Rewiring of the ubiquitinated proteome determines ageing in C. elegans.

Ageing is driven by a loss of cellular integrity1. Given the major role of ubiquitin modifications in cell function2, here we assess the link between ubiquitination and ageing by quantifying whole-proteome ubiquitin signatures in Caenorhabditis elegans. We find a remodelling of the ubiquitinated proteome during ageing, which is ameliorated by longevity paradigms such as dietary restriction and reduced insulin signalling. Notably, ageing causes a global loss of ubiquitination that is triggered by increased deubiquitinase activity. Because ubiquitination can tag proteins for recognition by the proteasome3, a fundamental question is whether deficits in targeted degradation influence longevity. By integrating data from worms with a defective proteasome, we identify proteasomal targets that accumulate with age owing to decreased ubiquitination and subsequent degradation. Lowering the levels of age-dysregulated proteasome targets prolongs longevity, whereas preventing their degradation shortens lifespan. Among the proteasomal targets, we find the IFB-2 intermediate filament4 and the EPS-8 modulator of RAC signalling5. While increased levels of IFB-2 promote the loss of intestinal integrity and bacterial colonization, upregulation of EPS-8 hyperactivates RAC in muscle and neurons, and leads to alterations in the actin cytoskeleton and protein kinase JNK. In summary, age-related changes in targeted degradation of structural and regulatory proteins across tissues determine longevity.

G3BP1-dependent mechanism suppressing protein aggregation in Huntington's models and its demise upon stress granule assembly.

Stress granules are membrane-less ribonucleoprotein organelles that assemble upon exposure to stress conditions, but rapidly disassemble upon removal of stress. However, chronic stress can lead to persistent stress granules, a feature of distinct age-related neurodegenerative disorders. Among them, Huntington's disease (HD), which is caused by mutant expansion of the polyglutamine (polyQ) repeats of huntingtin protein (HTT), leading to its aggregation. To identify modulators of mutant HTT aggregation, we define its interactome in striatal neurons differentiated from patient-derived induced pluripotent stem cells (HD-iPSCs). We find that HTT interacts with G3BP1, a characteristic component of stress granules. Knockdown of G3BP1 increases mutant HTT protein levels and abolishes the ability of iPSCs as well as their differentiated neural counterparts to suppress mutant HTT aggregation. Moreover, loss of G3BP1 hastens polyQ-expanded aggregation and toxicity in the neurons of HD C. elegans models. Likewise, the assembly of G3BP1 into stress granules upon distinct stress conditions also reduces its interaction with HTT in human cells, promoting mutant HTT aggregation. Notably, enhancing the levels of G3BP1 is sufficient to induce proteasomal degradation of mutant HTT and prevent its aggregation, whereas the formation of stress granules blocks these ameliorative effects. In contrast, a mutant G3BP1 variant that cannot accumulate into granules retains its capacity to prevent mutant HTT aggregation even when the cells assemble stress granules. Thus, our findings indicate a direct role of G3BP1 and stress granule assembly in mutant HTT aggregation that may have implications for HD.

Association between placental oxygen transport and fetal brain cortical development: a study in monochorionic diamniotic twins.

Normal cortical growth and the resulting folding patterns are crucial for normal brain function. Although cortical development is largely influenced by genetic factors, environmental factors in fetal life can modify the gene expression associated with brain development. As the placenta plays a vital role in shaping the fetal environment, affecting fetal growth through the exchange of oxygen and nutrients, placental oxygen transport might be one of the environmental factors that also affect early human cortical growth. In this study, we aimed to assess the placental oxygen transport during maternal hyperoxia and its impact on fetal brain development using MRI in identical twins to control for genetic and maternal factors. We enrolled 9 pregnant subjects with monochorionic diamniotic twins (30.03 ± 2.39 gestational weeks [mean ± SD]). We observed that the fetuses with slower placental oxygen delivery had reduced volumetric and surface growth of the cerebral cortex. Moreover, when the difference between placenta oxygen delivery increased between the twin pairs, sulcal folding patterns were more divergent. Thus, there is a significant relationship between placental oxygen transport and fetal brain cortical growth and folding in monochorionic twins.

Defining mesenchymal stem/stromal cell-induced myeloid-derived suppressor cells using single-cell transcriptomics.

Mesenchymal stem/stromal cells (MSCs) modulate the immune response through interactions with innate immune cells. We previously demonstrated that MSCs alleviate ocular autoimmune inflammation by directing bone marrow cell differentiation from pro-inflammatory CD11bhiLy6ChiLy6Glo cells into immunosuppressive CD11bmidLy6CmidLy6Glo cells. Herein, we analyzed MSC-induced CD11bmidLy6Cmid cells using single-cell RNA sequencing and compared them with CD11bhiLy6Chi cells. Our investigation revealed seven distinct immune cell types including myeloid-derived suppressor cells (MDSCs) in the CD11bmidLy6Cmid cells, while CD11bhiLy6Chi cells included mostly monocytes/macrophages with a small cluster of neutrophils. These MSC-induced MDSCs highly expressed Retnlg, Cxcl3, Cxcl2, Mmp8, Cd14, and Csf1r as well as Arg1. Comparative analyses of CSF-1RhiCD11bmidLy6Cmid and CSF-1RloCD11bmidLy6Cmid cells demonstrated that the former had a homogeneous monocyte morphology and produced elevated levels of interleukin-10. Functionally, these CSF-1RhiCD11bmidLy6Cmid cells, compared with the CSF-1RloCD11bmidLy6Cmid cells, inhibited CD4+ T cell proliferation and promoted CD4+CD25+Foxp3+ Treg expansion in culture and in a mouse model of experimental autoimmune uveoretinitis. Resistin-like molecule (RELM)-γ encoded by Retnlg, one of the highly upregulated genes in MSC-induced MDSCs, had no direct effects on T cell proliferation, Treg expansion, or splenocyte activation. Together, our study revealed a distinct transcriptional profile of MSC-induced MDSCs and identified CSF-1R as a key cell-surface marker for detection and therapeutic enrichment of MDSCs.

The dopamine analogue CA140 alleviates AD pathology, neuroinflammation, and rescues synaptic/cognitive functions by modulating DRD1 signaling or directly binding to Abeta.

BACKGROUND: We recently reported that the dopamine (DA) analogue CA140 modulates neuroinflammatory responses in lipopolysaccharide-injected wild-type (WT) mice and in 3-month-old 5xFAD mice, a model of Alzheimer's disease (AD). However, the effects of CA140 on Aß/tau pathology and synaptic/cognitive function and its molecular mechanisms of action are unknown. METHODS: To investigate the effects of CA140 on cognitive and synaptic function and AD pathology, 3-month-old WT mice or 8-month-old (aged) 5xFAD mice were injected with vehicle (10% DMSO) or CA140 (30 mg/kg, i.p.) daily for 10, 14, or 17 days. Behavioral tests, ELISA, electrophysiology, RNA sequencing, real-time PCR, Golgi staining, immunofluorescence staining, and western blotting were conducted. RESULTS: In aged 5xFAD mice, a model of AD pathology, CA140 treatment significantly reduced Aß/tau fibrillation, Aß plaque number, tau hyperphosphorylation, and neuroinflammation by inhibiting NLRP3 activation. In addition, CA140 treatment downregulated the expression of cxcl10, a marker of AD-associated reactive astrocytes (RAs), and c1qa, a marker of the interaction of RAs with disease-associated microglia (DAMs) in 5xFAD mice. CA140 treatment also suppressed the mRNA levels of s100ß and cxcl10, markers of AD-associated RAs, in primary astrocytes from 5xFAD mice. In primary microglial cells from 5xFAD mice, CA140 treatment increased the mRNA levels of markers of homeostatic microglia (cx3cr1 and p2ry12) and decreased the mRNA levels of a marker of proliferative region-associated microglia (gpnmb) and a marker of lipid-droplet-accumulating microglia (cln3). Importantly, CA140 treatment rescued scopolamine (SCO)-mediated deficits in long-term memory, dendritic spine number, and LTP impairment. In aged 5xFAD mice, these effects of CA140 treatment on cognitive/synaptic function and AD pathology were regulated by dopamine D1 receptor (DRD1)/Elk1 signaling. In primary hippocampal neurons and WT mice, CA140 treatment promoted long-term memory and dendritic spine formation via effects on DRD1/CaMKIIα and/or ERK signaling. CONCLUSIONS: Our results indicate that CA140 improves neuronal/synaptic/cognitive function and ameliorates Aß/tau pathology and neuroinflammation by modulating DRD1 signaling in primary hippocampal neurons, primary astrocytes/microglia, WT mice, and aged 5xFAD mice.

Altered development of structural MRI connectome hubs at near-term age in very and moderately preterm infants.

Preterm infants may exhibit altered developmental patterns of the brain structural network by endogenous and exogenous stimuli, which are quantifiable through hub and modular network topologies that develop in the third trimester. Although preterm brain networks can compensate for white matter microstructural abnormalities of core connections, less is known about how the network developmental characteristics of preterm infants differ from those of full-term infants. We identified 13 hubs and 4 modules and revealed subtle differences in edgewise connectivity and local network properties between 134 preterm and 76 full-term infants, identifying specific developmental patterns of the brain structural network in preterm infants. The modules of preterm infants showed an imbalanced composition. The edgewise connectivity in preterm infants showed significantly decreased long- and short-range connections and local network properties in the dorsal superior frontal gyrus. In contrast, the fusiform gyrus and several nonhub regions showed significantly increased wiring of short-range connections and local network properties. Our results suggested that decreased local network in the frontal lobe and excessive development in the occipital lobe may contribute to the understanding of brain developmental deviances in preterm infants.

Subconjunctival aflibercept inhibits corneal angiogenesis and VEGFR-3+CD11b+ cells.

PURPOSE: This study aimed to investigate the effects of subconjunctival injection of aflibercept, a soluble protein decoy for VEGFR-1 and VEGFR-2, on corneal angiogenesis and VEGFR-expressing CD11b+ cells in a mouse model of suture-induced corneal neovascularization. METHODS: Corneal neovascularization was induced in BALB/c mice by placing three sutures on the cornea. Immediately after surgery, either 200 µg aflibercept (5 µL) or an equal volume of phosphate-buffered saline (PBS) was administered into the subconjunctival space. Seven days after later, corneal new vessels were quantified through clinical examination and measurement of the CD31-stained area in corneal flat mounts. The levels of pro-angiogenic and inflammatory markers in the cornea were evaluated using RT-qPCR. The percentages of VEGFR-2+CD11b+ cells and VEGFR-3+CD11b+ cells were analyzed in the cornea, blood, and draining cervical lymph nodes (DLNs) using flow cytometry. RESULTS: Subconjunctival injection of aflibercept significantly reduced the growth of corneal new vessels compared to subconjunctival PBS injection. The mRNA levels of Cd31, vascular growth factors (Vegfc and Angpt1), and pro-angiogenic/inflammatory markers (Tek/Tie2, Mrc1, Mrc2, and Il6) in the cornea were downregulated by subconjunctival aflibercept. Also, the percentage of VEGFR-3+CD11b+ cells in the cornea, blood, and DLNs was decreased by aflibercept, whereas that of VEGFR-2+CD11b+ cells was unaffected. CONCLUSION: Subconjunctival aflibercept administration inhibits inflammatory angiogenesis in the cornea and reduces the numbers of cornea-infiltrating and circulating VEGFR-3+CD11b+ cells.

Mesenchymal Stem/Stromal Cells Induce Myeloid-Derived Suppressor Cells in the Bone Marrow via the Activation of the c-Jun N-Terminal Kinase Signaling Pathway.

Our previous study demonstrated that mesenchymal stem/stromal cells (MSCs) induce the differentiation of myeloid-derived suppressor cells (MDSCs) in the bone marrow (BM) under inflammatory conditions. In this study, we aimed to investigate the signaling pathway involved. RNA-seq revealed that the mitogen-activated protein kinase (MAPK) pathway exhibited the highest number of upregulated genes in MSC-induced MDSCs. Western blot analysis confirmed the strong phosphorylation of c-Jun N-terminal kinase (JNK) in BM cells cocultured with MSCs under granulocyte-macrophage colony-stimulating factor stimulation, whereas p38 kinase activation remained unchanged in MSC-cocultured BM cells. JNK inhibition by SP600125 abolished the expression of Arg1 and Nos2, hallmark genes of MDSCs, as well as Hif1a, a molecule mediating monocyte functional reprogramming toward a suppressive phenotype, in MSC-cocultured BM cells. JNK inhibition also abrogated the effects of MSCs on the production of TGF-ß1, TGF-ß2 and IL-10 in BM cells. Furthermore, JNK inhibition increased Tnfa expression, while suppressing IL-10 production, in MSC-cocultured BM cells in response to lipopolysaccharides. Collectively, our results suggest that MSCs induce MDSC differentiation and promote immunoregulatory cytokine production in BM cells during inflammation, at least in part, through the activation of the JNK-MAPK signaling pathway.

Myeloid cells protect corneal nerves against sterile injury through negative-feedback regulation of TLR2-IL-6 axis.

BACKGROUND: Mounting evidence suggests that the immune system plays detrimental or protective roles in nerve injury and repair. MAIN BODY: Herein we report that both CD11bhiLy6Ghi and CD11bhiLy6ChiLy6Glo myeloid cells are required to protect corneal nerves against sterile corneal injury. Selective depletion of CD11bhiLy6Ghi or CD11bhiLy6ChiLy6Glo cells resulted in aggravation of corneal nerve loss, which correlated with IL-6 upregulation. IL-6 neutralization preserved corneal nerves while reducing myeloid cell recruitment. IL-6 replenishment exacerbated corneal nerve damage while recruiting more myeloid cells. In mice lacking Toll-like receptor 2 (TLR2), the levels of IL-6 and myeloid cells were decreased and corneal nerve loss attenuated, as compared to wild-type and TLR4 knockout mice. Corneal stromal fibroblasts expressed TLR2 and produced IL-6 in response to TLR2 stimulation. CONCLUSION: Collectively, our data suggest that CD11bhiLy6Ghi and CD11bhiLy6ChiLy6Glo myeloid cells confer corneal nerve protection under sterile injury by creating a negative-feedback loop to suppress the upstream TLR2-IL-6 axis that drives corneal nerve loss.

MHC Class I Enables MSCs to Evade NK-Cell-Mediated Cytotoxicity and Exert Immunosuppressive Activity.

Allogeneic mesenchymal stem/stromal cells (MSCs) are frequently used in clinical trials due to their low expression of major histocompatibility complex (MHC) class I and lack of MHC class II. However, the levels of MHC classes I and II in MSCs are increased by inflammatory stimuli, raising concerns over potential adverse effects associated with allogeneic cell therapy. Also, it is unclear how the host immune response to MHC-mismatched MSCs affects the therapeutic efficacy of the cells. Herein, using strategies to manipulate MHC genes in human bone marrow-derived MSCs via the CRISPR-Cas9 system, plasmids, or siRNAs, we found that inhibition of MHC class I-not MHC class II-in MSCs lowered the survival rate of MSCs and their immunosuppressive potency in mice with experimental autoimmune uveoretinitis, specifically by increasing MSC vulnerability to natural killer (NK)-cell-mediated cytotoxicity. A subsequent survey of MSC batches derived from 6 human donors confirmed a significant correlation between MSC survival rate and susceptibility to NK cells with the potency of MSCs to increase MHC class I level upon stimulation. Our overall results demonstrate that MHC class I enables MSCs to evade NK-cell-mediated cytotoxicity and exert immunosuppressive activity.

Inhibition of CDK4/6 regulates AD pathology, neuroinflammation and cognitive function through DYRK1A/STAT3 signaling.

Repurposing approved drugs is an emerging therapeutic development strategy for Alzheimer's disease (AD). The CDK4/6 inhibitor abemaciclib mesylate is an FDA-approved drug for breast cancer treatment. However, whether abemaciclib mesylate affects Aß/tau pathology, neuroinflammation, and Aß/LPS-mediated cognitive impairment is unknown. In this study, we investigated the effects of abemaciclib mesylate on cognitive function and Aß/tau pathology and found that abemaciclib mesylate improved spatial and recognition memory by regulating the dendritic spine number and neuroinflammatory responses in 5xFAD mice, an Aß-overexpressing model of AD. Abemaciclib mesylate also inhibited Aß accumulation by enhancing the activity and protein levels of the Aß-degrading enzyme neprilysin and the α-secretase ADAM17 and decreasing the protein level of the γ-secretase PS-1 in young and aged 5xFAD mice. Importantly, abemaciclib mesylate suppressed tau phosphorylation in 5xFAD mice and tau-overexpressing PS19 mice by reducing DYRK1A and/or p-GSK3ß levels. In wild-type (WT) mice injected with lipopolysaccharide (LPS), abemaciclib mesylate rescued spatial and recognition memory and restored dendritic spine number. In addition, abemaciclib mesylate downregulated LPS-induced microglial/astrocytic activation and proinflammatory cytokine levels in WT mice. In BV2 microglial cells and primary astrocytes, abemaciclib mesylate suppressed LPS-mediated proinflammatory cytokine levels by downregulating AKT/STAT3 signaling. Taken together, our results support repurposing the anticancer drug, CDK4/6 inhibitor abemaciclib mesylate as a multitarget therapeutic for AD pathologies.

Brain age predicted using graph convolutional neural network explains neurodevelopmental trajectory in preterm neonates.

OBJECTIVES: Dramatic brain morphological changes occur throughout the third trimester of gestation. In this study, we investigated whether the predicted brain age (PBA) derived from graph convolutional network (GCN) that accounts for cortical morphometrics in third trimester is associated with postnatal abnormalities and neurodevelopmental outcome. METHODS: In total, 577 T1 MRI scans of preterm neonates from two different datasets were analyzed; the NEOCIVET pipeline generated cortical surfaces and morphological features, which were then fed to the GCN to predict brain age. The brain age index (BAI; PBA minus chronological age) was used to determine the relationships among preterm birth (i.e., birthweight and birth age), perinatal brain injuries, postnatal events/clinical conditions, BAI at postnatal scan, and neurodevelopmental scores at 30 months. RESULTS: Brain morphology and GCN-based age prediction of preterm neonates without brain lesions (mean absolute error [MAE]: 0.96 weeks) outperformed conventional machine learning methods using no topological information. Structural equation models (SEM) showed that BAI mediated the influence of preterm birth and postnatal clinical factors, but not perinatal brain injuries, on neurodevelopmental outcome at 30 months of age. CONCLUSIONS: Brain morphology may be clinically meaningful in measuring brain age, as it relates to postnatal factors, and predicting neurodevelopmental outcome. CLINICAL RELEVANCE STATEMENT: Understanding the neurodevelopmental trajectory of preterm neonates through the prediction of brain age using a graph convolutional neural network may allow for earlier detection of potential developmental abnormalities and improved interventions, consequently enhancing the prognosis and quality of life in this vulnerable population. KEY POINTS: •Brain age in preterm neonates predicted using a graph convolutional network with brain morphological changes mediates the pre-scan risk factors and post-scan neurodevelopmental outcomes. •Predicted brain age oriented from conventional deep learning approaches, which indicates the neurodevelopmental status in neonates, shows a lack of sensitivity to perinatal risk factors and predicting neurodevelopmental outcomes. •The new brain age index based on brain morphology and graph convolutional network enhances the accuracy and clinical interpretation of predicted brain age for neonates.

Risk Factors for Sudden Death Within 2 Days After Diagnosis of COVID-19 in Korea.

BACKGROUND: We aimed to analyze the risk factors for sudden death after diagnosis of coronavirus disease 2019 (COVID-19) in South Korea and to provide evidence for informing prevention and control interventions for patients at risk of sudden death. METHODS: We included 30,302 COVID-19 related deaths registered in the patient management information system (Central Disease Control Headquarters) between January 1, 2021, and December 15, 2022. We collected their epidemiological data recorded by the reporting city, province, or country. We performed multivariate logistic regression analysis to identify risk factors for sudden death after diagnosis of COVID-19. RESULTS: Among the 30,302 deaths, there were 7,258 (24.0%) and 23,044 (76.0%) sudden and non-sudden deaths, respectively. Sudden death means a person who died within 2 days of diagnosis and who did not receive inpatient treatment. Underlying condition, vaccination status, and place of death were significantly associated with the survival period in all age groups. Moreover, region, sex, and prescription were significantly associated with the survival period only in certain age groups. However, reinfection was not significantly associated with the survival period in any age group. CONCLUSION: To our knowledge, this is the first study on the risk factors for sudden death after a diagnosis of COVID-19, which included age, underlying condition, vaccination status, and place of death. Additionally, individuals aged < 60 years without an underlying condition were at high risk for sudden death. However, this group has relatively low interest in health, as can be seen from the high non-vaccination rate (16.1% of the general population vs. 61.6% of the corresponding group). Therefore, there is a possibility for the presence of an uncontrolled underlying disease in this population. In addition, many sudden deaths occurred due to delayed hospital visits to continue economic activities even after the onset of COVID-19 symptoms (7 days overall vs. 10 days average for the group). In conclusion, 'continued interest in health' is a key factor in avoiding sudden death in the economically active group (under 60 years of age).

Pediatric Deaths Associated With Coronavirus Disease 2019 (COVID-19) in Korea.

As of September 3, 2022, 5,388,338 coronavirus disease 2019 (COVID-19) cases and 46 deaths (3 in 2021 and 43 in 2022) were reported in children ≤ 18 years in Korea. Cumulative confirmed cases accounted for 67.3% of the population aged ≤ 18 years and case fatality rate was 0.85/100,000. Among 46 fatal cases, 58.7% were male and median age was 7 years. Underlying diseases were present in 47.8%; neurologic diseases (63.6%) and malignancy (13.6%) most common. Only four had history of COVID-19 immunization. COVID-19 associated deaths occurred at median 2 days from diagnosis (range: -1 to 21). Among COVID-19 deaths, 41.3% occurred before admission; 2 before hospital arrival and 17 in the emergency department. Among children whose cause was documented, myocarditis, respiratory and multiorgan failure were most common. COVID-19 associated death was seen early after diagnosis in children and public health policies to provide access to medical care for children with COVID-19 are essential during the pandemic.

Quantification of sulcal emergence timing and its variability in early fetal life: Hemispheric asymmetry and sex difference.

Human fetal brains show regionally different temporal patterns of sulcal emergence following a regular timeline, which may be associated with spatiotemporal patterns of gene expression among cortical regions. This study aims to quantify the timing of sulcal emergence and its temporal variability across typically developing fetuses by fitting a logistic curve to presence or absence of sulcus. We found that the sulcal emergence started from the central to the temporo-parieto-occipital lobes and frontal lobe, and the temporal variability of emergence in most of the sulci was similar between 1 and 2 weeks. Small variability (< 1 week) was found in the left central and postcentral sulci and larger variability (>2 weeks) was shown in the bilateral occipitotemporal and left superior temporal sulci. The temporal variability showed a positive correlation with the emergence timing that may be associated with differential contributions between genetic and environmental factors. Our statistical analysis revealed that the right superior temporal sulcus emerged earlier than the left. Female fetuses showed a trend of earlier sulcal emergence in the right superior temporal sulcus, lower temporal variability in the right intraparietal sulcus, and higher variability in the right precentral sulcus compared to male fetuses. Our quantitative and statistical approach quantified the temporal patterns of sulcal emergence in detail that can be a reference for assessing the normality of developing fetal gyrification.

Rapid Biodegradable Ionic Aggregates of Polyesters Constructed with Fertilizer Ingredients.

Synthetic biodegradable polyesters tend to undergo slow biodegradation under ambient natural conditions and, hence, have been rejected or even banned recently in ecofriendly applications. Here, we demonstrate the preparation of polyesters exhibiting enhanced biodegradability, which were generated through a combination of old controversial macromolecules and aggregate theories. H3PO4-catalyzed diacid/diol polycondensation afforded polyester chains bearing chain-end -CH2OP(O)(OH)2 and inner-chain (-CH2O)2P(O)(OH) groups, which were subsequently treated with M(2-ethylhexanoate)2 (M = Zn, Mg, Mn, and Ca) to form ionic aggregates of polyesters. The prepared ionic aggregates of polyesters, which were constructed with fertilizer ingredients (such as M2+ and phosphate), exhibit much faster biodegradability than that of the conventional polyesters under controlled soil conditions at 25 °C, while displaying comparable or superior rheological and mechanical properties.

Dapagliflozin attenuates diabetes-induced diastolic dysfunction and cardiac fibrosis by regulating SGK1 signaling.

BACKGROUND: Recent studies have reported improved diastolic function in patients administered sodium-glucose cotransporter 2 inhibitors (SGLT2i). We aimed to investigate the effect of dapagliflozin on left ventricular (LV) diastolic function in a diabetic animal model and to determine the molecular and cellular mechanisms underlying its function. METHODS: A total of 30 male New Zealand white rabbits were randomized into control, diabetes, or diabetes+dapagliflozin groups (n = 10/per each group). Diabetes was induced by intravenous alloxan. Cardiac function was evaluated using echocardiography. Myocardial samples were obtained for histologic and molecular evaluation. For cellular evaluation, fibrosis-induced cardiomyoblast (H9C2) cells were obtained, and transfection was performed for mechanism analysis (serum and glucocorticoid-regulated kinase 1 (SGK1) signaling analysis). RESULTS: The diabetes+dapagliflozin group showed attenuation of diastolic dysfunction compared with the diabetes group. Dapagliflozin inhibited myocardial fibrosis via inhibition of SGK1 and epithelial sodium channel (ENaC) protein, which was observed both in myocardial tissue and H9C2 cells. In addition, dapagliflozin showed an anti-inflammatory effect and ameliorated mitochondrial disruption. Inhibition of SGK1 expression by siRNA decreased and ENaC and Na+/H+ exchanger isoform 1 (NHE1) expression was confirmed as significantly reduced as siSGK1 in the diabetes+dapagliflozin group. CONCLUSIONS: Dapagliflozin attenuated left ventricular diastolic dysfunction and cardiac fibrosis via regulation of SGK1 signaling. Dapagliflozin also reduced macrophages and inflammatory proteins and ameliorated mitochondrial disruption.

Nilotinib modulates LPS-induced cognitive impairment and neuroinflammatory responses by regulating P38/STAT3 signaling.

BACKGROUND: In chronic myelogenous leukemia, reciprocal translocation between chromosome 9 and chromosome 22 generates a chimeric protein, Bcr-Abl, that leads to hyperactivity of tyrosine kinase-linked signaling transduction. The therapeutic agent nilotinib inhibits Bcr-Abl/DDR1 and can cross the blood-brain barrier, but its potential impact on neuroinflammatory responses and cognitive function has not been studied in detail. METHODS: The effects of nilotinib in vitro and in vivo were assessed by a combination of RT-PCR, real-time PCR, western blotting, ELISA, immunostaining, and/or subcellular fractionation. In the in vitro experiments, the effects of 200 ng/mL LPS or PBS on BV2 microglial cells, primary microglia or primary astrocytes pre- or post-treated with 5 µM nilotinib or vehicle were evaluated. The in vivo experiments involved wild-type mice administered a 7-day course of daily injections with 20 mg/kg nilotinib (i.p.) or vehicle before injection with 10 mg/kg LPS (i.p.) or PBS. RESULTS: In BV2 microglial cells, pre- and post-treatment with nilotinib altered LPS-induced proinflammatory/anti-inflammatory cytokine mRNA levels by suppressing AKT/P38/SOD2 signaling. Nilotinib treatment also significantly downregulated LPS-stimulated proinflammatory cytokine levels in primary microglia and primary astrocytes by altering P38/STAT3 signaling. Experiments in wild-type mice showed that nilotinib administration affected LPS-mediated microglial/astroglial activation in a brain region-specific manner in vivo. In addition, nilotinib significantly reduced proinflammatory cytokine IL-1ß, IL-6 and COX-2 levels and P38/STAT3 signaling in the brain in LPS-treated wild-type mice. Importantly, nilotinib treatment rescued LPS-mediated spatial working memory impairment and cortical dendritic spine number in wild-type mice. CONCLUSIONS: Our results indicate that nilotinib can modulate neuroinflammatory responses and cognitive function in LPS-stimulated wild-type mice.

Regulation of habenular G-protein gamma 8 on learning and memory via modulation of the central acetylcholine system.

Guanine nucleotide binding protein (G protein) gamma 8 (Gng8) is a subunit of G proteins and expressed in the medial habenula (MHb) and interpeduncular nucleus (IPN). Recent studies have demonstrated that Gng8 is involved in brain development; however, the roles of Gng8 on cognitive function have not yet been addressed. In the present study, we investigated the expression of Gng8 in the brain and found that Gng8 was predominantly expressed in the MHb-IPN circuit of the mouse brain. We generated Gng8 knockout (KO) mice by CRISPR/Cas9 system in order to assess the role of Gng8 on cognitive function. Gng8 KO mice exhibited deficiency in learning and memory in passive avoidance and Morris water maze tests. In addition, Gng8 KO mice significantly reduced long-term potentiation (LTP) in the hippocampus compared to that of wild-type (WT) mice. Furthermore, we observed that levels of acetylcholine (ACh) and choline acetyltransferase (ChAT) in the MHb and IPN of Gng8 KO mice were significantly decreased, compared to WT mice. The administration of nAChR α4ß2 agonist A85380 rescued memory impairment in the Gng8 KO mice, suggesting that Gng8 regulates cognitive function via modulation of cholinergic activity. Taken together, Gng8 is a potential therapeutic target for memory-related diseases and/or neurodevelopmental diseases.