Plasma membrane calcium ATPases (PMCAs) are essential ion pumps, which extrude calcium from cells. Although two isoforms, PMCA2 and PMCA3, are primarily expressed in neurons, their contribution to neuronal pathology is not well defined. Earlier studies in our laboratory indicated that the expression of PMCA2 is significantly decreased in spinal cord neurons at onset of experimental autoimmune encephalomyelitis (EAE)^1,2. EAE is an animal model of Multiple Sclerosis (MS), an inflammatory, demyelinating and neurodegenerative disease of the central nervous system. The aforementioned finding was of particular interest as it raised the possibility of an involvement of PMCA2 in neuronal injury and suggested a putative novel mechanism underlying cellular dysfunction in EAE. Subsequent studies established a causal relationship between inhibition of PMCA activity and neuronal pathology. Treatment of spinal cord neuronal cultures with carboxyeosin (CE), a PMCA inhibitor, delayed the clearance of depolarization induced calcium transients which was followed by abnormal expression of non-phosphorylated neurofilament H (NFH), neuritic beading, an increase in the number of activated caspase-3 positive cells and decreased survival³. To further unravel the mechanisms leading to neuronal injury in response to inhibition of PMCA, we evaluated activation of calpain by quantifying the breakdown of its substrate α-spectrin. We focused on calpain because it is a calcium dependent protease which has been implicated in MS pathology. There was a two-fold increase in the ~ 145 KDa α-spectrin breakdown product in cultures treated with CE as compared to controls. The augment in the α-spectrin degradation product preceded induction of caspase-3 and neuronal loss. The triggers that modulate PMCA2 levels in spinal cord neurons are not well defined. As activated microglia are abundant in the spinal cord during EAE and have been implicated in neuronal injury, we determined whether these cells secrete soluble factors that suppress PMCA2 expression. We found a significant but transient decrease in PMCA2 protein levels in neurons co-cultured with microglia. The reduction in PMCA2 correlated with an increase in the number of neurons expressing non-phosphorylated NFH, a change that persisted despite the return of PMCA2 levels to control values. Additional investigations were undertaken to determine whether factors which are believed to be present in the inflammatory milieu during EAE, modulate PMCA2 levels. One of these agents, glutamate, is released by activated microglia and damaged neurons. It has been reported that glutamate mediates EAE pathology by acting via the AMPA/kainate receptors^3,4. Treatment of pure spinal cord neuronal cultures with 4 µM kainic acid (KA) for 36 hours decreased PMCA2 protein levels without affecting cell viability. We concluded that continuous exposure to KA for extended periods can suppress PMCA2 protein expression even when the concentration is low. Higher KA concentrations (20 µM) decreased PMCA2 levels within 12 hours without inducing cell death. A further reduction in PMCA2 was observed after 24 hours but this was in part due to neuronal loss. To further analyze the role of PMCA2 in spinal cord neurons, in vivo, the consequences of a null mutation in the PMCA2 gene were examined in heterozygous and knockout mice. Our earlier investigations had shown a significant reduction in the number of motor neurons in the lumbar spinal cord of PMCA2-null mice. Motor unit number estimation (MUNE), a non-invasive, electrophysiological method that quantifies the approximate number of motor neurons innervating a single muscle or a small group of muscles, indicated a 60% decrease in the knockout as compared to the wild type mice. Although heterozygotes exhibit a phenotype which roughly appears similar to that of the wild type, a 30% reduction in MUNE was observed in these mice as compared to the wild type littermates. In contrast, sensory function was not affected in either PMCA2^-/- or PMCA2^+/- mice. The performance of heterozygotes in the rotarod test was significantly inferior to that of the wild type. Thus, even partial reductions in PMCA2 may be sufficient to induce motor deficits. In sum, PMCAs and in particular PMCA2 appear to play a critical role in the integrity and function of spinal cord neurons. In pathological conditions involving inflammation, a number of triggers, including those produced by microglia, may suppress PMCA activity or expression, leading to neuronal pathology and, in some cases, to cell death.