title: Sequestration of nNOS in neurofilamentous aggregate bearing neurons in vitro leads to enhanced NMDA-mediated calcium influx.
abstract: The significance of copper/zinc superoxide dismutase (SOD1) and neuronal nitric oxide synthase (nNOS) co-localization to neurofilamentous (NF) aggregates in amyotrophic lateral sclerosis (ALS) is unknown. In this study, we have used dissociated motor neurons from either C57BL/6 or mice that over-express the human low molecular weight neurofilament protein (hNFL+/+) to examine the relationship between NF aggregate formation, SOD1 and nNOS co-localization, and the regulation of NMDA-mediated calcium influx in vitro. The intracellular distribution of NF aggregates, SOD1 and nNOS was examined by confocal microscopy and NMDA-induced alterations in intracellular calcium levels using either Oregon green fluorescence or FURA-2 photometric imaging. Cell death was assessed using an antibody to activated caspase-3. C57 Bl/6 motor neurons expressed nNOS in a punctate manner, whereas SOD1 was distributed homogeneously throughout the cytosol. In contrast, hNFL+/+ motor neurons demonstrated co-localization of SOD1 and nNOS by day 9 post-plating, preceding the formation of NF aggregates. Both proteins co-localized to NF aggregates once formed. With NMDA stimulation, aggregate-bearing hNFL+/+ motor neurons demonstrated significant increases in intracellular calcium, whereas only a minimal alteration in intracellular calcium was observed in C57 Bl/6 neurons. Following stimulation with 100 microM NMDA, 75.5+/-5.5% of hNFL+/+ neurons became apoptotic, whereas only 16.3+/-5.3% of C57 Bl/6 were. These observations suggest that the presence of NF aggregates results in a failure of regulation of NMDA-mediated calcium influx, and that this occurs due to the sequestration of nNOS to the NF aggregate, preventing its down-regulation of the NMDA receptor.
authors.affiliation: Robarts Research Institute and Department of Pathology, The University of Western Ontario, London, Ontario, Canada.
authors.forename: Teresa R
authors.initials: TR
authors.lastname: Sanelli
authors.forename: Maggie M
authors.initials: MM
authors.lastname: Sopper
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Strong
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002449:Cell Aggregation / Q000187:drug effects / Q000502:physiology; D016923:Cell Death / Q000187:drug effects / Q000502:physiology; D004305:Dose-Response Relationship, Drug; D006801:Humans; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D016202:N-Methylaspartate / Q000378:metabolism* / Q000494:pharmacology; D016900:Neurofilament Proteins / Q000032:analysis / Q000096:biosynthesis*; D009474:Neurons / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D019001:Nitric Oxide Synthase / Q000032:analysis / Q000096:biosynthesis*; D052248:Nitric Oxide Synthase Type I
chemical_list: D016900:Neurofilament Proteins; C044882:neurofilament protein L; D016202:N-Methylaspartate; C496320:NOS1 protein, human; D019001:Nitric Oxide Synthase; D052248:Nitric Oxide Synthase Type I; C496321:Nos1 protein, mouse; D002118:Calcium