Spatiotemporal Proteomic Profiling of Human Cerebral Development

Abstract

Mass spectrometry (MS) analysis of human post-mortem central nervous system (CNS) tissue and induced pluripotent stem cell (iPSC)-based directed differentiations offer complementary avenues to define protein signatures of neurodevelopment. Methodological improvements of formalin-fixed, paraffin-embedded (FFPE) protein isolation now enable widespread proteomic analysis of well-annotated archival tissue samples in the context of development and disease. Here, we utilize a shotgun label-free quantification (LFQ) MS method to profile magnetically enriched human cortical neurons and neural progenitor cells (NPCs) derived from iPSCs. We use these signatures to help define spatiotemporal protein dynamics of developing human FFPE cerebral regions. We show that the use of high resolution Q Exactive mass spectrometers now allow simultaneous quantification of >2700 proteins in a single LFQ experiment and provide sufficient coverage to define novel biomarkers and signatures of NPC maintenance and differentiation. Importantly, we show that this abbreviated strategy allows efficient recovery of novel cytoplasmic, membrane-specific and synaptic proteins that are shared between both and neuronal differentiation. This study highlights the discovery potential of non-comprehensive high-throughput proteomic profiling of unfractionated clinically well-annotated FFPE human tissue from a diverse array of development and diseased states.

Complete Disruption of Autism-Susceptibility Genes by Gene-Editing Predominantly Reduces Functional Connectivity of Isogenic Human Neurons

“…For example, a recent study identified that CASK protein levels, not mRNA levels, were impacted by a deletion of the ASD and schizophrenia risk gene NRXN1 (Pak et al, 2015). Future proteomic studies may indeed be needed to identify similar mechanisms in our isogenic neuron lines, as demonstrated with different neurons (Djuric et al, 2017). Regardless, the reduced activity phenotype in our KO cell lines suggests a convergence between different signaling systems in neurons, indicating that pharmacological interventions that correct the synaptic phenotype could be applied to diverse genetic causes of ASD (Lai et al, 2014; Sahin and Sur, 2015).…”

New insights into the development of the human cerebral cortex

“…) and recently Djuric et al. () were able to demonstrate time‐specific protein abundance changes between proliferative and post‐mitotic layers of the cerebral cortex, and compare these with proteomic data derived from human induced pluripotent stem cell‐derived neural precursors induced to differentiate under defined culture conditions.…”

Synaptic Dysfunction in Human Neurons With Autism-Associated Deletions in PTCHD1-AS

“…For morphometric analyses, neurons were predifferentiated for 3 weeks, dissociated, reseeded on coverslips with mouse astrocytes, and analyzed 5 weeks later. For some gene expression analyses, NPCs and astrocytes were depleted by magnetic-activated cell sorting (MACS) as described elsewhere (30), and neurons were reseeded on Matrigel (Corning Life Sciences, Oneonta, NY). For details, see Supplemental Methods and Materials and Supplemental Table S1.…”