Overexpression of Sirtuin 1 protein in neurons prevents and reverses experimental diabetic neuropathy

Abstract

In diabetic neuropathy, there is activation of axonal and sensory neuronal degeneration pathways leading to distal axonopathy. The nicotinamide-adenine dinucleotide (NAD+)-dependent deacetylase enzyme, Sirtuin 1 (SIRT1), can prevent activation of these pathways and promote axonal regeneration. In this study, we tested whether increased expression of SIRT1 protein in sensory neurons prevents and reverses experimental diabetic neuropathy induced by a high fat diet (HFD). We generated a transgenic mouse that is inducible and overexpresses SIRT1 protein in neurons (nSIRT1OE Tg). Higher levels of SIRT1 protein were localized to cortical and hippocampal neuronal nuclei in the brain and in nuclei and cytoplasm of small to medium sized neurons in dorsal root ganglia. Wild-type and nSIRT1OE Tg mice were fed with either control diet (6.2% fat) or a HFD (36% fat) for 2 months. HFD-fed wild-type mice developed neuropathy as determined by abnormal motor and sensory nerve conduction velocity, mechanical allodynia, and loss of intraepidermal nerve fibres. In contrast, nSIRT1OE prevented a HFD-induced neuropathy despite the animals remaining hyperglycaemic. To test if nSIRT1OE would reverse HFD-induced neuropathy, nSIRT1OE was activated after mice developed peripheral neuropathy on a HFD. Two months after nSIRT1OE, we observed reversal of neuropathy and an increase in intraepidermal nerve fibre. Cultured adult dorsal root ganglion neurons from nSIRT1OE mice, maintained at high (30 mM) total glucose, showed higher basal and maximal respiratory capacity when compared to adult dorsal root ganglion neurons from wild-type mice. In dorsal root ganglion protein extracts from nSIRT1OE mice, the NAD+-consuming enzyme PARP1 was deactivated and the major deacetylated protein was identified to be an E3 protein ligase, NEDD4-1, a protein required for axonal growth, regeneration and proteostasis in neurodegenerative diseases. Our results indicate that nSIRT1OE prevents and reverses neuropathy. Increased mitochondrial respiratory capacity and NEDD4 activation was associated with increased axonal growth driven by neuronal overexpression of SIRT1. Therapies that regulate NAD+ and thereby target sirtuins may be beneficial in human diabetic sensory polyneuropathy.

Keywords: NAD+; NEDD4; diabetic neuropathy; mitochondria; sirtuins.

Figure 1

Expression and regulation of SIRT1 in brain and DRGs (n = 6). (A) SIRT1 protein expression was measured by western blot in protein extracts from DRGs using a polyclonal rabbit antibody that recognizes both the endogenous protein and the transgene. In contrast, anti-HA antibody recognizes only the SIRT1 transgene expression. GAPDH was used as loading control. nSIRT1 expression was induced by feeding the bigenic mice with normal diet or suppressed by feeding with DOX-containing diet. (B and C) Coronal hippocampal brain sections from nmito-eYFP (express eYFP in neuronal mitochondria) and nSIRTOE/mito-eYFP mice (express SIRT1 in neurons and eYFP in neuronal mitochondria) were incubated with primary rabbit polyclonal SIRT1 antibody. Sections were then incubated with goat anti-rabbit (Alexa Fluor® 594) secondary antibody, followed by Hoechst 33342 counterstain. Brain and DRG sections were imaged on a Keyence BZ-X800E fluorescence microscope using appropriate filters. Optical sectioning function was applied during image requisition to eliminate fluorescence blurring to produce a confocal-like image. (D and E) Sections of DRG from nmito-eYFP and nSIRTOE/mito-eYFP mice were immunostained with primary rabbit polyclonal SIRT1 antibody, then with goat anti-rabbit (Alexa Fluor® 594) secondary antibody, and finally with Hoechst 33342 dye. The images represent an overlay of all stains. Higher magnification is shown in the inserts. n = nucleus; m = mitochondrion; s = SIRT1. In DRG, the satellite glial cells that surround DRGs are intensely stained with Hoechst 33342 to mark the nuclei of satellite glial cells. In nSIRT1OE showed clear nuclear localization in hippocampal CA1 neurons and this localization was distinct from mito-eYFP fluorescence. In contrast, in DRG neurons, SIRT1 has a nuclear and cytoplasmic localization. Green = neuronal mitochondria; red = neuronal SIRT1; blue = neuronal nuclei. Scale bars = 50 µm. WT = wild-type.

Figure 2

nSIRT1OE prevents HFD-induced neuropathy (n = 6). IENFD in paw skin biopsy is preserved in nSIRT1OE HFD-fed mice. Images show 50-µm thick paw skin sections immunostained with anti-PGP9.5 antibody in wild-type (WT) and nSIRT1OE mice that were fed a control diet or HFD for 2 months. e = epidermis; d = dermis. In the insets, to delineate fibre crossing at the dermo-epidermal junction better, slides were counterstained by dipping in eosin (Sigma-Aldrich Eosin Y solution HT110316). ef = example of an epidermal fibre; df = example of a dermal fibre. Scale bars = 100 µm; insets = 50 µm. (E) Graphic representation of the IENFD. The large central black horizontal bar for each group indicates the mean. The smaller lower bar is the 25th percentile and the smaller upper bar is the 75th percentile. ***P < 0.001 WT+HFD compared to WT+CD; ⁺⁺P < 0.01 nSIRT1OE+CD compared to WT+CD and ^###P < 0.001 nSIRT1OE+HFD compared to WT+HFD.

Figure 3

nSIRT1 overexpression reverses neuropathy (n = 6). (A) Schematic representation of the reversal study. SIRT1 transgene expression was turned off by feeding the nSIRT1OE mice with DOX for 2 weeks before the start of the experiment. Zero-time refers to the time of onset of feeding the mice with a HFD. Group 1: nSIRT1OE Off + CD (4 months); Group 2: nSIRT1OE Off + HFD (4 months); Group 3: nSIRT1OE Off + HFD (same as Group 2 but up to 2 months only); Group 4: nSIRT1OE On + HFD (2–4 months). Group 4 are the same mice as in Group 3 with nSIRT1OE Off (0–2 months) but with nSIRT1OE On from 2 to 4 months. As the data were not statistically different for Groups 3 and 4 up to 2 months, the data has been combined in the graphs. (B) Weekly measurement of random blood glucose before and after turning on SIRT1OE. nSIRTOE did not significantly affect the blood glucose: when SIRT1 was turned on (nSIRT1 On + HFD, Group 4 at 4 months) compared to Group 2 at 4 months (nSIRT1 Off + HFD). However, there was a significant difference between Group 1 (SIRT1 Off + CD) at 4 months and Group 2 (SIRT1 Off + HFD) and Group 4 (SIRT1 On + HFD) (***P < 0.001). (C) Measurement of the sciatic-fibular motor nerve conduction velocity showing reversal in nSIRT1OE mice after turning on SIRT1 expression with the removal of DOX from the diet.***P < 0.001, Group 1 (SIRT1Off + CD) at 2 and 4 months and Group 2 (nSIRT1OE Off + HFD) at 2 months. ⁺⁺⁺P < 0.001, Group 2 (SIRT1 Off + HFD) at 4 months compared to Group 4 (SIRT1 On + HFD). (D) Paw withdrawal threshold measured using Von Frey filaments showing reversal in nSIRT1OE mice after turning on SIRT1 expression with the removal of DOX from the diet. ***P < 0.001, Group 1 (SIRT1 Off + CD) at 2 and 4 months compared with Group 2 (nSIRT1OE Off + HFD) at 2 months. ⁺⁺⁺P < 0.001, Group 2 (SIRT1 Off + HFD) at 4 months compared to Group 4 (SIRT1 On + HFD).

Figure 4

nSIRT1OE reverses HFD-induced loss of IENFD (n = 6). (A) Schematic representation of the reversal study representing the four groups (Groups 1–4). SIRT1 transgene expression was turned off by feeding the nSIRT1OE mice with DOX for 2 weeks before the start of the experiment. Zero-time refers to the time of onset of feeding the mice with a HFD. Intra-epidermal nerve fibre immunohistochemistry and fibre counts in paw skin biopsy from nSIRT1OE are reversed in HFD-fed mice by nSIRT1OE. Images show 50-µm thick paw skin sections immunostained with anti-PGP9.5 antibody (B, C, E and F). e = epidermis; d = dermis. In the insets, to delineate fibre crossing at the dermo-epidermal junction better, slides were counterstained by dipping in eosin (Sigma-Aldrich Eosin Y solution HT110316). ef = example of an epidermal fibre; df = example of a dermal fibre. Scale bars = 100 µm; insets = 50 µm. (D) Graph showing the combined IENFD for all groups. There is a reduction in IENFD in HFD-fed SIRT1 Off + HFD-fed mice (Groups 2 and 3) as compared with SIRT1 Off + CD-fed mice (Group 1) at 2 and 4 months. ***P < 0.001 nSIRT1 Off + HFD compared to nSIRT1 Off + CD at 4 months. ⁺⁺P < 0.01 nSIRT1 Off + HFD compared to nSIRT1 Off + CD at 2 months. Neuronal overexpression of SIRT1 protein (nSIRT1OE) by the removal of DOX in HFD-fed mice after 2 months (Group 4) reversed the loss of epidermal fibres and IENFD counts at 4 months. ^###P < 0.001 nSIRT1OE On + HFD at 4 months + HFD compared to nSIRT1 Off + HFD at 2 months.

Figure 5

Mitochondrial respiration (n = 6). Measurement of mitochondrial function in cultured DRG neurons using the XF24 analyser. Oxygen consumption rate was measured at basal level with the subsequent and sequential addition of oligomycin, FCCP and rotenone+antimycin A (AA) to DRG neurons cultured from (A) 3-month-old wild-type (WT) and nSIRT1OE mice. Levels of oxygen consumption rate were normalized per 4000 cells. DRG neurons were cultured for 24 h either in low (5.5 mM) or high (30 mM) glucose. Dotted line represents oxygen consumption rate measurements in DRG neurons cultures in 30 mM glucose, straight lines represent oxygen consumption rate measurements in DRG neurons cultures in 5 mM glucose. Red coloured lines are from nSIRT1OE DRG neurons and blue are from wild-type DRG neurons. From the oxygen consumption rate, basal respiration and maximal capacity respiration were calculated and are shown in B and C. The significance was calculated by ANOVA multiple comparison post hoc Tukey analysis. The statistical significance is indicated. The raw data are shown in Table 2.

Figure 6

NEDD4-1 deacetylation by nSIRT1OE (n = 6). Western blot analysis of NAD⁺-consuming enzymes and acetylated proteins in DRG protein extracts prepared from wild-type (WT) and nSIRT1OE mice fed either a control diet (CD) or HFD for 4 months. (A) Preparation of DRG protein extracts, blot analysis, the source and the dilution of the antibodies used are described in the ‘Materials and methods’ section. (B–E) Quantification of the intensity of the bands are shown. Significant decrease in SIRT1 protein, detected with the rabbit polyclonal anti-SIRT1 that recognized both endogenous and overexpressed SIRT1 protein (Millipore 07-131, 1:1000) (B), decrease in PGC-1α protein levels (C), increase in cleaved PARP1 protein (D) and increase in acetylated 118 kDa protein (E) were observed in WT+HFD samples, but not in nSIRT1OE DRG neurons with a control diet or HFD. ***P < 0.001 WT+HFD compared with nSIRT1OE+HFD, WT+CD, and nSIRT1OE+CD in B, C and E and WT+HFD compared with WT+CD, and nSIRT1OE + CD in D. ^###P = 0.008 (D) for a significant decrease in cleaved PARP1 protein in nSIRT1OE+HFD mice when compared with WT+HFD.