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International audience; Increasing loss of structure and function of neurons and decline in cognitive function is commonly seen during the progression of neurologic diseases, although the causes and initial symptoms of individual diseases are distinct. This observation suggests a convergence of common degenerative features. In myotonic dystrophy type 1 (DM1), the expression of expanded CUG RNA induces neurotransmission dysfunction before axon and dendrite degeneration and reduced MBNL2 expression associated with aberrant alternative splicing. The role of loss of function of MBNL2 in the pathogenesis of neurodegeneration and the causal mechanism of neurodegeneration-reduced expression of MBNL2 remain elusive. Here, we show that increased MBNL2 expression is associated with neuronal maturation and required for neuronal morphogenesis and the fetal to adult developmental transition of RNA processing. Neurodegenerative conditions including NMDA receptor (NMDAR)-mediated excitotoxicity and dysregulated calcium homeostasis triggered nuclear translocation of calpain-2, thus resulting in MBNL2 degradation and reversal of MBNL2-regulated RNA processing to developmental patterns. Nuclear expression of calpain-2 resembled its developmental pattern and was associated with MBNL2 degradation. Knock-down of calpain-2 expression or inhibition of calpain-2 nuclear translocation prevented neurodegeneration-reduced MBNL2 expression and dysregulated RNA processing. Increased calpain-2 nuclear translocation associated with reduced MBNL2 expression and aberrant RNA processing occurred in models for DM1 and Alzheimer's disease (AD) including EpA960/CaMKII-Cre mice of either sex and female APP/PS1 and THY-Tau22 mice. Our results identify a regulatory mechanism for MBNL2 downregulation and suggest that calpain-2-mediated MBNL2 degradation accompanied by re-induction of a developmental RNA processing program may be a converging pathway to neurodegeneration.SIGNIFICANCE STATEMENTNeurologic diseases share many features during disease progression, such as cognitive decline and brain atrophy, which suggests a common pathway for developing degenerative features. Here, we show that the neurodegenerative conditions glutamate-induced excitotoxicity and dysregulated calcium homeostasis induced translocation of the cysteine protease calpain-2 into the nucleus, resulting in MBNL2 degradation and reversal of MBNL2-regulated RNA processing to an embryonic pattern. Knock-down or inhibition of nuclear translocation of calpain-2 prevented MBNL2 degradation and maintained MBNL2-regulated RNA processing in the adult pattern. Models of myotonic dystrophy and Alzheimer's disease (AD) also showed calpain-2-mediated MBNL2 degradation and a developmental RNA processing program. Our studies suggest MBNL2 function disrupted by calpain-2 as a common pathway, thus providing an alternative therapeutic strategy for neurodegeneration.

Vascular rarefaction due to impaired angiogenesis is associated with contractile dysfunction and the transition from compensation to decompensation and heart failure. The regulatory mechanism controlling vascular rarefaction during the transition remains elusive. Increased expression of a nuclear RNA-binding protein CUGBP Elav-like family member 1 (CELF1) in the adult heart is associated with the transition from compensated hypertrophy to decompensated heart failure. Elevated CELF1 level resulted in degradation of the major cardiac gap junction protein, connexin 43, in dilated cardiomyopathy (DCM), the most common cause of heart failure. In the present study, we investigated the role of increased CELF1 expression in causing vascular rarefaction in DCM. CELF1 overexpression (CELF1-OE) in cardiomyocytes resulted in reduced capillary density. CELF1-OE mice administered hypoxyprobe showed immunoreactivity and increased mRNA levels of HIF1α, Glut-1, and Pdk-1, which suggested the association of a reduced capillary density-induced hypoxic condition with CELF1 overexpression. Vegfa mRNA level was downregulated in mouse hearts exhibiting DCM, including CELF1-OE and infarcted hearts. Vegfa mRNA level was also downregulated to a similar extent in cardiomyocytes isolated from infarcted hearts by Langendorff preparation, which suggested cardiomyocyte-derived Vegfa expression mediated by CELF1. Cardiomyocyte-specific depletion of CELF1 preserved the capillary density and Vegfa mRNA level in infarcted mouse hearts. Also, CELF1 bound to Vegfa mRNA and regulated Vegfa mRNA stability via the 3' untranslated region. These results suggest that elevated CELF1 level has dual effects on impairing the functions of cardiomyocytes and microvasculature in DCM.

The Muscleblind-like (MBNL) protein family plays an important role in regulating developmental RNA processing transition. Loss of MBNL2 function has been implicated in the neurodegeneration of myotonic dystrophy type 1 (DM1). However, the causal mechanism of neurodegeneration-induced MBNL2 loss of function remains elusive. Here, we show that neurodegenerative conditions including NMDAR-mediated excitotoxicity and dysregulated calcium homeostasis triggered nuclear translocation of calpain-2 resulting in MBNL2 degradation and reversion of MBNL2-regulated RNA processing to developmental patterns. The developmental stage featured nucleus-enriched distribution of calpain-2 and low expression of MBNL2. Increased MBNL2 expression during development is required for promoting developmental RNA processing transition and neuronal maturation. Knockdown of calpain-2 expression inhibited neurodegeneration-induced MBNL2 reduction and dysregulated RNA processing. Neurodegenerative disease mouse models including DM1 and Alzheimer’s disease showed nuclear translocation of calpain-2 associated with MBNL2 degradation and reversion of MBNL2-regulated RNA processing to the developmental pattern. Our results identify a novel regulatory mechanism for MBNL2 downregulation and suggest that reduced MBNL2 expression accompanied by the re-induction of a developmental RNA processing program may be a common feature of neurodegeneration.

Rationale: Downregulation of Cx43 (connexin 43), the major cardiac gap junction protein, is often associated with arrhythmia, dilated cardiomyopathy (DCM), and heart failure. However, the cause of the reduced expression remains elusive. Reinduction of a nuclear RNA-binding protein CELF1 (CUGBP Elav-like family member 1) in the adult heart has been implicated in the cardiac pathogenesis of myotonic dystrophy type 1. However, how elevated CELF1 level leads to cardiac dysfunction, such as conduction defect, DCM, and heart failure, remains unclear. Objective: We investigated the mechanism of CELF1-mediated Cx43 mRNA degradation and determined whether elevated CELF1 expression is also a shared feature of the DCM heart. Methods and Results: RNA immunoprecipitation revealed the involvement of CELF1-regulated genes, including Cx43, in controlling contractility and conduction. CELF1 mediated Cx43 mRNA degradation by binding the UG-rich element in the 3′ untranslated region of Cx43. Mutation of the nuclear localization signal in CELF1 abolished the ability to downregulate Cx43 mRNA, so nuclear localization was required for its function. We further identified a 3′ to 5′ exoribonuclease, RRP6 (ribosomal RNA processing protein 6), as a CELF1-interacting protein. The interaction of CELF1 and RRP6 was RNA-independent and nucleus specific. With knockdown of endogenous RRP6, CELF1 failed to downregulate Cx43 mRNA, which suggests that RRP6 was required for CELF1-mediated Cx43 mRNA degradation. In addition, increased CELF1 level accompanied upregulated RRP6, and reduced Cx43 level was detected in mouse models with DCM, including myotonic dystrophy type 1 and CELF1 overexpression models and a myocardial infarction model. Importantly, depletion of CELF1 in the infarcted heart preserved Cx43 mRNA level and ameliorated the cardiac phenotypes of the infarcted heart. Conclusions: Our results suggest a mechanism for increased CELF1 expression downregulating Cx43 mRNA level and a pathogenic role for elevated CELF1 level in the DCM heart.

Myotonic dystrophy type 1 (DM1) is caused by an expansion of CTG repeats in the 3' untranslated region (UTR) of the dystrophia myotonia protein kinase (DMPK) gene. Cognitive impairment associated with structural change in the brain is prevalent in DM1. How this histopathological abnormality during disease progression develops remains elusive. Nuclear accumulation of mutant DMPK mRNA containing expanded CUG RNA disrupting the cytoplasmic and nuclear activities of muscleblind-like (MBNL) protein has been implicated in DM1 neural pathogenesis. The association between MBNL dysfunction and morphological changes has not been investigated. We generated a mouse model for postnatal expression of expanded CUG RNA in the brain that recapitulates the features of the DM1 brain, including the formation of nuclear RNA and MBNL foci, learning disability, brain atrophy and misregulated alternative splicing. Characterization of the pathological abnormalities by a time-course study revealed that hippocampus-related learning and synaptic potentiation were impaired before structural changes in the brain, followed by brain atrophy associated with progressive reduction of axon and dendrite integrity. Moreover, cytoplasmic MBNL1 distribution on dendrites decreased before dendrite degeneration, whereas reduced MBNL2 expression and altered MBNL-regulated alternative splicing was evident after degeneration. These results suggest that the expression of expanded CUG RNA in the DM1 brain results in neurodegenerative processes, with reduced cytoplasmic MBNL1 as an early event response to expanded CUG RNA.

The genetic basis of myotonic dystrophy type 1 (DM1) is a CTG expansion in the 3' untranslated region (UTR) of DMPK. The pathogenic mechanism involves an RNA gain of function in which the repeat-containing transcripts accumulate in nuclei and alter the functions of RNA-binding proteins such as CUG-binding protein 1 (CUGBP1). CUGBP1 levels are increased in DM1 myoblasts, heart, and skeletal muscle tissues and in some DM1 mouse models. However, the molecular mechanisms for increased CUGBP1 in DM1 are unclear. Here, we demonstrate that expression of DMPK-CUG-repeat RNA results in hyperphosphorylation and stabilization of CUGBP1. CUGBP1 is hyperphosphorylated in DM1 tissues, cells, and a DM1 mouse model. Activation of PKC is required for CUGBP1 hyperphosphorylation in DM1 cells, and PKCalpha and betaII directly phosphorylate CUGBP1 in vitro. These results indicate that inappropriate activation of the PKC pathway contributes to the pathogenic effects of a noncoding RNA.

Human genes contain a dense array of diverse cis-acting elements that make up a code required for the expression of correctly spliced mRNAs. Alternative splicing generates a highly dynamic human proteome through networks of coordinated splicing events. Cis- and trans-acting mutations that disrupt the splicing code or the machinery required for splicing and its regulation have roles in various diseases, and recent studies have provided new insights into the mechanisms by which these effects occur. An unexpectedly large fraction of exonic mutations exhibit a primary pathogenic effect on splicing. Furthermore, normal genetic variation significantly contributes to disease severity and susceptibility by affecting splicing efficiency.

Tbr-1, a neuron-specific T-box transcription factor, plays a critical role in brain development. Here, we performed a computational search using the non-palindromic T-box binding sequence, namely the non-palindromic T-element, to determine the putative downstream target genes of Tbr-1. More than 20 identified genes containing the non-palindromic T-element in the 5' regulatory region were found expressed in brain. Luciferase reporter assays using cultured hippocampal neurons showed that overexpression of Tbr-1 and CASK-enhanced promoter activities of some of these putative target genes, including NMDAR subunit 2b (NR2b), glycine transporter, interleukin 7 receptor (IL-7R) and OX-2. Among these genes, NR2b promoter responded strongest to overexpression of Tbr-1 and CASK. Deletion of the non-palindromic T-elements from NR2b promoter impaired the induction by Tbr-1 and CASK. We also examined expression of these target genes in Tbr-1 knockout mice, it was found that NR2b expression was consistently downregulated. Similarly, both RNA and protein expression levels of NMDAR subunit 1 (NR1), which also contains the non-palindromic T-elements in its 5' regulatory region, were reduced in Tbr-1 knockout mice. We suggest that Tbr-1/CASK protein complex regulates expression of these downstream target genes and thus modulates neuronal activity and function.

Diabetic cardiomyopathy is one of the complications of diabetes that eventually leads to heart failure and death. Aberrant activation of PKC signaling contributes to diabetic cardiomyopathy by mechanisms that are poorly understood. Previous reports indicate that PKC is implicated in alternative splicing regulation. Therefore, we wanted to test whether PKC activation in diabetic hearts induces alternative splicing abnormalities. Here, using RNA sequencing we identified a set of 22 alternative splicing events that undergo a developmental switch in splicing, and we confirmed that splicing reverts to an embryonic pattern in adult diabetic hearts. This network of genes has important functions in RNA metabolism and in developmental processes such as differentiation. Importantly, PKC isozymes α/β control alternative splicing of these genes via phosphorylation and up-regulation of the RNA-binding proteins CELF1 and Rbfox2. Using a mutant of CELF1, we show that phosphorylation of CELF1 by PKC is necessary for regulation of splicing events altered in diabetes. In summary, our studies indicate that activation of PKCα/β in diabetic hearts contributes to the genome-wide splicing changes through phosphorylation and up-regulation of CELF1/Rbfox2 proteins. These findings provide a basis for PKC-mediated cardiac pathogenesis under diabetic conditions.

Cardiac complications are a common cause of death in individuals with the inherited multisystemic disease myotonic dystrophy type 1 (DM1). A characteristic molecular feature of DM1 is misregulated alternative splicing due to disrupted functioning of the splicing regulators muscleblind-like 1 (MBNL1) and CUG-binding protein 1 (CUGBP1). CUGBP1 is upregulated in DM1 due to PKC pathway activation and subsequent CUGBP1 protein hyperphosphorylation and stabilization. Here, we blocked PKC activity in a heart-specific DM1 mouse model to determine its pathogenic role in DM1. Animals given PKC inhibitors exhibited substantially increased survival that correlated with reduced phosphorylation and decreased steady-state levels of CUGBP1. Functional studies demonstrated that PKC inhibition ameliorated the cardiac conduction defects and contraction abnormalities found in this mouse model. The inhibitor also reduced misregulation of splicing events regulated by CUGBP1 but not those regulated by MBNL1, suggesting distinct roles for these proteins in DM1 cardiac pathogenesis. The PKC inhibitor did not reduce mortality in transgenic mice with heart-specific CUGBP1 upregulation, indicating that PKC inhibition did not have a general protective effect on PKC-independent CUGBP1 increase. Our results suggest that pharmacological blockade of PKC activity mitigates the DM1 cardiac phenotype and provide strong evidence for a role for the PKC pathway in DM1 pathogenesis.

Myotonic dystrophy type 1 (DM1) is caused by a CTG trinucleotide expansion in the 3' untranslated region (3' UTR) of DM protein kinase (DMPK). The key feature of DM1 pathogenesis is nuclear accumulation of RNA, which causes aberrant alternative splicing of specific pre-mRNAs by altering the functions of CUG-binding proteins (CUGBPs). Cardiac involvement occurs in more than 80% of individuals with DM1 and is responsible for up to 30% of disease-related deaths. We have generated an inducible and heart-specific DM1 mouse model expressing expanded CUG RNA in the context of DMPK 3' UTR that recapitulated pathological and molecular features of DM1 including dilated cardiomyopathy, arrhythmias, systolic and diastolic dysfunction, and mis-regulated alternative splicing. Combined in situ hybridization and immunofluorescent staining for CUGBP1 and CUGBP2, the 2 CUGBP1 and ETR-3 like factor (CELF) proteins expressed in heart, demonstrated elevated protein levels specifically in nuclei containing foci of CUG repeat RNA. A time-course study demonstrated that colocalization of MBNL1 with RNA foci and increased CUGBP1 occurred within hours of induced expression of CUG repeat RNA and coincided with reversion to embryonic splicing patterns. These results indicate that CUGBP1 upregulation is an early and primary response to expression of CUG repeat RNA.

CASK interacting nucleosome assembly protein (CINAP) modulates gene expression and its abundance in cultured neurons is regulated by synaptic activity. To further study the function of CINAP in vivo, we examined the temporal and spatial expression profiles of CINAP. CINAP was widely expressed in different regions of adult mouse brain, including the cerebral cortex, hippocampus, striatum, hypothalamus, cerebellum, and two adult brain regions known to generate progenitor neurons. During early development, CINAP was also expressed in regions where neuronal progenitor cells were actively dividing, the ventricular and subventricular zones, suggesting that in addition to regulating gene expression in mature neurons, CINAP may also play a role in dividing cells. Since the hypothalamus responds to several physiological responses, we examined whether CINAP protein levels in the paraventricular nucleus (PVN) of the hypothalamus are regulated by changes in osmolality achieved through oral administration of hypertonic saline. Compared with control mice, mice treated with hypertonic saline expressed higher CINAP protein levels in the PVN, supporting a role of CINAP in neural response in vivo. Using confocal microscopic analysis, a significant amount of CINAP was found in the cytoplasm of neurons. Biochemical fractionation further confirmed that CINAP was associated with synapses, suggesting a translocation of CINAP protein from synapse to the nucleus. Consistent with this speculation, nuclear CINAP levels in the PVN were higher in hypertonic saline-treated mice than those who drank water. CINAP may be regulated through changes in protein stability and nuclear translocation in neurons. J. Comp. Neurol. 494:606–619, 2006. © 2005 Wiley-Liss, Inc.

Tbr-1, a neuron-specific T-box transcription factor, plays a critical role in brain development. Here, we performed a computational search using the non-palindromic T-box binding sequence, namely the non-palindromic T-element, to determine the putative downstream target genes of Tbr-1. More than 20 identified genes containing the non-palindromic T-element in the 5' regulatory region were found expressed in brain. Luciferase reporter assays using cultured hippocampal neurons showed that overexpression of Tbr-1 and CASK-enhanced promoter activities of some of these putative target genes, including NMDAR subunit 2b (NR2b), glycine transporter, interleukin 7 receptor (IL-7R) and OX-2. Among these genes, NR2b promoter responded strongest to overexpression of Tbr-1 and CASK. Deletion of the non-palindromic T-elements from NR2b promoter impaired the induction by Tbr-1 and CASK. We also examined expression of these target genes in Tbr-1 knockout mice, it was found that NR2b expression was consistently downregulated. Similarly, both RNA and protein expression levels of NMDAR subunit 1 (NR1), which also contains the non-palindromic T-elements in its 5' regulatory region, were reduced in Tbr-1 knockout mice. We suggest that Tbr-1/CASK protein complex regulates expression of these downstream target genes and thus modulates neuronal activity and function.

CASK acts as a coactivator for Tbr-1, an essential transcription factor in cerebral cortex development. Presently, the molecular mechanism of the CASK coactivation effect is unclear. Here, we report that CASK binds to another nuclear protein, CINAP, which binds histones and facilitates nucleosome assembly. CINAP, via its interaction with CASK, forms a complex with Tbr-1, regulating expression of the genes controlled by Tbr-1 and CASK, such as NR2b and reelin. A knockdown of endogenous CINAP in hippocampal neurons reduces the promoter activity of NR2b. Moreover, NMDA stimulation results in a reduction in the level of CINAP protein, via a proteasomal degradation pathway, correlating with a decrease in NR2b expression in neurons. This study suggests that reduction of the CINAP protein level by synaptic stimulation contributes to regulation of the transcriptional activity of the Tbr-1/CASK/CINAP protein complex and thus modifies expression of the NR2b gene.

There are three subtypes of alpha2 adrenoceptor, i.e., alpha2A, alpha2B, and alpha2C, mediating the specific effect of epinephrine and norepinephrine in various tissues by means of G protein-coupled signal transduction pathways. In an attempt to delineate the regulatory mechanism of the alpha2B receptor subtype (encoded by subtype gene Adra2b) expression in the central nervous system (CNS), we have established transgenic (Tg) mice lines in which the transgene (NN-lacZ) was composed of the promoter region of Adra2b (NcoI fragment, 4.7 kb immediately upstream from receptor coding region) and a reporter gene lacZ (encoding beta-galactosidase). The selective expression of alpha2B in brain as indexed by beta-galactosidase, under the direction of this promoter region, may be traced in situ by using X-gal staining. The expression pattern of Adra2b-NN-lacZ in CNS of Tg mice during development was examined. The temporal course of examination was from gestation day 9.5 (E9.5) to postnatal day 28 (P28). Significant X-gal staining was detected in the dorsal root ganglion and cranial nerves V and VII at E12.5. By E18.5, expression was noted in the cerebral cortex, anterior olfactory nucleus, hypothalamus, brainstem, and cerebellar Purkinje cells, among others, and persisted through postnatal development. Adra2b-NN-directed reporter expression was detected in the hippocampal dentate gyrus first at P4. The temporal course of expression up to P28 in this area is in accordance with the developmental profiles of granule neurons of dentate gyrus. From P7 on, transgene expression was detected in additional brain areas, including the septum and thalamus. The expression correlates well with the noradrenergic innervations as evidenced by colocalization by using tyrosine hydroxylase or dopamine-beta-hydroxylase immunocytochemistry.

The effects of vitamin B(6) deficiency on metabolic activities of brain structures were studied. Male Sprague-Dawley weanling rats received one of the following diets: (1) 7 mg pyridoxine HCl/kg (control group); (2) 0 mg pyridoxine HCl/kg (vitamin B(6)-deficient group); or (3) 7 mg pyridoxine HCl/kg with food intake restricted in quantity to that consumed by the deficient group (pair-fed control group). After 8 weeks of dietary treatment, rats in all three groups received an intravenous injection of 2-deoxy-[(14)C] glucose (100 microCi/kg). Vitamin B(6) status was evaluated by plasma pyridoxal 5'-phosphate concentrations. The vitamin B(6)-deficient group had significantly lower levels of plasma pyridoxal 5'-phosphate than did the control and pair-fed groups. The local cerebral glucose utilization rates in structures of the limbic system, basal ganglia, sensory motor system, and hypothalamic system were determined. The local cerebral glucose utilization rates in each of the four brain regions in the deficient animals were approximately 50% lower (P0.05) than in the control group. Results of the present study suggest that serious cognitive deficit may occur in vitamin B(6)-deficient animals.