Maf links Neuregulin1 signaling to cholesterol synthesis in myelinating Schwann cells.

Cholesterol is a major constituent of myelin membranes, which insulate axons and allow saltatory conduction. Therefore, Schwann cells, the myelinating glia of the peripheral nervous system, need to produce large amounts of cholesterol. Here, we define a crucial role of the transcription factor Maf in myelination and cholesterol biosynthesis and show that Maf acts downstream from Neuregulin1 (Nrg1). Maf expression is induced when Schwann cells begin myelination. Genetic ablation of Maf resulted in hypomyelination that resembled mice with defective Nrg1 signaling. Importantly, loss of Maf or Nrg1 signaling resulted in a down-regulation of the cholesterol synthesis program, and Maf directly binds to enhancers of cholesterol synthesis genes. Furthermore, we identified the molecular mechanisms by which Nrg1 signaling regulates Maf levels. Transcription of Maf depends on calmodulin-dependent kinases downstream from Nrg1, whereas Nrg1-MAPK signaling stabilizes Maf protein. Our results delineate a novel signaling cascade regulating cholesterol synthesis in myelinating Schwann cells.

Dual and Direction-Selective Mechanisms of Phosphate Transport by the Vesicular Glutamate Transporter.

Vesicular glutamate transporters (VGLUTs) fill synaptic vesicles with glutamate and are thus essential for glutamatergic neurotransmission. However, VGLUTs were originally discovered as members of a transporter subfamily specific for inorganic phosphate (Pi). It is still unclear how VGLUTs accommodate glutamate transport coupled to an electrochemical proton gradient ΔµH+ with inversely directed Pi transport coupled to the Na+ gradient and the membrane potential. Using both functional reconstitution and heterologous expression, we show that VGLUT transports glutamate and Pi using a single substrate binding site but different coupling to cation gradients. When facing the cytoplasm, both ions are transported into synaptic vesicles in a ΔµH+-dependent fashion, with glutamate preferred over Pi. When facing the extracellular space, Pi is transported in a Na+-coupled manner, with glutamate competing for binding but at lower affinity. We conclude that VGLUTs have dual functions in both vesicle transmitter loading and Pi homeostasis within glutamatergic neurons.

Activation of MAPK overrides the termination of myelin growth and replaces Nrg1/ErbB3 signals during Schwann cell development and myelination.

Myelination depends on the synthesis of large amounts of myelin transcripts and proteins and is controlled by Nrg1/ErbB/Shp2 signaling. We developed a novel pulse labeling strategy based on stable isotope labeling with amino acids in cell culture (SILAC) to measure the dynamics of myelin protein production in mice. We found that protein synthesis is dampened in the maturing postnatal peripheral nervous system, and myelination then slows down. Remarkably, sustained activation of MAPK signaling by expression of the Mek1DD allele in mice overcomes the signals that end myelination, resulting in continuous myelin growth. MAPK activation leads to minor changes in transcript levels but massively up-regulates protein production. Pharmacological interference in vivo demonstrates that the effects of activated MAPK signaling on translation are mediated by mTOR-independent mechanisms but in part also by mTOR-dependent mechanisms. Previous work demonstrated that loss of ErbB3/Shp2 signaling impairs Schwann cell development and disrupts the myelination program. We found that activated MAPK signaling strikingly compensates for the absence of ErbB3 or Shp2 during Schwann cell development and myelination.

The NADPH oxidase Nox2 regulates VEGFR1/CSF-1R-mediated microglial chemotaxis and promotes early postnatal infiltration of phagocytes in the subventricular zone of the mouse cerebral cortex.

The phagocyte NADPH oxidase Nox2 generates superoxide ions implicated in the elimination of microorganisms and the redox control of inflammatory signaling. However, the role of Nox2 in phagocyte functions unrelated to immunity or pathologies is unknown. During development, oriented cell migrations insure the timely recruitment and function of phagocytes in developing tissues. Here, we have addressed the role of Nox2 in the directional migration of microglial cells during development. We show that microglial Nox2 regulates the chemotaxis of purified microglia mediated by the colony stimulating factor-1 receptor (CSF-1R) and the vascular endothelial growth factor receptor-1 (VEGFR1). Stimulation of these receptors triggers activation of Nox2 at the leading edge of polarized cells. In the early postnatal stages of mouse brain development, Nox2 is activated in macrophages / microglial cells in the lateral ventricle or the adjacent subventricular zone (SVZ). Fluorescent microglia injected into the lateral ventricle infiltrate the dorso-caudal SVZ through a mechanism that is blocked by pretreatment of the injected cells with an irreversible Nox inhibitor. Infiltration of endogenous microglia into the caudal SVZ of the cerebral cortex is prevented by (1) Nox2 gene deficiency, (2) treatment with a Nox2 inhibitor (apocynin), and (3) invalidation of the VEGFR1 kinase. We conclude that phagocytes move out of the lateral ventricle soon after birth and infiltrate the cortical SVZ through a mechanism requiring microglial Nox2 and VEGFR1 activation. Nox2 therefore modulates the migration of microglia and their development.

The transcription factor c-Maf controls touch receptor development and function.

The sense of touch relies on detection of mechanical stimuli by specialized mechanosensory neurons. The scarcity of molecular data has made it difficult to analyze development of mechanoreceptors and to define the basis of their diversity and function. We show that the transcription factor c-Maf/c-MAF is crucial for mechanosensory function in mice and humans. The development and function of several rapidly adapting mechanoreceptor types are disrupted in c-Maf mutant mice. In particular, Pacinian corpuscles, a type of mechanoreceptor specialized to detect high-frequency vibrations, are severely atrophied. In line with this, sensitivity to high-frequency vibration is reduced in humans carrying a dominant mutation in the c-MAF gene. Thus, our work identifies a key transcription factor specifying development and function of mechanoreceptors and their end organs.

The tyrosine phosphatase Shp2 (PTPN11) directs Neuregulin-1/ErbB signaling throughout Schwann cell development.

The nonreceptor tyrosine phosphatase Shp2 (PTPN11) has been implicated in tyrosine kinase, cytokine, and integrin receptor signaling. We show here that conditional mutation of Shp2 in neural crest cells and in myelinating Schwann cells resulted in deficits in glial development that are remarkably similar to those observed in mice mutant for Neuregulin-1 (Nrg1) or the Nrg1 receptors, ErbB2 and ErbB3. In cultured Shp2 mutant Schwann cells, Nrg1-evoked cellular responses like proliferation and migration were virtually abolished, and Nrg1-dependent intracellular signaling was altered. Pharmacological inhibition of Src family kinases mimicked all cellular and biochemical effects of the Shp2 mutation, implicating Src as a primary Shp2 target during Nrg1 signaling. Together, our genetic and biochemical analyses demonstrate that Shp2 is an essential component in the transduction of Nrg1/ErbB signals.

Neurotoxic activation of microglia is promoted by a nox1-dependent NADPH oxidase.

Reactive oxygen species (ROS) modulate intracellular signaling but are also responsible for neuronal damage in pathological states. Microglia, the resident CNS macrophages, are prominent sources of ROS through expression of the phagocyte oxidase which catalytic subunit Nox2 generates superoxide ion (O2(.-)). Here we show that microglia also express Nox1 and other components of nonphagocyte NADPH oxidases, including p22(phox), NOXO1, NOXA1, and Rac1/2. The subcellular distribution and functions of Nox1 were determined by blocking Nox activity with diphenylene iodonium or apocynin, and by silencing the Nox1 gene in microglia purified from wild-type (WT) or Nox2-KO mice. [Nox1-p22(phox)] dimers localized in intracellular compartments are recruited to phagosome membranes during microglial phagocytosis of zymosan, and Nox1 produces O2(.-) in zymosan-loaded phagosomes. In microglia activated with lipopolysaccharide (LPS), Nox1 produces O2(.-), which enhances cell expression of inducible nitric oxide synthase and secretion of interleukin-1beta. Comparisons of microglia purified from WT, Nox2-KO, or Nox1-KO mice indicate that both Nox1 and Nox2 are required to optimize microglial production of nitric oxide. By injecting LPS in the striatum of WT and Nox1-KO mice, we show that Nox1 also enhances microglial production of cytotoxic nitrite species and promotes loss of presynaptic proteins in striatal neurons. These results demonstrate the functional expression of Nox1 in resident CNS phagocytes, which can promote production of neurotoxic compounds during neuroinflammation. Our study also shows that Nox1- and Nox2-dependent oxidases play distinct roles in microglial activation and that Nox1 is a possible target for the treatment of neuroinflammatory states.

System Xc- and apolipoprotein E expressed by microglia have opposite effects on the neurotoxicity of amyloid-beta peptide 1-40.

Because senile plaques in Alzheimer's disease (AD) contain reactive microglia in addition to potentially neurotoxic aggregates of amyloid-beta (Abeta), we examined the influence of microglia on the viability of rodent neurons in culture exposed to aggregated Abeta 1-40. Microglia enhanced the toxicity of Abeta by releasing glutamate through the cystine-glutamate antiporter system Xc-. This may be relevant to Abeta toxicity in AD, because the system Xc(-)-specific xCT gene is expressed not only in cultured microglia but also in reactive microglia within or surrounding amyloid plaques in transgenic mice expressing mutant human amyloid precursor protein or in wild-type mice injected with Abeta. Inhibition of NMDA receptors or system Xc- prevented the microglia-enhanced neurotoxicity of Abeta but also unmasked a neuroprotective effect of microglia mediated by microglial secretion of apolipoprotein E (apoE) in the culture medium. Immunodepletion of apoE or targeted inactivation of the apoE gene in microglia abrogated neuroprotection by microglial conditioned medium, whereas supplementation by human apoE isoforms restored protection, which was potentiated by the presence of microglia-derived cofactors. These results suggest that inhibition of microglial system Xc- might be of therapeutic value in the treatment of AD. Its inhibition not only prevents glutamate excitotoxicity but also facilitates neuroprotection by apoE.