In Vitro Studies Of Trophic Factors For Central Neurons

The work to be presented here can be divided into four major parts: (1) Studies of the effects of factors produced by non-neuronal cells, and of other agents, on the activity of the enzyme choline acetyltransferase (CAT) in cultures of rat central neurons; (2) studies of the effects of factors from non-neuronal cells on neurite outgrowth from cultured neurons; (3) the partial purification of a factor (Kaufman and Barrett, 1983) from serum which supports the survival of neurons in culture; (4) experiments examining the binding of putative trophic factors from radiolabelled conditioned media to their target cells.The major results in each category were: (1) The 40,000 dalton factor from lung conditioned medium (LCM) which increases CAT activity in spinal cord cultures (Kaufman, et al., 1985) has an isoelectric point of about 8.0. The effect of LCM is exerted directly on neurons, and not by way of glial cells induced by the LCM in the cultures. LCM stimulates increased CAT activity in cultures of the basal forebrain (nucleus basalis/septum) as well as spinal cord. Medium conditioned by glial cells (GCM) increases CAT in spinal cord cultures, and the active factor has an apparent molecular weight of 50,000 daltons. (2) The component of LCM which stimulates neurite outgrowth from spinal cord explants when it is bound to the tissue culture substrate (Dribin and Barrett, 1982) is also active on cultures of neurons from many regions of the central nervous system (CNS). GCM has similar activity, as do extracts of cortex and hippocampus. The 50,000 dalton component of LCM which stimulates neurite outgrowth when present as a soluble component of the culture medium has an isoelectric point of about 8.0, and a second component with an isoelectric point of about 5.0. (3) The neuron survival-promoting factor NSF-55k can be purified several hundredfold by affinity chromatography, preparative isoelectric focusing, and preparative sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE). (4) When radiolabelled LCM is incubated with cells from spinal cord, brain, and skeletal muscle, the major protein that binds after extensive washing is one that appears similar to the protein designated B14 (Perry and Wilson, 1983) found in fast axonal transport in frog and rat sciatic nerves