Satoru Yamagishi

Involvement of caspase-4 in endoplasmic reticulum stress-induced apoptosis and Aβ-induced cell death

Recent studies have suggested that neuronal death in Alzheimers disease or ischemia could arise from dysfunction of the endoplasmic reticulum (ER). Although caspase-12 has been implicated in ER stress-induced apoptosis and amyloid-β (Aβ)–induced apoptosis in rodents, it is controversial whether similar mechanisms operate in humans. We found that human caspase-4, a member of caspase-1 subfamily that includes caspase-12, is localized to the ER membrane, and is cleaved when cells are treated with ER stress-inducing reagents, but not with other apoptotic reagents. Cleavage of caspase-4 is not affected by overexpression of Bcl-2, which prevents signal transduction on the mitochondria, suggesting that caspase-4 is primarily activated in ER stress-induced apoptosis. Furthermore, a reduction of caspase-4 expression by small interfering RNA decreases ER stress-induced apoptosis in some cell lines, but not other ER stress-independent apoptosis. Caspase-4 is also cleaved by administration of Aβ, and Aβ-induced apoptosis is reduced by small interfering RNAs to caspase-4. Thus, caspase-4 can function as an ER stress-specific caspase in humans, and may be involved in pathogenesis of Alzheimers disease.

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.

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.

p38 mitogen-activated protein kinase regulates low potassium-induced c-Jun phosphorylation and apoptosis in cultured cerebellar granule neurons.

Cultured rat cerebellar granule neurons are widely used as a model system for studying neuronal apoptosis. After maturation by culturing in medium containing 26 mmpotassium (high K+), changing to medium containing 5 mm potassium (low K+; LK) rapidly induces neuronal apoptosis. Then over 50% of granule cells die within 24 h. However, the molecular mechanisms by which the LK-induced apoptosis occurs in cultured cerebellar granule cells remain unclear. In the present study, we found that p38 MAP kinase (p38) was an important factor for LK-induced apoptosis. Three hours after changing to LK medium, p38 was markedly activated. In addition, SB203580, a specific inhibitor of p38, strongly inhibited the phosphorylation and expression of c-Jun in LK-induced apoptosis of cultured cerebellar granule cells.In vitro kinase assay using glutathioneS-transferase-c-Jun as a substrate showed that p38 directly phosphorylated c-Jun. Furthermore, in the presence of SB203580, about 80% of neurons survived. These results indicate that p38 regulates LK-induced apoptosis of cerebellar granule neurons.

Promotion of Axon Regeneration by Myelin-Associated Glycoprotein and Nogo through Divergent Signals Downstream of Gi/G

Several myelin-derived proteins have been identified as components of the CNS myelin that prevents axonal regeneration in the adult vertebrate CNS. Activation of RhoA has been shown to be an essential part of the signaling mechanism of these proteins. Here we report an additional signal, which determines whether these proteins promote or inhibit axon outgrowth. Myelin-associated glycoprotein (MAG) and Nogo trigger the intracellular elevation of Ca2+ as well as the activation of PKC, presumably mediated by Gi/G. Neurite outgrowth inhibition and growth cone collapse by MAG or Nogo can be converted to neurite extension and growth cone spreading by inhibiting conventional PKC, but not by inhibiting inositol 1,4,5-triphosphate (IP3). Conversely, neurite growth of immature neurons promoted by MAG is abolished by inhibiting IP3. Activation of RhoA is independent of PKC. Thus, a balance between PKC and IP3 is important for bidirectional regulation of axon regeneration by the myelin-derived proteins.

Inhibition of phosphatidylinositol 3-kinase activity elevates c-Jun N-terminal kinase activity in apoptosis of cultured cerebellar granule neurons.

Abstract Cerebellar granule neurons maintained in medium containing 26 mM potassium or in medium (5 mM potassium) with 50 ng/ml brain-derived neurotrophic factor (BDNF) undergo an apoptotic cell death when exposed to 10 μM LY294002, an inhibitor of phosphatidylinositol 3-kinase (PI3-K). To investigate the intracellular signaling mechanism of LY294002-induced apoptosis, the activities of Akt and c-Jun N-terminal kinase (JNK) were measured in cells in HK (26 mM potassium) medium or LK+ (5 mM potassium) medium containing BDNF, with or without 10 μM LY294002. Akt activity decreased following the addition of 10 μM LY294002. In addition, we found that LY294002 increased the JNK activity, which is known to mediate some types of cell death in cultured PNS neurons. We also observed elevated expression of c-Jun by LY294002 in HK+ or LK++BDNF. These findings demonstrated that apoptosis induced by inhibition of PI3-K activity involves suppression of the Akt activity and elevation of the JNK activity in cultured cerebellar granule neurons. Our results suggested that the PI3-K-Akt pathway suppresses the activation of JNK and c-Jun expression, and as a result prevents the neuronal cell death in cerebellar granule neurons.

Brain-derived neurotrophic factor-induced potentiation of glutamate and GABA release: different dependency on signaling pathways and neuronal activity.

The mechanisms underlying BDNF-modulated neurotransmitter release remain elusive. Here, we found that 24-h exposure of postnatal cortical neurons to BDNF potentiated depolarization-evoked glutamate and GABA release in a protein synthesis-dependent manner. BDNF-potentiated glutamate release occurred through the PLC-gamma and MAPK pathways. The expression of synapsin I, synaptotagmin, and synaptophysin, but not of syntaxin or SNAP25, increased through the PLC-gamma and MAPK pathways. In contrast, BDNF-up-regulated GABA release and GAD65/67 expression depended on MAPK. Furthermore, neuronal activity was necessary for the up-regulation of glutamate release and synapsin I, synaptotagmin, and synaptophysin expression, but not of GABA or GAD65/67. PLC-gamma inhibitor attenuated BDNF-stimulated long-lasting MAPK activation. As BDNF rapidly potentiates glutamatergic transmission through PLC-gamma (J. Biol. Chem. 277, (2002) 6520-6529), PLC-gamma-mediated neuronal activity might sustain MAPK activation, resulting in BDNF-potentiated glutamate release. In conclusion, BDNF potentiates the excitatory and inhibitory system separately, which may be important for the regulation of synaptic plasticity.

Multiple signals regulate axon regeneration through the Nogo receptor complex.

Several myelin-derived proteins have been identified as components of central nervous system (CNS) myelin, which prevents axonal regeneration in the adult vertebrate CNS. The discovery of the receptor for these proteins was a major step toward understanding the failure of axon regeneration. The receptor complex consists of at least three elements: the p75 receptor (p75NTR), the Nogo receptor and LINGO-1. Downstream from the receptor complex, RhoA activation has been shown to be a key element of the signaling mechanism of these proteins. Rho activation arrests axon growth, and blocking Rho activation promotes axon regeneration in vivo. Recent studies have identified conventional protein kinase C as an additional necessary component for axon growth inhibition. Possible crosstalk downstream of these signals should be explored to clarify all the inhibitory signals and may provide an efficient molecular target against injuries to the CNS.

FLRT2 and FLRT3 act as repulsive guidance cues for Unc5‐positive neurons

Netrin‐1 induces repulsive axon guidance by binding to the mammalian Unc5 receptor family (Unc5A–Unc5D). Mouse genetic analysis of selected members of the Unc5 family, however, revealed essential functions independent of Netrin‐1, suggesting the presence of other ligands. Unc5B was recently shown to bind fibronectin and leucine‐rich transmembrane protein‐3 (FLRT3), although the relevance of this interaction for nervous system development remained unclear. Here, we show that the related Unc5D receptor binds specifically to another FLRT protein, FLRT2. During development, FLRT2/3 ectodomains (ECDs) are shed from neurons and act as repulsive guidance molecules for axons and somata of Unc5‐positive neurons. In the developing mammalian neocortex, Unc5D is expressed by neurons in the subventricular zone (SVZ), which display delayed migration to the FLRT2‐expressing cortical plate (CP). Deletion of either FLRT2 or Unc5D causes a subset of SVZ‐derived neurons to prematurely migrate towards the CP, whereas overexpression of Unc5D has opposite effects. Hence, the shed FLRT2 and FLRT3 ECDs represent a novel family of chemorepellents for Unc5‐positive neurons and FLRT2/Unc5D signalling modulates cortical neuron migration.

JAB1 participates in unfolded protein responses by association and dissociation with IRE1

Recent papers have reported that neuronal death in patients with Alzheimers disease, Parkinsons disease, and cerebral ischemia has its origin in the endoplasmic reticulum (ER). IRE1alpha is one of the ER stress transducers that detect the accumulation of unfolded proteins in the ER. IRE1alpha mediates two major cellular responses, which are the unfolded protein response (UPR), a defensive response, and apoptosis that leads to cell death. However, little is known about the regulatory mechanisms that select between the UPR and apoptosis. We identified Jun activation domain-binding protein-1 (JAB1) as a molecule that interacts with IRE1alpha using a yeast two-hybrid system. We demonstrated that JAB1 binds to IRE1alpha in the absence of stress, but that binding is decreased by ER stress inducers. Moreover, mutant JAB1 down-regulates the UPR signaling pathway through tight binding with IRE1alpha. These results suggested that JAB1 may act as a key molecule in selecting the UPR or cell death by association and dissociation with IRE1alpha.