α-mangostin derivative 4e as a PDE4 inhibitor promote proteasomal degradation of alpha-synuclein in Parkinson's disease models through PKA activation.

BACKGROUND: Parkinson's disease (PD) is a multi-factorial neurodegenerative disease affecting motor function of patients. The hall markers of PD are dopaminergic neuron loss in the midbrain and the presence of intra-neuronal inclusion bodies mainly composed of aggregation-prone protein alpha-synuclein (α-syn). Ubiquitin-proteasome system (UPS) is a multi-step reaction process responsible for more than 80% intracellular protein degradation. Impairment of UPS function has been observed in the brain tissue of PD patients. PDE4 inhibitors have been shown to activate cAMP-PKA pathway and promote UPS activity in Alzheimer's disease model. α-mangostin is a natural xanthonoid with broad biological activities, such as antioxidant, antimicrobial and antitumour activities. Structure-based optimizations based on α-mangostin produced a potent PDE4 inhibitor, 4e. Herein, we studied whether 4e could promote proteasomal degradation of α-syn in Parkinson's disease models through PKA activation. METHODS: cAMP Assay was conducted to quantify cAMP levels in samples. Model UPS substrates (Ub-G76V-GFP and Ub-R-GFP) were used to monitor UPS-dependent activity. Proteasome activity was investigated by short peptide substrate, Suc-LLVY-AMC, cleavage of which by the proteasome increases fluorescence sensitivity. Tet-on WT, A30P, and A53T α-syn-inducible PC12 cells and primary mouse cortical neurons from A53T transgenic mice were used to evaluate the effect of 4e against α-syn in vitro. Heterozygous A53T transgenic mice were employed to assess the effect of 4e on the clearance of α-syn in vivo, and further validations were applied by western blotting and immunohistochemistry. RESULTS: Taken together, α-mangostin derivative 4e, a PDE4 inhibitor, efficiently activated the cAMP/PKA pathway in neuronal cells, and promoted UPS activity as evidenced by enhanced degradation of UPS substrate Ub-G76V-GFP and Ub-R-GFP, as well as elevated proteasomal enzyme activity. Interestingly, 4e dramatically accelerated degradation of inducibly-expressed WT and mutant α-syn in PC12 cells, in a UPS dependent manner. Besides, 4e consistently activated PKA in primary neuron and A53T mice brain, restored UPS inhibition and alleviated α-syn accumulation in the A53T mice brain. CONCLUSIONS: 4e is a natural compound derived highly potent PDE4 inhibitor. We revealed its potential effect in promoting UPS activity to degrade pathogenic proteins associated with PD.

Enhancing autophagy maturation with CCZ1-MON1A complex alleviates neuropathology and memory defects in Alzheimer disease models.

Rationale: Impairment of autophagy maturation has been implicated in Alzheimer's disease (AD) pathogenesis. However, the mechanism for this impairment has not been elucidated, and whether enhancing autophagy maturation is a viable therapeutic strategy for AD has not been verified. Methods: We examined the autophagosome maturation process in AD cell and mouse models by immunoblotting. To further understand the changes in autophagy in AD brains, we analyzed the transcriptome by RNA-sequencing and measured the expression of RAB7, CCZ1 and MON1A. We performed brain stereotaxic injections of AAV into 3xTg AD mouse brain and WT mouse brain to over-express MON1A/CCZ1 or knockdown MON1A. For in vitro studies, we purified autophagosomes, and determined GTP-RAB7 level in autophagosome fractions by GST-R7BD affinity-isolation assay. Results: We report that the active form of RAB7 was selectively decreased in autophagosome fractions isolated from cells and tissues of AD models, and that this decrease was accompanied by impaired activity of its guanine nucleotide exchange factor (GFE) CCZ1-MON1A. Overexpressing CCZ1-MON1A increased the active form of RAB7, enhanced autophagosome maturation, and promoted degradation of APP-CTFs, Aß and P-tau in an autophagy-dependent manner in cells and a mouse AD model. Conclusions: Our data reveals that CCZ1-MON1A-RAB7 complex dysfunction is a potential mechanism for autophagosome maturation defects in AD, and advances the possibility that enhancing autophagosome maturation is a novel therapeutic strategy against AD.

Amelioration of Alzheimer's disease pathology by mitophagy inducers identified via machine learning and a cross-species workflow.

A reduced removal of dysfunctional mitochondria is common to aging and age-related neurodegenerative pathologies such as Alzheimer's disease (AD). Strategies for treating such impaired mitophagy would benefit from the identification of mitophagy modulators. Here we report the combined use of unsupervised machine learning (involving vector representations of molecular structures, pharmacophore fingerprinting and conformer fingerprinting) and a cross-species approach for the screening and experimental validation of new mitophagy-inducing compounds. From a library of naturally occurring compounds, the workflow allowed us to identify 18 small molecules, and among them two potent mitophagy inducers (Kaempferol and Rhapontigenin). In nematode and rodent models of AD, we show that both mitophagy inducers increased the survival and functionality of glutamatergic and cholinergic neurons, abrogated amyloid-ß and tau pathologies, and improved the animals' memory. Our findings suggest the existence of a conserved mechanism of memory loss across the AD models, this mechanism being mediated by defective mitophagy. The computational-experimental screening and validation workflow might help uncover potent mitophagy modulators that stimulate neuronal health and brain homeostasis.

Lycorine, a natural alkaloid, promotes the degradation of alpha-synuclein via PKA-mediated UPS activation in transgenic Parkinson's disease models.

BACKGROUND: Parkinson's disease (PD) is one of the most common neurodegenerative motor disorders, and is characterized by the presence of Lewy bodies containing misfolded α-synuclein (α-syn) and by selective degeneration of midbrain dopamine neurons. Studies have shown that upregulation of ubiquitin-proteasome system (UPS) activity promotes the clearance of aggregation-prone proteins such as α-syn and Tau, so as to alleviate the neuropathology of neurodegenerative diseases. PURPOSE: To identify and investigate lycorine as a UPS enhancer able to decrease α-syn in transgenic PD models. METHODS: Dot blot was used to screen α-syn-lowering compounds in an inducible α-syn overexpression cell model. Inducible wild-type (WT) and mutant α-syn-overexpressing PC12 cells, WT α-syn-overexpressing N2a cells and primary cultured neurons from A53T transgenic mice were used to evaluate the effects of lycorine on α-syn degradation in vitro. Heterozygous A53T transgenic mice were used to evaluate the effects of lycorine on α-syn degradation in vivo. mCherry-GFP-LC3 reporter was used to detect autophagy-dependent degradation. Ub-R-GFP and Ub-G76V-GFP reporters were used to detect UPS-dependent degradation. Proteasome activity was detected by fluorogenic substrate Suc-Leu-Leu-Val-Tyr-AMC (Suc-LLVY-AMC). RESULTS: Lycorine significantly promoted clearance of over-expressed WT and mutant α-syn in neuronal cell lines and primary cultured neurons. More importantly, 15 days' intraperitoneal administration of lycorine effectively promoted the degradation of α-syn in the brains of A53T transgenic mice. Mechanistically, lycorine accelerated α-syn degradation by activating cAMP-dependent protein kinase (PKA) to promote proteasome activity. CONCLUSION: Lycorine is a novel α-syn-lowering compound that works through PKA-mediated UPS activation. This ability to lower α-syn implies that lycorine has the potential to be developed as a pharmaceutical for the treatment of neurodegenerative diseases, such as PD, associated with UPS impairment and protein aggregations.

Comprehensive Perspectives on Experimental Models for Parkinson's Disease.

Parkinson's disease (PD) ranks second among the most common neurodegenerative diseases, characterized by progressive and selective loss of dopaminergic neurons. Various cross-species preclinical models, including cellular models and animal models, have been established through the decades to study the etiology and mechanism of the disease from cell lines to nonhuman primates. These models are aimed at developing effective therapeutic strategies for the disease. None of the current models can replicate all major pathological and clinical phenotypes of PD. Selection of the model for PD largely relies on our interest of study. In this review, we systemically summarized experimental PD models, including cellular and animal models used in preclinical studies, to understand the pathogenesis of PD. This review is intended to provide current knowledge about the application of these different PD models, with focus on their strengths and limitations with respect to their contributions to the assessment of the molecular pathobiology of PD and identification of the therapeutic strategies for the disease.

PI3KC3 complex subunit NRBF2 is required for apoptotic cell clearance to restrict intestinal inflammation.

NRBF2, a regulatory subunit of the ATG14-BECN1/Beclin 1-PIK3C3/VPS34 complex, positively regulates macroautophagy/autophagy. In this study, we report that NRBF2 is required for the clearance of apoptotic cells and alleviation of inflammation during colitis in mice. NRBF2-deficient mice displayed much more severe colitis symptoms after the administration of ulcerative colitis inducer, dextran sulfate sodium salt (DSS), accompanied by prominent intestinal inflammation and apoptotic cell accumulation. Interestingly, we found that nrbf2-/- mice and macrophages displayed impaired apoptotic cell clearance capability, while adoptive transfer of nrbf2+/+ macrophages to nrbf2-/- mice alleviated DSS-induced colitis lesions. Mechanistically, NRBF2 is required for the generation of the active form of RAB7 to promote the fusion between phagosomes containing engulfed apoptotic cells and lysosomes via interacting with the MON1-CCZ1 complex and regulating the guanine nucleotide exchange factor (GEF) activity of the complex. Evidence from clinical samples further reveals the physiological role of NRBF2 in maintaining intestinal homeostasis. In biopsies of UC patient colon, we observed upregulated NRBF2 in the colon macrophages and the engulfment of apoptotic cells by NRBF2-positive cells, suggesting a potential protective role for NRBF2 in UC. To confirm the relationship between apoptotic cell clearance and IBD development, we compared TUNEL-stained cell counts in the UC with UC severity (Mayo Score) and observed a strong correlation between the two indexes, indicating that apoptotic cell population in colon tissue correlates with UC severity. The findings of our study reveal a novel role for NRBF2 in regulating apoptotic cell clearance to restrict intestinal inflammation.Abbreviation: ANOVA: analysis of variance; ATG14: autophagy related 14; ATG16L1: autophagy related 16-like 1 (S. cerevisiae); BMDM: bone marrow-derived macrophage; BSA: bovine serum albumin; CD: Crohn disease; CD68: CD68 molecule; CFP: cyan fluorescent protein; CMFDA: 5-chloromethylfluorescein diacetate; Co-IP, co-immunoprecipitation; CPR: C-reactive protein; Cy7: cyanine 7 maleimide; DAB: diaminobezidine 3; DAI: disease activity indexes; DAPI: 4'6-diamidino-2-phenylindole; DMEM: dulbecco's modified eagle's medium; DMSO: dimethyl sulfoxide; DOC: sodium deoxycholate; DSS: dextran sulfate sodium; EDTA: ethylenediaminetetraacetic acid; EGTA: ethylenebis (oxyethylenenitrilo) tetraacetic acid; FBS: fetal bovine serum; FITC: fluorescein isothiocyanate; FRET: Förster resonance energy transfer; GDP: guanine dinucleotide phosphate; GEF: guanine nucleotide exchange factor; GFP: green fluorescent protein; GTP: guanine trinucleotide phosphate; GWAS: genome-wide association studies; HEK293: human embryonic kidney 293 cells; HRP: horseradish peroxidase; IBD: inflammatory bowel disease; IgG: immunoglobin G; IL1B/IL-1ß: interleukin 1 beta; IL6: interleukin 6; IRGM: immunity related GTPase M; ITGAM/CD11b: integrin subunit alpha M; KO: knockout; LRRK2: leucine rich repeat kinase 2; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MOI: multiplicity of infection; MPO: myeloperoxidase; NaCl: sodium chloride; NEU: neutrophil; NOD2: nucleotide binding oligomerization domain containing 2; NP40: nonidet-P40; NRBF2: nuclear receptor binding factor 2; PBS: phosphate buffer saline; PCR: polymerase chain reaction; PE: P-phycoerythrin; PIK3C3/VPS34: phosphatidylinositol 3-kinase catalytic subunit type 3; PtdIns3P: phosphatidylinositol-3-phosphate; PTPRC/CD45: protein tyrosine phosphatase receptor type C; SDS-PAGE: sodium dodecylsulphate-polyacrylamide gel electrophoresis; TBST: tris-buffered saline Tween-20; Tris-HCl: trihydroxymethyl aminomethane hydrochloride; TUNEL: TdT-mediated dUTP nick-end labeling; UC: ulcerative colitis; ULK1: unc-51 like autophagy activating kinase 1; WB: western blotting; WT: wild type; YFP: yellow fluorescent protein.

NRBF2 is a RAB7 effector required for autophagosome maturation and mediates the association of APP-CTFs with active form of RAB7 for degradation.

NRBF2 is a component of the class III phosphatidylinositol 3-kinase (PtdIns3K) complex. Our previous study has revealed its role in regulating ATG14-associated PtdIns3K activity for autophagosome initiation. In this study, we revealed an unknown mechanism by which NRBF2 modulates autophagosome maturation and APP-C-terminal fragment (CTF) degradation. Our data showed that NRBF2 localized at autolysosomes, and loss of NRBF2 impaired autophagosome maturation. Mechanistically, NRBF2 colocalizes with RAB7 and is required for generation of GTP-bound RAB7 by interacting with RAB7 GEF CCZ1-MON1A and maintaining the GEF activity. Specifically, NRBF2 regulates CCZ1-MON1A interaction with PI3KC3/VPS34 and CCZ1-associated PI3KC3 kinase activity, which are required for CCZ1-MON1A GEF activity. Finally, we showed that NRBF2 is involved in APP-CTF degradation and amyloid beta peptide production by maintaining the interaction between APP and the CCZ1-MON1A-RAB7 module to facilitate the maturation of APP-containing vesicles. Overall, our study revealed a pivotal role of NRBF2 as a new RAB7 effector in modulating autophagosome maturation, providing insight into the molecular mechanism of NRBF2-PtdIns3K in regulating RAB7 activity for macroautophagy/autophagy maturation and Alzheimer disease-associated protein degradation..Abbreviations: 3xTg AD, triple transgenic mouse for Alzheimer disease; Aß, amyloid beta peptide; Aß1-40, amyloid beta peptide 1-40; Aß1-42, amyloid beta peptide 1-42; AD, Alzheimer disease; APP, amyloid beta precursor protein; APP-CTFs, APP C-terminal fragments; ATG, autophagy related; ATG5, autophagy related 5; ATG7, autophagy related 7; ATG14, autophagy related 14; CCD, coiled-coil domain; CCZ1, CCZ1 homolog, vacuolar protein trafficking and biogenesis associated; CHX, cycloheximide; CQ, chloroquine; DAPI, 4',6-diamidino-2-phenylindole; dCCD, delete CCD; dMIT, delete MIT; FYCO1, FYVE and coiled-coil domain autophagy adaptor 1; FYVE, Fab1, YGL023, Vps27, and EEA1; GAP, GTPase-activating protein; GDP, guanine diphosphate; GEF, guanine nucleotide exchange factor; GTP, guanine triphosphate; GTPase, guanosine triphosphatase; HOPS, homotypic fusion and vacuole protein sorting; ILVs, endosomal intralumenal vesicles; KD, knockdown; KO, knockout; LAMP1, lysosomal associated membrane protein 1; MAP1LC3/LC3, microtubule associated protein 1 light chain 3; MLVs, multilamellar vesicles; MON1A, MON1 homolog A, secretory trafficking associated; NRBF2, nuclear receptor binding factor 2; PtdIns3K, class III phosphatidylinositol 3-kinase; PtdIns3P, phosphatidylinositol-3-phosphate; RILP, Rab interacting lysosomal protein; SNARE, soluble N-ethylmaleimide-sensitive factor attachment protein receptor; SQSTM1/p62, sequestosome 1; UVRAG, UV radiation resistance associated; VPS, vacuolar protein sorting; WT, wild type.

Pharmacological enhancement of TFEB-mediated autophagy alleviated neuronal death in oxidative stress-induced Parkinson's disease models.

Autophagy, a conserved cellular degradation and recycling process, can be enhanced by nutrient depletion, oxidative stress or other harmful conditions to maintain cell survival. 6-Hydroxydopamine/ascorbic acid (6-OHDA/AA) is commonly used to induce experimental Parkinson's disease (PD) lesions by causing oxidative damage to dopaminergic neurons. Activation of autophagy has been observed in the 6-OHDA-induced PD models. However, the mechanism and exact role of autophagy activation in 6-OHDA PD model remain inconclusive. In this study, we report that autophagy was triggered via mucolipin 1/calcium/calcineurin/TFEB (transcription factor EB) pathway upon oxidative stress induced by 6-OHDA/AA. Interestingly, overexpression of TFEB alleviated 6-OHDA/AA toxicity. Moreover, autophagy enhancers, Torin1 (an mTOR-dependent TFEB/autophagy enhancer) and curcumin analog C1 (a TFEB-dependent and mTOR-independent autophagy enhancer), significantly rescued 6-OHDA/AA-induced cell death in SH-SY5Y cells, iPSC-derived DA neurons and mice nigral DA neurons. The behavioral abnormality of 6-OHDA/AA-treated mice can also be rescued by Torin 1 or C1 administration. The protective effects of Torin 1 and C1 can be blocked by autophagy inhibitors like chloroquine (CQ) or by knocking down autophagy-related genes TFEB and ATG5. Taken together, this study supports that TFEB-mediated autophagy is a survival mechanism during oxidative stress and pharmacological enhancement of this process is a neuroprotective strategy against oxidative stress-associated PD lesions.

Autophagy protein NRBF2 has reduced expression in Alzheimer's brains and modulates memory and amyloid-beta homeostasis in mice.

BACKGROUND: Dysfunctional autophagy is implicated in Alzheimer's Disease (AD) pathogenesis. The alterations in the expression of many autophagy related genes (ATGs) have been reported in AD brains; however, the disparity of the changes confounds the role of autophagy in AD. METHODS: To further understand the autophagy alteration in AD brains, we analyzed transcriptomic (RNAseq) datasets of several brain regions (BA10, BA22, BA36 and BA44 in 223 patients compared to 59 healthy controls) and measured the expression of 130 ATGs. We used autophagy-deficient mouse models to assess the impact of the identified ATGs depletion on memory, autophagic activity and amyloid-ß (Aß) production. RESULTS: We observed significant downregulation of multiple components of two autophagy kinase complexes BECN1-PIK3C3 and ULK1/2-FIP200 specifically in the parahippocampal gyrus (BA36). Most importantly, we demonstrated that deletion of NRBF2, a component of the BECN1-PIK3C3 complex, which also associates with ULK1/2-FIP200 complex, impairs memory in mice, alters long-term potentiation (LTP), reduces autophagy in mouse hippocampus, and promotes Aß accumulation. Furthermore, AAV-mediated NRBF2 overexpression in the hippocampus not only rescues the impaired autophagy and memory deficits in NRBF2-depleted mice, but also reduces ß-amyloid levels and improves memory in an AD mouse model. CONCLUSIONS: Our data not only implicates NRBF2 deficiency as a risk factor for cognitive impairment associated with AD, but also support the idea of NRBF2 as a potential therapeutic target for AD.

Neuroprotective effects of berberine in animal models of Alzheimer's disease: a systematic review of pre-clinical studies.

BACKGROUND: Berberine is an isoquinoline alkaloid extracted from various Berberis species which is widely used in East Asia for a wide range of symptoms. Recently, neuroprotective effects of berberine in Alzheimer's disease (AD) animal models are being extensively reported. So far, no clinical trial has been carried out on the neuroprotective effects of berberine. However, a review of the experimental data is needed before choosing berberine as a candidate drug for clinical experiments. We conducted a systematic review on AD rodent models to analyze the drug effects with minimal selection bias. METHODS: Five online literature databases were searched to find publications reporting studies of the effect of berberine treatment on animal models of AD. Up to March 2018, 15 papers were identified to describe the efficacy of berberine. RESULTS: The included 15 articles met our inclusion criteria with different quality ranging from 3 to 5. We analyzed data extracted from full texts with regard to pharmacological effects and potential anti-Alzheimer's properties. Our analysis revealed that in multiple memory defects animal models, berberine showed significant memory-improving activities with multiple mechanisms, such as anti-inflammation, anti-oxidative stress, cholinesterase (ChE) inhibition and anti-amyloid effects. CONCLUSION: AD is likely to be a complex disease driven by multiple factors. Yet, many therapeutic strategies based on lowering ß-amyloid have failed in clinical trials. This suggest that the threapy should not base on a single cause of Alzheimer's disease but rather a number of different pathways that lead to the disease. Overall we think that berberine can be a promising multipotent agent to combat Alzheimer's disease.

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.

Canthin-6-One Accelerates Alpha-Synuclein Degradation by Enhancing UPS Activity: Drug Target Identification by CRISPR-Cas9 Whole Genome-Wide Screening Technology.

Parkinson's disease (PD) is the second most common neurodegenerative disorder characterized by the accumulation of protein aggregates (namely Lewy bodies) in dopaminergic neurons in the substantia nigra region of the brain. Alpha-synuclein (α-syn) is the major component of Lewy bodies in PD patients, and impairment of the ubiquitin-proteasome system has been linked to its accumulation. In this work, we developed a tetracycline-inducible expression system, with simultaneous induced expression of α-syn-EGFP and a bright red fluorescent protein marker (mCherry) to screen for potential compounds for degrading α-syn. We identified canthin-6-one as an α-syn lowering compound which promoted both wild type and mutants α-syn degradation in an ubiquitin-proteasome-system (UPS) dependent manner. By CRISPR/Cas9 genome-wide screening technology, we identified RPN2/PSMD1, the 26S proteasome non-ATPase regulatory subunit 1, as the targeting gene for pharmacological activity of canthin-6-one. Finally, we showed that canthin-6-one up-regulates PSMD1 and enhances UPS function by activating PKA.

Selective autophagy: The new player in the fight against neurodegenerative diseases?

Autophagy is the lysosome-mediated bulk degradation of cellular components for material recycling to maintain cellular homeostasis. Autophagy was initially regarded as a nonselective process, however, recent evidence indicates that this process can in fact be highly selective, especially for targeting and degrading organelles, invading pathogens and protein aggregates. Recent studies have revealed an intrinsic connection between selective autophagy and neurodegenerative diseases, including Alzheimer's disease and Parkinson's disease. Given the vital roles of selective autophagy in these neurodegenerative diseases, modulation of this process is emerging as a new therapeutic strategy for neuroprotection. This review introduces the concept of selective autophagy, provides an overview of the pathological connection between selective autophagy and neurodegenerative diseases, and discusses approaches to modulate selective autophagy for therapeutic effects against neurodegenerative diseases.

Natural autophagy blockers, dauricine (DAC) and daurisoline (DAS), sensitize cancer cells to camptothecin-induced toxicity.

Autophagy is a cellular bulk degradation pathway implicated in various diseases. Inhibition of autophagy has been regarded as a new therapeutic strategy for cancer treatment, especially in combination with chemotherapy. In our study, we identified two natural compounds, dauricine (DAC) and daurisoline (DAS), as two potent autophagy blockers through a high-content screening. DAC and DAS are alkaloids isolated from traditional Chinese medicine Rhizoma Menispermi. We systematically examined the effects of DAC and DAS on autophagy function in HeLa cells and found that DAC and DAS induced massive formation of autophagic vacuoles and lipidation of LC3. The accumulation of autophagic vacuoles and LC3 lipidation are due to blockage of autophagosome maturation as evidenced by interrupted colocalization of autophagsosome and lysosome, increased GFP-LC3/RFP-LC3 ratio and accumulation of autophagic substrate p62. Moreover, DAC and DAS impaired lysosomal function, as indicated by reduced lysosomal protease activity and increased lysosomal pH values. Importantly, we showed that DAC and DAS strongly inhibited the lysosome V-type ATPase activity. For the therapeutic potential, we found that DAC and DAS blocked the campothecin (CPT)-induced protective autophagy in HeLa cells, and dramatically sensitized the multiple cancer cells to CPT-induced cell death. In conclusion, our result shows that DAC and DAS are autophagy inhibitors which inhibit the lysosomal degradation of auophagic vacuoles, and sensitize the CPT-induced cancer cell death. The study implies the therapeutic potential of DAC and DAS in the treatment of cancers in combination of chemotherapy by inhibiting autophagy.

NRBF2 is involved in the autophagic degradation process of APP-CTFs in Alzheimer disease models.

Alzheimer disease (AD) is the most common neurodegenerative disease characterized by the deposition of amyloid plaque in the brain. The autophagy-associated PIK3C3-containing phosphatidylinositol 3-kinase (PtdIns3K) complex has been shown to interfere with APP metabolism and amyloid beta peptide (Aß) homeostasis via poorly understood mechanisms. Here we report that NRBF2 (nuclear receptor binding factor 2), a key component and regulator of the PtdIns3K, is involved in APP-CTFs homeostasis in AD cell models. We found that NRBF2 interacts with APP in vivo and its expression levels are reduced in hippocampus of 5XFAD AD mice; we further demonstrated that NRBF2 overexpression promotes degradation of APP C-terminal fragments (APP-CTFs), and reduces Aß1-40 and Aß1-42 levels in human mutant APP-overexpressing cells. Conversely, APP-CTFs, Aß1-40 and Aß1-42 levels were increased in Nrbf2 knockdown or nrbf2 knockout cells. Furthermore, NRBF2 positively regulates autophagy in neuronal cells and NRBF2-mediated reduction of APP-CTFs levels is autophagy dependent. Importantly, nrbf2 knockout attenuates the recruitment of APP and APP-CTFs into phagophores and the sorting of APP and APP-CTFs into endosomal intralumenal vesicles, which is accompanied by the accumulation of the APP and APP-CTFs into RAB5-positive early endosomes. Collectively, our results reveal the potential connection between NRBF2 and the AD-associated protein APP by showing that NRBF2 plays an important role in regulating degradation of APP-CTFs through modulating autophagy.

Baicalein prevents 6-OHDA/ascorbic acid-induced calcium-dependent dopaminergic neuronal cell death.

6-OHDA plus ascorbic acid (AA) has long been used to induce Parkinson's disease in rodents, while only 6-OHDA is commonly used to induce cell damage in cellular PD models. AA was believed to act as an anti-oxidant to prevent the degradation of 6-OHDA; however, some studies suggested that AA dramatically enhanced the selectivity and toxicity of 6-OHDA. To understand the mechanisms by which 6-OHDA/AA induces cell death, we established a 6-OHDA/AA cell toxicity model in human dopaminergic neuroblastoma SH-SY5Y cells. We confirmed that the toxicity of 6-OHDA was dramatically increased in the presence of AA, and the toxicity can be prevented by a flavonoid, baicalein. Mechanistically, our research reveals that 6-OHDA/AA induces cell death mainly through the interruption of intracellular calcium homeostasis, which leads to calpain activation and mitochondrial damage. Baicalein prevents 6-OHDA/AA-induced intracellular calcium elevation as well as consequent mitochondria damage. Taken together, our study confirms that 6-OHDA/AA is a more sensitive model for inducing neuronal lesion in vitro and reveals the central role of intracellular calcium in 6-OHDA/AA-induced cell death. Our studies further show that baicalein prevents 6-OHDA/AA-induced cell death by inhibiting intracellular calcium elevation.

Autophagy modulators from traditional Chinese medicine: Mechanisms and therapeutic potentials for cancer and neurodegenerative diseases.

ETHNOPHARMACOLOGICAL RELEVANCE: Traditional Chinese medicine (TCM), an ancient yet still alive medicinal system widely used in East Asia, has played an essential role in health maintenance and diseases control, for a wide range of human chronic diseases like cancers and neurodegenerative diseases. TCM-derived compounds and extracts attract wide attention for their potential application as therapeutic agents against above mentioned diseases. AIM OF REVIEW: Recent years the enthusiasm in searching for autophagy regulators for human diseases has yielded many positive hits. TCM-derived compounds as important sources for drug discovery have been widely tested in different models for autophagy modulation. Here we summarize the current progress in the discovery of natural autophagy regulators from TCM for the therapeutic application in cancer and neurodegenerative disease models, aiming to provide the direct link from traditional use to new pharmacological application. METHODS: The present review collected the literature published during the recent 10 years which studied the effect of TCM-derived compounds or extracts on autophagy regulation from PubMed, Web of Science, Google Scholar and Science Direct. The names of chemical compounds studied in this article are corresponding to the information in journal plant list. RESULTS: In this review, we give a brief introduction about the autophagy and its roles in cancer and neurodegenerative disease models and describe the molecular mechanisms of autophagy modulation. We also make comprehensive lists to summarize the effects and underlying mechanisms of TCM-derived autophagy regulators in cancer and neurodegenerative disease models. In the end of the review, we discuss the current strategies, problems and future direction for TCM-derived autophagy regulators in the treatment of human diseases. CONCLUSIONS: A number of data from in vivo and in vitro models indicated TCM derived compounds and extracts hold great potential for the treatment of human diseases including cancers and neurodegenerative diseases. Autophagy, as a novel and promising drug target involved in a wide range of human diseases, can be modulated by many TCM derived agents, indicating autophagy modulation may be an important mechanism underlying the therapeutic effect of TCM in treating diseases. Furthermore, we look forward to seeing the discovery of ideal autophagy modulators from TCM with considerably higher selectivity for the treatment of human diseases.