Ceruloplasmin oxidized and deamidated by Parkinson's disease cerebrospinal fluid induces epithelial cells proliferation arrest and apoptosis

Abstract

In Parkinson's disease, the ferroxidase ceruloplasmin (Cp) is oxidized and deamidated by the pathological cerebrospinal fluid (CSF) environment. These modifications promote the gain of integrin binding properties, fostered by the deamidation of two NGR-motifs present in the Cp sequence that convert into the isoDGR-motif. Through isoDGR/integrin binding, the oxidized/deamidated-Cp (Cp-ox/de) mediates cell adhesion and transduces an intracellular signal in epithelial cells that seems to be addressed to regulate cell cycle, proliferation and cytoskeletal re-arrangement. However, the effect fostered on cells by integrins engagement via Cp-ox/de is not known. We found that in HaCaT epithelial cells, the incubation with Cp-ox/de resulted in proliferation inhibition mediated by isoDGR, cell cycle arrest and apoptosis induction. Similar proliferation inhibition was induced by treatment with purified Cp previously incubated in the CSF from Parkinson's disease patients, but not by Cp incubated in the CSF from healthy subjects. In human primary choroid plexus epithelial cells, a possible in vivo target of Cp-ox/de generated in pathological CSFs, we found that Cp-ox/de mediated cell adhesion via isoDGR/integrins binding and transduced an intracellular signal, which resulted in cell proliferation inhibition. Thus, the generation of Cp-ox/de in pathological CSFs and the consequent apoptosis induction of epithelial cells facing the liquor, might represent a novel mechanism that contributes to neurodegeneration.

Ceruloplasmin Deamidation in Neurodegeneration: From Loss to Gain of Function

“…Moreover, Cp-ox/de induced a series of protein phosphorylation events related to integrin-mediated signaling transduction, particularly those regulating MAPK signaling pathway and cell cycle [ 9 ]. The Cp-ox/de interaction with integrins caused cell proliferation arrest due to cell cycle blocking and apoptosis triggering, affecting the epithelial cell functionality [ 12 ]. The same effects of cell-adhesion promotion and proliferation inhibition were also observed with Cp modified ex vivo, by the incubation in the CSF from PD or AD patients, suggesting that cellular targeting by modified Cp might also occur in vivo [ 9 , 10 , 12 ].…”

“…The Cp-ox/de interaction with integrins caused cell proliferation arrest due to cell cycle blocking and apoptosis triggering, affecting the epithelial cell functionality [ 12 ]. The same effects of cell-adhesion promotion and proliferation inhibition were also observed with Cp modified ex vivo, by the incubation in the CSF from PD or AD patients, suggesting that cellular targeting by modified Cp might also occur in vivo [ 9 , 10 , 12 ]. These effects on HaCat epithelial cell functionality were mediated by the direct binding to integrins through the deamidated NGR motifs of Cp, as inferred by competitive binding with a peptide containing the isoDGR motif and by Cp-ox/de pre-treatment with PIMT enzyme, which convert the isoDGR integrin-binding motif to DGR, thus avoiding integrins engagement [ 9 , 10 , 12 , 20 , 41 ].…”

“…The possibility to target CPEpiCs is supported by evidence that, via integrin-binding, Cp-ox/de promotes cell adhesion of primary human choroid plexus epithelial cells (HCPEpiCs), which express αVβ3 and α5β1 integrins suitable for isoDGR binding, transducing an intracellular signaling that, as for HaCat, inhibited cell proliferation [ 12 ]. This might have consequences on the major physiological roles of these cells, namely secretion and barrier properties, which contribute to determining the CSF composition.…”

“…Protein deamidation occurs on two amino acid residues, namely asparagine and glutamine, and generally results in loss of protein function [ 11 ]. In the case of Cp, the deamidation involves asparagine residues at the level of two asparagine-glycine-arginine (Asn-Gly-Arg, NGR) sites of the protein sequence whose, upon deamidation, gain binding activity to integrins [ 9 , 10 , 12 ]. Integrins are a family of receptors that mediated cell-extracellular matrix adhesion [ 13 ].…”

“…Integrins are a family of receptors that mediated cell-extracellular matrix adhesion [ 13 ]. The binding of modified Cp to integrins triggers in vitro intracellular signaling on epithelial cells, altering the cell physiology with the induction of detrimental effects [ 9 , 12 ]. However, despite the presence of NGR motifs deamidation in the Cp from neurodegenerative diseases patients’ CSF, in vivo cellular targets and possible pathological effects are still unexplored topics.…”