Quantitative mass spectrometry-based proteomics in the study of iPSC-derived neurons and disease modeling: A study in neurodevelopment and neurological disorders

In this thesis we applied mass spectrometry-based proteomics to study neuronal differentiation derived from induced pluripotent stem cells (iPSCs) and to study neurodevelopmental disorders such as Spinal Muscular Atrophy (SMA) and Rett syndrome (RTT). In chapter one we give a general introduction to study human brain development. We introduce the generation of iPSCs that can be differentiated into neurons for several research areas. We give an overview of several neurological disorders that are being studied using this model system. Furthermore, we give an introduction to mass spectrometry-based proteomics describing general workflows and mass spectrometers. In addition, we describe several strategies used for quantitative mass spectrometry. In chapter two, we study the protein determinant during neuronal differentiation. Here, we used iPSCs and differentiated these cells into induced neurons (iN) and into small molecule-derived patterned motor neurons. We provide a mass spectrometry-based quantitative proteomic signature at temporat resolution. We identified key proteins showing significant expression changes during neuronal differentiation. Furthermore, we provide a rich source of information on several signaling pathways. Chapter three, describes the study of RTT syndrome using iPSC-based model with isogenic controls and a time-series of mass spectrometry-based proteomic analysis during early stages of neuronal development. We provide evidence of proteomic alterations long before symptoms of RTT syndrome become apparent. Such that proteins associated with dendrite morphology and synapses are differentially expressed at early neuronal stages of development. These changes increase from early to late neuronal phases giving awareness of the protein alterations at early onset of RTT syndrome. In chapter four, we again used quantitative mass spectrometry to study the proteomic changes during early stages of iPSC-derived motor neurons (MN) in SMA. Also here, we show altered proteins at early stages of MN differentiation that are associated with known SMA phenotypes such as defective ER to Golgi transport, mRNA splicing and protein ubiquitination. In addition, these proteins increase in differential expression in SMA towards later stages of MN differentiation. Furthermore, we highlight that differences in altered protein expression between SMA patients have similar biological functions. In this chapter we provide a rich source of molecular events during MN differentiation that might be interesting in the development of new biomarkers and therapeutic approaches. Chapter five is a future outlook describing the cross-talk between the multi-omic studies to better understand a biological mechanism. We introduce several challenges in the study of human brain 131 development associated with temporal and spatial resolution. Furthermore we discuss the latest studies done in SMA and RTT syndrome and provide a future perspective to further improve our understanding of these devastating disorders