NeuroCovid

Wider research context / theoretical framework

The pandemic of coronavirus disease 19 (COVID-19) marks the emergence of the highly contagious severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and has been associated with significant global morbidity and mortality. Recently, there has been increasing recognition of neurologic symptoms in COVID-19 patients. The pathophysiology underlying the encephalopathy induced by SARS-CoV-2 is yet to be elucidated.

Hypotheses / research questions / objectives

Analysis of the limited COVID-19 literature favors the hypothesis that SARS-CoV-2 might trigger an immune-mediated encephalopathy. Based on previous findings and our preliminary data, we hypothesise that upon SARS-CoV-2 infection, the immune system generates anti-spike antibodies. These antibodies may recognize and attack neurological autoantigens due to molecular mimicry. The research question of this project is: is auto-immunity responsible for the Neurocovid pathological manifestation? We aim at obtaining a detailed clinical characterization of Neurocovid patients, identifying the endogenous target(s) recognized by Neurocovid CSF autoantibodies, and scrutinizing the mechanism underlying Neurocovid pathological manifestation.

Approach / methods

We will analyse well characterised human Neurocovid samples (CSF and serum) for the presence of neurologically relevant auto-antibodies and pro-inflammatory cytokines. We will identify potential novel antigens exploiting a state-of-art forward genetic screen that combine GeCKO library and FACS. We will validate the targets using live cell-based assays, including immunofluorescence or flow cytometry. We will investigate the molecular mechanisms underlying Covid19-related neurophysiological dysfunctions in disease-relevant neuronal models, such as rodent primary neuronal cultures and human neurons derived from induced pluripotent stem cells (iPSC). We will profile neuronal response to Neurocovid samples by electrophysiology. Ultimately, we will take advantage of a proprietary CRISPR/Cas9 human iPSC line to achieve the final validation of Neurocovid target(s).

Background

The pandemic of coronavirus disease 19 (COVID-19) marks the emergence of the highly contagious severe
acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and has been associated with significant global
morbidity and mortality (1). Clinical presentations associated with COVID-19 are variable in severity, ranging
from asymptomatic cases to severe pneumonia and acute respiratory distress syndrome (ARDS) requiring
intensive care (2,3). In symptomatic individuals, COVID-19 typically manifests as an influenza-like respiratory illness, with fever, cough, dyspnea, and malaise/myalgias (4). Atypical presentations, however, are frequent and include extrapulmonary involvement such as gastrointestinal symptoms, multiorgan failure (liver, kidneys, heart), and neurologic manifestations (5). Recently, there has been increasing recognition of neuropsychiatric symptoms in the course of COVID-19 (6). One third of COVID-19 positive patients exhibit anosmia, hyposmia and hypogeusia, which also occur in otherwise asymptomatic patients. Among the patients who required hospitalization for COVID-19, 45% of those with severe respiratory illness and 85% of those with acute respiratory distress syndrome presented neurological abnormalities (3,7). The pathophysiology underlying the encephalopathy induced by SARS-CoV-2 is yet to be elucidated. The severity of systemic illness (and the associated metabolic derangements and inflammatory cascades) in some patients with COVID-19 is likely sufficient to cause the toxic-metabolic encephalopathy often seen in hospitalized patients. However, some features of SARS-CoV-2 and specifically the presentation of patients with severe confusional states in the absence of respiratory symptoms or other organ failure have raised questions about alternative mechanisms of CNS injury. Cases of SARS-CoV-2–associated encephalitis (8,9) and Guillain-Barré syndrome (GBS) have been recently described (10–12).

SARS-CoV-2 and the CNS
The mechanism of infection by SARS-CoV- 2 exploits the virus’ strong binding affinity to the angiotensinconverting enzyme-2 (ACE2) to gain entry into cells (13). This receptor is expressed in lungs, heart, kidneys, testicles, venous endothelial cells and small intestinal enterocytes (14). ACE-2 expression has recently been found in limited neuronal population in the choroid plexus and neocortical neurons (15). Current human evidence is strongest for its expression on endothelial and smooth muscle cells (16). Besides ACE2, SARS-CoV-2 may use other neuronal proteins, such as basigin (BSG; CD147)(17) and neuropilin-1 (NRP1)(18) as docking receptors. While systematic and experimental studies regarding the neurotropism of SARS-CoV-2 are lacking (19), several mechanisms such as the transcribial route (20,21), the axonal transport and trans-synaptic transfer (22–24), and the hematogenous and/or lymphatic route (25) are currently discussed as possible viral access routes to the brain. However, after several months in the worldwide COVID-19 pandemic, there is no substantial evidence for the presence of SARS-CoV-2 in the patients brain (26), with the exception of two patients that tested positive for COVID-19 in their cerebrospinal fluid (CSF)(8,27). Notably, not all neurological manifestations require direct infection the CNS. Analysis of the limited COVID-19 literature favors the hypothesis that SARS-CoV-2 might trigger an immune-mediated encephalopathy that is a more likely event than a direct viral infection of neurons and glia (26). SARS-CoV-2 induces the release and activation of several cytokines such as interleukin-1, interleukin-16, and TNF-alpha (“cytokine storm”) that are capable of causing injury to the blood-brain barrier (BBB) (28). With increasing damage to BBB, cytokines penetrate the brain parenchyma, especially in temporal lobes where BBB is weaker (29,30). Strong inflammatory response and entry of blood material into the brain results in seizures and encephalopathy (28). The cytokines activated by SARS-CoV-2 can also trigger vasculitis in and around nerves and muscles. Indeed, a direct invasion by the virus to the peripheral nerves can potentially occur, but the lack of any SARS-CoV-2 material in the CSF to date makes this unlikely (12,31). For now, the pattern of clinical presentations and rapid response to intra venous Ig favors an immune-mediated etiology for peripheral and cranial neuropathy in patients with COVID-19.

Our hypothesis: SARS-CoV-2 and molecular mimicry
There is strong epidemiological evidence for the association between acute pathogen infections and
neurological disorders. Post-infectious immune-mediated diseases in humans can be triggered by molecular
mimicry. This process entails the induction of autoantibodies and/or autoreactive T-cells that are initially
directed against the pathogen antigens upon infection. However, owing to the structural resemblance
between microbial and particular host antigens, the antibodies and T-cells not only destroy the invading
pathogen but also attack host tissue by triggering an autoimmune disease. At least five mechanisms may
underlie immunological cross-reactivity. First is the well-known sharing of identical or homologous amino
acid sequences or homologous structural epitopes. Second, since B-cell receptors (BCRs) and TCRs show a high level of degeneracy, they can recognize non-homologous peptide sequences (32). Third, it has recently been demonstrated that a single T-cell can recognize different peptides in the context of different human leukocyte antigen (HLA) molecules (33). Fourth, the immunological receptors can recognize structural similarity in complex molecular structures other than just proteins. Lastly, to complicate matters even further, so called mimotopes have been described. These are peptides that are bound by either antibodies or T-cells directed against unrelated antigens, for example, carbohydrates, suggesting that cross-reactivity can be induced by biochemically distinct molecules (34). The surface of SARS-CoV-2 virions is coated with the spike glycoprotein, whose proteolysis is key to the infection lifecycle. After the initial interaction of the spike protein with the ACE2 receptor (35), host cell entry is mediated by two key proteolytic steps. The S1 subunit of the spike protein engages ACE2, and viral entry into the host cell is facilitated by proteases that catalyze S1/ S2 cleavage at Arginine-667/Serine-668 (36,37). This is followed by cleavage of the S2 site that is required for fusion of viral-host cell membranes (35,38). SARS-CoV-2 has evolved a unique S1/S2 cleavage site, 683-RRARSVAS-690, absent in any previously sequenced coronavirus, resulting in the striking mimicry of an identical FURIN cleavable peptide on the human epithelial sodium channel alpha-subunit (ENaC-a) (39). Interestingly, ENaC-a is expressed in the nasal epithelial cells, type II alveolar cells of the lungs, tongue keratinocytes, colon enterocytes, neurons and glia (40). Potentially, a cell-mediated or antibody-mediated reactivity towards the RRARSVAS peptide, or overlapping peptide sequences, or structural epitopes shared with other proteins may cause a dangerous inflammatory response in several tissue, including the CNS.

Preliminary data
At the beginning of the pandemic, it was thought that COVID-19 could have few extra-pulmonary
manifestations, mainly driven by either hypoxic and metabolic alterations or prolonged critical illness; the
need for strict isolation and the high number of patients also impaired further evaluation and deeply limited
the availability of instrumental tools to assess neurological symptoms, likely leading to oversimplification of
a significant proportion of cases that were misinterpreted as delirious. Nevertheless, 15 subjects (11 males, 4 females, median age 66), accounting for 3% of overall COVID-19 patients, presented with unexpected acute and subacute neurological signs between March and May 2020 in the COVID-Hospital of Rovereto,
warranting detailed clinical evaluation and lumbar puncture in 8 cases. Neurological manifestation developed 2-30 days after the onset of typical respiratory features and 80% of the patients required intensive care either for ventilatory support or severe neurological symptoms. In particular, two patients were treated with immunoglobulins for GBS, one of which progressed to bulbar involvement despite mild and early viral respiratory involvement. However, most of the patients showed ambiguous signs of  encephalopathy, such as reduced consciousness (55%), confusion (63%), altered cognitive functions and speech problems (81%), definite or suspected seizures (36%), focal motor deficits (27%), dystonia (9%), and pupillary dysfunction (18%); only two patients had plausible explanations (new onset diabetes and presumptive tardive dyskinesia), while all the other cases had no clear correlation to concomitant metabolic disturbances or alternative causes despite extensive diagnostic workup. Indeed, even when brain MRI was available, it did not exhibit any relevant signal alteration, with the exception of 2 scans, disclosing a small splenial hemorrhage and a focal hippocampal T2-hyperintensity, respectively. Aside from the typical dissociation in patients with GBS, CSF analysis was also unremarkable, with no pleocytosis and CSF proteins only reaching the upper normal range (median 44 mg/dL); a mirror oligoclonal band pattern was identified in one case. PCR testing was negative for SARS-CoV-2 as well as for common neurotropic viruses. Similar data was collected in another hospital in Pavia, in northern Italy. In particular, immunomodulating treatment was started in 5/6 cases, owing to clinical and radiological features of acute disseminated encephalomyelitis (paraparesis with urinary retention), myelitis (paraparesis), cerebellitis and GBS with strict temporal association to COVID-19 onset, thus suggesting a possible role of the virus in the pathogenesis of neurological manifestations.

Frequency of antibodies to neuronal and glial autoantigens in non-Neurocovid patients
Accordingly, Prof. Reindl’s group at Medical University of Innsbruck (MUI) started to investigate whether
SARS-CoV-2 infection can lead to secondary autoimmune reactions against CNS antigens. In a first step we
established and validated serological assays for serum and CSF antibodies to SARS-CoV-2 spike protein in
our laboratory. We have used a combination of two commercial CE certified ELISA test kits developed by
the German biopharmaceutical company Euroimmun AG (SARS-CoV-2 spike protein specific IgG) and the
Chinese biopharmaceutical company Wantai (receptor binding domain of SARS-CoV-2 spike protein total
Ig). Using this combination assay sensitivity was 92.4% (95% CI 82.1 to 97.0%) in 53 cases with COVID-19
and specificity was 100% (95% CI 98.1 to 100%) in 200 historic controls. This combination of assays was
already used to analyze serum and CSF SARS-CoV-2 antibodies in a GBS case at MUI (11). Furthermore, we established an in-house cell-based assay using recombinant full-length SARS-CoV-2 spike protein expressed on the surface of HEK 293 cells, the preferred method for the detection of neurological autoantibodies (41–43). This assay showed an excellent agreement with the ELISAs and will be used for epitope mapping and analysis of immunological cross reactivity. In a next step we have analyzed serum samples from 50 COVID-19 patients with mild to moderate disease without neurological manifestations for IgG autoantibodies to myelin oligodendrocyte glycoprotein (MOG), aquaporin-4 (AQP4) and NMDAR-receptor (NMDAR) using well established in-house recombinant live cell-based assays (44–47). None of the 50 patients (0%) developed serum AQP4 antibodies, one of the 50 patients (2%) developed low-titer MOG antibodies and two patients (4%) low-titer serum NMDAR antibodies. This observation indicates a slightly increased autoimmune response against CNS targets in non-Neuro Covid-19 and calls for a more detailed analysis of Neurocovid patients. Thus, we gathered CSF from 13 of the Neurocovid patients described above and from two patients affected by normal pressure hydrocephalus without neurological symptoms (ctrl S and V). All CSF samples resulted negative by RT-qPCR for the presence of SARS-CoV-2. We diluted the CSF samples in dedicated incubation buffer. We stained HEK293 cells over-expressing cleaved SARS-CoV-2 spike protein with diluted CSF samples in not permeabilizing condition. We detected a strong immunoreactivity in specimen stained with CSF gathered from Neurocovid patients during the acute phase.
We monitored the reactivity against spike along the time. To this aim, we collected CSF from patient Trento
5 two months after the acute phase. We still observed a strong immuno-reactivity towards Spike-expressing
HEK293 cells. Rodent primary cultures are a convenient model to scrutinize the presence of auto-antibodies reacting with endogenous proteins. Thus, we performed an immunofluorescence staining for human IgG binding to murine cortical cultures and astrocytes with the CSF gathered from 13 Neurocovid patients, from one patient suffering from auto-immune encephalitis due to anti-NMDAR auto-antibodies (positive control) and from two patients affected by normal pressure hydrocephalus (control S and V). We noticed a strong immunoreactivity towards epitopes expressed in both neurons and astrocytes. Having observed such an immunoreactivity profile, we aimed to identify the potential target(s) recognized by IgG present in the Neurocovid CSFs. We immobilized 50 ul of control S, control V, Trento 5, and Trento 7 CSF on agarose beads decorated by protein G. Then we incubated the beads with different protein samples, namely lysate obtained from murine adult brain tissue, mouse cortical culture, membrane fraction prepared from cortical culture, and HEK293 cells. After extensive washing steps, we eluted bound proteins in Laemmli buffer. We analysed the samples by SDS-PAGE followed by silver staining. We excised and measured by MS/MS the protein bands appearing only in Neurocovid samples.

AIMS
Our clinical observations clearly indicate that an inflammatory process underlies neurological symptoms
observed in COVID-19 patients. Accordingly, our in vitro data suggest the presence of autoantibodies in the
CSF of Neurocovid patients that recognize viral spike protein and endogenous targets yet to be characterized.
We hypothesize that upon SARS-CoV-2 infection, the immune system generates anti spike Igs. These Igs
may recognize and attack CNS-expressed proteins due to molecular mimicry.
Given the prevalence of neurological manifestations following SARS-CoV-2 infection, our project aims to
1. obtain a detailed clinical characterization of Neurocovid patients
2. identify the endogenous target(s) recognized by Neurocovid CSF autoantibodies
3. scrutinize the mechanism underlying Neurocovid pathological manifestation

Aim 1. Clinical characterization - Unit Dr. Bruno Giometto APSS-Santa Chiara Hospital
Early descriptions of neurological syndromes in COVID-19 patients were undoubtedly biased by the limited
tools that were available to clinicians at the outbreak of the pandemic, leading to a wide array of syndromes
with inconsistent or partial evidence of direct or indirect infectious involvement under the guise of feeble
temporal association. However, those case reports were crucial to raise awareness about possible neurological complications in SARS-CoV-2 infection, thus prompting neurologists to apply a more rigorous
diagnostic workup in the face of a dynamic and complex framework, with critically ill patients and novel
experimental therapeutic protocols with their uncertain side effects. This new basis for clinical suspicion
even in non-consultants will therefore help the identification of a higher number of patients that were
previously missed. A systematic strategy will be needed for a solid clinical characterization of possible
Neurocovid phenotypes. We propose the application of a protocol in patients with central nervous system
manifestations and COVID-19, considering these steps:
- neurological evaluation during the acute phase of the clinical manifestation; clinical data will also
include age, sex, current pharmacological treatments and pathological anamnesis, with specific
regards to immune-mediated diseases and cancer history;
- lumbar puncture to collect CSF samples for cell count, protein level and virus exclusion; CSF will be
paired with serum samples to assess oligoclonal bands;
- immunological screening, including HLA testing and lymphocyte subset typing;
- samples' transfer to the research laboratories to screen for known autoantibodies and further testing
(vide infra);
- 1,5 T brain MRI with a standard imaging protocol comprising (pre- and post-contrast) T1 weighted,
T2-weighted, FLAIR and DWI sequences; FDG-PET will be considered as an additional diagnostic
imaging technique on the basis of strong clinical suspicion for encephalitis or paraneoplastic
syndromes;
- Neuropsychological assessment: patients will be evaluated during the acute phase if possible;
- Follow-up appointment: after 6 months patients will undergo clinical and neuropsychological
evaluation and will be asked for another blood sample.
The establishment of a network inside the hospital and among different neurological departments will be
fundamental to report new cases and obtain uniform information to increase reliability. The identification of
clinical phenotypes of Neurocovid will also help the interpretation of subtle or ambiguous neurological
symptoms, thus reaching ex-post diagnosis. Moreover, the longitudinal assessment of cognitive function
along with neurological examination may give clues about the potential for chronic complications of
COVID-19, since there has been speculation about cognitive decline and motor dysfunction (e.g.
development of extra-pyramidal features) following the infection.

Aim 2. Target identification – Unit Prof. Reindl, Medical University of Innsbruck

Task 1 - Screening for antibodies to CNS autoantigens
CSF and serum samples of patients with Neurocovid will be analyzed for antibodies against known CNS
antigens such as MOG, AQP4, NMDAR, LGI1, CASPR2, AMPAR, GABABR, DPPX, IGLON5 and GFAP
using recombinant cell-based assay well established in our laboratory (41–44,46–50). Due to the limited availability of CSF and the fact that these autoantibodies are usually also present in serum we will focus on the analysis of serum samples and confirm positive results in matched CSF samples. Furthermore, we will together with our long-lasting collaboration partner Prof. Höftberger from Medical University of Vienna, screen these samples for a broad range of autoantibodies against neuronal, glial, and endothelial antigens using an unbiased approach with a rat brain tissue-based assay optimized for the detection of known and novel autoantibodies (51,52). As controls we will use serum samples from a second cohort of COVID-19 patients without CNS manifestations from Innsbruck and Trento. Cytokines storm plays a relevant role in COVID-19 pathological cascade. Furthermore, recent data have linked the severity of COVID-19 symptoms to the level of INFs (53,54). Thus, we will perform a multiplex characterization of the CSF and serum cytokine and chemokine response using the Procartaplex system from Invitrogen which can measure up to 70 analytes simultaneously in a single sample as described before (55). This approach will tell us if the inflammatory response in Neurocovid resembles that of neurological autoimmune diseases which is dominated by a IL-6 driven Th17 reaction. Identified upregulated cytokines or chemokines can then identified for their effects on neuronal and glial cells in Aim 3.

Task 2 GecKo approach (in collaboration with prof. Piccoli, University of Trento)
Several auto-immune diseases arise from the presence of conformation-specific antibodies. Mostly, target
identification relies on immunoprecipitation followed by MS/MS measurement. However, this strategy
implies the use of detergent to extract the proteins that might harm the proper folding of the target. As such,
traditional biochemical approaches towards target identification may fail. To overcome such issue, we
propose a forward genetic screen combined to fluorescence activated cell sorting (FACS). The GeCKO
library consists of specific sgRNA sequences for all-gene knock-out. The library contains 122,417 unique
human sgRNAs targeting 19000 genes and 1800 miRNAs. The library thus contains 6 sgRNAs per gene and 4 per miRNA (56).

We will package the sgRNA library into lentiviral vectors for delivery into HEK293 cells for screening. We
will use low multiplicity of infection (MOI) to ensure that most cells receive only one stably integrated RNA
guide. In order to maintain sufficient coverage of the library (>500 cells per sgRNA), we will transduce 100
million cells. We will isolate infected cells by puromycin selection and perform subsequent experiment on a
pool of minimum 60 million cells. We will decorate HEK293 cells in suspension with a pool including about
10 serum or CSF from Neurocovid patients and anti-human Ig antibody coupled to FITC. Next we will
proceed with fluorescence-activated cell sorting on a FACS Aria™ II cell sorter. We aim at a negative
selection of HEK293 cells, i.e. to isolate cells that do not express the target and therefore will not bind the
patients IgG. We will gather and expand the negative pool. We will iterate the negative FACS sorting three
times. Lastly, we will harvest the DNA, isolate and amplify the sgRNA regions and identify sgRNA by nextgeneration sequencing followed by RIGER statistical analyses to identify candidate genes.

Task 3 - Target validation
To validate the target arising from task 1 and 2 as well as the ones appearing in our preliminary data, we will
generate two complementary in vitro models: over-expression and silencing of the target via CRISPR-Cas9
approaches in human and murine culture. We will gather eukaryotic expression vector and silencing
constructs for the protein of interest from commercially available sources (Addgene, Genescript,
Genecopeia) or thanks to the scientific community. We will monitor expression/silencing efficiency by RTqPCR and/or western-blotting. These cDNAs encoding the identified target proteins will be expressed in
mammalian expression vectors as fluorescent fusion proteins in Prof. Reindl’s lab. After expression in HEK
cells bound autoantibodies will be detected using recombinant cell-based assays using immunofluorescence
or flow cytometry. Live cell-based assays will be used for membrane proteins and fixed cell-based assays or
ELISA for soluble proteins. Sensitivity and specificity of these assays will be determined using sufficient
numbers of neurological and healthy controls from our biobank. Identified autoantibodies will be analyzed
for their cross-reactivity with SARS-CoV-2 spike protein expressed in our recombinant cell-based assays or
commercial ELISA systems (see preliminary findings above) using preabsorption experiments with both
targets. Moreover, we will perform epitope mappings using amino acid deletions and point mutations of
both, SARS-CoV-2 spike protein and target antigens. We will assess by imaging approaches the
immunoreactivity profile of each individual Neurocovid serum or CSFs in both models, the expected
outcome being increase signal upon target over-expression, decreased signal upon target down-regulation.
Alternatively, we will purify and embed in liposomes our target(s). We will exploit such proteoliposomes to
test CSF immunoreactivity.

Aim 3. Molecular mechanisms of Neurocovid- Unit Dr. Mattia Volta, EURAC
Task 1 Impact of Neurocovid biosamples.
First, we will assess the impact of disease relevant specimen gathered from Neurocovid patients on neuronal physiology. Our analysis will include CSF, purified IgG from serum and commercially available cytokines identified by our network in aim 2. We will directly assess the neurophysiological effects of SARS-CoV-2-induced immune response using electrophysiology on neurons. We will use in parallel mouse primary
cortical neurons, consistent with Aim 2. Our groups have already validated the use of frozen primary neuron
cultures to enhance reproducibility across different paradigms and even different labs (57). Thus, for electrophysiology we will use the same batches of mouse neurons employed in the rest of the project. After
incubation with CSF (from patients and controls), neurons will be recorded in whole-cell patch clamp
configuration to measure voltage and current properties. We will assess action potential generation and ionic currents to appreciate the fundamental working of each neuron and its capacity to maintain neurotransmission. Thus, we will study action potential properties and firing capacities, Na+-, K+- and
Ca2+-mediated currents. Firing will be assessed by measuring threshold and frequency upon injection of
increasing current intensities. Ionic currents will be analyzed by holding the cells at specific voltages that
keep the specific channels in open and closed states. These experiments will reveal if Covid-19 material
alters the abilities of neurons in elaborating transmission. In addition, we will investigate synaptic
transmission to detect alterations in the neuronal network through the analysis of miniature excitatory postsynaptic currents (mEPSCs). Patched neurons will be hold at Vh ~60mV and quantum release of glutamate measured through the frequency and amplitude of mEPSCs.These experiments will clarify if the immune response that invades the CNS during Covid-19 has a direct effect on the normal physiology of neurons, and thus provide a first explanation of the neurological comorbidities.
In parallel, we will employ human induced pluripotent stem cells (iPSCs) differentiated in dopamine (DA)
and cortical neurons. We have already experience in DA differentiation and genome editing (58), thus we will be able to prepare immediately neurons from iPSCs obtained from healthy subjects and analyze the
neurophysiological effects of CSF incubation. DA neurons constitute a valuable neuronal population to study with regard to Covid-19 given the common alterations to olfaction and their sensibility to SARS-CoV-2 (59). In addition, we will integrate these studies establishing cortical differentiation of the same iPSCs lines, taking advantage of close collaborators who developed reliable protocols to obtain human cortical neurons (60,61). Investigating cortical neurons will provide important complementary evidence, Covid-19 patients also develop psychiatric symptoms amongst other neurological manifestations. We will subject iPSC-derived
cortical neurons to the same treatments and electrophysiological experiments described above. Altogether,
our studies will provide the scientific and medical communities with unprecedented data on the neurophysiological consequences of the “cytokine storm” that characterizes Covid-19 and constitute a solid
basis for clinical practices and therapy development.

Task 2. Role of the target(s)
Based on the results obtained in Aim 2, we will apply CRISPR-Cas9 genome editing (for which we have
published expertise (58)) to silence the most promising 5 targets. Mouse primary neurons will be treated with AAV vectors containing both Cas9 and the specific sgRNA to knock down the gene of interest. AAVs will be applied at DIV4 and recordings obtained at DIV14-16. Neurons from the same cultures will be analyzed for successful silencing via qPCR, Western blotting and immunocytochemistry (ICC). For human neurons, we have already available in-house an iPSC line constitutively expressing Flag-Cas9 (generated via lentiviral transduction). These cells, upon differentiation, will be incubated with AAVs carrying the sgRNA for the target of interest and subjected to electrophysiology with the same procedure detailed above. Similar to
mouse neurons, qPCR, Western blotting and ICC will be carried out at the same time points to confirm the
silencing of the gene of interest. This set of experiments will yield data of paramount importance as: 1- it
will provide insights into specific molecular mechanisms underlying Covid-19-related neurophysiological
dysfunctions; 2- nominate targets that are effectively modulating Covid-19-induced neuronal deficits, paving
the way for novel, effective therapeutic approaches.