Proteomic profiling of neuronal and microglial cells using TurboID in inflammatory and homeostatic states for extension into in vivo systems. Público

Abstract

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 Rosa26^TurboID/wt/Camk2a-Cre mice capable of labeling excitatory neuronal proteomes in living mice. This thesis includes foundational experiments validating the genetic strategy underlying the Rosa26^TurboID/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.

Table of Contents

Table of Contents

I.              Introduction11

i.                Historical Background13

a.     The neuron doctrine and dawn of microglial research13

b.    The discovery of Alzheimer’s disease14

ii.              Pathological Characterization of Alzheimer’s disease15

a.     Histological characterizations of Alzheimer’s disease in post mortem human brain15

b.    Clinically-staging Alzheimer’s disease16

c.     Genetic Landscape of Alzheimer’s disease18

d.    Limitations to the Amyloid Cascade Hypothesis and amyloidogenic transgenic mouse models20

iii.            Inflammation in prodromal AD21

a.     The central nervous system presents unique challenges to canonical understandings of immunity21

b.    Inflammation in aging contributes to vulnerability in prodromal Alzheimer’s disease22

c.     The evolutionarily-conserved role of Toll Like Receptors as mediators of innate immunity23

d.    The relevance of lipopolysaccharide-induced inflammation in neurodegeneration 24

e.     LPS as a model of neuroinflammation in neurodegenerative disease; strengths and limitations26

f.      Introduction to microglia in Alzheimer’s disease29

Figures30

II.           Advances in proteomic phenotyping of microglia in neurodegeneration31

i.                Microglia are the resident macrophage of the central nervous system32

ii.              Genome Wide Association Studies implicate microglia in late onset Alzheimer’s disease33

iii.            Transcriptomic diversity of microglia in murine models of Alzheimer’s disease and human Alzheimer’s disease34

iv.             The impact of microglia signaling on Alzheimer’s disease pathogenesis37

v.               Bulk-brain proteomic studies41

a.     Bulk brain proteomics studies reveal metabolic re-programming in glial cells in AD brain42

b.    Bulk brain studies provide evidence for increased inflammasome activation in AD brain44

c.     Complement signaling in transition from mild cognitive impairment to AD dementia46

vi.             Isolation-based proteomic interrogations of microglia49

a.     What have we learned from isolation-based microglial proteomics in AD pathology?52

vii.           Emerging proteomic labeling approaches circumvent the need for isolation 56

a.     Nascent proteomic in vivo labeling using bio-orthogonal chemistry (BONCAT)57

b.    Proximity labeling methods for in vivo proteomic labeling58

viii.         Figures60

III.        Cellular proteomic profiling using proximity labeling by TurboID-NES in microglial and neuronal cell lines66

i.                Abstract67

ii.              Introduction68

iii.            Results72

a.     Generation and validation of stably-transduced microglial and neuronal TurboID-NES cell lines72

b.    TurboID-NES-based MS captures representative proteomes in mammalian microglial and neuronal cell lines73

c.     TurboID-NES over-expression has minimal impact on cellular proteomic and functional profiles of BV2 and N2A cells78

d.    TurboID-NES biotinylates a variety of subcellular compartments within BV2 and N2A cells including several neurodegenerative disease-relevant proteins82

e.     BV2 proteomes biotinylated by TurboID-NES capture Lipopolysaccharide driven changes, partially reflected in the whole-cell BV2 proteomes85

iv.             Discussion89

v.               Conclusion97

vi.             Materials and Methodology98

vii.           Figures119

IV.         Extension of TurboID into murine Camk2a neurons129

i.                Introduction129

ii.              Paving the way to in vivo extension of TurboID131

a.     In vitro validation studies confirm the Cre-lox genetic strategy131

iii.            In vivo biotinylation of murine neuronal proteomes mediated by Adeno Associated viral delivery133

a.     AAV9-TurboID-dsRES into to Camk2a-Cre^ERT2 cortex and striatum134

b.    AAV9-hSyn-Cre into hippocampi of TurboID floxed mice134

iv.             Creation and validation of the novel Rosa26^TurboID/wt mice135

v.               Methodology137

vi.             Figures143

V.            Impact of LPS on Rosa26^TurboID/Camk2a-Cre^ERT2 proteomes149

i.                Introduction149

ii.              Experimental design150

a.     Resolving the impact of LPS on excitatory neurons in vivo150

iii.            Methodology154

iv.             Preliminary conclusions158

v.               Figures162

VI.         Discussion172

i.                Integrated summary of findings & field implications172

a.     Establishing the proteomic breadth captured by TurboID-NES174

b.    TurboID-NES efficiently labels trafficking proteins and can be applied to capturing stress-induced changes in trafficking175

c.     TurboID-NES has minimal impacts on cellular physiology177

d.    TurboID-NES captures cellularly distinct proteins of disease relevance179

e.     Aspects of LPS-mediated inflammation captured by TurboID-NES labeling179

f.      Capturing LPS impacts on glutamatergic neuronal proteomes184

g.    Conclusion185

ii.              Future applications of TurboID186

a.     Resolving cell-autonomous and non-cell autonomous mechanisms of neuroinflammation186

b.    Applications of TurboID-NES to systems level analyses in diverse brain cell types 188

iii.            Figures189

VII.      Bibliography193

About this Dissertation

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School	Laney Graduate School
Department	Biological and Biomedical Sciences
Subfield / Discipline	Neuroscience
Degree	Ph.D.
Submission	Dissertation
Language	English
Research Field	Biology, Cell Biology, Bioinformatics Chemistry, Biochemistry
Palavra-chave	Neuroblastoma TurboID Microglia
Committee Chair / Thesis Advisor	Srikant Rangaraju, Emory University Nicholas T. Seyfried, Emory University
Committee Members	Ranjita Betarbet, Emory University Steven Sloan, Emory University Allan Levey, Emory University

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