Defective Mitochondrial Dynamics Underlie Manganese-Induced Neurotoxicity

Abstract

Perturbations in mitochondrial dynamics have been observed in most neurodegenerative diseases. Here, we focus on manganese (Mn)-induced Parkinsonism-like neurodegeneration, a disorder associated with the preferential of Mn in the basal ganglia where the mitochondria are considered an early target. Despite the extensive characterization of the clinical presentation of manganism, the mechanism by which Mn mediated mitochondrial toxicity is unclear. In this study we hypothesized whether Mn exposure alters mitochondrial activity, including axonal transport of mitochondria and mitochondrial dynamics, morphology, and network. Using primary neuron cultures exposed to 100 μM Mn (which is considered the threshold of Mn toxicity in vitro) and intraperitoneal injections of MnCl₂ (25mg/kg) in rat, we observed that Mn increased mitochondrial fission mediated by phosphorylation of dynamin-related protein-1 at serine 616 (p-s616-DRP1) and decreased mitochondrial fusion proteins (MFN1 and MFN2) leading to mitochondrial fragmentation, defects in mitochondrial respiratory capacity, and mitochondrial ultrastructural damage in vivo and in vitro. Furthermore, Mn exposure impaired mitochondrial trafficking by decreasing dynactin (DCTN1) and kinesin-1 (KIF5B) motor proteins and increasing destabilization of the cytoskeleton at protein and gene levels. In addition, mitochondrial communication may also be altered by Mn exposure, increasing the length of nanotunnels to reach out distal mitochondria. These findings revealed an unrecognized role of Mn in dysregulation of mitochondrial dynamics providing a potential explanation of early hallmarks of the disorder, as well as a possible common pathway with neurological disorders arising upon chronic Mn exposure.

Keywords: Cytoskeleton; Manganese; Mitochondrial dynamics; Neuron; Striatum.

Fig. 1

Mn induces mitochondrial fission but not fusion in primary neuron cultures. a Experimental workflow of primary neuron culture isolated from E15 embryo rats and exposed to Mn at DIV8. b Representative Western blot of DRP1 protein level from primary neuron cultures exposed to Mn (100, 500, and 1000 μM) for 1 h and 24 h. Bars expressed as mean ± SD show the protein quantification of DRP1 in control and primary neurons exposed to 100 μM Mn for 1 h (n=6). ACTB was used as a loading control for both control and Mn-exposed primary neurons. c Representative Western blot of p-616-DRP1 in control and primary neuron cultures exposed to 100 μM Mn for 1 h. Bars expressed as mean ± SD show the fold change of p-616-DRP1/DRP1 in control and Mn-exposed primary neurons (n=5). d Representative confocal microscopy images show immunostaining of p-616-DRP1 (red) together with neuron marker TU-20 (green) in control and primary neurons exposed to 100 μM Mn for 1 h. Scale bars 50 μm. e Quantification of fluorescent intensity of p-s616-DRP1 in control and Mn-exposed primary neurons exposed to 100 μM Mn for 1 h. Bars expressed as mean ± SD show fluorescent intensity per cells (n=3 independent experiments with ≥ 50 neurons each). f Representative Western blot of VPS35, MFN1, and MFN2 proteins in control and primary neurons exposed to 100 μM Mn for 1 h. Bars expressed as mean ± SD show fold change of VPS35, MFN1, and MFN2 proteins in control and Mn-exposed primary neurons (n=5 independent experiments). g Representative confocal microscopy images of control primary neurons exposed to 100 μM Mn for 1 h. Scale bars 10 μm. h Quantification of mitochondrial morphology in control and primary neurons after Mn treatment to overt fragmented or overt intermediate-to-fused mitochondria. Bars expressed as mean ± SD show the percentage of mitochondria (n=4 independent experiments with n≥ 50 neurons each). Statistical significance was analyzed by Student’s t-test (*p < 0.05; **p < 0.01; ***p < 0.001, **** p < 0.0001) compared to control. Filam, filamentous; Interm, intermediate; Frag, fragmented; DIV, days in vitro

Fig. 2

Mitochondrial respiration is decreased by Mn exposure in primary neurons. a Oxygen consumption rate (OCR) measurements in control and primary neurons exposed to Mn (100 μM, 1h) were obtained over time (min) using an extracellular flux analyzer (Seahorse Bioscience). OCR was measured at baseline and following sequential additions of 2 mM oligomycin, 1.5 mM FCCP, and 1 mM rotenone in both control and Mn-exposed primary neuron cultures. b–d Quantification of basal OCR, maximal OCR, and ATP-linked OCR in control and primary neurons exposed to Mn (100 μM, 1h). e Mitochondrial membrane potential was measured by fluorescence after labeling primary neurons with TMRE dye. Results are expressed as mean ± SD (n = 4 independent cultures). Statistical significance was analyzed by Student’s t-test (*p < 0.05;** p < 0.01; nd, no differences)

Fig. 3

Intraperitoneal administration of Mn intervenes in mitochondrial dynamics in rat striatum. a Schematic design of in vivo experiments. Rats received an intraperitoneal injection of vehicle (control) or MnCl₂ 4H₂O (at 25mg/kg) for 15 days, and the striatum was collected 24 h after the last Mn injection (day 16). b Representative Western blot of enriched mitochondrial and cytosolic fractions from striatum of control and Mn-exposed groups. Bars expressed as mean ± SD show the fold change of DRP1 from the mitochondrial fraction in the control and Mn-exposed groups (n=3). Cytoplasmic and mitochondrial fractions are normalized to ACTB and TOM20, respectively. c Representative Western blot of mitochondrial dynamic-related proteins (DRP1, p-ser616, VPS35, MFN1, and MFN2) in control and Mn-treated striatum. Bars expressed as mean ± SD show the fold change of p-ser616, VPS35, MFN1, and MFN2 proteins in control and Mn-exposed groups (n=6). Statistical significance was analyzed by Student’s t-test (*p < 0.05; **p < 0.01; **** p < 0.0001). d, e Representative transmission electron micrographs of mitochondria from striatum in control group showing typical tubular cristae and crista junctions. f Representative electron micrograph showing appearance of spaces without cristae in the mitochondrial matrix from striatum of Mn-exposed group. g Three-dimensional reconstruction of a donut-shaped mitochondrion from striatum in Mn-exposed group. Scale bars 500 nm, 200 nm, 200 nm, 300 nm, respectively

Fig. 4

Mn toxicity promotes mitochondrial fission in rat striatum. a Representative electron microscopy micrographs showing mitochondrial morphology in neuropils from striatum of control and Mn-exposed groups. Scale bars 2 μm. b–d Photoshop software was used for quantifying mitochondrial area (μm²) (b), perimeter (μm) (c), and circularity index (d) in the neuropils from striatum of control and Mn-exposed groups. e Electron microscopy micrographs showing mitochondrial morphology in the soma. f–h Bar graphs show quantification of mitochondrial area (ratio) (f), mitochondrial perimeter (ratio) (g), and circularity index (h) in the soma from striatum of control and Mn-exposed groups. i, j Mitochondrial density quantification (number of mitochondria per area) in the neuropils (i) and in the soma (j) from striatum of control and Mn-exposed groups. Results are expressed as mean ± SD (n=4). Statistical significance was analyzed by Student’s t-test (*p < 0.05; **p < 0.01; **** p < 0.0001; nd, no differences). Scale bars 1 μm

Fig. 5

Mitochondrial transport failure underlies Mn-induced neurotoxicity. a Representative Western blot of acetyl-K40-TUBA in primary neurons exposed to Mn (100 μM).Bar graphs show the protein quantification analysis (n=4). TUBA was used as a loading control for both control and Mn-exposed groups. b Representative confocal microscopy images of a primary neuron immunostained for TUBA (green). Scale bars 45 μm. c Representative electron microscopy micrographs showing the distribution of the cytoskeleton in the axon (left) and in the soma (right) of control and primary neurons treated with Mn. Scale bars 1 μm (left) and 0.5 μm (right), respectively. d Representative Western blot of KIF5B and DCTN1 levels obtained from primary neurons in control and Mn-exposed groups. Bars expressed as mean ± SD show fold change of KIF5B and DCTN1 proteins in control and Mn-exposed groups (n=4). TUBA was used as a loading control for both control and Mn-exposed groups. e Representative Western blot analysis of acetyl-K40-TUBA, KIF5B, and DCTN1 in rat striatum exposed to Mn. Bars expressed as mean ± SD show the fold change of acetyl-K40-TUBA and KIF5B proteins in control and Mn-exposed groups (n=6). TUBA and ACTB were used as a loading control for both control and Mn-exposed groups. f Analysis of mitochondrial displacement rates in control and primary neurons exposed to Mn. Results are expressed as mean ± SD (n=4 independent experiments with ≥ 15 neurons each). Statistical significance was analyzed by Student’s t-test (*p < 0.05; **p < 0.01; ***p < 0.001;**** p < 0.0001)

Fig. 6

RNA-seq analysis reveals alterations in cytoskeletal genes in striatum treated with Mn. a Principal component analysis (PCA) corresponding to all differentially expressed genes (DEG) from the striatum exposed to Mn and control (n=3). b Volcano plot of upregulated (green) and downregulated (blue) genes in striatum exposed to Mn when compared to the control after DESeq2 analysis. The differences were considered significant when adjusted p value < 0.05 and absolute log₂ fold change ≥ 1. c Heatmap representation of DE cytoskeleton-related genes in Mn-treated striatum when compared to the control group. Results are expressed as z-score. d Network analysis of differentially regulated cytoskeleton genes in striatum exposed to Mn. PC, principal component

Fig. 7

Mn alters mitochondrial communication in rat striatum. a Representative electron microscopy micrographs of mitochondrial contact sites in adjacent mitochondria illustrating cristae alignment. Scale bars 300 nm. b Z-stack at EM resolution from serial block face scanning electron microscopy (AT-SEM) of mitochondrial nanotunnel formation from the striatum in control and Mn-exposed groups. Scale bars 300 nm. c Three-dimensional reconstructions of mitochondria connected via nanotunnels in the striatum of control and Mn-exposed groups. Scale bars 300 nm. d, e Measurement of nanotunnel length (nm) (d) and diameter (nm) (e) in control and Mn-exposed groups assessed by IMOD image analysis software. Results are expressed as mean ± SD (n=3 with ≥ 10 nanotunnels each). Statistical significance was analyzed by Student’s t-test (*p < 0.05; nd, no differences). f Image stack from AT-SEM of mitochondria (colored in pink) in neuropils from striatum of control and Mn-exposed groups. Scale bars 800 nm