Role of Receptor Protein Tyrosine Phosphatase β/ζ in Neuron-Microglia Communication in a Cellular Model of Parkinson's Disease

Abstract

Pleiotrophin (PTN) is a neurotrophic factor that regulates glial responses in animal models of different types of central nervous system (CNS) injuries. PTN is upregulated in the brain in different pathologies characterized by exacerbated neuroinflammation, including Parkinson's disease. PTN is an endogenous inhibitor of Receptor Protein Tyrosine Phosphatase (RPTP) β/ζ, which is abundantly expressed in the CNS. Using a specific inhibitor of RPTPβ/ζ (MY10), we aimed to assess whether the PTN/RPTPβ/ζ axis is involved in neuronal and glial injury induced by the toxin MPP+. Treatment with the RPTPβ/ζ inhibitor MY10 alone decreased the viability of both SH-SY5Y neuroblastoma cells and BV2 microglial cultures, suggesting that normal RPTPβ/ζ function is involved in neuronal and microglial viability. We observed that PTN partially decreased the cytotoxicity induced by MPP+ in SH-SY5Y cells underpinning the neuroprotective function of PTN. However, MY10 did not seem to modulate the SH-SY5Y cell loss induced by MPP+. Interestingly, we observed that media from SH-SY5Y cells treated with MPP+ and MY10 decreases microglial viability but may elicit a neuroprotective response of microglia by upregulating Ptn expression. The data suggest a neurotrophic role of microglia in response to neuronal injury through upregulation of Ptn levels.

Keywords: MPP+; RPTP β/ζ; microglia; midkine; neuroinflammation; pleiotrophin.

Figure 1

PTN modulates MPP+-induced SH-SY5Y cell toxicity. MPP+ causes a significant decrease in SH-SY5Y viability which is partially prevented by co-incubation with PTN at 6 and 12 µM. **** p < 0.0001 vs. Control non-treated cells, # p < 0.05 vs. MPP+. Data are shown as % viability considering 100% viability of SH-SY5Y cells incubated with complete media (Control, black bar).

Figure 2

Pharmacological inhibition of RPTPβ/ζ does not modulate MPP+-induced SH-SY5Y cell toxicity but is involved in SH-SY5Y cell viability. MPP+ causes a significant decrease in SH-SY5Y cell viability, but to a lesser extent than that caused by MPP+ in the experiments performed for Figure 1. The toxic effect of MPP+ was not significantly modulated by MY10. However, treatment with MY10 alone caused a significant reduction of SH-SY5Y cell viability at 1 and 10 µM. * p < 0.05, *** p < 0.001, **** p < 0.0001 vs. Control, non-treated cells. Data are shown as % viability considering 100% viability of SH-SY5Y cells incubated with complete media (Control, black bar).

Figure 3

Pharmacological inhibition of RPTPβ/ζ does not modulate MPP+-induced microglia injury but significantly aggravates the loss of microglia viability induced by conditioned media from MPP+-treated SH-SY5Y cells. (a) MPP+ causes a significant decrease in BV2 cell viability, which is not significantly modulated by MY10. Treatment with MY10 alone caused a significant reduction of microglial viability. (b) MY10 significantly enhances the loss of BV2 cells viability when microglial cells were incubated with the media from MPP+/MY10-treated SH-SY5Y cells. Treatment with conditioned media from SH-SY5Y-treated cells with MY10 alone causes a significant reduction of BV2 cell viability. Data are shown as % viability considering 100% viability of BV2 microglial cells incubated with complete media or with conditioned media from untreated SH-SY5Y cells (Controls, black bar and striped white bar, respectively). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 vs. untreated cells (Control). ### p < 0.001 vs. MPP+.

Figure 4

Upregulation of Ptn mRNA in microglial cells injured with conditioned media from SH-SY5Y cells treated with MPP+ and MY10. (a) Real-time PCR analyses of SH-SY5Y cells incubated with MPP+ and increasing concentrations of MY10 did not reveal significant differences in PTN mRNA expression. GAPDH and RPL30 were used as housekeeping genes in human SH-SY5Y cells. (b) Real-time PCR analyses of BV2 cells treated with the conditioned media from SH-SY5Y cells incubated with MPP+ and increasing concentrations of MY10 showed Ptn mRNA upregulation in BV2 cells. Rpl13 and Hprt were used as housekeeping genes in mouse BV2 cells. * p < 0.05 vs. Control.

Figure 5

Microglial-induced neurotrophic response. Microglial injury caused by the treatment with conditioned media from SH-SY5Y cells treated with MPP+ is exacerbated when microglial cells are treated with the media from SH-SY5Y cells treated concomitantly with MY10 and MPP+. These treatments significantly induced the upregulation of Ptn mRNA in microglial cells. The data suggest a neuroprotective response of microglia through the increased secretion of microglial PTN and enhanced actions of this cytokine in proximal neurons expressing different PTN receptors. This figure was created with BioRender’s web-based software (https://biorender.com/ accessed 14 June 2021). SDC-3: Syndecan-3. ALK: Anaplastic lymphoma kinase. NCAM: Neural cell adhesion molecule. NGC: Neuroglycan C.