Intranasal Human NSC-Derived EVs Therapy Can Restrain Inflammatory Microglial Transcriptome, and NLRP3 and cGAS-STING Signalling, in Aged Hippocampus.

Intranasal hiPSC-NSC-EVs therapy reduced astrocyte hypertrophy, microglial clusters, and oxidative stress in the aged hippocampus.

• Late middle-aged (18-month-old) male and female C57BL6/J mice received two intranasal doses of hiPSC-NSC-EVs and were assessed at 20.5 months of age.
• Compared with vehicle-treated animals, hiPSC-NSC-EVs-treated animals exhibited reductions in astrocyte hypertrophy and microglial clusters.
• Oxidative stress was also reduced in the hippocampus of treated animals.
• Elevated expression of antioxidant proteins and genes that maintain mitochondrial respiratory chain integrity was observed in treated animals.

hiPSC-NSC-EVs therapy decreased levels of proteins involved in NLRP3 inflammasome activation in the aged hippocampus.

• Protein levels involved in NLRP3 inflammasome activation were reduced in hippocampi of hiPSC-NSC-EVs-treated animals compared to vehicle-treated controls.
• Reductions were also observed in p38/mitogen-activated protein kinase signalling pathway proteins.
• The therapy additionally decreased proteins involved in Janus kinase and signal transducer and activator of transcription (JAK-STAT) signalling pathways.

hiPSC-NSC-EVs therapy decreased activation of the cGAS-STING-IFN-1 signalling pathway in the aged hippocampus.

• Levels of various proteins involved in cGAS-STING-IFN-1 pathway activation were reduced in hiPSC-NSC-EVs-treated animals.
• The cGAS-STING pathway triggers type 1 interferon (IFN-1) signalling and contributes to neuroinflammaging.
• Reductions in this pathway were observed alongside reductions in NLRP3 and p38/MAPK signalling.

miRNA-30e-3p present in hiPSC-NSC-EVs significantly inhibits NLRP3 inflammasome activation.

• In vitro assays using genetically engineered RAW cells and hiPSC-NSC-EVs with or without targeted depletion of specific miRNAs were conducted.
• Targeted depletion of miRNA-30e-3p from hiPSC-NSC-EVs demonstrated that this specific miRNA mediates inhibition of NLRP3 inflammasome activation.
• miRNA-30e-3p is present in hiPSC-NSC-EVs and contributes to their anti-inflammatory therapeutic effects.

miRNA-181a-5p present in hiPSC-NSC-EVs significantly inhibits STING pathway activation.

• In vitro assays using genetically engineered RAW cells with targeted depletion of miRNA-181a-5p demonstrated that this miRNA mediates inhibition of STING pathway activation.
• miRNA-181a-5p is present in hiPSC-NSC-EVs alongside miRNA-30e-3p.
• These findings were established using a targeted miRNA depletion approach in genetically engineered RAW cells.

Single-cell RNA sequencing revealed widespread transcriptomic changes in microglia induced by hiPSC-NSC-EVs therapy.

• Single-cell RNA sequencing was conducted 7 days post-treatment.
• hiPSC-NSC-EVs induced increased expression of numerous genes that enhance oxidative phosphorylation in microglia.
• hiPSC-NSC-EVs reduced expression of abundant genes that drive multiple proinflammatory signalling pathways in microglia.
• The transcriptomic changes were widespread across the microglial population.

hiPSC-NSC-EVs therapy was associated with improved cognitive and memory function in aged mice.

• Cognitive and memory improvements were observed in animals treated with hiPSC-NSC-EVs compared to vehicle-treated controls.
• Animals were treated at 18 months of age and assessed at 20.5 months of age.
• Improvements in brain function were associated with the transcriptomic and signalling changes observed in the hippocampus.

Neuroinflammaging in the aged hippocampus involves activation of NLRP3 inflammasomes and the cGAS-STING pathway triggering type 1 interferon signalling.

• Neuroinflammaging is described as a moderate, chronic, and sterile inflammation in the hippocampus that contributes to age-related cognitive decline.
• The NLRP3 inflammasome involves the nucleotide-binding domain, leucine-rich repeat family, and pyrin domain-containing 3 protein.
• The cGAS-STING pathway activates type 1 interferon (IFN-1) signalling as part of the neuroinflammaging process.
• hiPSC-NSC-EVs contain therapeutic miRNAs that can alleviate neuroinflammation.