Synapse Formation in Monosynaptic Sensory-Motor Connections Is Regulated by Presynaptic Rho GTPase Cdc42.

UNLABELLED: Spinal reflex circuit development requires the precise regulation of axon trajectories, synaptic specificity, and synapse formation. Of these three crucial steps, the molecular mechanisms underlying synapse formation between group Ia proprioceptive sensory neurons and motor neurons is the least understood. Here, we show that the Rho GTPase Cdc42 controls synapse formation in monosynaptic sensory-motor connections in presynaptic, but not postsynaptic, neurons. In mice lacking Cdc42 in presynaptic sensory neurons, proprioceptive sensory axons appropriately reach the ventral spinal cord, but significantly fewer synapses are formed with motor neurons compared with wild-type mice. Concordantly, electrophysiological analyses show diminished EPSP amplitudes in monosynaptic sensory-motor circuits in these mutants. Temporally targeted deletion of Cdc42 in sensory neurons after sensory-motor circuit establishment reveals that Cdc42 does not affect synaptic transmission. Furthermore, addition of the synaptic organizers, neuroligins, induces presynaptic differentiation of wild-type, but not Cdc42-deficient, proprioceptive sensory neurons in vitro Together, our findings demonstrate that Cdc42 in presynaptic neurons is required for synapse formation in monosynaptic sensory-motor circuits. SIGNIFICANCE STATEMENT: Group Ia proprioceptive sensory neurons form direct synapses with motor neurons, but the molecular mechanisms underlying synapse formation in these monosynaptic sensory-motor connections are unknown. We show that deleting Cdc42 in sensory neurons does not affect proprioceptive sensory axon targeting because axons reach the ventral spinal cord appropriately, but these neurons form significantly fewer presynaptic terminals on motor neurons. Electrophysiological analysis further shows that EPSPs are decreased in these mice. Finally, we demonstrate that Cdc42 is involved in neuroligin-dependent presynaptic differentiation of proprioceptive sensory neurons in vitro These data suggest that Cdc42 in presynaptic sensory neurons is essential for proper synapse formation in the development of monosynaptic sensory-motor circuits.

Magmas expression in neoplastic human prostate.

Magmas, is a 13-kDa mitochondrial protein which is ubiquitously expressed in eukaryotic cells. It was identified as a granulocyte-macrophage-colony stimulating factor (GM-CSF) inducible gene in hematopoietic cells and has a key role in the transport of mitochondrial proteins in yeast. Because GM-CSF receptor levels are elevated in prostate cancer, Magmas expression was examined in normal and neoplastic tissue. Magmas protein levels were barely detectable in non-neoplastic prostate glands. Increased amounts were observed in some samples of intraepithelial neoplasia. Approximately one half of the adenocarcinoma samples examined had weak Magmas expression, while the remainder had intermediate to high levels. The increased Magmas observed in malignant tissue was a result of higher protein expression and not from changes in mitochondrial content. Interestingly, in some patients, the normal prostate tissue had more Magmas message than the malignant portion. The results indicated that Magmas expression in prostate cancer is heterogeneous and independent of clinical stage and Gleason score. Further studies are needed to determine if Magmas expression has prognostic significance in prostate cancer.

Nf1 loss and Ras hyperactivation in oligodendrocytes induce NOS-driven defects in myelin and vasculature.

Patients with neurofibromatosis type 1 (NF1) and Costello syndrome Rasopathy have behavioral deficits. In NF1 patients, these may correlate with white matter enlargement and aberrant myelin. To model these features, we induced Nf1 loss or HRas hyperactivation in mouse oligodendrocytes. Enlarged brain white matter tracts correlated with myelin decompaction, downregulation of claudin-11, and mislocalization of connexin-32. Surprisingly, non-cell-autonomous defects in perivascular astrocytes and the blood-brain barrier (BBB) developed, implicating a soluble mediator. Nitric oxide (NO) can disrupt tight junctions and gap junctions, and NO and NO synthases (NOS1-NOS3) were upregulated in mutant white matter. Treating mice with the NOS inhibitor NG-nitro-L-arginine methyl ester or the antioxidant N-acetyl cysteine corrected cellular phenotypes. CNP-HRasG12V mice also displayed locomotor hyperactivity, which could be rescued by antioxidant treatment. We conclude that Nf1/Ras regulates oligodendrocyte NOS and that dysregulated NO signaling in oligodendrocytes can alter the surrounding vasculature. The data suggest that antioxidants may improve some behavioral deficits in Rasopathy patients.

Perinatal or adult Nf1 inactivation using tamoxifen-inducible PlpCre each cause neurofibroma formation.

Plexiform neurofibromas are peripheral nerve sheath tumors initiated by biallelic mutation of the NF1 tumor suppressor gene in the Schwann cell lineage. To understand whether neurofibroma formation is possible after birth, we induced Nf1 loss of function with an inducible proteolipid protein Cre allele. Perinatal loss of Nf1 resulted in the development of small plexiform neurofibromas late in life, whereas loss in adulthood caused large plexiform neurofibromas and morbidity beginning 4 months after onset of Nf1 loss. A conditional EGFP reporter allele identified cells showing recombination, including peripheral ganglia satellite cells, peripheral nerve S100ß+ myelinating Schwann cells, and peripheral nerve p75+ cells. Neurofibromas contained cells with Remak bundle disruption but no recombination within GFAP+ nonmyelinating Schwann cells. Extramedullary lympho-hematopoietic expansion was also observed in PlpCre;Nf1fl/fl mice. These tumors contained EGFP+/Sca-1+ stromal cells among EGFP-negative lympho-hematopoietic cells indicating a noncell autonomous effect and unveiling a role of Nf1-deleted microenvironment on lympho-hematopoietic proliferation in vivo. Together these findings define a tumor suppressor role for Nf1 in the adult and narrow the range of potential neurofibroma-initiating cell populations.

Human dendritic cell activity against Histoplasma capsulatum is mediated via phagolysosomal fusion.

Histoplasma capsulatum is a fungal pathogen that requires the induction of cell-mediated immunity (CMI) for host survival. We have demonstrated that human dendritic cells (DC) phagocytose H. capsulatum yeasts and, unlike human macrophages (Mø) that are permissive for intracellular growth, DC killed and degraded the fungus. In the present study, we sought to determine whether the mechanism(s) by which DC kill Histoplasma is via lysosomal hydrolases, via the production of toxic oxygen metabolites, or both. Phagosome-lysosome fusion (PL-fusion) was quantified by using fluorescein isothiocyanate-dextran and phase and fluorescence microscopy and by electron microscopy with horseradish peroxidase colloidal gold to label lysosomes. Unlike Mphi, Histoplasma-infected DC exhibited marked PL-fusion. The addition of suramin to Histoplasma-infected DC inhibited PL-fusion and DC fungicidal activity. Incubation of Histoplasma-infected DC at 18 degrees C also concomitantly reduced PL-fusion and decreased the capacity of DC to kill and degrade H. capsulatum yeasts. Further, culture of Histoplasma-infected DC in the presence of bafilomycin, an inhibitor of the vacuolar ATPase, did not block DC anti-Histoplasma activity, indicating that phagosome acidification was not required for lysosome enzyme activity. In contrast, culture of Histoplasma-infected DC in the presence of inhibitors of the respiratory burst or inhibitors of NO synthase had little to no effect on DC fungicidal activity. These data suggest that the major mechanism by which human DC mediate anti-Histoplasma activity is through the exposure of yeasts to DC lysosomal hydrolases. Thus, DC can override one of the strategies used by H. capsulatum yeasts to survive intracellularly within Mø.

Rapid apoptosis induced by Shiga toxin in HeLa cells.

Apoptosis was induced rapidly in HeLa cells after exposure to bacterial Shiga toxin (Stx1 and Stx2; 10 ng/ml). Approximately 60% of HeLa cells became apoptotic within 4 h as detected by DNA fragmentation, terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) assay, and electron microscopy. Stx1-induced apoptosis required enzymatic activity of the Stx1A subunit, and apoptosis was not induced by the Stx2B subunit alone or by the anti-globotriaosylceramide antibody. This activity was also inhibited by brefeldin A, indicating the need for toxin processing through the Golgi apparatus. The intracellular pathway leading to apoptosis was further defined. Exposure of HeLa cells to Stx1 activated caspases 3, 6, 8, and 9, as measured both by an enzymatic assay with synthetic substrates and by detection of proteolytically activated forms of these caspases by Western immunoblotting. Preincubation of HeLa cells with substrate inhibitors of caspases 3, 6, and 8 protected the cells against Stx1-dependent apoptosis. These results led to a more detailed examination of the mitochondrial pathway of apoptosis. Apoptosis induced by Stx1 was accompanied by damage to mitochondrial membranes, measured as a reduced mitochondrial membrane potential, and increased release of cytochrome c from mitochondria at 3 to 4 h. Bid, an endogenous protein known to permeabilize mitochondrial membranes, was activated in a Stx1-dependent manner. Caspase-8 is known to activate Bid, and a specific inhibitor of caspase-8 prevented the mitochondrial damage. Although these data suggested that caspase-8-mediated cleavage of Bid with release of cytochrome c from mitochondria and activation of caspase-9 were responsible for the apoptosis, preincubation of HeLa cells with a specific inhibitor of caspase-9 did not protect against apoptosis. These results were explained by the discovery of a simultaneous Stx1-dependent increase in endogenous XIAP, a direct inhibitor of caspase-9. We conclude that the primary pathway of Stx1-induced apoptosis and DNA fragmentation in HeLa cells is unique and includes caspases 8, 6, and 3 but is independent of events in the mitochondrial pathway.