mechanisms distinct from the adult retention of embryonic splice patterns may make important contributions to the onset of age-associated pathologies in DM1

Muscleblind1, but Not Dmpk or Six5, Contributes to a Complex Phenotype of Muscular and Motivational Deficits in Mouse Models of Myotonic Dystrophy

Assessment of molecular defects that underlie cognitive deficits observed in mendelian disorders provides a unique opportunity to identify key regulators of human cognition. Congenital Myotonic Dystrophy 1 (cDM1), a multi-system disorder is characterized by both cognitive deficits and a spectrum of behavioral abnormalities, which include visuo-spatial memory deficits, anxiety and apathy. Decreased levels of DMPK (Dystrophia Myotonica-protein kinase), SIX5, a transcription factor or MBNL1 (Muscleblind-like 1), an RNA splice regulator have been demonstrated to contribute to distinct features of cDM1. Mouse strains in which either Dmpk, Six5 or Mbnl1 are inactivated were therefore studied to determine the relative contribution of each gene to these cognitive functions. The open field and elevated plus maze tasks were used to examine anxiety, sucrose consumption was used to assess motivation, whereas the water maze and context fear conditioning were used to examine spatial learning and memory. Cognitive and behavioral abnormalities were observed only in Mbnl1 deficient mice, which demonstrate behavior consistent with motivational deficits in the Morris water maze, a complex visuo-spatial task and in the sucrose consumption test for anhedonia. All three models of cDM1 exhibit normal spatial learning and memory. These data identify MBNL1 as a potential regulator of emotional state with decreased MBNL1 levels underlying the motivational deficits observed in cDM1

Gene co-expression network analysis for identifying modules and functionally enriched pathways in SCA2

Spinocerebellar ataxia type 2 (SCA2) is an autosomal dominant neurodegenerative disease caused by CAG repeat expansion in the ATXN2 gene. The repeat resides in an encoded region of the gene resulting in polyglutamine (polyQ) expansion which has been assumed to result in gain of function, predominantly, for the ATXN2 protein. We evaluated temporal cerebellar expression profiles by RNA sequencing of ATXN2Q127 mice versus wild-type (WT) littermates. ATXN2Q127 mice are characterized by a progressive motor phenotype onset, and have progressive cerebellar molecular and neurophysiological (Purkinje cell firing frequency) phenotypes. Our analysis revealed previously uncharacterized early and progressive abnormal patterning of cerebellar gene expression. Weighted Gene Coexpression Network Analysis revealed four gene modules that were significantly correlated with disease status, composed primarily of genes associated with GTPase signaling, calcium signaling and cell death. Of these genes, few overlapped with differentially expressed cerebellar genes that we identified in Atxn2−/− knockout mice versus WT littermates, suggesting that loss-of-function is not a significant component of disease pathology. We conclude that SCA2 is a disease characterized by gain of function for ATXN2

Antisense oligonucleotide therapy for spinocerebellar ataxia type 2

There are no disease-modifying treatments for adult human neurodegenerative diseases. Here we test RNA-targeted therapies1 in two mouse models of spinocerebellar ataxia type 2 (SCA2), an autosomal dominant polyglutamine disease2. Both models recreate the progressive adult-onset dysfunction and degeneration of a neuronal network that are seen in patients, including decreased firing frequency of cerebellar Purkinje cells and a decline in motor function3,4. We developed a potential therapy directed at the ATXN2 gene by screening 152 antisense oligonucleotides (ASOs). The most promising oligonucleotide, ASO7, downregulated ATXN2 mRNA and protein, which resulted in delayed onset of the SCA2 phenotype. After delivery by intracerebroventricular injection to ATXN2-Q127 mice, ASO7 localized to Purkinje cells, reduced cerebellar ATXN2 expression below 75% for more than 10 weeks without microglial activation, and reduced the levels of cerebellar ATXN2. Treatment of symptomatic mice with ASO7 improved motor function compared to saline-treated mice. ASO7 had a similar effect in the BAC-Q72 SCA2 mouse model, and in both mouse models it normalized protein levels of several SCA2-related proteins expressed in Purkinje cells, including Rgs8, Pcp2, Pcp4, Homer3, Cep76 and Fam107b. Notably, the firing frequency of Purkinje cells returned to normal even when treatment was initiated more than 12 weeks after the onset of the motor phenotype in BAC-Q72 mice. These findings support ASOs as a promising approach for treating some human neurodegenerative diseases

Altered Capicua expression drives regional Purkinje neuron vulnerability through ion channel gene dysregulation in spinocerebellar ataxia type 1

Selective neuronal vulnerability in neurodegenerative disease is poorly understood. Using the ATXN1[82Q] model of spinocerebellar ataxia type 1 (SCA1), we explored the hypothesis that regional differences in Purkinje neuron degeneration could provide novel insights into selective vulnerability. ATXN1[82Q] Purkinje neurons from the anterior cerebellum were found to degenerate earlier than those from the nodular zone, and this early degeneration was associated with selective dysregulation of ion channel transcripts and altered Purkinje neuron spiking. Efforts to understand the basis for selective dysregulation of channel transcripts revealed modestly increased expression of the ATXN1 co-repressor Capicua (Cic) in anterior cerebellar Purkinje neurons. Importantly, disrupting the association between ATXN1 and Cic rescued the levels of these ion channel transcripts, and lentiviral overexpression of Cic in the nodular zone accelerated both aberrant Purkinje neuron spiking and neurodegeneration. These findings reinforce the central role for Cic in SCA1 cerebellar pathophysiology and suggest that only modest reductions in Cic are needed to have profound therapeutic impact in SCA1

New function for the RNA helicase p68/DDX5 as a modifier of MBNL1 activity on expanded CUG repeats

Myotonic Dystrophy type I (DM1) is caused by an abnormal expansion of CTG triplets in the 3′ UTR of the dystrophia myotonica protein kinase (DMPK) gene, leading to the aggregation of the mutant transcript in nuclear RNA foci. The expanded mutant transcript promotes the sequestration of the MBNL1 splicing factor, resulting in the misregulation of a subset of alternative splicing events. In this study, we identify the DEAD-box RNA helicase p68 (DDX5) in complexes assembled onto in vitro-transcribed CUG repeats. We showed that p68 colocalized with RNA foci in cells expressing the 3′UTR of the DMPK gene containing expanded CTG repeats. We found that p68 increased MBNL1 binding onto pathological repeats and the stem–loop structure regulatory element within the cardiac Troponin T (TNNT2) pre-mRNA, splicing of which is misregulated in DM1. Mutations in the helicase core of p68 prevented both the stimulatory effect of the protein on MBNL1 binding and the colocalization of p68 with CUG repeats, suggesting that remodeling of RNA secondary structure by p68 facilitates MBNL1 binding. We also found that the competence of p68 for regulating TNNT2 exon 5 inclusion depended on the integrity of MBNL1 binding sites. We propose that p68 acts as a modifier of MBNL1 activity on splicing targets and pathogenic RNA

RNA steady-state defects in myotonic dystrophy are linked to nuclear exclusion of SHARP

We describe a new mechanism by which CTG tract expansion affects myotonic dystrophy (DM1). Changes to the levels of a panel of RNAs involved in muscle development and function that are downregulated in DM1 are due to aberrant localization of the transcription factor SHARP (SMART/HDAC1-associated repressor protein). Mislocalization of SHARP in DM1 is consistent with increased CRM1-mediated export of SHARP to the cytoplasm. A direct link between CTG repeat expression and SHARP mislocalization is demonstrated as expression of expanded CTG repeats in normal cells recapitulates cytoplasmic SHARP localization. These results demonstrate a role for the inactivation of SHARP transcription in DM1 biology

Identification of MBNL1 and MBNL3 domains required for splicing activation and repression

Muscleblind-like 1 (MBNL1) is a splicing regulator that controls developmentally regulated alternative splicing of a large number of exons including exon 11 of the Insulin Receptor (IR) gene and exon 5 of the cardiac Troponin T (cTNT) gene. There are three paralogs of MBNL in humans, all of which promote IR exon 11 inclusion and cTNT exon 5 skipping. Here, we identify a cluster of three binding sequences located downstream of IR exon 11 that constitute the MBNL1 response element and a weaker response element in the upstream intron. In addition, we used sequential deletions to define the functional domains of MBNL1 and MBNL3. We demonstrate that the regions required for splicing regulation are separate from the two pairs of zinc-finger RNA-binding domains. MBNL1 and MBNL3 contain core regulatory regions for both activation and repression located within an 80-amino-acid segment located downstream of the N-terminal zinc-finger pair. Deletions of these regions abolished regulation without preventing RNA binding. These domains have common features with the CUG-BP and ETR3-like Factor (CELF) family of splicing regulators. These results have identified protein domains required for splicing repression and activation and provide insight into the mechanism of splicing regulation by MBNL proteins

Cytoplasmic CUG RNA Foci Are Insufficient to Elicit Key DM1 Features

The genetic basis of myotonic dystrophy type I (DM1) is the expansion of a CTG tract located in the 3′ untranslated region of DMPK. Expression of mutant RNAs encoding expanded CUG repeats plays a central role in the development of cardiac disease in DM1. Expanded CUG tracts form both nuclear and cytoplasmic aggregates, yet the relative significance of such aggregates in eliciting DM1 pathology is unclear. To test the pathophysiology of CUG repeat encoding RNAs, we developed and analyzed mice with cardiac-specific expression of a beta-galactosidase cassette in which a (CTG)400 repeat tract was positioned 3′ of the termination codon and 5′ of the bovine growth hormone polyadenylation signal. In these animals CUG aggregates form exclusively in the cytoplasm of cardiac cells. A key pathological consequence of expanded CUG repeat RNA expression in DM1 is aberrant RNA splicing. Abnormal splicing results from the functional inactivation of MBNL1, which is hypothesized to occur due to MBNL1 sequestration in CUG foci or from elevated levels of CUG-BP1. We therefore tested the ability of cytoplasmic CUG foci to elicit these changes. Aggregation of CUG RNAs within the cytoplasm results both in Mbnl1 sequestration and in approximately a two fold increase in both nuclear and cytoplasmic Cug-bp1 levels. Significantly, despite these changes RNA splice defects were not observed and functional analysis revealed only subtle cardiac dysfunction, characterized by conduction defects that primarily manifest under anesthesia. Using a human myoblast culture system we show that this transgene, when expressed at similar levels to a second transgene, which encodes expanded CTG tracts and facilitates both nuclear focus formation and aberrant splicing, does not elicit aberrant splicing. Thus the lack of toxicity of cytoplasmic CUG foci does not appear to be a consequence of low expression levels. Our results therefore demonstrate that the cellular location of CUG RNA aggregates is an important variable that influences toxicity and support the hypothesis that small molecules that increase the rate of transport of the mutant DMPK RNA from the nucleus into the cytoplasm may significantly improve DM1 pathology