DISSECTING THE MOLECULAR MECHANISMS RELATED TO THE ONSET OF NEUROLOGICAL COMPLICATIONS IN THREE CELLULAR MODELS OF SARS-COV 2 INFECTION: IPSC-DERIVED MOTO R NEURONS, IPSC-DERIVED DOPAMINERGIC NEURONS, IPSC DERIVED-HUMAN CORTICAL ORGANOIDS.

Although COVID‐19 typically leads to respiratory disorders, recent evidence show that acute and sub-acute neurological complications are reported in patients not only with severe disease, but also in otherwise minimally symptomatic or asymptomatic people [1–3]. Notably, up to 65% of COVID-19 affected patients reported a wide range of neurological conditions, such as decreased sense of smell or hyposmia, dizziness, headache, nausea, vomiting, impaired consciousness, as well as cases of encephalopathy, but also peripheral disorders including Guillain-Barre syndrome and myositis-like muscle injury [2, 4–8], suggesting detrimental effects of SARS-CoV-2 on both the central and peripheral nervous system (CNS, PNS). Multiple studies have shown neuroinflammation in patients with COVID-19, which may underlie these neurological and neuropsychiatric symptoms [7, 9–17]. In addition, as with other viruses, the hypothesis that SARS-CoV-2 infection may accelerate neurodegeneration and the risk of neurodegenerative diseases is gaining increasing attention. [18–21]. However, the molecular mechanisms responsible for these disfunctions, as well as the potential neurotropism of SARS-CoV-2, are still under investigation. To date it remains unclear whether the neurological symptoms are a consequence of direct neural infection, para-infectious or post-infectious immune-mediated disease, or sequalae of systemic disease [1, 22]. It has been hypothesized that SARS-CoV-2 might affect the dopamine pathway [23–28] which in turn would interfere with neuronal activities. In this frame, it has been demonstrated both in-vivo and in-vitro that SARS-CoV-2 is able to infect different neuronal cell types with different degrees of success [29–37]. Further, considering that human brain tissue is difficult to access, particularly from patients with a contagious pathogen due to safety concerns [38], 2D and 3D in vitro models can provide a viable and safe alternative and represent a suitable model system to test the neurotoxic effects of SARS-CoV-2 [32, 34–37, 39]. In this context, during my PhD training I’ve been involved in different projects in collaboration with different research groups, whose general purpose was to assess the relationship existing between SARS-CoV-2 infection and the alteration of the nervous system. Thus, I was given the chance to work with three different neuronal models, including dopaminergic neurons, iPSC derived motor neurons, and human cortical organoids, in order to investigate this extremely current and concerning condition. Each of these projects, addressed below in the text, has independently contributed to broadening knowledge about the relationship between SARS-CoV-2 and the nervous system. From this scenario, using human iPSC differentiated to dopaminergic neurons (DA neurons) we found that. Infection with EU and Delta variants results in a reduced intracellular content and extracellular release of dopamine, while tyrosine hydroxylase was upregulated at the mRNA level and downregulated at the protein level. DOPA-decarboxylase and dopamine transporter were downregulated at both mRNA and protein levels, and in addition, in vitro SARS-CoV-2 infection was associated with altered MAP2 and TAU expression and increased neuronal stress markers. These results suggest that SARS-CoV-2 affects dopamine metabolism and production, partially explaining the neurological symptoms. In parallel, for the very first time, we found that SARS-CoV-2 can productively infect human iPSC-derived MNs probably by binding CD147 and NRP1 receptors. Furthermore, SARS-CoV-2 infection in iPSC-MNs significantly altered the expression of genes (IL-6, ANG, S1PR1, BCL2, BAX, Casp8, HLA-A, ERAP1, CD147, MX1) associated with cell survival and metabolism, as well as antiviral and inflammatory response. Such information will be important to unveil the biological bases of neuromuscular disorders characterizing SARS-CoV-2 infection and the so called long-COVID symptoms. At the same time, we observed that iPSC-Human Cortical Organoids (HCO) were productively infected by SARS-CoV-2, probably by binding CD147 receptor. Furthermore, SARS-CoV-2 infection as well as Spike exposure of MNs were accompanied by the activation of apoptotic and stress pathways (caspase 3, caspase8, Bcl2) (S100B), inflammatory process (CCL2, NLRP3) and induce expression of both Interferon Stimulated Genes (IFITM1, IFITM3, STAT1, NFkB) and the antigen presentation pathway (ERAP1, ERAP2, HLA-A and TAP). Overall, the results obtained suggest that HCO organoids may be infected by SARS-CoV-2 and their homeostasis may be significantly altered even following S-exposure. These three independent projects contributed to partially explain some of the neurological symptoms, characterizing the Long COVID symptomatology. Further analyses are ongoing to unveil the molecular mechanisms responsible for the neuronal disorders characterizing SARS-CoV-2 infection and the so called long-COVID symptoms in these neuronal models.