Toshihide Yamashita

Neurotrophin binding to the p75 receptor modulates Rho activity and axonal outgrowth

While the neurotrophin receptor p75NTR is expressed by many developing neurons, its function in cells escaping elimination by programmed cell death remains unclear. The lack of intrinsic enzymatic activity of p75NTR prompted a search for protein interactors expressed in the developing retina, which resulted in the identification of the GTPase RhoA. In transfected cells, p75NTR activated RhoA, and neurotrophin binding abolished RhoA activation. In cultured neurons, inactivation of Rho proteins mimicked the effect of neurotrophins by increasing the rate of neurite elongation. In vivo, axonal outgrowth was retarded in mice carrying a mutation in the p75NTR gene. These results indicate that p75NTR modulates in a ligand-dependent fashion the activity of intracellular proteins known to regulate actin assembly.

The p75 receptor acts as a displacement factor that releases Rho from Rho-GDI.

The neurotrophin receptor p75NTR is involved in the regulation of axonal elongation by neurotrophins as well as several myelin components, including Nogo, myelin-associated glycoprotein (MAG) and myelin oligodendrocyte glycoprotein (OMgp). Neurotrophins stimulate neurite outgrowth by inhibiting Rho activity, whereas myelin-derived proteins activate RhoA and thereby inhibit growth. Here we show that direct interaction of the Rho GDP dissociation inhibitor (Rho-GDI) with p75NTR initiates the activation of RhoA, and this interaction between p75NTR and Rho-GDI is strengthened by MAG or Nogo. We also found that p75NTR facilitates the release of prenylated RhoA from Rho-GDI. The peptide ligand that is associated with the fifth α helix of p75NTR inhibits the interaction between Rho-GDI and p75NTR, thus silencing the action mediated by p75NTR. This peptide has potential as a therapeutic agent against the inhibitory cues that block regeneration in the central nervous system.

The p75 receptor transduces the signal from myelin-associated glycoprotein to Rho

Myelin-associated glycoprotein (MAG) is a potent inhibitor of neurite outgrowth from a variety of neurons. The receptor for MAG or signals that elicit morphological changes in neurons remained to be established. Here we show that the neurotrophin receptor p75 (p75NTR) is the signal transducing element for MAG. Adult dorsal root ganglion neurons or postnatal cerebellar neurons from mice carrying a mutation in the p75NTR gene are insensitive to MAG with regard to neurite outgrowth. MAG activates small GTPase RhoA, leading to retarded outgrowth when p75NTR is present. Colocalization of p75NTR and MAG binding is seen in neurons. Ganglioside GT1b, which is one of the binding partners of MAG, specifically associates with p75NTR. Thus, p75NTR and GT1b may form a receptor complex for MAG to transmit the inhibitory signals in neurons.

Tumor necrosis factor inhibits neurite outgrowth and branching of hippocampal neurons by a rho-dependent mechanism

In response to injury and inflammation of the CNS, brain cells including microglia and astrocytes secrete tumor necrosis factor-α (TNF). This pro-inflammatory cytokine has been implicated in both neuronal cell death and survival. We now provide evidence that TNF affects the formation of neurites. Neurons cultured on astrocytic glial cells exhibited reduced outgrowth and branching of neurites after addition of recombinant TNF or prestimulation of glial cells to secrete TNF. This effect was absent in neurons of TNF receptor-deficient mice cultured on prestimulated glia of wild-type mice and was reverted by blocking TNF with soluble TNF receptor IgG fusion protein. TNF activated in neurons the small GTPase RhoA. By inactivating Rho with C3 transferase, the inhibitory effect of TNF on neurite outgrowth and branching was abolished. These results suggest that glia-derived TNF, as part of an injury or inflammatory process, can inhibit neurite elongation and branching during development and regeneration.

Layer V cortical neurons require microglial support for survival during postnatal development

Neurons require trophic support during neural circuit formation; however, how the cellular milieu contributes to neuronal survival remains unclear. We found that layer V cortical neurons require support from microglia for survival during postnatal development. Specifically, we found that microglia accumulated close to the subcerebral and callosal projection axons in the postnatal brain. Inactivation of microglia by minocycline treatment or transient ablation of microglia in CD11b-DTR transgenic mice led to increased apoptosis, specifically in layer V subcerebral and callosal projection neurons. CX3CR1 in microglia was required for the survival of layer V neurons. Microglia consistently promoted the survival of cortical neurons in vitro. In addition, we identified microglia-derived IGF1 as a trophic factor that maintained neuronal survival. Our results highlight a neuron-glia interaction that is indispensable for network formation during a specific period in the developing brain.

Induction of aquaporin-4 water channel mRNA after focal cerebral ischemia in rat.

Aquaporin-4 (AQP4) is a member of a water-selective channel aquaporin-family and mainly expressed in the several structures of the brain and in the collecting duct of the kidney. Here we show its functional involvement in the water homeostasis of the ischemic brain. The expression of AQP4-mRNA is increased in the peri-infarcted cortex during the observation period ( approximately 7 days) after MCA-occlusion, maximally on day 3. The change corresponds to the generation and resolution of brain edema monitored by MRI. The signals for the mRNA are predominantly observed in glial cells in the molecular and outer granular layer of the peri-infarcted cortex. These results indicate that AQP4 plays a role in post-ischemic edema formation.

RGMa inhibition promotes axonal growth and recovery after spinal cord injury

Repulsive guidance molecule (RGM) is a protein implicated in both axonal guidance and neural tube closure. We report RGMa as a potent inhibitor of axon regeneration in the adult central nervous system (CNS). RGMa inhibits mammalian CNS neurite outgrowth by a mechanism dependent on the activation of the RhoA–Rho kinase pathway. RGMa expression is observed in oligodendrocytes, myelinated fibers, and neurons of the adult rat spinal cord and is induced around the injury site after spinal cord injury. We developed an antibody to RGMa that efficiently blocks the effect of RGMa in vitro. Intrathecal administration of the antibody to rats with thoracic spinal cord hemisection results in significant axonal growth of the corticospinal tract and improves functional recovery. Thus, RGMa plays an important role in limiting axonal regeneration after CNS injury and the RGMa antibody offers a possible therapeutic agent in clinical conditions characterized by a failure of CNS regeneration.

miR-124a is required for hippocampal axogenesis and retinal cone survival through Lhx2 suppression

MicroRNA-124a (miR-124a) is the most abundant microRNA expressed in the vertebrate CNS. Despite past investigations into the role of miR-124a, inconsistent results have left the in vivo function of miR-124a unclear. We examined the in vivo function of miR-124a by targeted disruption of Rncr3 (retinal non-coding RNA 3), the dominant source of miR-124a. Rncr3−/− mice exhibited abnormalities in the CNS, including small brain size, axonal mis-sprouting of dentate gyrus granule cells and retinal cone cell death. We found that Lhx2 is an in vivo target mRNA of miR-124a. We also observed that LHX2 downregulation by miR-124a is required for the prevention of apoptosis in the developing retina and proper axonal development of hippocampal neurons. These results suggest that miR-124a is essential for the maturation and survival of dentate gyrus neurons and retinal cones, as it represses Lhx2 translation.

Cytoplasmic p21Cip1/WAF1 regulates neurite remodeling by inhibiting Rho-kinase activity

p21Cip1/WAF1 has cell cycle inhibitory activity by binding to and inhibiting both cyclin/Cdk kinases and proliferating cell nuclear antigen. Here we show that p21Cip1/WAF1 is induced in the cytoplasm during the course of differentiation of chick retinal precursor cells and N1E-115 cells. Ectopic expression of p21Cip1/WAF1 lacking the nuclear localization signal in N1E-115 cells and NIH3T3 cells affects the formation of actin structures, characteristic of inactivation of Rho. p21Cip1/WAF1 forms a complex with Rho-kinase and inhibits its activity in vitro and in vivo. Neurite outgrowth and branching from the hippocampal neurons are promoted if p21Cip1/WAF1 is expressed abundantly in the cytoplasm. These results suggest that cytoplasmic p21Cip1/WAF1 may contribute to the developmental process of the newborn neurons that extend axons and dendrites into target regions.

Myelin-associated Glycoprotein Inhibits Microtubule Assembly by a Rho-kinase-dependent Mechanism

Myelin-associated glycoprotein (MAG) and Nogo are potent inhibitors of neurite outgrowth from a variety of neurons, and they have been identified as possible components of the central nervous system myelin that prevents axonal regeneration in the adult vertebrate central nervous system. The activation of RhoA and Rho-kinase is reported to be an essential part of the signaling mechanism of these proteins. Here, we report that the collapsing response mediator protein-2 (CRMP-2) is phosphorylated by a Rho-kinase-dependent mechanism downstream of MAG or Nogo-66. The overexpression of the nonphosphorylated form of CRMP-2 at threonine 555, which is the phosphorylation site for Rho-kinase, counteracts the inhibitory effect of MAG on the postnatal cerebellar neurons. Additionally, the expression of the dominant negative form of CRMP-2 or knockdown of the gene using small interference RNA (siRNA) mimics the effect of MAG in vitro. Consistent with the function of CRMP-2, which promotes microtubule assembly, microtubule levels are down-regulated in the cerebellar neurons that are stimulated with MAG in vitro. Reduction in the density of microtubules is also observed in the injured axons following the spinal cord injury, and this effect depends on the Rho-kinase activity. Our data suggest the important roles of CRMP-2 and microtubules in the inhibition of the axon regeneration by the myelin-derived inhibitors.