Mitochondrial dysfunction driven by the LRRK2-mediated pathway is associated with loss of Purkinje cells and motor coordination deficits in diabetic rat model

Abstract

Diabetic neuropathy develops on a background of hyperglycemia and an entangled metabolic imbalance. There is increasing evidence of central nervous system involvement in diabetic neuropathy and no satisfactory treatment except maintenance of good glycemic control, thereby highlighting the importance of identifying novel therapeutic targets. Purkinje cells are a class of metabolically specialized active neurons, and degeneration of Purkinje cells is a common feature of inherited ataxias in humans and mice. However, whether Purkinje cells are implicated in diabetic neuropathy development under metabolic stress remains poorly defined. Here, we revealed a novel leucine-rich repeat kinase 2 (LRRK2)-mediated pathway in Purkinje cells that is involved in the pathogenesis of diabetic neuropathy from a 24-week long study of streptozotocin (STZ)-diabetic rats. We found that hyperglycemia, cerebellum proinflammatory cytokines, and chemokines increased markedly in 24-week STZ-diabetic rats. Furthermore, we demonstrated that degeneration of Purkinje cells is characterized by progressive swellings of axon terminals, no autophagosome formation, the reduction of LC3II/LC3I and Lamp2, and accumulation of p62 puncta in 24-week STZ-diabetic rats. Importantly, a higher expression level of LRRK2-mediated hyperphosphorylation of tau along with increased mitochondrial dynamin-like protein (mito-DLP1) was demonstrated in 24-week STZ-diabetic rats. This effect of LRRK2 overexpression induced mitochondrial fragmentation, and reduced mitochondrial protein degradation rates were confirmed in vitro. As a consequence, 24-week STZ-diabetic rats showed mitochondrial dysfunction in cerebellar Purkinje neurons and coordinated motor deficits evaluated by rotarod test. Our findings are to our knowledge the first to suggest that the LRRK2-mediated pathway induces mitochondrial dysfunction and loss of cerebellar Purkinje neurons and, subsequently, may be associated with motor coordination deficits in STZ-diabetic rats. These data may indicate a novel cellular therapeutic target for diabetic neuropathy.

Figure 1

The intake of food and water, body weights, blood glucose, and motor coordination function in STZ-diabetic rats. (a) The food intake and (b) water intake of the STZ-diabetic rats (n=40/group) compared with control rats (n=25/group). (c) The body weights and (d) blood glucose concentrations of the STZ-diabetic rats compared with control rats maintained for 24 weeks of observation (one-way ANOVA, *P<0.05, **P<0.01, significantly different from control rats). Error bars indicate mean±S.D. from three independent experiments. (e) Behavioral analysis of 24-week STZ-diabetic rats (n=9/group) and control rats (n=8/group) in rotarod test. STZ-diabetic and control rats were tested for three consecutive trials on the rotarod. Results were expressed as the time spent (in seconds) on the rotarod (one-way ANOVA, *P<0.05, significantly different from control rats). Data represent mean±S.D. from 3 to 4 independent experiments

Figure 2

The TNF-α, IL-6, and MCP-1 concentrations in the cerebella of STZ-diabetic rats. The concentration of tumor necrosis factor (TNF)-α, interleukin (IL)-6, and MCP-1 in STZ-diabetic rats at 4 and 24 weeks compared with controls (^#P<0.05, 24-week diabetic rats versus 4-week rats; *P<0.05, 24-week diabetic versus 24-week control rats). Error bars indicate mean±S.D. from three independent experiments. Two-way ANOVA followed by the Newman–Keuls post hoc testing for pair-wise comparison was used for analysis of diabetic and control rats at two time points (4 and 24 weeks)

Figure 3

Autophagic activity protein expression, anti-LC3-labeled autophagosomes, and p62 accumulation in Purkinje cells of 24-week STZ-diabetic rats. (a) Autophagy-associated proteins LC3I, LC3II, Lamp2, and p62 levels were analyzed by immunoblotting at 4 and 24 weeks of STZ-diabetic rats compared with controls (Ctl-4, 4-week control rats; Ctl-24, 24-week control rats; Dia-4, 4-week diabetic rats, Dia-24, 24-week diabetic rats). The relative expression of LC3II, Lamp2, and p62/β-actin is shown in the right panel (^#P<0.05, 24-week diabetic rats versus 4-week diabetic rats; *P<0.05, **P<0.01, 24-week diabetic versus 24-week control rats). Mean±S.D. from three independent experiments. Two-way ANOVA followed by the Newman–Keuls post hoc testing for pair-wise comparison was used for analysis of diabetic and control rats at two time points (4 and 24 weeks). (b) Representative confocal images showed the redistribution of anti-LC3 green foci in the DCN of control (n=7/group) (up) and 24-week STZ-diabetic rats (n=6/group) (down). (c) Levels of p62 were exhibited in dystrophic swellings of Purkinje cells in 24-week STZ-diabetic rats. Anti-p62 immunofluorescent staining (in green) showed p62 in Purkinje cell axonal dystrophic swellings (Calbindin labeling in red). (d) Quantified levels of p62. The levels of p62 were normalized against values from STZ-diabetic rats (one-way ANOVA, **P<0.01, 24-week diabetic versus 24-week control rats). Mean±S.D. from three independent experiments

Figure 4

The degeneration and loss of Purkinje cells in 24-week STZ-diabetic rats. (a) Hematoxylin and eosin-stained midsagittal cerebella. (b and e) Representative confocal images showed the redistribution of green foci in the Purkije cells of STZ-diabetic rats (n=6/group). (c) Immunofluorescent staining with monoclonal anti-NeuN antibody in STZ-diabetic rats. (f) Immunofluorescent staining with monoclonal anti-Calbindin antibody in STZ-diabetic rats. (d) Overlapping of green fluorescence with red immunofluorescent staining of monoclonal anti-NeuN in STZ-diabetic rats. (g) Overlapping of green fluorescence with red immunofluorescent staining of anti-Calbindin in STZ-diabetic rats. (h and k) Representative confocal images showed the redistribution of green foci in the Purkinje cells of control rats. (i) Immunofluorescent staining with monoclonal anti-NeuN antibody in control rats. (l) Immunofluorescent staining of monoclonal anti-Calbindin antibody in control rats. (j) Overlapping of green fluorescence with red immunofluorescent staining of monoclonal anti-NeuN in control rats. (m) Overlapping of green fluorescence with red immunofluorescent staining of anti-Calbindin in control rats. (n) Representive presence of Purkinje cell loss in the DCN in two groups by Calbindin/Alexafluor488 (green). (o) Quantification of the images of panel (n). Bar, 20 μm. The Purkinje cell number was normalized against values from control rats. Differences between means were analyzed by one-way ANOVA. **P<0.01, significantly different from control rats. Data represent mean±S.D. from 3 to 4 independent experiments

Figure 5

The distribution and expression of LRRK2 in the brain of STZ-diabetic rats. (a and b) Neuroanatomical distribution of LRRK2 mRNA was detected in Purkinje cells of 24-week STZ-diabetic and control rats, respectively. Cerebellar cortex rat sections were hybridized with a digoxigenin-labeled riboprobe complementary to rat LRRK2 and further processed as described in the Materials and Methods section. (c) LRRK2 expression of Purkinje cells from 4-week STZ-diabetic (n=6/group), 24-week STZ-diabetic (n=6/group), and control rats were detected by western blotting. Densitometry values represented the ratio of LRRK2/Actin was showed in the right panel (^#P<0.05, 24-week diabetic rats versus 4-week control rats; *P<0.05, 24-week diabetic versus 24-week control rats). Error bars indicate mean±S.D. from three independent experiments. Two-way ANOVA followed by the Newman–Keuls post hoc testing for pair-wise comparison was used for analysis of diabetic and control rats at two time points (4 and 24 weeks)

Figure 6

The effect of LRRK2 overexpression on phosphorylation of tau and mito-DLP1 levels in vivo, mitochondrial morphology, and protein degradation in vitro. (a) Proteins were analyzed using antibodies against LRRK2, phospho-tau, and non-phosphorylated tau. (b) The ratio of P-tau/tau is shown in the right panel (one-way ANOVA, *P<0.05). (c) Representative immunoblot and (d) quantification analysis levels of mito-DLP1 in diabetic rats compared with controls (one-way ANOVA, *P<0.05). (e) Representative electron microscopy analysis of multilamellar onion-like structures in cultured cerebellar Purkinje neurons transfected by LRRK2 compared with controls, arrows marked the mitophagy inside cells. At least 30 mitochondria were analyzed. (f) Mitochondrial proteins were measured in cultured cerebellar Purkinje neurons transfected by LRRK2-inserted pCMV-AC-GFP vectors. Level of the mitochondrial proteins was analyzed using antibodies against complex II (CII), ATP synthase (CV), porin, and Tom20. Golgi apparatus, cytosol, lysosomes, and endoplasmic reticulum were analyzed using Golgi58, Gapdh, Lamp2, and calregulin (calreg) antibodies. Quantification of protein level is shown in the right panel. Black bars are LRRK2, and gray bars are controls (one-way ANOVA, *P<0.05, significantly different from control cells). Data are presented as mean±S.D. from 3 to 4 independent experiments

Figure 7

The mitochondrial function in cerebellar Purkinje neurons of 24-week STZ-diabetic rats. (a) Immunofluorescence panels of cerebellar slice cultures from 24-week STZ-diabetic and control rats. Cultures were treated with JC-1, a cationic dye that exhibits green emission in weakly polarized mitochondria that shifts to red in more strongly depolarized mitochondria. Optical sections of 300–400 nm were taken, line-averaged twice to improve the signal-to-noise ratio, and image stacks were deconvolved by using the Huygens Essential software (scale bar: 10 μm). (b) Cerebellar Purkinje neurons from 24-week STZ-diabetic and control rats were stained with Mitotracker green and Mitotracker deep red or MitoSOX for 30 min and analyzed by flow cytometry