The ER is uniquely enriched in chaperones and folding enzymes that facilitate folding and unfolding reactions and ensure that only correctly folded and assembled proteins leave this compartment. Here we address the extent to which proteins that leave the ER and localize to distal sites in the secretory pathway are able to return to the ER folding environment during their lifetime. Retrieval of proteins back to the ER was studied using an assay based on the capacity of the ER to retain misfolded proteins. The lumenal domain of the temperature-sensitive viral glycoprotein VSVGtsO45 was fused to Golgi or plasma membrane targeting domains. At the nonpermissive temperature, newly synthesized fusion proteins misfolded and were retained in the ER, indicating the VSVGtsO45 ectodomain was sufficient for their retention within the ER. At the permissive temperature, the fusion proteins were correctly delivered to the Golgi complex or plasma membrane, indicating the lumenal epitope of VSVGtsO45 also did not interfere with proper targeting of these molecules. Strikingly, Golgi-localized fusion proteins, but not VSVGtsO45 itself, were found to redistribute back to the ER upon a shift to the nonpermissive temperature, where they misfolded and were retained. This occurred over a time period of 15 min–2 h depending on the chimera, and did not require new protein synthesis. Significantly, recycling did not appear to be induced by misfolding of the chimeras within the Golgi complex. This suggested these proteins normally cycle between the Golgi and ER, and while passing through the ER at 40°C become misfolded and retained. The attachment of the thermosensitive VSVGtsO45 lumenal domain to proteins promises to be a useful tool for studying the molecular mechanisms and specificity of retrograde traffic to the ER.
Mitochondrial dysfunction plays a central role in the selective vulnerability of dopaminergic neurons in Parkinson's disease (PD) and is influenced by both environmental and genetic factors. Expression of the PD protein α-synuclein or its familial mutants often sensitizes neurons to oxidative stress and to damage by mitochondrial toxins. This effect is thought to be indirect, since little evidence physically linking α-synuclein to mitochondria has been reported. Here, we show that the distribution of α-synuclein within neuronal and non-neuronal cells is dependent on intracellular pH. Cytosolic acidification induces translocation of α-synuclein from the cytosol onto the surface of mitochondria. Translocation occurs rapidly under artificially-induced low pH conditions and as a result of pH changes during oxidative or metabolic stress. Binding is likely facilitated by low pH-induced exposure of the mitochondria-specific lipid cardiolipin. These results imply a direct role for α-synuclein in mitochondrial physiology, especially under pathological conditions, and in principle, link α-synuclein to other PD genes in regulating mitochondrial homeostasis.
Long-term follow up is necessary to understand the natural history of treated Parkinson's disease (PD). The Longitudinal and Biomarker Study in PD (LABS-PD) is an observational study designed to prospectively measure the evolution of motor and non-motor features of PD and sample promising biomarkers from early to late stage illness. LABS-PD is organized on the premise that cohorts from completed clinical trials can be re-recruited for long-term follow up. LABS-PD will eventually contain multiple cohorts, but to test the feasibility of the strategy, we examined enrollment and biomarker sampling in the initial cohorts. The first PD cohort (PostCEPT) comes from the de novo clinical trial of a mixed lineage kinase inhibitor (PRECEPT). We assessed the recruitment from PRECEPT to PostCEPT, the ability to link data from the two studies, and sample collection for a variety of biomarkers. A total of 537 of 709 eligible PRECEPT subjects (76%) enrolled in PostCEPT; 509 (95%) had repeat dopamine transporter imaging. PRECEPT clinical and imaging data were successfully linked to PostCEPT, to provide 3 to 4 year follow-up. A biomarker sub-study enrolled over 100 PD cases from PostCEPT and 100 controls to measure olfaction and blood markers of gene expression, alpha-synuclein, and proteomic profiles. We were also successful in linking clinical and biomarker data to DNA samples that have been collected in the publicly accessible Coriell repository. The PostCEPT cohort and associated studies strongly support the feasibility of the LABS-PD approach of retaining and repurposing clinical trial cohorts to collect longitudinal clinical and biomarker data.
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