Microglia in Glia-Neuron Co-cultures Exhibit Robust Phagocytic Activity Without Concomitant Inflammation or Cytotoxicity.

A simple method to co-culture granule neurons and glia from a single brain region is described, and microglia activation profiles are assessed in response to naturally occurring neuronal apoptosis, excitotoxin-induced neuronal death, and lipopolysaccharide (LPS) addition. Using neonatal rat cerebellar cortex as a tissue source, glial proliferation is regulated by omission or addition of the mitotic inhibitor cytosine arabinoside (AraC). After 7-8 days in vitro, microglia in AraC(-) cultures are abundant and activated based on their amoeboid morphology, expressions of ED1 and Iba1, and ability to phagocytose polystyrene beads and the majority of neurons undergoing spontaneous apoptosis. Microglia and phagocytic activities are sparse in AraC(+) cultures. Following exposure to excitotoxic kainate concentrations, microglia in AraC(-) cultures phagocytose most dead neurons within 24 h without exacerbating neuronal loss or mounting a strong or sustained inflammatory response. LPS addition induces a robust inflammatory response, based on microglial expressions of TNF-α, COX-2 and iNOS proteins, and mRNAs, whereas these markers are essentially undetectable in control cultures. Thus, the functional effector state of microglia is primed for phagocytosis but not inflammation or cytotoxicity even after kainate exposure that triggers death in the majority of neurons. This model should prove useful in studying the progressive activation states of microglia and factors that promote their conversion to inflammatory and cytotoxic phenotypes.

Mild exercise reduces cerebral vasospasm after aneurysm subarachnoid hemorrhage: a retrospective clinical study and correlation with laboratory investigation.

BACKGROUND: Aneurysmal subarachnoid hemorrhage (SAH) is a leading cause of death and disability and is often complicated by cerebral vasospasm (CV). Conventional management to prevent CV includes bedrest; however, inactivity places the patient at risk for nonneurological complications. We investigated the effect of mild exercise after SAH in clinical and laboratory settings. METHODS: Clinical: Data from 80 patients with SAH were analyzed retrospectively. After aneurysms were secured, physical therapy was initiated as tolerated. CV and complications were compared by the timing of active physical therapy. Laboratory: 18 Rodents were divided into three groups: (1) control, (2) SAH without exercise, and (3) SAH plus mild exercise. On day 5, brainstems were removed and analyzed for the injury marker inducible nitric oxide synthase (iNOS). RESULTS: Clinical: Mild exercise before day 4 significantly lowered the incidence of symptomatic CV compared with the nonexercised group. There was no difference in the incidence of additional complications based upon exercise. Laboratory: Staining for iNOS was significantly higher in the SAH group than the control group, but there was no difference between exercised and nonexercised SAH groups, confirming that exercise did not promote neuronal injury. CONCLUSION: Early mobilization significantly reduced clinical CV. The relationship should be studied further in a prospective trial with defined exercise regimens.

Reduced blockade by extracellular Mg(2+) is permissive to NMDA receptor activation in cerebellar granule neurons that model a migratory phenotype.

NMDA receptors (NMDAR) contribute to neuronal development throughout the CNS. However, their mode(s) of activation preceding synaptic maturation is unclear, as they are not co-localized with alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate receptors (AMPARs) which normally provide sufficient depolarization to relieve voltage-dependent blockade by Mg(2+). We used cerebellar granule neurons (CGNs) cultured at a near-physiological KCl concentration to examine maturation-dependent changes in NMDAR responses. In contrast, most studies use KCl-supplemented medium to promote survival. At 2-4 days in vitro CGNs: (i) express developmental markers resembling the in vivo migratory phenotype; (ii) maintain a basal amount of calcium responsive element-binding protein phosphorylation that requires NMDARs and calcium/calmodulin-dependent kinases, but not AMPARs; (iii) exhibit NMDA-mediated Ca(2+) influx not effectively blocked by ambient Mg(2+) (0.75 mM) or AMPARs; (iv) maintain a more depolarized resting membrane potential and increased resistance compared to synaptically-connected CGNs. Moreover, migrating CGNs in explant cultures demonstrate NMDA-mediated Ca(2+) influx not effectively blocked by 0.75 mM Mg(2+), and NMDAR but not AMPAR antagonists slow migration. These data suggest the biophysical properties of immature CGNs render NMDARs less sensitive to Mg(2+) blockade, enhancing the likelihood of activation in the absence of AMPAR depolarization.

Depolarization and Ca(2+) down regulate CB1 receptors and CB1-mediated signaling in cerebellar granule neurons.

Presynaptic terminals of cerebellar granule neurons are primary targets of cannabinoids, which act through type 1 G alpha(i/o)-coupled cannabinoid receptors (CB1) to modulate glutamate release. To study CB1 signaling investigators use primary cultures of granule neurons, typically grown in medium supplemented with elevated KCl to improve long-term survival. Herein, we demonstrate that CB1 expression and signaling are perturbed under these conditions. Specifically, immunochemical and RT-PCR assays indicate that depolarizing growth conditions decrease CB1 protein, mRNA and CB1-mediated inhibition of adenylyl cyclase compared to cultures grown in physiologic medium containing 5mM KCl. Depolarization-dependent downregulation of CB1 mRNA, like survival, is attenuated by L-type VDCC antagonists but not the Na(+)-channel antagonist, tetrodotoxin. Comparison of oligonucleotide microarrays from cultures grown in 5mM versus 25 mM KCl confirms that depolarization reduces CB1 mRNA, but not mRNAs encoding several G-protein subunits or adenylyl cyclases. However, significant alterations in synaptic signaling proteins that likely lie downstream of CB1 are observed, including K(+) channels, alpha-neurexins, cAMP-GEFII, Munc13-3, secretogranin and synaptotagmin. These findings make a compelling argument to adopt cultures grown in 5mM KCl for future study of CB1 signaling in granule neurons. Further, they suggest that a depolarization and Ca(2+)-dependent signaling pathway represses CB1 gene transcription.

Transcriptional profiling of depolarization-dependent phenotypic alterations in primary cultures of developing granule neurons.

Rat cerebellar granule neurons cultured in medium supplemented with elevated KCl are extensively used as a model to examine the coupling between neural activity and Ca(2+)-dependent gene expression. Elevated (25 mM) KCl is believed to mimic endogenous neural activity because it promotes depolarization and Ca(+2)-dependent survival and some aspects of maturation. By comparison, at least half of the granule neurons grown in standard medium containing 5 mM KCl undergo apoptosis beginning approximately 4 days in vitro. However, accumulating evidence suggests that chronic depolarization induces phenotypic abnormalities whereas growth in chemically defined medium containing 5 mM KCl more closely resembles the constitutive phenotype. To examine this, oligonucleotide microarrays and RT-PCR of selected mRNAs were used to compare transcription profiles of cultures grown in 5 mM and 25 mM KCl. In some cases, N-methyl-D-aspartate (NMDA) which, like elevated KCl, promotes long-term survival was also tested. Robust changes in several gene groups were observed and indicated that growth in elevated KCl: induces expression of mRNAs that are not normally observed; represses expression of mRNAs that should be present; maintains expression of mRNAs that are markers of immature neurons. Supplementation of the growth medium with NMDA instead of elevated KCl produces similar abnormalities. Altogether, these data indicate that growth in 5 mM KCl more closely mimics survival and maturation of granule neurons in vivo and should therefore be adopted in future studies.

Role of protein kinases in neurodegenerative disease: cyclin-dependent kinases in Alzheimer's disease.

Cyclin-dependent kinases (Cdks) are serine/threonine kinases that regulate a number of cellular processes including the cell cycle and neuronal differentiation. Accumulating evidence indicates that two distinct Cdk pathways may have a role in the neuronal loss that is responsible for Alzheimer's disease. One pathway involves the aberrant reactivation of the cell cycle, a process believed to be incompatible with neuronal function. A second involves dysregulation of Cdk5, a member of this kinase family with no known cell cycle functions, but prominently expressed in postmitotic neurons. Reports supporting the involvement of both pathways are plentiful, but the story is not yet complete. In particular, difficulties incorporating the extended latency of AD into model approaches persist. Despite this, the theory that Cdks are involved in the pathogenesis of AD has generated considerable interest.

Roscovitine, olomoucine, purvalanol: inducers of apoptosis in maturing cerebellar granule neurons.

Cyclin-dependent kinases (CDKs) mediate proliferation and neuronal development, while aberrant CDK activity is associated with cancer and neurodegeneration. Consequently, pharmacologic inhibitors, such as 2,6,9-trisubstituted purines, which potently inhibit CDKs 1, 2, and 5, were developed to combat these pathologies. One agent, R-roscovitine (CYC202), has advanced to clinical trials as a potential cancer therapy. In primary neuronal cultures, these agents have been used to delineate the physiologic and pathologic functions of CDKs, and associated signaling pathways. Herein we demonstrate that three 2,6,9-trisubstituted purines: olomoucine, roscovitine, and purvalanol, used at concentrations ascribed by others to potently inhibit CDKs 1, 2, and 5, are powerful triggers of death in maturing cerebellar granule neurons, assessed by loss of mitochondrial reductive capacity and differential staining with fluorescent indicators of living/dead neurons. Based on several criteria, including delayed time course and establishment of an irreversible commitment point of death, pyknotic cell and nuclear morphology, and caspase-3 cleavage, the death process is apoptotic. However, pharmacological and biochemical data indicate that apoptosis is independent of CDK 1, 2, or 5 inhibition. This is based on the pattern of changes in c-jun mRNA, c-Jun protein, and Ca(2+)/cAMP response element binding protein (CREB) phosphorylation, and also, the ineffectiveness of structurally distinct CDK 1, 2, and 5 inhibitors butyrolactone-1 and PNU112445A to induce apoptosis. Collectively, our results, and those of others, indicate that the CDK regulation of transcription (CDKs 7 and 9) should be examined as a target of these agents, and as an indirect mediator of neuronal fate.

Roscovitine triggers excitotoxicity in cultured granule neurons by enhancing glutamate release.

Cerebellar granule neurons are highly susceptible to injury in vivo and in vitro, and primary cultures are widely used to characterize relevant receptors and signaling pathways. However, there are problems associated with their use. In particular, cultures are typically grown in medium supplemented with elevated KCl levels because it improves survival, but accumulating evidence indicates that this causes profound neuroadaptations. For example, growth in elevated KCl levels renders neurons electrically silent. Thus, they cannot be used to examine excitotoxicity of synaptic origins. On the other hand, cultures grown in physiological medium are rarely studied because a proportion undergoes apoptosis. Herein, we provide evidence that mature neurons cultured in physiological KCl develop spontaneous action potentials that support survival through N-methyl-D-aspartate (NMDA) receptor-mediated mechanisms. Furthermore, the cdk inhibitor roscovitine enhances the coupling between tetrodotoxin-sensitive action potentials and P/Q-type voltage-dependent calcium channels (VDCCs), thereby converting this survival program to excitotoxicity of synaptic origin. Therefore, roscovitine-triggered necrosis requires spontaneous Na+-based action potentials (tetrodotoxin inhibits, (+/-)-2-amino-4-phosphonobutyric acid enhances), P/Q-type VDCC currents (omega-agatoxin-IVA and omega-conotoxin-MVIIC inhibit, but not omega-conotoxin-GVIA), intact vesicle fusion processes (tetanus toxin inhibits), and transmitter-filled vesicles (concanamycin and bafilomycin inhibit). From a postsynaptic standpoint, roscovitine-mediated excitotoxicity requires the functionally linked activation of alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionate/kainate (AMPA/KA) and NMDA receptors, which is consistent with evidence that activated AMPA/KA receptors relieve the voltage-dependent Mg2+ block of NMDA receptors, resulting in excitotoxic Ca2+ influx. In the end, NMDA receptor-linked pathways transduce excitotoxicity. On the other hand, L-type VDCC blockers are not protective. Further characterization of this new model is expected to provide important insights about excitotoxicity of synaptic origins and about roscovitine as a selective modulator of this process.

Nuclear calpain regulates Ca2+-dependent signaling via proteolysis of nuclear Ca2+/calmodulin-dependent protein kinase type IV in cultured neurons.

Accumulating evidence indicates that calpains can reside in or translocate to the cell nucleus, but their functions in this compartment remain poorly understood. Dissociated cultures of cerebellar granule cells (GCs) demonstrate improved long-term survival when their growth medium is supplemented with depolarizing agents that stimulate Ca(2+) influx and activate calmodulin-dependent signaling cascades, notably 20 mm KCl. We previously observed Ca(2+)-dependent down-regulation of Ca(2+)/calmodulin-dependent protein kinase (CaMK) type IV, which was attenuated by calpain inhibitors, in GCs supplemented with 20 mm KCl (Tremper-Wells, B., Mathur, A., Beaman-Hall, C. M., and Vallano, M. L. (2002) J. Neurochem. 81, 314-324). CaMKIV is highly enriched in the nucleus and thought to be critical for improved survival. Here, we demonstrate by immunolocalization/confocal microscopy and subcellular fractionation that the regulatory and catalytic subunits of m-calpain are enriched in GC nuclei, including GCs grown in medium containing 5 mm KCl. Calpain-mediated proteolysis of CaMKIV is selective, as several other nuclear and non-nuclear calpain substrates were not degraded under chronic depolarizing culture conditions. Depolarization and Ca(2+)-dependent down-regulation of CaMKIV were associated with significant alterations in other components of the Ca(2+)-CaMKIV signaling cascade: the ratio of phosphorylated to total cAMP response element-binding protein (a downstream CaMKIV substrate) was reduced by approximately 10-fold, and the amount of CaMK kinase (an upstream activator of CaMKIV) protein and mRNA was significantly reduced. We hypothesize that calpain-mediated CaMKIV proteolysis is an autoregulatory feedback response to sustained activation of a Ca(2+)-CaMKIV signaling pathway, resulting from growth of cultures in medium containing 25 mm KCl. This study establishes nuclear m-calpain as a regulator of CaMKIV and associated signaling molecules under conditions of sustained Ca(2+) influx.

Trophic agents that prevent neuronal apoptosis activate calpain and down-regulate CaMKIV.

CaMKIV is enriched in neuronal nuclei and mediates Ca2+-dependent survival via transcription factor phosphorylation. Cultured cerebellar granule neurons were used to examine whether distinct modes of Ca2+ signaling differentially modulate CaMKIV expression and function. For long-term survival, these neurons require 25 mm KCl or NMDA, which stimulates Ca2+ entry through voltage-sensitive Ca2+ channels or NMDA receptors (NRs). Lower levels of Ca2+ entry through NRs support survival of a neuronal subpopulation grown in 5 mm KCl media. Several effects were demonstrated: (i) sustained exposure to 25 mM KCl or 140 microM NMDA produced CaMKIV down-regulation, compared to 5 mM KCl cultures; (ii) CaMKIV down-regulation was attenuated by nifedipine, APV and CaM kinase inhibitors, indicating that it is Ca2+ dependent and reversible; (iii) down-regulation was both selective for nuclear substrates and calpain-mediated; (iv) proteolysis was exacerbated by leptomycin B, a nuclear export inhibitor. Although CaMKIV proteolysis by trophic agents seems paradoxical in light of evidence supporting its critical role in survival, the CaMKIV/CREB signal transduction pathway was preserved, as assessed by CaM kinase-mediated CREB phosphorylation, and the ability of CaM kinase inhibitors to interfere with KCl-mediated survival. We hypothesize that limited calpain-mediated proteolysis of CaMKIV is a negative feedback response to the sustained activation of a Ca2+ and CaMKIV signaling pathway by these agents.