Recent Advances in Extracellular Vesicles in Amyotrophic Lateral Sclerosis and Emergent Perspectives

Amyotrophic lateral sclerosis (ALS) is a severe and incurable neurodegenerative disease characterized by the progressive death of motor neurons, leading to paralysis and death. It is a rare disease characterized by high patient-to-patient heterogeneity, which makes its study arduous and complex. Extracellular vesicles (EVs) have emerged as important players in the development of ALS. Thus, ALS phenotype-expressing cells can spread their abnormal bioactive cargo through the secretion of EVs, even in distant tissues. Importantly, owing to their nature and composition, EVs’ formation and cargo can be exploited for better comprehension of this elusive disease and identification of novel biomarkers, as well as for potential therapeutic applications, such as those based on stem cell-derived exosomes. This review highlights recent advances in the identification of the role of EVs in ALS etiopathology and how EVs can be promising new therapeutic strategies.

two or more genes may be required for the disease [6,7]. People with a history of ALS in their family and those carrying ALS-related genes are more likely to develop the disease (familial, fALS), representing 5-10% of all cases. For the remaining 90% to 95%, the illness can occur spontaneously, without a family history (sporadic ALS) [6], and still be linked to ALS-related genes. Currently, ALS is difficult to diagnose due to the absence of a test that can solely lead to its identification unless it is a familial form. In the absence of a family history, a battery of examinations is often performed to exclude other possible pathologies. Currently, ALS remains cureless, and the available treatments are sparse and mostly palliative. Two approved medications are currently prescribed to patients, Riluzole and Edaravone, with the latter only being approved in some countries. However, they only present small benefits in delaying ALS progression, usually only by a few months [8]. Therefore, the discovery of new and effective drugs is of utmost importance.

Risk Factors for ALS Onset and Progression
The likelihood of developing ALS and its progression are influenced by numerous factors, including genetic and non-genetic origins. One important non-genetic factor is age, as individuals who develop ALS in early adulthood tend to experience slower disease progression rates [9,10]. Another factor is gender, with men being about 1.3 times more likely to develop ALS than women and earlier in life [11]. Gender also plays a role in ALS onset type, with spinal onset more common in men, while women are more likely to present bulbar onset [10]. In addition to genetic factors, exposure to certain modifiers throughout an individual's life may also contribute to the risk of developing ALS [12]. Several environmental and lifestyle factors have been identified as potential risk factors for ALS onset, including hazardous smoking habits [13], higher lipid levels [14], prolonged exposure to pollutants [15], heavy metals [16], chemicals [17], electromagnetic fields [17], a history of electric shock [18], and head trauma [19]. Other factors linked to an increased risk of ALS include military service [20], participation in professional sports [21,22], and occupations that involve repetitive physical work [12,23,24]. However, some of these factors have been contested, owing to studies with inconclusive results [25]. These factors can eventually lead to epigenetic and genomic changes that may contribute to ALS onset, such as the occurrence of C9ORF72  EVs are produced by different cell types in the central nervous system and neuromuscular junctions. In the context of ALS, EVs may carry disease-related biological molecules (proteins and miRNAs) involved in the transformation and degeneration of the brain and neuromuscular elements, thus contributing to the spread of the pathology between different cell types. Furthermore, EVs can reach long distances in the body, contributing to the exchange of harmful molecules between the brain and neuromuscular junction. Considering this, the molecules transported by EVs circulating in the bloodstream and cerebrospinal fluid are considered potential biomarkers for the diagnosis and prognosis of ALS. Finally, EVs have a therapeutic potential. Blocking the exchange of EVs carrying harmful molecules and administering EVs with neuroprotective cargo may slow the progression of ALS or revert its pathological effects (right-greenish side). ALS, amyotrophic lateral sclerosis, EV, extracellular vesicle; miRNA, microRNA. Figure created using BioRender.com (accessed on 6 June 2023).

EVs in ALS Disease Progression and Pathological Mechanisms
EVs have emerged as significant players in ALS progression, with increasing evidence pointing to their role in the dissemination of detrimental biocargo. EVs allow for the hypothetical prion-like propagation of ALS-related mutant misfolded proteins and dysregulated miRNAs [81], which are believed to contribute to disease severity and progression [127][128][129]. The most common cargos found in EVs from patients with ALS include misfolded proteins such as mSOD1, FUS, TDP43, and C9orf72 expansions DPRs and other neurotoxic elements [64,81]. These harmful cargos have been screened in both astrocytes and neuron-derived exosomes in different ALS disease models, such as the SOD1-G93A mouse model, which is one of the most commonly used animal models for studying ALS. In this model, the mutated SOD1 gene harbors a glycine-to-alanine substitution at codon 93. Recently, ref. [130] demonstrated that mutant SOD1 (mSOD1) accumulation occurs in cellular vacuoles, which may be constituted by different portions of organelles, and once released, leads to the existence of different types of EVs, particularly mitoEVs. The formation and type of vacuoles and the resulting EVs appear to be related to the stage of ALS pathology in this mouse model. Interestingly, before the onset of motor symptoms, these vacuoles are already present and are mainly of mitochondrial origin, with a high content of mSOD1, ultimately resulting in the release of mSOD1-containing EVs [130]. The authors of this study hypothesized that these EVs, derived from damaged neurons, may be responsible for the initiation of a sequence of signaling cascades that contribute to In the context of ALS, EVs may carry disease-related biological molecules (proteins and miRNAs) involved in the transformation and degeneration of the brain and neuromuscular elements, thus contributing to the spread of the pathology between different cell types. Furthermore, EVs can reach long distances in the body, contributing to the exchange of harmful molecules between the brain and neuromuscular junction. Considering this, the molecules transported by EVs circulating in the bloodstream and cerebrospinal fluid are considered potential biomarkers for the diagnosis and prognosis of ALS. Finally, EVs have a therapeutic potential. Blocking the exchange of EVs carrying harmful molecules and administering EVs with neuroprotective cargo may slow the progression of ALS or revert its pathological effects (right-greenish side). ALS, amyotrophic lateral sclerosis, EV, extracellular vesicle; miRNA, microRNA. Figure created using BioRender.com (accessed on 6 June 2023).

EVs in ALS Disease Progression and Pathological Mechanisms
EVs have emerged as significant players in ALS progression, with increasing evidence pointing to their role in the dissemination of detrimental biocargo. EVs allow for the hypothetical prion-like propagation of ALS-related mutant misfolded proteins and dysregulated miRNAs [81], which are believed to contribute to disease severity and progression [127][128][129]. The most common cargos found in EVs from patients with ALS include misfolded proteins such as mSOD1, FUS, TDP43, and C9orf72 expansions DPRs and other neurotoxic elements [64,81]. These harmful cargos have been screened in both astrocytes and neuron-derived exosomes in different ALS disease models, such as the SOD1-G93A mouse model, which is one of the most commonly used animal models for studying ALS. In this model, the mutated SOD1 gene harbors a glycine-to-alanine substitution at codon 93. Recently, ref. [130] demonstrated that mutant SOD1 (mSOD1) accumulation occurs in cellular vacuoles, which may be constituted by different portions of organelles, and once released, leads to the existence of different types of EVs, particularly mitoEVs. The formation and type of vacuoles and the resulting EVs appear to be related to the stage of ALS pathology in this mouse model. Interestingly, before the onset of motor symptoms, these vacuoles are already present and are mainly of mitochondrial origin, with a high content of mSOD1, ultimately resulting in the release of mSOD1-containing EVs [130].
The authors of this study hypothesized that these EVs, derived from damaged neurons, may be responsible for the initiation of a sequence of signaling cascades that contribute to neuroinflammation, glial-mediated neurotoxicity, and prion-like spreading of the disease. The muscle-specific expression of mutant SOD1(G93A) can also have a negative effect on neurons, as it has been reported to alter and dismantle neuromuscular junctions through a PKCθ-dependent mechanism [131]. The expression of mutant SOD1 induces the upregulation of PKCθ, and its colocalization with acetylcholine receptors (AChR) leads to a decrease in mitochondrial function, alterations in redox signaling, and neuromuscular junctions hindering transmission [131]. Moreover, and as noted in Peggion et al. [132], myocytes from hSOD1(G93A) mice are susceptible to reactive oxygen species that lead to an unbalanced mitochondrial redox state and changes in Ca 2+ homeostasis. This ultimately triggers a reactive glial response and the release of proinflammatory cytokines that affect both motor neurons and neuromuscular junctions (NMJs). To support this finding, cytokines such as IL-1b, IFN-y, and IL-6 were found in circulating EVs of the spinal cord from SOD1(G93A) mice [133]. The existence of different vacuole/EV phenotypes and associated cell death pathways may have different roles in the onset and severity of symptoms, as well as in the heterogeneity and progression of the disease.
Exosomal TDP-43 is another significant cargo that plays a crucial role in ALS progression. A longitudinal study conducted on ALS patients demonstrated an increase in the exosomal TDP-43 ratio in peripheral blood during the course of the disease, particularly in the early stages [134]. This increase in the TDP-43 ratio is associated with elevated levels of neurofilament light chain (NFL) in the plasma of these patients, which is more prevalent in individuals with rapid disease progression [134].  [135]. Although in vivo studies are required, this previous work suggests that EVs may act as vehicles for the spread of TDP-43 aggregates in the context of ALS.
EVs and their toxic payloads not only damage neurons but also spread pathological signaling by transferring them between different cell types, including neurons, astrocytes, and muscle cells. Evidence of these interactions was provided by a study showing that the EV-mediated transfer of DRPs occurred between MNs-like NSC34 cells and rat cortical neurons and, then, from those to rat cortical astrocytes [64]. This transfer is relevant to ALS, as EVs carrying C9orf72-encoded DPRs were identified to be involved in the exchange between human C9orf72-induced pluripotent stem cell-derived motor neurons (hiPSC-MNs) and control iPSC-derived spinal MNs [64]. In NSC34 cells transfected with mutant SOD1(G93A) (hSOD1-G93A NSC34 cells), miR-124 was found to be upregulated and transferred to EVs. When these cells were cocultured with N9-microglial cells, miR-124 contained in mSOD1 exosomes was translocated to N9-microglial cells, resulting in phenotypic alterations, such as a reduction in their phagocytic capability and activation of neuroinflammation pathways [136]. Exosomes released by mouse astrocytes overexpressing G93A SOD1 were also previously shown to be responsible for the transfer of mutant SOD1 to mouse spinal neurons and to induce MN death [137]. Moreover, astrocytic-derived exosomes from the plasma of sALS patients were found to transport inflammation-related cargo, including IL-6, a proinflammatory interleukin, which was increased in these vesicles and positively associated with the rate of disease progression [138]. The negative impact of EVs and their cargo on the interaction of affected muscle cells with MNs was further demonstrated by evidence that multivesicular bodies released from ALS muscle cells are neurotoxic to healthy MNs [139]. In this study, EVs derived from muscle cells obtained from biopsies of sALS patients were exposed to healthy hiPSC-MNs and were shown to be neurotoxic through increased FUS expression, resulting in shorter and less branched neurites, atrophic myotubes, and enhanced cell death [139]. The observed cell death was greatly reduced by immunoblocking the vesicle uptake by MNs with anti-CD63. Finally, a study by Anakor et al. supported the cause and effect relationship between muscle cell vesicles and MNs. The exposure of MNs to skeletal muscle cell-derived exosome-like vesicles (MuVs) in ALS patients resulted in reduced neurite length, number of neurite branches, and reduced MN survival and myotubes by 31% and 18%, respectively. Moreover, adding ALS-derived MuVs to healthy astrocytes led to an increase in the proportion of stellate astrocytes and, thus, mild activation of these cells [140].
Despite recent advances in the understanding of the role of miRNAs associated with EVs in driving the progression of ALS, this field is still in its early stages. An analysis of miRNA expression profiles suggests that the current knowledge is insufficient to predict their involvement in the pathological mechanisms of ALS [286]. In a study that analyzed the results of research from 2013 to 2018, Foggin et al. (2019) reported that most dysregulated miRNAs were either upregulated or downregulated. This outcome may be due to intrinsic differences in the methodologies used for miRNA detection or other factors, such as differences in miRNA expression across different tissues and sample extraction protocols. Interestingly, eight of the nine most commonly dysregulated miRNAs were predicted to target at least one of the most commonly mutated genes in ALS, but a random sample of unrelated miRNAs that were not found to be dysregulated in ALS patients also yielded a similar prediction [286]. Nonetheless, the search for miRNAs as potential biomarkers for ALS remains promising because of their good preservation in different types of biological samples, such as CSF and blood, often with an advantage over several proteins in allowing for a more reliable and faster diagnosis and closer classification and understanding of each case. In this scope, and as suggested by Rizzuti and colleagues [162], it is important to analyze miRNAs isolated from different human biological samples (e.g., MNs, exosomes, and CSF) in different ALS types. Likewise, miR-206 has been proposed as a potential biomarker in a study by Toivonen et al. (2014), since it displayed consistent changes towards its upregulation in ALS disease progression in SOD1 mice [287]. miR-206 is a microRNA that has been identified as a tumor suppressor involved in regulating the transforming growth factor-β (TGF-β) signaling pathway [288]. Importantly, it is one of the canonical myomiR due to its high expression in skeletal muscle [289], where it is involved in myogenesis and skeletal tissue regeneration [290][291][292]. In several studies related to ALS pathology, consistent expression levels of miR-206 have been observed across different biological samples. For example, miR-206 was found to be overexpressed in the serum of sALS patients [293] and in both plasma and skeletal muscles of spinal onset ALS patients [294]. In a study performed with the SOD1-G93A ALS mouse model, miR-206 overexpression was found to be associated with the onset of neurological symptoms, which may be attributed to skeletal muscle denervation [290]. Indeed, the downregulation of miR-206 restored neuromuscular synapses, indicating the potential of miR-206 as a therapeutic target for ALS [290]. In a recent study performed on SOD1-G93A mice, the pivotal role of miR-206 and miR-133a in skeletal muscle remodeling was evidenced [292]. This skeletal muscle remodeling by miR-133a seems to be related to myoblast proliferation by inhibiting the serum response factor (SRF) [295]. The study reported a significant decrease in the levels of both miR-206 and miR-133a in the serum of these animals 2 and 10 days after surgical-induced nerve dissection. However, after 30 days post-surgery, the miR-206 levels returned to normal, indicating its critical involvement in skeletal muscle reinnervation. Furthermore, the study found that the miR-206 expression levels in the serum could serve as an indicator of ALS disease progression, as a significant upregulation of this miRNA was observed during the late symptomatic phase of ALS (at 220 days). It is worth noting that miR-206 has been detected in EVs in ALS [275], suggesting its potential release into the bloodstream and contribution to the disease progression.
In addition to miRNAs, the protein cargo of EVs associated with ALS may also hold potential as a novel biomarker (summarized in Table 2). In a study by Vassileff and colleagues [296], 12 proteins were identified as being exclusive to EVs derived from the postmortem motor cortex tissue of ALS patients, including CD177, CHMP4B, CSPG5, DYNC1I2, IGHV3-43, LBP, RPS29, S100A9, SAA1, SCAMP4, SCN2B, and SLC16A1. Additionally, Pasetto and collaborators [297] discovered a potential new method for patient stratification based on the levels of cyclophilin A, a protein involved in TDP-43 trafficking and function, in combination with the EV size distribution in plasma-derived EVs from ALS patients. This approach can be used to distinguish between slow and fast disease progression. Recently, Sjoqvist and Otake [298] conducted a pilot study comparing CSF and CSF-EVs from patients with ALS and matched control subjects to search for novel ALS biomarker candidates. They found four differentially expressed proteins in the CSF of ALS patients, including downregulated MB and upregulated JAM-A, TNF-R2, and CHIT1. Although no proteins were differentially expressed in CSF-EVs, there was a trend for the downregulation of perlecan, a proteoglycan of the extracellular matrix involved in cell proliferation, differentiation, adhesion, migration, and tissue repair and regeneration [299]. Conversely, Thompson et al. (2020) found no significant differences in CSF-EV concentration and size distribution between the control and ALS groups. However, they identified altered protein homeostatic mechanisms in ALS patients, including the downregulation of bleomycin hydrolase [300], a cytosolic cysteine protease that has been associated with the release of chemokines in inflammation and wound healing processes [301]. These data, together with those indicating the involvement of EVs in aggregated protein spreading, suggest that the analysis of EV protein contents is mandatory for the development of innovative diagnostic and prognostic tools and for the identification of new therapeutic targets for ALS. Overall, while these studies have provided promising results, further research is needed to understand the role of miRNAs and proteins transported by EVs in ALS development and progression and their possible use as biomarkers.

HSP90
Chaperone protein involved in protein folding [307] EVs from ALS patients and symptomatic SOD1G93A and TDP-43Q331K ALS mice models plasma Downregulation in EVs from sALS patients [297] JAM-A Regulation of several processes including paracellular permeability, platelet activation, angiogenesis and the modulation of junctional tightness in the blood brain barrier (BBB) [308] EVs from ALS patients CSF

MB
Oxygen-binding molecule expressed in skeletal and cardiac muscle tissue [310,311] EVs from ALS patients CSF Downregulation in ALS patients CSF-derived EVs [298] mfSOD1 Antioxidant enzyme, protects cells from ROS [312] Vacuoles and EVs from spinal cord samples of SOD1 G93A mice model Accumulation of mfSOD1-vacuoles in degenerating MNs, released into the extracellular space in the form of extracellular vesicles [130] NIR Translocates from the nucleolus to the nucleoplasm in response to the nucleolar stress [313] Exos from sALS patients CSF and anterior horn postmortem tissue sections Upregulation in sALS patients CSF-derived exos [314] Nrf2 Antioxidant factor [315] EVs from spinal cord tissue of SOD1 G93A mice model Upregulation after exposure to MSCs-derived EVs, with consequent reduction of ROS [316] pCREB Involved in the synthesis of proteins required for LTP [317] Exos from SOD1 G93A mice model SVZ-derived NSCs, differentiated into G93A neuronal cells Downregulation in G93A cells, normalized with ADSC-exos treatment [318] PGC-1α Involved in the regulation cell metabolism [319] Exos from SOD1 G93A mice model SVZ-derived NSCs, differentiated into G93A neuronal cells Downregulation levels in G93A cells, normalized with ADSC-exos treatment [318] Phenylalanine Precursor for tyrosine [320], the monoamine neurotransmitters dopamine, norepinephrine, and epinephrine lEVs and sEVs from sALS patients' plasma Downregulation in EVs from sALS patients [321] pMLKL Effector of necroptotic pathways [322] Vacuoles and EVs from spinal cord samples of SOD1 G93A mice model

Therapeutic Perspectives with EVs in ALS
In recent years, several studies have proposed innovative next-generation EV-related therapies that hold great promise for the treatment of human diseases [329]. EVs present several therapeutic advantages owing to their higher biocompatibility and reduced immunogenicity compared to alternative carriers, such as synthetic nanocarriers, which may also be prone to accumulation in the liver and spleen [330,331]. As natural nanoparticles, EVs can be easily isolated from various biofluids and can cross biological barriers to deliver potential therapeutics [332]. Despite some uncertainty regarding their functional mechanisms, it is becoming clear that these bioparticles shuttle diverse cargos capable of recapitulating the benefits of "whole-cell therapy", either by preventing or mitigating abnormal cellular functions [333].
One emerging therapeutic approach related to EVs is the use of stem cell-derived EVs. These EVs have a positive impact on the pathophysiology of various neurodegenerative diseases [334]. In ALS, these EVs may achieve beneficial effects by modulating the immune system, addressing mitochondrial dysfunction, and boosting neuroprotection in MNs [335][336][337]. For example, exosomes derived from adipose-derived stem cells (ASCs) obtained from SOD1-G93A mice were shown to have a neuroprotective effect by reducing oxidative stress-related damage in MN-like NSC-34 cells overexpressing ALS-associated mutations, including SOD1(G93A), SOD1(G37R), and SOD1(A4V) [336]. Furthermore, the same research group observed that NSC-34(G93A) cells internalized ASC-derived exosomes, leading to the downregulation of proapoptotic proteins (Bax and cleaved caspase-3) and the upregulation of antiapoptotic proteins (Bcl-2α), ultimately improving neuronal survival [338]. In a more recent study, ASC-derived exosomes obtained from SOD1-G93A mice were used to slow the progression of ALS by reducing glial cell activation and improving motor performance. Interestingly, these exosomes showed an affinity towards the lesioned areas of the brain, suggesting targeted delivery, although the exact mechanisms behind this phenomenon require further elucidation [339]. Similarly, ASC-derived exosomes were found to increase the expression levels of phospho-CREB/CREB and PGC-1α in neurons derived from the neural stem cells of SOD1-G93A mice. This results in a reduction in cytosolic SOD1 aggregates and rescues mitochondrial dysfunction [318]. Additionally, the same type of exosomes was shown to rescue the inherent impairment in oxidative phosphorylation (OXPHOS) specifically linked to mitochondrial complex I in NSC-34(G93A) cells [340]. In their study, human bone marrow endothelial progenitor cell (hBM-EPC)-derived exosomes were shown to restore mouse brain endothelial cells previously damaged through in vitro exposure to SOD1-G93A mutant male mouse plasma. These results indicated a significant reduction in microvascular endothelial damage. Interestingly, blocking the β1 integrin of exosomes using an anti-CD29 blocking antibody prevented their internalization by recipient cells, thereby increasing the percentage of brain endothelial cell death. These findings suggest that hBM-EPC-derived exosomes have the potential to repair endothelial damage in ALS and that their internalization by recipient cells may play a critical role in their therapeutic effects. Garbuzova-Davis et al. (2020) investigated the potential therapeutic role of exosomes derived from human hBM-EPCs in the repair of endothelial damage in ALS. To induce damage, researchers exposed a mouse brain endothelial cell line to plasma from SOD1-G93A male mice. They found that plasma-derived exosomes treatment significantly increased endothelial cell death. However, a significant reduction in cell death was obtained by supplementing the brain endothelium, previously exposed to ALS plasma-derived exosomes, with 1 µg/mL of hBM-EPC-derived exosomes for 24 h. Moreover, when these EVs were pretreated with an anti-CD29 blocking antibody to block β1 integrin, they were prevented from being internalized by recipient cells, resulting in a significant increase in brain endothelial cell death. These findings suggest that hBM-EPC-derived exosomes have the potential to reduce the number of damaged endothelial cells in ALS, but their beneficial effects may be dependent on proper cellular internalization [341]. In contrast, the negative effects of ALS-related EVs were reversed by Varcianna et al. (2019). The authors isolated EVs from human-induced astrocytes derived from C9ORF72-ALS sALS patients (C9ORF72-ALS iAstrocyte-derived EVs) and found that they originally compromised both neurite network maintenance and MN survival in HB9-GFP+ mouse-cultured MNs (Hb9-GFP + MNs). This effect was related to the downregulation of the miR-494-3p content in these EVs. Nevertheless, following treatment with C9ORF72-ALS iAstrocyte-derived EVs, where the miR-494-3p levels were intentionally upregulated, HB9-GFP + mouse-cultured motor neurons presented neurite network restoration and decreased MN death. These beneficial effects of miR-494-3p overexpression may be related to its function as a negative regulator of semaphorin 3A (SEMA3A) and other targets involved in axonal maintenance [205].
In addition to the described therapeutic possibilities, EVs have emerged as promising drug carriers with the potential to deliver synthetic drugs to the brain. This is especially important because many proteic and small-molecule neurological drugs may fail to bypass the blood-brain barrier (BBB), which can hinder their effectiveness [342]. The encapsulation of these drugs within EVs could help overcome this limitation by allowing them to cross the BBB and improve drug targeting and efficiency [343]. While EVs are not currently used to deliver drugs for ALS treatment, they have been employed in the treatment of other brain diseases, such as brain tumors, using doxorubicin-loaded exosomes [344]. Therefore, this approach may also be a promising avenue for future ALS research.
Although previous findings demonstrated promise for the potential application of EV-based therapies in ALS, it is important to acknowledge the existing challenges and limitations in this field, which align with the obstacles encountered in ALS research. The intricate nature of ALS, coupled with the substantial heterogeneity among patients, adds to the complexity. Moreover, obtaining patient samples that involve brain and nervous tissue poses invasive methodologies, making it arduous. Consequently, researchers resort to animal models or in vitro systems. However, these alternatives have inherent limitations and fail to capture the full disease complexity and phenotypic variations across the entire population, thereby lacking translation to real clinical scenarios. Innovative approaches involving EVs, like genome therapy [345] or immunomodulation, hold the potential to establish more robust foundations for advancing ALS therapeutics. An intriguing avenue for investigation lies in harnessing EVs in conjunction with CRISPR/CAS9 gene editing technologies [346,347]. It is worth noting that, to the best of our knowledge, no clinical studies have been conducted or proposed involving the use of EV-based therapies in ALS with human patients, which demonstrates that research in this field is strongly needed.

Conclusions
ALS is a fatal neurodegenerative disease with a complex and unclear etiopathology that strongly affects patient health and well-being. With no cure available thus far, searching for an effective treatment that can improve patients' life expectancy and quality of life is paramount. ALS presents several important challenges and hurdles to the research because of the intrinsic complexity and heterogeneity of the disease. Nevertheless, important advances have been made in recent years. Among these are the recent advances in ALSrelated EV research, which are emerging as key players in the surfacing and development of the disease by allowing for the transport of biomolecular cargo from cell to cell, thus spreading anomalies across the system. EVs also have the potential to be employed as a source of potential biomarkers for the early detection of ALS and personalized prognostic purposes. Furthermore, they may also be exploitable to tackle existing altered mechanisms and for applications in a variety of therapeutic strategies, such as being employed for drug delivery, as they can carry different types of molecules, both natural and artificial. Specifically, within the ALS research area, stem cell-derived EV use is increasing for therapeutic purposes, which is of higher relevance given the disease heterogeneity and allows for a precision-based approach.
While the use of stem cell-derived EVs for therapeutic purposes is promising, further innovative and consensual approaches are needed to reverse the disease's biopathologic mechanisms and translate this knowledge into real-life applications that can bring hope to both patients and their families.