Alzheimer's disease (AD) is characterized by beta-amyloid (Aβ) plaques in the brain and widespread neuronal damage. Because of the high drug attrition rates in AD, there is increased interest in characterizing neuroimmune responses to Aβ plaques. In response to AD pathology, microglia are innate phagocytotic immune cells that transition into a neuroprotective state and form barriers around plaques. We seek to understand the role of microglia in modifying Aβ dynamics and barrier formation. To quantify the influence of individual microglia behaviors (activation, chemotaxis, phagocytosis, and proliferation) on plaque size and barrier coverage, we developed an agent-based model to characterize the spatiotemporal interactions between microglia and Aβ. Our model qualitatively reproduces mouse data trends where the fraction of microglia coverage decreases as plaques become larger. In our model, the time to microglial arrival at the plaque boundary is significantly negatively correlated (p < 0.0001) with plaque size, indicating the importance of the time to microglial activation for regulating plaque size. In addition, in silico behavioral knockout simulations show that phagocytosis knockouts have the strongest impact on plaque size, but modest impacts on microglial coverage and activation. In contrast, the chemotaxis knockouts had a strong impact on microglial coverage with a more modest impact on plaque volume and microglial activation. These simulations suggest that phagocytosis, chemotaxis, and replication of activated microglia have complex impacts on plaque volume and coverage, whereas microglial activation remains fairly robust to perturbations of these functions. Thus, our work provides insights into the potential and limitations of targeting microglial activation as a pharmacological strategy for the treatment of AD.

Elevated expression of the complement component 4A (C4A) protein has been linked to an increased risk of schizophrenia (SCZ). However, there are few human models available to study the mechanisms by which C4A contributes to the development of SCZ. In this study, we established a C4A overexpressing neuroimmune cortical organoid (NICO) model, which includes mature neuronal cells, astrocytes, and functional microglia. The C4A NICO model recapitulated several neuroimmune endophenotypes observed in SCZ patients, including modulation of inflammatory genes and increased cytokine secretion. C4A expression also increased microglia-mediated synaptic uptake in the NICO model, supporting the hypothesis that synapse and brain volume loss in SCZ patients may be due to excessive microglial pruning. Our results highlight the role of C4A in the immunogenetic risk factors for SCZ and provide a human model for phenotypic discovery and validation of immunomodulating therapies.

Tissue clearing and Light sheet fluorescence microscopy (LSFM) provide spatial information at a subcellular resolution in intact organs and tumors which is a significant advance over tools that limit imaging to a few representative tissue sections. The spatial distribution of drugs, targets, and biomarkers can help inform relationships between exposure at the site of action, efficacy, and safety during drug discovery. We demonstrate the use of LSFM to investigate distribution of an oncolytic virus (OV) and vasculature in xenograft tumors, as well as brain Aβ pathology in an Alzheimer’s disease (AD) mouse model. Machine learning-based image analysis tools developed to segment vasculature in tumors showed that random forest and deep learning methods provided superior segmentation accuracy vs intensity-based thresholding. Sub-cellular resolution enabled detection of punctate and diffuse intracellular OV distribution profiles. LSFM investigation in the brain in a TgCRND8 AD mouse model at 6.5 months of age enabled evaluation of Aβ plaque density in different brain regions. The utility of LSFM data to support quantitative systems pharmacology (QSP) and physiology-based pharmacokinetics (PBPK) modeling to inform drug development are also discussed. In summary, we showcase how LSFM can expand our understanding of macromolecular drug and biomarker distribution to advance drug discovery and development.

Journal Article High-Resolution Ex Vivo Tissue Clearing, Lightsheet Imaging, and Data Analysis to Support Macromolecular Drug and Biomarker Distribution in Whole Organs and Tumors Get access Niyanta Kumar, Niyanta Kumar ADME & Discovery Toxicology, Merck & Co. Inc., West Point, PA, United States Corresponding author: niyanta.kumar@merck.com Search for other works by this author on: Oxford Academic Google Scholar Petr Hrobař, Petr Hrobař Data Science & Scientific Informatics, MSD, Prague, Czech Republic Search for other works by this author on: Oxford Academic Google Scholar Martin Vagenknecht, Martin Vagenknecht Data Science & Scientific Informatics, MSD, Prague, Czech Republic Search for other works by this author on: Oxford Academic Google Scholar Jindrich Soukup, Jindrich Soukup Data Science & Scientific Informatics, MSD, Prague, Czech Republic Search for other works by this author on: Oxford Academic Google Scholar Peter Bloomingdale, Peter Bloomingdale Quantitative Pharmacology and Pharmacometrics, Merck & Co. Inc., Boston, MA, United States Search for other works by this author on: Oxford Academic Google Scholar Tomoko Freshwater, Tomoko Freshwater Quantitative Pharmacology and Pharmacometrics, Merck & Co. Inc., Rahway, NJ, United States Search for other works by this author on: Oxford Academic Google Scholar Sophia Bardehle, Sophia Bardehle Neuroscience, Merck & Co. Inc., Boston, MA, United States Search for other works by this author on: Oxford Academic Google Scholar Roman Peter, Roman Peter Data Science & Scientific Informatics, MSD, Prague, Czech Republic Search for other works by this author on: Oxford Academic Google Scholar Nadia Patterson, Nadia Patterson ADME & Discovery Toxicology, Merck & Co. Inc., West Point, PA, United States Search for other works by this author on: Oxford Academic Google Scholar Ruban Mangadu, Ruban Mangadu Immuno-Oncology, Merck & Co. Inc., San Francisco, CA, United States Search for other works by this author on: Oxford Academic Google Scholar ... Show more Cinthia Pastuskovas, Cinthia Pastuskovas ADME & Discovery Toxicology, Merck & Co. Inc., San Francisco, CA, United States Search for other works by this author on: Oxford Academic Google Scholar Mark Cancilla Mark Cancilla ADME & Discovery Toxicology, Merck & Co. Inc., West Point, PA, United States Search for other works by this author on: Oxford Academic Google Scholar Microscopy and Microanalysis, Volume 28, Issue S1, 1 August 2022, Pages 1436–1437, https://doi.org/10.1017/S1431927622005840 Published: 01 August 2022

Advances in our understanding of cellular functions and phenotypes in the brain rely on technically robust experimental in vivo models with face validity towards human disease. The cuprizone toxin-induced demyelination model is widely used to investigate pathophysiological mechanisms of demyelinating and remyelinating phases of multiple sclerosis. The C57BL6 mouse is a common inbred strain used as the genetic background for genetically engineered and congenic mice. Substrains of C57BL6 mice sourced from distinct vendors are often treated as equivalent in research studies. Here, we demonstrated that an alternative dosing approach via oral gavage with a well-tolerated, lower dose of cuprizone resulted in significant differences in C57BL/6NTac (Taconic) over C57BL/6J (Jax) mice. With consistent dosing of cuprizone for 5 weeks, body weights were significantly affected in C57BL/6NTac versus C57BL/6J mice. DT-MRI showed significant demyelination in white matter regions in the C57BL/6NTac mice. Concomitantly, histology analysis illustrated increased microgliosis and proliferation in C57BL/6NTac compared with C57BL/6J mice. These observations suggest that the C57BL/6NTac substrain of C57BL6 mice is more vulnerable to cuprizone challenge. Genetic factors along with breeder source appear to influence susceptibility to cuprizone toxin. Thus, the awareness of the limitations of in vivo models in addition to informed decision making on the appropriate background substrain can greatly improve sensitivity and reproducibility of results and use for evaluating investigational therapeutics.

Microglial surveillance is a key feature of brain physiology and disease. Here, we found that G i -dependent microglial dynamics prevent neuronal network hyperexcitability. By generating Mg PTX mice to genetically inhibit G i in microglia, we show that sustained reduction of microglia brain surveillance and directed process motility induced spontaneous seizures and increased hypersynchrony after physiologically evoked neuronal activity in awake adult mice. Thus, G i -dependent microglia dynamics may prevent hyperexcitability in neurological diseases.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Activation of innate immunity and deposition of blood-derived fibrin in the central nervous system (CNS) occur in autoimmune and neurodegenerative diseases, including multiple sclerosis (MS) and Alzheimer’s disease (AD). However, mechanisms linking blood-brain barrier (BBB) disruption with neurodegeneration are poorly understood, and exploration of fibrin as a therapeutic target has been limited by its beneficial clotting functions. Here we report the generation of monoclonal antibody 5B8 targeted against the cryptic fibrin epitope γ 377–395 to selectively inhibit fibrin-induced inflammation and oxidative stress without interfering with clotting. 5B8 suppressed fibrin-induced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and proinflammatory gene expression. In animal models of MS and AD, 5B8 entered the CNS and bound to parenchymal fibrin, and its therapeutic administration reduced innate immune activation and neurodegeneration. Thus, fibrin-targeting immunotherapy inhibits autoimmune- and amyloid-driven neurotoxicity and may have clinical benefit without globally suppressing innate immunity or interfering with coagulation in diverse neurological diseases. neurotoxicity in autoimmune and amyloid-driven neurodegeneration. Although innate immune activation is an attractive candidate for therapeutic intervention, selective therapies to inhibit neurotoxic effects of innate immune responses are not widely available. By targeting the γ 377–395 cryptic fibrin epitope, we developed the first fibrin immunotherapy (monoclonal antibody 5B8) to selectively target the inflammatory form of fibrin without interfering with clotting or activation of innate immune cells by other ligands, such as lipopolysaccharide (LPS). 5B8 selectively bound to fibrin, but not soluble fibrinogen, and inhibited binding of fibrin to CR3 without interfering with fibrin polymerization, in vivo clotting time, or partial thromboplastin time (aPTT) in human plasma. 5B8 reduced NADPH oxidase activation, ROS release, microglial activation, and neurodegeneration in MS and AD animal models. These studies identify fibrin as a blood-derived signal that activates NADPH oxidase to promote innate immune-driven neurotoxicity, and identify fibrin-targeted immunotherapy as a novel therapeutic strategy to suppress innate immune-driven neurodegeneration at sites of increased vascular permeability without interfering with clotting or globally suppressing innate immunity. mice, transcriptomic and signal transduction studies establish fibrin as a dual inflammatory and oxidative stress signal in the CNS and suggest that fibrin immunotherapy could prevent neurodegeneration and other complications in conditions associated with fibrin deposits at sites of BBB disruption. Fibrin immunotherapy may hold promise as a new therapeutic strategy for neurological and inflammatory disorders with vascular damage. n.s., not significant. g , ROS production measured by DHE in murine BMDMs 24 h after fibrin stimulation in the presence of apocynin. Data are mean ± s.e.m. of 3 independent experiments. A.U.-- P

Alzheimer's disease (AD), one of the most common cause of dementia, is a complex neurodegenerative disease marked by amyloid-β (Aβ) plaques and hyperphosphorylated tau tangles. Genome-wide association studies have identified rare variants of genes that implicate novel biological underpinnings of AD, unearthing untapped insights into modulation of innate immune pathways. Recent studies have implicated crucial functions of microglia (brain's resident immune cells) clustering around Aβ plaques, such as plaque compaction and containment, suggesting a beneficial impact on limiting the extent of neuronal damage. In order to test this hypothesis, extraction of neuronal damage characteristics in correlation with microglia coverage is required on a single plaque level. We utilized immunohistochemistry and confocal microscopy to collect 3D image data sets from an AD mouse model. For the quantitative correlative assessment of the heterogeneity of microglia clustering and plaque-associated neuronal damage, we developed a multi-step image analysis pipeline consisting of (a) U-Net based automated region of interest (ROI) detection algorithm (96 % true positive rate), (b) FIJI-based custom-built image profiling tool that creates biologically meaningful image features from ROIs (plaques), and (c) Spotfire-based data visualization dashboard. Our proof-of-concept data set shows that plaque-associated microglia clustering correlates with lower neuronal damage in a disease stage and plaque size-dependent manner. This novel platform has validated our working hypothesis on protective functions of microglia during AD pathology. Future applications of the plaque profiling pipeline will enable unbiased quantitative assessment of potential neuroprotective effects by pharmacological or genetic interventions in preclinical AD models with amyloid pathology.

Activation of innate immunity and deposition of blood-derived fibrin in the central nervous system (CNS) occur in autoimmune and neurodegenerative diseases, including multiple sclerosis (MS) and Alzheimer’s disease (AD). However, the mechanisms that link disruption of the blood–brain barrier (BBB) to neurodegeneration are poorly understood, and exploration of fibrin as a therapeutic target has been limited by its beneficial clotting functions. Here we report the generation of monoclonal antibody 5B8, targeted against the cryptic fibrin epitope γ377–395, to selectively inhibit fibrin-induced inflammation and oxidative stress without interfering with clotting. 5B8 suppressed fibrin-induced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activation and the expression of proinflammatory genes. In animal models of MS and AD, 5B8 entered the CNS and bound to parenchymal fibrin, and its therapeutic administration reduced the activation of innate immunity and neurodegeneration. Thus, fibrin-targeting immunotherapy inhibited autoimmunity- and amyloid-driven neurotoxicity and might have clinical benefit without globally suppressing innate immunity or interfering with coagulation in diverse neurological diseases. Fibrin deposition occurs after the blood–brain barrier is breached. Akassoglou and colleagues generate a therapeutic monoclonal antibody that targets a cryptic fibrin epitope to suppress activation of innate immune responses in the CNS and diminish neuroinflammation.

Blood-brain barrier (BBB) disruption alters the composition of the brain microenvironment by allowing blood proteins into the CNS. However, whether blood-derived molecules serve as extrinsic inhibitors of remyelination is unknown. Here we show that the coagulation factor fibrinogen activates the bone morphogenetic protein (BMP) signaling pathway in oligodendrocyte progenitor cells (OPCs) and suppresses remyelination. Fibrinogen induces phosphorylation of Smad 1/5/8 and inhibits OPC differentiation into myelinating oligodendrocytes (OLs) while promoting an astrocytic fate in vitro. Fibrinogen effects are rescued by BMP type I receptor inhibition using dorsomorphin homolog 1 (DMH1) or CRISPR/Cas9 activin A receptor type I (ACVR1) knockout in OPCs. Fibrinogen and the BMP target Id2 are increased in demyelinated multiple sclerosis (MS) lesions. Therapeutic depletion of fibrinogen decreases BMP signaling and enhances remyelination in vivo. Targeting fibrinogen may be an upstream therapeutic strategy to promote the regenerative potential of CNS progenitors in diseases with remyelination failure.

The deubiquitinase USP15 acts with the ubiquitin ligase TRIM25 to activate a type I interferon response and exacerbate microbial and autoimmune neuroinflammation.

Blood proteins at the neurovascular unit (NVU) are emerging as important molecular determinants of communication between the brain and the immune system. Over the past two decades, roles for the plasminogen activation (PA)/plasmin system in fibrinolysis have been extended from peripheral dissolution of blood clots to the regulation of central nervous system (CNS) functions in physiology and disease. In this review, we discuss how fibrin and its proteolytic degradation affect neuroinflammatory, degenerative and repair processes. In particular, we focus on novel functions of fibrin the final product of the coagulation cascade and the main substrate of plasmin in the activation of immune responses and trafficking of immune cells into the brain. We also comment on the suitability of the coagulation and fibrinolytic systems as potential biomarkers and drug targets in diseases, such as multiple sclerosis (MS), Alzheimer's disease (AD) and stroke. Studying coagulation and fibrinolysis as major molecular pathways that regulate cellular functions at the NVU has the potential to lead to the development of novel strategies for the detection and treatment of neurologic diseases.

Blood proteins at the neurovascular unit (NVU) are emerging as important molecular determinants of communication between the brain and the immune system. Over the past two decades, roles for the plasminogen activation (PA)/plasmin system in fibrinolysis have been extended from peripheral dissolution of blood clots to the regulation of central nervous system (CNS) functions in physiology and disease. In this review, we discuss how fibrin and its proteolytic degradation affect neuroinflammatory, degenerative and repair processes. In particular, we focus on novel functions of fibrin-the final product of the coagulation cascade and the main substrate of plasmin-in the activation of immune responses and trafficking of immune cells into the brain. We also comment on the suitability of the coagulation and fibrinolytic systems as potential biomarkers and drug targets in diseases, such as multiple sclerosis (MS), Alzheimer's disease (AD) and stroke. Studying coagulation and fibrinolysis as major molecular pathways that regulate cellular functions at the NVU has the potential to lead to the development of novel strategies for the detection and treatment of neurologic diseases.

Astrocytes are thought to have important roles after brain injury, but their behavior has largely been inferred from postmortem analysis. To examine the mechanisms that recruit astrocytes to sites of injury, we used in vivo two-photon laser-scanning microscopy to follow the response of GFP-labeled astrocytes in the adult mouse cerebral cortex over several weeks after acute injury. Live imaging revealed a marked heterogeneity in the reaction of individual astrocytes, with one subset retaining their initial morphology, another directing their processes toward the lesion, and a distinct subset located at juxtavascular sites proliferating. Although no astrocytes actively migrated toward the injury site, selective proliferation of juxtavascular astrocytes was observed after the introduction of a lesion and was still the case, even though the extent was reduced, after astrocyte-specific deletion of the RhoGTPase Cdc42. Thus, astrocyte recruitment after injury relies solely on proliferation in a specific niche.

Multiphoton microscopy (MPM) offers a unique approach for addressing both the function and structure of an organ in near-real time in the live animal. The method however is limited by the tissue-specific penetration depth of the excitation laser. In the kidney, structures in the range of 100 µm from the surface are accessible for MPM. This limitation of MPM aggravates the investigation of the function of structures located deeper in the renal cortex, like the glomerulus and the juxtaglomerular apparatus. In view of the relevance of gene-targeted mice for investigating the function of these structures, we aimed to identify a mouse strain with a high percentage of superficially located glomeruli. The mean distance of the 30 most superficial glomeruli from the kidney surface was determined in 10 commonly used mouse strains. The mean depth of glomeruli was 118.4±3.4, 123.0±2.7, 133.7±3.0, 132.3±2.6, 141.0±4.0, 145.3±4.3, 148.9±4.2, 151.6±2.7, 167.7±3.9, and 207.8±3.2 µm in kidney sections from 4-week-old C3H/HeN, BALB/cAnN, SJL/J, C57BL/6N, DBA/2N, CD1 (CRI), 129S2/SvPas, CB6F1, FVB/N and NMRI (Han) mice, respectively (n = 5 animals from each strain). The mean distance from the kidney surface of the most superficial glomeruli was significantly lower in the strains C3H/HeN Crl, BALB/cAnN, DBA/2NCrl, and C57BL/6N when compared to a peer group consisting of all the other strains (p<.0001). In 10-week-old mice, the most superficial glomeruli were located deeper in the cortex when compared to 4-week-old animals, with BALB/cAnN and C57BL/6N being the strains with the highest percentage of superficial glomeruli (25% percentile 116.7 and 121.9 µm, respectively). In summary, due to significantly more superficial glomeruli compared to other commonly used strains, BALB/cAnN and C57BL/6N mice appear to be particularly suitable for the investigation of glomerular function using MPM.

It is generally suggested that astrocytes play important restorative functions after brain injury, yet little is known regarding their recruitment to sites of injury, despite numerous in vitro experiments investigating astrocyte polarity. Here, we genetically manipulated one of the proposed key signals, the small RhoGTPase Cdc42, selectively in mouse astrocytes in vitro and in vivo. We used an in vitro scratch assay as a minimal wounding model and found that astrocytes lacking Cdc42 (Cdc42Δ) were still able to form protrusions, although in a nonoriented way. Consequently, they failed to migrate in a directed manner toward the scratch. When animals were injured in vivo through a stab wound, Cdc42Δ astrocytes developed protrusions properly oriented toward the lesion, but the number of astrocytes recruited to the lesion site was significantly reduced. Surprisingly, however, lesions in Cdc42Δ animals, harboring fewer astrocytes contained significantly higher numbers of microglial cells than controls. These data suggest that impaired recruitment of astrocytes to sites of injury has a profound and unexpected effect on microglia recruitment.