Disruption of neuronal actin barrier promotes the entry of disease-implicated proteins to exacerbate amyotrophic lateral sclerosis pathology

Amyotrophic lateral sclerosis (ALS) is a devastating neurological disease characterized by the accumulation of aberrant proteins in motor neurons of the brain and spinal cord. Patients with ALS develop skeletal muscle weakness, resulting in death from respiratory paralysis, which usually occurs 2–4 years after clinical onset (Goutman et al., 2022). Although the precise pathological mechanisms remain elusive, several processes, including aberrant ribonucleic acid metabolism, altered proteostasis/autophagy, mitochondrial dysfunction, and compromised DNA repair, are reportedly involved in the onset and progression of ALS. Recently, increasing evidence suggests that disruption of cytoskeletal dysregulation could be attributed to ALS (Goutman et al., 2022). Profilin-1, an actin monomer-binding protein essential for the regulation of actin polymerization, has been identified as a causative gene of familial ALS. Profilin-1 mutations have been reported to result not only in a decreased F-actin arrangement (Fil et al., 2017) but also in altered stress granule dynamics and TAR DNA-binding protein of 43kD (TDP-43) aggregation (Fil et al., 2017). Transgenic mice with mutated profilin-1 not only recapitulate paralysis and motor neuron degeneration similar to ALS, but also show TDP-43 aggregation pathology, suggesting that actin dynamics are intrinsically involved in the pathogenesis of ALS. Cofilin, which is essential for actin depolymerization, has also been associated with the pathology of ALS. A previous report showed that cofilin is associated with the reduction of F-actin in induced pluripotent stem cell-derived motor neurons in patients with ALS with GGGGCC intronic repeat expansion in C9orf72, a common genetic form of familial ALS. Therefore, disruption of actin dynamics, including alterations in profilin-1 and cofilin, has been implicated in the pathogenesis of ALS, contributing to motor neuron degeneration and disease progression. In this perspective, we will focus on the role of actin dynamics as a neuronal barrier inhibiting aberrant protein deposition. Additionally, we will discuss on the potential of axon guidance molecules regulating actin dynamics as a novel therapeutic target.

Actin barrier collapse promotes the uptake of extracellular pathogenic proteins: Most neurodegenerative diseases are characterized by the accumulation of misfolded proteins in insoluble aggregates in the central nervous system, accompanied by a progressive loss of neurons in the affected regions (Peng et al., 2020). Protein aggregates involved vary between diseases; for example, amyloid-beta and tau aggregates in Alzheimer’s disease, misfolded alpha-synuclein in Parkinson’s disease, tau accumulation in frontotemporal dementia, and TDP43 and superoxide dismutase 1 (SOD1) pathology in ALS. The toxic effects of these protein aggregates on the central nervous system and the molecular mechanisms underlying the resulting neuronal dysfunction have been investigated. Interestingly, recent studies have suggested that many neurodegenerative disease-related pathological proteins undergo cell-to-cell transmission, and this propagation drives the progression of the disease pathology. In fact, some previous reports showed that the administration of anti-human SOD1 antibody improved disease symptoms, prolonged their lifespan, and reduced the aggregation of misfolded SOD1 protein and motor neuron degeneration in mSOD1 mice (Maier et al., 2018). These results suggest that the removal of extracellular SOD1 induced interference with the prion-like spread of SOD1 aggregates. Moreover, in a study determining the structure of TDP-43 aggregates extracted from the frontal and motor cortices of two ALS patients with frontotemporal lobar degeneration using electron cryo-microscopy, identical TDP-43 structures were observed in both brain regions and individuals. The detection of the same TDP-43 structure in spatially distant regions suggests that TDP-43 filaments act as seeds, inducing seed-dependent aggregation of TDP-43 in a self-templating manner (Arseni et al., 2022). These results reveal that extracellular SOD1 and TDP-43 released from dying cells or living cells induced prion-like spread of aggregates.

Endocytosis, modulated through actin dynamics, promoted the uptake of extracellular molecules, which served as the key mechanism of propagation (Zhong et al., 2018). Additionally, some previous reports showed that endocytosis is modulated by axon guidance molecules, such as semaphorin 3A, which alter actin dynamics (Kabayama et al., 2011). Furthermore, dephosphorylated cofilin, which is essential for actin depolymerization, results in actin disassembly and impairs the actin barrier to exploit the entry of protein aggregates, such as mutated SOD1, tau, and alpha-synuclein (Zhong et al., 2018). These results suggest that actin depolymerization, leading to the collapse of the actin barrier, could promote the uptake of extracellular pathogenic proteins, such as SOD1 and TDP-43, and could be involved in the propagation of ALS pathology.

Axon guidance molecule: Axon guidance proteins were originally identified as instructive signals for guiding embryonic axons. Specialized receptor proteins at the growth-cone-cell surface detect axon guidance proteins and trigger intracellular signaling cascades that cause axon steering through the induction of changes in the growth-cone cytoskeleton, such as actin or tubulin, to guide them toward or away from specific regions of the developing brain or embryo. Researchers have identified five families of canonical guidance proteins: semaphorins, netrins, slits, repulsive guidance molecules, and ephrins.

Repulsive guidance molecule A (RGMa) is expressed not only in neurons but also in glial cells and is shed from the cell surface. The ectodomain of RGMa provides three forms of soluble fragments that exert their functions. These three forms consist of N1, N2, and C variants, the last of which contains a C-terminal domain capable of binding to NEO1 or a receptor for RGMa and activating downstream signaling cascades. The binding of RGMa to NEO1 induces stabilization and dimerization of its ectodomain to activate downstream signaling cascades, one of which is the regulation of small guanosine triphosphatases (GTPases), such as Ras, Rho, and Rac, thus controlling actin dynamics (Siebold et al., 2017). RGMa was originally identified as an axon guidance molecule in the visual system of the embryo (Monnier et al., 2002), but in adults, it plays various roles in physiological and pathological conditions, including axon guidance through alteration of actin dynamics, T cell activation, apoptosis induction, synapse formation, and angiogenesis (Figure 1).

Figure 1.

The RGMa/NEO1 axis plays multiple roles to various targets.

The binding of RGMa to NEO1 induces stabilization and dimerization of its ectodomain to activate downstream signaling cascades, one of which is the regulation of small GTPases such as Ras, Rho, and Rac, thus controlling actin dynamics. Moreover, RGMa plays various roles in physiological and pathological conditions through not only the alteration of actin dynamics but also repulsive axon guidance, T cell activation, apoptosis induction, synapse formation, and angiogenesis. This figure was generated originally by the authors based on the cited literature (Monnier et al., 2002; Siebold et al., 2017; Nakagawa et al., 2019; Jacobson et al., 2023). Created with BioRender.com. GTP: Guanosine triphosphate; NEO1: neogenin1; RGMa: repulsive guidance molecule A.

These multifunctional roles of RGMa have been demonstrated in both physiological and pathological conditions of the nervous system. For example, neutralizing antibodies against RGMa promoted recovery from impaired manual dexterity in monkey models of spinal cord injury by promoting the sprouting of corticospinal tract fibers from the motor cortex and improved functional recovery in a rat spinal cord injury model through axonal growth of the corticospinal tract (Nakagawa et al., 2019). Additionally, a human anti-RGMa monoclonal antibody, elezanumab, ameliorated neuromotor function and modulated neuroinflammatory cell activation in a rabbit stroke model (Jacobson et al., 2023). Furthermore, RGMa is highly expressed in CD4⁺ T cells in experimental autoimmune encephalomyelitis mice, an animal model of multiple sclerosis, and therapeutic inhibition of RGMa improved the clinical symptoms of diseased mice. Importantly, RGMa upregulation was reported in an animal model of neurodegenerative diseases, such as Parkinson’s disease, in which anti-RGMa antibodies have therapeutic potential. These reports suggest that RGMa could be a therapeutic target for neurological disorders. However, its role in ALS remains unclear. Therefore, our study revealed the relationship between RGMa and ALS from the viewpoint of actin dynamics (Shimizu et al., 2023).

Roles of RGMa roles in the pathology of ALS: Our study first revealed that RGMa levels were elevated in cerebrospinal fluid from patients with ALS, which correlates with disease severity, suggesting its potential as a diagnostic and prognostic biomarker. Specifically, variant C of RGMa, capable of activating the NEO1 signaling pathway, was increased in the cerebrospinal fluid of patients with ALS, accompanied by aberrant localization of NEO1 in motor neurons of the autopsied ALS spinal cord.

These findings were mirrored in mSOD1 mice, an ALS model. Elevated RGMa levels were found in the cerebrospinal fluid of mSOD1 mice, and NEO1 expression was upregulated in anterior horn cells of mSOD1 mice, indicating RGMa/NEO1 axis dysregulation in ALS model mice. Treatment with anti-RGMa antibodies in mSOD1 mice extended lifespan, preserved motor function, and reduced protein aggregation. Immunohistochemical analysis, quantitative polymerase chain reaction analysis, and western blot assay of the spinal cord of mSOD1 mice revealed that anti-RGMa treatment did not show any effect on astrogliosis, microgliosis, or neuroinflammation, but played a key role in the promotion of cofilin phosphorylation and actin polymerization. Moreover, western blotting analysis showed that Smad1, Smad2, and Smad3 phosphorylation did not change among wild-type and mSOD1 mice treated with anti-RGMa and control antibodies. NEO1 cleavage, which was induced upstream of LIM domain only 4 activation and regulates apoptosis induction, was also not observed in wild-type or mSOD1 mice. Additionally, immunohistochemical analysis of the gastrocnemius muscle revealed that anti-RGMa antibodies did not affect the number of innervated neuromuscular junctions in both groups, suggesting that anti-RGMa antibody treatment did not have a positive impact on NMJ of mSOD1 mice. These results suggested that anti-RGMa therapy stops neurodegeneration in ALS by reinforcing actin polymerization through cofilin phosphorylation.

Furthermore, in vitro experiments showed that RGMa facilitated the entry of extracellular pathogenic proteins into neurons through the RGMa/NEO1 axis, suggesting its role in the progression of ALS by compromising the actin barrier through cofilin dephosphorylation (Shimizu et al., 2023). Overall, these results underscore the therapeutic potential of targeting the RGMa/NEO1 axis in ALS by modulating actin dynamics to prevent disease progression and reduce protein aggregation (Figure 2).

Figure 2.

The RGMa/NEO1 axis plays a pivotal role in promoting pathogenic protein uptake via the collapse of the actin barrier.

(A) The RGMa ectodomain shed from the cell surface binds to pre-clustered NEO1, leading to stabilization and dimerization of the NEO1 ectodomain, and subsequently activates downstream signaling, such as the small GTPase cascade. (B) The small GTPase cascade dephosphorylates cofilin and enhances actin depolymerization. (C) Actin depolymerization induces the collapse of the actin barrier and promotes the entry of extracellular pathogenic proteins, leading to an exacerbated neurodegeneration. This figure was generated originally by the authors based on the cited literature (Shimizu et al., 2023). Created with BioRender.com. GTP: Guanosine triphosphate; NEO1: neogenin1; RGMa: repulsive guidance molecule A.

Conclusion and perspectives: This work demonstrated that the RGMa/NEO1 axis plays an important role in the collapse of the actin barrier to promote the propagation of pathogenic proteins in ALS, serving not only as a diagnostic and prognostic biomarker but also as a potential therapeutic target. Findings from this perspective paper reveal that the disruption of the neuronal actin barrier induced by axon guidance molecules promotes the cell-to-cell transmission of pathogenic proteins and exacerbates the pathology of the disease. This mechanism may be common not only in ALS, but also in other neurodegenerative diseases attributed to protein aggregation. Therefore, future work could focus on whether therapies targeting actin dynamics with axonal guidance factors, such as RGMa, can improve other diseases, including Alzheimer’s disease, Parkinson’s disease, and frontotemporal dementia.

This work was supported in part by the JSPS KAKENHI (grant number 22K07539 to MS). TO is funded by Mitsubishi Tanabe Pharma Corporation.

Presentation at a meeting: Annual Meeting of the Japanese Society of Neurochemistry, Kobe, Hyogo, Japan, July 6–8, 2023.

Additional file: Open peer review report 1 ^{(78.7KB, pdf)} .