Autocrine and immune cell-derived BDNF in human skeletal muscle: implications for myogenesis and...

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Autocrine and immune cell derived BDNF in human skeletal muscle:

implications for myogenesis and tissue regeneration

Emanuela Colombo1, Francesco Bedogni2, Isabella Lorenzetti1, Nicoletta Landsberger2,3,

Stefano C. Previtali1 and Cinthia Farina1*.

1Institute of Experimental Neurology (INSpe), Division of Neuroscience, San Raffaele

Scientific Institute, Milan, Italy

2 San Raffaele Rett Research Center, San Raffaele Scientific Institute, Milan, Italy

3 Laboratory of Genetic and Epigenetic Control of Gene Expression, Department of

Theoretical and Applied Sciences, University of Insubria, Busto Arsizio, Italy

∗ Corresponding author: Institute of Experimental Neurology (INSpe), Division of

Neuroscience, San Raffaele Scientific Institute, Via Olgettina, 58, 20132 Milan – Italy.

Phone.: +39 02 2643 6180 E-mail: [email protected]

No conflicts of interest to be declared

This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1002/path.4228


Abstract

The neurotrophin system has a role in skeletal muscle biology. Conditional depletion of

BDNF in mouse muscle precursor cells alters myogenesis and regeneration in vivo. However,

the expression, localisation and function of BDNF in human skeletal muscle tissue is not

known so the relevance of the rodent findings for human muscle are unknown. Here we

address this by combining ex vivo histological investigations on human biopsies with in vitro

analyses of human primary myocytes. We found that BDNF was expressed by precursor and

differentiated cells both in vitro and in vivo. Differential analysis of BDNF receptors showed

expression of p75NTR and not of TrkB in myocytes, suggesting that the BDNF-p75NTR axis

is predominant in human skeletal muscle cells. Several in vitro functional experiments

demonstrated that BDNF gene silencing or protein blockade in myoblast cultures hampered

myogenesis. Finally, histological investigations of inflammatory myopathy biopsies revealed

that infiltrating immune cells localised preferentially near p75NTR positive regenerating

fibers and that they produced BDNF. In conclusion, BDNF is an autocrine factor for skeletal

muscle cells and may regulate human myogenesis. Furthermore, the preferential localisation

of BDNF producing immune cells near p75NTR positive regenerating myofibers suggests

that immune cell derived BDNF may sustain tissue repair in inflamed muscle.

Keywords: BDNF, inflammation, human skeletal muscle, neurotrophin, myogenesis, immune

cells


Introduction

Postnatal growth and repair of skeletal muscle after damage require the activation of satellite

cells, resident muscle precursor cells located between the cell membrane and the basal lamina

of muscle fibers [1]. Several regulatory factors control the myogenic properties of satellite

cells and thereby muscle regeneration. For example, while IGF-1, FGF and VEGF are

positive regulators of myogenesis, TGF-β and myostatin reduce myoblast differentiation

[2,3]. Moreover, TGF-β is a potent regulator of extracellular matrix deposition, thus favoring

fibrosis [2,3].

Recent evidence suggests a role for neurotrophins (NT) in skeletal muscle physiology and

repair. The family of neurotrophins consists of nerve growth factor (NGF), NT-3, NT-4 and

brain derived neurotrophic factor (BDNF). Although initially described as factors specific for

the central nervous system [4], NT have been detected also in skeletal muscle where they

regulate motor neuron survival, enhance presynaptic release of neurotransmitter and promote

maintenance of postsynaptic region on skeletal myofibers [5-9]. Two different types of

receptors mediate neurotrophin signaling: the tropomyosin related kinase (Trk) receptors and

the p75 neurotrophin receptor (p75NTR) [4]. Recently, attention has been devoted to

p75NTR. A study from our group demonstrated that p75NTR is a marker for human skeletal

muscle precursor cells at high differentiation potential, and that it positively regulates

myogenesis and myofiber maturation [10]. Furthermore, blockade of p75NTR signaling in

vivo in an injury mouse model hampers muscle regeneration [11].

Regarding p75NTR ligands, some studies suggest the expression of all neurotrophins in

skeletal muscle [12-15], albeit the sources of neurotrophins in human tissues have not been

clearly characterized. Here we focused our attention on BDNF, as low levels of BDNF are


detected in developing and postnatal avian and rodent skeletal muscle [14,16-19], and its

production is maintained by myogenic progenitor cells in adult rodent muscle [20-23].

Further, BDNF transcript levels increase in rat muscle after exercise [24,25] and after

denervation [12,26], indicating that BDNF expression may be regulated in skeletal muscle.

Beyond its function as a retrograde trophic factor supporting motor neuron survival and

neuromuscular transmission [8,19], BDNF may support skeletal muscle regeneration. In fact,

transgenic mice carrying selective depletion of BDNF in skeletal muscle precursor cells

display reduced numbers of regenerating fibers after injury [27]. However, implications of

these findings for the human skeletal muscle physiology are unknown as no studies

performed an accurate characterization of BDNF sources in human skeletal muscle.

Furthermore, it is still not clear whether human pathological conditions are associated with

changes in BDNF levels or distribution. An in situ hybridization study on post-mortem

tissues showing enhanced BDNF mRNA in amyotrophic lateral sclerosis muscle suggests

that expression of the neurotrophin may change in human skeletal muscle under disease [28].

Finally, the evidence of a role for this neurotrophin in human skeletal muscle biology is not

yet available. The aim of our study was therefore to assess BDNF expression and localization

in striated muscle under physiological and pathological conditions and to illustrate its

involvement in human myogenesis.

Methods

Patients and tissues

Muscle biopsies were performed for diagnostic reasons and stored in the institutional tissue

bank. Informed consent for biopsy and its storage for research purposes was obtained in all

cases. Biopsies were taken from quadriceps femoris (n=19), deltoid (n=3), biceps (n=2) or

rectus femoris (n=1) muscles. We selected specimens with clear diagnosis, based on clinical,

electromyographic and histological findings [29,30]. A total of 17 patients suffered from


idiopathic inflammatory myopathies (IIM): polymyositis n=8, dermatomyositis n=4 or

inclusion body myositis n=5 (mean age 65±12, 10 male and 7 female subjects). Finally, 8

muscle specimens from adult individuals (mean age 37±13, 6 male and 2 female subjects)

initially suspected of neuromuscular disease whose biopsies were histologically normal were

included as controls.

Myoblast cell culture

Human primary myoblasts were selected for CD56/NCAM expression, cultured and

differentiated as previously described [10,31,32]. Fusion index was calculated as the

percentage of myonuclei within myotubes (with more than two nuclei) on total nuclei

number. Each experiment was performed in triplicates, each figure shows representative data

out of minimum three performed experiments. Data were confirmed in two primary cell lines.

Immunohistochemistry and double immunofluorescence

The following primary antibodies were used: two distinct anti-human BDNF, anti-human

NGFR, anti-human TrkB (all from R&D Systems, Milan, Italy), anti-human CD56 (BD

Biosciences, Buccinasco, Italy), anti-human CD68, anti-human CD4, anti-human CD8, anti-

human desmin (all from Dako, Milan, Italy), anti-human CD3, anti-Ki67 (both from Lab

Vision, Fremont, California), anti-human perforin (BD Biosciences), anti-dystrophin

(provided by Dr. Mora), purified mouse IgG1 isotype (Sigma-Aldrich, Gallarate, Italy),

polyclonal rabbit Ig (Dako). Detection was perfomed as in [10,31].

The Zenon technique was used to perform double staining with multiple mouse monoclonal

antibodies according to the manufacturer’s protocol (Life Technologies, Monza, Italy).


Fluorescence images were captured with confocal laser-scanning microscopes (Leica

Microsystems, Milan, Italy). Images from immunohistochemistry sections were acquired

using light microscopes (Leica Microsystems). The ImageProPlus software (Media

Cybernetics, Silver Spring, MD, USA) was used for image analysis. For the quantification of

immune cell distribution, firstly we assessed the total number of regenerating (CD56

positive) or mature (CD56 negative) myofibers in each tissue section. Mature fibers in

contact with regenerating fibers were not included in the quantification. Infiltrating immune

cells were identified as basophilic small nuclei by haematoxylin staining. Nuclei positioned

within myofibers were not considered for the quantification. Then, the number of immune

cells localized in the proximity of regenerating or mature myofibers was calculated as the

ratio between the number of immune cells and the total number of regenerating or mature

myofibers respectively.

In Situ Hybridization

Digoxygenin BDNF exon 5 in situ was performed as previously described [33]. After pre-

hybridization buffer, sections were hybridized overnight at 60°C with a a proper dilution of

riboprobe in hybridization buffer. Sections were then washed four times at 60°C in Washing

Solution, blocked in Blocking Solution at r.t. (Roche, Monza, Italy) then incubated overnight

at 4°C in 1:1000 Anti-DIG (Roche). After several washings and brief incubation in

Levamisole (0,1M, Sigma Aldrich), BCIP/NBT colorimetric reaction (Roche) was then

carried out in the dark at r.t.


Flow cytometry

Labelling with mouse monoclonal anti-human TrkB (R&D Systems) or with the

corresponding isotype controls (BD Biosciences) was followed by detection with PE-labeled

F(ab’)2 fragments goat anti-mouse Ig (Dako). Cytofluorimetric analyses were performed on

FACSCalibur (BD Biosciences). CellQuest software (BD Biosciences) was used for

acquisition and FlowJo (Tree Star Inc, Ashland, Oregon, USA) for data analysis. Thresholds

for positivity were set on isotype control.

BDNF RNA interference

Small interfering RNA fragments (siRNA) for BDNF and non-specific control (47% GC

content) were purchased at Eurofins MWG (M-Medical, Cornaredo, Italy). siRNA were

diluted at 20nM in Optimem (Life Technologies). Transfection was obtained by Interferin

(Polyplus, Celbio, Milan, Italy). Three days after siRNA transfection myoblasts were induced

to differentiate and silencing efficiency was monitored at day 3 after differentiation

induction.

Treatment with anti-BDNF blocking antibody

Myoblasts were seeded in 4-well chamber slides and induced to differentiate for 7 days in

medium supplemented with 10μg/ml rabbit polyclonal anti-human BDNF blocking antibody

(Millipore, Vimodrone, Italy) [34] or purified rabbit Ig (Dako). Antibody treatment was

repeated for three consecutive days.

RNA extraction, cDNA synthesis and Real-Time PCR

RNA extraction, cDNA synthesis and Real time PCR were performed as in [34]. To remove

contaminating DNA, RNA was treated with DNase I enzyme (Life Technologies).


Amplification sets for BDNF, p75NTR, TrkB were described in [35]. mRNA levels of target

genes were graphically reported as percentage of the housekeeping gene PPIA (Life

Technologies).

Statistical Analysis

Normality of the distribution was assessed by Kolmogorov–Smirnov statistics. Student’s T-

test was used to compare means. All P-values were two-sided and subjected to a significance

level of 0.05.

Results

BDNF is strongly expressed in myoblasts and in differentiating cells in vitro

First, we analyzed BDNF expression in primary cultures of human muscle precursor cells, the

myoblasts. Quantitative RT-PCR revealed robust transcript levels for the neurotrophin in

these undifferentiated cells under normal growth conditions (Figure 1A, first column).

Double immunofluorescence performed with a specific antibody showed BDNF protein in all

CD56/NCAM positive myoblasts (Figure 1B). Staining with another anti-BDNF antibody

confirmed this observation (not shown). Then, we induced myoblast differentiation and

fusion into multinucleated myotubes by the addition of specific culture medium, and

monitored BDNF mRNA levels over two weeks. Neurotrophin expression remained high

during the first 10 days and slightly increased afterwards (Figure 1C). Double

immunofluorescence confirmed the presence of BDNF in cultured myotubes (Figure 1D). As

BDNF action may be mediated by two distinct receptors, we investigated the expression of

TrkB and p75NTR at mRNA levels in parallel to BDNF. As already described [10], p75NTR

mRNA was expressed in undifferentiated myoblasts (Figure 1A, second column) and

increased during myogenesis (Figure 1C). In contrast, TrkB transcript levels were barely


detectable both in myoblasts and in differentiated cultures (Figure 1A third column and

Figure 1C). Moreover, cytofluorimetric and double immunofluorescence experiments

confirmed the lack of TrkB at protein level in cultured myoblasts (Figure 1E-F). Therefore

human muscle cells produce BDNF in vitro and may respond to it through p75NTR.

BDNF is displayed by satellite cells and mature myofibers in vivo

As p75NTR is expressed in satellite cells in vivo [10] and myoblasts strongly produced

BDNF in vitro, we checked expression of the neurotrophin in satellite cells in situ. Double

immunofluorescence on non-myopathic tissues showed that satellite cells stained positive for

BDNF and p75NTR (Figure 1G-H). Accurate quantification revealed that more than 80% of

CD56/NCAM-positive satellite cells expressed the neurotrophin (Figure 1I). Furthermore, in

accordance with in vitro observations on myotubes, in situ hybridization and

immunohistochemistry experiments on skeletal muscle tissues showed that all mature

myofibers were positive for BDNF (Figure 1J-K). In contrast, the receptor TrkB was not

displayed by myofibers but its expression was restricted to some vessels (Figure 1L, black

arrow). These in vitro and in vivo observations suggest that BDNF may play a physiological

role in human skeletal muscle and that p75NTR is the only available receptor for BDNF

signalling in human muscle precursor cells.

BDNF regulates human myogenesis in vitro

We performed functional in vitro studies to evaluate the involvement of BDNF in human

myogenesis. For this purpose we induced myoblast differentiation while hampering BDNF

expression or activity. First, we reduced BDNF mRNA availability by specific RNA


interference in undifferentiated myoblasts (0.33±0.01 vs. 0.72±0.07 in BDNF-silenced and

control cultures respectively ; p value = 0.011; degrees of freedom = 2; t Stat = 9.48; t

Critical = 4.3; Figure 2A), and then induced in vitro differentiation for 6 days. Importantly,

myoblasts transfected with specific BDNF siRNA showed lower fusion index than control

cultures (23.3±3.3 vs. 32.4±2.4 in BDNF-silenced and control cultures respectively ; p value

= 0.02; degrees of freedom = 4; t Stat = 3.79; t Critical = 2.78; Figure 2B-C). Second, we

blocked BDNF protein activity in differentiating cultures by adding a specific neutralizing

antibody [34]. Parallel cultures where myoblasts were induced to differentiate in the presence

of an isotype antibody were used as controls. At day 6 myogenesis was significantly reduced

in cultures exposed to the anti-BDNF antibody (23.6±0.9 vs. 34±1 in anti-BDNF antibody-

treated and control cultures respectively; p value = 0.0002; degrees of freedom = 4; t Stat =

12.98; t Critical = 2.78; Figure 2D-E). In conclusion, the functional assays showed that

blockade of BDNF either at transcript or at protein level impairs human myogenesis.

Immune cells in inflammatory myopathies are preferentially located near regenerating

fibers and produce BDNF

We extended analysis for BDNF to muscle biopsies from patients affected by idiopathic

inflammatory myopathies (IIM). First of all, we checked BDNF transcript levels in healthy

and diseased tissues by quantitative PCR experiments and found detectable and comparable

expression of BDNF in the two groups (0.97±0.21 in control samples vs. 0.78±0.16 in IIM; p

value = 0.82; Figure 3A). Then, immunohistochemical experiments in pathological tissues

confirmed that mature (CD56 negative, asterisks in Figure 3B-E) myofibers were positive for

BDNF under inflamed settings. Moreover a number of CD56 positive regenerating fibers

(triangles, Figure 3B-E) displayed enhanced BDNF immunoreactivity. Importantly, the same

fibers expressed p75NTR (triangles, Figure 3D), suggesting that autocrine usage of BDNF


supports in vivo regeneration. Further, as expected, inflamed tissues were characterized by

massive immune cell infiltration. We noticed that immune cells were not homogenously

distributed within the tissue, but were mostly organized into clusters. Such clusters were

topologically juxtaposed near CD56 positive regenerating fibers (Figure 3F-G). To quantify

this observation we counted the number of immune cells in the vicinity of regenerating fibers

or of mature myofibers in 8 biopsies from IIM patients. Such analysis confirmed that immune

cells were preferentially located near newly forming fibers (5.7±4.7 immune

cells/regenerating fiber vs. 1±1.7 immune cells/mature fiber; p value = 0.027; degrees of

freedom = 9; t Stat = 2.64; t Critical = 2.26; Figure 3H). IIM are considered autoimmune

disorders of skeletal muscle [36]. It was in fact shown that some immune cells, mainly

located within myofibers, are activated and secrete cytolytic factors [37]. To further

characterize the inflammatory infiltrates in inflamed muscle we checked the expression of the

proliferation marker Ki-67 and of the cytolytic factor perforin. We found that perforin

releasing T cells were extremely rare, located near mature myofibers and not in the vicinity of

CD56 positive regenerating fibers (Supplementary Figure 1A-B). Similar distributions were

observed for Ki-67-positive macrophages (Supplementary Figure 1C-D) or T cells (not

shown). In contrast, immunohistochemistry for BDNF revealed several immune cells strongly

positive for the neurotrophin (white arrows, Figure 3E) and frequently localized near

p75NTR-expressing regenerating myofibers (triangles, Figure 3C-D). Accurate quantification

of this observation confirmed that, while immune cells located near mature myofibers rarely

showed staining for the neurotrophin, those cells in the vicinity of regenerating fibers often

expressed BDNF (1.37±1.39 BDNF positive immune cells/regenerating fiber vs. 0.021±0.018

BDNF positive immune cells/mature fiber; p value = 0.029; degrees of freedom = 7; t Stat =

2.74; t Critical = 2.36; Figure 3I). Fine histological characterization by double

immunofluorescence and confocal microscopy showed that BDNF was produced by T


lymphocytes (both CD4+ and CD8+ cells Figure 3J-K) and macrophages (Figure 3L) but not

by B cells (not shown). In summary, in inflamed tissue both myocytes and immune cells

contribute to BDNF production. The preferential distribution of BDNF producing immune

cells around p75NTR positive regenerating fibers strongly suggests that such immune cells

are not pathogenic but rather sustain skeletal muscle repair.

Discussion

In this study we identified the neurotrophin BDNF as a factor endogenously produced by

both skeletal muscle precursor cells and mature myofibers that may positively regulate

human myogenesis. In IIM several immune cells infiltrating skeletal muscle may contribute

to BDNF production and preferentially accumulate near p75NTR positive regenerating fibers.

Furthermore, BDNF positively regulates human myogenesis, identifying this factor as a

candidate ligand triggering p75NTR action in human skeletal muscle.

Human skeletal muscle regeneration requires the activation of satellite cells which is

regulated by stimuli derived from the microenvironment, such as growth factors. Recent

reports on rodent muscle highlight the role of the neurotrophin BDNF in this process [20,27].

In fact, BDNF is expressed in rodent satellite cells [20] and regulates myogenesis in vivo

[27]. In our study the fine characterization of the sources of BDNF in human skeletal muscle

revealed that this neurotrophin is expressed by both precursor and differentiated cells.

Quantification of satellite cells expressing the neurotrophin showed that the great majority of

precursors is BDNF positive. This observation is in harmony with the in vivo expression of

p75NTR. Consistent with in vivo data, high levels of BDNF were found in primary myoblasts

and in multinucleated myotubes in vitro. Regarding the receptor TrkB, it has been detected at

mRNA or protein level in total extracts from animal muscle [12,14,24], however its presence

in muscle cells has not been described. In contrast, p75NTR is expressed by skeletal muscle


and regulates myogenesis via modulation of RhoA-GTP signaling in differentiating cells

[10,11,31]. In fact, we described recently that p75NTR positive precursor cells display a

repertoire of gene products that accounts for their enhanced differentiation properties and that

p75NTR blockade either at the transcript or protein level impairs myogenesis [10]. However,

the precise downstream signaling activated by NT binding to p75NTR in myocytes remains

largely unknown, and future studies are needed to resolve this issue.

Based on our data shown here and in previous publications [10,31,32], the receptor TrkB is

absent in human myocytes in vitro and in vivo, while p75NTR appears on BDNF-expressing

satellite cells and regenerating fibers as well as in cultured cells. These evidences indicate

that the neurotrophin BDNF is commonly produced by human muscle cells and that, due to

the expression of p75NTR, muscle cells may be not only the source but also the target of

BDNF. As the neurotrophin receptor p75NTR is a key regulator of human myogenesis [10],

we hypothesized that myoblast responses to BDNF could modulate skeletal muscle cell

differentiation. Functional experiments in the in vitro model of myocyte differentiation

showed that the reduction in BDNF transcript levels or in protein availability hampered cell

fusion. This is in accordance with the in vivo data obtained in a transgenic mouse model

where BDNF depletion was achieved in muscle precursor cells [27].

Overall, these observations demonstrate that BDNF is essential for skeletal muscle

differentiation and that its action is presumably mediated by p75NTR. A limitation of the

study is that the molecular mechanisms triggered by BDNF during myogenesis have not been

characterized and require further investigations.

The characterization of muscle tissues from patients affected by IIM for BDNF protein

expression led to novel additional observations. First of all, mature myofibers consistently

expressed BDNF also in IIM. Interestingly, we described recently upregulation of p75NTR in


mature muscle fibers in IIM [31]. In vitro blocking experiments of p75NTR expression by

RNA interference in myotubes showed that p75NTR-silenced myotubes were more

susceptible to apoptosis after exposure to IL-1 than control myotubes [31]. These evidences

suggest that BDNF-p75NTR axis activates a tissue protective response. Moreover, we

showed here that in IIM immune cells are not evenly distributed within the tissue, but that

they accumulate in regenerating areas. This might indicate an exacerbated immune response

against newly-forming fibers. However, these immune cells were not in a proliferative state

or did not produce cytolytic factors, dampening the hypothesis of a pathogenic immune

response in such regions.

Opposed to the evidence about the involvement of immunity in the etiopathogenesis of

inflammatory myopathies, some authors demonstrated a crosstalk between immune and

muscle cells that positively regulates muscle homeostasis and repair. For example,

macrophages seem to play a primary role in muscle regeneration as they may regulate re-

activation of muscle satellite cells. An in vitro study showed that human myoblasts attract

monocytes producing specific chemoattractants such as monocyte chemoattractant protein-1,

macrophages-derived chemokine and VEGF, and that co-culture with macrophages increases

myoblast proliferation [38]. Further, after cardiotoxin-induced damage, skeletal muscle

regeneration is severely impaired if mice are treated with a specific antibody blocking

macrophage infiltration into the damaged tissues. In particular, muscles show enhanced

fibrosis, low numbers of satellite cells and of newly forming myotubes [39], indicating a clear

role for macrophages in sustaining muscle regeneration after injury. Different activation

states for macrophages have been described in vitro [40]. Classical activation (M1) is

characterized by production of proinflammatory cytokines and represents the primary

response of macrophages to tissue injury. Interestingly, macrophages with an inflammatory

phenotype are found in human dystrophic muscle [41], where they are prominently located in


necrotic foci [42]. Conversely, the anti-inflammatory profile (M2) has been associated with

muscle repair [41,43].

Our data are consistent with a model where BDNF acts as a crosstalk factor between immune

and muscle cells. We demonstrated the preferential accumulation of BDNF-releasing immune

cells near regenerating fibers. Immunofluorescence experiments combined with confocal

microscopy revealed that both (CD4+ and CD8+) T lymphocytes and macrophages

contributed to BDNF production. These findings are not surprising as BDNF production by

human thymocytes and circulating immune cells has been previously described [35,44,45]. It

becomes important however to consider that the localization of BDNF producing immune

cells was preferentially within regeneration areas and that such cells were not activated and

did not produce cytolytic factors. These observations support the hypothesis that protective

immune responses may also take place in inflamed skeletal muscle and that BDNF may be a

mediator for regeneration.

Acknowledgements

We acknowledge the EurobioBank and the Italian Telethon Network of Genetic Biobanks

(GTB07001F) for providing myoblast cell lines, and Dr. M. Mora from Neurological Institute

Carlo Besta for anti-dystrophin antibody. This work was supported by the Association

Française contre les Myopathies (AFM).

Author contributions

EC carried out experiments, analysed and interpreted the data, wrote the manuscript. FB

carried out ISH experiments. IL and NL contributed reagents/material/analysis tool. SP

provided tissue specimens. CF designed the study, conceived the experiments, analysed and

interpreted the data, wrote the manuscript. All authors approved the submitted manuscript.


Online Supporting Information

Figure S1. Perforin expressing T cells or Ki67 positive macrophages are localized near

mature fibers.

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Figure legends

Figure 1. BDNF is expressed in human muscle precursor cells and in mature myofibers. (A)

BDNF, p75NTR and TrkB mRNA levels in cultured myoblasts. (B) Double

immunofluorescence for BDNF and CD56/NCAM in cultured myoblasts. (C) Regulation of

BDNF, p75NTR and TrkB mRNA levels upon differentiation. Quantitative real-time PCR

was performed at distinct time points. In (A) and (C) mRNA levels of target genes were

graphically reported as percentage of the housekeeping gene PPIA. (D) Double

immunofluorescence for BDNF and dystrophin in cultured myotubes. (E) Cytofluorimetric

analysis of cultured myoblasts for TrkB surface expression. (F) Double immunofluorescence

for TrkB and CD56/NCAM in cultured myoblasts. (G-H) BDNF immunoreactivity in a

CD56/NCAM (G) or p75NTR (H) positive satellite cell of adult skeletal muscle. (I)

Percentage of CD56/NCAM positive satellite cells expressing BDNF in human adult skeletal

muscle. Circles represent distinct tissue samples, red bar represents mean. At least 40 CD56

positive satellite cells in at least 2 sections per sample were counted. (J) In situ hybridization

for BDNF in human adult skeletal muscle. (K-L) Immunohistochemistry for BDNF (K) and

TrkB (L) in human adult skeletal muscle. Black arrow indicates TrkB positive vessel. In (A)

and (C) error bars represent standard deviations. Scale bar 30 μm in (B), (D), (F) and (L), 3,5

μm in (G) and (H), 50 μm in (J), 100 μm in (K).



Figure 2. BDNF regulates myogenesis in vitro. (A-C) BDNF silencing in myoblasts and

effects on myogenesis. (A) BDNF silencing efficiency monitored 3 days after differentiation

induction by quantitative RT-PCR. BDNF mRNA levels were graphically reported as

percentage of the housekeeping gene PPIA. (B-C) Immunofluorescence for desmin and DAPI

(B) and fusion index evaluation at day 6 after differentiation induction (C) in cultures treated

with BDNF or control siRNA. (D-E) Immunofluorescence for desmin and DAPI (D) and

fusion index quantification (E) in a blocking experiment with anti-BDNF antibody or isotype

control administered during cell differentiation. Arrows indicate myotubes (cells with more

than 2 nuclei). Error bars represent standard deviations. Experiments were performed in

triplicates and at least 1000 nuclei per replicate were counted. Similar observations were

obtained in at least three independent experiments in two primary cell lines. Scale bar 50 μm.

* P< 0.05, *** P<0.001



Figure 3. BDNF is produced by immune cells located near regenerating fibers in

inflammatory myopathies. (A) Quantitative PCR for BDNF mRNA tissue levels in control

biopsies (n=7) and in idiopathic inflammatory myopathies specimens (n=14). BDNF mRNA

levels were graphically reported as percentage of the housekeeping gene PPIA. Error bars

represent SEM. (B-E) Newly forming fibers are basophilic at H&E (B), and stain positively

for CD56 (C), p75NTR (D) and BDNF (E). Several immune cells localized near p75NTR and

CD56/NCAM positive regenerating fibers, and were positive for BDNF. Stainings were

performed on serial sections. Triangles, asterisks and white arrows indicate regenerating

myofibers, mature myofibers and groups of BDNF-positive immune cells respectively. (F-G)

haematoxylin-eosin and CD56/NCAM stained serial sections of IIM muscle. Right panels

show enlarged areas with mature (asterisks, upper) or regenerating (triangles, lower) fibers.

Asterisks and triangles point out fibers included in the quantification. (H) Numbers of

immune cells localized in the proximity of regenerating or mature myofibers. (I) Numbers of

BDNF positive immune cells localized in the proximity of regenerating or mature myofibers.

Dots represent distinct tissue samples, red bars represent means. In (H) and (I) all fields of at

least 2 sections per sample were analyzed; mature fibers near regenerating fibers were not

included in the quantification. Double immunofluorescence for BDNF and CD4 (J), CD8 (K)

or CD68 (L) in IIM muscle. Left panels represent single stainings. Arrows indicate BDNF

positive cells. Scale bar 30 μm in (B-E), 100 μm in (F-G), 20 μm in (J-L). * P < 0.05.



Supplementary Figure. 1 Perforin expressing T cells or Ki67 positive macrophages are

localized near mature fibers. Left panels show immunohistochemistry for CD56. Right panels

show triple immunofluorescence for CD3 and perforin (A and B) or CD68 and Ki67 (C and

D) in combination with desmin. Middle panels represent single stainings. Arrows indicate

perforin or Ki67 positively stained cells. Asterisks indicate the same fibers in left and right

panels. Scale bar 50 μm.

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