Human SEMAPHORIN 5A (SEMA5A) is an autism susceptibility gene; however, its function in brain dev... more Human SEMAPHORIN 5A (SEMA5A) is an autism susceptibility gene; however, its function in brain development is unknown. In this study, we show that mouse Sema5A negatively regulates synaptogenesis in early, developmentally born, hippocampal dentate granule cells (GCs). Sema5A is strongly expressed by GCs and regulates dendritic spine density in a cell-autonomous manner. In the adult mouse brain, newly born Sema5A −/− GCs show an increase in dendritic spine density and increased AMPA-type synaptic responses. Sema5A signals through PlexinA2 co-expressed by GCs, and the PlexinA2-RasGAP activity is necessary to suppress spinogenesis. Like Sema5A −/− mutants, PlexinA2 −/− mice show an increase in GC glutamatergic synapses, and we show that Sema5A and PlexinA2 genetically interact with respect to GC spine phenotypes. Sema5A −/− mice display deficits in social interaction, a hallmark of autism-spectrum-disorders. These experiments identify novel intra-dendritic Sema5A/PlexinA2 interactions that inhibit excitatory synapse formation in developmentally born and adult-born GCs, and they provide support for SEMA5A contributions to autism-spectrum-disorders.
Mitochondrial Ca 2+ uptake and poly(ADP-ribose) polymerase-1 (PARP-1) activation are both require... more Mitochondrial Ca 2+ uptake and poly(ADP-ribose) polymerase-1 (PARP-1) activation are both required for glutamate-induced excitotoxic neuronal death. Since activation of the glutamate receptors can induce increased levels of reactive oxygen species (ROS), we investigated the relationship of mitochondrial Ca 2+ uptake and ROS generation, and the possibility that ROS increase is a required signal for PARP-1 activation in cultured striatal neurons. Based on the spatial profile of NMDA-induced ROS generation, we found that only mitochondria showed a significant ROS increase within 30 min after NMDA receptor activation. This ROS increase was inhibited by the mitochondrial complex inhibitors rotenone and oligomycin, but not by the cytosolic phospholipase A 2 or xanthine oxidase inhibitors. Mitochondrial ROS generation was also inhibited by both removal of Ca 2+ from extracellular medium and blockage of mitochondrial Ca 2+ uptake by either a mitochondrial uncoupler or a Ca 2+ uniporter inhibitor. Furthermore, both DNA damage and PARP-1 activation induced by NMDA treatment was inhibited by blocking mitochondrial Ca 2+ uptake or by antioxidants. Our results demonstrate that ROS production during the early stage of acute excitotoxicity derives primarily from mitochondria and is Ca 2+ -dependent. More importantly, the increase of mitochondrial ROS serves as a signal for PARP-1 activation, suggesting that concomitant mitochondrial Ca 2+ uptake and PARP-1 activation constitute a unified mechanism for excitotoxic neuronal death.
In the adult mammalian CNS, the growth inhibitors oligodendrocyte-myelin glycoprotein (OMgp) and ... more In the adult mammalian CNS, the growth inhibitors oligodendrocyte-myelin glycoprotein (OMgp) and the reticulon RTN4 (Nogo) are broadly expressed in oligodendrocytes and neurons. Nogo and OMgp complex with the neuronal cell surface receptors Nogo receptor-1 (NgR1) and paired Ig-like receptor-B (PirB) to regulate neuronal morphology. In the healthy CNS, NgR1 regulates dendritic spine shape and attenuates activity-driven synaptic plasticity at Schaffer collateral-CA1 synapses. Here, we examine whether Nogo and OMgp influence functional synaptic plasticity, the efficacy by which synaptic transmission occurs. In acute hippocampal slices of adult mice, Nogo-66 and OMgp suppress NMDA receptor-dependent long-term potentiation (LTP) when locally applied to Schaffer collateral-CA1 synapses. Neither Nogo-66 nor OMgp influences basal synaptic transmission or paired-pulse facilitation, a form of short-term synaptic plasticity. PirB Ϫ/Ϫ and NgR1 Ϫ/Ϫ single mutants and NgR1 Ϫ/Ϫ ;PirB Ϫ/Ϫ double mutants show normal LTP, indistinguishable from wild-type controls. In juvenile mice, LTD in NgR1 Ϫ/Ϫ , but not PirB Ϫ/Ϫ , slices is absent. Mechanistic studies revealed that Nogo-66 and OMgp suppress LTP in an NgR1-dependent manner. OMgp inhibits LTP in part through PirB but independently of p75. This suggests that NgR1 and PirB participate in ligand-dependent inhibition of synaptic plasticity. Loss of NgR1 leads to increased phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2), signaling intermediates known to regulate neuronal growth and synaptic function. In primary cortical neurons, BDNF elicited phosphorylation of AKT and p70S6 kinase is attenuated in the presence of myelin inhibitors. Collectively, we provide evidence that mechanisms of neuronal growth inhibition and inhibition of synaptic strength are related. Thus, myelin inhibitors and their receptors may coordinate structural and functional neuronal plasticity in CNS health and disease.
Myelin-associated glycoprotein (MAG) is a sialic acid binding Ig-family lectin that functions in ... more Myelin-associated glycoprotein (MAG) is a sialic acid binding Ig-family lectin that functions in neuronal growth inhibition and stabilization of axon-glia interactions. The ectodomain of MAG is comprised of five Ig-like domains and employs neuronal cell-type specific mechanisms to signal growth inhibition. We show that the first three Ig-like domains of MAG bind with high-affinity and in a sialic acid dependent manner to the Nogo-66 receptor-1 (NgR1) and its homologue NgR2. Domains Ig3-Ig5 of MAG are sufficient to inhibit neurite outgrowth but fail to associate with NgR1 or NgR2. Nogo receptors are sialoglycoproteins comprised of 8.5 canonical leucine-rich repeats (LRR) flanked by LRR N-terminal (NT) and C-terminal (CT)-cap domains. The LRR cluster is connected through a stalk region to a membrane lipid anchor. The CT-cap domain and stalk region of NgR2, but not NgR1, are sufficient for MAG binding, and when expressed in neurons, exhibit constitutive growth inhibitory activity. The LRR cluster of NgR1 supports binding of Nogo-66, OMgp and MAG. Deletion of disulfide loop Cys 309 -Cys 336 of NgR1 selectively increases its affinity for Nogo-66 and OMgp. A chimeric Nogo receptor variant (NgR OMNI ) in which Cys 309 -Cys 336 is deleted and followed by a 13 amino acid MAG binding motif of the NgR2 stalk, shows superior binding of OMgp, Nogo-66, and MAG compared to wild-type NgR1 or NgR2. Soluble NgR OMNI (NgR OMNI -Fc) binds strongly to membrane bound inhibitors and promotes neurite outgrowth on both MAG and CNS myelin substrates. Thus, NgR OMNI -Fc may offer therapeutic opportunities following nervous system injury or disease where myelin inhibits neuronal regeneration.
It has been known for more than two decades that chondroitin sulfate proteoglycans (CSPGs) inhibi... more It has been known for more than two decades that chondroitin sulfate proteoglycans (CSPGs) inhibit axonal growth and regeneration. In the adult nervous system, CSPGs are enriched in perineuronal nets, and their abundance is increased in reactive astrocytes following injury to brain or spinal cord. Degradation of chondroitin sulfate (CS) sugar moieties by the local infusion of the bacterial enzyme chondroitinase ABC (ChaseABC) enhances experience-dependent neuronal plasticity in the adult visual cortex and results in substantially improved behavioral outcomes after spinal cord injury (SCI). Although the positive effects of ChaseABC treatment on neuronal plasticity have been known for some time, the underlying mechanisms remained enigmatic. The receptor protein tyrosine phosphatase sigma (RPTPsigma) has now been identified as a receptor for inhibitory CSPGs. Similarly to ChaseABC treatment, functional ablation of Ptprs, the gene encoding RPTPsigma, promotes neurite outgrowth in the presence of CSPGs in vitro and enhances axonal growth into CSPG-rich scar tissue following SCI in vivo. The discovery of neuronal RPTPsigma as a receptor for inhibitory CSPGs not only provides important mechanistic clues about CSPG function, but also identifies a potential new target for enhancing axonal growth and plasticity after nervous system injury.
The downstream of kinase (Dok) family of adapter proteins consists of at least five members struc... more The downstream of kinase (Dok) family of adapter proteins consists of at least five members structurally characterized by an NH 2 -terminal tandem of conserved pleckstrin homology and phosphotyrosine binding domains linked to a unique COOH-terminal region. To determine the role of the novel adapter protein Dok-4 in endothelial cells, we first investigated the cell localization of Dok-4. Most surprisingly, immunofluorescence microscopy, cell fractionation studies, and studies with enhanced green fluorescent protein chimeras showed that wild type Dok-4 (Dok-4-WT) specifically localized in mitochondria. An NH 2 -terminal deletion mutant of Dok-4 (Dok-4-(⌬N11-29)), which lacks the mitochondrial targeting sequence, could not accumulate in mitochondria.
Human SEMAPHORIN 5A (SEMA5A) is an autism susceptibility gene; however, its function in brain dev... more Human SEMAPHORIN 5A (SEMA5A) is an autism susceptibility gene; however, its function in brain development is unknown. In this study, we show that mouse Sema5A negatively regulates synaptogenesis in early, developmentally born, hippocampal dentate granule cells (GCs). Sema5A is strongly expressed by GCs and regulates dendritic spine density in a cell-autonomous manner. In the adult mouse brain, newly born Sema5A −/− GCs show an increase in dendritic spine density and increased AMPA-type synaptic responses. Sema5A signals through PlexinA2 co-expressed by GCs, and the PlexinA2-RasGAP activity is necessary to suppress spinogenesis. Like Sema5A −/− mutants, PlexinA2 −/− mice show an increase in GC glutamatergic synapses, and we show that Sema5A and PlexinA2 genetically interact with respect to GC spine phenotypes. Sema5A −/− mice display deficits in social interaction, a hallmark of autism-spectrum-disorders. These experiments identify novel intra-dendritic Sema5A/PlexinA2 interactions that inhibit excitatory synapse formation in developmentally born and adult-born GCs, and they provide support for SEMA5A contributions to autism-spectrum-disorders.
Mitochondrial Ca 2+ uptake and poly(ADP-ribose) polymerase-1 (PARP-1) activation are both require... more Mitochondrial Ca 2+ uptake and poly(ADP-ribose) polymerase-1 (PARP-1) activation are both required for glutamate-induced excitotoxic neuronal death. Since activation of the glutamate receptors can induce increased levels of reactive oxygen species (ROS), we investigated the relationship of mitochondrial Ca 2+ uptake and ROS generation, and the possibility that ROS increase is a required signal for PARP-1 activation in cultured striatal neurons. Based on the spatial profile of NMDA-induced ROS generation, we found that only mitochondria showed a significant ROS increase within 30 min after NMDA receptor activation. This ROS increase was inhibited by the mitochondrial complex inhibitors rotenone and oligomycin, but not by the cytosolic phospholipase A 2 or xanthine oxidase inhibitors. Mitochondrial ROS generation was also inhibited by both removal of Ca 2+ from extracellular medium and blockage of mitochondrial Ca 2+ uptake by either a mitochondrial uncoupler or a Ca 2+ uniporter inhibitor. Furthermore, both DNA damage and PARP-1 activation induced by NMDA treatment was inhibited by blocking mitochondrial Ca 2+ uptake or by antioxidants. Our results demonstrate that ROS production during the early stage of acute excitotoxicity derives primarily from mitochondria and is Ca 2+ -dependent. More importantly, the increase of mitochondrial ROS serves as a signal for PARP-1 activation, suggesting that concomitant mitochondrial Ca 2+ uptake and PARP-1 activation constitute a unified mechanism for excitotoxic neuronal death.
In the adult mammalian CNS, the growth inhibitors oligodendrocyte-myelin glycoprotein (OMgp) and ... more In the adult mammalian CNS, the growth inhibitors oligodendrocyte-myelin glycoprotein (OMgp) and the reticulon RTN4 (Nogo) are broadly expressed in oligodendrocytes and neurons. Nogo and OMgp complex with the neuronal cell surface receptors Nogo receptor-1 (NgR1) and paired Ig-like receptor-B (PirB) to regulate neuronal morphology. In the healthy CNS, NgR1 regulates dendritic spine shape and attenuates activity-driven synaptic plasticity at Schaffer collateral-CA1 synapses. Here, we examine whether Nogo and OMgp influence functional synaptic plasticity, the efficacy by which synaptic transmission occurs. In acute hippocampal slices of adult mice, Nogo-66 and OMgp suppress NMDA receptor-dependent long-term potentiation (LTP) when locally applied to Schaffer collateral-CA1 synapses. Neither Nogo-66 nor OMgp influences basal synaptic transmission or paired-pulse facilitation, a form of short-term synaptic plasticity. PirB Ϫ/Ϫ and NgR1 Ϫ/Ϫ single mutants and NgR1 Ϫ/Ϫ ;PirB Ϫ/Ϫ double mutants show normal LTP, indistinguishable from wild-type controls. In juvenile mice, LTD in NgR1 Ϫ/Ϫ , but not PirB Ϫ/Ϫ , slices is absent. Mechanistic studies revealed that Nogo-66 and OMgp suppress LTP in an NgR1-dependent manner. OMgp inhibits LTP in part through PirB but independently of p75. This suggests that NgR1 and PirB participate in ligand-dependent inhibition of synaptic plasticity. Loss of NgR1 leads to increased phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2), signaling intermediates known to regulate neuronal growth and synaptic function. In primary cortical neurons, BDNF elicited phosphorylation of AKT and p70S6 kinase is attenuated in the presence of myelin inhibitors. Collectively, we provide evidence that mechanisms of neuronal growth inhibition and inhibition of synaptic strength are related. Thus, myelin inhibitors and their receptors may coordinate structural and functional neuronal plasticity in CNS health and disease.
Myelin-associated glycoprotein (MAG) is a sialic acid binding Ig-family lectin that functions in ... more Myelin-associated glycoprotein (MAG) is a sialic acid binding Ig-family lectin that functions in neuronal growth inhibition and stabilization of axon-glia interactions. The ectodomain of MAG is comprised of five Ig-like domains and employs neuronal cell-type specific mechanisms to signal growth inhibition. We show that the first three Ig-like domains of MAG bind with high-affinity and in a sialic acid dependent manner to the Nogo-66 receptor-1 (NgR1) and its homologue NgR2. Domains Ig3-Ig5 of MAG are sufficient to inhibit neurite outgrowth but fail to associate with NgR1 or NgR2. Nogo receptors are sialoglycoproteins comprised of 8.5 canonical leucine-rich repeats (LRR) flanked by LRR N-terminal (NT) and C-terminal (CT)-cap domains. The LRR cluster is connected through a stalk region to a membrane lipid anchor. The CT-cap domain and stalk region of NgR2, but not NgR1, are sufficient for MAG binding, and when expressed in neurons, exhibit constitutive growth inhibitory activity. The LRR cluster of NgR1 supports binding of Nogo-66, OMgp and MAG. Deletion of disulfide loop Cys 309 -Cys 336 of NgR1 selectively increases its affinity for Nogo-66 and OMgp. A chimeric Nogo receptor variant (NgR OMNI ) in which Cys 309 -Cys 336 is deleted and followed by a 13 amino acid MAG binding motif of the NgR2 stalk, shows superior binding of OMgp, Nogo-66, and MAG compared to wild-type NgR1 or NgR2. Soluble NgR OMNI (NgR OMNI -Fc) binds strongly to membrane bound inhibitors and promotes neurite outgrowth on both MAG and CNS myelin substrates. Thus, NgR OMNI -Fc may offer therapeutic opportunities following nervous system injury or disease where myelin inhibits neuronal regeneration.
It has been known for more than two decades that chondroitin sulfate proteoglycans (CSPGs) inhibi... more It has been known for more than two decades that chondroitin sulfate proteoglycans (CSPGs) inhibit axonal growth and regeneration. In the adult nervous system, CSPGs are enriched in perineuronal nets, and their abundance is increased in reactive astrocytes following injury to brain or spinal cord. Degradation of chondroitin sulfate (CS) sugar moieties by the local infusion of the bacterial enzyme chondroitinase ABC (ChaseABC) enhances experience-dependent neuronal plasticity in the adult visual cortex and results in substantially improved behavioral outcomes after spinal cord injury (SCI). Although the positive effects of ChaseABC treatment on neuronal plasticity have been known for some time, the underlying mechanisms remained enigmatic. The receptor protein tyrosine phosphatase sigma (RPTPsigma) has now been identified as a receptor for inhibitory CSPGs. Similarly to ChaseABC treatment, functional ablation of Ptprs, the gene encoding RPTPsigma, promotes neurite outgrowth in the presence of CSPGs in vitro and enhances axonal growth into CSPG-rich scar tissue following SCI in vivo. The discovery of neuronal RPTPsigma as a receptor for inhibitory CSPGs not only provides important mechanistic clues about CSPG function, but also identifies a potential new target for enhancing axonal growth and plasticity after nervous system injury.
The downstream of kinase (Dok) family of adapter proteins consists of at least five members struc... more The downstream of kinase (Dok) family of adapter proteins consists of at least five members structurally characterized by an NH 2 -terminal tandem of conserved pleckstrin homology and phosphotyrosine binding domains linked to a unique COOH-terminal region. To determine the role of the novel adapter protein Dok-4 in endothelial cells, we first investigated the cell localization of Dok-4. Most surprisingly, immunofluorescence microscopy, cell fractionation studies, and studies with enhanced green fluorescent protein chimeras showed that wild type Dok-4 (Dok-4-WT) specifically localized in mitochondria. An NH 2 -terminal deletion mutant of Dok-4 (Dok-4-(⌬N11-29)), which lacks the mitochondrial targeting sequence, could not accumulate in mitochondria.
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Papers by Yuntao Duan