Indigenous gut microbes modulate neural cell state and neurodegenerative disease susceptibility Público
Blackmer-Raynolds, Lisa (Summer 2025)
Abstract
The human gastrointestinal tract is home to trillions of microorganisms—collectively referred to as the gut microbiome—that maintain a symbiotic relationship with their host. This diverse community of microbes grows and changes as we do, with developmental, lifestyle, and environmental factors all shaping microbiome community structure. Increasing evidence suggests this relationship is bidirectional, with the microbiome also influencing a wide range of host physiological processes, including various aspects of neurological health. However, the ways in which the native microbiome concurrently impacts diverse brain cell types remain poorly understood. Therefore, this thesis begins by characterizing microbiome-dependent transcriptional changes across hippocampal cell types using single nucleus RNA sequencing (snRNA-seq) of wild-type germ-free (GF) mice, born and raised in a sterile environment. Simultaneous profiling of all major cell types allowed for direct comparison of transcriptional changes occurring within specific cell populations, identifying cell-type-specific and conserved microbiome-dependent transcriptional changes. This analysis highlighted an increase in adaptive immune and neurodegenerative disease-associated pathways across cell types in GF mice, highlighting a potential link between microbial signals and disease susceptibility. Therefore, to explore the sufficiency of specific indigenous microbes to mediate neuroimmune outcomes, wildtype GF mice were mono-colonized with select taxa associated with human neurological disease. RNA sequencing of brain myeloid cells from mice mono-colonized with Escherichia coli, Clostridium celatum, Bacteroides thetaiotaomicron, and Lactobacillus johnsonii each displayed their own unique phenotypes, highlighting species-specific effects of the microbiome on neuroinflammatory tone. One organism, E. coli, induced a unique adaptive immune and neurodegenerative disease-associated state, suggesting an increased disease susceptibility. SnRNA-seq of the hippocampus of E. coli mono-colonized mice demonstrated time and cell-type-dependent effects of E. coli on the brain, with changes in adaptive immune and neurodegenerative disease pathways occurring across cell types and time points. Further highlighting the importance of these transcriptional changes for disease outcomes, exposure of the 5xFAD beta-amyloidosis mouse model to E. coli resulted in exacerbated cognitive decline and amyloid pathology, demonstrating that this bacterium is sufficient to worsen AD-relevant outcomes. Together, these results emphasize the wide-reaching, species-specific, microbiome-dependent consequences on neurological functions, highlighting the capacity of specific microbes to modulate brain health and disease susceptibility.
Table of Contents
Chapter 1: Introduction to the Microbiome................……………….………………….….…………….……..……………….…………………………….….…………….……..……………….….……............ 1
1.1 Abstract..……………………….……………………………….…………………………….….…………….……..……………….…………………………….….…………………….….…………….……..……. 2
1.2 Introduction..…………………………………….…………………………….….…………….……..……………….…………………………….….…………….……………….……………………….…………. 2
1.3 The gut microbiome through development and aging…………………….…………………………….….…………….……..……………….…………………………….….…………….……..….….…. . 4
1.4 Perturbations to the gut microbiome………………………….…………………………….…………………………….….…………….……..……………….…………………………….….….…..…………. 7
1.5 Contributions of the gut microbiome to brain health throughout the lifespan…..…………..………….….…………….……..……………….…………………………….….…………….……..….. 11
1.6 Association between neurological diseases and gut microbiome composition………………………….…………………………….….…………….……..……………….………………………….…. 13
1.7 Experimental impacts of the microbiome on neurological disease……………….………………….…………………………….….…………….……..……………….…………………………….……. 17
1.8 Microbiome-based therapeutics…………………………….…………………………….….…………….……..……………….…………………………….….………………….…………………………….… 20
1.9 Future outlooks……………………………………….…………………………….….…………….……..……………….…………………………….….………….…………………….……………….……….... 22
1.10 Figures..…….………………………………….…………………………….….…………….……..……………….…………………………….….………..….…………….…………………………..……….……. 24
Chapter 2: Alzheimer’s Disease and the Microbiome.......……………….…………………………….….…………….……..……………….…………………………….….…………….……..……………….…... 26
2.1 Introduction to Alzheimer’s disease…………………………….…………………………….….…………….……..……………….…………………………….….……………………………………………. 27
2.2 Alzheimer’s disease symptoms and pathology…………………………….…………………………….….…………….……..……………….…………………………….….…………….…………………. 27
2.3 Alzheimer’s disease etiology……………….……………….…………………………….….…………….……..……………….…………………………….….….….…………………………………….…….. 28
2.4 Alzheimer’s disease treatments……….…………………………….…………………………….….…………….……..……………….…………………………….….……….………………..…….………… 29
2.5 The microbiome in Alzheimer’s disease ……………………………….…………………………….….…………….……..……………….…………………………….….…..………………...……………… 29
2.6 Alzheimer’s disease, neuroinflammation, and the microbiome ….………………….…………………………….….…………….……..……………….……………………………..……………….…… 31
2.7 Dissertation aims……………..…..………….…………………….………………………….…………………………….….…………….……..……………….…………………………….….……….…..……. 33
Chapter 3: Indigenous gut microbes modulate neural cell state and neurodegenerative disease susceptibility.........……………….…………………………….….…………….……..………………..... 35
3.1 Abstract……………...………….…………………….…………….……………………………….…………………………….….…………….……..……………….…………………………….….………..……. 36
3.2 Introduction ……………...………….……………………….…………………………….….…………….……..……………….…………………………….….……….…………….………….…………………. 36
3.3 Results ……………...………….…………………………….…………………………….….…………….……..……………….…………………………….….………..……….…………….…………………….. 39
3.3.1 A complex microbiome is necessary for the steady-state transcriptional landscape across many brain-resident cell types ………………….…………………………….…….……….………. 39
3.3.2 Select gut microbes differentially and specifically modulate the brain-resident myeloid cell transcriptome ……………..………………………….……...…………………….…...……………. 40
3.3.3 E. coli elicits a temporal transcriptional response across CNS-resident cells .………………….…………………………….….…………….……..……………….…………………………….…..…… 43
3.3.4 E. coli modulates cognitive impairment in an animal model of amyloid pathology……………….…………………………….….…………….……..……………….…………………………….….…46
3.4 Discussion ……………..………….………………….…………….……………………….…………………………….….…………….……..……………….…………………………….….………..……………. 48
3.5 Methods …………….………….…………………….……………………….…………………………….….…………….……..……………….…………………………….….……………….……………………. 52
3.5.1 Experimental model and study participant details………………………….…………………………….….…………….……..……………….…………………………….….……….……….………….… 52
3.5.1.1 Animals …….………………………………….…………………………….….…………….……..……………….…………………………….….…………….……..….….…………….……………………. 52
3.5.1.2 Bacteria ………………………….…………………………….….…………….……..……………….…………………………….….…………….……..….….….………….…………….…………………… 53
3.5.2 Method details …………………………….…………………………….….…………….……..……………….…………………………….….…………….……….…………………….………..………………… 54
3.5.2.1 Bacterial enrichment …………………….…………………………….….…………….……..……………….…………………………….….…………….……..…….…………………...…………………. 54
3.5.2.2 Behavioral testing ….…….…………………….…………………………….….…………….……..……………….…………………………….….…………….……..………….…….……………..……… 54
3.5.2.3 Tissue collection and processing ………………….…………………………….….…………….……..……………….…………………………….….…………….…..…………….………….…………. 57
3.5.2.4 Brain myeloid cell enrichment ……….………………………….…………………………….….…………….……..……………….…………………………….….…………….…….…………………… 54
3.5.2.5 Single nucleus preparation and sequencing …………………….…………………………….….…………….……..……………….…………………………….….…………….…….………….…….. 58
3.5.2.6 Single nucleus RNA-seq analysis ….…….………………….…………………………….….…………….……..……………….…………………………….….…………….….……….…..………..…… 59
3.5.2.7 Bulk RNA-seq preparation and sequencing .…………………….…………………………….….…………….……..……………….…………………………….….……………....………..……….…. 60
.3.5.2.8 Bulk RNA-seq analysis ….……….………………….…………………………….….…………….……..……………….…………………………….….…………….…….…….……....…….……….…… 60
3.5.2.9 Protein extraction and ELISA ……………………….…………………………….….…………….……..……………….…………………………….….…………….…..……...………...……………….. 61
3.5.2.10 Immunohistochemistry ….……….…..………………….…………………………….….…………….……..……………….…………………………….….…………….…….….……..…….……….…… 61
3.5.3 Overview of statistical tests ….……….………..….…………………….…………………………….….…………….……..……………….…………………………….….…………..….……….…….….…… 62
3.6 Acknowledgments….……….……………….…………………………….….…………….……..……………….…………………………….….……..……….……….……….…...……….……….……….…… 63
3.7 Figures ….……….……….……….………………….…………………………….….…………….……..……………….…………………………….….…………….……….…….……….….……….…..….…… 63
3.8 Supplementary Figures .………..…….……….……….…………………….…………………………….….…………….……..……………….…………………………….….….….……...…….…….….…… 71
Chapter 4: Discussion .............................………………………….….…………….……..……………….…………………………….….…………….……..……………….………………………….................... 75
4.1 Implications for the field ….……….…..……….……….………………….…………………………….….…………….……..……………….…………………………….….….…….……….…….……….… 76
4.2 Limitations in the identification of an Alzheimer’s disease-associated microbiome .………………….…………………………….….…………….……..……………….……………………………. 77
4.3 The promise of microbes for personalized medicine ….……….……….….…..….………………….…………………………….….…………….……..……………….……………………………...……. 78
References .....................................……………….…………………………….….…………….……..….…….…………..……….…………………………….….…………….……......................................... 82
Appendix 1: Longitudinal characterization reveals behavioral impairments in aged APP knock in mouse models ......……………….…………………………….….…………….……..…………....... 103
A1.1 Abstract ….……….……….……….……….……….…….…………………….…………………………….….…………….……..……………….…………………………….….……….…...…..….………....… 103
A1.2 Introduction …….………….………………….…………………………….….…………….……..……………….…………………………….….………………….………….………….…….…..………….…… 103
A1.3 Materials and methods …….………………….…………………………….….…………….……..……………….…………………………….….……..……….………….………….……...…..………….…… 105
A1.4 Results …….………………………….…………………………….….…………….……..……………….…………………………….….…………..………….…….……….………….…………..…………..…… 109
A1.5 Discussion …….………….………….…………………….…………………………….….…………….……..……………….…………………………….….………….…….………….………….….……….…… 113
A1.6 Acknowledgments …….………….………….…………………….…………………………….….…………….……..……………….…………………………….….………..…….….………….….……….…… 117
A1.7 Figures …….………….………..……………….…………………………….….…………….……..……………….…………………………….….………….…….………….………….………….…….…….…… 118
A1.8 References …….………….……………………….…………………………….….…………….……..……………….…………………………….….………….….………….………….…………..………….…… 122
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