SUPPLEMENTARY FIGURES
HDAC6 inhibitors reverse axonal loss in a mouse model of mutant HSPB1-‐induced Charcot-‐
Marie-‐Tooth disease
Constan'n d’Ydewalle, Jyothsna Krishnan, Driss M. Chiheb, Philip Van Damme, Joy Irobi, Alan P.
Kozikowski, Pieter Vanden Berghe, Vincent Timmerman, Wim Robberecht, Ludo Van Den Bosch
1
Nature Medicine doi:10.1038/nm.2396
Supplementary figure 1
Absence of expression in non-‐neuronal 'ssue and Thy1.2.-‐driven neuronal expression of human
HSPB1 in spinal cords of transgenic animals.
(a) Western blot analysis of liver and kidney from 2 months old mice demonstra8ng the absence of
HA-‐tagged HSPB1 in non-‐neuronal 8ssues. Glyceraldehyde-‐3-‐phosphate dehydrogenase (Gapdh)
was used as loading control. (b—c) Representa8ve fluorescent micrographs of ventral horn in
spinal cord of 2 months old transgenic animals (shown here is HSPB1WT). Nuclei are stained with
DAPI (blue). HA-‐tagged HSPB1 (red) co-‐localized with a specific marker for neurons (neurofilament
Smi32, green, b), while there was no co-‐localiza8on with glial cells (stained with glial fibrillary
acidic protein Gfap, green, c). Scale bar: 50 μm
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Nature Medicine doi:10.1038/nm.2396
Supplementary figure 2
No effect on survival and more severe motor phenotype in mutant HSPB1P182L
(a) Kaplan-‐Meier curve of transgenic WT (black), S135F (red) and P182L (blue) HSPB1 mice. n = 5
mice in each group. Log-‐rank test. P > 0.05. (b) Linear curve fi[ng of the averaged data points of
the rotarod test over 8me of both mutant HSPB1 (S135F in red; P182L in blue) mice. Linear
regression. R2S135F = 0.95; R2P182L = 0.94; P = 0.02. (c) Linear fit of averaged data points of muscle
force of all four paws together in func8on of age for both mutant (S135F in red; P182L in blue)
HSPB1 mice. Linear regression. R2S135F = 0.86; R2P182L = 0.97; P < 0.0001. (d) Linear fi[ng of
averaged data points over 8me of muscle force of forepaws only for both mutant (S135F in red;
P182L in blue) HSPB1 mice. Linear regression. R2S135F = 0.90; R2P182L = 0.82; P = 0.005.
3
Nature Medicine doi:10.1038/nm.2396
Supplementary figure 3
Mutant HSPB1-‐induced neuropathy caused no proximal axonal loss, but is characterized by muscle
denerva'on.
(a) Tolduine blue staining of semi-‐thin proximal scia8c nerve sec8ons of 10 months old HSPB1WT
(lec panel) and mutant HSPB1 (middle and right panel) mice showing no axonal loss. No signs of
demyelina8on were observed. Scale bar 40 μm. (b) Correla8on of myelin thickness and axonal
diameter confirming the absence of demyelina8on in HSPB1WT (lec panel) and mutant HSPB1
(middle and right panel) mice. (c) Quan8fica8on of the number of axons in proximal parts of the
scia8c nerve. One-‐way ANOVA. P > 0.05. (d) Fluorescent micrograph of acetylcholine-‐receptor
clusters stained with α-‐bungarotoxin (in red) and terminal axon branch stained with neurofilament
heavy chain (Nf200; in green) from a 25 μm thick longitudinal sec8on of gastrocnemius muscle of
a 10 months old HSPB1S135F animal. Scale bar: 20 μm. (e) Quan8fica8on of the number of
acetylcholine-‐receptor clusters per terminal axon branch visible within a field-‐of-‐view. One-‐way
ANOVA. * P < 0.05; ** P < 0.001; *** P < 0.0001.
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Nature Medicine doi:10.1038/nm.2396
Supplementary figure 4
Mutant HSPB1-‐induced neuropathy caused neurogenic changes in the gastrocnemius muscle
(a) Haematoxillin-‐Eosin (H&E) staining of 15 μm thick transversal sec8ons of the gastrocnemius
muscle isolated from 10 months old HSPB1WT and mutant HSPB1S135F animals. Mutant HSPB1
muscle demonstrated pykno8c nuclear clumps, atrophic and angular muscle fibres while this was
never seen in HSPB1WT animals. Scale bar: 20 μm. (b) Nico8namide Adenine Dinucleo8de (NADH)
staining of 15 μm thick transversal sec8ons of the gastrocnemius muscle isolated from 10 months
old HSPB1WT and mutant HSPB1S135F animals. HSPB1WT animals showed a “checkerboard” panern
of type 1 and type 2 muscle fibres, while fibre type grouping was observed in mutant HSPB1 mice.
Scale bar: 40 μm.
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Nature Medicine doi:10.1038/nm.2396
Supplementary figure 5
Top-‐down views of Tubasta'n A docked to the ac've site of an HDAC6 homology model.
Tubasta8n A was docked to a previously reported homology model of HDAC6 using FlexX (Sankt
Augus8n) by specifying an essen8al metal-‐ligand interac8on and leaving other parameters at their
default se[ngs1. The different panels represent various points of view to illustrate the perfect fit
of Tubasta8n A to HDAC6 surface. Leners A-‐D correspond to the boundary regions of the HDAC6
cataly8c channel rim. The yellow arrow indicates the distance between the two boundary regions
in which the γ-‐carboline cap group of Tubasta8n A is accommodated.
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Nature Medicine doi:10.1038/nm.2396
Supplementary figure 6
Tubasta'n A dose-‐dependently rescued axonal transport and increased acetyl-‐tubulin levels in
vitro.
(a,b) Axonal transport of mitochondria was assessed in DRG neurons isolated from symptoma8c (8
months old) HSPB1S135F mice acer 12 h incuba8on with various concentra8ons (0, 0.25, 0.50 or
1.00 μM) of Tubasta8n A. (a) Quan8fica8on of the total number of mitochondria at various
concentra8ons of Tubasta8n A. One-‐way ANOVA. P > 0.05. (b) Quan8fica8on of the number of
moving mitochondria at different concentra8ons of Tubasta8n A. One-‐way ANOVA. * P < 0.05; **
P < 0.001; P < 0.0001. (c) Quan8fica8on of the integrated density of the acetylated tubulin signal
along neurites at various concentra8ons of Tubasta8n A. n = 25-‐30 cells/condi8on. One-‐way
ANOVA. * P < 0.05.
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Nature Medicine doi:10.1038/nm.2396
References
1. Butler, K.V., et al. Ra8onal design and simple chemistry yield a superior, neuroprotec8ve
HDAC6 inhibitor, tubasta8n A. J. Am. Chem. Soc. 132, 10842—10846 (2010).
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Nature Medicine doi:10.1038/nm.2396
1
SUPPLEMENTARY METHODS
HDAC6 inhibitors reverse axonal loss in a mouse model of mutant HSPB1-‐induced Charcot-‐
Marie-‐Tooth disease
Constantin d’Ydewalle, Jyothsna Krishnan, Driss M. Chiheb, Philip Van Damme, Joy Irobi, Alan P.
Kozikowski, Pieter Vanden Berghe, Vincent Timmerman, Wim Robberecht, Ludo Van Den Bosch
Nature Medicine doi:10.1038/nm.2396
2
Genotyping of transgenic mice
Genotyping of transgenic animals was performed using PureTaq Ready-‐To-‐Go PCR beads
(GE Healthcare Bio-‐Sciences) according to manufacturer’s instructions with two primer sets. The
first primer set was designed to amplify the transgene construct: forward primer 5’-‐
CAgCTggCTgACCTgTAgC-‐3’; reverse primer 5’-‐ CTTggCggCAgTCTCATCg-‐3’. The second primer set
was used to amplify the mouse Interleukin-‐2 gene as an internal positive control: forward primer
5’-‐CTAggCCACAgAATTGAAAgATCT-‐3’; reverse primer 5’-‐gTAgTggAAATTCTAgCATCATCC-‐3’.
The transgenic founders were identified and subsequently transferred to the animal facility
of the K.U.Leuven.
Behavioural assessment of mice
General motor performance was assessed using an accelerating rotarod (Ugo Basile)
rotating from 4 to 40 rpm on 5 min ramp duration. At different ages, each animal was given three
consecutive trials with a 1 min resting interval without prior training sessions. The average time
spent on the rotarod was used as a measure of motor performance.
Muscle force of the animals was measured using a Grip Strength Meter (Columbus
Instruments) with either a grid (all paws) or a triangular bar (forepaws) as probe. The average of
three trials per animal was determined at different ages using the different probes and was used
as a measure for grip strength.
Gait analysis was performed using the Catwalk system (Noldus) as described.1,2 Briefly,
each mouse was given three trials during which the animal had to cross the pressure-‐sensitive
plate of the Catwalk system without any interruption. At different ages, paw angle, stride length
and paw print area were recorded and averaged over the three trials.
To measure sensory deficits, animals were placed on a hot plate kept digitally at a constant
temperature of 55 °C during maximally 30 s. The latency to the first response (either a paw lick, a
paw flick or jump) was recorded for the mutant HSPB1 mice and was normalized to the
measurements obtained for the wild type HSPB1 mice. For every age, a new set of mice was used.
Western blotting and ELISA
Protein concentrations were determined using the microBCA kit (Thermo Fisher Scientific
Inc.) according to the manufacturer’s instructions. Western blotting was performed as described
before.3 Optical densities were determined using the integrated density measurement tool of
Nature Medicine doi:10.1038/nm.2396
3
ImageJ (NIH). Glyceraldehyde-‐3-‐phosphate dehydrogenase (Gapdh; Covance) and hemagglutinin-‐
tag (HA-‐tag; Roche Diagnostics) were the antibodies used.
For ELISA, the colorimetric Pathscan Sandwich ELISA kit (Cell Signaling Technology Inc.) for
acetylated tubulin was used according to manufacturer’s instructions with minor adaptations.
Briefly, every well was coated with an anti-‐tubulin antibody. For each genotype, 1 mg ml—1 sciatic
nerve or spinal cord homogenized in RIPA buffer was loaded into the wells and incubated
overnight. We used a horseradish peroxidase (HRP)-‐linked antibody against acetylated lysine or
against α-‐tubulin (Cell Signaling Technology Inc.), followed by incubation with HRP substrate for
detection. Absorbance was measured at 450 nm. We normalized acetylated α-‐tubulin signal to
total α-‐tubulin levels.
Immunohistochemistry and histology
Sections of spinal cords, nerves and gastrocnemius muscles were washed in phosphate-‐
buffered saline (PBS) and blocked with 5% normal donkey serum in 0.1% Triton X100/PBS for 1 h.
Smi32-‐R (Covance), Gfap (Sigma-‐Aldrich), acetylated tubulin (Sigma-‐Aldrich), Pmp22 (Abcam), HA-‐
tag (Roche Diagnostics or Cell Signalling Technology), Nf200 (Millipore) were diluted in 0.1% Triton
X-‐100/PBS and incubated for 2—3 h. Alexa-‐conjugated secondary antibodies (Invitrogen) and/or
α-‐bungarotoxin conjugated to Alexa-‐555 fluorophore were diluted in 0.1% Triton X-‐100/PBS and
incubated for 1 h. Sections were mounted with DAPI-‐containing Vectashield (Vectorlabs Inc.) to
visualize nuclei. Innervation level of morphologically normal neuromuscular junctions (NMJs) was
defined as full or not present when there was complete or no overlap of Nf200 and α-‐
bungarotoxin, respectively. Innervation level of NMJs was determined on every 10th slide (50 in
total).
Whole sciatic nerves were washed in a phosphate buffered solution (pH 7.4) containing
170 mM NaH2PO4 (Sigma-‐Aldrich) and 100 mM NaOH (Sigma-‐Aldrich), and incubated for 2 h in 2%
OsO4 (Sigma-‐Aldrich). After OsO4 incubation, samples were washed and dehydrated using
subsequent steps of 50%, 70%, 90% and 100% ethanol, in which the samples were incubated for
60 min. Next, nerves were incubated in propylene oxide and embedded in epoxy resin diluted in
propylene oxide. The resin hardened during 3 days at 60 °C. Semi-‐thin (1 μm) transverse sections
were cut using a Leica ultra-‐microtome (Leica Microsystems) and stained with 1% toluidine blue at
80 °C for 30—45 s. On every 10th slide, myelin thickness and the number of axons were measured
using ImageJ software.
Nature Medicine doi:10.1038/nm.2396
4
Haematoxilin-‐Eosin (H&E) staining was performed by incubating sections in 1% formol-‐
calcium for 10 min, 3 min in Harris’ haemtoxylin (Sigma-‐Aldrich), 3 min in water and 3 min in eosin.
Nicotinamide adenine dinucleotide (NADH) staining was performed on transverse sections by
incubating the samples for 45 min at 37°C in a Tris-‐buffered staining solution containing 200 mM
Tris (Sigma-‐Aldrich), 2 mg ml—1 Nitro-‐tetrazolium Blue Chloride (Sigma-‐Aldrich), 17% HCl (Sigma-‐
Aldrich) and 1 mg ml—1 NADH (Sigma-‐Aldrich). Samples were fixed for 30 min in 1% formol-‐calcium
and washed subsequently in 30, 60 and 30% acetone, and distilled water. Finally, H&E and NADH
stained muscle sections were dehydrated in methanol, ethanol and toluene and mounted using
Pertex (Histolab).
Fluorescent and brightfield micrographs were captured using a Zeiss Axio Imager M1
microscope (Carl Zeiss) equipped with an AxioCam MRc5 (brightfield; Carl Zeiss) or a monochrome
AxioCam Mrm camera (fluorescence; Carl Zeiss).
DRG neuron cultures
Dorsal Root Ganglion (DRG) neurons were dissociated by incubation with 0.5% collagenase
and 1.3% trypsin at 37 °C for 45 min. Unless indicated otherwise, all culture media and
supplements were from Invitrogen. Cell suspensions were washed with DRGPREP medium
[containing DMEM medium supplemented with bovine serum (Greiner Bio-‐One; 10%), non-‐
essential amino acids (1%), sodium bicarbonate (0.14%) and L-‐glutamine (200 nM)], and
centrifuged at 800 g for 5 min. The pellet was resuspended in DRGPREP medium and incubated in
fetal calf serum (Greiner Bio-‐One) for 50 min at 37 °C. Next, the cell suspension was centrifuged at
800 g for 5 min and the pellet was resuspended in DRG medium containing 1:1 mix of DMEM and
F12 medium supplemented with L-‐glutamax (4 mM), non-‐essential amino acids (1%), fetal calf
serum (10%), penicillin (50 U ml—1), streptomycin (50 μg ml—1), nerve growth factor (NGF; 10 ng
ml—1) (Millipore) and NaHCO3 (0.045%)]. Subsequently, DRG neurons were seeded at a density of
100 cells per well coated with poly-‐L-‐ornithine (Sigma-‐Aldrich) and laminin (Sigma-‐Aldrich). After
24 h, DRG medium was replaced by NGF-‐deprived DRG medium.
For rescue experiments, DRG neurons were treated with either 0.4 μM TSA, 2 μM Tubacin
or 0.25, 0.50 and 1 μM Tubastatin A or an equivalent amount of DMSO for 12 h in NGF-‐deprived
DRG medium to exclude outgrowth effects by NGF.
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5
Image acquisition and analysis of axonal transport
Neurons were selected under differential interference optics (DIC) based on normal DRG
neuron morphology consisting of a cell body and neurites that have at least three times the length
of the cell body. Mitotracker-‐RED (Invitrogen) was excited at 570/15 nm using a TILL Poly V light
source (TILL Photonics) and image sequences were recorded (200 images at 1 Hz) onto a cooled
CCD camera (PCO Sensicam-‐QE) using TillVisION (TILL Photonics) software. A heated gravity-‐fed
perfusion system was used to keep cells at 36 ± 0.5 oC during the recordings. After recording, DRG
neurons were fixed and stained immunocytochemically to confirm the expression of HA-‐tagged
human HSPB1.
All image analysis was performed in Igor Pro (Wavemetrics) using custom-‐written routines
based on a previously described analysis algorithm.4 In brief, kymographs or spatio-‐temporal maps
were constructed for each of the neuronal processes. In these maps, stationary mitochondria
appear as vertical lines and moving mitochondria generate tilted lines. Proportions of moving and
stationary mitochondria as well as transport velocity were extracted from the maps by marking
and analyzing the properties (deflections, changes in direction, etc.) of each of the mitochondrial
trajectories.
Acetylated tubulin levels in neurites of fixed DRG neurons were assessed by measuring the
intensity the fluorescent signal after incubation with an anti-‐acetyl-‐tubulin antibody (Sigma
Aldrich, 1:5000, 1 h) followed by incubation with a secondary antibody conjugated to Alexa-‐488
(Invitrogen, 1:5000, 1 h) using the integrated density measurement tool of ImageJ (NIH).
References
1. Hamers, F.P., Koopmans, G.C. & Joosten, E.A. CatWalk-‐assisted gait analysis in the
assessment of spinal cord injury. J. Neurotrauma 23, 537—548 (2006).
2. Vandeputte, C., et al. Automated quantitative gait analysis in animal models of movement
disorders. BMC Neurosci. 11, 92 (2010).
3. Krishnan, J. et al. Over-‐expression of Hsp27 does not influence disease in the mutant
SOD1(G93A) mouse model of amyotrophic lateral sclerosis. J. Neurochem. 106, 2170—2183
(2008).
4. 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).
Nature Medicine doi:10.1038/nm.2396