Mechanistic Insights into a Rare Mutation in Nacad as a Possible Cause of Covid Orchitis

ABSTRACT

Objective: Aiming to uncover causal mechanisms of COVID-19 sequela outside of pulmonary symptomology, a recent study from our group identified a variant of the NACAD protein (120bp coding sequence deletion) associated with COVID-19 orchitis, a risk factor for male infertility. Interestingly, these patients had decreased ACE2 serum levels. NACAD is proposed to prevent inappropriate targeting of non-secreted peptides to the endoplasmic reticulum. We hypothesized that a defect in NACAD function/processing will decrease ACE2 serum levels by disrupting extracellular transport and endoplasmic reticulum interaction. We explored whether intracellular levels of ACE levels were altered and if cell membrane protein deposition was increased, which may result in more severe effects to SARS-CoV-2 infection. Material(s) and Method(s) We obtained testis biopsies from men undergoing sperm retrieval for infertility. After siRNA NACAD knockdown, we analyzed total ACE2 expression using Western blot and qPCR as well as immunofluorescence/H&E staining for co-localization (with NACAD). Membrane-anchored ACE2 (mACE2) levels were quantified using immunostaining and flowcytometry while secreted ACE2 (sACE2) was analyzed by ELISA and Western blot. Result(s) NACAD and ACE2 co-localize in both the Leydig and germ cells. When NACAD is knocked down, primary testes cells show 80% decreased mRNA and 50% total protein levels of ACE2. The secreted ACE2 was also reduced by nearly 50%. However, when we specifically measured the level of mACE2 on the cell surface, we found that the knockdown of NACAD did not lead to reduction. Conclusion(s) NACAD likely specifically affects the level of sACE2, which is speculated to act as a protection factor by retaining the virus in serum and reducing cellular entry. While the exact reason for the differential effects of NACAD on mACE2 and sACE2 remain unclear, our data is consistent to other studies, which found a reduction of total ACE2 expression and sACE2 in patients is coupled with worse outcomes in COVID-19. We hope that establishing a relationship between NACAD and ACE2 expression will prove useful not only to elucidate why some men with COVID develop orchitis but, ultimately, why some men develop multiorgan failure with COVID infection whereas most do not. Impact Statement We expect that men with a history of orchitis and an increased level of ACE2 receptors will present with a higher and prolonged risk of impaired semen parameters. By identifying the impact of COVID-19 infection on male fertility, we can determine whether recommendations are warranted for sperm cryopreservation for men at high risk of contracting the infection, such as healthcare workers. Copyright © 2022