Antagonizing pathological α-synuclein-mediated neurodegeneration by J24335 via the activation of immunoproteasome.

The aggregation of misfolded proteins, such as α-synuclein in Parkinson's disease (PD), occurs intracellularly or extracellularly in the majority of neurodegenerative diseases. The immunoproteasome has more potent chymotrypsin-like activity than normal proteasome. Thus, degradation of α-synuclein aggregation via immunoproteasome is an attractive approach for PD drug development. Herein, we aimed to determine if novel compound, 11-Hydroxy-1-(8-methoxy-5-(trifluoromethyl)quinolin-2-yl)undecan-1-one oxime (named as J24335), is a promising candidate for disease-modifying therapy to prevent the pathological progression of neurodegenerative diseases, such as PD. The effects of J24335 on inducible PC12/A53T-α-syn cell viability and cytotoxicity were evaluated by MTT assay and LDH assay, respectively. Evaluation of various proteasome activities was done by measuring the luminescence of enzymatic activity after the addition of different amounts of aminoluciferin. Immunoblotting and real-time PCR were employed to detect the expression of various proteins and genes, respectively. We also used a transgenic mouse model for behavioral testing and immunochemical analysis, to assess the neuroprotective effects of J24335. J24335 inhibited wild-type and mutant α-synuclein aggregation without affecting the growth or death of neuronal cells. The inhibition of α-synuclein aggregation by J24335 was caused by activation of immunoproteasome, as mediated by upregulation of LMP7, and increased cellular chymotrypsin-like activity in 20S proteasome. J24335-enhanced immunoproteasome activity was mediated by PKA/Akt/mTOR pathway activation. Moreover, animal studies revealed that J24335 treatment markedly mitigated both the loss of tyrosine hydroxylase-positive (TH-) neurons and impaired motor skill development. This is the first report to use J24335 as an immunoproteasome enhancing agent to antagonize pathological α-synuclein-mediated neurodegeneration.

A novel agent attenuates cardiotoxicity and improves antitumor activity of doxorubicin in breast cancer cells.

Doxorubicin (Dox) is a well-known chemotherapeutic agent used in the treatment of various cancers. However, Dox-induced cardiotoxicity limits its further clinical use. We have previously reported a small molecular named biotin-conjugated ADTM analog (BAA) that exhibits cytoprotective effects against oxidative stress-induced cell injury in cardiomyoblast H9c2 cells. Here, the protective effects of BAA, indexed by attenuation of the cardiotoxicity induced by Dox as well as synergistic antitumor activity that increases the chemotherapeutic efficacy of Dox were investigated. Our results demonstrated that BAA significantly ameliorated Dox-induced toxicity in the H9c2 cells and zebrafish models. In addition, BAA attenuated Dox-induced endoplasmic reticulum (ER) stress in H9c2 cells. An ER stress inhibitor, 4-phenylbutyric acid, reversed the protective effect of BAA in H9c2 cells. In contrast, in human breast tumor MDA-MB-231 cells, BAA significantly enhanced Dox-induced cytotoxicity through upregulating Dox-induced ER stress response. Taken together, our findings indicate that Dox combined with BAA can significantly enhance its antitumor activity in breast cancer cells and reduce its cardiotoxicity, at least in part, by mediating ER stress activation.

Schisantherin A Attenuates Neuroinflammation in Activated Microglia: Role of Nrf2 Activation Through ERK Phosphorylation.

BACKGROUND/AIMS: In the present study, we investigated whether schisantherin A (StA) had anti-inflammatory effects under neuroinflammatory conditions. METHODS: The effects of StA and its underlying mechanisms were examined in lipopolysaccharide (LPS)-activated BV-2 microglial cells by ELISA, qPCR, EMSA, Western blot, and IHC. RESULTS: Firstly, we found that StA inhibited the inflammatory response in LPS-activated BV-2 microglia. Secondly, we found that StA suppressed LPS-induced activation of NF-κB via interfering with degradation of IκB and phosphorylation of IκB, IKK, PI3K/Akt, JNK, and p38 MAPK. Thirdly, StA conferred indirect antioxidative effects via quenching ROS and promoted expression of antioxidant enzymes, including HO-1 and NQO-1, via stimulating activation of Nrf2 pathways. Finally, we demonstrated that anti-neuroinflammatory actions of StA were dependent on ERK phosphorylation-mediated Nrf2 activation. CONCLUSION: StA induced ERK phosphorylation-mediated Nrf2 activation, which contributed to its anti-inflammation and anti-oxidation. The anti-neuroinflammatory and anti-oxidative effects of StA may show preventive therapeutic potential for various neuroinflammatory disorders.

Evaluation in zebrafish model of the toxicity of rhodamine B-conjugated crotamine, a peptide potentially useful for diagnostics and therapeutics.

Crotamine is defensin-like cationic peptide from rattlesnake venom that possesses anticancer, antimicrobial, and antifungal properties. Despite these promising biological activities, toxicity is a major concern associated with the development of venom-derived peptides as therapeutic agents. In the present study, we used zebrafish as a system model to evaluate the toxicity of rhodamine B-conjugated (RhoB) crotamine derivative. The lethal toxic concentration of RhoB-crotamine was as low as 4 µM, which effectively kill zebrafish larvae in less than 10 min. With non-lethal concentrations (<1 µM), crotamine caused malformation in zebrafish embryos, delayed or completely halted hatching, adversely affected embryonic developmental programming, decreased the cardiac functions, and attenuated the swimming distance of zebrafish. The RhoB-crotamine translocated across vitelline membrane and accumulated in zebrafish yolk sac. These results demonstrate the sensitive responsivity of zebrafish to trial crotamine analogues for the development of novel therapeutic peptides with improved safety, bioavailability, and efficacy profiles.

Transcriptome analysis of leaves, roots and flowers of Panax notoginseng identifies genes involved in ginsenoside and alkaloid biosynthesis.

BACKGROUND: Panax notoginseng (Burk.) F.H. Chen is one of the most highly valued medicinal plants in the world. The major bioactive molecules are triterpene saponins, which are also known as ginsenosides. However, its large genome size has hindered the assembly of a draft genome by whole genome sequencing. Hence, genomic and transcriptomic details about P. notoginseng, especially its biosynthetic pathways and gene expression in different parts of the plant, have remained largely unknown until now. RESULTS: In this study, RNA sequencing of three different P. notoginseng tissues was performed using next generation DNA sequencing. After assembling the high quality sequencing reads into 107,340 unigenes, biochemical pathways were predicted and 9,908 unigenes were assigned to 135 KEGG pathways. Among them, 270 unigenes were identified to be involved in triterpene saponin biosynthesis. In addition, 350 and 342 unigenes were predicted to encode cytochrome P450s and glycosyltransferases, respectively, based on the annotation results, some of which encode enzymes responsible for the conversion of the triterpene saponin backbone into different ginsenosides. In particular, one unigene predominantly expressed in the root was annotated as CYP716A53v2, which probably participates in the formation of protopanaxatriol from protopanaxadiol in P. notoginseng. The differential expression of this gene was further confirmed by real-time PCR. CONCLUSIONS: We have established a global transcriptome dataset for P. notoginseng and provided additional genetic information for further genome-wide research and analyses. Candidate genes involved in ginsenoside biosynthesis, including putative cytochrome P450s and glycosyltransferases were obtained. The transcriptomes in different plant tissues also provide invaluable resources for future study of the differences in physiological processes and secondary metabolites in different parts of P. notoginseng.

J24335 exerts neuroprotective effects against 6-hydroxydopamine-induced lesions in PC12 cells and mice.

Parkinson's disease is the second most prevalent age-related neurodegenerative disease and disrupts the lives of people aged >60 years. Meanwhile, single-target drugs becoming inapplicable as PD pathogenesis diversifies. Mitochondrial dysfunction and neurotoxicity have been shown to be relevant to the pathogenesis of PD. The novel synthetic compound J24335 (11-Hydroxy-1-(8-methoxy-5-(trifluoromethyl)quinolin-2-yl)undecan-1-one oxime), which has been researched similarly to J2326, has the potential to be a multi-targeted drug and alleviate these lesions. Therefore, we investigated the mechanism of action and potential neuroprotective function of J24335 against 6-OHDA-induced neurotoxicity in mice, and in PC12 cell models. The key target of action of J24335 was also screened. MTT assay, LDH assay, flow cytometry, RT-PCR, LC-MS, OCR and ECAR detection, and Western Blot analysis were performed to characterize the neuroprotective effects of J24335 on PC12 cells and its potential mechanism. Behavioral tests and immunohistochemistry were used to evaluate behavioral changes and brain lesions in mice. Moreover, bioinformatics was employed to assess the drug-likeness of J24335 and screen its potential targets. J24335 attenuated the degradation of mitochondrial membrane potential and enhanced glucose metabolism and mitochondrial biosynthesis to ameliorate 6-OHDA-induced mitochondrial dysfunction. Animal behavioral tests demonstrated that J24335 markedly improved motor function and loss of TH-positive neurons and dopaminergic nerve fibers, and contributed to an increase in the levels of dopamine and its metabolites in brain tissue. The activation of both the CREB/PGC-1α/NRF-1/TFAM and PKA/Akt/GSK-3ß pathways was a major contributor to the neuroprotective effects of J24335. Furthermore, bioinformatics predictions revealed that J24335 is a low toxicity and highly BBB permeable compound targeting 8 key genes (SRC, EGFR, ERBB2, SYK, MAPK14, LYN, NTRK1 and PTPN1). Molecular docking suggested a strong and stable binding between J24335 and the 8 core targets. Taken together, our results indicated that J24335, as a multi-targeted neuroprotective agent with promising therapeutic potential for PD, could protect against 6-OHDA-induced neurotoxicity via two potential pathways in mice and PC12 cells.

[Preparation and characterization of upconversion phosphor based on AlF3-YbF3 : Er3+].

In the present paper, AlF3-YbF3 : Er3+ was prepared by high temperature solid phase reaction, and the concentration effect of Er3+ on luminous intensity of phosphors was studied. The crystal structures of the phosphors were characterized by means of X-ray diffraction (XRD), and the upconversion luminescence properties of phosphor were studied by fluorescence emission spectra. Upon 980 nm excitation, when the Er3+ concentration was fixed to be 0.7 mol%, the maximum red emission intensities can be obtained in the sample. Furthermore, the research results showed that the fitted slope for red transition emission was 2.24, indicating that red emission is due to a two-photon excitation process.

Oral Delivery of Transformable Bilirubin Self-Assembled System for Targeted Therapy of Colitis.

Ulcerative colitis (UC) is a high incidence disease worldwide and clinically presents as relapsing and incurable inflammation of the colon. Bilirubin (BR), a natural antioxidant with significant anti-colitic effects, is utilized in preclinical studies as an intestinal disease therapy. Due to their water-insolubility, the design of BR-based agents usually involves complicated chemosynthetic processes, introducing various uncertainties in BR development. After screening numerous materials, it is identified that chondroitin sulfate can efficiently mediate the construction of BR self-assembled nanomedicine (BSNM) via intermolecular hydrogen bonds between dense sulfate and carboxyl of chondroitin sulfate and imino groups of BR. BSNM exhibits pH sensitivity and reactive oxygen species responsiveness, enabling targeted delivery to the colon. After oral administration, BSNM significantly inhibits colonic fibrosis and apoptosis of colon and goblet cells; it also reduces the expression of inflammatory cytokines. Moreover, BSNM maintains the normal level of zonula occludens-1 and occludin to sustain the integrity of intestinal barrier, regulates the macrophage polarization from M1 to M2 type, and promotes the ecological recovery of intestinal flora. Collectively, the work provides a colon-targeted and transformable BSNM that is simple to prepare and is useful as an efficient targeted UC therapy.

[Investigation of synthesis and luminescent properties of the Sr2CeO4: Dy3+].

In the present article, Sr2CeO4:Dy3+ was synthesized with N,N-methylene bisacrylamide (MBAA) as the net agent. The structure, morphology and luminescent properties were also characterized. It is indicated that Sr2CeO4:Dy3+ was single-phased without other phase existing and also had good dispersion The UV-visible absorption spectra suggested that the absorption bands were almost at 480 nm. The excitation spectrum for 270 nm emission has several excitation bands; The emission spectrum of Sr2 CeO4:Dy3+ shows two broad bands at 292 and 338 nm under the 370 nm excitation. The effects of Dy3+ doping concentration on the emission spectrum intensity of Sr2 CeO4:Dy3+ were also studied, the results showed that the ratio of yellow emission to blue emission increases with increasing the D/3+ doping concentration, but with increasing the Dy3+ doping concentration, the emission intensity firstly increased, then decreased, and the maximal emission intensity was at 0.4 mol% Dy3+ concentration.

Aucubin Exerts Anticancer Activity in Breast Cancer and Regulates Intestinal Microbiota.

Aucubin, a natural compound isolated from herbal medicine, has been reported to possess multiple beneficial properties. In this study, we aimed to verify the anticancer effect of aucubin on breast cancer and investigate the effect of cancer on the intestinal flora and whether aucubin has a therapeutic effect on intestinal problems caused by cancer. We established the breast cancer model with mouse 4T1 cell line and BALB/c mice. Aucubin was given once a day by gavage for 14 days. The results showed that aucubin suppress the growth of tumor in vivo by inducing tumor cell apoptosis. The tumor suppression rate of aucubin could reach 51.31 ± 4.07%. Organ histopathology was evaluated by tissue staining, which demonstrated that aucubin could alleviate the organ inflammatory damage caused by breast cancer without visible side effects. Moreover, aucubin could increase the expression of colonic tight junction protein occluding and adjust the gut microbiome to normal level according to 16S rDNA high-throughput sequencing. Herein, our results provide evidence for developing aucubin as an alternative and safe therapeutic for breast cancer treatment.

Targeted delivery of Chinese herb pair-based berberine/tannin acid self-assemblies for the treatment of ulcerative colitis.

INTRODUCTION: Ulcerative colitis (UC) is a chronic recurrent idiopathic disease characterized by damage to the colonic epithelial barrier and disruption of inflammatory homeostasis. At present, there is no curative therapy for UC, and the development of effective and low-cost therapies is strongly advocated. OBJECTIVES: Multiple lines of evidence support that tannic acid (TA) and berberine (BBR), two active ingredients derived from Chinese herb pair (Rhei Radix et Rhizoma and Coptidis Rhizoma), have promising therapeutic effects on colonic inflammation. This study aims to develop a targeted delivery system based on BBR/TA-based self-assemblies for the treatment of UC. METHODS: TA and BBR self-assemblies were optimized, and hyaluronic acid (HA) was coated to achieve targeted colon delivery via HA-cluster of differentiation 44 (CD44) interactions. The system was systematically characterized and dextran sodium sulfate (DSS)-induced mouse colitis model was further used to investigate the biodistribution behavior, effect and mechanism of the natural system. RESULTS: TA and BBR could self-assemble into stable particles (TB) and HA-coated TB (HTB) further increased cellular uptake and accumulation in inflamed colon lesions. Treatment of HTB inhibited pro-inflammatory cytokine levels, restored expression of tight junction-associated proteins and recovered gut microbiome alteration, thereby exerting anti-inflammatory effects against DSS-induced acute colitis. CONCLUSION: Our targeted strategy may provide a convenient and powerful platform for UC and reveal new modes of application of herbal combinations.

Ginger polysaccharides relieve ulcerative colitis via maintaining intestinal barrier integrity and gut microbiota modulation.

Ulcerative colitis (UC) is an autoimmune disease afflicting an increasing number of patients and increasing demands towards the development of efficacious and safe drugs. Recently, with increasing interest in alternative medicines, natural resources have become a hotspot for drug discovery against UC. In addition to being consumed as a food and spice, ginger is also widely used as a well-recognized gastrointestinal herbal medicine. With a long history in the treatment of digestive disorders, the potential of ginger in alleviating UC has been documented in several experimental models and clinical trials. However, as a major active constituent of ginger, ginger polysaccharides (GP) and its effect on UC has yet to be reported. In this study, GP was firstly separated and characterized. In a dextran sulfate sodium (DSS)-induced colitis mouse model, GP alleviated UC symptoms by inhibiting pro-inflammatory cytokines levels to regulate intestinal inflammation, repairing the intestinal barrier as indicated by occludin-1 and ZO-1, as well as regulating gut microbiota. Taking these results together, we believe GP could be an innovative option in developing functional foods or therapeutic agents for UC management.

Boosted EfficientNet: Detection of Lymph Node Metastases in Breast Cancer Using Convolutional Neural Networks.

(1) Purpose: To improve the capability of EfficientNet, including developing a cropping method called Random Center Cropping (RCC) to retain the original image resolution and significant features on the images' center area, reducing the downsampling scale of EfficientNet to facilitate the small resolution images of RPCam datasets, and integrating attention and Feature Fusion (FF) mechanisms with EfficientNet to obtain features containing rich semantic information. (2) Methods: We adopt the Convolutional Neural Network (CNN) to detect and classify lymph node metastasis in breast cancer. (3) Results: Experiments illustrate that our methods significantly boost performance of basic CNN architectures, where the best-performed method achieves an accuracy of 97.96% ± 0.03% and an Area Under the Curve (AUC) of 99.68% ± 0.01% on RPCam datasets, respectively. (4) Conclusions: (1) To our limited knowledge, we are the only study to explore the power of EfficientNet on Metastatic Breast Cancer (MBC) classification, and elaborate experiments are conducted to compare the performance of EfficientNet with other state-of-the-art CNN models. It might provide inspiration for researchers who are interested in image-based diagnosis using Deep Learning (DL). (2) We design a novel data augmentation method named RCC to promote the data enrichment of small resolution datasets. (3) All of our four technological improvements boost the performance of the original EfficientNet.

Identification of dihydrotanshinone I as an ERp57 inhibitor with anti-breast cancer properties via the UPR pathway.

Salvia miltiorrhiza (Danshen) is a well-known traditional Chinese medicine for treating various diseases, such as breast cancer. However, knowledge regarding its mechanisms is scant. Herein, the active ingredient dihydrotanshinone I (DHT) in Salvia miltiorrhiza extract (SME), which binds ERp57 was identified and verified by an enzymatic solid-phase method combined with LC-MS/MS. DHT potentially inhibited ERp57 activity and suppressed ERp57 expression at both the RNA and protein levels. Molecular docking simulation indicated that DHT could form a hydrogen bond with catalytic site of ERp57. Moreover, ERp57 overexpression decreased DHT-induced cytotoxicity in MDA-MB-231 cells. Thereafter, the signaling pathway downstream of ERp57 was investigated by Western blot analysis. The mechanistic study revealed that DHT treatment resulted in activation of endoplasmic reticulum (ER) stress, the unfolded protein response (UPR), and cellular apoptosis. In conclusion, our data implied that DHT targeted ERp57 for inhibition and induced ER stress and UPR activation, which in turn triggered breast cancer cell apoptosis.

[Synthesis and luminescence properties of europium (III) complexes].

Three types of europium complexes were synthesized by introducing benzoylacetone as the first ligand and 1, 10-phenanthroline, triphenylphosphine oxide, 2,2'-bipyridyl as the second ligand, respectively. The properties of above materials were characterized by infrared absorption spectra, UV-Vis absorption spectra and fluorescence spectra. Then, it was discussed that the different second ligands of europium complexes can affect their luminescence properties, and their intramolecular energy transfer models had been set up. The results indicated that ligands and complexes have a strong absorption of UV light and the three types of europium complexes exhibit characteristic luminescence of europium ion when excited by UV light. In addition, it is suggested that the fluorescence yield of europium complexes mostly depend on both the energy difference between the second ligand and the Eu3+ ion and the energy difference between the second ligand and the first ligand.

[Spectral analysis and band gap of RbVO3].

In the present paper, RbVO3 was prepared by wet chemistry synthesis from hybrid precursor. The structure of RbVO3 was characterized by X-ray diffraction (XRD) pattern and FTIR spectra. Photoluminescent (PL) properties were investigated by UV-Vis absorption spectrum and PL spectrum. In addition, the band gap of RbVO3 was calculated by using the CASTEP code with density-functional theory (DFT) method. The results indicated that RbVO3 could emit intense green-white fluorescence with peak wavelength at 525 nm and the color coordinates was (0.318 0, 0.430 9) under UV excitation at 357 nm. It was demonstrated that the theoretical result of band gap, which is 2.67 eV, agreed well with the experiment.

Oxyphylla A Promotes Degradation of α-Synuclein for Neuroprotection via Activation of Immunoproteasome.

Parkinson's disease (PD), the second most common neurodegenerative disorder, is neuropathologically characterized by the loss of dopaminergic neurons in the substantia nigra pars compacta (SNc) and the presence of Lewy bodies in surviving neurons. α-synuclein (α-syn) is the major component of Lewy bodies and its deposition in neurons is critical pathological event in the pathogenesis of PD. Herein, we reported that Oxyphylla A, a novel lead compound from the fruit of Alpinia oxyphylla, significantly promoted α-syn degradation in a cellular PD model. When exploring the molecular pathways, we found that Oxyphylla A promoted α-syn degradation in a ubiquitin proteasome system (UPS)-dependent and autophagy-independent manner. We further confirmed that Oxyphylla A enhanced UPS activity by upregulating 20S subunit PSMB8 expression. A mechanism study revealed that Oxyphylla A activated the PKA/Akt/mTOR pathway to trigger PSMB8 expression and enhance UPS activity. Finally, we illustrated that Oxyphylla A alleviated the accumulation of both Triton-soluble and Triton-insoluble forms of α-syn and protected against α-syn-induced neurotoxicity in A53T α-syn transgenic mice. These findings suggest that the activation of UPS, via small molecular UPS enhancers including Oxyphylla A, may be a therapeutic strategy for intervention against PD and related diseases.

Tetramethylpyrazine Analogue T-006 Promotes the Clearance of Alpha-synuclein by Enhancing Proteasome Activity in Parkinson's Disease Models.

Parkinson's disease (PD) is the second most common neurodegenerative disorder worldwide and is characterized in part by the degeneration of dopaminergic neurons in the substantia nigra pars compacta (SNc). The main pathological hallmark of PD is the intraneuronal accumulation of misfolded α-synuclein (α-syn) aggregates. Mutations in the SNCA gene (encoding α-syn) and variations in its copy number are associated with some forms of familial PD. In the present study, T-006, a new tetramethylpyrazine (TMP) derivative with recently reported anti-Alzheimer activity, is shown to significantly promote α-syn degradation in a cellular PD model. Moreover, we illustrate that T-006 inhibits the accumulation of both Triton-soluble and -insoluble forms of α-syn and protects against α-syn-induced neurotoxicity in A53T-α-syn transgenic mice. The mechanism of action of T-006 was verified by evaluation of a potential protein degradation pathway. We found that T-006 promotes α-syn degradation in a proteasome-dependent and autophagy-independent manner. We further confirmed that T-006 enhances proteasome activity by upregulating 20S proteasome subunit ß5i (LMP7) protein expression. A functional study revealed that T-006 activates the PKA/Akt/mTOR/p70S6K pathway to trigger LMP7 expression and enhance chymotrypsin-like proteasomal activity. These findings indicate that T-006 is a potent proteasome activator and a potential therapeutic agent for the prevention and treatment of PD and related diseases.

Tetramethylpyrazine Analogue T-006 Exerts Neuroprotective Effects against 6-Hydroxydopamine-Induced Parkinson's Disease In Vitro and In Vivo.

Parkinson's disease (PD) is a neurodegenerative disorder characterized by the progressive loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc), and there is no cure for it at present. We have previously reported that the tetramethylpyrazine (TMP) derivative T-006 exhibited beneficial effects in Alzheimer's disease (AD) models. However, its effect on PD remains unclear. In the present study, we investigated the neuroprotective effects and underlying mechanisms of T-006 against 6-hydroxydopamine- (6-OHDA-) induced lesions in in vivo and in vitro PD models. Our results demonstrated that T-006 alleviated mitochondrial membrane potential loss and restored the energy metabolism and mitochondrial biogenesis that were induced by 6-OHDA in PC12 cells. In addition, animal experiments showed that administration of T-006 significantly attenuated the 6-OHDA-induced loss of tyrosine hydroxylase- (TH-) positive neurons in the SNpc, as well as dopaminergic nerve fibers in the striatum, and also increased the concentration of dopamine and its metabolites (DOPAC, HVA) in the striatum. Functional deficits were restored following T-006 treatment in 6-OHDA-lesioned mice, as demonstrated by improved motor coordination and rotational behavior. In addition, we found that the neuroprotective effects of T-006 were mediated, at least in part, by the activation of both the PKA/Akt/GSK-3ß and CREB/PGC-1α/NRF-1/TFAM pathways. In summary, our findings demonstrate that T-006 could be developed as a novel neuroprotective agent for PD, and the two pathways might be promising therapeutic targets for PD.

Natural alkaloid harmine promotes degradation of alpha-synuclein via PKA-mediated ubiquitin-proteasome system activation.

BACKGROUND: Parkinson's disease (PD) is an age-dependent progressive movement disorder characterized by a profound and selective loss of nigrostriatal dopaminergic neurons. Accumulation of -synuclein (-syn) positive protein aggregates in the substantia nigra is a pathological hallmark of PD, indicating that protein turnover defect is implicated in PD pathogenesis. PURPOSE: This study aims to identify neuroprotective compounds which can alleviate the accumulation of -syn in neuronal cells and dissect the underlying mechanisms. METHODS: High throughput screening was performed by dot blot assay. The degradation of different forms of -syn by candidate compounds were assessed by western blot. The autophagy lysosome pathway and ubiquitin-proteasome system were examined to dissect the degradation pathway. The UPS activity was assessed by cellular UPS substrates degradation assay and biochemical proteasome activity assay. Q-PCR was performed to test the mRNA level of different proteasome subunits. Furthermore, Neuroprotective effect of candidate compound was tested by LDH assay and PI staining. RESULTS: Through the high throughput screening, harmine was identified as a potent -syn lowering compound. The time-dependent and dose-dependent effects of harmine on the degradation of different forms of -syn were further confirmed. Harmine could dramatically promote the degradation of UPS substrates GFP-CL1, Ub-R-GFP and Ub-G76V-GFP, and activate cellular proteasome activity. Mechanistically, harmine dramatically enhanced PKA phosphorylation to enhance proteasome subunit PSMD1 expression. PKA inhibitor blocked the effects of harmine in activating UPS, up regulating PSMD1 and promoting -syn degradation, indicating that harmine enhances UPS function via PKA activation. Moreover, harmine efficiently rescued cell death induced by over-expression of -syn, via UPS-dependent manner. CONCLUSION: Harmine, as a new proteasome enhancer, may have potential to be developed into therapeutic agent against neurodegenerative diseases associated with UPS dysfunction and aberrant proteins accumulation.