App Knock-in Mouse Research Articles

Humanization of the entire murine Mapt gene provides a murine model of pathological human tau propagation

In cortical regions of brains from individuals with preclinical or clinical Alzheimer's disease (AD), extracellular β-amyloid (Aβ) deposition precedes the aggregation of pathological intracellular tau (the product of the gene microtubule-associated protein tau (MAPT)). To our knowledge, current mouse models of tauopathy reconstitute tau pathology by overexpressing mutant human tau protein. Here, through a homologous recombination approach that replaced the entire murine Mapt gene with the human ortholog, we developed knock-in mice with humanized Mapt to create an in vivo platform for studying human tauopathy. Of note, the humanized Mapt expressed all six tau isoforms present in humans. We next cross-bred the MAPT knock-in mice with single amyloid precursor protein (App) knock-in mice to investigate the Aβ-tau axis in AD etiology. The double-knock-in mice exhibited higher tau phosphorylation than did single MAPT knock-in mice but initially lacked apparent tauopathy and neurodegeneration, as observed in the single App knock-in mice. We further observed that tau humanization significantly accelerates cell-to-cell propagation of AD brain-derived pathological tau both in the absence and presence of Aβ-amyloidosis. In the presence of Aβ-amyloidosis, tau accumulation was intensified and closely associated with dystrophic neurites, consistently showing that Aβ-amyloidosis affects tau pathology. Our results also indicated that the pathological human tau interacts better with human tau than with murine tau, suggesting species-specific differences between these orthologous pathogenic proteins. We propose that the MAPT knock-in mice will make it feasible to investigate the behaviors and characteristics of human tau in an animal model.

A NOVEL DROSOPHILA MODEL OF ALZHEIMER’S DISEASE

Alzheimer's disease (AD) is a progressive neurodegenerative disease and is the main cause of senile dementia. AD is characterized by two pathological hallmarks, senile plaques composed by amyloid β peptides (Aβ) and neurofibrillary tangles composed by hyper-phosphorylated tau proteins. The accumulation of Aβ42 in the brain is thought to be the primary event in AD pathogenesis. To understand pathological roles of Aβ42 in vivo, we previously established a Drosophila model of AD expressing Aβ42 peptides in the secretory pathway of neurons (Iijima, K. et al. 2004, PNAS). This fly model developed several key pathological phenotypes in an age-dependent manner. However, substantial amount of Aβ42 was accumulated intracellularly and caused chronic ER stress, which may not be common pathologies in AD patients. To overcome this issue, we generated novel transgenic flies that efficiently secrete Aβ42 peptides from neurons to extracellular space in the brains. To examine the effects of and metabolism of Aβ42 peptides in the extracellular space of fly brains, we generated transgenic fly lines carrying engineered Aβ42 construct that efficiently secrete Aβ42 from neurons in the brains. We then systematically characterized their phenotypes including Aβ42 accumulation, locomotor functions, neurodegeneration and life span during aging and compared those in our previous Aβ42 fly model. As reported, our previous Aβ42 fly model showed age-dependent Aβ42 accumulation, locomotor defects, neurodegeneration and premature death. In contrast, despite high expression levels of Aβ42 in the brains, the new Aβ42 fly model did not show any significant phenotypes including Aβ42 deposits, suggesting that secreted Aβ42 did not accumulate and aggregate in the extracellular space even in aged fly brains. Our data indicate that overexpression of Aβ42 in the extracellular space of fly brain is not sufficient to produce Aβ42 deposits and neurotoxicity in flies. This result might be similar to recent observation in App knock-in mouse of AD carrying only Swedish mutation (Salas, IH. et al. 2018, J Alzheimers Dis. & Sakakibara, Y. et al. 2019, BMC Neurosci.). Our new Aβ42 fly model can be used as an efficient genetic model system to provide significant insights into pathomechanisms of an early phase of AD.

An App knock-in mouse inducing the formation of a toxic conformer of Aβ as a model for evaluating only oligomer-induced cognitive decline in Alzheimer's disease

Irie and colleagues identified a “toxic conformer”, which possesses a turn structure at positions 22-23, among various conformations of Aβ and have been reporting its potent oligomeric capacity and neurotoxicity. This toxic conformer was detected in the brains of AD patients and AD model mice (Tg2576 line), and passive immunization targeting this conformer ameliorated the cognitive dysfunction in an AD model. In this study, we developed a novel AD mouse model (AppNL-P-F/NL-P-F) with Swedish mutation (NL), Iberian mutation (F), and mutation (P) overproducing E22P-Aβ, a mimic of the toxic conformer, utilizing the knock-in technique that well recapitulates the Aβ pathology of AD patients in mice and avoids the artificial phenotype observed in transgenic-type model mice. We confirmed that AppNL-P-F/NL-P-F mice produce Aβ by ELISA and accumulate senile plaques by immunohistochemistry at eight months of age. In WB, we observed a potential trimer band and high molecular-weight oligomer bands without a monomeric band in the TBS-soluble fraction of AppNL-P-F/NL-P-F mice at six months of age. In the novel object recognition test, cognitive impairment was observed at six months of age in these mice. These findings suggest that the toxic conformer of Aβ induces cognitive dysfunction mediated by its oligomer formation in this mouse brain. AppNL-P-F/NL-P-F mice may be a useful model for evaluating Aβ oligomer-induced cognitive impairment in AD and will aid in exploring therapeutic targets for AD pathology.

Amyloid-\u03b2 plaque formation and reactive gliosis are required for induction of cognitive deficits in App knock-in mouse models of Alzheimer\u2019s disease

BackgroundKnock-in (KI) mouse models of Alzheimer’s disease (AD) that endogenously overproduce Aβ without non-physiological overexpression of amyloid precursor protein (APP) provide important insights into the pathogenic mechanisms of AD. Previously, we reported that AppNL-G-F mice, which harbor three familial AD mutations (Swedish, Beyreuther/Iberian, and Arctic) exhibited emotional alterations before the onset of definitive cognitive deficits. To determine whether these mice exhibit deficits in learning and memory at more advanced ages, we compared the Morris water maze performance of AppNL-G-F and AppNL mice, which harbor only the Swedish mutation, with that of wild-type (WT) C57BL/6J mice at the age of 24 months. To correlate cognitive deficits and neuroinflammation, we also examined Aβ plaque formation and reactive gliosis in these mice.ResultsIn the Morris water maze, a spatial task, 24-month-old AppNL-G-F/NL-G-F mice exhibited significantly poorer spatial learning than WT mice during the hidden training sessions, but similarly to WT mice during the visible training sessions. Not surprisingly, AppNL-G-F/NL-G-F mice also exhibited spatial memory deficits both 1 and 7 days after the last training session. By contrast, 24-month-old AppNL/NL mice had intact spatial learning and memory relative to WT mice. Immunohistochemical analyses revealed that 24-month-old AppNL-G-F/NL-G-F mice developed massive Aβ plaques and reactive gliosis (microgliosis and astrocytosis) throughout the brain, including the cortex and hippocampus. By contrast, we observed no detectable brain pathology in AppNL/NL mice despite overproduction of human Aβ40 and Aβ42 in their brains.ConclusionsAβ plaque formation, followed by sustained neuroinflammation, is necessary for the induction of definitive cognitive deficits in App-KI mouse models of AD. Our data also indicate that introduction of the Swedish mutation alone in endogenous APP is not sufficient to produce either AD-related brain pathology or cognitive deficits in mice.

Behavioral characterization of APP knock-in mice model in touchscreen-based tests aiming early detection of Alzheimer's disease.

Behavioral characterization of APP knock-in mice model in touchscreen-based tests aiming early detection of Alzheimer's disease.

Pridopidine stabilizes mushroom spines in mouse models of Alzheimer's disease by acting on the sigma-1 receptor

There is evidence that cognitive decline in Alzheimer's disease (AD) results from deficiencies in synaptic communication (e.g., loss of mushroom-shaped ‘memory spines’) and neurodegenerative processes. This might be treated with sigma-1 receptor (S1R) agonists, which are broadly neuroprotective and modulate synaptic plasticity. For example, we previously found that the mixed muscarinic/S1R agonist AF710B prevents mushroom spine loss in hippocampal cultures from APP knock-in (APP-KI) and presenilin-1-M146 V knock-in (PS1-KI) mice. We also found that the “dopaminergic stabilizer” pridopidine (structurally similar to the S1R agonist R(+)-3-PPP), is a high-affinity S1R agonist and is synaptoprotective in a mouse model of Huntington disease. Here we tested whether pridopidine and R(+)-3-PPP are synaptoprotective in models of AD and whether this requires S1R. We also examined the effects of pridopidine on long-term potentiation (LTP), endoplasmic reticulum calcium and neuronal store-operated calcium entry (nSOC) in spines, all of which are dysregulated in AD, contributing to synaptic pathology. We report here that pridopidine and 3-PPP protect mushroom spines from Aβ42 oligomer toxicity in primary WT hippocampal cultures from mice. Pridopidine also reversed LTP defects in hippocampal slices resulting from application of Aβ42 oligomers. Pridopidine and 3-PPP rescued mushroom spines in hippocampal cultures from APP-KI and PS1-KI mice. S1R knockdown from lenti-viral shRNA expression destabilized WT mushroom spines and prevented the synaptoprotective effects of pridopidine in PS1-KI cultures. Knockout of PS1/2 destabilized mushroom spines and pridopidine was unable to prevent this. Pridopidine lowered endoplasmic reticulum calcium levels in WT, PS1-KO, PS1-KI and PS2 KO neurons, but not in PS1/2 KO neurons. S1R was required for pridopidine to enhance spine nSOC in PS1-KI neurons. Pridopidine was unable to rescue PS1-KI mushroom spines during pharmacological or genetic inhibition of nSOC. Oral pridopidine treatment rescued mushroom spines in vivo in aged PS1-KI-GFP mice. Pridopidine stabilizes mushroom spines in mouse models of AD and this requires S1R, endoplasmic reticulum calcium leakage through PS1/2 and nSOC. Thus, pridopidine may be useful to explore for the treatment of AD.

Cognitive and emotional alterations in App knock-in mouse models of A\u03b2 amyloidosis

BackgroundAlzheimer’s disease (AD), the most common cause of dementia, is characterized by the progressive deposition of amyloid-β (Aβ) peptides and neurofibrillary tangles. Mouse models of Aβ amyloidosis generated by knock-in (KI) of a humanized Aβ sequence provide distinct advantages over traditional transgenic models that rely on overexpression of amyloid precursor protein (APP). In App-KI mice, three familial AD-associated mutations were introduced into the endogenous mouse App locus to recapitulate Aβ pathology observed in AD: the Swedish (NL) mutation, which elevates total Aβ production; the Beyreuther/Iberian (F) mutation, which increases the Aβ42/Aβ40 ratio; and the Arctic (G) mutation, which promotes Aβ aggregation. AppNL-G-F mice harbor all three mutations and develop progressive Aβ amyloidosis and neuroinflammatory response in broader brain areas, whereas AppNL mice carrying only the Swedish mutation exhibit no overt AD-related pathological changes. To identify behavioral alterations associated with Aβ pathology, we assessed emotional and cognitive domains of AppNL-G-F and AppNL mice at different time points, using the elevated plus maze, contextual fear conditioning, and Barnes maze tasks.ResultsAssessments of emotional domains revealed that, in comparison with wild-type (WT) C57BL/6J mice, AppNL-G-F/NL-G-F mice exhibited anxiolytic-like behavior that was detectable from 6 months of age. By contrast, AppNL/NL mice exhibited anxiogenic-like behavior from 15 months of age. In the contextual fear conditioning task, both AppNL/NL and AppNL-G-F/NL-G-F mice exhibited intact learning and memory up to 15–18 months of age, whereas AppNL-G-F/NL-G-F mice exhibited hyper-reactivity to painful stimuli. In the Barnes maze task, AppNL-G-F/NL-G-F mice exhibited a subtle decline in spatial learning ability at 8 months of age, but retained normal memory functions.ConclusionAppNL/NL and AppNL-G-F/NL-G-F mice exhibit behavioral changes associated with non-cognitive, emotional domains before the onset of definitive cognitive deficits. Our observations consistently indicate that AppNL-G-F/NL-G-F mice represent a model for preclinical AD. These mice are useful for the study of AD prevention rather than treatment after neurodegeneration.

Histological and Behavioral Phenotypes of a Novel Mutated APP Knock-In Mouse.

Gene mutations within amyloid precursor protein (APP or AβPP) and/or presenilin 1 (PS1) genes are determinants of familial Alzheimer's disease (fAD) and remain fundamental for experimental models. Here, we generated a neuronal knock-in mouse (PLB2APP) with mutated human APPSwe/Lon and investigated histopathology and behavioral phenotypes. Additionally, PLB2APP mice were cross-bred with a presenilin (PS1A246E) line to assess the impact of this gene combination. Immunohistochemistry determined amyloid-β (Aβ) pathology, astrogliosis (via GFAP labelling), and neuronal densities in hippocampal and cortical brain regions. One-year old PLB2APP mice showed higher levels of intracellular Aβ in CA1, dentate gyrus, and cortical regions compared to PLBWT controls. Co-expression of PS1 reduced hippocampal but elevated cortical Aβ build-up. Amyloid plaques were sparse in aged PLB2APP mice, and co-expression of PS1 promoted plaque formation. Heightened GFAP expression followed the region-specific pattern of Aβ in PLB2APP and PLB2APP/PS1 mice. Behaviorally, habituation to a novel environment was delayed in 6-month-old PLB2APP mice, and overall home-cage activity was reduced in both lines at 6 and 12 months, particularly during the dark phase. Spatial learning in the water maze was impaired in PLB2APP mice independent of PS1 expression and associated with reduced spatial navigation strategies. Memory retrieval was compromised in PLB2APP mice only. Our data demonstrate that low expression of APP is sufficient to drive histopathological and cognitive changes in mice without overexpression or excessive plaque deposition. AD-like phenotypes were altered by co-expression of PS1, including a shift from hippocampal to cortical Aβ pathology, alongside reduced deficits in spatial learning.

Generation of App knock-in mice reveals deletion mutations protective against Alzheimer\u2019s disease-like pathology

Although, a number of pathogenic mutations have been found for Alzheimer’s disease (AD), only one protective mutation has been identified so far in humans. Here we identify possible protective deletion mutations in the 3′-UTR of the amyloid precursor protein (App) gene in mice. We use an App knock-in mouse model carrying a humanized Aβ sequence and three AD mutations in the endogenous App gene. Genome editing of the model zygotes using multiple combinations of CRISPR/Cas9 tools produces genetically mosaic animals with various App 3′-UTR deletions. Depending on the editing efficiency, the 3′-UTR disruption mitigates the Aβ pathology development through transcriptional and translational regulation of APP expression. Notably, an App knock-in mouse with a 34-bp deletion in a 52-bp regulatory element adjacent to the stop codon shows a substantial reduction in Aβ pathology. Further functional characterization of the identified element should provide deeper understanding of the pathogenic mechanisms of AD.

Spatial reversal learning defect coincides with hypersynchronous telencephalic BOLD functional connectivity in APPNL-F/NL-F knock-in mice

Amyloid pathology occurs early in Alzheimer’s disease (AD), and has therefore been the focus of numerous studies. Transgenic mouse models have been instrumental to study amyloidosis, but observations might have been confounded by APP-overexpression artifacts. The current study investigated early functional defects in an APP knock-in mouse model, which allows assessing the effects of pathological amyloid-beta (Aβ) without interference of APP-artifacts. Female APPNL/NL knock-in mice of 3 and 7 months old were compared to age-matched APPNL-F/NL-F mice with increased Aβ42/40 ratio and initial Aβ-plaque deposition around 6 months of age. Spatial learning was examined using a Morris water maze protocol consisting of acquisition and reversal trials interleaved with reference memory tests. Functional connectivity (FC) of brain networks was assessed using resting-state functional MRI (rsfMRI). The Morris water maze data revealed that 3 months old APPNL-F/NL-F mice were unable to reach the same reference memory proficiency as APPNL/NL mice after reversal training. This cognitive defect in 3-month-old APPNL-F/NL-F mice coincided with hypersynchronous FC of the hippocampal, cingulate, caudate-putamen, and default-mode-like networks. The occurrence of these defects in APPNL-F/NL-F mice demonstrates that cognitive flexibility and synchronicity of telencephalic activity are specifically altered by early Aβ pathology without changes in APP neurochemistry.

The Disease-modifying Drug Candidate, SAK3 Improves Cognitive Impairment and Inhibits Amyloid beta Deposition in App Knock-in Mice

Alzheimer’s disease (AD) is a progressive neurodegenerative disease and the most common form of elderly dementia in the world. At present, acetylcholine inhibitors, such as donepezil, galantamine and rivastigmine, are used for AD therapy, but the therapeutic efficacy is limited. We recently proposed T-type voltage-gated Ca2+ channels’ (T-VGCCs) enhancer as a new therapeutic candidate for AD. In the current study, we confirmed the pharmacokinetics of SAK3 in the plasma and brain of mice using ultra performance liquid chromatography-tandem mass spectrometry. We also investigated the effects of SAK3 on the major symptoms of AD, such as cognitive dysfunction and amyloid beta (Aβ) accumulation, in AppNL-F knock-in (NL-F) mice, which have been established as an AD model. Chronic SAK3 (0.5 mg/kg/day) oral administration for 3 months from 9 months of age improved cognitive function and inhibited Aβ deposition in 12-month-old NL-F mice. Using microarray and real-time PCR analysis, we discovered serum- and glucocorticoid-induced protein kinase 1 (SGK1) as one of possible genes involved in the inhibition of Aβ deposition and improvement of cognitive function by SAK3. These results support the idea that T-VGCC enhancer, SAK3 could be a novel candidate for disease-modifying therapeutics for AD.

NEPRILYSIN IS THE MAJOR ENZYME TO DEGRADE EXTRACELLULAR Aβ IN NOVEL AD ANIMAL MODELS

Alzheimer's disease (AD) is the most common cause of dementia, which is pathologically characterized by the presence of senile plaques composed of amyloid β peptide (Aβ). In sporadic AD, it is hypothesized that an age-related decrease in extracellular Aβ catabolism causes Aβ accumulation. Neprilysin (NEP) and insulin degrading enzyme (IDE) are two major candidates considered to degrade extracellular Aβ in brain. They have, however, never been compared to each other in a side-by-side manner in vivo. In order to achieve objective comparison, we crossbred our single App knock-in mice, AppNL-F mice (NL-F mice) with NEP-knockout (KO) and IDE-KO mice. We observed an increase in the insoluble Aβ42 in NL-F x NEP-KO mice and, by contrast, a decrease in NL-F x IDE-KO mice by ELISA. NEP deficiency significantly increased pathological Aβ deposition in NL-F mice, whereas IDE deficiency decreased Aβ deposition. Expression of NEP was increased in NL-F x IDE-KO mice compared to NL-F mice, suggesting that the decrease of Aβ in NL-F x IDE-KO mice was likely a consequence of compensatory NEP upregulation. Expression of presenilin 1, BACE-1 and C-terminal fragment-β (CTF-β) remained unchanged in NL-F x IDE-KO mice, indicating that the decrease of Aβ in the double mutant mice was not due to alteration of intracellular APP processing. These observations establish NEP as the major Aβ-degrading enzyme in vivo. We conclude that NEP can become an ideal therapeutic target for reducing Aβ burdens in the preclinical stage of AD patients and that our new generation AD mice are relevant models for AD prevention study. Using a similar strategy, we are now trying to generate a novel non-human primate model of AD.

THE EFFECT OF AMYLOID β-PEPTIDE ON ER-MITOCHONDRIA CONTACT IN ALZHEIMER’S DISEASE

Cellular organelles are in close proximity at specialized contact sites where important cellular processes take place. For example, Ca2+-homeostasis, lipid synthesis, apoptosis and autophagy are regulated at the ER-mitochondria axis and interestingly all these processes are altered in Alzheimer's disease (AD). We have previously shown that modulation of ER-mitochondria contacts affect the assembly and maturation of the γ-secretase complex as well as generation of amyloid β-peptide (Aβ) (Leal et al 2016). Here we have investigated the effect of Aβ on the structure and dynamics of ER-mitochondria contacts in human tissue and AD mouse models. Human brain biopsies were collected from patients undergoing neurosurgery due to idiopathic normal pressure hydrocephalus (iNPH). The biopsies were stained for amyloid plaques (Aβ) and neurofibriallary tangles (tau). Mouse brain tissue was obtained from wt and tgAPPswe/lon mice of different ages. ER-mitochondria contact length and number were analyzed by structural electron microscopy. In addition, ER-mitochondria contact is being currently analyzed by proximity ligation assay (PLA) in primary neurons derived from a novel APP-knockin mouse model, APPNL-F. From brain biopsies we were able to characterise the structure of ER-mitochondria contacts in human brain. EM analysis of mouse brain material shows that ER-mitochondria apposition is affected by Aβ accumulation. Increased length of ER-mitochondria contacts were detected in tgAPPswe/lon mice at 6 months of age. Using PLA we can also visualize and quantify contact sites in APPNL-F mice. On-going analysis will reveal if Aβ accumulation affects ER-mitochondria apposition at an early stage before plaque pathology. The mechanisms underlying synapse loss and neurodegeneration in AD are not yet clarified. Here we show a close relationship between Aβ accumulation and regulation of ER-mitochondria contact and discuss its role in AD pathogenesis.

Store-Operated Calcium Channel Complex in Postsynaptic Spines: A New Therapeutic Target for Alzheimer's Disease Treatment

Mushroom dendritic spine structures are essential for memory storage and the loss of mushroom spines may explain memory defects in Alzheimer's disease (AD). This study demonstrated that Transient Receptor Potential Canonical 6 (TRPC6) and Orai2 form stromal interaction molecule 2 (STIM2)-regulated neuronal-store-operated Ca2+ influx (nSOC) channel complex in hippocampal synapse and the resulting Ca2+ influx is critical for long-term maintenance of mushroom spines in hippocampal neurons. A novel nSOC-positive modulator, NSN21778 (NSN), rescues mushroom spine loss and synaptic plasticity impairment in AD mice models. The TRPC6/Orai2 nSOC channel complex is a new therapeutic target and NSN is a potential candidate molecule for therapeutic intervention in brain aging and AD.

Cognitive deficits in single App knock-in mouse models

Transgenic mouse models of Alzheimer’s disease (AD) with nonphysiologic overexpression of amyloid precursor protein (APP) exhibit various unnatural symptoms/dysfunctions. To overcome this issue, mice with single humanized App knock-in (KI) carrying Swedish (NL), Beyreuther/Iberian (F), and Arctic (G) mutations in different combinations were recently developed. The validity of these mouse models of AD from a behavioral viewpoint, however, has not been extensively evaluated. Thus, using an automated behavior monitoring system, we analyzed various behavioral domains, including executive function, and learning and memory. The App-KI mice carrying NL-G-F mutations showed clear deficits in spatial memory and flexible learning, enhanced compulsive behavior, and reduced attention performance. Mice carrying NL-F mutations exhibited modest abnormalities. The NL-G-F mice had a greater and more rapid accumulation of Aβ deposits and glial responses. These findings reveal that single pathologic App-KI is sufficient to produce deficits in broad cognitive domains and that App-KI mouse lines with different levels of pathophysiology are useful models of AD.

Single App knock-in mouse models of Alzheimer's disease.

Experimental studies of Alzheimer's disease have largely depended on transgenic mice overexpressing amyloid precursor protein (APP). These mice, however, suffer from artificial phenotypes because, in addition to amyloid β peptide (Aβ), they overproduce other APP fragments. We generated knock-in mice that harbor Swedish and Beyreuther/Iberian mutations with and without the Arctic mutation in the APP gene. The mice showed typical Aβ pathology, neuroinflammation and memory impairment in an age-dependent manner.