SARS-CoV-2 infection in hiPSC-derived neurons is cathepsin-dependent and causes accumulation of HIF1ɑ and phosphorylated tau
Authors: Pinja Kettunen , Janika Ruuska , Tania Quirin , Ravi Ojha , Saber Saber H , Shaker Mohamed , Morrison Sean , Wolvetang Ernst , Joensuu Merja , Jari Koistinaho , Taisia Rolova , Giuseppe Balistreri 
Published: November 2024

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been shown to infect the human brain and a subset of human neuronsin vitro. We have previously demonstrated that the virus enters the human induced pluripotent stem cell (hiPSC)-derived neurons via an endosomal-lysosomal pathway, which is dependent on low levels of angiotensin-converting enzyme 2 (ACE2) and independent of transmembrane serine protease 2 (TMPRSS2). Here, we use hiPSC-derived neurons overexpressing ACE2 in co-culture with human astrocytes to show that the infection with both SARS-CoV-2 Wuhan and Omicron XBB.1.5 variants is dependent on cathepsins and can be efficiently blocked by an inhibitor of cathepsin B (CA-074-ME). The result was reproducible in non-transgenic hiPSC-derived cortical organoids. The cathepsin L inhibitor SB412515 was less effective against the Wuhan strain but equally effective against the Omicron variant. Using PCR and reinfection assays, we show that SARS-CoV-2 can replicate in neurons in 2D co-cultures. Interestingly, the infectivity of the newly produced virions declined at 24 hours post-infection despite a further increase in released viral RNA at later time points, suggesting the possible activation of an antiviral response in neurons and/or astrocytes, which is supported by a correspondent increase in the levels of secreted cytokines. Furthermore, the number of infected neurons decreased within five days, suggesting that SARS-CoV-2 infection eventually leads to the death of the target neuronal cellin vitro. The infection also caused the accumulation of the hypoxia-inducible stress factor HIF1-α in infected neurons under normoxia. Finally, we confirm and expand the previous finding that in SARS-CoV-2 infected neurons, the microtubule-associated protein tau is hyperphosphorylated at multiple loci, including S202/T205, and mislocalized to the soma of the infected neurons. Hyperphosphorylation and mislocalization of tau are hallmarks of Alzheimer’s disease (AD) and other ‘tauopathies’. Our data provides further evidence supporting the neurodegenerative potential of SARS-CoV-2 infection.

The recent COVID-19 pandemic has raised concerns about the potential for SARS-CoV-2 to infect the brain and worsen brain diseases like Alzheimer’s disease. Research has shown that SARS-CoV-2 can indeed infect the human brain, including a small number of neurons and other brain cells in laboratory settings.

In our previous studies, we identified the endosomal pathway as the route the virus uses to enter neuronal cells. In this study, we build on that work by demonstrating that inhibitors of endo-lysosomal cathepsin proteases can block this neuronal infection. We also found that infectious progeny virions are released from the infected neuronal cells.

Importantly, the infection proves harmful to the host cells, as evidenced by a decrease in the number of infected cells in experimental cultures over a five-day period. Additionally, we confirm and expand on earlier findings that SARS-CoV-2 infection leads to the phosphorylation and altered localization of the tau protein, a process associated with brain diseases like Alzheimer’s.

Finally, we observed an increase in the production of inflammatory cytokines following neuronal infection with SARS-CoV-2, along with an accumulation of the stress marker protein HIF-1α in neurons. This protein has been linked to other viral infections and Alzheimer’s disease. Overall, our data suggest that SARS-CoV-2 exhibits neurodegenerative characteristics.