NOX4 and its association with myeloperoxidase and osteopontin in regulating endochondral ossification.

IMPORTANCE: Endochondral ossification plays an important role in skeletal development. Recent studies have suggested a link between increased intracellular reactive oxygen species (ROS) and skeletal disorders. Moreover, previous studies have revealed that increasing the levels of myeloperoxidase (MPO) and osteopontin (OPN) while inhibiting NADPH oxidase 4 (NOX4) can enhance bone growth. This investigation provides further evidence by showing a direct link between NOX4 and MPO, OPN in bone function. OBJECTIVE: This study investigates NOX4, an enzyme producing hydrogen peroxide, in endochondral ossification and bone remodeling. NOX4's role in osteoblast formation and osteogenic signaling pathways is explored. METHODS: Using NOX4-deficient (NOX4-/-) and ovariectomized (OVX) mice, we identify NOX4's potential mediators in bone maturation. RESULTS: NOX4-/- mice displayed significant differences in bone mass and structure. Compared to the normal Control and OVX groups. Hematoxylin and eosin staining showed NOX4-/- mice had the highest trabecular bone volume, while OVX had the lowest. Proteomic analysis revealed significantly elevated MPO and OPN levels in bone marrow-derived cells in NOX4-/- mice. Immunohistochemistry confirmed increased MPO, OPN, and collagen II (COLII) near the epiphyseal plate. Collagen and chondrogenesis analysis supported enhanced bone development in NOX4-/- mice. CONCLUSIONS AND RELEVANCE: Our results emphasize NOX4's significance in bone morphology, mesenchymal stem cell proteomics, immunohistochemistry, collagen levels, and chondrogenesis. NOX4 deficiency enhances bone development and endochondral ossification, potentially through increased MPO, OPN, and COLII expression. These findings suggest therapeutic implications for skeletal disorders.

Atypical Gradenigo's syndrome in a pediatric case: A critical review of neuroimaging.

Gradenigo's syndrome, a rare but serious complication of otitis media, encompasses a triad of symptoms including otalgia, facial palsy, and abducens nerve palsy, pointing to the involvement of the petrous apex. This case report presents an 11-year-old boy with an atypical manifestation of Gradenigo's syndrome, characterized by the absence of classic features such as abducens nerve palsy and purulent otorrhea. MRI findings were significant for petrous apicitis extending to Meckel's cave and the cavernous sinus, along with abscess formation and clivus osteomyelitis. The report highlights the critical role of advanced neuroimaging, particularly MRI, in the diagnosis and management of this condition. It underscores the importance of recognizing atypical presentations of Gradenigo's syndrome and the effectiveness of imaging-guided conservative treatment strategies in pediatric otological cases.

SARM1 is responsible for calpain-dependent dendrite degeneration in mouse hippocampal neurons.

Sterile alpha and toll/interleukin receptor motif-containing 1 (SARM1) is a critical regulator of axon degeneration that acts through hydrolysis of NAD+ following injury. Recent work has defined the mechanisms underlying SARM1's catalytic activity and advanced our understanding of SARM1 function in axons, yet the role of SARM1 signaling in other compartments of neurons is still not well understood. Here, we show in cultured hippocampal neurons that endogenous SARM1 is present in axons, dendrites, and cell bodies and that direct activation of SARM1 by the neurotoxin Vacor causes not just axon degeneration, but degeneration of all neuronal compartments. In contrast to the axon degeneration pathway defined in dorsal root ganglia, SARM1-dependent hippocampal axon degeneration in vitro is not sensitive to inhibition of calpain proteases. Dendrite degeneration downstream of SARM1 in hippocampal neurons is dependent on calpain 2, a calpain protease isotype enriched in dendrites in this cell type. In summary, these data indicate SARM1 plays a critical role in neurodegeneration outside of axons and elucidates divergent pathways leading to degeneration in hippocampal axons and dendrites.

BCG Signal Quality Assessment Based on Time-Series Imaging Methods.

This paper describes a signal quality classification method for arm ballistocardiogram (BCG), which has the potential for non-invasive and continuous blood pressure measurement. An advantage of the BCG signal for wearable devices is that it can easily be measured using accelerometers. However, the BCG signal is also susceptible to noise caused by motion artifacts. This distortion leads to errors in blood pressure estimation, thereby lowering the performance of blood pressure measurement based on BCG. In this study, to prevent such performance degradation, a binary classification model was created to distinguish between high-quality versus low-quality BCG signals. To estimate the most accurate model, four time-series imaging methods (recurrence plot, the Gramain angular summation field, the Gramain angular difference field, and the Markov transition field) were studied to convert the temporal BCG signal associated with each heartbeat into a 448 × 448 pixel image, and the image was classified using CNN models such as ResNet, SqueezeNet, DenseNet, and LeNet. A total of 9626 BCG beats were used for training, validation, and testing. The experimental results showed that the ResNet and SqueezeNet models with the Gramain angular difference field method achieved a binary classification accuracy of up to 87.5%.

Effects of Saffron Extract (Affron®) with 100 mg/kg and 200 mg/kg on Hypothalamic-Pituitary-Adrenal Axis and Stress Resilience in Chronic Mild Stress-Induced Depression in Wistar Rats.

Stress-related symptoms are a global concern, impacting millions of individuals, yet effective and safe treatments remain scarce. Although multiple studies have highlighted the stress- alleviating properties of saffron extract, the underlying mechanisms remain unclear. This study employs the unpredictable chronic mild stress (CMS) animal model to investigate the impact of a standardized saffron extract, Affron® (AFN), on hypothalamic-pituitary-adrenal (HPA) axis regulation and neuroplasticity in Wistar rats following repeated oral administration. The research evaluates AFN's effects on various stress-related parameters, including hypothalamic gene expression, stress hormone levels, and the sucrose preference test. In animals subjected to continuous unpredictable CMS, repetitive administration of AFN at doses of 100 mg/kg and 200 mg/kg effectively normalized HPA axis dysregulation and enhanced neuroplasticity. Increased concentrations of AFN demonstrated greater efficacy. Following AFN oral administration, adrenocorticotropic and corticosterone hormone levels exhibited significant or nearly significant reductions in comparison to subjects exposed to stress only. These changes align with the alleviation of stress and the normalization of the HPA axis. These findings elucidate AFN's role in stress mitigation, affirm its health benefits, validate its potential as a treatment for stress-related symptoms, confirm its physiological effectiveness, and emphasize its therapeutic promise.

Mechanical Properties of Cement Mortar Containing Ground Waste Newspaper as Cementitious Material.

In recent years, several studies have reported the recycling of by-products generated by the paper industry and their application to the construction industry. A majority of the existing studies used waste paper sludge ash, and considerable energy is consumed in such incineration processes. This may further contribute to air pollution. In this study, we used waste newspaper (WNP), which underwent a simple crushing process without a separate high-temperature treatment process, and we integrated it in cement mortar. We prepared mortars containing 0%, 0.2%, 0.4%, 0.6%, 0.8%, and 1.0% ground WNP as a cement substitute. Subsequently, the fluidity, compressive strength, tensile strength, carbonation depth, drying shrinkage, and microstructure of the mortars were compared and analyzed. The 28-day compressive strength of the mortar samples with WNP was approximately 3.2-16.1% higher than that of the control sample. The 28-day accelerated carbonation depth of the samples with WNP was approximately 1.03-1.61 mm. Furthermore, their carbonation resistance was approximately 5.2-39.4% higher than that of the control sample. Compressive strength, tensile strength, and carbonation resistance were improved by appropriately using ground WNP as a cement substitute in cement mortar. In this study, the appropriate amount of WNP according to the mechanical properties of cement mortar was found to be 0.4-0.8%, and considering the durability characteristics, the value 0.6 was the most ideal.

Survey on the Status of Breastfeeding in Korean Medical Institution Workers.

BACKGROUND: Human breast milk is essential and provides irreplaceable nutrients for early humans. However, breastfeeding is not easy for various reasons in medical institution environments. Therefore, in order to improve the breastfeeding environment, we investigated the difficult reality of breastfeeding through questionnaire responses from medical institution workers. METHODS: A survey was conducted among 179 medical institution workers with experience in childbirth within the last five years. The survey results of 175 people were analyzed, with incoherent answers excluded. RESULTS: Of the 175 people surveyed, a total of 108 people (61.7%) worked during the day, and 33 people (18.9%) worked in three shifts. Among 133 mothers who stayed with their babies in the same nursing room, 111 (93.3%) kept breastfeeding for more than a month, but among those who stayed apart, only 10 (71.4%) continued breastfeeding for more than a month (P = 0.024). Ninety-five (88.0%) of daytime workers, 32 (94.1%) two-shift workers, and 33 (100%) three-shift workers continued breastfeeding for more than a month (P = 0.026). Workers in general hospitals tended to breastfeed for significantly longer than those that worked in tertiary hospitals (P = 0.003). A difference was also noted between occupation categories (P = 0.019), but a more significant difference was found in the comparison between nurses and doctors (P = 0.012). Longer breastfeeding periods were noted when mothers worked three shifts (P = 0.037). Depending on the period planned for breastfeeding prior to childbirth, the actual breastfeeding maintenance period after birth showed a significant difference (P = 0.002). Of 112 mothers who responded to the question regarding difficulties in breastfeeding after returning to work, 87 (77.7%) mentioned a lack of time caused by being busy at work, 82 (73.2%) mentioned the need for places and appropriate circumstances. CONCLUSION: In medical institutions, it is recommended that environmental improvements in medical institutions, the implementation of supporting policies, and the provision of specialized education on breastfeeding are necessary to promote breastfeeding.

Antenatal Betamethasone Induces Increased Surfactant Proteins and Decreased Foxm1 Expressions in Fetal Rabbit Pups.

Introduction: Antenatal steroid improves respiratory distress syndrome in preterm infants. The molecular mechanism of the process is not well established. The aim of this study is to investigate the possible association between antenatal steroid and fetal Forkhead box M1(Foxm1) expression. Materials and methods: An animal study using mated pregnant New Zealand white rabbits and their fetuses was designed. Fourteen mother rabbits were assigned to four groups to undergo a cesarean section. In groups 1, 2, and 3, preterm pups were harvested on day 27 of gestation. In group 4, term pups were harvested on day 31. Antenatal maternal intramuscular injection was performed in groups 2 (normal saline) and 3 (betamethasone). Using qRT-PCR and Western blot, mRNA transcription and protein expression of surfactant protein (SP) A, B, C, and Foxm1 were compared between the pups of those four groups. Results: Sixty two fetal rabbits were harvested. One-way ANOVA test showed higher mRNA transcription of SPs in groups 3 and 4 than groups 1 and 2. Significantly lower Foxm1 mRNA transcription and protein expression were observed in group 3 or 4 compared with group 1 or 2. Conclusion: Decreased Foxm1 expression was associated in an antenatal betamethasone animal model.

Neuronal Apolipoprotein E4 Expression Results in Proteome-Wide Alterations and Compromises Bioenergetic Capacity by Disrupting Mitochondrial Function.

Apolipoprotein (apo) E4, the major genetic risk factor for Alzheimer's disease (AD), alters mitochondrial function and metabolism early in AD pathogenesis. When injured or stressed, neurons increase apoE synthesis. Because of its structural difference from apoE3, apoE4 undergoes neuron-specific proteolysis, generating fragments that enter the cytosol, interact with mitochondria, and cause neurotoxicity. However, apoE4's effect on mitochondrial respiration and metabolism is not understood in detail. Here we used biochemical assays and proteomic profiling to more completely characterize the effects of apoE4 on mitochondrial function and cellular metabolism in Neuro-2a neuronal cells stably expressing apoE4 or apoE3. Under basal conditions, apoE4 impaired respiration and increased glycolysis, but when challenged or stressed, apoE4-expressing neurons had 50% less reserve capacity to generate ATP to meet energy requirements than apoE3-expressing neurons. ApoE4 expression also decreased the NAD+/NADH ratio and increased the levels of reactive oxygen species and mitochondrial calcium. Global proteomic profiling revealed widespread changes in mitochondrial processes in apoE4 cells, including reduced levels of numerous respiratory complex subunits and major disruptions to all detected subunits in complex V (ATP synthase). Also altered in apoE4 cells were levels of proteins related to mitochondrial endoplasmic reticulum-associated membranes, mitochondrial fusion/fission, mitochondrial protein translocation, proteases, and mitochondrial ribosomal proteins. ApoE4-induced bioenergetic deficits led to extensive metabolic rewiring, but despite numerous cellular adaptations, apoE4-expressing neurons remained vulnerable to metabolic stress. Our results provide insights into potential molecular targets of therapies to correct apoE4-associated mitochondrial dysfunction and altered cellular metabolism.

Amyloid ß-induced elevation of O-GlcNAcylated c-Fos promotes neuronal cell death.

Alzheimer's disease (AD) is an age-related neurodegenerative disease characterized by progressive memory loss resulting from cumulative neuronal cell death. O-linked ß-N-acetyl glucosamine (O-GlcNAc) modification of the proteins reflecting glucose metabolism is altered in the brains of patients with AD. However, the link between altered O-GlcNAc modification and neuronal cell death in AD is poorly understood. Here, we examined the regulation of O-GlcNAcylation of c-Fos and the effects of O-GlcNAcylated c-Fos on neuronal cell death during AD pathogenesis. We found that amyloid beta (Aß)-induced O-GlcNAcylation on serine-56 and 57 of c-Fos was resulted from decreased interaction between c-Fos and O-GlcNAcase and promoted neuronal cell death. O-GlcNAcylated c-Fos increased its stability and potentiated the transcriptional activity through higher interaction with c-Jun, resulting in induction of Bim expression leading to neuronal cell death. Taken together, Aß-induced O-GlcNAcylation of c-Fos plays an important role in neuronal cell death during the pathogenesis of AD.

miR-27a and miR-27b regulate autophagic clearance of damaged mitochondria by targeting PTEN-induced putative kinase 1 (PINK1).

BACKGROUND: Loss-of-function mutations in PINK1 and PARKIN are the most common causes of autosomal recessive Parkinson's disease (PD). PINK1 is a mitochondrial serine/threonine kinase that plays a critical role in mitophagy, a selective autophagic clearance of damaged mitochondria. Accumulating evidence suggests mitochondrial dysfunction is one of central mechanisms underlying PD pathogenesis. Therefore, identifying regulatory mechanisms of PINK1 expression may provide novel therapeutic opportunities for PD. Although post-translational stabilization of PINK1 upon mitochondrial damage has been extensively studied, little is known about the regulation mechanism of PINK1 at the transcriptional or translational levels. RESULTS: Here, we demonstrated that microRNA-27a (miR-27a) and miR-27b suppress PINK1 expression at the translational level through directly binding to the 3'-untranslated region (3'UTR) of its mRNA. Importantly, our data demonstrated that translation of PINK1 is critical for its accumulation upon mitochondrial damage. The accumulation of PINK1 upon mitochondrial damage was strongly regulated by expression levels of miR-27a and miR-27b. miR-27a and miR-27b prevent mitophagic influx by suppressing PINK1 expression, as evidenced by the decrease of ubiquitin phosphorylation, Parkin translocation, and LC3-II accumulation in damaged mitochondria. Consequently, miR-27a and miR-27b inhibit lysosomal degradation of the damaged mitochondria, as shown by the decrease of the delivery of damaged mitochondria to lysosome and the degradation of cytochrome c oxidase 2 (COX2), a mitochondrial marker. Furthermore, our data demonstrated that the expression of miR-27a and miR-27b is significantly induced under chronic mitophagic flux, suggesting a negative feedback regulation between PINK1-mediated mitophagy and miR-27a and miR-27b. CONCLUSIONS: We demonstrated that miR-27a and miR-27b regulate PINK1 expression and autophagic clearance of damaged mitochondria. Our data further support a novel negative regulatory mechanism of PINK1-mediated mitophagy by miR-27a and miR-27b. Therefore, our results considerably advance our understanding of PINK1 expression and mitophagy regulation and suggest that miR-27a and miR-27b may represent potential therapeutic targets for PD.

Inhibition of Cholesterol Biosynthesis Reduces γ-Secretase Activity and Amyloid-ß Generation.

Amyloid-ß (Aß) is one of major molecules contributing to the pathogenesis of Alzheimer's disease (AD). Aß is derived from amyloid-ß protein precursor (AßPP) through sequential cleavages by ß- and γ-secretases. Regulation of these components is thought to be an important factor in Aß generation during the pathogenesis of AD. AßPP, ß-secretase, and γ-secretase reside in lipid rafts, where cholesterol regulates the integrity and flexibility of membrane proteins and Aß is generated. However, the relationship between cholesterol and Aß generation is controversial. In this study, we aimed to elucidate the direct effects of cholesterol depletion on AßPP processing using AY9944, which blocks the last step of cholesterol biosynthesis and thus minimizes the unknown side effects of upstream inhibitors, such as HMG-CoA reductase inhibitors. Treatment with AY9944 decreased γ-secretase activity and Aß generation. These results suggested that changes in membrane composition by lowering cholesterol with AY9944 affected γ-secretase activity and Aß generation, which is associated with AD pathogenesis.

The novel RAGE interactor PRAK is associated with autophagy signaling in Alzheimer's disease pathogenesis.

BACKGROUND: The receptor for advanced glycation end products (RAGE) has been found to interact with amyloid ß (Aß). Although RAGE does not have any kinase motifs in its cytosolic domain, the interaction between RAGE and Aß triggers multiple cellular signaling involved in Alzheimer's disease (AD). However, the mechanism of signal transduction by RAGE remains still unknown. Therefore, identifying binding proteins of RAGE may provide novel therapeutic targets for AD. RESULTS: In this study, we identified p38-regulated/activated protein kinase (PRAK) as a novel RAGE interacting molecule. To investigate the effect of Aß on PRAK mediated RAGE signaling pathway, we treated SH-SY5Y cells with monomeric form of Aß. We demonstrated that Aß significantly increased the phosphorylation of PRAK as well as the interaction between PRAK and RAGE. We showed that knockdown of PRAK rescued mTORC1 inactivation induced by Aß treatment and decreased the formation of Aß-induced autophagosome. CONCLUSIONS: We provide evidence that PRAK plays a critical role in AD pathology as a key interactor of RAGE. Thus, our data suggest that PRAK might be a potential therapeutic target of AD involved in RAGE-mediated cell signaling induced by Aß.

Mitochondrial ATP synthase activity is impaired by suppressed O-GlcNAcylation in Alzheimer's disease.

Glycosylation with O-linked ß-N-acetylglucosamine (O-GlcNAc) is one of the protein glycosylations affecting various intracellular events. However, the role of O-GlcNAcylation in neurodegenerative diseases such as Alzheimer's disease (AD) is poorly understood. Mitochondrial adenosine 5'-triphosphate (ATP) synthase is a multiprotein complex that synthesizes ATP from ADP and Pi. Here, we found that ATP synthase subunit α (ATP5A) was O-GlcNAcylated at Thr432 and ATP5A O-GlcNAcylation was decreased in the brains of AD patients and transgenic mouse model, as well as Aß-treated cells. Indeed, Aß bound to ATP synthase directly and reduced the O-GlcNAcylation of ATP5A by inhibition of direct interaction between ATP5A and mitochondrial O-GlcNAc transferase, resulting in decreased ATP production and ATPase activity. Furthermore, treatment of O-GlcNAcase inhibitor rescued the Aß-induced impairment in ATP production and ATPase activity. These results indicate that Aß-mediated reduction of ATP synthase activity in AD pathology results from direct binding between Aß and ATP synthase and inhibition of O-GlcNAcylation of Thr432 residue on ATP5A.

Acute ER stress regulates amyloid precursor protein processing through ubiquitin-dependent degradation.

Beta-amyloid (Aß), a major pathological hallmark of Alzheimer's disease (AD), is derived from amyloid precursor protein (APP) through sequential cleavage by ß-secretase and γ-secretase enzymes. APP is an integral membrane protein, and plays a key role in the pathogenesis of AD; however, the biological function of APP is still unclear. The present study shows that APP is rapidly degraded by the ubiquitin-proteasome system (UPS) in the CHO cell line in response to endoplasmic reticulum (ER) stress, such as calcium ionophore, A23187, induced calcium influx. Increased levels of intracellular calcium by A23187 induces polyubiquitination of APP, causing its degradation. A23187-induced reduction of APP is prevented by the proteasome inhibitor MG132. Furthermore, an increase in levels of the endoplasmic reticulum-associated degradation (ERAD) marker, E3 ubiquitin ligase HRD1, proteasome activity, and decreased levels of the deubiquitinating enzyme USP25 were observed during ER stress. In addition, we found that APP interacts with USP25. These findings suggest that acute ER stress induces degradation of full-length APP via the ubiquitin-proteasome proteolytic pathway.

Common Pesticide, Dichlorodiphenyltrichloroethane (DDT), Increases Amyloid-ß Levels by Impairing the Function of ABCA1 and IDE: Implication for Alzheimer's Disease.

While early-onset familial Alzheimer's disease (AD) is caused by a genetic mutation, the vast majority of late-onset AD is likely caused by the combination of genetic and environmental factors. Unlike genetic studies, potential environmental factors affecting AD pathogenesis have not yet been thoroughly investigated. Among environmental factors, pesticides seem to be one of critical environmental contributors to late-onset AD. Recent studies reported that the serum and brains of AD patients have dramatically higher levels of a metabolite of dichlorodiphenyltrichloroethane (DDT). While these epidemiological studies provided initial clues to the environmental risks potentially contributing to disease pathogenesis, a functional approach is required to determine whether they actually have a causal role in disease development. In our study, we addressed this critical knowledge gap by investigating possible mechanisms by which DDT affects amyloid-ß (Aß) levels. We treated H4-AßPPswe or H4 cells with DDT to analyze its effect on Aß metabolism using Aß production, clearance, and degradation assays. We found that DDT significantly increased the levels of amyloid-ß protein precursor (AßPP) and ß-site AßPP-cleaving enzyme1 (BACE1), affecting Aß synthesis pathway in H4-AßPPswe cells. Additionally, DDT impaired the clearance and extracellular degradation of Aß peptides. Most importantly, we identified for the first time that ATP-binding cassette transporter A1 (ABCA1) and insulin-degrading enzyme (IDE) are the downstream target genes adversely affected by DDT. Our findings provide insight into the molecular mechanisms by which DDT exposure may increase the risk of AD, and it further supports that ABCA1 and IDE may be potential therapeutic targets.

Analysis of extracellular RNA by digital PCR.

The transfer of extracellular RNA is emerging as an important mechanism for inter-cellular communication. The ability for the transfer of functionally active RNA molecules from one cell to another within vesicles such as exosomes enables a cell to modulate cellular signaling and biological processes within recipient cells. The study of extracellular RNA requires sensitive methods for the detection of these molecules. In this methods article, we will describe protocols for the detection of such extracellular RNA using sensitive detection technologies such as digital PCR. These protocols should be valuable to researchers interested in the role and contribution of extracellular RNA to tumor cell biology.

Synapsin-1 and tau reciprocal O-GlcNAcylation and phosphorylation sites in mouse brain synaptosomes.

O-linked N-acetylglucosamine (O-GlcNAc) represents a key regulatory post-translational modification (PTM) that is reversible and often reciprocal with phosphorylation of serine and threonine at the same or nearby residues. Although recent technical advances in O-GlcNAc site-mapping methods combined with mass spectrometry (MS) techniques have facilitated study of the fundamental roles of O-GlcNAcylation in cellular processes, an efficient technique for examining the dynamic, reciprocal relationships between O-GlcNAcylation and phosphorylation is needed to provide greater insights into the regulatory functions of O-GlcNAcylation. Here, we describe a strategy for selectively identifying both O-GlcNAc- and phospho-modified sites. This strategy involves metal affinity separation of O-GlcNAcylated and phosphorylated peptides, ß-elimination of O-GlcNAcyl or phosphoryl functional groups from the separated peptides followed by dithiothreitol (DTT) conjugation (BEMAD), affinity purification of DTT-conjugated peptides using thiol affinity chromatography, and identification of formerly O-GlcNAcylated or phosphorylated peptides by MS. The combined metal affinity separation and BEMAD approach allows selective enrichment of O-GlcNAcylated peptides over phosphorylated counterparts. Using this approach with mouse brain synaptosomes, we identified the serine residue at 605 of the synapsin-1 peptide, 603QASQAGPGPR612, and the serine residue at 692 of the tau peptide, 688SPVVSGDTSPR698, which were found to be potential reciprocal O-GlcNAcylation and phosphorylation sites. These results demonstrate that our strategy enables mapping of the reciprocal site occupancy of O-GlcNAcylation and phosphorylation of proteins, which permits the assessment of cross-talk between these two PTMs and their regulatory roles.

O-linked ß-N-acetylglucosaminidase inhibitor attenuates ß-amyloid plaque and rescues memory impairment.

Deposition of ß-amyloid (Aß) as senile plaques and disrupted glucose metabolism are two main characteristics of Alzheimer's disease (AD). It is unknown, however, how these two processes are related in AD. Here we examined the relationship between O-GlcNAcylation, which is a glucose level-dependent post-translational modification that adds O-linked ß-N-acetylglucosamine (O-GlcNAc) to proteins, and Aß production in a mouse model of AD carrying 5XFAD genes. We found that 1,2-dideoxy-2'-propyl-α-d-glucopyranoso-[2,1-D]-Δ2'-thiazoline (NButGT), a specific inhibitor of O-GlcNAcase, reduces Aß production by lowering γ-secretase activity both in vitro and in vivo. We also found that O-GlcNAcylation takes place at the S708 residue of nicastrin, which is a component of γ-secretase. Moreover, NButGT attenuated the accumulation of Aß, neuroinflammation, and memory impairment in the 5XFAD mice. This is the first study to show the relationship between Aß generation and O-GlcNAcylation in vivo. These results suggest that O-GlcNAcylation may be a suitable therapeutic target for the treatment of AD.

Asp664 cleavage of amyloid precursor protein induces tau phosphorylation by decreasing protein phosphatase 2A activity.

Caspase cleavage of amyloid precursor protein (APP) has been reported to be important in amyloid beta protein (Aß)-mediated neurotoxicity. However, the underlying mechanisms are not clearly understood. In this study, we explored the effect of caspase cleavage of APP on tau phosphorylation in relation to Aß. We found that Asp664 cleavage of APP increased tau phosphorylation at Thr212 and Ser262 in N2A cells and primary cultured hippocampal neurons. Compared with wild-type APP, protein phosphatase 2A (PP2A) activity was significantly increased when Asp664 cleavage was blocked by the D664A point mutation. Furthermore, we found that over-expression of C31 reduced PP2A activity. C31 binds directly to the PP2A catalytic subunit, through the asparagine, proline, threonine, tyrosine (NPTY) motif, which is essential for C31-induced tau hyperphosphorylation. However, it appears that the other fragment produced by Asp664 cleavage, Jcasp, modulates neither PP2A activity nor tau hyperphosphorylation. Asp664 cleavage and accompanying tau hyperphosphorylation were remarkably diminished by blockage of Aß production using a γ-secretase inhibitor. Taken together, our results suggest that Asp664 cleavage of APP leads to tau hyperphosphorylation at specific epitopes by modulating PP2A activity as a downstream of Aß. Direct binding of C31 to PP2A through the C31-NPTY domain was identified as a mechanism underlying this effect.