Prolyl endopeptidase is revealed following SILAC analysis to be a novel mediator of human microglial and THP‐1 cell neurotoxicity

Abstract

Reactive microglial cells may exacerbate the pathology in some neurodegenerative disorders. Supernatants of stimulated human microglial cells, or their surrogate THP-1 cells, are lethal to cultured human neuroblastoma SH-SY5Y cells. To explore this neurotoxicity, we examined the spectrum of proteins generated by THP-1 cells using the technique of stable isotope labeling by amino acids in cell culture (SILAC). Unstimulated cells were grown in medium with light L-[(12)C(6)] arginine while cells stimulated by lipopolysaccharide (LPS) plus interferon-gamma (IFN-gamma) were grown in medium with heavy L-[(13)C(6)] arginine. Proteins isolated from the media were digested with trypsin, and relative concentrations of generated peptides determined by mass spectrometry. More than 1,500 proteins or putative proteins were identified. Of these, 174 were increased and 189 decreased by more than twofold in the stimulated cell supernatant. We selected one upregulated protein, prolyl endopeptidase (PEP), for further investigation of its potential contribution to neurotoxicity. We first confirmed its upregulation by comparing its enzymatic activity in stimulated and unstimulated cell supernatants. We then evaluated two specific PEP inhibitors, Boc-Asn-Phe-Pro-aldehyde and Z-Pro-Pro-aldehyde-dimethyl acetal, for their potential to reduce toxicity of stimulated THP-1 cell and human microglia supernatants towards SH-SY5Y cells. We found both to be partially protective in a concentration-dependent manner. Inhibition of PEP may be a therapeutic approach to neurodegenerative disorders including Alzheimer and Parkinson diseases.

Prolyl Oligopeptidase: A Rising Star on the Stage of Neuroinflammation Research

“…The enzymes may also release pro-inflammatory cytokines and participate in regulating the expression of the cell death signalling molecule FasL [70]. Several MMPs including MMP-2, 3 and 9 contribute to microglial toxicity [68]. In MS brain tissues MMP-2, -7, -9, and -12 are reportedly elevated [59,69,70].…”

“…Based on this fact, Klegeris and coworkers [68] investigated microglia and their surrogate, THP-1 cells, in order to identify the proteins with potential of microglial toxicity. In these studies POP mRNA was found also to be significantly increased in both cell types.…”

“…Also, various proteolytic enzymes have been identified as candidate neurotoxins [67]. In the course of MS, MMPs are involved in degrading the extracellular matrix, thus facilitating immune cell transmigration into the CNS [60,68,69]. In addition, MMPs have been reported to degrade proteins of the myelin sheath.…”

Prolyl endopeptidase is involved in the degradation of neural cell adhesion molecules in vitro

“…Recent in vitro studies demonstrated that PREP contributes to the toxic effects of reactive microglial cells, as it was demonstrated that activated microglia cells expressed high levels of PREP and the supernatant of these cells demonstrated toxic effects on SH-SY5Y neuroblastoma cells. This toxic effect was reduced by selective PREP inhibitors in a dose-dependent matter (Klegeris et al, 2008). Moreover, studies in PREP-knockout mice have demonstrated the association of PREP in the processes modulating neuroplasticity through inflammatory response.…”

The Microtubule-Dissociating Tau in Neurological Disorders

“…This is a huge limitation for the study of CSF and human brain tissue taking into account that neurons are postmitotic cells (Bantscheff M et al, 2007). Despite this handicap SILAC is a powerful tool to study cellular pathways as polyubiquitin involment in the aetiology of AD (Dammer EB et al, 2011), neuroinflamation (McGeer EG andMcGeer PL, 2010), reactive microglia (Klegeris A et al, 2008), neurotrophin signaling (Zhang G et al, 2011), oxidative stress (Akude E et al, 2011), TDP-43 proteinopathy in frontotemporal lobar degeneration and amyotrophic lateral sclerosis (Seyfried NT et al, 2010) mitochondrial alterations in dopaminergic cells (Jin J et al, 2007) and modulation of ion channels by phosphorylation (Park KS et al, 2006). Other methods for protein quantification are multiple reaction monitoring (MRM) that has been successfully used for low abundant proteins in plasma (Anderson L and Hunter CL, 2006) and phosphopeptides quantification (Lange V et al, 2008).…”