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Quantification of microglial activation through morphometric analysis has long been a staple of the neuroimmunologist’s toolkit. Microglial morphological phenomics can be conducted through either manual classification or constructing a digital skeleton and extracting morphometric data from it. Multiple open-access and paid software packages are available to generate these skeletons via semi-automated and/or fully automated methods with varying degrees of accuracy. Despite advancements in methods to generate morphometrics (quantitative measures of cellular morphology), there has been limited development of tools to analyze the datasets they generate, in particular those containing parameters from tens of thousands of cells analyzed by fully automated pipelines. In this review, we compare and critique the approaches using cluster analysis and machine learning driven predictive algorithms that have been developed to tackle these large datasets, and propose improvements for these methods. In particular, we highlight the need for a commitment to open science from groups developing these classifiers. Furthermore, we call attention to a need for communication between those with a strong software engineering/computer science background and neuroimmunologists to produce effective analytical tools with simplified operability if we are to see their wide-spread adoption by the glia biology community.

Complement is involved in developmental synaptic pruning and pathological synapse loss in Alzheimer’s disease. It is posited that C1 binding initiates complement activation on synapses; C3 fragments then tag them for microglial phagocytosis. However, the precise mechanisms of complement-mediated synaptic loss remain unclear, and the role of the lytic membrane attack complex (MAC) is unexplored. We here address several knowledge gaps: (i) is complement activated through to MAC at the synapse? (ii) does MAC contribute to synaptic loss? (iii) can MAC inhibition prevent synaptic loss? Novel methods were developed and optimised to quantify C1q, C3 fragments and MAC in total and regional brain homogenates and synaptoneurosomes from WT and App NL−G−F Alzheimer’s disease model mouse brains at 3, 6, 9 and 12 months of age. The impact on synapse loss of systemic treatment with a MAC blocking antibody and gene knockout of a MAC component was assessed in Alzheimer’s disease model mice. A significant increase in C1q, C3 fragments and MAC was observed in App NL−G−F mice compared to controls, increasing with age and severity. Administration of anti-C7 antibody to App NL−G−F mice modulated synapse loss, reflected by the density of dendritic spines in the vicinity of plaques. Constitutive knockout of C6 significantly reduced synapse loss in 3xTg-AD mice. We demonstrate that complement dysregulation occurs in Alzheimer’s disease mice involving the activation (C1q; C3b/iC3b) and terminal (MAC) pathways in brain areas associated with pathology. Inhibition or ablation of MAC formation reduced synapse loss in two Alzheimer’s disease mouse models, demonstrating that MAC formation is a driver of synapse loss. We suggest that MAC directly damages synapses, analogous to neuromuscular junction destruction in myasthenia gravis.

The gut-brain-axis is increasingly recognised as a mediator of neurodegenerative processes, with the gut microbiota emerging as a potential target for intervention. The Lab4 probiotic has demonstrated neuroprotective activity and, here, we have investigated its impact on aspects of neurodegeneration in the 3xTg Alzheimer's disease (AD) murine model. Male 3xTg-AD mice were fed a high fat diet (to accelerate neurodegeneration) with or without daily Lab4 probiotic supplementation for 84 days. Endpoints included hippocampal neuronal spine density, novel object recognition, whole-brain gene expression, plasma cytokines/lipids, body weight, and faecal microbiota composition. Lab4 Probiotic supplementation preserved the neuronal spine density, particularly thin spines, and improved recognition memory. Gene expression analysis of whole brain extracts detected reductions in pro-inflammatory markers (IL-5 and Caspase-1) and plasma analysis revealed reduced levels of pro-inflammatory TNF- . The probiotic also mitigated weight gain, though plasma lipid profiles were unchanged. Microbiota analysis indicated increased abundance of and decreased in probiotic-supplemented mice, alongside reduced numbers of viable yeast. These preliminary findings highlight a neuroprotective impact in 3xTg-AD mice receiving the Lab4 probiotic and warrant more extensive assessments in murine models and/or human subjects.

Neuronal dendritic and synaptic pruning are early features of neurodegenerative diseases, including Alzheimer’s disease. In addition to brain pathology, amyloid plaque deposition, microglial activation, and cell loss occur in the retinas of human patients and animal models of Alzheimer’s disease. Retinal ganglion cells, the output neurons of the retina, are vulnerable to damage in neurodegenerative diseases and are a potential opportunity for non-invasive clinical diagnosis and monitoring of Alzheimer’s progression. However, the extent of retinal involvement in Alzheimer’s models and how well this reflects brain pathology is unclear. Here we have quantified changes in retinal ganglion cells dendritic structure and hippocampal dendritic spines in three well-studied Alzheimer’s mouse models, Tg2576, 3xTg-AD and APP NL-G-F . Dendritic complexity of DiOlistically labelled retinal ganglion cells from retinal explants was reduced in all three models in an age-, gender-, and receptive field-dependent manner. DiOlistically labelled hippocampal slices showed spine loss in CA1 apical dendrites in all three Alzheimer’s models, mirroring the early stages of neurodegeneration as seen in the retina. Morphological classification showed that loss of thin spines predominated in all. The demonstration that retinal ganglion cells dendritic field reduction occurs in parallel with hippocampal dendritic spine loss in all three Alzheimer’s models provide compelling support for the use of retinal neurodegeneration. As retinal dendritic changes are within the optical range of current clinical imaging systems (for example optical coherence tomography), our study makes a case for imaging the retina as a non-invasive way to diagnose disease and monitor progression in Alzheimer’s disease.

Aging and metabolic syndrome are associated with neurodegenerative pathologies including Alzheimer’s disease (AD) and there is growing interest in the prophylactic potential of probiotic bacteria in this area. In this study, we assessed the neuroprotective potential of the Lab4P probiotic consortium in both age and metabolically challenged 3xTg-AD mice and in human SH-SY5Y cell culture models of neurodegeneration. In mice, supplementation prevented disease-associated deteriorations in novel object recognition, hippocampal neurone spine density (particularly thin spines) and mRNA expression in hippocampal tissue implying an anti-inflammatory impact of the probiotic, more notably in the metabolically challenged setting. In differentiated human SH-SY5Y neurones challenged with β-Amyloid, probiotic metabolites elicited a neuroprotective capability. Taken together, the results highlight Lab4P as a potential neuroprotective agent and provide compelling support for additional studies in animal models of other neurodegenerative conditions and human studies.

Optical coherence tomography (OCT) is an imaging technique based on interferometry of backscattered lights from materials and biological samples. For the quantitative evaluation of an OCT system, artificial optical samples or phantoms are commonly used. They mimic the structure of biological tissues and can provide a quality standard for comparison within and across devices. Phantoms contain medium matrix and scattering particles within the dimension range of target biological structures such as the retina. The aim was to determine if changes in speckle derived optical texture could be employed to classify the OCT phantoms based on their structural composition. Four groups of phantom types were prepared and imaged. These comprise different concentrations of a medium matrix (gelatin solution), different sized polystyrene beads (PBs), the volume of PBs and different refractive indices of scatterers (PBs and SiO2). Texture analysis was applied to detect subtle optical differences in OCT image intensity, surface coarseness and brightness of regions of interest. A semi-automated classifier based on principal component analysis (PCA) and support vector machine (SVM) was applied to discriminate the various texture models. The classifier detected correctly different phantom textures from 82% to 100%, demonstrating that analysis of the texture of OCT images can be potentially used to discriminate biological structure based on subtle changes in light scattering.

Background Neuroinflammation is a critical factor of Alzheimer’s Disease (AD). Dysregulation of complement leads to excessive inflammation, direct damage to self‐cells and propagation of injury. This is likely of particular relevance in the brain where inflammation is poorly tolerated and brain cells are vulnerable to direct damage by complement. Membrane attack complex (MAC) is highly pro‐inflammatory product of the complement cascade killing cells by lysis and/or causing ‘bystander’ damage initiating NLRP3 inflammasome activation and provoking other immune damaging responses leading to death of the vulnerable nerve cells. Method The role of MAC in AD was investigated in MAC‐deficient animals and by using a newly developed anti‐C7 monoclonal antibody (mAb) that efficiently inhibits formation of the MAC in vitro and in vivo. Impact of C7 deficiency on brain complement dysregulation, synapse loss, amyloid load and cognitive decline was examined by comparing APPNL‐G‐F mice back‐crossed to C7 deficiency (APPNL‐G‐FxC7) with unmodified APPNL‐G‐F mice. To assess the effect of therapeutic C7 blockade, unmodified APPNL‐G‐F mice were treated systemically (for four weeks) with anti‐C7 mAb or control IgG and the same set of parameters of complement dysregulation, pathology and cognition measured. Result C7 deficiency in AppNL−G−F mice reduced levels of complement activation markers, reduced amyloid load and increased synapse density with a commensurate improvement in cognitive test performance. Systemic treatment of AppNL−G−F mice with a blocking anti‐C7 mAb reduced brain levels of complement activation markers, amyloid load and increased neuronal spine density in treated mice in peri‐plaque areas when compared to controls. in AppNL−G−F mice. APPNL‐G‐FxC7 performed significanlty better in behavioural cognitive tests. Conclusion We demonstrate that complement dysregulation occurs in brain in mouse models of AD. C7 deficiency reduced brain complement dysregulation, improved pathological parameters and cognitive function; systemic anti‐C7 therapy reduced complement dysregulation and protected from synapse loss in the model. Modification for brain delivery of the anti‐C7 mAb will enhance efficacy in the model. The findings highlight the potential for complement inhibition at the level of MAC as a therapy in AD.

Background In the brain as in other organs, complement contributes to immune defence and housekeeping to maintain homeostasis. Sources of complement may include local production by brain cells and influx from the periphery, the latter severely restricted by the blood brain barrier (BBB) in healthy brain. Dysregulation of complement leads to excessive inflammation, direct damage to self‐cells and propagation of injury. This is likely of particular relevance in the brain where inflammation is poorly tolerated and brain cells are vulnerable to direct damage by complement. Method We have developed novel anti‐C7 antibodies (mAb) that efficiently inhibit formation of the pro‐inflammatory membrane attack complex (MAC) in vitro and in vivo. Here we describe recombinant fusion proteins (FP) that replicate the MAC‐blocking action of the mAb, and are designed to access the brain utilising “Trojan horse” shuttles. The Alzheimer model APPNL‐G‐F mice were treated systemically with native mAb to swamp peripheral C7 followed by the FP. Immunohistochemistry and ELISA were used to demonstrate FP entry into brain and show impact on the disease pathology. Result The recombinant FP showed complement inhibitory activity in vitro equivalent to their parent mAb and were able to cross an artificial BBB in transwells. The presence of the FP in brain homogenates of peripherally dosed animals was confirmed by ELISA. Treatment with the FP caused reduced levels of complement activation products C3b and terminal complement complex (TCC) in brain. Diolistics analysis showed significant increased neuronal spine density in treated mice compared to controls, demonstrating a protective effect of the FP on synaptic function. Mice treated with the drug showed significant improvements in cognition. Conclusion The FP described are able to cross BBB and are potent inhibitors of complement in brain; impact on brain pathology was detected after just one week of treatment. The findings highlight the potential for complement inhibition as a therapy in Alzheimer’s disease.

A rare coding missense variant (rs72824905; P522R) in PLCG2 decreases the risk of late-onset Alzheimer’s disease, but how this protective effect is mediated is unclear. Here we demonstrate a mechanism for this protection, the R522 variant of PLCγ2 alters microglial activity leading to a marked preservation of synaptic integrity and reduced peri-plaque microglial engulfment of synapses independently of amyloid burden. Our data advocate for a direct central role of PLCγ2 in mediating synaptic loss as part of the pathological process of Alzheimer’s disease (AD), prioritising it as a therapeutic target and modulator of disease.

Infections have long been implicated as causative factors in Alzheimer’s disease (AD). Multiple studies have further suggested a key role for herpesviruses, such as cytomegalovirus (CMV). Using transgenic 3xTg-AD mice, we demonstrate that systemic infection with the β-herpesvirus murine cytomegalovirus (MCMV) accelerates the development of cognitive decline, tauopathy, and synaptic loss in the hippocampus, all of which are key features of AD. Accelerated disease progression after infection was associated with substantial lymphocyte infiltration into the brain dominated by CD8+ T cells specific for MCMV. Moreover, T cell receptor analyses revealed that these responses were clonally diverse, suggesting that multiple viral antigens were targeted in the brain during chronic infection with MCMV. T cell depletion during virus chronicity rescued infection-induced cognitive decline. In addition, antiviral drug treatment reduced lymphocytic infiltrates in the brain and reversed cognitive decline, suggesting potential clinical utility. These data provide a mechanistic link between chronic viral infections and the development of AD.