Significance of miRNA-based therapeutics in neurological disorders

Due to their distinct regulatory roles in gene expression and their potential as highly focused and specific therapeutic agents, miRNA-based therapies show tremendous promise in the treatment of neurological illnesses. Table 1 outlines several significant factors that highlight the importance of miRNA-based treatments in neurological disorders.

Key players in miRNA biogenesis

Some important factors are necessary for the appropriate processing and maturation of miRNA molecules during the complicated and tightly regulated process of miRNA biogenesis. In Figure 1, we have depicted the key factors involved in the biogenesis and function of miRNA. These important factors have been discussed below.

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Figure 1. The factors involved in the biogenesis and function of miRNA. AGO-2: Argonaute 2; miRNA: microRNA; RNA pol II/III: RNA polymerase II/III; TRBP: TAR RNA-binding protein; RISC: RNA-induced silencing complex; mRNA: messenger RNA

Role of miRNA processing in neuronal development and function

miRNA processing plays a vital role in orchestrating the intricate processes of neuronal development and function in the central nervous system (CNS). As small non-coding RNA molecules, miRNAs regulate gene expression at the post-transcriptional level, influencing various aspects of neural development and synaptic plasticity [48]. The tightly controlled miRNA processing ensures the precise regulation of target gene expression, enabling miRNAs to act as crucial regulators of neural circuitry and function [50]. During neuronal development, miRNA processing influences the differentiation of neural progenitor cells into mature neurons. Specific miRNAs promote or inhibit neuronal differentiation by targeting genes involved in neural fate determination and differentiation processes [51]. Additionally, miRNAs guide axon outgrowth, neuronal migration, and neurite formation, influencing the establishment of neural circuits and proper connectivity in the developing brain [50]. In mature neurons, miRNA processing continues to play a pivotal role in synaptic plasticity, a cellular mechanism underlying learning and memory [48]. miRNAs regulate the expression of synaptic proteins and receptors, modulating synaptic strength and plasticity in response to neuronal activity. They contribute to the fine-tuning of synaptic transmission, optimizing neural communication and adaptive responses to environmental stimuli.

Moreover, miRNA processing is involved in the maintenance of neuronal homeostasis. miRNAs help regulate the balance of excitatory and inhibitory inputs in neural networks, contributing to stable and functional neural circuitry [50]. They also influence neuronal survival and apoptosis, impacting the overall health and viability of neurons. Altered miRNA expression profiles can disrupt normal neural development and synaptic function, contributing to the pathogenesis of these disorders. Understanding the role of miRNA processing in neuronal development and function provides valuable insights into the complex regulatory mechanisms underlying brain development, plasticity, and neural network dynamics. Moreover, the therapeutic modulation of miRNA processing holds great potential for developing targeted interventions for neurological disorders, offering new avenues for the treatment and management of these challenging conditions.

Neurodegenerative diseases and miRNA dysregulation

A set of conditions known as neurodegenerative diseases is characterized by the progressive death of nerve cells in the brain or spinal cord. Normal outcomes of these illnesses include the slow deterioration of autonomic, motor, and/or cognitive abilities. AD, PD, Huntington’s disease (HD), and ALS are a few examples of neurodegenerative illnesses. Small non-coding RNA molecules called miRNAs are essential for post-transcriptional gene control. They work by attaching to mRNA molecules, which serve as the blueprints for protein synthesis [52]. This causes the mRNA to degrade or the creation of proteins to be inhibited. Alterations in miRNA expression profiles have been linked to disease progression and may help to explain the underlying causes of various disorders, according to research. The expression of genes related to neuronal survival, synaptic plasticity, inflammation, and other related processes can be influenced by miRNAs [53].

For instance, in AD, miRNA dysregulation has been seen, and some miRNAs may have a role in amyloid-beta metabolism, tau protein phosphorylation, and neuroinflammation [54]. In PD, it has been discovered that miRNAs regulate genes involved in neuroinflammation, protein handling, and mitochondrial activity in PD [55]. In AD, miR-107 has been shown to regulate amyloid-beta metabolism by targeting beta-site amyloid precursor protein cleaving enzyme 1 (BACE-1), a key enzyme in amyloid-beta production. Similarly, miR-132 dysregulation is associated with tau hyperphosphorylation, contributing to neurofibrillary tangle formation [56]. In PD, miR-155 has been implicated in modulating neuroinflammation by targeting inflammatory cytokines, while miR-34b/c downregulation affects mitochondrial function, contributing to neuronal apoptosis [57]. These findings underscore the pivotal roles of miRNAs in the molecular cascades underlying neurodegenerative disorders. In ALS, miRNAs have been linked to motor neuron degeneration and glial cell activation, two hallmarks of ALS. In ALS, miR-218 has been identified as a key regulator of motor neuron degeneration, with its downregulation contributing to impaired neuromuscular signaling [58, 59]. Additionally, miR-155 is upregulated in activated glial cells, promoting neuroinflammation and exacerbating motor neuron damage [60]. A developing field of study is figuring out how miRNA dysregulation affects neurodegenerative disorders. It may shed light on illness mechanisms, reveal possible biomarkers, and inspire the creation of potential therapeutic approaches. The complex picture of neurodegenerative illnesses, which are influenced by a combination of genetic, environmental, and epigenetic variables, is complicated, and miRNA dysregulation is just one component of it.

Stroke and altered miRNA profiles

A stroke is a neurological condition that happens when the blood flow to a portion of the brain is cut off, causing oxygen and nutrient deprivation and damage or cell death in the affected area of the brain. For a better understanding of their involvement in illness etiology and perhaps to discover possible therapeutic targets, altered miRNA profiles have been investigated in the context of stroke and other neurological disorders. The progression and recovery from neurological illnesses like stroke have been linked to cellular processes like inflammation, oxidative stress, apoptosis, and neuroplasticity, according to research. Both the brain tissue injured by a stroke and the blood in circulation exhibit altered miRNA expression patterns, making them suitable biomarkers for diagnostic and predictive purposes [61]. Stroke and its consequences have been linked to particular miRNAs. miRNAs, including miR-21, miR-23a, miR-29a, miR-126, and miR-210, for instance, have been examined for their functions in pathways connected to stroke [62]. These miRNAs are thought to target genes that, among other things, regulate apoptosis, inflammation, and blood vessel integrity.

miRNAs can potentially affect the characteristics and operations of cellular membranes. They can control the expression of genes related to membrane transport, ion channels, and receptor signaling, all of which are crucial for intercellular communication and overall cellular function [63]. It’s crucial to remember that research in this area is continuing and that we are constantly learning more about the precise methods by which miRNA profiles alter cellular membranes in neurological illnesses like stroke. Even while miRNAs show promise as possible therapeutic targets or diagnostic markers, further research is required to completely understand their functions and prospective uses.

Psychiatric disorders and aberrant miRNA expression

Small non-coding RNA molecules called miRNAs are essential for the post-transcriptional control of gene expression. They are recognized to play a role in several cellular processes, such as differentiation, proliferation, and apoptosis. Psychiatric diseases have been linked to aberrant miRNA expression in some medical situations. A category of mental health diseases known as psychiatric disorders has an impact on a person’s mood, thinking, and behavior. Depression, bipolar illness, schizophrenia, anxiety disorders, and ASDs are a few examples of psychiatric disorders [64]. These complicated illnesses are assumed to be the result of a confluence of genetic, environmental, and epigenetic variables. The expression of genes involved in neural development, synaptic plasticity, and neurotransmitter signaling, all of which are essential for maintaining healthy brain function, can be affected by miRNAs [65]. People with psychiatric diseases have abnormal miRNA expression patterns in their brains. The delicate balance of gene expression can be upset by these miRNA changes, which can also hasten the onset or progression of many illnesses [66]. Psychiatric diseases have been associated with abnormal miRNA expression. Studies on people with depression have found that some miRNAs express themselves differently. These miRNAs might target genes related to synaptic plasticity, neurotransmitter metabolism, and neurotrophic signaling. The neurobiological changes connected to depression may be influenced by altered miRNA expression [67].

Schizophrenia is a complex condition characterized by disturbances in thinking, feeling, and behavior. Schizophrenia has been linked to aberrant miRNA expression [68]. Some miRNAs may control genes involved in synaptic function, dopaminergic signaling, and neuronal migration, all of which are important in the pathophysiology of schizophrenia [69]. miRNAs have been discovered to be involved in the brain circuits and behaviors associated with anxiety disorders. The development of anxiety disorders may be influenced by the dysregulation of miRNAs that control the hypothalamic-pituitary-adrenal (HPA) axis and stress responses [70]. A neurodevelopmental illness with a significant hereditary component is known as ASD. Individuals with ASD have been found to have altered miRNA expression patterns, and these miRNAs may have an impact on the genes involved in synapse function, neural connection, and neurodevelopment [71].

The potential of dysregulated miRNAs as diagnostic and prognostic biomarkers

Table 2 highlights key miRNA biomarkers associated with various neurological disorders, emphasizing their diagnostic and prognostic potential. miRNAs such as miR-21-5p in AD, miR-133b in PD, and miR-181c in MS demonstrate significant expression changes linked to disease progression (Figure 2). Their roles in neuronal function, inflammation, and pathology suggest their utility as biomarkers for early detection and disease monitoring.

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Figure 2. Expression of miRNA in disease progression. AD: Alzheimer’s disease; PD: Parkinson’s disease; MS: multiple sclerosis; ALS: amyotrophic lateral sclerosis; HD: Huntington’s disease

Viral vectors as delivery vehicles

miRNAs and other genetic material can be delivered to target cells via viral vectors, which are frequently utilized in gene therapy and molecular medicine. These vectors are altered forms of naturally occurring viruses that have been created to transport and deliver certain therapeutic payloads, like miRNAs, to host cells [97]. Viral vectors are useful tools for a variety of applications because they are highly effective at transmitting genetic material into cells. However, there are certain difficulties and things to think about when using them. Here is a summary of viral vectors used as delivery systems:

Adenoviral vectors: Adenoviruses are huge DNA-containing, non-enveloped viruses. Cells that divide and those that do not can both be effectively infected by adenoviral vectors. Because of their immunogenicity, they are frequently utilized for short-term expression [98]. Advexin and Gendicine are adenoviral vectors expressing the p53 tumor suppressor gene, showing promise in treating cancers like HNSCC and colorectal cancer [99]. Phase I/II trials demonstrated safety and anti-tumor activity, with Advexin administered intratumorally at doses up to 2.5 × 10¹¹ viral particles, highlighting its potential as a targeted cancer therapy [100].

Adeno-associated viral (AAV) vectors: The single-stranded DNA-based AAV virus is a tiny, non-pathogenic virus. Long-lasting gene expression can be established by AAV vectors in both dividing and non-dividing cells [101]. Comparatively, they are less immunogenic than adenoviruses. A clinical trial of AAV9-SMN1 gene therapy for spinal muscular atrophy (SMA) type 1 demonstrated significant motor function improvement, milestone achievement, and a 94% two-year survival rate. Long-term follow-up confirmed sustained SMN protein expression and durable benefits [102]. The therapy was well-tolerated, with mostly mild adverse effects, marking a promising advance in SMA treatment.

Lentiviruses: Viruses having an RNA genome that, upon infection, is reverse-transcribed into DNA. To provide stable and long-lasting expression, lentiviral vectors can incorporate their genetic material into the DNA of the host cell [103]. They are especially helpful for delivering genes to dividing and non-dividing cells, such as specific kinds of stem cells. A study using lentiviral vectors to deliver the FAH gene via portal vein administration in HT1-affected pigs showed stable, long-term FAH expression, normalizing activity within weeks. Liver histology revealed no fibrosis or tumorigenicity, indicating safety and effectiveness [104].

Exosomes for targeted miRNA delivery

Exosomes have shown promise as natural nanocarriers for the delivery of tailored miRNAs. Exosomes are tiny vesicles that cells release. By transporting bioactive chemicals, such as miRNAs, between cells, they aid in cell-to-cell communication. They are the subject of intensive investigation in the area of RNA-based therapies because they have various benefits as miRNA delivery mechanisms. Exosomes are naturally biocompatible because they are made of lipid bilayers, which are extremely biocompatible and well-tolerated by the body [105]. Depending on the parent cell type, exosomes may be naturally enriched with particular miRNAs. To deliver drugs precisely to particular cell types or regions, they can also be designed to show targeting ligands on their surface. Exosomes enhance the stability of miRNAs in circulation by shielding them from enzymatic activity and destruction [106]. Exosomes have a limited ability to elude immune identification, which lowers the likelihood of immunological reactions and possible negative effects. Exosome-based delivery systems offer a transformative approach for transporting therapeutic miRNAs across the blood-brain barrier (BBB) with enhanced precision and reduced immunogenicity. Recent advances in nanotechnology have enabled self-assembling nanoparticles as effective miRNA delivery vehicles for neurological disorders. These nanoparticles form stable structures via electrostatic interactions and can be functionalized with ligands like transferrin or RVG peptide to cross the BBB. Engineering strategies, including LAMP2B modification for LDLR targeting and RVG peptide functionalization for neuronal uptake, enable receptor-mediated transcytosis and cell-specific delivery. These exosomes protect miRNAs (e.g., miR-124-3p, miR-132-5p, miR-384-5p) from degradation, support scalable loading, and improve dose efficiency. By combining BBB penetration with neuronal specificity, exosome engineering holds significant clinical promise for treating neurological disorders through targeted modulation of gene expression, synaptic plasticity, and neuroinflammation, paving the way for next-generation RNA-based neurotherapeutics [107]. Exosomes have the potential to traverse biological barriers, including the BBB. This makes it easier to distribute miRNAs to hard-to-reach target areas. Exosomes can be loaded with specific miRNAs by utilizing viral vectors to express them or by directly transfecting parent cells with artificial miRNAs. The exosome synthesis then results in the miRNAs becoming enclosed within them [108].

Cell-penetrating peptides (CPPs) in miRNA delivery

Short amino acid sequences called CPPs can go across cell membranes and help deliver diverse payloads, such as miRNAs, into cells [109]. As prospective strategies for improving the intracellular delivery of miRNAs and other therapeutic compounds, CPPs have drawn a lot of interest. They provide a flexible and successful method for breaking down the cellular membrane barrier and facilitating effective miRNA distribution [110]. The interaction of CPPs with cell membranes and subsequent internalization via endocytosis or direct translocation constitutes the mechanism by which they promote cellular uptake. Depending on the CPP sequence, cargo, and cell type, the precise method may change. While some CPPs may interact with negatively charged elements of the cell membrane because they are positively charged, others may be taken up by receptors. CPPs offer several benefits for miRNA delivery in therapeutic applications. Firstly, CPPs enhance cellular uptake, enabling the effective transfer of miRNAs into various cell types, including non-dividing cells, which conventional delivery methods may struggle to achieve. Secondly, CPPs are versatile as they can be chemically synthesized and easily modified to carry various payloads, such as miRNAs, proteins, peptides, and nanoparticles. Additionally, CPPs improve the stability of miRNAs in the extracellular environment, protecting them from enzymatic degradation. They also allow for tissue-specific targeting by incorporating targeting ligands, ensuring the precise delivery of miRNAs to desired cells or tissues [111]. Finally, CPPs demonstrate minimal immunogenicity, reducing the likelihood of triggering immune responses.

Cellular uptake mechanisms and intracellular trafficking

miRNA uptake and intracellular trafficking are intricately regulated by cell-type-specific endocytic pathways, with significant implications for immune function, metabolism, and therapeutic delivery. In macrophages, actin-dependent phagocytosis enables efficient internalization of miRNA-loaded nanoparticles, a process fully inhibited by cytochalasin D. miRNAs such as miR-142-3p modulate this process by downregulating PKCα, reducing phagocytic activity and inflammatory cytokine production. Interestingly, miRNAs like miR-24 and miR-30b exert dual regulatory roles—controlling phagocytosis through transcriptional and post-translational mechanisms while also being internalized via the same pathways [112]. Pinocytosis and macropinocytosis serve as primary uptake routes for miRNA nanoparticles in non-phagocytic cells. Fluid-phase uptake dominates in hepatocytes and HEK293T cells, while receptor-mediated endocytosis via clathrin and caveolin governs uptake in epithelial cells. A recently characterized macropinocytosis-like pathway utilizes serum cationic proteins to form miRNA nanoparticles that bypass lysosomal degradation and target mitochondria via the PNPT1 transporter, enhancing mitochondrial CYB translation and increasing ATP production. Uptake mechanisms vary across cell types: macrophages rely on flotillin-1-rich domains for phagocytosis and macropinocytosis; epithelial cells depend predominantly on clathrin-mediated endocytosis; and stromal cells exhibit hybrid uptake patterns [113]. These mechanistic differences dictate miRNA bioavailability, with inefficient internalization leading to therapeutic sequestration, immune imbalance, or altered metabolic states.

Clathrin-mediated endocytosis: By producing clathrin-coated vesicles, clathrin-coated pits on the cell membrane enable the uptake of particular ligands, such as growth factors or nutrients. Some carriers may be made to take advantage of the clathrin-mediated endocytosis pathway, which enables the internalization of miRNAs into clathrin-coated vesicles [114]. Following their fusion with early endosomes, these vesicles may release miRNA into the cytoplasm. Cells can take up miRNAs, which are tiny RNA molecules that control gene expression, through endocytosis. miRNAs that are supplied exogenously can be contained in a variety of carriers, including nanoparticles, liposomes, or exosomes. Endocytic vesicles may hold onto certain miRNAs, blocking their release and subsequent action. Endocytosed miRNAs can occasionally break free from the vesicles and enter the cytoplasm, where they can then participate in the processes that silence genes. The release of miRNAs from endocytic vesicles may be accelerated by specific carriers or alterations, enhancing their bioavailability for gene regulation.

Endocytosis mediated by caveolae: Endocytosis mediated by caveolae holds significant potential for miRNA drug delivery due to the unique properties of caveolae as specialized lipid rafts in the plasma membrane. Caveolae are flask-shaped invaginations enriched in caveolin proteins, making them distinct sites for cargo uptake and intracellular trafficking. Caveolae-mediated endocytosis presents several advantages in miRNA drug delivery. First, it enables efficient cellular uptake, allowing miRNA-loaded nanocarriers or exosomes to be targeted to caveolae-rich regions of the plasma membrane, enhancing internalization [115]. Additionally, caveolae can be selectively targeted, facilitating cell-specific delivery of miRNAs, which is particularly beneficial in neurological disorders that require precise targeting within the brain. This process also offers protection from degradation, as internalization within caveolae shields miRNAs from extracellular nucleases, improving their stability and bioavailability. Furthermore, caveolae are involved in intracellular trafficking pathways, promoting the efficient delivery of miRNAs to their intracellular targets, which enhances miRNA-mediated gene regulation.

Intracellular trafficking of miRNAs

miRNAs migrate throughout cells to reach particular subcellular compartments where they carry out their regulatory tasks. This process is known as intracellular trafficking. Small RNA molecules called miRNAs are essential for post-transcriptional gene control. For miRNAs to interact with their target mRNAs and modify gene expression, proper intracellular trafficking is required. Recent research has deepened our understanding of cytoplasmic miRNA dynamics, highlighting complex regulatory mechanisms that govern their localization, stability, and function. Transport of mature miRNAs is mediated by exportin-5 for nuclear export and Importin-8, which partners with AGO-2, enabling selective nuclear re-entry [116]. RNA-binding proteins influence miRNA activity through competitive binding, chaperone-like coordination of miRNA-AGO-2 complexes, and reciprocal regulation of processing and expression. AGO-2 itself plays dual roles in gene silencing—facilitating mRNA target repression in the cytoplasm and engaging in transcriptional regulation within the nucleus. Emerging evidence also underscores spatial regulation, including miRNA sequestration in nucleoli during stress and mitochondrial localization that may modulate metabolic gene expression.

Endosomal escape and cytoplasmic delivery of miRNAs

Exosomes and synthetic nanoparticles are two prominent methods for delivering miRNAs into target cells, both primarily relying on endocytosis for cellular entry. However, miRNAs must escape the endosomal compartment and reach the cytoplasm to exert their gene-regulatory functions. Endosomal escape is, therefore, a critical determinant of delivery efficiency. Once internalized, endocytic vesicles can fuse with lysosomes, exposing miRNAs to enzymatic degradation. To avoid this fate, some delivery systems are designed to promote fusion with non-degradative compartments, such as early or late endosomes and multivesicular bodies [117]. These pathways allow time for release mechanisms to activate before degradation occurs. Multiple endosomal escape strategies have been developed to enhance cytoplasmic delivery. pH-responsive polymers and ionizable lipid formulations are engineered to sense the acidic environment of endosomes and disrupt the membrane, enabling release. Some systems use enzyme-cleavable linkers activated by tumor-specific proteases, while others exploit the proton sponge effect, inducing osmotic swelling and rupture of the endosome. Carrier degradation strategies, such as the glutathione-mediated dissolution of gold nanoparticles, also contribute to controlled release within the cytoplasm. In the case of extracellular vesicle-mediated delivery, fusion of the vesicle membrane with endosomal membranes or directly with the plasma membrane enables miRNA release [118]. Modifications like CD63 surface targeting have been shown to improve fusion efficiency and boost release by up to 40% in cancer models. Additionally, alternative entry routes bypass endocytosis entirely. CPPs and other direct translocation mechanisms allow certain miRNA complexes to cross the plasma membrane and localize directly in the cytoplasm, avoiding endosomal entrapment. Once in the cytoplasm, regulatory protein interactions further modulate miRNA availability. Complexes such as TNRC6-GW182 facilitate the dissociation of miRNAs from carriers by recruiting deadenylases, while AGO-2 phosphorylation at Ser387 enhances release efficiency in neuronal systems. Molecular chaperones like HSP90 stabilize miRNA-carrier complexes during trafficking and dissociate upon release to allow regulatory activity [119]. In contrast, endogenous miRNAs synthesized by the cell follow natural processing and export pathways, reaching the cytoplasm without the need for artificial carriers. Regardless of the source, efficient cytoplasmic release remains essential for effective gene silencing. Failure to escape the endosome can lead to therapeutic resistance, prolonged oncogenic signaling, or inflammatory responses due to carrier accumulation. As a result, optimizing endosomal escape and cytoplasmic delivery remains a central goal in miRNA-based therapeutic design.