Amyotrophic Lateral Sclerosis (ALS) is a devastating neuromuscular disorder characterized by progressive motor neuron death and skeletal muscle wasting. Remarkably, extraocular muscles (EOMs) exhibit superior preservation of structure, neuromuscular junction (NMJ) integrity and function in ALS patients and rodent models [[1], [2], [3], [4], [5]]. Beside EOMs, animal model studies also show that limb and body muscles respond to ALS progression differently, with fast-twitch muscles generally more susceptible to degeneration than in slow-twitch muscles [[6], [7], [8]]. Factors underlying this phenomenon can be multi-faceted and could hold clues for identifying novel therapeutic targets against muscle degeneration in ALS or other neuromuscular disorders.

Reactive aldehydes resulting from oxidative stress are involved in the pathological process of multiple neurodegenerative disorders including ALS [9]. One major route for intracellular production of reactive aldehydes is the peroxidation of polyunsaturated fatty acids (PUFA), which are major component of cellular membranous structures [10,11]. Well-known examples include 4-hydroxynonenal (4-HNE), malondialdehyde (MDA) and acrolein [11], which can form adducts with protein through Michael addition or Schiff base formation [[12], [13], [14]]. These adducts contribute to protein crosslinking and aggregation [11], leading to broad pathological consequences including disrupted cell signaling, altered gene expression, inhibited enzyme activity, compromised mitochondrial function and disformed cytoskeleton [15]. Reactive aldehydes can also form adducts with DNA, resulting in inter-strand crosslinks, base substitution, mutation and fragmentation [[16], [17], [18], [19], [20], [21], [22]].

4-HNE most commonly reacts with deoxyguanosine (dG) residues to form bulky exocyclic DNA adducts. These adducts distort the DNA double helix and can stall DNA polymerase during replication, leading to single-strand and double-strand breaks [23]. In addition, 4-HNE forms adducts with mitochondrial proteins such as the adenine nucleotide translocase, which disrupts the mitochondrial inner membrane potential that triggers the release of Cytochrome c. This activates the Caspase cascade, leading to the activation of Caspase-Activated DNase (CAD) to cleave genomic DNA between nucleosomes [24]. 4-HNE also forms adducts with pivotal DNA repair proteins involved in Nucleotide Excision Repair (NER), reducing their repair capacity. The persistent single-strand breaks eventually reach the threshold that activates the p53-mediated apoptotic response and subsequent fragmentation [24].

In the aspect of plasma membrane integrity, 4-HNE is known to form adducts with phosphatidylethanolamine and certain ion channel proteins, causing altered ionic homeostasis and cellular osmolarity [25,26]. The deprivation of local antioxidants like glutathione by 4-HNE can promote further lipid peroxidation at the plasma membrane, creating a vicious cycle that leads to cell death [27,28]. Elevated levels of lipid peroxidation markers, including 4-HNE adducts, have been detected in cells of central nervous system and body fluids from patients and/or models of amyotrophic lateral sclerosis (ALS), Alzheimer disease (AD), Parkinson disease (PD) and Huntington disease (HD) [9].

To neutralize reactive aldehydes, the human body has deployed a series of detoxification enzymes. Aldehyde dehydrogenases (ALDHs) aimed to oxidize the carbonyl group into corresponding acids, while aldo-keto reductases (AKRs) reduce aldehydes into corresponding alcohols [12]. Aldh2, Aldh1a1 and Aldh3a1 are the three most studied ALDHs [29]. Aldh2 is a mitochondrial ALDH abundantly present in liver, brain, heart and muscle [30]. Aldh1a1 and Aldh3a1 are primarily cytosolic and are extremely abundant in mammalian corneal and lens to protect against ultraviolet radiation (UVR) induced generation of reactive aldehydes and their pathological consequences [20,31,32]. Aldh3a1 knockout mice and Aldh1a1/Aldh3a1 double knockout mice develop cataracts by 1 month of age [32]. It is also worth noticing that a kinetics study of these three ALDHs reported that Aldh2 was irreversibly inactivated by 4-HNE and acrolein at above 10 μM. Aldh1a1 was inactivated by acrolein at concentrations higher than 1 mM, while no inactivation of Aldh3a1 was observed by either 4-HNE, acrolein or MDA even at 20 mM [29]. Thus, Aldh3a1 is the most inactivation-resistant isoform of the three and the focus of the current study.

It is unknown whether aldehyde dehydrogenases are involved in varied susceptibility of different muscles to degeneration under ALS. In this study, we examined the expression of Aldh2, Aldh1a1 and Aldh3a1 in EDL, soleus, diaphragm and EOMs of end-stage hSOD1G93A (G93A) mice, a well-established ALS rodent model, as well as their wildtype (WT) littermates [33]. Only Aldh3a1 exhibited dramatically higher expression in EOMs compared to other muscles in WT mice. Meanwhile it was prominently upregulated in G93A soleus and diaphragm compared to WT controls. However, the upregulation was less pronounced in G93A EDL muscle, which suffers the most severe NMJ degeneration in these four muscle types in G93A mice [5]. Indeed, the commonly used denervation marker Ankrd1 [34,35] was induced most in G93A EDL muscle, suggesting that the expression level of Aldh3a1 is inversely linked to the severity of muscle pathological remodeling. The distinct expression pattern of Aldh3a1 gene was also confirmed at the protein level. In EDL and soleus muscles of WT mice with sciatic nerve transection (SNT), Ankrd1 expression was quickly elevated post operation, especially in EDL muscles and gradually decreased over time. In contrast, the upregulation of Aldh3a1 occurred with a multi-day delay in soleus, while no significant upregulation of Aldh3a1 occurred in EDL muscles even after 14 days. Thus, Ankrd1 and Aldh3a1 exhibit inverse induction pattern over muscle type and time post denervation.

Whole muscle RNA-seq analysis and pharmacological tests revealed potential mechanisms underlying the differential regulations of Aldh3a1 expression in various muscles involving Nrf2 signaling activation. Importantly, transduction of EDL and soleus-SC derived myotubes with adeno-associated virus (AAV) vector expressing human Aldh3a1 markedly decreased 4-HNE-induced DNA fragmentation. Furthermore, MG53 is a muscle specific tripartite motif family protein nucleating the assembly of the repair machinery on injured plasma membrane [36]. We previously reported abnormal MG53 intracellular aggregation and compromised membrane repair in ALS muscles [37]. Here, we demonstrated that enforced expression of Aldh3a1 in cultured myotubes protected against 4-HNE-induced plasma membrane damage and restored MG53 mediated membrane repair. Thus, the differential expression level of Aldh3a1 in muscles in response to ALS progression likely reflects a varying protective role of Aldh3a1 against reactive aldehyde cytotoxicity in different muscles.