Philippe, B. | bioRxiv Science

bioRxiv2023

Higher Angiotensin I Converting Enzyme 2 (ACE2) levels in the brain of individuals with Alzheimer's disease.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a major cause of death in the elderly. Cognitive decline due to Alzheimers disease (AD) is frequent in the geriatric population disproportionately affected by the COVID-19 pandemic. Interestingly, central nervous system (CNS) manifestations have been reported in SARS-CoV-2-infected patients. In this study, we investigated the levels of Angiotensin I Converting Enzyme 2 (ACE2), the main entry receptor of SARS-COV-2 in cells, in postmortem parietal cortex samples from two independent AD cohorts, totalling 142 persons. Higher concentrations of ACE2 protein and mRNA were found in individuals with a neuropathological diagnosis of AD compared to age-matched healthy control subjects. Brain levels of soluble ACE2 were inversely associated with cognitive scores (p = 0.02), markers of pericytes (PDGFR{beta}, p=0.02 and ANPEP, p = 0.007) and caveolin1 (p = 0.03), but positively correlated with soluble amyloid-{beta} peptides (A{beta}) concentrations (p = 0.01) and insoluble phospho- tau (S396/404, p = 0.002). No significant differences in ACE2 were observed in the 3xTgAD mouse model of tau and A{beta} neuropathology. Results from immunofluorescence and Western blots showed that ACE2 protein is mainly localized in neurons in the human brain but predominantly in microvessels in the mouse brain. The present data show that an AD diagnosis is associated with higher levels of soluble ACE2 in the human brain, which might contribute to a higher risk of CNS SARS-CoV-2 infection.

neuroscience↗

bioRxiv2020

Enzymatic analysis of yeast cell wall-resident GAPDH and its secretion

In yeast, many proteins are found both in the cytoplasmic and extracellular compartments, and consequently it can be difficult to distinguish non-conventional secretion from cellular leakage. We therefore monitored extracellular glyceraldehyde-3-phosphate dehydrogenase (GAPDH) activity of intact cells as a specific marker for non-conventional secretion. Extracellular GAPDH activity was proportional to the number of cells assayed, increased with incubation time, and was dependent on added substrates. Preincubation of intact cells with 100M dithiothreitol increased the reaction rate, consistent with increased access of the enzyme after reduction of cell wall disulfide crosslinks. Such treatment did not increase cell permeability to propidium iodide, in contrast to effects of higher concentrations of reducing agents. An amine-specific membrane-impermeant biotinylation reagent specifically inactivated extracellular GAPDH. The enzyme was secreted again after a 30-60-minute lag following the inactivation, and there was no concomitant increase in propidium iodide staining. There were about 4 x 104 active GAPDH molecules per cell at steady state, and secretion studies showed replenishment to that level one hour after inactivation. These results establish conditions for specific quantitative assays of cell wall proteins in the absence of cytoplasmic leakage and for subsequent quantification of secretion rates in intact cells. ImportanceEukaryotic cells secrete many proteins, including many proteins that do not follow the classical secretion pathway. Among these, the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is unexpectedly found in the walls of yeasts and other fungi, and in extracellular space in mammalian cell cultures. It is difficult to quantify extracellular GAPDH, because leakage of just a little of the very large amount of cytoplasmic enzyme can invalidate the determinations. We used enzymatic assays of intact cells, while also maintaining membrane integrity. The results lead to estimates of the amount of extracellular enzyme, and its rate of secretion to the wall in intact cells. Therefore, enzyme assays under controlled conditions can be used to investigate non-conventional secretion more generally.

cell biology↗