Amyloid-like aggregation or fibrillization of alpha-synuclein (alpha-Syn) and the filamentous deposits in Lewy bodies are believed to be closely associated with several fatal neurodegenerative disorders, including Parkinson's disease and Alzheimer's disease. Here, we report the importance of a nine-residue peptide motif, (66)VGGAVVTGV(74), in the fibrillization and cytotoxicity of human alpha-Syn. Mutagenesis combined with thioflavin T fluorescence detection, atomic force microscopic imaging, and cytotoxicity assays reveal that deletion of this sequence completely eliminates alpha-Syn fibrillization and cell toxicity. However, deletion of the (71)VTGV(74) sequence decreases the fibrillization rate while the cytotoxicity remains unchanged. Incorporation of charged residues within this region slows aggregation and even impedes filament formation. In addition, substitution of Gly68 with Ala or C-terminal truncations of alpha-Syn accelerate the fibrillization processes. Circular dichroism studies suggest that beta-sheet formation is often concomitant with filament formation. Thus, this segment, namely, the GAV motif, is responsible for aggregation or fibrillization of alpha-Syn and perhaps other amyloidogenic proteins. The oligomers formed during fibrillogenesis might be associated with the cytotoxicities of various alpha-Syn species. This finding may provide further insight into the understanding of the molecular mechanism underlying the fibrillogenesis implicated in neurodegeneration as well as aid in drug design and development of transgenic models.

Parkinson's disease (PD) is a common movement disorder characterized by degeneration of dopaminergic neurons and deposition of fibrillar Lewy bodies comprising primarily a-synuclein (a-Syn) in the substantia nigra [1][2][3][4]. A growing body of evidence strongly supports the theory that formation of a-Syn fibrils and dopamine (DA) metabolism are closely associated with the pathogenesis of this fatal disease [5][6][7]. These findings imply an intrinsic link between the presynaptic a-Syn protein and the DA molecule [8], a synaptic neurotransmitter that functions in signal transmission. Recent research has focused on oxidative stress in brain, DA metabolism and dysfunction of a-Syn in synapses, in an attempt to elucidate an overview linking these important biological processes. Conway et al. [9] reported that DA stabilized the a-Syn protofibrils by forming a DA-a-Syn adduct. They proposed that DA could react with the phenol Fibrillization of a-synuclein (a-Syn) is closely associated with the formation of Lewy bodies in neurons and dopamine (DA) is a potent inhibitor for the process, which is implicated in the causative pathogenesis of Parkinson's disease (PD). To elucidate any molecular mechanism that may have biological relevance, we tested the inhibitory abilities of DA and several analogs including chemically synthetic and natural polyphenols in vitro. The MS and NMR characterizations strongly demonstrate that DA and its analogs inhibit a-Syn fibrillization by a mechanism where the oxidation products (quinones) of DA analogs react with the amino groups of a-Syn chain, generating a-Syn-quinone adducts. It is likely that the amino groups of a-Syn undergo nucleophilic attack on the quinone moiety of DA analogs to form imino bonds. The covalently cross-linked a-Syn adducts by DA are primarily large molecular mass oligomers, while those by catechol and p-benzoquinone (or hydroquinone) are largely monomers or dimers. The DA quinoprotein retains the same cytotoxicity as the intact a-Syn, suggesting that the oligomeric intermediates are the major elements that are toxic to the neuronal cells. This finding implies that the reaction of a-Syn with DA is relevant to the selective dopaminergic loss in PD.Abbreviations Ab, amyloid b-protein; AFM, atomic force microscopy; CA, catechol; DA, dopamine; DAQ, dopamine-quinone; EGCG, (-)-epigallocatechin gallate; GAV, a peptide motif with a homologous sequence of VGGAVVAGV; HQ, hydroquinone; HSQC, heteronuclear single quantum coherence; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; NAC, nonamyloid component; NBT, nitroblue tetrazolium chloride; pNP, p-nitrophenol; PrP, prion protein; pXG, p-xylylene glycol; Q, p-benzoquinone; a-Syn, human a-synuclein; ThT, thioflavin T.

Aims: Angiotensin II (AngII), a vasoconstrictive peptide of the renin–angiotensin system (RAS), promotes hepatic fibrogenesis and induces microRNA-21(mir-21) expression. Angiotensin-(1–7) [Ang-(1–7)] is a peptide of the RAS, which attenuates liver fibrosis. Recently, it was reported that the NOD-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome participated in liver fibrosis. However, it remains unclear how mir-21 mediates AngII-induced NLRP3 inflammasome activation. We investigate the role of AngII-induced mir-21 in the regulation of NLRP3 inflammasome/IL-1β axis in liver fibrosis.Results: In vivo, circulating mir-21 was upregulated in patients with liver fibrosis and was positively correlated with liver fibrosis and oxidation. Treatment with Ang-(1–7) inhibited mir-21, NLRP3 inflammasome, and liver fibrosis after bile duct ligation (BDL) or AngII infusion. Inhibition of mir-21 suppressed the Smad7/Smad2/3/NOX4, Spry1/ERK/NF-κB pathway, NLRP3 inflammasome, and liver fibrosis induced by AngII infusion. In vitro, AngII upregulated mir-21 expression via targeting Smad7 and Spry1 in primary hepatic stellate cells (HSCs). In contrast, Ang-(1–7) suppressed mir-21 expression and oxidation induced by AngII. Overexpression of mir-21 promoted oxidation, and collagen production enhanced the effect of AngII on NLRP3 inflammasome activation via the Spry1/ERK/NF-κB, Smad7/Smad2/3/NOX4 pathways. However, downregulation of mir-21 exerted the opposite effects.Innovation and Conclusions: Mir-21 mediates AngII-activated NLRP3 inflammasome and resultant HSC activation via targeting Spry1 and Smad7. Ang-(1–7) protected against BDL or AngII infusion-induced hepatic fibrosis and inhibited mir-21 expression. Antioxid. Redox Signal. 27, 1–20.

Apomorphine (APO), a potent D1/D2 dopamine receptor agonist, is currently used as an antiparkinsonian drug. We have shown previously that APO stimulates synthesis and release of multiple trophic factors, such as brain-derived neurotrophic factor (BDNF) and glial cell line-derived neurotrophic factor (GDNF), in both mesencephalic and striatal neurons, thereby effectively preventing dopaminergic neuron loss in vitro. The present study was designed to investigate the effects of APO on fibroblast growth factor-2 (FGF-2) expression and regulation in astrocytes, and furthermore, to identify signaling mechanisms underlying these effects. Here, we show that FGF-2 expression is robustly induced in cultured astrocytes in response to APO. FGF-2 expression was proportional to APO concentration and time-dependent. Conversely, treatment with S-APO, a derivative of R-APO lacking DA receptor agonist activity, did not alter FGF-2 levels. APO treatment resulted in enhanced cytosol FGF-2 immunoreactivity, export of high MW forms of FGF-2 to the cytoplasm from the nucleus and increased extracellular release of FGF-2. Interestingly, both high and low MW forms of FGF-2 were detectable in conditioned medium of APO-treated cultures. This APO-induced effect was correlated with activation of D1 and D2 receptors, as it could be either mimicked by dopamine receptor agonists (SKF38393, quinpirole) or partially blocked by antagonists (SCH23390, SKF83566, haloperidol). Activation of the D1 receptor preferentially increased PKA activity, whereas activation of the D2 receptor only promoted phosphorylation of MAPK. Importantly, APO-modulated FGF-2 expression was independent of Akt/phosphoinositide 3-kinase signaling. These data suggest that APO can enhance biosynthesis and release of FGF-2 through activation of dopamine receptors in striatal astrocytes. Both cAMP/PKA and MEK/MAPK signaling cascades are major steps mediating this process.

A series of tetrabutylphosphonium ([Bu4P]+)-based ionic liquids (ILs) with multiple-site for CO2 capture and activation in their anions, which could efficiently catalyze the cyclization reaction of propargylic alcohols with CO2 at ambient conditions, are reported. Especially, the IL, [Bu4P]3[2,4-OPym-5-Ac], which has three interaction sites for attracting CO2 together with a pK a1 value of 9.13, exhibited the best performance, affording a series of α-alkylidene cyclic carbonates in moderate to good yields. The mechanism exploration demonstrated that IL served as a bifunctional catalyst with anion simultaneously activating CO2 via multiple-site cooperative interactions and the CC triple bond in propargylic alcohol via inductive effect, thus resulting in the production of α-alkylidene cyclic carbonates.

Aldosterone, with pro-oxidation and pro-autophagy capabilities, plays a key role in liver fibrosis. However, the mechanisms underlying aldosterone-promoted liver sinusoidal endothelial cells (LSECs) defenestration remain unknown. Caveolin 1 (Cav1) displays close links with autophagy and fenestration. Hence, we aim to investigate the role of Cav1-related autophagy in LSECs defenestration. We found the increase of aldosterone/MR (mineralocorticoid receptor) level, oxidation, autophagy, and defenestration in LSECs in the human fibrotic liver, BDL or hyperaldosteronism models; while antagonizing aldosterone or inhibiting autophagy relieved LSECs defenestration in BDL-induced fibrosis or hyperaldosteronism models. In vitro, fenestrae of primary LSECs gradually shrank, along with the down-regulation of the NO-dependent pathway and the augment of the AMPK-dependent autophagy; these effects were aggravated by rapamycin (an autophagy activator) or aldosterone treatment. Additionally, aldosterone increased oxidation mediated by Cav1, reduced ATP generation, and subsequently induced the AMPK-dependent autophagy, leading to the down-regulation of the NO-dependent pathway and LSECs defenestration. These effects were reversed by MR antagonist spironolactone, antioxidants or autophagy inhibitors. Besides, aldosterone enhanced the co-immunoprecipitation of Cav1 with p62 and ubiquitin, and induced Cav1 co-immunofluorescence staining with LC3, ubiquitin, and F-actin in the perinuclear area of LSECs. Furthermore, aldosterone treatment increased the membrane protein level of Cav1, whereas decrease the cytoplasmic protein level of Cav1, indicating that aldosterone induced Cav1-related selective autophagy and F-actin remodeling to promote defenestration. Consequently, Cav1-related selective autophagy initiated by aldosterone-induced oxidation promotes LSECs defenestration via activating the AMPK-ULK1 pathway and inhibiting the NO-dependent pathway.

Autophagy, interacting with actin cytoskeleton and the NO-dependent pathway, may affect the phenotype and function of endothelial cells. Moreover, caveolin-1 (Cav-1), as a structure protein in liver sinusoidal endothelial cells (LSECs), is closely related to autophagy. Hence, we aim to explore the role of autophagic degradation of Cav-1 in LSECs defenestration. In vivo, we found the increase of autophagy in liver sinusoidal endothelium in human fibrotic liver. Furthermore, autophagy, degradation of Cav-1, and actin filament (F-actin) remodeling were triggered during the process of CCl4-induced LSECs defenestration; in contrast, autophagy inhibitor 3MA diminished the degradation of Cav-1 to maintain fenestrae and relieve CCl4-induced fibrosis. In vitro, during LSECs defenestration, the NO-dependent pathway was down-regulated through the reduction of the PI3K–AKT–MTOR pathway and initiation of autophagic degradation of Cav-1; while, these effects were aggravated by starvation. However, VEGF inhibited autophagic degradation of Cav-1 and F-actin remodeling to maintain LSECs fenestrae via activating the PI3K–AKT–MTOR pathway. Additionally, inhibiting autophagy, such as 3MA, bafilomycin, or ATG5-siRNA, could attenuate the depletion of Cav-1 and F-actin remodeling to maintain LSECs fenestrae and improve the NO-dependent pathway; in turn, eNOS-siRNA and L-NAME, for blocking the NO-dependent pathway, could elevate autophagic degradation of Cav-1 to aggravate defenestration. Finally, overexpressed Cav-1 rescued rapamycin-induced autophagic degradation of Cav-1 to maintain LSECs fenestrae; whereas knockdown of Cav-1 facilitated defenestration due to the activation of the AMPK-dependent autophagy. Consequently, autophagic degradation of Cav-1 promotes LSECs defenestration via inhibiting the NO-dependent pathway and F-actin remodeling.

Reductive amination/cyclization of levulinic acid was presented to selectively produce pyrrolidones versus pyrrolidines by switching the catalyst from AlCl3 to RuCl3 under mild conditions.