Graduation Date

Spring 5-5-2018

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Programs

Pharmacology and Experimental Neuroscience

First Advisor

R. Lee Mosley

Abstract

Increasing evidence suggests that neurotoxic inflammatory activities affect the pathogenesis and progression of PD. Neuroinflammatory processes also produce oxidized and modified self-central nervous system (CNS) proteins which lead to dysfunction, mis-folding, aggregation, and retention of those oxidized products. In PD, nitrated α-synuclein (N-α-syn) is found aggregated within the cytoplasm and Lewy bodies of dopaminergic neurons within the substantia nigra and is released to the extraneuronal environment by dying and damaged neurons. Previous studies have shown that after 1-methyl 4-phenyl 1,2,3,6-tetrahydropyridine (MPTP) intoxication, adoptive transfer of effector T cells (Teff) exacerbates microglial-mediated neuroinflammation and amplifies dopaminergic neurodegeneration with accelerated and prolonged neuropathology. Additionally, Teffs that secrete IL-17 (Th17 type) exacerbate neurodegeneration to greater levels than those Teff types that secrete IFN-ɣ (Th1 type). Thus, we hypothesize that N-α-syn specific Th17 effector T cells exacerbate neurodegeneration indirectly via activation of microglial-mediated neurotoxicity or alternatively by direct cytotoxicity to neurons.

To test this hypothesis we first established the long-term culture of CD4+ effector T cell lines of the Th17, Th1, and Th17.1 phenotype. These cell lines stably produced their respective classical cytokines while retaining phenotype, without de-differentiation to other phenotypic subsets, and were functional both in vitro and in vivo. The development of a cloned Th17, Th1, and Th17.1 cell lines allowed their utilization to test in vitro and in vivo effects on microglia and cytotoxicity to dopaminergic neurons through direct and indirect mechanisms.

With the generation of cloned Th17 effector T cell line we sought to test its toxicity to neuronal cells in vitro through the use of the BV-2 microglial cell line and MES23.5 neuronal cell line. In vitro culture of MES23.5 neuronal cells with Th17 cells or supernatants increased neuronal cell toxicity while decreasing viability. Moreover, the supernatant from activated Th17 cells proved more neurotoxic to MES23.5 cells than cells. While Th17 cells demonstrated direct toxicity to neuronal cells, they also exhibited the ability to induce microglial-mediated neurotoxicity through apoptotic mechanisms, which were ablated with the use of a Z-VAD-FMK pan caspase inhibitor.

Growing evidence supports the notion that not all Teff cells are created equally, and some are more pathogenic than others, with IFN-γ/IL-17 dual expressing TH17.1 cells sufficient for heightened pathogenicity. In vitro analysis of Th17, Th1, and Th17.1 cell line pathogenicity indicated that compared to other cell lines, Th17.1 cells or supernatants exhibit the most pathogenic nature upon co-culture with neuronal cells (direct toxicity) as well as through microglial-mediated cell death (indirect toxicity). In fact, the Th17.1 cell line caused the largest increase in apoptosis, and induced the most extensive neuronal cell death directly via T cell/T cell supernatant interactions, and indirectly via microglial-mediated cellular death. Moreover, the Th17.1 cell line also demonstrated similar effects in vivo, as adoptive transfer of Th17.1 cells produced the greatest loss of TH+ dopaminergic neurons, and the most exacerbation of microgliosis compared to the Th17 and Th1 cell lines.

Taken together, this research has shown the ability of effector T cell types to exacerbate neurodegeneration in vitro and in vivo, possibly through apoptotic mechanism(s). Additionally, this work has detailed the generation and long-term culture of effector T cell lines in vitro through the use of specific cytokine and antibody milieus, which affects effector T cell type pathogenicity. Further investigations into the mechanistic details behind exacerbation of neurodegeneration induced by CD4+ effector T cells in PD models, for instance by TNFα or Fas/FasL interactions, have the potential to advance therapeutic strategies that target neurotoxic mechanisms to attenuate neuroinflammation and ameliorate neuronal loss in PD.

Available for download on Thursday, April 25, 2019

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