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. Author manuscript; available in PMC: 2017 Mar 1.
Published in final edited form as: Exp Neurol. 2016 Jan 13;277:275–282. doi: 10.1016/j.expneurol.2016.01.008

Macrophage-mediated inflammation and glial response in the skeletal muscle of a rat model of familial amyotrophic lateral sclerosis (ALS)

Jonathan M Van Dyke 1, Ivy M Smit-Oistad 1, Corey Macrander 1, Dan Krakora 1, Michael G Meyer 1, Masatoshi Suzuki 1
PMCID: PMC4762214  NIHMSID: NIHMS754598  PMID: 26775178

Abstract

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by progressive motor dysfunction and loss of large motor neurons in the spinal cord and brain stem. While much research has focused on mechanisms of motor neuron cell death in the spinal cord, degenerative processes in skeletal muscle and neuromuscular junctions (NMJs) are also observed early in disease development. Although recent studies support the potential therapeutic benefits of targeting the skeletal muscle in ALS, relatively little is known about inflammation and glial responses in skeletal muscle and near NMJs, or how these responses contribute to motor neuron survival, neuromuscular innervation, or motor dysfunction in ALS. We recently showed that human mesenchymal stem cells modified to release glial cell line-derived neurotrophic factor (hMSC-GDNF) extend survival and protect NMJs and motor neurons in SOD1G93A rats when delivered to limb muscles. In this study, we evaluate inflammatory and glial responses near NMJs in the limb muscle collected from a rat model of familial ALS (SOD1G93A transgenic rats) during disease progression and following hMSC-GDNF transplantation. Muscle samples were collected from pre-symptomatic, symptomatic, and end-stage animals. A significant increase in the expression of microglial inflammatory markers (CD11b and CD68) occurred in the skeletal muscle of symptomatic and end-stage SOD1G93A rats. Inflammation was confirmed by ELISA for inflammatory cytokines interleukin-1 β (IL-1β) and tumor necrosis factor-α (TNF-α) in muscle homogenates of SOD1G93A rats. Next, we observed active glial responses in the muscle of SOD1G93A rats, specifically near intramuscular axons and NMJs. Interestingly, strong expression of activated glial markers, glial fibrillary acidic protein (GFAP) and nestin, was observed in the areas adjacent to NMJs. Finally, we determined whether ex vivo trophic factor delivery influences inflammation and terminal Schwann cell (TSC) response during ALS. We found that intramuscular transplantation of hMSC-GDNF tended to exhibit less inflammation and significantly maintained TSC association with NMJs. Understanding cellular responses near NMJs is important to identify suitable cellular and molecular targets for novel treatment of ALS and other neuromuscular diseases.

Keywords: Amyotrophic lateral sclerosis (ALS), SOD1G93A rats, inflammation, glial activation, skeletal muscle

Introduction

Amyotrophic lateral sclerosis (ALS) is a fatal, rapidly progressing neurodegenerative disease caused by the selective loss of motor neurons in the spinal cord and brain stem (Ajroud-Driss and Siddique, 2015; Peters et al., 2015). The cause and process by which motor neurons die as ALS progresses is complex and incompletely understood. Nearly 90% of all ALS cases arise sporadically while the remaining 10% follow familial lines. Of the familial cases, approximately 20% can be attributed to one of over 160 mutations within the gene encoding the ubiquitously expressed human superoxide dismutase 1 (SOD1), the first gene linked to ALS neurotoxicity (Rosen et al., 1993). Other important genes in which ALS-causing mutations can arise have since been described including C9ORF72 and TDP-43 (Ajroud-Driss and Siddique, 2015). Overexpression of mutated human SOD1 gene in rodents produces a neuropathic phenotype similar to the ALS disease state in humans (Gurney et al., 1994; Howland et al., 2002; Nagai et al., 2001; Philips and Rothstein, 2015). While the exact mechanism of pathology remains elusive, multiple pathologies have been implicated as contributing factors to motor neuron death during ALS. These include protein misfolding and aggregation (Blokhuis et al., 2013), defects in axonal transport, glutamate excitotoxicity (Bogaert et al., 2010), oxidative stress (Barber and Shaw, 2010), mitochondrial dysfunction (Duffy et al., 2011), and abnormal astrocyte activation (Hall et al., 1998; Radford et al., 2015).

Despite the enigmatic nature of the ALS mechanism of motor neuron pathology, previous studies have demonstrated that direct muscle delivery of neuroprotective trophic/growth factors is effective to support neuromuscular connections, axon integrity, and motor neuron survival (Azzouz et al., 2004; Kaspar et al., 2003; Mohajeri et al., 1999). Specifically, our group has demonstrated the therapeutic benefits of ex vivo gene therapy (stem cell-based growth/trophic factor delivery) targeting the skeletal muscles in a rat model of familial amyotrophic lateral sclerosis (SOD1G93A transgenic rats) (Krakora et al., 2013; Suzuki et al., 2008). Human mesenchymal stem cells (hMSCs) constitutively secreting glial cell line-derived neurotrophic factor (GDNF) prevented degeneration of motor neurons and associated neuromuscular junctions (NMJs), and slowed ALS progression when delivered to skeletal muscle of SOD1G93A transgenic rats (Suzuki et al., 2008). Most recently, we delivered a combination of GDNF and vascular endothelial growth factor (VEGF) to muscle using hMSCs which further slowed disease progression in SOD1G93A rats (Krakora et al., 2013). While these studies demonstrated a significant ability of GDNF and VEGF to slow motor neuron degeneration and preserve skeletal muscle function, the question of how these growth factors and/or grafted hMSCs protect the motor endplate neuromuscular connection and motor neuron remains. To answer this question, it is important to understand how growth factors and hMSCs influence skeletal muscle degeneration during disease progression and it is logical to expect that the NMJs are the central affected structures.

The NMJ is a structure made up of the motor axon terminals, the muscle, and other supporting cells including terminal Schwann cells (TSCs). TSCs, also known as peri-synaptic Schwann cells, are glial cells found at the NMJ with known functions in synaptic transmission, synaptogenesis, and nerve regeneration (Moloney et al., 2014). NMJ dissociation (the separation of the TSC and motor axon from the motor endplate of the muscle) is a hallmark process of ALS and precedes symptom onset in ALS rodent models and human patients (Dupuis and Loeffler, 2009; Fischer et al., 2004; Krakora et al., 2012). While it is unclear whether NMJ dissociation occurs prior to or after motor neuron death, mounting evidence suggests that it plays a larger role in the progression of ALS than previously thought. Furthermore, little is known about the role of TSCs at the NMJs during ALS progression and pathology. Normally, TSCs play an important role supporting the synapse by taking up excess neurotransmitter, modulating neurotransmitter release, and lending trophic support. This role is analogous to the glial cells of the central nervous system (Feng and Ko, 2008). However, in the limb muscles of end-stage ALS patients, TSCs exhibit abnormal expressions of glial markers such as glial fibrillary acidic protein (GFAP), p75 neurotrophin receptor, and S100β (as known as S100 calcium binding protein B) (Liu et al., 2013). It is possible that progressive distal degeneration of the NMJs occurs early and is followed by axonal degeneration and motor neuron degeneration which would support a “dying back” hypothesis (Krakora et al., 2012).

Inflammation could play a role in NMJ dissociation during ALS progression but the exact role and mechanism is relatively unknown. Inflammation is recognized to play a role in motor neuron death and has been shown to accompany motor neuron degeneration in the central nervous system (Evans et al., 2013; Philips and Robberecht, 2011). As ALS progresses, pro- and anti-inflammatory cytokines increase in the cerebrospinal fluid of patients (Evans et al., 2013; Mitchell et al., 2009). The source of inflammatory factors is thought to be glial. High levels of microglial activation have been observed in the spinal cord of ALS rodent models (Beers et al., 2011; Boillee et al., 2006), as well as the spinal cord and brain stem of ALS patients (Evans et al., 2013). Inflating and activated macrophages are increased in the ventral root and sciatic nerve of ALS mice as the disease progresses (Chiu et al., 2009; Dibaj et al., 2011). Interestingly, macrophage activation is observed in the peripheral sciatic nerve before symptom onset and then steadily increased through end stage (Graber et al., 2010). Furthermore, the presence of inflammatory responses in degenerating peripheral nerve axons is also reported as an early event that occurs prior to the onset of clinical signs of motor weakness.

Inflammation and abnormal glial activation in the spinal cord and peripheral nerve fibers are both early events that occur prior to clinical signs of motor weakness in ALS; however, it is unknown whether inflammation and glial responses occur within the skeletal muscle and near NMJs, and how these responses contribute to motor neuron survival and neuromuscular innervation in ALS. A previous study using endpoint ALS mice revealed an increase in macrophage presence in innervating motor axon fascicles (Chiu et al., 2009); however, it is unclear whether these macrophages are causal or resolving (cleaning up debris from the degenerating axon). In this study, we first evaluate the time course of inflammatory and glial responses in the skeletal muscle near neuromuscular connections in the limb muscle of a rat model of familial ALS (SOD1G93A transgenic). We also ask how intramuscular GDNF delivery using hMSCs influences inflammation and glial responses in the skeletal muscle of SOD1G93A rats.

Materials and Methods

SOD1G93A transgenic rats

Female SOD1G93A transgenic rats exhibiting slow disease progression were used in this study (Suzuki et al., 2007b). The SOD1G93A transgenic male founders, originally obtained from Taconic (Hudson, NY) (Howland et al., 2002), were crossed with wild type female Sprague-Dawley rats to maintain colonies. While colony drift was previously observed in this transgenic rat line (Suzuki et al., 2007b), we have developed a genomic PCR screen and breeding schedule and now maintain stable lines that show similar disease onset and survival. Only animals showing disease onset before 150 days were used as breeders in order to minimize genetic drift in the colony that could increase variability in onset age of symptoms and end-stage age. Care was taken initially to select rats from sires that exhibited only hindlimb symptom onset. Rats exhibiting forelimb symptom onset were excluded from this study. Rats were maintained in a room with controlled illumination, temperature (23±1 °C), and humidity. All animals were given free access to food and water. Heterozygous SOD1G93A progeny were identified by genomic PCR screen using tail snip DNA and primers specific for human SOD1. All animal work in the present study was carried out in accordance with the guidelines for University of Wisconsin-Madison and National Institutes of Health standards of animal care.

Muscle section preparation

ALS rats were sacrificed at pre-symptomatic (<80 days old), symptomatic (100–120 days old), and endpoint stages (n=4–5 for each time point). Hind limb muscles (tibalis anterior; TA) were dissected and flash-frozen in super-cooled isopentane. The muscles were sectioned at 20 μm using a cryostat and placed on glass slides for staining. We confirmed that all symptomatic animals exhibited the Basso-Beattie-Bresnahan (BBB) locomotor rating score of 17 or lower (Krakora et al., 2013). Rats were considered at end-stage (endpoint) when they no longer exhibited reflexes allowing them to right themselves within 30 seconds. Our recent data from this colony indicates the median to reach disease onset and end-stage is 100 and 161 days, respectively (Hayes-Punzo et al., 2012).

Immunohistochemistry

The muscle sections were fixed with ice-cold methanol for 10 min or 4% paraformaldehyde-phosphate-buffered saline (PFA-PBS) for 20 min, and then incubated with primary antibodies at 4 °C for overnight. The following primary antibodies were used to probe for macrophages: anti-CD11b (OX-42, 1:500, mouse monoclonal, MorphoSys, Planegg, Germany), or anti-CD68 (ED1, 1:500, mouse monoclonal, AbD Serotec, Raleigh, NC). The following antibodies were used to visualize and probe markers of TSCs and activated glial cells: anti-glial fibrillary acidic protein (GFAP, 1:200, rabbit polyclonal, Dako, Glostrup, Denmark), anti-nestin (1:50, rabbit polyclonal, Sigma-Aldrich), anti-S100β (1:100, mouse monoclonal, Sigma-Aldrich), anti-4E2 (3G2) (neuromuscular junction and reactive Schwann cell associated antigen; 1:400, mouse monoclonal, Developmental Studies Hybridoma Bank, Iowa City, IA), and anti-GDNF family receptor alpha 1 (GFRα1, 1:50, rabbit polyclonal, Sigma-Aldrich) antibodies. To identify endplate distribution, the muscle sections were incubated with α-bungarotoxin (α-BTX) conjugated with fluorescence marker Alexa Fluor 594 (1:1000, Life Technologies, Carlsbad, CA) or CF405S (1:500, Biotium, Hayward, CA) with primary antibodies. After incubation with the primary antibodies, the sections were rinsed in PBS and incubated at room temperature for 1 hour with secondary antibodies conjugated to Alexa Fluor 488 or Cy3 (1:1000, Jackson ImmunoResearch Laboratories, West Grove, PA). All images were optimized by using a Nikon Eclipse 80i fluorescence microscope with a digital camera (DS-QiIMC, Nikon, Tokyo, Japan). The relative intensity of CD11b-positive signals was densitometrically evaluated using NIH ImageJ software. The number of S100β-positive endplates was presented as a percentage of the total number of counted NMJs. Approximately 50 endplates were exhaustively analyzed.

IL-1β and TNF-α ELISA

TA muscle was dissected and flash-frozen in super-cooled isopentane as described above. The tissues (approximately 50–80 mg) were then homogenized using a tissue homogenizer in 0.5 ml of ice-cold homogenization buffer containing protease inhibitors (40 mM Tris-HCl pH 7.5, 1 mM EDTA, 5 mM EGTA, 0.5% Triton X-100, 1 mM PMSF, and 10 μg/ml leupeptin). Protein concentrations were determined using a DC Protein Assay kit (Bio-Rad, Hercules, CA). The concentrations of rat IL-1β and TNF-α protein were measured by ELISA (DuoSet, R&D Systems, Minneapolis, MN).

Western blotting

Muscle homogenates (n=3–4 for each group) were prepared as describe above and loaded into wells of a 10% polyacrylamide gel and subjected to SDS-PAGE. Proteins were electrotransferred to a PVDF membrane and incubated with anti-GFAP (1:500, mouse monoclonal, Covance, Princeton, NJ) and anti-GAPDH antibodies (1:5,000, rabbit monoclonal, Cell Signaling Technology Inc,, Danvers, MA). Following secondary incubation with horse anti-mouse peroxidase-tagged antibody (1:5000, Vector Laboratories, Burlingame, CA), and incubation with enhanced chemiluminescence incubation (Thermo Scientific, Rockford, IL), the membrane was exposed to film. The protein band signals on the film were scanned and densitometrically analyzed using ImageJ 1.42q software.

Ex vivo GDNF delivery in SOD1G93A rats

Preparation of hMSCs expressing GDNF (hMSC-GDNF) and intramuscular transplantation of hMSCs in SOD1G93A rats were performed as described previously (Krakora et al., 2013; Suzuki et al., 2008). Briefly, hMSC-GDNF were prepared by lentiviral infection for constitutive expression of human GDNF (Krakora et al., 2013; Suzuki et al., 2008). Pre-symptomatic female SOD1G93A rats (90 days old; n = 4–5 animals/group) were administered intramuscular injections of a local anesthetic, bupivacaine hydrochloride (BVC; Sensorcaine-MPF, AstraZeneca, London, UK), to induce focal muscle injury and enhance hMSC integration and survival in the limb muscle prior to transplantation (Krakora et al., 2013; Suzuki et al., 2008). BVC was injected unilaterally into the TA muscle. At time-points of 24 hours, 1 week, and 2 weeks after the BVC injection, wild-type hMSC (hMSC-WT) or hMSC-GDNF (150,000 cells in 50 μl) were injected into the same muscle. All animals were immunosuppressed with cyclosporine which was necessary to prevent rejection of the transplanted hMSCs (10 mg/kg/day, Sandimmun; Novartis, Basel, Switzerland) (Borel et al., 1994). The animals were sacrificed at symptomatic stage (≅153 days old or 9 weeks after the first hMSC injection). The muscle sections were prepared as described above.

Statistical analysis

Prizm software (Graphpad software Inc., La Jolla, CA) was used for all statistical analyses. All ELISA data were analyzed by one-way functional ANOVA using Newman-Keuls post hoc test. Differences were considered significant when P<0.05.

Results

Inflammation is increased in the skeletal muscle of symptomatic and end-stage SOD1G93A rats

In this study, we first evaluated the time course of the inflammatory response found in the skeletal muscle of SOD1G93A rats (Fig. 1). Muscle samples were collected from early (40 days of age) or late pre-symptomatic (80 days), symptomatic (approximately 120 days old), and end-stage animals. The samples were sectioned and stained for CD11b, a glycoprotein known also known as integrin alpha-M (Robinson et al., 1986). CD11b is implicated in various adhesive interactions of monocytes, macrophages and granulocytes. In this study, we used the OX-42 antibody, a mouse monoclonal clone against rat CD11b (Robinson et al., 1986). At early pre-symptomatic stage (40 days of age), there were few CD11b-positive macrophages located in the skeletal muscle (Fig. 1A). The presence of CD11b-positive cells gradually increased as the disease progressed through late pre-symptomatic (80 days), symptomatic (120 days), and end-stage (Fig. 1A). Specifically, most of CD11b-positive cells resided in the laminar layer of the myofibers. Densitometirc analysis revealed that the level of inflammation in symptomatic and endpoint SOD1G93A rats were 6 and 10 times higher compared to 40-day-old animals, respectively (Fig. 1B). To determine how CD11b-expressing macrophages were distributed near endplates, alpha-bungarotoxin (α-BTX) conjugated with a fluorescence marker was used to identify acetylcholine receptor of the muscle endplates. We found that many CD11b-postive cells were located near endplates in the muscle of symptomatic SOD1G93A rats (Fig. 1C). Additionally, we performed immunostaining against CD68 (ED1 clone), another marker for cells of the macrophage lineage (Holness and Simmons, 1993), and confirmed activated immune cells in the muscle of end-stage SOD1G93A rats (Fig. 1D).

Figure 1. Active inflammatory responses occur near degenerating neuromuscular junctions (NMJs) and axons in symptomatic and end-stage SOD1G93A rats.

Figure 1

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(A) CD11b-positive macrophages were identified in the hindlimb muscle of symptomatic and end-stage SOD1G93A transgenic rats. OX-42, a mouse monoclonal antibody clone against rat CD11b, was used for immunostaining. (B) Densitometric analyses indicated that the level of inflammation in symptomatic and endpoint SOD1G93A rats was higher compared to 40-day-old animals. (C) In symptomatic SOD1G93A rats, CD11b-postive signal was identified near AChR-positive endplates. (D) Immunostaining against another macrophage marker CD68 (ED1 clone) showed inflammation in the muscle of end-stage SOD1G93A rats. (E) The concentration of inflammatory cytokines IL1-β and TNF-α was increased in the muscle of end-stage animals. Scale bars: 50 μm in A and D; 20 μm in C. *: P<0.05 vs. pre-symptomatic and symptomatic.

We next measured the levels of inflammatory cytokines, interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α). Muscle homogenates were prepared from the TA of SOD1G93A rats (n=4 for each group) at pre-symptomatic (Day 60), symptomatic (Day 120) and end-stage and analyzed by ELISA for IL-1β and TNF-α expression. Although the concentration of IL-1β was unchanged between pre-symptomatic and symptomatic rats, the homogenates from end-stage animals contained approximately three times more IL-1β (28.5 ± 3.2 pg/mg) compared to pre-symptomatic (10.3 ± 2.7 pg/mg) and symptomatic animals (8.5 ± 1.4 pg/mg) (Fig. 1E; P<0.05). There was a similar trend in TNF-α levels (10.0 ± 2.6 pg/mg at Day 60; 13.9 ± 3.3 pg/mg at Day 120; 32.8 ± 12.0 pg/mg at end-stage). However, due to high variance among the end-stage samples we could not conclusively claim significance (Fig. 1E). Together, these results demonstrate a significant increase in inflammation occurred in the skeletal muscle of symptomatic and end-stage SOD1G93A rats.

Increased expression of GFAP and nestin near endplates of SOD1G93A rats follows ALS disease progression

Adjacent serial sections to those used in Fig. 1 were stained for GFAP, a glial intermediate filament component considered to be a marker of active glial cells (including TSCs, astrocytes, and Schwann cells) (Yang and Wang, 2015). At the early pre-symptomatic period (40 days of age), GFAP protein was tightly expressed in the pre-synaptic compartments adjacent to AChR-positive endplates (Fig. 2A and B). The expression of GFAP protein gradually increased following disease progression from late pre-symptomatic (80 days old) (Fig. 2C and D), symptomatic (Fig. 2E and F) to end stage (Fig. 2G and H). Notably, strong GFAP expression was observed in a wide area around endplates and extended along with axons in end-stage animals. Furthermore, western blotting for GFAP protein confirmed a steady and significant increase in relative GFAP expression (Fig. 2I and J).

Figure 2. GFAP expression is increased in the muscle of SOD1G93A rats following disease progression.

Figure 2

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(A–H) GFAP immunostaining in the hindlimb muscle of SOD1G93A rats. GFAP expression was limited near AChR-positive endplates at 40 days (A, B) and 80 days (C, D) of age. However, GFAP expression gradually increased in symptomatic (E, F) and end stage (G, H). (I) Representative band images of western blotting for GFAP and GAPDH expression. (J) Densitometric analyses of western blotting data supported that GFAP was increased in the muscle homogenates of end-stage rats. Scale bar: 100 μm in A, C, E, G; 50 μm in B, D, F, H. *: P<0.05 vs. pre-symptomatic and symptomatic.

Nestin is an intermediate filament that localizes to the muscle fiber underneath the NMJs of innervated muscle (Kang et al., 2007). Upon denervation, muscle fiber nestin expression decreases. Simultaneously, TSCs associated with the denervated NMJs begin to express nestin. We examined nestin expression patterns in the NMJs of SOD1G93A rats during ALS progression (Fig. 3). Nestin was found to overlap endplates in pre-symptomatic muscles (Fig. 3A–C) and was not altered at the symptomatic stage (Fig. 3D) compared to the pre-symptomatic animals. In end-stage animals, nestin was upregulated specifically in the pre-synaptic area of NMJs and took on morphology similar to that of activated Schwann cells (Fig. 3E and F). Together, the shifts in expression patterns of GFAP and nestin proteins follow the expected pattern of denervation associated with disease progression in SOD1G93A rats.

Figure 3. An intermediate filament nestin is detected near NMJs of SOD1G93A rat muscle at end-stage.

Figure 3

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Nestin protein expression overlapped with endplates in pre-symptomatic (40 day in A, B; 80 days in C) and symptomatic stage muscles (D). Nestin was upregulated specifically in the pre-synaptic area of NMJs at end-stage (E, F). Scale bar: 100 μm.

TSCs dissociate from NMJ as ALS progresses

We next analyzed TSC localization relative to the NMJ by co-labeling for a TSC marker (S100β) and motor endplates. In pre-symptomatic SOD1G93A rats, S100β signals overlapped the pre-synaptic endplates (Fig. 4A). Approximately 80% of endplates were S100β positive at this time point (Day 60) (Fig. 4B). The number of S100β-positive endplates was then gradually decreased in symptomatic and end-stage ALS rats (Fig. 4B). This dissociation correlated with a loss in endplate innervation in serially adjacent muscle sections (Fig. 4C and D). Immunostaining with 4E2 (another marker of TSCs) (Astrow et al., 1994) also showed a reduction of positive signals associated with endplates in the end-stage animals (Fig. 4A).

Figure 4. TSC survival is associated with the level of NMJ innervation.

Figure 4

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(A) Immuno-labeling of TSCs using anti-S100β and 4E2 (Schwann cell associated antigen) antibodies. S100β and 4E2-positive TSCs overlapped with pre-synaptic area of endplates (arrows) in pre-symptomatic rats. However, these positive signals were weak or undetectable in end-point animals (arrowheads). (B) The number of S100β-positive endplates was significantly decreased at symptomatic (Sympt) and end-stage compared to pre-symptomatic stage (Pre-sympt). (C) The reduction of S100β-positive endplates was associated with the level of innervated endplates. Scale bar: 20 μm. *: P<0.05 vs. pre-symptomatic. n.d.: not detected.

Intramuscular delivery of GDNF using hMSCs enhanced TSC association with NMJs in SOD1G93A rats

We tested whether GDNF delivery using hMSCs influenced inflammation and TSC response during ALS. hMSCs genetically modified to secrete GDNF (hMSC-GDNF) were unilaterally injected in pre-injured TA of pre-symptomatic SOD1G93A rats (90 days old). Control groups received wild-type hMSCs (naïve cells with no GDNF over-expression; hMSC-WT) or vehicle. The animals were sacrificed at the symptomatic stage (≅153 days old). CD11b staining intensity was reduced in TA muscle sections by approximately 36% and 34% when transplanted with hMSC-WT and hMSC-GDNF respectively (Fig. 5A and B). Further, we hypothesized that direct delivery of GDNF via hMSCs could increase the percent of NMJ-associated TSCs. In adjacent sections, a subunit of GDNF receptor GFRα1 was detected in the S100β+ pre-synaptic area of NMJs in pre-symptomatic SOD1G93A rats (Fig. 5C and D). We also counted S100β+ endplates in the TA muscle of hMSC-transplanted SOD1G93A rats and found that hMSC-GDNF significantly increased the number of TSC-positive endplates (P<0.05 vs. Control and hMSC-WT; Fig. 5E).

Figure 5. Direct delivery of GDNF using hMSCs influences inflammation and TSC survival in SOD1G93A rats.

Figure 5

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hMSCs genetically modified to secrete GDNF, were transplanted in the hindlimb muscle of pre-symptomatic SOD1G93A rats. (A) Representative images of CD11b in symptomatic muscle injected with vehicle control or GDNF-secreting hMSCs. (B) Relative intensity of CD11b-positive signals in the muscle sections. TSCs labeled with S100β (C), and GFRα1 (D) at the pre-synaptic area of neuromuscular connections. (E) Direct delivery of GDNF using hMSCs significantly increased TSCs. Scale bars: 100 μm in A; 10 μm in D. *: P<0.05 vs. Control.

Discussion

In this study, we demonstrate that activated inflammation and abnormal glial responses occur in the limb muscle of familial ALS model rats, specifically near denervated NMJs. We previously reported that while over 80% of NMJs were innervated in pre-symptomatic SOD1G93A rats up to 80 days old, this number gradually decreased to the point where all NMJs were denervated by end-stage (Suzuki et al., 2007a). Although most ALS research has focused on mechanisms of motor neuron cell death, degeneration is also observed in skeletal muscle, particularly at the neuromuscular connection. While recent studies potentially support the therapeutic benefits targeting the skeletal muscle in ALS (Krakora et al., 2013; Suzuki et al., 2008), it has not been fully determined how inflammation and glial responses proceed in skeletal muscle and near NMJs in ALS model animals at different time points. Although further study is necessary to determine the possible roles of inflammation and glial responses in ALS patients, this animal study provides useful insight into the biological events occurring within the skeletal muscle during ALS.

Inflammation is recognized to play a role in ALS-related motor neuron pathology and has been shown to accompany motor neuron degeneration in the central and peripheral nervous systems of rodent models of ALS and human patients. In the spinal cord, this upregulation of inflammation is thought to be due to activation of microglia primed for activity whose subsequent activation plays a role in ALS pathology (Dibaj et al., 2011). In this study, we examine a time course of inflammatory responses in the skeletal muscle near NMJs in the limbs of transgenic rats carrying the SOD1G93A mutation. Markers of inflammation are significantly increased in the limb muscle as ALS progressed until end stage. The progressive increase in CD11b expression is similar to what has been previously observed in the ventral nerve root and sciatic nerve of SOD1G93A rats (Graber et al., 2010). Similarly, we found that CD68 expression is elevated in the TA near the NMJs at end stage. CD68 has also been observed in the ventral nerve roots of SOD1G93A rats before the onset of symptoms (Graber et al., 2010), in the peripheral nerves of SOD1G93A mice (Chiu et al., 2009; Dibaj et al., 2011) and around the innervating axon terminals of SOD1G93A mouse diaphragm (Chiu et al., 2009). Inflammation markers IL1-β and TNF-α are also elevated in end stage muscle in this study. IL1-β is secreted by activated microglia and speeds motor neuron pathology in ALS. Also, the systemic blockade of the IL-1 receptor has been shown to slow disease progression in SOD1G93A mice (Meissner et al., 2010). TNF-α is secreted by activated microglia and promotes an imbalance of AMPA and NMDA excitatory receptors compared to inhibitory GABA receptors in the motor neuron. This imbalance is thought to increase susceptibility of the motor neurons to excitotoxicity which is one possible mechanism involved in motor neuron pathology (Olmos and Llado, 2014). While inflammatory cytokines are observed at the NMJ in this study, it is important to note that inflammatory responses can vary by region and exhibit heterogeneous expression as exhibited in the spinal and cortical tissue of SOD1G93A rats (Nikodemova et al., 2014). It is possible that inflammatory responses at the NMJ may also be heterogeneous and regional.

We also characterize the response of resident glial cells adjacent to axons and TSCs adjacent to NMJs as ALS progresses and motor control is lost. Specifically, we monitored the expression of the intermediate filament proteins GFAP and nestin. GFAP is commonly used as a marker of astrocyte activation in the central nervous system (Yang and Wang, 2015). GFAP expression has been shown to be increased in sciatic nerves damaged by crush or cut and in rodent models of ALS preceding and during symptom onset through end stage (Keller et al., 2009). Similarly, GFAP has been found to be elevated in the serum of patients with motor and sensory neuropathies (Notturno et al., 2009). These reports support the idea that GFAP expression correlates with the degeneration of motor axons and denervation of NMJs. Further, we observed a marked increase in NMJ-associated nestin expression as ALS progressed. Nestin is known to be expressed near the post-synaptic nuclei of innervated muscle fibers but not in the presynaptic axon or TSCs. Following denervation, post-synaptic nestin expression drops off while nestin expression in TSCs increases dramatically (Kang et al., 2007).

In adults, TSCs play an important role in maintaining the NMJ (Feng and Ko, 2008). We examined TSCs in SOD1G93A rat muscle as ALS progressed. As expected, colocalization of AChRs and TSCs was lost as exhibited by S100β and 4E2 staining. We also observed small increases in nestin and GFAP in pre-symptomatic muscle that increased dramatically as symptoms progressed toward end stage. An increase in glial GFAP expression has been observed in spinal cord and peripheral nerves of SOD1G93A mice with spikes of increased expression at distinct time points beginning before symptom onset (Keller et al., 2009). The data we present here also demonstrate a general increase in GFAP expression at the NMJ beginning before gross loss of motor function develops. TSCs do not normally express appreciable levels of GFAP. In cases of denervation or axonal damage, TSCs enter a dedifferentiated state during which they upregulate GFAP as part of the repair/reinnervation process (Cheng and Zochodne, 2002; Kang et al., 2014). Indeed nerve crush and cut can induce elevated GFAP expression at the NMJ (Keller et al., 2009). Despite multiple studies describing GFAP expression increase in the central nervous system of ALS animal models and patients, GFAP expression increase has not been observed in either the limb muscles or extra-ocular muscles of human ALS patients who have reached end stage (Liu et al., 2013). The combination of the steady increase in nestin and GFAP expressions in TSC as ALS progresses and simultaneous loss of TSC colocalization with AChRs matches previous findings in SOD1G93A rodent models and human ALS patients (Hegedus et al., 2008; Pun et al., 2006) and correlates with loss of motor function.

We next used hMSCs to deliver GDNF to muscle of SOD1G93A rats and analyzed the effect on inflammation and glial response. We previously described a significant delay in symptom onset and an increase in overall survival of SOD1G93A rats following intramuscular transplantation of hMSCs overexpressing GDNF (Krakora et al., 2013; Suzuki et al., 2008). Interestingly, we found that both hMSC-GDNF and hMSC-WT tended to exhibit less inflammation, suggesting that GDNF may not be directly linked to inflammation regulation. Several studies have demonstrated that hMSCs show significant immunomodulatory effects following transplantation (De Miguel et al., 2012; Vercelli et al., 2008). Because inflammation severity correlates with disease progression, it is possible that the trend toward less inflammation following hMSC transplantation is due to an overall slowing of disease progression as we have previously observed. The increase in inflammation markers at the NMJs may indicate inflammation-based activation of peripheral macrophages similar to microglial activation in the central nervous system as ALS progresses. Macrophage activity may play a role in NMJ and axonal degeneration during ALS. Our study suggests a correlation between an increase in inflammatory markers and markers of TSCs dissociation from NMJs; however, it has also been suggested that the elevated macrophage presence and activity found in the peripheral nerves as ALS progresses, may be due to increased cellular debris as motor axons degenerate and that macrophages are simply present to clean up (Chiu et al., 2009; Dibaj et al., 2011). Indeed, the possibility exists that the preservation of NMJ integrity may result in reduced inflammation. In this study, an immunosuppressive reagent cyclosporine was administered to all animals to prevent the rejections of grafted hMSCs. Based on our preliminary observations, cyclosporine itself did not cause obvious differences in disease progression and animal survival in our ALS rat models (Suzuki et al., 2007a). However, it is still necessary to elucidate how cyclosporine influences inflammation in the muscle and the effects of hMSC-based GDNF delivery following disease progression.

Finally, we found that hMSC-GDNF, but not hMSC-WT, prevented TSC dissociation from the NMJs in SOD1G93A animals. Motor neurons express GDNF family receptor alpha (GFRα1) and GDNF is a known critical factor for motor neuron survival and development (Henderson et al., 1994; Tovar et al., 2014). Interestingly, we found that GFRα1 is also expressed in TSCs and observed a significant preservation of TSC overlap with endplates in the hMSC-GDNF transplanted muscles compared to those transplanted with the hMSC-WT. These data further support that GDNF preserves tripartite NMJ integrity and suggest that GDNF plays a direct role in preserving TSC association with the NMJ. Subsequently, the preservation of NMJ integrity would potentially lead to further motor neuron protection and could be the mechanism by which delivered GDNF delays ALS symptom onset. Further studies are necessary to fully explore this possibility and to determine underlying mechanisms of NMJ and motor neuron protection by hMSC-GDNF.

Highlights.

  • Macrophage-mediated inflammation is increased at the NMJs in ALS rats following disease progression

  • Strong expressions of reactive glial markers are observed near NMJs

  • TSCs dissociate when NMJ integrity is lost

  • Ex vivo GDNF delivery alters inflammation and maintains TSC-NMJ association

Acknowledgments

This work was supported by grants from NIH (R01NS091540 to M.S.), US Department of Defense, the ALS Association, and the University of Wisconsin Foundation.

Abbreviations

α-BTX

alpha-bungarotoxin

AChR

acetylcholine receptor

ALS

amyotrophic lateral sclerosis

ANOVA

analysis of variance

BBB

Basso-Beattie-Bresnahan hindlimb locomotor rating

BVC

bupivacaine hydrochloride

CD11b

cluster of differentiation 11b

CD68

cluster of differentiation 68

C9ORF72

chromosome 9 open reading frame 72

EDTA

Ethylenediaminetetraacetic acid

EGTA

ethylene glycol tetraacetic acid

ELISA

enzyme-linked immunosorbent assay

GAPDH

Glyceraldehyde-3-phosphate dehydrogenase

GDNF

glial cell line-derived neurotrophic factor

GFAP

glial fibrillary acidic protein

GFRα1

GDNF family receptor α 1

hMSC

human mesenchymal stem cell

IL-1β

interleukin-1β

NMJ

neuromuscular junction

PBS

phosphate buffered saline

PCR

polymerase chain reaction

PFA

paraformaldehyde

PMSF

phenylmethylsulfonyl fluoride

SDS-PAGE

sodium dodecyl-sulfate polyacrylamide gel electrophoresis

SOD1

superoxide dismutase 1

S100

calcium binding protein

TA

tibialis anterior

TDP-43

TAR DNA binding protein 43

TNF-α

tumor necrosis factor-α

TSC

terminal Schwann cell

VEGF

vascular endothelial growth factor

Footnotes

The authors declared no conflict of interest.

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