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

## TL;DR

This study explores how α-enolase, γ-enolase, and cathepsin X influence the differentiation of SH-SY5Y cells into distinct neuronal-like and epithelial-like phenotypes.

## Contribution

The study reveals a regulatory role of cathepsin X in modulating enolase isoform expression and neuronal differentiation in SH-SY5Y cells.

## Key findings

- γ-enolase is specifically expressed in N-type cells, while α-enolase is not phenotype-specific.
- Cathepsin X inhibition with AMS36 promotes differentiation and alters enolase and protein expression.
- AMS36 activates ERK1/2 and enhances γ-enolase interaction with tyrosine receptor kinase in N-type cells.

## 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.

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.

The online version contains supplementary material available at 10.1007/s12035-025-04898-2.