Demyelination is considered to be the principal cause of functional deficits in multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE). However, recent reports indicate that neuronal and axonal pathology are important disease determinants, even at the earliest clinical stages. The molecular events leading to neuronal/axonal injury remain elusive. The overall goal of our studies is to define the molecular mechanisms underlying neuronal and axonal damage during EAE. Our investigations indicated that transcript and protein levels of plasma membrane calcium ATPase 2 (PMCA2), an ion pump expressed exclusively in gray matter and an essential component of the Ca2+ extrusion machinery in neurons, were dramatically decreased in the lumbar spinal cord (SC) of the Lewis rat coincident with the onset of clinical symptoms during acute EAE. Moreover, we found that kainic acid significantly reduces PMCA2 expression in SC slice cultures. Glutamate excitotoxicity has recently been implicated in neuronal/axonal damage during EAE. Activated microglia/macrophages are a potent source of glutamate and other neurotoxins in MS. In view of our findings and the aforementioned studies, we hypothesize that reductions in neuronal PMCA2 levels and activity lead to Ca2+ overload which, in turn, induces neuronal/axonal damage during EAE. We also postulate that PMCA2 levels are modulated by microglial agents, including glutamate acting through AMPA/kainate receptors.
Our aims are to assess 1) neuronal dysfunction, axonal injury and susceptibility to EAE in a mouse with a functionally null PMCA2 mutation 2) the regulation of PMCA2 levels by glutamate acting through AMPA/kainate receptors 3) the role of activated microglia in axonal damage and in the regulation of neuronal PMCA2 and Ca2+ levels. Neuronal dysfunction in the mutant mouse will be investigated by evaluating intracellular calcium levels utilizing ratiometric calcium imaging on SC slices and by delineating the differential gene expression profiles in the SC. Axonal damage will be analyzed by quantifying dephosphorylated neurofilament H (NH), amyloid precursor protein (APP) and terminal axonal ovoids. The effects of endogenous glutamate on PMCA2 levels will be assessed by treatment of diseased rats with AMPA/kainate antagonists. SC microglia-neuron co-cultures will be used to determine the effects of microglial signals on neuronal PMCA2 expression, Ca2+ levels and axonal damage. Our long-term goal is to define the critical determinants of neuronal damage during MS in order to identify therapeutic targets that can attenuate disease severity and prevent progression.
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