Loss of synaptic contacts, formation of neurofibrillary tangles and neuronal death are characteristic features of Alzheimer's disease. Oxidative stress causes neurodegeneration and affected neurons in AD demonstrate biochemical footprints of the past presence of oxidative stress. We reported that apolipoprotein E (apoE) regulates NO production, a key reactive oxygen/nitrogen species that causes oxidative stress. Our additional new data demonstrate that apolipoprotein E alters the neuronal uptake of L-arginine, the substrate* converted by nitric oxide synthase (NOS) into NO. We hypothesize that apoE regulates the function of cationic amino acid (CAT) transporters in an isoform specific manner, thereby altering the entry of L- arginine into the neuron which is then converted to NO by the action of NOS. We will test this hypothesis by measuring the functional activity of the CAT transporter in murine primary neuronal cultures exposed to human apoE and its isoforms and in primary neuronal cultures from mouse models expressing isoforms of human APOE and its gene products. Using semi-quantitative and quantitative measures, we will determine the effect of apoE and APOE genotype on CAT transporter mRNA and protein expression. Translation to NO production will be measured both directly by determining supernatant nitrite levels produced by NOS -containing neurons and indirectly by measuring indices of past-presence of NO (nitrotyrosine and hemeoxygenase 1). The validity of the mouse model will be tested by determining the apoE isoform dependency of arginine transporters in post-mortem human brain from individuals with and without previous clinical signs of dementia due to Alzheimer's disease.
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