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Proteomic profiling of brain cell types using isolation-based strategies pose limitations in resolving cellular phenotypes representative of their native state. We describe a mouse line for cell type-specific expression of biotin ligase TurboID, for in vivo biotinylation of proteins. Using adenoviral and transgenic approaches to label neurons, we show robust protein biotinylation in neuronal soma and axons throughout the brain, allowing quantitation of over 2000 neuron-derived proteins spanning synaptic proteins, transporters, ion channels and disease-relevant druggable targets. Next, we contrast Camk2a-neuron and Aldh1l1-astrocyte proteomes and identify brain region-specific proteomic differences within both cell types, some of which might potentially underlie the selective vulnerability to neurological diseases. Leveraging the cellular specificity of proteomic labeling, we apply an antibody-based approach to uncover differences in neuron and astrocyte-derived signaling phospho-proteins and cytokines. This approach will facilitate the characterization of cell-type specific proteomes in a diverse number of tissues under both physiological and pathological states. Current isolation-based approaches for cell type-specific proteomics pose several challenges. Here, the authors present an approach for in vivo cell type-specific protein labeling to characterize proteomic differences between neurons and astrocytes in their native state in adult mouse brain.

The brain is a cellularly complex organ possessing glia, neurons, and vascular cells. Each cell type supports distinct physiological roles in homeostatic states to orchestrate higher-order cognitive processes. Likewise, each cell type expresses unique proteomic profiles capable of emerging physiological phenotypes with distinct vulnerabilities in neurodegenerative disease. Alzheimer’s disease (AD) is the most common neurodegenerative disease, and ongoing systems-level analyses continue to highlight the importance of cellular complexity with disease progression. Bulk brain analyses provide a broad picture of global molecular transformations occurring in the brain that correlate with disease pathology and other traits. However, these bulk methods cannot directly resolve molecular changes occurring in distinct brain cell types. Cellular isolation upstream of mass-spectrometry poses important challenges ranging from contamination from other cell types, reliance on well-validated surface markers which can alter in disease states, and the inability to purify adult neurons. The recent development of proximity-based biotin ligases including TurboID, have made it possible to label and purify cellular proteomes in living cells and animals without the need for cellular isolation. This thesis includes the foundational in vitro studies validating the use of TurboID-based proximity labeling to resolve proteomic differences between two brain cell types (neurons and microglia) under both homeostatic and neuroinflammatory contexts. Additionally, these studies interrogated the proteomic breadth captured by cytosolic TurboID-mediated biotinylation, the impact of TurboID expression on homeostatic phenotypes, the cellular-distinction of proteins labeled by cytosolic TurboID, and the propensity of agnostically-directed cytosolic TurboID to label proteins of disease relevance in homeostatic and neuroinflammatory conditions. Our proteomic analyses demonstrate that cytosolic TurboID and streptavidin-based affinity purification capture >50% of microglial and neuroblastoma proteomes. Cytosolic expression of TurboID minimally impacted cellular proteome abundances, and did not significantly impact cellular respiration or microglial cytokine release profiles with inflammatory challenge. TurboID-NES captured proteins of relevance to neurodegeneration in both microglia and neuroblastoma cell lines, and successfully captured a portion of microglial proteomic changes in response to inflammatory challenge. These in vitro experiments laid the foundation for the generation of novel Rosa26TurboID/wt/Camk2a-Cre mice capable of labeling excitatory neuronal proteomes in living mice. This thesis includes foundational experiments validating the genetic strategy underlying the Rosa26TurboID/wt/Camk2a-Cre mice, as well as experiments assessing the impact of inflammation specifically on the proteomes of excitatory neurons, which are not apparent at the bulk proteome level. Using TurboID as a discovery tool, our findings show that neuroinflammation is associated with an increase in glutamatergic post-synaptic proteins in Camk2a neurons which may be indicative of neuronal hyper-excitability.

Isolation and proteomic profiling of brain cell types, particularly neurons, pose several technical challenges which limit our ability to resolve distinct cellular phenotypes in neurological diseases. Therefore, we generated a novel mouse line that enables cell type-specific expression of a biotin ligase, TurboID, via Cre-lox strategy for in vivo proximity-dependent biotinylation of proteins. Using adenoviral-based and transgenic approaches, we show striking protein biotinylation in neuronal cell bodies and axons throughout the mouse brain. We quantified more than 2,000 neuron-derived proteins following enrichment that mapped to numerous subcellular compartments. Synaptic, transmembrane transporters, ion channel subunits, and disease-relevant druggable targets were among the most significantly enriched proteins. Remarkably, we resolved brain region-specific proteomic profiles of Camk2a neurons with distinct functional molecular signatures and disease associations that may underlie regional neuronal vulnerability. Leveraging the neuronal specificity of this in vivo biotinylation strategy, we used an antibody-based approach to uncover regionally unique patterns of neuron-derived signaling phospho-proteins and cytokines, particularly in the cortex and cerebellum. Our work provides a proteomic framework to investigate cell type-specific mechanisms driving physiological and pathological states of the brain as well as complex tissues beyond the brain. Competing Interest Statement The authors have declared no competing interest.

Understanding synapse-specific effects of neuroinflammation can provide mechanistic and therapeutically relevant insights across the spectrum of neurological diseases. We applied neuron-specific proteomic biotinylation , differential centrifugation of brain for crude synaptosome enrichment (P2 fraction) and mass spectrometry (MS) analysis of biotinylated proteins to derive native-state proteomes of Camk2a-positive neurons and their corresponding P2 synaptic compartments. Next, in an model of systemic lipopolysaccharide (LPS) dosing, we examined the effects of neuroinflammation on whole neuron and synaptic compartments using a combination of MS, network analysis, confirmatory biochemical and ultrastructural assays and integrative approaches across our mouse-derived and existing human datasets. Ultrastructural and biochemical analyses of P2 fractions verified enrichment in synaptic elements, including synaptic vesicles and mitochondria. MS of biotinylated proteins from Camk2a-specific bulk brain homogenates (whole neuron) and P2 fractions (synaptosome) showed enrichment of >1000 proteins, consistent with neuron-specific biotinylation, also confirmed by immunofluorescence microscopy. Camk2a-specific synaptic proteome revealed molecular signatures related to mitochondrial function, synaptic transmission, protein translation. LPS-treated mice displayed body weight loss and neuroinflammation, characterized by glial activation, increased pro-inflammatory cytokine levels and upregulated expression of Alzheimer's disease (AD)-related microglial genes. LPS-induced neuroinflammation exerted distinct effects on the synaptic proteome, including increased mitochondrial and reduced cytoskeletal-synaptic proteins, while suppressed synaptic MAPK signaling. Importantly, these changes were not observed at the whole neuron level, indicating unique vulnerability of the synapse to neuroinflammation. In line with synapse proteomic and signaling changes, LPS altered the ultrastructure of asymmetric synapses, suggesting dysregulation of excitatory neurotransmission. Co-expression network analysis of Camk2a neuronal proteins further resolved mitochondria- and synapse-specific protein modules, some of which were neuroinflammation-dependent. Neuroinflammation increased levels of a mitochondria-enriched module, and decreased levels of a pre-synaptic vesicle module, without impacting a post-synaptic membrane module. LPS-dependent mitochondrial and LPS-independent post-synaptic modules in mouse neurons mapped to post-mortem human AD brain proteomic modules which were decreased in cases with AD dementia and positively correlated to cognitive function, including pro-resilience markers for AD. Our findings using native-state proteomics of Camk2a neurons combined with synaptosome enrichment identify proteome-level mechanisms of early synaptic vulnerability to neuroinflammation relevant to AD.

Different brain cell types play distinct roles in brain development and disease. Molecular characterization of cell-specific mechanisms using cell type-specific approaches at the protein (proteomic) level, can provide biological and therapeutic insights. To overcome the barriers of conventional isolation-based methods for cell type-specific proteomics, in vivo proteomic labeling with proximity dependent biotinylation of cytosolic proteins using biotin ligase TurboID, coupled with mass spectrometry (MS) of labeled proteins, has emerged as a powerful strategy for cell type-specific proteomics in the native state of cells without need for cellular isolation. To complement in vivo proximity labeling approaches, in vitro studies are needed to ensure that cellular proteomes using the TurboID approach are representative of the whole cell proteome, and capture cellular responses to stimuli without disruption of cellular processes. To address this, we generated murine neuroblastoma (N2A) and microglial (BV2) lines stably expressing cytosolic TurboID to biotinylate the cellular proteome for downstream purification and analysis using MS. TurboID-mediated biotinylation captured 59% of BV2 and 65% of N2A proteomes under homeostatic conditions. TurboID expression and biotinylation minimally impacted homeostatic cellular proteomes of BV2 and N2A cells, and did not affect cytokine production or mitochondrial respiration in BV2 cells under resting or lipopolysaccharide (LPS)-stimulated conditions. These included endo-lysosome, translation, vesicle and signaling proteins in BV2 microglia, and synaptic, neuron projection and microtubule proteins in N2A neurons. The effect of LPS treatment on the microglial proteome was captured by MS analysis of biotinylated proteins (>500 differentially-abundant proteins) including increased canonical pro-inflammatory proteins (Il1a, Irg1, Oasl1) and decrease anti-inflammatory proteins (Arg1, Mgl2). Competing Interest Statement The authors have declared no competing interest. Footnotes * Updated abstract.