Alzheimer's disease (AD) is a devastating neurodegenerative disorder characterized by progressive memory loss and cognitive decline. A key pathological hallmark of AD is mitochondrial dysfunction, with accumulating evidence suggesting its crucial role in disease initiation and progression. Damaged mitochondria accumulate in both sporadic and familial AD, as well as in animal models. This mitochondrial dysfunction contributes to energetic stress, leading to the formation of amyloid-β (Aβ) oligomers and hyper-phosphorylated tau (pTau) – key hallmarks of AD pathology. Impaired mitochondrial biogenesis, potentially due to decreased PGC-1α expression, further exacerbates synaptic dysfunction and neuronal degeneration. Mitochondrial dysfunction also disrupts cytosolic calcium homeostasis, contributing to neuronal death. Critically, mitochondrial impairment has been shown to precede Aβ accumulation in AD mouse models, suggesting its early involvement in the disease process. Therefore, bolstering mitochondrial health is considered a promising therapeutic strategy for AD. Mitophagy, a selective autophagy pathway, is responsible for removing damaged or dysfunctional mitochondria. Emerging research indicates that mitophagy is impaired in AD, resulting in the accumulation of dysfunctional mitochondria and contributing to cognitive decline. Conversely, enhancing mitophagy has been shown to reduce Aβ plaques and tau tangles in cellular and animal models of AD, and improve memory function. Several mitophagy receptors, including ubiquitin-binding receptors (OPTN, p62, NDP52, NBR1) and ubiquitin-independent receptors (NIX/BNIP3L, FUNDC1, AMBRA1, Bcl-2-L-13, PHB2), mediate this process. These receptors bind to LC3 family proteins via LIR motifs to facilitate the engulfment of mitochondria by autophagosomes. This study aimed to identify novel mitophagy activators and investigate their therapeutic potential in AD. A high-throughput screen of 2024 FDA-approved drugs and drug candidates was conducted to discover compounds capable of inducing mitophagy.

Extensive research has linked mitochondrial dysfunction to the pathogenesis of Alzheimer's disease. Studies have shown that impaired mitochondrial function leads to increased oxidative stress, altered calcium homeostasis, and ultimately, neuronal death. The accumulation of damaged mitochondria further contributes to the production of amyloid-beta plaques and neurofibrillary tangles, the characteristic hallmarks of AD. Moreover, the role of mitophagy, the selective autophagy of mitochondria, has gained significant attention in recent years. Mitophagy is essential for maintaining mitochondrial quality control and removing damaged mitochondria, thereby preventing the accumulation of dysfunctional mitochondria and mitigating oxidative stress. Several studies have demonstrated that impaired mitophagy contributes to AD pathogenesis, while enhancing mitophagy has shown promising therapeutic effects in preclinical models of AD, leading to a reduction in Aβ plaques, tau tangles and improved cognitive function. The identification and characterization of mitophagy receptors and the mechanisms regulating mitophagy are critical to developing effective AD therapies.

This study employed a multi-faceted approach combining high-throughput drug screening, cellular and molecular biology techniques, and in vivo studies using an AD mouse model. **High-Throughput Drug Screening:** A library of 2024 FDA-approved drugs or drug candidates was screened using a HEK293T cell line stably expressing mt-Keima, a fluorescent reporter of mitophagy. The screen identified compounds that increased mitophagy levels by >1.5-fold compared to controls. **Cellular Assays:** The effects of identified mitophagy inducers, particularly UMI-77, were further investigated in various cell lines (HEK293T, HeLa, SH-SY5Y, U2OS) using a range of techniques. These included assessing mitochondrial membrane potential using JC-1, evaluating mitophagy using mt-Keima and LysoTracker Green staining, monitoring mitochondrial protein degradation by western blotting, and examining autophagic flux. Transmission electron microscopy (TEM) was used to visualize autophagosomes containing mitochondria. The role of apoptosis was assessed using a pan-caspase inhibitor (Z-VAD-fmk) and caspase-3 activity assays. The involvement of specific mitophagy pathways was explored by inhibiting lysosomes (E64D, NH4Cl/Leupeptin) and proteasomes (MG-132). Knockdown experiments using siRNA and shRNA were performed to investigate the role of key proteins in UMI-77-induced mitophagy. Proximity ligation assays (PLA) were used to examine the interaction between MCL-1 and LC3A. Site-directed mutagenesis was used to analyze the role of specific LIR motifs in MCL-1. **In Vivo Studies:** The effects of UMI-77 were investigated in an APP/PS1 mouse model of AD. Mice were treated with UMI-77 (10 mg/kg) via intraperitoneal injection and assessed for cognitive function using the Morris water maze. Brain tissues were analyzed for Aβ levels using ELISA and immunohistochemistry (IHC) to visualize Aβ plaques and astrocyte activation (GFAP). Cytokine levels (TNFα, IL-6, IL-10) were measured by ELISA to assess neuroinflammation. Electron microscopy was used to evaluate mitochondrial morphology. **Molecular Cloning and other techniques:** Site-directed mutagenesis was employed to create MCL-1 mutants lacking specific LIR motifs. Immunoprecipitation assays were used to investigate protein-protein interactions, particularly between MCL-1 and LC3 family proteins. A GST-pull down assay was performed to confirm the direct interaction between purified MCL-1 and LC3A proteins. Stable cell lines with doxycycline-inducible MCL-1 overexpression were generated to investigate the effects of MCL-1 overexpression on mitophagy. AAV-mediated gene delivery was used for MCL-1 overexpression in the hippocampus of APP/PS1 mice.

This research revealed several key findings: 1. **UMI-77 as a Mitophagy Inducer:** A high-throughput screen identified UMI-77, an MCL-1 inhibitor, as a potent mitophagy inducer. Importantly, UMI-77 induced mitophagy independently of mitochondrial damage and apoptosis, even at sub-lethal doses. The combination of UMI-77 and CCCP (a mitochondrial uncoupler) synergistically increased mitophagy levels. 2. **MCL-1 as a Mitophagy Receptor:** Further mechanistic studies demonstrated that MCL-1, the target of UMI-77, is a crucial mitophagy receptor. Knockdown of MCL-1 abolished UMI-77-induced mitophagy, while MCL-1 overexpression induced mitophagy and mitochondrial fragmentation. MCL-1 contains two LIR motifs (LIR261-264 and LIR318-321) that are crucial for interaction with LC3A and the induction of mitophagy. The LIR261-264 motif was found to be particularly important for this interaction, as demonstrated by site-directed mutagenesis experiments. 3. **UMI-77-induced Mitophagy is ATG5-dependent, but independent of other mitophagy receptors:** UMI-77-induced mitophagy was shown to be dependent on ATG5, but independent of other canonical mitophagy receptors (NDP52, p62, NBR1, TAX1BP1, FUNDC1, BNIP3, NIX) and Bax. The interaction between MCL-1 and LC3A was enhanced in both wild-type and quadruple knockout cells (NBR1, TAX1BP1, p62, NDP52). Knockdown of Bax surprisingly enhanced UMI-77-induced mitophagy. 4. **MCL-1 in OGD-induced Mitophagy:** MCL-1 was also found to be crucial for oxygen-glucose deprivation (OGD)-induced mitophagy. Knockdown of MCL-1 blocked OGD-induced mitophagy and mitochondrial fragmentation. The LIR261-264 motif of MCL-1 was necessary for OGD-induced mitophagy but not for mitochondrial fragmentation. 5. **In Vivo Efficacy in APP/PS1 Mice:** In vivo studies using APP/PS1 mice showed that UMI-77 treatment significantly improved cognitive function (Morris water maze), reduced insoluble Aβ1-42 levels, decreased Aβ plaque size, inhibited astrocyte activation, reduced neuroinflammation, and improved mitochondrial morphology in neurons. Overexpression of MCL-1 in the hippocampus of APP/PS1 mice similarly improved cognitive function and reduced Aβ plaques.

This study provides compelling evidence for a novel role of MCL-1 as a mitophagy receptor and demonstrates the therapeutic potential of targeting MCL-1 to enhance mitophagy for treating Alzheimer's disease. The findings that UMI-77, an MCL-1 inhibitor, induces mitophagy independently of apoptosis and mitochondrial damage is particularly significant, as it suggests a potential therapeutic window where mitophagy can be selectively enhanced without inducing detrimental apoptotic cell death. The identification of the LIR motifs in MCL-1 that mediate the interaction with LC3A provides a detailed mechanistic understanding of this process. The observation that UMI-77-induced mitophagy is ATG5-dependent, but independent of other known mitophagy receptors, highlights the unique nature of this pathway. The in vivo data in the APP/PS1 mouse model strongly supports the therapeutic potential of UMI-77 and MCL-1 targeting for Alzheimer's disease. The observed improvements in cognitive function, reduction in Aβ plaques and neuroinflammation, and restoration of mitochondrial morphology demonstrate a significant therapeutic effect. The data further suggests that MCL-1 may play a broader role in neuronal health and function, beyond its established role in apoptosis.

This study identifies MCL-1 as a novel mitophagy receptor and demonstrates that pharmacological inhibition of MCL-1 using UMI-77 promotes mitophagy and reverses Alzheimer's disease pathology in an APP/PS1 mouse model. These findings provide strong evidence for MCL-1 as a promising drug target for AD and support the therapeutic potential of mitophagy induction. Future research could focus on optimizing UMI-77 or developing more selective MCL-1 inhibitors for AD therapy, as well as exploring the potential interplay between MCL-1-mediated mitophagy and other cellular pathways involved in AD pathogenesis. Furthermore, investigating the role of MCL-1's distinct LIR motifs in different mitophagy contexts may reveal potential avenues for more precise therapeutic intervention.

While this study provides strong evidence for the therapeutic potential of MCL-1 inhibition in Alzheimer's disease, several limitations should be considered. The study primarily used an APP/PS1 mouse model, which may not fully capture the complexity of human AD. Further studies in other AD models and potentially in human cells are needed to confirm these findings. Although the study demonstrated that UMI-77 induced mitophagy independently of apoptosis, the possibility of off-target effects cannot be completely ruled out. Future studies should investigate the specificity of UMI-77 for MCL-1 and its potential impact on other cellular processes. Moreover, the long-term effects of UMI-77 treatment and its potential toxicity require further investigation before clinical translation.