bafilomycin-a1 and Neurodegenerative-Diseases

Beta-propeller protein-associated neurodegeneration (BPAN, OMIM 300894) is an X-linked neurodegenerative disorder caused by mutations in WDR45. WDR45 is required for autophagy, defect in WDR45 impaired autophagy which contributes for the pathogenesis of BPAN. Previously, we reported a novel de novo mutation (c.1040_1041del, p.Glu347GlyfsTer7) in WDR45 (NM_007075) in a 3-year-old Chinese girl with BPAN.. The protein structure was constructed using SWISS-MODEL and the isoelectric point (pI) was predicted by the online pI/Mw tool at ExPASy. The functional effects of this mutation were predicted by two online software programs: PROVEN and MutationTaster. Stable overexpression of Flag-tagged wild-type or mutant WDR45 in HeLa cells was constructed. Protein levels of LC3 and p62 were analyzed by western blot upon treatment with/without autophagy inhibitor Bafilomycin A1, the formation of LC3 puncta were analyzed in HeLa cells transfected with mCherry-LC3 by confocal microscopy.. The mutation resulted in a shift of pI from 6.74 to 8.84 and was predicted to be pathogenic. The protein levels of LC3-II and p62 were increased in cells overexpression of wild-type and mutant WDR45 while the protein levels were not increased in cells overexpression of mutant WDR45 upon treatment with autophagy inhibitor Bafilomycin A1. Results from confocal microscopy revealed that LC3-positive puncta were increased in cells expressing both wild-type and mutant WDR45 while the number of LC3-positive puncta was not increased in cells expressing mutant WDR45 upon treatment with Bafilomycin A1.. Our study evidenced that this novel mutation in WDR45 impaired autophagy in cells thus this mutation is the cause for BPAN in this patient.

Topics: Asian People; Autophagy; Brain; Carrier Proteins; Child, Preschool; Female; Genes, X-Linked; HeLa Cells; Humans; Macrolides; Microtubule-Associated Proteins; Models, Molecular; Mutation; Neurodegenerative Diseases; Protein Conformation; RNA-Binding Proteins

Trinucleotide CAG repeat expansion in the coding region of genes has a propensity to form polyglutamine (polyQ) aggregates that contribute to neuronal disorders. Strategies in elevating autophagy to disintegrate the insoluble aggregates without injuring cells have become a major goal for therapy. In this work, a triazole derivative, OC-13, was found accelerating autophagic clearance of polyQ aggregation in human neuroblastoma cells following induction of the enhanced green fluorescence-conjugated chimeric protein that enclosed 79 polyQ repeats (Q79-EGFP). OC-13 accelerated autophagy development and removed nuclear Q79-EGFP aggregates. The increase of Beclin-1, turnover of LC3-I to LC3-II and degradation of p62 supported autophagy activation. Pretreatment of autophagy inhibitor, bafilomycin A1, not only suppressed autophagolysome fusion, but also impeded aggregate eradication. The study also showed that c-Jun N-terminal kinase/Beclin-1 pathway was activated during OC-13 treatment and c-Jun N-terminal kinase inhibitor impaired autophagy and final breakdown. Autophagic clearance of the insoluble aggregates demonstrated the feasibility of OC-13 in alleviating neuronal disorders because of expanded glutamine stretches.

Topics: Autophagy; Green Fluorescent Proteins; Humans; Macrolides; Nerve Tissue Proteins; Neurodegenerative Diseases; Peptides; Triazoles

Accelerating the clearance of intracellular protein aggregates through elevation of autophagy represents a viable approach for the treatment of neurodegenerative diseases. In our earlier report, we have demonstrated the enhanced degradation of mutant huntingtin protein aggregates through autophagy process induced by europium hydroxide nanorods [EHNs: Eu(III)(OH)3], but the underlying molecular mechanism of EHNs mediated autophagy was unclear. The present report reveals that EHNs induced autophagy does not follow the classical AKT-mTOR and AMPK signaling pathways. The inhibition of ERK1/2 phosphorylation using the specific MEK inhibitor U0126 partially abrogates the autophagy as well as the clearance of mutant huntingtin protein aggregates mediated by EHNs suggesting that nanorods stimulate the activation of MEK/ERK1/2 signaling pathway during autophagy process. In contrast, another mTOR-independent autophagy inducer trehalose has been found to induce autophagy without activating ERK1/2 signaling pathway. Interestingly, the combined treatment of EHNs and trehalose leads to more degradation of mutant huntingtin protein aggregates than that obtained with single treatment of either nanorods or trehalose. Our results demonstrate the rational that further enhanced clearance of intracellular protein aggregates, needed for diverse neurodegenerative diseases, may be achieved through the combined treatment of two or more autophagy inducers, which stimulate autophagy through different signaling pathways.

Topics: Adenine; Androstadienes; Animals; Autophagy; Autophagy-Related Protein 5; Butadienes; Cell Line, Tumor; Cell Survival; Chloroquine; Europium; Extracellular Signal-Regulated MAP Kinases; Green Fluorescent Proteins; HeLa Cells; Humans; Huntingtin Protein; Hydroxides; Lysosomes; Macrolides; Mice; Microscopy, Fluorescence; Microtubule-Associated Proteins; Nanotubes; Nerve Tissue Proteins; Neurodegenerative Diseases; Nitriles; Phagosomes; Phosphorylation; RNA, Small Interfering; Signal Transduction; TOR Serine-Threonine Kinases; Trehalose; Wortmannin