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The cholinesterase inhibitor donepezil increases brain acetylcholine levels and improves Alzheimer\u2019s disease (AD)-associated learning disabilities. Acetylcholine activates striatal\/nucleus accumbens dopamine receptor D2-expressing medium spiny neurons (D2R-MSNs), which regulate aversive learning through muscarinic receptor M1 (M1R). However, how acetylcholine stimulates learning beyond M1Rs remains unresolved. Here, we found that acetylcholine stimulated protein kinase C (PKC) in mouse striatal\/nucleus accumbens. Our original kinase-oriented phosphoproteomic analysis revealed 116 PKC substrate candidates, including Rac1 activator \u03b2-PIX. Acetylcholine induced \u03b2-PIX phosphorylation and activation, thereby stimulating Rac1 effector p21-activated kinase (PAK). Aversive stimulus activated the M1R-PKC-PAK pathway in mouse D2R-MSNs. D2R-MSN-specific expression of PAK mutants by the Cre-Flex system regulated dendritic spine structural plasticity and aversive learning. Donepezil induced PAK activation in both accumbal D2R-MSNs and in the CA1 region of the hippocampus and enhanced D2R-MSN-mediated aversive learning. These findings demonstrate that acetylcholine stimulates M1R-PKC-\u03b2-PIX-Rac1-PAK signaling in D2R-MSNs for aversive learning and imply the cascade\u2019s therapeutic potential for AD as aversive learning is used to preliminarily screen AD drugs.<\/jats:p>","DOI":"10.1038\/s41380-022-01643-2","type":"journal-article","created":{"date-parts":[[2022,6,3]],"date-time":"2022-06-03T06:03:01Z","timestamp":1654236181000},"page":"3479-3492","update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":21,"title":["Phosphoproteomic of the acetylcholine pathway enables discovery of the PKC-\u03b2-PIX-Rac1-PAK cascade as a stimulatory signal for aversive learning"],"prefix":"10.1038","volume":"27","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-3697-0861","authenticated-orcid":false,"given":"Yukie","family":"Yamahashi","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"You-Hsin","family":"Lin","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Akihiro","family":"Mouri","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sho","family":"Iwanaga","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kazuhiro","family":"Kawashima","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yuya","family":"Tokumoto","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yo","family":"Watanabe","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Md. 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