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. 2018 Jan 19;9(1):6.
doi: 10.1186/s13293-018-0164-z.

Axonal transport in a peripheral diabetic neuropathy model: sex-dimorphic features

Marzia Pesaresi  1 , Silvia Giatti  1 , Roberto Spezzano  1 , Simone Romano  1 , Silvia Diviccaro  1 , Tiziana Borsello  1   2 , Nico Mitro  1 , Donatella Caruso  1 , Luis Miguel Garcia-Segura  3 , Roberto Cosimo Melcangi  4
Affiliations

Axonal transport in a peripheral diabetic neuropathy model: sex-dimorphic features

Marzia Pesaresi et al. Biol Sex Differ. .

Abstract

Background: Disruption of axonal transport plays a pivotal role in diabetic neuropathy. A sex-dimorphism exists in the incidence and symptomatology of diabetic neuropathy; however, no studies so far have addressed sex differences in axonal motor proteins expression in early diabetes as well as the possible involvement of neuroactive steroids. Interestingly, recent data point to a role for mitochondria in the sexual dimorphism of neurodegenerative diseases. Mitochondria have a fundamental role in axonal transport by producing the motors' energy source, ATP. Moreover, neuroactive steroids can also regulate mitochondrial function.

Methods: Here, we investigated the impact of short-term diabetes in the peripheral nervous system of male and female rats on key motor proteins important for axonal transport, mitochondrial function, and neuroactive steroids levels.

Results: We show that short-term diabetes alters mRNA levels and axoplasm protein contents of kinesin family member KIF1A, KIF5B, KIF5A and Myosin Va in male but not in female rats. Similarly, the expression of peroxisome proliferator-activated receptor γ co-activator-1α, a subunit of the respiratory chain complex IV, ATP levels and the key regulators of mitochondrial dynamics were affected in males but not in females. Concomitant analysis of neuroactive steroid levels in sciatic nerve showed an alteration of testosterone, dihydrotestosterone, and allopregnanolone in diabetic males, whereas no changes were observed in female rats.

Conclusions: These findings suggest that sex-specific decrease in neuroactive steroid levels in male diabetic animals may cause an alteration in their mitochondrial function that in turn might impact in axonal transport, contributing to the sex difference observed in diabetic neuropathy.

Keywords: Dorsal root ganglia; Female; Male; Mitochondria; Neuroactive steroids; Sciatic nerve; Streptozotocin.

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Conflict of interest statement

Ethics approval

The care and husbandry of the animals were approved by the Institutional Review Committee and followed institutional guidelines that are in compliance with national (D.L. No. 26, March 4, 2014, G.U. No. 61 March 14, 2014) and international laws and policies (EEC Council Directive 2010/63, September 22, 2010: Guide for the Care and Use of Laboratory Animals, United States National Research Council, 2011).

Consent for publication

Not applicable

Competing interests

The authors declare that they have no competing interests.

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Figures

Fig. 1
Fig. 1
Effect of short-term diabetes on KIF5A. a RNA was extracted from DRG of control (Ctrl) and diabetic (streptozotocin, STZ) male (M) and female (F) rats. RT-PCR was used to analyze mRNA levels of KIF5A. b KIF5A protein levels analyzed by immunoblotting in DRG total extracts of Ctrl and STZ M and F rats. c KIF5A protein levels analyzed by immunoblotting in isolated axoplasm of Ctrl and STZ M and F rats. Representative Western blots are presented above the graphs, with the respective loading controls (GAPDH). The densitometry of each band was analyzed, and the results are expressed as percentage of M Ctrl. The columns represent the mean ± SEM, n = 6 animals for each group. The effects of sex, diabetes, and the interaction sex by diabetes were analyzed using two-way ANOVA (significance: #p < 0.05; ###p < 0.001) followed by Bonferroni post hoc test (significance: *p < 0.05)
Fig. 2
Fig. 2
Effect of short-term diabetes on KIF5B. a RNA was extracted from DRG of control (Ctrl) and diabetic (streptozotocin, STZ) male (M) and female (F) rats. RT-PCR was used to analyze mRNA levels of KIF5B. b KIF5B protein levels analyzed by immunoblotting in DRG total extracts of Ctrl and STZ M and F rats. c KIF5B protein levels analyzed by immunoblotting in isolated axoplasm of Ctrl and STZ M and F rats. Representative Western blots are presented above the graphs, with the respective loading controls (GAPDH). The densitometry of each band was analyzed and the results are expressed as percentage of M Ctrl. The columns represent the mean ± SEM, n = 6 animals for each group. The effects of sex, diabetes, and the interaction sex by diabetes were analyzed using two-way ANOVA followed by Bonferroni post hoc test (significance: *p < 0.05; **p < 0.01)
Fig. 3
Fig. 3
Effect of short-term diabetes on KIF1A. a RNA was extracted from DRG of control (Ctrl) and diabetic (streptozotocin, STZ) male (M) and female (F) rats. RT-PCR was used to analyze mRNA levels of KIF1A. b KIF1A protein levels analyzed by immunoblotting in DRG total extracts of Ctrl and STZ M and F rats. c KIF1A protein levels analyzed by immunoblotting in isolated axoplasm of Ctrl and STZ M and F rats. Representative Western blots are presented above the graphs, with the respective loading controls (GAPDH). The densitometry of each band was analyzed, and the results are expressed as percentage of M Ctrl. The columns represent the mean ± SEM, n = 6 animals for each group. The effects of sex, diabetes, and the interaction sex by diabetes were analyzed using two-way ANOVA (significance: #p < 0.05; ##p < 0.01) followed by Bonferroni post hoc test (significance: *p < 0.05)
Fig. 4
Fig. 4
Effect of short-term diabetes on Myosin Va. a RNA was extracted from DRG of control (Ctrl) and diabetic (streptozotocin, STZ) male (M) and female (F) rats. RT-PCR was used to analyze mRNA levels of Myosin Va. b Myosin Va protein levels analyzed by immunoblotting in DRG total extracts of Ctrl and STZ M and F rats. c Myosin Va protein levels analyzed by immunoblotting in isolated axoplasm of Ctrl and STZ M and F rats. Representative Western blots are presented above the graphs, with the respective loading controls (GAPDH). The densitometry of each band was analyzed, and the results are expressed as percentage of M Ctrl. The columns represent the mean ± SEM, n = 6 animals for each group. The effects of sex, diabetes, and the interaction sex by diabetes were analyzed using two-way ANOVA (significance: #p < 0.05) followed by Bonferroni post hoc test (significance: *p < 0.05)
Fig. 5
Fig. 5
Effect of short-term diabetes on Dynein. a RNA was extracted from DRG of control (Ctrl) and diabetic (streptozotocin, STZ) male (M) and female (F) rats. RT-PCR was used to analyze mRNA levels of Dynein. b Dynein protein levels analyzed by immunoblotting in DRG total extracts of Ctrl and STZ M and F rats. c Dynein protein levels analyzed by immunoblotting in isolated axoplasm of Ctrl and STZ M and F rats. Representative Western blots are presented above the graphs, with the respective loading controls (GAPDH). The densitometry of each band was analyzed, and the results are expressed as percentage of M Ctrl. The columns represent the mean ± SEM, n = 6 animals for each group. The effects of sex, diabetes, and the interaction sex by diabetes were analyzed using two-way ANOVA (significance: ###p < 0.001) followed by Bonferroni post hoc test
Fig. 6
Fig. 6
Effect of short-term diabetes on PGC-1α gene expression, respiratory chain complex IV expression and mitochondrial DNA. a RNA was extracted from DRG of control (Ctrl) and diabetic (streptozotocin, STZ) male (M) and female (F) rats. RT-PCR was used to analyze mRNA levels of PGC1-α. b Immunoblot of mitochondrial OXPHOS Complex IV from DRG total extracts of Ctrl and STZ M and F rats. Representative Western blots are presented above the graph, with the respective loading control (GAPDH). The densitometry of each band was analyzed, and the results are expressed as percentage of M Ctrl. c Total DNA was isolated from non-diabetic Ctrl and STZ rats and run for quantitative real-time PCR to obtain a relative ratio of mt-CoxII (a gene coded on the mitochondrial genome) over 36B4 (a gene coded on nuclear genome), as indicator for relative mt-DNA copy number. The columns represent the mean ± SEM, n = 6 animals for each group. The effects of sex, diabetes, and the interaction sexby diabetes were analyzed using two-way ANOVA (significance: ##p < 0.01; ###p < 0.001) followed by Bonferroni post hoc test (significance: *p < 0.05; **p < 0.01)
Fig. 7
Fig. 7
Effect of short-term diabetes on proteins involved in mitochondrial fission/fusion: MFN2, Drp1 and OPA1. a Immunoblot of DRP1 phosphorylation from DRG total extracts of control (Ctrl) and diabetic (streptozotocin, STZ) male (M) and female (F) rats. The bar graph represents densitometric analysis and statistical evaluation of DRP1 phosphorylation over total DRP1 (DRP1P/DRP1 tot); representative western blot of phosphorylated and total DRP1 is presented above the graph. b Immunoblot of MFN2 from DRG total extracts of Ctrl and STZ M and F rats. c Immunoblot of OPA1 from DRG total extracts of Ctrl and STZ M and F rats. Representative Western blots are presented above the graphs, with the respective loading controls (GAPDH). The densitometry of each band was analyzed and the results are expressed as percentage of M Ctrl. The columns represent the mean ± SEM, n = 6 animals for each group. The effects of sex, diabetes, and the interaction sex by diabetes were analyzed using two-way ANOVA (significance: #p < 0.05) followed by Bonferroni post hoc test (significance: *p < 0.05)
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