A rogue microRNA sabotages the neuron’s mitochondrial cleanup crew in Alzheimer’s disease

Deep inside the neurons of an aging brain, mitochondria are quietly failing. These organelles, which supply the enormous energy demands of neural firing and calcium buffering, accumulate damage in Alzheimer’s disease earlier than the classic plaques and tangles appear. Normally, cells dispose of broken mitochondria through mitophagy, a specialized form of autophagy that tags defective organelles for lysosomal destruction. When that cleanup crew falters, damaged mitochondria pile up, spewing reactive oxygen species and draining ATP, which accelerates neurodegeneration. A new study published in Frontiers in Molecular Neuroscience has now identified a molecular saboteur at the heart of this failure: a small RNA molecule called miR-615-3p that suppresses a key mitochondrial quality-control protein, effectively jamming the neuron’s ability to initiate mitophagy.

The research team, led by Yang Jing and colleagues at Nanchong Psychosomatic Hospital in China, focused on miR-615-3p because it has repeatedly surfaced in Alzheimer’s disease datasets. MicroRNAs are tiny non-coding RNAs, roughly 22 nucleotides long, that fine-tune gene expression by binding to the 3′-untranslated region of target messenger RNAs, either blocking translation or promoting mRNA degradation. Previous work had shown that miR-615-3p is abnormally elevated in the brains and biofluids of Alzheimer’s patients, and that it appears among a small set of microRNA markers with detection accuracy exceeding 84 percent. It is also one of the few microRNAs found dysregulated in both Alzheimer’s and Parkinson’s disease. Yet its precise contribution to mitochondrial dysfunction remained unclear until now.

To dissect the mechanism, the researchers used HT22 cells, a mouse hippocampal neuronal cell line, and treated them with amyloid-β1-42, the peptide that forms the hallmark plaques of Alzheimer’s disease. This created an in vitro model that recapitulates key features of the disease, including amyloid accumulation, oxidative stress, and mitochondrial damage. The team then manipulated miR-615-3p levels using synthetic mimics to overexpress it and inhibitors to knock it down, while simultaneously measuring cell viability, apoptosis, ATP production, mitochondrial membrane potential, reactive oxygen species, lactate dehydrogenase release, and inflammatory cytokines such as IL-1β and IL-18.

The results were striking. When the researchers overexpressed miR-615-3p in amyloid-stressed neurons, cell viability and ATP levels dropped significantly, while apoptosis, amyloid-β accumulation, lactate dehydrogenase release, and reactive oxygen species production all increased. Mitochondrial membrane potential deteriorated, and inflammatory cytokine levels climbed. Crucially, the team observed elevated levels of mitophagy markers, including the LC3-II to LC3-I ratio, Parkin, PINK1, and p62. At first glance, this might suggest that mitophagy was being activated. But a closer look revealed something more insidious.

Using bafilomycin A1, a lysosomal inhibitor that blocks autophagic degradation, the researchers performed an autophagic flux assay. In control and model cells, bafilomycin A1 treatment caused LC3-II and p62 levels to accumulate further, indicating that the autophagic machinery was still functional and that the lysosomal blockage was being detected. But in miR-615-3p-overexpressing cells, bafilomycin A1 failed to further increase LC3-II and p62 levels. This meant that autophagic flux was already attenuated or completely blocked in these cells. The accumulation of mitophagy-related proteins was not a sign of increased autophagosome formation but rather of impaired autophagosome-lysosome degradation, a state the researchers describe as autophagy stress.

Having established that miR-615-3p disrupts mitophagy, the team turned to bioinformatics to identify its molecular target. Using the TargetScan and miRDB databases, they predicted that miR-615-3p binds to a conserved site within the 3′-untranslated region of SESN2 messenger RNA. SESN2, also known as Sestrin2, is a stress-inducible protein that regulates cellular metabolism, oxidative stress responses, and autophagy. The researchers confirmed this interaction using a dual-luciferase reporter assay, in which co-transfection of miR-615-3p mimics with a wild-type SESN2 3′-untranslated region construct significantly reduced luciferase activity, while a mutant construct lacking the binding site showed no such reduction. This proved that miR-615-3p directly targets SESN2.

The mechanistic picture became clearer when the team examined the AMPK/ULK1 signaling axis. SESN2 is known to activate AMP-activated protein kinase, or AMPK, which in turn phosphorylates ULK1 at the Ser555 site, triggering the autophagic machinery. The researchers found that miR-615-3p overexpression significantly reduced phosphorylation of both AMPK at Thr172 and ULK1 at Ser555, indicating genuine suppression of the cascade rather than an inhibitory phosphorylation event. Co-transfection with SESN2 siRNA produced similar reductions, while SESN2 overexpression or miR-615-3p inhibition restored phosphorylation levels. This established a clear regulatory chain: miR-615-3p downregulates SESN2, which suppresses AMPK/ULK1 phosphorylation, which blocks the initiation of mitophagy.

The therapeutic implications became apparent when the researchers overexpressed SESN2 in the amyloid-stressed neurons. SESN2 overexpression reversed nearly all the damage caused by amyloid-β1-42 treatment, restoring mitochondrial membrane potential and ATP levels, reducing reactive oxygen species and lactate dehydrogenase release, lowering LC3-II, Parkin, PINK1, and p62 levels, and suppressing inflammatory cytokines. Cell viability improved and apoptosis decreased. Conversely, knocking down SESN2 with siRNA exacerbated the damage, confirming that SESN2 is functionally protective in this context. The findings align with previous reports that SESN2 promotes mitophagy through AMPK activation in hypoxic nerve injury models, and that SESN2 deletion weakens mitophagy signaling and aggravates neuronal damage.

The study also highlights a paradox in mitochondrial quality control that has been gaining attention in Alzheimer’s research. Simply upregulating mitophagy-related proteins without restoring the complete autophagic flow may lead to autophagy accumulation and increased cytotoxicity. The activation of Parkin, for instance, has been shown to reduce amyloid load and improve synaptic plasticity in mouse models, and exercise intervention can enhance mitophagy by upregulating Parkin. But the new findings suggest that the real therapeutic goal is not merely to increase the expression of mitophagy markers but to restore the full autophagic flux from autophagosome formation through lysosomal degradation. This distinction matters because lysosomal dysfunction is increasingly recognized as a primary trigger of autophagic build-up and neuronal death in Alzheimer’s disease.

The researchers acknowledge several limitations. The study was conducted only in the HT22 cell line, which does not fully recapitulate the molecular features of primary or human neurons. Validation in primary neurons and in vivo Alzheimer’s models, such as APP/PS1 transgenic mice, will be necessary to confirm the pathological relevance of the miR-615-3p/SESN2/AMPK/ULK1 axis. The team also focused exclusively on the PINK1/Parkin-mediated mitophagy pathway and did not examine whether receptor-mediated mitophagy participates in the same regulatory network. Additionally, miR-615-3p may have multiple target genes beyond SESN2, and future studies using RNA sequencing or proteomics will be needed to identify other potential targets. Despite these caveats, the study provides a compelling mechanistic framework and suggests that developing miR-615-3p antagonists or SESN2 agonists could represent a promising therapeutic strategy for Alzheimer’s disease, one that targets the mitochondrial quality-control failure at the root of neurodegeneration rather than its downstream consequences.

Subject of Research: miR-615-3p targets SESN2 to inhibit neuronal mitophagy in an in vitro Alzheimer’s disease model by regulating the AMPK/ULK1 axis

Article Title: miR-615-3p targets SESN2 to inhibit neuronal mitophagy in an in vitro Alzheimer’s disease model by regulating the AMPK/ULK1 axis

Article References: Jing, Y., Li, D., Chen, L., Li, S., & Yuan, X. (2026). miR-615-3p targets SESN2 to inhibit neuronal mitophagy in an in vitro Alzheimer’s disease model by regulating the AMPK/ULK1 axis. Frontiers in Molecular Neuroscience, 19, Article 1873619. https://doi.org/10.3389/fnmol.2026.1873619

Image Credits: AI Generated

DOI: 10.3389/fnmol.2026.1873619

Keywords: miR-615-3p, targets, SESN2, inhibit, neuronal, mitophagy, vitro, Alzheimer, disease, model, regulating, AMPK