Pharmacology Of Neurotoxins

National Institute Of Mental Health

Investigators

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Abstract

The release of soluble neurotoxins and growth promoting factors by activated macrophages/microglia has been reported following chronic HIV infection and various types of brain injury. A neuronal cultured cell bioassay using neonatal rat was refined for quantitative measurements. The isolation and characterization of soluble factors isolated from conditioned media from activated human peripheral blood monocytes or HIV-infected macrophages was monitored with this bioassay. Extracts of conditioned media were separated into component classes (lipids vs. non-lipids, low versus high molecular weight). Neuronal toxicity was demonstrated in six of eleven batches of conditioned media from HIV-infected-LPS-stimulated macrophages. Activity was highest in a lipid-free fraction containing substances less than 3000 Da. The neurotoxicity was trypsin insensitive. Glutamate concentrations were elevated in neurotoxic media. Three tests demonstrated that glutamate concentrations are alone sufficient to account for the observed neurotoxicity. Glutamate concentrations were lowered to control levels and neurotoxicity was abolished by treatment of the conditioned media with glutamate pyruvate transaminase. Pretreatment of neurotoxic media by pre-incubation with astrocytes also reduced glutamate concentrations and abolished neurotoxicity. Finally, addition of glutamate to control conditioned media in concentrations matching those in the neurotoxic HIV/LPS media replicated the observed extent and morphology of the neurotoxic effect. Two other neurotoxicology investigations have are in progress. In the first, we are testing the hypothesis that recruitment of peripheral cells into the CNS following inflammation is mediated by leukotrienes. Biochemical events following CNS inflammation are been studied in gerbils and rats using stereotaxically micro-injected solutions containing an immuno-stimulant such as lipopolysaccharide or an excitatory amino acid. Previously, we quantified the phenomenon of increased tryptophan metabolism as characteristic of immune mediated human neurologic disease in gerbils. Gerbils exhibit the same biochemical response as humans, providing a means to study the role of this pathway in an animal model. The activity of leukotriene receptor antagonists and lipoxygenase inhibitors will be studied to determine whether the secondary damage due to peripheral blood cell infiltration can be ameliorated with reduction of neurodegeneration. In a second study, we are testing the hypothesis that there are specific proteome changes accompanying stem cell phenotype development. Investigating the differentiation process of stem cells could elucidate the complex events that occur during normal human brain development. Rat bone marrow stromal cells (BMSCs) are multipotent stem cells that are self-renewing with broad potential. When grown in culture, BMSCs can be induced to a neuronal phenotype expressing neuronal marker proteins. Neuronal phenotype expression parallels terminal differentiation, halting cell division. The objective of these studies is to profile nuclear proteome changes characteristic of the phenotypic changes. The DNA synthesomes and nuclear proteins from the stromal cells will be characterized using two-dimensional gel and mass spectrometric techniques.