supplementaryfigures - nature research · 2011-08-04 · supplementaryfigures...

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

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



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.

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.

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.

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.

7


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).

8


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


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


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.


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.


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).


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