Ting-Jia Lu

Differential Roles of NMDA Receptor Subtypes in Ischemic Neuronal Cell Death and Ischemic Tolerance

Background and Purpose— Activation of NMDA subtypes of glutamate receptors is implicated in cell damage induced by ischemia as well as for the establishment of ischemic tolerance after ischemic preconditioning in animal models. We investigated the contributions of NR2A- and NR2B-containing NMDA receptors to ischemic cell death and ischemic tolerance in a rat model of transient global ischemia. Methods— Transient global ischemia was produced in rats by 4-vessel occlusion. Neuronal injury was analyzed by Fluoro-Jade B and Nissl staining. Phosphorylation of CREB was detected by Western blotting and immunohistochemistry. In situ hybridization and reverse transcriptase–polymerase chain reaction were used to evaluate the mRNA level of cpg15 and bdnf. Results— NR2A subtype-specific antagonist NVP-AAM077 enhanced neuronal death after transient global ischemia and abolished the induction of ischemic tolerance. In contrast, NR2B subtype-specific antagonist ifenprodil attenuated ischemic cell death and enhanced preconditioning-induced neuroprotection. Furthermore, selectively blocking NR2A-, but not NR2B-, containing NMDA receptors inhibited ischemia-induced phosphorylation of CREB and the subsequent upregulation of CREB target genes such as cpg15 and bdnf. Conclusions— We found that NR2A- and NR2B-containing NMDA receptor subtypes play differential roles in ischemic neuronal death and ischemic tolerance, suggesting attractive new strategies for the development of drugs for patients with stroke.

Clathrin-dependent Endocytosis Is Required for TrkB-dependent Akt-mediated Neuronal Protection and Dendritic Growth

Endocytosis of Trk (tropomyosin-related kinase) receptors is critical for neurotrophin signal transduction and biological functions. However, the mechanism governing endocytosis of TrkB (tropomyosin-related kinase B) and the specific contributions of TrkB endocytosis to downstream signaling are unknown. In this study, we report that blocking clathrin, dynamin, or AP2 in cultured neurons of the central nervous system inhibited brain-derived neurotrophic factor (BDNF)-induced activation of Akt but not ERK. Treating neurons with the clathrin inhibitor monodansylcadaverine or a peptide that blocks dynamin function specifically abrogated Akt pathway activation in response to BDNF but did not affect the response of other downstream effectors or the up-regulation of immediate early genes neuropeptide Y and activity-regulated cytoskeleton-associated protein. Similar effects were found in neurons expressing small interfering RNA to silence AP2 or a dominant negative form of dynamin that inhibits clathrin-mediated endocytosis. In PC12 cells, ERK but not Akt activation required TrkA endocytosis following stimulation with nerve growth factor, whereas the opposite was true when TrkA-expressing neurons were stimulated with nerve growth factor in the central nervous system. Thus, the specific effects of internalized Trk receptors probably depend on the presence of cell type-specific modulators of neurotrophin signaling and not on differences inherent to Trk receptors themselves. Endocytosis-dependent activation of Akt in neurons was found to be critical for BDNF-supported survival and dendrite outgrowth. Together, these results demonstrate the functional requirement of clathrin- and dynamin-dependent endocytosis in generating the full intracellular response of neurons to BDNF in the central nervous system.

Conditional Deletion of NRSF in Forebrain Neurons Accelerates Epileptogenesis in the Kindling Model

Neuron-restrictive silencer factor (NRSF), also known as repressor element-1 silencing transcription factor, is a transcriptional repressor that plays important roles in embryonic development and neurogenesis. Recent findings show that NRSF is upregulated after seizures activity however, the link between NRSF and epileptogenesis remains poorly understood. To investigate the role of NRSF in epilepsy, we employed a Cre-loxp system to specifically delete NRSF in excitatory neurons of the postnatal mouse forebrain. In the kindling model of epileptogenesis, conditional NRSF knockout (NRSF-cKO) mice exhibited dramatically accelerated seizure progression and prolonged afterdischarge duration compared with control mice. Moreover, seizures activity-induced mossy fiber sprouting was enhanced in the NRSF-cKO mice. The degree of upregulation of Fibroblast growth factor 14 and Brain-derived neurotrophic factor (BDNF) following kainic acid-induced status epilepticus was significantly increased in the cortex of NRSF-cKO mice compared with control mice. Furthermore, the derepression of BDNF was associated by activation of PLCγ and PI(3)K signaling pathways. These findings indicate that NRSF functions as an intrinsic repressor of limbic epileptogenesis.

Limbic Epileptogenesis in a Mouse Model of Fragile X Syndrome

Fragile X syndrome (FXS), caused by silencing of the Fmr1 gene, is the most common form of inherited mental retardation. Epilepsy is reported to occur in 20–25% of individuals with FXS. However, no overall increased excitability has been reported in Fmr1 knockout (KO) mice, except for increased sensitivity to auditory stimulation. Here, we report that kindling increased the expressions of Fmr1 mRNA and protein in the forebrain of wild-type (WT) mice. Kindling development was dramatically accelerated in Fmr1 KO mice, and Fmr1 KO mice also displayed prolonged electrographic seizures during kindling and more severe mossy fiber sprouting after kindling. The accelerated rate of kindling was partially repressed by inhibiting N-methyl-D-aspartic acid receptor (NMDAR) with MK-801 or mGluR5 receptor with 2-methyl-6-(phenylethynyl)-pyridine (MPEP). The rate of kindling development in WT was not effected by MPEP, however, suggesting that FMRP normally suppresses epileptogenic signaling downstream of metabolic glutamate receptors. Our findings reveal that FMRP plays a critical role in suppressing limbic epileptogenesis and predict that the enhanced susceptibility of patients with FXS to epilepsy is a direct consequence of the loss of an important homeostatic factor that mitigates vulnerability to excessive neuronal excitation.

Erythropoietin prevents zinc accumulation and neuronal death after traumatic brain injury in rat hippocampus: In vitro and in vivo studies

Erythropoietin (Epo) has been gaining great interest for its potential neuroprotective effect in various neurological insults. However, the molecular mechanism underlying how Epo exerts the function is not clear. Recent studies have indicated that Zn(2+) may have a key role in selective cell death in excitotoxicity after injury. In the present study, we studied the effect of recombinant human Epo (rhEpo) in zinc-induced neurotoxicity both in vitro and in vivo. Exposure of cultured hippocampal neurons to 200 muM ZnC1(2) for 20 min resulted in remarkable neuronal injury, revealed by assessing neuronal morphology. By measuring mitochondrial function using MTT assay, we found that application of rhEpo (0.1 U/ml) 24 h before zinc exposure resulted in a significant increase of neuronal survival (0.6007+/-0.2280 Epo group vs 0.2333+/-0.1249 in control group; n=4, p<0.01). Furthermore, we demonstrated that administration of rhEpo (5,000 IU/kg, intraperitoneal) 30 min after traumatic brain injury (TBI) in rats dramatically protected neuronal death indicated by ZP4 staining, a new zinc-specific fluorescent sensor which has been widely used to indicate neuronal damage after excitotoxic injury (n=5/group, p<0.05). Neuronal damage was also assessed by Fluoro-Jade B (FJB) staining, a highly specific fluorescent marker for the degenerating neurons. Consistent with ZP4 staining, we found the beneficial effects of rhEpo on neuronal survival in hippocampus after TBI (n=5/group, p<0.05). Our results suggest that rhEpo can significantly reduce the pathological Zn(2+) accumulation in rat hippocampus after TBI as well as zinc-induced cell death in cultured cells, which may potentially contribute to its neuronal protection after excitotoxic brain damage.

Zinc neurotoxicity to hippocampal neurons in vitro induces ubiquitin conjugation that requires p38 activation

There is increasing evidence showing that zinc plays a key role in inducing neuronal death during central nervous system injury. However, the underlying mechanisms are poorly understood. Here we assessed the effect of zinc on ubiquitin conjugation and subsequent neurodegeneration using cultured hippocampal cells. We report that cultured neurons are vulnerable to increased level of extracellular Zn²⁺. Zn²⁺-induced poly-ubiquitination in cultured neurons is in a concentration- and time-dependent manner. Furthermore our data demonstrated that Zn²⁺-induced ubiquitination requires p38 activation. These findings indicate that excessive zinc could impair the protein degradation pathway and may be a crucial factor mediating neuronal death following traumatic brain injury.

X-linked microtubule-associated protein, Mid1, regulates axon development

Significance The gene responsible for the X-linked form of Opitz syndrome (OS), Midline-1 (MID1), encodes an E3 ubiquitin ligase and was reported to guide the degradation of the catalytic subunit of protein phosphatase 2A (PP2Ac). But whether and how it is involved in neural development is unclear. We demonstrate here that Mid1-dependent PP2Ac turnover is involved in axon development. Knocking down or knocking out Mid1 not only promotes axon growth and branching in vitro, but also accelerates axon elongation and disrupts the pattern of callosal projection in mouse cortex. These defects can be reversed by down-regulating the accumulated PP2Ac in Mid1-depleted cells. Dysfunction of this Mid1–PP2Ac pathway may underlie neural symptoms of OS patients. Opitz syndrome (OS) is a genetic neurological disorder. The gene responsible for the X-linked form of OS, Midline-1 (MID1), encodes an E3 ubiquitin ligase that regulates the degradation of the catalytic subunit of protein phosphatase 2A (PP2Ac). However, how Mid1 functions during neural development is largely unknown. In this study, we provide data from in vitro and in vivo experiments suggesting that silencing Mid1 in developing neurons promotes axon growth and branch formation, resulting in a disruption of callosal axon projections in the contralateral cortex. In addition, a similar phenotype of axonal development was observed in the Mid1 knockout mouse. This defect was largely due to the accumulation of PP2Ac in Mid1-depleted cells as further down-regulation of PP2Ac rescued the axonal phenotype. Together, these data demonstrate that Mid1-dependent PP2Ac turnover is important for normal axonal development and that dysregulation of this process may contribute to the underlying cause of OS.

Zn2+mediates ischemia-induced impairment of the ubiquitin-proteasome system in the rat hippocampus

Deposition of ubiquitinated protein aggregates is a hallmark of neurodegeneration in both acute neural injuries, such as stroke, and chronic conditions, such as Parkinson’s disease, but the underlying mechanisms are poorly understood. In the present study, we examined the role of Zn2+ in ischemia‐induced impairment of the ubiquitin‐proteasome system in the CA1 region of rat hippocampus after transient global ischemia. We found that scavenging endogenous Zn2+ reduced ischemia‐induced ubiquitin conjugation and free ubiquitin depletion. Furthermore, exposure to zinc chloride increased ubiquitination and inhibited proteasomal enzyme activity in cultured hippocampal neurons in a concentration‐ and time‐dependent manner. Further studies of the underlying mechanisms showed that Zn2+‐induced ubiquitination required p38 activation. These findings indicate that alterations in Zn2+ homeostasis impair the protein degradation pathway.

Zinc as mediator of ubiquitin conjugation following traumatic brain injury

Previous studies have shown that pathological zinc accumulation and deposition of ubiquitinated protein aggregates are commonly detected in many acute neural injuries, such as trauma, epilepsy and ischemia. However, the underlying mechanisms are poorly understood. Here we assessed the effect of zinc on ubiquitin conjugation and subsequent neurodegeration following traumatic brain injury (TBI). First, we found that scavenging endogenous Zn(2+) reduced trauma-induced ubiquitin conjugation and protected neurons from TBI insults in rat hippocampus. Second, we detected both zinc accumulation and increased ubiquitin conjugated protein following brain trauma in human cortical neurons. Our previous study has shown that zinc can induce ubiquitin conjugation in cultured hippocampal neurons. All these findings indicate that alterations in Zn(2+) homeostasis may impair the protein degradation pathway and ultimately cause neuronal injury following traumatic brain injury.

NGF-dependent retrograde signaling: survival versus death.

Nerve growth factor (NGF) was first discovered more than 5 decades ago as a molecule that promotes the survival and maturation of developing neurons in the peripheral nervous system [1]. NGF released from target cells activates tropomyosin-related kinase A (TrkA) on axon terminals and triggers activation of PI3K/Akt, MEK/ ERK, and PLCg signaling pathways. The signal then travels retrogradely along axon to cell body to promote neuronal survival [2]. However, the nature of the retrograde signal remains mysterious. Several distinct types of retrograde signals could derive from axon terminals [3]. First, NGF itself could undergo retrograde transport from terminals to cell body thus activates intracellular TrkA receptor. Indeed, 125I-labeled NGF applied to axon terminals in vivo was found to be retrogradely transported to the neuronal cell bodies [4]. Second, NGF triggers TrkA endocytosis through binding to TrkA, and the endocytic TrkA might serve as the retrograde signal [5]. In supporting of this notion, both NGF and phosphorylated TrkA are found in endosomes [6]. Finally, NGF activates TrkA downstream signal molecules, which could also provide as the retrograde signals [7]. These hypotheses are not mutually exclusive, and multiple retrograde signals may exist. In this issue, Mok and colleagues describe a fundamentally different retrograde mechanism in which NGF suppresses an apoptotic signal in distal axons [8]. Campenot’s group developed compartmentalized cultures of sympathetic neurons which could segregate the distal axons from cell bodies and proximal axons by a distance of about 1 mm. By employing this system with advantages of segregating cellular compartments, researchers could dissect the molecular mechanisms underlying NGF-dependent retrograde signaling. Previous studies from Campenot’s laboratory demonstrated that NGF applied to distal axons of sympathetic neurons supports neuronal survival without transport of NGF towards the cell bodies or TrkA phosphorylation in the cell bodies, suggesting that NGF binding to TrkA in distal axons triggers its downstream signaling cascades locally; afterwards the signals travel retrogradely to the cell bodies and communicate with soma [9]. In the current study, the authors found that neutralizing NGF in distal segment of axons but not in the cell bodies led to activation of c-jun