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As current treatments for Alzheimer's disease (AD) lack disease‐modifying interventions, novel therapies capable of restraining AD progression and maintaining better brain function have great significance. Anti‐inflammatory extracellular vesicles (EVs) derived from human induced pluripotent stem cell (hiPSC)‐derived neural stem cells (NSCs) hold promise as a disease‐modifying biologic for AD. This study directly addressed this issue by examining the effects of intranasal (IN) administrations of hiPSC‐NSC‐EVs in 3‐month‐old 5xFAD mice. IN administered hiPSC‐NSC‐EVs incorporated into microglia, including plaque‐associated microglia, and encountered astrocyte soma and processes in the brain. Single‐cell RNA sequencing revealed transcriptomic changes indicative of diminished activation of microglia and astrocytes. Multiple genes linked to disease‐associated microglia, NOD‐, LRR‐, and pyrin domain‐containing protein 3 (NLRP3)‐inflammasome and interferon‐1 (IFN‐1) signalling displayed reduced expression in microglia. Adding hiPSC‐NSC‐EVs to cultured human microglia challenged with amyloid‐beta oligomers resulted in similar effects. Astrocytes also displayed reduced expression of genes linked to IFN‐1 and interleukin‐6 signalling. Furthermore, the modulatory effects of hiPSC‐NSC‐EVs on microglia in the hippocampus persisted 2 months post‐EV treatment without impacting their phagocytosis function. Such effects were evidenced by reductions in microglial clusters and inflammasome complexes, concentrations of mediators, and end products of NLRP3 inflammasome activation, the expression of genes and/or proteins involved in the activation of p38/mitogen‐activated protein kinase and IFN‐1 signalling, and unaltered phagocytosis function. The extent of astrocyte hypertrophy, amyloid‐beta plaques, and p‐tau were also reduced in the hippocampus. Such modulatory effects of hiPSC‐NSC‐EVs also led to better cognitive and mood function. Thus, early hiPSC‐NSC‐EV intervention in AD can maintain better brain function by reducing adverse neuroinflammatory signalling cascades, amyloid‐beta plaque load, and p‐tau. These results reflect the first demonstration of the efficacy of hiPSC‐NSC‐EVs to restrain neuroinflammatory signalling cascades in an AD model by inducing transcriptomic changes in activated microglia and reactive astrocytes. [ABSTRACT FROM AUTHOR]

Background: Alzheimer's disease (AD) is characterized by the accumulation of amyloid‐beta (Aβ) in the extracellular space, which leads to various adverse effects such as oxidative stress, neuroinflammation, mitochondrial dysfunction, tau phosphorylation, synapse loss, and neurodegeneration. Therefore, therapeutic interventions that can reduce Aβ‐toxicity and slow down the progression of cognitive dysfunction in AD have significance. One promising approach is to use extracellular vesicles (EVs) that are released by neural stem cells (NSCs) derived from human induced pluripotent stem cells (hiPSCs). These EVs contain therapeutic proteins and miRNAs that can help protect against Aβ‐induced pathological changes in the brain, providing a potential avenue for neuroprotection. Method: This study first isolated and characterized EVs secreted from hiPSC‐NSCs by chromatographic methods. Next, mature human neurons generated from 2 different hiPSC lines were exposed to 1‐µM Aβ42 oligomers with different concentrations of hNSC‐EVs. The protective effects of EVs on neurons exposed to Aβ were quantified using MTT and live/dead cell assays and counting of surviving MAP‐2+ neurons. The extent of oxidative stress was investigated via measurements of malondialdehyde (MDA) and protein carbonyls (PCs) and the expression of genes encoding iNos and Cox2. Furthermore, the expression of genes and/or proteins linked to pro‐apoptotic (Bad, Bax) and antiapoptotic (Bcl2) effects, mitochondrial respiratory chain (Nudfs6, Nudfs7, Sdha, Sdhb, Cyc1, Bcs11, Cox6a1, Atp6ap1), autophagy (Beclin 1 and MAPILC3B) were quantified. Total tau and p‐tau were also measured. Result: The addition of hiPSC‐NSC‐EVs to mature human neuronal cultures exposed to Aβ reduced the extent of Aβ‐induced neurodegeneration. When human neurons were exposed to Aβ‐42 alone, it increased pro‐apoptotic genes (Bad, Bax) and reduced the expression of an antiapoptotic gene (BCL‐2). However, adding hNSC‐EVs to Aβ‐42 exposed neurons normalized the expression of pro‐ and antiapoptotic genes to control levels, reduced oxidative stress markers, prevented Aβ‐42‐induced hyperactivation of mitochondria and decreased autophagy, reduced the phosphorylation of tau. Conclusion: Treating human neurons with hiPSC‐NSC‐EVs can significantly decrease the various pathological changes caused by Aβ exposure. Supported by a grant from the National Institute for Aging (1RF1AG074256‐01A1 to A.K.S.) [ABSTRACT FROM AUTHOR]

Background: Current treatments for Alzheimer's disease (AD) lack disease‐modifying interventions. Hence, novel therapies capable of restraining AD progression and maintaining better brain function for extended periods after the initial diagnosis have great significance. Extracellular vesicles (EVs) from human induced pluripotent stem cell (hiPSC)‐derived neural stem cells (NSCs) are attractive in this context due to their robust antiinflammatory properties. Method: Three‐month‐old 5x familial AD (5xFAD) mice received IN administration of hiPSC‐NSC‐EVs (∼30 billion EVs) or the vehicle (weekly for two weeks). Seventy‐two hours later, a cohort of mice was euthanized, fresh microglia were isolated from the brain, and the expression of genes linked to DAM and NLRP3‐inflammasome signaling was examined via scRNA‐sequencing. Another cohort of 5xFAD mice was investigated for cognitive and mood function commencing one‐month post‐EV/Veh administration and euthanized two months post‐EV/Veh administration to assess the effects on long‐term progressive increases in neuroinflammation, amyloid‐beta plaques, and phosphorylated tau (p‐tau). Result: Microglia in all brain regions, including plaque‐associated microglia, internalized hiPSC‐NSC‐EVs entering the brain. Single‐cell RNA sequencing at 72 hours post‐EV administration revealed microglia with transcriptomic changes indicative of diminished activation. Multiple genes linked to disease‐associated microglia, NOD‐, LRR‐ and pyrin domain‐containing protein 3 (NLRP3)‐inflammasome and interferon‐1 signaling displayed reduced expression. Adding hiPSC‐NSC‐EVs to cultured human microglia challenged with amyloid‐beta oligomers also resulted in similar effects. Furthermore, the modulatory effects of hiPSC‐NSC‐EVs on microglia in the hippocampus persisted even two months post‐EV treatment, evidenced by reductions in microglial clusters and inflammasome complexes, concentrations of mediators, and end products of NLRP3 inflammasome activation, the expression of genes and proteins involved in p38/mitogen‐activated protein kinase hyperactivation, and proteins linked to activation of interferon‐1 signaling. The hippocampus also exhibited reduced amyloid‐beta plaques and p‐tau. Such modulatory effects of hiPSC‐NSC‐EVs also led to better cognitive and mood function. Conclusion: Early hiPSC‐NSC‐EV intervention in AD can maintain better brain function by reducing adverse neuroinflammatory signaling cascades, amyloid‐beta plaque load, and p‐tau. Supported by a grant from the National Institute for Aging (1RF1AG074256‐01A1 to A.K.S.) [ABSTRACT FROM AUTHOR]

Background: Chronic neuroinflammation is one of the most conspicuous features of Alzheimer's disease (AD). While microglia play a vital role in phagocytosing amyloid beta (Aβ) and restraining plaque extension, disease‐associated microglia are known to perpetuate neuroinflammation. Previous studies have shown that ∼80% microglia elimination through a month‐long colony‐stimulating factor 1 receptor (CSF1R) inhibition in the advanced stage of AD in 5X Familial AD (5XFAD) mice does not reduce Aβ levels or plaques but restrains synapse loss and neurodegeneration. On the other hand, prolonged CSF1R inhibition has been shown to prevent parenchymal plaque development. However, the immediate effects of transient CSF1R inhibition in the early stage of AD on residual microglial morphology and their metabolic fitness are unknown. Method: We investigated the impact of transient CSF1R inhibition in three‐month‐old 5XFAD mice, a stage at which neuroinflammation has commenced, but Aβ plaques were minimal. Microglia from the hippocampus and the somatosensory cortex (SSC) of 5XFAD mice receiving a diet containing either the CSF1R inhibitor PLX5622 or a standard diet for ten days were examined for morphology, markers of NOD‐, LRR‐, and pyrin domain‐containing protein 3 (NLRP3) inflammasome complex, autophagy, and mechanistic target of rapamycin (mTOR) signaling. Result: Ten days of CSF1R inhibition resulted in 67‐70% depletion of microglia in the hippocampus and SSC. The residual microglia displayed a noninflammatory phenotype, typified by highly branched and ramified processes, reduced incidence of NLRP3 inflammasome complexes, and increased autophagy. Interestingly, Aβ plaque load was reduced, and mTOR signaling was unaltered in microglia but reduced in neurons. However, transient CSF1R inhibition did not impact astrocyte hypertrophy or neurogenesis in the hippocampus. Conclusion: The results showed that transient CSF1R inhibition in the early stage of AD promotes the retention of homeostatic microglia with reduced inflammasomes and better metabolic fitness but with no changes in mTOR signaling, which can restrain the perpetuation of neuroinflammation and improve phagocytosis of Aβ by microglia. [ABSTRACT FROM AUTHOR]

While moderately activated microglia in Alzheimer's disease (AD) are pivotal in clearing amyloid beta (Aβ), hyperactivated microglia perpetuate neuroinflammation. Prior investigations reported that the elimination of ~80% of microglia through inhibition of the colony‐stimulating factor 1 receptor (CSF1R) during the advanced stage of neuroinflammation in 5xFamilial AD (5xFAD) mice mitigates synapse loss and neurodegeneration. Furthermore, prolonged CSF1R inhibition diminished the development of parenchymal plaques. Nonetheless, the effects of short‐term CSF1R inhibition during the early stages of neuroinflammation on residual microglia are unknown. Therefore, we investigated the effects of 10‐day CSF1R inhibition using PLX5622 in three‐month‐old female 5xFAD mice, a stage characterized by the onset of neuroinflammation and minimal Aβ plaques. We observed ~65% microglia depletion in the hippocampus and cerebral cortex. The leftover microglia displayed a noninflammatory phenotype with reduced NOD‐, LRR‐, and pyrin domain‐containing protein 3 (NLRP3) inflammasome complexes. Moreover, plaque‐associated microglia were reduced with diminished Clec7a expression. Additionally, phosphorylated S6 ribosomal protein and the protein sequestosome 1 analysis suggested reduced mechanistic targets of rapamycin (mTOR) signaling and autophagy in microglia and neurons within the hippocampus and cerebral cortex. Biochemical assays validated the inhibition of NLRP3 inflammasome activation, decreased mTOR signaling in the hippocampus and cerebral cortex, and enhanced autophagy in the hippocampus. However, short‐term CSF1R inhibition did not influence Aβ plaques, soluble Aβ‐42 levels, astrocyte hypertrophy, or hippocampal neurogenesis. Thus, short‐term CSF1R inhibition during the early stages of neuroinflammation in 5xFAD mice promotes the retention of homeostatic microglia with diminished inflammasome activation and mTOR signaling, alongside increased autophagy. [ABSTRACT FROM AUTHOR]