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Mouse models of human diseases are invaluable tools for studying pathogenic mechanisms and testing interventions and therapeutics. For disorders such as Alzheimer’s disease in which numerous models are being generated, a challenging first step is to identify the most appropriate model and age to effectively evaluate new therapeutic approaches. Here we conducted a detailed phenotypic characterization of the 5xFAD model on a congenic C57BL/6 J strain background, across its lifespan – including a seldomly analyzed 18-month old time point to provide temporally correlated phenotyping of this model and a template for characterization of new models of LOAD as they are generated. This comprehensive analysis included quantification of plaque burden, Aβ biochemical levels, and neuropathology, neurophysiological measurements and behavioral and cognitive assessments, and evaluation of microglia, astrocytes, and neurons. Analysis of transcriptional changes was conducted using bulk-tissue generated RNA-seq data from microdissected cortices and hippocampi as a function of aging, which can be explored at the MODEL-AD Explorer and AD Knowledge Portal. This deep-phenotyping pipeline identified novel aspects of age-related pathology in the 5xFAD model. Measurement(s) Protein Expression • gene expression • electrophysiology data • protein measurement • behavior Technology Type(s) immunofluorescence microscopy assay • RNA sequencing • electrophysiology assay • Electrochemiluminescence Immunoassay • animal activity monitoring system Factor Type(s) genotype • age • sex Sample Characteristic - Organism Mus musculus Machine-accessible metadata file describing the reported data: https://doi.org/10.6084/m9.figshare.15176109

Animal models of disease are valuable resources for investigating pathogenic mechanisms and potential therapeutic interventions. However, for complex disorders such as Alzheimer’s disease (AD), the generation and availability of innumerous distinct animal models present unique challenges to AD researchers and hinder the success of useful therapies. Here, we conducted an in-depth analysis of the 3xTg-AD mouse model of AD across its lifespan to better inform the field of the various pathologies that appear at specific ages, and comment on drift that has occurred in the development of pathology in this line since its development 20 years ago. This modern characterization of the 3xTg-AD model includes an assessment of impairments in long-term potentiation followed by quantification of amyloid beta (Aβ) plaque burden and neurofibrillary tau tangles, biochemical levels of Aβ and tau protein, and neuropathological markers such as gliosis and accumulation of dystrophic neurites. We also present a novel comparison of the 3xTg-AD model with the 5xFAD model using the same deep-phenotyping characterization pipeline and show plasma NfL is strongly driven by plaque burden. The results from these analyses are freely available via the AD Knowledge Portal (https://admodelexplorer.synapse.org). Our work demonstrates the utility of a characterization pipeline that generates robust and standardized information relevant to investigating and comparing disease etiologies of current and future models of AD.

Pompe disease, an autosomal recessive disorder caused by deficient lysosomal acid α-glucosidase (GAA), is characterized by accumulation of intra-lysosomal glycogen in skeletal and oftentimes cardiac muscle. The c.1935C>A (p.Asp645Glu) variant, the most frequent GAA pathogenic mutation in people of Southern Han Chinese ancestry, causes infantile-onset Pompe disease (IOPD), presenting neonatally with severe hypertrophic cardiomyopathy, profound muscle hypotonia, respiratory failure, and infantile mortality. We applied CRISPR-Cas9 homology-directed repair (HDR) using a novel dual sgRNA approach flanking the target site to generate a Gaa em1935C > A knock-in mouse model and a myoblast cell line carrying the Gaa c.1935C>A mutation. Herein we describe the molecular, biochemical, histological, physiological, and behavioral characterization of 3-month-old homozygous Gaa em1935C > A mice. Homozygous Gaa em1935C > A knock-in mice exhibited normal Gaa mRNA expression levels relative to wild-type mice, had near-abolished GAA enzymatic activity, markedly increased tissue glycogen storage, and concomitantly impaired autophagy. Three-month-old mice demonstrated skeletal muscle weakness and hypertrophic cardiomyopathy but no premature mortality. The Gaa em1935C > A knock-in mouse model recapitulates multiple salient aspects of human IOPD caused by the GAA c.1935C>A pathogenic variant. It is an ideal model to assess innovative therapies to treat IOPD, including personalized therapeutic strategies that correct pathogenic variants, restore GAA activity and produce functional phenotypes.

Variants in the mitochondrial complex I assembly factor, NUBPL are associated with a rare cause of complex I deficiency mitochondrial disease. Patients affected by complex I deficiency harboring homozygous NUBPL variants typically have neurological problems including seizures, intellectual disability, and ataxia associated with cerebellar hypoplasia. Thus far only 19 cases have been reported worldwide, and no treatment is available for this rare disease. To investigate the pathogenesis of NUBPL-associated complex I deficiency, and for translational studies, we generated a knock-in mouse harboring a patient-specific variant Nubpl c.311T>C; p. L104P reported in three families. Similar to Nubpl global knockout mice, the Nubpl p. L104P homozygous mice are lethal at embryonic day E10.5, suggesting that the Nubpl p. L104P variant is likely a hypomorph allele. Given the recent link between Parkinson's disease and loss-of-function NUBPL variants, we also explored aging-related behaviors and immunocytochemical changes in Nubpl hemizygous mice and did not find significant behavioral and pathological changes for alpha-synuclein and oxidative stress markers . Our data suggest that homozygotes with Nubpl variants, similar to the null mice, are lethal, and heterozygotes are phenotypically and neuropathologically normal. We propose that a tissue-specific knockout strategy is required to establish a mouse model of Nubpl-associated complex I deficiency disorder for future mechanistic and translational studies.

IntroductionLeaky gut has been linked to autoimmune disorders including lupus. We previously reported upregulation of anti-flagellin antibodies in the blood of lupus patients and lupus-prone mice, which led to our hypothesis that a leaky gut drives lupus through bacterial flagellin-mediated activation of toll-like receptor 5 (TLR5).MethodsWe created MRL/lpr mice with global Tlr5 deletion through CRISPR/Cas9 and investigated lupus-like disease in these mice.ResultContrary to our hypothesis that the deletion of Tlr5 would attenuate lupus, our results showed exacerbation of lupus with Tlr5 deficiency in female MRL/lpr mice. Remarkably higher levels of proteinuria were observed in Tlr5-/- MRL/lpr mice suggesting aggravated glomerulonephritis. Histopathological analysis confirmed this result, and Tlr5 deletion significantly increased the deposition of IgG and complement C3 in the glomeruli. In addition, Tlr5 deficiency significantly increased renal infiltration of Th17 and activated cDC1 cells. Splenomegaly and lymphadenopathy were also aggravated in Tlr5-/- MRL/lpr mice suggesting impact on lymphoproliferation. In the spleen, significant decreased frequencies of regulatory lymphocytes and increased germinal centers were observed with Tlr5 deletion. Notably, Tlr5 deficiency did not change host metabolism or the existing leaky gut; however, it significantly reshaped the fecal microbiota.ConclusionGlobal deletion of Tlr5 exacerbates lupus-like disease in MRL/lpr mice. Future studies will elucidate the underlying mechanisms by which Tlr5 deficiency modulates host-microbiota interactions to exacerbate lupus.

Background Apolipoprotein E ε4 ( APOE4 ) is the strongest genetic risk factor for late-onset Alzheimer’s disease (LOAD). A recent case report identified a rare variant in APOE, APOE3 -R136S (Christchurch), proposed to confer resistance to autosomal dominant Alzheimer’s Disease (AD). However, it remains unclear whether and how this variant exerts its protective effects. Methods We introduced the R136S variant into mouse Apoe ( ApoeCh ) and investigated its effect on the development of AD-related pathology using the 5xFAD model of amyloidosis and the PS19 model of tauopathy. We used immunohistochemical and biochemical analysis along with single-cell spatial omics and bulk proteomics to explore the impact of the ApoeCh variant on AD pathological development and the brain’s response to plaques and tau. Results In 5xFAD mice, ApoeCh enhances a Disease-Associated Microglia (DAM) phenotype in microglia surrounding plaques, and reduces plaque load, dystrophic neurites, and plasma neurofilament light chain. By contrast, in PS19 mice, ApoeCh suppresses the microglial and astrocytic responses to tau-laden neurons and does not reduce tau accumulation or phosphorylation, but partially rescues tau-induced synaptic and myelin loss. We compared how microglia responses differ between the two mouse models to elucidate the distinct DAM signatures induced by ApoeCh . We identified upregulation of antigen presentation-related genes in the DAM response in a PS19 compared to a 5xFAD background, suggesting a differential response to amyloid versus tau pathology that is modulated by the presence of ApoeCh . Bulk proteomics show upregulated mitochondrial protein abundance with ApoeCh in 5xFAD mice, but reductions in mitochondrial and translation associated proteins in PS19 mice. Conclusions These findings highlight the ability of the ApoeCh variant to modulate microglial responses based on the type of pathology, enhancing DAM reactivity in amyloid models and dampening neuroinflammation to promote protection in tau models. This suggests that the Christchurch variant's protective effects likely involve multiple mechanisms, including changes in receptor binding and microglial programming. Graphical Abstract

The TREM2 R47H variant is one of the strongest genetic risk factors for late-onset Alzheimer's Disease (AD). Unfortunately, many current Trem2 mouse models are associated with cryptic mRNA splicing of the mutant allele that produces a confounding reduction in protein product. To overcome this issue, we developed the Trem2 (Normal Splice Site) mouse model in which the Trem2 allele is expressed at a similar level to the wild-type Trem2 allele without evidence of cryptic splicing products. Trem2 mice were treated with the demyelinating agent cuprizone, or crossed with the 5xFAD mouse model of amyloidosis, to explore the impact of the TREM2 R47H variant on inflammatory responses to demyelination, plaque development, and the brain's response to plaques. Trem2 mice display an appropriate inflammatory response to cuprizone challenge, and do not recapitulate the null allele in terms of impeded inflammatory responses to demyelination. Utilizing the 5xFAD mouse model, we report age- and disease-dependent changes in Trem2 mice in response to development of AD-like pathology. At an early (4-month-old) disease stage, hemizygous 5xFAD/homozygous Trem2 (5xFAD/Trem2 ) mice have reduced size and number of microglia that display impaired interaction with plaques compared to microglia in age-matched 5xFAD hemizygous controls. This is associated with a suppressed inflammatory response but increased dystrophic neurites and axonal damage as measured by plasma neurofilament light chain (NfL) level. Homozygosity for Trem2 suppressed LTP deficits and loss of presynaptic puncta caused by the 5xFAD transgene array in 4-month-old mice. At a more advanced (12-month-old) disease stage 5xFAD/Trem2 mice no longer display impaired plaque-microglia interaction or suppressed inflammatory gene expression, although NfL levels remain elevated, and a unique interferon-related gene expression signature is seen. Twelve-month old Trem2 mice also display LTP deficits and postsynaptic loss. The Trem2 mouse is a valuable model that can be used to investigate age-dependent effects of the AD-risk R47H mutation on TREM2 and microglial function including its effects on plaque development, microglial-plaque interaction, production of a unique interferon signature and associated tissue damage.

Background Genome‐Wide Association Studies (GWAS) identified ApoE4 and Trem2*R47H as two of the strongest genetic risk factors for late‐onset Alzheimer’s Disease (LOAD). As part of our efforts to develop mouse models that better recapitulate LOAD, at Model Organism Development & Evaluation for Late‐Onset Alzheimer’s Disease (MODEL‐AD) consortium at University of California – Irvine, we have created a triple homozygous mouse model that combines our previously developed hAb‐KIloxP mice (Jackson Lab #031050), Trem2R47H NSS (Jackson Lab #034036) and a humanized ApoE4 (Jackson Lab #027894), to evaluate the interactions between aging, hAPOE4, TREM2*R47H, and hAb. Method By breeding the hAb‐KIloxP, hApoE4 and Trem2R47H NSS, we obtained triple homozygous (HO) mice and we then generated four different groups: WT (C57BL6/J), hAb‐KIloxP HO, hAb‐KIloxP HO;hApoE4 HO and hAb‐KIloxP HO;hApoE4 HO;Trem2R47H NSS HO. All four groups were then aged to 4, 12, 18 and 24 months of age, when coronal hippocampal slices were prepared, and long‐term potentiation recordings were obtained. Additionally, we also measured soluble and insoluble Ab40 and Ab42 as well as plasma neurofilament light chain (NfL). Result hAb‐KIloxP mice showed a significant reduction in mean potentiation 50‐60 minutes post TBS, when compared to WT mice at 4, 12, 18 and 24 months of age, indicative of an LTP deficit associated to human Ab. Interestingly, both hAPOE4 and Trem2R47H NSS variants completely prevent the LTP deficits observed on the hAb‐KIloxP mice from 4 months of age. In addition, cortical soluble Ab40, Ab42 and the Ab42/Ab40 ratio were significantly decreased in hAb‐KIloxP;hAPOE4 and hAb‐KIloxP;hAPOE4;Trem2R47H NSS mice at 24 m of age, when compared to hAb‐KIloxP. However, plasma NfL levels were significantly increased in hAb‐KIloxP;hAPOE4;Trem2R47H NSS mice at 24 months of age, indicative of axonal damage in this mouse model. Conclusion hAb induces robust LTP deficits that are prevent by either hAPOE4 or TREM2*R47H, contrary to expectations. However, hAPOE4 and Trem2R47H NSS variants have a cumulative detrimental effect on axonal damage when combined with aging.

Genome-Wide Association Studies (GWAS) implicate SPI1 (PU.1) as a risk factor for late-onset Alzheimer's Disease (LOAD). Within the brain, SPI1 encodes a microglia-specific transcription factor, necessary for microglial proliferation and activation. SPI1 rs1377416 has been identified as a non-coding GWAS risk variant for AD. We have developed a novel mouse model with the SPI1 rs1377416 variant to explore its impacts on AD pathogenesis. By using CRISPR/Cas9, we have generated SPI1*rs1377416 mice that carry a non-coding mutation corresponding to the rs1377416 SNP found in human SPI1. We crossed the SPI1 mice with the 5xFAD mouse model of AD and aged them to 4 and 12 months of age. Coronal brain sections were then obtained and immunolabeled with several markers to visualize amyloid plaques, glial cells and assess axonal and neuritic damage. Confocal images were then obtained and quantified in subiculum and cortex. We found an age-related increase in dense core plaque number and size in the subiculum of 5xFAD;SPI1 mice. Microglial volume as well as astrocytic activation were reduced in 5xFAD;SPI1 compared to 5xFAD mice in both brain areas at 12 months. Correspondingly, neuritic dystrophy and axonal damage were also diminished. Significant sex difference was observed in different analyses with males being affected less than females (with the exception of plaque deposition), and mainly detected in subiculum. Our results indicate that the rs1377416 variant of SPI1 induces an age-dependent increase in amyloid deposition in the 5xFAD model of AD. Moreover, this SPI1 variant exerts a protective effect by suppressing astrocytic response and preventing neuritic and axonal damage. There is a strong sex difference observed between males and females when the variant is present and requires further investigation.