Le, V. Q. | bioRxiv Science

bioRxiv2018

Characteristics of the mitochondrial and cellular uptake of MPP+, as probed by the fluorescent mimic, 4′I-MPP+

The discovery that 1-methyl-4-phenylpyridinium (MPP+) selectively destroys dopaminergic neurons and causes Parkinsons disease (PD) symptoms in mammals has strengthened the environmental hypothesis of PD. The current model for the dopaminergic toxicity of MPP+ is centered on the uptake into dopaminergic neurons, accumulation into the mitochondria, inhibition of the complex-I leading to ATP depletion, increased reactive oxygen species (ROS) production, and apoptotic cell death. However, some aspects of this mechanism and the details of the cellular and mitochondrial accumulation of MPP+ are still poorly understood. The aim of this study was to characterize a structural and functional MPP+ mimic which is suitable to study the cellular distribution and mitochondrial uptake of MPP+ in live cells and use it to identify the molecular details of these processes to advance the understanding of the mechanism of the selective dopaminergic toxicity of MPP+. Here we report the characterization of the fluorescent MPP+ derivative, 1-methyl-4-(4-iodophenyl)pyridinium (4I-MPP+), as a suitable candidate for this purpose. Using this novel probe, we show that cytosolic/mitochondrial Ca2+ play a critical role through sodium-calcium exchanger (NCX) in the mitochondrial and cellular accumulation of MPP+ suggesting for the first time that MPP+ and related mitochondrial toxins may also exert their toxic effects through the perturbation of Ca2+ homeostasis in dopaminergic cells. We also found that the specific mitochondrial NCX (mNCX) inhibitors protect dopaminergic cells from the MPP+ and 4I-MPP+ toxicity, most likely through the inhibition of the mitochondrial uptake, which could potentially be exploited for the development of pharmacological agents to protect the central nervous system (CNS) dopaminergic neurons from PD-causing environmental toxins.

neuroscience↗

bioRxiv2017

Tolloid cleavage activates latent GDF8 by priming the pro-complex for dissociation

Growth differentiation factor 8 (GDF8)/Myostatin is a latent TGF{-}{beta} family member that potently inhibits skeletal muscle growth. Here, we compared the conformation and dynamics of precursor, latent, and Tolloid{-}cleaved GDF8 pro{-}complexes to understand structural mechanisms underlying latency and activation of GDF8. Negative stain electron microscopy (EM) of precursor and latent pro{-}complexes reveals a V{-}shaped conformation that is unaltered by furin cleavage and sharply contrasts with the ring{-}like, cross{-}armed conformation of latent TGF{-}{beta}1. Surprisingly, Tolloid{-}cleaved GDF8 does not immediately dissociate, but in EM exhibits structural heterogeneity consistent with partial dissociation. Hydrogen-deuterium exchange was not affected by furin cleavage. In contrast, Tolloid cleavage, in the absence of prodomain-growth factor dissociation, increased exchange in regions that correspond in pro-TGF-{beta}1 to the 1-helix, latency lasso, and {beta}1 strand in the prodomain and to the {beta}6-7 strands in the growth factor. Thus, these regions are important in maintaining GDF8 latency. Our results show that Tolloid cleavage activates latent GDF8 by destabilizing specific prodomain-growth factor interfaces and primes the growth factor for release from the prodomain.

biochemistry↗