PLCγ2 regulates TREM2 signalling and integrin-mediated adhesion and migration of human iPSC-derived macrophages

Human genetic studies have linked rare coding variants in microglial genes, such as TREM2, and more recently PLCG2 to Alzheimer’s disease (AD) pathology. The P522R variant in PLCG2 has been shown to confer protection for AD and to result in a subtle increase in enzymatic activity. PLCγ2 is a key component of intracellular signal transduction networks and induces Ca2+ signals downstream of many myeloid cell surface receptors, including TREM2. To explore the relationship between PLCγ2 and TREM2 and the role of PLCγ2 in regulating immune cell function, we generated human induced pluripotent stem cell (iPSC)- derived macrophages from isogenic lines with homozygous PLCG2 knockout (Ko). Stimulating TREM2 signalling using a polyclonal antibody revealed a complete lack of calcium flux and IP1 accumulation in PLCγ2 Ko cells, demonstrating a non-redundant role of PLCγ2 in calcium release downstream of TREM2. Loss of PLCγ2 led to broad changes in expression of several macrophage surface markers and phenotype, including reduced phagocytic activity and survival, while LPS-induced secretion of the inflammatory cytokines TNFα and IL-6 was unaffected. We identified additional deficits in PLCγ2- deficient cells that compromised cellular adhesion and migration. Thus, PLCγ2 is key in enabling divergent cellular functions and might be a promising target to increase beneficial microglial functions

Inhibition of IL-34 Unveils Tissue-Selectivity and Is Sufficient to Reduce Microglial Proliferation in a Model of Chronic Neurodegeneration

The proliferation and activation of microglia, the resident macrophages in the brain, is a hallmark of many neurodegenerative diseases such as Alzheimer´s disease (AD) and prion disease. Colony stimulating factor 1 receptor (CSF1R) is critically involved in regulating microglial proliferation, and CSF1R blocking strategies have been recently used to modulate microglia in neurodegenerative diseases. However, CSF1R is broadly expressed by many cell types and the impact of its inhibition on the innate immune system is still unclear. CSF1R can be activated by two independent ligands, CSF-1 and interleukin 34 (IL-34). Recently, it has been reported that microglia development and maintenance depend on IL-34 signalling. In this study, we evaluate the inhibition of IL-34 as a novel strategy to reduce microglial proliferation in the the ME7 model of prion disease. Selective inhibition of IL-34 showed no effects on peripheral macrophage populations in healthy mice, avoiding the side effects observed after CSF1R inhibition on the systemic compartment. However, we observed a reduction in microglial proliferation after IL-34 inhibition in prion-diseased mice, indicating that microglia could be more specifically targeted by reducing IL-34. Overall, our results highlight the challenges of targeting the CSF1R/IL34 axis in the systemic and central compartments, important for framing any therapeutic effort to tackle microglia/macrophage numbers during brain disease

CSF1R inhibitor JNJ-40346527 attenuates microglial proliferation and neurodegeneration in P301S mice.

Neuroinflammation and microglial activation are significant processes in Alzheimer's disease pathology. Recent genome-wide association studies have highlighted multiple immune-related genes in association with Alzheimer's disease, and experimental data have demonstrated microglial proliferation as a significant component of the neuropathology. In this study, we tested the efficacy of the selective CSF1R inhibitor JNJ-40346527 (JNJ-527) in the P301S mouse tauopathy model. We first demonstrated the anti-proliferative effects of JNJ-527 on microglia in the ME7 prion model, and its impact on the inflammatory profile, and provided potential CNS biomarkers for clinical investigation with the compound, including pharmacokinetic/pharmacodynamics and efficacy assessment by TSPO autoradiography and CSF proteomics. Then, we showed for the first time that blockade of microglial proliferation and modification of microglial phenotype leads to an attenuation of tau-induced neurodegeneration and results in functional improvement in P301S mice. Overall, this work strongly supports the potential for inhibition of CSF1R as a target for the treatment of Alzheimer's disease and other tau-mediated neurodegenerative diseases.Funded by a grant from the Wellcome Trust (Grant number: 104025/Z/14/Z), and by the NIHR Oxford Health Biomedical Research Centre

CSF1R inhibitor JNJ-40346527 attenuates microglial proliferation and neurodegeneration in P301S mice

Neuroinflammation and microglial activation are significant processes in Alzheimer’s disease pathology. Recent genome-wide association studies have highlighted multiple immune-related genes in association with Alzheimer’s disease, and experimental data have demonstrated microglial proliferation as a significant component of the neuropathology. In this study, we tested the efficacy of the selective CSF1R inhibitor JNJ-40346527 (JNJ-527) in the P301S mouse tauopathy model. We first demonstrated the anti-proliferative effects of JNJ-527 on microglia in the ME7 prion model, and its impact on the inflammatory profile, and provided potential CNS biomarkers for clinical investigation with the compound, including pharmacokinetic/pharmacodynamics and efficacy assessment by TSPO autoradiography and CSF proteomics. Then, we showed for the first time that blockade of microglial proliferation and modification of microglial phenotype leads to an attenuation of tau-induced neurodegeneration and results in functional improvement in P301S mice. Overall, this work strongly supports the potential for inhibition of CSF1R as a target for the treatment of Alzheimer’s disease and other tau-mediated neurodegenerative diseases

Einfluss des IL-12 / IL-23 Signalweges auf die β-Amyloid Pathologie der Alzheimerschen Krankheit

Alzheimer’s disease (AD) is a progressive neurodegenerative disorder which displays an inflammatory component characterized by the presence of pro- inflammatory cytokines. Previously, the hetero-dimeric cytokines interleukin- (IL-) 12 and IL-23 have been found to be up-regulated by brain-intrinsic microglia cells in Alzheimer’s disease APPPS1 mice, while the genetic deficiency or pharmacological blockade of p40, the common essential subunit of both IL-12 and IL-23, resulted in a strong reduction of amyloid-β (Aβ) plaque burden. Based on these findings, this thesis aimed to investigate possible mechanisms that lead to the observed modulation of Aβ pathology, to identify downstream effector cells of p40 signaling and to evaluate whether pharmacological blockade affects Aβ pathology and cognitive function in two AD-like mouse models with established disease phenotype. As a potential mechanism of Aβ modulation, the production of Aβ by amyloid precursor protein (APP) processing was found to be unaltered in the absence of p40. In contrast, microglial phagocytosis activity as a possible Aβ clearance mechanism was improved upon p40 deficiency in AD-prone APPPS1 brain slices. While microglia constitute the cell type producing p40 in the context of AD, accumulating evidence points towards astrocytes being the cells responding to p40 and facilitating downstream effector function. Astrocytes were found to be expressing the common intracellular downstream mediator of both IL-12 and IL-23 signaling, namely STAT4, in the diseased brain and proved to be able to express IL-12 receptor subunits in vitro. Pharmacological blockade of p40 signaling using anti-p40 antibodies was not sufficient to reduce Aβ pathology in aged AD-like APP23 mice, whereas aged APPPS1 mice demonstrated reduced soluble Aβ levels and improved cognitive function after anti-p40 treatment. These findings suggest that reducing p40 by anti-p40 antibodies appears to be a novel preventive or therapeutic strategy to combat AD pathology in prospective clinical trials.Die Alzheimersche Krankheit ist eine fortschreitende neurodegenerative Erkrankung, die durch entzündliche Prozesse und die Anwesenheit proinflammatorischer Zytokine gekennzeichnet ist. In früheren Studien wurde eine Hochregulation der Zytokine Interleukin- (IL-) 12 und IL-23 durch die ortsständigen Mikrogliazellen im Gehirn von APPPS1 Mäusen, einem Tiermodell der Alzheimer Pathologie, gezeigt. Darüber hinaus wurde dargelegt, dass die genetische Defizienz oder pharmakologische Inhibition von p40, der gemeinsamen Untereinheit von IL-12 und IL-23, zu einer ausgeprägten Reduktion der Amyloid-β (Aβ) Plaque-Last im Gehirn von APPPS1 Mäusen führt. Basierend auf die genannten Ergebnisse zielt diese Arbeit darauf aus, mögliche Mechanismen zu untersuchen, die zu der beobachteten Modulation der Aβ Pathologie führen könnten, die Zellen im Gehirn zu definieren, die als Zielzellen von p40 nachgeschaltete Effekte bewirken, sowie zu bewerten, ob pharmakologische Inhibition von p40 die Aβ Pathologie sowie kognitive Funktionen in zwei Mausmodellen der Alzheimer Erkrankung mit bestehendem Krankheits-Phänotyp beeinflusst. Als möglicher Mechanismus der Aβ Modulation wurde die Produktion von Aβ durch Prozessierung des Amyloid-Vorläuferproteins (APP) untersucht, die keine Veränderung in Abwesenheit von p40 zeigte. Im Gegensatz dazu war die Phagozytose-Aktivität der Mikrogliazellen als potenzieller Mechanismus der Aβ- Beseitigung in Gehirnschnitten von APPPS1-Tieren in der Abwesenheit von p40 erhöht. Während Mikroglia den Zelltyp darstellen, der p40 im Alzheimer-Kontext produziert, mehren sich Hinweise, die zeigen, dass Astrozyten der Zelltyp im Gehirn ist, der auf p40 reagiert und nachgeschaltete Effektor-Funktionen ausübt. So produzieren Astrozyten den gemeinsamen intrazellulären Mediator von IL-12 und IL-23, STAT4, im Gehirn von Alzheimer-Mäusen und waren fähig, IL-12-Rezeptoruntereinheiten in vitro zu exprimieren. Pharmakologische Inhibition des p40-Signalweges mithilfe von neutralisierenden Antikörpern wies zwar keinen Effekt auf die Aβ Pathologie im Alzheimer-Mausmodell APP23 auf, jedoch zeigten APPPS1-Tiere eine Reduktion von löslichem Aβ im Gehirn sowie eine Verbesserung der kognitiven Leistungen nach der Behandlung mit p40-neutralisierenden Antikörpern. Diese Ergebnisse unterstützen anti-p40 Antikörper als neue präventive oder therapeutische Strategie gegen die Alzheimer Krankheit in zukünftigen klinischen Studien anzuwenden

The evolving dialogue of microglia and neurons in Alzheimer's disease: microglia as necessary transducers of pathology

The understanding of the contribution of microglial cells to the onset and/or progression chronic neurodegenerative diseases is key to identify disease-modifying therapies, given the strong neuroimmune component of these disorders. In this review, we dissect the different pathways by which microglia can affect, directly or indirectly, neuronal function and dysfunction associated with diseases like Alzheimer’s. We here present the rationale for proposing a model to explain the contribution of microglia to the pathophysiology of Alzheimer’s disease, defining microglial cells as necessary transducers of pathology and ideal targets for intervention

The Role of Microglia in Prion Diseases: A Paradigm of Functional Diversity

Inflammation is a major component of neurodegenerative diseases. Microglia are the innate immune cells in the central nervous system (CNS). In the healthy brain, microglia contribute to tissue homeostasis and regulation of synaptic plasticity. Under disease conditions, they play a key role in the development and maintenance of the neuroinflammatory response, by showing enhanced proliferation and activation. Prion diseases are progressive chronic neurodegenerative disorders associated with the accumulation of the scrapie prion protein PrPSc, a misfolded conformer of the cellular prion protein PrPC. This review article provides the current knowledge on the role of microglia in the pathogenesis of prion disease. A large body of evidence shows that microglia can trigger neurotoxic pathways contributing to progressive degeneration. Yet, microglia are also crucial for controlling inflammatory, repair and regenerative processes. This dual role of microglia is regulated by multiple pathways and evidences the ability of these cells to polarize into distinct phenotypes with characteristic functions. The awareness that the neuroinflammatory response is inextricably involved in producing tissue damage as well as repair in neurodegenerative disorders, opens new perspectives for the modulation of the immune system. A better understanding of this complex process will be essential for developing effective therapies for neurodegenerative diseases, in order to improve the quality of life of patients and mitigating the personal, economic and social consequences derived from these diseases

The role of microglia in prion diseases: a paradigm of functional diversity

Inflammation is a major component of neurodegenerative diseases. Microglia are the innate immune cells in the central nervous system (CNS). In the healthy brain, microglia contribute to tissue homeostasis and regulation of synaptic plasticity. Under disease conditions, they play a key role in the development and maintenance of the neuroinflammatory response, by showing enhanced proliferation and activation. Prion diseases are progressive chronic neurodegenerative disorders associated with the accumulation of the scrapie prion protein PrPSc, a misfolded conformer of the cellular prion protein PrPC. This review provides the current knowledge on the role of microglia in the pathogenesis of prion disease. A large body of evidence shows that microglia can trigger neurotoxic pathways contributing to progressive degeneration. Yet, microglia are also crucial for controlling inflammatory, repair and regenerative processes. This dual role of microglia is regulated by multiple pathways and evidences the ability of these cells to polarize into distinct phenotypes with characteristic functions. The awareness that the neuroinflammatory response is inextricably involved in producing tissue damage as well as repair in neurodegenerative disorders, opens new perspectives for the modulation of the immune system. A better understanding of this complex process will be essential for developing effective therapies for neurodegenerative diseases, in order to improve the quality of life of patients and mitigating the personal, economic and social consequences derived from these diseases

PD-1 deficiency is not sufficient to induce myeloid mobilization to the brain or alter the inflammatory profile during chronic neurodegeneration

Chronic inflammation is a major driver of neurodegenerative disease and immune regulatory pathways could be potential targets for therapeutic intervention. Recently, Programmed cell death-1 (PD-1) immune checkpoint inhibition has been proposed to mount an IFN-γ-dependent systemic immune response, leading to the recruitment of peripheral myeloid cells to the brain and neuropathological and functional improvements in mice with Alzheimer’s disease- like β-amyloid pathology. Here we investigate the impact of PD-1 deficiency on murine prion disease (ME7 strain), a model of chronic neurodegeneration. Although PD-1 was found to be increased in the brain of prion mice, the absence of PD-1 did not cause myeloid cell infiltration into the brain or major changes in the inflammatory profile. However, we observed a slight exacerbation of the behavioural phenotype of ME7 mice upon PD-1 deficiency. These results do not support the possibility of using immune checkpoint blockade as a therapeutic strategy in neurodegenerative disease

Replicative senescence dictates the emergence of disease-associated microglia and contributes to Aβ pathology

The sustained proliferation of microglia is a key hallmark of Alzheimer’s disease (AD), accelerating its progression. Here, we sought to understand the long-term impact of the early and prolonged microglial proliferation observed in AD, hypothesising that extensive and repeated cycling would engender a distinct transcriptional and phenotypic trajectory. We found that the early and sustained microglial proliferation seen in an AD-like model promotes replicative senescence, characterised by increased βgal activity, a senescence-associated transcriptional signature and telomere shortening, correlating with the appearance of disease-associated microglia (DAM) and senescent microglial profiles in human post-mortem AD cases. Prevention of early microglial proliferation hindered the development of senescence and DAM, impairing the accumulation of Aβ and associated neuritic damage. Overall, our results support that excessive microglial proliferation leads to the generation of senescent DAM, which contribute to early Aβ pathology in AD