MicroRNA Alteration, Application as Biomarkers, and Therapeutic Approaches in Neurodegenerative Diseases

MicroRNAs (miRNAs) are essential post-transcriptional gene regulators involved in various neuronal and non-neuronal cell functions and play a key role in pathological conditions. Numerous studies have demonstrated that miRNAs are dysregulated in major neurodegenerative diseases, such as Alzheimer’s disease, Parkinson’s disease, multiple sclerosis, amyotrophic lateral sclerosis, or Huntington’s disease. Hence, in the present work, we constructed a comprehensive overview of individual microRNA alterations in various models of the above neurodegenerative diseases. We also provided evidence of miRNAs as promising biomarkers for prognostic and diagnostic approaches. In addition, we summarized data from the literature about miRNA-based therapeutic applications via inhibiting or promoting miRNA expression. We finally identified the overlapping miRNA signature across the diseases, including miR-128, miR-140-5p, miR-206, miR-326, and miR-155, associated with multiple etiological cellular mechanisms. However, it remains to be established whether and to what extent miRNA-based therapies could be safely exploited in the future as effective symptomatic or disease-modifying approaches in the different human neurodegenerative disorders.

Neurodegenerative diseases (NDs) affect millions of people worldwide, causing significant societal, emotional, and economic burdens [4,5]. Most NDs are based on multicomplex pathological mechanisms. Due to the impact of NDs on human health and the lack of definitive therapies for almost all of them, early detection before disease onset and effective therapeutic interventions can helpfully reduce cost and time efforts. Thus, scientists investigated miRNAs as sensitive diagnostic and prognostic biomarkers [4], and miRNA-based scientists investigated miRNAs as sensitive diagnostic and prognostic biomarkers [4], and miRNA-based therapeutic approaches by regulating miRNA expressions via miRNA activity enhancement (miRNA mimics or agomirs) or inhibition (miRNA inhibitors or antagomirs) were also analysed [5]. This article aims to provide a comprehensive overview of miRNA alterations in NDs, their contribution as potential biomarkers, and possible therapeutic applications. To this purpose, we evaluated the most recent studies related to miRNA dysregulations in ND, the pathogenic pathways in vitro and in vivo in animal models and humans, the promising miRNA role as biomarkers, the novel miRNA-based therapies, the delivery to CNS techniques, and their advantages and limitations. Finally, we identified the cross-over of some miRNAs among different NDs.

Biology of miRNAs
Since the first miRNAs, lin-4 and let-7, were discovered in Caenorhabditis elegans in 1993, over 2000 miRNAs have been to date reported on http://www.mirbase.org (accessed on 22 February 2022) [6,7]. Due to the number of miRNAs, 30-80% of the human genes are possibly under miRNA regulation [2,8]. Each miRNA can interfere with multiple functions of a single cell type, and several miRNAs can interact to target the same mRNA [1]. Most miRNAs are located in the intronic gene portion, whereas others are localized in the coding position [8]. Furthermore, numerous investigations have shown that miRNA expression differs between tissues and cell lines [4]. Therefore, the interaction between a given miRNA and its target genes depends on many factors, such as the miRNA's location, miRNA-mRNA quantities, and affinity [2]. miRNAs are assumed to have critical roles in many biological processes in physiological and pathological conditions [1,2]. Indeed, miRNA dysregulation has been associated with several neurological disorders [1]. In addition, both mature miRNAs and their precursors are secreted into extracellular fluids; thus, they can be considered signaling molecules for cell-to-cell communication or potential biomarkers for various diseases [2,8]. This article aims to provide a comprehensive overview of miRNA alterations in NDs, their contribution as potential biomarkers, and possible therapeutic applications. To this purpose, we evaluated the most recent studies related to miRNA dysregulations in ND, the pathogenic pathways in vitro and in vivo in animal models and humans, the promising miRNA role as biomarkers, the novel miRNA-based therapies, the delivery to CNS techniques, and their advantages and limitations. Finally, we identified the cross-over of some miRNAs among different NDs.

Biology of miRNAs
Since the first miRNAs, lin-4 and let-7, were discovered in Caenorhabditis elegans in 1993, over 2000 miRNAs have been to date reported on http://www.mirbase.org (accessed on 22 February 2022) [6,7]. Due to the number of miRNAs, 30-80% of the human genes are possibly under miRNA regulation [2,8]. Each miRNA can interfere with multiple functions of a single cell type, and several miRNAs can interact to target the same mRNA [1]. Most miRNAs are located in the intronic gene portion, whereas others are localized in the coding position [8]. Furthermore, numerous investigations have shown that miRNA expression differs between tissues and cell lines [4]. Therefore, the interaction between a given miRNA and its target genes depends on many factors, such as the miRNA's location, miRNA-mRNA quantities, and affinity [2]. miRNAs are assumed to have critical roles in many biological processes in physiological and pathological conditions [1,2]. Indeed, miRNA dysregulation has been associated with several neurological disorders [1]. In addition, both mature miRNAs and their precursors are secreted into extracellular fluids; thus, they can be considered signaling molecules for cell-to-cell communication or potential biomarkers for various diseases [2,8].
In normal conditions, Aβ is generated in neurons and released to the extracellular space, where it becomes a target of microglia and astrocytes for degradation. Initially, in the brain, the large molecule amyloid precursor protein (APP) can be cleaved under the action of β-secretases, with BACE1 being the major β-secretase species, to form Aβ40 and Aβ42 [23]. Soluble Aβ40 is more abundant than Aβ42; however, Aβ42 has a higher propensity for aggregation to generate amyloid plaques that show neurotoxic effects in AD [27]. Tau is a microtubule-associated protein that contributes to microtubule stability and its hyperphosphorylation is present in the brain of AD patients [24]. Furthermore, this hyperphosphorylation causes Tau detachment from microtubules and subsequent microtubule instability, self-aggregation, and neurofibrillary tangle formation. Many protein kinases and phosphatases regulate the phosphorylation status of Tau in phosphorylationsite-dependent manners [24,27]. In addition, the acetylation of Tau (Ac-Tau) promotes Tau aggregation, which suggests that Ac-Tau plays a role in Tau's pathologic transformation [24,27]. Besides, the detrimental effects may come from the synergistic interaction between Aβ and Tau that triggers neurodegeneration in AD [24,26].
Among the complex multifactorial mechanisms, miRNA alterations may have a role in AD pathogenesis [27,28]. Consequently, miRNAs have been considered potential biomarkers and therapeutic agents in counteracting the disease [28,29].
Notably, proinflammatory cytokines, such as tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6), and Il-10 released by reactive astrocytes and microglia, are involved in AD pathology [29,33]. In the HEK 293T AD cell model and in the in vivo AD rat model, miR-132 (considered a protective agent in AD) inhibited mitogen-activated protein kinase 1 (MAPK) and inducible nitric oxide synthase (iNOS), reduced oxidative stress, and improved cognitive function via the p38 signaling pathway, a member of MAPK family involved in inflammation and apoptosis [60]. By targeting gasdermin D, the executing protein of pyroptosis of glial cells, miR-22 negatively correlated with IL-18, IL-1β, and TNF-α levels in AD patients' peripheral blood and enhanced the memory ability in APP/PS1 mice [61]. miR-331-3p was the direct target of the von Hippel-Lindau tumor suppressor that has neuroprotective effects. It was downregulated in AD patients' serum and Aβ40-treated SH-SY5Y cells, and negatively correlated with IL-1β, IL-6, and TNF-α. The overexpression of miR-331-3p enhanced cell viability and inhibited inflammatory responses in Aβ40treated SH-SY5Y, thus supporting its neuroprotective role [62]. In contrast, miR-485-3p promoted AD severity by targeting AKT3, a gene regulating cell proliferation, apoptosis, and inflammatory response, in Aβ40-treated SH-SY5Y and BV2 cells, positively correlating with the inflammatory response triggered by IL-1β, IL-6, and TNF-α [58].
Overall, this growing evidence demonstrates the involvement of miRNAs in multiple pathophysiological mechanisms of AD.

Therapeutic Implications of miRNA in Alzheimer's Disease
miRNA-based therapeutic approaches have been broadly evaluated [55,[85][86][87]. miR-181a inhibitors decreased soluble and synaptosome-enriched Tau in the hippocampus from 3xTg-AD mice [55]. The administration of miR-124 antagomir attenuated Tau hyperphosphorylation and rescued learning and memory impairments in the P301S mouse model of AD [85]. The treatment with miR-1233-5p, downregulated in Aβ(+)MCI patients' platelets and megakaryocytes MEG-01 cells, reduced Aβ-increased platelet adhesion to fibronectin and expression of P-selectin [86]. Injecting lentivirus encoding miR-31 into the hippocampus of 3xTg-AD mice reduced Aβ and Vesicular glutamate transporter 1containing puncta and improved cognitive deficits. In addition, miR-31 overexpression also decreased APP and BACE1 expression in vitro and in vivo [87]. In AD rat models, miR-592 was upregulated and, consequently, its blocking rescued oxidative stress, promoting cell viability by activating the Keap1/Nrf2/ARE antioxidant signaling pathway and upregulating KIAA0319 (targeted gene of miR-592) [88]. miR-204-3p was downregulated in APP/PS1 mice and its overexpression reduced neurotoxicity by inhibiting NADPH oxidase 4, one of its targets, enhanced synaptic and memory functions, and decreased oxidative stress in the hippocampus [89]. In addition, a microRNA-based multitargeted therapeutic was also developed as MG-6267-the dual inhibitor of acetylcholinesterase and miR-15b biogenesis [90]. These data highlight the promising potential of miRNAs in the cure of AD.

Parkinson's Disease
Parkinson's disease (PD) is the second most common neurological disorder after AD, characterized by progressive loss of neurons in the brain, especially dopaminergic (DA) ones, in the substantia nigra pars compacta (SNpc), resulting in cognitive and behavioral dysfunctions [91][92][93][94][95]. The literature reports that 1% of people above 60 years old suffer from PD and approximately nine million individuals worldwide will develop PD by 2030 [91]. The loss of DA neurons and decrease in DA signaling result in motor dysfunction and clinical symptoms such as resting tremor, bradykinesia, rigidity, and postural instability [91]. Besides, the intracellular inclusions of Lewy bodies, enriched with aggregated α-synuclein (α-syn), are also identified in neurons of PD patients, and impair various pathways and activate neuroinflammation [92]. Apart from the SNpc, neuron loss occurs in several other brain regions, such as the amygdala, the vagus nerve's dorsal motor nucleus, the hypothalamus, cortex, and thalamus [93]. First motor dysfunctions develop after about a 70% loss of DA neurons in the SNpc. The preclinical phase is estimated to last 8-17 years, indicating the existence of complex mechanisms in the early PD phases [96]. Therefore, the availability of preclinical PD biomarkers is essential to design future neuroprotective strategies for high-risk patients.

The Biomarker Value of miRNAs in Parkinson's Disease
The diagnostic criteria for PD are based on clinical signs of motor functions, but the main issue is that PD can only be diagnosed once the DA neuron loss reaches up to 70% [115]. Therefore, the need for molecular biomarkers as potential clinical tools to diagnose PD is obvious. The biomarkers for PD could be the PD-related proteins in the CSF and brain tissues, such as α-syn for protein aggregation and Lewy body formation or protein Deglycase 1 (DJ-1) for mitochondrial dysfunction [116]. Blood and plasma samples are the ideal biomarker source, and miRNAs obtained from plasma are more abundant, tissue-specific, and stable. Circulating miRNAs can be used as noninvasive biomarkers, promoting the early PD detection and controlling the progression of the pathology [117]. Table 2 presents some miRNAs recently proposed as promising PD biomarkers.

Therapeutic Implications of miRNA in Parkinson's Disease
Treatments for PD include several approved medications (Levodopa, dopamine receptor agonists, catechol-O-methyl transferase inhibitors, and monoamine oxidase B inhibitors) [95], but there is also a variety of potentially effective compounds of natural origin under investigation (e.g., Mucuna pruriens [122]; ursolic acid [123]; chlorogenic acid [124]). More recently, different miRNA-based approaches are being investigated to cure PD. miRNA mimics and anti-miRNAs may represent useful tools to re-establish the physiological level of miRNAs in PD models, thus being promising as novel therapeutic tools. miR-150 levels in serums of PD patients were downregulated compared to healthy controls (HC) and its concentration negatively correlated with the proinflammatory cytokine levels (IL-1β, IL-6, and TNF-α) [109]. The restoration of miR-150 by mimics in lipopolysaccharide (LPS)-treated BV2 cells reduced the above-reported inflammatory cytokines via targeting the AKT3 gene [109]. miR-29c-3p mimics inhibited microglia activation and suppressed NLRP3 inflammasome in in vitro PD mouse models through directly targeting the nuclear factor of activated T cells 5 (NFAT5) [108], and miR-135b mimics attenuated pyroptosis [101]. The injection of AAV2 or AAV8-miR-30 human α-syn mimics into the SN rescued TH-positive dopamine neuron loss and reduced the forelimb deficits in PD rat models [104]. On the other side, the injection of antagomiR-421 into SNpc protected DA neurons in 6-OHDA-treated PD mice [99]. The intracerebral administration of agomiR-425 into SNpc reduced MPTP-induced necroptosis, restored locomotor impairments, and increased dopamine levels in the striatum in a PD mouse model [103]. The treatment with lentivirus-containing antisense miR-543-3p into SN locally and unilaterally in PD mice reduced the DA neuronal injury and α-syn aggregation levels, increased TH-positive cell numbers, and improved motor performance [107]. The injection of miR-3473b antagomir into the midbrain of PD mice enhanced autophagy and inhibited microglia activation via targeting TREM2/ULK1 [98]. Moreover, its inhibition also attenuated LPS-induced BV2 microglial activation [98]. These results are promising for a future potential therapeutic approach in PD treatment.

Multiple Sclerosis
Multiple sclerosis (MS) is a progressive autoimmune CNS disease characterized by inflammatory demyelination. It is the leading cause of nontraumatic neurological disability in young adults, and it is more common in women than men [125]. The most affected areas of the CNS are periventricular white matter, optic nerve, spinal cord, brain stem, and cerebellum. The main clinical symptoms include muscle weakness, blurred vision, dizziness, fatigue, and gate problems [126]. Several factors are responsible for the pathogenesis of MS and include genetic, epigenetic, microbial, and environmental causes [127]. Therefore, the aetiology and mechanisms of the disease are still not clear. Furthermore, there is no cure for this disease, although there are several effective disease-modifying treatments [128]. Current research on the pathophysiological changes occurring in MS reports an increase in proinflammatory miRNAs and related pathogenic biomarkers, pointing out that there is a great need for MS treatment as well as for understanding the mechanisms of disease progression [129].

Therapeutic Implications of miRNA in Multiple Sclerosis
RNA interference technology plays an important role in regulating miRNA content in MS [132]. The injection of miRNA-467b mimics in mouse-spleen-derived CD4 + T cells led to the downregulation of Th17 differentiation by targeting eukaryotic initiation factor 4 F (eIF4E), preventing infiltration of inflammatory cells into CNS, and delaying disease progression in the EAE mouse model of the disease [132]. Moreover, a neutral lipid emulsion containing miR-146a mimics were shown to cross the blood-brain barrier (BBB), increasing the M2 microglia/macrophage phenotype, rescuing OPC differentiation, enhancing remyelination, and improving the neurological in vivo outcomes via negatively affecting toll-like receptor 2/interleukin-1 receptor-associated kinase 1 signaling pathway [150]. miR-223 directly targets the autophagy related 16-like 1 (Atg16l1) and its deficiency augmented autophagy in the EAE mouse brain microglial cells. Overexpression of miR-223 decreased the cellular level of Atg16l1 in the LPS-induced autophagy model in BV2 cells [139]. In EAE mice, the administration of miR-219-5p through the tail vein negatively regulated fibronectin 1 expression, blocked bladder fibrosis, and controlled smooth bladder muscle tone [138]. In contrast, antagomiR-125a-3p stimulated oligodendrocyte maturation in vitro since miR-125a-3p targets Neuregulin1, Tyrosine kinase protein Fyn, the small GTPase Ras homolog family member A (RhoA), and p38, regulating myelin basic protein mainly expressed in mature/myelinating oligodendrocytes [151]. Obstacles to the miRNA-based therapeutic approach in ND in general and MS are the off-target effects due to multiple target genes and difficulty in crossing the BBB. Therefore, the development of novel delivering methods, such as nanosystems, biomaterials, EVs, gene therapy (lentivirus vectors), and stem cell implants, deserves to be investigated [152].

Huntington's Disease
Huntington's disease (HD) is a neurodegenerative disease caused by CAG repeat expansion in the Huntingtin gene (HTT), including a complex net of pathogenic mechanisms [159][160][161]. HD is the most common of the nine polyglutamine diseases [162], with a prevalence of~12 per 100,000 individuals in European populations [163]. The motor onset occurs from childhood to old age, with a mean age around 45 years [164]. Currently, there is no effective treatment, and patients usually die 10-20 years after illness onset [165]. HD symptoms include progressive involuntary choreiform movements, behavioral and psychiatric disturbances, and dementia [161]. Recently, miRNA-expression dysregulation has been reported in many studies using different HD human samples [166][167][168][169] and animal models [170][171][172][173].
In animal models, CAG length-dependent microRNA expression was altered in the mouse brain. In particular, 159 microRNAs were altered in the striatum, 102 in the cerebellum, 51 in the hippocampus, and 45 in the cortex [170]. Among them, miR-212, miR-132, miR-218, and miR-128, associated with aspects of neuronal development and survival, were found to be downregulated [171]. In a monkey model, miR-194 level was upregulated, whereas miR-181c, miR-128, and miR-133 expressions were downregulated in the frontal cortex region [172]. In addition, this study also confirmed HD-signaling genes regulated by miR-128a, including HTT and Huntingtin interaction protein 1, have a crucial role in the disease.

The Biomarker Value of miRNAs in Huntington's Disease
There is currently an urgent need for biomarker measure methods consistent with HD pathology, and the development of miRNA biomarker assays may contribute as a significant indicator for HD progression diagnostic [166]. Some studies focused on detecting specific miRNA [166,167]; others figured out several miRNA-signature alterations [161,168,169]. miR-9* was downregulated in peripheral leukocytes of HD patients and supposed to increase the expression of the corepressor of repressor element 1-silencing transcription factor [166]. miR-34b was elevated in mHTT-expressing NT2-derived neurons and in plasma samples of HD patients [167]. Moreover, the elevated expression of miR-34b appeared prior to symptom onset that was affordable for early detection of HD, needing a sample volume as small as 10 µL [167]. The circulating miRNAs from plasma or CSF samples were investigated to explore miRNA signatures [168,169]. Table 5 reports miRNAs as potential biomarkers in HD. However, the general limitations of these studies are the sample size, the unknown interactions of extrinsic factors, such as nutrition, medications, ethnicity, or race, as well as technical issues, such as accurate detection methods or internal reference for miRNA expression [161,[166][167][168][169]. Therefore, additional analysis of larger cohorts during disease progression will undoubtedly improve the efficacy of these measures.

Therapeutic Implications of miRNA in Huntington's Disease
Currently, miRNA-based therapeutics are being developed to target mutant-HTT [170,175,[179][180][181]. By injecting miRNA-124 in mice, the two neuroprotective molecules peroxisome proliferator-activated receptor-coactivator-1 alpha (PGC-1α) and BDNF were increased, while the SRY-related HMG box transcription factor 9, a repressor of cell differentiation, was downregulated [170]. The role of miR-196a was examined in cultured primary cortical neurons isolated from FVB mouse embryos and miR-196a-overexpressing transgenic mice [175]. The results showed that miR-196a improved neuronal morphology by suppressing the expression of RAN-binding protein 10 and increasing β-tubulin polymerization, and ameliorated intracellular transport, synaptic plasticity, learning, and memory abilities [175].
Despite the improved knowledge about miRNA alterations in HD, only some studies on miRNA-based therapeutic delivering strategies have been conducted in different in vivo models. An exosome-based delivery method was developed to inject miRNA-124 into the striatum of R6/2 transgenic HD mice, and it reduced the target protein RE1-silencing transcription factor, a regulator of the neurogenesis [179]. However, in that study, the behavioral performances were not improved due to the critical issues of the delivery method. Recently, many other studies have shown that artificial miRNAs can reduce mutant HTT in small and large HTT animal models [180,181]. An AAV5-encoded miRNA targeting human HTT was recently administrated into the striatal region of the Hu128/21 mouse model to lower the different HTT isoform expression [180]. The outcomes of that study showed a behavioral improvement and a long-lasting reduction of wild-type HTT [180]. Pfister and Coll. (2018) also applied a single administration of scAAV9-miRHTT into HD sheep striatum and recorded a reduction of the human mutant HTT mRNA in caudate and putamen at 1 and 6 months postinjection [181]. We can conclude that miRNA-mediated gene therapy is promising in treating of HD.

Amyotrophic Lateral Sclerosis
Amyotrophic lateral sclerosis (ALS) is the most frequent motor neuron (MN) disease that affects motor neurons in the motor cortex, brainstem, and spinal cord [182,183]. Approximately 90% of ALS cases are sporadic (sALS), while 10% are familial (fALS), defined by the occurrence of ALS in more than one family member [145]. Around 30 different genes and more than 100 mutations are linked to ALS. The most frequent gene mutations are chromosome 9 open reading frame 72 (C9orf72), Superoxide dismutase type 1 (SOD1), TAR DNA-binding (TARDBP), and fused in sarcoma (FUS) [184][185][186][187]. The pathophysiological mechanisms of MN degeneration remain largely unknown. ALS is a complex disease in which multiple cell types, such as astrocytes, microglia, oligodendrocytes, Schwann cells, and skeletal muscle cells, have important roles in the pathology [188,189]. Different cellular and molecular mechanisms contributing to ALS include protein misfolding and aggregation, mitochondrial dysfunction, neuroinflammation, oxidative stress, axonal transport deficits, glutamate excitotoxicity, RNA dysfunction, neuromuscular junction abnormalities, cytoskeletal derangements, dysregulation of growth factors, and abnormal calcium metabolism [188]. In this context, several studies have investigated the dysregulation of miRNAs, thus pointing out that the miRNA signature could be a valuable tool to identify ALS biomarkers and therapeutic targets [190].
Overexpression of miR-129-5p in NSC-34, SOD1 G93A , and SH-SY5Y/SOD1 G93A cells decreased HuD level, a crucial protein for neuronal development and maturation [194]. miR-129-5p also inhibited neurite outgrowth in SH-SY5Y/SOD1 G93A cells [194]. miR-5572 is a recently discovered molecule in humans, and its function is still unclear [194]. Moreover, miR-5572 binds the 3 -UTR of the targeted SLC30A3 gene and is increased in the spinal cord of sALS patients [196]. miR-142-3p was altered in some NDs, such as AD or MS, and non-NDs, such as diabetes or heart failure. In ALS, miR-142-3p is associated with neuroinflammation and microglial activation and was predicted to target both TDP-43 and C9orf72 genes. Moreover, it increased in serum of the SOD1 G86R and TDP43 A315T mouse models of the disease and sALS patients [195]. A clinical study in C9orf72 patients demonstrated miR-34a-5p and miR-345-5p overexpression, while miR-200c-3p and miR-10a-3p were downregulated in correlation with the disease stage [197]. Table 6 summarizes the miRNAs altered in ALS patients.

The Biomarker Value of miRNAs in Amyotrophic Lateral Sclerosis
miRNAs are secreted in the CSF and their analysis in this fluid could be used for clinical diagnosis. In addition, miRNAs are also muscle-specific and, therefore, they may have a broad application as biomarkers in ALS [190,202]. In a cohort of 20 ALS/motor neuron disease patients and 20 controls, some miRNAs were isolated from a neural-enriched subpopulation of EVs from total plasma samples and confirmed eight miRNAs differently expressed with respect to controls. In detail, miR-146a-5p, miR-199a-3p, miR-151a-3p, miR-151a-5p, and miR-199a-5p were upregulated in ALS patients, while miR-4454, miR-10b-5p, miR-29b-3p, and miR-151a-5p were downregulated [203]. In a study including 14 ALS patients, 9 nonALS neurological disease controls, and 9 healthy controls, CSF samples showed evidence of a positive correlation between EV-derived miR-124 levels and the disease severity (indicated by ALSFRS-R score) of male patients [204]. Another study collected muscle biopsy samples from 19 ALS patients to validate miRNAs and showed that only miR-206 levels negatively correlated with the muscle strength, assessed using a medical research council grading scale [205]. However, due to the limitation of the sample size, biological sources, and mixed hereditary causes, further studies are needed before using these miRNAs for clinical diagnosis [206].

Therapeutic Implications of miRNA in Amyotrophic Lateral Sclerosis
The pharmacological treatment based on miRNAs as a novel therapeutic approach in ALS has been exploited in numerous preclinical studies by stimulating or inhibiting miRNA production via different delivering techniques, such as adeno-associated virus vectors (AAV), EVs, or antisense oligonucleotides [202]. miR-494-3p secreted in EVs from inducible neural pluripotent cell-derived astrocytes was downregulated in astrocytes prepared from patients carrying the C9orf72 mutation and healthy controls. Treating HB9-GFP + mouse MNs with an miR-494-3p mimic rescued the neurite length and number of nodes per cell and increased MN survival [206].
Two miRNAs, miR-101 and miR-451, delivered by AVV5 and targeting C9orf72 to silence its expression, reduced the C9orf72 mRNA expression in both the nucleus and cytoplasm in two ALS cell models, namely HEK293T and induced pluripotent stem cell (iPSC)-derived frontal brainlike neurons from a patient affected by frontotemporal dementia (FTD). They also inhibited the formation of nuclear RNA foci in (G 4 C 2 ) 44 -expressing HEK293T cells [207]. These data would support the feasibility of miRNA-based and AAVdelivered gene therapy to reduce the gain of toxicity in ALS and FTD patients.
The dysregulation of the hsa-miR-17~92 cluster/nuclear PTEN pathway was evidenced in SOD1 G93A mice before the disease onset. Overexpressing miR-17~92 via selfcomplementary AAV9 delivering prolonged the survival of SOD1 G93A mice and ameliorated the neuromuscular function; besides, the hsa-miR-17~92 deletion provoked severe loss of MNs in the lateral motor column in the spinal cord. Finally, the survival of human iPSC-derived SOD1 +/L144F MNs was extended [208]. Therefore, miR-17~92 may be valuable as a prognostic marker of MN degeneration and a therapeutic target in SOD1-linked ALS. On the other hand, genetic ablation of one or two miR-155 alleles in SOD1 G93A mice reduced the expression of the proinflammatory genes Tnf, Fasl, Ccl2, and Nos2 in the spinal cord microglia and Tnf, Il1b, Fasl, Nos2, and CCR2 in Ly6C Hi splenic monocytes. Partial or total miR-155 deletion reversed the expression of abnormal proteins in the spinal cord and preserved the phagocytic function of microglia in vivo. Moreover, antimiR-155 administration to SOD1 G93A mice increased rotarod performance, delayed disease onset, and extended survival [209]. In SOD1 G93A mice, miR-29a-antagomirs, administered in vivo ICV, maintained muscular strength longer than vehicle-treated mice and tended to improve lifespan [210]. The available evidence suggests that miRNAs may represent a promising tool for ALS treatment. However, further studies are needed to evaluate efficacy and safety, figure out effective delivering methods, deepen knowledge of the molecular pathways related to disease, and verify the results in patients.

miRNA Engagement Overlapping in Neurodegenerative Diseases
Several studies have identified some miRNA dysregulation in one specific ND, whereas others have focused on the influence of one miRNA in different NDs. However, the miR-NAs' role across several NDs still needs further study. Figure 2 represents specific miRNAs shared among NDs.
As mentioned above, there was a diversified regulation of miR-128 levels related to the oxidative stress mechanism in AD, PD, and HD [212], involving the TrkC.T1 receptor and the TNF-α level in astrocytes in ALS [213], and regulation of pleiotropic cytokine TGFβ related to T-helper 17 (Th17) cells in immunological effects in MS [214].
As mentioned above, there was a diversified regulation of miR-128 levels related to the oxidative stress mechanism in AD, PD, and HD [212], involving the TrkC.T1 receptor and the TNF-α level in astrocytes in ALS [213], and regulation of pleiotropic cytokine TGFβ related to T-helper 17 (Th17) cells in immunological effects in MS [214].

Conclusions
Numerous studies have described miRNA functions and their aberrant expression affecting neuronal and non-neuronal mechanisms in AD, PD, MS, HD, and ALS. The emerging data also outlined the overlapping functions across the NDs. The extensive research results have improved our knowledge on the remarkable potential value for diagnosis, prognosis, prevention, and treatment of NDs based on up-or downregulated miRNAs expressions. However, there are still significant challenges to surmount since most miRNA-based therapeutic data are on preclinical models, and further studies are needed to increase human safety and efficacy [10,11]. One single miRNA may display several mechanisms and interact with other miRNAs, increasing the complexity of the cellular mechanisms affected in ND, thus leading to unwanted side effects and reducing the efficacy of the treatment. Moreover, miRNA-delivering-improvements are required to efficiently access the target during therapy [11]. To conclude, future identification and characterization of novel miRNAs involved in NDs are highly desired to improve the potential of this novel and up-and-coming research field.