Somatostatin-evoked Aß catabolism in the brain: Mechanistic involvement of α-endosulfine-KATP channel pathway.

Alzheimer's disease (AD) is characterized by the deposition of amyloid ß peptide (Aß) in the brain. The neuropeptide somatostatin (SST) regulates Aß 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 Aß levels altered in the brain. Pharmacological intervention targeting the particular KATP channel attenuated Aß 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.

APP mouse models for Alzheimer's disease preclinical studies.

Animal models of human diseases that accurately recapitulate clinical pathology are indispensable for understanding molecular mechanisms and advancing preclinical studies. The Alzheimer's disease (AD) research community has historically used first-generation transgenic (Tg) mouse models that overexpress proteins linked to familial AD (FAD), mutant amyloid precursor protein (APP), or APP and presenilin (PS). These mice exhibit AD pathology, but the overexpression paradigm may cause additional phenotypes unrelated to AD Second-generation mouse models contain humanized sequences and clinical mutations in the endogenous mouse App gene. These mice show Aß accumulation without phenotypes related to overexpression but are not yet a clinical recapitulation of human AD In this review, we evaluate different APP mouse models of AD, and review recent studies using the second-generation mice. We advise AD researchers to consider the comparative strengths and limitations of each model against the scientific and therapeutic goal of a prospective preclinical study.

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 ß-amyloid (Aß) 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 Aß-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 Aß-amyloidosis. In the presence of Aß-amyloidosis, tau accumulation was intensified and closely associated with dystrophic neurites, consistently showing that Aß-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.

Endoplasmic reticulum stress responses in mouse models of Alzheimer's disease: Overexpression paradigm versus knockin paradigm.

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-ß (Aß) peptide without overexpressing APP to investigate whether the ER stress response is heightened because of Aß 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 Aß 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.

Protective effects of dietary kaempferol glycoside components from unripe soybean (Edamame, Glycine max L. Merrill. 'Jindai') leaves and their serous metabolite on carbon tetrachloride-induced liver injury mice.

The study investigated the protective effects of kaempferol galactoside (KG) components in mice, which were separated from Jindai soybean leaves (JDL) and mainly composed by two kaempferol galactosides. Further, KG-related metabolites in serum of mice were identified by Tof-MS. Results showed that both JDL and KG prevented the CCl4-induced increases in serum aspartate aminotransferase and serum alanine aminotransferase. Additionally, mice treated with KG had significantly decreased TBARS and TNF-alpha levels, compared to CCl4-treated mice. Serous analysis showed that kaempferol, glucuronidated kaempferol and methylated kaempferol with a glucuronic acid moiety were identified in the serum of mice fed unripe soybean leaves or kaempferol galactosides isolated from the leaves. The results indicated that kaempferol 3-O-galactoside connected to other glycosides via galactose might be hydrolyzed in the gastro-intestinal tract and/or epithelium cells to release kaempferol, followed by glucuronidation and/or methylation in the liver to contribute to a reduction in liver injury. The use of raw leaves containing kaempferol galactosides as food materials may contribute to a reduction in oxidation-related diseases.

Calpain Activation in Alzheimer's Model Mice Is an Artifact of APP and Presenilin Overexpression.

UNLABELLED: Intraneuronal calcium stimulates the calpain-dependent conversion of p35 to p25, a CDK5 activator. It is widely believed that amyloid ß peptide (Aß) induces this conversion that, in turn, has an essential role in Alzheimer's disease pathogenesis. However, in vivo studies on p25 generation used transgenic mice overexpressing mutant amyloid precursor protein (APP) and presenilin (PS). Here, using single App knock-in mice, we show that p25 generation is an artifact caused by membrane protein overexpression. We show that massive Aß42 accumulation without overexpression of APP or presenilin does not produce p25, whereas p25 generation occurred with APP/PS overexpression and in postmortem mouse brain. We further support this finding using mice deficient for calpastatin, the sole calpain-specific inhibitor protein. Thus, the intracerebral environment of the APP/PS mouse brain and postmortem brain is an unphysiological state. SIGNIFICANCE STATEMENT: We recently estimated using single App knock-in mice that accumulate amyloid ß peptide without transgene overexpression that 60% of the phenotypes observed in Alzheimer's model mice overexpressing mutant amyloid precursor protein (APP) or APP and presenilin are artifacts (Saito et al., 2014). The current study further supports this estimate by invalidating key results from papers that were published in Cell These findings suggest that more than 3000 publications based on APP and APP/PS overexpression must be reevaluated.

Inhibition of the functional interplay between endoplasmic reticulum (ER) oxidoreduclin-1α (Ero1α) and protein-disulfide isomerase (PDI) by the endocrine disruptor bisphenol A.

Bisphenol A (BPA) is an endocrine disruptor that may have adverse effects on human health. We recently isolated protein-disulfide isomerase (PDI) as a BPA-binding protein from rat brain homogenates and found that BPA markedly inhibited PDI activity. To elucidate mechanisms of this inhibition, detailed structural, biophysical, and functional analyses of PDI were performed in the presence of BPA. BPA binding to PDI induced significant rearrangement of the N-terminal thioredoxin domain of PDI, resulting in more compact overall structure. This conformational change led to closure of the substrate-binding pocket in b' domain, preventing PDI from binding to unfolded proteins. The b' domain also plays an essential role in the interplay between PDI and ER oxidoreduclin 1α (Ero1α), a flavoenzyme responsible for reoxidation of PDI. We show that BPA inhibited Ero1α-catalyzed PDI oxidation presumably by inhibiting the interaction between the b' domain of PDI and Ero1α; the phenol groups of BPA probably compete with a highly conserved tryptophan residue, located in the protruding ß-hairpin of Ero1α, for binding to PDI. Consistently, BPA slowed down the reoxidation of PDI and caused the reduction of PDI in HeLa cells, indicating that BPA has a great impact on the redox homeostasis of PDI within cells. However, BPA had no effect on the interaction between PDI and peroxiredoxin-4 (Prx4), another PDI family oxidase, suggesting that the interaction between Prx4 and PDI is different from that of Ero1α and PDI. These results indicate that BPA, a widely distributed and potentially harmful chemical, inhibits Ero1-PDI-mediated disulfide bond formation.

Protein-disulfide isomerase regulates the thyroid hormone receptor-mediated gene expression via redox factor-1 through thiol reduction-oxidation.

Protein-disulfide isomerase (PDI) is a dithiol/disulfide oxidoreductase that regulates the redox state of proteins. We previously found that overexpression of PDI in rat pituitary tumor (GH3) cells suppresses 3,3',5-triiodothyronine (T(3))-stimulated growth hormone (GH) expression, suggesting the contribution of PDI to the T(3)-mediated gene expression via thyroid hormone receptor (TR). In the present study, we have clarified the mechanism of regulation by which TR function is regulated by PDI. Overexpression of wild-type but not redox-inactive mutant PDI suppressed the T(3)-induced GH expression, suggesting that the redox activity of PDI contributes to the suppression of GH. We considered that PDI regulates the redox state of the TR and focused on redox factor-1 (Ref-1) as a mediator of the redox regulation of TR by PDI. Interaction between Ref-1 and TRß1 was detected. Overexpression of wild-type but not C64S Ref-1 facilitated the GH expression, suggesting that redox activity of Cys-64 in Ref-1 is involved in the TR-mediated gene expression. Moreover, PDI interacted with Ref-1 and changed the redox state of Ref-1, suggesting that PDI controls the redox state of Ref-1. Our studies suggested that Ref-1 contributes to TR-mediated gene expression and that the redox state of Ref-1 is regulated by PDI. Redox regulation of PDI via Ref-1 is a new aspect of PDI function.

Endoplasmic reticulum protein (ERp) 29 binds as strongly as protein disulfide isomerase (PDI) to bisphenol A.

Bisphenol A (BPA), which is used in polycarbonate and epoxy resins, affects the development or function of the central nervous system. Previously, we isolated a BPA-binding protein from rat brain, identified it as protein disulfide isomerase (PDI), and found that BPA binds to the b' domain of PDI and inhibits its activity. There are 20 kinds of PDI family proteins in mammalian endoplasmic reticulum. The member proteins each have a different length and domain arrangement. Here we investigated the binding of BPA and T3 to ERp29, ERp57, and ERp72, which each have the b or b' domain. BPA/T3 binding of ERp57 and that of ERp72 were lower than that of PDI, and BPA did not inhibit the oxidase or reductase activity of these proteins. On the other hand, BPA and T3 bound to ERp29 as strongly as to PDI. The CD spectrum of PDI was changed in the presence of BPA in a dose-dependent manner, while that of ERp29 was not, suggesting that BPA did not affect the conformation of ERp29. We found that PDI suppresses GH expression in rat GH3 cells stimulated by thyroid hormone (T3) overexpression of PDI and that ERp57 reduced the GH level, but overexpression of ERp29 did not change GH expression. These results suggested that affinity to T3 does not involve the reduction of the T3 response. In this study, ERp29 was first identified as a BPA-binding protein but is not involved in the T3 response of GH3 cells.

Neuronal glutathione depletion elevates the Aß42/Aß40 ratio and tau aggregation in Alzheimer's disease mice.

Alzheimer's disease (AD) involves reduced glutathione levels, causing oxidative stress and contributing to neuronal cell death. Our prior research identified diminished glutamate-cysteine ligase catalytic subunit (GCLC) as linked to cell death. However, the effect of GCLC on AD features such as amyloid and tau pathology remained unclear. To address this, we investigated amyloid pathology and tau pathology in mice by combining neuron-specific conditional GCLC knockout mice with amyloid precursor protein (App) knockin (KI) or microtubule-associated protein tau (MAPT) KI mice. Intriguingly, GCLC knockout resulted in an increased Aß42/40 ratio. Additionally, GCLC deficiency in MAPT KI mice accelerated the oligomerization of tau through intermolecular disulfide bonds. These findings suggest that the decline in glutathione levels, due to aging or AD pathology, may contribute to the progression of AD.

Metabolic resistance of Aß3pE-42, a target epitope of the anti-Alzheimer therapeutic antibody, donanemab.

The amyloid ß peptide (Aß), starting with pyroglutamate (pE) at position 3 and ending at position 42 (Aß3pE-42), predominantly accumulates in the brains of Alzheimer's disease. Consistently, donanemab, a therapeutic antibody raised against Aß3pE-42, has been shown to be effective in recent clinical trials. Although the primary Aß produced physiologically is Aß1-40/42, an explanation for how and why this physiological Aß is converted to the pathological form remains elusive. Here, we present experimental evidence that accounts for the aging-associated Aß3pE-42 deposition: Aß3pE-42 was metabolically more stable than other Aßx-42 variants; deficiency of neprilysin, the major Aß-degrading enzyme, induced a relatively selective deposition of Aß3pE-42 in both APP transgenic and App knock-in mouse brains; Aß3pE-42 deposition always colocalized with Pittsburgh compound B-positive cored plaques in APP transgenic mouse brains; and under aberrant conditions, such as a significant reduction in neprilysin activity, aminopeptidases, dipeptidyl peptidases, and glutaminyl-peptide cyclotransferase-like were up-regulated in the progression of aging, and a proportion of Aß1-42 may be processed to Aß3pE-42. Our findings suggest that anti-Aß therapies are more effective if given before Aß3pE-42 deposition.

Neuronal glutathione loss leads to neurodegeneration involving gasdermin activation.

Accumulating evidence suggests that glutathione loss is closely associated with the progression of neurodegenerative disorders. Here, we found that the neuronal conditional-knockout (KO) of glutamyl-cysteine-ligase catalytic-subunit (GCLC), a rate-limiting enzyme for glutathione synthesis, induced brain atrophy accompanied by neuronal loss and neuroinflammation. GCLC-KO mice showed activation of C1q, which triggers engulfment of neurons by microglia, and disease-associated-microglia (DAM), suggesting that activation of microglia is linked to the neuronal loss. Furthermore, gasdermins, which regulate inflammatory form of cell death, were upregulated in the brains of GCLC-KO mice, suggesting the contribution of pyroptosis to neuronal cell death in these animals. In particular, GSDME-deficiency significantly attenuated the hippocampal atrophy and changed levels of DAM markers in GCLC-KO mice. Finally, we found that the expression of GCLC was decreased around amyloid plaques in AppNL-G-F AD model mice. AppNL-G-F mouse also exhibited inflammatory events similar to GCLC-KO mouse. We propose a mechanism by which a vicious cycle of oxidative stress and neuroinflammation enhances neurodegenerative processes. Furthermore, GCLC-KO mouse will serve as a useful tool to investigate the molecular mechanisms underlying neurodegeneration and in the development of new treatment strategies to address neurodegenerative diseases.

Effects of polybrominated diphenyl ethers (PBDEs) and their derivatives on protein disulfide isomerase activity and growth hormone release of GH3 cells.

Polybrominated diphenyl ethers (PBDEs) have been used in a variety of consumer products such as flame retardants and recently have been known to be widespread environmental pollutants, which probably affect biological functions of mammalian cells. However, the risk posed by PBDE metabolites has not been clarified. Our previous study suggested that bisphenol A (BPA), an endocrine-disrupting chemical, binds to protein disulfide isomerase (PDI) and inhibits its activity. PDI is an isomerase enzyme in the endoplasmic reticulum and facilitates the formation or cleavage of disulfide bonds. PDI consists of a, b, b', and a' domains and the c region, with the a and a' domains having isomerase active sites. In the present study, we tested the effects of 10 kinds of PBDE compounds and their metabolites on PDI. OH-PBDEs specifically inhibited the isomerase activity of PDI, with 4'-OH-PBDE more effective than 2' (or 2)-OH-PBDEs. 4'-OH-PBDE inhibited the isomerase activity of the b'a'c fragment but not that of ab and a'c, suggesting that the b' domain of PDI is essential for the inhibition by 4'-OH-PBDE. We also investigated the effects of these chemicals on the production of growth hormone (GH) in GH3 cells. In GH3 cells, levels of mRNA and protein of GH stimulated by T(3) were reduced by 4'-OH-PBDE and 4'-MeO-PBDE. The reduction in GH expression caused by these compounds was not changed by the overexpression or knockdown of PDI in GH3 cells, while these manipulations of PDI levels significantly suppressed the expression of GH. These results suggest that the biological effects of PBDEs differed depending on their brominated and hydroxylated positions.

Crystallization and preliminary crystallographic analysis of the complex between triiodothyronine and the bb' fragment of rat protein disulfide isomerase.

Protein disulfide isomerase (PDI) is a multifunctional protein that catalyzes the formation of a disulfide bond in nascent and misfolded proteins and is also known to bind to the thyroid hormone triiodothyronine (T3). When T3 is bound to PDI its catalytic activity is inhibited, but the biological function of this binding is not well understood. In previous studies, it was found that T3 binds to the bb' fragment of PDI. Therefore, to clarify the structure of the complex consisting of PDI bound to T3, a crystallographic analysis of the three-dimensional structure of the T3-rat PDI bb' complex was performed. Native bb' crystals and T3-bb' complex crystals were both obtained using the hanging-drop vapour-diffusion technique with 1.6 M trisodium citrate pH 6.2 as a precipitant. The space group of the native bb' crystals was found to be C222, with unit-cell parameters a = 94.8, b = 114.9, c = 182.9 Å, while the space group of the T3-bb' complex crystals was P2(1)2(1)2(1), with unit-cell parameters a = 99.9, b = 184.5, c = 232.2 Å. Diffraction data for the native and complex crystals were collected to resolutions of 3.06 and 3.00 Å, respectively.

Tau-binding protein PRMT8 facilitates vacuole degeneration in the brain.

Amyloid-ß and tau pathologies are important factors leading to neurodegeneration in Alzheimer's disease (AD); however, the molecular mechanisms that link these pathologies remain unclear. Assuming that important though as yet unidentified factors inhibit/accelerate tau pathology and neuronal cell death under amyloid pathology, we sought to isolate and identify tau-interacting proteins from mouse brains with or without amyloid pathology. Among the proteins that were identified, we focused on protein arginine methyltransferase 8 (PRMT8), which interacts with tau specifically in the absence of amyloid pathology. To investigate the role of PRMT8 in the pathogenesis of AD, we conducted Prmt8 gene deletion and overexpression experiments in AppNL-G-F/MAPT double knock-in mice and analysed the resulting pathological alterations. PRMT8-knockout did not alter the AD pathology in double knock-in mice, whereas PRMT8-overexpression promoted tau phosphorylation, neuroinflammation and vacuole degeneration. To evaluate if such a PRMT8-induced vacuole degeneration depends on tau pathology, PRMT8 was overexpressed in tau-KO mice, which were consequently found to exhibit vacuole degeneration. In addition, proteomic analyses showed that PRMT8 overexpression facilitated the arginine methylation of vimentin. Abnormal protein methylation could be involved in PRMT8-induced brain pathologies. Taken together, PRMT8 may play an important role in the formation of tau pathology and vacuole degeneration.

Peritoneal natural killer cell chemotaxis is decreased in women with pelvic endometriosis.

PROBLEM: NK cell and macrophage function are decreased in endometriosis, and the disease may involve reduced immune surveillance in the peritoneal cavity. NK cell cytotoxicity and migration ability (chemotaxis) are considered important; the former has been investigated, but the latter has not. METHOD OF STUDY: We compared chemotaxis of immunocompetent cells (NK cells, macrophages, T cells) in peritoneal fluid obtained during laparoscopy in 27 women with and 13 without endometriosis. Peripheral blood NK cells were also obtained by the peripheral blood antibody beads method. Micro-cultured cells were examined by time-lapse photography, and the mean migration speed per cell was calculated as the chemotaxis. We investigated the relationship between chemotaxis and endometriosis. RESULTS: NK cell chemotaxis was significantly lower in the endometriosis group. Macrophages and lymphocytes were not significantly different between the groups. During menstruation, NK cell chemotaxis decreased in both groups. Postmenstrual chemotaxis was increased significantly in women without endometriosis but remained low in women with endometriosis. The Revised-American Society for Reproductive Medicine score was not correlated with chemotaxis; in women with endometriosis, chemotaxis was decreased even at early stages. Peripheral blood NK cells showed no significant differences. CONCLUSIONS: In women with endometriosis, not only cytotoxicity but also chemotaxis by NK cells in peritoneal cavity is significantly decreased, and particularly chemotaxis is decreased throughout the menstrual cycle. Therefore, antigens in retrograde menstrual blood that enters the peritoneal cavity might be left unprocessed. Repetition of this immune process in the peritoneal cavity may lead to the onset and subsequent progression of endometriosis.

An isogenic panel of App knock-in mouse models: Profiling ß-secretase inhibition and endosomal abnormalities.

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 (AppNL-G-F and AppNL-F mice). We have now generated App knock-in mice devoid of the Swedish mutations (AppG-F mice) and evaluated its characteristics. Amyloid ß peptide (Aß) pathology was exhibited by AppG-F mice from 6 to 8 months of age and was accompanied by neuroinflammation. Aß-secretase inhibitor, verubecestat, attenuated Aß 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 ß-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 ß (CTF-ß) and humanization of Aß 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.

Early memory deficits and extensive brain network disorganization in the AppNL-F/MAPT double knock-in mouse model of familial Alzheimer's disease.

A critical challenge in current research on Alzheimer's disease (AD) is to clarify the relationship between network dysfunction and the emergence of subtle memory deficits in itspreclinical stage. The AppNL-F/MAPT double knock-in (dKI) model with humanized ß-amyloid peptide (Aß) and tau was used to investigate both memory and network dysfunctions at an early stage. Young male dKI mice (2 to 6 months) were tested in three tasks taxing different aspects of recognition memory affected in preclinical AD. An early deficit first appeared in the object-place association task at the age of 4 months, when increased levels of ß-CTF and Aß were detected in both the hippocampus and the medial temporal cortex, and tau pathology was found only in the medial temporal cortex. Object-place task-dependent c-Fos activation was then analyzed in 22 subregions across the medial prefrontal cortex, claustrum, retrosplenial cortex, and medial temporal lobe. Increased c-Fos activation was detected in the entorhinal cortex and the claustrum of dKI mice. During recall, network efficiency was reduced across cingulate regions with a major disruption of information flow through the retrosplenial cortex. Our findings suggest that early perirhinal-entorhinal pathology is associated with abnormal activity which may spread to downstream regions such as the claustrum, the medial prefrontal cortex and ultimately the key retrosplenial hub which relays information from frontal to temporal lobes. The similarity between our findings and those reported in preclinical stages of AD suggests that the AppNL-F/MAPT dKI model has a high potential for providing key insights into preclinical AD.

Recent Advances in the Modeling of Alzheimer's Disease.

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 ß peptide (Aß) 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 Aß 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 Aß metabolism and clearance because the Aß 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-Aß 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 Aß pathology by genome editing, to revealing the differential roles of neprilysin and insulin-degrading enzyme in Aß metabolism, and to identifying somatostatin receptor subtypes involved in Aß 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 Psen1 P117L/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 Aß. Furthermore, a derivative AppG-F line devoid of Swedish mutations which can be utilized for preclinical studies of ß-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-Aß immunotherapy. Use of the App knock-in mice also led to identification of the α-endosulfine-K ATP channel pathway as components of the somatostatin-evoked physiological mechanisms that reduce Aß 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.

Clinical profiles of hyperglycemic crises: A single-center retrospective study from Japan.

AIMS/INTRODUCTION: The aim of the present study was to clarify the pathophysiologies of hyperglycemic crises in Japanese patients. MATERIALS AND METHODS: This was a retrospective study of patients with hyperglycemic crises admitted to Kumamoto Medical Center, Kumamoto, Japan, between 2012 and 2019. Patients were classified as having diabetic ketoacidosis (DKA), hyperglycemic hyperosmotic syndrome (HHS) or a mixed state of the two conditions (MIX), and laboratory data and levels of consciousness at hospital admission, as well as the rates of mortality and coagulation disorders, were compared. RESULTS: The diagnostic criteria for hyperglycemic crisis were met in 144 cases, comprising 87 (60.4%), 38 (26.4%) and 19 (13.2%) cases of DKA, HHS and MIX, respectively. Type 1 diabetes was noted in 46.0 and 26.3% of patients in the DKA and MIX groups, respectively. Fibrin degradation product and D-dimer levels were significantly higher in the HHS group than in the DKA group (DKA and HHS groups: fibrin degradation product 7.94 ± 8.43 and 35.54 ± 51.80 µg/mL, respectively, P < 0.01; D-dimer 2.830 ± 2.745 and 14.846 ± 21.430 µg/mL, respectively, P < 0.01). Mortality rates were 5.7, 13.2 and 5.3% in the DKA, HHS and MIX groups, respectively. Seven patients (4.9%), four of whom were in the MIX group, had acute arterial occlusive diseases. CONCLUSIONS: The low frequency of type 1 diabetes in DKA and MIX might be responsible for reduced insulin secretion in Japanese populations. Patients with hyperglycemic crises have increased coagulability, and acute arterial occlusion needs to be considered, particularly in MIX.