Inhibition of PirB Activity by TAT-PEP Improves Mouse Motor Ability and Cognitive Behavior.

Paired immunoglobulin-like receptor B (PirB), a functional receptor for myelin-associated inhibitory proteins, plays an important role in axon regeneration in injured brains. However, its role in normal brain function with age has not been previously investigated. Therefore in this study, we examined the expression level of PirB in the cerebral cortex, hippocampus and cerebellum of mice at 1 month, 3 months and 18 months of age. The results showed that the expression of PirB increased with age. We further demonstrated that overexpression of PirB inhibited neurite outgrowth in PC12 cells, and this inhibitory activity of PirB could be reversed by TAT-PEP, which is a recombinant soluble PirB ectodomain fused with TAT domain for blood-brain barrier penetration. In vivo study, intraperitoneal administration of TAT-PEP was capable of enhancing motor capacity and spatial learning and memory in mice, which appeared to be mediated through regulation of brain-derived neurotrophic factor (BDNF) secretion. Our study suggests that PirB is associated with aging and TAT-PEP may be a promising therapeutic agent for modulation of age-related motor and cognitive dysfunctions.

PirB Overexpression Exacerbates Neuronal Apoptosis by Inhibiting TrkB and mTOR Phosphorylation After Oxygen and Glucose Deprivation Injury.

Previous studies have proven that paired immunoglobulin-like receptor B (PirB) plays a crucial suppressant role in neurite outgrowth and neuronal plasticity after central nervous system injury. However, the role of PirB in neuronal survival after cerebral ischemic injury and its mechanisms remains unclear. In the present study, the role of PirB is investigated in the survival and apoptosis of cerebral cortical neurons in cultured primary after oxygen and glucose deprivation (OGD)-induced injury. The results have shown that rebarbative PirB exacerbates early neuron apoptosis and survival. PirB gene silencing remarkably decreases early apoptosis and promotes neuronal survival after OGD. The expression of bcl-2 markedly increased and the expression of bax significantly decreased in PirB RNAi-treated neurons, as compared with the control- and control RNAi-treated ones. Further, phosphorylated TrkB and mTOR levels are significantly downregulated in the damaged neurons. However, the PirB silencing markedly upregulates phosphorylated TrkB and mTOR levels in the neurons after the OGD. Taken together, the overexpression of PirB inhibits the neuronal survival through increased neuron apoptosis. Importantly, the inhibition of the phosphorylation of TrkB and mTOR may be one of its mechanisms.

A link between the nuclear-localized srGAP3 and the SWI/SNF chromatin remodeler Brg1.

The Slit-Robo GTPase activating protein 3 (srGAP3) is an important modulator of actin cytoskeletal dynamics and has an important influence on a variety of neurodevelopmental processes. Mutations in the SRGAP3 gene on chromosome 3p25 have been found in patients with intellectual disability. Genome-wide association studies and behavioral assays of knockout mice had also revealed SRGAP3 as a risk gene for schizophrenia. We have recently shown that srGAP3 protein undergoes regulated shuttling between the cytoplasm and the nucleus during neuronal development. It is shown here that nuclear-localized srGAP3 interacts with the SWI/SNF remodeling factor Brg1. This interaction is mediated by the C-terminal of srGAP3 and the ATPase motif of Brg1. In the primary cultured rat cortical neurons, the levels of nuclear-localized srGAP3 and its interaction with Brg1 have a significant impact on dendrite complexity. Furthermore, the interaction between srGAP3 and Brg1 was also involved in valproic acid (VPA) -induced neuronal differentiation of Neuro2a cells. We then show that GTP-bound Rac1 and GAP-43 may be potential mediators of nuclear srGAP3 and Brg1. Our results not only indicate a novel signaling pathway that contributes to neuronal differentiation and dendrite morphology, but also implicate a novel molecular mechanism underlying srGAP3 regulation of gene expression.

M9, a novel region of amino-Nogo-A, attenuates cerebral ischemic injury by inhibiting NADPH oxidase-derived superoxide production in mice.

AIMS: In acute stroke, neurological damage is due to oxidative stress and neuronal apoptotic death. This study investigated whether Nogo-A 290-562 residues region (M9), fused to the transduction domain of the HIV trans-activator (TAT) protein, is neuroprotective against cerebral ischemia and the mechanisms. METHODS: Transient focal cerebral ischemia was induced by middle cerebral artery occlusion in male C57BL/6J mice. TAT-M9, its mutation or vehicle was applied via intraperitoneal injection at the onset of reperfusion. The neurobehavioral scores, infarction volumes, neuronal apoptosis, and the ratio of Bax/Bcl-2 were evaluated. Malondialdehyde (MDA), reactive oxygen species (ROS) levels, and NADPH oxidase activation were measured in the presence or absence of the NADPH oxidase inhibitor apocynin or activator tetrabromocinnamic acid (TBCA). RESULTS: Immunofluorescence results confirmed that TAT-M9 was transduced into brain parenchyma, and it significantly improved neurological behavior, reduced infarct volumes, protected neuronal cells from apoptosis, inhibited activation of NADPH oxidase, and decreased MDA and ROS contents. Furthermore, apocynin imitated the beneficial effects of TAT-M9, while TBCA abolished them. CONCLUSIONS: Our results demonstrate that TAT-M9 administration attenuates cerebral ischemia by inhibiting NADPH oxidase-mediated oxidative damage and neuronal apoptosis in mice. TAT-M9 may be a potential treatment for cerebrovascular disease.

The inverse F-BAR domain protein srGAP2 acts through srGAP3 to modulate neuronal differentiation and neurite outgrowth of mouse neuroblastoma cells.

The inverse F-BAR (IF-BAR) domain proteins srGAP1, srGAP2 and srGAP3 are implicated in neuronal development and may be linked to mental retardation, schizophrenia and seizure. A partially overlapping expression pattern and highly similar protein structures indicate a functional redundancy of srGAPs in neuronal development. Our previous study suggests that srGAP3 negatively regulates neuronal differentiation in a Rac1-dependent manner in mouse Neuro2a cells. Here we show that exogenously expressed srGAP1 and srGAP2 are sufficient to inhibit valporic acid (VPA)-induced neurite initiation and growth in the mouse Neuro2a cells. While ectopic- or over-expression of RhoGAP-defective mutants, srGAP1(R542A) and srGAP2(R527A) exert a visible inhibitory effect on neuronal differentiation. Unexpectedly, knockdown of endogenous srGAP2 fails to facilitate the neuronal differentiation induced by VPA, but promotes neurite outgrowth of differentiated cells. All three IF-BAR domains from srGAP1-3 can induce filopodia formation in Neuro2a, but the isolated IF-BAR domain from srGAP2, not from srGAP1 and srGAP3, can promote VPA-induced neurite initiation and neuronal differentiation. We identify biochemical and functional interactions of the three srGAPs family members. We propose that srGAP3-Rac1 signaling may be required for the effect of srGAP1 and srGAP2 on attenuating neuronal differentiation. Furthermore, inhibition of Slit-Robo interaction can phenocopy a loss-of-function of srGAP3, indicating that srGAP3 may be dedicated to the Slit-Robo pathway. Our results demonstrate the interplay between srGAP1, srGAP2 and srGAP3 regulates neuronal differentiation and neurite outgrowth. These findings may provide us new insights into the possible roles of srGAPs in neuronal development and a potential mechanism for neurodevelopmental diseases.

The mental retardation associated protein, srGAP3 negatively regulates VPA-induced neuronal differentiation of Neuro2A cells.

The Slit-Robo GTPase-activating proteins (srGAPs) are important multifunctional adaptor proteins involved in various aspects of neuronal development, including axon guidance, neuronal migration, neurite outgrowth, dendritic morphology and synaptic plasticity. Among them, srGAP3, also named MEGAP (Mental disorder-associated GTPase-activating protein), plays a putative role in severe mental retardation. SrGAP3 expression in ventricular zones of neurogenesis indicates its involvement in early stage of neuronal development and differentiation. Here, we show that overexpression of srGAP3 inhibits VPA (valproic acid)-induced neurite initiation and neuronal differentiation in Neuro2A neuroblastoma cells, whereas knockdown of srGAP3 facilitates the neuronal differentiation in this cell line. In contrast to the wild type, overexpression of srGAP3 harboring an artificially mutation R542A within the functionally important RhoGAP domain does not exert a visible inhibitory effect on neuronal differentiation. The endogenous srGAP3 selectively binds to activated form of Rac1 in a RhoGAP pull-down assay. We also show that constitutively active (CA) Rac1 can rescue the effect of srGAP3 on attenuating neuronal differentiation. Furthermore, change in expression and localization of endogenous srGAP3 is observed in neuronal differentiated Neuro2A cells. Together, our data suggest that srGAP3 could regulate neuronal differentiation in a Rac1-dependent manner.