Research PaperMacrophage-mediated inflammation and glial response in the skeletal muscle of a rat model of familial amyotrophic lateral sclerosis (ALS)
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.
References (54)
- et al.
Sporadic and hereditary amyotrophic lateral sclerosis (ALS)
Biochim. Biophys. Acta
(2015) - et al.
Oxidative stress in ALS: key role in motor neuron injury and therapeutic target
Free Radic. Biol. Med.
(2010) - et al.
Neuroinflammation modulates distinct regional and temporal clinical responses in ALS mice
Brain Behav. Immun.
(2011) - et al.
In vivo proliferation, migration and phenotypic changes of Schwann cells in the presence of myelinated fibers
Neuroscience
(2002) - et al.
Neuromuscular junction destruction during amyotrophic lateral sclerosis: insights from transgenic models
Curr. Opin. Pharmacol.
(2009) - et al.
Inflammation and neurovascular changes in amyotrophic lateral sclerosis
Mol. Cell. Neurosci.
(2013) - et al.
Amyotrophic lateral sclerosis is a distal axonopathy: evidence in mice and man
Exp. Neurol.
(2004) - et al.
Molecular cloning of CD68, a human macrophage marker related to lysosomal glycoproteins
Blood
(1993) - et al.
Synergistic effects of GDNF and VEGF on lifespan and disease progression in a familial ALS rat model
Mol. Ther.
(2013) - et al.
Spinal but not cortical microglia acquire an atypical phenotype with high VEGF, galectin-3 and osteopontin, and blunted inflammatory responses in ALS rats
Neurobiol. Dis.
(2014)
Neuroinflammation in amyotrophic lateral sclerosis: role of glial activation in motor neuron disease
Lancet Neurol.
Direct muscle delivery of GDNF with human mesenchymal stem cells improves motor neuron survival and function in a rat model of familial ALS
Mol. Ther.
Human mesenchymal stem cell transplantation extends survival, improves motor performance and decreases neuroinflammation in mouse model of amyotrophic lateral sclerosis
Neurobiol. Dis.
Differential neural regulation of a neuromuscular junction-associated antigen in muscle fibers and Schwann cells
J. Neurobiol.
VEGF delivery with retrogradely transported lentivector prolongs survival in a mouse ALS model
Nature
Protein aggregation in amyotrophic lateral sclerosis
Acta Neuropathol.
Amyotrophic lateral sclerosis and excitotoxicity: from pathological mechanism to therapeutic target
CNS Neurol. Disord. Drug Targets
Onset and progression in inherited ALS determined by motor neurons and microglia
Science
Biological effects of cyclosporin A: a new antilymphocytic agent. 1976
Agents Actions
Activation of innate and humoral immunity in the peripheral nervous system of ALS transgenic mice
Proc. Natl. Acad. Sci. U. S. A.
Immunosuppressive properties of mesenchymal stem cells: advances and applications
Curr. Mol. Med.
In vivo imaging reveals distinct inflammatory activity of CNS microglia versus PNS macrophages in a mouse model for ALS
PLoS One
Review: the role of mitochondria in the pathogenesis of amyotrophic lateral sclerosis
Neuropathol. Appl. Neurobiol.
The role of glial cells in the formation and maintenance of the neuromuscular junction
Ann. N. Y. Acad. Sci.
Progressive changes in microglia and macrophages in spinal cord and peripheral nerve in the transgenic rat model of amyotrophic lateral sclerosis
J. Neuroinflammation
Motor neuron degeneration in mice that express a human Cu,Zn superoxide dismutase mutation
Science
Relationship of microglial and astrocytic activation to disease onset and progression in a transgenic model of familial ALS
Glia
Cited by (60)
Transcriptome analysis using patient iPSC-derived skeletal myocytes: Bet1L as a new molecule possibly linked to neuromuscular junction degeneration in ALS
2021, Experimental NeurologyCitation Excerpt :In more matured myocytes at 6 weeks of terminal differentiation, Bet1L primarily had a linear localization along the length of the myocytes (Fig. 2C). Next, we used a rat model of familial ALS (SOD1G93A transgenic rat)(Suzuki et al., 2007a; Van Dyke et al., 2016; Krakora et al., 2013; Klein et al., 2005; Suzuki et al., 2008; Suzuki et al., 2007b) to see if the four genes of interest were also downregulated in an additional model of ALS pathology. Hindlimb muscles (tibialis anterior, TA) were harvested from SOD1G93A rats at the presymptomatic, symptomatic, and end point stages of the disease.
Macrophage roles in peripheral nervous system injury and pathology: Allies in neuromuscular junction recovery
2021, Molecular and Cellular NeuroscienceMuscle fiber-type specific terminal Schwann cell pathology leads to sprouting deficits following partial denervation in SOD1<sup>G93A</sup> mice
2020, Neurobiology of DiseaseCitation Excerpt :Inflammatory responses, including intramuscular infiltration of skeletal muscles with macrophages, have also been documented outside the CNS in animal models of ALS and in patients with the disease (Beers and Appel, 2019; Martinez-Muriana et al., 2016; Trias et al., 2017; Van Dyke et al., 2016; Wang et al., 2017). Furthermore, interventions reducing intramuscular macrophage infiltration attenuate the rate of endplate denervation (Martinez-Muriana et al., 2016; Trias et al., 2018, 2017; Van Dyke et al., 2016; Wang et al., 2017) and improve the overall sprouting capacity caused by the loss of TSCs at type IIb fibers (present study). While little is known about macrophage-SC interactions in ALS, a great deal is known about the role of macrophages in Wallerian degeneration.
Skeletal muscle as a molecular and cellular biomarker of disease progression in amyotrophic lateral sclerosis: A narrative review
2024, Neural Regeneration Research