Simei Long

Scutellarin Attenuates Hypertension-Induced Expression of Brain Toll-Like Receptor 4/Nuclear Factor Kappa B

Hypertension is associated with low-grade inflammation, and Toll-like receptor 4 (TLR4) has been shown to be linked to the development and maintenance of hypertension. This study aimed to investigate the effects of scutellarin (administered by oral gavage daily for 2 weeks) on brain TLR4/nuclear factor kappa B-(NF-κB-) mediated inflammation and blood pressure in renovascular hypertensive (using the 2-kidney, 2-clip method) rats. Immunofluorescence and western immunoblot analyses revealed that hypertension contributed to the activation of TLR4 and NF-κB, accompanied by significantly enhanced expression of proinflammatory mediators, such as tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interleukin-18 (IL-18). Furthermore, expression of the antiapoptotic protein, myeloid cell leukemia-1 (Mcl1), was decreased, and the pro-apoptotic proteins, Bax and cleavedcaspase-3 p17 were increased in combined cerebral cortical/striatal soluble lysates. Scutellarin significantly lowered blood pressure and attenuated the number of activated microglia and macrophages in brains of hypertensive rats. Furthermore, scutellarin significantly reduced the expression of TLR4, NF-κB p65, TNF-α, IL-1β, IL-18, Bax and cleaved-caspase-3 p17, and increased the expression of Mcl1. Overall, these results revealed that scutellarin exhibits anti-inflammatory and anti-apoptotic properties and decreases blood pressure in hypertensive rats. Therefore, scutellarin may be a potential therapeutic agent in hypertension-associated diseases.

Deletion of aquaporin-4 is neuroprotective during the acute stage of micro traumatic brain injury in mice.

Micro traumatic brain injury (TBI) is the most common type of brain injury, but the mechanisms underlying it are poorly understood. Aquaporin-4 (AQP4) is a water channel expressed in astrocyte end-feet, which plays an important role in brain edema. However, little is known about the role of AQP4 in micro TBI. Here, we examined the role of AQP4 in the pathogenesis of micro TBI in a closed-skull brain injury model, using two-photon microscopy. Our results indicate that AQP4 deletion reduced cell death, water content, astrocyte swelling and lesion volume during the acute stage of micro TBI. Our data revealed that astrocyte swelling is a decisive pathophysiological factor in the acute phase of this form of micro brain injury. Thus, treatments that inhibit AQP4 could be used as a neuroprotective strategy for micro TBI.

Marine Compound Catunaregin Inhibits Angiogenesis through the Modulation of Phosphorylation of Akt and eNOS in vivo and in vitro

Angiogenesis is the formation of blood vessels from pre-existing vasculature. Excessive or uncontrolled angiogenesis is a major contributor to many pathological conditions whereas inhibition of aberrant angiogenesis is beneficial to patients with pathological angiogenesis. Catunaregin is a core of novel marine compound isolated from mangrove associate. The potential anti-angiogenesis of catunaregin was investigated in human umbilical vein endothelial cells (HUVECs) and zebrafish. HUVECs were treated with different concentrations of catunaregin in the presence or absence of VEGF. The angiogenic phenotypes including cell invasion cell migration and tube formation were evaluated following catunaregin treatment in HUVECs. The possible involvement of AKT, eNOS and ERK1/2 in catunaregin-induced anti-angiogenesis was explored using Western blotting. The anti-angiogenesis of catunaregin was further tested in the zebrafish embryo neovascularization and caudal fin regeneration assays. We found that catunaregin dose-dependently inhibited angiogenesis in both HUVECs and zebrafish embryo neovascularization and zebrafish caudal fin regeneration assays. In addition, catunaregin significantly decreased the phosphorylation of Akt and eNOS, but not the phosphorylation of ERK1/2. The present work demonstrates that catunaregin exerts the anti-angiogenic activity at least in part through the regulation of the Akt and eNOS signaling pathways.

Protective effects of puerarin against Aß40-induced vascular dysfunction in zebrafish and human endothelial cells.

Aß40-induced vascular dysfunction has been implicated in the pathogenesis of Alzheimer׳s disease (AD). In the present study, we investigated the possible protective effects of puerarin against Aß40-induced vascular damage and impairment to angiogenesis in transgenic TG (fli1:EGFP) zebrafish and human endothelial cells. Aß40 peptides at 5μM caused an obvious reduction of vessel branches in the subintestinal vein basket, induced NADPH oxidase-derived reactive oxygen species and impaired vascular endothelial growth factor (VEGF)-dependent angiogenesis. Pretreatment with puerarin attenuated Aβ40-induced vessel reduction and impairment to angiogenesis in a dose-dependent manner. In addition, Aß40 decreased VEGF-dependent phosphorylation of Akt and eNOS, whereas puerarin treatment attenuated these detrimental effects. Furthermore, the restoration of Aß40-induced-angiogenesis impairment by puerarin was abolished by either the PI3 kinase inhibitor LY294002 (10μM) or eNOS inhibitor L-NAME. The present study suggests that puerarin exerts its protective action probably through reduction of NADPH oxidase-derived reactive oxygen species overproduction and activation of the PI3K/Akt/eNOS pathways.

Identification of marine neuroactive molecules in behaviour-based screens in the larval zebrafish.

High-throughput behavior-based screen in zebrafish is a powerful approach for the discovery of novel neuroactive small molecules for treatment of nervous system diseases such as epilepsy. To identify neuroactive small molecules, we first screened 36 compounds (1–36) derived from marine natural products xyloketals and marine isoprenyl phenyl ether obtained from the mangrove fungus. Compound 1 demonstrated the most potent inhibition on the locomotor activity in larval zebrafish. Compounds 37–42 were further synthesized and their potential anti-epilepsy action was then examined in a PTZ-induced epilepsy model in zebrafish. Compound 1 and compounds 39, 40 and 41 could significantly attenuate PTZ-induced locomotor hyperactivity and elevation of c-fos mRNA in larval zebrafish. Compound 40 showed the most potent inhibitory action against PTZ-induced hyperactivity. The structure-activity analysis showed that the OH group at 12-position played a critical role and the substituents at the 13-position were well tolerated in the inhibitory activity of xyloketal derivatives. Thus, these derivatives may provide some novel drug candidates for the treatment of epilepsy.

Iron promotes α-synuclein aggregation and transmission by inhibiting TFEB-mediated autophagosome-lysosome fusion

Recent studies have strongly shown that cell‐to‐cell transmission of neuropathogenic proteins is a common mechanism for the development of neurodegenerative diseases. However, the underlying cause is complex and little is known. Although distinct processes are involved in the pathogenesis of various diseases, they all share the common feature of iron accumulation, an attribute that is particularly prominent in synucleinopathies. However, whether iron is a cofactor in facilitating the spread of α‐synuclein remains unclear. Here, we constructed a cell‐to‐cell transmission model of α‐synuclein using SN4741 cell line based on adenovirus vectors. Cells were treated with FeCl2, and α‐synuclein aggregation and transmission were then evaluated. In addition, the possible mechanisms were investigated through gene knockdown or over‐expression. Our results demonstrated that iron promoted α‐synuclein aggregation and transmission by inhibiting autophagosome‐lysosome fusion. Furthermore, iron decreased the expression of nuclear transcription factor EB (TFEB), a master transcriptional regulator of autophagosome‐lysosome fusion, and inhibited its nuclear translocation through activating AKT/mTORC1 signaling. After silencing TFEB, ratios of α‐synuclein aggregation and transmission were not significantly altered by the presence of iron; on the other hand, when TFEB was over‐expressed, the transmission of α‐synuclein induced by iron was obviously reversed; suggesting the mechanism by which iron promotes α‐synuclein transmission may be mediated by TFEB. Taken together, our data reveal a previously unknown relationship between iron and α‐synuclein, and identify TFEB as not only a potential target for preventing α‐synuclein transmission, but also a critical factor for iron‐induced α‐synuclein aggregation and transmission. Indeed, this newly discovered role of iron and TFEB in synucleinopathies may provide novel targets for developing therapeutic strategies to prevent α‐synuclein transmission in Parkinsons disease.

Xyloketal-derived small molecules show protective effect by decreasing mutant Huntingtin protein aggregates in Caenorhabditis elegans model of Huntington’s disease

Huntington’s disease is an autosomal-dominant neurodegenerative disorder, with chorea as the most prominent manifestation. The disease is caused by abnormal expansion of CAG codon repeats in the IT15 gene, which leads to the expression of a glutamine-rich protein named mutant Huntingtin (Htt). Because of its devastating disease burden and lack of valid treatment, development of more effective therapeutics for Huntington’s disease is urgently required. Xyloketal B, a natural product from mangrove fungus, has shown protective effects against toxicity in other neurodegenerative disease models such as Parkinson’s and Alzheimer’s diseases. To identify potential neuroprotective molecules for Huntington’s disease, six derivatives of xyloketal B were screened in a Caenorhabditis elegans Huntington’s disease model; all six compounds showed a protective effect. Molecular docking studies indicated that compound 1 could bind to residues GLN369 and GLN393 of the mutant Htt protein, forming a stable trimeric complex that can prevent the formation of mutant Htt aggregates. Taken together, we conclude that xyloketal derivatives could be novel drug candidates for treating Huntington’s disease. Molecular target analysis is a good method to simulate the interaction between proteins and drug compounds. Further, protective candidate drugs could be designed in future using the guidance of molecular docking results.

Circadian Rhythm Dysfunction Accelerates Disease Progression in a Mouse Model With Amyotrophic Lateral Sclerosis

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease caused by interactions between environmental factors and genetic susceptibility. Circadian rhythm dysfunction (CRD) is a significant contributor to neurodegenerative conditions such as Alzheimer’s disease and Parkinson’s disease. However, whether CRD contributes to the progression of ALS remains little known. We performed behavioral and physiological tests on SOD1G93A ALS model mice with and without artificially induced CRD, and on wild-type controls; we also analyzed spinal cord samples histologically for differences between groups. We found that CRD accelerated the disease onset and progression of ALS in model mice, as demonstrated by aggravated functional deficits and weight loss, as well as increased motor neuron loss, activated gliosis, and nuclear factor κB-mediated inflammation in the spinal cord. We also found an increasing abundance of enteric cyanobacteria in the ALS model mice shortly after disease onset that was further enhanced by CRD. Our study provides initial evidence on the CRD as a risk factor for ALS, and intestinal cyanobacteria may be involved.

G2019S LRRK2 increases stress susceptibility through inhibition of DAF-16 nuclear translocation in a 14-3-3 associated-manner in Caenorhabditis elegans

Mutations in leucine-rich repeat kinase 2 (LRRK2) are common causes of familial Parkinson’s disease (PD). Oxidative stress plays a key role in the pathogenesis of PD. Mutations in LRRK2 have been shown to increase susceptibility to oxidative stress. To explore mechanisms underlying susceptibility to oxidative stress in LRRK2 mutants, we generated stable Caenorhabditis elegans (C. elegans) strains in which human LRRK2 proteins including wild type LRRK2 (WT), G2019S LRRK2 (G2019S), and G2019S-D1994A kinase-dead LRRK2 (KD) were expressed in all neurons. Human 14-3-3 β was injected into LRRK2 transgenic worms to allow co-expression of 14-3-3 β and LRRK2 proteins. We found that G2019S transgenic worms had increased sensitivity to stress (heat and juglone treatment) and impaired stress-induced nuclear translocation of DAF-16. In addition, G2019S inhibited ftt2 (a 14-3-3 gene homolog in C. elegans) knockdown-associated nuclear translocation of DAF-16. Comparably, overexpression of human 14-3-3 β could attenuate G2019S-associated toxicity in response to stress and rescued G2019S-mediated inhibition of sod-3 and dod-3 expression. Taken together, our study provides evidence suggesting that 14-3-3-associated inhibition of DAF-16 nuclear translocation could be a mechanism for G2019S LRRK2-induced oxidative stress and cellular toxicity. Our findings may give a hint that the potential of 14-3-3 proteins as neuroprotective targets in PD patients carrying LRRK2 mutations.

High expression levels of the D686N Parkinson's disease mutation in VPS35 induces α-synuclein-dependent toxicity in yeast

Parkinsons disease (PD) is a common neurodegenerative disorder that affects ~2% of the human population aged >65. α-synuclein serves a role in the pathogenesis of PD as it is a primary component of Lewy bodies, a pathological feature of PD. Endosomal-lysosomal dysfunction may be a key factor involved in the pathophysiology of PD, and may cause PD-associated neurodegeneration via α-synuclein-dependent and -independent mechanisms. The D620N mutation in the endosomal-lysosomal gene, vacuolar protein sorting-associated protein 35 (VPS35), has been linked to PD. To clarify the underlying cellular mechanism of the VPS35 D620N mutation in PD, cell growth and endosomal-lysosomal functions were investigated in Saccharomyces cerevisiae (sc) yeast cells that exhibited various expression levels of sc VPS35, in the presence or absence of non-toxic expression levels of α-synuclein. Overexpression of the sc VPS35 D686N mutation (the yeast equivalent of D620N) did not lead to toxicity in yeast. However, the co-expression of high copy numbers of sc VPS35 D686N and low copy numbers of α-synuclein caused toxicity, whereas the co-expression of sc VPS35 wild-type and α-synuclein did not. In addition, the sc VPS35 D686N mutant enhanced α-synuclein aggregation. Fragmentation of vacuoles and subsequent inhibition of lysosome function was evident in yeast cells bearing the sc VPS35 mutant. The results of the present study suggested that α-synuclein and sc VPS35 were interlinked via the endosomal-lysosome pathway, which is important for the pathogenesis of PD.