Browsing by Subject "Parkinson Disease"
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Item Behavioral, Neurochemical, and Histological Characterization of Mice Deficient for Parkin, DJ-1, and Antioxidant Proteins(2011-08-26T17:35:01Z) Seamans, Katherine Webster; Goldberg, Matthew S.Parkinson’s disease is a progressive neurodegenerative disease characterized by a loss of dopaminergic neurons in the substantia nigra. The cause of Parkinson’s disease remains uncertain, however, evidence implicates mitochondrial dysfunction and oxidative stress with selective vulnerability of dopaminergic neurons. Although most cases of Parkinson’s disease are sporadic, 5-10% of cases are caused by mutations in a single gene. Loss-of-function mutations in parkin and DJ-1 were the first to be linked to recessively inherited parkinsonism. Surprisingly, mice bearing similar loss-of-function mutations in parkin and DJ-1 do not show age-dependent loss of nigral dopaminergic neurons or depletion of dopamine in the striatum. Although the normal cellular functions of Parkin and DJ-1 remain unclear, we hypothesized that Parkin and DJ-1 protect cells from oxidative stress and that loss-of-function mutations in these genes cause neurodegeneration in Parkinson’s disease by rendering cells more sensitive to mitochondrial dysfunction and oxidative stress. We crossed mice deficient for Parkin and DJ-1 with mice deficient for the major mitochondrial antioxidant protein Mn-superoxide dismutase or Cu/Zn-superoxide dismutase. Previous studies have shown that mice with reduced levels of Cu/Zn-superoxide dismutase or Mn-superoxide dismutase are more sensitive to dopaminergic neurotoxins whereas mice with increased levels of superoxide dismutase are more resistant to dopaminergic neurotoxins. We predicted that reducing levels of antioxidant proteins in parkin-/-DJ-1-/- mice would result in age-dependent nigral cell loss, striatal dopamine depletion or behavioral abnormalities. Characterization of these mice for behavioral abnormalities, neurotransmitter defects and neuropathology, revealed significant behavioral abnormalities in the mutant mice even in the absence of significant changes to dopamine levels in the striatum, dopamine receptor density, or dopaminergic neuron numbers. Aged parkin-/-DJ-1-/- and Mn-superoxide dismutase triple deficient mice have a surprising enhanced rotorod performance without the presence of an anxiety phenotype or hyperactivity. Cu/Zn-superoxide dismutase and Mn-superoxide dismutase triple deficient mice have elevated levels of dopamine in the striatum, however none of the mice present with nigral cell loss. Levels of D1-like and D2-like dopamine receptors in the striatum were unchanged. It is evident from our studies that on a parkin/DJ-1 null background, additional loss of major antioxidant proteins does not lead to a progressive loss of dopaminergic neurons in mice.Item Metabolism of Alpha-Synuclein by the 20S Proteasome(2009-06-18) Lewis, Karen Adell; Rizo, JosepParkinson's disease is one of several common neurodegenerative disorders that are related by the intracellular aggregation of the neuronal protein alpha-synuclein (alphaSyn) that normally associates with synaptic vesicles. Within the aggregates, a fraction of alphaSyn is truncated at the C-termini, and these truncations are hypothesized to participate in disease pathogenesis. The prevailing model for cytotoxicity of the aggregated protein proposes that oligomeric forms cause dysfunction of the ubiquitin-proteasome pathway of protein degradation, thereby enhancing an alternative pathway that involves ubiquitin-independent degradation by the 20S core particle of the proteasome. The 20S proteasome is known to degrade proteins with regions of intrinsic disorder, which figure prominently in a wide range of neurodegenerative disorders, including alphaSyn in Parkinson's disease. Importantly, some forms of 20S exert an endoproteolytic activity that produces highly amyloidogenic truncations of alphaSyn. An in vitro system containing liposomes and the 20S proteasome was developed to identify the mechanism by which the three point mutations in alphaSyn associated with familial Parkinson's disease exert cytotoxicity. The proteasome produced truncations from all three mutants in the presence of liposomes, but not wildtype alphaSyn. Additionally, the putative cytotoxic oligomer was formed most rapidly in the presence of both 20S proteasome and liposomes. Polyclonal antibodies specific for the C-termini of a set of truncated alphaSyn proteins were successfully developed to detect cleavage products of alphaSyn in vitro and in vivo. To better understand the physiological role of 20S-mediated degradation of intrinsically disordered proteins, the mechanism of recognition and interaction between substrate and enzyme was studied. While the presence of disorder appears to be necessary for 20S degradation, it was found to be insufficient to define a protein as a 20S substrate. Furthermore, a novel correlation was identified between the endoproteolytic activity of 20S and a modification of the alpha6 subunit. The data support a role for the 20S proteasome in Lewy body disease pathogenesis, where it accelerates the formation of cytotoxic species by endoproteolysis of an intrinsically disordered protein. In that regard, a form of the 20S proteasome was identified that may be responsible for the endoproteolytic cleavage of intrinsically disordered proteins in vivo.Item Neuroinflammation, TNF, and Ceramide Signaling: Putative Pathways for Neurotoxicity in Parkinson's Disease(2010-05-14) Martinez, Terina Nichole; Tansey, Malu G.Parkinson’s disease is a progressive neurodegenerative disorder that is characterized by the loss of dopaminergic neurons in the substantia nigra that innervate the striatum, and it is the loss of these neurons that causes the motor dysfunction that is associated with the disease. However, the mechanisms that contribute to the induction and perpetuation of dopaminergic neuronal cell death in Parkinson’s disease are multifaceted and poorly understood. Inflammation has been shown to contribute to cytotoxicity in animal models of Parkinson’s disease, and increased levels of inflammatory cytokines have been observed in the cerebral spinal fluid and striatum of Parkinson’s disease patients. We have previously demonstrated that blocking soluble tumor necrosis factor (TNF) signaling with dominant-negative TNF inhibitors attenuates the loss of dopaminergic neurons in models of Parkinson’s disease, but which signaling pathways downstream of TNF mediate this effect remain undetermined. Here, I show that TNF-dependent ceramide signaling contributes to dopamine neuron cytotoxicity by compromising mitochondrial membrane potential, inducing endoplasmic reticulum stress and activating caspase signaling in vitro. My data demonstrate that TNF-induced cytotoxicity is partially ceramide-dependent, as TNF-induced cytotoxic effects are attenuated with two different pharmacological inhibitors of sphingomyelinase, an enzyme that hydrolyzes active ceramide from inactive sphingomyelin pools. Collectively, my data support a model whereby low-dose TNF and concomitant low TNF receptor1 occupancy activates downstream ceramide signaling and metabolism, culminating in caspasedependent cytotoxic cell death of dopaminergic neurons. My data and the data associating ceramide biology and metabolism with Parkinson’s disease warrants future studies examining the potential neuroprotective effects of inhibition of sphingomyelinase in animal models of Parkinson’s disease, and may eventually lead to improved therapy for patients who suffer from Parkinson’s disease.Item The Role of Tumor Necrosis Factor (TNF) in Microglial Activation and Progressive Degeneration of Dopaminergic Neurons(2010-05-14) Harms, Ashley Nicole Simpson; Tansey, Malu G.Parkinson’s disease (PD) is a progressive neurodegenerative disorder characterized by a loss of dopaminergic (DA) neurons in the substantia nigra pars compacta (SNpc). A number of studies have implicated chronic inflammation in the pathophysiology of PD; however, it is unclear which inflammatory mechanisms directly contribute to neuronal loss in PD. A number of cytokines, including TNF, are elevated in post-mortem brain and cerebrospinal fluid of patients with PD. Previous studies from our group have shown that blocking solTNF signaling at the time of a unilateral 6-OHDA striatal lesion attenuated behavioral deficits and the acute loss of dopaminergic neuron loss by 50%. However, a critical question of clinical relevance is whether delayed solTNF signaling inhibition can prevent the progressive loss of DA neurons that occurs after a CNS insult. I report here that a single intranigral injection of a lentivirus encoding a dominant negative TNF inhibitor delivered 2 weeks after an intrastriatal 6-OHDA lesion attenuated microgliosis in SNpc and halted the progressive loss of nigral DA neurons and the associated locomotor deficits. Given the potential contribution of microglial activation to PD and the suggestion that anti-TNF therapies in the CNS may exert neuroprotective effects on vulnerable dopaminergic neuron populations, I also investigated the role of TNF in regulating microglia effector functions to gauge the potential detrimental effects of anti-TNF therapies on the microglia functional response. I found that microglia from TNF-null mice produced reduced protein levels of cytokines and chemokines in response to LPS stimulation and they displayed no cytotoxic effects on dopaminergic neuroblastoma MN9D cells when activated in vitro. I also demonstrated that microglia isolated from TNF-null mice failed to display the expected morphological changes in response to LPS stimulation, including enhanced perinuclear expression of the activation marker CD45. My results suggest that TNF plays a critical role in microglia activation, regulation of several microglia effector functions and is the primary microglia-derived inflammatory factor that compromises survival of dopaminergic neurons. Furthermore, these studies suggest that TNF-dependent neuroinflammation directly contributes to the delayed and progressive degeneration of nigral DA neurons after neurotoxic injury and further validates solTNF as a potential therapeutic target in PD.Item Tower of California performance early in Parkinson's disease(2008-09-19) Vega, Martin C.; Silver, Cheryl H.Cognitive impairment associated with Parkinson's disease (PD) is widely described in the literature. Executive dysfunction has been reported even when the patients are not experiencing dementia. A significant (24% - 50+%) number of PD patients display cognitive impairment from the onset of the disease and progressively worsen. However, executive dysfunction in newly diagnosed patients often escapes clinical detection. This paper describes a study designed to test both early and late PD patients (0-5 years disease duration and 5-10 years disease duration, respectively) vs. controls on a novel tower task, the Tower of California (TOC, Delis, Kaplan,&Kramer, 2001). Use of the TOC with PD patients has not been published. The TOC is designed to be more difficult and may be more sensitive to subtle executive impairment, specifically in the areas of planning and spatial working memory. It is predicted that the early PD group will perform worse than the control group but better than the late PD group in the number of successful towers (ST) built. The early PD group is also expected to have a longer time to first move on ST built even when corrected for bradykinesia, but not as long as the late PD group, which is anticipated to be the slowest. Implications of the possible outcomes of this study are then discussed.Item Tumor Necrosis Factor Dependent Mechanisms and Neuroprotective Strategies in Models of Parkinson's Disease(2008-5-13) McCoy, Melissa Kay; Tansey, Mal󠇮Parkinson's disease is a chronic, progressive, neurodegenerative disorder characterized by the loss of dopaminergic neurons in the substantia nigra that innervate the striatum. Although the nigral cell loss that causes motor dysfunction in Parkinson's disease has been identified for some time, the mechanisms that lead to this dopaminergic neuron loss are unclear. Elevated levels of the cytokine tumor necrosis factor in cerebrospinal fluid and postmortem brains of Parkinson's patients and in animal models of the disease implicate tumor necrosis factor in contributing to disease pathology; but a specific role for this cytokine in mediating loss of dopaminergic neurons in Parkinson's disease has not been clearly established. Here I demonstrate that neutralization of soluble tumor necrosis factor in vivo with a recombinant dominant-negative tumor necrosis factor inhibitor reduced 6-hydroxydopamine-induced nigral degeneration by 50% and attenuated amphetamine-induced rotational behavior, indicative of striatal dopamine preservation in a rodent model of Parkinson's disease. Similar protective effects were observed with in vivo chronic co-infusion of dominant-negative tumor necrosis factor inhibitor with a proinflammatory initiator, low-dose lipopolysaccharide, into the substantia nigra of rodents, confirming a role for soluble tumor necrosis factor inhibitor-dependent neuroinflammation in contributing to nigral degeneration. In rat embryonic midbrain neuron/glia mixed cell cultures exposed to lipopolysaccharide, delayed administration of dominant-negative tumor necrosis factor inhibitor prevented degeneration of dopaminergic neurons despite sustained microglia activation. Addition of a dominant-negative tumor necrosis factor inhibitor also attenuated 6-hydroxydopamine-induced dopaminergic neuron toxicity in vitro. In this dissertation the ability of lentiviral-encoded dominant-negative tumor necrosis factor inhibitor to provide neuroprotection was also investigated. Intranigral delivery of lentiviral dominant-negative tumor necrosis factor inhibitor in a hemiparkinsonian rat 6-hydroxydopamine model attenuated nigral dopaminergic neuron loss and reduced behavior deficits when compared to control lentiviral-infected animals. Collectively, these data identify tumor necrosis factor signaling in contributing to dopaminergic neuron loss in vitro and in vivo in two chronic rat models of Parkinson's disease, and provide evidence that delaying the progressive degeneration of the nigrostriatal pathway in the early stages of Parkinson's disease in humans may be therapeutically feasible with agents that block soluble tumor necrosis factor signaling.