Manganese exposure exacerbates progressive motor deficits and neurodegeneration in the MitoPark mouse model of Parkinson's disease: Relevance to gene and environment interactions in metal neurotoxicity

Abstract

Parkinson's disease (PD) is now recognized as a neurodegenerative condition caused by a complex interplay of genetic and environmental influences. Chronic manganese (Mn) exposure has been implicated in the development of PD. Since mitochondrial dysfunction is associated with PD pathology as well as Mn neurotoxicity, we investigated whether Mn exposure augments mitochondrial dysfunction and neurodegeneration in the nigrostriatal dopaminergic system using a newly available mitochondrially defective transgenic mouse model of PD, the MitoPark mouse. This unique PD model recapitulates key features of the disease including progressive neurobehavioral changes and neuronal degeneration. We exposed MitoPark mice to a low dose of Mn (10mg/kg, p.o.) daily for 4 weeks starting at age 8 wks and then determined the behavioral, neurochemical and histological changes. Mn exposure accelerated the rate of progression of motor deficits in MitoPark mice when compared to the untreated MitoPark group. Mn also worsened olfactory function in this model. Most importantly, Mn exposure intensified the depletion of striatal dopamine and nigral TH neuronal loss in MitoPark mice. The neurodegenerative changes were accompanied by enhanced oxidative damage in the striatum and substantia nigra (SN) of MitoPark mice treated with Mn. Furthermore, Mn-treated MitoPark mice had significantly more oligomeric protein and IBA-1-immunoreactive microglia cells, suggesting Mn augments neuroinflammatory processes in the nigrostriatal pathway. To further confirm the direct effect of Mn on impaired mitochondrial function, we also generated a mitochondrially defective dopaminergic cell model by knocking out the TFAM transcription factor by using a CRISPR-Cas9 gene-editing method. Seahorse mitochondrial bioenergetic analysis revealed that Mn decreases mitochondrial basal and ATP-linked respiration in the TFAM KO cells. Collectively, our results reveal that Mn can augment mitochondrial dysfunction to exacerbate nigrostriatal neurodegeneration and PD-related behavioral symptoms. Our study also demonstrates that the MitoPark mouse is an excellent model to study the gene-environment interactions associated with mitochondrial defects in the nigral dopaminergic system as well as to evaluate the contribution of potential environmental toxicant interactions in a slowly progressive model of Parkinsonism.

Keywords: Animal model; Dopamine; Gene-environment interaction; Manganese; MitoPark; Mitochondria; Neuroinflammation; Parkinson’s disease; TFAM.

Figure 1. Mn accelerates and exacerbates progressive behavioral deficits in MitoPark mice

A, Exposure schedule showing C57 and MitoPark mice orally administered water or Mn (10 mg/kg) from ages 8-12 wks. B, VersaPlot showing horizontal activity (lines) and rearing activity (red dots) during a 10-min open-field test. Horizontal (C), vertical (D), and rearing (G) activities and distance traveled (F) during open-field test. E, Time spent on RotaRod. H, Time spent sniffing scented zone during 3-min social discrimination test. Graphical results represented as the mean±SEM (n=7-10 mice/group). *, p<0.05, and ns, p>0.05 versus water-treated C57 Control.

Figure 2. Mn exacerbates striatal DA depletion and TH neuronal loss MitoPark mice

HPLC with electrochemical detection of the neurotransmitters dopamine (A), DOPAC (B), and HVA (C) in the striatum. DAB immunostaining (D) was performed in striatum and substantia nigra of vehicle-treated C57 (top), Mn-treated C57 (second row), vehicle-treated MitoPark (MP) (third row) and Mn-treated MP mice (bottom). Graphical results represented as the mean±SEM (n=7-10 mice/group). ns, p>0.05, **, p<0.01 and ***, p<0.001 versus water-treated C57 Control.

Figure 3. Behavioral deficits correlate with neurochemical depletion in MitoPark mice

Correlation of horizontal activity with dopamine (A, R²= 0.29, F_1,32=13.3, p=0.0010), DOPAC (B, R²=0.20, F_1,32=8.2, p=0.0074), and HVA (C, R²=0.13, F_1,32=4.6, p=0.040). Correlation of vertical activity with dopamine (D, R²=0.33, F_1,32=16.2, p=0.0003), DOPAC (E, R²=0.088, F_1,32=3.1, p=0.088), and HVA (F, R²=0.093, F_1,32=3.3, p=0.079). Correlation of time spent on Rotarod with dopamine (G, R²=0.14, F_1,32=5.2, p=0.030), DOPAC (H, R²=0.053, F_1,32=1.8, p=0.19), and HVA (I, R²=0.43, F_1,32=24.7, p<0.0001). Regression lines are displayed with the 95% confidence bands of possible regression lines.

Figure 4. Mn increases oxidative stress in the brains of MitoPark mice

Representative Western blots and densitometric analysis of 4-HNE protein in the striatum (A) and substantia nigra (B). Immunohistochemistry of 12-wk mouse substantia nigra (C) reveals co-localization of TH and 4-HNE in dopaminergic neurons of Mn-treated MitoPark mice. Graphical results represented as the mean±SEM (n=3-4 mice/group). Ns, p>0.05, *, p<0.05, and **, p<0.01 versus water-treated C57 Control.

Figure 5. Mitochondrial dysfunction and neuronal cell death in Mn-treated MitoPark mice

Representative Western blots and densitometric analysis of MTCO1 protein in the striatum (A) and substantia nigra (B). Fluoro-Jade C staining of 12-wk mouse substantia nigra (C) reveals increased neuronal cell death in Mn-treated MitoPark mice. Graphical results represented as the mean±SEM (n=3-4 mice/group). Ns, p>0.05, and **, p<0.01 versus water-treated C57 Control.

Figure 6. Neuroinflammatory changes in Mn-treated MitoPark mice

A, Western blot and corresponding densitometric analysis of IBA-1 protein in the SN. B, IBA-1 DAB immunostained sections from 12-wk mouse substantia nigra of vehicle-treated C57 (top), Mn-treated C57 (second row), vehicle-treated MitoPark (MP) (third row) and Mn-treated MP mice (bottom) show more IBA-1⁺ microglia in Mn-treated MitoPark mice when compared to vehicle-treated C57 Control mice. Graphical results represented as the mean±SEM (n=3-4 mice/group). Ns, p>0.05, and *, p<0.05 versus water-treated C57 Control.

Figure 7. Mn increases protein aggregation in MitoPark mice

A, Representative slot blot analysis for oligomeric protein-specific antibody (A11) shows more oligomeric protein present in the substantia nigra of Mn-treated MitoPark mice. B, Quantification of A11 slot blot. Graphical results represented as the mean±SEM (n=3 mice/group). Ns, p>0.05, and *, p<0.05 versus water-treated C57 Control.

Figure 8. Mn exposure potentiated mitochondrial deficits in TFAM-KO neuronal cells

A, CRISPR/Cas9-based TFAM-KO and Control N27 cells were treated with or without 100 μM Mn for 24 h and mitochondrial dynamics were measured using Seahorse XF24 analyzer. A, Quantification of basal respiration rate prior to MitoStressor injections. B, Quantification of ATP-linked respiration following oligomycin injection. Graphical results represented as the mean±SEM (n=3-4 /group). Ns, p>0.05, **, p<0.01, ***, p<0.001, ****, p<0.0001, #, p<0.05, versus untreated control from same cell type.