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Microglia-mediated inflammation can lead to central nervous system disorders, and more than one TLR might be involved in these pathological processes. The cysteine peptidase cathepsin X has been recognized as a pathogenic factor for inflammation-induced neurodegeneration. Here, we hypothesized that simultaneous TLR3 and TLR4 activation induces synergized microglia responses and that these phenotype changes affect cathepsin X expression and activity. Murine microglia BV2 cells and primary murine microglia were exposed to the TLR3 ligand polyinosinic-polycytidylic acid (poly(I:C)) and the TLR4 ligand lipopolysaccharide (LPS), individually and simultaneously. TLR3 and TLR4 co-activation resulted in increased inflammatory responses compared to individual TLR activation, where poly(I:C) and LPS induced distinct patterns of proinflammatory factors together with different patterns of cathepsin X expression and activity. TLR co-activation decreased intracellular cathepsin X activity and increased cathepsin X localization at the plasma membrane with concomitant increased extracellular cathepsin X protein levels and activity. Inhibition of cathepsin X in BV2 cells by AMS36, cathepsin X inhibitor, significantly reduced the poly(I:C)- and LPS-induced production of proinflammatory cytokines as well as apoptosis. Additionally, inhibiting the TLR3 and TLR4 common signaling pathway, PI3K, with LY294002 reduced the inflammatory responses of the poly(I:C)- and LPS-activated microglia and recovered cathepsin X activity. We here provide evidence that microglial cathepsin X strengthens microglia activation and leads to subsequent inflammation-induced neurodegeneration. As such, cathepsin X represents a therapeutic target for treating neurodegenerative diseases related to excess inflammation.<\/jats:p>","DOI":"10.1007\/s12035-021-02694-2","type":"journal-article","created":{"date-parts":[[2022,1,23]],"date-time":"2022-01-23T00:04:21Z","timestamp":1642896261000},"page":"2258-2276","update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":14,"title":["Cysteine Peptidase Cathepsin X as a Therapeutic Target for Simultaneous TLR3\/4-mediated Microglia Activation"],"prefix":"10.1007","volume":"59","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-1159-1024","authenticated-orcid":false,"given":"Anja","family":"Pi\u0161lar","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Biljana Bo\u017ei\u0107","family":"Nedeljkovi\u0107","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mina","family":"Peri\u0107","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Tanja","family":"Jako\u0161","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Nace","family":"Zidar","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Janko","family":"Kos","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2022,1,23]]},"reference":[{"issue":"2","key":"2694_CR1","doi-asserted-by":"publisher","first-page":"137","DOI":"10.1093\/jb\/mvm032","volume":"141","author":"T Kawai","year":"2007","unstructured":"Kawai T, Akira S (2007) Antiviral signaling through pattern recognition receptors. 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