Combined LRRK2 mutation, aging and chronic low dose oral rotenone as a model of Parkinson’s disease

published_or_final_versio

Identification of “sarsasapogenin-aglyconed” timosaponins as novel Aβ-lowering modulators of amyloid precursor protein processing

published_or_final_versio

A novel synthetic epitope tag and its monoclonal antibody for specific and efficient protein detection and purification

Organizer: Cambridge Healthtech Institute (CHI)With a rapid growth of the global protein purification and isolation market, there is a huge demand for developing new ligands and kits to facilitate rapid extraction and purification of target protein samples. Affinity tag is one of the most important tools for protein purification at high quality and purity in both research laboratories and biotechnology industries. Here we introduce a novel 18-residue epitope tag carrying the peptide sequence TKENPRSNQEESYDDNES [MW: 2142.07 g/mol; Isoelectric point: pH(I) 3.84] and its highly specific monoclonal antibody for detection and affinity purification of tagged recombinant proteins. We name this new tag system the “TrackenProbeTM” as inspired from part of its peptide sequence. This synthetic peptide is a unique epitope tag designed to give stringent specificity in detection and minimal structural perturbation to target recombinant proteins being tagged. The sequence of this tag shares no significant conformational and sequence homology to any known protein in nature, thus offering maximum immuno-specificity for antibody recognition. Moreover, the inertness of this peptide tag is unlikely to cause any significant interference with natural protein functions or activities in living cells when incorporated into its target protein. Regarding solubility, computational prediction demonstrated little ordered secondary structure of this highly hydrophilic peptide tag, with optimal solvent accessibility for antibody recognition in aqueous solution. Strong hydrophilicity also enables optimal protein hydration to minimize denaturation of the target recombinant proteins. The high specificity and efficient immuno-labeling make TrackenProbeTM-antibody system an attractive tool for a wide variety of protein detection and purification strategies, e.g. Western blotting, in situ immuno-labeling, affinity chromatography, etc. (Technical details: http://www.versitech.hku.hk/reagents/ne/). We welcome biomedical research and business partnership for advanced solutions in the field

Activation of chaperone-mediated autophagy prevents accumulation of oligomeric a-synuclein in LRRK2 R1441G knockin mice of Parkinson's disease

OBJECTIVE: Explore effects of LRRK2 mutation and aging, on a-synuclein accumulation and lysosomal chaperone-mediated autophagy (CMA) and disease-modification strategy for Parkinson’s disease (PD). BACKGROUND: Leucine-rich-repeat kinase-2 (LRRK2) mutations are the commonest genetic cause of PD. a-synuclein accumulation with aging is a key pathogenic process. Its accumulation enhances formation of its toxic oligomers. Unwanted protein clearance by CMA is essential for neurons. CMA dysfunction was linked to PD. DESIGN/METHODS: a-synuclein levels in striatal lysates of LRRK2(R1441G) knockin (KI) mice and wild-type (WT) from 3 to 18 months of age were determined by Western and dot-blotting. Primary cortical neurons were incubated with recombinant a-synuclein to determine its clearance rate. Levels of lysosomal lamp2a and hsc70 levels relative to lamp1, and multimeric lamp2a complexes in cerebral cortices of 3 and 18-month-old mice were assayed to determine CMA activity. Primary cortical neurons were treated with AR7 [specific retinoic acid receptor alpha-antagonist and CMA activator] at 10 and 20μM, for 72-hr or 12 days in culture. a-synuclein levels were determined in cell lysates and conditioned medium. RESULTS: Greater age-dependent increase of striatal oligomeric a-synuclein in KI mice compared with WT, was apparent by 15 months, reaching significance at 18 months of age. KI neurons had higher maximal monomeric a-synuclein levels with slower decline indicating impaired a-synuclein clearance. Unlike young mice, aged KI mice had greater reduction of lamp2a and hsc70 levels, with increased multimeric lamp2a complexes compared to WT mice indicating greater age-dependent decrease in CMA activity. AR7 significantly decreased monomeric a-synuclein levels, and prevented oligomeric a-synuclein accumulation in KI neuronal lysates and conditioned medium. AR7 did not show cytotoxic effects by LDH release assay. CONCLUSIONS: Aged related accumulation of striatal oligomeric a-synuclein was due to impaired clearance of a-synuclein in KI mice, associated with impaired CMA activity. CMA activation prevented oligomeric a-synuclein accumulation

Impaired Stress-induced Mitophagy In Parkinsonian LRRK2(R1441G) Knockin Mutant Mice

Basic Science Abstracts - Session Title: Parkinson's Disease: Pathophysiology: abstract no. 518Objective: 1. Morphological changes and accumulation of ubiquitinated mitochondria in the striatum of aged leucine-rich-repeat kinase 2 (LRRK2) R1441G knockin mutant mice (Liu et al., 2014, 2016); 2. Mechanism of LRRK2 mutation in defective mitochondria turnover in mutant mouse embryonic fibroblasts (MEFs). Background: Autophagy and ubiquitin-proteasome system maintain mitochondria homeostasis and turnover. Autophagic stress, a key pathological feature of Parkinson’s disease (PD), perturbs mitochondrial quality and energy homeostasis in neurons. LRRK2 mutation is the most common genetic risk of PD. Studies suggested a putative role for LRRK2 in macroautophagy. We hypothesize that LRRK2 mutation disrupts mitophagy process causing defective mitochondria accumulation in aged mutant brains. Methods: For mitochondria morphology, aged mouse striatal sections were fixed and examined under transmission electron microscopy. Total number of mitochondria were quantified and compared with wildtype (WT) controls (20 randomized photomicrographs x 3 animals). Freshly isolated striatal mitochondria were immuno-labeled by antiubiquitin antibody and MitoTracker™. The degree of ubiquitination in total mitochondria pool was determined by flow cytometry. MEF cultures isolated from LRRK2 mutant mice or their WT littermates were treated with rotenone (250uM) or FCCP (10uM) for 0, 30 and 120 min. Autophagic response was determined by levels of autophagic markers (LC3B-I/II and Lamp-1) and immunocytochemistry. Results: Smaller but higher total number of mitochondria was seen in aged LRRK2 mutant mouse striatum as compared to their age-matched WT controls. The relative proportion of ubiquitinated mitochondria was higher in the mutant mice. Abnormal perinuclear clustering of enlarged lysosomes were observed in mutant but not WT MEFs under normal culture condition. Activation of autophagy was observed in both WT and mutant MEFs after exposure to rotenone or FCCP. However, mutant MEFs have significantly lower levels of total LC3B (LC3B-I/II) than the WT controls suggesting autophagosome depletion. Conclusions: Accumulation of ubiquitinated mitochondria in aged LRRK2 mutant mice indicated impaired mitochondria turnover, possibly due to depletion of LC3B for autophagosome maturation and abnormal clustering of lysosomes

Identification of timosaponins as novel A lowering modulators of amyloid precursor protein processing

Abstracts from AAIC 2016 will be published in a future issue of Alzheimer's & Dementia: The Journal of the Alzheimer's Associatio