The amyloid cascade hypothesis is one of the leading principles in our current understanding of the development and progression of neurodegeneration in AD (Ricciarelli and Fedele, 2017). The amyloid precursor protein (APP), a key mediator of neuronal growth and repair, is normally broken down by α-secretase and γ-secretase into soluble peptides that can be recycled. However, cleavage of APP at aberrant sites can yield insoluble Aβ peptides of varying lengths, with Aβ40 and Aβ42 being the most common (Ricciarelli and Fedele, 2017). The increased formation of Aβ is usually due to the imbalance, mutation, or genetic dysregulation of several key enzymes: (1) PSEN, the catalytic protein subunit of γ-secretase, (2) ADAM10, the major α-secretase in neurons, and (3) BACE, the major β-secretase in neurons. If not effectively cleared, Aβ accumulation ultimately leads to NFT formation, synaptic dysfunction, and neuronal apoptosis. Hence, it becomes clear that miRNAs that regulate the production and clearance of Aβ contribute to the disease and can serve as potential CSF biomarkers for AD.

Compared to healthy controls, CSF levels of miR-103, miR-16, miR-19b-3p, miR-328-3p, miR-340-5p, and miR-361-5p were all decreased in AD patients (Hébert et al., 2008; Denk et al., 2015; Sørensen et al., 2016; Gong et al., 2017; Lusardi et al., 2017; Zhong et al., 2018; Ji Y. et al., 2019; Zhang et al., 2020; Liu et al., 2022). These miRNAs modulate Aβ metabolism by directly targeting BACE1. Since alterations to the CSF often reflect brain changes, the downregulation of these miRNAs and the subsequent loss of inhibition on BACE1 expression promotes the deposition of Aβ. For example, the expression of miR-15b was reported to be decreased in the frontal cortices of sporadic AD patients by qRT-PCR analysis and was negatively correlated with BACE1 mRNA levels (Gong et al., 2017). Furthermore, the induced overexpression of miR-15b in transfected SH-SY5Y cells was observed to significantly downregulate BACE1 expression and protect against Aβ-induced neuronal apoptosis (Gong et al., 2017). Some miRNAs such as miR-1273g-3p can also regulate BACE1 indirectly. In contrast with the aforementioned miRNAs, CSF levels of miR-1273g-3p were reported to be constantly elevated in patients with early stage AD (Kim et al., 2021). MiR-1273g-3p is a key regulator of numerous molecular targets and primarily inhibits the mitochondrial genes TIMM13, MTCH1, and GLRX5. TIMM13 is of special interest because its knockdown in H4-APPswe cells not only compromised mitochondrial function but was also discovered to directly upregulate BACE1 expression and promote Aβ42 production (Kim et al., 2021). Although the altered expression of miR-1273g-3p was not tested for in post-mortem examination of AD brain tissue, TIMM13 was confirmed to be downregulated in the hippocampus.

ADAM10 is primarily responsible for the normal proteolytic cleavage of APP into soluble peptides. However, loss-of-function mutations or deficiencies of ADAM10 force APP to undergo amyloidogenic processing which ultimately promotes the formation of Aβ (Sarkar et al., 2019). In a recent study by Sarkar et al. (2019), the induced overexpression of miR-34a in transgenic mice was associated with a decrease in ADAM10 levels and rapid cognitive decline. Immunohistochemistry also revealed significant intraneural accumulation of Aβ and the development of AD-like neuropathology across several regions of the mice’s brains. When measured in human AD subjects, CSF levels of miR-34a were found to be elevated (Liu et al., 2022). Both miR-221-3p and miR-30a-5p were also significantly upregulated in the CSF and negatively correlated with the expression of ADAM10 (Sørensen et al., 2016; Manzine et al., 2018; Sun et al., 2022). In contrast, the current evidence on the precise expression levels of miR-140-5p in AD remains conflicting. MiR-140-5p is similar to miR-34a, miR-221-3p, and miR-30a-5p in that it is overexpressed in AD hippocampal tissue and inhibits the translation of ADAM10 (Manzine et al., 2018). However, it is reported that miR-140-5p CSF levels are remarkably decreased (Lusardi et al., 2017). Since the reasons for this discrepancy are not fully elucidated, further replication of these studies is needed to establish a clearer understanding of the role miR-140-5p plays in AD progression.

Amyloid precursor protein itself is the last major notable target of differentially expressed CSF miRNAs that have been discovered to play a role in amyloidogenesis. For example, miR-101-3p, miR-106a, and miR-384 have all been demonstrated to directly downregulate the expression of APP by binding the 3′UTR sequence of its mRNA transcript (Liu et al., 2014; Wang et al., 2019; Zhou et al., 2019). If this function is lost, the unregulated overexpression of APP will in turn increase activation of amyloidogenic pathways that are closely related with the occurrence of sporadic AD (Zhou et al., 2019). When measured in the CSF, miR-384 levels were reduced in patients with AD compared to normal controls. The expression of miR-384, in particular, was further shown to be decreased in the hippocampal tissue of APP/PSI transgenic mice as well as the serum and plasma of AD patients (Liu et al., 2014). Interestingly, these patients also displayed a significant negative correlation between CSF levels of miR-384 and Aβ42. These findings suggest that some miRNAs possess therapeutic and neuroprotective properties that can be used to slow AD development.

The aberrant hyperphosphorylation of tau is another major hallmark of AD. Tau, a microtubule-associated protein (MAP), is most abundantly expressed by mature neurons in the hippocampus and cerebral cortex of human brains. The phosphorylation of tau, under normal physiologic conditions, is essential for its activation and biological function: promotion of microtubule assembly and stability, preservation of cytoskeletal integrity, and maintenance of axonal transport (Iqbal et al., 2005). Although the exact underlying mechanisms are not yet fully understood, the extracellular deposition of Aβ and the imbalance between tau kinases/phosphatases have been shown to abnormally promote tau hyperphosphorylation. Hyperphosphorylated tau proteins generally exhibit lower binding affinity to axonal microtubules and are more prone to polymerize forming cytotoxic aggregates called neurofibrillary tangles. Several miRNAs have been implicated in the regulation of tau phosphorylation and are believed to significantly contribute to tau pathology in AD.

Of the major tau kinases, miRNAs are currently known to modulate the expression of ERK1/2, CDK5, and GSK-3β. MiR-15a-5p, a potent negative regulator of ERK1, is significantly decreased in the CSF and brains of AD patients (Hébert et al., 2010; Liu et al., 2022). Without the regulation of miR-15a-5p, increased ERK1 activity has been directly associated with tau hyperphosphorylation in primary cortical neurons and is believed to equally contribute to neurofibrillary pathology in humans (Hébert et al., 2010). In sharp contrast to miR-15a-5p, miR-125b overexpression increased ERK1/2 activation and subsequently promoted the phosphorylation of tau (Banzhaf-Strathmann et al., 2014). It is reported that, compared to healthy controls, the expression of miR-125b is elevated by approximately 1.6-fold in AD brains and is similarly high in the CSF (Banzhaf-Strathmann et al., 2014; Liu et al., 2022). Interestingly, the induced overexpression of miR-125b in primary hippocampal neurons was also seen to alter the levels of tau phosphatases such as Bcl-W, DUSP-6, and PP1CA. Translational inhibition of these phosphatases, which normally regulate ERK1/2 by negative feedback loops, significantly increased the phosphorylation of tau by fivefold compared with ERK1/2 upregulation alone (Banzhaf-Strathmann et al., 2014). Taken together, the concomitant inhibition of phosphatases and promotion of kinases by elevated miR-125b suggests that some miRNAs can modulate different targets that produce additive effects. Indeed, a recent study by Liu and Zhang (2019) reveals that the overexpression of miR-132 in AD upregulates CDK5 but downregulates GTDC-1, with both changes promoting the phosphorylation of tau. Similarly, miR-34a can inhibit the translation of PTPA and SIRT1 resulting in tau hyperphosphorylation (Sarkar et al., 2019). PTPA is normally an activator of protein phosphatase 2A (PP2A), the major serine/threonine phosphatase expressed in the brain. When activated, the PP2A complex enhances dephosphorylation of tau and consequently preserves synaptic function (Luo et al., 2013). However, PP2A has been shown to be downregulated in the brains of AD patients promoting tau phosphorylation, and this effect is perhaps further compounded by translational inhibition of PTPA via miR-34a (Wei et al., 2020). On the other hand, Sirtuin-1 is a NAD-dependent deacetylase that normally reverses the pathogenic acetylation of hyperphosphorylated tau proteins making them more prone to degradation by ubiquitin ligases and less likely to accumulate (Manjula et al., 2021). Even if aggregates do form, Sirtuin-1 was demonstrated to suppress the cell-to-cell propagation of abnormal tau limiting the spread of tauopathy into surrounding brain parenchyma (Min et al., 2018). In tauP3018 mice, the brain-specific deletion of Sirt1 was observed to directly result in tau-mediated synaptic loss causing behavioral and cognitive deficits (Min et al., 2018). In parallel, Sirtuin-1 is downregulated by miR-132, miR-30a-5p, and miR-34a, all of which are significantly elevated in the CSF of AD patients (Hernandez-Rapp et al., 2016; Sarkar et al., 2019; Sun et al., 2022). The last major kinase of interest is GSK-3β. MiR-23b-3p can directly bind and inhibit the translation of GSK-3β mRNA. Overexpression of miR-23b-3p in vitro reversed GSK-3β-mediated tau hyperphosphorylation and was described to be largely neuroprotective (Jiang et al., 2022). However, CSF levels of miR-23b-3p are downregulated in AD subjects and were found to be negatively correlated with tau phosphorylation as AD progressed into different stages (Jiang et al., 2022). Recent research also reveals that Caveolin-1 promotes the activity of GSK-3β by preventing its phosphorylation via the PI3K/Akt pathway (Kang et al., 2017). Thus, the decreased translational inhibition of Caveolin-1, as seen by low CSF miR-124-3p, allows for the continued activation of GSK-3β and phosphorylation of tau in AD (Kang et al., 2017).

Chronic low-grade inflammation of the brain parenchyma has long been demonstrated to play a central role in the development of AD. Neuroinflammation is characterized by the marked increase in the production and release of pro-inflammatory cytokines (such as IL-1β and TNF-α), chemokines (such as CCL1, CCL2, and CCL5), and reactive oxygen species (ROS). Although the primary mediators of neuroinflammation are innate immune cells, recent evidence indicates that peripheral blood cells can cross the blood-brain barrier (BBB) and contribute to the immune response seen in AD (Leng and Edison, 2021).

Microglia, resident macrophages of the brain, normally protect against neuronal loss by phagocytosing and preventing the accumulation of neurotoxic substances such as Aβ. In CNS injury or infection, microglia can migrate to the site of insult and become activated to one of two phenotypes, M1 (pro-inflammatory) or M2 (anti-inflammatory/phagocytic), whose different but balanced functions help facilitate tissue repair and healing (Onyango et al., 2021). However, with aging and in AD, microglia have been observed to assume a predominantly M1 phenotype (Onyango et al., 2021). The consequent decrease in amyloid clearance and increase in pro-inflammatory cytokine release are believed to accelerate AD pathogenesis and nerve damage. Moreover, the accumulation of Aβ has been linked to increased activation and recruitment of microglia further exacerbating neuroinflammation (Onyango et al., 2021). MiR-146a is believed to be a key regulator of the inflammatory response and microglial function. In a recent study by Liang et al. (2021), the overexpression of miR-146a in APP/PS1 transgenic mice was seen to reverse microglial polarization (i.e., phenotype transition) in favor of the M2 phenotype, which was associated with enhanced phagocytic clearance of Aβ. Moreover, miR-146a overexpression was observed to significantly reduce levels of pro-inflammatory cytokines (IL-1β, IL-6, TNF-α) and inflammasome markers (NLRP, ASC, caspase-1) indicating decreased NLRP3 activation (Liang et al., 2021). Collectively, these changes reduced Aβ accumulation, attenuated neuroinflammation, prevented neuronal apoptosis, and, most importantly, rescued cognition. Similar results were reported in a study where the intranasal administration of miR-146a in AD mice relieved hippocampal inflammation and ameliorated cognitive impairment (Mai et al., 2019). Although the exact molecular mechanisms have not yet been elucidated, the neuroprotective effects of miR-146a are currently thought to be mediated by negative regulation of Nkd2 (Liang et al., 2021). The knockdown of Nkd2 in vivo was reported to induce microglial phenotype switching and was negatively correlated with levels of miR-146a. Moreover, the 3′UTR sequence of its mRNA has been confirmed to be a target of miR-146a. In human AD subjects, CSF levels of miR-146a are significantly decreased (Liu et al., 2022). Without microglial phenotype switching and anti-inflammatory mediator release, the downregulation of miR-146a serves as a risk factor for sustained neuroinflammation in AD.

MiRNAs can also contribute to the AD inflammatory response by other mechanisms. MiR-100-5p, which is upregulated in the CSF of AD patients, can directly bind and activate endosomal TLR7/8 promoting cell-autonomous degeneration in neurons and pro-inflammatory cytokine release by microglia (Wallach et al., 2021). Similarly, increased activation of extracellular TLR4 by elevated CSF miR-let-7i-5p is associated with microglial ROS production and widespread neuronal damage (Sørensen et al., 2016). MiR-19b-3p and miR-29c-3p, both of which are downregulated in the CSF, lose their translational inhibition on STAT3 which in turn induces reactive gliosis and impaired cognitive function (Wu et al., 2017). MiR-497-5p, a direct negative regulator of Endophilin-1 expression, is decreased in AD allowing for Endophilin-1-mediated downregulation of tight junction proteins such as ZO-1, occludin, and claudin-5 (Zhu et al., 2019). The subsequent increase in BBB permeability augments the infiltration of peripheral blood cells further exacerbating neuroinflammation. Lastly, the downregulation of miR-223-3p and miR-373-5p in AD patients has been associated with increased NLRP3 inflammasome formation (Bauernfeind et al., 2012; Taşdelen et al., 2022).

Maintaining synapse structure is essential for the normal functioning of the brain. Synaptic plasticity refers to the ability of neuronal tissue to modify or strengthen synaptic connections in response to new information and stimuli. The neurochemical processes underlying synaptic plasticity are integral to learning and memory, and their derangement results in cognitive decline. In fact, the loss of synaptic transmission and activity are now recognized as the earliest events in AD pathology that precede clinical presentation. Thus, miRNAs that regulate synaptic plasticity and neuronal function are believed to greatly contribute to the cognitive impairment seen in AD patients.

Changes to synaptic plasticity, particularly in brain regions responsible for learning and memory, have been correlated with altered expression of neurotrophins, a group of regulatory proteins that promote the survival, growth, and function of neurons (Ng et al., 2019). BDNF is a potent neurotrophic factor that plays a key role in hippocampal long-term potentiation (LTP) and memory consolidation (Croce et al., 2013; Ng et al., 2019). When BDNF forms complexes with cell-surface tyrosine kinase B receptors (TrkB), major intraneural signaling pathways such as PI3K/Akt, MAPK, and PLC-γ are activated (Kowiański et al., 2017). Albeit by different mechanisms, these pathways will ultimately induce the synthesis of cytoskeletal proteins, polymerization of actin filaments, and branching of dendritic trees (Kowiański et al., 2017). The BDNF/TrkB complex can also modify the expression of AMPA and GABA receptors on post-synaptic membranes to promote LTP (Kowiański et al., 2017). MiR-206, miR-30a-5p, and miR-613, all of which are elevated in the CSF of AD patients, have been found to target and downregulate BDNF (Lee et al., 2012; Croce et al., 2013; Li et al., 2016). Without the effects of BDNF, the aging brain’s capacity for memory consolidation and synaptic plasticity is likely to become severely limited. Weinstein et al. (2014) found that in older adults, the risk for AD decreased by 33% for every increase in BDNF levels by one standard deviation increment. In parallel, the treatment of Tg2576 mice by a miR-206 antagomir restored brain levels of BDNF with direct improvements in hippocampal synaptic density and memory function (Lee et al., 2012).

Similar to BDNF, some miRNAs can directly alter the expression of surface receptors, channel proteins, or synaptic vesicle proteins involved in neurotransmission. For instance, elevated miR-34a has been shown to downregulate NR2A, a subunit of post-synaptic NMDA receptors, affecting LTP and memory formation (Sarkar et al., 2016). MiR-34a also affects pre-synaptic membranes by targeting VAMP2 and SYT1, key synaptic vesicle proteins involved in calcium-dependent neurotransmitter exocytosis (Sarkar et al., 2016). MiR-92a-3p, which is elevated in AD, also inhibits the translation and integration of AMPA receptors into post-synaptic membranes further contributing to synaptic dysfunction (Letellier et al., 2014).

The high metabolic demands of neural tissue make the brain particularly susceptible to injury following mitochondrial dysfunction. The energy generated by healthy mitochondria helps fuel vital intraneural processes such as ion transport, axonal development, and neurotransmitter biosynthesis. Mitochondria are also essential for intracellular calcium homeostasis and the generation of reactive oxygen species, both of which heavily impact cell integrity and viability. In AD, the derangement of mitochondrial function and the subsequent death of neural tissue have already been associated with the accumulation of amyloid-beta. However, increasing evidence suggests miRNAs that regulate the expression of mitochondrial proteins may also contribute to the development of AD.

miRNAs can cause mitochondrial damage by directly disrupting the oxidant-antioxidant balance. MiR-9-5p, an activator of the NRF2/KEAP1 pathway, plays a critical role in suppressing cancer and inflammation by promoting the expression of antioxidants such as HO-1, SOD-1, and GCLC (Liu et al., 2020). MiR-9-5p can also directly downregulate expression of GSK-3 β which has been linked to caspase activation and subsequent mitochondria-dependent neuronal apoptosis (Yang et al., 2017; Liu et al., 2020). In the CSF and frontal cortex of AD patients, however, miR-9-5p levels are reported to be decreased (Sørensen et al., 2016; Liu et al., 2020). Without the antioxidant and cytoprotective properties of miR-9-5p, oxidative stress can result in significant neuronal damage. This is further supported by the study of Liu et al. (2020) where the overexpression of miR-9-5p in an Aβ25-35-induced HT22 cell model reversed mitochondrial dysfunction and decreased cell apoptosis. MiR-146a-5p, on the other hand, is elevated in AD and has a pro-apoptotic effect as it targets TIGAR, a fructose-2,6-bisphosphatase (Lei et al., 2021). TIGAR’s activity normally shifts cellular glucose metabolism in favor of the pentose phosphate pathway which ultimately increases the availability of reduced glutathione (GSH), a potent antioxidant against mitochondrial hydrogen peroxide (Lei et al., 2021).

The dysregulation of miRNAs in AD may also compromise neuron survival by affecting mitochondrial function. For instance, miR-423-5p, which is upregulated in AD, has been observed to directly inhibit the translation of the mitochondrial cytochrome C mRNA, COX6A2, halting ATP production by the electron transport chain (Siengdee et al., 2015). Similarly, miR-34a downregulates the expression of NDUFC2, SHDC, and COX10 which have been correlated with severely decreased oxidative phosphorylation (Sarkar et al., 2016). Decreased miR-195 alters the expression of outer mitochondrial membrane mitofusin-2, a GTPase responsible for coordinating mitochondrial fusion-fission reactions (Zhang R. et al., 2016). Elevated miR-204-5p increases downregulation of TRPML1, or mucolipin-1, which is a lysosomal protein responsible for the removal of damaged or aged mitochondria by autophagy (Zhang X. et al., 2016; Zhang L. et al., 2021). Lastly, some miRNAs such as miR-29b-3p can promote neuronal apoptosis by inhibiting the anti-apoptotic factors BCL2L2 and MCL-1 (Lungu et al., 2013).