Collapse of late endosomal pH elicits a rapid Rab7 response via V-ATPase and RILP.

Endosomal-lysosomal trafficking is accompanied by the acidification of endosomal compartments by the H+-V-ATPase to reach low lysosomal pH. Disruption of proper pH impairs lysosomal function and the balance of protein synthesis and degradation (proteostasis). We used the small dipeptide LLOMe, which is known to permeabilize lysosomal membranes, and find that LLOMe also impacts late endosomes (LEs) by neutralizing their pH without causing membrane permeabilization. We show that LLOMe leads to hyper-activation of Rab7 and disruption of tubulation and mannose-6-phosphate receptor (CI-M6PR) recycling on pH-neutralized LEs. Either pH neutralization (NH4Cl) or Rab7 hyper-active mutants alone can phenocopy the alterations in tubulation and CI-M6PR trafficking. Mechanistically, pH neutralization increases the assembly of the V1G1 subunit of the V-ATPase on endosomal membranes, which stabilizes GTP-bound Rab7 via RILP, a known interactor of Rab7 and V1G1. We propose a novel pathway by which V-ATPase and RILP modulate LE pH and Rab7 activation in concert. This pathway might broadly contribute to pH control during physiologic endosomal maturation or starvation and during pathologic pH neutralization, which occurs via lysosomotropic compounds or in disease states.

Nestin in immature embryonic neurons affects axon growth cone morphology and Semaphorin3a sensitivity.

Correct wiring in the neocortex requires that responses to an individual guidance cue vary among neurons in the same location, and within the same neuron over time. Nestin is an atypical intermediate filament expressed strongly in neural progenitors and is thus used widely as a progenitor marker. Here we show a subpopulation of embryonic cortical neurons that transiently express nestin in their axons. Nestin expression is thus not restricted to neural progenitors, but persists for 2-3 d at lower levels in newborn neurons. We found that nestin-expressing neurons have smaller growth cones, suggesting that nestin affects cytoskeletal dynamics. Nestin, unlike other intermediate filament subtypes, regulates cdk5 kinase by binding the cdk5 activator p35. Cdk5 activity is induced by the repulsive guidance cue Semaphorin3a (Sema3a), leading to axonal growth cone collapse in vitro. Therefore, we tested whether nestin-expressing neurons showed altered responses to Sema3a. We find that nestin-expressing newborn neurons are more sensitive to Sema3a in a roscovitine-sensitive manner, whereas nestin knockdown results in lowered sensitivity to Sema3a. We propose that nestin functions in immature neurons to modulate cdk5 downstream of the Sema3a response. Thus, the transient expression of nestin could allow temporal and/or spatial modulation of a neuron's response to Sema3a, particularly during early axon guidance.

Antisense transcription in the mammalian transcriptome.

Antisense transcription (transcription from the opposite strand to a protein-coding or sense strand) has been ascribed roles in gene regulation involving degradation of the corresponding sense transcripts (RNA interference), as well as gene silencing at the chromatin level. Global transcriptome analysis provides evidence that a large proportion of the genome can produce transcripts from both strands, and that antisense transcripts commonly link neighboring "genes" in complex loci into chains of linked transcriptional units. Expression profiling reveals frequent concordant regulation of sense/antisense pairs. We present experimental evidence that perturbation of an antisense RNA can alter the expression of sense messenger RNAs, suggesting that antisense transcription contributes to control of transcriptional outputs in mammals.

Distinct glutamate receptors govern differential levels of nitric oxide production in a layer-specific manner in the rat cerebellar cortex.

To evaluate roles of nitric oxide (NO) in neural functions, it is critical to know how neural inputs activate neuronal NO synthase in individual sites. Although NMDA receptor-dependent mechanism well explains postsynaptic, robust NO production, this sole mechanism does not explain some aspects of NO production in the brain, such as the low-level production of NO and the mechanism for presynaptic NO production. We hypothesized that the glutamate receptor involved in NO production is site-specific and controls the initial NO concentration in each site. We visualized NO production mediated by NMDA, AMPA and type-1 metabotropic glutamate (mGlu-1) receptors in rat cerebellar slices and granule cells in culture, with an NO-specific fluorescent indicator, diaminofluorescein-2. AMPA receptor, but not NMDA or mGlu-1 receptor, was responsible for NO production at parallel fiber terminals, which was blocked by CNQX, tetrodotoxin or voltage-dependent calcium channel blockers. More numbers of electrical stimulation were required for NO production in the molecular layer than in other layers, suggesting that AMPA receptor activation generates NO at lower concentrations through a remote interaction with NO synthase. Although Purkinje cell does not express NO synthase, we detected NO production in Purkinje cell layer following electrical stimulation in the white matter at 50 Hz, but not at 10 Hz. This NO production was tetrodotoxin-sensitive, suggesting occurrence in the basket cell terminals, and required synergistic activation of mGlu-1 and NMDA receptors. In the granule cell layer, activation of AMPA or mGlu-1 receptor produced NO uniformly, while NMDA receptor activation produced NO in discontinuous areas of this layer. Thus, distinct glutamate receptors, including non-NMDA receptors, govern occurrence and level of NO production in a layer-specific manner.

Sast124, a novel splice variant of syntrophin-associated serine/threonine kinase (SAST), is specifically localized in the restricted brain regions.

Syntrophin is an adaptor protein that binds signaling molecules to the dystrophin-associated protein complex, which connects extracellular matrix to intracellular cytoskeleton for construction and maintenance of the postsynaptic structures in the neuromuscular junction and the CNS. Among these signaling molecules, a family of microtubule-associated serine/threonine kinases has a unique structural feature with a serine/threonine kinase domain and a postsynaptic density protein-95/discs large/zona occludens-1 domain. In the present study, we identified syntrophin-associated serine/threonine kinase-124, a novel splice variant of the syntrophin-associated serine/threonine kinase which is a member of the microtubule-associated serine/threonine kinases family. Comparing to the original clone (syntrophin-associated serine/threonine kinase-170), syntrophin-associated serine/threonine kinase-124 is truncated just downstream of the postsynaptic density protein-95/discs large/zona occludens-1 domain. Using a monoclonal antibody specifically recognizing syntrophin-associated serine/threonine kinase-124, strong expression of the protein was observed in neurons of the subventricular zone and granule cells of the olfactory bulb, Islands of Calleja, hippocampal dentate gyrus and cerebellum. syntrophin-associated serine/threonine kinase-124 is selectively localized in the nuclei of neurons and distinct from syntrophin-associated serine/threonine kinase-170, which is interacting with syntrophin on the cell surface. Considering the tissue and subcellular distributions of syntrophin-associated serine/threonine kinase-124, it is suggested that syntrophin-associated serine/threonine kinase-124 may have functions in transcriptional regulation for the features commonly shared by these neurons. On the other hand, syntrophin-associated serine/threonine kinase-124 was also localized in glia-like cell bodies in the corpus callosum and fiber bundles in the spinal trigeminal and solitary tracts, suggesting syntrophin-associated serine/threonine kinase-124 may have other functions in these types of cells.

Expression of hepatitis C virus NS5B protein: characterization of its RNA polymerase activity and RNA binding.

The nonstructural protein 5B (NS5B) of hepatitis C virus (HCV) is considered to possess RNA-dependent RNA polymerase (RdRp) activity and to play an essential role for the viral replication. In this study, we expressed the NS5B protein of 65 kd by a recombinant baculovirus. With the highly purified NS5B protein, we established an in vitro system for RdRp activity by using poly(A) as a template and a 15-mer oligo(U) (oligo(U)15) as a primer. Optimal conditions of temperature and pH for primer-dependent polymerase activity of the NS5B were 32 degrees C and pH 8.0. The addition of 10 mmol of Mg2+ increased the activity. The importance of three motifs conserved in RdRp among other positive-strand RNA viruses was confirmed by introduction of an Ala residue to every amino acid of the motifs by site-directed mutagenesis. All mutants lost RdRp activity, but retained the RNA binding activity, except one mutant at Thr287/Asn291. Deletion mutant analysis indicated that the N-terminal region of NS5B protein was critical for the RNA binding. Inhibition of RdRp activity by (-)beta-L-2', 3'-dideoxy-3'-thiacytidine 5'-triphosphate (3TC; lamivudine triphosphate) and phosphonoacetic acid (PAA) was observed after screening of nucleoside analogs and known polymerase inhibitors. These data provide us not only important clues for understanding the mechanism of HCV replication, but also a new target of antiviral therapy.

Expression of target genes by coinfection with replication-deficient viral vectors.

An in vivo transcription system was developed by coinfection of cells with replication-deficient viral vectors. Recombinant baculovirus (AcT7HCVLuc) and fowlpox virus (FPVT7HCVLuc) carrying a cDNA of the hepatitis C virus (HCV) minigene encoding the HCV 5' untranslated region (UTR), a luciferase gene and the 3' UTR, including the 98 nt extra sequence, under the control of the T7 promoter were constructed. The HCV minigene was synthesized in various cells by coinfection with one of these two viruses and recombinant baculovirus (AcCAT7) or adenovirus (AdexCAT7) expressing T7 RNA polymerase under the control of a mammalian promoter. Only a low level of luciferase expression was obtained in cells coinfected with AcT7HCVLuc and either AcCAT7 or AdexCAT7. In contrast, high-level luciferase expression was detected when the same cells were coinfected with FPVT7HCVLuc and either AcCAT7 or AdexCAT7. We further constructed a recombinant fowlpox virus with its HCV minigene extended to contain the whole HCV core protein region. Significantly high levels of expression of HCV core protein were detected in MT-2, COS7 and Vero cells by coinfection with the recombinant fowlpox virus and AdexCAT7. A coinfection system consisting of recombinant fowlpox virus and AdexCAT7 was established for high level of expression of a target gene in various cells.

A hybrid baculovirus-T7 RNA polymerase system for recovery of an infectious virus from cDNA.

We established a hybrid baculovirus-T7 RNA polymerase system for transient expression in mammalian cells. Two recombinant baculoviruses carrying cDNA of bacteriophage T7 RNA polymerase, with or without a nuclear localization signal, under the control of a mammalian promoter were constructed. High level expression of T7 RNA polymerase was observed in various mammalian cell lines after infection with the recombinant baculoviruses. After transfection of plasmids containing the luciferase gene under the control of the T7 promoter, high luciferase activity was detected in cells infected with the recombinant baculoviruses. We also constructed a plasmid containing an entire cDNA clone of type 1 poliovirus under the T7 promoter. Two days after transfection of the plasmid into the cells infected with the recombinant baculoviruses, a high titer of poliovirus was recovered. The use of the recombinant baculoviruses did not cause any cytopathic effects even at a high multiplicity of infection. The lack of replication ability and low toxicity are the advantageous features of the hybrid baculovirus-T7 polymerase system in comparison with the widely used vaccinia-T7 polymerase system for gene expression and recovery of infectious viruses from its cDNA.