BACE1 modulates filopodia-like protrusions induced by sodium channel beta4 subunit

Processing of APP by BACE1 plays a crucial role in the pathogenesis of Alzheimer disease (AD). Recently, the voltage-gated sodium channel (Na_v) β4 subunit (β4), an auxiliary subunit of Na_v that is supposed to serve as a cell adhesion molecule, has been identified as a substrate for BACE1. However, the biological consequence of BACE1 processing of β4 remains illusive. Here, we report the biological effects of β4 processing by BACE1. Overexpression of β4 in Neuro2a cells promoted neurite extension and increased the number of F-actin rich filopodia-like protrusions. While coexpression of BACE1 together with β4 further accelerated neurite extension, the number of filopodia-like protrusions was reduced. Overexpression of C-terminal fragment of β4 that was generated by BACE1 (β4-CTF) partially recapitulated the results obtained with BACE1 overexpression. These results suggest that the processing of β4 by BACE1 regulates neurite length and filopodia-like protrusion density in neurons.     

Exogenous neuritin treatment improves survivability and functions of Schwann cells with improved outgrowth of neurons in rat diabetic neuropathy

Pathogenesis and treatment for diabetic neuropathy are still complex. A deficit of neurotrophic factors affecting Schwann cells is a very important cause of diabetic neuropathy. Neuritin is a newly discovered potential neurotrophic factor. In this study, we explored the effect of exogenous neuritin on survivability and functions of diabetic Schwann cells of rats with experimental diabetic neuropathy. Diabetic neuropathy was induced in rats. 12‐week diabetic rats contrasted with non‐diabetic normal rats had decreased levels of serum neuritin and slowed nerve conduction velocities (NCVs). Schwann cells isolated from these diabetic rats and cultured in high glucose showed reduced cell neuritin mRNA and protein and supernatant neuritin protein, increased apoptosis rates, increased caspase‐3 activities and progressively reduced viability. In contrast, exogenous neuritin treatment reduced apoptosis and improved viability, with elevated Bcl‐2 levels (not Bax) and decreased caspase‐3 activities. Co‐cultured with diabetic Schwann cells pre‐treated with exogenous neuritin in high glucose media, and diabetic DRG neurons showed lessened decreased neurite outgrowth and supernatant NGF concentration occurring in co‐culture of diabetic cells. Exogenous neuritin treatment ameliorated survivability and functions of diabetic Schwann cells of rats with diabetic neuropathy. Our study may provide a new mechanism and potential treatment for diabetic neuropathy.     

Polycistronic lentivirus induced pluripotent stem cells from skin biopsies after long term storage,blood outgrowth endothelial cells and cells from milk teeth

The generation of human induced pluripotent stem cells (hiPSCs) requires the collection of donor tissue, but clinical circumstances in which the interests of patients have highest priority may compromise the quality and availability of cells that are eventually used for reprogramming. Here we compared (i) skin biopsies stored in standard physiological salt solution for up to two weeks (ii) blood outgrowth endothelial cells (BOECs) isolated from fresh peripheral blood and (iii) children's milk teeth lost during normal replacement for their ability to form somatic cell cultures suitable for reprogramming to hiPSCs. We derived all hiPSC lines using the same reprogramming method (a conditional (FLPe) polycistronic lentivirus) and under similar conditions (same batch of virus, fetal calf serum and feeder cells). Skin fibroblasts could be reprogrammed robustly even after long-term biopsy storage. Generation of hiPSCs from juvenile dental pulp cells gave similar high efficiencies, but that of BOECs was lower. In terms of invasiveness of biopsy sampling, biopsy storage and reprogramming efficiencies skin fibroblasts appeared best for the generation of hiPSCs, but where non-invasive procedures are required (e.g. for children and minors) dental pulp cells from milk teeth represent a valuable alternative.     

TRPC6 channel‐mediated neurite outgrowth in PC12 cells and hippocampal neurons involves activation of RAS/MEK/ERK,PI3K,and CAMKIV signaling

The non‐selective cationic transient receptor canonical 6 (TRPC6) channels are involved in synaptic plasticity changes ranging from dendritic growth, spine morphology changes and increase in excitatory synapses. We previously showed that the TRPC6 activator hyperforin, the active antidepressant component of St. John's wort, induces neuritic outgrowth and spine morphology changes in PC12 cells and hippocampal CA1 neurons. However, the signaling cascade that transmits the hyperforin‐induced transient rise in intracellular calcium into neuritic outgrowth is not yet fully understood. Several signaling pathways are involved in calcium transient‐mediated changes in synaptic plasticity, ranging from calmodulin‐mediated Ras‐induced signaling cascades comprising the mitogen‐activated protein kinase, PI3K signal transduction pathways as well as Ca²⁺/calmodulin‐dependent protein kinase II (CAMKII) and CAMKIV. We show that several mechanisms are involved in TRPC6‐mediated synaptic plasticity changes in PC12 cells and primary hippocampal neurons. Influx of calcium via TRPC6 channels activates different pathways including Ras/mitogen‐activated protein kinase/extracellular signal‐regulated kinases, phosphatidylinositide 3‐kinase/protein kinase B, and CAMKIV in both cell types, leading to cAMP‐response element binding protein phosphorylation. These findings are interesting not only in terms of the downstream targets of TRPC6 channels but also because of their potential to facilitate further understanding of St. John's wort extract‐mediated antidepressant activity.

     

Gadd45a, the gene induced by the mood stabilizer valproic acid, regulates neurite outgrowth through JNK and the substrate paxillin in N1E-115 neuroblastoma cells

Valproic acid (VPA), a mood stabilizer and anticonvulsant, has a variety of neurotrophic functions; however, less is known about how VPA regulates neurite outgrowth. Here, using N1E-115 neuroblastoma cells as the model, we show that VPA upregulates Gadd45a to trigger activation of the downstream JNK cascade controlling neurite outgrowth. VPA induces the phosphorylation of c-Jun N-terminal kinase (JNK) and the substrate paxillin, while VPA induction of neurite outgrowth is inhibited by JNK inhibitors (SP600125 and the small JNK-binding peptide) or a paxillin construct harboring a Ser 178-to-Ala mutation at the JNK phosphorylation. Transfection of Gadd45a, acting through the effector MEKK4, leads to the phosphorylation of the JNK cascade. Conversely, knockdown of Gadd45a with siRNA reduces the effect of VPA. Taken together, these results suggest that upregulation of Gadd45a explains one of the mechanisms whereby VPA induces the neurotrophic effect, providing a new role of Gadd45a in neurite outgrowth.     

The roles of cyclin-dependent kinase 5 in dendrite and synapse development

Since the isolation of cyclin-dependent kinase 5 (Cdk5), this proline-directed serine/threonine kinase has been demonstrated as an important regulator of neuronal migration, neuronal survival and synaptic functions. Recently, a number of players implicated in dendrite and synapse development have been identified as Cdk5 substrates. Neurite extension, synapse and spine maturation are all modulated by a myriad of extracellular guidance cues or trophic factors. Cdk5 was recently demonstrated to regulate signaling downstream of some of these extracellular factors, in addition to modulating Rho GTPase activity, which regulates cytoskeletal dynamics. In this communication, we summarize our existing knowledge on the pathways and mechanisms through which Cdk5 affects dendrite, synapse and spine development.     

Studies on the mechanism of the osmoresistance of spores of Bacillus subtilis

AIMS: To determine the reason that spores of Bacillus species, in particular Bacillus subtilis, are able to form colonies with high efficiency on media with very high salt concentrations. METHODS AND RESULTS: Spores of various Bacillus species have a significantly higher plating efficiency on media with high salt concentration (termed osmoresistance) than do log or stationary phase cells. This spore osmoresistance is higher on richer media. Bacillus subtilis spores lacking various small, acid-soluble spore proteins (SASP) were generally significantly less osmoresistant than were wild-type spores, as shown previously (Ruzal et al. 1994). Other results included: (a) spore osmoresistance varied significantly between species; (b) the osmoresistance of spores lacking SASP was not restored well by amino acid osmolytes added to plating media, but was completely restored by glucose; (c) the osmoresistance of spores lacking SASP was restored upon brief germination in the absence of salt in a process that did not require protein synthesis; (d) significant amounts of amino acids generated by SASP degradation were retained within spores upon germination in a medium with high but not low salt; (e) slowing but not abolishing SASP degradation by loss of the SASP-specific germination protease (GPR) did not affect spore osmoresistance; (f) sporulation at higher temperatures produced less osmoresistant spores; and (g) spore osmoresistance was not decreased markedly by the absence of the stress sigma factor for RNA polymerase, sigmaB. CONCLUSIONS: Spore osmoresistance appears as a result of three major factors: (1) specific characteristics of spores and cells of individual species; (2) the precise sporulation conditions that produce the spores; and (3) sufficient energy generation by the germinating and outgrowing spore to allow the spore to adapt to conditions of high osmotic strength; the substrates for this energy generation can come from either the endogenous generation of amino acids by SASP degradation or from the spore's environment, in the form of a readily taken up and metabolized energy source such as glucose. SIGNFICANCE AND IMPACT OF STUDY: These results provide information on the mechanisms of spore osmoresistance, a spore property that can be of major applied significance given the use of high osmotic strength with or without high salt as a means of food preservation.     

Lipoproteins produced by ApoE-/- astrocytes infected with adenovirus expressing human ApoE

We have developed an astrocyte cell culture system that is attractive for the study of apoE structure and its impact on astrocyte lipoproteins and neuronal function. Primary astrocytes from apoE-/- mice were infected with adenovirus expressing apoE3 or apoE4 and the nascent lipoproteins secreted were characterized. The nascent apoE-containing astrocyte particles were predominantly the size of plasma high density lipoprotein (HDL). ApoE4, in contrast to apoE3, appeared to be distributed in two distinct lipoprotein peaks and the apoE4-containing lipoproteins contained significantly more radiolabeled triglyceride. On electron micrographs the astrocyte particles were both discoidal and spherical in shape with a prevalence of stacked discs in apoE3 particles, but single discs and larger spheres in apoE4 particles. The apoE4 discs were significantly wider than apoE3 discs. These properties of the astrocyte lipoproteins are similar to those obtained from apoE isoform transgenic mice. Astrocyte lipoproteins containing apoE3, but not apoE4, stimulated neurite outgrowth in Neuro-2a cells. These studies suggest that the isoform-specific effects of apoE lipoproteins may involve differences in particle size and composition. Finally we demonstrate the usefulness of this system by expressing a truncated apoE3 (delta202-299) mutant and show preliminary data indicating that a liver X receptor agonist promotes HDL output by the astrocytes without an increase in apoE in the media. This cell culture system is more flexible and allows for more rapid expression of apoE mutants.     

Development of single blastomeres from four- and eight-cell mouse embryos fused into the enucleated half of a two-cell embryo

Single blastomeres from four- and eight-cell mouse embryos were fused into the enucleated halves of two-cell embryos, and the ability of these reconstituted embryos to develop in vitro and in vivo was examined. The proportion of these reconstituted embryos developing to blastocysts was 74% (60/81) when four-cell embryo blastomeres were used as nuclei donors and 31% (57/182) when eight-cell embryo blastomeres were used. Eight complete sets of the quadruplet-reconstituted embryos developed to blastocysts, and five live young (9%, 5/57) were obtained after transfer; however, none of the live young were clones. Although when using blastomeres from eight-cell embryos no complete set of eight developed to blastocysts, sextuplets were obtained. The blastocysts, however, failed to produce live young after transfer. In assessing the outgrowths, it was found that 43% of those derived from reconstituted embryos using blastomeres from four-cell embryos had an inner cell mass (ICM); however, outgrowths derived from reconstituted embryos using blastomeres from eight-cell embryos lacked an ICM. These results suggest that the genomes of four- and eight-cell nuclei introduced into the enucleated halves of two-cell embryos are reversed to support the development of the reconstituted embryo.