α‑enolase and γ‑enolase expression in enriched S- and N-type SH-SY5Y cells : regulatory role of cathepsin X

Horvat, Selena (author), Zidar, Nace (author), Kos, Janko (author), Pišlar, Anja (author)

Enolase is well-known for its role in glycolysis but also plays other roles in the central nervous system, including neuronal survival, differentiation, and axonal regeneration. Here, we investigated α- and γ-enolase expression patterns and their association with cathepsin X in distinct SH-SY5Y cell phenotypes. Enriched substrate-adherent S-type cells are characterized by large, flat morphology with extensive cytoplasm and higher expression of vimentin, while neuroblastic N-type are recognized by neurite extensions and higher expression of B-cell lymphoma 2 (Bcl-2) and growth-associated protein-43. We demonstrated that γ-enolase expression was specific to N-type cells, whereas α-enolase expression was not phenotype-specific. Moreover, a shift from ubiquitously expressed α-enolase to neuron-specific γ-enolase was observed during the enrichment and differentiation. Additionally, cathepsin X exhibited higher proteolytic activity in S-type cells. Inhibition of cathepsin X with AMS36 promoted differentiated cell morphology and increased expression of the active form of γ-enolase. Furthermore, AMS36 altered the expression of vimentin and Bcl-2, indicating a regulatory role in neuronal differentiation. Furthermore, AMS36 activated extracellular signal-regulated kinase 1/2 in N-type cells and enhanced the association between γ-enolase and tyrosine receptor kinase in both, suggesting a link between cathepsin X/γ-enolase and the key signaling pathways of differentiation. Our findings underscore the multifaceted role of enolase isoforms in SH-SY5Y cell differentiation, with α-enolase and γ-enolase showing distinct expression patterns in S- and N-type cells. The expression and activity of cathepsin X in S-type cells, along with its regulatory impact on γ-enolase in N-type cells, highlight the importance of these proteins in neuronal differentiation.

• Language: English
• Keywords: neuron-like cells, neuronal diferentiation, SH-SY5Y cell phenotypes, enolase isoforms, cathepsin X inhibition
• Work type: Article
• Typology: 1.01 - Original Scientific Article
• Organization: FFA - Faculty of Pharmacy
• Publication status: Published
• Publication version: Version of Record
• Year: 2025
• Number of pages: Str. 10006–10019
• Numbering: Vol. 62, iss. 8
• PID: 20.500.12556/RUL-178431
• UDC: 577.152.34
• ISSN on article: 1559-1182
• DOI: 10.1007/s12035-025-04898-2
• COBISS.SI-ID: 231412483
• Publication date in RUL: 27.01.2026

Record is a part of a journal

• Title: Molecular neurobiology
• Shortened title: Mol. neurobiol.
• Publisher: Springer Nature
• ISSN: 1559-1182
• COBISS.SI-ID: 513709593

Licences

• License: CC BY 4.0, Creative Commons Attribution 4.0 International
• Link: http://creativecommons.org/licenses/by/4.0/
• Description: This is the standard Creative Commons license that gives others maximum freedom to do what they want with the work as long as they credit the author.

Secondary language

• Language: Slovenian
• Keywords: nevronom podobne celice, nevronska diferenciacija, fenotipi celic SH-SY5Y, izoforme enolaze, katepsin X zaviralci, katepsin

Projects

• Funder: ARRS - Slovenian Research Agency
• Project number: P4-0127
• Name: Farmacevtska biotehnologija: znanost za zdravje

• Funder: ARRS - Slovenian Research Agency
• Project number: P1-0208
• Name: Farmacevtska kemija: načrtovanje, sinteza in vrednotenje učinkovin