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Transient global cerebral ischemia induces profound changes in the transcriptome of brain cells,which is partially associated with the induction or repression of genes that influence the ischemic response. However,the mechanisms responsible for the selective vulnerability of hippocampal neurons to global ischemia remain to be clarified. To identify molecular changes elicited by ischemic insults,we subjected hippocampal primary cultures to oxygen-glucose deprivation (OGD),an in vitro model for global ischemia that resulted in delayed neuronal death with an excitotoxic component. To investigate changes in the transcriptome of hippocampal neurons submitted to OGD,total RNA was extracted at early (7 h) and delayed (24 h) time points after OGD and used in a whole-genome RNA microarray. We observed that at 7 h after OGD there was a general repression of genes,whereas at 24 h there was a general induction of gene expression. Genes related with functions such as transcription and RNA biosynthesis were highly regulated at both periods of incubation after OGD,confirming that the response to ischemia is a dynamic and coordinated process. Our analysis showed that genes for synaptic proteins,such as those encoding for PICK1,GRIP1,TARPγ3,calsyntenin-2/3,SAPAP2 and SNAP-25,were down-regulated after OGD. Additionally,OGD decreased the mRNA and protein expression levels of the GluA1 AMPA receptor subunit as well as the GluN2A and GluN2B subunits of NMDA receptors,but increased the mRNA expression of the GluN3A subunit,thus altering the composition of ionotropic glutamate receptors in hippocampal neurons. Together,our results present the expression profile elicited by in vitro ischemia in hippocampal neurons,and indicate that OGD activates a transcriptional program leading to down-regulation in the expression of genes coding for synaptic proteins,suggesting that the synaptic proteome may change after ischemia. View Publication

Dynamics of GABAergic synaptic components have been studied previously over milliseconds to minutes,revealing mobility of postsynaptic scaffolds and receptors. Here we image inhibitory synapses containing fluorescently tagged postsynaptic scaffold Gephyrin,together with presynaptic vesicular GABA transporter (VGAT) or postsynaptic GABA(A) receptor γ2 subunit (GABA(A)Rγ2),over seconds to days in cultured rat hippocampal neurons,revealing modes of inhibitory synapse formation and remodeling. Entire synapses were mobile,translocating rapidly within a confined region and exhibiting greater nonstochastic motion over multihour periods. Presynaptic and postsynaptic components moved in unison,maintaining close apposition while translocating distances of several micrometers. An observed flux in the density of synaptic puncta partially resulted from the apparent merging and splitting of preexisting clusters. De novo formation of inhibitory synapses was observed,marked by the appearance of stably apposed Gephyrin and VGAT clusters at sites previously lacking either component. Coclustering of GABA(A)Rγ2 supports the identification of such new clusters as synapses. Nascent synapse formation occurred by gradual accumulation of components over several hours,with VGAT clustering preceding that of Gephyrin and GABA(A)Rγ2. Comparing VGAT labeling by active uptake of a luminal domain antibody with post hoc immunocytochemistry indicated that recycling vesicles from preexisting boutons significantly contribute to vesicle pools at the majority of new inhibitory synapses. Although new synapses formed primarily on dendrite shafts,some also formed on dendritic protrusions,without apparent interconversion. Altogether,the long-term imaging of GABAergic presynaptic and postsynaptic components reveals complex dynamics and perpetual remodeling with implications for mechanisms of assembly and synaptic integration. View Publication

UNLABELLED Herpes simplex virus 1 (HSV-1) establishes lifelong latent infections in the sensory neurons of the trigeminal ganglia (TG),wherein it retains the capacity to reactivate. The interferon (IFN)-driven antiviral response is critical for the control of HSV-1 acute replication. We therefore sought to further investigate this response in TG neurons cultured from adult mice deficient in a variety of IFN signaling components. Parallel experiments were also performed in fibroblasts isolated concurrently. We showed that HSV-1 replication was comparable in wild-type (WT) and IFN signaling-deficient neurons and fibroblasts. Unexpectedly,a similar pattern was observed for the IFN-sensitive vesicular stomatitis virus (VSV). Despite these findings,TG neurons responded to IFN-β pretreatment with STAT1 nuclear localization and restricted replication of both VSV and an HSV-1 strain deficient in γ34.5,while wild-type HSV-1 replication was unaffected. This was in contrast to fibroblasts in which all viruses were restricted by the addition of IFN-β. Taken together,these data show that adult TG neurons can mount an effective antiviral response only if provided with an exogenous source of IFN-β,and HSV-1 combats this response through γ34.5. These results further our understanding of the antiviral response of neurons and highlight the importance of paracrine IFN-β signaling in establishing an antiviral state. IMPORTANCE Herpes simplex virus 1 (HSV-1) is a ubiquitous virus that establishes a lifelong latent infection in neurons. Reactivation from latency can cause cold sores,blindness,and death from encephalitis. Humans with deficiencies in innate immunity have significant problems controlling HSV infections. In this study,we therefore sought to elucidate the role of neuronal innate immunity in the control of viral infection. Using neurons isolated from mice,we found that the intrinsic capacity of neurons to restrict virus replication was unaffected by the presence or absence of innate immunity. In contrast,neurons were able to mount a robust antiviral response when provided with beta interferon,a molecule that strongly stimulates innate immunity,and that HSV-1 can combat this response through the γ34.5 viral gene. Our results have important implications for understanding how the nervous system defends itself against virus infections. View Publication

Neuronal morphology is highly sensitive to ischemia,although some re-organization may promote neuroprotection. In this study we investigate the role of actin regulating proteins (ARP2,ARP3 and WAVE-1) and their role in rapid ischemic tolerance. Using an established in vitro model of rapid ischemic tolerance,we show that WAVE-1 protein levels are stabilized following brief tolerance inducing ischemia (preconditioning). The stabilization appears to be due to a reduction in the ubiquitination of WAVE-1. Levels of ARP2,ARP3 and N-WASP were not affected by ischemic preconditioning. Immunocytochemical studies show a relocalization of ARP2 and ARP3 proteins in neurons following preconditioning ischemia,as well as a re-organization of actin. Blocking the protein kinase CK2 using emodin blocks ischemic tolerance,and our data suggests CK2 binds to WAVE-1 in neurons. We observe an increase in binding of the ARP2 subunit with WAVE-1. The neuroprotection observed following preconditioning is inhibited when cells are transduced with an N-WASP CA domain that blocks the activation of ARP2/3. Together these data show that ischemia affects actin regulating enzymes,and that the ARP2/3 pathway plays a role in rapid ischemic tolerance induced neuroprotection. View Publication

BACKGROUND Type 2 diabetes (T2D) is a recognized risk factor for the development of cognitive impairment (CI) and/or dementia,although the exact nature of the molecular pathology of T2D-associated CI remains obscure. One link between T2D and CI might involve decreased insulin signaling in brain and/or neurons in either animal or postmortem human brains as has been reported as a feature of Alzheimer's disease (AD). Here we asked if neuronal insulin resistance is a cell autonomous phenomenon in a familial form of AD. METHODS We have applied a newly developed protocol for deriving human basal forebrain cholinergic neurons (BFCN) from skin fibroblasts via induced pluripotent stem cell (iPSC) technology. We generated wildtype and familial AD mutant PSEN2 N141I (presenilin 2) BFCNs and assessed if insulin signaling,insulin regulation of the major AD proteins Abeta$ and/or tau,and/or calcium fluxes is altered by the PSEN2 N141I mutation. RESULTS We report herein that wildtype,PSEN2 N141I and CRISPR/Cas9-corrected iPSC-derived BFCNs (and their precursors) show indistinguishable insulin signaling profiles as determined by the phosphorylation of canonical insulin signaling pathway molecules. Chronic insulin treatment of BFCNs of all genotypes led to a reduction in the Abeta$42/40 ratio. Unexpectedly,we found a CRISPR/Cas9-correctable effect of PSEN2 N141I on calcium flux,which could be prevented by chronic exposure of BFCNs to insulin. CONCLUSIONS Our studies indicate that the familial AD mutation PSEN2 N141I does not induce neuronal insulin resistance in a cell autonomous fashion. The ability of insulin to correct calcium fluxes and to lower Abeta$42/40 ratio suggests that insulin acts to oppose an AD-pathophysiology. Hence,our results are consistent with a potential physiological role for insulin as a mediator of resilience by counteracting specific metabolic and molecular features of AD. View Publication

Global cerebral ischemia induces selective acute neuronal injury of the CA1 region of the hippocampus. The type of cell death that ensues may include different programmed cell death mechanisms namely apoptosis and necroptosis,a recently described type of programmed necrosis. We investigated whether necroptosis contributes to hippocampal neuronal death following oxygen-glucose deprivation (OGD),an in vitro model of global ischemia. We observed that OGD induced a death receptor (DR)-dependent component of necroptotic cell death in primary cultures of hippocampal neurons. Additionally,we found that this ischemic challenge upregulated the receptor-interacting protein kinase 3 (RIP3) mRNA and protein levels,with a concomitant increase of the RIP1 protein. Together,these two related proteins form the necrosome,the complex responsible for induction of necroptotic cell death. Interestingly,we found that caspase-8 mRNA,a known negative regulator of necroptosis,was transiently decreased following OGD. Importantly,we observed that the OGD-induced increase in the RIP3 protein was paralleled in an in vivo model of transient global cerebral ischemia,specifically in the CA1 area of the hippocampus. Moreover,we show that the induction of endogenous RIP3 protein levels influenced neuronal toxicity since we found that RIP3 knock-down (KD) abrogated the component of OGD-induced necrotic neuronal death while RIP3 overexpression exacerbated neuronal death following OGD. Overexpression of RIP1 also had deleterious effects following the OGD challenge. Taken together,our results highlight that cerebral ischemia activates transcriptional changes that lead to an increase in the endogenous RIP3 protein level which might contribute to the formation of the necrosome complex and to the subsequent component of necroptotic neuronal death that follows ischemic injury. View Publication

General anesthetics produce a reversible coma-like state through modulation of excitatory and inhibitory synaptic transmission. Recent evidence suggests that anesthetic exposure can also lead to sustained cognitive dysfunction. However,the subcellular effects of anesthetics on the structure of established synapses are not known. We investigated effects of the widely used volatile anesthetic isoflurane on the structural stability of hippocampal dendritic spines,a postsynaptic structure critical to excitatory synaptic transmission in learning and memory. Exposure to clinical concentrations of isoflurane induced rapid and non-uniform shrinkage and loss of dendritic spines in mature cultured rat hippocampal neurons. Spine shrinkage was associated with a reduction in spine F-actin concentration. Spine loss was prevented by either jasplakinolide or cytochalasin D,drugs that prevent F-actin disassembly. Isoflurane-induced spine shrinkage and loss were reversible upon isoflurane elimination. Thus,isoflurane destabilizes spine F-actin,resulting in changes to dendritic spine morphology and number. These findings support an actin-based mechanism for isoflurane-induced alterations of synaptic structure in the hippocampus. These reversible alterations in dendritic spine structure have important implications for acute anesthetic effects on excitatory synaptic transmission and synaptic stability in the hippocampus,a locus for anesthetic-induced amnesia,and have important implications for anesthetic effects on synaptic plasticity. View Publication

Induced pluripotent stem cell (iPSC)-based technologies offer an unprecedented opportunity to perform high-throughput screening of novel drugs for neurological and neurodegenerative diseases. Such screenings require a robust and scalable method for generating large numbers of mature,differentiated neuronal cells. Currently available methods based on differentiation of embryoid bodies (EBs) or directed differentiation of adherent culture systems are either expensive or are not scalable. We developed a protocol for large-scale generation of neuronal stem cells (NSCs)/early neural progenitor cells (eNPCs) and their differentiation into neurons. Our scalable protocol allows robust and cost-effective generation of NSCs/eNPCs from iPSCs. Following culture in neurobasal medium supplemented with B27 and BDNF,NSCs/eNPCs differentiate predominantly into vesicular glutamate transporter 1 (VGLUT1) positive neurons. Targeted mass spectrometry analysis demonstrates that iPSC-derived neurons express ligand-gated channels and other synaptic proteins and whole-cell patch-clamp experiments indicate that these channels are functional. The robust and cost-effective differentiation protocol described here for large-scale generation of NSCs/eNPCs and their differentiation into neurons paves the way for automated high-throughput screening of drugs for neurological and neurodegenerative diseases. View Publication

Increasing evidence suggests that cellular stress may underlie mood disorders such as bipolar disorder and major depression,particularly as lithium and its targets can protect against neuronal cell death. Here we describe N-methyl-D-aspartate (NMDA)-induced filamentous actin reorganization (NIFAR) as a useful in-vitro model for studying acute neurocellular stress and investigating the effects of mood stabilizers. Brief incubation of cultured neurons with NMDA (50 µM,5 min) induces marked reorganization of F-actin within the somatodendritic domain of a majority of neurons. During NIFAR,F-actin is rapidly depleted from dendritic spines and aberrantly aggregates within the dendrite shaft. The widely used mood stabilizer lithium chloride prevented NIFAR in a time-dependent and dose-dependent manner,consistent with its known efficacy in treating bipolar disorder. Inhibitors of the lithium target glycogen synthase kinase 3 and its upstream activator phosphoinositide-3-kinase also prevented NIFAR. The antidepressant compounds imipramine and fluoxetine also attenuated NIFAR. These findings have potential relevance to neuropsychiatric diseases characterized by excessive glutamate receptor activity and synaptotoxicity. We propose that protection of the dendritic actin cytoskeleton may be a common mechanism shared by various mood stabilizers. View Publication