Prostaglandin I2 upregulates the expression of anterior pharynx‐defective‐1α and anterior pharynx‐defective‐1β in amyloid precursor protein/presenilin 1 transgenic mice

Cyclooxygenase‐2 (COX‐2) has been recently identified to be involved in the pathogenesis of Alzheimer's disease (AD). Yet, the role of an important COX‐2 metabolic product, prostaglandin (PG) I₂, in the pathogenesis of AD remains unknown. Using human‐ and mouse‐derived neuronal cells as well as amyloid precursor protein/presenilin 1 (APP/PS1) transgenic mice as model systems, we elucidated the mechanism of anterior pharynx‐defective (APH)‐1α and pharynx‐defective‐1β induction. In particular, we found that PGI₂ production increased during the course of AD development. Then, PGI₂ accumulation in neuronal cells activates PKA/CREB and JNK/c‐Jun signaling pathways by phosphorylation, which results in APH‐1α/1β expression. As PGI₂ is an important metabolic by‐product of COX‐2, its suppression by NS398 treatment decreases the expression of APH‐1α/1β in neuronal cells and APP/PS1 mice. More importantly, β‐amyloid protein (Aβ) oligomers in the cerebrospinal fluid (CSF) of APP/PS1 mice are critical for stimulating the expression of APH‐1α/1β, which was blocked by NS398 incubation. Finally, the induction of APH‐1α/1β was confirmed in the brains of patients with AD. Thus, these findings not only provide novel insights into the mechanism of PGI₂‐induced AD progression but also are instrumental for improving clinical therapies to combat AD.     

Disrupted‐in‐schizophrenia‐1 protects synaptic plasticity in a transgenic mouse model of Alzheimer’s disease as a mitophagy receptor

Mitochondrial dysfunction is an early feature of Alzheimer's disease (AD). Accumulated damaged mitochondria, which are associated with impaired mitophagy, contribute to neurodegeneration in AD. We show levels of Disrupted‐in‐schizophrenia‐1 (DISC1), which is genetically associated with psychiatric disorders and AD, decrease in the brains of AD patients and transgenic model mice and in Aβ‐treated cultured cells. Disrupted‐in‐schizophrenia‐1 contains a canonical LC3‐interacting region (LIR) motif (²¹⁰FSFI²¹³), through which DISC1 directly binds to LC3‐I/II. Overexpression of DISC1 enhances mitophagy through its binding to LC3, whereas knocking‐down of DISC1 blocks Aβ‐induced mitophagy. We further observe overexpression of DISC1, but not its mutant (muFSFI) which abolishes the interaction of DISC1 with LC3, rescues Aβ‐induced mitochondrial dysfunction, loss of spines, suppressed long‐term potentiation (LTP). Overexpression of DISC1 via adeno‐associated virus (serotype 8, AAV8) in the hippocampus of 8‐month‐old APP/PS1 transgenic mice for 4 months rescues cognitive deficits, synaptic loss, and Aβ plaque accumulation, in a way dependent on the interaction of DISC1 with LC3. These results indicate that DISC1 is a novel mitophagy receptor, which protects synaptic plasticity from Aβ accumulation‐induced toxicity through promoting mitophagy.     

Haploinsufficiency of SGO1 results in deregulated centrosome dynamics,enhanced chromosomal instability and colon tumorigenesis

Chromosome instability (CIN) is found in 85% of colorectal cancers. Defects in mitotic processes are implicated in high CIN and may be critical events in colorectal tumorigenesis. Shugoshin-1 (SGO1) aids in the maintenance of chromosome cohesion and prevents premature chromosome separation and CIN. In addition, integrity of the centrosome may be compromised due to the deficiency of Cohesin and Sgo1 through the disengagement of centrioles. We report here the generation and characterization of SGO1-mutant mice and show that haploinsufficiency of SGO1 leads to enhanced colonic tumorigenesis. Complete disruption of SGO1 results in embryonic lethality, whereas SGO1^+/- mice are viable and fertile. Haploinsufficiency of SGO1 results in genomic instability manifested as missegregation of chromosomes and formation of extra centrosomal foci in both murine embryonic fibroblasts and adult bone marrow cells. Enhanced CIN observed in SGO1-deficient mice resulted in an increase in formation of aberrant crypt foci (ACF) and accelerated development of tumors after exposure to azoxymethane (AOM), a colon carcinogen. Together, these results suggest that haploinsufficiency of SGO1 causes enhanced CIN, colonic preneoplastic lesions and tumorigenesis in mice. SGO1 is essential for the suppression of CIN and tumor formation.     

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 Aβ 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.     

Proliferation of functional human natural killer cells with anti‐HIV‐1 activity in NOD/SCID/Jak3null mice

Natural killer cells, a critical component of the innate immune system, eradicate both virus‐infected cells and tumor cells through cytotoxicity and secretion of cytokines. Human NK cell research has largely been based on in vitro studies because of the lack of appropriate animal models. In this study, a selective proliferation model of functional human NK cells was established in NOD/SCID/Jak3^null (NOJ) mice transplanted with peripheral blood mononuclear cells (PBMC) and K562 cells. The antiviral effects of NK cells were evaluated by challenging this mouse model with HIV‐1. The percentage of intracellular p24⁺ T cells and the amount of plasma p24 was decreased compared with NOJ mice transplanted with PBMC. Our findings indicate that NK cells have an anti‐HIV‐1 effect through direct cytotoxicity against HIV‐1‐infected cells. These mice provide an important model for evaluating human NK function against human infectious diseases such as HIV‐1 and malignancies.     

Nurr1 (NR4A2) regulates Alzheimer’s disease‐related pathogenesis and cognitive function in the 5XFAD mouse model

The orphan nuclear receptor Nurr1 (also known as NR4A2) is critical for the development and maintenance of midbrain dopaminergic neurons, and is associated with Parkinson's disease. However, an association between Nurr1 and Alzheimer's disease (AD)‐related pathology has not previously been reported. Here, we provide evidence that Nurr1 is expressed in a neuron‐specific manner in AD‐related brain regions; specifically, it is selectively expressed in glutamatergic neurons in the subiculum and the cortex of both normal and AD brains. Based on Nurr1’s expression patterns, we investigated potential functional roles of Nurr1 in AD pathology. Nurr1 expression was examined in the hippocampus and cortex of AD mouse model and postmortem human AD subjects. In addition, we performed both gain‐of‐function and loss‐of‐function studies of Nurr1 and its pharmacological activation in 5XFAD mice. We found that knockdown of Nurr1 significantly aggravated AD pathology while its overexpression alleviated it, including effects on Aβ accumulation, neuroinflammation, and neurodegeneration. Importantly, 5XFAD mice treated with amodiaquine, a highly selective synthetic Nurr1 agonist, showed robust reduction in typical AD features including deposition of Aβ plaques, neuronal loss, microgliosis, and impairment of adult hippocampal neurogenesis, leading to significant improvement of cognitive impairment. These in vivo and in vitro findings suggest that Nurr1 critically regulates AD‐related pathophysiology and identify Nurr1 as a novel AD therapeutic target.     

Nitric oxide‐mediated regulation of ‐amyloid clearance via alterations of MMP‐9/TIMP‐1

Fibrillar amyloid plaques are largely composed of amyloid‐beta (Aβ) peptides that are metabolized into products, including Aβ1‐16, by proteases including matrix metalloproteinase 9 (MMP‐9). The balance between production and degradation of Aβ proteins is critical to amyloid accumulation and resulting disease. Regulation of MMP‐9 and its endogenous inhibitor tissue inhibitor of metalloproteinase (TIMP)‐1 by nitric oxide (NO) has been shown. We hypothesize that nitric oxide synthase (NOS2) protects against Alzheimer's disease pathology by increasing amyloid clearance through NO regulation of MMP‐9/TIMP‐1 balance. We show NO‐mediated increased MMP‐9/TIMP‐1 ratios enhanced the degradation of fibrillar Aβ in vitro, which was abolished when silenced for MMP‐9 protein translation. The in vivo relationship between MMP‐9, NO and Aβ degradation was examined by comparing an Alzheimer's disease mouse model that expresses NOS2 with a model lacking NOS2. To quantitate MMP‐9 mediated changes, we generated an antibody recognizing the Aβ1‐16 fragment, and used mass spectrometry multi‐reaction monitoring assay for detection of immunoprecipitated Aβ1‐16 peptides. Aβ1‐16 levels decreased in brain lysates lacking NOS2 when compared with strains that express human amyloid precursor protein on the NOS2 background. TIMP‐1 increased in the APPSwDI/NOS2^?/? mice with decreased MMP activity and increased amyloid burden, thereby supporting roles for NO in the regulation of MMP/TIMP balance and plaque clearance.     

Reduced amyloid‐β degradation in early Alzheimer's disease but not in the APPswePS1dE9 and 3xTg‐AD mouse models

Alzheimer's disease (AD) is hallmarked by amyloid‐β (Aβ) peptides accumulation and aggregation in extracellular plaques, preceded by intracellular accumulation. We examined whether intracellular Aβ can be cleared by cytosolic peptidases and whether this capacity is affected during progression of sporadic AD (sAD) in humans and in the commonly used APPswePS1dE9 and 3xTg‐AD mouse models. A quenched Aβ peptide that becomes fluorescent upon degradation was used to screen for Aβ‐degrading cytoplasmic peptidases cleaving the aggregation‐prone KLVFF region of the peptide. In addition, this quenched peptide was used to analyze Aβ‐degrading capacity in the hippocampus of sAD patients with different Braak stages as well as APPswePS1dE9 and 3xTg‐AD mice. Insulin‐degrading enzyme (IDE) was found to be the main peptidase that degrades cytoplasmic, monomeric Aβ. Oligomerization of Aβ prevents its clearance by IDE. Intriguingly, the Aβ‐degrading capacity decreases already during the earliest Braak stages of sAD, and this decline correlates with IDE protein levels, but not with mRNA levels. This suggests that decreased IDE levels could contribute to early sAD. In contrast to the human data, the commonly used APPswePS1dE9 and 3xTg‐AD mouse models do not show altered Aβ degradation and IDE levels with AD progression, raising doubts whether mouse models that overproduce Aβ peptides are representative for human sAD.     

Mutant presenilin‐1 deregulated peripheral immunity exacerbates Alzheimer‐like pathology

Mutations in the presenilin‐1 (PS1) gene are independent causes of familial Alzheimer's disease (AD). AD patients have dysregulated immunity, and PS1 mutant mice exhibit abnormal systemic immune responses. To test whether immune function abnormality caused by a mutant human PS1 gene (mhPS1) could modify AD‐like pathology, we reconstituted immune systems of AD model mice carrying a mutant human amyloid precursor protein gene (mhAPP; Tg2576 mice) or both mhAPP and mhPS1 genes (PSAPP mice) with allo‐geneic bone marrow cells. Here, we report a marked reduction in amyloid‐β (Aβ) levels, β‐amyloid plaques and brain inflammatory responses in PSAPP mice following strain‐matched wild‐type PS1 bone marrow reconstitution. These effects occurred with immune switching from pro‐inflammatory T helper (Th) 1 to anti‐inflammatory Th2 immune responses in the periphery and in the brain, which likely instructed microglia to phagocytose and clear Aβ in an ex vivo assay. Conversely, Tg2576 mice displayed accelerated AD‐like pathology when reconstituted with mhPS1 bone marrow. These data show that haematopoietic cells bearing the mhPS1 transgene exacerbate AD‐like pathology, suggesting a novel therapeutic strategy for AD based on targeting PS1 in peripheral immune cells.     

Hyperactive and anxiolytic‐like behaviors result from loss of COUP‐TFI/Nr2f1 in the mouse cortex

The nuclear receptor COUP TFI (also known as Nr2f1) plays major roles in specifying distinct neuronal subtypes during patterning of the neocortical motor and somatosensory cortex, as well as in regulating the longitudinal growth of the hippocampus during development. In humans, mutations in the NR2F1 gene lead to a global developmental delay and intellectual disabilities. While more than 30% of patients show behavioral features of autism spectrum disorder, 16% of haploinsufficient children show signs of hyperactivity and impulsivity. Loss of COUP‐TFI in the cortical mouse primordium results in altered area organization and serotonin distribution, abnormal coordination of voluntary movements and learning and memory deficits. Here, we asked whether absence of COUP‐TFI affects locomotor activity, anxiety, as well as depression. Mice mutant for COUP‐TFI have normal motor coordination, but significant traits of hyperactivity, which does not seem to respond to N‐Methyl‐D‐aspartate (NMDA) antagonists. However, no changes in anxiety, despite increased locomotor performances, were observed in the open field task. On the contrary, elevated plus maze and dark‐light test explorations indicate a decreased anxiety‐like behavior in COUP‐TFI mutant mice. Finally, significantly reduced immobility in the forced swim test and no changes in anhedonia in the sucrose preference task suggest no particular depressive behaviors in mutant mice. Taken together, our study shows that loss of COUP‐TFI leads to increased locomotor activity but less anxiety and contributes in further deciphering the pathophysiology of patients haploinsufficient for NR2F1.     

Toll‐like receptor 4 regulates spontaneous intestinal tumorigenesis by up‐regulating IL‐6 and GM‐CSF

Inflammation is as an important component of intestinal tumorigenesis. The activation of Toll‐like receptor 4 (TLR4) signalling promotes inflammation in colitis of mice, but the role of TLR4 in intestinal tumorigenesis is not yet clear. About 80%–90% of colorectal tumours contain inactivating mutations in the adenomatous polyposis coli (Apc) tumour suppressor, and intestinal adenoma carcinogenesis in familial adenomatous polyposis (FAP) is also closely related to the germline mutations in Apc. The Apc^Min/+ (multiple intestinal neoplasia) model mouse is a well‐utilized model of FAP, an inherited form of intestinal cancer. In this study, Apc^Min/+ intestinal adenoma mice were generated on TLR4‐sufficient and TLR4‐deficient backgrounds to investigate the carcinogenic effect of TLR4 in mouse gut by comparing mice survival, peripheral blood cells, bone marrow haematopoietic precursor cells and numbers of polyps in the guts of Apc^Min/+ WT and Apc^Min/+ TLR4^?/? mice. The results revealed that TLR4 had a critical role in promoting spontaneous intestinal tumorigenesis. Significant differential genes were screened out by the high‐throughput RNA‐Seq method. After combining these results with KEGG enrichment data, it was determined that TLR4 might promote intestinal tumorigenesis by activating cytokine‐cytokine receptor interaction and pathways in cancer signalling pathways. After a series of validation experiments for the concerned genes, it was found that IL6, GM‐CSF (CSF2), IL11, CCL3, S100A8 and S100A9 were significantly decreased in gut tumours of Apc^Min/+ TLR4^?/? mice compared with Apc^Min/+ WT mice. In the functional study of core down‐regulation factors, it was found that IL6, GM‐CSF, IL11, CCL3 and S100A8/9 increased the viability of colon cancer cell lines and decreased the apoptosis rate of colon cancer cells with irradiation and chemical treatment.     

Genetic inactivation of the allograft inflammatory factor‐1 locus

Allograft inflammatory factor‐1 (Aif‐1) is a 17 kDa EF hand motif‐bearing protein expressed primarily in developing spermatids and cells of monocyte/macrophage lineage. Increased Aif‐1 expression has been identified in clinically important conditions, including rheumatoid arthritis, systemic sclerosis, endometriosis, and transplant‐associated arteriosclerosis. Largely similar gene products arising from the same locus are known as ionized Ca²⁺ binding adapter‐1 (Iba1), microglial response factor‐1 (MRF1), and daintain; Iba1 in particular has emerged as a histologic marker of microglia and their activation in pathologic CNS conditions, including the response to facial nerve axotomy and stroke, uveitis, and experimental autoimmune neuritis and encephalomyelitis. Nevertheless, how aif‐1 gene products affect cellular function is only partly understood, and the physiologic significance of these products for male fertility, immune system development, and inflammation has not been described. To permit such investigations, we generated a mouse line with targeted deletion of the coding regions of the aif‐1 gene. Here we report that mice lacking Aif‐1 breed well and show normal post‐natal growth, but show resistance to disease in a model of collagen‐induced arthritis. We anticipate that these mice will be useful for studies of Aif‐1 function in a variety of immune and inflammatory disease models. genesis 51:734–740. © 2013 Wiley Periodicals, Inc.     

Genetic depletion of Polo‐like kinase 1 leads to embryonic lethality due to mitotic aberrancies

Polo‐like kinase 1 (PLK1) is a serine/threonine kinase that plays multiple and essential roles during the cell division cycle. Its inhibition in cultured cells leads to severe mitotic aberrancies and cell death. Whereas previous reports suggested that Plk1 depletion in mice leads to a non‐mitotic arrest in early embryos, we show here that the bi‐allelic Plk1 depletion in mice certainly results in embryonic lethality due to extensive mitotic aberrations at the morula stage, including multi‐ and mono‐polar spindles, impaired chromosome segregation and cytokinesis failure. In addition, the conditional depletion of Plk1 during mid‐gestation leads also to severe mitotic aberrancies. Our data also confirms that Plk1 is completely dispensable for mitotic entry in vivo. On the other hand, Plk1 haploinsufficient mice are viable, and Plk1‐heterozygous fibroblasts do not harbor any cell cycle alterations. Plk1 is overexpressed in many human tumors, suggesting a therapeutic benefit of inhibiting Plk1, and specific small‐molecule inhibitors for this kinase are now being evaluated in clinical trials. Therefore, the different Plk1 mouse models here presented are a valuable tool to reexamine the relevance of the mitotic kinase Plk1 during mammalian development and animal physiology.     

Silencing of ATP2B1‐AS1 contributes to protection against myocardial infarction in mouse via blocking NFKBIA‐mediated NF‐κB signalling pathway

Myocardial infarction (MI) is an acute coronary syndrome that refers to tissue infarction of the myocardium. This study aimed to investigate the effect of long intergenic non‐protein‐coding RNA (lincRNA) ATPase plasma membrane Ca²⁺ transporting 1 antisense RNA 1 (ATP2B1‐AS1) against MI by targeting nuclear factor‐kappa‐B inhibitor alpha (NFKBIA) and mediating the nuclear factor‐kappa‐B (NF‐κB) signalling pathway. An MI mouse model was established and idenepsied by cardiac function evaluation. It was determined that ATP2B1‐AS1 was highly expressed, while NFKBIA was poorly expressed and NF‐κB signalling pathway was activated in MI mice. Cardiomyocytes were extracted from mice and introduced with a series of mouse ATP2B1‐AS1 vector, NFKBIA vector, siRNA‐mouse ATP2B1‐AS1 and siRNA‐NFKBIA. The expression of NF‐κBp50, NF‐κBp65 and IKKβ was determined to idenepsy whether ATP2B1‐AS1 and NFKBIA affect the NF‐κB signalling pathway, the results of which suggested that ATP2B1‐AS1 down‐regulated the expression of NFKBIA and activated the NF‐κB signalling pathway in MI mice. Based on the data from assessment of cell viability, cell cycle, apoptosis and levels of inflammatory cytokines, either silencing of mouse ATP2B1‐AS1 or overexpression of NFKBIA was suggested to result in reduced cardiomyocyte apoptosis and expression of inflammatory cytokines, as well as enhanced cardiomyocyte viability. Our study provided evidence that mouse ATP2B1‐AS1 silencing may have the potency to protect against MI in mice through inhibiting cardiomyocyte apoptosis and inflammation, highlighting a great promise as a novel therapeutic target for MI.     

Early‐onset aging and mitochondrial defects associated with loss of histone acetyltransferase 1 (Hat1)

Histone acetyltransferase 1 (Hat1) is responsible for the acetylation of newly synthesized histone H4 on lysines 5 and 12 during the process of chromatin assembly. To understand the broader biological role of Hat1, we have generated a conditional mouse knockout model of this enzyme. We previously reported that Hat1 is required for viability and important for mammalian development and genome stability. In this study, we show that haploinsufficiency of Hat1 results in a significant decrease in lifespan. Defects observed in Hat1^+/? mice are consistent with an early‐onset aging phenotype. These include lordokyphosis (hunchback), muscle atrophy, minor growth retardation, reduced subcutaneous fat, cancer, and paralysis. In addition, the expression of Hat1 is linked to the normal aging process as Hat1 mRNA and protein becomes undetectable in many tissues in old mice. At the cellular level, fibroblasts from Hat1 haploinsufficient embryos undergo early senescence and accumulate high levels of p21. Hat1^+/? mouse embryonic fibroblasts (MEFs) display modest increases in endogenous DNA damage but have significantly higher levels of reactive oxygen species (ROS). Consistently, further studies show that Hat1^?/? MEFs exhibit mitochondrial defects suggesting a critical role for Hat1 in mitochondrial function. Taken together, these data show that loss of Hat1 induces multiple hallmarks of early‐onset aging.     

Rho‐associated coiled‐coil kinase 1 activation mediates amyloid precursor protein site‐specific Ser655 phosphorylation and triggers amyloid pathology

Rho‐associated coiled‐coil kinase 1 (ROCK1) is proposed to be implicated in Aβ suppression; however, the role for ROCK1 in amyloidogenic metabolism of amyloid precursor protein (APP) to produce Aβ was unknown. In the present study, we showed that ROCK1 kinase activity and its APP binding were enhanced in AD brain, resulting in increased β‐secretase cleavage of APP. Furthermore, we firstly confirmed that APP served as a substrate for ROCK1 and its major phosphorylation site was located at Ser655. The increased level of APP Ser655 phosphorylation was observed in the brain of APP/PS1 mice and AD patients compared to controls. Moreover, blockade of APP Ser655 phosphorylation, or inhibition of ROCK1 activity with either shRNA knockdown or Y‐27632, ameliorated amyloid pathology and improved learning and memory in APP/PS1 mice. These findings suggest that activated ROCK1 targets APP Ser655 phosphorylation, which promotes amyloid processing and pathology. Inhibition of ROCK1 could be a potential therapeutic approach for AD.     

Dyrk1 inhibition improves Alzheimer's disease‐like pathology

There is an urgent need for the development of new therapeutic strategies for Alzheimer's disease (AD). The dual‐specificity tyrosine phosphorylation‐regulated kinase‐1A (Dyrk1a) is a protein kinase that phosphorylates the amyloid precursor protein (APP) and tau and thus represents a link between two key proteins involved in AD pathogenesis. Furthermore, Dyrk1a is upregulated in postmortem human brains, and high levels of Dyrk1a are associated with mental retardation. Here, we sought to determine the effects of Dyrk1 inhibition on AD‐like pathology developed by 3xTg‐AD mice, a widely used animal model of AD. We dosed 10‐month‐old 3xTg‐AD and nontransgenic (NonTg) mice with a Dyrk1 inhibitor (Dyrk1‐inh) or vehicle for eight weeks. During the last three weeks of treatment, we tested the mice in a battery of behavioral tests. The brains were then analyzed for the pathological markers of AD. We found that chronic Dyrk1 inhibition reversed cognitive deficits in 3xTg‐AD mice. These effects were associated with a reduction in amyloid‐β (Aβ) and tau pathology. Mechanistically, Dyrk1 inhibition reduced APP and insoluble tau phosphorylation. The reduction in APP phosphorylation increased its turnover and decreased Aβ levels. These results suggest that targeting Dyrk1 could represent a new viable therapeutic approach for AD.