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Alzheimer’s disease (AD) is characterized by the deposition of amyloid β peptide (Aβ) in the brain. The neuropeptide somatostatin (SST) regulates Aβ catabolism by enhancing neprilysin (NEP)-catalyzed proteolytic degradation. However, the mechanism by which SST regulates NEP activity remains unclear. Here, we identified α-endosulfine (ENSA), an endogenous ligand of the ATP-sensitive potassium (KATP) channel, as a negative regulator of NEP downstream of SST signaling. The expression of ENSA is significantly increased in AD mouse models and in patients with AD. In addition, NEP directly contributes to the degradation of ENSA, suggesting a substrate-dependent feedback loop regulating NEP activity. We also discovered the specific KATP channel subtype that modulates NEP activity, resulting in the Aβ levels altered in the brain. Pharmacological intervention targeting the particular KATP channel attenuated Aβ deposition, with impaired memory function rescued via the NEP activation in our AD mouse model. Our findings provide a mechanism explaining the molecular link between KATP channel and NEP activation, and give new insights into alternative strategies to prevent AD.

We previously developed single App knock-in mouse models of Alzheimer’s disease (AD) that harbor the Swedish and Beyreuther/Iberian mutations with or without the Arctic mutation ( App NL-G-F and App NL-F mice). We have now generated App knock-in mice devoid of the Swedish mutations ( App G-F mice) and evaluated its characteristics. Amyloid β peptide (Aβ) pathology was exhibited by App G-F mice from 6 to 8 months of age and was accompanied by neuroinflammation. Aβ-secretase inhibitor, verubecestat, attenuated Aβ production in App G-F mice, but not in App NL-G-F mice, indicating that the App G-F mice are more suitable for preclinical studies of β-secretase inhibition given that most patients with AD do not carry the Swedish mutations. Comparison of isogenic App knock-in lines revealed that multiple factors, including elevated C-terminal fragment β (CTF-β) and humanization of Aβ might influence endosomal alterations in vivo. Thus, experimental comparisons between different isogenic App , knock-in mouse lines will provide previously unidentified insights into our understanding of the etiology of AD.

Since 1995, more than 100 transgenic (Tg) mouse models of Alzheimer’s disease (AD) have been generated in which mutant amyloid precursor protein (APP) or APP/presenilin 1 (PS1) cDNA is overexpressed (1st generation models). Although many of these models successfully recapitulate major pathological hallmarks of the disease such as amyloid β peptide (Aβ) deposition and neuroinflammation, they have suffered from artificial phenotypes in the form of overproduced or mislocalized APP/PS1 and their functional fragments, as well as calpastatin deficiency-induced early lethality, calpain activation, neuronal cell death without tau pathology, endoplasmic reticulum stresses, and inflammasome involvement. Such artifacts bring two important uncertainties into play, these being (1) why the artifacts arise, and (2) how they affect the interpretation of experimental results. In addition, destruction of endogenous gene loci in some Tg lines by transgenes has been reported. To overcome these concerns, single App knock-in mouse models harboring the Swedish and Beyreuther/Iberian mutations with or without the Arctic mutation (AppNL–G–F and AppNL–F mice) were developed (2nd generation models). While these models are interesting given that they exhibit Aβ pathology, neuroinflammation, and cognitive impairment in an age-dependent manner, the model with the Artic mutation, which exhibits an extensive pathology as early as 6 months of age, is not suitable for investigating Aβ metabolism and clearance because the Aβ in this model is resistant to proteolytic degradation and is therefore prone to aggregation. Moreover, it cannot be used for preclinical immunotherapy studies owing to the discrete affinity it shows for anti-Aβ antibodies. The weakness of the latter model (without the Arctic mutation) is that the pathology may require up to 18 months before it becomes sufficiently apparent for experimental investigation. Nevertheless, this model was successfully applied to modulating Aβ pathology by genome editing, to revealing the differential roles of neprilysin and insulin-degrading enzyme in Aβ metabolism, and to identifying somatostatin receptor subtypes involved in Aβ degradation by neprilysin. In addition to discussing these issues, we also provide here a technical guide for the application of App knock-in mice to AD research. Subsequently, a new double knock-in line carrying the AppNL–F and Psen1P117L/WT mutations was generated, the pathogenic effect of which was found to be synergistic. A characteristic of this 3rd generation model is that it exhibits more cored plaque pathology and neuroinflammation than the AppNL–G–F line, and thus is more suitable for preclinical studies of disease-modifying medications targeting Aβ. Furthermore, a derivative AppG–F line devoid of Swedish mutations which can be utilized for preclinical studies of β-secretase modifier(s) was recently created. In addition, we introduce a new model of cerebral amyloid angiopathy that may be useful for analyzing amyloid-related imaging abnormalities that can be caused by anti-Aβ immunotherapy. Use of the App knock-in mice also led to identification of the α-endosulfine-KATP channel pathway as components of the somatostatin-evoked physiological mechanisms that reduce Aβ deposition via the activation of neprilysin. Such advances have provided new insights for the prevention and treatment of preclinical AD. Because tau pathology plays an essential role in AD pathogenesis, knock-in mice with human tau wherein the entire murine Mapt gene has been humanized were generated. Using these mice, the carboxy-terminal PDZ ligand of neuronal nitric oxide synthase (CAPON) was discovered as a mediator linking tau pathology to neurodegeneration and showed that tau humanization promoted pathological tau propagation. Finally, we describe and discuss the current status of mutant human tau knock-in mice and a non-human primate model of AD that we have successfully created.

Neprilysin (NEP) and insulin-degrading enzyme (IDE) are considered the two major catabolic enzymes that degrade amyloid β peptide (Aβ), the primary cause of Alzheimer’s disease (AD). However, their roles in Aβ metabolism in vivo have never been compared in an impartial and side-by-side manner. Here, we crossbred single App knock-in mice with NEP (Mme) KO mice and with IDE (Ide) KO mice to generate double mutants that were analyzed for their biochemical and Aβ pathology properties. We found that NEP is responsible for the metabolism of amyloidogenic insoluble Aβ whereas IDE affects soluble Aβ. A deficiency of NEP, but not of IDE, augmented the formation of Aβ plaques, dystrophic neurites, and astrocytic and microglial activation, all of which are key pathological events in the development of AD. In addition, a deficiency of NEP had no significant impact on the levels of various neuropeptides (somatostatin, substance P, cholecystokinin, and neuropeptide Y), well known to be in vitro substrates for NEP, presumably because NEP is expressed in secretory vesicles and on the presynaptic membranes of excitatory neurons while most if not all neuropeptides are secreted from inhibitory neurons. This argues against the concern that NEP up-regulation for treatment of preclinical AD would reduce the levels of these neuropeptides. These findings indicate that NEP relatively selectively degrades Aβ in the brain. Whereas familial AD (FAD) is unambiguously caused by an increased anabolism of Aβ, and of Aβ 42 and Aβ 43 in particular, the anabolism of Aβ appears unaffected before its deposition in the brain that subsequently leads to the onset of sporadic AD (SAD). These observations thus suggest that NEP-sensitive amyloidogenic Aβ likely plays a primary pathogenic role in the etiology of SAD. Our findings are consistent with the aging-dependent decline of NEP expression in human brain and with recent genome-wide association studies (GWAS) indicating that variants of the gene encoding NEP (MME) are associated with the risk of SAD development. Taken together, our results imply that the aging-associated decrease in NEP expression is a primary cause of SAD and could thus be a target for the treatment of preclinical AD once other factors such as apolipoprotein E genotypes have also been considered.

SUMMARYWe previously developed single App knock-in mouse models of Alzheimer’s disease (AD) that harbor the Swedish and Beyreuther/Iberian mutations with or without the Arctic mutation (AppNL- G-F and AppNL-F mice). These models showed the development of amyloid β peptide (Aβ) pathology, neuroinflammation and cognitive impairment with aging. We have now generated App knock-in mice devoid of the Swedish mutations (AppG-F mice) and some additional mutants to address the following two questions: [1] Do the Swedish mutations influence the mode of β-secretase inhibitor action in vivo? [2] Does the quantity of C-terminal fragment of amyloid precursor protein (APP) generated by β-secretase (CTF-β) affect endosomal properties as previously reported as well as other pathological events? Aβ pathology was exhibited by AppG-F mice from 6 to 8 months of age, and was accompanied by microglial and astrocyte activation. We found that a β-secretase inhibitor, verubecestat, inhibited Aβ production in AppG-F mice, but not in AppNL-G-F mice, indicating that the AppG-F mice are more suitable for preclinical studies of β-secretase inhibition given that most AD patients do not carry Swedish mutations. We also found that the quantity of CTF-β generated by various App knock-in mutants failed to correlate with endosomal alterations or enlargement, implying that CTF-β, endosomal abnormalities, or both are unlikely to play a major role in AD pathogenesis. This is the first AD mouse model ever described that recapitulates amyloid pathology in the brain without the presence of Swedish mutations and without relying on the overexpression paradigm. Thus, experimental comparisons between different App knock-in mouse lines will potentially provide new insights into our understanding of the etiology of AD.

Most mouse models for preclinical research into Alzheimer's disease (AD) rely on the overexpression paradigm, in which familial AD (FAD)-related genes linked to amyloid precursor protein (APP) and presenilin-1 (PSEN1) are overexpressed. Such mice have been used for over two decades as the first-generation transgenic lines for AD, with animals exhibiting AD pathologies along with additional phenotypes, leading to the serious artifacts. To overcome the intrinsic drawbacks of the overexpression paradigm, we previously developed second-generation mouse models that incorporate humanized amyloid β (Aβ) sequences and several FAD-related mutations on the mouse endogenous App gene. Such models show AD pathologies in an age-dependent manner. In addition, our group recently generated additional lines of mice harboring multiple mutations without gene overexpression; these third-generation models exhibit an accelerated AD pathology compared to earlier generations. In this review, we describe the development and future prospects of AD mouse models in terms of their scientific properties and therapeutic perspectives in the context of the preclinical study of AD.

We previously developed single App knock-in mouse models of Alzheimer’s disease (AD), harboring the Swedish and Beyreuther/Iberian mutations with or without the Arctic mutation (AppNL-G-F and AppNL-F mice). These models showed amyloid β peptide (Aβ) pathology, neuroinflammation and cognitive impairment in an age-dependent manner. The former line exhibits extensive pathology as early as 6 months but is unsuitable for investigating Aβ metabolism and clearance because the Arctic mutation renders Aβ resistant to proteolytic degradation and prone to aggregation. In particular, it is inapplicable to preclinical immunotherapy studies due to its discrete affinity for anti-Aβ antibodies. The weakness of the latter model is that it may take as long as 18 months for the pathology to become prominent. We have thus generated a new model that exhibits early deposition of wild-type human Aβ by crossbreeding the AppNL-F line with the Psen1P117L/WT line. We show that the effects of the pathogenic mutations in the App and Psen1 genes are additive or synergistic. This new mouse model showed more cored plaque pathology and neuroinflammation than AppNL-G-F mice and will help accelerate the development of disease-modifying therapies to treat preclinical AD.

Alzheimer's disease (AD) is characterized by the deposition of amyloid β peptide (Aβ) in the brain. The neuropeptide somatostatin (SST) regulates Aβ catabolism by enhancing neprilysin (NEP)-catalyzed proteolytic degradation. However, the mechanism by which SST regulates NEP activity remains unclear. Here, we identified α-endosulfine (ENSA), an endogenous ligand of the ATP-sensitive potassium (K

The neuropeptide somatostatin (SST) regulates amyloid β peptide (Aβ) catabolism by enhancing neprilysin (NEP)-catalyzed proteolytic degradation. However, the mechanism by which SST regulates NEP activity remains unclear. Here we report the identification by differential proteomics of α-endosulfine (ENSA), an endogenous ligand of the ATP-sensitive potassium (KATP) channel, as a negative regulator of NEP activity downstream of SST signaling. Genetic deficiency of ENSA resulted in enhanced NEP activity and decreased Aβ deposition in the brains of wild-type and Alzheimer’s disease (AD) model mice. Pharmacological intervention to increase the probability of KATP channel opening reduced Aβ deposition in AD model mice. Our findings provide new insights into possible mechanisms to prevent AD.

Alzheimer’s disease (AD) brains are characterized by increased levels of the pathogenic amyloid beta (Aβ) peptide, which accumulates into extracellular plaques. Finding a way to lower Aβ levels is fundamental for the prevention and treatment of AD. Neprilysin is the major Aβ degrading enzyme which is regulated by the neuropeptide somatostatin. Here we used a combination of in vitro and in vivo approaches to identify the subtype specificity of the five somatostatin receptors (SSTs) expressed in the brain, involved in the regulation of neprilysin. Using a battery of Sst double knockout (dKO) mice we show that neprilysin is regulated by SST1 and SST4 in a redundant manner. Sst1 and Sst4 dKO mice exhibit a specific decrease of presynaptic neprilysin in the Lacunosum molecular layer. Moreover, a genetic deficiency of Sst1 and Sst4 in amyloid beta precursor protein (App) knock-in mice, an AD mouse model, aggravates the Aβ pathology in the hippocampus. As a first proof of concept towards an Aβ-lowering strategy involving neprilysin, we demonstrate that treatment with an agonist selective for SST1 and SST4 ameliorates the Aβ pathology and improves cognition in the App knock-in AD mouse model.

Endoplasmic reticulum (ER) stress is believed to play an important role in the etiology of Alzheimer's disease (AD). The accumulation of misfolded proteins and perturbation of intracellular calcium homeostasis are thought to underlie the induction of ER stress, resulting in neuronal dysfunction and cell death. Several reports have shown an increased ER stress response in amyloid precursor protein (APP) and presenilin1 (PS1) double-transgenic (Tg) AD mouse models. However, whether the ER stress observed in these mouse models is actually caused by AD pathology remains unclear. APP and PS1 contain one and nine transmembrane domains, respectively, for which it has been postulated that overexpressed membrane proteins can become wedged in a misfolded configuration in ER membranes, thereby inducing nonspecific ER stress. Here, we used an App-knockin (KI) AD mouse model that accumulates amyloid-β (Aβ) peptide without overexpressing APP to investigate whether the ER stress response is heightened because of Aβ pathology. Thorough examinations indicated that no ER stress responses arose in App-KI or single APP-Tg mice. These results suggest that PS1 overexpression or mutation induced a nonspecific ER stress response that was independent of Aβ pathology in the double-Tg mice. Moreover, we observed no ER stress in a mouse model of tauopathy (P301S-Tau-Tg mice) at various ages, suggesting that ER stress is also not essential in tau pathology–induced neurodegeneration. We conclude that the role of ER stress in AD pathogenesis needs to be carefully addressed in future studies.

We previously developed single App knock-in mouse models of Alzheimer's disease (AD) harboring the Swedish and Beyreuther/Iberian mutations with or without the Arctic mutation (AppNL-G-F and AppNL-F mice, respectively). These models showed Aβ pathology, neuroinflammation, and cognitive impairment in an age-dependent manner. The former model exhibits extensive pathology as early as 6 months, but is unsuitable for investigating Aβ metabolism and clearance because the Arctic mutation renders Aβ resistant to proteolytic degradation and prone to aggregation. In particular, it is inapplicable to preclinical immunotherapy studies due to its discrete affinity for anti-Aβ antibodies. The latter model may take as long as 18 months for the pathology to become prominent, which leaves an unfulfilled need for an Alzheimer's disease animal model that is both swift to show pathology and useful for antibody therapy. We thus utilized mutant Psen1 knock-in mice into which a pathogenic mutation (P117L) had been introduced to generate a new model that exhibits early deposition of wild-type human Aβ by crossbreeding the AppNL-F line with the Psen1P117L/WT line. We show that the effects of the pathogenic mutations in the App and Psen1 genes are additive or synergistic. This new third-generation mouse model showed more cored plaque pathology and neuroinflammation than AppNL-G-F mice and will help accelerate the development of disease-modifying therapies to treat preclinical AD.

Alzheimer's disease (AD) is the most common neurodegenerative disorder characterized by cognitive impairment with neuronal loss. The number of patients suffering from AD has increased, but none of the present therapies stops the progressive symptoms in patients with AD. It has been reported that the activation of microglial cells induces harmful chronic inflammation, leading to neuronal death. Furthermore, the impairment of adult neurogenesis in the hippocampus has been observed earlier than amyloid plaque formation. Inflammatory response may lead to impaired adult neurogenesis in patients with AD. This study examines the relationship between adult neurogenesis and neuroinflammation using APPswe/PS1M146V/tauP301L (3 × Tg) mice. We observed a decline in the proliferation of neural stem cells and the occurrence of severe inflammation in the hippocampus of 3 × Tg mouse brains at 12 months of age. Previously, our research had shown an anti-inflammatory effect of all-trans retinoic acid (ATRA) in the 3 × Tg mouse brain. We found that ATRA has effects on the recovery of proliferative cells along with suppression of activated microglia in the hippocampus. These results suggest that the inhibition of microglial activation by ATRA leads to recovery of adult neurogenesis in the hippocampus in an AD mouse model. © 2016 Wiley Periodicals, Inc.

Alzheimer's disease (AD) is one of the best known neurodegenerative diseases; it causes dementia and its pathological features include accumulation of amyloid β (Aβ) and neurofibrillary tangles (NFTs) in the brain. Elevated Cdk5 activity and CRMP2 phosphorylation have been reported in the brains of AD model mice at the early stage of the disease, but the significance thereof in human AD remains unelucidated. We have recently reported that Aβ accumulation in the cerebellum of AD model APPswe/PS1dE9 (APP/PS1) mice, and cerebellar dysfunctions, such as impairment of motor coordination ability and long-term depression (LTD) induction, at the pre-Aβ accumulation stage. In the present study, we found increased phosphorylation levels of CRMP2 as well as increased p35 protein levels in the cerebellum of APP/PS1 mice. Interestingly, we show that pioglitazone, an agonist of peroxisome proliferator-activated receptor γ, normalized the p35 protein and CRMP2 phosphorylation levels in the cerebellum. Impaired motor coordination ability and LTD in APP/PS1 mice were ameliorated by pioglitazone treatment at the pre-Aβ accumulation stage. These results suggest a correlation between CRMP2 phosphorylation and AD pathophysiology, and indicate the effectiveness of pioglitazone treatment at the pre-Aβ accumulation stage in AD model mice.

Endoplasmic reticulum (ER) stress is believed to play an important role in the etiology of Alzheimer's disease (AD). The accumulation of misfolded proteins and perturbation of intracellular calcium homeostasis are thought to underlie the induction of ER stress, resulting in neuronal dysfunction and cell death. Several reports have shown an increased ER stress response in amyloid precursor protein (APP) and presenilin1 (PS1) double-transgenic (Tg) AD mouse models. However, whether the ER stress observed in these mouse models is actually caused by AD pathology remains unclear. APP and PS1 contain one and nine transmembrane domains, respectively, for which it has been postulated that overexpressed membrane proteins can become wedged in a misfolded configuration in ER membranes, thereby inducing nonspecific ER stress. Here, we used an App-knockin (KI) AD mouse model that accumulates amyloid-β (Aβ) peptide without overexpressing APP to investigate whether the ER stress response is heightened because of Aβ pathology. Thorough examinations indicated that no ER stress responses arose in App-KI or single APP-Tg mice. These results suggest that PS1 overexpression or mutation induced a nonspecific ER stress response that was independent of Aβ pathology in the double-Tg mice. Moreover, we observed no ER stress in a mouse model of tauopathy (P301S-Tau-Tg mice) at various ages, suggesting that ER stress is also not essential in tau pathology–induced neurodegeneration. We conclude that the role of ER stress in AD pathogenesis needs to be carefully addressed in future studies.

Alzheimer's disease (AD) is the most common neurodegenerative disorder characterized by cognitive impairment with neuronal loss. The number of patients suffering from AD has increased, but none of the present therapies stops the progressive symptoms in patients with AD. It has been reported that the activation of microglial cells induces harmful chronic inflammation, leading to neuronal death. Furthermore, the impairment of adult neurogenesis in the hippocampus has been observed earlier than amyloid plaque formation. Inflammatory response may lead to impaired adult neurogenesis in patients with AD. This study examines the relationship between adult neurogenesis and neuroinflammation using APPswe/PS1M146V/tauP301L (3 × Tg) mice. We observed a decline in the proliferation of neural stem cells and the occurrence of severe inflammation in the hippocampus of 3 × Tg mouse brains at 12 months of age. Previously, our research had shown an anti-inflammatory effect of all-trans retinoic acid (ATRA) in the 3 × Tg mouse brain. We found that ATRA has effects on the recovery of proliferative cells along with suppression of activated microglia in the hippocampus. These results suggest that the inhibition of microglial activation by ATRA leads to recovery of adult neurogenesis in the hippocampus in an AD mouse model. © 2016 Wiley Periodicals, Inc.

Alzheimer's disease (AD) is the most common type of dementia among the elderly. Neurofibrillary tangles (NFTs), a major pathological hallmark of AD, are composed of tau protein that is hyperphosphorylated by cyclin-dependent kinase 5 (Cdk5) and glycogen synthase kinase 3β (GSK3β). NFTs also contain Wiskott-Aldrich syndrome protein family verprolin-homologous protein 1 (WAVE1) and collapsin response-mediator protein 2 (CRMP2). Although Cdk5 is known to phosphorylate tau, WAVE1, and CRMP2, the significance of this with respect to NFT formation remains to be elucidated. This study examines the involvement of phosphorylated (p-) CRMP2 and WAVE1 in p-tau aggregates using a triple-transgenic (3×Tg; APPswe/PS1M146V/tauP301L) AD mouse model. First, we verified the colocalization of p-WAVE1 and p-CRMP2 with aggregated hyperphosphorylated tau in the hippocampus at 23 months of age. Biochemical analysis revealed the inclusion of p-WAVE1, p-CRMP2, and tau in the sarkosyl-insoluble fractions of hippocampal homogenates. To test the significance of phosphorylation of these proteins further, we administered all-trans-retinoic acid (ATRA) to the 3×Tg mice, which downregulates Cdk5 and GSK3β activity. In ATRA-treated mice, fewer and smaller tau aggregates were observed compared with non-ATRA-treated mice. These results suggest the possibility of novel therapeutic target molecules for preventing tau pathology.

Alzheimer's disease (AD) is a neurodegenerative disorder that represents the most common type of dementia among elderly people. Amyloid beta (Aβ) peptides in extracellular Aβ plaques, produced from the amyloid precursor protein (APP) via sequential processing by β- and γ-secretases, impair hippocampal synaptic plasticity, and cause cognitive dysfunction in AD patients. Here, we report that Aβ peptides also impair another form of synaptic plasticity; cerebellar long-term depression (LTD). In the cerebellum of commonly used AD mouse model, APPswe/PS1dE9 mice, Aβ plaques were detected from 8 months and profound accumulation of Aβ plaques was observed at 18 onths of age. Biochemical analysis revealed relatively high levels of APP protein and Aβ in the cerebellum of APPswe/PS1dE9 mice. At pre-Aβ accumulation stage, LTD induction, and motor coordination are disturbed. These results indicate that soluble Aβ oligomers disturb LTD induction and cerebellar function in AD mouse model.