AMPK Preferentially Depresses Retrograde Transport of Axonal Mitochondria during Localized Nutrient Deprivation.

Mitochondrial clusters are found at regions of high-energy demand, allowing cells to meet local metabolic requirements while maintaining neuronal homeostasis. AMP-activated protein kinase (AMPK), a key energy stress sensor, responds to increases in AMP/ATP ratio by activating multiple signaling cascades to overcome the energetic deficiency. In many neurologic conditions, the distal axon experiences energetic stress independent of the soma. Here, we used microfluidic devices to physically isolate these two neuronal structures and to investigate whether localized AMPK signaling influenced axonal mitochondrial transport. Nucleofection of primary cortical neurons, derived from E16-18 mouse embryos (both sexes), with mito-GFP allowed monitoring of the transport dynamics of mitochondria within the axon, by confocal microscopy. Pharmacological activation of AMPK at the distal axon (0.1 mm 5-aminoimidazole-4-carboxamide riboside) induced a depression of the mean frequency, velocity, and distance of retrograde mitochondrial transport in the adjacent axon. Anterograde mitochondrial transport was less sensitive to local AMPK stimulus, with the imbalance of bidirectional mitochondrial transport resulting in accumulation of mitochondria at the region of energetic stress signal. Mitochondria in the axon-rich white matter of the brain rely heavily on lactate as a substrate for ATP synthesis. Interestingly, localized inhibition of lactate uptake (10 nm AR-C155858) reduced mitochondrial transport in the adjacent axon in all parameters measured, similar to that observed by 5-aminoimidazole-4-carboxamide riboside treatment. Coaddition of compound C restored all parameters measured to baseline levels, confirming the involvement of AMPK. This study highlights a role of AMPK signaling in the depression of axonal mitochondrial mobility during localized energetic stress.SIGNIFICANCE STATEMENT As the main providers of cellular energy, the dynamic transport of mitochondria within the neuron allows for clustering at regions of high-energy demand. Here we investigate whether acute changes in energetic stress signal in the spatially isolated axon would alter mitochondrial transport in this local region. Both direct and indirect activation of AMP-activated protein kinase isolated to the distal axon induced a rapid, marked depression in local mitochondrial transport. This work highlights the ability of acute localized AMP-activated protein kinase signaling to affect mitochondrial mobility within the axon, with important implications for white matter injury, axonal growth, and axonal degeneration.

Increased A20-E3 ubiquitin ligase interactions in bid-deficient glia attenuate TLR3- and TLR4-induced inflammation.

BACKGROUND: Chronic pro-inflammatory signaling propagates damage to neural tissue and affects the rate of disease progression. Increased activation of Toll-like receptors (TLRs), master regulators of the innate immune response, is implicated in the etiology of several neuropathologies including amyotrophic lateral sclerosis, Alzheimer's disease, and Parkinson's disease. Previously, we identified that the Bcl-2 family protein BH3-interacting domain death agonist (Bid) potentiates the TLR4-NF-κB pro-inflammatory response in glia, and specifically characterized an interaction between Bid and TNF receptor associated factor 6 (TRAF6) in microglia in response to TLR4 activation. METHODS: We assessed the activation of mitogen-activated protein kinase (MAPK) and interferon regulatory factor 3 (IRF3) inflammatory pathways in response to TLR3 and TLR4 agonists in wild-type (wt) and bid-deficient microglia and macrophages, using Western blot and qPCR, focusing on the response of the E3 ubiquitin ligases Pellino 1 (Peli1) and TRAF3 in the absence of microglial and astrocytic Bid. Additionally, by Western blot, we investigated the Bid-dependent turnover of Peli1 and TRAF3 in wt and bid-/- microglia using the proteasome inhibitor Bortezomib. Interactions between the de-ubiquitinating Smad6-A20 and the E3 ubiquitin ligases, TRAF3 and TRAF6, were determined by FLAG pull-down in TRAF6-FLAG or Smad6-FLAG overexpressing wt and bid-deficient mixed glia. RESULTS: We elucidated a positive role of Bid in both TIR-domain-containing adapter-inducing interferon-ß (TRIF)- and myeloid differentiation primary response 88 (MyD88)-dependent pathways downstream of TLR4, concurrently implicating TLR3-induced inflammation. We identified that Peli1 mRNA levels were significantly reduced in PolyI:C- and lipopolysaccharide (LPS)-stimulated bid-deficient microglia, suggesting disturbed IRF3 activation. Differential regulation of TRAF3 and Peli1, both essential E3 ubiquitin ligases facilitating TRIF-dependent signaling, was observed between wt and bid -/- microglia and astrocytes. bid deficiency resulted in increased A20-E3 ubiquitin ligase protein interactions in glia, specifically A20-TRAF6 and A20-TRAF3, implicating enhanced de-ubiquitination as the mechanism of action by which E3 ligase activity is perturbed. Furthermore, Smad6-facilitated recruitment of the de-ubiquitinase A20 to E3-ligases occurred in a bid-dependent manner. CONCLUSIONS: This study demonstrates that Bid promotes E3 ubiquitin ligase-mediated signaling downstream of TLR3 and TLR4 and provides further evidence for the potential of Bid inhibition as a therapeutic for the attenuation of the robust pro-inflammatory response culminating in TLR activation.

Time-lapse imaging of p65 and IκBα translocation kinetics following Ca2+-induced neuronal injury reveals biphasic translocation kinetics in surviving neurons.

The transcription factor nuclear factor-κB (NF-κB) regulates neuronal differentiation, plasticity and survival. It is well established that excitatory neurotransmitters such as glutamate control NF-κB activity. Glutamate receptor overactivation is also involved in ischemic- and seizure-induced neuronal injury and neurodegeneration. However, little is known at the single cell-level how NF-κB signaling relates to neuronal survival during excitotoxic injury. We found that silencing of p65/NF-κB delayed N-methyl-d-aspartate (NMDA)-induced excitotoxic injury in hippocampal neurons, suggesting a functional role of p65 in excitotoxicity. Time-lapse imaging of p65 and its inhibitor IκBα using GFP and Cerulean fusion proteins revealed specific patterns of excitotoxic NF-κB activation. Nuclear translocation of p65 began on average 8±3min following 15min of NMDA treatment and was observed in up to two thirds of hippocampal neurons. Nuclear translocation of IκBα preceded that of p65 suggesting independent translocation processes. In surviving neurons, the onset of p65 nuclear export correlated with mitochondrial membrane potential recovery. Dying neurons exhibited persistent nuclear accumulation of p65-eGFP until plasma membrane permeabilization. Our data demonstrate an important role for p65 activation kinetics in neuronal cell death decisions following excitotoxic injury.

Caspase 6 has a protective role in SOD1(G93A) transgenic mice.

In amyotrophic lateral sclerosis (ALS), it has been suggested that the process of neurodegeneration starts at the neuromuscular junction and is propagated back along axons towards motor neurons. Caspase-dependent pathways are well established as a cause of motor neuron death, and recent work in other disease models indicated a role for caspase 6 in axonal degeneration. Therefore we hypothesised that caspase 6 may be involved in motor neuron death in ALS. To investigate the role of caspase 6 in ALS we profiled protein levels of caspase-6 throughout disease progression in the ALS mouse model SOD1(G93A); this did not reveal differences in caspase 6 levels during disease. To investigate the role of caspase 6 further we generated a colony with SOD1(G93A) transgenic mice lacking caspase 6. Analysis of the transgenic SOD1(G93A); Casp6(-/-) revealed an exacerbated phenotype with motor dysfunction occurring earlier and a significantly shortened lifespan when compared to transgenic SOD1(G93A); Casp6(+/+) mice. Immunofluorescence analysis of the neuromuscular junction revealed no obvious difference between caspase 6(+/+) and caspase 6(-/-) in non-transgenic mice, while the SOD1(G93A) transgenic mice showed severe degeneration compared to non-transgenic mice in both genotypes. Our data indicate that caspase-6 does not exacerbate ALS pathogenesis, but may have a protective role.

The BCL-2 family protein Bid is critical for pro-inflammatory signaling in astrocytes.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the loss of motoneurons in the spinal cord, brainstem and motor cortex. Mutations in the superoxide dismutase 1 (SOD1) gene represent a frequent genetic determinant and recapitulate a disease phenotype similar to ALS when expressed in mice. Previous studies using SOD1(G93A) transgenic mice have suggested a paracrine mechanism of neuronal loss, in which cytokines and other toxic factors released from astroglia or microglia trigger motoneuron degeneration. Several pro-inflammatory cytokines activate death receptors and may downstream from this activate the Bcl-2 family protein, Bid. We here sought to investigate the role of Bid in astrocyte activation and non-cell autonomous motoneuron degeneration. We found that spinal cord Bid protein levels increased significantly during disease progression in SOD1(G93A) mice. Subsequent experiments in vitro indicated that Bid was expressed at relatively low levels in motoneurons, but was enriched in astrocytes and microglia. Bid was strongly induced in astrocytes in response to pro-inflammatory cytokines or exposure to lipopolysaccharide. Experiments in bid-deficient astrocytes or astrocytes treated with a small molecule Bid inhibitor demonstrated that Bid was required for the efficient activation of transcription factor nuclear factor-κB in response to these pro-inflammatory stimuli. Finally, we found that conditioned medium from wild-type astrocytes, but not from bid-deficient astrocytes, was toxic when applied to primary motoneuron cultures. Collectively, our data demonstrate a new role for the Bcl-2 family protein Bid as a mediator of astrocyte activation during neuroinflammation, and suggest that Bid activation may contribute to non-cell autonomous motoneuron degeneration in ALS.

Fibroblast growth factor homologous factor 1 interacts with NEMO to regulate NF-κB signaling in neurons.

Neuronal survival and plasticity critically depend on constitutive activity of the transcription factor nuclear factor-κB (NF-κB). We here describe a role for a small intracellular fibroblast growth factor homologue, the fibroblast growth factor homologous factor 1 (FHF1/FGF12), in the regulation of NF-κB activity in mature neurons. FHFs have previously been described to control neuronal excitability, and mutations in FHF isoforms give rise to a form of progressive spinocerebellar ataxia. Using a protein-array approach, we identified FHF1b as a novel interactor of the canonical NF-κB modulator IKKγ/NEMO. Co-immunoprecipitation, pull-down and GAL4-reporter experiments, as well as proximity ligation assays, confirmed the interaction of FHF1 and NEMO and demonstrated that a major site of interaction occurred within the axon initial segment. Fhf1 gene silencing strongly activated neuronal NF-κB activity and increased neurite lengths, branching patterns and spine counts in mature cortical neurons. The effects of FHF1 on neuronal NF-κB activity and morphology required the presence of NEMO. Our results imply that FHF1 negatively regulates the constitutive NF-κB activity in neurons.

AMP-activated protein kinase (AMPK)-induced preconditioning in primary cortical neurons involves activation of MCL-1.

Neuronal preconditioning is a phenomenon where a previous exposure to a sub-lethal stress stimulus increases the resistance of neurons towards a second, normally lethal stress stimulus. Activation of the energy stress sensor, AMP-activated protein kinase (AMPK) has been shown to contribute to the protective effects of ischaemic and mitochondrial uncoupling-induced preconditioning in neurons, however, the molecular basis of AMPK-mediated preconditioning has been less well characterized. We investigated the effect of AMPK preconditioning using 5-aminoimidazole-4-carboxamide riboside (AICAR) in a model of NMDA-mediated excitotoxic injury in primary mouse cortical neurons. Activation of AMPK with low concentrations of AICAR (0.1 mM for 2 h) induced a transient increase in AMPK phosphorylation, protecting neurons against NMDA-induced excitotoxicity. Analysing potential targets of AMPK activation, demonstrated a marked increase in mRNA expression and protein levels of the anti-apoptotic BCL-2 family protein myeloid cell leukaemia sequence 1 (MCL-1) in AICAR-preconditioned neurons. Interestingly, over-expression of MCL-1 protected neurons against NMDA-induced excitotoxicity while MCL-1 gene silencing abolished the effect of AICAR preconditioning. Monitored intracellular Ca²âº levels during NMDA excitation revealed that MCL-1 over-expressing neurons exhibited improved bioenergetics and markedly reduced Ca²âº elevations, suggesting a potential mechanism through which MCL-1 confers neuroprotection. This study identifies MCL-1 as a key effector of AMPK-induced preconditioning in neurons.

Bone morphogenetic protein 3 controls insulin gene expression and is down-regulated in INS-1 cells inducibly expressing a hepatocyte nuclear factor 1A-maturity-onset diabetes of the young mutation.

Inactivating mutations in the transcription factor hepatocyte nuclear factor (HNF) 1A cause HNF1A-maturity-onset diabetes of the young (HNF1A-MODY), the most common monogenic form of diabetes. To examine HNF1A-MODY-induced defects in gene expression, we performed a microarray analysis of the transcriptome of rat INS-1 cells inducibly expressing the common hot spot HNF1A frameshift mutation, Pro291fsinsC-HNF1A. Real-time quantitative PCR (qPCR), Western blotting, immunohistochemistry, reporter assays, and chromatin immunoprecipitation (ChIP) were used to validate alterations in gene expression and to explore biological activities of target genes. Twenty-four hours after induction of the mutant HNF1A protein, we identified a prominent down-regulation of the bone morphogenetic protein 3 gene (Bmp-3) mRNA expression. Reporter assays, qPCR, and Western blot analysis validated these results. In contrast, inducible expression of wild-type HNF1A led to a time-dependent increase in Bmp-3 mRNA and protein levels. Moreover, reduced protein levels of BMP-3 and insulin were detected in islets of transgenic HNF1A-MODY mice. Interestingly, treatment of naïve INS-1 cells or murine organotypic islet cultures with recombinant human BMP-3 potently increased their insulin levels and restored the decrease in SMAD2 phosphorylation and insulin gene expression induced by the HNF1A frameshift mutation. Our study suggests a critical link between HNF1A-MODY-induced alterations in Bmp-3 expression and insulin gene levels in INS-1 cells and indicates that the reduced expression of growth factors involved in tissue differentiation may play an important role in the pathophysiology of HNF1A-MODY.

TGF-{beta}1 activates two distinct type I receptors in neurons: implications for neuronal NF-{kappa}B signaling.

Transforming growth factor-betas (TGF-betas) are pleiotropic cytokines involved in development and maintenance of the nervous system. In several neural lesion paradigms, TGF-beta1 exerts potent neuroprotective effects. Neurons treated with TGF-beta1 activated the canonical TGF-beta receptor I/activin-like kinase receptor 5 (ALK5) pathway. The transcription factor nuclear factor-kappaB (NF-kappaB) plays a fundamental role in neuroprotection. Treatment with TGF-beta1 enhanced NF-kappaB activity in gelshift and reporter gene analyses. However, ectopic expression of a constitutively active ALK5 failed to mimic these effects. ALK1 has been described as an alternative TGF-beta receptor in endothelial cells. Interestingly, we detected significant basal expression of ALK1 and its injury-induced up-regulation in neurons. Treatment with TGF-beta1 also induced a pronounced increase in downstream Smad1 phosphorylation. Overexpression of a constitutively active ALK1 mimicked the effect of TGF-beta1 on NF-kappaB activation and neuroprotection. Our data suggest that TGF-beta1 simultaneously activates two distinct receptor pathways in neurons and that the ALK1 pathway mediates TGF-beta1-induced NF-kappaB survival signaling.

p53 upregulated mediator of apoptosis (Puma) deficiency increases survival of adult neural stem cells generated physiologically in the hippocampus, but does not protect stem cells generated in surplus after an excitotoxic lesion.

OBJECTIVES: Neurogenesis occurs in the mammalian brain throughout adulthood and increases in response to metabolic, toxic or traumatic insults. To remove potentially superfluous or unwanted neural stem cells/neuronal progenitors, their rate of proliferation and differentiation is fine-tuned against their rate of apoptosis. Apoptosis requires the transcriptional and posttranslational activation of Bcl-2-homolgy domain 3 (BH3)-only proteins. Previously, we demonstrated that the BH3-only protein p53-upregulated mediator of apoptosis (Puma) controls the physiological rate of apoptosis of neural precursor cells in the adult mouse hippocampus. Puma's role in controlling a lesion-induced increase in neural stem cells is currently not known. METHODS: We employed a model of local, N-methyl-D-asparte (NMDA)-induced excitotoxic injury to the CA1 hippocampal subfield and immunofluorescence labelling to produce increased neural stem cell proliferation/ neurogenesis in the dentate gyrus at two survival times following the excitotoxic lesion. RESULTS: Deletion of puma failed to rescue any NMDA-induced increase in adult born cells as assessed by BrdU or Doublecortin labelling in the long-term. No difference in the proportion of BrdU/NeuN-positive cells comparing the different genotypes and treatments suggested that the phenotypic fate of the cells was preserved regardless of the genotype and the treatment. CONCLUSIONS: While neurogenesis is up-regulated in puma-deficient animals following NMDA-induced excitotoxicity to the hippocampal CA1 subfield, puma deficiency could not protect this surplus of newly generated cells from apoptotic cell death.

A constitutively-active IKK-complex at the axon initial segment.

BACKGROUND: Previous studies provided evidence for an accumulation of IκB-kinase (IKK) α/ß at the axon initial segment (AIS), a neuronal compartment defined by ankyrin-G expression. Here we explored whether the presence of the IKK-complex at the AIS was associated with the activation of IKK signaling at this site. METHODS AND RESULTS: Proximity-ligation assays (PLAs) using pan-IKKα/ß, phospho-IKKα/ß-specific as well as ankyrin-G specific antibodies validated their binding to proximal epitopes in the AIS, while antibodies to other phosphorylated signaling proteins showed no preference for the AIS. Small-hairpin mediated silencing of IKKß significantly reduced anti-phospho-IKKα/ß-immunoreactivities in the AIS. ank3 gene-deficient cerebellar Purkinje cells also exhibited no phosphorylated IKKα/ß at the proximal region of their axons. Transient ankyrin-G overexpression in PC12 cells augmented NF-κB transactivation in an ankyrin-G death-domain dependent manner. Finally, small molecule inhibitors of IKK-activity, including Aspirin, inhibited the accumulation of activated IKK proteins in the AIS. CONCLUSION: Our data suggest the existence of a constitutively-active IKK signaling complex in the AIS.

Full length Bid is sufficient to induce apoptosis of cultured rat hippocampal neurons.

BACKGROUND: Bcl-2 homology domain (BH) 3-only proteins are pro-apoptotic proteins of the Bcl-2 family that couple stress signals to the mitochondrial cell death pathways. The BH3-only protein Bid can be activated in response to death receptor activation via caspase 8-mediated cleavage into a truncated protein (tBid), which subsequently translocates to mitochondria and induces the release of cytochrome-C. Using a single-cell imaging approach of Bid cleavage and translocation during apoptosis, we have recently demonstrated that, in contrast to death receptor-induced apoptosis, caspase-independent excitotoxic apoptosis involves a translocation of full length Bid (FL-Bid) from the cytosol to mitochondria. We induced a delayed excitotoxic cell death in cultured rat hippocampal neurons by a 5-min exposure to the glutamate receptor agonist N-methyl-D-aspartate (NMDA; 300 microM). RESULTS: Western blot experiments confirmed a translocation of FL-Bid to the mitochondria during excitotoxic apoptosis that was associated with the release of cytochrome-C from mitochondria. These results were confirmed by immunofluorescence analysis of Bid translocation during excitotoxic cell death using an antibody raised against the amino acids 1-58 of mouse Bid that is not able to detect tBid. Finally, inducible overexpression of FL-Bid or a Bid mutant that can not be cleaved by caspase-8 was sufficient to induce apoptosis in the hippocampal neuron cultures. CONCLUSION: Our data suggest that translocation of FL-Bid is sufficient for the activation of mitochondrial cell death pathways in response to glutamate receptor overactivation.

NF-κB regulates neuronal ankyrin-G via a negative feedback loop.

The axon initial segment (AIS) is a neuronal compartment defined by ankyrin-G expression. We here demonstrate that the IKK-complex co-localizes and interacts with the cytoskeletal anchor protein ankyrin-G in immunoprecipitation and proximity-ligation experiments in cortical neurons. Overexpression of the 270 kDa variant of ankyrin-G suppressed, while gene-silencing of ankyrin-G expression increased nuclear factor-κB (NF-κB) activity in primary neurons, suggesting that ankyrin-G sequesters the transcription factor in the AIS. We also found that p65 bound to the ank3 (ankyrin-G) promoter sequence in chromatin immunoprecipitation analyses thereby increasing ank3 expression and ankyrin-G levels at the AIS. Gene-silencing of p65 or ankyrin-G overexpression suppressed ank3 reporter activity. Collectively these data demonstrate that p65/NF-κB controls ankyrin-G levels via a negative feedback loop, thereby linking NF-κB signaling with neuronal polarity and axonal plasticity.

Bid Promotes K63-Linked Polyubiquitination of Tumor Necrosis Factor Receptor Associated Factor 6 (TRAF6) and Sensitizes to Mutant SOD1-Induced Proinflammatory Signaling in Microglia.

Mutations in the superoxide dismutase 1 (SOD1) gene contribute to motoneuron degeneration and are evident in 20% of familial amyotrophic lateral sclerosis cases. Mutant SOD1 induces microglial activation through a stimulation of Toll-like receptors 2 and 4 (TLR2 and TLR4). In the present study, we identified the proapoptotic Bcl-2 family protein Bid as a positive regulator of mutant SOD1-induced TLR-nuclear factor-κB (NF-κB) signaling in microglia. bid-deficient primary mouse microglia showed reduced NF-κB signaling in response to TLR4 activation or exposure to conditioned medium derived from SOD1 (G93A) expressing NSC-34 cells. Attenuation of NF-κB signaling in bid-deficient microglia was associated with lower levels of phosphorylated IKKα/ß and p65, with a delayed degradation of IκBα and enhanced degradation of Peli1. Upstream of IKK, we found that Bid interacted with, and promoted, the K63-linked polyubiquitination of the E3 ubiquitin ligase tumor necrosis factor receptor associated factor 6 (TRAF6) in microglia. Our study suggests a key role for Bid in the regulation of TLR4-NF-κB proinflammatory signaling during mutant SOD1-induced disease pathology. Bid promotes TLR4-NF-κB signaling by interacting with TRAF6 and promoting TRAF6 K63-linked polyubiquitination in microglia.

BID Mediates Oxygen-Glucose Deprivation-Induced Neuronal Injury in Organotypic Hippocampal Slice Cultures and Modulates Tissue Inflammation in a Transient Focal Cerebral Ischemia Model without Changing Lesion Volume.

The BH3 interacting-domain death agonist (BID) is a pro-apoptotic protein involved in death receptor-induced and mitochondria-mediated apoptosis. Recently, it has also been suggested that BID is involved in the regulation of inflammatory responses in the central nervous system. We found that BID deficiency protected organotypic hippocampal slice cultures in vitro from neuronal injury induced by oxygen-glucose deprivation. In vivo, BID-knockout (KO) mice and wild type (WT) mice were subjected to 60 min of transient middle cerebral artery occlusion (tMCAO) to induce focal cerebral ischemia, and allowed to recover for 24 h. Infarct volumes and functional outcome were assessed and the inflammatory response was evaluated using immunofluorescence, Western blotting, quantitative PCR (qPCR) and Mesoscale multiplex analysis. We observed no difference in the infarct volume or neurological outcome between BID-KO and WT mice. The inflammatory response was reduced by BID deficiency as indicated by a change in microglial/leukocyte response. In conclusion, our data suggest that BID deficiency is neuroprotective in an in vitro model and modulates the inflammatory response to focal cerebral ischemia in vivo. However, this is not translated into a robust neuroprotection in vivo.

Vascular endothelial growth factor protects cultured rat hippocampal neurons against hypoxic injury via an antiexcitotoxic, caspase-independent mechanism.

The authors investigated the effect of vascular endothelial growth factor (VEGF) on hypoxic injury of cultured rat hippocampal neurons. Treatment with glutamate receptor antagonists prevented hypoxic neuron death. The same magnitude of protection was observed in cultures treated with VEGF, which also reduced excitotoxic neuron death induced directly by an exposure to -methyl-d-aspartate. Vascular endothelial growth factor did not alter the activation of the transcription factor nuclear factor-kappaB during hypoxia and protected cells in a PI-3-kinase-independent manner. Vascular endothelial growth factor failed to protect against staurosporine-induced, caspase-dependent apoptosis. These data suggest that VEGF-induced protection against hypoxic injury primarily involves the inhibition of excitotoxic processes.

NMDA-induced injury of mouse organotypic hippocampal slice cultures triggers delayed neuroblast proliferation in the dentate gyrus: an in vitro model for the study of neural precursor cell proliferation.

We present a model for the study of injury-induced neurogenesis in the dentate gyrus (DG) in murine organotypic hippocampal slice cultures (OHCs). A brief exposure of 8-day-old hippocampal slice cultures to the glutamate receptor agonist N-methyl-d-aspartate (NMDA; 20-50µM for 30 min) caused a selective excitotoxic injury in the CA1 subfield of the hippocampus that matured over a period of 24h. The insult resulted in a prominent up-regulation of proliferating nuclei within the OHC dentate gyrus (DG), and a corresponding increase in Ki67/doublecortin double-positive cells in the SGZ of the dentate gyrus. 5-bromo-2-deoxyuridine (BrdU)-labelling of the OHCs for three days subsequent to the NMDA exposure revealed significantly increased BrdU incorporation within the DG (SGZ and GCL) of the hippocampus. Doublecortin immunofluorescence indicated a concurrent up-regulation of neuronal precursor cells specifically in the SGZ and GCL. Significantly increased BrdU incorporation could be detected up to 6-9 days after termination of the NMDA exposure. The model presented here enables easy manipulation and follow-up of injury-induced neuroblast proliferation in the DG that is amenable to the study of transgenic mice.

Induction of transcription factor CEBP homology protein mediates hypoglycaemia-induced necrotic cell death in human neuroblastoma cells.

Oxygen and glucose deprivation are direct consequences of tissue ischaemia. We explored the interaction of hypoxia and hypoglycaemia on cell survival and gene expression in the absence of glutamatergic signalling using human SH-SY5Y neuroblastoma cells as a model. In agreement with previous investigations in non-neural cells, prolonged hypoxia (0.5% O(2)) failed to induce significant cell death in this system. In contrast, exposure to hypoglycaemia induced significant necrotic cell death (> 80% after 72 h). Interestingly, hypoglycaemia-induced cell death was completely abrogated by simultaneous exposure to hypoxia, suggesting strong cytoprotective effects of hypoxia. Subsequent microarray analysis of the underlying transcriptional responses revealed that the transcription factor CEBP homology protein (CHOP) was strongly induced by hypoglycaemia, and suppressed by simultaneous hypoxia. RNA interference against CHOP significantly protected cells from glucose deprivation-induced cell death. Hypoxia-induced vascular endothelial growth factor (VEGF) activation also protected cells against hypoglycaemia-induced cell death, but VEGF failed to modify hypoglycaemia-induced CHOP induction. Our data suggest that hypoglycaemia-induced necrotic cell death of neuroblastoma cells is an active process mediated via the induction of the transcription factor CHOP, and that hypoxia counteracts this cell death via at least two distinct mechanisms: repression of CHOP and induction of VEGF.

Coincident enrichment of phosphorylated IkappaBalpha, activated IKK, and phosphorylated p65 in the axon initial segment of neurons.

Phosphorylation of the inhibitory protein IkappaBalpha by the activated IkappaB kinase (IKK) is a crucial step in the activation of the transcription factor NF-kappaB. In neurons of the mammalian central nervous system, constitutive activation of NF-kappaB has been previously documented. The cellular compartments involved in this activation have not yet been fully identified. Here we document a striking enrichment of several molecules involved in NF-kappaB activation in the axon initial segment (AIS) of neurons: Phosphorylated-IkappaBalpha (pIkappaBalpha), activated IKK, and p65 phosphorylated at serine 536 were found to be enriched in the AIS in vivo as well as in vitro. Both, pIkappaBalpha and activated IKK, were associated with cytoskeletal components of the AIS. Activated IKK was associated with the membrane cytoskeleton, whereas pIkappaBalpha was sequestered to microtubules of the AIS. Colchicine-induced depolymerization of microtubules resulted in the loss of pIkappaBalpha in the AIS, demonstrating that the integrity of the axonal cytoskeleton is essential for the clustering of this NF-kappaB pathway component. These data provide the first evidence for a compartmentalized clustering of NF-kappaB pathway components in the AIS and implicate this neuronal compartment in the activation of NF-kappaB.

p75 neurotrophin receptor is required for constitutive and NGF-induced survival signalling in PC12 cells and rat hippocampal neurones.

We have previously shown that nerve growth factor (NGF)-induced activation of nuclear factor-kappaB increased neuronal expression of Bcl-xL, an anti-apoptotic Bcl-2 family protein. In the present study we determined the role of the p75 neurotrophin receptor in constitutive and NGF-induced survival signalling. Treatment of rat pheochromocytoma (PC12) cells with a blocking anti-rat p75 antibody or inhibition of p75 expression by antisense oligonucleotides reduced constitutive and NGF-induced bcl-xL expression. Treatment with the blocking anti-p75 antibody also inhibited NGF-induced activation of the survival kinase Akt. Inhibition of phosphatidylinositol-3-kinase (PI3 kinase) activity or overexpression of a dominant-negative mutant of Akt kinase inhibited NGF-induced nuclear factor-kappaB activation. Activation of Akt kinase by NGF was also observed in PC12nnr5 cells and cultured rat hippocampal neurones which both lack significant TrkA expression. Treatment of hippocampal neurones with the blocking anti-p75 antibody inhibited constitutive and NGF-induced Bcl-xL expression, activation of Akt, and blocked the protective effect of NGF against excitotoxic and apoptotic injury. Our data suggest that the p75 neurotrophin receptor mediates constitutive and NGF-induced survival signalling in PC12 cells and hippocampal neurones, and that these effects are mediated via the PI3-kinase pathway.