Dysregulated miRNAs as Biomarkers and Therapeutical Targets in Neurodegenerative Diseases

Alzheimer’s disease (AD), Parkinson’s disease (PD), and Amyotrophic Lateral Sclerosis (ALS) are representative neurodegenerative diseases (NDs) characterized by degeneration of selective neurons, as well as the lack of effective biomarkers and therapeutic treatments. In the last decade, microRNAs (miRNAs) have gained considerable interest in diagnostics and therapy of NDs, owing to their aberrant expression and their ability to target multiple molecules and pathways. Here, we provide an overview of dysregulated miRNAs in fluids (blood or cerebrospinal fluid) and nervous tissue of AD, PD, and ALS patients. By emphasizing those that are commonly dysregulated in these NDs, we highlight their potential role as biomarkers or therapeutical targets and describe the use of antisense oligonucleotides as miRNA therapies.

AD represents the most common ND of aging and the leading cause of dementia worldwide and is characterized by the accumulation of amyloid-β (Aβ) and tau aggregates in different brain areas [2,5]. PD is the most common neurodegenerative movement disorder and is characterized by the loss of dopaminergic neurons (DNs) in substantia nigra pars compacta (SNpc) and the accumulation of toxic amyloid structures made up of α-synuclein aggregates [3,6]. ALS, also known as Lou Gehrig's disease, represents a progressive neurodegenerative disease of adulthood and is due to the progressive degeneration of upper and/or lower motor neurons (MNs) and, in some cases, by ubiquitinated protein aggregates [4,7]. Even if some treatments are able to alleviate symptoms or prolong life expectancy, there is still no cure for these NDs [8][9][10][11][12][13] and the primary goal today is the identification of effective therapies. The development of new treatment options requires a better understanding of the molecular basis underlying these pathological conditions and the identification of sensitive and specific disease biomarkers to aid early diagnosis and monitor disease progression and response to treatment.
Different non-coding RNAs have been proposed as biomarkers of neurodegeneration and, among them, microRNAs (miRNAs) have attracted the scientific community's attention thanks to their role as key regulators of gene expression [14][15][16][17]. MiRNAs are short molecules (20-22 nucleotides) able to degrade or inhibit the translation of their multiple 2. AD AD represents the most common age-related neurodegenerative disorder and is characterized by the presence of β-amyloid-containing plaques and tau-containing neurofibrillary tangles (NFTs) in different brain districts. The majority of cases manifest as a late-onset sporadic form (sAD), whereas familial forms (fAD) are mainly due to pathogenic variants in APP, PSEN1, and PSEN2 [22]. From a molecular perspective, AD is characterized by extracellular deposits of Aβ peptides, generated in the amyloidogenic pathway from the cleavage of APP by BACE1 and γ-secretase, and by the intracellular accumulation of strings of hyperphosphorylated Tau proteins known as neurofibrillary tangles (NFTs) [23]. In particular, Aβ peptides accumulation is due to the unbalanced synthesis and clearance of Aβ oligomers, and the mechanisms involved in Aβ clearance include ubiquitin-proteasome system (UPS), autophagic processes, proteolytic regulation and clearance of blood-brain barrier (BBB) [24].

PD
PD is a severely debilitating neurodegenerative disease associated with motor symptoms such as slowness of movement, stiffness, tremor, and postural instability [100,101]. It is characterized by the accumulation of α-synuclein in neuronal perikarya (Lewy bodies) and neuronal processes (Lewy neurites), and the selective loss of DNs in substantia nigra, which results in striatal dopaminergic deficiency [101]. Current treatments aimed at preserving DNs or compensating dopamine deficit (such as levodopa and deep brain stimulation) can relieve motor symptoms but are not effective in halting or slowing disease progression [100,101].
Although the molecular mechanisms underlying PD are not fully elucidated, the progressive deterioration of vulnerable DNs arises from several cellular disturbances, including protein misfolding and aggregation, synaptic damages, apoptosis, mitochondrial dysfunctions, oxidative stress, impairment of the UPS, and neuroinflammation [102].
Multiple genetic and environmental causes of PD have been described and clarified in the last decades. Approximately 5-10% of all patients suffer from a monogenic form of PD caused by mutations in autosomal-dominant (AD)-SNCA, LRRK2, and VPS35-or autosomal recessive (AR)-PINK1, DJ-1, and PARK2-genes [103,104]. The majority of PD cases are sporadic and result from a combination of common genetic risk loci in concert with environmental factors (lifestyle, exposure to toxins, physical activity) [101].

ALS
ALS is a progressive neurodegenerative disease characterized by selective degeneration of upper and lower MNs, resulting in muscle weakness and atrophy, with respiratory failure and ultimately death 3-5 years after the first clinical manifestation [197]. Only a fraction of ALS cases (approximately 10%) is familiar (fALS), because of mutations in genes involved in a wide range of cellular functions, whereas the vast majority of ALS cases are sporadic (sALS) [197]. Rilutek (riluzole) and Radicava (edaravone) are the only two drugs approved for ALS, which only slightly slow disease progression [198].
Understanding the etiopathogenesis of ALS is crucial for the implementation of effective therapies that are urgently needed. ALS is considered to have a complex etiology involving multiple genes and environmental factors. Among the implicated pathological processes are protein aggregation, glutamate excitotoxicity, defects in stress response, mitochondrial dysfunction, protein aggregation, altered axonal transport, and aberrant RNA metabolism [199][200][201]. The role of this last, in particular, seems particularly central when considering that several ALS-linked genes, such as TARDBP or FUS, are key components of coding and noncoding RNA processing machinery [17,[202][203][204][205][206][207][208].
MiR-142 is an important regulator of neuronal viability and apoptosis. Its inhibition produces neuroprotective effects by reducing neuronal injury and oxidative stress via the IL-6 and Nrf2/ARE signaling pathways and modulates axonal transport and mitochondrial activity in MNs by targeting vimentin and other intermediate filament types [232][233][234][235]258,259].

Common Dysregulated miRNAs in AD, PD, and ALS
In the previous sections, we reported the altered expression of specific miRNA molecules in nervous tissue and fluids of patients with AD, PD, and ALS. Although each of these NDs has its own unique clinical aspects, they share common pathological features and etiopathogenetic mechanisms such as inflammation or apoptosis. Identification of commonly dysregulated miRNAs may provide useful insights into the implicated molecular pathways thus unrevealing novel potential drug targets.
Using the lists of commonly dysregulated miRNAs in human post-mortem nervous tissues and circulating fluids of AD, PD, and ALS patients (Tables 1-3), we identified 7 commonly dysregulated miRNAs (miR-9, miR-124, miR-218, miR-132, miR-133b, miR-338, miR-146a) (Figure 1). In particular, altered expression of miR-124 and miR-218 was reported in all the three NDs (Figure 1a). MiR-133b and miR-338 were dysregulated in PD and ALS, miR-132 in both PD and AD, while miR-9 and miR-146a in AD and ALS (Figure 1a). The regulatory interaction network among these overlapping miRNAs and their corresponding disease-associated targets shows a high level of interconnectedness, with miR-124 as the most interconnected node (hub) in the network and commonly dysregulated miRNA for the three NDs pathologies (Figure 2). This suggests the possibility to target a single miRNA and affect multiple pathogenic pathways. and ALS, miR-132 in both PD and AD, while miR-9 and miR-146a in AD and ALS ( Figure  1a). The regulatory interaction network among these overlapping miRNAs and their corresponding disease-associated targets shows a high level of interconnectedness, with miR-124 as the most interconnected node (hub) in the network and commonly dysregulated miRNA for the three NDs pathologies (Figure 2). This suggests the possibility to target a single miRNA and affect multiple pathogenic pathways.  Interaction network of dysregulated miRNAs and their targets. The network was constructed using miRNet [260] and the miRNAs identified in this review as dysregulated in AD, PD, and ALS as an input list together with their disease-associated targets shown in Tables 1-3. Network visualization was obtained using the Cytoscape tool [261]. The most interconnected node (hub) is represented by miR-124 with a degree of connection of 36, while a degree of connection of 16 has been calculated for miR-218 which is also common to the three NDs pathologies. The blue diamond icons represent the dysregulated miRNAs, while ellipses represent target genes and are colored based on their disease association (yellow = PD; purple = ALS; light blue = AD).
In the next sections, we will describe these commonly dysregulated miRNAs and review their potential role and main targets. Interaction network of dysregulated miRNAs and their targets. The network was constructed using miRNet [260] and the miRNAs identified in this review as dysregulated in AD, PD, and ALS as an input list together with their disease-associated targets shown in Tables 1-3. Network visualization was obtained using the Cytoscape tool [261]. The most interconnected node (hub) is represented by miR-124 with a degree of connection of 36, while a degree of connection of 16 has been calculated for miR-218 which is also common to the three NDs pathologies. The blue diamond icons represent the dysregulated miRNAs, while ellipses represent target genes and are colored based on their disease association (yellow = PD; purple = ALS; light blue = AD).
In the next sections, we will describe these commonly dysregulated miRNAs and review their potential role and main targets.

Dysregulated miRNAs in AD, PD, and ALS
Several studies reported dysregulation of miR-124 in AD, PD, and ALS [225,262] ( Tables 1-3). This represents one of the most abundant miRNAs in CNS and plays an important role in neuronal survival, autophagy, mitochondrial dysfunction, synapse morphology, oxidative damage, and neuroinflammation by modulating the activity of downstream factors [263] (Tables 1-3, Figure 1). Specifically, in AD miR-124 modulates both Aβ production by targeting BACE1 [58,60] APP [59] and tau phosphorylation levels through PTPN1 signaling [62], and its decrease was detected in the CSF of patients with AD, supporting its role as a potential diagnostic biomarker in AD [43] (Table 1, Figure 2). Reduced plasma miR-124 levels support its potential utility as a diagnostic biomarker in the early stage of PD [136] (Table 2). In particular, aberrant expression of miR-124 in DNs leads to mitochondrial damage and cell death by targeting many key components of AMPK/mTOR, NF-κB, and p25/CDK5 pathways, including p62/p38, STAT3, KPNB1, and Calpains 1-2 [107,136,153,158,159,162,164,[264][265][266] (Table 2, Figure 2). In addition, miR-124 interacts with the modulator of BCL2-interacting mediator of cell death (Bim), whose suppression leads to reduction of Bax translocation to mitochondria and lysosomes, attenuating apoptosis and autophagosome accumulation [154] (Table 2, Figure 2). In ALS, miR-124 exerts a neuroprotective role in transgenic mice, by targeting Sox2 and Sox9, which encode two important regulators of neuronal and glial differentiation (Table 3, Figure 2) [223,225]. Differential expression of this miRNA can also be detected in both the spinal cord and leukocytes of sALS patients (Table 3) [222,227,228].
In addition to PD, AD, and ALS (Tables 1-3, Figure 1), miR-218, has been associated with neuropsychiatric disorders and other NDs [135,249,267,268]. In AD it is considered a potential peripheral biomarker [95] and was shown to regulate learning and memory in a mice AD model [94] and to affect the homeostasis between phosphorylated and dephosphorylated tau proteins [93] (Table 1, Figure 2). In PD models, miR-218 plays a role in modulating the NF-κB inflammatory signaling pathway, by influencing the activity of three importins, KPNB1, KPNA3, and KPNA4 [107], and interacts with the PD related gene PRKN [269], leading to mitochondrial dysfunction through the autophagic pathway [188] (Table 2, Figure 2). In addition, altered levels of miR-218 were found in brain regions and blood of PD patients [145] and were also associated with therapeutic brain stimulation [134,135] (Table 2). Dysregulation of miR-218 was also observed in ALS patients and animal models [212,229,248,251] (Table 3). A direct target of miR-218 in MNs is the voltage-gated potassium channel Kv10.1, whose upregulation was associated with an abnormal neuronal activity and excitability of MNs [249] (Table 3, Figure 2). It also targets EAAT2 (encoded by SLC1A2), an astrocytic glutamate excitatory amino acid transporter, that carries glutamate back into the cell after neurotransmission [248] and, when mutated, leads to impairment of glutamate levels, promoting post-synaptic neuronal cell death [270] (Table 3, Figure 2).

Dysregulated miRNAs in AD and PD
MiR-132 has been linked to several neurophysiological processes such as neuronal differentiation, migration and maturation, synaptic transmission, plasticity, and neuroprotection [271,272]. In particular, it represents one of the most-studied miRNAs in AD and, together with its downstream molecular targets (HDAC3, ITPKB, p250GAP, HN-RNPU, PTBP2, and SIRT1), is involved in the regulation of two AD pathological hallmarks: tau and Aβ [72][73][74][75][76][77][78] (Table 1, Figure 2). Dysregulated expression levels of this miRNA were found in the brain and CSF of AD patients and correlated with disease progression, supporting its use as an early biomarker (Table 1) [43]. MiR-132 was also proposed as a good candidate for monitoring PD progression as well as response to various therapeutic approaches [125,143,152] (Table 2). Upregulation of this miRNA was associated with neuroinflammation, microglial activation, and DNs neurodegeneration [117,148] (Table 2, Figure 2).

Dysregulated miRNAs in AD and ALS
Among miRNAs differentially expressed in brain tissues and fluids of AD and ALS patients, miR-9 is a brain-specific miRNA that has demonstrated great potential as a biomarker (Tables 1 and 3, Figure 1). Its levels were reduced in the blood of LOAD patients [42] and correlated with disease severity [43] as was ell response to treatment in primary neurons (Table 1). In particular, the synapse-enriched miR-9 [40] regulates different AD-related genes (BACE1, CREB, OPTN, and CAMKK2) Figure 2). MiR-9 plays an important role in regulating MNs development and its differential expression in ALS leukocytes supports its role as a diagnostic biomarker [218][219][220] (Table 3). Since it is known to interact with the 3 -UTRs of NEFL and PRPH and Pak4, its dysregulation may affect cell-cell junctions and axonal transport, leading to MN degeneration [17,218,220,274] (Table 3, Figure 2). Similar pathogenic mechanisms may follow the dysregulation of the NF-κB-sensitive miR-146a, implicated in the formation of pathological neurofilamentous aggregates [215,216,229], neuroinflammation, and immune response [55,65,[85][86][87][88] (Tables 1 and 3, Figure 1). Differential expression of this miRNA in plasma and CSF of AD and ALS patients [65,85] supports its role as a potential biomarker [242] (Tables 1 and 3).
In PD, miR-338 has been functionally linked to DNs survival and its decrease in plasma extracellular vesicles has been proposed as a potential diagnostic biomarker [139] (Table 2). In ALS, this miRNA was found differentially expressed in blood, CFS, serum, and spinal cord, and its use as an effective early biomarker has been considered [222,251,252,254,255] (Table 3). From a functional point of view, miR-338 modulates the expression of COXIV and ATP synthase [281], as well as the ALS-related genes ARHGEF28 (involved in the aggregation of low molecular weight neurofilaments) and VAPB (involved in protein misfolding and ER-associated aggregates) [282,283]. Moreover, ectopic expression of miR-338 mediated by FoxO3a may play a critical role in reducing cell survival by directly suppressing the expression of NRP1 [284] (Table 3, Figure 2).

ASOs-Based miRNA Therapies
The leading approach against inappropriate miRNA expression is based on ASOs. ASOs-therapies are used to directly modulate the expression of mRNAs or miRNAs. They are based on single-stranded oligonucleotides forming a complementary heteroduplex with the targeted mRNA, complementary double-stranded oligonucleotides miming endogenous miRNAs, or single-stranded that inhibit miRNAs [285]. These molecules can be used to mimic (agomir) or, more often, inhibit (antagomir) specific miRNAs [285], and simultaneously affect the expression of multiple proteins [13,286]. To allow adequate bio-distribution of therapeutic ASOs to the brain and circumvent the BBB, they can be directly delivered to the CSF (ICV or intrathecal) [20,285]. Taking advantage of their ability to regulate the expression of multiple genes, therapies involving miRNAs offer this peculiar opportunity to be used in different pathologies.
Although no miRNA-based ASOs have yet entered the clinical phase in AD, PD, or ALS, some miRNA-based therapies have been pre-clinically tested in vitro or in vivo, and showed promising results either in AD [13,285], PD [269,277], or ALS [205,287,288]. One of the most interesting examples is miR-124, which is dysregulated in all three pathologies (Figure 1). In AD, miR-124 mimic was used to regulate BACE1 and alleviate cell death induced by Aβ neurotoxicity [289], and reduce APP gene expression [59], while the use of a miR-124 antagomir resulted in the attenuation of tau phosphorylation and increased PTPN1 levels [62]. In MPTP-induced mouse models of PD, the use of a miR-124 mimic promotes neuronal proliferation and suppression of neuronal apoptosis via the Hedgehog signaling pathway [157]. The over-expression of miR-124 significantly reverses the loss of DNs and striatal DA, and reduces autophagosome accumulation and lysosomal depletion in MPP(+)-intoxicated SH-SY5Y cells [154]. Exogenous delivery of miR-124 attenuates microglia activation in SN and apoptotic cell death in midbrain DA of MPTP-treated mice in vivo [153,158]. In addition, polymeric nanoparticles (NPs) have been used to deliver miR-124 to specific regions of the brain [290,291]. Normalization of miR-124 level in ALS cellular models by using miR-124-targeting drugs attenuates inflammatory responses by inhibiting the NF-kB signaling pathway and preventing neuronal death [225,226].
Neuroprotective effects were obtained with antagomir inhibition of miR-218, a miRNA dysregulated in AD, PD, and ALS patients. In vivo ASO-mediated inhibition of miR-218 has anti-inflammatory, anti-apoptotic, and antioxidant effects in ALS model mice by attenuating the loss of a key glutamate transporter, the excitatory amino acid transporter Slc1a2 [248].

Conclusions
The recognition that inappropriate production of individual miRNAs may contribute to NDs has invigorated interest in these molecules and hope for new diagnostic methods and therapeutical approaches. While the pathogenic role of inappropriate miRNA expression is being characterized, different strategies to mimic or inhibit these miRNAs by ASOs have been effectively tested in pre-clinical models of NDs. Although delivery of these ASOs therapies to brain cells remains a key obstacle, the successful translation from in vitro and experimental animal studies into clinical practice may soon allow the development of effective drugs.

Supplementary Materials:
The following supporting information can be downloaded at: https://www. mdpi.com/article/10.3390/jpm12050770/s1, Table S1. GOs enrichment for dysregulated miRNAs in AD human nervous tissues and circulating fluids. Table S2. GOs enrichment for dysregulated miR-NAs in PD human nervous tissues and circulating fluids. Table S3. GOs enrichment for dysregulated miRNAs in ALS human nervous tissues and circulating fluids.