Proteomic analysis of the response of cultured neurons to chemical excitation

Abstract

Mammalian cells secrete a large number of proteins into the extracellular space, the composition and levels of which change as result of environmental factors. However, the identity of most of these secreted proteins has yet to be determined. Recent advances in proteomic technology now allow the isolation and identification of these secreted proteins. Neuronal cell death and injury as a result of excessive and prolonged activation of excitatory amino acid receptors has been termed excitotoxicity, and has been implicated in mechanisms leading to a variety of disease states, such as Alzheimer’s disease. Available evidence suggests that under excitotoxic stress, neurons secrete signals that can alter the function of neurons with which they are connected via synapses or to which they are proximate. Many of these signals act via volume transfer, diffusing via the ECF to act on populations of cells, to induce slow long-lasting effects. The successful isolation and in vitro culture of rat cortical cells is reported here, These cultures are 95 % pure for neurons, as determined by imunocytochemistry. They are viable for more than 14 days under B27-supplemented culturing conditions, and are viable for up to a further 3 days under protein-free culturing conditions. Live dead/assays, utilising fluorescein diacetate and propidium iodide, demonstrated that cell survival remained high even after 72 h exposure to 50 µM kainic acid. Analysis of conditioned medium using 2D-PAGE combined with MALDI-TOF MS demonstrated significant proteolysis of the bovine serum albumin protein. This proteolysis only occurred in the presence of the cortical cells. In addition, immunocytochemistry confirmed that both neurons and glia had taken up the bovine serum albumin. Increasing evidence implicates cerebrovascular dysfunction, in particular the breakdown of the blood-brain barrier, as an early event in the development of Alzheimer’s disease. Elevated intracellular Cu2+ levels are also seen in the Alzheimer’s disease brain, and are thought to be responsible for neuronal cell death through metal-mediated oxidative stress. It is proposed here that the albumin molecule, which binds Cu2+with high-affinity, gains entry to the CNS as a result of BBB dysfunction. Neuronal uptake and processing of the albumin molecule, as demonstrated here, resulting in the intracellular release of Cu2+, may be the mechanism by which the elevated Cu2+ levels seen in Alzheimer’s disease occur. In addition, initial analysis of the conditioned medium of cortical neurons stimulated with 50 µM KA for 72 h, suggests that processing of the bovine serum albumin is altered, with some fragments up-regulated and others down-regulated.