Proteomics and phosphoproteomics profiling in glutamatergic neurons and microglia in an iPSC model of Jansen de Vries Syndrome

Abstract

BackgroundJansen de Vries Syndrome (JdVS) is a rare neurodevelopmental disorder (NDD) caused by gain-of-function (GOF) truncating mutations inPPM1Dexons 5 or 6. PPM1D is a serine/threonine phosphatase that plays an important role in the DNA damage response (DDR) by negatively regulating TP53 (P53). JdVS-associated mutations lead to the formation of a truncated PPM1D protein that retains catalytic activity and has a GOF effect because of reduced degradation. SomaticPPM1Dexons 5 and 6 truncating mutations are well-established factors in a number of cancers, due to excessive dephosphorylation and reduced function of P53 and other substrates involved in DDR. Children with JdVS have a variety of neurodevelopmental, psychiatric, and physical problems. In addition, a small fraction has acute neuropsychiatric decompensation apparently triggered by infection or severe non-infectious environmental stress factors.MethodsTo understand the molecular basis of JdVS, we developed an induced pluripotent stem cell (iPSC) model system. iPSCs heterozygous for the truncating variant (PPM1D+/tr), were made from a patient, and control lines engineered using CRISPR-Cas9 gene editing. Proteomics and phosphoprotemics analyses were carried out on iPSC-derived glutamatergic neurons and microglia from three control and threePPM1D+/triPSC lines. We also analyzed the effect of the TLR4 agonist, lipopolysaccharide, to understand how activation of the innate immune system in microglia could account for acute behavioral decompensation.ResultsOne of the major findings was the downregulation of POGZ in unstimulated microglia. Since loss-of-function variants in thePOGZgene are well-known causes of autism spectrum disorder, the decrease inPPM1D+/trmicroglia suggests this plays a role in the neurodevelopmental aspects of JdVS. In addition, neurons, baseline, and LPS-stimulated microglia show marked alterations in the expression of several E3 ubiquitin ligases, most notably UBR4, and regulators of innate immunity, chromatin structure, ErbB signaling, and splicing. In addition, pathway analysis points to overlap with neurodegenerative disorders.LimitationsOwing to the cost and labor-intensive nature of iPSC research, the sample size was small.ConclusionsOur findings provide insight into the molecular basis of JdVS and can be extrapolated to understand neuropsychiatric decompensation that occurs in subgroups of patients with ASD and other NDDs.