The role of skeletal muscle in ALS: “dying-back” or “dying-forward” phenomenon?

Stavroula Tsitkanou1*, Angus Lindsay1, Paul Della Gatta1

1
Institute for Physical Activity and Nutrition, School of Exercise and Nutrition Sciences,
Deakin University, Geelong, Victoria, Australia.

* Correspondence:
Stavroula Tsitkanou, MSc, PhD candidate
Institute for Physical Activity and Nutrition, School of Exercise and Nutrition Sciences,
Deakin University, 221 Burwood Highway, Burwood 3125, Victoria, Australia.
Email address: stsitkan@deakin.edu.au

Amyotrophic lateral sclerosis (ALS) is a fatal motor neuron disease. Motor neurons that send
signals from the brain to skeletal muscles of the upper and lower body are gradually
degenerated during ALS progression. Motor neuron degeneration results in severe muscle
atrophy and eventually death from respiratory insufficiency in ALS patients. ALS manifests
as either familial ALS (FALS; 5-10% of cases) or sporadic ALS (SALS; 90-95% of cases).
Mutations in the copper/zinc (CuZn) superoxide dismutase (SOD1) gene, a ubiquitously-
expressed free-radical defense enzyme, account for approximately 20% of FALS cases and
the mutant SOD1 mouse model has been used extensively to help understand the ALS
pathology.

ALS is considered a “multi-systemic” disease. Changes in cell structure, physiology and

metabolism act mutually and synergistically in ALS which complicates the identification of
the primary pathogenic mechanism. Neuronal defects, including excitotoxicity, deficits in
axonal transport and neurofilament aggregation are proposed as major contributors to ALS
pathophysiology. Based on the predominant “dying-forward” hypothesis, degeneration of
upper and lower motor neurons precedes neuromuscular junction (NMJ) degeneration and
skeletal muscle atrophy in ALS. However, therapeutics focused on rescuing motor neurons
have shown limited success in SOD1G93A mice or ALS patients.

Skeletal muscle dysfunction resulting from disequilibrium in mitochondrial function, satellite

cell proliferation, differentiation, and microRNA expression is considered an important
This is an Accepted Article that has been peer-reviewed and approved for publication in the The
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mechanism in the pathogenesis of ALS. Based on the “dying-back” phenomenon, ALS is a
distal axonopathy, whereby the disease originates in the peripheral tissues (including skeletal
muscle) and a retrograde signaling cascade leads to motor neuron death. The basis for the
“dying-back” theory resides in previous literature that shows defects in skeletal muscle occur
early in life of SOD1G93A mice and prior to motor neuron death (Loeffler et al., 2016).
However, a more holistic approach is required to define whether ALS is indeed a “dying-
back” or “dying-forward” disease, evaluating brain, spinal cord, peripheral nerve and skeletal
muscle both prior to disease initiation and following the onset of symptoms.

In a recent study, in the Journal of Physiology, Cheng et al. (2019) suggest that skeletal
muscle weakness is attributed to motor neuron degeneration and not intrinsic muscle fibre
impairments. To determine whether muscle weakness observed in SOD1G93A mice can be
explained by intrinsic skeletal muscle impairments or motor neuron degeneration, they used
male and female SOD1G93A mice with a high-copy number of the mutated SOD1G93A
transgene. The animals were assigned into two age groups: pre-symptomatic (50 days of age)
and symptomatic (125 days of age). Motor neuron retrograde labeling with subcutaneous
injections of cholera toxin B subunit was performed to investigate the level of motor neuron
denervation in the predominantly fast-twitch flexor digitorum brevis (FDB) muscle. Skeletal
muscle health of SOD1G93A mice was also evaluated using whole muscle and single fibre
experiments. The whole muscle experiment included assessment of in vitro force-generating
capacity and myosin-heavy chain (MHC) isoform distribution in FDB muscle, protein
expression of mitochondrial markers and citrate synthase activity in tibialis anterior muscle
(TA), as well as mRNA expression of mitochondrial biogenesis regulators, autophagy- and
apoptosis-related genes in extensor digitorum longus (EDL) muscle. The single muscle
experiments included quantification of specific force, tetanic [Ca2+], fatigue resistance and
fibre cross-sectional area.

Cheng et al. (2019) showed that the number of motor neurons innervating FDB muscle was
lower in late stage SOD1G93A mice compared to WT mice, indicating denervation of muscle
fibres. In vitro force production of FDB muscle was also lower in SOD1G93A mice at the late
stage of disease only. The lower FDB force production in late symptomatic SOD1G93A mice
can be explained by the loss of FDB muscle mass accompanied by an increased expression of
autophagy- and apoptosis-related markers (Bnip3 and Casp3/Casp7, respectively) in EDL
muscle, because no change was measured in muscle fibre MHC isoform distribution at the

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late disease stage. However, when intact single FDB fibres were analysed, their cross-
sectional area was similar between late stage SOD1G93A and WT mice. Furthermore, no
difference was found in the sarcolemmal membrane excitability of intact single fibres through
assessment of the voltage threshold to elicit a tetanic contraction, as well as the specific force,
tetanic [Ca2+] (150 Hz electrical stimulation) and resting [Ca2+] between SOD1G93A and WT
mice in early and late disease stages. The fatigue-induced decline in force and tetanic [Ca2+],
and the increase in resting [Ca2+] in intact single fibres were similar between pre-
symptomatic SOD1G93A and WT mice. However, the late stage SOD1G93A mice had a
surprisingly increased fatigue resistance compared to age-matched WT mice, displaying
improved preservation of force, tetanic [Ca2+] and resting [Ca2+] during 150 repeated
contractions. Although these adaptations cannot be explained by a shift in MHC isoform
distribution in the FDB muscle, they may imply an improved skeletal muscle oxidative
energy metabolism in the late stage SOD1G93A mice. The improved oxidative metabolism was
confirmed by the increased expression of myoglobin and mitochondrial electron transport
chain proteins in TA muscles of late stage SOD1G93A mice. Moreover, the increased mRNA
expression of mitochondrial biogenesis genes (Pcg1a1, Nrf1, Tfam, Cox5b, mt-Co3) in EDL
muscle at the early (except from Pcg1a1) and the late disease stage of SOD1G93A mice may
also explain the improved oxidative metabolism observed in the late stage SOD1G93A mice.
However, although the FDB, TA and EDL muscles are categorised as fast-twitch muscles
and, by extension, their force production and fatigue resistance may be similar, the
morphology, physiology and metabolism are different between hindlimb muscles of mice
(Terry et al., 2018). Therefore, molecular and cellular adaptations measured by Cheng et al.
(2019) across multiple muscles may be not translatable.

These results suggest that compensatory adaptive mechanisms, such as muscle-fibre

apoptosis, mitochondrial biogenesis and an increased fatigue resistance, occur simultaneously
within the muscle tissue of SOD1G93A mice. These compensatory adaptations may be
activated to delay disease progression and maintain skeletal muscle homeostasis. Specifically,
the increased expression of mitochondrial biogenesis genes, myoglobin and mitochondrial
electron transport chain proteins in skeletal muscle of SOD1G93A mice could be an attempt to
provide more energy for reversing ALS-induced muscle atrophy. The phenomenon of
compensatory adaptations has also been supported by previous studies, which found
hypertrophied fibres in skeletal muscle of end-stage SOD1G93A mice (Atkin et al., 2005).

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Although scientific evidence indicates skeletal muscle contributes to ALS pathophysiology
through the “dying back” phenomenon (Loeffler et al., 2016), it still remains unknown
whether ALS is indeed a “dying back” or “dying forward” disease. The study by Cheng et al.
(2019) suggests motor neuron degeneration precedes muscle atrophy in ALS which is in
agreement with previous studies (Atkin et al., 2005). Measurements at both the motor neuron
and skeletal muscle level were performed by Cheng et al. (2019); however, the functional
assessment of FDB muscle could be considered a methodological limitation of this study.
First, specific force of FDB muscle cannot be normalised by cross sectional area because of
the complex pennate architecture as well as increased fibrosis, fibre atrophy and loss of fibres
in skeletal muscle of ALS mice. However, the limitation of normalising the force production
in the whole FDB muscle was eliminated by measuring specific force in intact single muscle
fibres because force can be normalised by cross-sectional area. Secondly, the characterisation
of the FDB muscle as “fast” may not be entirely accurate when comparing it to other fast
muscles. The FDB muscle is composed of ~50% type IIa fibres and 50% type IIx fibres
(Tarpey et al., 2018). It is widely known that Type IIb muscle fibres are preferentially
affected in SOD1G93A mice. This preferential degeneration of type IIb fibres has further
implications for the gene and protein results presented by Cheng et al. (2019), as they were
measured in the EDL and TA muscles, both of which are predominantly comprised of Type
IIb fibres. Irrespective of this limitation, the use of the FDB muscle is of incredible interest.
Based on Cheng et al’s results, it would seem that FDB is a fast muscle that is somewhat
protected from the ALS-like pathology of the SOD1G93A mouse. Human skeletal muscle does
not contain Type IIb fibres, so investigation of the pathology of the FDB muscle may be
more akin to investigating muscle pathology in humans. Further investigation of the FDB
muscle and other muscles that are more representative of human muscle, such as the peroneus
longus, which is a mixed muscle that can be analysed in an ex vivo muscle preparation set-up
(Lindsay et al. 2019), may help us to better understand the pathogenesis of ALS in human
muscle.

In conclusion, whether ALS is a “dying-back” or “dying-forward” disease still remains a

subject of intense debate in understanding the pathogenic mechanisms of ALS. The study by
Cheng et al. (2019) provides evidence indicating that muscle fibres in isolation are not a
major site of ALS pathology, elegantly demonstrating the adaptive mechanisms present in
various fast muscles to combat denervation and muscle atrophy. Although motor neuron
death is considered as leading candidate of ALS pathophysiology, further investigations using

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more holistic approaches to ALS pathology including the sampling of multiple organ sites
(e.g. the brain, spinal cord, sciatic nerve and muscle) and muscles of various fibre type
composition are required to determine the true origin of ALS pathology.

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Additional information
Competing interests
All authors listed declare that they have no conflict of interest.

Author contributions
All authors listed have made substantial, direct and intellectual contribution to the work, and
approved it for publication. They agree to be accountable for all aspects of the work in
ensuring that questions related to the accuracy or integrity of any part of the work are
appropriately investigated and resolved. All persons designated as authors qualify for
authorship, and all those who qualify for authorship are listed.

Funding
S.T. is supported by an International Postgraduate Research Scholarship, awarded by the
Deakin University (Victoria, Australia) and a Greek Scholarship for doctoral studies abroad,
awarded by the Onassis Foundation (Greece).
A.L. is supported by an Alfred Deakin Postdoctoral Research Fellowship by the Deakin
University (Victoria, Australia).

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