Proteomics and network analysis identify common and specific pathways of neurodegeneration

Neurodegenerative disorders, such as Parkinson's disease (PD), Alzheimer's disease (AD) and amyotrophic lateral sclerosis (ALS) are multi-factorial in nature, involving several genetic mutations (in coding or regulatory regions) and epigenetic and environmental factors. The main clinical manifestation (movement disorders, cognitive impairment and/or psychiatric disturbances) depends on the neuron population being primarily affected. Complex and multifactorial neurodegenerative diseases can be investigated using a holistic approach that can give a global view about the pathogenetic process and shed light on specific and generic pathways of neurodegeneration. Proteomics offers a global molecular snapshot of proteins and consequently of processes that may influence neuronal death. The proteome in fact provides a dynamic view of what is happening in the system under investigation, because the expression of proteins, their abundance, their localization in tissues or cells, the type and amount of their post-translational changes depend from the environment and from the cellular physiological state. Therefore, all the projects presented in this thesis, by combining bioinformatics tools with proteomics, aimed at highlighting biochemical processes shared by different neurodegenerative diseases and diseasespecific pathways, which may justify the degeneration of dopaminergic neurons in PD. Finally, a focus on the mitochondrial interactome and proteome intended to elucidate important specific steps of the degenerative process in PD

Identification of Amyotrophic Lateral Sclerosis Disease Mechanisms by Cerebrospinal Fluid Proteomic Profiling

Amyotrophic lateral sclerosis (ALS) is the most common form of adult-onset motor neuron disease. Heterogeneity in clinical, genetic, and pathological features of ALS suggest the disease is a spectrum of disorders each resulting in motor neuron degeneration. Molecular profiling of ALS patients is, therefore, a useful means of characterizing and stratifying the ALS population. To this end, mass spectrometric proteomic profiling was performed on cerebrospinal fluid (CSF) from ALS, healthy control (HC), and other neurological disease (OND) subjects. This resulted in the identification of 1,712 CSF proteins, 123 of which exhibited altered relative abundance in ALS CSF. Biological processes related to these 123 proteins included synaptic activity, extracellular matrix, and inflammation. The application of feature selection and machine learning methods to these CSF proteomic profiles resulted in a classifier that used relative levels of WDR63, APLP1, SPARCL1, and CADM3 to predict independent ALS, HC, and OND samples with 83% sensitivity and 100% specificity. To aid in the validation of selected CSF proteins, a Western blot loading control method was developed and validated using a reversible, iodine-based total protein stain. This method improves the accuracy and sensitivity of the relative quantification of CSF proteins via Western blot. As RNA binding protein (RBP) pathology/dysfunction is common to several forms of ALS, the largest CSF RBP alteration, that of RNA binding motif 45 (RBM45) protein, was validated externally. The results demonstrated that RBM45 pathology is common to several forms of ALS, frontotemporal lobar degeneration (FTLD), and Alzheimer’s disease. To further understand the biological functions of RBM45, immunoprecipitation coupled to mass spectrometry was performed to identify RBM45 protein-protein interactions (PPIs). RBM45 PPIs and associated pathways were most strongly associated with hnRNP proteins, RNA processing, and cytoplasmic translation. RBM45 also participates in the general cellular response to stress via association with nuclear stress bodies. This association is dependent on RNA binding, is upregulated in ALS/FTLD, and is sufficient to induce the aggregation of the protein. Collectively, these results illustrate the utility of CSF proteomic profiling for characterizing mechanisms of neurological disease and provide new insights into the contributions of RNA binding protein dysregulation to ALS/FTLD

Metabolic Imaging and Applications in Protein and Lipid Homeostasis

Metabolic activity is an important functional parameter of a living cell. Microscopic techniques are demanded to resolve the heterogeneity of metabolic activity from cell to cell and among subcellular compartments. Towards this, work in the Min lab has been dedicated to developing a prevailing metabolic imaging platform that couples chemical imaging by stimulated Raman scattering (SRS) microscopy with small vibrational tags on precursor molecules. This thesis describes efforts along metabolic imaging by SRS microscopy, with focus on visualizing protein and lipid homeostasis. Chapter 1 describes the design principle of metabolic imaging, including selection of vibrational tags and setup of SRS microscopy, and an overview of successful demonstrations of metabolic imaging in protein and lipid metabolism. Chapter 2 describes adoption of such principle to visualize protein turnover with 13C-phenylalanine metabolic labeling under steady-state condition and various perturbations. The rest of this thesis (Chapters 3-6) switches focus to fatty acid metabolism and cellular lipid homeostasis. As the minimal tagging in vibrational imaging preserves the physicochemical property of lipid molecules to the largest extent, it motivated me to revisit fatty acid metabolism from a biophysical perspective. Bearing the question in mind whether the non-equilibrium metabolic activity could drive phase separation in biological membranes, I thus look into the principle of membrane organization and its implication in biological membranes in Chapter 3. Then in Chapter 4, I describe the discovery and characterization of previously unknown phase separation in endoplasmic reticulum (ER) membrane caused by lipid synthesis. In this case, metabolic imaging by SRS enables identification of solid-like domains formed by saturated fatty acid (SFA) metabolites. This observation further raises the question whether phase separation bears any functional roles in the adverse effects of SFAs (or lipotoxicity). Towards this, Chapter 5 introduces the background of lipotoxicity including its definition and models. Then I review proposed mechanisms for lipotoxicity, which point to the central role of ER in mediating the stress transduction. In Chapter 6, I present our findings that suggest the association of the observed solid-like domains with ER structural remodeling and local autophagic arrest. Together, these efforts demonstrate the valuable capability of SRS imaging to reveal metabolic heterogeneity and how this aids in the investigation of metabolic stress