Recipient Organization
IOWA STATE UNIVERSITY
S. AND 16TH ELWOOD
AMES,IA 50011
Performing Department
Veterinary Medicine
Non Technical Summary
Environmental exposure to excessive manganese impairs basal ganglia function, resulting in a neurological disorder relatively similar to Parkinsonism commonly known as Manganism. Manganese exposure is of serious concern due to the increased incidences of extrapyramidal neurological symptoms among miners and industrial workers, including welders. Manganese predominantly accumulates in the basal ganglia structures and causes mitochondrial dysfunction, oxidative stress, and apoptosis. However, cellular and molecular mechanisms underlying manganese neurotoxicity are poorly understood. The proposed study will elucidate the neurotoxic mechanisms underlying manganese induced upregulation of a proapoptotic kinase PKC and its functional relevance to manganese neurotoxicity.
Animal Health Component
(N/A)
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Goals / Objectives
The main goal of our competitive renewal proposal is to study novel cellular mechanisms of upregulation of a proapoptotic kinase PKCdelta during manganese (Mn) exposure and to determine its functional role in manganese neurotoxicity. Hypothesis I: Manganese exposure upregulates proapoptotic kinase PKC in cell culture and animal models of manganese neurotoxicity. Specific Aim I: To characterize upregulation of an oxidative stress-sensitive proapoptotic kinase PKCdelta in mouse primary neuronal cultures and animal models following Mn exposure. Hypothesis II: Manganese-induced PKCdelta upregulation is mediated by the redox transcription factor NFkB and SP1 activation during manganese exposure. Specific Aim II: To investigate molecular mechanisms of manganese-induced upregulation of PKCdelta by examining its transcriptional regulation of PKCdelta promoter. Hypothesis III: Suppression of NFkB and SP1 activation will dampen manganese-induced PKCdelta upregulation and neuronal apoptosis in animal models of Mn neurotoxicity. Specific Aim III: To further confirm the functional role of NFkB and Sp1-dependent PKCdelta upregulation in manganese-induced neuronal damage during chronic manganese exposure in animal models.
Project Methods
A. Approach: i) Determine dose-response and time course of PKC mRNA and protein levels in primary striatal and nigral cultures following exposure to manganese, ii) Demonstrate PKC upregulation sensitizes neurons to Mn toxicity, iii) Examine time course and dose-response of PKC mRNA and protein levels in various brain regions in a mouse model of chronic manganese exposure, and iv) Characterize the relative expression of PKC upregulation in neurons, astrocytes and microglia in a chronic animal model of Mn neurotoxicity. B. Approach: i) Determine the activation of NFkB and Sp1 transcription factors during manganese exposure by gel-shift and chromatin immunoprecipitation (ChIP) assays, ii) Evaluate whether NFkB and Sp1 activation contributes to Mn-induced PKC upregulation by promoter analysis, and iii) Examine whether pharmacological inhibition of NFkB and Sp1 or overexpression of NFkB and Sp1 mutants suppresses Mn-induced PKC upregulation and neurotoxicity in primary striatal cultures. C. Approach: i) Examine whether NFkB subunit p50 Knockout (KO) animals suppress Mn-induced PKC upregulation and neuronal damage during chronic manganese exposure, and ii) Evaluate whether expression of dominant negative sp1 via adenoassociated (AAV) vector dampens Mn-induced PKC upregulation and neuronal damage during chronic manganese exposure.