Effekte des MAP Kinase Inhibitors CNI-1493 auf Amyloid beta behandelte primäre mikrogliäre und neuronale Zellen

Der Morbus Alzheimer (AD) ist eine fortschreitende neurodegenerative Erkrankung des zentralen Nervensystems, die vor allem ältere Bevölkerungsgruppen betrifft (mit einer Prävalenz von ca. 6% bei den über 65-Jährigen) und schließlich zu schwerer Behinderung führt. Lösliche und unlösliche Aggregate von Amyloid β (Aβ) und Tau-Protein spielen bei der Pathogenese der Erkrankung eine wesentliche Rolle. Sie verursachen zunächst eine Störung der Neuronenfunktion und führen im weiteren Verlauf zur Neurodegeneration. Dabei kommt es in den betroffenen Hirnabschnitten zu einer sekundären Aktivierung glialer Zellen – der sogenannten Neuroinflammation. Diese trägt im weiteren Verlauf zusätzlich zur Neurodegeneration bei. Die vorliegende Arbeit untersucht die Effekte von CNI-1493 auf Aβ-behandelte primäre Neuronen und Mikroglia. CNI-1493 ist ein tetravalentes Guanylhydrazon, das die Phosphorylierung der p38-MAP Kinase inhibiert. Dabei wirkt es entzündungshemmend, indem es unter anderem die Freisetzung von Zytokinen aus Monozyten und Makrophagen inhibiert. CNI-1493 wurde bereits in einem transgenen Alzheimer-Mausmodell getestet, wo es eine Verbesserung kognitiver Leistungen und Abnahme von Aβ-Plaques in den behandelten Tieren bewirkte. An primären neuronalen Mischkulturen, die mit Aβ behandelt wurden, konnte nach Vorbehandlung mit CNI-1493 die Hochregulation des purinergen Nucleotidrezeptors der Klasse P2Y2 (P2Y2R) nachgewiesen werden. Dieser Rezeptor ist inflammationsregulierend. Mehrere Publikationen konnten zeigen, dass vor allem erhöhte IL-1β Spiegel die Expression des P2Y2R induzieren. Seine Aktivierung kann mehrere positive Effekte auf die AD-Pathologie bedeuten. Desweiteren wurde die mikrogliäre Ausschüttung von Zytokinen nach Behandlung von primärer Mikroglia mit Aβ mittels ELISA bestimmt. Eine Behandlung mit CNI-1493 (2,5 µM) führte dabei zu einer Abnahme der Aβ-bedingten proinflammatorischen Zytokine Interleukin 6 (IL-6) und Tumor Nekrose Faktor α (TNF α). Gleichzeitig war ein deutlicher Anstieg des ebenfalls proinflammatorischen Interleukin-1β (IL-1β) zu beobachten. Die durch CNI-1493 bedingte Abnahme von TNF α und IL-6 bei gleichzeitiger Zunahme des IL-1β ist ein bisher nicht beschriebener Effekt. Wir vermuten, dass die von uns zuvor im Mausmodell beschriebenen neuroprotektiven Effekte von CNI-1493 nicht nur auf der Suppression von TNF α und IL-6, sondern auch auf der Induktion der neuroprotektiven Wirkung von IL-1β beruhen. Diese Ergebnisse stehen in einer Reihe mit Publikationen über eine protektive Wirkung von IL-1β im Rahmen der Alzheimer-Pathologie

Multiomic spatial landscape of innate immune cells at human central nervous system borders

The innate immune compartment of the human central nervous system (CNS) is highly diverse and includes several immune-cell populations such as macrophages that are frequent in the brain parenchyma (microglia) and less numerous at the brain interfaces as CNS-associated macrophages (CAMs). Due to their scantiness and particular location, little is known about the presence of temporally and spatially restricted CAM subclasses during development, health and perturbation. Here we combined single-cell RNA sequencing, time-of-flight mass cytometry and single-cell spatial transcriptomics with fate mapping and advanced immunohistochemistry to comprehensively characterize the immune system at human CNS interfaces with over 356,000 analyzed transcriptomes from 102 individuals. We also provide a comprehensive analysis of resident and engrafted myeloid cells in the brains of 15 individuals with peripheral blood stem cell transplantation, revealing compartment-specific engraftment rates across different CNS interfaces. Integrated multiomic and high-resolution spatial transcriptome analysis of anatomically dissected glioblastoma samples shows regionally distinct myeloid cell-type distributions driven by hypoxia. Notably, the glioblastoma-associated hypoxia response was distinct from the physiological hypoxia response in fetal microglia and CAMs. Our results highlight myeloid diversity at the interfaces of the human CNS with the periphery and provide insights into the complexities of the human brain's immune system.</p

IGF1R expression by adult oligodendrocytes is not required in the steady-state but supports neuroinflammation.

In the central nervous system (CNS), insulin-like growth factor 1 (IGF-1) regulates myelination by oligodendrocyte (ODC) precursor cells and shows anti-apoptotic properties in neuronal cells in different in vitro and in vivo systems. Previous work also suggests that IGF-1 protects ODCs from cell death and enhances remyelination in models of toxin-induced and autoimmune demyelination. However, since evidence remains controversial, the therapeutic potential of IGF-1 in demyelinating CNS conditions is unclear. To finally shed light on the function of IGF1-signaling for ODCs, we deleted insulin-like growth factor 1 receptor (IGF1R) specifically in mature ODCs of the mouse. We found that ODC survival and myelin status were unaffected by the absence of IGF1R until 15 months of age, indicating that IGF-1 signaling does not play a major role in post-mitotic ODCs during homeostasis. Notably, the absence of IGF1R did neither affect ODC survival nor myelin status upon cuprizone intoxication or induction of experimental autoimmune encephalomyelitis (EAE), models for toxic and autoimmune demyelination, respectively. Surprisingly, however, the absence of IGF1R from ODCs protected against clinical neuroinflammation in the EAE model. Together, our data indicate that IGF-1 signaling is not required for the function and survival of mature ODCs in steady-state and disease

Patentability, R&D direction, and cumulative innovation

We present a model of cumulative innovation where firms can conduct R&D in both a safe and a risky direction. Innovations in the risky direction produce quality improvements with higher expected sizes and variances. As patentability standards rise, an innovation in the risky direction is less likely to receive a patent that replaces the current technology, which decreases the static incentive for new entrants to conduct risky R&D, but increases their dynamic incentive because of the longer duration---and hence higher reward---for incumbency. These, together with a strategic substitution and a market structure effect, result in an inverted-U shape in the risky direction but a U shape in the safe direction for the relationship between R&D intensity and patentability standards. There exists a patentability standard that induces the efficient innovation direction, whereas R&D is biased towards (against) the risky direction under lower (higher) standards. The optimal patentability standard may distort the R&D direction to increase the industry innovation rate that is socially deficient

Effekte des MAP Kinase Inhibitors CNI-1493 auf Amyloid beta behandelte primäre mikrogliäre und neuronale Zellen

Der Morbus Alzheimer (AD) ist eine fortschreitende neurodegenerative Erkrankung des zentralen Nervensystems, die vor allem ältere Bevölkerungsgruppen betrifft (mit einer Prävalenz von ca. 6% bei den über 65-Jährigen) und schließlich zu schwerer Behinderung führt. Lösliche und unlösliche Aggregate von Amyloid β (Aβ) und Tau-Protein spielen bei der Pathogenese der Erkrankung eine wesentliche Rolle. Sie verursachen zunächst eine Störung der Neuronenfunktion und führen im weiteren Verlauf zur Neurodegeneration. Dabei kommt es in den betroffenen Hirnabschnitten zu einer sekundären Aktivierung glialer Zellen – der sogenannten Neuroinflammation. Diese trägt im weiteren Verlauf zusätzlich zur Neurodegeneration bei. Die vorliegende Arbeit untersucht die Effekte von CNI-1493 auf Aβ-behandelte primäre Neuronen und Mikroglia. CNI-1493 ist ein tetravalentes Guanylhydrazon, das die Phosphorylierung der p38-MAP Kinase inhibiert. Dabei wirkt es entzündungshemmend, indem es unter anderem die Freisetzung von Zytokinen aus Monozyten und Makrophagen inhibiert. CNI-1493 wurde bereits in einem transgenen Alzheimer-Mausmodell getestet, wo es eine Verbesserung kognitiver Leistungen und Abnahme von Aβ-Plaques in den behandelten Tieren bewirkte. An primären neuronalen Mischkulturen, die mit Aβ behandelt wurden, konnte nach Vorbehandlung mit CNI-1493 die Hochregulation des purinergen Nucleotidrezeptors der Klasse P2Y2 (P2Y2R) nachgewiesen werden. Dieser Rezeptor ist inflammationsregulierend. Mehrere Publikationen konnten zeigen, dass vor allem erhöhte IL-1β Spiegel die Expression des P2Y2R induzieren. Seine Aktivierung kann mehrere positive Effekte auf die AD-Pathologie bedeuten. Desweiteren wurde die mikrogliäre Ausschüttung von Zytokinen nach Behandlung von primärer Mikroglia mit Aβ mittels ELISA bestimmt. Eine Behandlung mit CNI-1493 (2,5 µM) führte dabei zu einer Abnahme der Aβ-bedingten proinflammatorischen Zytokine Interleukin 6 (IL-6) und Tumor Nekrose Faktor α (TNF α). Gleichzeitig war ein deutlicher Anstieg des ebenfalls proinflammatorischen Interleukin-1β (IL-1β) zu beobachten. Die durch CNI-1493 bedingte Abnahme von TNF α und IL-6 bei gleichzeitiger Zunahme des IL-1β ist ein bisher nicht beschriebener Effekt. Wir vermuten, dass die von uns zuvor im Mausmodell beschriebenen neuroprotektiven Effekte von CNI-1493 nicht nur auf der Suppression von TNF α und IL-6, sondern auch auf der Induktion der neuroprotektiven Wirkung von IL-1β beruhen. Diese Ergebnisse stehen in einer Reihe mit Publikationen über eine protektive Wirkung von IL-1β im Rahmen der Alzheimer-Pathologie

GPCRomics of Homeostatic and Disease-Associated Human Microglia

G-protein-coupled receptors (GPCRs) are critical sensors affecting the state of eukaryotic cells. To get systematic insight into the GPCRome of microglia, we analyzed publicly available RNA-sequencing data of bulk and single cells obtained from human and mouse brains. We identified 17 rhodopsin and adhesion family GPCRs robustly expressed in microglia from human brains, including the homeostasis-associated genes CX3CR1, GPR34, GPR183, P2RY12, P2RY13, and ADGRG1. Expression of these microglial core genes was lost upon culture of isolated cells ex vivo but could be acquired by human induced pluripotent stem cell (iPSC)-derived microglial precursors transplanted into mouse brains. CXCR4 and PTGER4 were higher expressed in subcortical white matter compared to cortical grey matter microglia, and ADGRG1 was downregulated in microglia obtained from normal-appearing white and grey matter tissue of multiple sclerosis (MS) brains. Single-cell RNA sequencing of microglia from active lesions, obtained early during MS, revealed downregulation of homeostasis-associated GPCR genes and upregulation of CXCR4 expression in a small subset of MS-associated lesional microglia. Functional presence of low levels of CXCR4 on human microglia was confirmed using flow cytometry and transwell migration towards SDF-1. Microglia abundantly expressed the GPCR down-stream signaling mediator genes GNAI2 (αi2), GNAS (αs), and GNA13 (α13), the latter particularly in white matter. Drugs against several microglia GPCRs are available to target microglia in brain diseases. In conclusion, transcriptome profiling allowed us to identify expression of GPCRs that may contribute to brain (patho)physiology and have diagnostic and therapeutic potential in human microglia

Commensal microbiota divergently affect myeloid subsets in the mammalian central nervous system during homeostasis and disease

The immune cells of the central nervous system (CNS) comprise parenchymal microglia and at the CNS border regions meningeal, perivascular, and choroid plexus macrophages (collectively called CNS-associated macrophages, CAMs). While previous work has shown that microglial properties depend on environmental signals from the commensal microbiota, the effects of microbiota on CAMs are unknown. By combining several microbiota manipulation approaches, genetic mouse models, and single-cell RNA-sequencing, we have characterized CNS myeloid cell composition and function. Under steady-state conditions, the transcriptional profiles and numbers of choroid plexus macrophages were found to be tightly regulated by complex microbiota. In contrast, perivascular and meningeal macrophages were affected to a lesser extent. An acute perturbation through viral infection evoked an attenuated immune response of all CAMs in germ-free mice. We further assessed CAMs in a more chronic pathological state in 5xFAD mice, a model for Alzheimer's disease, and found enhanced amyloid beta uptake exclusively by perivascular macrophages in germ-free 5xFAD mice. Our results aid the understanding of distinct microbiota-CNS macrophage interactions during homeostasis and disease, which could potentially be targeted therapeutically

Commensal microbiota divergently affect myeloid subsets in the mammalian central nervous system during homeostasis and disease.

The immune cells of the central nervous system (CNS) comprise parenchymal microglia and at the CNS border regions meningeal, perivascular, and choroid plexus macrophages (collectively called CNS-associated macrophages, CAMs). While previous work has shown that microglial properties depend on environmental signals from the commensal microbiota, the effects of microbiota on CAMs are unknown. By combining several microbiota manipulation approaches, genetic mouse models, and single-cell RNA-sequencing, we have characterized CNS myeloid cell composition and function. Under steady-state conditions, the transcriptional profiles and numbers of choroid plexus macrophages were found to be tightly regulated by complex microbiota. In contrast, perivascular and meningeal macrophages were affected to a lesser extent. An acute perturbation through viral infection evoked an attenuated immune response of all CAMs in germ-free mice. We further assessed CAMs in a more chronic pathological state in 5xFAD mice, a model for Alzheimer's disease, and found enhanced amyloid beta uptake exclusively by perivascular macrophages in germ-free 5xFAD mice. Our results aid the understanding of distinct microbiota-CNS macrophage interactions during homeostasis and disease, which could potentially be targeted therapeutically

The Multi-target Effects of CNI-1493: Convergence of Antiamylodogenic and Antiinflammatory Properties in Animal Models of Alzheimer’s Disease

Abstract After several decades of Alzheimer’s disease (AD) research and failed clinical trials, one can speculate that targeting a single pathway is not sufficient. However, a cocktail of novel therapeutics will constitute a challenging clinical trial. A more plausible approach will capitalize on a drug that has relevant and synergistic multiple-target effects in AD. We have previously demonstrated the efficacy of CNI-1493 in the CRND8 transgenic AD mouse model. Similar to many antiinflammatory drugs that were tested in preclinical models of AD, it was speculated that the significant effect of CNI-1493 is due to its established antiinflammatory properties in rodents and humans. In the present study, we set out to elucidate the protective mechanism of CNI-1493 as a drug simultaneously targeting several aspects of AD pathology. Using C1213, a highly similar analogue of CNI-1493 that lacks antiinflammatory properties, we show that both compounds directly interact with soluble and insoluble amyloid β (Aβ) aggregates and attenuate Aβ cytotoxicity in vitro. Additionally, CNI-1493 and C1213 ameliorates Aβ-induced behavioral deficits in nematodes. Finally, C1213 reduces Aβ plaque burden and cognitive deficits in transgenic CRND8 mice to a similar extent as previously shown with CNI-1493. Taken together, our findings suggest antiamyloidogenic activity as a relevant component of the in vivo efficacy of CNI-1493 and its analogue C1213. Thus, CNI-1493, a drug with proven safety in humans, is a viable candidate for novel multitarget therapeutic approaches to AD

Transcriptome Analysis Identifies Accumulation of Natural Killer Cells with Enhanced Lymphotoxin-&beta; Expression during Glioblastoma Progression

Glioblastomas are the most common primary brain tumors. Despite extensive clinical and molecular insights into these tumors, the prognosis remains dismal. While targeted immunotherapies have shown remarkable success across different non-brain tumor entities, they failed to show efficacy in glioblastomas. These failures prompted the field to reassess the idiosyncrasies of the glioblastoma microenvironment. Several high-dimensional single-cell RNA sequencing studies generated remarkable findings about glioblastoma-associated immune cells. To build on the collective strength of these studies, we integrated several murine and human datasets that profiled glioblastoma-associated immune cells at different time points. We integrated these datasets and utilized state-of-the-art algorithms to investigate them in a hypothesis-free, purely exploratory approach. We identified a robust accumulation of a natural killer cell subset that was characterized by a downregulation of activation-associated genes with a concomitant upregulation of apoptosis genes. In both species, we found a robust upregulation of the Lymphotoxin-&beta; gene, a cytokine from the TNF superfamily and a key factor for the development of adaptive immunity. Further validation analyses uncovered a correlation of lymphotoxin signaling with mesenchymal-like glioblastoma regions in situ and in TCGA and CGGA glioblastoma cohorts. In summary, we identify lymphotoxin signaling as a potential therapeutic target in glioblastoma-associated natural killer cells