Alzheimer’s disease

AD is a progressive neurodegenerative disorder characterized by cognitive decline and behavioral impairments. ^{Reference Lane, Hardy and Schott32} It is a common form of dementia in the elderly and a major global concern. A survey in the USA alone states that an estimated 6.7 million elderly people currently live with Alzheimer’s dementia, a number that is expected to double by 2060 in the absence of medical advancements. ³³ The main pathological hallmarks of AD are Aβ deposition and tau hyperphosphorylation, resulting in amyloid plaques and neurofibrillary tangles (NFTs), respectively. Moreover, synaptic dysfunction and neuroinflammation also contribute to disease progression. ^{Reference Wei, Wang, Ma, Zhang, Cao and Li34} Despite advances in understanding AD, early diagnosis and effective treatments remained challenging, and an urgent need for early biomarkers carved a path for miRNAs. Various findings have illustrated the role of miRNAs in AD due to their involvement in Aβ metabolism and tau hyperphosphorylation.

The accumulation of Aβ plaques is controlled by the cleavage pathway of amyloid precursor protein (APP), a large transmembrane glycoprotein. In the non-amyloidogenic pathway, APP is sequentially cleaved by α-secretase and γ-secretase, generating neuroprotective fragments (sAPPα, p3 and amyloid intracellular domain), while in the amyloidogenic pathway, β-secretase (BACE1) and γ-secretase process APP into Aβ40 and Aβ42 (Figure 2). Aβ42 is more prone to aggregation and produces neurotoxic plaques. ^{Reference Vu Nguyen35} Various miRNAs are responsible for the regulation of this process by varying BACE1 expression. miR-339-5p, miR-29c, miR-15b, miR-195 and miR-124 suppress BACE1 activity, leading to a reduction in Aβ production, ^{Reference Das, Wang and Ganguly36,Reference Selkoe and Hardy37} but the downregulation of miR-339-5p in AD leads to excessive Aβ accumulation and accelerates plaque formation, ^{Reference Long, Ray and Lahiri38} whereas miR-135b and miR-29c enhance Aβ clearance and act as neuroprotective factors. ^{Reference Zhang, Xing, Guo, Zheng, Wang and Xu39}

Figure 2.

Schematic representation of amyloidogenic and non-amyloidogenic pathway of amyloid precursor protein. sAPPβ = soluble amyloid precursor protein beta; sAPPα = soluble amyloid precursor protein alpha; AICD = amyloid intracellular domain; Aβ = amyloid-beta. Model generated with icons and templates from BioRender.com.

Another important feature of AD is tau hyperphosphorylation. Tau is a microtubule-associated protein, which stabilizes the neuronal cytoskeleton, but in AD, it undergoes abnormal hyperphosphorylation by kinases like CDK5 and GSK-3β, due to which it detaches from microtubules and aggregates as NFTs. Studies have shown that miRNAs such as mir-219-5p and mir-124-3p target the kinases responsible for tau phosphorylation and regulate tau metabolism, ^{Reference Li, Chen, Yi and Tong40,Reference Zhou, Deng, Chu, Zhao and Guo41} whereas upregulation of miR-146a promotes tau hyperphosphorylation. ^{Reference Wang, Huang and Wang42} Moreover, dysregulation of several other miRNAs contributes to AD pathology, like mir-132, a consistently altered miRNA in AD that influences tau stability and neural apoptosis, ^{Reference Salta, Sierksma, Vanden Eynden and De Strooper10} and mir-9, a highly conserved brain-enriched miRNA linked to tau acetylation and aggregation, as it is involved in neurofilament regulation. ^{Reference Schonrock, Humphreys, Preiss and Götz43}

Beyond these key pathological hallmarks, neuroinflammation and synaptic dysregulation are also main contributors to AD progression. Mir-146a modulates inflammatory pathways by targeting key immune regulators that amplify neuroinflammation, promoting neuronal damage, and simultaneously plays an important role in Aβ metabolism. Beyond plaque formation, the soluble Aβ oligomers enhance glutamate release, disrupting synaptic signaling and leading to excitotoxicity and neuronal death. ^{Reference Edwards44} The neuronal function is preserved by miRNAs like mir-431 and mir-188-5p, which protect synapses by regulating Wnt/β-catenin signaling. ^{Reference Lee, Kim and An45}

There are several miRNAs that are promising biomarkers for early diagnosis. Mir-125b, mir-146a and mir-34a are consistently altered in AD patients, while mir-9, mir-118 and mir-1256 show increased expression in AD brains, suggesting their potential in identifying disease progression (Table 1). A comprehensive meta-analysis by Yoon et al. (2022) further emphasized the diagnostic value of miRNAs in AD. ^{Reference Yoon, Kim and Ko46} It covered a wide range of samples, including blood and CSF and identified 56 miRNAs – 40 upregulated and 16 downregulated with significant differential expression in Alzheimer’s patients compared to healthy. Moreover, a study by Swarbrick et al. (2019) identified 10 miRNAs that are consistently dysregulated in AD. ^{Reference Swarbrick, Wragg, Ghosh and Stolzing47} These miRNAs were reported to show altered expression up to 20 years before symptoms appear, which highlighted their potential as early biomarkers. Other than diagnostics, miRNA-based therapies, including miRNA mimics (which restore neuroprotective miRNA levels) and miRNA oligonucleotides (which inhibit pathogenic miRNAs), offer a novel strategy for AD treatment. ^{Reference Bouchie48,Reference Rupaimoole and Slack49}

Table 1.

Dysregulation of various miRNAs in different NDDs

AD = Alzheimer’s disease; PD = Parkinson’s disease; ALS = amyloid lateral sclerosis; BACE1 = beta-site amyloid precursor protein ceaving enzyme 1; APP = amyloid precursor protein; ERK1/2 = extracellular signal-regulated kinase 1 and 2; MFN2 = mitofusin 2; ER = endoplasmic reticulum; BAG2 = Bcl-2-associated athanogene 2; GSK-3β = glycogen synthase kinase 3 beta; CDK5 = cyclin-dependent kinase 5; HSPB8 = heat shock protein family B member 8; NF-κB = nuclear factor kappa-light-chain-enhancer of activated B cells; PINK1 = PTEN-induced putative kinase 1; PBMCs = peripheral blood mononuclear cells; ELAVL4 = embryonic lethal abnormal vision-like protein 4; COX IV = cytochrome c oxidase subunit IV; PGC-1 α = peroxisome proliferator-activated receptor gamma coactivator 1 α; HDAC4 = histone deacetylase 4; Bcl-2 = B-cell lymphoma 2; Mcl-1 = myeloid cell leukemia 1; Snc = substantia nigra pars compacta; SNCA = synuclein alpha; LRRK2 = leucine-rich repeat kinase 2; SOCS1 = suppressor of cytokine signaling 1; FADD = Fas-associated protein with death domain; IKK = IkB kinase; MMP14 = matrix metalloproteinase 14; LRRN3 = leucine-rich repeat neuronal protein.

Amyotrophic lateral sclerosis

ALS, also known as Lou Gehrig’s disease, is a fatal NDD that primarily affects the upper and lower motor neurons in the brain and spinal cord. Upper and lower motor neurons are key components of the motor system, controlling voluntary and involuntary muscle movements. ^{Reference Hardiman, Al-Chalabi and Chio50} Any damage to these neurons can lead to muscle weakness, atrophy, respiratory failure and eventual death within 2–5 years after diagnosis. ALS predominantly affects individuals aged 40–60 years. The annual incidence is approximately 2.6 cases per 100,000 people, with males exhibiting a higher risk with 1:350 compared to females with 1:400. ^{Reference Wijesekera and Leigh51,Reference Al-Chalabi and Hardiman52} There are two types of ALS: familial ALS (accounts for approximately 10% ALS cases), which has a family history and sporadic ALS without any family history (accounts for approximately 90% ALS cases). ^{Reference R., A. and J.53} Clinically, symptoms of familial and sporadic ALS are very similar, which include dysarthria, dysphagia, laryngeal dysfunction, abnormal neurons, pseudobulbar effects, abnormal muscles, etc. ^{Reference Abe, Itoyama and Sobue54}

The etiology of ALS is not yet well understood. Several studies indicate that the disease is associated with the aggregation of pathological proteins such as superoxide dismutase 1 (SOD1), TAR DNA-binding protein 43 (TARDBP) and fused in sarcoma (FUS). ^{Reference F., J., Cooper-Knock, J. and Kirby55} Additionally, defining features of ALS include neuronal intracytoplasmic inclusion bodies and a hexanucleotide repeat expansion in the C9ORF72 gene located on chromosome 9. ^{Reference Al-Chalabi, Jones, Troakes, King, Al-Sarraj and van den Berg56–Reference Tomar, Sharma, Siddhanta and Deep58} In addition to these genetic factors, several hypothetical mechanisms may contribute to the disease’s progression (Figure 3). These include defects in nucleocytoplasmic transport, altered RNA metabolism, protein aggregation, abnormal DNA repair processes, dysfunctional mitochondria, issues with axonal transport, irregular vesicle transport, neuron inflammation, excitotoxicity and dysfunctional oligodendrocytes. ^{Reference Morgan and Orrell59–Reference Mejzini, Flynn, Pitout, Fletcher, Wilton and Akkari61}

Figure 3.

Factors leading to motor neuron degeneration in amyotrophic lateral sclerosis (ALS) patients. FUS = fused in sarcoma; SOD1 = superoxide dismutase 1; C9orf72 = chromosome 9 open reading frame 72; TDP43 = TAR DNA-binding protein 43. Model generated with icons and templates from BioRender.com.

Recent advances in RNA biology have highlighted the role of miRNAs in the pathogenesis of ALS. Research has revealed a link between the dysfunctional activity of miRNAs and ALS-related proteins. ALS-related proteins like FUS and TDP-43 have been found to influence miRNA regulation, while miRNAs, in turn, regulate both ALS-related proteins and associated genes. ^{Reference Ricci, Marzocchi and Battistini62,Reference Freischmidt, Müller, Ludolph and Weishaupt63} Several studies have shown that specific miRNAs are altered in patients with ALS (Table 1), suggesting that these small ncRNAs may influence ALS pathology.

Parisi et al. (2016) highlighted the role of upregulated miR-125b in enhancing NF-kB signaling in microglia, promoting neuroinflammation and motor neuron damage in ALS patients. Although miR-155 upregulation impairs microglial function, miRNAs like miR-335-5p, miR-27b-3p and miR-34a-5p impair mitochondrial function by disrupting mitophagy, leading to oxidative stress and neuronal death. ^{Reference Parisi, Napoli and Amadio64–Reference Nguyen, Kumar, Fedele, Bonanno and Bonifacino67} MiR-129-5p disrupts neuronal mRNA regulation by downregulating ELAVL4, impairing motor neuron function. ^{Reference Bronicki and Jasmin68,Reference Loffreda, Nizzardo and Arosio69} MiR-206’s overexpression may reflect a compensatory response to muscle degeneration by promoting neuromuscular regeneration, potentially delaying disease progression. ^{Reference Przanowska, Sobierajska and Su70} Additionally, miR-181 promotes apoptosis and neuroinflammation by inhibiting mitophagy and targeting anti-apoptotic proteins. ^{Reference Ouyang, Lu, Yue and Giffard71}

Several studies have identified potential miRNA biomarkers for the early diagnosis of ALS. Liu et al. (2022) highlighted dysregulated miRNAs including miR-181c-5p, miR-27b-3p, miR-194, miR-b2122, miR-126-5p, miR-425-5p, miR-146b, miR-124a, miR-18b-5p and miR-155. ^{Reference Liu, Zhou and Guan72} Similarly, Ruffo et al. (2023) observed significant downregulation of miR-132-5p, miR-132-3p and miR-143-3p and upregulation of miR-143-5p and miR-574-5p in sporadic ALS patients or those with mutations like TARDBP, FUS and C9ORF72. ^{Reference Ruffo, Catalano, La Bella and Conforti73} For individuals with sporadic ALS or specific gene mutations such as TARDBP, FUS and C9ORF72, the levels of miR-132-5p and miR-132-3p (two forms of miR-132) are significantly lower. However, in cases where the patient has a mutation in the SOD1 gene, the levels of miR-132 remain unchanged. ^{Reference Freischmidt, Müller, Ludolph and Weishaupt63} Additionally, the presence of certain miRNAs might act as early biomarkers for ALS, such as miR-132-5p, miR-132-3p, miR-124-3p and miR-133a-3p in the blood act as early biomarkers for ALS in people with a specific gene mutation (G376D-TARDBP). ^{Reference Ruffo, Catalano, La Bella and Conforti73} Moreover, systematic analysis by Liu et al. (2023) speculated the increased expression levels of miR-206, miR113b and miR338-p as potential promising biomarkers for ALS, ^{Reference Liu, Lan and Shi74} whereas Casado Gama et al. (2024) have identified consistently dysregulated miRNAs in various biological fluids of ALS patients. ^{Reference Casado Gama, Amorós and Andrade de Araújo75} These include hsa-miR-3665, hsa-miR-4530, hsa-miR-4745-5p and hsa-miR-206 in serum; hsa-miR-338-3p and hsa-miR-183-5p in blood; hsa-miR-34a-3p in CSF; hsa-miR-206 in plasma; and hsa-miR-146a-5p, hsa-miR-151a-5p, hsa-miR-10b-5p, hsa-miR-29b-3p and hsa-miR-4454 in neural-enriched extracellular vesicles from plasma. The upregulation of hsa-miR-206, hsa-miR-338-3p, hsa-miR-146a-5p and hsa-miR-151a-5p and the downregulation of hsa-miR-183-5p, hsa-miR-10b-5p, hsa-miR-29b-3p and hsa-miR-4454 are reliable biomarkers for ALS across independent studies.

Parkinson’s disease

Parkinson’s disease (PD) is one of the most common neurodegenerative disorders, affecting more than 6 million people around the globe, caused by loss of dopaminergic neurons (Figure 4) in the substantia nigra pars compacta (SNc), a part of the midbrain that plays a crucial role in movement regulation, dopamine production and supporting the brain’s reinforcement network. ^{Reference Dorsey and Bloem76,Reference Lees, Hardy and Revesz77} Degeneration of these dopaminergic neurons in the SNc can lead to characteristic symptoms of PD such as tremors, muscle inflexibility and impaired motor function. ^{Reference Kalia and Lang78,Reference Parkinson79}

Figure 4.

Schematic representation of dopamine level at synaptic cleft. The level is considerably higher in (a) healthy person compared to (b) Parkinson patient.

The ailment is defined by the intracellular accumulation of α-synuclein (α-syn), manifested as Lewy bodies and Lewy neurites. ^{Reference Jahan, Ahmad and Deep80,Reference Xu and Pu81} The precise etiology of PD remains unidentified; however, age is considered the most substantial risk factor. It is influenced by both environmental and genetic variables. ^{Reference Pang, Ho and Liu82} α-synuclein (SNCA) and leucine-rich repeat kinase 2 (LRRK2) are the genes linked to PD; for early onset, these genes are Parkin (PARK2), oncogene DJ1 (DJ1) and PTEN-induced putative kinase 1 (PINK1). ^{Reference Coppedè83} The pathophysiology of PD has been shown to be closely related to the dysregulation of these genes and their corresponding proteins, which are regulated by various miRNAs that ultimately promote α-synuclein accumulation, mitochondrial dysfunction, oxidative stress, calcium homeostasis, iron accumulation and neuroinflammation. ^{Reference Poewe, Seppi and Tanner84}

Recent studies have identified specific miRNA signatures that correlate with PD progression (Table 1). For example, the upregulation of miR-7 and miR-153 can repress SNCA expression, which leads to decrease in α-synuclein aggregation. ^{Reference McMillan, Murray and Bengoa-Vergniory85–Reference Wu, Xu and Zhou88} In contrast, the downregulation of miR-34b and miR-34c also suppresses DJ-1/Parkin, impairs the ubiquitin-proteasome system and reduces clearance of damaged proteins, leading to mitochondrial dysfunction and neuronal apoptosis. Additionally, the upregulation of hsa-miR-4639 5p can also inhibit DJ-1, promoting cell death. ^{Reference Kabaria, Choi, Chaudhuri, Mouradian and Junn89–Reference Rezaei, Nateghinia, Estiar, Taheri and Ghafouri-Fard91} MiR-205 supresses LRRK2, a kinase associated with both familial and sporadic PD that influences mitochondrial dynamics when mutated. It is found that miR-205 is downregulated in PD patients. ^{Reference Mortiboys, Johansen, Aasly and Bandmann92–Reference Cho, Liu and Jin94} Pro-inflammatory cytokines are released by activated microglia due the upregulation of mir-155, which causes neuroinflammation. Thome et al. (2016) highlighted that the absence of miR-155 reduces the release of α-Syn, thereby alleviating microgliosis in a sequential manner. ^{Reference Kim, Jou and Joe95–Reference Thome, Harms, Volpicelli-Daley and Standaert97} Upregulation of miR-30e reduces neuroinflammation. ^{Reference Li, Yang, Ma, Luo, Chen and Gu98,Reference Miñones-Moyano, Porta and Escaramís90} Oxidative stress plays a central role in dopaminergic neuron death in PD. ^{Reference Sanders and Timothy Greenamyre99} MiR-7, miR-9 and miR-29b protect against oxidative stress by targeting genes, which regulate stress responses such as Nrf2 and Sirtuin (silent mating type information regulation 2 homolog), ^{Reference Hou, Zuo, Li, Zhang and Teng100,Reference Kabaria, Choi, Chaudhuri, Jain, Li and Junn101} whereas miR-124 enhances autophagy by targeting Bim. ^{Reference Wang, Ye and Zhu102} Additionally, miR-181a influences autophagy by regulating the PTEN/AKT pathway. miR-29c-3p, miR-19b-3p and miR-29a-3p show decreased expression in serum from PD patients, with levels correlating with disease severity. ^{Reference Bai, Tang and Yu103,Reference Fernández-Santiago, Iranzo and Gaig104} MiR-24 and miR-485-5p are differentially expressed in the CSF of PD patients compared to controls. ^{Reference Marques, Kuiperij and Bruinsma105–Reference Wang, Li, Wang and Su107} Notably, miR-200a-3p levels in CSF could distinguish PD patients from healthy controls with high sensitivity and specificity. A combined panel of upregulated miRNAs (miR-1275, miR-23a-5p, miR-432-5p, miR-4433b-3p, miR-4443) and downregulated miRNAs (miR-142-5p, miR-143-3p, miR-374a-3p, miR-542-3p, miR-99a-5p) achieved remarkable diagnostic accuracy. ^{Reference Salemi, Marchese and Lanza108}

Systematic studies like Santos-Lobato et al. (2021) identified approximately 99 differentially expressed miRNAs in brain samples from patients with PD, comprising 60 upregulated and 39 downregulated miRNAs, which regulate around 135 target genes; notably, hsa-miR-144 is the sole miRNA exhibiting both upregulation and downregulation in PD. ^{Reference Santos-Lobato, Vidal and Ribeiro-dos-Santos109} Salemi et al. (2022) identified a dysregulation of the miRNAs hsa-miR-1275, hsa-miR-23a-5p, hsa-miR-432-5p, hsa-miR-4433b-3p, hsa-miR-4443, hsa-miR-142-5p, hsa-miR-143-3p, hsa-miR-374a-3p, hsa-miR-542-3p and hsa-miR-99a-5p for the first time in a study involving patients with Parkinson’s disease. ^{Reference Salemi, Marchese and Lanza108}

The alteration of miRNAs presents intriguing therapeutic methods for PD. For instance, miR-146a, miR-124 and miR-21 can inhibit inflammatory processes, miR-195 inhibits the release of pro-inflammatory cytokines and enhances the release of anti-inflammatory cytokines and miR-let-7a mitigates α-synuclein-induced microglial inflammation. ^{Reference Slota and Booth96–Reference Dorval and Hébert111} MiR-150 functions as a diagnostic biomarker by inhibiting AKT3, hence diminishing neuroinflammation and apoptosis, ^{Reference Li, Yu and Li112} whereas miR-7, miR-7116-5p, miR-29c/30e and miR-425 also offer promising therapeutic targets for PD. ^{Reference Li, Bi, Han and Huang113–Reference Cao, Wang, Qu, Kang and Yang117} These miRNAs serve a dual purpose as biomarkers for early diagnosis and as therapeutic agents to influence critical pathways in Parkinson’s disease, such as neuroinflammation, mitochondrial failure and protein aggregation.

Therapeutic use of miRNAs

Scientists are currently exploring two main ways to use miRNAs for therapy:

1. 1. Inhibiting harmful miRNAs that are found at high levels in disease, using tools like antagomirs or sponges.

2. 2. Restoring helpful miRNAs that are reduced in disease by giving synthetic copies called miRNA mimics.

For example, miR-124, which is mostly found in the brain, supports neuron health and reduces inflammation. In diseases like Alzheimer’s, levels of miR-124 drop. Studies in mice have shown that bringing back miR-124 using mimics can protect brain cells and improve memory. ^{Reference An, Gong, Wang, Bian, Yu and Wei143} Another miRNA, miR-155, increases during brain inflammation and can worsen damage. Blocking miR-155 has been shown to lower immune cell activity in the brain and help protect neurons. ^{Reference Cardoso, Guedes, Pereira de Almeida and Pedroso de Lima144}

However, a major challenge is delivering these miRNAs to the right cells in the brain. To solve this, researchers are testing viral vectors, like adeno-associated viruses, and nanoparticles, which can carry miRNAs across the BBB. ^{Reference Saraiva, Praça, Ferreira, Santos, Ferreira and Bernardino145}

miRNAs as biomarkers

One of the most promising roles of miRNAs is in diagnosis. Since they are stable in blood, CSF and saliva, they can be used as noninvasive markers to detect brain diseases early or track their progress. For instance, in AD, higher levels of miR-125b and lower levels of miR-29a have been found in patient blood samples. These changes are linked to how severe the disease is. ^{Reference Tan, Yu and Tan146} In Parkinson’s disease, miR-153 and miR-223 are often reduced, and this may reflect nerve cell damage and brain inflammation. ^{Reference Cardo, Coto and Mena147} Instead of using just one miRNA, scientists often combine several into a panel to improve accuracy. Some teams have even used machine learning to analyze miRNA patterns along with clinical data, which helps doctors make better predictions. ^{Reference Parveen, Mustafa, Yadav and Kumar148,Reference Ling, Aldoghachi and Chong149}

Emerging technologies: systems biology, AI and CRISPR

New technologies are changing the way we study miRNAs and use them in medicine. Three major tools are helping in this area: systems biology, artificial intelligence (AI) and CRISPR gene editing.

Systems biology

Rather than focusing on one gene at a time, systems biology looks at how everything is connected. It helps researchers see the big picture – how miRNAs fit into complex networks of genes, proteins and cell pathways. Using this method, scientists have linked changes in miRNA levels to problems like inflammation, energy failure in cells and loss of communication between neurons in diseases like Alzheimer’s. ^{Reference Roth, Hecker and Fava150}

Artificial intelligence

There’s so much biological data today that it’s hard to analyze manually. That’s where AI and machine learning come in. These tools can scan thousands of miRNA profiles and predict which ones are linked to disease. AI-based tools like miRNet and deepTarget help identify new miRNA targets and improve diagnostic tools. ^{Reference Panwar, Omenn and Guan151}

CRISPR/Cas gene editing

CRISPR, a gene-editing tool, has opened new doors in miRNA research. A version called CRISPR/Cas13 can edit RNA directly, including miRNAs, without changing the DNA. This allows scientists to silence or boost specific miRNAs safely in the lab. For example, researchers removed miR-132 in mice and found it led to memory loss and poor brain function, suggesting this miRNA is important for learning and memory. ^{Reference Hansen, Karelina, Sakamoto, Wayman, Impey and Obrietan152–Reference Zhang and Bian154}