LEVs are identified as a critical vesicle subtype mediating the effects of SIRT2 knockdown and support a cell-free therapeutic strategy for AD centered on EVs-driven metabolic reprogramming of microglia.
Abstract
Background
Current therapies for Alzheimer's disease (AD) offer only symptomatic relief, highlighting the urgent need for disease-modifying approaches capable of halting or reversing neurodegeneration. Extracellular vesicles (EVs) have attracted growing interest as therapeutic vehicles owing to their inherent capacity to bypass the blood-brain barrier and deliver complex biological cargo to the central nervous system.
Methods
Here, we examined whether large EVs (LEVs) derived from microglia with stable Sirtuin-2 knockdown (SIRT2-KD) confer the neuroprotective effects associated with SIRT2 inhibition. LEVs harvested from SIRT2-KD microglia were administered intranasally to APP/PS1 mice. We assessed microglial uptake of LEVs, along with subsequent changes in cellular metabolism, migration toward amyloid-beta (Aβ) plaques, phagocytic activity, and downstream pathological and behavioral outcomes. Proteomic and acetylomic profiling were employed to characterize the molecular cargo of LEVs-SIRT2-KD.
Results
LEVs-SIRT2-KD were readily internalized by microglia in vivo following intranasal delivery. Uptake of these vesicles markedly enhanced microglial bioenergetics, driving coordinated upregulation of both oxidative phosphorylation and glycolysis. This metabolic shift was accompanied by improved microglial recruitment to Aβ plaques and increased phagocytic clearance. Consequently, treated mice showed reduced Aβ plaque deposition, restored synaptic integrity, and reversal of cognitive deficits. Proteomic and acetylomic analyses revealed that LEVs-SIRT2-KD are selectively enriched in proteins and acetylation modifications linked to energy metabolism and phagocytic function, offering a mechanistic basis for the observed metabolic reprogramming.
Conclusion
Together, these results identify LEVs as a critical vesicle subtype mediating the effects of SIRT2 knockdown and support a cell-free therapeutic strategy for AD centered on EVs-driven metabolic reprogramming of microglia.
BACKGROUND
Alzheimer's disease (AD) is a significant global health challenge characterized as a multifactorial neurodegenerative disorder, involving amyloid-β (Aβ) and Tau aggregation, neuroinflammation and progressive neuronal injury. While Amyloid-targeted therapies have achieved a breakthrough in prevention of Aβ aggregation, the strategies face notable limitations in achieving curative outcomes and management of amyloid-independent central nervous system (CNS) dysfunction. Consequently, targeting microglia, the central immune cells of the brain, has emerged as a promising strategy to enhance the specificity and efficacy of AD interventions.
MAIN BODY
Accumulating evidence indicates microglial dysfunction is not a passive immune bystander of AD, but serves as a critical mechanistic nexus linking Aβ accumulation and AD symptomatic phenotype. This review critically examines the "next generation" of microglial therapeutics, moving beyond broad immunosuppression to precision phenotype modulation. We highlight breakthrough strategies in recent years including immune reconstitution, metabolic reprogramming, nanomaterial-mediated drug delivery, and the revolutionary potential of iPSC-derived microglia replacement. By elucidating the rationale underlying the specific strategies based on microglial biofunction and potential molecular mechanism in AD pathology, we provide an overview of current development of clinical trials and cutting-edge modalities aimed at restoring microglial homeostasis, affording an opportunity to alter the AD trajectory.
CONCLUSION
This review aims to delineate the path from bench to bedside and propose promising pathways to overcome current bottlenecks in AD drug development.
Alzheimer’s disease (AD) remains a major neurodegenerative challenge with limited therapeutic options. Microglia, the resident immune cells of the central nervous system, shape key pathological processes in AD, including amyloid-beta (Aβ) clearance, neuroinflammation, tau pathology, and synaptic homeostasis. Accordingly, microglial receptors that regulate microglial sensing, phagocytosis, and inflammatory signaling have emerged as candidates for disease-modifying interventions. However, translation from preclinical discovery to clinical benefit is impeded by multiple barriers, including stage-dependent receptor functions, peripheral off-target effects, compensatory signaling within interconnected receptor networks, biomarker deficiencies, and species differences, all of which are key bottlenecks detailed in this review. We summarize preclinical and clinical progress in therapeutics targeting microglial receptors; analyze these critical translational bottlenecks; and discuss potential strategies including precision delivery, humanized experimental systems, and biomarker-forward trial designs, with the goal of supporting rigorously designed and biomarker-informed clinical translation.
Alzheimer’s disease (AD) is characterized by progressive metabolic failure, impaired mitochondrial function, and diminished adaptive stress responses, highlighting the need for disease-modifying therapies that restore cellular resilience rather than target downstream pathology. Here, we report the discovery and preclinical validation of C273, a translationally optimized, brain-penetrant mitochondrial complex I (mtCI) modulator developed through medicinal chemistry optimization of our first-generation compounds. C273 retained nanomolar neuroprotective activity against Aβ-induced toxicity while exhibiting favorable drug-like properties, including high oral bioavailability, efficient brain penetration, microsomal stability, minimal CYP and off-target pharmacology liabilities, and selective mild modulation of mtCI. Mechanistic studies demonstrated that C273 activated AMP-activated protein kinase (AMPK) and coordinated antioxidant, autophagic, anti-inflammatory, and mitochondrial quality-control pathways in cultured cells and mouse brain. These responses were absent in AMPKα1/α2-deficient cells, establishing AMPK as an essential mediator, while rotenone pretreatment abolished C273-mediated neuroprotection, supporting engagement of the mtCI quinone-binding site. Repeated administration to wild-type mice for 30 days produced no detectable cardiac or hepatic toxicity. Importantly, C273 activated the same neuroprotective pathways and reduced Aβ and p-Tau levels in induced pluripotent stem cell-derived cerebral organoids from patients with sporadic AD. Together, these findings establish mild modulation of mtCI as a therapeutic strategy to restore metabolic resilience and identify C273 as a promising disease-modifying candidate for AD treatment.
Sergey Trushin, Thi Kim Oanh Nguyen, Mark Ostroot et al.· npj Drug Discovery· 0 citations
Alzheimer's disease (AD) is a progressive neurodegenerative disorder characterized by the accumulation of amyloid‐β (Aβ) plaques and tau (τ) ‐related neurofibrillary tangles, often exacerbated by dysfunctional cellular clearance mechanisms. This manuscript explores the pivotal role of autophagy impairment in AD pathogenesis, with a specific focus on the AMPK/mTOR signaling axis as a primary regulatory pathway. Findings revealed that while mTOR overactivation suppresses autophagic flux and promotes the buildup of toxic protein aggregates, the activation of AMPK serves to restore homeostatic degradation processes. The review highlights that various pharmacological agent including rapamycin, metformin, trehalose, and curcumin, as well as repurposed drugs like lithium and statins can effectively enhance autophagy to ameliorate cognitive decline and neuroinflammation. Furthermore, herbal formulations such as Danggui Shaoyao San and phytoconstituents like Icariin demonstrate significant neuroprotective potential by modulating these same molecular pathways. Targeting autophagy represents a translationally viable approach for combating AD progression, with drug repurposing offering a time‐efficient and cost‐effective strategy. To advance these findings, future research should prioritize large‐scale clinical trials to validate the efficacy of autophagy‐inducing agents in human subjects. Additionally, investigating synergistic combinations of traditional bioactives with synthetic drugs and utilizing innovative delivery systems, such as intranasal nanotechnology‐based platforms to bypass the blood‐brain barrier, represents a promising frontier for developing effective, multi‐targeted treatments against AD.
Ayush Dubey, Ayush Chaurasia, Zeeshan Ansari et al.· Drug development research (P...· 0 citations
Growing evidence suggests that impaired phagocytosis/autophagy and histone deacetylase 3 (HDAC3)‐mediated microglial activation contribute significantly to Alzheimer's disease (AD) pathogenesis by promoting pathological protein aggregation. The ketogenic diet (KD) has demonstrated neuroprotective effects in AD, potentially through modulation of phagocytosis, autophagy, and inflammation. However, the underlying mechanisms remain unclear. Here, we report that a 12‐week KD treatment ameliorated cognitive deficits, reduced amyloid‐β (Aβ) deposition and tau hyperphosphorylation, and attenuated neuroinflammation in male APP/PS1 mice. Mechanistic studies revealed that these effects were associated with the modulation of microglial HDAC3. Specifically, KD downregulated microglial HDAC3, thereby enhancing Aβ phagocytosis, activating AMPK–ULK1‐dependent autophagy, and restraining NLRP3 inflammasome activation, thereby promoting pathologic protein clearance. Given that β‐hydroxybutyrate (BHB) is a major circulating ketone body produced during KD, we tested whether BHB recapitulates these effects in vitro. In Aβ‐induced BV2 microglia, BHB mimicked the neuroprotective effects by downregulating HDAC3, which markedly enhanced Aβ clearance and suppressed inflammatory responses; these neuroprotective effects were largely abrogated by HDAC3 overexpression. To further confirm the causal role of microglial HDAC3 in mediating KD effects, we performed microglia‐specific HDAC3 knockdown via AAV11‐Iba1‐shHDAC3. Notably, this manipulation enhanced both Aβ phagocytosis and autophagic degradation while inhibiting NLRP3 activation and contributed to neuroprotection. Overall, our findings suggest a novel mechanism by which KD exerts neuroprotective effects through modulation of the microglial HDAC3–AMPK–ULK1 axis and highlight microglia‐specific HDAC3 inhibition as a promising therapeutic strategy for AD that simultaneously enhances Aβ clearance and attenuates neuroinflammation.
Mingxiao Zheng, Gabriele Loers, Sheng-Nan Lin et al.· Food Frontiers· 0 citations
Microglia are central mediators of Alzheimer’s disease (AD) pathogenesis, yet the mechanisms driving disease-associated microglial states and their therapeutic modulation remain poorly understood. Here, we integrated single-nucleus transcriptomic datasets across the AD spectrum and identified disease- and lipid-associated microglia (DLaM) as a major AD-enriched population linked to genetic risk, neuropathology and cognitive decline. To model this state experimentally, we screened AD-relevant perturbations in human induced pluripotent stem cell (hiPSC)-derived microglia and found that ferric ammonium citrate (FAC) reproducibly induced a DLaM-like state characterized by lipid accumulation, lysosomal dysfunction and impaired Aβ phagocytosis. Using a transcriptomics-based state-reversion screen, we identified LY2090314 as a potent modulator that restored microglial function and induced a distinct lysosomal-metabolic state. These findings establish a framework for transcriptomic disease-state-guided therapeutic discovery in AD.
Gerardo Garcia-Diaz Barriga, Daniel Rosebrock, Henrik Renner et al.· bioRxiv· 0 citations
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