No abstract

Cellular cholesterol homeostasis is a balance of influx, catabolism and synthesis, and efflux. Unlike vascular lipoprotein cholesterol transport, intracellular cholesterol trafficking is only beginning to be resolved. Exogenous cholesterol and cholesterol ester enter cells via the low-density lipoprotein (LDL) receptor! lysosomal and less so by nonvesicular, high-density lipoprotein (HDL) receptor!caveolar pathways. However, the mechanism(s) whereby cholesterol enters the lysosomal membrane, translocates, and transfers out of the lysosome to the cell interior are unknown. likewise, the steps whereby cholesterol enters the cytofacial leaflet of the plasma membrane caveolae, rapidly translocates, leaves the exofacial leaflet, and transfers to extracellular HDL are unclear. Increasing evidence obtained with model and isolated cell membranes, transfected cells, genetic mutants, and gene-ablated mice suggests that proteins such as caveolin, sterol carrier proteln-2 (SCP-2), Niemann-Pick C1 protein, steroidogenic acute regulatory protein (StAR), and other intracellular proteins mediate intracellular cholesterol transfer. While these proteins bind cholesterol and/or Interact with cholesterol-rich membrane microdomains (e.g., caveolae, rafts, and annuli), their relative contributions to direct molecular versus vesicular cholesterol transfer remain to be resolved. The formation, regulation, and role of membrane microdomalns in regulating cholesterol uptake/efflux and trafficking are unclear. Some cholesterol-binding proteins exert opposing effects on cellular cholesterol uptake/efflux, transfer of cholesterol out of This manuscript is an update of a previously published minireview entitled, "Recent Advances in Membrane Cholesterol Domain Dynamics and Intracellular Cholesterol Trafficking".

Inclusion body myopathy with Paget disease of bone and frontotemporal dementia (IBMPFD) is an autosomal dominant disorder characterized by progressive myopathy that is often accompanied by bone weakening and/or frontotemporal dementia. Although it is known to be caused by mutations in the gene encoding valosin-containing protein (VCP), the underlying disease mechanism remains elusive. Like IBMPFD, neurofibromatosis type 1 (NF1) is an autosomal dominant disorder. Neurofibromin, the protein encoded by the NF1 gene, has been shown to regulate synaptogenesis. Here, we show that neurofibromin and VCP interact and work together to control the density of dendritic spines. Certain mutations identified in IBMPFD and NF1 patients reduced the interaction between VCP and neurofibromin and impaired spinogenesis. The functions of neurofibromin and VCP in spinogenesis were shown to correlate with the learning disability and dementia phenotypes seen in patients with IBMPFD. Consistent with the previous finding that treatment with a statin rescues behavioral defects in Nf1 +/-mice and providing further support for our hypothesis that there is crosstalk between neurofibromin and VCP, statin exposure neutralized the effect of VCP knockdown on spinogenesis in cultured hippocampal neurons. The data presented here demonstrate that there is a link between IBMPFD and NF1 and indicate a role for VCP in synapse formation.

Long-term memory requires activity-dependent synthesis of plasticity-related proteins (PRPs) to strengthen synaptic efficacy and consequently consolidate memory. Cytoplasmic polyadenylation element binding protein (CPEB)3 is a sequence-specific RNA-binding protein that regulates translation of several PRP RNAs in neurons. To understand whether CPEB3 plays a part in learning and memory, we generated CPEB3 knock-out (KO) mice and found that the null mice exhibited enhanced hippocampus-dependent, short-term fear memory in the contextual fear conditioning test and long-term spatial memory in the Morris water maze. The basal synaptic transmission of Schaffer collateral-CA1 neurons was normal but long-term depression evoked by paired-pulse low-frequency stimulation was modestly facilitated in the juvenile KO mice. Molecular and cellular characterizations revealed several molecules in regulating plasticity of glutamatergic synapses are translationally elevated in the CPEB3 KO neurons, including the scaffolding protein PSD95 and the NMDA receptors along with the known CPEB3 target, GluA1. Together, CPEB3 functions as a negative regulator to confine the strength of glutamatergic synapses by downregulating the expression of multiple PRPs and plays a role underlying certain forms of hippocampusdependent memories.

Abbreviations used in this paper: CaMK, calcium/calmodulin-dependent protein kinase; CASK, calcium/calmodulin-dependent serine protein kinase; DIV, day in vitro; KS, Kolmogorov-Smirnov; MAGUK, membrane-associated guanylate kinase; MEF2, myocyte enhancer factor 2; shRNA, short hairpin RNA; SUMO, small ubiquitin-like modifi er; SynCAM, synaptic cell adhesion molecule; TTX, tetrodotoxin.The online version of this article contains supplemental material.

Acyl-CoA-binding protein (ACBP) 1 is a ubiquitous intracellular lipid-binding protein whose physiological function remains to be determined (reviewed in Refs. 1-3). Until recently, ACBP was thought to be primarily cytosolic and unique among the soluble intracellular lipid proteins in exhibiting very high affinity (K d values of 0.5-10 nM) and exclusive specificity for long chain fatty acyl-CoAs (LCFA-CoAs) (1, 4 -7). Based on the fact that ACBP exclusively binds LCFA-CoAs, it may participate in several aspects of LCFA-CoA metabolism.First, ACBP may be involved in directly presenting LCFACoAs as substrates for lipid metabolic enzymes. A variety of studies in vitro suggest that ACBP extracts LCFA-CoAs from membranes (6) to increase the soluble LCFA-CoA pool available for intracellular transport (reviewed in Ref.2). The cytosolic ACBP⅐LCFA-CoA complexes then interact with and present LCFA-CoA to acyltransferase enzymes involved in phospholipid synthesis in the endoplasmic reticulum (8, 9), lysophosphatidic acid synthesis in mitochondria (10), cholesteryl ester synthesis in the endoplasmic reticulum (11), and oxidation in mitochondria (10).Second, ACBP may control the level of unbound LCFA-CoA available for interaction with regulatory sites on metabolic enzymes (e.g. acetyl-CoA carboxylase) and intracellular signaling proteins (e.g. protein kinase C) in the cytosol (reviewed in Refs. 12 and 13).Third, recent data demonstrating the presence of significant amounts of ACBP in the nuclei of transfected cells overexpressing ACBP suggest that ACBP may also be involved in direct or ligand (LCFA-CoA)-dependent regulation of nuclear proteins that activate transcription of genes involved in lipid and glucose metabolism (14,15). Several members of the nuclear receptor superfamily including the hepatocyte nuclear receptor 4␣ (HNF-4␣) (16 -18), thyroid hormone receptor (TR) (19), and peroxisome proliferator-activated receptor-␣ and -␦ (PPAR-␣ and -␦) (20, 21) interact with LCFA-CoAs. The relative order of affinities of these nuclear receptors for LCFA-CoAs is HNF-4␣ (K d of 1.5-4 nM) Ͼ Ͼ TR (K d of 120 nM) Ͼ Ͼ PPAR␣ (displaces Wy14643). On this basis, it appears that only HNF-4␣ binds LCFA-CoAs with affinities in the physiological range of LCFACoA levels in the nucleus, Ͻ10 nM (17, 18). HNF-4␣ is a nuclear receptor with major roles in hepatocyte differentiation during liver development and in regulating the transcription of nu-

G-protein-coupled receptors (GPCRs) participate in a broad range of physiological functions. A priority for fundamental and clinical research, therefore, is to decipher the function of over 140 remaining orphan GPCRs. The suprachiasmatic nucleus (SCN), the brain's circadian pacemaker, governs daily rhythms in behaviour and physiology. Here we launch the SCN orphan GPCR project to (i) search for murine orphan GPCRs with enriched expression in the SCN, (ii) generate mutant animals deficient in candidate GPCRs, and (iii) analyse the impact on circadian rhythms. We thereby identify Gpr176 as an SCN-enriched orphan GPCR that sets the pace of circadian behaviour. Gpr176 is expressed in a circadian manner by SCN neurons, and molecular characterization reveals that it represses cAMP signalling in an agonist-independent manner. Gpr176 acts independently of, and in parallel to, the Vipr2 GPCR, not through the canonical Gi, but via the unique G-protein subclass Gz.

Cytoplasmic polyadenylation element-binding protein (CPEB)3 is a nucleocytoplasm-shuttling RNA-binding protein and predominantly resides in the cytoplasm where it represses target RNA translation. When translocated into the nucleus, CPEB3 binds to Stat5b and downregulates Stat5b-dependent transcription. In neurons, the activation of N-methyl-d-aspartate receptors (NMDARs) accumulates CPEB3 in the nucleus and redistributes CPEB3 in the nucleocytoplasmic compartments to control gene expression. Nonetheless, it is unclear which karyopherin drives the nuclear import of CPEB3 and which transport direction is most affected by NMDA stimulation to increase the nuclear pool of CPEB3. Here, we have identified that the karyopherins, IPO5 and CRM1, facilitate CPEB3 translocation by binding to RRM1 and a leucine-containing motif of CPEB3, respectively. NMDAR signaling increases RanBP1 expression and reduces the level of cytoplasmic GTP-bound Ran. These changes enhance CPEB3–IPO5 interaction, which consequently accelerates the nuclear import of CPEB3. This study uncovers a novel NMDA-regulated import pathway to facilitate the nuclear translocation of CPEB3.