The symptoms of Parkinson's Disease (PD) result from reduced striatal dopamine (DA) secondary to degeneration of the DA - producing cells of the substantia nigra pars compacta (SNpc). Although genetic and environmental factors are thought to be involved, the etiology of PD remains elusive. Recent preliminary findings in the investigators' laboratory show that maternal administration of lipopolysaccharide (LPS), derived from gram(-) bacteria, at embryonic (E) day 10.5 induces DA neuron loss in the fetal brain by E15. This loss is still present in the pups at postnatal (P) day 10. This suggests that prenatal gram (-) infection attenuates fetal DA neuron development. In addition, in vitro culture data as well as in vivo results demonstrate that the pro-inflammatory cytokines tumor necrosis factor (TNF-alpha) and interleukin lB IL-1beta, which are normally induced by LPS, mediate this cytotoxic effect. This is particularly relevant since postmortem studies on PD brains reveal increased pro-inflammatory cytokines. The investigators hypothesize that gram (-) infection during pregnancy attenuates DA neuron development leaving the offspring with fewer DA neurons at birth and therefore at increased risk for PD in later life. This heretofore unexamined risk factor for PD will be explored first in a rat model (specific aim 1) and the mechanistic issues associated with LPS-induced cell death will be studied in primary mesencephalic cultures (PMCs;
specific aim 2). LPS will be injected into the gravid female at E1O.5 and the embryos harvested at various times thereafter through P40. TNF-alpha and IL-1Beta, IFN-gamma will be measured (ELISA) in fetal brain, and maternal blood to establish the relationship between systemic infection and fetal brain cytokine elevation. DA function (DA biochemistry by HPLC) and DA neuron counts (quantitative stereology) will be the primary outcome variables for correlational studies with the presumed cytokine alterations. Striatal glial cell line-derived neurotrophic factor (GDNF) will also be measured (ELISA) in striatal tissue. The role of TNF- alpha and IL-1Beta, and IFN-gamma in the presumed apoptotic death of DA neurons will be explored in PMCs using confocal microscopy (TH+ cells and apoptotic nuclei) as the primary outcome variable and the measurement of the pro-apoptotic Bax and the anti-apoptotic Bcl-2/Bcl-xL proteins as well as the production of nitric oxide as secondary outcome variables. The investigators anticipate that maternal LPS injection will produce a profound elevation of fetal brain cytokines that outlasts the maternal elevation and this elevation will be associated with diminution of fetal brain GDNF, DA function, and cell number. Moreover, the pro-inflammatory cytokines will kill DA neurons through apoptosis in tissue culture by decreasing Bcl-2 related proteins and by increasing nitric oxide production and that the toxic effects of LPS in culture will be attenuated by TNF-alpha and IL-1Beta antagonists. The successful implementation of these two specific aims will provide strong support for a larger proposal designed to fully explore the possible impact of gram (-) infections on DA neuron development.
