Introduction: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 in vitro and, here, we have investigated its impact on aspects of neurodegeneration in the 3xTg Alzheimer's disease (AD) murine model. Method: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. Results: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-α. The probiotic also mitigated weight gain, though plasma lipid profiles were unchanged. Microbiota analysis indicated increased abundance of Blautia and decreased Muribaculaceae in probiotic-supplemented mice, alongside reduced numbers of viable yeast. Discussion: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.

IntroductionThe 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 in vitro and, here, we have investigated its impact on aspects of neurodegeneration in the 3xTg Alzheimer’s disease (AD) murine model.MethodMale 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.ResultsLab4 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-α. The probiotic also mitigated weight gain, though plasma lipid profiles were unchanged. Microbiota analysis indicated increased abundance of Blautia and decreased Muribaculaceae in probiotic-supplemented mice, alongside reduced numbers of viable yeast.DiscussionThese 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.

AIMS:Microglia undergo profound structural and functional changes during Alzheimer's disease, yet the earliest stages of morphological remodelling that occur prior to amyloid deposition remain poorly defined. We hypothesised that microglia in the hippocampus of AppNL-G-F mice would exhibit early, region-specific structural adaptations before local plaque formation, reflecting an initial phase of disease-associated structural remodelling. METHODS:Two-month-old AppNL-G-F and wildtype mice were examined using high-resolution confocal microscopy of Iba1-labelled microglia in the dorsal CA1 apical field. Automated three-dimensional reconstructions were generated in Imaris, and quantitative morphometric analyses quantified cell density, Iba1 coverage, process topology and Sholl-based arbor complexity. Statistical analyses were performed using linear mixed-effects models incorporating sex as a fixed factor in all analyses. RESULTS:Microglial density and total Iba1 coverage were unaffected in AppNL-G-F mice at this age. In contrast, Sholl analysis revealed significant genotype-dependent reductions in process intersections and total process length, accompanied by reduced individual-cell territorial coverage, indicating an early contraction of the surveillance arbor independent of cell number. CONCLUSIONS:These findings demonstrate that hippocampal microglia in AppNL-G-F mice undergo an early, coordinated structural remodelling before local amyloid deposition becomes apparent. This preplaque adaptation defines an early structural remodelling of hippocampal microglia prior to evident local amyloid deposition, providing new insight into the earliest structural adaptations associated with neuroimmune engagement in AD pathogenesis.

The Alzheimer's disease protective P522R PLCG2 coding variant (rs72824905) is downstream of TREM2, but how it confers disease protection is poorly understood. Using a Plcg2-R522 knock-in mouse and Plcg2-P522 control on both wildtype and Alzheimer's disease-like AppNL-G-F amyloidosis mouse backgrounds, aged mice were assayed for amyloid load, microglial activity, and synaptic integrity. In the absence of Alzheimer's disease-like pathology, the R522 variant increased microglial coverage and was associated with reduced ramification complexity, fewer terminal points, and elevated lysosomal CD68 expression. On the AppNL-G-F background, total amyloid burden was unaffected, but expression of the R522 variant led to increased plaque compaction compared to the P522 common variant. The protective R522 variant was also associated with: enhanced microglial engagement with less compact amyloid plaques; reduced microglial localisation around highly compacted plaques; protection from amyloid-induced synapse loss; and decreased engulfment of synaptic material by microglia. Our data indicate a significant direct PLCγ2 role in controlling microglial-plaque interactions and synaptic protection downstream of amyloid deposition, prioritizing it as a therapeutic target, potentially as an adjunct to other approaches, such as those targeting amyloid.

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 CMV (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 MCMV-specific effector memory CD8+ T cells expressing CXCR3. T cell receptor analyses revealed that clonally diverse virus-specific CD8+ T cells were selectively recruited into the brain during the development of AD. T cell depletion or treatment with the antiviral drug valganciclovir during chronic infection reduced lymphocytic infiltrates in the brain and reversed cognitive decline. These data provide a mechanistic link between chronic viral infections and the development of AD.

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. 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 APP NL-G-F mice back-crossed to C7 deficiency (APP NL-G-F xC7) with unmodified APP NL-G-F mice. To assess the effect of therapeutic C7 blockade, unmodified APP NL-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. C7 deficiency in App NL−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 App NL−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 App NL−G−F mice. APP NL-G-F xC7 performed significanlty better in behavioural cognitive tests. 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.

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. 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 APP NL-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. 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. 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.

The detection of selective retinal ganglion cell damage in glaucoma has been a long sought-after goal, not just for the development of clinical tests for the early detection of glaucoma but for the elucidation of potential mechanisms underlying retinal ganglion cell loss. Early reports of the selective vulnerability of larger retinal ganglion cells (RGCs) in human studies did not translate simply to the loss of a particular class of RGC but more likely reflected shrinkage and degeneration across all RGC classes. Subsequent studies of nonhuman primate (NHP) models of glaucoma indicated some selectivity with great damage to the magnocellular vs parvocellular pathways. More recently, rodent models of experimental glaucoma have highlighted a selective vulnerability of OFF-centered RGCs-particularly those with transient responses. Selectivity for OFF pathway damage is also seen as a trend in a rat model of glaucoma. These data support the concept that some RGCs are more vulnerable to the effects of glaucoma damage. This chapter covers some of the methods to elucidate RGC damage and the relevance of model selection to mimic human glaucoma rather than just RGC death.

Complement activation is implicated in driving brain inflammation, self-cell damage and progression of injury in Alzheimer's disease and other neurodegenerative diseases. Here, we investigate the impact of brain delivery of a complement-blocking antibody on neurodegeneration in an Alzheimer's mouse model. We engineered a brain-penetrant recombinant antibody targeting the pro-inflammatory membrane attack complex. Systemic administration of this antibody in APPNL-G-F mice reduced brain levels of complement activation products, demonstrating successful brain entry and target engagement. Prolonged treatment decreased synapse loss, amyloid burden and brain inflammatory cytokine levels, concomitant with cognitive improvement compared to controls. These results underscore the potential of brain-penetrant complement-inhibiting drugs as promising therapeutics, targeting downstream of amyloid plaques in Alzheimer's disease.

Background: Retinal ganglion cell (RGC) loss is crucial in eye diseases like glaucoma. Axon damage and dendritic degeneration precede cell death, detectable within optical coherence tomography (OCT) resolution, indicating their correlation with neuronal degeneration. The purpose of this study is to evaluate the optical changes of early retinal degeneration. Methods: The detection of optical changes in the axotomised retinal explants was completed in six C57BL/6J mice. OCT images were acquired up to 120 min from enucleation. A grey-level co-occurrence-based texture analysis was performed on the inner plexiform layer (IPL) to monitor changes in the optical speckles using a principal component analysis (PCA) and a support vector machine (SVM). In parallel tests, retinal transparency was confirmed by a comparison of the modulation transfer functions (MTFs) at 0 and 120 min. Results: Quantitative confirmation by analysis of the MTFs confirmed the non-degradation of optical transparency during the imaging period: MTF (fx) = 0.267 ± 0.02. Textural features in the IPL could discriminate between the optical signals of RGC degeneration. The mean accuracy of the SVM classification was 86.3%; discrimination was not enhanced by the combination of the SVM and PCA (81.9%). Conclusions: Optical changes in the IPL can be detected using OCT following RGC axotomy. High-resolution OCT can provide an index of retinal neuronal integrity and its degeneration.

Complement is dysregulated in the brain in Alzheimer's Disease and in mouse models of Alzheimer's disease. Each of the complement derived effectors, opsonins, anaphylatoxins and membrane attack complex (MAC), have been implicated as drivers of disease but their relative contributions remain unclarified. Here we have focussed on the MAC, a lytic and pro-inflammatory effector, in the AppNL-G-F mouse amyloidopathy model. To test the role of MAC, we back-crossed to generate AppNL-G-F mice deficient in C7, an essential MAC component. C7 deficiency ablated MAC formation, reduced synapse loss and amyloid load and improved cognition compared to complement-sufficient AppNL-G-F mice at 8-10 months age. Adding back C7 caused increased MAC formation in brain and an acute loss of synapses in C7-deficient AppNL-G-F mice. To explore whether C7 was a viable therapeutic target, a C7-blocking monoclonal antibody was administered systemically for one month in AppNL-G-F mice aged 8-9 months. Treatment reduced brain MAC and amyloid deposition, increased synapse density and improved cognitive performance compared to isotype control-treated AppNL-G-F mice. The findings implicate MAC as a driver of pathology and highlight the potential for complement inhibition at the level of MAC as a therapy in Alzheimer's disease.

DiOlistic labelling is a robust, unbiased ballistic method that utilises lipophilic dyes to morphologically label neurons. While its efficacy on freshly dissected tissue specimens is well-documented, applying DiOlistic labelling to stored, fixed brain tissue and its use in polychromatic multi-marker studies poses significant technical challenges. Here, we present an improved, step-by-step protocol for DiOlistic labelling of dendrites and dendritic spines in fixed mouse tissue. Our protocol encompasses the five key stages: Tissue Preparation, Dye Bullet Preparation, DiOlistic Labelling, Confocal Imaging, and Image Analysis. This method ensures reliable and consistent labelling of dendritic spines in fixed mouse tissue, combined with increased throughput of samples and multi-parameter staining and visualisation of tissue, thereby offering a valuable approach for neuroscientific research.

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.

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.

Gene gun DiOlistic labelling enables the detailed visualization of retinal ganglion cells (RGCs) dendritic structure. Since the level of labelling is independent of cellular health, it is useful for the characterization of neuronal structure in degenerating neurons where expressed reporters may be inadequate. The method uses compressed helium gas to fire tungsten or gold microparticles coated in carbocyanine dyes (DiD, DiI, DiO) into flat mounted retinas. Here we describe the methods to optimize labelling and ensure a high yield of adequately labelled cells, with a focus on retinal ganglion cells.

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.

Brain degenerative disorders such as Alzheimer's disease (AD) can be exacerbated by aberrant metabolism. Supplementation with probiotic bacteria is emerging as a promising preventative strategy for both neurodegeneration and metabolic syndrome. In this study, we assess the impact of the Lab4b probiotic consortium on (i) cognitive and pathological markers of AD progression and (ii) metabolic status in 3xTg-AD mice subjected to metabolic challenge with a high fat diet. The group receiving the probiotic performed better in the novel object recognition test and displayed higher hippocampal neuronal spine density than the control group at the end of the 12 weeks intervention period. These changes were accompanied by differences in localised (brain) and systemic anti-inflammatory responses that favoured the Probiotic group together with the prevention of diet induced weight gain and hypercholesterolaemia and the modulation of liver function. Compositional differences between the faecal microbiotas of the study groups included a lower Firmicutes:Bacteroidetes ratio and less numbers of viable yeast in the Probiotic group compared to the Control. The results illustrate the potential of the Lab4b probiotic as a neuroprotective agent and encourage further studies with human participants.

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.