Mechanistic insights into a rare genetic cause of covid orchitis

Abstract Introduction To date, biomedical efforts against the SARS-CoV-2 virus have mainly focused on mitigating spread and primary infection mortality. While significant strides have been made in community response and vaccine production, post-acute COVID-19 syndromes (Long COVID) are growing in prevalence and becoming an increasing burden on public health. Current evidence suggests COVID-19 can affect the testis and cases of orchitis (testicular pain) appear to present in up to 20% of men with COVID-19. With an aim of uncovering causal mechanisms of COVID-19 sequela outside of pulmonary symptomology, a recent study from our group has identified a nonsynonymous non-frameshift mutation in the NACAD protein associated with COVID-19 orchitis and decreased ACE2 serum levels. NACAD is thought to prevent inappropriate targeting of non-secreted peptides to the endoplasmic reticulum. Objective 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 ACE2 as well as TMPRSS2 were altered and if cell membrane protein deposition was increased, which may result in more severe effects to SARS-CoV-2 infection. Methods 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 colocalization (with NACAD). Membrane-anchored ACE2 (mACE2) levels were quantified using immunostaining and flowcytometry while secreted ACE2 (sACE2) was analyzed by ELISA and Western blot. Results 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. Conclusions 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. Disclosure No