This application will focus on a hypothesis that impaired regulation of the inhibitory G protein:calcium channel complex occurs in diabetes mellitus and contributes to the pathogenesis of diabetic neuropathy via enhancement in calcium influx and concomitant induction of apoptosis (programmed cell death). PI will also examine the hypothesis that a factor present in the sum of diabetic humans and rats with neuropathy contributes to the alterations in neuronal calcium signaling.
Specific aims i nclude: 1. Determine whether the increase in calcium influx observed in diabetic neurons is associated with impaired regulation of the inhibitory G-protein:calcium channel complex; a) Evaluate whether DRG neurons from diabetic BB/W rats demonstrate altered inhibitory G protein function compared to age-matched controls; b) Evaluate whether treatment of DRG neurons from non-diabetic rats or culture SH-SY5Y neurons with serum obtained from diabetic humans or diabetic rats reproduces the altered calcium signaling and G protein function observed in DRG neurons from diabetic rats; 2. Assess whether DRG neurons from diabetic BB/W rats with neuropathy demonstrate an increase in apoptosis compared to age-matched, non-diabetic controls; a) Examine whether exposure of DRG neurons from non-diabetic rats or cultures SH-SY5Y neurons to serum from diabetic humans or diabetic mice is associated with induction of apoptosis or necrosis and assess the role of altered calcium homeostasis as a mediator of these events; and 3. Perform the initial characterization of the serum factor(s) that produces the abnormalities in calcium signaling and induction of apoptosis.
The specific aims will be examined using a variety of methodological approaches including: a. patch-clamp electrophysiologic recordings and direct measurements of cytosolic calcium levels using a calcium-sensitive dye; 2. Quantitative Western blot assessment of G protein expression and calcium channel binding studies in conjunction with Scatchard analysis to quantify calcium channel expression in controls and neurons from diabetic animals; 3. Direct assessment of G protein function using a GTPase assay; 4. Assessment of neuronal apoptosis and necrosis using histology and immunofluorescence techniques in conjunction with flow cytometry and direct visualization; 5. Characterization of putative serum factor(s) using immunoprecipitation, heat- and trypsin-sensitivity, molecular weight assessment with sizing filters, immunohistochemistry, and enrichment with immunoaffinity columns.
