The broad, long-term objective of this proposal is to understand the pathogenesis of Lyme neuroborreliosis of the central nervous system (CNS). Inflammation in the CNS is thought to play a primary role in the pathogenesis of neurodegenerative diseases such as Alzheimer's disease and the AIDS-dementia complex. Lyme borreliosis (LB), caused by the spirochete Borrelia burgdorferi, is primarily an inflammatory disease, and neuroborreliosis, i.e. LB of the central and peripheral nervous systems, may manifest as a mild neurodegenerative disease. The central hypothesis to be explored in this grant proposal is that 1) B. burgdorferi spirochetes can cause inflammatory responses in CNS cells and tissues, and 2) these inflammatory responses may lead to neural injury, and ultimately to loss of glial and neuronal cells. Limited loss of these cells is viewed as a determinant of the neurologic impairment seen in Lyme neuroborreliosis. The secondary hypothesis is that spirochetal lipoproteins can induce, in part or in toto, the inflammatory effects of B. burgdorferi. We will use tissues and cells from the brain of rhesus macaques, a model developed under this Cooperative Agreement.
Specific Aims; SA-I: Experiments ex vivo. SA-la: Assessment of inflammation and glial/neuronal loss in freshly obtained brain tissues fr6m rhesus macaques after exposure to lipoprotein and spirochetes. Local cytokine secretion (IL-6, TNF-alpha, and IL-1 beta) as well as expression of iNOS and COX-2 will be assessed by confocal microscopy using a novel procedure whereby cytokines and other inflammatory mediators are stained intracellularly with appropriate antibodies in fixed brain sections. Freshly obtained rhesus brain slices from normal animals will be stimulated ex vivo with lipoprotein or live spirochetes in the presence of the protein-secretion blocker Brefeldin A. After fixation and cutting of frozen sections, the cytokine/mediator-producing cell phenotype will be identified with appropriate antibodies, Toll-like receptor (TLR) expression determined, and glial/neuronal apoptosis verified in situ by the TUNEL assay. Spirochetes will be localized with an anti-lipoprotein antibody. Unstimulated rhesus brain slices will be used as negative controls. SA-lb: Effect of mitogen-activated protein-kinase inhibitors upon inflammation and glial/neuronal loss induced by lipoprotein in freshly obtained brain tissues from rhesus macaques. SA-lc: Assessment of inflammation and glial/neuronal loss in archival fixed samples. Archival samples of B. burgdorferi-infected rhesus meninges and brain parenchyma will be assessed for presence of inflammatory infiltrate (meninges), association of similar infiltrates present in brain specimens or areas of gliosis with foci of glial/neuronal apoptosis, and the overall correlation of such findings with presence of B. burgdorferi lipoprotein. Cumulative neuronal loss will be quantified relative to equivalent tissue sections from uninfected control animals using image analysis software. SA-2: Experiments in vitro. SA-2a will assess the production of inflammatory cytokines and mediators in single-cell-type and mixed primary cultures of rhesus glia stimulated with live B. burgdorferi and lipoprotein. The human neuroblastoma cell lines SK-N-SH and SH-SY5Y also will be employed, alone and in combinations with rhesus glia, as well as rhesus neuronal-glial primary cultures. SA-2b will assess the role of glial cells and cytokines in eliciting glial/neuronal apoptosis. SA-2c will assess the role of innate CNS responses to B. burgdorferi and lipoprotein in inflammation and neurodegeneration, specifically, the expression and involvement of pertinent TLR in mediating cytokine production and apoptosis in the different cell combinations.
