The effect of manganese exposure in Atp13a2-deficient mice

Abstract

Loss of function mutations in the P₅-ATPase ATP13A2 are associated with Kufor-Rakeb Syndrome and Neuronal Ceroid Lipofuscinosis. While the function of ATP13A2 is unclear, in vitro studies suggest it is a lysosomal protein that interacts with the metals manganese (Mn) and zinc and the presynaptic protein alpha-synuclein. Loss of ATP13A2 function in mice causes sensorimotor deficits, enhanced autofluorescent storage material, and accumulation of alpha-synuclein. The present study sought to determine the effect of Mn administration on these same outcomes in ATP13A2-deficient mice. Wildtype and ATP13A2-deficient mice received saline or Mn at 5-9 or 12-19 months for 45days. Sensorimotor function was assessed starting at day 30. Autofluorescence was quantified in multiple brain regions and alpha-synuclein protein levels were determined in the ventral midbrain. Brain Mn, iron, zinc, and copper concentrations were measured in 5-9 month old mice. The results show Mn enhanced sensorimotor function, increased autofluorescence in the substantia nigra, and increased insoluble alpha-synuclein in the ventral midbrain in older ATP13A2-deficient mice. In addition, the Mn regimen used increased Mn concentration in the brain and levels were higher in Mn-treated mutants than controls. These results indicate loss of ATP13A2 function leads to increased sensitivity to Mn in vivo.

Keywords: Alpha-synuclein; Lipofuscin; Manganese; Mice; Parkinson’s disease; Sensorimotor function.

Figure 1

WT and 13a2 mice received daily injections of saline (WT=8, 13a2=7) or MnCl (5mg/kg, ip; WT=8, 13a2=8) for 45 days at 12–19 months of age. Starting on day 30 sensorimotor function was assessed in the cylinder (A: hindlimb steps), on the challenging beam (B: Errors), and in gait (C: maximum stride difference). * represents p≤0.05 compared to WT-Saline, Δ represents p≤0.05 compared to 13a2-Saline.

Figure 2

Concentration (μg/g) of Mn (A) and Fe (B) in sagittal half brain of WT and 13a2 mice at 5–9 months of age. Δ represents p≤0.05 compared to saline treatment in the same genotype. * represents p≤0.05 compared to MnCl-treated WT. Mean ± SEM shown.

Figure 3

Autofluorescent storage material was quantified in the prefrontal cortex (A), hippocampus (B), and cerebellum (C) in 12–19 month old mice treated with saline (WT=4, 13a2=4) or MnCl (WT=4, 13a2=4). Mean ± SEM relative to saline-treated WT values shown. ** represents p≤0.01 compared WT receiving the same treatment.

Figure 4

Autofluorescent storage material quantified in the substantia nigra in 12–19 month old mice treated with saline (WT=4, 13a2=4) or MnCl (WT=4, 13a2=4). Mean ± SEM relative to saline-treated WT values shown. Representative photomicrographs (right) of autofluorescent storage material in substantia nigra in saline-treated 13a2 mice (top panel) and MnCl-treated 13a2 mice (bottom panel), Scale bar= 50μm, Δ represents p≤0.05 compared to saline-treated 13a2 mice.

Figure 5

Representative images of tyrosine hydroxylase immunoreactivity (TH-IR) overlap with autofluorescence storage material in the substantia nigra of 12–19 month WT and 13a2 mice treated with saline or MnCl. Brightfield (TH-IR) and Texas Red (Autofluorescence) images were taken at 10x magnification for each condition. Images were then superimposed. The white box on each superimposed image marks the location of the 20x magnification. The white arrows indicate an example of where overlap of TH-IR with autofluorescence occurs, the blue arrow indicates where there is no overlap. Scale bar= 200μm.

Figure 6

Representative images of alpha-synuclein immunoreactivity (aSyn-IR) overlap with autofluorescence storage material in the substantia nigra of 12–19 month old mice treated with saline or MnCl. Brightfield (aSyn-IR) and Texas Red (Autofluorescence) images were all taken at 10x magnification for each condition. Images were then superimposed. The white box on each superimposed image marks the location of the 20x. The white arrows indicate an example of where overlap of aSyn-IR with autofluorescence occurs. Scale bar= 200μm.

Figure 7

Triton-soluble and triton-insoluble aSyn protein levels were measured in the ventral midbrain of 5–9 (A and C) and 12–19 (B and D) month old mice treated with saline (WT=3–4, 13a2=4) or MnCl (WT=4, 13a2=4). aSyn protein levels were normalized to β-actin and calculated as relative to WT-Saline. Representative blots of aSyn (14kDa) and the loading control β-actin (42 kDa) are shown, WS=WT-saline, WM=WT-MnCl, AS=13a2-saline, and AM=13a2-MnCl treatment. * represents p≤0.05 compared to WT same treatment, Δ represents p≤0.05 compared to 13a2-saline.