From the Department of Neuroscience Karolinska Institutet, Stockholm, Sweden GUT MICROBES AND THE DEVELOPING BRAIN Tim Ben Frans Arentsen Stockholm 2017 Cover Illustration by Laleh Maleki All previously published papers were reproduced with permission from the publisher. Published by Karolinska Institutet. Printed by E-Print AB, 2017 © Arentsen, 2017 ISBN 978-91-7676-613-2 Gut Microbes and the Developing Brain THESIS FOR DOCTORAL DEGREE (Ph.D.) ACADEMIC DISSERTATION For the degree of PhD at Karolinska Institutet This thesis will be defended in public at the Hillarp Lecture Hall, Retzius väg 8, Karolinska Institutet, Stockholm, Sweden Friday the 7th of April, 2017, 09:30 By Tim Ben Frans Arentsen Principal Supervisor: Opponent: Assoc. Prof. Rochellys Diaz Heijtz Prof. John Cryan Karolinska Institutet University College Cork Department of Neuroscience Department of Neuroscience/Anatomy Co-supervisor: Examination Board: Prof. Hans Forssberg Assoc. Prof. Elisabeth Norin Karolinska Institutet Karolinska Institutet Department of Women’s and Children’s Health Department of Microbiology, Tumor, and Cell Neuropediatric Unit Biology Prof. Jon Laman University of Groningen Department of Neuroscience Prof. Klas Blomgren Karolinska Institutet Department of Women’s and Children’s Health Dedicated to my beloved mother, father, and sister and to my dearest wife Laleh. ABSTRACT The discovery that commensal gut microbiota can influence host development and physiology beyond the gastrointestinal (GI) tract has triggered a paradigm shift in our conceptualization of the origin of human diseases. A growing body of preclinical research has demonstrated that gut microbiota exert a modulatory role on the development and function of brain circuits involved in motor control, emotion and cognition. These findings have lent support to the hypothesis that gut bacteria may play a role in the etiology and/or pathophysiology of human brain disorders. The current challenge is to understand the precise mechanisms mediating the communication between the microbiota and the brain. In the present thesis, we used a combination of mouse models (e.g., germ-free; GF, antibiotic treated, and transgenic mice), molecular, biochemical, and behavioral approaches to gain a deeper insight into the role of gut microbiota on brain development and behavior. A major goal was to explore whether microbial products from the commensal gut microbiota can be translocated into the developing brain and be sensed by pattern recognition receptors (PRRs) of the innate immune system. In Paper I, we took advantage of the GF mouse model (mice raised throughout development in an environment devoid of bacteria) to study the influence of gut microbiota on social behavior. Using the three-chamber social approach task, we demonstrated significant differences in social approach behavior between GF and conventionally raised mice (specific pathogen-free, SPF). Adult GF Swiss-Webster mice displayed higher levels of sociability than SPF mice, as indicated by a stronger preference for time spent close to the unfamiliar stimulus mouse versus the novel object. In addition, they showed reduced expression levels of total BDNF and BDNF exon-containing transcripts I-, IV-, VI-, and IX in the amygdala, a brain region involved in the processing of social stimuli. These findings suggest that alterations in the expression of specific BDNF exon transcripts within the amygdala may contribute to the abnormal development of social behavior in GF mice. In Paper II, we investigated whether antibiotic-induced perturbation of the maternal gut microbiota during pregnancy influences brain development and behavior of the offspring. The juvenile offspring of antibiotic-treated dams showed hyperactivity and sex-specific changes in social behavior (similar to that observed in GF mice), without changes in body weight. In addition, the male juvenile offspring had reduced BDNF mRNA and protein expression in the amygdala. Interestingly, we found a negative correlation between time spent interacting with an unfamiliar stimulus mouse and levels of BDNF protein. These findings in mice indicate that antibiotic-induced perturbation of the maternal gut microbiota during pregnancy has profound effects on brain development leading to abnormal motor and social development of the offspring. In Paper III, we examined the possibility that fragments of bacterial peptidoglycan (PGN), a major component of the bacterial cell wall, derived from commensal gut microbiota can cross the blood brain barrier under normal conditions and influence the developing brain via activation of PRRs. Using various expression-profiling techniques (i.e., qRT-PCR, Western Blot and immunohistochemistry), we showed that two families of PRRs that specifically detect PGN and its derivates (PGN recognition proteins and NOD-like receptors), and the PGN transporter PepT1 are highly expressed in the developing brain during critical windows of postnatal development. In addition, we demonstrated that the expression of several of these PGN-sensing molecules are sensitive to manipulation of the gut microbiota (i.e., GF conditions and antibiotic exposure in early life). Finally, we demonstrated that the absence of PGN recognition protein 2 (Pglyrp2; which is an N-acetylmuramyl-L-alanine amidase that hydrolyzes bacterial PGN between the sugar backbone and the peptide chain) leads to sex- specific changes in social behavior in the prepubertal period. However, we did not observe changes in motor or anxiety-like behavior at this age. These novel findings support the notion that central activation of PRRs by bacterial PGN fragments could be one of the signaling pathways mediating the communication between the gut microbiota and the developing brain. In Paper IV, we tested the hypothesis that the modulatory role of PGN recognition proteins (PGRPs) on behavior changes with age, by using Pglyrp2 knockout (KO) mice. Using a battery of behavioral tests, we demonstrated sex-dependent alterations in motor and anxiety-like behavior in 15-month-old Pglyrp2 KO mice, as well as mild changes in the expression of synaptophysin (a presynaptic marker) and gephyrin (a protein associated with inhibitory synapses) in key brain regions implicated in the processing of emotional stimuli. These observations indicate that the mammalian Pglyrp2 plays an important role in the modulation of brain circuits involved in motor control and anxiety in later life. In summary, this thesis provides conceptually novel evidence that the central activation of PRRs by bacterial PGN fragments could be one of the signaling pathways mediating the communication between the gut microbiota and the developing brain. This new signaling pathway may be a new entry point for the exploration of the role of gut microbiota on brain development, function and behavior. Finally, we propose that alterations within different components of this signaling pathway could lead to deviations in brain developmental trajectories, thus increasing risk for neurodevelopmental and psychiatric disorders. LIST OF SCIENTIFIC PAPERS I. Arentsen T, Raith H, Qian Y, Forssberg H, Diaz Heijtz R. Host microbiota modulates development of social preference in mice. Microbial Ecology in Health & Disease, 2015, volume 26: 29719 II. Arentsen T, Mina D, St. Pierre J, Foster J, Forssberg H, Diaz Heijtz R. Perturbation of maternal gut microbiota during pregnancy influences offspring brain development and behavior. Submitted manuscript. III. Arentsen T, Qian Y, Gkotzis S, Femenia T, Wang T, Udekwu K, Forssberg H, Diaz Heijtz R. The bacterial peptidoglycan-sensing molecule Pglyrp2 modulates brain development and behavior. Molecular Psychiatry (2017) 22, 257–266; doi:10.1038/mp.2016.182 published online 15 November 2016. IV. Arentsen T, Khalid R, Qian Y, Diaz Heijtz R. Sex-dependent alterations in motor and anxiety-like behavior of aged bacterial peptidoglycan sensing molecule 2 knockout mice. Submitted manuscript.