HDAC6 is a therapeutic target in mutant GARS-induced Charcot-Marie-Tooth disease

Abstract

Peripheral nerve axons require a well-organized axonal microtubule network for efficient transport to ensure the constant crosstalk between soma and synapse. Mutations in more than 80 different genes cause Charcot-Marie-Tooth disease, which is the most common inherited disorder affecting peripheral nerves. This genetic heterogeneity has hampered the development of therapeutics for Charcot-Marie-Tooth disease. The aim of this study was to explore whether histone deacetylase 6 (HDAC6) can serve as a therapeutic target focusing on the mutant glycyl-tRNA synthetase (GlyRS/GARS)-induced peripheral neuropathy. Peripheral nerves and dorsal root ganglia from the C201R mutant Gars mouse model showed reduced acetylated α-tubulin levels. In primary dorsal root ganglion neurons, mutant GlyRS affected neurite length and disrupted normal mitochondrial transport. We demonstrated that GlyRS co-immunoprecipitated with HDAC6 and that this interaction was blocked by tubastatin A, a selective inhibitor of the deacetylating function of HDAC6. Moreover, HDAC6 inhibition restored mitochondrial axonal transport in mutant GlyRS-expressing neurons. Systemic delivery of a specific HDAC6 inhibitor increased α-tubulin acetylation in peripheral nerves and partially restored nerve conduction and motor behaviour in mutant Gars mice. Our study demonstrates that α-tubulin deacetylation and disrupted axonal transport may represent a common pathogenic mechanism underlying Charcot-Marie-Tooth disease and it broadens the therapeutic potential of selective HDAC6 inhibition to other genetic forms of axonal Charcot-Marie-Tooth disease.

Figure 1

HDAC6 co-immunoprecipitates with GlyRS. (A) N2a cells were transiently transfected with either pCMV-DDK-tagged human cDNA encoding GlyRS or pCMV-dsRed-tagged human cDNA encoding HDAC6. Co-immunoprecipitation was performed using agarose beads conjugated to an antibody directed against the DDK peptide. Transfected N2a cells were pre-treated with 1 µM tubastatin A (TubA) or vehicle (DMSO). Western blot analysis was performed to check for equal protein expression and co-immunoprecipitation of HDAC6. (B) N2a cells were transiently transfected with either pCMV-DDK-tagged human cDNA encoding wild-type or C175R mutant GlyRS or pCMV-dsRed-tagged human cDNA encoding HDAC6. Co-immunoprecipitation was performed using agarose beads conjugated to an antibody directed against the DDK peptide. Transfected N2a cells were pretreated with 1 µM tubastatin A or vehicle (DMSO). Western blot analysis was performed to check for equal protein expression and co-immunoprecipitation of HDAC6.

Figure 2

RNA interference-mediated knock down of GlyRS affects HDAC6 expression. (A) N2a cells were transfected with siRNA molecules directed against Gars (GlyRS) mRNA or a control siRNA. At 72 h post transfection, cells were collected to check protein expression levels by western blot analysis. GAPDH was used as a loading control. (B) Using densitometry, the GlyRS protein expression level was determined, relative to GAPDH. All values were normalized to control siRNA samples within each experiment: control siRNA 1.00 ± 0.00, n = 3 versus siRNA 1 GlyRS 0.11 ± 0.01, n = 3 versus siRNA 2 GlyRS 0.23 ± 0.05, n = 3 versus siRNA 3 GlyRS 0.09 ± 0.02; one-way ANOVA, F(3,8) = 1.935, P < 0.0001. (C) The HDAC6 expression levels were analysed using densitometry, relative to the gapdh expression levels and normalized to control siRNA values: control siRNA 1.0 ± 0, n = 3 versus siRNA 1 GlyRS 0.57 ± 0.11, n = 3 and siRNA 2 GlyRS 0. 45 ± 0.21, n = 3 and siRNA 3 GlyRS 0.61 ± 0.16, n = 3; One-way ANOVA, F(3,8) = 8.327, P = 0.0076; Dunnett’s multiple comparisons test. (D) pCMV-dsRed plasmids containing either a scrambled shRNA or short hairpin RNA (shRNA) directed against Hdac6 mRNA were transfected in N2a cells. At 72 h post transfection, cells were collected to check protein expression levels by western blot analysis. (E) HDAC6 expression was checked by western blot analysis after 72 h shRNA-mediated knockdown. Densitometry was used to calculate HDAC6 expression levels, relative to GAPDH and all values were normalized to scrambled (SCR) shRNA samples: scrambled shRNA 1.00 ± 0.00, n = 3 versus shRNA Hdac6 0.24 ± 0.22, n = 3; one-sample t-test, t = 6.088, P = 0.0259. (F) The relative GlyRS expression levels were calculated using densitometry, relative to GAPDH levels and normalized to scrambled shRNA values: scrambled shRNA 1.0 ± 0.0, n = 3 versus shRNA Hdac6 0.75 ± 0.09, n = 3; one sample t-test, t = 4.353, P = 0.0489.

Figure 3

Motor and sensory deficits reminiscent of CMT2D are present in 1-year old Gars^C201R/+ mice. (A) The motor performance of Gars^C201R/+ mice and littermate controls (non-transgenic, NTG) was assessed by a rotarod test which accelerates from 4 to 40 rpm for 5 min: non-transgenic 268 ± 43.68 s, n = 5 mice versus Gars^C201R/+ 172 ± 75.51 s, n = 7 mice; unpaired t-test t = 2.531, P = 0.0298. B. Grip strength was measured in all paws using a grid in combination with a dynamometer: non-transgenic 2.55 ± 0.26 N, n = 4 mice versus Gars^C201R/+ 1.54 ± 0.08 N, n = 5 mice; unpaired t-test t = 8.211, P < 0.0001. (C) Mice were tested for the ability to hold on to a grid in the hanging wire test and the time spent hanging was measured with a maximum time limit of 60 s: non-transgenic 58.08 ± 3.84 s, n = 4 mice versus Gars^C201R/+ 17.45 ± 8.96 s, n = 6 mice; unpaired t-test t = 8.436, P < 0.0001. (D) CMAP amplitudes were recorded in the gastrocnemius muscle: non-transgenic 82.68 ± 5.79 mV, n = 4 mice versus Gars^C201R/+ 20.84 ± 3.14 mV, n = 5 mice; unpaired t-test t = 20.61, P < 0.0001. (E) SNAP amplitudes were measured in the tail nerve: non-transgenic 26.75 ± 7.263 µV, n = 4 mice versus Gars^C201R/+ 12.42 ± 4.53 µV, n = 5 mice; unpaired t-test t = 3.645, P = 0.0082. (F) The innervation of neuromuscular junctions was evaluated by quantifying the percentage of neuromuscular junctions demonstrating overlap of neurofilament light, NEFL/synaptic vesicle glycoprotein 2A, SV2 and α-bungarotoxin staining: non-transgenic 3.57 ± 1.43%, n = 3 mice versus Gars^C201R/+ 18.81 ± 8.98 %, n = 6 mice; unpaired t-test t = 2.826, P = 0.0255. (G and H) Innervated (G) and denervated (H) neuromuscular junctions (NMJs) in the gastrocnemius muscle were visualized by immunofluorescence (green: NEFL + SV2; red: α-bungarotoxin). Confocal images are represented. Scale bar = 20 µm. For colour blind corrected figure, see Supplementary Fig. 2F and G.

Figure 4

Acetylated α-tubulin levels are altered in peripheral nerve tissue from Gars^C201R/+ mice. (A) Western blot analysis was used to determine the acetylation status of α-tubulin in tissue homogenates from Gars^C201R/+ and littermate control mice (non-transgenic, NTG). An antibody directed against GlyRS was used to assess GlyRS expression levels. GAPDH expression levels were used as a control for equal sample loading. (B) The amount of acetylated α-tubulin compared to the total α-tubulin levels was quantified by densitometry and the values were normalized to the non-transgenic samples: non-transgenic 1.00 ± 0.00, n = 3 samples versus Gars^C201R/+ 0.60 ± 0.15 ms, n = 3 samples; unpaired t-test: t = 4.518, P = 0.0107. (C) The level of acetylated α-tubulin was detected by western blot analysis in tissue homogenates from spinal cord from Gars^C201R/+ and littermate control (non-transgenic) mice. GlyRS expression levels were checked and GAPDH was used as a loading control. (D) The ratio between acetylated α-tubulin and total α-tubulin was quantified by densitometry and values were normalized to non-transgenic samples. n = 3 mice.

Figure 5

Altered α-tubulin acetylation and disturbances in mitochondrial axonal transport in DRGs from Gars^C201R/+ mice are rescued by selective HDAC6 inhibition. (A) Western blot analysis was used to determine the acetylation status of α-tubulin in DRG homogenates from Gars^C201R/+ and littermate control mice (non-transgenic, NTG). GAPDH and calnexin were used as loading control. (B) Densitometry was used to quantify the ratio of acetylated α-tubulin to GAPDH levels. Values were normalized to non-transgenic samples. Non-transgenic 1.00 ± 0.17, n = 6 mice versus Gars^C201R/+ 0.75 ± 0.14, n = 5 mice; unpaired t-test t = 2.574, P = 0.0300. (C) Representative kymographs from the analysis of mitochondrial movement in non-transgenic (top) and Gars^C201R/+ (middle and bottom) DRG neurons. Stationary mitochondria are represented by vertical lines. Lines deflecting to the right or left are anterograde or retrograde moving mitochondria, respectively. Horizontal scale bar = 30 μm. Vertical scale bar = 80 s. DRG neurons were treated with vehicle or 1 µM tubastatin A (TubA). (D) Mitochondrial movement was quantified within one neurite per cell, relative to the total number of mitochondria per 100 µm. Gars^C201R/+ DRG neurons were treated with vehicle or with 1 µm tubastatin A (TubA): non-transgenic 19.14 ± 2.48% moving mitochondria, n = 6 different DRG primary cultures versus Gars^C201R/+ + vehicle 7.53 ± 5.69% moving mitochondria, n = 7 different DRG primary cultures versus Gars^C201R/+ + tubastatin A 21.21 ± 9.07% moving mitochondria, n = 5 different DRG primary cultures; Kruskal-Wallis test, P = 0.0015. (E) Anterograde and retrograde moving mitochondria were quantified from the kymographs obtained from non-transgenic, vehicle-treated and tubastatin A-treated Gars^C201R/+ DRG neurons: antero 0.95 ± 0.46 mitochondria, n = 3 versus retro −1.66 ± 1.10 mitochondria, n = 3 different DRG primary cultures; two-way ANOVA, treatment F(2,18) = 3.898, P = 0.0392, direction of transport F(1,18) = 81.20, P < 0.0001. (F) The number of stationary mitochondria in the neurites from non-transgenic or Gars^C201R/+ DRG neurons was quantified relative to the total number of mitochondria, in the absence or presence of tubastatin A was assessed. Non-transgenic 80.87 ± 2.48% stationary mitochondria, n = 6 different DRG primary cultures versus Gars^C201R/+ + vehicle 92.57 ± 5.75% stationary mitochondria, n = 7 different DRG primary cultures versus Gars^C201R/+ + tubastatin A 79.09 ± 9.34, n = 5 different DRG primary cultures; one-way ANOVA, F(2,15) = 1.515, P = 0.0030. (G) The acetylation of α-tubulin was determined by immunocytochemistry in Gars^C201R/+ DRG neuron cultures on tubastatin A treatment. (H) Quantification of the intensity of acetylated α-tubulin in neurites of DRG neuron cultures normalized to the fluorescence length: Gars^C201R/++vehicle 1.00 ± 0.0, n = 6 different DRG primary cultures versus Gars^C201R/+ + tubastatin A 1.44 ± 0.34, n = 6 cells from six different DRG primary cultures; Mann-Whitney test, P = 0.0022.

Figure 6

Treatment of Gars^C201R/+ mice with a HDAC6 inhibitor improves motor behaviour and stimulates regeneration of motor and sensory nerves in 4-month-old mice. (A) After daily intraperitoneal injections during 40 consecutive days with tubastatin A (50 mg/kg), the grip strength in all paws of vehicle- versus tubastatin A-treated Gars^C201R/+ mice was assessed: vehicle 1.80 ± 0.09 N, n = 9 mice versus tubastatin A 1.96 ± 0.16 N, n = 12 mice; unpaired t-test: t = 2.697, P = 0.0159. (B) The motor behaviour after treatment was measured by the accelerated rotarod test: vehicle 174 ± 40.47 s, n = 12 mice versus tubastatin A 211 ± 63.96 s, n = 12 mice; unpaired t-test: t = 1.706, P = 0.1021. (C) The time spent hanging (hanging wire) as well as the number of attempts to climb back on top of a grid (turning grid) was measured in vehicle versus tubastatin A-treated mice: turning grid test: vehicle 22.40 ± 7.70, n = 5 mice versus tubastatin A 36.83 ± 8.42, n = 5 mice; unpaired t-test t = 2.939, P = 0.0165. (D) The CMAP amplitudes were measured in the sciatic nerve in vehicle and tubastatin A-treated mice: vehicle 15.98 ± 3.61 mV, n = 13 mice versus tubastatin A 28.28 ± 5.09 mV, n = 13 mice; unpaired t-test t = 7.110, P < 0.0001. (E) SNAP amplitudes were measured in the tail nerve in vehicle and tubastatin A-treated mice: vehicle 14.45 ± 6.98 µV, n = 13 mice versus tubastatin A 27.45 ± 6.18 µV, n = 13 mice; unpaired t-test t = 5.027, P < 0.0001. (F) Quantification of the percentage of innervated neuromuscular junctions (NMJs) in the gastrocnemius muscle after treatment: vehicle 23.57 ± 3.58%, n = 5 mice (one muscle per mouse) versus tubastatin A 13.27 ± 2.38 %, n = 5 mice (one muscle per mouse); unpaired t-test t = 5.366, P = 0.0007.

Figure 7

Tubastatin A treatment increases the acetylation of α-tubulin in sciatic nerve and DRG homogenates from Gars^C201R/+ mice. (A and D) The levels of acetylated of α-tubulin and Gars expression in sciatic nerve homogenates was determined by western blot. Densitometry was used to assess the ratio of acetylation α-tubulin to total α-tubulin levels in sample from vehicle and tubastatin A-treated mice. All values were normalized to samples from vehicle treated mice: vehicle 1.00 ± 0.18, n = 3 mice versus tubastatin A 1.35 ± 0.29, n = 3 mice; unpaired t-test t = 7.636, P = 0.0016. (B and E) The acetylation status of α-tubulin and Gars expression in DRGs from vehicle- and tubastatin A-treated mice was determined using western blot. The ratio between the acetylation of α-tubulin and total α-tubulin levels was quantified by densitometry. All values were normalized to samples from vehicle-treated mice: vehicle 1.00 ± 0.30, n = 3 mice versus tubastatin A 2.13 ± 0.15, n = 3 mice; unpaired t-test t = 5.799, P = 0.0044. (C and F) Spinal cord samples were collected from vehicle- and tubastatin A-treated mice to assess the acetylation status of α-tubulin. Densitometry was used to quantify the ratio between acetylated and total α-tubulin levels. All values were normalized to samples from vehicle-treated mice: vehicle 1.00 ± 0.18, n = 3 mice versus tubastatin A 1.35 ± 0.29, n = 3 mice; unpaired t-test t = 1.8, P = 0.1463.