Skeletal muscle as a molecular and cellular biomarker of disease progression in amyotrophic lateral sclerosis: a narrative review

Peter H.King

Abstract Amyotrophic lateral sclerosis is a fatal multisystemic neurodegenerative disease with motor neurons being a primary target.Although progressive weakness is a hallmark feature of amyotrophic lateral sclerosis,there is considerable heterogeneity,including clinical presentation,progression,and the underlying triggers for disease initiation.Based on longitudinal studies with families harboring amyotrophic lateral sclerosis-associated gene mutations,it has become apparent that overt disease is preceded by a prodromal phase,possibly in years,where compensatory mechanisms delay symptom onset.Since 85-90% of amyotrophic lateral sclerosis is sporadic,there is a strong need for identifying biomarkers that can detect this prodromal phase as motor neurons have limited capacity for regeneration.Current Food and Drug Administration-approved therapies work by slowing the degenerative process and are most effective early in the disease.Skeletal muscle,including the neuromuscular junction,manifests abnormalities at the earliest stages of the disease,before motor neuron loss,making it a promising source for identifying biomarkers of the prodromal phase.The accessibility of muscle through biopsy provides a lens into the distal motor system at earlier stages and in real time.The advent of “omics” technology has led to the identification of numerous dysregulated molecules in amyotrophic lateral sclerosis muscle,ranging from coding and non-coding RNAs to proteins and metabolites.This technology has opened the door for identifying biomarkers of disease activity and providing insight into disease mechanisms.A major challenge is correlating the myriad of dysregulated molecules with clinical or histological progression and understanding their relevance to presymptomatic phases of disease.There are two major goals of this review.The first is to summarize some of the biomarkers identified in human amyotrophic lateral sclerosis muscle that have a clinicopathological correlation with disease activity,evidence of a similar dysregulation in the SOD1G93A mouse during presymptomatic stages,and evidence of progressive change during disease progression.The second goal is to review the molecular pathways these biomarkers reflect and their potential role in mitigating or promoting disease progression,and as such,their potential as therapeutic targets in amyotrophic lateral sclerosis.

Key Words: amyotrophic lateral sclerosis;biomarkers;clinicopathological correlation;disease progression;muscle biomarkers;neurogenic atrophy;neuromuscular junction;non-coding RNAs;presymptomatic stages;skeletal muscle;SOD1G93A mouse model

Introduction

Amyotrophic lateral sclerosis (ALS) is a progressive multisystemic disease involving the degeneration of motor neurons which leads to progressive weakness and eventually respiratory failure (Hardiman et al.,2011;Kazamel et al.,2013).A hallmark of ALS is the extensive heterogeneity both clinically and in the wide array of disease-initiating or disease-modifying gene mutations with more than 50 identified thus far (Hardiman et al.,2011;Mejzini et al.,2019).Disease onset is variable and usually begins focally in the arms,legs,or bulbar region,and definitive diagnosis is often delayed up to 15 months after symptom onset.While survival is 3 to 5 years,some patients have rapid progression,dying within a year of onset,whereas a small subset (～4%) lives beyond 10 years (Turner et al.,2003;Kazamel et al.,2013).Surprisingly,despite this heterogeneity,the pattern of disease progression,once triggered,is remarkably consistent and ultimately fatal (Ravits and La Spada,2009).

The main target of ALS,the motor pathway,is complex,with a central nervous system (CNS) “unit” including Betz cells (upper motor neurons),corticospinal tracts,anterior horn cells,and a peripheral neuromuscular unit comprised of motor axons,neuromuscular junction (NMJ) and skeletal muscle.Since Charcot’s first description of neuropathology over 150 years ago,ALS has been considered a disease of the CNS unit with evidence of pathological changes at all levels (Charcot,1874;King and Mitsumoto,1996;Eisen,2021;Verma et al.,2022).More recently,a “dying back” hypothesis has emerged due to the very early pathological changes occurring at the NMJ and in skeletal muscle before motor neuron loss (reviewed in Moloney et al.,2014).The NMJ is complex with ongoing crosstalk between muscle fibers,motor neurons,and terminal Schwann cells to maintain the health of each component (Darabid et al.,2014;Li et al.,2018).This includes electrical input from the motor neuron and release of trophic factors by myofibers,motor neurons,and terminal Schwann cells such as fibroblast growth factors (FGFs),transforming growth factor β1 (TGF-β1),glial cell line-derived neurotrophic factor (GDNF),WNT ligands,and neuroregulin 1.Although there continues to be debate about the role of the peripheral neuromuscular unit in disease initiation,there is growing evidence from human and animal studies that it can provide insight into molecular pathways linked to ALS disease progression.The distal portion of the peripheral neuromuscular unit is accessible by muscle biopsy,allowing insight into dysregulated molecular and cellular patterns in earlier stages of the disease (as opposed to post-mortem tissue).Since muscle is the “end” organ of the motor system,it may also be a source of biomarkers for tracking the clinical progression of the disease.The revised Amyotrophic Lateral Sclerosis functional rating scale (ALSFRS-R),although a validated and standardized tool for measuring clinical progression (Kimura et al.,2006),lacks sensitivity for detecting early disease and also requires long periods to assess disease progression (Benatar et al.,2016).These limitations translate into longer clinical trials,greater financial costs,and more importantly,delays in identifying promising new treatments.In light of the limited regenerative capacity of motor neurons,early recognition of ALS is paramount to maximize the therapeutic benefit of those interventions that slow disease progression such as with the current Food and Drug Administration-approved drugs riluzole,edaravone,and sodium phenylbutyrate/taurursodiol (Eisen et al.,2014;Ketabforoush et al.,2023).In this review,I will focus on studies using human ALS muscle samples as the driver for biomarker discovery,with an emphasis on those that might be useful in detecting early,presymptomatic stages based on correlation with clinicopathological features or with orthologs in the mutant SOD1 rodent model of ALS.Finally,I will discuss how the discovery of these biomarkers has provided insight into disease mechanisms and potential new therapeutic targets.

Search Strategy

The following search platforms were used between January 2023 and April 2023: 1) PubMed https://pubmed.ncbi.nlm.nih.gov/;2) Scite https://scite.ai/home;3) Google;4) Bing (https://www.bing.com/).

Discovery of Biomarkers in Amyotrophic Lateral Sclerosis Muscle: Promises and Challenges

While progressive muscle weakness is a clinical hallmark of ALS,most studies reporting aberrant molecular patterns in skeletal muscle are cross-sectional,with only time point examined,and with the biopsied muscles having variable degrees of pathology (independent of disease duration or degree of overall functional impairment of the patient).Since motor neurons destined for fasttwitch (type-II) muscle fibers are more vulnerable to the degenerative process in ALS (Frey et al.,2000;Allodi et al.,2021),results may be skewed in muscle samples with a disproportionately higher or lower population of these fiber types.The impact of normal aging on gene expression in muscle also comes into play as ALS is an age-associated disease with a typical onset between 55-61 years of age (Kazamel et al.,2013;Tumasian et al.,2021).This effect also applies to rodent models as was shown with miRNA sequencing,for example,in wild-type mice (Si et al.,2018).

These caveats notwithstanding,the muscle biopsy provides a direct view of the distal neuromuscular unit and its microenvironment (as opposed to iPSCderived human cells,for example).With the rapid advancement of “omics” technology,the number of aberrantly regulated molecules in human ALS muscle has grown large,ranging from coding and non-coding RNAs to proteins and metabolites (Pradat et al.,2012;Bernardini et al.,2013;Conti et al.,2014;Elf et al.,2014;Si et al.,2018;Lanznaster et al.,2022;Tsitsipatis et al.,2022).To understand the potential utility of these molecules as biomarkers of disease progression,a number of approaches can be taken which combine clinicopathological correlation and the use of the SOD1G93Arodent model of ALS (Figure 1).These correlates,which can mitigate limitations of crosssectional muscle samples,include bedside assessment of muscle power using the Medical Research Council grading scale,electrophysiological assessment of acute and chronic denervation in muscle by electromyography,quantitation of neurogenic atrophy which is a nearly universal histopathological feature of ALS muscle (Pradat et al.,2012;Al-Sarraj et al.,2014),and the rate of decline in the ALSFRS-R score.Finally,assessment of orthologs in the SOD1G93Arodent model can be very helpful in determining temporal patterns during disease progression,including presymptomatic stages.Even though mutations in SOD1 account for only～2% of all ALS (Brown et al.,2021),the mouse model recapitulates a number of pathological features of ALS,particularly in the neuromuscular unit,where fast-twitch muscle fibers show a 40% reduction in NMJ innervation by postnatal day 30 (Vinsant et al.,2013;Morrice et al.,2018).While very subtle motor symptoms appear in this time frame,overt motor symptoms as measured by grip strength or rotarod testing occur after postnatal day 70 (Heiman-Patterson et al.,2005;Vinsant et al.,2013;Si et al.,2014;Allodi et al.,2021).The mouse model,therefore,provides a window into altered molecular pathways in the prodromal phase of the disease.In humans,this clinically asymptomatic phase may be up to years in duration (Eisen et al.,2014;Benatar et al.,2019).The prodromal phase in humans is now being characterized in studies by Benatar et al.(2018) who follow asymptomatic carriers of known ALS-associated mutations such as C9ORF72 or SOD1 through phenoconversion.

Figure 1｜Clinicopathological correlations used for characterizing ALS muscle biomarkers.

Promising Muscle Biomarkers that Detect Early Disease

As mentioned earlier in this review,the scientific literature is replete with studies using “omics” to identify aberrantly expressed molecules in ALS muscle,either in humans or rodent models of ALS (mainly the SOD1G93Amouse).Applying the criteria described inFigure 1,I have focused on biomarkers identified in human muscle and with corresponding mouse orthologs appearing in presymptomatic stages (Table 1).These biomarkers were divided into three tiers (1-3) based on the extent of correlation with clinicopathological phenotypes in human disease and their pattern of progression in the SOD1G93Arodent model.Tier 1 biomarkers show a correlation with denervation in ALS patients,either clinically or histologically,or a correlation with fast progression as determined by changes in the ALSFRS-R scores.Five biomarkers,TGF-β3,FGF23,frizzled-related protein (FRZB),GDNF,and phospho-Smad1,5,8 correlated positively with myofiber atrophy in human ALS muscle (several examples are shown inFigure 2) (Lie and Weis,1998;Pradat et al.,2012;Si et al.,2014,2021;Kwan et al.,2020).TGF-β3 was the most consistent,showing an inverse correlation with muscle weakness as measured by the Medical Research Council scale on clinical exam and a positive correlation with the degree of histological denervation (Pradat et al.,2012;Si et al.,2015).TGF-β1 had a similar clinical correlation and immunohistochemical pattern (Si et al.,2015).

Table 1｜ Skeletal muscle biomarkers in ALS

Figure 2｜Immunoreactive patterns of four tier 1 biomarkers in human ALS muscle samples.

Two tier 1 biomarkers,HDAC4 and CYP27B1,correlated with fast progression as determined clinically by the ALSFRS-R (Bruneteau et al.,2013;Si et al.,2020).Perhaps the most convincing studies for determining a correlation with disease progression used serial biopsies in the same patient and same muscle: GDNF (over 12 weeks) and CYP27B1 (over 1 year) (Jensen et al.,2016;Si et al.,2020).In those studies,both biomarkers increased with disease progression,and CYP27B1 showed a correlation with rapid progression.Orthologs for tier 1 biomarkers were detected in the SOD1G93Amouse in the presymptomatic stage (except for GDNF) with evidence of progressive change throughout the disease course.

Tier 2 biomarkers lacked clinical or histopathological correlations in ALS patients (or were not reported/investigated) but demonstrated consistently altered patterns in the SOD1G93Amouse beginning in presymptomatic stages.Likewise,these biomarkers showed progressive changes throughout the disease course.Two biomarkers,hsa_circ_0007099 and hsa_circ_ 0005171,were recently identified by circRNA sequencing of human ALS muscle (Tsitsipatis et al.,2022).Many of the myomiRs (miR-1,miR-133a,miR-133b,and miR-206) that have been associated with ALS fell into tier 2.Multiple studies have investigated miR-206 and have consistently observed high levels in skeletal muscle of the SOD1G93Amouse starting in the presymptomatic stages and increasing with disease progression.In human muscle samples,however,there is variability with some studies showing increases (Bruneteau et al.,2013;Russell et al.,2013;Di Pietro et al.,2017) and others no change (Jensen et al.,2016;Si et al.,2018;Pegoraro et al.,2020).This may relate to the subtype of ALS (Pegoraro et al.,2020) but also variable expression in different muscles.For example,we observed a～2.5-fold increase in miR-206 in vastus lateralis versus deltoid muscles in normal controls (Si et al.,2018).Differences in methodology may add to variability,such as the use of different internal controls (e.g.,spiked-in miRNA versus endogenous small RNAs).Tier 3 candidates were all non-coding RNAs and had consistent dysregulation (suppression) between human samples and the SOD1G93Amouse and were detected in presymptomatic ages but without progressive changes.This stability over time,as with the neurofilament light chain (Benatar et al.,2020),may be a favorable feature in the clinic if a therapeutic intervention reverses the dysregulation.

Interestingly,several biomarkers reverted to control levels at end-stage in the SOD1G93Amouse,including FRZB,GDNF,and amyloid precursor protein (APP) (Yamamoto et al.,1996;Lie and Weis,1998;Stanga et al.,2018;Kwan et al.,2020).With GDNF,there was a similar pattern in human muscle where high levels were observed in muscle biopsies with active denervation but not in end-stage muscle from post-mortem samples (Lie and Weis,1998;Stanga et al.,2018).For FRZB,there was a progressive decline in expression in wild-type mouse controls indicating an effect of development/aging on gene expression.These patterns provide supportive evidence that the biomarkers are active in acutely denervating muscle but then decline as surviving motor neurons and myofibers wane.

Detecting Muscle Biomarkers in Blood

Biomarkers described inTable 1have favorable features for tracking disease onset and progression.To be useful in the clinic,however,biomarkers should be readily measurable,ideally with blood testing.Lacking a blood barrier like the CNS unit,dysregulated molecules in the peripheral neuromuscular unit may be more likely to enter the circulatory system.Consistently,miR-206 is elevated in ALS patient blood samples across many studies with some correlation to clinical profiles (Vijayakumar et al.,2019;Dobrowolny et al.,2021;Malacarne et al.,2021).Serum levels of miR-133a and miR-133b are also increased in ALS patients with higher levels of miR-133a appearing to correlate with slower disease progression (Raheja et al.,2018;Dobrowolny et al.,2021).TGF-β1 is increased in the serum of ALS patients toward the end stage of the disease (Ilzecka et al.,2002),which may come from muscle as CNS tissues showed no significant change (Peters et al.,2017).Serum TGF-β2 was not significantly changed (Peters et al.,2017).FRZB and FGF23,both secreted factors,were not altered in ALS serum although immunohistochemistry showed strong immunoreactivity in the endomysial connective tissue (Figure 2;Kwan et al.,2020;Si et al.,2021).It is possible that these factors and others get trapped in this compartment due to an increase in extracellular matrix deposition found in ALS muscle (Gonzalez et al.,2017) and an enrichment of negatively charged heparan sulfate glycosaminoglycans in the basal lamina (Jenniskens et al.,2000).Lastly,the quantity of the biomarker,such as the circRNAs (tier 2 and 3 biomarkers) may be too low for detection (Tsitsipatis et al.,2022).Taken together,muscle biomarkers detectable in serum have not yet been used in the clinic because of their variability and lack of consistency in clinical correlation.This may relate to methodological challenges for consistent measurement,particularly for non-coding RNAs,and our limited insight into the heterogeneity of ALS.

Muscle Biomarkers Identify Novel and Connected Pathways in Amyotrophic Lateral Sclerosis Pathophysiology

In addition to providing utility for tracking disease progression,biomarkers described inTable 1provide insight into altered and connected molecular pathways in the neuromuscular unit and possibly new therapeutic targets in ALS (Figure 3).A major challenge is determining whether the dysregulated pathways are part of the pathological process and detrimental,or are compensatory and beneficial.Some pathways may be salutary in acute injury but detrimental in chronic denervating disease,further complicating the therapeutic potential of a target.Below is a brief overview of these pathways,their connections,and their potential relevance to ALS pathophysiology and therapeutics.

Figure 3｜Signaling pathways of tier 1,2,and 3 muscle biomarkers show interconnectivity and potential mitigating or detrimental effects on ALS disease progression.

TGF-β/Smad/myomiR signaling

In ALS muscle,there is evidence that the phosphorylated (active) form of Smad1,5,8 is detected in non-atrophic myofibers in human ALS and the presymptomatic SOD1G93Amouse,in parallel with increases in all three TGF-β isoforms (Si et al.,2014,2015).Smad8 is one of the most consistently altered biomarkers in human ALS muscle (Si et al.,2014;Tsitsipatis et al.,2022).A link between TGF-β and Smad8 was made by our group where we found that stimulation of muscle cells with each TGF-β isoform markedly induced Smad8 mRNA (and to a lesser extent Smad1 and Smad5),and also activated p-Smad1,5,8 (Si et al.,2015).These findings are in line with prior observations that TGF-β ligands can activate receptors classically linked to bone morphogenetic protein (BMP) ligands as well as Smad2 and Smad3 (Schmierer and Hill,2007).BMP4 is induced in SOD1G93Amouse muscle later in the symptomatic stage (Si et al.,2015).Our group subsequently showed that knockdown of Smad8 led to a derepression of myomiRs miR-1,miR-133a,and miR-133b (Lopez et al.,2022).Smad8-silenced cells showed increased muscle differentiation consistent with the function of myomiRs to promote myogenesis (Horak et al.,2016;Giagnorio et al.,2021).Other physiological processes linked to these myomiRs include muscle regeneration,maintenance of fast-twitch fibers,and mitochondrial biogenesis all of which are critical compensatory pathways to the degenerative process of ALS.TGF-β signaling promotes fibrosis in ALS muscle via Smad3 activation in PDGFRα+fibro/adipogenic progenitor cells (FAPs) (Gonzalez et al.,2017).Often produced by macrophages (Lemos et al.,2015;Nawaz et al.,2022),TGF-β1 induces a pro-fibrotic activation of FAPs (Nawaz et al.,2022) whereas Smad2 and Smad3 activation in muscle cells suppresses myogenesis and leads to muscle atrophy (Sartori et al.,2009).Increased interleukin-6/STAT3 signaling in FAPs is also detected in human ALS muscle and late-stage SOD1G93Amouse muscle and likewise promotes muscle fibrosis and atrophy (Madaro et al.,2018).Secretion of interleukin-6 by FAPs is postulated to be the main driver of these phenotypes,but this cytokine has not been identified as a clinical muscle biomarker in human ALS.

Taken together,there is good evidence supporting a deleterious role of TGF-β and BMP signaling in ALS muscle via Smads by suppressing myomiRs and promoting atrophy and fibrosis.On the other hand,TGF-β plays an important role in the development and maintenance of the NMJ and has neuroprotective effects on motor neurons which extensively express TGF-β receptors (McLennan and Koishi,2002;Si et al.,2015;Galbiati et al.,2020).These pleiotropic effects may come into play in the therapeutic targeting of these signaling pathways in ALS.

CYP27B1

CYP27B1 is the primary enzyme that catalyzes the biosynthesis of 1,25(OH)2D,which is the active form of vitamin D.Immunohistochemistry of ALS patient muscle samples indicates that the enzyme is expressed within myofibers,especially those with features of denervation (Si et al.,2020).Classically,this enzyme is concentrated in the kidney and tightly regulated,but ectopic expression can be induced in other organs.Interestingly,TGF-β1 (which parallels CYP27B1 with disease progression) is an inducer of this ectopic expression.Other cytokines produced by mast cells and neutrophils that are present in ALS muscle,including TNF-α and interferon-γ,can induce CYP27B1 (Tecchio et al.,2014;Jones et al.,2018;Mukai et al.,2018;Trias et al.,2018).Vitamin D signaling may play a role in muscle regeneration,including myoblast differentiation,NMJ innervation,and suppressing cytokine expression in muscle (Pojednic and Ceglia,2014;Gifondorwa et al.,2016).The relative contribution of circulating versus locally produced vitamin D in these processes is unclear.The role of circulating vitamin D as a biomarker of ALS disease progression is clouded by a number of conflicting reports and has not been useful in the clinic (Si et al.,2020).Taken together,enhanced vitamin D signaling likely plays a compensatory rather than pathogenic role in ALS.

FGF23

A tier 1 biomarker,FGF23 was enriched in the endomysial connective tissue within areas of myofiber atrophy (Figure 2;Si et al.,2021).FGF23 has been associated with aging,frailty,and muscle wasting but the mechanism(s) remains obscure (Cardoso et al.,2018).The source of FGF23 in muscle is not clear but has been attributed to infiltrating macrophages in other tissues in response to inflammatory cytokines (David et al.,2017).Further amplification of the inflammatory milieu in ALS muscle may tie into TGF-β2 (tier 2 biomarker) which can induce FGF23 (Feger et al.,2017).FGF23 itself can trigger the production of inflammatory cytokines.In addition to its effect on inflammation,FGF23 was also shown to suppress mesenchymal stem cells in skeletal muscle and promote their senescence (Sato et al.,2016).Interestingly,in cardiac muscle,FGF23 promotes fibrosis by activation of fibroblasts via TGFβ1 signaling (Leifheit-Nestler and Haffner,2018).Taken together,FGF23 may promote disease progression and thus represents a potential therapeutic target.

FRZB

FRZB,a tier 1 biomarker,is a secreted Wnt antagonist that accumulates in the endomysial connective tissue space predominantly surrounding atrophic muscle fibers in ALS muscle (Kwan et al.,2020).Wnt signaling is a complicated pathway with different and sometimes opposing effects in skeletal muscle.It promotes myogenesis,the formation of NMJs,and axon guidance,all essential components for muscle regeneration in ALS or other denervating diseases (von Maltzahn et al.,2012;Cisternas et al.,2014).One study found a disproportionally higher expression of Wnt isoforms in extraocular muscles which are typically spared in ALS,suggesting that Wnt signaling is protective (McLoon et al.,2014).A recent study using iPSC-induced motor neurons and astrocytes from ALS patients harboring the FUS mutation found that the motor neurons had defects in neurite outgrowth and NMJ formation related to reduced Wnt signaling (Stoklund Dittlau et al.,2023).Interestingly,riluzole,one of the Food and Drug Administration-approved ALS drugs that slow disease progression,enhances Wnt/β-catenin signaling (Biechele et al.,2010).In contrast to the potential mitigating effects,Wnt signaling can promote fibrosis through crosstalk with TGF-β signaling (Cisternas et al.,2014).This has been observed in aging muscle and other muscle diseases like Duchenne’s muscular dystrophy.TGF-β can stimulate β-catenin signaling in part by suppressing the destruction of β-catenin.Findings from our group and another group provide support for that possibility,showing prominent increases in β-catenin immunoreactivity in human ALS myofibers (McLoon et al.,2014;Kwan et al.,2020).As a Wnt antagonist,FRZB may come into play in these opposing effects,potentially inhibiting reinnervation and recovery,or compensating against a pro-fibrotic program triggered by TGF-β.

GDNF

GDNF is a soluble neuronal trophic factor produced by glial cells,including Schwann cells,and skeletal muscle,where it promotes motor neuron survival,sprouting,and NMJ remodeling (Cintron-Colon et al.,2022).Its increase in ALS muscle,therefore,suggests a compensatory effect on the denervation process.The potential therapeutic effect of GDNF is supported by several studies showing that exogenous delivery to muscle in the SOD1G93Amouse delayed disease onset and extended survival (Verma et al.,2022).One report showed that GDNF reduced NMJ loss in SOD1G93Arats,preserved terminal Schwann cell association with NMJs,and reduced CD11b expression (a marker of infiltrating inflammatory cells) (Van Dyke et al.,2016).GDNF also has neuroprotective effects on motor neurons centrally in ALS and is the focus of a clinical trial using stem cells engineered to express GDNF (Laperle et al.,2023).

HDAC4 and miR-206

HDAC4 is a tier 1 muscle biomarker and correlates with rapid ALS progression in humans,raising the possibility that it plays a role in disease pathology (Bruneteau et al.,2013).HDAC4 was first linked to ALS in the SOD1G93Amouse where it was found to suppress FGFBP1 expression in muscle cells (Williams et al.,2009).FGFBP1 is a soluble growth factor that is enriched near NMJs and promotes their regeneration after denervation.FGFBP1 is reduced in the presymptomatic SOD1G93Amouse and its deletion leads to accelerated NMJ loss and shortened lifespan (Taetzsch et al.,2017).MicroRNA-206 targets HDAC4 to suppress its expression,leading to increased FGFBP1.Deletion of miR-206 reduces the lifespan in the SOD1G93Amouse,increases muscle atrophy,and reduces NMJ innervation.TGF-β1 inhibits FGFBP1 expression in myotubes thereby linking these two biomarkers in a deleterious role in ALS pathology.Interestingly,loss of FGFBP1 increases NMJ defects in wildtype aging mice suggesting an ongoing requirement of this growth factor for NMJ maintenance.The correlation of FGFBP1 with human disease is unclear,however,as it is increased by～8-fold in human ALS muscle (Bruneteau et al.,2013).

Interleukin-1β and aCasp1

These tier 2 biomarkers reflect an increasing inflammatory milieu in the neuromuscular unit,starting in the presymptomatic stages,and driven by progressive infiltration of macrophages,neutrophils,and mast cells (Chiu et al.,2009;Van Dyke et al.,2016;Trias et al.,2017,2018;Wang et al.,2017;Lehmann et al.,2018).This infiltration parallels disease progression and spans the entire peripheral neuromuscular unit,including muscle,nerve,and ventral root.Although immune cells such as macrophages can play an important role in recovery after nerve or muscle injury (Cintron-Colon et al.,2022;Nawaz et al.,2022),recent studies suggest a harmful effect.In a study by Trias et al.(2018),for example,neutrophil extracellular traps were identified near motor endplates in human ALS muscle indicative of neutrophil hyperactivation and high cytotoxic potential.In the SOD1G93Arat,neutrophils accumulate at the NMJ with disease progression along with activated and degranulating mast cells (Trias et al.,2017,2018).In further support for a pathogenic role,treatment of SOD1G93Arats with the c-Kit inhibitor,masitinib,which blocks mast cell and macrophage activation and migration,significantly reduced muscle infiltration of both cell populations.Masitinib treatment ameliorated NMJ denervation and preserved motor function (Trias et al.,2017).Reduced infiltration was observed in other components of the peripheral neuromuscular unit including sciatic nerve and ventral roots and a concomitant reduction in axonal and myelin degeneration (Trias et al.,2018).Masitinib is now in a Phase III study with riluzole in human ALS and has shown promise in slowing disease progression (Mora et al.,2021).

PDK4

Mitochondrial dysfunction in skeletal muscle is one of the earliest abnormalities detected in muscle in the presymptomatic stages of ALS and leads to defects in energy production (Vandoorne et al.,2018).In these early stages,there is a metabolic switch from glycolytic to oxidative-based pathways of energy production in parallel with a shift from fast-to-slow muscle fiber type (Palamiuc et al.,2015;Scaricamazza et al.,2020).PDK4 is a biomarker of this early switch,functioning as an inhibitor of the pyruvate dehydrogenase complex,a major driver of glycolytic metabolism in muscle (Palamiuc et al.,2015).The progressive increase in PDK4 with disease progression reflects this ongoing switch and is likely linked to NMJ denervation.In parallel with PDK4 upregulation,there is an increase in the use of lipids for energy production via β-oxidation which exacerbates mitochondrial dysfunction via oxidative stress.A detrimental effect was implicated in the SOD1G85Rrat where pharmacological inhibition of PDK4 led to the preservation of muscle strength,increased myofiber size and mitochondrial biogenesis,and a reduction of atrophyassociated markers (Palamiuc et al.,2015).

APP

APP is most often associated with Alzheimer’s disease as the source of the pathogenic β-amyloid peptide (Muller and Zheng,2012).APP is aberrantly detected in human ALS muscle and the SOD1G93Amouse beginning in the presymptomatic phase where expression is increased in atrophic type-II muscle fibers (Koistinen et al.,2006;Bryson et al.,2012).Increased APP levels may lead to destabilization of the NMJ (Moloney et al.,2014) and genetic deletion in the SOD1G93Amouse improved motor neuron survival,NMJ innervation,and delayed the loss of motor function (Bryson et al.,2012).Lifespan,however,was not extended.In vitrostudies of cultured muscle cells showed that overexpression of APP impaired NMJ formation (McFerrin et al.,1998;Koistinen et al.,2006).On the other hand,APP (perhaps at lower levels) is important in NMJ formation during development and its cleaved product,sAPP-α,may contribute to NMJ stabilization (Wang et al.,2005;Moloney et al.,2014).Thus,it remains unclear whether APP upregulation is beneficial or detrimental.

Circular RNAs

Circular RNAs (circRNA) are long non-coding RNAs that are covalently closed and generated during the splicing of pre-mRNAs (Memczak et al.,2013).Using circRNA sequencing on human ALS muscle,our group recently found a distinct population of differentially expressed circRNAs (Tsitsipatis et al.,2022).Three (hsa-Circ_0000033 hsa-Circ_0007099 and hsa_circ_0005171) were not altered in disease controls and thus appeared specific for ALS.Many functions have been attributed to circRNAs including transcriptional,posttranscriptional,and post-translational regulation of gene expression.While the role of dysregulated circRNAs in ALS muscle is unknown,we assessed their expression pattern in the spinal cord and brain of ALS patients as well as iPSC-derived motor neurons from patients with C9ORF72 repeat expansions.Five circRNAs which included the tier 2 biomarker,hsa_circ_0005171,were significantly increased in C9ORF72-derived motor neurons.Yet these circRNAs were reduced in ALS spinal cord tissue.The gradient could not be explained by the end-stage nature of the tissue or the degradation of RNA as the linear counterparts were unchanged compared to controls.These findings raise the hypothesis that some of the circRNAs mobilize to the distal (intramuscular) portion of the motor neuron as part of the disease process.Additional investigations into the biological function of these dysregulated circRNAs will be necessary to determine whether they are compensatory or pathogenic.Their utility as biomarkers may be limited due to the generally low copy numbers of circRNAs.As such,we were not able to detect them in the patient’s plasma.

Conclusions

In this review,I have highlighted studies identifying human ALS muscle biomarkers which show a correlation with clinicopathological features and are detectable in presymptomatic phases of the disease in the SOD1G93Amouse.The prodromal phase of ALS may be in years,so a biomarker that can define this phase will facilitate earlier diagnosis and treatment initiation.Unfortunately,many of the muscle biomarkers were either intracellular or not detectable in blood,limiting their immediate utility in the clinic.Muscle biopsy is invasive and not practical for multiple samplings.With advancements in imaging and radiotracer technology,it may be possible in the future to detect and quantify biomarkers such as p-Smads or active vitamin Din vivo.The discovery of these biomarkers also reveals novel disease mechanisms that could be targeted therapeutically to promote compensatory processes or block pathogenic ones.

Author contributions:PHK completed manuscript writing,data search,collection,and analysis and approved the final version of the manuscript.

Conflicts of interest:The author declares no conflicts of interest.

Data availability statement:Not applicable.

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