Suppression of TGFß-Induced Interleukin-6 Secretion by Sinulariolide from Soft Corals through Attenuation of the p38-NF-kB Pathway in Carcinoma Cells.

Sinulariolide (SC-1) is a natural product extracted from the cultured-type soft coral Sinularia flexibilis and possesses anti-inflammation, anti-proliferative, and anti-migratory in several types of cancer cells. However, the molecular pathway behind its effects on inflammation remains poorly understood. Since inflammatory cytokines such as TGFß, TNFα, IL-1, IL-6, and IL-8 activate transcription factors such as Smads, NF-κB, STAT3, Snail, Twist, and Zeb that drive the epithelial-to-mesenchymal transition (EMT), in this study, we focus on the investigation in effects of SC-1 on TGFß-induced interleukin-6 (IL-6) releases in an in vitro cell culture model. We showed that both intracellular IL-6 expression and secretion were stimulated by TGFß and associated with strong upregulation of IL-6 mRNA and increased transcription in A549 cells. SC-1 blocked TGFß-induced secretion of IL-6 while showing no effect on the induction of fibronectin and plasminogen activator inhibitor-1 genes, indicating that SC-1 interferes with only a subset of TGFß activities. In addition, SC-1 inhibits TGFß-induced IL-6 by suppressing p38 MAPK signaling and subsequently inhibits NF-κB and its nuclear translocation without affecting the canonical Smad pathway and receptor turnover. Overall, these data suggest that p38 may involve in the inhibition of SC-1 in IL-6 release, thus illustrating an inhibitory effect for SC-1 in the suppression of inflammation, EMT phenotype, and tumorigenesis.

Cerebral Semaphorin3D is a novel risk factor for age-associated cognitive impairment.

BACKGROUND: We previously reported that miR-195 exerts neuroprotection by inhibiting Sema3A and cerebral miR-195 levels decreased with age, both of which urged us to explore the role of miR-195 and miR-195-regulated Sema3 family members in age-associated dementia. METHODS: miR-195a KO mice were used to assess the effect of miR-195 on aging and cognitive functions. Sema3D was predicted as a miR-195 target by TargetScan and then verified by luciferase reporter assay, while effects of Sema3D and miR-195 on neural senescence were assessed by beta-galactosidase and dendritic spine density. Cerebral Sema3D was over-expressed by lentivirus and suppressed by si-RNA, and effects of over-expression of Sema3D and knockdown of miR-195 on cognitive functions were assessed by Morris Water Maze, Y-maze, and open field test. The effect of Sema3D on lifespan was assessed in Drosophila. Sema3D inhibitor was developed using homology modeling and virtual screening. One-way and two-way repeated measures ANOVA were applied to assess longitudinal data on mouse cognitive tests. RESULTS: Cognitive impairment and reduced density of dendritic spine were observed in miR-195a knockout mice. Sema3D was identified to be a direct target of miR-195 and a possible contributor to age-associated neurodegeneration as Sema3D levels showed age-dependent increase in rodent brains. Injection of Sema3D-expressing lentivirus caused significant memory deficits while silencing hippocampal Sema3D improved cognition. Repeated injections of Sema3D-expressing lentivirus to elevate cerebral Sema3D for 10 weeks revealed a time-dependent decline of working memory. More importantly, analysis of the data on the Gene Expression Omnibus database showed that Sema3D levels were significantly higher in dementia patients than normal controls (p < 0.001). Over-expression of homolog Sema3D gene in the nervous system of Drosophila reduced locomotor activity and lifespan by 25%. Mechanistically, Sema3D might reduce stemness and number of neural stem cells and potentially disrupt neuronal autophagy. Rapamycin restored density of dendritic spines in the hippocampus from mice injected with Sema3D lentivirus. Our novel small molecule increased viability of Sema3D-treated neurons and might improve autophagy efficiency, which suggested Sema3D could be a potential drug target. Video Abstract CONCLUSION: Our results highlight the importance of Sema3D in age-associated dementia. Sema3D could be a novel drug target for dementia treatment.

Alternative role of glucagon-like Peptide-1 receptor agonists in neurodegenerative diseases.

Aging is a crucial risk factor for common neurodegenerative diseases, such as Alzheimer's disease (AD) and Parkinson's disease (PD). Limited options are available for the treatment of age-related, multiple pathogenic mechanism-contributed diseases that usually advance to irreversible conditions with severe neurological deficits and result in a heavy socioeconomic burden on patients, families, and society. A therapy that decelerates disease progression and reduces the socioeconomic burden stemming from these diseases is required. Glucagon-like peptide-1 receptor (GLP-1R) is an important class of medication for type 2 diabetes mellitus (T2DM). Through pancreatic effects, GLP-1R agonists can stimulate insulin secretion, increase ß-cell proliferation, reduce ß-cell apoptosis, and inhibit glucagon secretion in patients with T2DM. Currently, seven clinically approved GLP-1R agonists are used for T2DM: exenatide, liraglutide, lixisenatide, extended-release exenatide, albiglutide, dulaglutide, and semaglutide. Besides the pancreas, GLP-1Rs are also expressed in organs, such as the gastrointestinal tract, heart, lung, kidney, and brain, indicating their potential use in diseases other than T2DM. Emerging evidence reveals that GLP-1R agonists possess pleiotropic effects that enrich neurogenesis, diminish apoptosis, preclude neurons from oxidative stress, and reduce neuroinflammation in various neurological conditions. These favorable effects may also be employed in neurodegenerative diseases. Herein, we reviewed the recent progress, both in preclinical studies and clinical trials, regarding these clinically used GLP-1R agonists in aging-related neurodegenerative diseases, mainly AD and PD. We stress the pleiotropic characteristics of GLP-1R agonists as repurposing drugs to target multiple pathological mechanisms and for use in the future for these devastating neurodegenerative conditions.

Fluoroquinolones Suppress TGF-ß and PMA-Induced MMP-9 Production in Cancer Cells: Implications in Repurposing Quinolone Antibiotics for Cancer Treatment.

BACKGROUND: Fluoroquinolones (FQs) are potent antimicrobials with multiple effects on host cells and tissues. Although FQs can attenuate cancer invasion and metastasis, the underlying molecular mechanisms remain unclear. Matrix metalloproteinase-9 (MMP-9) has functional roles in tumor angiogenesis, invasion, and metastasis, and is associated with cancer progression and poor prognosis, suggesting that inhibitors of MMP-9 activity and transcription are prime candidates for cancer therapy. Despite numerous preclinical data supporting the use of MMP-9 inhibitors as anticancer drugs, the few available examples are not therapeutically useful due to low specificity and off-target effects. We examined the effects of FQs on MMP-9 production in cancer cells following transforming growth factor beta (TGF-ß) and phorbol 12-myristate 13-acetate (PMA) stimulation. EXPERIMENTAL APPROACHES: Using confluent cultures of HepG2 and A549 cells, the effects of FQs (ciprofloxacin, levofloxacin, clinafloxacin, gatifloxacin, and enrofloxacin) on TGF-ß and PMA-induced MMP-9 mRNA expression and production were studied in RNA extracts and culture supernatants, respectively. FQs specifically abrogated TGF-ß and PMA-induced MMP-9 levels and activity in a concentration and time-dependent manner, without affecting other MMPs or proteins involved in epithelial-mesenchymal transition. Additionally, FQs inhibited TGF-ß and PMA-induced cell migration via p38 and cyclic AMP signaling pathways. CONCLUSIONS AND IMPLICATIONS: Overall, we demonstrated that FQs inhibit cancer cell migration and invasion by downregulating MMP-9 expression and revealed the cellular mechanisms underlying their potential value in cancer treatment.

Empagliflozin Ameliorates Free Fatty Acid Induced-Lipotoxicity in Renal Proximal Tubular Cells via the PPARγ/CD36 Pathway in Obese Mice.

High serum levels of free fatty acids (FFAs) could contribute to obesity-induced nephropathy. CD36, a class B scavenger receptor, is a major receptor mediating FFA uptake in renal proximal tubular cells. Empagliflozin, a new anti-diabetic agent, is a specific inhibitor of sodium-glucose co-transporter 2 channels presented on renal proximal tubular cells and inhibits glucose reabsorption. In addition, empagliflozin has shown renoprotective effects. However, the mechanism through which empagliflozin regulates CD36 expression and attenuates FFA-induced lipotoxicity remains unclear. Herein, we aimed to elucidate the crosstalk between empagliflozin and CD36 in FFA-induced renal injury. C57BL/6 mice fed a high-fat diet (HFD) and palmitic acid-treated HK-2 renal tubular cells were used for in vivo and in vitro assessments. Empagliflozin attenuated HFD-induced body weight gain, insulin resistance, and inflammation in mice. In HFD-fed mice, CD36 was upregulated in the tubular area of the kidney, whereas empagliflozin attenuated CD36 expression. Furthermore, empagliflozin downregulated the expression of peroxisome proliferator-activated receptor (PPAR)-γ. Treatment with a PPARγ inhibitor (GW9662) did not further decrease PPARγ expression, whereas a PPARγ antagonist reversed this effect; this suggested that empagliflozin may, at least partly, decrease CD36 by modulating PPARγ. In conclusion, empagliflozin can ameliorate FFA-induced renal tubular injury via the PPARγ/CD36 pathway.

Potential Roles of Sestrin2 in Alzheimer's Disease: Antioxidation, Autophagy Promotion, and Beyond.

Alzheimer's disease (AD) is the most common age-related neurodegenerative disease. It presents with progressive memory loss, worsens cognitive functions to the point of disability, and causes heavy socioeconomic burdens to patients, their families, and society as a whole. The underlying pathogenic mechanisms of AD are complex and may involve excitotoxicity, excessive generation of reactive oxygen species (ROS), aberrant cell cycle reentry, impaired mitochondrial function, and DNA damage. Up to now, there is no effective treatment available for AD, and it is therefore urgent to develop an effective therapeutic regimen for this devastating disease. Sestrin2, belonging to the sestrin family, can counteract oxidative stress, reduce activity of the mammalian/mechanistic target of rapamycin (mTOR), and improve cell survival. It may therefore play a crucial role in neurodegenerative diseases like AD. However, only limited studies of sestrin2 and AD have been conducted up to now. In this article, we discuss current experimental evidence to demonstrate the potential roles of sestrin2 in treating neurodegenerative diseases, focusing specifically on AD. Strategies for augmenting sestrin2 expression may strengthen neurons, adapting them to stressful conditions through counteracting oxidative stress, and may also adjust the autophagy process, these two effects together conferring neuronal resistance in cases of AD.

Development and Validation of KASP Assays for the Genotyping of Racing Performance-Associated Single Nucleotide Polymorphisms in Pigeons.

Pigeon racing's recent upturn in popularity can be attributed in part to the huge prize money involved in these competitions. As such, methods to select pigeons with desirable genetic characteristics for racing or for selective breeding have also been gaining more interest. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) for genotyping-specific genes is one of the most commonly used molecular techniques, which can be costly, laborious and time consuming. The present study reports the development of an alternative genotyping method that employs Kompetitive Allele Specific Polymerase Chain Reaction (KASP) technology with specifically designed primers to detect previously reported racing performance-associated polymorphisms within the LDHA, MTYCB, and DRD4 genes. To validate, KASP assays and PCR-RFLP assays results from 107 samples genotyped for each of the genes were compared and the results showed perfect (100%) agreement of both methods. The developed KASP assays present an alternative rapid, reliable, and cost-effective method to identify polymorphisms in pigeons.

The AST-120 Recovers Uremic Toxin-Induced Cognitive Deficit via NLRP3 Inflammasome Pathway in Astrocytes and Microglia.

Chronic kidney disease (CKD) is characterized by the progressive loss of renal function; moreover, CKD progression commonly leads to multiple comorbidities, including neurological dysfunction and immune disorders. CKD-triggered neuroinflammation significantly contributes to cognitive impairment. This study aimed to investigate the contribution of uremic toxins to cognitive impairment. Serum creatinine, blood urea nitrogen (BUN), indoxyl sulfate (IS), and p-cresol sulfate (PCS) levels were measured using an enzyme-linked immunosorbent assay and high-performance liquid chromatography. The creatinine, BUN, IS, and PCS levels were increased from 4 weeks after 5/6-nephrectomy in mice, which suggested that 5/6-nephrectomy could yield a CKD animal model. Further, CKD mice showed significantly increased brain and serum indoxyl sulfate levels. Immunohistochemistry analysis revealed hippocampal inflammation and NLRP3-inflammasomes in astrocytes. Further, the Y-maze and Morris water maze tests revealed learning and memory defects in CKD mice. AST-120, which is also an IS absorbent, effectively reduced serum and hippocampal IS levels as well as reversed the cognitive impairment in CKD mice. Additionally, NLRP3-knockout mice that underwent 5/6-nephrectomy showed no change in cognitive function. These findings suggested that IS is an important uremic toxin that induces NLRP3 inflammasome-mediated not only in microglia, but it also occurred in astrocytic inflammation, which subsequently causes cognitive impairment.

Inhibition of PAI-1 Blocks PD-L1 Endocytosis and Improves the Response of Melanoma Cells to Immune Checkpoint Blockade.

Immune checkpoint molecules, especially PD-1 and its ligand PD-L1, act as a major mechanism of cancer immune evasion. Although anti-PD-1/PD-L1 monotherapy increases therapeutic efficacy in melanoma treatment, only a subset of patients exhibits long-term tumor remission, and the underlying mechanism of resistance to PD-1/PD-L1 inhibitors remains unclear. In this study, we demonstrated that cell surface retention of PD-L1 is inversely correlated with PAI-1 expression in vitro, in vivo, and in clinical specimens. Moreover, extracellular PAI-1 induced the internalization of surface-expressed PD-L1 by triggering clathrin-mediated endocytosis. The endocytosed PD-L1 was transported to lysosomes for degradation by endolysosomal systems, resulting in the reduction of surface PD-L1. Notably, inhibition of PAI-1 by pharmacological inhibitor with tiplaxtinin led to elevated PD-L1 expression on the plasma membrane, both in vitro and in vivo. Strikingly, targeting PAI-1 by tiplaxtinin treatment synergizes with anti-PD-L1 immune checkpoint blockade therapy in a syngeneic murine model of melanoma. Our findings demonstrate a role for PAI-1 activity in immune checkpoint modulation by promoting surface PD-L1 for lysosomal degradation and provides an insight into the combination of PAI-1 inhibition and anti-PD-L1 immunotherapy as a promising therapeutic regimen for melanoma treatment.

Effects of Early Rehydration on Brain Perfusion and Infarct Core after Middle Cerebral Artery Occlusion in Rats.

Imaging evidence for the effect of rehydration on cerebral perfusion and brain ischemia has never been proposed in the literature. This study aimed to test the hypothesis that early rehydration treatment can improve cerebral perfusion and decrease infarct volume, consequently reducing mortality of dehydrated stroke animals. METHODS: Thirty dehydrated experimental rats were randomly assigned to either a rehydration or control group after middle cerebral artery occlusion (MCAO). Diffusion-weighted imaging and dynamic contrast enhancement perfusion imaging were performed at 30 min and 6 h after MCAO using a 9.4T MR imaging scanner to measure the infarct volume and brain perfusion. RESULTS: The survival rates after the first MRI scan were 91.7% for the rehydration group and 58.3% for the control group (p = 0.059). The survival rates after the second MRI scan were 66.7% for the rehydration group, and 8.3% of the control group survived (p = 0.003). The infarct volume of the rehydration group was significantly smaller than control group at 30 min after MCAO (p = 0.007). The delay time and time to maximum were significantly shorter in the rehydration group at 30 min (p = 0.004 and 0.035, respectively). CONCLUSIONS: The findings suggest that early rehydration therapy can decrease the infarct volume, shorten the delay time of cerebral perfusion, and increase survival of dehydrated ischemic-stroke rats. This preliminary study provided imaging evidence that more intensive early hydration therapies and reperfusion strategies may be necessary for acute stroke patients with dehydrated status.

Plasminogen Activator Inhibitor-1 Secretion by Autophagy Contributes to Melanoma Resistance to Chemotherapy through Tumor Microenvironment Modulation.

Autophagy plays a crucial role in maintenance of cellular homeostasis via intracellular signaling pathways, lysosomal degradation of selective cargo and mediating protein secretion. Dysregulation of autophagy has been implicated in tumorigenesis, tumor progression, and resistance to therapy. However, the mechanism of autophagy-dependent secretion involved in the responsiveness to chemotherapy is poorly understood. In this study, we showed that mitoxantrone (MitoX), a chemotherapeutic agent used for treating various cancers but not melanoma, induced autophagy in melanoma cells in vitro and in vivo. We also found that plasminogen activator inhibitor (PAI)-1 secretion by MitoX-induced autophagy modulated the pro-tumoral microenvironment. Attenuation of PAI-1 activity using a specific inhibitor, tiplaxtinin (TPX), or by targeting the autophagy gene, Becn1, induced efficient antitumor immunity, thereby overcoming the resistance to MitoX in vivo. Of note, the therapeutic efficacy of TPX was abolished in MitoX-treated Becn1-defective tumors. Collectively, our results demonstrate that tumor autophagy-dependent PAI-1 secretion impairs the therapeutic efficacy of MitoX and highlight targeting of tumor autophagy or its secretory cargo, PAI-1, as a novel strategy to repurpose MitoX-based chemotherapy for melanoma treatment.

Group 2 innate lymphoid cells contribute to IL-33-mediated alleviation of cardiac fibrosis.

Rationale: The major cause of heart failure is myocardium death consequent to detrimental cardiac remodeling and fibrosis following myocardial infarction. The cardiac protective cytokine interleukin (IL)-33, which signals by ST2 receptor binding, is associated with group 2 innate lymphoid cell (ILC2) activation and regulates tissue homeostasis and repair following tissue injury in various tissues. However, the distribution and role of IL-33-responsive ILC2s in cardiac fibrosis remain unclear. In this study, we elucidated the roles of IL-33-responsive cardiac-resident ILC2s and IL-33-mediated immunomodulatory functions in cardiac fibrosis. Methods: We examined the distribution of cardiac ILC2s by using flow cytometry. The roles of IL-33-mediated ILC2 expansion in cardiac fibrosis was evaluated in the mouse model of catecholamine-induced cardiac fibrosis. ILC-deficient Rag2‒/‒IL2Rγc‒/‒ mice were implemented to determine the contribution of endogenous ILC in the progression of cardiac fibrosis. Histopathological assessments, speckle tracking echocardiography, and transcriptome profile analysis were performed to determine the effects of IL-33-mediated cardiac protective functions. Results: We identified the resident cardiac ILC2s, which share similar cell surface marker and transcriptional factor expression characteristics as peripheral blood and lung tissue ILC2s. IL-33 treatment induced ILC2 expansion via ST2. In vivo, ILC-deficient Rag2‒/‒IL2Rγc‒/‒ mice developed exacerbated cardiac fibrosis following catecholamine-induced stress cardiac injury. IL-33 treatment expanded cardiac ILC2s and revealed protective effects against cardiac tissue damage with reduced cardiomyocyte death, immune cell infiltration, tissue fibrosis, and improved myocardial function. Transcriptome analysis revealed that IL-33 attenuated extracellular matrix synthesis- and fibroblast activation-associated gene expressions. IL13-knockout or epidermal growth factor receptor (EGFR) inhibition abolished IL-33-mediated cardiac protective function, confirming IL-13 and EGFR signaling as crucial for IL-33-mediated cardioprotective responses. Moreover, ILC2-produced BMP-7 served as a novel anti-fibrotic factor to inhibit TGF-ß1-induced cardiac fibroblast activation. Conclusion: Our findings indicate the presence of IL-33-responsive ILC2s in cardiac tissue and that IL-33-mediated ILC2 expansion affords optimal cardioprotective function via ILC2-derived factors. IL-33-mediated immunomodulation is thus a promising strategy to promote tissue repair and alleviate cardiac fibrosis following acute cardiac injury.

Melatonin ameliorates methamphetamine-induced cognitive impairments by inhibiting neuroinflammation via suppression of the TLR4/MyD88/NFκB signaling pathway in the mouse hippocampus.

Methamphetamine (METH) is a highly addictive psychostimulant that causes significant health issues due to high prevalence of its illegal use. Chronic use of METH is associated with cognitive impairments in both human and animal studies, but the underlying mechanism remains unclear. METH-induced neuroinflammation is, potentially, one of the factors that causes cognitive impairments. Therefore, the present study aimed to assess whether melatonin could provide protection against inflammation, in a manner comparable to the anti-inflammatory agent, minocycline, with consequent improvements of METH-induced cognitive impairments and associated abnormalities in the mouse hippocampus. Results from the Morris water maze (MWM) test and the novel object recognition test (NORT) showed that melatonin given after METH injections could ameliorate both METH-induced spatial and recognition memory impairments. These memory impairments are associated with changes in the neuroinflammatory profiles, including IL-6, IL-1ß, and TNF-α, both in the blood serum and hippocampus of adult mice. METH-treated mice also exhibited reactive astrocytes and activated microglia in the hippocampus. METH-induced activation of glial cells is associated with the activation of the TLR4/MyD88/NFκB signaling pathway. Moreover, melatonin administration led to recovery of these METH-induced markers to control levels. Thus, we conclude that melatonin could potentially be used as a cognitive enhancer and anti-inflammatory agent in the treatment of METH use disorder in humans.

Melatonin reverts methamphetamine-induced learning and memory impairments and hippocampal alterations in mice.

AIMS: Methamphetamine (METH) has become a major public health problem because of its abuse and profound neurotoxic effects, causing alterations in brain structure and function, and impairing cognitive functions, including attention, decision making, emotional memory, and working memory. This study aimed to determine whether melatonin (MEL), the circadian-control hormone, which has roles beyond circadian rhythm regulation, could restore METH-induced cognitive and neuronal impairment. MAIN METHODS: Mice were treated with either METH (1 mg/kg) or saline for 7 days, followed by MEL (10 mg/kg) or saline for another 14 days. The Morris water maze (MWM) test was performed one day after the last saline or MEL injection. The hippocampal neuronal density, synaptic density, and receptors involved in learning and memory, along with downstream signaling molecules (NMDA receptor subunits GluN2A, GluN2B, and CaMKII) were investigated by immunoblotting. KEY FINDINGS: METH administration significantly extended escape latency in learning phase and reduced the number of target crossings in memory test-phase as well as decreased the expression of BDNF, NMDA receptors, TrkB receptors, CaMKII, ßIII tubulin, and synaptophysin. MEL treatment significantly ameliorated METH-induced increased escape latency, decreased the number of target crossings and decreased expression of BDNF, NMDA receptors, TrkB receptors, CaMKII, ßIII tubulin and synaptophysin. SIGNIFICANCE: METH administration impairs learning and memory in mice, and MEL administration restores METH-induced neuronal impairments which is probably through the changes in BDNF, NMDA receptors, TrkB receptors, CaMKII, ßIII tubulin and synaptophysin. Therefore, MEL is potentially an innovative and promising treatment for learning and memory impairment of humans.

miR-195 reduces age-related blood-brain barrier leakage caused by thrombospondin-1-mediated selective autophagy.

Blood-brain barrier (BBB) disruption contributes to neurodegenerative diseases. Loss of tight junction (TJ) proteins in cerebral endothelial cells (ECs) is a leading cause of BBB breakdown. We recently reported that miR-195 provides vasoprotection, which urges us to explore the role of miR-195 in BBB integrity. Here, we found cerebral miR-195 levels decreased with age, and BBB leakage was significantly increased in miR-195 knockout mice. Furthermore, exosomes from miR-195-enriched astrocytes increased endothelial TJ proteins and improved BBB integrity. To decipher how miR-195 promoted BBB integrity, we first demonstrated that TJ proteins were metabolized via autophagic-lysosomal pathway and the autophagic adaptor p62 was necessary to promote TJ protein degradation in cerebral ECs. Next, proteomic analysis of exosomes revealed miR-195-suppressed thrombospondin-1 (TSP1) as a major contributor to BBB disruption. Moreover, TSP1 was demonstrated to activate selective autophagy of TJ proteins by increasing the formation of claudin-5-p62 and ZO1-p62 complexes in cerebral ECs while TSP1 impaired general autophagy. Delivering TSP1 antibody into the circulation showed dose-dependent reduction of BBB leakage by 20%-40% in 25-month-old mice. Intravenous or intracerebroventricular injection of miR-195 rescued TSP1-induced BBB leakage. Dementia patients with BBB damage had higher levels of serum TSP1 compared to those without BBB damage (p = 0.0015), while the normal subjects had the lowest TSP1 (p < 0.0001). Taken together, the study implies that TSP1-regulated selective autophagy facilitates the degradation of TJ proteins and weakens BBB integrity. An adequate level of miR-195 can suppress the autophagy-lysosome pathway via a reduction of TSP1, which may be important for maintaining BBB function.

The Neurotrophic Function of Glucagon-Like Peptide-1 Promotes Human Neuroblastoma Differentiation via the PI3K-AKT Axis.

 Background: Neurons are terminally-differentiated cells that generally develop from neuronal stem cells stimulated by various neurotrophic factors such as NGF, BDNF, NT3, and NT-4. Neurotrophic factors have multiple functions for neurons, including enabling neuronal development, growth, and protection. Glucagon-like peptide-1 (GLP-1) is an intestinal-secreted incretin that enhances cellular glucose up-take to decrease blood sugar levels. However, many studies suggest that the function of GLP-1 is not limited to the regulation of blood sugar levels. Instead, it may also act as a neurotrophic factor with a role in ensuring neuronal survival and neurite outgrowth, as well as protecting synaptic plasticity and memory formation. Methods: The SH-SY5Y cells were differentiated by sequential treatments of retinoic acid and GLP-1 treatment within polyethylenimine-coated dishes under serum-free Neurobasal medium. PI3K inhibitor (LY294002) and MEK inhibitor (U0126) were used to determine the signaling pathway in regulation of neuronal differentiation. Neuronal marker (TUJ1) and synaptic markers (synapsin 1, synaptophysin, and PSD95) as well as single cell patch-clamp were applied to determine maturity of neurons. Antibodies of AMPA receptor, NMDA receptor subunit 2A, dopamine receptor D1, muscarinic acetylcholine receptor 2, and nicotinic acetylcholine receptor α4 were used to examine the types of differentiated neurons. Results: Our study's results demonstrated that the treatment with GLP-1 of SH-SY5Y human neuroblastoma cells increased the expression of AMPA receptors, NMDA receptors, dopamine receptors, synaptic proteins-synapsin 1, synaptophysin, and postsynaptic density protein 95, but not muscular and nicotinic acetylcholine receptors. In addition, the biomarker of dividing neuronal cells, vimentin, was decreased after treatment with GLP-1. Tuj1 immunostaining images showed that GLP-1 induced neurite processes and the development of neuronal morphologies. The GLP-1-differentiated neurons were able to be induced to generate action potentials by single cell patch-clamp. Our study also suggested that the PI3K-AKT axis is the dominant signaling pathway promoting the differentiation of SH-SY5Y cells into mature and functional neurons in response to GLP-1 receptor activation. Conclusions: The sequential treatment of retinoic acid and GLP-1 within a serum-free medium is able to trigger the differentiation of SH-SY5Y cells into morphologically and physiologically mature glutamatergic and dopaminergic neurons.

Circulating MicroRNAs from Serum Exosomes May Serve as a Putative Biomarker in the Diagnosis and Treatment of Patients with Focal Cortical Dysplasia.

Focal cortical dysplasia (FCD) is a congenital malformation of cortical development where the cortical neurons located in the brain area fail to migrate in the proper formation. Epilepsy, particularly medically refractory epilepsy, is the most common clinical presentation for all types of FCD. This study aimed to explore the expression change of circulating miRNAs in patients with FCD from serum exosomes. A total of nine patients with FCD and four healthy volunteers were enrolled in this study. The serum exosomes were isolated from the peripheral blood of the subjects. Transmission electron microscopy (TEM) was used to identify the exosomes. Both exosomal markers and neuronal markers were detected by Western blotting analysis to prove that we could obtain central nervous system-derived exosomes from the circulation. The expression profiles of circulating exosomal miRNAs were assessed using next-generation sequencing analysis (NGS). We obtained a total of 107 miRNAs with dominant fold change (>2-fold) from both the annotated 5p-arm and 3p-arm of 2780 mature miRNAs. Based on the integrated platform of HMDD v3.2, miRway DB and DIANA-miRPath v3.0 online tools, and confirmed by MiRBase analysis, four potentially predicted miRNAs from serum exosomes in patients with FCD were identified, including miR194-2-5p, miR15a-5p, miR-132-3p, and miR-145-5p. All four miRNAs presented upregulated expression in patients with FCD compared with controls. Through Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis and pathway category of four target miRNAs, we found eight possible signaling pathways that may be related to FCD. Among them, we suggest that the mTOR signaling pathway, PI3K-Akt signaling pathway, p53 signaling pathway, and cell cycle regulation and TGF-beta signaling pathway are high-risk pathways that play a crucial role in the pathogenesis of FCD and refractory epilepsy. Our results suggest that the circulating miRNAs from exosomes may provide a potential biomarker for diagnostic, prognostic, and therapeutic adjuncts in patients with FCD and refractory epilepsy.

Emerging Roles of Inhibitor of Differentiation-1 in Alzheimer's Disease: Cell Cycle Reentry and Beyond.

Inhibitor of DNA-binding/differentiation (Id) proteins, a family of helix-loop-helix (HLH) proteins that includes four members of Id1 to Id4 in mammalian cells, are critical for regulating cell growth, differentiation, senescence, cell cycle progression, and increasing angiogenesis and vasculogenesis, as well as accelerating the ability of cell migration. Alzheimer's disease (AD), the most common neurodegenerative disease in the adult population, manifests the signs of cognitive decline, behavioral changes, and functional impairment. The underlying mechanisms for AD are not well-clarified yet, but the aggregation of amyloid-beta peptides (Aßs), the major components in the senile plaques observed in AD brains, contributes significantly to the disease progression. Emerging evidence reveals that aberrant cell cycle reentry may play a central role in Aß-induced neuronal demise. Recently, we have shown that several signaling mediators, including Id1, hypoxia-inducible factor-1 (HIF-1), cyclin-dependent kinases-5 (CDK5), and sonic hedgehog (Shh), may contribute to Aß-induced cell cycle reentry in postmitotic neurons; furthermore, Id1 and CDK5/p25 mutually antagonize the expression/activity of each other. Therefore, Id proteins may potentially have clinical applications in AD. In this review article, we introduce the underlying mechanisms for cell cycle dysregulation in AD and present some examples, including our own studies, to show different aspects of Id1 in terms of cell cycle reentry and other signaling that may be crucial to alter the neuronal fates in this devastating neurodegenerative disease. A thorough understanding of the underlying mechanisms may provide a rationale to make an earlier intervention before the occurrence of cell cycle reentry and subsequent apoptosis in the fully differentiated neurons during the progression of AD or other neurodegenerative diseases.

Emerging Roles of Interleukin-33-responsive Kidney Group 2 Innate Lymphoid Cells in Acute Kidney Injury.

Interleukin (IL)-33, a member of the IL-1 family of cytokines, is involved in innate and adaptive immune responses. IL-33 triggers pleiotropic immune functions in multiple types of immune cells, which express the IL-33 receptor, ST2. Recent studies have revealed the potential applications of IL-33 for treating acute kidney injury in preclinical animal models. However, IL-33 and IL-33-responding immune cells are reported to exhibit both detrimental and beneficial roles. The IL-33-mediated immunomodulatory functions have been investigated using loss-of-function approaches, such as IL33-deficient mice, IL-33 antagonists, or administration of exogenous IL-33 recombinant protein. This review will discuss the key findings on IL-33-mediated activation of kidney resident group 2 innate lymphoid cells (ILC2s) and summarize the current understanding of the differential functions of endogenous IL-33 and exogenous IL-33 and their potential implications in treating acute kidney injury.

The potential of drug repurposing combined with reperfusion therapy in cerebral ischemic stroke: A supplementary strategy to endovascular thrombectomy.

Stroke is the major cause of adult disability and the second or third leading cause of death in developed countries. The treatment options for stroke (thrombolysis or thrombectomy) are restricted to a small subset of patients with acute ischemic stroke because of the limited time for an efficacious response and the strict criteria applied to minimize the risk of cerebral hemorrhage. Attempts to develop new treatments, such as neuroprotectants, for acute ischemic stroke have been costly and time-consuming and to date have yielded disappointing results. The repurposing approved drugs known to be relatively safe, such as statins and minocycline, may provide a less costly and more rapid alternative to new drug discovery in this clinical condition. Because adequate perfusion is thought to be vital for a neuroprotectant to be effective, endovascular thrombectomy (EVT) with advanced imaging modalities offers the possibility of documenting reperfusion in occluded large cerebral vessels. An examination of established medications that possess neuroprotective characters using in a large-vessel occlusive disorder with EVT may speed the identification of new and more broadly efficacious medications for the treatment of ischemic stroke. These approaches are highlighted in this review along with a critical assessment of drug repurposing combined with reperfusion therapy as a supplementary means for halting or mitigating stroke-induced brain damage.