SUMMARY A critical feature of neural networks is that they balance excitation and inhibition to prevent pathological dysfunction. How this is achieved is largely unknown, though deficits in the balance contribute to many neurological disorders. We show here that a microRNA (miR-101) is a key orchestrator of this essential feature, shaping the developing network to constrain excitation in the adult. Transient early blockade of miR-101 induces long-lasting hyper-excitability and persistent memory deficits. Using target-site blockers in vivo, we identify multiple developmental programs regulated in parallel by miR-101 to achieve balanced networks. Repression of one target, NKCC1, initiates the switch in GABA signaling, limits early spontaneous activity, and constrains dendritic growth. Kif1a and Ank2 are targeted to prevent excessive synapse formation. Simultaneous de-repression of these three targets completely phenocopies major dysfunctions produced by miR-101 blockade. Our results provide new mechanistic insight into brain development and suggest novel candidates for therapeutic intervention.

Endoplasmic reticulum (ER) stress has been implicated as an initiator or contributing factor in neurodegenerative diseases. The mechanisms that lead to ER stress and whereby ER stress contributes to the degenerative cascades remain unclear but their understanding is critical to devising effective therapies. Here we show that knockdown of Herp (Homocysteine-inducible ER stress protein), an ER stress-inducible protein with an ubiquitin-like (UBL) domain, aggravates ER stress-mediated cell death induced by mutant α-synuclein (αSyn) that causes an inherited form of Parkinson's disease (PD). Functionally, Herp plays a role in maintaining ER homeostasis by facilitating proteasome-mediated degradation of ER-resident Ca(2+) release channels. Deletion of the UBL domain or pharmacological inhibition of proteasomes abolishes the Herp-mediated stabilization of ER Ca(2+) homeostasis. Furthermore, knockdown or pharmacological inhibition of ER Ca(2+) release channels ameliorates ER stress, suggesting that impaired homeostatic regulation of Ca(2+) channels promotes a protracted ER stress with the consequent activation of ER stress-associated apoptotic pathways. Interestingly, sustained upregulation of ER stress markers and aberrant accumulation of ER Ca(2+) release channels were detected in transgenic mutant A53T-αSyn mice. Collectively, these data establish a causative link between impaired ER Ca(2+) homeostasis and chronic ER stress in the degenerative cascades induced by mutant αSyn and suggest that Herp is essential for the resolution of ER stress through maintenance of ER Ca(2+) homeostasis. Our findings suggest a therapeutic potential in PD for agents that increase Herp levels or its ER Ca(2+)-stabilizing action.

The endoplasmic reticulum (ER) is a key organelle regulating intracellular Ca 2؉ homeostasis. Oxidants and mitochondria-derived free radicals can target ER-based Ca 2؉ regulatory proteins and cause uncontrolled Ca 2؉ release that may contribute to protracted ER stress and apoptosis. Several ER stress proteins have been suggested to counteract the deregulation of ER Ca 2؉ homeostasis and ER stress. Here we showed that knockdown of Herp, an ubiquitin-like domain containing ER stress protein, renders PC12 and MN9D cells vulnerable to 1-methyl-4-phenylpyridinium-induced cytotoxic cell death by a mechanism involving up-regulation of CHOP expression and ER Ca 2؉ depletion. Conversely, Herp overexpression confers protection by blocking 1-methyl-4-phenylpyridinium-induced CHOP upregulation, ER Ca 2؉ store depletion, and mitochondrial Ca 2؉ accumulation in a manner dependent on a functional ubiquitinproteasomal protein degradation pathway. Deletion of the ubiquitin-like domain of Herp or treatment with a proteasomal inhibitor abolished the central function of Herp in ER Ca 2؉ homeostasis. Thus, elucidating the underlying molecular mechanism(s) whereby Herp counteracts Ca 2؉ disturbances will provide insights into the molecular cascade of cell death in dopaminergic neurons and may uncover novel therapeutic strategies to prevent and ameliorate Parkinson disease progression.Parkinson disease (PD) 2 is the second most common agerelated neurodegenerative disorder that results in the selective degeneration of dopaminergic neurons of the substantia nigra pars compacta (1, 2). The proximate cause of selective degeneration of dopaminergic neurons in PD has not been clearly elucidated. Several mechanisms are inferred to play a role in the pathogenesis of PD based on studies from animals or in vitro studies using dopaminergic neurotoxins. These include mitochondrial dysfunction, oxidative stress, and impairment of the ubiquitin-proteasomal pathway (UPP) (1-3). It has been shown that several genes that are mutated in familial PD encode for proteins that have functions linked to UPP and mitochondria (1-3). The UPP plays a critical role in ER-associated protein degradation (ERAD), a protein quality control system of the ER that eliminates misfolded proteins in the ER lumen (4). UPP dysfunction results in the accumulation of misfolded or unfolded proteins within the ER, which induces ER stress (5).Important roles for ER stress and ER stress-induced cell death have been reported in a broad spectrum of pathological conditions (6). To alleviate ER stress and enhances cell survival, cells launch the unfolded protein response (UPR), an adaptive response to minimize accumulation of misfolded proteins that would otherwise be toxic to the cell (7). The biological objectives of the UPR are to reduce the overall protein translation, increase the production of ER localized chaperones, and increase the clearance of unfolded proteins by UPP (7). Although short time UPR activation serves to reduce the unfolded protein load, a protracted activation o...

The Notch signaling pathway plays an essential role in the regulation of cell specification by controlling differentiation, proliferation, and apoptosis. Numb is an intrinsic regulator of the Notch pathway and exists in four alternative splice variants that differ in the length of their phosphotyrosine-binding domain (PTB) and proline-rich region domains. The physiological relevance of the existence of the Numb splice variants and their exact regulation are still poorly understood. We previously reported that Numb switches from isoforms containing the insertion in PTB to isoforms lacking this insertion in neuronal cells subjected to trophic factor withdrawal (TFW). The functional relevance of the TFW-induced switch in Numb isoforms is not known. Here we provide evidence that the TFW-induced switch in Numb isoforms regulates Notch signaling strength and Notch target gene expression. PC12 cells stably overexpressing Numb isoforms lacking the PTB insertion exhibited higher basal Notch activity and Notch-dependent transcription of the transient receptor potential channel 6 (TRPC6) when compared with those overexpressing Numb isoforms with the PTB insertion. The differential regulation of TRPC6 expression is correlated with perturbed calcium signaling and increased neuronal vulnerability to TFW-induced death. Pharmacological inhibition of the Notch pathway or knockdown of TRPC6 function ameliorates the adverse effects caused by the TFW-induced switch in Numb isoforms. Taken together, our results indicate that Notch and Numb interaction may influence the sensitivity of neuronal cells to injurious stimuli by modulating calcium-dependent apoptotic signaling cascades.The Notch pathway is required for the embryonic and postnatal development of many organs by regulating cell proliferation, differentiation, and apoptosis through direct cell to cell contact (1). Notch consists of a family of single-pass transmembrane receptors (Notch1-4), which can be activated by interaction with membrane-tethered ligands of the Delta/Serrate/ Lag2 family expressed on neighboring cells (2). Upon activation, Notch is cleaved by ␥-secretase (3), releasing from the membrane the Notch intracellular domain (NICD) 3 that translocates to the nucleus. NICD associates with CSL (an acronym for the mammalian CBF-1, Drosophila Suppressor of Hairless and Caenorhabditis elegans Lag-1 transcription factors (4)) to control the expression of target genes, most notably the basic helix-loop-helix transcription factors belonging to the Hes (hairy and enhancer of split) (5) and Herp (Hes-related protein) family (6). These transcription factors in turn regulate the ability of the cells to respond to environmental clues. In the absence of Notch signaling, CSL represses transcription of Notch target genes, and following activation by Notch, CSL is converted into a transcriptional activator and activates transcription of the same genes (7). Disruption of the Notch gene in mice results in severe developmental defects and embryonic lethality, supporting a major rol...

Chronic intake of nicotine can impair hippocampal plasticity, but the underlying mechanism is poorly understood. Here, we demonstrate that chronic nicotine administration in adult rats inactivates the cyclic AMP-response element binding protein (CREB), a transcription factor that regulates neurogenesis and other plasticity-related processes necessary for learning and memory. Consequently, we showed that impaired CREB signaling is associated with a significant decline in the production of new neurons in the dentate gyrus. Combining retrovirus labeling with gene expression approaches, we found that chronic nicotine administration reduces the number of adult-generated granule neurons by decreasing the survival of newborn cells but not the proliferation of progenitor cells. Additionally, we found that retroviral-mediated expression of a constitutively active CREB in the dentate gyrus rescues survival of newborn cells and reverses the nicotine-induced decline in the number of mature granule neurons. Prolonged nicotine exposure also compromises CREB activation and reduces the viability of progenitor cells in vitro, thereby suggesting that nicotine may exert its adverse effects directly on immature cells in vivo. Taken together, these data demonstrate that inhibition of CREB activation is responsible for the nicotine-induced impairment of hippocampal plasticity.