Pagano, P. J. | bioRxiv Science

bioRxiv2021

Cooperation between CYB5R3 and NOX4 via coenzyme Q mitigates endothelial inflammation

NADPH oxidase 4 (NOX4) regulates endothelial inflammation by producing reactive oxygen species. Since coenzyme Q (CoQ) mimics affect NOX4 activity, we hypothesize that cytochrome b5 reductase 3 (CYB5R3), a CoQ reductase abundant in vascular endothelial cells, modulates inflammatory activation. Mice lacking endothelial CYB5R3 (R3 KO), under lipopolysaccharides (LPS) challenge, showed exacerbated hypotension, decreased acetylcholine-induced vasodilation, and elevated vascular adhesion molecule 1 (Vcam-1) mRNA in aorta. In vitro, silencing Cyb5r3 enhanced LPS-induced VCAM-1 protein in a NOX4 dependent manner. APEX2- based electron microscopy and proximity biotinylation demonstrated CYB5R3s localization on the mitochondrial outer membrane and its interaction with NOX4, which was further confirmed by the proximity ligation assay. Notably, Cyb5r3 silenced HAECs had less total H2O2 but more mitochondrial O2*-. Using inactive or non-membrane bound active CYB5R3, we found CYB5R3 activity and membrane translocation were needed for optimal generation of H2O2 by NOX4. Lastly, CoQ deficient cells showed decreased NOX4-derived H2O2, indicating a requirement for endogenous CoQ in NOX4 activity. In conclusion, CYB5R3 mitigates endothelial inflammatory activation by assisting in NOX4-dependent H2O2 generation via CoQ. NOVELTY AND SIGNIFICANCEO_ST_ABSWhat Is Known?C_ST_ABSNADPH oxidase 4 (NOX4) reportedly produces primarily hydrogen peroxide (H2O2) and, to a lesser extent, superoxide (O2*-) and has been shown to have both beneficial and deleterious effects in the cardiovascular system. NOX4 activity can be affected by NAD(P)H quinone oxidoreductase 1 (NQO1), a CoQ reductase, and synthetic quinone compounds used to mimic CoQ. Cytochrome b5 reductase 3 (CYB5R3) is known to reduce CoQ and is highly expressed in endothelial cells. What New Information Does This Article Contribute?In vivo, the lack of endothelial CYB5R3 causes exacerbated lipopolysaccharides (LPS)-induced inflammatory signaling, endothelial dysfunction, and hypotension. Endothelial CYB5R3 mitigates inflammatory signaling by LPS and tumor necrosis factor (TNF-) in a NOX4 dependent manner. In endothelial cells, CYB5R3 and NOX4 reside in close proximity on the mitochondrial outer membrane. NOX4s ability to generate H2O2 depends on the membrane translocation and activity of CYB5R3 and the presence of endogenous CoQ. NONSTANDARD Abbreviations and Acronyms [Table 1] Protein names are abbreviated as capital letters (e.g., CYB5R3), while the corresponding gene names are annotated as in italic lower cases (e.g., Cyb5r3).

cell biology↗

bioRxiv2021

Neuronal NADPH oxidase in Parkinson disease pathogenesis

Mitochondrial dysfunction and oxidative stress are strongly implicated in the pathogenesis of Parkinsons disease (PD) and there is evidence that mitochondrially-generated superoxide can activate NADPH oxidase 2 (NOX2), which is a major enzymatic generator of superoxide. Although NOX2 has been examined in the context of PD, previous studies have focused on microglial function; the role of neuronal NOX2 in PD pathogenesis remains to be defined. Here we devised and validated a proximity ligation assay for NOX2 activity and demonstrated that in human PD and 2 models thereof, neuronal NOX2 is highly active in substantia nigra dopamine neurons. Further, NOX2 activity is responsible for accumulation, post-translational modification and oligomerization of -synuclein as well as activation of leucine-rich repeat kinase 2 (LRRK2). Administration of a brain-penetrant, specific NOX2 inhibitor prevented NOX2 activation and its downstream effects in vivo in a rat model of PD. We conclude that neuronal NOX2 is a major contributor to oxidative stress in PD, to -synuclein pathology and to LRRK2 activation in idiopathic PD. In this context, NOX2 inhibitors hold potential as a disease-modifying therapy in PD. SummaryIn dopamine neuron, NADPH oxidase isoform 2 amplifies the oxidative stress-related pathogenic cascade in Parkinsons disease

neuroscience↗