Mortality in Levodopa-Treated Parkinson\u27s Disease

Parkinson’s disease (PD) is associated with increased mortality despite many advances in treatment. Following the introduction of levodopa in the late 1960’s, many studies reported improved or normalized mortality rates in PD. Despite the remarkable symptomatic benefits provided by levodopa, multiple recent studies have demonstrated that PD patients continue to die at a rate in excess of their peers. We undertook this retrospective study of 211 deceased PD patients to determine the factors associated with mortality in levodopa-treated PD. Our findings confirm that PD is associated with increased mortality in both men and women. Unlike the majority of other mortality studies, we found that women have a greater reduction in lifespan compared to men. We also found that patients with early onset PD (onset at the age of 50 or before) have reduced survival relative to PD patients with later ages of onset. A final important finding is that survival is equal in PD patients treated with levodopa early (within 2 years or less of PD onset) versus later

Bioenergetic Phenotyping of DEN-Induced Hepatocellular Carcinoma Reveals a Link Between Adenylate Kinase Isoform Expression and Reduced Complex I-Supported Respiration

Hepatocellular carcinoma (HCC) is the most common form of liver cancer worldwide. Increasing evidence suggests that mitochondria play a central role in malignant metabolic reprogramming in HCC, which may promote disease progression. To comprehensively evaluate the mitochondrial phenotype present in HCC, we applied a recently developed diagnostic workflow that combines high-resolution respirometry, fluorometry, and mitochondrial-targeted nLC-MS/MS proteomics to cell culture (AML12 and Hepa 1-6 cells) and diethylnitrosamine (DEN)-induced mouse models of HCC. Across both model systems, CI-linked respiration was significantly decreased in HCC compared to nontumor, though this did not alter ATP production rates. Interestingly, CI-linked respiration was found to be restored in DEN-induced tumor mitochondria through acute in vitro treatment with P1, P5-di(adenosine-5′) pentaphosphate (Ap5A), a broad inhibitor of adenylate kinases. Mass spectrometry-based proteomics revealed that DEN-induced tumor mitochondria had increased expression of adenylate kinase isoform 4 (AK4), which may account for this response to Ap5A. Tumor mitochondria also displayed a reduced ability to retain calcium and generate membrane potential across a physiological span of ATP demand states compared to DEN-treated nontumor or saline-treated liver mitochondria. We validated these findings in flash-frozen human primary HCC samples, which similarly displayed a decrease in mitochondrial respiratory capacity that disproportionately affected CI. Our findings support the utility of mitochondrial phenotyping in identifying novel regulatory mechanisms governing cancer bioenergetics

Bioenergetic Phenotyping of DEN-Induced Hepatocellular Carcinoma Reveals a Link Between Adenylate Kinase Isoform Expression and Reduced Complex I-Supported Respiration

Hepatocellular carcinoma (HCC) is the most common form of liver cancer worldwide. Increasing evidence suggests that mitochondria play a central role in malignant metabolic reprogramming in HCC, which may promote disease progression. To comprehensively evaluate the mitochondrial phenotype present in HCC, we applied a recently developed diagnostic workflow that combines high-resolution respirometry, fluorometry, and mitochondrial-targeted nLC-MS/MS proteomics to cell culture (AML12 and Hepa 1-6 cells) and diethylnitrosamine (DEN)-induced mouse models of HCC. Across both model systems, CI-linked respiration was significantly decreased in HCC compared to nontumor, though this did not alter ATP production rates. Interestingly, CI-linked respiration was found to be restored in DEN-induced tumor mitochondria through acute in vitro treatment with P1, P5-di(adenosine-5′) pentaphosphate (Ap5A), a broad inhibitor of adenylate kinases. Mass spectrometry-based proteomics revealed that DEN-induced tumor mitochondria had increased expression of adenylate kinase isoform 4 (AK4), which may account for this response to Ap5A. Tumor mitochondria also displayed a reduced ability to retain calcium and generate membrane potential across a physiological span of ATP demand states compared to DEN-treated nontumor or saline-treated liver mitochondria. We validated these findings in flash-frozen human primary HCC samples, which similarly displayed a decrease in mitochondrial respiratory capacity that disproportionately affected CI. Our findings support the utility of mitochondrial phenotyping in identifying novel regulatory mechanisms governing cancer bioenergetics

Macular carotenoid supplementation in subjects with atypical spatial profiles of macular pigment

This study was designed to investigate the impact of macular carotenoid supplementation on the spatial profile of macular pigment (MP) in subjects where the profile does not exhibit the typical central peak (i.e. peaked MP at foveal epicentre). Thirty one healthy subjects with such atypical MP spatial profiles were assigned to one of three intervention groups: Group 1: (n = 10), 20 mg/day lutein (L), 2 mg/day zeaxanthin (Z); Group 2: (n = 10), 10 mg/day meso-zeaxanthin (MZ), 10 mg/day L, 2 mg/day Z; Group 3: (n = 10), 17 mg/day MZ, 3 mg/day L, 2 mg/day Z. Subjects were instructed to take one capsule daily over an 8-week period. MP at 0.25°, 0.5°, 1°, 1.75° and 3° was measured using customized-heterochromatic flicker photometry at baseline, four weeks and 8 weeks. Over the study period, we report no statistically significant increase in MP at any eccentricity in Group 1 (p > 0.05, for all eccentricities). There was a trend towards an increase in MP at all eccentricities in Group 2, with a significant increase found at 0.25° and 0.50° (p = 0.000 and p = 0.016, respectively). There was a statistically significant increase evident in MP at 0.25° in Group 3 (p = 0.005), but at no other eccentricity (p > 0.05, for all other). We report that the typical central peak of MP can be realised in subjects with atypical spatial profiles, following supplementation with a preparation containing all three macular carotenoids, but not with a supplement lacking MZ. The implications of our findings, in terms of visual performance and/or a (photo)-protective effect, warrant additional study

Spent Culture Medium from Virulent Borrelia burgdorferi Increases Permeability of Individually Perfused Microvessels of Rat Mesentery

Lyme disease is a common vector-borne disease caused by the spirochete Borrelia burgdorferi (Bb), which manifests as systemic and targeted tissue inflammation. Both in vitro and in vivo studies have shown that Bb-induced inflammation is primarily host-mediated, via cytokine or chemokine production that promotes leukocyte adhesion/migration. Whether Bb produces mediators that can directly alter the vascular permeability in vivo has not been investigated. The objective of the present study was to investigate if Bb produces a mediator(s) that can directly activate endothelial cells resulting in increases in permeability in intact microvessels in the absence of blood cells.The effects of cell-free, spent culture medium from virulent (B31-A3) and avirulent (B31-A) B. burgdorferi on microvessel permeability and endothelial calcium concentration, [Ca(2+)](i), were examined in individually perfused rat mesenteric venules. Microvessel permeability was determined by measuring hydraulic conductivity (Lp). Endothelial [Ca(2+)](i), a necessary signal initiating hyperpermeability, was measured in Fura-2 loaded microvessels. B31-A3 spent medium caused a rapid and transient increase in Lp and endothelial [Ca(2+)](i). Within 2-5 min, the mean peak Lp increased to 5.6+/-0.9 times the control, and endothelial [Ca(2+)](i) increased from 113+/-11 nM to a mean peak value of 324+/-35 nM. In contrast, neither endothelial [Ca(2+)](i) nor Lp was altered by B31-A spent medium.A mediator(s) produced by virulent Bb under culture conditions directly activates endothelial cells, resulting in increases in microvessel permeability. Most importantly, the production of this mediator is associated with Bb virulence and is likely produced by one or more of the 8 plasmid(s) missing from strain B31-A

Coaggregation of RNA-Binding Proteins in a Model of TDP-43 Proteinopathy with Selective RGG Motif Methylation and a Role for RRM1 Ubiquitination

TAR DNA-binding protein 43 (TDP-43) is a major component within ubiquitin-positive inclusions of a number of neurodegenerative diseases that increasingly are considered as TDP-43 proteinopathies. Identities of other inclusion proteins associated with TDP-43 aggregation remain poorly defined. In this study, we identify and quantitate 35 co-aggregating proteins in the detergent-resistant fraction of HEK-293 cells in which TDP-43 or a particularly aggregate prone variant, TDP-S6, were enriched following overexpression, using stable isotope-labeled (SILAC) internal standards and liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). We also searched for differential post-translational modification (PTM) sites of ubiquitination. Four sites of ubiquitin conjugation to TDP-43 or TDP-S6 were confirmed by dialkylated GST-TDP-43 external reference peptides, occurring on or near RNA binding motif (RRM) 1. RRM-containing proteins co-enriched in cytoplasmic granular structures in HEK-293 cells and primary motor neurons with insoluble TDP-S6, including cytoplasmic stress granule associated proteins G3BP, PABPC1, and eIF4A1. Proteomic evidence for TDP-43 co-aggregation with paraspeckle markers RBM14, PSF and NonO was also validated by western blot and by immunocytochemistry in HEK-293 cells. An increase in peptides from methylated arginine-glycine-glycine (RGG) RNA-binding motifs of FUS/TLS and hnRNPs was found in the detergent-insoluble fraction of TDP-overexpressing cells. Finally, TDP-43 and TDP-S6 detergent-insoluble species were reduced by mutagenesis of the identified ubiquitination sites, even following oxidative or proteolytic stress. Together, these findings define some of the aggregation partners of TDP-43, and suggest that TDP-43 ubiquitination influences TDP-43 oligomerization