MicroRNA-Ferroptosis-Spinal Cord Injury: A Complex Interplay in Neurodegeneration and Repair

1. Introduction

Spinal cord injury (SCI) is a devastating insult to the central nervous system (CNS) that results in profound and often permanent loss of sensory, motor, and autonomic function below the level of injury [1]. This injury imposes severe functional deficits and immense financial and psychological burdens on affected individuals and society [2]. Figure 1 shows the pathophysiology of SCI is characterized by a biphasic process: the primary injury involving direct mechanical damage, followed by a secondary injury cascade of biochemical and cellular events that exacerbate initial damage over time [3].

The hallmarks of SCI include neuronal loss, axonal degeneration, reactive gliosis, and a pronounced neuroinflammatory response [4]. A significant challenge in treating SCI lies in the CNS's inherently limited regenerative capacity, making spontaneous neuronal recovery rare after injury [5]. This lack of intrinsic repair mechanisms underscores the need to explore novel therapeutic strategies that protect the remaining neural tissue or actively promote the survival and regeneration of damaged neurons.

MicroRNAs (miRNAs) are a class of small, non-coding RNA molecules, typically 20-24 nucleotides in length, that play a critical role in the post-transcriptional regulation of gene expression [7]. They exert their regulatory effects by binding to the 3'-untranslated region (3'-UTR) of target messenger RNAs (mRNAs), leading to either the inhibition of protein translation or the degradation of the mRNA transcript [7]. These small RNA molecules are involved in a wide array of biological processes that are highly relevant to SCI pathology, including inflammation, oxidative stress, apoptosis, and neurodevelopment [8]. Given their broad regulatory roles in processes critical to SCI, miRNAs are recognized as key players in the disease and potential therapeutic targets.

Ferroptosis represents a distinct form of regulated cell death, characterized as non-apoptotic and dependent on iron, with a primary feature being the peroxidation of lipids [9,10]. Key factors in this cell death mechanism include reliance on ferric ions, accumulation of reactive oxygen species (ROS), iron overload, and lipid peroxidation [2,11]. Morphologically, cells undergoing ferroptosis exhibit characteristic changes such as mitochondrial shrinkage, increased membrane density, and a reduction in or absence of mitochondrial cristae [12]. The process of ferroptosis is tightly regulated by key molecules, including glutathione peroxidase 4 (GPX4) and ferroptosis suppressor protein 1 (FSP1), which function to inhibit this form of cell death [13]. Fsp1, previously referred to as apoptosis-inducing factor 2 (Aifm2), has been highlighted in studies [14] and [15] as a key component that works in conjunction with GPX4. Given its unique mechanisms and morphological features, which are distinct from those of other cell death pathways, ferroptosis represents a specific and potentially crucial target for therapeutic intervention in SCI.

Emerging research suggests a potential interplay between miRNAs and ferroptosis in the context of SCI, indicating that these two biological processes may be interconnected and contribute to both neurodegeneration and the potential for repair following SCI. This review aims to explore this complex interaction, synthesizing current findings from various research articles to provide a thorough understanding of the existing knowledge in this field. The scope of this report includes an in-depth analysis of the molecular mechanisms involved in this interplay, the evidence supporting their interaction in the context of SCI, and the therapeutic implications of targeting this axis for the treatment of SCI.

2. Extracellular Vesicles, MicroRNA, and Ferroptosis

Extracellular vesicles (EVs) have emerged as critical, highly coordinated vehicles for cell-to-cell communication within the CNS [16]. These membrane-bound structures encapsulate diverse bioactive molecular payloads, including genomic DNA, messenger RNAs (mRNAs), microRNAs (miRNAs), structural lipids, and signaling proteins [17].By serving as protective envelopes, EVs transport these regulatory molecules safely through the extracellular space to recipient cells. miRNAs are critical regulators of fundamental cellular processes, including proliferation, differentiation, signaling, cell growth, and cell death (Figure 2).

Among the distinct subpopulations of EVs, exosomes nanosized vesicles ranging from approximately 30 to 150 nm in diameter—play highly specialized roles in maintaining neural tissue homeostasis and coordinating multicellular responses to pathological stress [18]. Secreted dynamically by neurons, astrocytes, microglia, and oligodendrocytes, exosomes cross biological barriers to modulate synaptic plasticity, direct immunomodulatory pathways, stimulate local angiogenesis, and promote neurogenesis [19]. Under physiological conditions, their cargo carefully regulates critical signaling cascades required for neural development. However, following trauma, these exosomal communication networks shift, often delivering altered instructions that modulate inflammation, autophagy, oxidative stress, and cell death pathways within the surrounding injury microenvironment [20].

MicroRNAs transferred via EVs act as post-transcriptional master regulators of recipient cell transcriptomes. Under standard physiological constraints, stable baseline miRNA expression is essential for orchestrating vital cellular processes, including proliferation, structural differentiation, and cell survival [21]. Conversely, abnormal variations in miRNA synthesis, sorting, or activity are fundamentally linked to the development and progression of diverse neurodegenerative disorders and systemic diseases [21].

Mechanistically, once an EV or exosome fuses with internalized by a recipient cell, its regulatory miRNA cargo is released directly into the cytoplasm [22]. These mature single-stranded miRNAs track and bind sequence-specifically to the 3′-untranslated regions (3′-UTRs) of complementary target messenger RNAs (mRNAs) [23]. Depending on the degree of sequence complementarity, this binding recruits the RNA-induced silencing complex (RISC), resulting in either translational repression or direct mRNA degradation [22]. Because a single miRNA can target multiple genes across interconnected metabolic pathways, exosomal miRNA delivery offers a powerful mechanism to simultaneously silence detrimental pro-death machinery or upregulate protective survival networks within injured tissues (Figure 2).

Importantly, research indicates that the abnormal expression of miRNAs is associated with the development and progression of many diseases [24,25]. Therefore, modulating the production and activity of these miRNAs holds potential for therapeutic intervention in a wide range of diseases. The cellular response to oxidative stress is a crucial factor in determining cell fate. ROS, oxygen-derived free radicals generated during metabolism, can induce lipid peroxidation, ultimately leading to ferroptosis [26]. Ferroptosis is a distinct, iron-dependent cell death characterized by excessive lipid peroxidation caused by agents like ROS, erastin, and RAS-selective lethal compound 3 (RSL-3). It occurs when the cellular antioxidant system is insufficient to counteract the increased peroxidation, activating genes and pathways that promote ferroptosis [27,28]. Recognizing that EVs encapsulate various molecules, including miRNAs, which are important regulators of cellular functions to the connection between miRNAs present in EVs and pyroptosis, another form of cell death [29]. In a study by Xia et al., EVs from adipose-derived stem cells (ADSCs) overexpressing miR-19b-3p were shown to reduce ferroptosis and neuronal damage following intracerebral hemorrhage. In essence, EVs loaded with regulatory miRNAs play a critical role in mediating intercellular communication. Given that miRNAs regulate key cellular functions and their dysregulation contributes to disease pathogenesis, they represent promising therapeutic targets.

3. Role of MicroRNAs in Pathways Related Spinal Cord Injury-Induced Ferroptosis

Numerous miRNAs show altered expression levels in animal models following SCI [30]. These changes are not static; certain miRNAs are upregulated, downregulated, or show bidirectional changes at different time points after injury [31]. This dynamic regulation suggests that miRNAs are involved in various phases of the injury and repair processes, highlighting the importance of considering the temporal context when exploring them as therapeutic targets.

Several specific miRNAs have been identified as playing critical roles in the pathological events that unfold following SCI. In the context of inflammation and immune responses, miRNA-543-5p and miRNA-940 have been shown to regulate inflammation [32,33]. Notably, miRNA-155 acts as a pro-inflammatory miRNA, contributing to the M1 polarization of microglia, a phenotype associated with the release of pro-inflammatory cytokines [34]. Conversely, the downregulation of miRNA-24-3p after SCI has been observed, and its upregulation or the knockdown of MAPK-activated protein kinase 2 (MK2) has been shown to reduce microglial cell activation and the expression of inflammatory cytokines such as TNF-α and IL-1β [3]. Furthermore, miRNA-146a is upregulated following spinal cord contusion [3], and miR-92a is induced in inflamed spinal cord neurons, where it targets cytoplasmic polyadenylation element-binding protein 3 (Cpeb3) [35].

Additionally, miRNA-125a and miRNA-216a-5p regulate the polarization of microglia towards the M1 or M2 phenotype, thereby inhibiting the overall inflammatory response after SCI [12]. miR-129-5p has also been shown to alleviate SCI by suppressing inflammation through the High-Mobility Group Protein B1 (HMGB1)/Toll-like receptor 4 (TLR4)/Nuclear factor-κB (NF-κB) pathway [36]. These findings demonstrate that specific miRNAs act as key regulators of the inflammatory response following SCI, presenting potential targets for modulating neuroinflammation and its detrimental effects (Table 1).

Oxidative stress represents another critical, destructive component of the secondary injury cascade in SCI, and altered miRNA profiles have been consistently linked to this process [3]. Specifically, miRNA-99a and miRNA-672-3p have been shown to regulate oxidative stress induced after SCI in rat models [40,41]. This evidence suggests that specific miRNAs actively participate in the complex cellular redox imbalance that occurs post-SCI, directly influencing the accumulation of damaging ROS.

Furthermore, miRNAs are intricately involved in the regulation of various forms of cell death after SCI, including apoptosis and ferroptosis [3]. For instance, miRNA-9-5p and miRNA-488 are involved in the regulation of neuronal apoptosis [42], and miR-21 has been shown to regulate both proliferation and apoptosis in neural progenitor cells [43]. Notably, miRNA-124 has demonstrated the ability to suppress apoptosis of neuronal cells, leading to improved functional recovery [44]. In contrast, miRNA-372 has been implicated in inhibiting SCI recovery, potentially through its involvement in inflammatory signaling [45]. These findings highlight that miRNAs are crucial regulators of cell death pathways following SCI, making them attractive targets for neuroprotective strategies.

Beyond inflammation and cell death, miRNAs also play a role in the structural remodeling of the spinal cord after injury, influencing axonal regeneration and the formation of the glial scar [5]. For example, miR-219 has the potential to promote the differentiation of oligodendrocytes, which are essential for the process of remyelination [46]. This indicates that miRNAs can affect the structural changes occurring in the SCI, impacting the regeneration of nerve fibers and the glial scar, which can either facilitate or impede recovery depending on its characteristics.

Given their specific actions and involvement in these key pathological processes, miRNAs represent promising potential therapeutics for the treatment of SCI. The concept of miRNA replacement therapy aims to deliver miRNAs to diseased cells to promote repair processes [5]. However, the clinical translation of miRNA-based therapies faces significant challenges, including the need to optimize dosages, delivery mechanisms to the target site in the spinal cord, and to ensure long-term safety profiles.

3.1. MicroRNA Landscape in SCI: Recent Advances

The miRNA landscape in SCI is highly dynamic, with expression profiles shifting throughout acute and chronic stages [20]. Beyond well-known regulators, recent research has identified new candidates:

1. miR-5627-5p: Delivered via MSC-derived EVs, this miRNA inhibits neuronal ferroptosis through the miR-5627-5p/FSP1 axis, enhancing cellular resistance to oxidative death [47].

2. miR-9-3p: Recently identified in cerebrospinal fluid (CSF) EVs as a biomarker and therapeutic target that adapts neuronal energy metabolism and reduces stress responses post-SCI [48].

3. miR-133b: A gold standard for promoting axonal regeneration by targeting the RhoA pathway. Recent trials using miRNA-loaded EVs at 200 μg dosage showed optimal functional recovery in spinal cord contusion models [49].

4. Ferroptosis as a Critical Factor in Spinal Cord Injury

Ferroptosis, a unique form of regulated cell death, is governed by a complex interplay of several key mechanisms. Iron dependency is a central characteristic, where iron overload can enhance lipid peroxidation and ROS production, potentially triggering ferroptosis [13]. Following SCI, iron derived from red blood cells and damaged cells accumulates at the injury site [50]. This iron, particularly within the labile iron pool (LIP), can participate in the Fenton reaction, generating highly reactive hydroxyl radicals that initiate lipid peroxidation [51]. The homeostasis of iron within cells is tightly controlled by proteins such as transferrin receptor (TfR) and ferritin, and this regulation is influenced by iron regulatory proteins (IRPs) [51].

SCI can cause brain iron buildup, activating microglia via NF-κB, which releases inflammatory substances that damage neurons and contribute to central pain. Reducing iron or inhibiting NF-κB/microglia might ease SCI-induced central pain [52]. In Feng et al. studied the activated microglia in the motor cortex cause iron overload in motor neurons, triggering ferroptosis and impeding motor recovery following SCI [53]. Targeting microglial activation and iron overload presents potential therapeutic strategies for SCI [53].

A primary driver of ferroptosis is the excessive accumulation of intracellular lipid peroxides. Polyunsaturated fatty acids (PUFAs) present in cell membranes are particularly vulnerable to this process [13]. Enzymes, such as acyl-CoA synthetase long-chain family member 4 (ACSL4) and lipoxygenases (LOXs), play a role in promoting this lipid peroxidation [54]. The regulation of ferroptosis involves key proteins. GPX4, a critical antioxidant enzyme, protects cells from ferroptosis by reducing lipid peroxides, utilizing glutathione (GSH) as a necessary cofactor [55]. FSP1 provides an alternative, glutathione-independent pathway to inhibit ferroptosis by reducing ubiquinone (CoQ10) [56]. The intricate balance between iron metabolism, lipid peroxidation, and these antioxidant defense mechanisms ultimately determines the susceptibility of cells to ferroptosis, offering multiple potential targets for therapeutic intervention in SCI.

A substantial amount of evidence supports the involvement of ferroptosis in the pathogenesis of SCI, derived from both in vitro and in vivo research [2]. The presence of iron overload, the accumulation of ROS, and lipid peroxidation, all hallmarks of ferroptosis, have been consistently observed in the context of SCI [13]. Ferroptosis is considered a major contributor to the secondary injury that occurs after the initial spinal cord trauma [57]. Studies have indicated that ferroptosis occurs in distinct phases following SCI, with an acute phase occurring within the first two days post-injury and a subacute phase extending from three to fourteen days after the injury [58]. Table 2 summarizes the temporal profile of ferroptosis after SCI, including key molecular events and biomarkers observed at different post-injury stages. The temporal profile suggests that ferroptosis plays a critical role in the early stages of secondary damage.

The process of ferroptosis significantly contributes to cell death following SCI, impacting both neurons and oligodendrocytes [61]. The ferroptosis-induced mitochondrial atrophy and functional decline further disrupt the intricate electrical and chemical signaling processes within neurons [62]. Given the significant role of ferroptosis in the detrimental processes after SCI, therapeutic strategies targeting this specific form of cell death have been explored. Inhibiting ferroptosis after SCI has been shown to aid in functional recovery in preclinical studies. Several ferroptosis inhibitors, including ferrostatin-, deferoxamine, and SRS16-86, have demonstrated neuroprotective effects and improved outcomes in animal models of SCI [13]. Furthermore, natural compounds such as proanthocyanidins, epigallocatechin gallate (EGCG), carnosic acid, and trehalose have also shown promise in inhibiting ferroptosis and promoting recovery following SCI [63]. These findings highlight the potential of targeting ferroptosis as a therapeutic approach for mitigating the damage caused by SCI and enhancing functional outcomes.

5. Delivery Methods for Ferroptosis Inhibitors in Spinal Cord Injury Treatment

5.1. Localized Injection at the Traumatized Spinal Cord

In preclinical studies, researchers have directly injected ferroptosis inhibitors, such as Ferrostatin-1, into the injured spinal cord of rats. This approach has successfully reduced iron and ROS accumulation and improved functional recovery [64]. Similarly, injecting sodium selenite locally in SCI rat models has lowered iron, MDA, and 4-HNE levels, leading to better recovery [65]. While precise, this method’s clinical application is challenging due to manipulation difficulties and the risk of causing further injury [66].

5.2. Intraperitoneal Injection

Some studies have shown that intraperitoneal injections of DFO (iron chelator deferoxamine) [67] and SRS16-86 (a third-generation ferroptosis inhibitor) [68] in SCI rat models can inhibit ferroptosis by upregulating the Xc-GSH-GPX4 axis, which in turn improves functional recovery. However, more research is needed to determine if these drugs can effectively cross the BBB or blood spinal cord barrier (BSCB) for clinical use.

5.3. Intranasal Administration

Intranasal administration offers a promising route as it can bypass the BBB/BSCB and deliver drugs directly to the CNS in animal models [69]. For instance, intranasal Liproxstatin-1 and Ferrostatin-1 significantly reduced infarct size in a mouse stroke model [70], suggesting its potential for clinical application in SCI treatment.

5.4. Intravenous Injection

Intravenous injection is a highly practical option for emergency clinical treatment due to its ease of use, ability to deliver large drug volumes, and low risk. However, its effectiveness in crossing the blood-brain barrier (BBB) for CNS diseases remains a subject of debate. For example, intravenous SRS16-86 (a third-generation ferroptosis inhibitor) did not cross the BBB in mice, as it was undetectable in cerebrospinal fluid and brain tissue fluid after the injection [68].

6. Mitigating Ferroptosis: The Role of Extracellular Vesicles in Spinal Cord Injury Recovery

EVs present a promising alternative to MSCs in therapeutic applications, offering advantages in safety, controllability, and logistical handling [71]. In SCI, EVs exert therapeutic effects through diverse mechanisms, including neuroprotection [72], stimulation of nerve tissue regeneration [73,74], attenuation of scar formation [75,76], and regulation of oxidative stress and angiogenesis [77]. Notably, MSC-derived EVs have been shown to reduce oxidative stress and restore the BBB [78], as well as inhibit neuronal cell death pathways, apoptosis [79], and ferroptosis [80]. Furthermore, EVs can function as efficient drug carriers due to their inherent properties of small size, high tissue penetration, and avoidance of immune clearance [81]. These therapeutic actions are mediated by suppressing detrimental pathological responses and activating regenerative signaling pathways in neural and vascular tissues [78]. The future direction of SCI therapy is leaning towards combination approaches [79], where the integration of EVs with degradable bio-scaffolds has demonstrated synergistic effects, enhancing treatment efficacy and facilitating nutrient delivery to the injury site [82].

Exosomes, tiny vesicles secreted by cells, are being extensively studied as natural carriers for therapeutic molecules like miRNAs (Table 3). Their ability to cross the BBB and protect their cargo from degradation makes them ideal for SCI treatment [83]. These are known to play crucial roles in the complex processes following SCI, including inflammation, oxidative stress, apoptosis, and neuronal regeneration. Modulating specific miRNAs can influence lesion size, cellular dynamics, and functional recovery [84]. Preclinical studies have identified several exosomal miRNAs that can inhibit ferroptosis in SCI models. For example, MSC-derived exosomes carrying miR-219-5p have shown the ability to reduce ferroptosis in neuronal cells and improve motor function in SCI rats by targeting pathways like UBE2Z/NRF2. Other miRNAs, such as miR-125a-3p, have been shown to alleviate SCI by regulating neutrophil extracellular trap formation, which contributes to inflammation and hinders recovery [57].

Exosomal miRNAs can exert their beneficial effects through various pathways beyond direct ferroptosis inhibition, including reducing inflammation, promoting neuroprotection, enhancing axonal regeneration, and influencing macrophage/microglia polarization [85]. Overall, EVs may carry molecules that either inhibit factors that promote ferroptosis or enhance those that prevent it. This suggests that EV therapy could potentially mitigate damage caused by ferroptosis in SCI. Research indicates that EVs can deliver factors that help regulate iron homeostasis and lipid peroxidation, directly influencing the mechanisms involved in ferroptosis.

7. Unraveling MicroRNA-Ferroptosis Crosstalk in Spinal Cord Injury

Emerging evidence suggests a crucial link between miRNAs and ferroptosis in SCI [86]. Several miRNAs have been identified as regulators of key components of the ferroptosis pathway, including iron metabolism, GSH synthesis, GPX4 expression, and lipid peroxidation [86]. For instance, some miRNAs can directly target genes involved in iron uptake and storage, thereby influencing cellular iron levels and susceptibility to ferroptosis [50]. Others can modulate the expression of genes involved in GSH synthesis or GPX4 activity, impacting the cellular antioxidant capacity and resistance to lipid peroxidation [68]. Furthermore, miRNAs can indirectly influence ferroptosis by targeting other signaling pathways involved in oxidative stress and inflammation, which are closely intertwined with ferroptosis [67].

One notable example is miR-672-3p, which is upregulated after SCI. Studies have demonstrated that this miRNA promotes ferroptosis by downregulating the expression of FSP1 [3]. FSP1 normally functions to inhibit ferroptosis, and its downregulation by miR-672-3p impairs this protective mechanism, leading to increased lipid peroxidation and ferroptotic cell death in the injured spinal cord. Conversely, inhibiting miR-672-3p has been shown to suppress ferroptotic cell death and alleviate the damage that occurs after SCI [41]. This suggests that miR-672-3p plays a significant role in promoting ferroptosis in SCI by targeting FSP1, and its inhibition could represent a potential therapeutic strategy.

Another important miRNA is miR-6315, which is also significantly upregulated following spinal cord injury. Research indicates that miR-6315 negatively regulates Smoothened (Smo), a key factor in the axonal growth cone pathway, as well as components of the anti-ferroptosis pathway, including xCT, GSH, and GPX4 [38]. Silencing miR-6315 in animal models of SCI has been shown to improve functional recovery, promote neuronal regeneration and migration, and attenuate both cell apoptosis and ferroptosis [38]. This suggests that miR-6315 has a detrimental role in SCI by suppressing neuroprotective pathways and promoting ferroptosis, making its silencing a promising therapeutic approach.

In addition, let-7b-5p has been identified as part of a ferroptosis-related mRNA-miRNA-lncRNA network in SCI [3]. A previous study indicated that let-7b-5p could induce ferroptosis by upregulating the expression of p53 [87]. Similarly, miR-15b-5p has also been found within this ferroptosis regulatory network in SCI [39]. One more study revealed the exosomes derived from iPSC-NSCs can package and deliver let-7b-5p to downregulate LRIG3, thereby alleviating microglia/macrophage pyroptosis and improving motor function in mice post-SCI [88]. These findings underscore the therapeutic potential of combining iPSC-NSC-derived exosomes with let-7b-5p to enhance functional recovery and reduce inflammation after SCI [88]. These findings suggest that both let-7b-5p and miR-15b-5p may play roles in modulating ferroptosis in the context of SCI, although further research is needed to fully elucidate their specific targets and functions within this network. Shao et al. investigated alternative cell death pathways, including ferroptosis, using MSC-derived EVs. They demonstrated that exosomal lncGm36569 acts as a competing endogenous RNA for miR-5627-5p, thereby inhibiting neuronal ferroptosis via the miR-5627-5p/FSP1 axis and reducing neuronal dysfunction [47].

Beyond these, several miRNAs implicated in SCI (such as miR-21, miR-124, miR-940, and miR-26a) may indirectly influence ferroptosis through their established roles in regulating inflammation, apoptosis, and oxidative stress [3]. The interplay between miRNAs and ferroptosis in SCI is likely a complex and multifaceted process involving numerous miRNAs with diverse targets and functions. A comprehensive understanding of the entire network of miRNAs and their interactions with the ferroptosis pathway is essential for developing effective therapeutic strategies (Table 4). Importantly, the relationship between miRNAs and ferroptosis is likely bidirectional. While miRNAs regulate ferroptosis, ferroptotic stress may also alter miRNA expression profiles in the injured spinal cord. Ferroptosis-induced cellular damage can trigger the release of damage-associated molecular patterns (DAMPs) and promote inflammation, both of which can influence miRNA expression. Investigating this reciprocal regulation may provide deeper insights into SCI pathophysiology

Long non-coding RNAs (LncRNAs) further add complexity to this regulatory landscape. LncRNAs are key modulators of gene expression and are implicated in various human diseases [95]. Recent studies indicate that LncRNAs play critical roles in regulating ferroptosis and are dysregulated in SCI [96]. For instance, lncRNA OIP5-AS1 has been shown to suppress ferroptosis and protect neural cells via the miR-128-3p/Nrf2 axis, highlighting its therapeutic potential [97]. Supporting evidence from cancer studies demonstrates that OIP5-AS1 can inhibit ferroptosis through the miR-128-3p/SLC7A11 pathway [98]. In vivo experiments using lentiviral-mediated overexpression of OIP5-AS1 in SCI rat models further confirmed its neuroprotective and anti-ferroptotic effects [97].

Overall, the combined effects of miRNA dysregulation and ferroptosis on neurodegeneration and repair following SCI are highly complex and context-dependent. Certain miRNAs may exacerbate injury by promoting ferroptosis, whereas others may confer neuroprotection by inhibiting it. The balance of these opposing interactions ultimately determines functional outcomes after SCI. Therefore, elucidating the precise roles of miRNA-ferroptosis interactions is crucial for designing targeted therapeutic strategies aimed at promoting neuroprotection and regeneration.

8. Therapeutic Potential of Targeting the microRNA-Ferroptosis Regulatory Axis in Spinal Cord Injury

Given the significant roles of both miRNAs and ferroptosis in the pathology of SCI, targeting the interplay between these two processes holds considerable therapeutic potential [5]. Several strategies are being explored to modulate miRNA expression to influence ferroptosis and promote neuroprotection and repair after SCI [99]. One approach involves the use of miRNA inhibitors, such as antagomirs or miRNA sponges, to block the action of miRNAs that promote ferroptosis, including miR-672-3p and miR-6315 [43]. Conversely, miRNA mimics could be employed to enhance the levels of miRNAs that are found to inhibit ferroptosis or promote neuroprotective effects (Figure 3; Table 5).

Effective delivery of these miRNA modulators to the injured spinal cord is crucial for their therapeutic efficacy [99]. Several studies have shown that modulating miRNA expression can attenuate ferroptosis and improve functional outcomes in animal models of SCI [99]. For example, administration of miRNA mimics targeting pro-ferroptosis factors or inhibitors aiming anti-ferroptosis factors has been shown to reduce neuronal death and promote functional recovery [5]. Various delivery methods are under investigation, including viral vectors, nanoparticles, and EVs, which offer the potential for targeted delivery [5]. For instance, the implantation of scaffolds enriched with miRNA-loaded EVs has shown promise [46], and neuronal EVs containing specific miRNAs have been found to promote SCI repair [3]. MSC-derived EVs have also demonstrated the ability to modulate ferroptosis and enhance neurological recovery in preclinical models [104]. Furthermore, researchers are developing engineered EVs that can respond to the specific microenvironment of the injury site, such as being responsive to ROS, for more targeted therapeutic action [105]. The development of safe and efficient delivery systems remains a critical aspect of translating miRNA-based therapies to the clinic.

In addition to modulating miRNA expression, directly targeting the ferroptosis pathway with specific inhibitors is another promising therapeutic strategy for SCI. Applying iron chelators, such as deferoxamine, and lipid peroxidation inhibitors, like ferrostatin-1 and SRS-16-86, has shown beneficial effects in preclinical SCI models [12]. Treatment with these ferroptosis inhibitors has demonstrated improved recovery and reduced tissue damage in animal studies [13]. Despite the promising preclinical findings, significant challenges remain in translating these therapeutic strategies to human clinical trials. Further research is necessary to fully elucidate the complex interplay between miRNAs and ferroptosis in the context of SCI. It is also crucial to investigate post-SCI miRNA expression profiles in human cell lines or non-human primates to establish a more robust foundation for clinical applications [3]. Addressing the limited understanding of the long-term effects of miRNA modulation and potential off-target effects is also essential [5]. Additionally, the potential problems that may be encountered during the clinical translation of ferroptosis inhibitors need careful consideration [13]. However, significant challenges remain in translating these promising preclinical findings to human clinical trials, including the need for further research into the complex interplay of miRNAs and ferroptosis in SCI, understanding long-term and off-target effects of miRNA modulation, and addressing potential issues with clinical translation of ferroptosis inhibitors.

9. Ferritinophagy in Spinal Cord Neurons: Mechanisms and Therapeutic Implications

Ferritinophagy is a selective autophagic process that regulates iron availability by targeting ferritin, the major intracellular iron-storage protein, for lysosomal degradation [106]. Following SCI-induced cellular stress, ferritin binds to the autophagy receptor nuclear receptor coactivator 4 (NCOA4), which mediates the recruitment of ferritin to the autophagic machinery and promotes the formation of ferritin-containing autophagosomes. These autophagosomes subsequently fuse with lysosomes, where ferritin is degraded and iron is released in a controlled manner to maintain iron homeostasis (Figure 4).

Ferritinophagy plays a critical role in regulating iron homeostasis and ferroptosis susceptibility following SCI. By reducing the labile iron pool and limiting iron-driven lipid peroxidation, ferritinophagy may function as a protective mechanism that mitigates ferroptosis and supports neuronal survival [107]. By facilitating the removal of excess iron, it can decrease substrate availability for the Fenton reaction, thereby reducing oxidative damage and lipid peroxidation [108]. Consequently, induction of ferritinophagy has been proposed as a potential therapeutic strategy to protect neurons and other cells from ferroptosis-induced damage after SCI.

However, the role of ferritinophagy is context-dependent. Excessive or dysregulated activation may disrupt iron homeostasis, leading to uncontrolled iron release into the cytoplasm and contributing to cellular damage if sequestration or export mechanisms are insufficient [109]. Despite this possibility, current evidence more commonly supports its protective role rather than a direct contribution to pathology in SCI. The interaction between ferritinophagy and ferroptosis is dynamic. SCI-induced cellular stress promotes iron accumulation and oxidative damage, thereby enhancing ferroptosis. In response, ferritinophagy may be activated as a compensatory mechanism to restore iron balance and limit ferroptotic injury [110]. This interplay highlights ferritinophagy as a context-dependent regulator of neuronal fate and a promising therapeutic target for reducing ferroptosis-mediated damage and promoting recovery after SCI.

Overall, ferroptosis is a key contributor to neuronal damage in SCI, ferritinophagy appears to play a predominantly protective role by modulating iron levels and oxidative stress. Nonetheless, its precise function remains complex and incompletely understood, emphasizing the need for more specific tools and detailed temporal and cell type-specific studies to fully elucidate its therapeutic potential.

10. Challenges and Future Directions

Despite significant progress in understanding the role of ferroptosis and its regulation in spinal cord injury, several critical challenges remain that hinder effective clinical translation.

10.1. Limited Understanding of Context-Dependent Ferritinophagy

Ferritinophagy exhibits a dual role in SCI, functioning as both a protective and potentially detrimental process depending on cellular context and timing. While moderate activation may reduce iron overload and oxidative stress, excessive or dysregulated ferritinophagy could increase cytosolic iron levels and exacerbate ferroptosis. A major challenge lies in defining the precise conditions under which ferritinophagy shifts from neuroprotective to neurotoxic. Future studies should focus on temporal and cell type-specific regulation of ferritinophagy in neurons, astrocytes, and microglia.

10.2. Incomplete Characterization of miRNA Networks

Although numerous miRNAs have been implicated in regulating ferroptosis, the overall regulatory network remains incompletely understood. Most studies focus on individual miRNAs, while the combinatorial and synergistic effects of multiple miRNAs are largely unexplored. In addition, inconsistencies between studies highlight the need for standardized experimental models and validation across different SCI conditions. Systems-level approaches and multi-omics analyses are required to construct comprehensive miRNA-ferroptosis regulatory networks.

10.3. Challenges in miRNA Delivery and Stability

The therapeutic application of miRNAs is limited by challenges related to delivery, stability, and off-target effects. Efficient delivery across the BBB/BSCB, targeted cell specificity, and sustained expression remain major obstacles. While emerging delivery systems such as exosomes, nanoparticles, and viral vectors show promise, their safety, immunogenicity, and long-term effects require further investigation before clinical application.

10.4. Lack of Translational and Clinical Evidence

Despite these promising findings, research on miRNAs targeting ferroptosis in SCI remains largely preclinical, with most evidence derived from animal models. Clinical studies directly miRNA regulation of ferroptosis in human SCI are currently very limited and remain in the early stages of investigation. There is an urgent need for well-designed preclinical studies using clinically relevant models, followed by carefully controlled clinical trials to evaluate the safety and efficacy of ferroptosis-targeted therapies.

10.5. Interaction with Other Cell Death Pathways

Ferroptosis does not occur in isolation but interacts with other forms of regulated cell death, including apoptosis, necroptosis, and autophagy. The crosstalk between these pathways in SCI remains poorly understood. Future research should aim to elucidate how ferroptosis integrates into the broader cell death network and whether combinatorial therapeutic strategies targeting multiple pathways may yield improved outcomes.

10.6. Need for Biomarkers and Therapeutic Windows

Reliable biomarkers for detecting ferroptosis and monitoring treatment response in SCI are currently lacking. Identifying specific molecular or imaging biomarkers would greatly enhance early diagnosis and therapeutic intervention. Additionally, defining the optimal therapeutic window for targeting ferroptosis and ferritinophagy is essential for maximizing neuroprotection.

11. Conclusion and Future Perspectives

Accumulating evidence demonstrates that ferroptosis is a key contributor to secondary injury following SCI, driving neuronal and oligodendroglial loss through iron-dependent lipid peroxidation and oxidative stress. MiRNAs emerge as pivotal regulators within this process, orchestrating multiple ferroptosis-related pathways that influence cell survival, inflammation, and regenerative capacity. The growing recognition of the miRNA–ferroptosis axis provides new mechanistic insight into the molecular complexity of SCI pathophysiology. Despite significant progress, several challenges remain. Most current evidence is derived from preclinical models, and the temporal and cell type-specific roles of individual miRNAs in regulating ferroptosis after SCI require further clarification. In addition, effective and targeted delivery of miRNA-based therapeutics to injured spinal cord tissue remains a major translational barrier. Future studies integrating high-resolution transcriptomics, ferroptosis biomarkers, and functional outcomes will be essential to define clinically relevant targets. In summary, targeting the miRNA-ferroptosis regulatory network represents a promising avenue for mitigating secondary injury and enhancing neural regeneration after SCI. Continued mechanistic investigation and translational refinement may ultimately facilitate the development of novel therapeutic strategies aimed at improving functional recovery in patients with SCI.