Cerebral Cortical Neurons Research Articles

Tissue-type plasminogen activator triggers the synaptic vesicle cycle in cerebral cortical neurons.

The active zone (AZ) is a thickening of the presynaptic membrane where exocytosis takes place. Chemical synapses contain neurotransmitter-loaded synaptic vesicles (SVs) that at rest are tethered away from the synaptic release site, but after the presynaptic inflow of Ca(+2) elicited by an action potential translocate to the AZ to release their neurotransmitter load. We report that tissue-type plasminogen activator (tPA) is stored outside the AZ of cerebral cortical neurons, either intermixed with small clear-core vesicles or in direct contact with the presynaptic membrane. We found that cerebral ischemia-induced release of neuronal tPA, or treatment with recombinant tPA, recruits the cytoskeletal protein βII-spectrin to the AZ and promotes the binding of SVs to βII-spectrin, enlarging the population of SVs in proximity to the synaptic release site. This effect does not require the generation of plasmin and is followed by the recruitment of voltage gated calcium channels (VGCC) to the presynaptic terminal that leads to Ca(+2)-dependent synapsin I phosphorylation, freeing SVs to translocate to the AZ to deliver their neurotransmitter load. Our studies indicate that tPA activates the SV cycle and induces the structural and functional changes in the synapse that are required for successful neurotransmission.

The mitochondrial uncoupler DNP triggers brain cell mTOR signaling network reprogramming andCREB pathway up-regulation.

Mitochondrial metabolism is highly responsive to nutrient availability and ongoing activity in neuronal circuits. The molecular mechanisms by which brain cells respond to an increase in cellular energy expenditure are largely unknown. Mild mitochondrial uncoupling enhances cellular energy expenditure in mitochondria and can be induced with 2,4-dinitrophenol (DNP), a proton ionophore previously used for weight loss. We found that DNP treatment reduces mitochondrial membrane potential, increases intracellular Ca(2+) levels and reduces oxidative stress in cerebral cortical neurons. Gene expression profiling of the cerebral cortex of DNP-treated mice revealed reprogramming of signaling cascades that included suppression of the mammalian target of rapamycin (mTOR) and insulin--PI3K - MAPK pathways, and up-regulation of tuberous sclerosis complex 2, a negative regulator of mTOR. Genes encoding proteins involved in autophagy processes were up-regulated in response to DNP. CREB (cAMP-response element-binding protein) signaling, Arc and brain-derived neurotrophic factor, which play important roles in synaptic plasticity and adaptive cellular stress responses, were up-regulated in response to DNP, and DNP-treated mice exhibited improved performance in a test of learning and memory. Immunoblot analysis verified that key DNP-induced changes in gene expression resulted in corresponding changes at the protein level. Our findings suggest that mild mitochondrial uncoupling triggers an integrated signaling response in brain cells characterized by reprogramming of mTOR and insulin signaling, and up-regulation of pathways involved in adaptive stress responses, molecular waste disposal, and synaptic plasticity. Physiological bioenergetic challenges such as exercise and fasting can enhance neuroplasticity and protect neurons against injury and neurodegeneration. Here, we show that the mitochondrial uncoupling agent 2,4-dinitrophenol (DNP) elicits adaptive signaling responses in the cerebral cortex involving activation of Ca(2+) -CREB and autophagy pathways, and inhibition of mTOR and insulin signaling pathways. The molecular reprogramming induced by DNP, which is similar to that of exercise and fasting, is associated with improved learning and memory, suggesting potential therapeutic applications for DNP.

Up-Regulation of Corticocerebral NKD2 in Lipopolysaccharide-Induced Neuroinflammation.

Naked2 (NKD2), one member of Naked family, has been shown to negatively regulate Wnt/β-catenin signaling pathway. It has been recognized that NKD2 plays a vital role in cell homeostasis and prevention of tumorigenesis. However, NKD2 expression and its functional role in the brain in neuroinflammatory processes remain unclear. In our study, we investigated NKD2 distribution and role in lipopolysaccharide (LPS)-induced neuroinflammation rat model. The data indicated that NKD2 was up-regulated in LPS-injected brain, and the cellular localization of NKD2 was predominantly in cerebral cortical neurons. Furthermore, we treated primary neurons with conditioned media (CM) collected from LPS-stimulated mixed glial cultures (MGC). We detected that the up-regulation of NKD2 might be associated with the subsequent apoptosis in neurons. We also found knockdown NKD2 partially depressed the increase of cleaved caspase-3 and increased the reduction of β-catenin stimulated by MGC-CM. Taken together, these results suggested that NKD2 might be involved in neuronal apoptosis via the Wnt/β-catenin pathway during neuroinflammation in CNS. Our findings might provide a new therapeutic target for the prevention of neuroinflammation-involved neurological disorders.

Axonal elongation and dendritic branching is enhanced by adenosine A2A receptors activation in cerebral cortical neurons

Axon growth and dendrite development are key processes for the establishment of a functional neuronal network. Adenosine, which is released by neurons and glia, is a known modulator of synaptic transmission but its influence over neuronal growth has been much less investigated. We now explored the action of adenosine A2A receptors (A2AR) upon neurite outgrowth, discriminating actions over the axon or dendrites, and the mechanisms involved. Morphometric analysis of primary cultures of cortical neurons from E18 Sprague-Dawley rats demonstrated that an A2AR agonist, CGS 21680, enhances axonal elongation and dendritic branching, being the former prevented by inhibitors of phosphoinositide 3-kinase, mitogen-activated protein kinase and phospholipase C, but not of protein kinase A. By testing the influence of a scavenger of BDNF (brain-derived neurotrophic factor) over the action of the A2AR agonist and the action of a selective A2AR antagonist over the action of BDNF, we could conclude that while the action of A2ARs upon dendritic branching is dependent on the presence of endogenous BDNF, the influence of A2ARs upon axonal elongation is independent of endogenous BDNF. In consonance with the action over axonal elongation, A2AR activation promoted a decrease in microtubule stability and an increase in microtubule growth speed in axonal growth cones. In conclusion, we disclose a facilitatory action of A2ARs upon axonal elongation and microtubule dynamics, providing new insights for A2ARs regulation of neuronal differentiation and axonal regeneration.

Potent KCNQ2/3-Specific Channel Activator Suppresses In Vivo Epileptic Activity and Prevents the Development of Tinnitus

Voltage-gated Kv7 (KCNQ) channels are voltage-dependent potassium channels that are activated at resting membrane potentials and therefore provide a powerful brake on neuronal excitability. Genetic or experience-dependent reduction of KCNQ2/3 channel activity is linked with disorders that are characterized by neuronal hyperexcitability, such as epilepsy and tinnitus. Retigabine, a small molecule that activates KCNQ2-5 channels by shifting their voltage-dependent opening to more negative voltages, is an US Food and Drug Administration (FDA) approved anti-epileptic drug. However, recently identified side effects have limited its clinical use. As a result, the development of improved KCNQ2/3 channel activators is crucial for the treatment of hyperexcitability-related disorders. By incorporating a fluorine substituent in the 3-position of the tri-aminophenyl ring of retigabine, we synthesized a small-molecule activator (SF0034) with novel properties. Heterologous expression of KCNQ2/3 channels in HEK293T cells showed that SF0034 was five times more potent than retigabine at shifting the voltage dependence of KCNQ2/3 channels to more negative voltages. Moreover, unlike retigabine, SF0034 did not shift the voltage dependence of either KCNQ4 or KCNQ5 homomeric channels. Conditional deletion of Kcnq2 from cerebral cortical pyramidal neurons showed that SF0034 requires the expression of KCNQ2/3 channels for reducing the excitability of CA1 hippocampal neurons. Behavioral studies demonstrated that SF0034 was a more potent and less toxic anticonvulsant than retigabine in rodents. Furthermore, SF0034 prevented the development of tinnitus in mice. We propose that SF0034 provides, not only a powerful tool for investigating ion channel properties, but, most importantly, it provides a clinical candidate for treating epilepsy and preventing tinnitus.

Deletion of Gαq in the telencephalon alters specific neurobehavioral outcomes.

G(αq) -coupled receptors are ubiquitously expressed throughout the brain and body, and it has been shown that these receptors and associated signaling cascades are involved in a number of functional outputs, including motor function and learning and memory. Genetic alterations to G(αq) have been implicated in neurodevelopmental disorders such as Sturge-Weber syndrome. Some of these associated disease outcomes have been modeled in laboratory animals, but as G(αq) is expressed in all cell types, it is difficult to differentiate the underlying circuitry or causative neuronal population. To begin to address neuronal cell type diversity in G(αq) function, we utilized a conditional knockout mouse whereby G(αq) was eliminated from telencephalic glutamatergic neurons. Unlike the global G(αq) knockout mouse, we found that these conditional knockout mice were not physically different from control mice, nor did they exhibit any gross motor abnormalities. However, similarly to the constitutive knockout animal, G(αq) conditional knockout mice demonstrated apparent deficits in spatial working memory. Loss of G(αq) from glutamatergic neurons also produced enhanced sensitivity to cocaine-induced locomotion, suggesting that cortical G(αq) signaling may limit behavioral responses to psychostimulants. Screening for a variety of markers of forebrain neuronal architecture revealed no obvious differences in the conditional knockouts, suggesting that the loss of G(αq) in telencephalic excitatory neurons does not result in major alterations in brain structure or neuronal differentiation. Taken together, our results define specific modulation of spatial working memory and psychostimulant responses through disruptions in G(αq) signaling within cerebral cortical glutamatergic neurons.

Cadmium-induced autophagy promotes survival of rat cerebral cortical neurons by activating class III phosphoinositide 3-kinase/beclin-1/B-cell lymphoma 2 signaling pathways.

Autophagy is an evolutionarily conserved response that can be activated in response to heavy metal. Thus, the present study investigated the effect of autophagy on neurotoxic damage caused by cadmium (Cd) in rat cerebral cortical neurons. The results indicated that the viability of cortical neurons treated with Cd was markedly decreased in a dose-and time-dependent manner. The present study provided evidence that cortical neurons treated with Cd underwent autophagy: The conversion of microtubule-associated protein 1A/1B-light chain 3 (LC3) to LC3-II, an increase in the punctate distribution of endogenous LC3-II and the presence of autophagosomes were identified. Combined treatment with Cd and chloroquine, an autophagy inhibitor, reduced the amount of autophagocytosis and cell activity, whereas rapamycin, an autophagy inducer, reduced Cd-mediated cytotoxicity. Furthermore, it was found that beclin-1 and class III phosphoinositide 3 kinase (PI3K) levels were increased, while levels of the anti-apoptotic protein B-cell lymphoma 2 (Bcl-2) were decreased after Cd treatment. LY294002, a specific inhibitor of PI3K, prevented the decline in Bcl-2 production and the increase in levels of beclin-1, class III PI3K and autophagy following Cd treatment. In conclusion, the results of the present study suggested that Cd can induce cytoprotective autophagy by activating the class III PI3K/beclin-1/Bcl-2 signaling pathway, and that the autophagy pathway can serve as a sensitive biomarker for nervous system injury after exposure to Cd.

Characteristics of Postsynaptic Currents in Primary Cultures of Rat Cerebral Cortical Neurons

The features of the generation of postsynaptic currents in primary cultures of cortical neurons from rat brains were studied at 7–20 days in vitro (DIV). Use of specific blockers of postsynaptic ion channels showed that from 10 DIV, neurons generated all types of electrical activity typical of the adult cortex: spontaneous miniature inhibitory (mIPSP) and excitatory (mEPSP) currents, as well as giant excitatory currents and action potentials (AP). The frequency of mEPSP increased exponentially from DIV 7 to 20 in parallel with a change in the nature of AP generation. mEPSP generated by activation of NMDA and AMPA or only AMPA receptors were identified. The role of inhibition of presynaptic NMDA receptors by magnesium ions or AP5 in modulating the frequency and amplitude of mEPSP was demonstrated; this was a characteristic of neurons cultured from brain slices and appeared to linked with the lack of glial control of synaptic transmission.

Olfactory ensheathing cell–neurite alignment enhances neurite outgrowth in scar-like cultures

The regenerative capacity of adult CNS neurons after injury is strongly inhibited by the spinal cord lesion site environment that is composed primarily of the reactive astroglial scar and invading meningeal fibroblasts. Olfactory ensheathing cell (OEC) transplantation facilitates neuronal survival and functional recovery after a complete spinal cord transection, yet the mechanisms by which this recovery occurs remain unclear. We used a unique multicellular scar-like culture model to test if OECs promote neurite outgrowth in growth-inhibitory areas. Astrocytes were mechanically injured and challenged by meningeal fibroblasts to produce key inhibitory elements of a spinal cord lesion. Neurite outgrowth of postnatal cerebral cortical neurons was assessed on three substrates: quiescent astrocyte control cultures, reactive astrocyte scar-like cultures, and scar-like cultures with OECs. Initial results showed that OECs enhanced total neurite outgrowth of cortical neurons in a scar-like environment by 60%. We then asked if the neurite growth-promoting properties of OECs depended on direct alignment between neuronal and OEC processes. Neurites that aligned with OECs were nearly three times longer when they grew on inhibitory meningeal fibroblast areas and twice as long on reactive astrocyte zones compared to neurites not associated with OECs. Our results show that OECs can independently enhance neurite elongation and that direct OEC–neurite cell contact can provide a permissive substrate that overcomes the inhibitory nature of the reactive astrocyte scar border and the fibroblast-rich spinal cord lesion core.

Bacurd2 is a novel interacting partner to Rnd2 which controls radial migration within the developing mammalian cerebral cortex.

BackgroundDuring fetal brain development in mammals, newborn neurons undergo cell migration to reach their appropriate positions and form functional circuits. We previously reported that the atypical RhoA GTPase Rnd2 promotes the radial migration of mouse cerebral cortical neurons (Nature 455(7209):114–8, 2008; Neuron 69(6):1069–84, 2011), but its downstream signalling pathway is not well understood.ResultsWe have identified BTB-domain containing adaptor for Cul3-mediated RhoA degradation 2 (Bacurd2) as a novel interacting partner to Rnd2, which promotes radial migration within the developing cerebral cortex. We find that Bacurd2 binds Rnd2 at its C-terminus, and this interaction is critical to its cell migration function. We show that forced expression or knockdown of Bacurd2 impairs neuronal migration within the embryonic cortex and alters the morphology of immature neurons. Our in vivo cellular analysis reveals that Bacurd2 influences the multipolar-to-bipolar transition of radially migrating neurons in a cell autonomous fashion. When we addressed the potential signalling relationship between Bacurd2 and Rnd2 using a Bacurd2-Rnd2 chimeric construct, our results suggest that Bacurd2 and Rnd2 could interact to promote radial migration within the embryonic cortex.ConclusionsOur studies demonstrate that Bacurd2 is a novel player in neuronal development and influences radial migration within the embryonic cerebral cortex.Electronic supplementary materialThe online version of this article (doi:10.1186/s13064-015-0032-z) contains supplementary material, which is available to authorized users.

Telmisartan promotes potential glucose homeostasis in stroke-resistant spontaneously hypertensive rats via peroxisome proliferator-activated receptor γ activation.

An angiotensin 2 type 1 receptor blocker (ARB) telmisartan possesses not only an anti-hypertensive effect but also an anti-metabolic syndrome effect due to peroxisome proliferator-activated receptor γ (PPAR-γ) activation. In the present study, we examined the effects of telmisartan on the angiotensin 2 type 1 receptor (AT1R), PPAR-γ, and insulin receptor (IR) in stroke-resistant spontaneously hypertensive rats (SHR-SR), comparing them with Wistar rats. Three-months-old SHR-SR rats were divided into three treatment groups, i.e., vehicle (SHR/Ve), low-dose telmisartan (0.3 mg/kg/day, SHR/Low), and high-dose telmisartan (3 mg/kg/day, SHR/High). Compared with Wistar rats, SHR/Ve increased the staining of AT1R, PPAR-γ and IR in the cerebral cortical neurons. On the other hand, telmisartan dose-dependently suppressed the excessive expression of AT1R and IR, but enhanced PPAR-γ activation. Low-dose telmisartan showed these effects even without lowering blood pressure (BP), while high-dose telmisartan lowered BP and showed further effects. The present study suggests that even a low dose of telmisartan decreased AT1R and IR, and increased PPAR-γ in the cerebral cortex of SHR-SR without lowering BP, probably by improving glucose homeostasis. The high dose of telmisartan showed further decreases in AT1R and IR, and further PPAR-γ activation while lowering BP, suggesting an additive benefit to lowering BP, namely the improvement of glucose homeostasis.

Effect of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine on Phosphorylated Neurofilament in Brain Cortex of Rat Model for Parkinson’s Disease

Parkinson’s disease (PD) is a progressive neurodegenerative disorder affecting people over 60 years old. Although many studies on PD focused on basal ganglia, it has been reported that other brain regions might be involved. The current work investigated the effect of 1-methyl-4-phenyl1,2,3,6-tetrahydropyridine (MPTP) injection on the brain cortex of PD rat model using Western blotting, immunohistochemistry and immunofluorescence techniques against phophorylated neurofilament antibody (pNF). A highly significant increase of pNF protein level in the treated rats was recorded using western blot as compared to the control rats. At the same time, Immunohistochemical and immunofluorescence analyses showed that the neuronal perikarya of the cerebral cortex of MPTP-treated rats were strongly immunopositive towards pNF. Meanwhile, axons and dendrites of the cerebral cortical neurons were selectively immunoreactive for pNF in Original Research Article Fathy and Addelkader; ARRB, 6(6): 409-415, 2015; Article no.ARRB.2015.100 410 the control rats. This study suggested that MPTP injection increased the level of pNF protein and its perikaryal accumulation with axonal reduction in the cortex of PD rat model. These variations might have an important role in the understanding of the pathologic mechanism of PD.

Activation of α7 nicotinic acetylcholine receptor alleviates cerebral cortical neuron injury induced by oxygen-glucose deprivation

Objective To investigate the effect of activating α7 nicotinic acetylcholine receptor(α7n AchR) on cerebral cortical neurons injury induced by oxygen-glucose deprivation(OGD) and the possible mechanism.Methods Cerebral cortical neurons cultured for 7 d were randomly divided into three groups: control group,OGD group(Cells experienced a 12 h oxygen-glucose deprivation) and OGD group treated with PNU-282987(Cells experienced a 12 h oxygen-glucose deprivation with PNU-282987 pretreatment for 24 h).Cell viability was determined by CCK-8 assay,lactate dehydrogenase(LDH) was examined to reflect cell injury,apoptosis and reactive oxygen species(ROS) production were analyzed by flow cytometry,and expression of hemeoxygenase-1(HO-1) and hypoxia inducible factor-1α(HIF-1α) were detected by Western blotting analysis.Results OGD resulted in cell death,LDH increase,and cell apoptosis.Compared with the OGD group,PNU-282987 pretreated group had significantly increased cell survival(P 0.05),significantly decreased LDH level and ROS production(P 0.05),and significantly inhibited cell apoptosis(P 0.05).Meanwhile,HO-1 protein expression was significantly increased and HIF-1α protein expression was significantly reduced in PNU-282987 pretreated group compared with the OGD group(P 0.05).Conclusion Activation of α7n AchR can protect cerebral cortical neurons against OGD-induced injury,which may be related to the anti-oxidative stress.

Curcumin pretreatment and post-treatment both improve the antioxidative ability of neurons with oxygen-glucose deprivation.

Recent studies have shown that induced expression of endogenous antioxidative enzymes thr-ough activation of the antioxidant response element/nuclear factor erythroid 2-related factor 2 (Nrf2) pathway may be a neuroprotective strategy. In this study, rat cerebral cortical neurons cultured in vitro were pretreated with 10 μM curcumin or post-treated with 5 μM curcumin, respectively before or after being subjected to oxygen-glucose deprivation and reoxygenation for 24 hours. Both pretreatment and post-treatment resulted in a significant decrease of cell injury as indicated by propidium iodide/Hoechst 33258 staining, a prominent increase of Nrf2 protein expression as indicated by western blot analysis, and a remarkable increase of protein expression and enzyme activity in whole cell lysates of thioredoxin before ischemia, after ischemia, and after reoxygenation. In addition, post-treatment with curcumin inhibited early DNA/RNA oxidation as indicated by immunocytochemistry and increased nuclear Nrf2 protein by inducing nuclear accumulation of Nrf2. These findings suggest that curcumin activates the expression of thioredoxin, an antioxidant protein in the Nrf2 pathway, and protects neurons from death caused by oxygen-glucose deprivation in an in vitro model of ischemia/reperfusion. We speculate that pharmacologic stimulation of antioxidant gene expression may be a promising approach to neuroprotection after cerebral ischemia.

Regulation of Extrasynaptic GABAA 4 Receptors by Ethanol-Induced Protein Kinase A, but Not Protein Kinase C Activation in Cultured Rat Cerebral Cortical Neurons

Regulation of Extrasynaptic GABAA 4 Receptors by Ethanol-Induced Protein Kinase A, but Not Protein Kinase C Activation in Cultured Rat Cerebral Cortical Neurons

Actions of the pyrethroid insecticide bifenthrin on sodium channels expressed in rat cerebral cortical neurons

Voltage-gated sodium channels are important sites for the neurotoxic actions of pyrethroid insecticides in mammals. Here, we studied the mode of action of bifenthrin on the native sodium channels in cerebral cortical neurons prepared from newborn rat brain, where the toxic effects are largely generated. Bifenthrin caused a pronounced late current that persisted at the end of a depolarizing pulse, a slowly-decaying tail current following repolarization and significant resting modification (25.3% modification at 10 μM). No significant bifenthrin-induced effect was observed at the peak current. Bifenthrin also caused a concentration-dependent hyperpolarizing shift in steady-state activation and inactivation as well as slowed recovery from channel inactivation. Repetitive depolarization increased the potency of bifenthrin with high frequency. There was approximately 64% inhibition of modification upon repetitive activation by 10-Hz trains of depolarizing pulses. These results suggest that bifenthrin binds to and modifies sodium channels in both the closed and open states and exhibits the behavior between type I and type II.

Nicotinic acetylcholine receptors regulate type 1 inositol 1,4,5-trisphosphate receptor expression via calmodulin kinase IV activation.

Type 1 inositol 1,4,5-trisphosphate receptors (IP3 R-1) are among the important calcium channels regulating intracellular Ca(2+) concentration in the central nervous system. In a previous study, we showed that drugs of abuse, such as cocaine, methamphetamine, and ethanol, induced IP3 R-1 upregulation via the calcium signal transduction pathway in psychological dependence. Although nicotine, a major component in tobacco smoke, participates in psychological and/or physical dependence, it has not yet been clarified how nicotine alters IP3 R-1 expression. The present study, therefore, seeks to clarify the mechanism bgy which nicotine modifies IP3 R-1 expression by using mouse cerebral cortical neurons in primary culture. Nicotine induced dose- and time-dependent upregulation of IP3 R-1 protein following its mRNA increase, and the latter was significantly suppressed by a nonselective nicotinic acetylcholine receptors (nAChR) antagonist, mecamylamine. Both cFos and phosphorylated-cJun (p-cJun) were immediately increased in the nucleus, together with an increase of calmodulin kinase (CaMK) IV but not CaMKII expression after nicotine exposure. A nonselective inhibitor of CaMKs, KN-93, and a calcium chelating regent, BAPTA-AM, completely suppressed the expression of cFos and p-cJun in the nucleus as well as the nicotine-induced IP3 R-1 upregulation. These results indicate that nAChR activation by nicotine upregulates IP3 R-1 via increase of activator protein-1, which is a cFos and cJun dimmer, in the nucleus, with activation of Ca(2+) signaling transduction processes.

Chemokine-like factor 1 promotes the migration of rat primary cortical neurons by the induction of actin polymerization.

Chemokine-like factor 1 (CKLF1), a newly cloned chemotactic cytokine, plays an essential role in immune cell migration. However, the potential role of CKLF1 in the cortical neuronal migration remains unclear. In the present research, by measuring the distance between the margin of brain slices and the leading population of migrating cells, we showed that the extent of cell migration was markedly enhanced in response to CKLF1 treatment, which was significantly inhibited by the simultaneous addition of anti-CKLF1 antibody. By immunofluorescence staining, it was found that CKLF1 induced actin polymerization in primary cerebral cortical neurons. By wound-healing assays, it was found that CKLF1 stimulated the migration of cortical neurons in a dose-dependent manner. In conclusion, our data suggest that CKLF1 promotes the migration of rat primary cerebral cortical neurons by the induction of actin polymerization.

Urokinase-type plasminogen activator promotes dendritic spine recovery and improves neurological outcome following ischemic stroke.

Spines are dendritic protrusions that receive most of the excitatory input in the brain. Early after the onset of cerebral ischemia dendritic spines in the peri-infarct cortex are replaced by areas of focal swelling, and their re-emergence from these varicosities is associated with neurological recovery after acute ischemic stroke (AIS). Urokinase-type plasminogen activator (uPA) is a serine proteinase that plays a central role in tissue remodeling via binding to the urokinase plasminogen activator receptor (uPAR). We report that cerebral cortical neurons release uPA during the recovery phase from ischemic stroke in vivo or hypoxia in vitro. Although uPA does not have an effect on ischemia- or hypoxia-induced neuronal death, genetic deficiency of uPA (uPA(-/-)) or uPAR (uPAR(-/-)) abrogates functional recovery after AIS. Treatment with recombinant uPA after ischemic stroke induces neurological recovery in wild-type and uPA(-/-) but not in uPAR(-/-) mice. Diffusion tensor imaging studies indicate that uPA(-/-) mice have increased water diffusivity and decreased anisotropy associated with impaired dendritic spine recovery and decreased length of distal neurites in the peri-infarct cortex. We found that the excitotoxic injury induces the clustering of uPAR in dendritic varicosities, and that the binding of uPA to uPAR promotes the reorganization of the actin cytoskeleton and re-emergence of dendritic filopodia from uPAR-enriched varicosities. This effect is independent of uPA's proteolytic properties and instead is mediated by Rac-regulated profilin expression and cofilin phosphorylation. Our data indicate that binding of uPA to uPAR promotes dendritic spine recovery and improves functional outcome following AIS.

Astrocytic exportin-7 responds to ischemia through mediating LKB1 translocation from the nucleus to the cytoplasm.

The superfamily of importin-β-related proteins is the largest class of nuclear transport receptors and can be generally divided into importins and exportins according to their transport directions. Eleven importins and seven exportins have been identified, and the expression patterns of both classes are important for their functions in nucleocytoplasmic transport activities. This study demonstrates that all of the importins (importin-β; transportin-1, -2, and -3; and importin-4, -5, -7, -8, -9, -11, and -13) and all the exportins (exportin-1, -2, -4, -5, -6, -7, and -t) are differentially expressed in the cerebral cortex, cerebellum, hippocampus, and brainstem and in primary cultures of cerebral cortical astrocytes and neurons. For astrocytes, we observed that different importins and exportins displayed different expression changes during 0-6 hr of ischemia treatment, especially an increase of both the mRNA and the protein of exportin-7. Immunostaining showed that exportin-7 accumulated inside the nucleus and around the nuclear envelope. In addition, we noticed an increased cytoplasmic distribution of one of the cargo proteins of exportin-7, LKB1, an important element in maintaining energy homeostasis. This increased cytoplasmic distribution was accompanied by an increased expression of exportin-7 under ischemia in astrocytes. We demonstrate that exportin-7 responds to ischemia in astrocytes and that this response involves translocation of LKB1, a protein that plays important roles during metabolic stress, from the nucleus to the cytoplasm.