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CMT affects approximately one in 2,500 individuals and is the most common inherited disorder of the peripheral nervous system1,2. Clinically, individuals with CMT show slowly progressing distal muscle weakness and atrophy, foot deformities, steppage gait, distal sensory loss, and decreased or absent deep-tendon reflexes1,2. Electrophysiologically, CMT is divided into three groups: type 1 CMT (CMT1), with predominantly demyelination, type 2 CMT (CMT2), characterized primarily by axonal loss, and intermediate forms demonstrating signs of demyelination and axonal loss. In cases in which the disease exclusively affects motor axons, the condition is referred to as distal HMN. Thus far, approximately 40 causative genes (http://www.molgen.ua.ac.be/CMTMutations/) have been associated with CMT and distal HMN, and the pattern of inheritance can be autosomal dominant, autosomal recessive or X linked1,2. The underlying pathogenic mechanisms for most of the mutated genes remain unknown.

We previously identified dominant mutations in the gene encod-ing HSPB1 (HSPB1, also called HSP27) on chromosome 7q11.23 as a cause of CMT type 2F and distal HMN type 2B3,4. HSPB1 is a member of the small heat-shock proteins that contain a highly conserved α-crystallin domain. HSPB1 acts as a chaperone by binding misfolded or denatured proteins, preventing them from forming toxic aggregates5,6. In addition, HSPB1 also has a role in diverse cellular processes, such as modulation of the intracellular redox state, assembly of cytoskeletal structures, cell differentiation and inhibition of apoptosis5–7.

So far, 11 missense mutations have been identified in HSPB1 that are all associated with CMT2F or distal HMN2B. Seven of these muta-tions are located in the α-crystallin domain (including S135F), two

in the N-terminal part of the protein and two targeting the same amino acid (P182L/S) in the short C-terminal tail of the protein4,8–11. Depending on the mutation, affected individuals show either CMT2 or distal HMN symptoms. The S135F mutation is the only one that causes both CMT2 and distal HMN4.

The exact pathogenic mechanism by which mutations in HSPB1 cause CMT2F or distal HMN2B is unknown. In vitro, mutant HSPB1 forms intracellular aggregates, inhibits cell division and causes neuronal death4,12,13. Moreover, mutant HSPB1 disrupts the neuro-filament network, thereby disturbing the intracellular distribution of specific cargoes12,13.

To elucidate the pathological mechanisms of HSPB1 mutations, we developed and characterized transgenic mouse models for mutant HSPB1–induced CMT2F and distal HMN2B. We found that mice expressing mutant HSPB1 in neurons recapitulate all key features of CMT2F or distal HMN2B, depending on the mutation. We provide evidence that mutant HSPB1 leads to severe axonal transport defects induced by a decrease in acetylated tubulin abundance in peripheral nerves. We could rescue the phenotype of mice expressing mutant HSPB1 and the axonal transport defects by inhibiting HDAC6, a major tubulin-deacetylating enzyme, using small drug-like molecules. This indicates that HDAC6 plays a key part in the pathology of mutant HSPB1–induced peripheral neuropathies.

RESULTSCreation and characterization of human HSPB1–expressing miceWe created transgenic mice using a Thy1.2 expression cassette in which we cloned the human wild-type (WT) or mutant (S135F and

1Vesalius Research Center, VIB and K.U. Leuven, Campus Gasthuisberg, Leuven, Belgium. 2Laboratory for Neurobiology, K.U. Leuven, Leuven, Belgium. 3Department of Neurology, University Hospital Leuven, Leuven, Belgium. 4Peripheral Neuropathy Group, Department of Molecular Genetics, VIB and University of Antwerp, Antwerp, Belgium. 5Neurogenetics Laboratory, Institute Born Bunge, Antwerp, Belgium. 6Drug Discovery Program, Department of Medicinal Chemistry and Pharmacognosy, University of Illinois at Chicago, Chicago, Illinois, USA. 7Laboratory for Enteric Neuroscience, Translational Research Center for Gastrointestinal Disorders, K.U. Leuven, Leuven, Belgium. Correspondence should be addressed to L.V.D.B. (ludo.vandenbosch@vib-kuleuven.be).

Received 1 February; accepted 10 May; published online 24 July 2011; doi:10.1038/nm.2396

HDAC6 inhibitors reverse axonal loss in a mouse model of mutant HSPB1–induced Charcot-Marie-Tooth diseaseConstantin d’Ydewalle1,2, Jyothsna Krishnan1,2, Driss M Chiheb1,2, Philip Van Damme1–3, Joy Irobi4,5, Alan P Kozikowski6, Pieter Vanden Berghe7, Vincent Timmerman4,5, Wim Robberecht1–3 & Ludo Van Den Bosch1,2

Charcot-Marie-Tooth disease (CMT) is the most common inherited disorder of the peripheral nervous system. Mutations in the 27-kDa small heat-shock protein gene (HSPB1) cause axonal CMT or distal hereditary motor neuropathy (distal HMN). We developed and characterized transgenic mice expressing two different HSPB1 mutations (S135F and P182L) in neurons only. These mice showed all features of CMT or distal HMN dependent on the mutation. Expression of mutant HSPB1 decreased acetylated a-tubulin abundance and induced severe axonal transport deficits. An increase of a-tubulin acetylation induced by pharmacological inhibition of histone deacetylase 6 (HDAC6) corrected the axonal transport defects caused by HSPB1 mutations and rescued the CMT phenotype of symptomatic mutant HSPB1 mice. Our findings demonstrate the pathogenic role of a-tubulin deacetylation in mutant HSPB1–induced neuropathies and offer perspectives for using HDAC6 inhibitors as a therapeutic strategy for hereditary axonopathies.

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P182L) HSPB1 cDNA coupled to an N-terminal hemagglutinin (HA) tag. Western blot analysis revealed expression of only human HSPB1 in neuronal tissues (Fig. 1a and Supplementary Fig. 1a). We observed no differences in expression levels of HSPB1 between the different transgenic mice.

In transgenic mice, human WT or mutant HSPB1 was present only in the gray matter and in extended processes in the white matter (Supplementary Fig. 1b,c). At the cellular level, HSPB1 was exclu-sively expressed in neurons (Supplementary Fig. 1b,c). In line with other studies using the Thy1.2 expression cassette, we did not observe any difference in transgene expression between DRG and motor neurons (data not shown)14–16.

All transgenic mice were normal at birth and showed normal weaning and grooming behavior. The frequency of birth of all trans-genic lines followed the normal Mendelian inheritance pattern, and we observed no significant differences in survival (P > 0.05; Supplementary Fig. 2a). From 6 months on, mutant (S135F and P182L) HSPB1–expressing mice showed limb-clasping behavior when suspended by the tail, whereas we did not observe this in nontrans-genic or HSPB1WT-expressing mice (Fig. 1b).

Compromised motor performance and decreased muscle strengthFrom 6 months of age on, both mutant HSPB1 lines started to fail on an accelerating rotarod with progressive worsening over time (P < 0.0001;

Figure 1 Neuronal expression of human mutant HSPB1 in mice leads to progressive motor defects and decreased muscle strength. (a) Western blots of sciatic nerve, DRG, spinal cord and brain homogenates isolated from 2-month-old nontransgenic (Nontg) mice or mice expressing WT or mutant (S135F or P182L) HSPB1 probed with an antibody to the HA tag of the 27-kDa human HSPB1. Glyceraldehyde-3-phosphate dehydrogenase (Gapdh) staining confirmed equal loading. MW, molecular weight. (b) Six-month-old mice expressing WT or mutant HSPB1 lifted by the tail showing normal spreading of the limbs (WT) or limb-clasping behavior (S135F and P182L). (c) Monthly testing of the general motor performance of the different HSPB1-transgenic mice using an accelerating rotarod. n = 25 mice per genotype. Two-way analysis of variance (ANOVA) for repeated measures. ***P < 0.0001. (d,e) Age-dependent measurements of grip strength normalized to body weight of all paws combined (d) or forepaws only (e) using a dynamometer with a grid or a triangular bar, respectively. AU, arbitrary units. n = 25 mice per genotype. Two-way ANOVA for repeated measures. Blue asterisks indicate differences between P182L and WT; red asterisks indicate differences between S135F and WT. The line represents the span in which data points are significantly different compared to WT. Tukey’s HSD test was used for post hoc analysis. *P < 0.05; ***P < 0.0001. Data are presented as means ± s.e.m.

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Figure 2 Mice expressing mutant HSPB1 show steppage gait and clawed hindpaws. (a) Typical gait patterns of 8-month-old transgenic mice monitored with the semiautomated Catwalk system. Colored bars represent the time a paw makes contact with the floor plate. LH, left hindpaw; LF, left forepaw; RH, right hindpaw; RF, right forepaw. Asterisks mark hesitations in the gait pattern observed for both mutant HSPB1 mouse lines. Scale bar, 10 cm. (b–f) Quantification of various parameters obtained from the gait analysis with the Catwalk. Stride length (b), forepaw angle (c), hindpaw angle (d), forepaw print area (e) and hindpaw print area (f) were measured as a function of age in mice expressing HSPB1WT, HSPB1S135F or HSPB1P182L. n = 15 mice per genotype. Two-way ANOVA for repeated measures. **P < 0.001; ***P < 0.0001. Blue asterisks indicate differences between P182L and WT. Red asterisks indicate differences between S135F and WT. The lines represent the span in which the data points are significantly different. Tukey’s HSD test was used for post hoc analysis. Data are presented as means ± s.e.m. (g) Typical example of a mutant HSPB1P182L-expressing mouse showing clawed hindpaws.

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Fig. 1c). HSPB1P182L-expressing mice showed a more pronounced decline compared to HSPB1S135F-expressing mice (Supplementary Fig. 2b).

We observed a progressive decline in the grip strength of all paws for both mutant HSPB1–expressing mouse lines (P < 0.0001; Fig. 1d). This decline was more severe for HSPB1P182L than for HSPB1S135F (Supplementary Fig. 2c). Grip strength of forepaws only showed a significant decrease from the age of 10 months on (P = 0.001; Fig. 1e). Again, this decrease was most pronounced for HSPB1P182L-expressing mice (Supplementary Fig. 2d).

Disturbed gait and mutant-dependent sensory abnormalitiesFrom 6 months on, both mutant HSPB1–expressing mouse lines needed on average twice the number of step cycles to cross the walkway of the Catwalk17,18 system (Fig. 2a). This resulted in a 50% reduction of the stride length (P < 0.0001; Fig. 2b). Moreover, both mutant HSPB1–expressing mouse lines showed hesitations in placing their paws and had increased hindpaw angles and decreased hindpaw print areas, whereas forepaws were unaffected (P < 0.0001; Fig. 2a–f). The decrease in hindpaw print area is in line with the clawed hindpaws observed in mutant HSPB1–expressing mice (Fig. 2g). These clawed hindpaws were more pronounced in mice expressing HSPB1P182L than in those expressing HSPB1S135F and were not observed in mice expressing HSPB1WT or nontransgenic mice.

Compared to HSPB1WT-expressing mice, mice expressing HSPB1S135F showed increased response latencies in the hot-plate test as a function of age, indicative of progressive sensory defects (P = 0.01; Fig. 3a). In contrast, HSPB1P182L-expressing mice did not show any differences (Fig. 3a), indicating that HSPB1P182L-expressing mice have only impaired motor performance.

Mutation-dependent electrophysiological alterationsNerve conduction studies are routinely done on individuals with CMT to discriminate demyelinating from axonal forms of disease, as well as to assess whether the individuals show either a mixed sensorimotor or a pure motor neuropathy. We used subdermal electrodes either at the level of the gastrocnemius muscle to measure compound muscle action potentials (CMAPs) or in the tail to record sensory nerve action potentials (SNAPs).

HSPB1P182L-expressing and HSPB1S135F-expressing mice showed a significant decrease in peak-to-peak amplitude of CMAPs at 6 and 8 months of age, respectively (P < 0.0001; Fig. 3b). The decrease in CMAP amplitudes as a function of age was most pronounced in HSPB1P182L-expressing mice (P = 0.004; Fig. 3c). CMAP laten-cies were unaffected (Fig. 3d), confirming the axonal nature of the motor neuropathy in both mutant HSPB1 lines. From 6 months on, HSPB1S135F-expressing mice also showed a significant decrease in

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Figure 3 Mutation-dependent pure motor or sensorimotor axonal loss and denervation of neuromuscular junctions in mice expressing mutant HSPB1. (a) Relative difference in response latencies on a hot plate between mice expressing HSPB1WT, HSPB1S135F and HSPB1P182L. (b–d) Determination of the amplitude and the latency of the CMAPs in mice expressing HSPB1WT, HSPB1S135F or HSPB1P182L. (b) CMAP amplitudes as a function of age. (c) Linear curve fitting of the CMAP data points (R2

S135F: 0.99 and R2P182L: 0.94). (d) CMAP latencies as a function of age. (e,f) Age-dependent

measurements of amplitudes (e) and latencies (f) of SNAPs. Blue asterisks indicate difference between P182L and WT. Red asterisks indicate difference between S135F and WT. Line represents the span in which the data points are significantly different compared to WT. Tukey’s HSD test was used to analyze post hoc. (g) Toluidine blue staining of semithin distal sciatic nerve sections of 10-month-old HSPB1WT-expressing (left) and mutant HSPB1–expressing (middle and right) mice, showing axonal loss in the mutant lines. No signs of demyelination were observed. Scale bars, 40 µm. (h) Correlation between myelin thickness and axonal diameter confirming the absence of demyelination in HSPB1WT-expressing (left) and mutant HSPB1–expressing (middle and right) mice. (i) Quantification of the number of axons in distal parts of the sciatic nerve. (j) Left, example of a completely denervated neuromuscular junction stained with α-bungarotoxin (red) and negative for neurofilament heavy chain. Scale bar, 20 µm. Right, quantification of these denervated neuromuscular junctions in 10-month-old transgenic HSPB1 mice. In a–f, n = 15 mice per genotype. Two-way ANOVA for repeated measures was used in a–f. One-way ANOVA was used in g–j. In g–j, n = 3 mice per genotype. *P < 0.05; ** P < 0.001; *** P < 0.0001. Data are presented as means ± s.e.m.

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baseline-to-peak amplitude of SNAPs (P < 0.0001; Fig. 3e). Again, SNAP latencies were unaltered, indicating that the sensory loss was due to an axonal neuropathy (Fig. 3f). In contrast, HSPB1P182L-expressing mice did not show any reduction in SNAP amplitudes, confirming that this line has no sensory deficits (Fig. 3e).

No demyelination, but distal axonal lossWe found no histological evidence for demyelination in both proximal and distal parts of the sciatic nerve (Fig. 3g,h and Supplementary Fig. 3a,b) or axonal loss in the proximal part of the sciatic nerve (P > 0.05; Supplementary Fig. 3c). In contrast, there was a significant decrease in the number of axons in distal parts of the sciatic nerve in both HSPB1S135F-expressing and HSPB1P182L-expressing mice (P = 0.002 and P = 0.001, respectively; Fig. 3i).

In the gastrocnemius muscle, we found less acetylcholine recep-tor clusters per terminal axon in mice expressing mutant HSPB1 compared to HSPB1WT-expressing mice (Supplementary Fig. 3d,e). HSPB1WT-expressing mice had almost no visibly denervated neuro-muscular junctions, whereas, of the remaining neuromuscular junc-tions, HSPB1S135F-expressing and HSPB1P182L-expressing mice showed a marked increase of denervated neuromuscular junctions (Fig. 3j).

Both mutant HSPB1–expressing mouse lines showed atrophic muscle fibers in the gastrocnemius muscle accompanied by pyknotic nuclear clumps (Supplementary Fig. 4a). Fiber type grouping was observed in muscles from both mutant HSPB1–expressing mouse lines, whereas muscle fibers showed a normal ‘checkerboard’ appear-ance in HSPB1WT-expressing mice (Supplementary Fig. 4b).

Disturbance of axonal transport in cultured DRG neuronsWe studied the effect of mutant HSPB1 on axonal transport of mito-chondria in isolated dorsal root ganglion (DRG) neurons from adult transgenic mice. We used kymographs to quantify the total number of mitochondria and to discriminate antero- and retrogradely moving mitochondria from stationary mitochondria (Fig. 4a).

DRG neurons isolated from symptomatic HSPB1S135F-expressing mice showed a reduction of the number of mitochondria compared to mice expressing HSPB1WT (Fig. 4a,b). In addition, significantly fewer moving mitochondria were present in their neurites (P < 0.0001; Fig. 4a,c). DRG neurons from mice expressing HSPB1P182L did not show any difference in either total number of mitochondria or mov-ing mitochondria, consistent with the pure motor phenotype of these mice (Fig. 4b,c), and mitochondrial transport was also unaffected in DRG neurons isolated from presymptomatic mutant HSPB1S135F-expressing mice (Fig. 4d,e).

Reduced acetylation of a-tubulin in mutant HSPB1 nervesMitochondria are mainly transported along microtubules consist-ing of polymerized tubulin19. Tubulin can undergo a variety of post- translational modifications20. We focused on acetylation of α-tubulin, as this process is considered to be a recognition signal for the anchor-ing of molecular motors19–21. In addition, decreased levels of acetylated α-tubulin have been associated with neurodegenerative diseases22,23.

We observed a marked decrease in acetylated α-tubulin abundance in peripheral nerves of symptomatic mutant HSPB1–expressing mice (P < 0.0001; Fig. 4f,g). We found no differences in acetylated

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Figure 4 Mutant HSPB1 mice show axonal transport defects and decreased acetylated tubulin levels. (a) Representative fluorescent micrograph of a cultured DRG neuron loaded with a selective mitochondrial marker (MitoTracker-Red; left) and typical kymographs obtained from DRG neurons isolated from 10-month-old mice (top right, HSPB1WT; bottom right, HSPB1S135F). Stationary mitochondria are visible as straight vertical lines, whereas moving mitochondria are deflected either to the left (retrograde) or to the right (anterograde). Left scale bar, 40 µm. In right images, time (t) scale bar: 50 s; distance (d) scale bar: 25 µm. (b–e) Quantification of total and moving (per 200 s and per 100 µm) mitochondria in DRG neurons isolated from different transgenic lines and at different ages. Total number (b,d) and number of moving (c,e) mitochondria were determined in DRG neurons from 10-month-old (b,c) or 2-month-old (d,e) transgenic mice. n = 25–35 cells isolated from three different mice per genotype. *P < 0.05; ***P < 0.0001. (f) Western blots from sciatic nerves of 10-month-old mice expressing HSPB1WT or mutant HSPB1 probed with antibodies to acetylated tubulin. We confirmed equal loading by staining for α-tubulin. n = 3. (g,h) Quantification of the optical densities of acetylated tubulin bands on western blots of sciatic nerve (g) and spinal cord (h) extracts. Signals are normalized to total α-tubulin levels. n = 3. (i) Acetylated α-tubulin in sciatic nerve and spinal cord of 10-month-old transgenic mice, as determined by ELISA. Signals are normalized to total α-tubulin levels. n = 3. One-way ANOVA. ***P < 0.0001. (j) Immunostaining of longitudinal sections of the sciatic nerve from 10-month-old mice expressing HSPB1WT (top) or HSPB1S135F (bottom) using an antibody to acetylated tubulin (green) and to peripheral myelin protein 22 (Pmp22, red). Scale bar, 40 µm. Statistical analysis of b, c, g and h was done with one-way ANOVA. Statistical analysis of d and e was done with Student’s t test. Statistical analysis of i was done with Fisher’s exact test. Data are presented as means ± s.e.m.

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α-tubulin amounts in spinal cord (Fig. 4f,h). Using ELISA, we con-firmed the decrease in acetylated α-tubulin levels in sciatic nerve of mutant HSPB1 mice (Fig. 4i). Longitudinal sections of sciatic nerves showed less acetylated α-tubulin in mutant HSPB1–expressing mice compared to HSPB1WT-expressing mice (Fig. 4j).

Restoration of axonal transport by HDAC6 inhibitionAcetylation and deacetylation of tubulin occurs at Lys40 of α-tubulin24. HDAC6, a class II histone deacetylase, is the major enzyme with α-tubulin deacetylating activity25,26. Moreover, HDAC6 can regulate the axonal transport of mitochondria in cultured hippocampal neurons21.

We examined the link between HDAC6 and the pathological mechanism underlying mutant HSPB1–induced CMT2 by incubating DRG neurons with several HDAC6 inhibitors. Trichostatin A (TSA)

is a pan-HDAC inhibitor for class I and II HDACs, whereas both tubacin and tubastatin A (Supplementary Figs. 5 and 6) are highly selective HDAC6 inhibitors27,28. Treatment of DRG neurons isolated from symptomatic HSPB1S135F-expressing mice with TSA, tubacin or tubastatin A increased total numbers of mitochondria (Fig. 5a) and restored the number of moving mitochondria (Fig. 5b). Tubacin and tubastatin A were more effective compared to TSA (Fig. 5a,b).

Reversal of the CMT2 phenotype by HDAC6 inhibitionNext, we treated symptomatic HSPB1S135F-expressing mice for 21 d with either a nonspecific or a specific HDAC6 inhibitor. TSA and tubastatin A treatment resulted in a significant increase of acetylated α-tubulin amounts in peripheral nerves (P = 0.009 and P < 0.008, respectively; Fig. 5c–e). TSA, but not tubastatin A, also increased acetylated α-tubulin amounts in spinal cord (Fig. 5c,d,f). Both TSA and tubastatin A did not affect overall α-tubulin abundance (Fig. 5c,d).

We further discovered that TSA or tubastatin A treatment signifi-cantly increased the motor performance of HSPB1S135F-expressing mice (P < 0.0001; Fig. 5g). Treatment with either drug also increased electrophysiological parameters such as CMAP amplitudes as well as SNAP amplitudes (Fig. 5h,i). We also observed that the loss of acetylcholine-receptor clusters at the end plates in gastrocnemius muscle was restored (Fig. 6a) and that the level of denervation of the remaining neuromuscular junctions was decreased (Fig. 6b). These observations indicate that increasing acetylated α-tubulin abundance in peripheral nerves leads to re-innervation of muscles.

Finally, we measured ex vivo axonal transport in DRG neurons isolated from treated mice. In contrast to TSA, tubastatin A increased the total number of mitochondria (Fig. 6c). Moreover, the number of moving mitochondria was increased significantly after TSA or tubastatin A treatment (P < 0.0001; Fig. 6d). Tubastatin A was more effective than TSA and resulted in a complete rescue of mitochondrial motility (Fig. 6c,d).

DISCUSSIONOur data show that neuronal expression of human HSPB1 with the S135F mutation in transgenic mice mimics the CMT2 phenotype, whereas the P182L mutation leads to a more distal HMN phenotype.

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Figure 5 HDAC6 inhibition rescues axonal transport defects and restores the CMT2 phenotype. (a,b) Axonal transport of mitochondria measured in DRG neurons isolated from 10-month-old HSPB1S135F-expressing mice after 12 h of treatment with TSA (0.4 µM), tubacin (2 µM), tubastatin A (1 µM) or an equivalent amount of DMSO. Total number of (a) and number of moving (b) mitochondria (per 200 s and per 100 µm) were quantified. n = 20–30 cells isolated from three different mice for each condition. *P < 0.05; **P < 0.01; ***P < 0.0001. (c,d) Western blots of sciatic nerve and spinal cord extracts from symptomatic 8-month-old HSPB1S135F-expressing mice treated daily for 3 weeks with TSA (10 mg per kg body weight; c) or tubastatin A (25 mg per kg body weight; d) probed against acetylated tubulin. Equal loading was confirmed with α-tubulin staining. n = 3. (e,f) Quantifications of optical density (OD) of acetylated tubulin on western blots of sciatic nerve (e) and spinal cord (f) extracts of symptomatic HSPB1S135F-expressing mice treated with TSA or tubastatin A. n = 3. (g) Motor performance on an accelerating rotarod of HSPB1S135F-expressing mice treated for 3 weeks with TSA or tubastatin A. (h,i) Effect of the TSA or tubastatin A treatments of HSPB1S135F-expressing mice on the amplitudes of CMAPs (h) or SNAPs (i). n = 3–6 mice per group. *P < 0.05; **P < 0.001; ***P < 0.0001. One-way ANOVA was used to analyze the data in a,b,e–i. Data are presented as means ± s.e.m.

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Figure 6 TSA or tubastatin A treatment leads to muscle reinnervation and rescues axonal transport defects. (a,b) Effect of vehicle (DMSO), TSA or tubastatin A treatment on the innervation level of the gastrocnemius muscle in symptomatic, 8-month-old HSPB1S135F-expressing mice. The number of visible neuromuscular junctions (NMJs) per axon in each field of view (a) and the relative quantity of denervated NMJs (b) were determined after staining gastrocnemius muscle with antibodies against α-bungarotoxin and neurofilament heavy chain. n = 2 or 3 mice per condition. ***P < 0.0001. (c,d) Axonal transport of mitochondria measured in cultured DRG neurons isolated from symptomatic HSPB1S135F-expressing mice after 3 weeks of treatment with TSA or tubastatin A. Total number (c) and number of moving mitochondria (d) were quantified (per 200 s and 100 µm). n = 20–25 cells isolated from three different mice in each condition. *P < 0.05; ***P < 0.0001. One-way ANOVA was used to analyze the data. Tukey’s HSD test was used for post hoc analysis in a–d. Data are presented as means ± s.e.m.

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Both HSPB1S135F-expressing and HSPB1P182L-expressing mice develop an adult-onset, slowly progressing disease without an effect on survival. This is reminiscent of what is observed in CMT2 and distal HMN diseases, which start in adulthood, progress slowly and have no major impact on the lifespan of the affected individual3,4,8–11. Both mutant HSPB1–expressing mouse lines also show several other key features of these axonal neuropathies. The altered gait pattern resembles the steppage gait of the affected humans, and the mice have clawed hindpaws. Reduced CMAP amplitudes and unaffected motor nerve latencies are also observed in individuals with CMT2 and distal HMN4,8–11. Both mutant HSPB1–expressing mouse lines show distal axonal loss in motor nerve fibers and a decrease in innervated neuromuscular junctions. Pathological changes in the gastrocnemius muscle such as pyknotic nuclear clumps, atrophic muscle fibers and fiber type grouping seem to have arisen from the neuronal defects caused by the expression of mutant HSPB1.

A notable difference between the two mutant HSPB1 mice is the severity of the motor phenotype. General motor coordination, mus-cle force, hindpaw angle and CMAP amplitudes are most affected in HSPB1P182L-expressing mice. This is in agreement with the more severe clinical symptoms in individuals with the P182L mutation compared to people with other HSPB1 mutations29.

Another difference between both mouse models is the involvement of the sensory system. The HSPB1S135F-expressing mice have a mixed sensorimotor polyneuropathy, whereas the HSPB1P182L-expressing mice have a pure motor neuropathy. This phenotypic distinction was conserved in vitro, as DRG neurons from symptomatic HSPB1P182L-expressing mice did not show axonal transport deficits. This situation mimics the human condition, as humans carrying the S135F mutation can have a sensorimotor polyneuropathy, whereas individuals with the P182L mutation have a pure motor neuropathy1,3,4,8–11. The fact that both mutations are situated in different domains of the HSPB1 protein implies that different cell types show varying sensitivity to the negative effects of these mutated proteins. Notably, in humans, mutations situated in the C-terminal part of the protein mainly affect motor axons, whereas mutations in the N-terminal region and in the α-crystallin domain affect both motor and sensory neurons.

Furthermore, our results indicate that mutant HSPB1 can cause the phenotypes in a cell-autonomous way, as expression limited to neurons is sufficient to induce the phenotypes. The cell-autonomous pathology of mutant HSPB1–induced CMT2 and distal HMN is in line with findings in other CMT2 models. Mice with neuron-specific expression of mutant mitofusin 2 (MFN2) showed reduced mass of anterior hind limb muscles, ultimately resulting in limp hindpaws30. Another mouse model with neuronal expression of mutant MFN2 showed locomotor impairment and gait defects31. In a tetracycline-responsive gene system, the selective expression of mutant neuro-filament light chain (NF-L) in the nervous system leads to features of axonal CMT, including anomalous hind limb posture and loss of muscle innervation32.

The phenotypes in our transgenic mice are probably caused by a gain-of-function mechanism. This is in line with the dominant inheritance patterns of mutant HSPB1–induced CMT2 and distal HMN in humans. Furthermore, Hspb1-knockout mice are viable and do not show any overt phenotype33. Our transgenic HSPB1 mouse models are a unique tool to study the pathological mechanism responsible for the observed pheno-types. We found that mitochondrial transport was severely affected in DRG neurons isolated from symptomatic mutant HSPB1S135F-expressing mice. This is in agreement with the observation that the presence of HSPB1P182L disturbs the intracellular distribution of specific proteins and

organelles in transfected cortical neurons12. Alterations in mitochon-drial transport were also observed in other CMT2 models. Mutations in mitochondrial proteins such as MFN2 and ganglioside-induced differentiation-associated protein 1 (GDAP1) affected mitochondrial motility in transfected cell lines34,35. Mutant MFN2 caused an increase in tethering to the endoplasmic reticulum, which impaired axonal transport of mitochondria in transfected DRG neurons36,37. Mutant GDAP1 also affected mitochondrial dynamics, partially excluding mitochondria from axons35. Mutant NF-L led to mitochondrial dysfunction before neuro-filament network disruption38. Furthermore, in humans with CMT2 or distal HMN, other proteins involved in axonal transport are mutated, including the small GTPase RAB7, emphasizing the central role of axonal transport defects in CMT2 pathology2.

Intracellular transport along axons requires motor proteins that move their cargoes using guidance cues19,24,39. Acetylated α-tubulin is one of these cues24,39,40. This is especially true for mitochondrial transport, as moving mitochondria preferentially localize to acetylated microtubules41. Moreover, disturbance of α-tubulin acetylation has a role in neurodegenerative diseases such as familial dysautonomia and increasing acetylated α-tubulin abundance induces an increase in axonal transport21,22,42. We observed that total acetylated α-tubulin amounts were dramatically decreased in peripheral nerve from mutant HSPB1–expressing mice. This reduction of α-tubulin acetylation is a distal phenomenon, as acetylated α-tubulin levels were not affected in the spinal cord of mutant HSPB1–expressing mice. The cause of this decrease is unknown. However, restoring acetylated α-tubulin levels by inhibiting HDAC6 rescued the axonal transport defects, suggesting a key role for deacetylation in the mechanism of mutant HSPB1–induced CMT. Similar observations were made in other neuro-degenerative disorders, including Huntington’s disease and multiple sclerosis, as well as in peripheral nerve injuries42–46.

Treatment of mutant HSPB1–expressing mice with HDAC6 inhibi-tors also partially restored the CMT2 phenotype both at the behav-ioral and at the electrophysiological level, further indicating that reduced acetylated α-tubulin amounts have a major role in mutant HSPB1-induced CMT2 pathology. Our study thus indicates that the phenotype of inherited peripheral neuropathies can be halted and even reversed by pharmacological inhibition of deacetylation, at least in animal models. Such reversibility was also found in mice condition-ally expressing mutant NF-L with CMT2 symptoms, a phenotype that could be restored by switching off the expression of mutant NF-L in symptomatic mice32.

In conclusion, we developed transgenic mice expressing human mutant HSPB1 in neurons only. These mice develop phenotypes that replicate the human symptoms of CMT2 and distal HMN. Mutant HSPB1 cell-autonomously affects axonal transport and decreases acetylated α-tubulin levels. This reduction in acetylated α-tubulin can be reversed by HDAC6 inhibition. Treatment with these inhibi-tors restores the defects in axonal transport and rescues the CMT2 phenotype. As a consequence, inhibition of HDAC6 may be a new therapeutic approach for peripheral neuropathies.

METHODSMethods and any associated references are available in the online version of the paper at http://www.nature.com/naturemedicine/.

Note: Supplementary information is available on the Nature Medicine website.

ACKnoWLeDgMenTsWe thank L. Almeida-Souza and S. Janssens for their constructive comments and the Molecular Small Animal Imaging Centre (MoSAIC, K.U. Leuven) for

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974 VOLUME 17 | NUMBER 8 | AUGUST 2011 nAture medicine

the use of the Catwalk system. We are grateful to B. Weynants and N. Hersmus for the technical assistance. We thank R. Mazitschek and J. Bradner (Dana-Farber Cancer Institute, Harvard Medical School) for kindly giving us tubacin. The research to create tubastatin A was supported by an American Chemical Society Fellowship and by the International Rett Syndrome Foundation. We thank J. H. Kalin for helping us with the creation of the HDAC6 homology model. This work was supported by grants from the Fund for Scientific Research Flanders (FWO-Vlaanderen), the University of Leuven (K.U. Leuven, GOA/12/014 and OT/10/046), the Belgian government (Interuniversity Attraction Poles, programme P6/43 of the Belgian Federal Science Policy Office), the Association Belge contre les Maladies neuro-Musculaires, the Association Française contre les Myopathies (projects 13169 and 14471), the Frick Foundation for Amyotrophic Lateral Sclerosis Research, the Muscular Dystrophy Association and the European Community’s Health Seventh Framework Programme (FP7/2007-2013 under grant agreement 259867). C.d.Y. is supported by the Agency for Innovation by Science and Technology in Flanders. P.V.D. is a clinical researcher, and J.I. is a postdoctoral fellow of the FWO-Vlaanderen. W.R. is supported through the E. von Behring Chair for Neuromuscular and Neurodegenerative Disorders.

AUTHoR ConTRIBUTIonsC.d.Y. planned and performed all the experiments. J.K. developed the transgenic mice and did the initial characterization of the phenotype. D.M.C. provided technical support. P.V.D. assisted with the electrophysiological experiments, J.I. provided the original HSPB1 constructs, A.P.K. provided tubastatin A and P.V.B. helped with the axonal transport measurements. P.V.B., P.V.D., J.I., A.P.K., V.T. and W.R. provided ideas for the project and participated in writing the paper. L.V.D.B. planned and supervised the experiments. C.d.Y. and L.V.D.B. wrote the paper.

CoMPeTIng FInAnCIAL InTeResTsThe authors declare no competing financial interests.

Published online at http://www.nature.com/naturemedicine/. Reprints and permissions information is available online at http://www.nature.com/reprints/index.html.

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nAture medicinedoi:10.1038/nm.2396

ONLINE METHODSCreation, genotyping and treatment of transgenic mice. We cloned HA-tagged human WT, S135F or P182L HSPB1 cDNA using the XhoI restriction endonuclease into the Thy1.2 expression cassette (kind gift from Novartis Pharma). We created the transgenic mice by pronuclear microinjections (out-sourced to Polygene). We excised a 7.4-kb DNA fragment of the Thy1.2–HA-HSPB1 (WT, S135F or P182L) vector with the restriction endonuclease EcoRI. We purified the fragment from SeaKem GTG agarose with the Qbiogene Geneclean Spin kit, dialyzed for 24 h against microinjection buffer (10 mM Tris-HCl, pH 7.2, 0.1 mM EDTA) and diluted to a concentration of 3.5 ng µl−1. We injected the DNA into FvB/N-derived zygotes. We kept injected zygotes overnight and transferred them into pseudopregnant B6CBAF1 females. We kept the mice in individually ventilated cages. We performed genotyping as described in the Supplementary Methods.

For TSA (Sigma-Aldrich) and tubastatin A treatment, we dissolved the com-pounds in DMSO (Sigma-Aldrich). We further diluted the compounds in saline to the final doses. We treated transgenic mice for 21 consecutive days by daily intraperitoneal injection of 10 mg per kg body weight TSA or 25 mg per kg body weight tubastatin A or an equivalent amount of vehicle.

Behavioral assessment of mice. The Ethical Committee of the K.U. Leuven approved all mouse experiments. At regular time points, we lifted each mouse by its tail and scored its behavior. Rotarod performance, grip strength, gait analysis and the hot-plate test are described in the Supplementary Methods.

Nerve conduction studies. We performed nerve conduction studies using a Medelec EMG monitoring set-up (Medelec Vickers/Modul USA). We used subdermal 0.4-mm electrodes (Technomed Europe) for stimulation and recording on anesthetized mice (10 mg per kg body weight Nembutal; CEVA Sante Animale). We measured CMAPs by placing the stimulating elec-trodes at the sciatic notch; we placed recording electrodes at the level of the gastrocnemius muscle. We derived SNAPs by stimulating the base of the tail and recording 4 cm more distally. We measured both CMAPs and SNAPs at supramaximal stimulation.

Western blotting and ELISA. Mice were killed with CO2, and flash-frozen tissue samples were maintained at −80 °C until processed further. We homogenized samples in RIPA buffer (containing 50 mM Tris, 150 mM NaCl, 1% (vol/vol) NP40, 0.5% sodium deoxycholate (wt/vol), 0.1% SDS (wt/vol) complemented with protease inhibitors (Complete, Roche Diagnostics), pH 7.6) using Lysing

Matrix A (MP Biomedicals). Western blotting and ELISA procedures are described in the Supplementary Methods.

Immunohistochemistry and histology. After intracardiac perfusion with 4% para-formaldehyde, we isolated and incubated spinal cords and sciatic nerves (longitudi-nal sections) overnight in a 30% sucrose gradient and embedded samples in optimal cutting compound (Takara Bio). We kept sciatic nerves (transverse semithin sec-tions) for 48 h in 2.5% glutaraldehyde solution before further processing. We froze gastrocnemius muscles immediately in liquid-nitrogen–cooled isopentane. For immunofluorescence and histological experiments, we used a Leica microtome (Leica Microsystems) to cut tissues in sections 15 µm (spinal cord and nerve) or 25 µm (muscle) thick. For further details, see Supplementary Methods.

Axonal transport of mitochondria. The protocol for the isolation and culturing of DRG neurons is described in the Supplementary Methods.

We loaded DRG neurons with MitoTracker-Red (50 nM, 30 min, Invitrogen), washed them and left them to equilibrate (30 min) in their original DRG medium, before we transferred them to a HEPES buffered salt solution (pH 7.4, composition in mM: NaCl 150, KCl 5, MgCl2 1, CaCl2 2, glucose 10, HEPES 10). Measurements were performed on an inverted Zeiss Axiovert 200M microscope (Carl Zeiss) with a 40× water immersion lens as described else-where47. Detailed information on image acquisition and analysis47 is available in the Supplementary Methods.

Statistical analyses. Statistical analysis was performed using Graphpad Prism version 5.0b. The level of significance was set to 0.05. We tested differences between genotypes over different ages by two-way ANOVA for repeated mea-sures. We tested differences in one parameter between genotypes by one-way ANOVA. We tested individual differences with the post hoc Tukey Honestly Significant Difference (HSD) test. We assessed the severity of the phenotype and correlation between myelin thickness and axonal diameter by linear regres-sion for the slopes of the averaged data points. We tested frequency differences between genotypes with Fisher’s exact test.

Additional methods. Detailed methodology is described in the Supplementary Methods.

47. Vanden Berghe, P., Hennig, G.W. & Smith, T.K. Characteristics of intermittent mitochondrial transport in guinea pig enteric nerve fibers. Am. J. Physiol. Gastrointest. Liver Physiol. 286, G671–G682 (2004).