Publisher Correction: LifeTime and improving European healthcare through cell-based interceptive medicine.

A Correction to this paper has been published: https://doi.org/10.1038/s41586-021-03287-8.</jats:p

Beeinflussung der Phagozytoseaktivität und Migration von Mikrogliazellen durch Monoaminneurotransmitter oder Amyloid beta Peptide

Contents 3 Table of contents 3 Table of Figures 6 Table of Tables 7 1 Introduction 1 1.1 Microglia - Immune defense of the brain 1 1.1.1 Origin of microglial cells 1 1.1.2 Microglial phagocytosis 3 1.1.3 Microglial motility 6 1.2 Microglia in neurodegenerative diseases 7 1.2.1 General aspects and examples 7 1.2.2 Microglia in Alzheimers disease 9 1.3 Microglia-Neuron interaction 13 1.3.1 Neurotransmitters and glia-transmission 15 2 Material and Methods 19 2.1 Materials 19 2.1.1 Reagents and dyes 19 2.1.2 Media and solutions 21 2.1.3 Antibodies 22 2.1.4 Tools 23 2.1.5 Commercial Kits 23 2.1.6 Devices 24 2.1.7 Software 24 2.2 Methods 25 2.2.1 Animals 25 2.2.2 Genotyping Cx3cr1-GFP mouse strain 25 2.2.3 Induction of a stab wound injury to activate microglia in vivo 27 2.2.4 Microglial cell culture 27 2.2.5 Microglia isolation from adult mouse brain 28 2.2.6 RNA Isolation and PCR 29 2.2.7 Microchemotaxis assay 32 2.2.8 ELISA for cytokine release 33 2.2.9 Nitric oxide release assay 34 2.2.10 Preparation of acute brain slice 34 2.2.11 Phagocytosis experiment in vitro 35 2.2.12 Phagocytosis experiment in situ 36 2.2.13 Phagocytic Index calculation 37 2.2.14 Immunohistochemistry 37 2.2.15 Fluorescent microscopy 38 2.2.16 Confocal microscopy 38 2.2.17 Two-photon imaging and laser lesion 38 2.2.18 Statistical analysis 40 3 Results 41 3.1 Modulation of microglial properties by neurotransmitter stimulation 41 3.1.1 Microglial baseline activity is age dependent 41 3.1.2 Microglial phagocytic activity is reduced upon stimulation with neurotransmitters of the monoamine family in vitro 43 3.1.3 Neurotransmitter application leads to a decrease in phagocytic activity of amoeboid microglia in situ 45 3.1.4 Microglial phagocytic activity in acute brain slice from adult mice is only modulated by high concentrations of dopamine 47 3.1.5 Adult microglial phagocytic activity in slices is not changed by dopaminergic or adrenergic receptor agonists 48 3.1.6 Serotonin facilitates ATP induced microglial migration in vitro and in situ 50 3.1.7 Norepinephrine but not dopamine or adrenergic and dopaminergic agonists modify microglial response towards laser induced acute injury 52 3.1.8 Serotonin does not alter LPS induced release of cytokines and nitric oxide 54 3.2 Neurotransmitter receptor expression in microglial cells 55 3.2.1 The serotonin receptor repertoire differs in neonatal versus adult microglial preparations 55 3.2.2 Microglial cells do not express the essential enzyme for serotonin synthesis 57 3.3 Microglial properties in a mouse model of Alzheimer’s Disease 58 3.3.1 Plaque load in APPPS1 mice differs with age and brain region 58 3.3.2 Phagocytic activity is increased in a model of acute injury 60 3.3.3 Microglial phagocytic activity is changed in cortex in different mouse models of AD 62 3.3.4 Phagocytic activity is impaired by plaque presence 64 3.3.5 Microglial response to a laser induced lesion in cortical brain slices is reduced in 10 months but not 4 months old APPPS1 mice 66 4 Discussion 68 4.1 Impact of neurotransmitters on microglial properties 68 4.1.1 Microglial phagocytosis and migration in different microglial preparations depend on age 69 4.1.2 Monoamine modulation of phagocytic activity in different microglia preparations 70 4.1.3 Serotonin does not alter LPS induced cytokine release 71 4.1.4 Modulation of migration by monoamines and receptor agonists 72 4.1.5 Serotonin receptor are differentially expressed in the investigated microglial preparations 73 4.2 Microglial properties in AD 75 4.2.1 Microglial cells around senile plaques do not represent phagocytic phenotype found in acute activation 75 4.2.2 Microglial phagocytic activity in AD 76 4.2.3 Microglial response to laser lesion is only reduced in AD 77 5 Summary 80 6 Zusammenfassung 82 7 Eidestattliche Erklärung 84 8 Danksagung 85 9 Bibliography 86 10 Appendix 96 10.1 Curriculum Vitae 96 10.2 Communications 98 10.2.1 Publications 98 10.2.2 Abstracts/Talks 99 10.2.3 Selected Abstracts/Posters 99Microglia are the immune cells of the brain and are involved in developmental as well as regenerative processes of the brain. As being part of the immune system they survey their surroundings by constant process extension and retraction for any insult and can act as phagocytes engulfing cellular debris and pathological invaders of the brain. In this project I studied the phagocytic activity as well as process motility 1\. upon stimulation with different monoamine neurotransmitters like serotonin, norepinephrine and dopamine to investigate microglia neuron communication and 2\. in the context of Aβ plaque deposition in mouse models of Alzheimer’s disease. Therefore I used different microglial preparations, namely primary neonatal microglia, amoeboid microglia on top of acute brain slices of P6-9 mice and ramified microglia in acute brain slices of adult mice from healthy C57BL/6 mice or mutated APPPS1 and APP23 mice, mouse models for Alzheimer’s disease, respectively. In the first part of my work I found phagocytic uptake of fluorescent microsphere to be reduced by monoamine neurotransmitters mainly in young microglia including neonatal as well as amoeboid microglia whereas adult microglia only respond to high concentrations of dopamine with reduced particle uptake. In contrast to adult microglia where application of specific dopaminergic or adrenergic receptor agonists did not show the same reduction, application of the specific 5HT2 receptor agonist DOI leads in neonatal microglia to a concentration dependent decrease of particle engulfment. Moreover, serotonin was found to be a potent stimulator of ATP induced microglial migration as it not only triggers microglial migration towards an applied ATP gradient in vitro but also enhances process motility towards a laser induced injury that was suggested to be a source for ATP released from damaged cells. Secondly, my data shows that microglial phagocytic activity is lowered in the cortex of diseased APPPS1 as well as APP23 mice. Experiments on microglial microsphere uptake in unaffected brain regions revealed fully functional microglial phenotype providing evidence that microglial impairment is dependent on the presence of Aβ plaques. In addition, microglial response towards acute injury induced by a cortical laser lesion increases during aging comparing 2, 4 and 10 months old mice which is attenuated in aged APPPS1 mice but not in younger, plaque bearing mice supporting functional impairment of microglia by the presence of Aβ plaques. In conclusion, this work provides data that microglia express functional neurotransmitter receptors for serotonin, norepinephrine and dopamine whose activation leads to modification of substantial effector functions involved in synapse rearrangement during development as well as regeneration in pathology. Moreover, I can show that these functions are impaired upon Aβ plaque presence supporting the view of a dysfunctional microglial phenotype during neurodegenerative disease progression.Mikrogliazellen sind eingebunden in verschiedenste Vorgänge im Gehirn, sowohl während der Entwicklung als auch bei Regeneration nach Verletzung. Als Teil des Immunsystems sind sie in der Lage ihre Umgebung mittels ihrer feinen Fortsätze ständig abzutasten, aber auch als Phagozyten Zelltrümmer und Pathogene aufzunehmen. In diesem Projekt sollte sowohl die Phagozytoseaktivität als auch die Bewegung der Zellfortsätze 1\. Nach Stimulation mit verschiedenen Monoaminneurotransmittern wie Serotonin, Noradrenalin und Dopamin zur Charakterisierung der Mikroglia Neuron Interaktion untersucht werden. 2\. Im Zusammenhang mit Aβ-Ablagerungen in verschiedenen Mausmodellen der Alzheimer Krankheit untersucht werden. Für meine Experimente nutzte ich verschiedene Mikrogliapräparationen, wie neonatale Mikroglia, amöboide Mikroglia auf der Oberfläche akuter Hirnschnitte von 6-9 Tage alten Mäusen und ramifizierte Mikroglia in akuten Hirnschnitten adulter Mäuse jeweils von gesunden C57BL/6 Mäusen oder mutierten APPPS1 bzw. APP23 Mäusen, als Mausmodell der Alzheimer Krankheit. Im ersten Teil dieser Arbeit konnte gezeigt werden, dass vor allem junge Mikroglia, also neonatale und amöboide Mikroglia, mit einer reduzierten Aufnahmefähigkeit von Mikropartikeln nach Neurotransmitterapplikation reagieren, während adulte Mikroglia nur bei hohen Konzentrationen von Dopamin diesen Effekt zeigen. Applikation von spezifischen dopaminergen oder adrenergen Rezeptoragonisten führte allerdings nicht zu einer reduzierten Phagozytoseaktivität im Gegensatz zu neonatalen Mikroglia, die aufgrund der Applikation des 5HT2-spezifischen Rezeptoragonisten DOI mit einer konzentrationsabhängigen Reduktion der Phagozytoseaktivität reagierten. Darüber hinaus konnte Serotonin als potenter Stimulator ATP-induzierter Mikrogliabewegung identifiziert werden, da es nicht nur die ATP-induzierte Migration von Mikroglia in vitro, sondern auch die Bewegung mikroglialer Fortsätze zu einer akuten Verletzung steigert. Es wurde bereits gezeigt, dass akute Verletzung mittels Laserläsion zur Freisetzung von ATP aus verletzten Zellen führt. Der zweite Teil meiner Arbeit zeigt, dass die Phagzytoseaktivität kortikaler Mikroglia in erkrankten APPPS1 und APP23 Mäusen reduziert ist. Experimente zur Partikelaufnahme durch Mikroglia in plaquefreien Regionen erkrankter Mäuse ergaben eine vollständige Funktionsfähigkeit der Mikroglia. Demzufolge scheint die Phagozytoseaktivität von Mikroglia abhängig von Aβ-Ablagerungen reduziert zu sein. Zusätzlich konnte gezeigt werden, dass beim Vergleich von 2, 4 und 10 Monate alten Mäusen die mikrogliale Antwort auf kortikale akute Verletzungen im Alter ansteigt. In gealterten APPPS1 Mäusen zeigen plaque-assoziierte Mikroglia allerdings eine reduzierte Bewegung der Fortsätze. Dies unterstützt die Ansicht, dass Mikroglia durch Aβ-Ablagerungen funktionell beeinträchtigt werden. Diese Arbeit verdeutlicht, dass Mikrogliazellen funktionelle Neurotransmitter- rezeptoren für Serotonin, Noradrenalin und Dopamine besitzen, dessen Stimulation zu Veränderungen substantieller Effektorfunktionen führt, die sowohl während der Entwicklung in den Umbau neuronaler Synapsen, als auch in Regenerations-prozesse nach Verletzung involviert sind. Darüber hinaus können diese Mikrogliafunktionen durch Aβ-Ablagerungen beeinträchtigt sein. Dies unterstützt den Ansatz, dass Mikroglia einen dysfunktionellen Phänotyp im Verlauf neurodegenerativer Erkrankungen ausbilden

Activation of serotonin receptors promotes microglial injury-induced motility but attenuates phagocytic activity

Microglia, the brain immune cell, express several neurotransmitter receptors which modulate microglial functions. In this project we studied the impact of serotonin receptor activation on distinct microglial properties as serotonin deficiency not only has been linked to a number of psychiatric disease like depression and anxiety but may also permeate from the periphery through blood-brain barrier openings seen in neurodegenerative disease. First, we tested the impact of serotonin on the microglial response to an insult caused by a laser lesion in the cortex of acute slices from Cx3Cr1-GFP mice. In the presence of serotonin the microglial processes moved more rapidly towards the laser lesion which is considered to be a chemotactic response to ATP. Similarly, the chemotactic response of cultured microglia to ATP was also enhanced by serotonin. Quantification of phagocytic activity by determining the uptake of microspheres showed that the amoeboid microglia in slices from early postnatal animals or microglia in culture respond to serotonin application with a decreased phagocytic activity whereas we could not detect any significant change in ramified microglia in situ. The presence of microglial serotonin receptors was confirmed by patch-clamp experiments in culture and amoeboid microglia and by qPCR analysis of RNA isolated from primary cultured and acutely isolated adult microglia. These data suggest that microglia express functional serotonin receptors linked to distinct microglial properties

Reducing inflammation and rescuing FTD-related behavioral deficits in progranulin-deficient mice with α7 nicotinic acetylcholine receptor agonists.

Mutations in the progranulin gene cause frontotemporal dementia (FTD), a debilitating neurodegenerative disease that involves atrophy of the frontal and temporal lobes and affects personality, behavior, and language. Progranulin-deficient mouse models of FTD exhibit deficits in compulsive and social behaviors reminiscent of patients with FTD, and develop excessive microgliosis and increased release of inflammatory cytokines. Activation of nicotinic acetylcholine receptors (nAChRs) by nicotine or specific α7 nAChR agonists reduces neuroinflammation. Here, we investigated whether activation of nAChRs by nicotine or α7 agonists improved the excessive inflammatory and behavioral phenotypes of a progranulin-deficient FTD mouse model. We found that treatment with selective α7 agonists, PHA-568487 or ABT-107, strongly suppressed the activation of NF-κB in progranulin-deficient cells. Treatment with ABT-107 also reduced microgliosis, decreased TNFα levels, and reduced compulsive behavior in progranulin-deficient mice. Collectively, these data suggest that targeting activation of the α7 nAChR pathway may be beneficial in decreasing neuroinflammation and reversing some of the behavioral deficits observed in progranulin-deficient FTD

Reducing inflammation and rescuing FTD-related behavioral deficits in progranulin-deficient mice with α7 nicotinic acetylcholine receptor agonists.

Mutations in the progranulin gene cause frontotemporal dementia (FTD), a debilitating neurodegenerative disease that involves atrophy of the frontal and temporal lobes and affects personality, behavior, and language. Progranulin-deficient mouse models of FTD exhibit deficits in compulsive and social behaviors reminiscent of patients with FTD, and develop excessive microgliosis and increased release of inflammatory cytokines. Activation of nicotinic acetylcholine receptors (nAChRs) by nicotine or specific α7 nAChR agonists reduces neuroinflammation. Here, we investigated whether activation of nAChRs by nicotine or α7 agonists improved the excessive inflammatory and behavioral phenotypes of a progranulin-deficient FTD mouse model. We found that treatment with selective α7 agonists, PHA-568487 or ABT-107, strongly suppressed the activation of NF-κB in progranulin-deficient cells. Treatment with ABT-107 also reduced microgliosis, decreased TNFα levels, and reduced compulsive behavior in progranulin-deficient mice. Collectively, these data suggest that targeting activation of the α7 nAChR pathway may be beneficial in decreasing neuroinflammation and reversing some of the behavioral deficits observed in progranulin-deficient FTD

Progranulin protects against amyloid β deposition and toxicity in Alzheimer's disease mouse models.

Haploinsufficiency of the progranulin (PGRN) gene (GRN) causes familial frontotemporal lobar degeneration (FTLD) and modulates an innate immune response in humans and in mouse models. GRN polymorphism may be linked to late-onset Alzheimer's disease (AD). However, the role of PGRN in AD pathogenesis is unknown. Here we show that PGRN inhibits amyloid β (Aβ) deposition. Selectively reducing microglial expression of PGRN in AD mouse models impaired phagocytosis, increased plaque load threefold and exacerbated cognitive deficits. Lentivirus-mediated PGRN overexpression lowered plaque load in AD mice with aggressive amyloid plaque pathology. Aβ plaque load correlated negatively with levels of hippocampal PGRN, showing the dose-dependent inhibitory effects of PGRN on plaque deposition. PGRN also protected against Aβ toxicity. Lentivirus-mediated PGRN overexpression prevented spatial memory deficits and hippocampal neuronal loss in AD mice. The protective effects of PGRN against Aβ deposition and toxicity have important therapeutic implications. We propose enhancing PGRN as a potential treatment for PGRN-deficient FTLD and AD

Proximal recolonization by self-renewing microglia re-establishes microglial homeostasis in the adult mouse brain.

Microglia are resident immune cells that play critical roles in maintaining the normal physiology of the central nervous system (CNS). Remarkably, microglia have an intrinsic capacity to repopulate themselves after acute ablation. However, the underlying mechanisms that drive such restoration remain elusive. Here, we characterized microglial repopulation both spatially and temporally following removal via treatment with the colony stimulating factor 1 receptor (CSF1R) inhibitor PLX5622. We show that microglia were replenished via self-renewal, with no contribution from nonmicroglial lineages, including Nestin+ progenitors and the circulating myeloid population. Interestingly, spatial analyses with dual-color labeling revealed that newborn microglia recolonized the parenchyma by forming distinctive clusters that maintained stable territorial boundaries over time, indicating the proximal expansive nature of adult microgliogenesis and the stability of microglia tiling. Temporal transcriptome profiling at different repopulation stages revealed that adult newborn microglia gradually regain steady-state maturity from an immature state that is reminiscent of the neonatal stage and follow a series of maturation programs, including nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) activation, interferon immune activation, and apoptosis. Importantly, we show that the restoration of microglial homeostatic density requires NF-κB signaling as well as apoptotic egress of excessive cells. In summary, our study reports key events that take place from microgliogenesis to homeostasis reestablishment

Functional Impairment of Microglia Coincides with Beta-Amyloid Deposition in Mice with Alzheimer-Like Pathology

<div><p>Microglial cells closely interact with senile plaques in Alzheimer’s disease and acquire the morphological appearance of an activated phenotype. The significance of this microglial phenotype and the impact of microglia for disease progression have remained controversial. To uncover and characterize putative changes in the functionality of microglia during Alzheimer’s disease, we directly assessed microglial behavior in two mouse models of Alzheimer’s disease. Using <i>in vivo</i> two-photon microscopy and acute brain slice preparations, we found that important microglial functions - directed process motility and phagocytic activity - were strongly impaired in mice with Alzheimer’s disease-like pathology compared to age-matched non-transgenic animals. Notably, impairment of microglial function temporally and spatially correlated with Aβ plaque deposition, and phagocytic capacity of microglia could be restored by interventionally decreasing amyloid burden by Aβ vaccination. These data suggest that major microglial functions progressively decline in Alzheimer’s disease with the appearance of Aβ plaques, and that this functional impairment is reversible by lowering Aβ burden, e.g. by means of Aβ vaccination.</p></div

SIRT1 deficiency in microglia contributes to cognitive decline in aging and neurodegeneration via epigenetic regulation of IL-1β.

Aging is the predominant risk factor for neurodegenerative diseases. One key phenotype as the brain ages is an aberrant innate immune response characterized by proinflammation. However, the molecular mechanisms underlying aging-associated proinflammation are poorly defined. Whether chronic inflammation plays a causal role in cognitive decline in aging and neurodegeneration has not been established. Here we report a mechanistic link between chronic inflammation and aging microglia and a causal role of aging microglia in neurodegenerative cognitive deficits. We showed that SIRT1 is reduced with the aging of microglia and that microglial SIRT1 deficiency has a causative role in aging- or tau-mediated memory deficits via IL-1β upregulation in mice. Interestingly, the selective activation of IL-1β transcription by SIRT1 deficiency is likely mediated through hypomethylating the specific CpG sites on IL-1β proximal promoter. In humans, hypomethylation of IL-1β is strongly associated with chronological age and with elevated IL-1β transcription. Our findings reveal a novel epigenetic mechanism in aging microglia that contributes to cognitive deficits in aging and neurodegenerative diseases