Mary Lou Vallano

Glia Modulate NMDA‐Mediated Signaling in Primary Cultures of Cerebellar Granule Cells

Abstract: Excessive activation of N‐methyl‐d‐aspartate (NMDA) receptor channels (NRs) is a major cause of neuronal death associated with stroke and ischemia. Cerebellar granule neurons in vivo, but not in culture, are relatively resistant to toxicity, possibly owing to protective effects of glia. To evaluate whether NR‐mediated signaling is modulated when developing neurons are cocultured with glia, the neurotoxic responses of rat cerebellar granule cells to applied NMDA or glutamate were compared in astrocyte‐rich and astrocyte‐poor cultures. In astrocyte‐poor cultures, significant neurotoxicity was observed in response to NMDA or glutamate and was inhibited by an NR antagonist. Astrocyte‐rich neuronal cultures demonstrated three significant differences, compared with astrocyte‐poor cultures: (a) Neuronal viability was increased; (b) glutamate‐mediated neurotoxicity was decreased, consistent with the presence of a sodium‐coupled glutamate transport system in astrocytes; and (c) NMDA‐ but not kainate‐mediated neurotoxicity was decreased, in a manner that depended on the relative abundance of glia in the culture. Because glia do not express NRs or an NMDA transport system, the mechanism of protection is distinct from that observed in response to glutamate. No differences in NR subunit composition (evaluated using RT‐PCR assays for NR1 and NR2 subunit mRNAs), NR sensitivity (evaluated by measuring NR‐mediated changes in intracellular Ca2+ levels), or glycine availability as a coagonist (evaluated in the presence and absence of exogenous glycine) were observed between astrocyte‐rich and astrocyte‐poor cultures, suggesting that glia do not directly modulate NR composition or function. Nordihydroguaiaretic acid, a lipoxygenase inhibitor, blocked NMDA‐mediated toxicity in astrocyte‐poor cultures, raising the possibility that glia effectively reduce the accumulation of highly diffusible and toxic arachidonic acid metabolites in neurons. Alternatively, glia may alter neuronal development/phenotype in a manner that selectively reduces susceptibility to NR‐mediated toxicity.

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 Alzheimers 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.

Ca2+ and pH modulate alternative splicing of exon 5 in NMDA receptor subunit 1.

RT-PCR and intracellular Ca2+ measurements were used to identify factors that modulate alternative splicing of exon 5 in the NMDA receptor transcript encoding NR1, in cultured cerebellar granule neurons. Although cells grown in media containing 5 mM KCl demonstrate compromised survival, they show the predicted developmental transition from NR1a (-exon 5) to NR1b (+exon 5) mRNA expression. This transition was blocked under culture conditions that promote survival; inclusion or exclusion of exon 5 is a reversible process that is sensitive to alterations in Ca2+ and pH. We conclude that alternative splicing of NR1 pre-mRNA transcripts may be regulated by developmental cues that modulate the degree of glutamate receptor activation.

2,2', 3,3', 4,4'-Hexahydroxy-1,1'-biphenyl-6,6'-dimethanol dimethyl ether (HBDDE)-induced neuronal apoptosis independent of classical protein kinase C α or γ inhibition

Abstract Protein kinase C (PKC) isozymes constitute a family of at least 12 structurally related serine–threonine kinases that are differentially regulated and localized, and are presumed to mediate distinct intracellular functions. To explore their roles in intact cells, investigators are developing cell-permeable, isoform-selective inhibitors. 2,2′,3,3′,4,4′-Hexahydroxy-1,1′-biphenyl-6,6′-dimethanol dimethyl ether (HBDDE) is reported to be a selective inhibitor of PKC α and γ with ic 50 values of 43 and 50 μM, respectively, using an in vitro assay. However, data examining the potency and selectivity of HBDDE in intact cells are lacking. Employing rodent cerebellar granule neurons as a model system, we investigated the effects of HBDDE using cell survival as a functional end-point. HBDDE induced an apoptotic form of cell death that was dependent upon protein synthesis and included activation of a terminal executioner of apoptosis, caspase 3. The concentration of HBDDE required for half-maximal cell death was less than 10 μM (∼5-fold less than the reported ic 50 values for PKC α and γ in vitro ). Furthermore, HBDDE induced apoptosis even after phorbol-ester-mediated down-regulation of PKC α and γ, indicating that this effect is independent of these isoforms. Consistent with this, 2-[1-(3-dimethylaminopropyl) indol-3-yl]-3-(indol-3-yl)-maleimide (GF 109203X), a general inhibitor of all classical and some novel PKCs, did not interfere with survival. Thus, HBDDE should not be used as an isoform-selective inhibitor of PKC α or γ in intact cells. Nevertheless, identification of its target in granule neurons will provide valuable information about survival pathways.

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.

Structural properties of the NMDA receptor and the design of neuroprotective therapies.

NMDA receptors are linked to neuronal loss in stroke and neurodegeneration because their activation can trigger excitotoxic Ca(2+) dysregulation. Accordingly, NMDA receptor antagonists are neuroprotective, providing a rationale for their clinical application. However, side effects often outweigh benefits. Herein we highlight structural properties in receptors that are used in drug development.

Reduced blockade by extracellular Mg2+ is permissive to NMDA receptor activation in cerebellar granule neurons that model a migratory phenotype

J. Neurochem. (2010) 114, 191–202.

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.