Establishment of a Novel Fluorescence-Based Method to Evaluate Chaperone-Mediated Autophagy in a Single Neuron

Background: Chaperone-mediated autophagy (CMA) is a selective autophagy-lysosome protein degradation pathway. The role of CMA in normal neuronal functions and in neural disease pathogenesis remains unclear, in part because there is no available method to monitor CMA activity at the single-cell level. Methodology/Principal Findings: We sought to establish a single-cell monitoring method by visualizing translocation of CMA substrates from the cytosol to lysosomes using the HaloTag (HT) system. GAPDH, a CMA substrate, was fused to HT (GAPDH-HT); this protein accumulated in the lysosomes of HeLa cells and cultured cerebellar Purkinje cells (PCs) after labeling with fluorescent dye-conjugated HT ligand. Lysosomal accumulation was enhanced by treatments that activate CMA and prevented by siRNA-mediated knockdown of LAMP2A, a lysosomal receptor for CMA, and by treatments that inactivate CMA. These results suggest that lysosomal accumulation of GAPDH-HT reflects CMA activity. Using this method, we revealed that mutant cPKC, which causes spinocerebellar ataxia type 14, decreased CMA activity in cultured PCs. Conclusion/Significance: In the present study, we established a novel fluorescent-based method to evaluate CMA activity in a single neuron. This novel method should be useful and valuable for evaluating the role of CMA in various neurona

Origin of Secretin Receptor Precedes the Advent of Tetrapoda: Evidence on the Separated Origins of Secretin and Orexin

At present, secretin and its receptor have only been identified in mammals, and the origin of this ligand-receptor pair in early vertebrates is unclear. In addition, the elusive similarities of secretin and orexin in terms of both structures and functions suggest a common ancestral origin early in the vertebrate lineage. In this article, with the cloning and functional characterization of secretin receptors from lungfish and X. laevis as well as frog (X. laevis and Rana rugulosa) secretins, we provide evidence that the secretin ligand-receptor pair has already diverged and become highly specific by the emergence of tetrapods. The secretin receptor-like sequence cloned from lungfish indicates that the secretin receptor was descended from a VPAC-like receptor prior the advent of sarcopterygians. To clarify the controversial relationship of secretin and orexin, orexin type-2 receptor was cloned from X. laevis. We demonstrated that, in frog, secretin and orexin could activate their mutual receptors, indicating their coordinated complementary role in mediating physiological processes in non-mammalian vertebrates. However, among the peptides in the secretin/glucagon superfamily, secretin was found to be the only peptide that could activate the orexin receptor. We therefore hypothesize that secretin and orexin are of different ancestral origins early in the vertebrate lineage