Neurogenesis continues in the adult forebrain subventricular zone (SVZ) and the dentate gyrus of the hippocampal formation. Degeneration of dopaminergic projections in Parkinson's disease and animals reduces, whereas ciliary neurotrophic factor (CNTF) promotes, neurogenesis. We tested whether the dopaminergic system promotes neurogenesis through CNTF. Astrocytes of the SVZ and dentate gyrus expressed CNTF and were close to dopaminergic terminals. Dopaminergic denervation in adult mice reduced CNTF mRNA by ϳ60%, whereas systemic treatment with the D 2 agonist quinpirole increased CNTF mRNA in the SVZ and hippocampal formation, and in cultured astrocytes by 1.5-5 fold. The effect of quinpirole in vitro was blocked by the D 2 antagonist eticlopride and did not cause astroglial proliferation or hypertrophy. Systemic quinpirole injections increased proliferation in wild-type mice by ϳ25-75% but not in CNTF

Washington 98195-7234 and the §Nencki Institute and the ¶International Institute of Molecular and Cellular Biology, Ksiecia Trojdena 4, Poland Inhibition of glycogen synthase kinase-3␤ (GSK3␤) is one of the mechanisms by which phosphatidylinositol 3-kinase (PI3K) activation protects neurons from apoptosis. Here, we report that inhibition of ERK1/2 increased the basal activity of GSK3␤ in cortical neurons and that both ERK1/2 and PI3K were required for brainderived neurotrophic factor (BDNF) suppression of GSK3␤ activity. Moreover, cortical neuron apoptosis induced by expression of recombinant GSK3␤ was inhibited by coexpression of constitutively active MKK1 or PI3K. Activation of both endogenous ERK1/2 and PI3K signaling pathways was required for BDNF to block apoptosis induced by expression of recombinant GSK3␤. Furthermore, cortical neuron apoptosis induced by LY294002-mediated activation of endogenous GSK3␤ was blocked by expression of constitutively active MKK1 or by BDNF via stimulation of the endogenous ERK1/2 pathway. Although both PI3K and ERK1/2 inhibited GSK3␤ activity, neither had an effect on GSK3␤ phosphorylation at Tyr-216. Interestingly, PI3K (but not ERK1/2) induced the inhibitory phosphorylation of GSK3␤ at Ser-9. Significantly, coexpression of constitutively active MKK1 (but not PI3K) still suppressed neuronal apoptosis induced by expression of the GSK3␤(S9A) mutant. These data suggest that activation of the ERK1/2 signaling pathway protects neurons from GSK3␤-induced apoptosis and that inhibition of GSK3␤ may be a common target by which ERK1/2 and PI3K protect neurons from apoptosis. Furthermore, ERK1/2 inhibits GSK3␤ activity via a novel mechanism that is independent of Ser-9 phosphorylation and likely does not involve Tyr-216 phosphorylation.

N-methyl-D-aspartate receptors (NMDARs) are critical for neuronal plasticity and survival, whereas their excessive activation produces excitotoxicity and may accelerate neurodegeneration. Here, we report that stimulation of NMDARs in cultured rat hippocampal or cortical neurons and in the adult mouse brain in vivo disinhibited glycogen synthase kinase 3␤ (GSK3␤) by protein phosphatase 1(PP1)-mediated dephosphorylation of GSK3␤ at the serine 9 residue. NMDA-triggered GSK3␤ activation was mediated by NMDAR that contained the NR2B subunit. Interestingly, GSK3␤ inhibition reduced inhibitory phosphorylation of the PP1 inhibitor 2 (I2) and attenuated serine 9 dephosphorylation by PP1. These data suggest existence of a feedback loop between GSK3␤ and PP1 that results in amplification of PP1 activation by GSK3␤. In addition, GSK3␤ inhibition decreased PP1-mediated dephosphorylation of the cAMPresponse element-binding protein (CREB) at the serine 133 residue in NMDA-stimulated neurons. Conversely, overexpression of GSK3␤ abolished non-NR2B-mediated activation of CRE-driven transcription. These data suggest that cross-talk between GSK3␤ and PP1 contributes to NR2B NMDAR-induced inhibition of CREB signaling by non-NR2B NMDAR. The excessive activation of NR2B-PP1-GSK3␤-PP1 circuitry may contribute to the deficits of CREBdependent neuronal plasticity in neurodegenerative diseases.

During cortical development, when NR2B subunit is the major component of the NMDA glutamate receptors (NMDARs), moderate NMDAR activity supports neuronal survival at least in part by regulating gene transcription. We report that, in cultured cortical neurons from newborn rats, the NMDARs activated the calcium-responsive transcription regulator nuclear factor of activated T cells (NFAT). Moreover, in developing rat cortex, the NFAT isoforms c3 and c4 (NFATc3 and NFATc4) were expressed at relatively higher levels at postnatal day 7 (P7) than P21, overlapping with the period of NMDAR-dependent survival. In cultured cortical neurons, NFATc3 and NFATc4 were regulated at least in part by the NR2B NMDAR. Conversely, knockdown of NFATc4 but not NFATc3 induced cortical neuron apoptosis. Likewise, NFATc4 inhibition prevented antiapoptotic neuroprotection in response to exogenous NMDA. Expression of the brain-derived neurotrophic factor (BDNF) was reduced by NFATc4 inhibition. NFATc4 regulated transcription by the NMDARresponsive bdnf promoter IV. In addition, NMDAR blockers including NR2B-selective once reduced BDNF expression in P7 cortex and cultured cortical neurons. Finally, exogenous BDNF rescued from the proapoptotic effects of NFATc4 inhibition. These results identify bdnf as one of the target genes for the antiapoptotic signaling by NMDAR-NFATc4. Thus, the previously unrecognized NMDARNFATc4 -BDNF pathway contributes to the survival signaling network that supports cortical development.

Neurons are exposed to damaging stimuli that can trigger cell death and subsequently cause serious neurological disorders. Therefore, it is important to define defense mechanisms that can be activated in response to damage to reduce neuronal loss. Here we report that cisplatin (CPDD), a neurotoxic anticancer drug that damages DNA, triggered apoptosis and activated the extracellular signal-regulated kinase 1/2 (ERK1/2) pathway in cultured rat cortical neurons. Inhibition of ERK1/2 activation using either pharmacological inhibitors or a dominant-negative mutant of the ERK1/2 activator, mitogen-activated protein kinase kinase 1, increased the toxicity of CPDD. Interestingly, N-methyl-Daspartate (NMDA) receptor (NMDAR) antagonists reduced the ERK1/2 activation and exacerbated apoptosis in CPDD-treated neurons. Pre-treatment with CPDD increased ERK1/2 activation triggered by exogenous NMDA, suggesting that CPDD augmented NMDAR responsiveness. CPDD-enhanced response of NMDAR and CPDD-mediated ERK1/2 activation were both decreased by inhibition of poly(ADP-ribose) polymerase (PARP). Interestingly, PARP activation did not produce ATP depletion, suggesting involvement of a non-energetic mechanism in NMDAR regulation by PARP. Finally, CPDD toxicity was reduced by brain-derived neurotrophic factor, and this protection required ERK1/2. In summary, our data identify a novel compensatory circuit in central nervous system neurons that couples the DNA injury, through PARP and NMDAR, to the defensive ERK1/2 activation.

To identify the intracellular signaling pathways that mediate the pro-survival activity of NMDA receptors (NMDARs), we studied effects of exogenous NMDA on cultured rat cortical and hippocampal neurons that were treated with a phosphatidylinositol-3-kinase (PI3K) inhibitor, LY294002. NMDA at 5 or 10 lM protected against LY294002-induced apoptosis, suggesting NMDAR-mediated activation of a survival signaling pathway that is PI3K-independent. NR2B-specific NMDAR blockers antagonized anti-apoptotic effects of NMDA, indicating a critical role of NR2B NMDARs in the neuroprotection. NMDA at 10 lM suppressed LY294002-induced activation of a pro-apoptotic kinase, glycogen synthase kinase 3b (GSK3b). GSK3b activation by LY294002 was associated with decreased levels of inhibitory GSK3b phosphorylation at the Ser9 residue. However, NMDA did not prevent the LY294002-mediated decline of phospho-Ser9 levels. In addition, NMDA inhibited cortical neuron apoptosis induced by the overexpression of either wild type (wt) or Ser9Ala mutant form of GSK3b, suggesting that NMDA suppressed GSK3b in a Ser9-independent manner. Finally, inhibition of NR2B NMDARs reduced the NMDA protection against overexpression of GSK3bwt. These data indicate that moderate stimulation of NR2B NMDAR protects against inhibition of PI3K by a Ser9-independent inhibition of the pro-apoptotic activity of GSK3b. Hence, the activation of NR2B and the Ser9-independent inhibition of GSK3b are two newly identified elements of the signaling network that mediates the pro-survival effects of NMDA. Keywords: cell death, extrasynaptic, glutamate receptors, N-methyl-D-aspartate receptor subunit 2B, survival. During development, neuronal survival is supported by multiple signals, including peptide trophic factors and neurotransmitters (Oppenheim 1991;Snider 1994;Verhage et al. 2000). In the central nervous system (CNS), an important pro-survival signal is delivered to neurons by the excitatory neurotransmitter, glutamate. N-Methyl-D-aspartate (NMDA) subtype of ionotropic glutamate receptors (NMDAR) is the principal mediator of glutamate trophic activity. The role of NMDAR in survival support was initially reported in cultured cerebellar granule neurons (CGN). Exogenous NMDA prevented the reduction of CGN survival that was triggered by exposure to suboptimal KCl concentrations (Balazs et al. 1988a;Balazs et al. 1988b). NMDA suppressed CGN apoptosis that followed placement in low KCl media (Yan et al. 1994

Active CREB (cAMP responsive element-binding protein) transcription factor is crucial for neuronal survival. Several members of the CREM/ICER (cAMP responsive element modulator/inducible cAMP early repressor) protein family may act as endogenous CREB antagonists. However, their involvement in a process of programmed cell death remains unexplored. Here we report that ICER may play such a role in neuronal apoptosis because it is upregulated in apoptotic neurons in vitro, and overexpression of ICER, delivered in adenoviral vector, evokes programmed cell death of three different kinds of cultured neurons, namely those derived from hippocampal dentate gyrus, cerebral cortex, and superior cervical ganglion. Reporter gene assay with a promoter containing a CREB-responsive sequence revealed a decrease in both basal and induced CRE-dependent gene expression in neurons overexpressing ICER. Finally, the level of expression of the anti-apoptotic protein Bcl-2, a well known CREB target, was markedly diminished in ICER-treated neurons. We suggest that the naturally occurring CREB functional antagonist ICER may have a specific function in programmed cell death of neurons, probably by silencing the expression of anti-apoptotic genes.

Nuclear factor erythroid 2-related factor 2 (Nrf2), the transcriptional master regulator of the stress-induced antioxidant response, plays a key role in neuronal resistance to oxidative stress and glutamateinduced excitotoxicity. Nrf2-mediated neuroprotection is primarily conferred by astrocytes both in vitro and in vivo, but little is known about physiologic signals that regulate neuronal and astrocytic Nrf2 signaling. Here, we report that activity of the Nrf2 pathway in the brain is fine-tuned through a regulatory loop between neurons and astrocytes: elevated neuronal activity leads to secretion of glutamate and other soluble factors, which activate the astrocytic Nrf2 pathway through a signaling cascade that involves group I metabotropic glutamate receptors and intracellular Ca 2+ . Therefore, regulation of endogenous antioxidant signaling is one of the functions of the neuron-astrocyte tripartite synapse; by matching the astrocyte neuroprotective capacity to the degree of activity in adjacent neuronal synapses, this regulatory mechanism may limit the physiologic costs associated with Nrf2 activation.