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Abstract <p>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.</p> Graphical Abstract <p><b>The roles of α-enolase, γ-enolase, and cathepsin X in enriched and differentiated SH-SY5Y cell populations.</b> The enrichment and differentiation of SH-SY5Y cells resulted in two distinct cell phenotypes: S-type and N-type cells. S-type cells were characterized by an epithelial-like morphology, the presence of vimentin, lower γ-enolase expression, and higher cathepsin X expression. N-type cells were characterized by a neuron-like morphology, GAP-43 and Bcl-2 expression, higher γ-enolase expression, and lower cathepsin X expression. Both phenotypes expressed α-enolase. The cathepsin X inhibitor AMS36 promoted SH-SY5Y cell differentiation and enrichment into S- and N-type cells. AMS36-treated S-type cells exhibited decreased vimentin levels and increased active γ-enolase levels, indicating enhanced differentiation. AMS36-treated N-type cells exhibited decreased Bcl-2 levels, indicating further differentiation. These results highlight the differential protein expression and activity between S- and N-type cells. Furthermore, they highlight the modulatory effects of AMS36, emphasizing its potential role in promoting differentiation and altering protein expression profiles.</p> <p></p>

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α-Enolase and γ-Enolase Expression in Enriched S- and N-Type SH-SY5Y Cells: Regulatory Role of Cathepsin X

  • 期刊论文

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  • 发表日期：

    2025年4月3日

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  • Selena Horvat,
  • Nace Zidar,
  • Janko Kos,
  • Anja Pišlar

摘要

Abstract

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.

Graphical Abstract

The roles of α-enolase, γ-enolase, and cathepsin X in enriched and differentiated SH-SY5Y cell populations. The enrichment and differentiation of SH-SY5Y cells resulted in two distinct cell phenotypes: S-type and N-type cells. S-type cells were characterized by an epithelial-like morphology, the presence of vimentin, lower γ-enolase expression, and higher cathepsin X expression. N-type cells were characterized by a neuron-like morphology, GAP-43 and Bcl-2 expression, higher γ-enolase expression, and lower cathepsin X expression. Both phenotypes expressed α-enolase. The cathepsin X inhibitor AMS36 promoted SH-SY5Y cell differentiation and enrichment into S- and N-type cells. AMS36-treated S-type cells exhibited decreased vimentin levels and increased active γ-enolase levels, indicating enhanced differentiation. AMS36-treated N-type cells exhibited decreased Bcl-2 levels, indicating further differentiation. These results highlight the differential protein expression and activity between S- and N-type cells. Furthermore, they highlight the modulatory effects of AMS36, emphasizing its potential role in promoting differentiation and altering protein expression profiles.