Mild in vitro trauma induces rapid Glur2 endocytosis, robustly augments calcium permeability and enhances susceptibility to secondary excitotoxic insult in cultured Purkinje cells

Abstract

Mild brain trauma results in a wide range of neurological symptoms that are not easily explained by the primary pathology. Purkinje neurons of the cerebellum are selectively vulnerable to brain trauma, including indirect remote trauma to the forebrain. This vulnerability manifests itself as a selective and delayed cell loss, for which the underlying mechanisms are poorly understood. Alterations to the surface expression of calcium impermeable AMPA receptors (GluR2-containing) may mediate post-traumatic calcium overload, and initiate biochemical cascades that ultimately cause progressive cell death. Our current study examined this hypothesis using an in vitro model of mild Purkinje trauma, delivered by an elastic stretch at 2.5-2.9 pounds per square inch (psi). This mild trauma alone did not increase cell loss as measured by propidium iodide (PI) uptake (at 20 h) compared to uninjured controls. However, there was a marked increase in cell loss, when cells following mild trauma, were exposed to 10 microM AMPA for 1 h compared to either mild trauma or AMPA exposure alone. Mild injury rendered Purkinje neurons significantly more permeable to AMPA-stimulated (4 microM) calcium influx at 15 min post-injury, including a sustained calcium plateau. This effect was eliminated by inhibiting protein kinase C-dependent GluR2 endocytosis with 2 microM Go6976 or blocking the calcium pore of GluR1/3 containing AMPARs with 500 nM 1-naphthylacetyl spermine (Naspm). Nifedipine (2 microM) eliminated the calcium plateau following mild injury but not the initial spike of Ca2+ increase. These results suggest that mild injuries resulted in a rapid AMPA receptor subtype switch (GluR2 was replaced by GluR1/3), which in turn resulted in an enhanced Ca2+ permeability. We further confirmed this by immunocytochemistry. Dendritic GluR2 co-localization with the pre-synaptic marker synaptophysin was markedly down-regulated at 15 min following mild stretch (P < 0.01), indicative of a rapid decrease in the synaptic expression of receptors containing this subunit. Carboxyfluorescence (CBF) assays revealed that mild stretch did not alter membrane integrity. Finally, we demonstrated that the combination of 500 nM Naspm and 5 nM Go6976 conferred a powerful neuroprotective effect on Purkinje cells by effectively eliminating the effects of mild stretch combined with AMPA in 95% of cells. These results represent a newly described mechanism rendering neurons susceptible to secondary injuries following trauma. Prevention of GluR2 endocytosis may be critical in the development of pharmacotherapies aimed at mild, seemingly inconsequential trauma, to avoid ensuing secondary damage.