To improve clinical translatability of non-clinical in-vivo Alzheimer’s disease (AD) models, a humanized APP knock-in mouse model (APP SAA ) was recently created (Xia, D. et al., 2022). The genetic modifications lead to increased Aβ42/40 ratios in AD relevant tissues, resulting in an age-dependent amyloid deposition. Here we assess the value of this model for non-clinical efficacy studies of experimental treatments with diverse mechanisms of action to facilitate the development of novel AD therapeutics. APP SAA mice and WT controls were aged and sacrificed at various time points. In addition, APP SAA mice were treated for 3 months from the age of 3 months onward with an NLRP3-inhibitor, an in-house developed calcium homeostasis modulator or vehicle. The major pathological hallmarks were investigated with biochemical and immunohistological assays. Additionally a preliminary screen for AD biomarkers was conducted in CSF samples. Since APP SAA mice lack an obvious behavioral phenotype, synaptic plasticity was investigated to serve as a functional readout. Cortical Aβ40, Aβ42 and pyroglutamate modified Aβ42 (N3pE-42) levels were shown to be affected with age. In addition, cortical amyloid plaques, surrounded by activated microglia, were shown to be present already at an age of 3 months, albeit at a low level. With age, the number of cortical plaques increased. In addition, we were able to confirm the presence of endogenous phospho-Tau positive dystrophic neurites within these plaques, clearly demonstrating the translational relevance of this model. Interestingly, several of the observed hallmarks were counteracted by compound intervention, including phospho-Tau pathology. Finally, we show that several CNS biomarkers can be detected in mouse CSF. These data lead us to conclude that this knock-in model is a broadly applicable tool to investigate efficacy of disease-modifying drugs with diverse mechanisms of action even without directly targeting protein aggregates.

Abnormal calcium signaling is a central pathological component of Alzheimer’s disease (AD). Here, we describe the identification of a class of compounds called ReS19-T, which are able to restore calcium homeostasis in cell-based models of tau pathology. Aberrant tau accumulation leads to uncontrolled activation of store-operated calcium channels (SOCCs) by remodeling septin filaments at the cell cortex. Binding of ReS19-T to septins restores filament assembly in the disease state and restrains calcium entry through SOCCs. In amyloid-β and tau-driven mouse models of disease, ReS19-T agents restored synaptic plasticity, normalized brain network activity, and attenuated the development of both amyloid-β and tau pathology. Our findings identify the septin cytoskeleton as a potential therapeutic target for the development of disease-modifying AD treatments.

To improve clinical translatability of non-clinical in-vivo Alzheimer’s disease (AD) models, a humanized APP knock-in mouse model (APP SAA ) was recently created (Xia, D. et al., 2022). This homozygous APP knock-in model carries a humanized Aβ 1-42 sequence and 3 disease causing mutations ( S wedish, A rctic, and A ustrian). These modifications lead to increased Aβ 42/40 ratios in AD relevant tissues, resulting in an age-dependent amyloid deposition in the brain. The highest plaque density is found in cortical and hippocampal regions. APP SAA mice also display clear neuroinflammation and an increase in fluid biomarkers of neurodegeneration (NF-L and total Tau in CSF). Here we assess the value of this model as a tool for non-clinical efficacy studies of experimental drugs with diverse mechanisms of action to facilitate the development of novel AD therapeutics. APP SAA mice and WT controls were aged and sacrificed at various time points. In addition, APP SAA mice were treated with drug or vehicle, on a daily basis for 3 months from the age of 3 months onward. The major pathological hallmarks of this model and the treatment effect will be investigated with biochemical and immunohistological assays. Monthly blood sampling will allow the follow up of pathology progression in individual mice by assessing plasma-based biomarkers. Since APP SAA mice lack an obvious behavioral phenotype, synaptic plasticity will be investigated to serve as a functional readout. The treatment is well tolerated by the mice in study. Body weights were not altered between compound and vehicle dosing during the first 2 months of the treatment. Data on pathological outcomes and synaptic plasticity are not yet available but will be shared during the presentation. A humanized knock-in model for AD is hypothesized to be a broadly applicable tool to investigate disease-modifying drugs with diverse modes of actions.

Alzheimer's disease, progressive supranuclear palsy and frontotemporal dementia are characterized by neuronal expression of aberrant tau protein, tau hyperphosphorylation (pTAU), tau aggregation and neurofibrillary tangle formation sequentially culminating into neuronal cell death, a process termed tauopathy. Our aim was to address at which tauopathy stage neuroinflammation starts and to study the related microglial phenotype. We used Thy1-hTau.P301S (PS) mice expressing human tau with a P301S mutation specifically in neurons. Significant levels of cortical pTAU were present from 2 months onwards. Dystrophic morphological complexity of cortical microglia arose after pTAU accumulation concomitant with increased microglial lysosomal volumes and a significant loss of homeostatic marker Tmem119. Interestingly, we detected increases in neuronal pTAU and postsynaptic structures in the lysosomes of PS microglia. Moreover, the overall cortical postsynaptic density was decreased in 6-month-old PS mice. Together, our results indicate that microglia adopt a pTAU-associated phenotype, and are morphologically and functionally distinct from wild-type microglia after neuronal pTAU accumulation has initiated.

As human Tau undergoes pathologically relevant post-translational modifications when expressed in yeast, the use of humanized yeast models for the generation of novel Tau monoclonal antibodies has previously been proven to be successful. In this study, human Tau2N4R-ΔK280 purified from yeast was used for the immunization of mice and subsequent selection of high affinity Tau-specific monoclonal antibodies. The characterization of four novel antibodies in different Tau model systems yielded a phosphorylation-dependent antibody (15A10), an antibody directed to the first microtubule-binding repeat domain (16B12), a carboxy-terminal antibody (20G10) and an antibody targeting an epitope on the hinge of the first and second amino-terminal insert (18F12). The latter was found to be conformation-dependent, suggesting structural differences between the Tau splicing isoforms and allowing insight in the roles played by the amino-terminal inserts. As this monoclonal antibody also has the capacity to detect tangle-like structures in different transgenic Tau mice and neurofibrillary tangles in brain sections of patients diagnosed with Alzheimer's disease, we also tested the diagnostic potential of 18F12 in a pilot study and found this monoclonal antibody to have the ability to discriminate Alzheimer's disease patients from control individuals based on increased Tau levels in the cerebrospinal fluid.

This study assessed the development of motor deficits in female hTau.P301S transgenic mice from 1.5 to 5.5 months of age. The test battery included clasping reflex, grid hanging, Rotarod test, spontaneous explorative activity, Catwalk gait analysis, and nest building. Starting from the age of 2-3 months the mice showed marked hyperactivity, abnormal placing of weight on the hindlimbs and defective nest building in their home cage. These behavioral impairments did not progress with age. In addition, there was a progressive development of hindlimb clasping, inability to stay on a rotating rod or hang on a metal grid, and gait impairment. Depending on the measured output parameter, the motor impairment became significant from 3 to 4 months onwards and rapidly worsened until the age of 5.5 months with little inter-individual variation. The progressive motor impairment was paralleled by a robust increase in AT8 p-tau positive neurons in deep cerebellar nuclei and pontine brainstem between 3 and 5.5 months of age. The quick and steadily progressive motor impairment between 3 and 5.5 months of age accompanied by robust development of tau pathology in the hindbrain makes this mouse well suited for preclinical studies aiming at slowing down tau pathology associated with primary or secondary tauopathies.

Chronic inflammation represents a central component in the pathogenesis of Alzheimer's disease (AD). Recent work suggests that breaking immune tolerance by Programmed cell Death‐1 (PD1) checkpoint inhibition produces an IFN‐γ‐dependent systemic immune response, with infiltration of the brain by peripheral myeloid cells and neuropathological as well as functional improvements even in mice with advanced amyloid pathology (Baruch et al., ( ): Nature Medicine, 22:135–137). Immune checkpoint inhibition was therefore suggested as potential treatment for neurodegenerative disorders when activation of the immune system is appropriate. Because a xenogeneic rat antibody (mAb) was used in the study, whether the effect was specific to PD1 target engagement was uncertain. In the present study we examined whether PD1 immunotherapy can lower amyloid‐β pathology in a range of different amyloid transgenic models performed at three pharmaceutical companies with the exact same anti‐PD1 isotype and two mouse chimeric variants. Although PD1 immunotherapy stimulated systemic activation of the peripheral immune system, monocyte‐derived macrophage infiltration into the brain was not detected, and progression of brain amyloid pathology was not altered. Similar negative results of the effect of PD1 immunotherapy on amyloid brain pathology were obtained in two additional models in two separate institutions. These results show that inhibition of PD1 checkpoint signaling by itself is not sufficient to reduce amyloid pathology and that additional factors might have contributed to previously published results (Baruch et al., ( ): Nature Medicine, 22:135–137). Until such factors are elucidated, animal model data do not support further evaluation of PD1 checkpoint inhibition as a therapeutic modality for Alzheimer's disease.

Transcriptomic and proteomic analyses of multiple sclerosis (MS) lesions indicate alterations in the gamma-aminobutyric acid (GABA) inhibitory system, suggesting its involvement in the disease process. To further elucidate the role of GABA in central nervous system (CNS) inflammation in vivo, the chronic myelin oligodendrocyte glycoprotein (MOG)(35-55) experimental autoimmune encephalomyelitis (EAE) model was used. Daily GABA injections (200mg/kg) from day 3 onwards significantly augmented disease severity, which was associated with increased CNS mRNA expression levels of tumor necrosis factor alpha (TNF-α) and interleukin (IL)-6. GABA-treated mice showed enhanced MOG-dependent proliferation and were skewed towards a T helper 1 phenotype. Moreover, in vitro, the lipopolysaccharide (LPS)-induced increase in interleukin (IL)-6 production by macrophages was enhanced at low GABA concentrations (0.03-0.3mM). In sharp contrast to exogenous GABA administration, endogenous GABA increment by systemic treatment with the GABA-transaminase inhibitor vigabatrin (250mg/kg) had prophylactic as well as therapeutic potential in EAE. Together, these results indicate an immune amplifying role of GABA in neuroinflammatory diseases like MS.

Glycine, an important inhibitory neurotransmitter in the mammalian central nervous system (CNS), has been shown to modulate peripheral immune cell responses. In that respect, glycine levels are increased in several neuroinflammatory disorders, such as amyotrophic lateral sclerosis (ALS) and multiple sclerosis (MS). In this study, we show that glycine modulates macrophage effector functions implicated in CNS inflammation and in other, related inflammatory conditions. We demonstrate that glycine does not affect the production of reactive oxygen species but stimulates myelin phagocytosis and the production of the proinflammatory mediators nitric oxide (NO) and tumor necrosis factor (TNF)‐α by rat macrophages. These effects of glycine are not mediated by the glycine receptor (GlyR) or by glycine transporters (GlyTs), as neither the GlyR antagonist strychnine nor the antagonist of GlyT1 (ALX5407) reverses the observed effects. In contrast, 2‐aminoisobutyric acid, a substrate of neutral amino acid transporters (NAATs), inhibits the glycine‐mediated enhancement of myelin phagocytosis as well as of NO and TNF‐α production. In conclusion, our findings demonstrate that glycine modulates macrophage function through activation of NAATs. Glycine may thereby influence immunological processes in inflammatory diseases involving macrophage activation and demyelination, including MS and related conditions associated with altered glycine levels. © 2010 Wiley‐Liss, Inc.

Glycine is an inhibitory neurotransmitter acting mainly in the caudal part of the central nervous system. Besides this neurotransmitter function, glycine has cytoprotective and modulatory effects in different non-neuronal cell types. Modulatory effects were mainly described in immune cells, endothelial cells and macroglial cells, where glycine modulates proliferation, differentiation, migration and cytokine production. Activation of glycine receptors (GlyRs) causes membrane potential changes that in turn modulate calcium flux and downstream effects in these cells. Cytoprotective effects were mainly described in renal cells, hepatocytes and endothelial cells, where glycine protects cells from ischemic cell death. In these cell types, glycine has been suggested to stabilize porous defects that develop in the plasma membranes of ischemic cells, leading to leakage of macromolecules and subsequent cell death. Although there is some evidence linking these effects to the activation of GlyRs, they seem to operate in an entirely different mode from classical neuronal subtypes.

Leukemia inhibitory factor (LIF) promotes survival of glial cells and neurons during autoimmune and injury responses in the central nervous system (CNS). While various studies indicate that LIF also modulates ongoing inflammatory responses, data on underlying events are lacking. In this study we demonstrate that LIF modulates macrophage function. LIF inhibits the production of oxygen radicals and TNFalpha and stimulates myelin uptake by macrophages. These effects of LIF are accompanied by activation of the JAK/STAT3 signalling pathway. Our findings demonstrate that LIF has anti-inflammatory properties and enhances myelin clearance, implicating that LIF may be an important factor in CNS inflammatory disease.