Elsevier

Experimental Neurology

Volume 277, March 2016, Pages 275-282
Experimental Neurology

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

https://doi.org/10.1016/j.expneurol.2016.01.008Get rights and content

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.

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.

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 (also 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.

Section snippets

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

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 also known as integrin alpha-M (Robinson et al., 1986). CD11b is implicated in various adhesive interactions of monocytes, macrophages and

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

Acknowledgments

This work was supported by grants from NIH (R01NS091540 to M.S.), US Department of Defense (W81XWH-14-1-0189), the ALS Association (J10IZ9 and 15-IIP-201), and the University of Wisconsin Foundation. The authors declared no conflict of interest.

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