Roscovitine Triggers Excitotoxicity in Cultured Granule Neurons by Enhancing Glutamate Release

Abstract

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 ϩ -based action potentials (tetrodotoxin inhibits, (Ϯ)-2-amino-4-phosphonobutyric acid enhances), P/Q-type VDCC currents (-agatoxin-IVA and -conotoxin-MVIIC inhibit, but not -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 ␣-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 Mg 2ϩ block of NMDA receptors, resulting in excitotoxic Ca 2ϩ 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.Glutamate-mediated excitotoxicity is extensively studied in cell cultures and animals to model key events underlying neuronal loss in human neurodegeneration (Aarts and Tymianski, 2004). Numerous experimental paradigms support the theory that sustained NMDA receptor activation leads to toxic intracellular Ca 2ϩ deregulation and death by necrosis and/or apoptosis. Therefore, NMDA receptor antagonists afford protection and provide a rationale for their use in clinical trials in humans. Unfortunately, these trials were not correspondingly successful, prompting investigators to re-evaluate the predictive value of existing models and stimulating renewed efforts to identify novel therapeutic targets.The "source-specificity hypothesis" of excitotoxicity posits that discrete Ca 2ϩ microdomains, as opposed to alteration...

Cdk5 Modulation of Mitogen-activated Protein Kinase Signaling Regulates Neuronal Survival

“…Can the release of channel activity by roscovitine inhibition of Cdk5 elicit sustained Erk1/2 activity and cell death? Cerebellar granule neurons treated with roscovitine become either apoptotic or necrotic, the latter correlated with an excitotoxicity at the synapse involving functionally linked ␣-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid and N-methyl-d-aspartate (NMDA) receptors also coupled to Ca 2ϩ influx via P/Q-type VDCCs (Monaco and Vallano, 2005). How does this relate to sustained activated Erk1/2 and neuronal cell death?…”

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

“…1984), including mature CGNs (Losi et al. 2002; Monaco and Vallano 2005). However, synapses are not present in immature CGNs (2–4 DIV).…”

“…1984), and this functional linkage is demonstrable in synaptically‐connected CGNs (Losi et al. 2002; Monaco and Vallano 2005). However, AMPARs may not provide such a depolarizing stimulus prior to synapse formation.…”

Gabapentin is neuroprotective through glutamate receptor-independent mechanisms in staurosporine-induced apoptosis of cultured rat cerebellar neurons

“…Moreover, from other studies it could have been predicted that blockade of glutamate receptors in CGN cultured in physiological medium would not result in neuroprotection [36,38].…”

“…Rat CGN cultures represent a well-described and valuable model to the study of the mechanisms of neurodegeneration and test molecules for neuroprotection [22]. These cultures contain glutamatergic cells which express ionotropic glutamate and GABA A receptors [31] and are characterized by spontaneous synapse formation and electrical activity when CGN are cultured in physiological medium [36]. In CGN cultures a proportion of naturally occurring apoptosis is documented 3-5 days after plating, which can be rescued by elevated KCl concentration through the activation of voltage-gated Ca 2+ channels and depolarization [32].…”