VAS-2870

Neuron-specific enzyme CYP46A1 converts cholesterol to 24-hydroxycholesterol (24-HC), which crosses the brain blood barrier, entering the systemic circulation. Production of 24-HC depends on synaptic and metabolic activity and changes significantly during aging and neurodegenerative diseases. Previously, it was shown that prolonged application of 24-HC (0.4 µM) suppressed recruitment of synaptic vesicles to exocytosis during 20 Hz nerve stimulation acting via elevation of NO synthesis at the mouse neuromuscular junctions (NMJs). Here, using microelectrode recording of postsynaptic responses and fluorescent trackers for endo-exocytosis, NO and reactive oxygen species (ROS) production, the effect of 24-HC on neuromuscular transmission at 10 Hz and 70 Hz nerve firing was studied. At 10 Hz stimulation, 24-HC decreased neurotransmitter release and synaptic vesicle involvement in exocytosis. This was associated with elevation of NO synthesis without marked changes in ROS generation. However, at 70 Hz activity, 24-HC increased the recruitment of synaptic vesicles in exocytosis in combination with attenuation of NO synthesis and enhancement of ROS production. 24-HC-mediated increase in ROS production was suppressed by NADPH-oxidase inhibitor VAS2870, and antioxidant N-acetylcysteine completely prevented 24-HC-dependent potentiation of neurotransmission and suppression of NO synthesis during 70 Hz activity. Similarly, protein kinase C inhibitor chelerythrine blocked 24-HC-mediated enhancement of exocytosis and attenuation of NO generation at 70 Hz stimulation. Thus, 24-HC suppresses neurotransmission at moderate-frequency activity, probably via elevation of NO synthesis, but it potentiates neurotransmitter release and synaptic vesicle recruitment into exocytosis during high-frequency nerve firing via an NADPH oxidase/ROS/protein kinase C pathway.

Ozone-induced inflammation has been linked to the development of skin ailments including atopic dermatitis, acne vulgaris, eczema and psoriasis, mainly through a redox-inflammatory pathway. While ozone cannot penetrate the cutaneous layers, it is able to damage the skin through oxinflammatory reactions in the epidermis that lead to the generation of lipid-peroxides, aldehydes, and H₂O₂. When the production of these bioactive oxidative molecules overwhelms the cutaneous redox defenses, cutaneous damage incurs. Antimicrobial peptides (AMPs) are effector molecules that regulate a variety of cutaneous immune responses. Increased AMPs levels have also been detected in active lesions of inflammatory skin diseases. Our previous research has shown that exposure to either ozone induced the expression of cutaneous AMPs (LL-37, β-defensin 2, and β-defensin 3) levels in ex vivo skin explants, corroborating the hypothesis that ozone exposure might worsen inflammatory skin conditions via AMPs de-regulation. In the present work, to further assess the cutaneous AMPs responses in a more physiological setting, skin models cultured under physiological tension (TenBio) were expose to ozone. As a proof of concept, cutaneous models were pre-treated with a variety of redox inhibitors (catalase, deferoxamine (DFO) and VAS2870 (VAS)) before ozone exposure to better understand the involvement of a redox signaling. Our data demonstrates that even in the most realistic cutaneous ex vivo model, ozone induces LL-37, hBD2, and hBD3 protein levels through a redox mechanism. This study lays the basis to uncover the mechanisms of ozone dysregulation of cutaneous AMPs, a fundamental step to understanding the development/worsening of pollution-linked inflammatory skin conditions.