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Integration of inputs by a neuron depends on dendritic arborization patterns. In mammals, the genetic programs that regulate dynamic remodeling of dendrites during development and in response to activity are incompletely understood. Here we report that knockdown of the transcription factor Sp4 led to an increased number of highly branched dendrites during maturation of cerebellar granule neurons in dissociated cultures and in cerebellar cortex. Time-course analysis revealed that depletion of Sp4 led to persistent generation of dendritic branches and a failure in resorption of transient dendrites. Depolarization induced a reduction in the number of dendrites, and knockdown of Sp4 blocked depolarization-induced remodeling. Furthermore, overexpression of Sp4 wild type, but not a mutant lacking the DNA-binding domain, was sufficient to promote dendritic pruning in nondepolarizing conditions. These findings indicate that the transcription factor Sp4 controls dendritic patterning during cerebellar development by limiting branch formation and promoting activitydependent pruning.branching ͉ dendrite ͉ depolarization ͉ pruning ͉ neuron T he nervous system is a complex and well coordinated network that depends on the formation of proper connections among diverse types of neurons. Dendritic arborization patterns determine the way a neuron integrates inputs. Defects in dendritic patterning correlate with severe neurodevelopmental disorders (1-3). Although proper dendritic arborizations are crucial for correct function of the nervous system, the genetic programs that govern dendritic morphology in mammals remain poorly described. Dendritic development is a highly dynamic process that involves many events, including neurite outgrowth, branching, stabilization, and pruning of dendrites (4-6). There is a delicate balance between addition and elimination of neuronal projections (7,8). During development, transient overproduction of branched dendrites appears in most neurons, after which some neurites are eliminated, whereas other neurites are stabilized to achieve the mature pattern (9-11). Recent studies have described intracellular signaling pathways that operate locally to regulate cytoskeletal elements important for branch formation or elimination (12)(13)(14).Extrinsic factors are coordinated with cell-intrinsic, gene expression programs to determine dendritic morphology. Genetic studies in Drosophila and Caenorhabditis elegans have identified transcription factors that regulate diverse aspects of dendritic morphology (15-18). Transcriptional control of the fine balance between formation and elimination of processes is illustrated by the finding that, in Drosophila, the homeodomain protein, Cut, promotes branching, whereas the BTB/POZ Zinc finger transcription, Abrupt, limits branch formation (18,19). In mammals, the transcriptional regulators CREB, NeuroD, and CREST have been identified to promote dendritic growth and branching (20-23). Transcriptional regulators that balance these activities to restrict dendritic branchin...

Objectives Regulation of gene expression is important for the development and function of the nervous system. However, the transcriptional programs altered in psychiatric diseases are not completely characterized. Human gene association studies and analysis of mutant mice suggest that the transcription factor specificity protein 4 (SP4) may be implicated in the pathophysiology of psychiatric diseases. We hypothesized that SP4 levels may be altered in the brain of bipolar disorder (BD) subjects and regulated by neuronal activity and drug treatment. Methods We analyzed messenger RNA (mRNA) and protein levels of SP4 and SP1 in the postmortem prefrontal cortex and cerebellum of BD subjects (n = 10) and controls (n = 10). We also examined regulation of SP4 mRNA and protein levels by neuronal activity and lithium in rat cerebellar granule neurons. Results We report a reduction of SP4 and SP1 proteins, but not mRNA levels, in the cerebellum of BD subjects. SP4 protein and mRNA levels were also reduced in the prefrontal cortex. Moreover, we found in rat cerebellar granule neurons that under non-depolarizing conditions SP4, but not SP1, was polyubiquitinated and degraded by the proteasome while lithium stabilized SP4 protein. Conclusions Our study provides the first evidence of altered SP4 protein in the cerebellum and prefrontal cortex in BD subjects supporting a possible role of transcription factor SP4 in the pathogenesis of the disease. In addition, our finding that SP4 stability is regulated by depolarization and lithium provides a pathway through which neuronal activity and lithium could control gene expression suggesting that normalization of SP4 levels could contribute to treatment of affective disorders.

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SummaryLithium is widely used in the treatment of bipolar disorder, but despite its proven therapeutic efficacy, the molecular mechanisms of action are not fully understood. The present study was undertaken to explore lithium effects of the MEK/ERK cascade of protein kinases in astrocytes and neurons. In asynchronously proliferating rat cortical astrocytes, lithium decreased time-and dose-dependently the phosphorylation of MEK and ERK, with 1 mM concentrations achieving 60 and 50% inhibition of ERK and MEK, respectively, after a 7-day exposure. Lithium also inhibited [ 3 H]thymidine incorporation into DNA and induced a G2/M cell cycle arrest. In serumdeprived, quiescent astrocytes, pre-exposure to lithium resulted in the inhibition of cell cycle re-entry as stimulated by the mitogen endothelin-1: under this experimental setting, lithium did not affect the rapid, peak phosphorylation of MEK taking place after 3-5 min, but was effective in inhibiting the long-term, sustained phosphorylation of MEK. Lithium inhibition of the astrocyte MEK/ERK pathway was independent of inositol depletion. Further, compound SB216763 inhibited Tau phosphorylation at Ser396 and stabilized cytosolic b-catenin, consistent with the inhibition of glycogen synthase kinase-3b (GSK-3b), but failed to reproduce lithium effects on MEK and ERK phosphorylation and cell cycle arrest. In cerebellar granule neurons, millimolar concentrations of lithium enhanced MEK and ERK phosphorylation in a concentrationdependent manner, again through an inositol and GSK-3b independent mechanism. These opposing effects in astrocytes and neurons make lithium treatment a promising strategy to favour neural repair and reduce reactive gliosis after traumatic injury. Lithium has been used in the treatment of bipolar mood disorder for decades, but despite its therapeutic efficacy, the molecular mechanisms underlying its actions remain unclear (Jope 1999;Phiel and Klein 2001). In this regard, based on the observation that lithium inhibits inositol monophosphatase and inositol polyphosphate 1-phosphatase, thereby blocking inositol 1,4,5-trisphosphate recycling to inositol, the inositol depletion hypothesis considered that persistent activation of phosphoinositide phospholipase C in the presence of lithium would lower the cellular inositol concentration, leading eventually to the depletion of phosphatidylinositol 4,5-bisphosphate and the impairment of calcium signalling (Berridge et al. 1989;Atack 1996). This hypothesis has received some support after the finding that lithium, carbamazepine, and valproic acid, all three drugs used in the treatment of bipolar disorder, increase growth cone area in sensory neurons, in a manner that was counteracted by inositol addition, and mediated probably by inhibition of prolyl oligopeptidase by an as yet unknown mechanism (Williams et al. 2002).In the past few years, however, lithium has emerged as a remarkable neuroprotective agent: it was first shown to protect cerebellar granule neurons from undergoing apoptotic cell death when cul...

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Apoptosis induced by antitumor phospholipid analogs takes place after the inhibition of the CTP:phosphocholine cytidylyltransferase (CCT; EC 2.7.7.15) catalyzed step of phosphatidylcholine (PtdCho) biosynthesis. Exposure of cells to synthetic short-chain ceramide analogs also triggers apoptosis concomitant with decreased PtdCho biosynthesis, and the present study was undertaken to ascertain whether C 2 -ceramide inhibition of PtdCho synthesis is direct or secondary to other ceramide-mediated cellular responses. The exposure of COS-7 cells to either C 2 -ceramide, ET-18-OCH 3 , or farnesol resulted in time-and dose-dependent apoptotic cell death. Cells treated with C 2 -ceramide or ET-18-OCH 3 selectively and immediately accumulated phosphocholine, whereas CDP-choline increased with farnesol treatment. In vitro assays of CCT activity demonstrated that C 2 -ceramide directly inhibited CCT. Comparison of different N-linked sphingosine derivatives suggests an inverse relationship between the length of the N-linked carbon chain and the derivatives ability to trigger apoptosis and inhibit CCT. Taken together, our results suggest CCT as a primary target for C 2 -ceramide inhibition that accounts for its cytotoxic effects.