Chronic Vanadium Exposure Promotes Aggregation of Alpha-Synuclein, Tau and Amyloid Beta in Mouse Brain

Abstract

The interaction of toxic environmental metals and metalloids with brain proteins and polypeptides can result in pathological aggregations and formation of toxic oligomers, which are key features of many neurodegenerative diseases. Occupational and environmental exposure to vanadium is connected to a neurological syndrome that includes psychiatric symptoms, cognitive decline, and neurodegeneration. In this study, we hypothesized that prolonged vanadium exposure may be a potential risk factor for Alzheimer's and Parkinson's diseases. A total of 72 male BALB/c mice, each 4 weeks' old, were used. Vanadium-treated groups received intraperitoneal injections of 3 mg/kg body weight of vanadium three times a week for 6, 12, or 18 months. The control group received sterile water while the withdrawal group were given vanadium injection for 3 months, followed by withdrawal of the metal, but treatment with sterile water only, and were sacrificed at 3-, 9-, or 15-months post vanadium exposure. Sagittal sections of brain paraffin-embedded tissue were prepared and analyzed using immunofluorescence to study the immunoreactivity and cellular localization of α-synuclein (α-syn), amyloid-β (Aβ), and tau proteins. Our findings revealed pathological aggregation of these proteins in the frontoparietal cortices and the dorsal CA1 and CA3 regions. Double immunolabeling with glial cells and neurons showed neuronal degeneration, functional gliosis, and activation of astrocytes and microglia at sites of α-synuclein immunoreactivity. We observed increased immunoreactivity of phosphorylated nuclei tau both in the parietal cortices and corpus callosum white matter while we observed intraneuronal accumulation of Aβ deposits in the cortex and dorsal hippocampal regions in vanadium treated brains. These cellular changes and proteinopathies, although persisting, were significantly attenuated after vanadium withdrawal. Our findings show that prolonged vanadium exposure promotes abnormal accumulation of neurodegeneration-associated proteins (α-syn, Tau, and Aβ) in the brain, which is further exacerbated by aging in the context of extended exposure to the metal.

Keywords: aging; neurodegeneration; protein aggregation; proteinopathies; vanadium.

FIGURE 1

Immunofluorescent staining showing the cellular localization of alpha‐synuclein (α‐syn) in the mice brains following chronic vanadium exposure and subsequent withdrawal. α‐Syn aggregation was detected in the frontoparietal cortices, FPC (A), hippocampal CA1 (B), and hippocampal CA3 (C) after chronic vanadium treatments. At each exposure period (6, 12, and 18 months), there was a significant increase in α‐syn aggregation in vanadium exposed compared to controls. However, a reduction was observed in the withdrawal brains compared to vanadium exposed brains. Densitometric quantification confirmed a trend of increasing α‐syn aggregation with advancing age and the duration of vanadium exposure. (D): α‐syn expressions in the cortex at 6, 12, and 18 months respectively; (E, F): Mean α‐syn expressions in the hippocampus (CA1 and CA3) at 6, 12, and 18 months respectively. [n = 8 mice/group; intensity analysis = 6 mice; Scale bar: 50 μm; p > 0.05; Normality test (Shapiro–Wilk) = FPC (6 months: p value 0.8498; 12 months: p value 0.0758; 18 months: p value 0.7509). CA1 (6 months: p value 0.1519; 12 months: p value 0.2002; 18 months: p value 0.48811). CA3 (6 months: p value 0.0926; 12 months: p value 0.6613; 18 months: p value 0.1903) (*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001)].

FIGURE 2

NeuN/α‐syn double immunolabeling of the mice brains following chronic vanadium exposure revealed alpha‐synuclein (α‐syn) positive aggregates in the frontoparietal cortical (FPC) pyramidal cells (A), hippocampal CA1 region (B), and hippocampal CA3 region (C). The level of α‐syn positive protein aggregates increased progressively from 6 to 18 months of vanadium exposure (A–C), which strongly correlated with the significant neuropathology observed in these regions. Colocalization of α‐syn immunolabeling with NeuN indicated neuronal pathology characterized by the accumulation of α‐syn positive aggregates and cytoplasmic toxicity in cortical pyramidal cells. Quantification of neuronal count indicated marked cell loss that increased with the chronicity of vanadium exposure in the cortex (D) and hippocampus (E, F). [n = 8 mice/group; intensity analysis = 6 mice; Scale bar: 50 μm; p > 0.05; Normality test (Shapiro–Wilk) = Neuronal loss (FPC: p value 0.3565, CA1: p value 0.1365, CA3: p value 0.2838); α‐syn aggregation (FPC: p value 0.0185, CA1: p value 0.5664, CA3: p value 0.9021) (*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001)].

FIGURE 3

GFAP/α‐syn double immunolabeling of the mice brains after chronic vanadium exposure revealed astrocytic activation in the frontoparietal cortical (FPC) pyramidal cells (A), hippocampal CA1 region (B), and hippocampal CA3 region (C). Astrogliosis increased progressively from 6 to 18 months of vanadium exposure (A–C), with corresponding increase in α‐syn immunoreactivity in these regions. Colocalization of α‐syn immunolabeling with the astrocytic marker glial fibrillary acidic protein (GFAP) revealed the presence of α‐syn positive aggregates in activated astrocytes (see insets). Quantitative analysis of astrocyte counts and α‐syn protein aggregation showed significant activation, correlating α‐syn protein aggregation with the chronicity of vanadium exposure in the cortex (D) and hippocampus (E, F). [n = 8 mice/group; intensity analysis = 6 mice; Scale bar: 50 μm, p > 0.05; Normality test (Shapiro–Wilk) = Astrocytic count (FPC: p value 0.4718, CA1: p value 0.1239, CA3: p value 0.3329), α‐syn (FPC: p value 0.0185, CA1: p value 0.5664, CA3: p value 0.9021) (**p < 0.01; ***p < 0.001; ****p < 0.0001)].

FIGURE 4

IBA1/α‐syn double immunolabeling of the mouse brain after chronic vanadium exposure revealed microglial activation in the frontoparietal cortical (FPC) pyramidal cells (A), hippocampal CA1 region (B), and hippocampal CA3 region (C). Microglial activation increased progressively from 6 to 18 months of vanadium exposure (A–C), with a corresponding increase in α‐syn immunoreactivity observed in these regions. Colocalization of α‐syn immunolabeling with the microglial marker ionized calcium‐binding adapter molecule 1 (IBA1) revealed α‐syn positive microglia and phagocytosis (see insets). Quantitative analysis indicated significant microglial activation and α‐syn protein aggregation with chronicity of vanadium exposure in the cortex (D) and hippocampus (E, F). [n = 8 mice/group; intensity analysis = 6 mice; Scale bar: 50 μm; p > 0.05; Normality test (Shapiro–Wilk) = Microglial count (FPC: p value 0.9358, CA1: p value 0.1385, CA3: p value 0.2707), α‐syn (FPC: p value 0.0185, CA1: p value 0.5664, CA3: p value 0.9021) (*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001)].

FIGURE 5

Tau phosphorylation and aggregation in the genu of corpus callosum of mice brains after 6 months (A–C), 12 months (D–F) and 18 months (G–I). Throughout the exposure periods, there were significant and progressive increases in the level of phosphorylated tau aggregates in the white tract of the corpus callosum in vanadium‐dosed groups (B, E, H) compared to age‐matched controls (A, D, G). However, a reduction in Tau immunoreactivity was observed in the withdrawal brains (C, F, I) compared to the vanadium‐treated brains. [n = 8 mice/group; intensity analysis = 6 mice; Scale bar: 50 μm; p < 0.05; Normality test (Shapiro–Wilk) = Corpus callosum (6 months: p value 0.8757, 12 months: p value 0.6270, 18 months: p value 0.0468) (*p < 0.05; ***p < 0.01; ****p < 0.0001)].

FIGURE 6

Tau phosphorylation and aggregation in the fronto‐parietal cortices (FPC) in mice brains after 6 months (A–C), 12 months (D–F) and 18 months (G–I). Throughout the exposure periods, there was a significant and progressive increase in the level of phosphorylated tau aggregates in the cortex of vanadium‐dosed groups (B, E, H) compared to age‐matched controls (A, D, G). However, a reduction in Tau immunoreactivity observed in the withdrawal brains (C, F, I) compared to the vanadium‐treated brains. [(n = 8 mice/group; intensity analysis = 6 mice; Scale bar: 50 μm; p < 0.05). Inset: Higher magnification; Normality test (Shapiro–Wilk) = FPC (6 months: p value 0.8043, 12 months: p value 0.8181, 18 months: p value 0.5703) (*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001)].

FIGURE 7

Immuno‐fluorescent staining showing amyloid beta (Aβ) aggregation/deposition in mice brains after 6–18 months of chronic vanadium treatment. Aβ accumulation was detected in the frontoparietal cortices, FPC (A), hippocampal CA1 region (B) and hippocampal CA3 region (C). At each period of exposure (6, 12, and 18 months), there was a significant increase in the level of Aβ deposition in the vanadium dosed group relative to control. However, a significant reduction was observed in the withdrawal brains relative to the vanadium animals. Densitometric quantification also confirmed increasing Aβ accumulation with advancing age and chronicity of vanadium exposure in the cortex (D), hippocampal CA1 (E) and CA 3 (F). [n = 8 mice/group; intensity analysis = 6 mice; Scale bar: 50 μm; p < 0.05; Normality test (Shapiro–Wilk) = FPC (6 months: p value 0.0356, 12 months: p value 0.0092, 18 months: p value 0.2071), CA1 (6 months: p value 0.9740, 12 months: p value 0.7442, 18 months: p value 0.0611), CA3 (6 months: p value 0.0064, 12 months: p value 0.2327, 18 months: p value 0.0116) (*p < 0.05; ***p < 0.001; ****p < 0.0001)].

FIGURE 8

Amyloid beta (Aβ) and NeuN co‐labelling of the frontoparietal cortices after 6 months (A–C), 12 months (D–F) and 18 months (G–I) in mice brains. Controls (A, D, G) and withdrawal (C, F, I) groups displayed had very little to almost no Aβ deposits. Marked Aβ deposits were seen in vanadium exposed mice after 6–18 months of chronic vanadium exposure (B, E, H). Colocalization of Aβ immunolabeling with neuronal marker (NeuN) revealed diffuse Aβ deposits and pyramidal cell pathology including pyknosis, cytoplasmic loss and vacuolation. Quantitative analysis of intracellular Aβ aggregates intensities revealed significant levels of Aβ protein aggregation in vanadium exposed compared to control. Aβ aggregates intensities in withdrawal were similar to control group [n = 8 mice/group; intensity analysis = 6 mice; Scale bar, 50 μm; p < 0.05; Normality test (Shapiro–Wilk) = FPC (6 months: p value 0.0356, 12 months: p value 0.0092, 18 months: p value 0.2071) (****p < 0.0001)].

FIGURE 9

Amyloid beta (Aβ) and NeuN co‐labelling of the hippocampal CA3 after 6 months (AC), 12 months (D–F) and 18 months (G–I) in mice brains. Controls (A, D, G) and withdrawal (C, F, I) groups displayed very little to almost no Aβ deposits. Marked Aβ deposits were seen in vanadium‐exposed mice after 6–18 months of chronic vanadium exposure (B, E, H). Colocalization of Aβ immunolabeling with neuronal marker (NeuN) revealed diffuse Aβ deposits and pyramidal cell pathology including pyknosis, cytoplasmic loss, and vacuolation. Quantitative analysis of intracellular Aβ aggregate intensities revealed significant levels of Aβ protein aggregation in vanadium‐exposed compared to control. Aβ aggregate intensities in withdrawal were similar to the control group [n = 8 mice/group; intensity analysis = 6 mice; Scale bar, 50 μm; p < 0.05; Normality test (Shapiro–Wilk) = FPC (6 months: p value 0.0064, 12 months: p value 0.2327, 18 months: p value 0.0116) (****p < 0.0001)].

FIGURE 10

Intraneuronal amyloid beta (Aβ) and NeuN co‐labelling of the hippocampal CA1 after 6 months (A–C), 12 months (D–F) and 18 months (G–I) in mice brains. Controls (A, D, G) and withdrawal (C, F, I) groups displayed very little to almost no Aβ deposits. Diffused intraneuronal Aβ accumulation was seen in vanadium‐exposed mice after 6–18 months of chronic vanadium exposure (B, E, H). Colocalization of Aβ immunolabeling with the neuronal marker (NeuN) revealed pyramidal cell pathology including pyknosis and cytoplasmic loss at the anatomical site of Aβ deposition [(n = 8 mice/group; intensity analysis = 6 mice; Scale bar, 50 μm; p < 0.05) (*p < 0.05; ***p < 0.001; ****p < 0.0001)].