Transcriptional regulation is a critical mechanism in the birth, specification, and differentiation of granule neurons in the adult hippocampus. One of the first negative-acting transcriptional regulators implicated in vertebrate development is REST/NRSF - thought to regulate hundreds of neuron-specific genes - yet its function in the adult brain remains elusive. Here we report that REST/NRSF is required to maintain the adult neural stem cell (NSC) pool and orchestrate stage-specific differentiation. REST/NRSF recruits CoREST and mSin3A corepressors to stem cell chromatin for the regulation of pro-neuronal target genes to prevent precocious neuronal differentiation in cultured adult NSCs. Moreover, mice lacking REST/NRSF specifically in NSCs display a transient increase in adult neurogenesis that leads to a loss in the neurogenic capacity of NSCs and eventually diminished granule neurons. Our work identifies REST/NRSF as a master negative regulator of adult NSC differentiation and offers a potential molecular target for neuroregenerative approaches.

Repetitive stimulation potentiates contractile tension of fasttwitch skeletal muscle. We examined the role of myosin regulatory light chain (RLC) phosphorylation in this physiological response by ablating Ca 2؉ ͞calmodulin-dependent skeletal muscle myosin light chain kinase (MLCK) gene expression. Western blot and quantitative-PCR showed that MLCK is expressed predominantly in fasttwitch skeletal muscle fibers with insignificant amounts in heart and smooth muscle. In contrast, smooth muscle MLCK had a more ubiquitous tissue distribution, with the greatest expression observed in smooth muscle tissue. Ablation of the MYLK2 gene in mice resulted in loss of skeletal muscle MLCK expression, with no change in smooth muscle MLCK expression. In isolated fast-twitch skeletal muscles from these knockout mice, there was no significant increase in RLC phosphorylation in response to repetitive electrical stimulation. Furthermore, isometric twitch-tension potentiation after a brief tetanus (posttetanic twitch potentiation) or low-frequency twitch potentiation (staircase) was attenuated relative to responses in muscles from wild-type mice. Interestingly, the site of phosphorylation of the small amount of monophosphorylated RLC in the knockout mice was the same site phosphorylated by MLCK, indicating a potential alternative signaling pathway affecting contractile potentiation. Loss of skeletal muscle MLCK expression had no effect on cardiac RLC phosphorylation. These results identify myosin light chain phosphorylation by the dedicated skeletal muscle Ca 2؉ ͞calmodulin-dependent MLCK as a primary biochemical mechanism for tension potentiation due to repetitive stimulation in fast-twitch skeletal muscle.calcium ͉ calmodulin ͉ twitch S keletal muscle contraction depends on a voltage-driven conformational change in the L-type Ca 2ϩ channel in the transverse tubule that triggers Ca 2ϩ release from the sarcoplasmic reticulum through the intracellular ryanodine receptor (1, 2). The Ca 2ϩ binds to troponin in thin filaments, thereby allowing myosin cross bridges to bind actin and generate muscle tension (3). However, muscle contractions involve more complex mechanisms that affect performance. Since Ranke noted in 1865 (4) that, with stimuli uniform in strength the later twitch contractions were stronger than the first, there has been considerable interest in identifying the mechanisms involved in isometric twitch potentiation during trains of stimuli at low frequency (staircase) or after a tetanus (posttetanic potentiation). Considerations have been given to changes in compliance of the series elastic elements, to activation of more fibers within a muscle, to increased Ca 2ϩ release within a single fiber to activate fully the contractile proteins, and to changes in excitation-contraction coupling processes (5-8).Ca 2ϩ released during muscle contraction can also activate the dedicated protein kinase Ca 2ϩ ͞calmodulin-dependent skeletal muscle myosin light chain kinase (skMLCK) to initiate myosin regulatory light chain (RLC) phosphorylat...

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In contrast to studies on skeletal and smooth muscles, the identity of kinases in the heart that are important physiologically for direct phosphorylation of myosin regulatory light chain (RLC) is not known. A Ca 2؉ /calmodulin-activated myosin light chain kinase is expressed only in cardiac muscle (cMLCK), similar to the tissue-specific expression of skeletal muscle MLCK and in contrast to the ubiquitous expression of smooth muscle MLCK. We have ablated cMLCK expression in male mice to provide insights into its role in RLC phosphorylation in normally contracting myocardium. The extent of RLC phosphorylation was dependent on the extent of cMLCK expression in both ventricular and atrial muscles. Attenuation of RLC phosphorylation led to ventricular myocyte hypertrophy with histological evidence of necrosis and fibrosis. Echocardiography showed increases in left ventricular mass as well as end-diastolic and end-systolic dimensions. Cardiac performance measured as fractional shortening decreased proportionally with decreased cMLCK expression culminating in heart failure in the setting of no RLC phosphorylation. Hearts from female mice showed similar responses with loss of cMLCK associated with diminished RLC phosphorylation and cardiac hypertrophy. Isoproterenol infusion elicited hypertrophic cardiac responses in wild type mice. In mice lacking cMLCK, the hypertrophic hearts showed no additional increases in size with the isoproterenol treatment, suggesting a lack of RLC phosphorylation blunted the stress response. Thus, cMLCK appears to be the predominant protein kinase that maintains basal RLC phosphorylation that is required for normal physiological cardiac performance in vivo.Sarcomeric proteins in myocytes account for contraction of the heart that depends on the molecular motor myosin in the thick filaments binding to actin in thin filaments to initiate shortening and force development (1-3). Myosin cross-bridges contain an actin-binding surface and ATP pocket in the motor domain that taper to an ␣-helical neck connecting to the myosin rod region responsible for the self-assembly into thick filaments. Two small protein subunits, the essential light chain and the phosphorylatable RLC, 2 wrap around each ␣-helical neck region providing mechanical stability (4). RLC is necessary for assembly of thick filaments in cardiac myocytes, and mutations in RLC are linked to inherited hypertrophic cardiomyopathy (5, 6). There are two types of cardiac RLCs, a ventricular myosin light chain, MLC2v, and an atrium-specific form, MLC2a (7).In heart and skeletal muscle Ca 2ϩ binds to troponin in the actin thin filament, thereby allowing myosin heads to attach to actin for sarcomeric force development and shortening (8). Additionally, phosphorylation of RLC in fast-twitch skeletal muscle fibers by a skeletal muscle-specific Ca 2ϩ /calmodulindependent MLCK modulates the contractile response by potentiating frequency-dependent force development (9 -11). In the heart, phosphorylation of multiple sarcomeric proteins adjusts myofilamen...

Accumulating reports demonstrate that apoptosis does not explain all the forms of programmed cell death (PCD), particularly in individual development and neurodegenerative disease. Recently, a novel type of PCD, designated `paraptosis', was described. Here, we show that overexpression of TAJ/TROY, a member of the tumor necrosis factor receptor superfamily, induces non-apoptotic cell death with paraptosis-like morphology in 293T cells. Transmission electron microscopy studies reveal extensive cytoplasmic vacuolation and mitochondrial swelling in some dying cells and no condensation or fragmentation of the nuclei. Characteristically, cell death triggered by TAJ/TROY was accompanied by phosphatidylserine externalization, loss of the mitochondrial transmembrane potential and independent of caspase activation. In addition, TAJ/TROY suppressed clonogenic growth of HEK293 and HeLa cells. Interestingly, overexpression of Programmed cell death 5 (PDCD5), an apoptosis-promoting protein, enhanced TAJ/TROY-induced paraptotic cell death. Moreover, cellular endogenous PDCD5 protein was significantly upregulated in response to TAJ/TROY overexpression. These results provide novel evidence that TAJ/TROY activates a death pathway distinct from apoptosis and that PDCD5 is an important regulator in both apoptotic and non-apoptotic PCD.