ß-Arrestin 2 and Epac2 Cooperatively Mediate DRD1-Stimulated Proliferation of Human Neural Stem Cells and Growth of Human Cerebral Organoids.

G-protein-coupled receptors (GPCRs) reportedly relay specific signals, such as dopamine and serotonin, to regulate neurogenic processes although the underlying signaling pathways are not fully elucidated. Based on our previous work, which demonstrated dopamine receptor D1 (DRD1) effectively induces the proliferation of human neural stem cells, here we continued to show the knockout of ß-arrestin 2 by CRISPR/Cas9 technology significantly weakened the DRD1-induced proliferation and neurosphere growth. Furthermore, inhibition of the downstream p38 MAPK by its specific inhibitors or small hairpin RNA mimicked the weakening effect of ß-arrestin 2 knockout. In addition, blocking of Epac2, a PKA independent signal pathway, by its specific inhibitors or small hairpin RNA also significantly reduced DRD1-induced effects. Simultaneous inhibition of ß-arrestin 2/p38 MAPK and Epac2 pathways nearly abolished the DRD1-stimulated neurogenesis, indicating the cooperative contribution of both pathways. Consistently, the expansion and folding of human cerebral organoids as stimulated by DRD1 were also mediated cooperatively by both ß-arrestin 2/p38 MAPK and Epac2 pathways. Taken together, our results reveal that GPCRs apply at least 2 different signal pathways to regulate neurogenic processes in a delicate and balanced manners.

Galangin Rescues Alzheimer's Amyloid-ß Induced Mitophagy and Brain Organoid Growth Impairment.

Dysfunctional mitochondria and mitophagy are hallmarks of Alzheimer's disease (AD). It is widely accepted that restoration of mitophagy helps to maintain cellular homeostasis and ameliorates the pathogenesis of AD. It is imperative to create appropriate preclinical models to study the role of mitophagy in AD and to assess potential mitophagy-targeting therapies. Here, by using a novel 3D human brain organoid culturing system, we found that amyloid-ß (Aß1-42,10 µM) decreased the growth level of organoids, indicating that the neurogenesis of organoids may be impaired. Moreover, Aß treatment inhibited neural progenitor cell (NPC) growth and induced mitochondrial dysfunction. Further analysis revealed that mitophagy levels were reduced in the brain organoids and NPCs. Notably, galangin (10 µM) treatment restored mitophagy and organoid growth, which was inhibited by Aß. The effect of galangin was blocked by the mitophagy inhibitor, suggesting that galangin possibly acted as a mitophagy enhancer to ameliorate Aß-induced pathology. Together, these results supported the important role of mitophagy in AD pathogenesis and suggested that galangin may be used as a novel mitophagy enhancer to treat AD.

Rescue of Mitochondrial SIRT3 Ameliorates Ischemia-like Injury in Human Endothelial Cells.

Structural and functional alterations of vasculature caused by age-related factors is critically involved in the pathogenesis of ischemic stroke. The longevity genes sirtuins (SIRTs) are extensively investigated in aging-associated pathologies, but their distinct roles in ischemic stroke still remain to be clarified. To address this question, we applied oxygen and glucose deprived/reperfusion (OGD/R) to induce ischemic injury in human endothelial cells (ECs), which are the main component of vasculature in the brain. The results showed that OGD/R led to various damages to ECs, including compromised cell viability, increased LDH release, overproduced ROS, enhanced apoptosis and caspase activity. Meanwhile, the expression of mitochondrial SIRT3 was robustly decreased in ECs after OGD/R treatment. Consistently, rescue of SIRT3 by ectopic expression, but not nuclear SIRT1, in ECs reversed the OGD/R-induced cell damage. Interestingly, some front-line drugs for ischemic stroke, including clopidogrel, aspirin and dl-3-n-butylphthalide (NBP), also rescued SIRT3 and reduced OGD/R-induced endothelial injury, suggesting that the recovery of SIRT3 expression was critical for the protection of these drugs. Moreover, our results demonstrated that 10-hydroxy-NBP (OHNBP), a major metabolite of NBP, showed better blood-brain barrier crossing capability than NBP, but still retained the effects on SIRT3 by NBP. Together, our results suggested that SIRT3 may serve as a potential novel target for treatment of ischemic stroke.

MicroRNA miR-326 regulates TH-17 differentiation and is associated with the pathogenesis of multiple sclerosis.

Interleukin 17 (IL-17)-producing T helper cells (T(H)-17 cells) are increasingly recognized as key participants in various autoimmune diseases, including multiple sclerosis. Although sets of transcription factors and cytokines are known to regulate T(H)-17 differentiation, the role of noncoding RNA is poorly understood. Here we identify a T(H)-17 cell-associated microRNA, miR-326, whose expression was highly correlated with disease severity in patients with multiple sclerosis and mice with experimental autoimmune encephalomyelitis (EAE). In vivo silencing of miR-326 resulted in fewer T(H)-17 cells and mild EAE, and its overexpression led to more T(H)-17 cells and severe EAE. We also found that miR-326 promoted T(H)-17 differentiation by targeting Ets-1, a negative regulator of T(H)-17 differentiation. Our data show a critical role for microRNA in T(H)-17 differentiation and the pathogenesis of multiple sclerosis.

Analysis and Improvement of a Dual-Core Photonic Crystal Fiber Sensor.

The characteristics of the dual-core photonic crystal fiber (PCF) sensor are studied using the finite element method (FEM), and the structure is improved according to the numerical simulation results. The results show that whether or not the four large air holes far away from the geometry center of the PCF are filled with analyte has no influence on the wavelength sensitivity of the sensor which means those holes can be replaced by small air holes. The wavelength sensitivity can be tuned by adjusting the sizes of the other large air holes which are as for liquid holes. The dynamic detection range of the refractive index (RI) is from 1.33 to 1.51. In particular, high linearity is obtained in the range of 1.44 to 1.51. The sensitivity is as high as 6021 nm/RIU when the liquid holes are the smallest. When liquid holes are tangential with the envelope of first layer air holes, the wavelength sensitivity is 4028 nm/RIU, and the coefficient of determination (R²) is 0.99822 when the RI of the analyte varies from 1.44 to 1.51 which shows that high sensitivity and good linearity are both obtained.

Deficiency of ß-arrestin1 ameliorates collagen-induced arthritis with impaired TH17 cell differentiation.

Rheumatoid arthritis (RA) is an inflammatory disease in which interleukin 17 (IL-17)-producing T helper 17 (T(H)17) cells have been critically involved. We show that in patients with RA, the expression of a multifunctional regulator ß-arrestin1 was significantly up-regulated in peripheral and synovial CD4(+) T cells, which correlated well with active phases of RA. In collagen-induced arthritis, deficiency of ß-arrestin1 ameliorated disease with decreased T(H)17 cell differentiation, proinflammatory cytokine production, synovitis, and cartilage and bone destruction. Further mechanistic study reveals that ß-arrestin1 promoted signal transducer and activator of transcription 3 (STAT3) activation required for T(H)17 cell differentiation through scaffolding the interaction of Janus kinase 1 and STAT3. These findings indicate a critical role for ß-arrestin1 in the pathogenesis of collagen-induced arthritis and T(H)17 cell differentiation and suggest ß-arrestin1 as a potential diagnostic biomarker and therapeutic target for RA.

The anti-spasticity drug baclofen alleviates collagen-induced arthritis and regulates dendritic cells.

Baclofen is used clinically as a drug that treats spasticity, which is a syndrome characterized by excessive contraction of the muscles and hyperflexia in the central nervous system (CNS), by activating GABA(B) receptors (GABA(B)Rs). Baclofen was recently reported to desensitize chemokine receptors and to suppress inflammation through the activation of GABA(B)Rs. GABA(B)Rs are expressed in various immune cells, but the functions of these receptors in autoimmune diseases remain largely unknown. In this study, we investigated the effects of baclofen in murine collagen-induced arthritis (CIA). Oral administration of baclofen alleviated the clinical development of CIA, with a reduced number of IL-17-producing T helper 17 (T(H)17) cells. In addition, baclofen treatment suppressed dendritic cell (DC)-primed T(H)17 cell differentiation by reducing the production of IL-6 by DCs in vitro. Furthermore, the pharmacological and genetic blockade of GABA(B)Rs in DCs weakened the effects of baclofen, indicating that GABA(B)Rs are the molecular targets of baclofen on DCs. Thus, our findings revealed a potential role for baclofen in the treatment of CIA, as well as a previously unknown signaling pathway that regulates DC function.

Flavonoid chrysin activates both TrkB and FGFR1 receptors while upregulates their endogenous ligands such as brain derived neurotrophic factor to promote human neurogenesis.

Neurogenesis is the process of generating new neurons from neural stem cells (NSCs) and plays a crucial role in neurological diseases. The process involves a series of steps, including NSC proliferation, migration and differentiation, which are regulated by multiple pathways such as neurotrophic Trk and fibroblast growth factor receptors (FGFR) signalling. Despite the discovery of numerous compounds capable of modulating individual stages of neurogenesis, it remains challenging to identify an agent that can regulate multiple cellular processes of neurogenesis. Here, through screening of bioactive compounds in dietary functional foods, we identified a flavonoid chrysin that not only enhanced the human NSCs proliferation but also facilitated neuronal differentiation and neurite outgrowth. Further mechanistic study revealed the effect of chrysin was attenuated by inhibition of neurotrophic tropomyosin receptor kinase-B (TrkB) receptor. Consistently, chrysin activated TrkB and downstream ERK1/2 and AKT. Intriguingly, we found that the effect of chrysin was also reduced by FGFR1 blockade. Moreover, extended treatment of chrysin enhanced levels of brain-derived neurotrophic factor, as well as FGF1 and FGF8. Finally, chrysin was found to promote neurogenesis in human cerebral organoids by increasing the organoid expansion and folding, which was also mediated by TrkB and FGFR1 signalling. To conclude, our study indicates that activating both TrkB and FGFR1 signalling could be a promising avenue for therapeutic interventions in neurological diseases, and chrysin appears to be a potential candidate for the development of such treatments.

Alzheimer's Amyloid-ß Accelerates Cell Senescence and Suppresses SIRT1 in Human Neural Stem Cells.

As a lifelong source of neurons, neural stem cells (NSCs) serve multiple crucial functions in the brain. The senescence of NSCs may be associated with the onset and progression of Alzheimer's disease (AD). Our study reveals a noteworthy finding, indicating that the AD-associated pathogenic protein amyloid-ß (Aß) substantially enhances senescence-related characteristics of human NSCs. These characteristics encompass the enhanced expression of p16 and p21, the upregulation of genes associated with the senescence-associated secretory phenotype (SASP), increased SA-ß-gal activity, and the activation of the DNA damage response. Further studies revealed that Aß treatment significantly downregulates the SIRT1 protein which plays a crucial role in regulating the aging process and decreases downstream PGC-1α and FOXO3. Subsequently, we found that SIRT1 overexpression significantly alleviates a range of Aß-induced senescent markers in human NSCs. Taken together, our results uncover that Aß accelerates cellular senescence in human NSCs, making SIRT1 a highly promising therapeutic target for senescent NSCs which may contribute to age-related neurodegenerative diseases, including AD.

Dietary Flavonoid Chrysin Functions as a Dual Modulator to Attenuate Amyloid-ß and Tau Pathology in the Models of Alzheimer's Disease.

Growing evidence indicates that healthy diets are associated with a slower progression of Alzheimer's disease (AD). Flavonoids are among the most abundant natural products in diets beneficial to AD, such as the Mediterranean diet. However, the effect and mechanism of these dietary flavonoids on AD remains incompletely understood. Here, we found that a representative dietary natural flavonoid, chrysin (Chr), significantly ameliorated cognitive impairment and AD pathology in APP/PS1 mice. Furthermore, mechanistic studies showed that Chr significantly reduced the levels of amyloid-ß (Aß) and phosphorylated tau (p-tau), along with dual inhibitory activity against ß-site amyloid precursor protein cleaving enzyme 1 (BACE1) and glycogen synthase kinase 3ß (GSK3ß). Moreover, the effect of Chr was further confirmed by EW233, a structural analog of Chr that exhibited an improved pharmacokinetic profile. To further verify the role of Chr and EW233, we utilized our previously established chimeric human cerebral organoid (chCO) model for AD, in which astrogenesis was promoted to mimic the neuron-astrocyte ratio in human brain tissue, and similar dual inhibition of Aß and p-tau was also observed. Altogether, our study not only reveals the molecular mechanisms through which dietary flavonoids, such as Chr, mitigate AD pathology, but also suggests that identifying a specific constituent that mimics some of the benefits of these healthy diets could serve as a promising approach to discover new treatments for AD.

Psychedelic LSD activates neurotrophic signal but fails to stimulate neural stem cells.

Accumulating evidence has shown that some hallucinogens, such as LSD, have fast and persistent effects on anxiety and depression. According to a proposed mechanism, LSD activates the TrkB and HTR2A signaling pathways, which enhance the density of neuronal dendritic spines and synaptic function, and thus promote brain function. Moreover, TrkB signaling is also known to be crucial for neural stem cell (NSC)-mediated neuroregeneration to repair dysfunctional neurons. However, the impact of LSD on neural stem cells remains to be elucidated. In this study, we observed that LSD and BDNF activated the TrkB pathway in human NSCs similarly to neurons. However, unlike BDNF, LSD did not promote NSC proliferation. These results suggest that LSD may activate an alternative mechanism to counteract the effects of BDNF-TrkB signaling on NSCs. Our findings shed light on the previously unrecognized cell type-specificity of LSD. This could be crucial for deepening our understanding of the mechanisms underlying the effects of LSD.

Tsaokoflavanols A1-J1: Flavanol-fatty alcohol hybrids with HPL inhibitory activity from Amomum tsao-ko.

Ten previously undescribed compounds were isolated from the fruits of Amomum tsao-ko (Zingiberaceae), including nine undescribed flavanol-fatty alcohol hybrids (1-6, 10-11, 13), and a flavanol-monoterpenoid hybrid (14), along with seven known flavanol hybrids (7-9, 12, 15-17). The structures of these compounds were determined using various analyses, such as HRESIMS, 1D/2D NMR, and ECD calculations. In terms of biological activity, compounds 1, 2, 5, and 6 exhibited inhibitions of human pancreatic lipase (HPL), with IC50 values ranging from 0.017 to 0.193 mM. Some of these values were found to be stronger than that of the positive control, orlistat (IC50, 0.067 mM). Molecular docking studies were also conducted to investigate the interactions between these compounds and HPL. The docking simulations revealed the importance of the orientation of the 3,4-dihydroxyphenyl in binding with HPL. Additionally, compound 9 demonstrated cytotoxicity against HepG2, with a CC50 value of 14.96 ± 0.62 µM as determined by the MTT assay. Flow cytometry analysis indicated that compound 9 induced apoptosis in HepG2 cells. Western blot results showed an up-regulation of apoptosis-related proteins, such as p53 protein, Bax and Caspase-3 proteins, while the expression of Bcl-2 protein was down-regulated.

[Application of ultraviolet spectroscopy for rapid analysis in extraction process of danhong injection].

In this work, a rapid analysis method basing on ultraviolet spectroscopy was established for the determination of danshensu, protocatechuic aldehyde, rosmarinci acid, lithospermic acid and salvianolic acid B in the extraction process of Danhong injection. In the extraction process of Danshen and Honghua crude drugs, 44 extraction solution samples were collected and the contents of the five components were determined by HPLC analysis. The ultraviolet spectra of the samples were collected. Partial least square regression was used to establish the multivariate calibration models between the ultraviolet spectra and the contents of the five components. The results showed that the established models could predict the contents of the five components in the extraction solution accurately. The ultraviolet spectroscopy method established in this work can be used for rapid analysis of the intermediates of Danhong injection, which may be applied for the quality control in the manufacturing process.

Rhamnose Displays an Anti-Obesity Effect Through Stimulation of Adipose Dopamine Receptors and Thermogenesis.

The imbalance between energy intake and energy expenditure leads to the prevalence of obesity worldwide. A strategy to simultaneously limit energy intake and promote energy expenditure would be an important new obesity treatment. Here, we identified rhamnose as a nonnutritive sweetener to promote adipose thermogenesis and energy expenditure. Rhamnose promotes cAMP production and PKA activation through dopamine receptor D1 in adipose tissue. As a result, rhamnose administration promotes UCP1-dependent thermogenesis and ameliorates obesity in mice. Thus, we have demonstrated a rhamnose-dopamine receptor D1-PKA axis critical for thermogenesis, and that rhamnose may have a role in therapeutic molecular diets against obesity.

A single amino acid substitution confers enhanced methylation activity of mammalian Dnmt3b on chromatin DNA.

Dnmt3a and Dnmt3b are paralogous enzymes responsible for de novo DNA methylation but with distinguished biological functions. In mice, disruption of Dnmt3b but not Dnmt3a causes global DNA hypomethylation, especially in repetitive sequences, which comprise the large majority of methylated DNA in the genome. By measuring DNA methylation activity of Dnmt3a and Dnmt3b homologues from five species, we found that mammalian Dnmt3b possessed significantly higher methylation activity on chromatin DNA than Dnmt3a and non-mammalian Dnmt3b. Sequence comparison and mutagenesis experiments identified a single amino acid substitution (I662N) in mammalian Dnmt3b as being crucial for its high chromatin DNA methylation activity. Further mechanistic studies demonstrated this substitution markedly enhanced the binding of Dnmt3b to nucleosomes and hence increased the chromatin DNA methylation activity. Moreover, this substitution was crucial for Dnmt3b to efficiently methylate repetitive sequences, which increased dramatically in mammalian genomes. Consistent with our observation that Dnmt3b evolved more rapidly than Dnmt3a during the emergence of mammals, these results demonstrated that the I662N substitution in mammalian Dnmt3b conferred enhanced chromatin DNA methylation activity and contributed to functional adaptation in the epigenetic system.

Alzheimer's Amyloid-ß Accelerates Human Neuronal Cell Senescence Which Could Be Rescued by Sirtuin-1 and Aspirin.

Cellular senescence is a major biological process related to aging. Neuronal cell senescence contributes to the pathogenesis of many aging-related neurodegenerative diseases including Alzheimer's disease (AD). In this study, we showed that amyloid-ß42 oligomers (Aß), one of the core pathological players of AD, significantly upregulated the expression of senescence markers, p21, plasminogen activator inhibitor-1 (PAI-1), and SA-ß-gal (senescence-associated ß-galactosidase) in multiple human neuronal cells, including SK-N-SH cells, SH-SY5Y cells, and neural stem cell (NSC)-derived neuronal cells. Moreover, it was consistently observed among the cells that Aß promoted senescence-associated DNA damage as the levels of 8-OHdG staining, histone variant H2AX phosphorylation (γ-H2AX), and genomic DNA lesion increased. Mechanism study revealed that the exposure of Aß markedly suppressed the expression of sirtuin-1 (SIRT1), a critical regulator of aging, and the exogenous expression of SIRT1 alleviated Aß-induced cell senescence phenotypes. To our surprise, a widely used cardiovascular drug aspirin considerably rescued Aß-induced cellular senescence at least partially through its regulation of SIRT1. In conclusion, our findings clearly demonstrate that exposure of Aß alone is sufficient to accelerate the senescence of human neuronal cells through the downregulation of SIRT1.

Alzheimer's Amyloid-ß Accelerates Cell Senescence and Suppresses the SIRT1/NRF2 Pathway in Human Microglial Cells.

Microglia play important roles in maintenance of brain homeostasis, while due to some pathological stimuli in aging-related neurodegenerative diseases including Alzheimer's disease, they are malfunctioning. Here, we demonstrated that amyloid-ß (Aß) accelerated cell senescence characterized by the upregulation of p21 and PAI-1 as well as senescence-associated beta-galactosidase (SA-ß-gal) in human microglial cells. Consistently, Aß induced the senescence-associated mitochondrial dysfunctions such as repression of ATP production, oxygen consumption rate (OCR), and mitochondrial membrane potential and enhancement of ROS production. Furthermore, Aß was found to significantly suppress mRNA expression and protein level of Sirtuin-1 (SIRT1), a key regulator of senescence, and inhibit mRNA expression and translocation of NRF2, a critical transcription factor in inflammatory responses, leading to impairment of phagocytosis. Rescue of SIRT1, as expected, could counteract the pathological effects of Aß. In summary, our findings revealed that Aß accelerates human microglial senescence mainly through its suppression of the SIRT1/NRF2 pathway and suggested that genetic and pharmaceutical rescue of SIRT1 may provide a potential alternative treatment.

Chimeric cerebral organoids reveal the essentials of neuronal and astrocytic APOE4 for Alzheimer's tau pathology.

The apolipoprotein E4 (APOE4) genotype is one of the strongest genetic risk factors for Alzheimer's disease (AD), and is generally believed to cause widespread pathological alterations in various types of brain cells. Here, we developed a novel engineering method of creating the chimeric human cerebral organoids (chCOs) to assess the differential roles of APOE4 in neurons and astrocytes. First, the astrogenic factors NFIB and SOX9 were introduced into induced pluripotent stem cells (iPSCs) to accelerate the induction of astrocytes. Then the above induced iPSCs were mixed and cocultured with noninfected iPSCs under the standard culturing condition of cerebral organoids. As anticipated, the functional astrocytes were detected as early as 45 days, and it helped more neurons matured in chCOs in comparation of the control human cerebral organoids (hCOs). More interestingly, this method enabled us to generate chCOs containing neurons and astrocytes with different genotypes, namely APOE3 or APOE4. Then, it was found in chCOs that astrocytic APOE4 already significantly promoted lipid droplet formation and cholesterol accumulation in neurons while both astrocytic and neuronal APOE4 contributed to the maximum effect. Most notably, we observed that the co-occurrence of astrocytic and neuronal APOE4 were required to elevate neuronal phosphorylated tau levels in chCOs while Aß levels were increased in chCOs with neuronal APOE4. Altogether, our results not only revealed the essence of both neuronal and astrocytic APOE4 for tau pathology, but also suggested chCOs as a valuable pathological model for AD research and drug discovery.

In vivo development and single-cell transcriptome profiling of human brain organoids.

OBJECTIVES: Human brain organoids can provide not only promising models for physiological and pathological neurogenesis but also potential therapies in neurological diseases. However, technical issues such as surgical lesions due to transplantation still limit their applications. MATERIALS AND METHODS: Instead of applying mature organoids, we innovatively developed human brain organoids in vivo by injecting small premature organoids into corpus striatum of adult SCID mice. Two months after injection, single-cell transcriptome analysis was performed on 6131 GFP-labeled human cells from transplanted mouse brains. RESULTS: Eight subsets of cells (including neuronal cells expressing striatal markers) were identified in these in vivo developed organoids (IVD-organoids) by unbiased clustering. Compared with in vitro cultured human cortical organoids, we found that IVD-organoids developed more supporting cells including pericyte-like and choroid plexus cells, which are important for maintaining organoid homeostasis. Furthermore, IVD-organoids showed lower levels of cellular stress and apoptosis. CONCLUSIONS: Our study thus provides a novel method to generate human brain organoids, which is promising in various applications of disease models and therapies.

PL201, a Reported Rhamnoside Against Alzheimer's Disease Pathology, Alleviates Neuroinflammation and Stimulates Nrf2 Signaling.

Neuroinflammation induced by overactivated glia cells is believed to be a major hallmark of Alzheimer's disease (AD) and a hopeful target against AD. A rhamnoside PL201 was previously reported to promote neurogenesis and ameliorate AD, and in this study, we revealed that PL201 also significantly reduced accumulation of the activated microglia and proinflammatory cytokines in APP/PS1 mice. In vitro, PL201 consistently suppressed the microglia induction of proinflammatory cytokines after stimulation with lipopolysaccharides and Aß42. Further mechanistic studies demonstrated that PL201 considerably enhanced the expression level and the nuclear translocation of Nrf2, a key regulator of neuroinflammation. Moreover, PL201 effectively stimulated Nrf2 signaling cascade, including upregulation of HO-1 and downregulation of NF-κB pathway. Thus, our findings indicated the anti-neuroinflammatory effect by PL201 in vivo and suggested that PL201 or the like, with multiple functions such as neurogenesis, mitochondria maintenance, and anti-neuroinflammation, could be a promising candidate in AD treatment.