Proteomic and transcriptomic signatures of microglia-derived extracellular vesicles and extension into in vivo systems. Open Access
Santiago, Juliet (Fall 2023)
Abstract
Microglia are resident immune cells of the brain that play important roles in mediating inflammatory responses in several neurological diseases via direct and indirect mechanisms. In neurodegenerative diseases, microglia can convert from homeostatic to disease-associated-microglial (DAM) states which play dual roles in disease pathogenesis, resulting in disease-promoting as well as protective responses. Insights into microglial functions in neurodegenerative disease and neuroinflammation can be gained by comprehensive molecular characterization of microglia, at both the proteomic and transcriptomic level. Although microglia-mediated neuroinflammation has emerged as a key pathological mechanism in neurodegenerative diseases, there is a gap in knowledge of how different microglial states contribute to disease pathogenesis. One indirect mechanism may involve extracellular vesicle (EV) release, such that the molecular cargo transported by microglia-derived EVs can have functional effects by facilitating intercellular communication with recipient cells. The molecular composition of microglia-derived EVs, and how microglial activation states impact EV composition and EV-mediated effects in neuroinflammation, remain poorly understood. This thesis includes the foundational in vitro studies identifying unique molecular profiles of microglia-derived EVs that are determined by microglial activation state. By combining proteomic and transcriptomic methods, we obtained comprehensive molecular profiles of BV2 microglia-derived EVs under homeostatic and stimulated conditions. Our analyses revealed novel state-specific proteomic and transcriptomic signatures of microglia-derived EVs. Particularly, lipopolysaccharide (LPS) activation had the most profound impact on proteomic and transcriptomic compositions of microglia-derived EVs. Additionally, we found that EVs derived from LPS-activated microglia were able to induce pro-inflammatory transcriptomic changes in resting responder microglia, confirming the ability of microglia-derived EVs to relay functionally-relevant inflammatory signals. These comprehensive molecular datasets represent important resources for the neuroscience and omics communities, and provide novel insights into the role of microglia-derived EVs in neuroinflammation. These in vitro experiments were fundamental for developing a novel method to characterize the proteome of microglia and their EVs in vivo using TurboID-based proximity labeling. Our findings reveal that the microglial proteome cannot be labeled in vivo using TurboID-based proximity labeling with the Rosa26-floxSTOP-TurboID;Tmem119-CreERT2 mice, despite successful labeling of the cell proteome and EV proteome in TurboID-BV2 microglia and astrocytic Rosa26-floxSTOP-TurboID;Aldh1l1-CreERT2 mice. This thesis includes foundational experiments along with newly developed techniques to interrogate the possibility of characterizing cell-type specific EVs in vivo.
Table of Contents
Table of Contents
I. Introduction1
i. Microglia and neuroinflammation1
a. The discovery of microglia1
b. Microglia origin and development1
c. Heterogeneity of microglia2
d. Microglia activation 3
e. Innate immunity and molecules that mediate microglia inflammatory responses 3
f. The relevance of lipopolysaccharide-induced inflammation4
g. LPS as a model of neuroinflammation in neurodegenerative disease; strengths and limitations5
ii. Neuroinflammation in Alzheimer’s disease 6
a. Alzheimer’s disease prevalence and genetic landscape 7
b. Alzheimer’s disease pathophysiology 8
c. Inflammation in aging contributes to vulnerability in prodromal Alzheimer’s disease 10
d. Genome Wide Association Studies implicate microglia in late onset Alzheimer’s disease 11
e. Transcriptomic diversity of microglia in murine models of Alzheimer’s disease and human Alzheimer’s disease 12
f. Microglia signaling in Alzheimer’s disease13
Figures15
iii. Extracellular vesicles in neuroinflammation and neurodegeneration15
a. The discovery of extracellular vesicles16
b. Extracellular vesicles biogenesis, composition, and secretion18
c. Extracellular vesicle transport and uptake20
d. Methods of isolating extracellular vesicles21
e. The role of extracellular vesicles in neurodegenerative disease23
f. Microglia-derived extracellular vesicles24
Figures26
iv. Proteomic interrogations of glia27
a. Bulk brain proteomic studies27
b. Isolation-based proteomic strategy limitations28
c. Emerging proteomic labeling strategies29
d. Cell-type specific biotin labeling in vivo31
v. Summary31
II. Identification of state-specific proteomic and transcriptomic signatures of microglia-derived extracellular vesicles32
i. Abstract32
ii. Introduction33
iii. Results37
a. Verification of EV purification from BV2 cell culture medium 37
b. Identification of proteomic signatures of microglia-derived EVs by quantitative mass spectrometry37
c. Microglial activation state impacts proteomic characteristics of EVs39
d. Novel transcriptomic signatures microglia-derived EVs in resting and pro-inflammatory states41
e. Integrative proteomic and transcriptomic analysis of BV2-dervied EVs reveal enrichment of pathways involved in RNA binding and translation43
f. Microglia-derived EVs exhibit unique microRNA signatures under resting and LPS-treated conditions44
g. EVs derived from LPS-treated microglia induce pro-inflammatory changes in responder microglia45
iv. Discussion47
v. Conclusion51
vi. Materials and Methodology 52
vii. Figures65
III. Cell-type specific biotin labeling in vivo to resolve glial-derived extracellular vesicle proteomes in the mouse brain 77
i. Introduction77
ii. Paving the way to in vivo extensions of TurboID to label microglia and extracellular vesicle proteomes 79
iii. Preliminary results82
a. TurboID labels extracellular vesicles derived from TurboID transduced BV2 microglia cells83
b. Rosa26-floxSTOP-TurboID;Tmem119-CreERT2 mice reveal insufficient biotin labeling of microglia 83
c. Rosa26-floxSTOP-TurboID;Aldh1l1-CreERT2 mice reveal sufficient biotin labeling of astrocytes and brain derived-EVs86
iv. Preliminary conclusions and study limitations86
v. Methodology89
vi. Figures95
IV. Discussion102
i. Integrated summary of findings & field implications102
a. Proteomic signatures of BV2 microglia-derived EVs 103
b. Microglia activation state impacts proteomic and transcriptomic characteristics of BV2 microglia-derived EVs 104
c. Potential role of BV2 microglia-derived EVs in RNA binding and translation 105
d. BV2 microglia-derived EVs exhibit unique microRNA signatures under pro-inflammatory conditions 105
e. EVs derived from LPS activated BV2 microglia can elicit pro-inflammatory changes in responder microglia 106
f. TurboID proximity labeling as a promising approach to label the proteome of EVs in vivo with cell-type specificity106
g. Conclusion107
ii. Future applications108
a. Investigating the potential of BV2 microglia-derived EVs from pro-inflammatory microglia to initiate translation of mRNAs in recipient cells108
b. Possible ways to resolve the inability to label microglia in vivo using TurboID proximity labeling 109
c. Future applications of TurboID proximity labeling to investigate the proteome of EVs from specific cell types110
V. Bibliography111
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