γ-Enolase, a glycolytic enzyme, is expressed specifically in neurons. It exerts neurotrophic activity and has been suggested to regulate growth, differentiation, survival and regeneration of neurons. In the present study, we investigated the involvement of γ-enolase in PI3K (phosphoinositide 3-kinase)/Akt and MAPK (mitogen-activated protein kinase)/ERK (extracellular-signal-regulated kinase) signalling, the two pathways triggered predominantly by neurotrophic factors. Whereas the PI3K/Akt pathway, rather than the MAPK/ERK pathway, is involved in γ-enolase-enhanced cell survival, γ-enolase-stimulated neurite outgrowth requires both pathways, i.e. the activation of both PI3K and ERK1/2, leading to subsequent expression of the growth-cone-specific protein GAP-43 (growth-associated protein of 43 kDa). MEK (MAPK/ERK kinase) and PI3K inhibition blocked or attenuated the neurite outgrowth associated with dynamic remodelling of the actin-based cytoskeleton. We show that γ-enolase-mediated PI3K activation regulates RhoA kinase, a key regulator of actin cytoskeleton organization. Moreover, the inhibition of RhoA downstream effector ROCK (Rho-associated kinase) results in enhanced γ-enolase-induced neurite outgrowth, accompanied by actin polymerization and its redistribution to growth cones. Our results show that γ-enolase controls neuronal survival, differentiation and neurite regeneration by activating the PI3K/Akt and MAPK/ERK signalling pathways, resulting in downstream regulation of the molecular and cellular processes of cytoskeleton reorganization and cell remodelling, activation of transcriptional factors and regulation of the cell cycle.

Summaryc-Enolase is a neurotrophic-like factor promoting growth, differentiation, survival and regeneration of neurons. Its neurotrophic activity is regulated by cysteine protease cathepsin X which cleaves the C-terminal end of the molecule. We have investigated the expression and colocalization of c-enolase and cathepsin X in brains of Tg2576 mice overexpressing amyloid precursor protein.In situ hybridization of c-enolase and cathepsin X revealed that mRNAs for both enzymes were expressed abundantly around amyloid plaques. Immunostaining demonstrated that the C-terminally cleaved form of c-enolase was present in the immediate plaque vicinity, whereas the intact form, exhibiting neurotrophic activity, was observed in microglia cells in close proximity to senile plaque. The upregulation of c-enolase in microglial cells in response to amyloid-b peptide (Ab) was confirmed in mouse microglial cell line EOC 13.31 and primary microglia and medium enriched with c-enolase proved to be neuroprotective against Ab toxicity; however, the effect was reversed by cathepsin X proteolytic activity. These results demonstrate an upregulation of c-enolase in microglia cells surrounding amyloid plaques in Tg2576 transgenic mice and demonstrate its neuroprotective role in amyloid-b-related neurodegeneration.

Syntrophins are scaffold proteins that can bind several signaling molecules and localize them to the plasma membrane. We demonstrate here that in neuroblastoma SH-SY5Y cells, brain-specific γ₁-syntrophin binds the neurotrophic factor γ-enolase through its PDZ domain, and translocates it to the plasma membrane, as shown by immunoprecipitation, surface plasmon resonance, fluorescence colocalization and flow cytometry. Extensive colocalization of γ₁-syntrophin and γ-enolase was observed in neurite growth cones in differentiated SH-SY5Y cells. Silencing of the γ₁-syntrophin gene by RNA interference significantly reduced the re-distribution of γ-enolase to the plasma membrane and impaired its neurotrophic effects. We demonstrated that an intact C-terminal end of γ-enolase is essential for its γ₁-syntrophin-assisted trafficking. The cleavage of two amino acids at the C-terminal end of γ-enolase by the carboxypeptidase cathepsin X prevents binding with the γ₁-syntrophin PDZ domain. Collectively, these data demonstrate that γ₁-syntrophin participates in γ-enolase translocation towards the plasma membrane, a pre-requisite for its neurotrophic activity. By disrupting this γ₁-syntrophin-guided subcellular distribution, cathepsin X reduces γ-enolase-induced neurotrophic signaling.

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