Loss of MBNL1-mediated retrograde BDNF signaling in the myotonic dystrophy brain.

Reduced brain volume including atrophy in grey and white matter is commonly seen in myotonic dystrophy type 1 (DM1). DM1 is caused by an expansion of CTG trinucleotide repeats in the 3' untranslated region (UTR) of the Dystrophia Myotonica Protein Kinase (DMPK) gene. Mutant DMPK mRNA containing expanded CUG RNA (DMPK-CUGexp) sequesters cytoplasmic MBNL1, resulting in morphological impairment. How DMPK-CUGexp and loss of MBNL1 cause histopathological phenotypes in the DM1 brain remains elusive. Here, we show that BDNF-TrkB retrograde transport is impaired in neurons expressing DMPK-CUGexp due to loss of cytoplasmic MBNL1 function. We reveal that mature BDNF protein levels are reduced in the brain of the DM1 mouse model EpA960/CaMKII-Cre. Exogenous BDNF treatment did not rescue impaired neurite outgrowth in neurons expressing DMPK-CUGexp, whereas overexpression of the cytoplasmic MBNL1 isoform in DMPK-CUGexp-expressing neurons improved their responsiveness to exogenous BDNF. We identify dynein light chain LC8-type 2, DYNLL2, as an MBNL1-interacting protein and demonstrate that their interaction is RNA-independent. Using time-lapse imaging, we show that overexpressed MBNL1 and DYNLL2 move along axonal processes together and that MBNL1-knockdown impairs the motility of mCherry-tagged DYNLL2, resulting in a reduced percentage of retrograde DYNLL2 movement. Examination of the distribution of DYNLL2 and activated phospho-TrkB (pTrkB) receptor in EpA960/CaMKII-Cre brains revealed an increase in the postsynaptic membrane fraction (LP1), indicating impaired retrograde transport. Finally, our neuropathological analysis of postmortem DM1 tissue reveals that reduced cytoplasmic MBNL1 expression is associated with an increase in DYNLL2 and activated pTrkB receptor levels in the synaptosomal fraction. Together, our results support that impaired MBNL1-mediated retrograde BDNF-TrkB signaling may contribute to the histopathological phenotypes of DM1.

Calpain-2 Mediates MBNL2 Degradation and a Developmental RNA Processing Program in Neurodegeneration.

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 STATEMENT Neurologic 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.

CELF1 promotes vascular endothelial growth factor degradation resulting in impaired microvasculature in heart failure.

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.

Reduced cytoplasmic MBNL1 is an early event in a brain-specific mouse model of myotonic dystrophy.

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.

Calreticulin, an endoplasmic reticulum-resident protein, is highly expressed and essential for cell proliferation and migration in oral squamous cell carcinoma.

OBJECTIVES: Oral squamous cell carcinoma (OSCC) has emerged as one of the major malignant tumors of the head and neck cancers. However, the molecular mechanism behind tumorigenesis of OSCC is not fully understood. The aim of this study was to investigate the role of calreticulin (CRT), an endoplasmic reticulum-resident protein, in OSCC cells. MATERIALS AND METHODS: Sixteen paired samples of tumor and non-cancerous matched tissue (NCMT), six OSCC cell lines and normal human oral keratinocytes (NHOKs), and oral tissue microarray were used to reveal the expression of CRT by Western blotting and immunohistochemistry. Later, shRNA-mediated stable knockdown of CRT in OSCC cells was generated. The knockdown cell line was used to analyze cell proliferation, colony formation, anchorage-independent growth and cell migration in vitro. RESULTS: CRT was differentially expressed in fresh tumor samples and six OSCC cell lines but not adjacent NCMTs and NHOKs. In oral tissue microarray, we showed that there was positive CRT staining in the vast majority of tumor cases (99/103), in sharp contrast to that in NCMT cases (29/92) (p<0.001). Stable knockdown of CRT in oral cancer cells resulted in significantly reduced growth rate, colony-forming capacity and anchorage-independent growth. This may be attributed to the induction of G0/G1 cell cycle arrest when CRT was depleted in the cells. Both horizontal and vertical movements of the CRT-knockdown stable line were markedly impaired. The phosphorylation levels of focal adhesion kinase (FAK), paxillin and ERK1/2 and the activity of matrix metalloproteinase-2 and -9 (MMP-2 and MMP-9) were decreased in the CRT-knockdown cells. These results suggest that CRT can regulate oral cancer cell migration through activation of the FAK signaling pathway accompanied with proteolytic degradation of the extracellular matrix (ECM) by MMP-2 and MMP-9. CONCLUSION: Together, this study has defined a novel biological role for CRT in oral cancer. CRT is a potential biomarker and may contribute to the malignant phenotypes of OSCC cells.