Severe acute respiratory syndrome coronavirus 2 infection leads to Tau pathological signature in neurons

COVID-19 has represented an issue for global health since its outbreak in March 2020. It is now evident that the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection results in a wide range of long-term neurological symptoms and is worryingly associated with the aggravation of Alzheimer’s disease. Little is known about the molecular basis of these manifestations. Here, several strain variants were used to infect SH-SY5Y neuroblastoma cells and K18-hACE C57BL/6J mice. The Tau phosphorylation profile and aggregation propensity upon infection were investigated on cellular extracts, subcellular fractions, and brain tissue. The viral proteins spike, nucleocapsid, and membrane were overexpressed in SH-SY5Y cells, and the direct interaction and effect on Tau phosphorylation were checked using immunoblot experiments. Upon infection, Tau is phosphorylated at several pathological epitopes associated with Alzheimer’s disease and other tauopathies. Moreover, this event increases Tau’s propensity to form insoluble aggregates and alters its subcellular localization. Our data support the hypothesis that SARS-CoV-2 infection in the central nervous system triggers downstream effects altering Tau function, eventually leading to the impairment of neuronal function

Ruolo della proteina Tau nelle Tauopatie: studio dell'alterazione della sintesi proteica

Le tauopatie come l’Alzheimer (AD), la paralisi sopranucleare progressiva (PSP) e la demenza frontotemporale (FTDP), sono malattie neurodegenerative caratterizzate da inclusioni neuronali e/o gliali composte dalla proteina Tau. Questi accumuli neuropatologici contribuiscono allo sviluppo di deficit sinaptici e perdita neuronale, portando così ad un progressivo declino delle funzioni motorie e cognitive. Il guadagno tossico di funzione della proteina Tau modula diversi pathway molecolari; la via di segnalazione PERK/eIF2a risulta iperattiva nei cervelli AD. Lo scopo del mio lavoro di tesi è focalizzato sullo studio della proteina nell'alterazione del pathway. Tauopathies such as Alzheimer's (AD), progressive supranuclear palsy (PSP) and frontotemporal dementia (FTDP), are neurodegenerative diseases characterized by neuronal and / or glial inclusions composed of the Tau protein. These neuropathological accumulations contribute to the development of synaptic deficits and neuronal loss, thus leading to a progressive decline in motor and cognitive functions. The toxic gain-of-function of the Tau protein modulates several molecular pathways; the PERK / eIF2a signaling pathway is overactive in AD brains. The purpose of my thesis work is focused on the study of the protein in the alteration of the pathway

SARS-CoV-2 Infection Alters the Phenotype and Gene Expression of Adipocytes

Epidemiological evidence emphasizes that excess fat mass is associated with an increased risk of severe COVID-19 disease. Nevertheless, the intricate interplay between SARS-CoV-2 and adipocytes remains poorly understood. It is crucial to decipher the progression of COVID-19 both in the acute phase and on long-term outcomes. In this study, an in vitro model using the human SGBS cell line (Simpson-Golabi-Behmel syndrome) was developed to investigate the infectivity of SARS-CoV-2 in adipocytes, and the effects of virus exposure on adipocyte function. Our results show that SGBS adipocytes expressing ACE2 are susceptible to SARS-CoV-2 infection, as evidenced by the release of the viral genome into the medium, detection of the nucleocapsid in cell lysates, and positive immunostaining for the spike protein. Infected adipocytes show remarkable changes compared to uninfected controls: increased surface area of lipid droplets, upregulated expression of genes of inflammation (Haptoglobin, MCP-1, IL-6, PAI-1), increased oxidative stress (MnSOD), and a concomitant reduction of transcripts related to adipocyte function (leptin, fatty acid synthase, perilipin). Moreover, exogenous expression of spike protein in SGBS adipocytes also led to an increase in lipid droplet size. In conclusion using the human SGBS cell line, we detected SARS-CoV-2 infectivity in adipocytes, revealing substantial morphological and functional changes in infected cells

Selection and characterization of human scFvs targeting the SARS-CoV-2 nucleocapsid protein isolated from antibody libraries of COVID-19 patients

<p>The Data Set is a collection of the Fasta of sequencing of 6 human library of scFvs (VH-linker VL) obtain from the IgM repertoire and of 6 human libraries of scFvs (VH-linker VL) obtain from the IgG_IgA repertoire of 6 patients that recover from COVID19.</p> <p>Each of the 12 libraries (6 for the IgM and 6 for the IaG/IgA repertoire) were sequenced by ION TORRENT technology by Genomnia. For each scFv library an independently VH and a VL Ion Torrent library was constructed as follows. VH and VL were amplified using primers (VH_fus_F- VH_fus_R and VL_fus_F - VL_fus_R_BXX) having, at the 3’end, the region presents in the pLinker220 plasmid flanking the VH or VL and, at the 5’ end, the Ion Torrent adaptors. In this step, Unique Molecular Identifiers (UMI) consisting of 13 degenerate bases are also introduced in the reverse primer used to amplify the VH and VH regions.  The amplified VH and VL were then subjected to a second PCR using the primers A_fus – trP1_fus to build the fusion library. Sequencing was performed on an Ion Torrent platform (IonS5) with 400bp chemistry on a 530 chip. Both types of sequencing libraries were constructed by Genomnia. Each VH library was individually sequenced to a 400bp read on a 530 chip, yielding approximately 20-25 million reads per sample. The VL libraries, on the other hand, were barcoded and sequenced in a pool of three libraries from different patients, aiming to obtain 6-10 million reads per library. All sequencing runs were of good quality, with an average of 73% of the reads mapping to immunoglobulin genes used for clonotype identification (i.e. less than 30% polyclonal reads) and less than 20% low-quality sequences</p> <p> </p> <p><span> The correspondence between the library number (#) and the # of the patient in the sequencing  files name is as follows:</span></p> <table> <tbody> <tr> <td> <p><strong><span>Library #</span></strong></p> </td> <td> <p><strong><span>Patient # in the file name</span></strong></p> </td> </tr> <tr> <td> <p><span>lib 1 </span></p> </td> <td> <p><span>pz24</span></p> </td> </tr> <tr> <td> <p><span>lib 2 </span></p> </td> <td> <p><span>pz26</span></p> </td> </tr> <tr> <td> <p><span>lib 3 </span></p> </td> <td> <p><span>pz27</span></p> </td> </tr> <tr> <td> <p><span>lib 4 </span></p> </td> <td> <p><span>pz39</span></p> </td> </tr> <tr> <td> <p><span>lib 5 </span></p> </td> <td> <p><span>pz42</span></p> </td> </tr> <tr> <td> <p><span>lib 6 </span></p> </td> <td> <p><span>pz44</span></p> </td> </tr> </tbody> </table> <p><span> </span></p> <p> </p> <p>The correspondence between the library number (#) and the file name for each of the VH and  sequencing is as follows:</p> <table> <tbody> <tr> <td> <p><strong><span>Library #</span></strong></p> </td> <td> <p><strong><span>File name VH Sequences</span></strong></p> </td> </tr> <tr> <td> <p><span>lib 1 IgM VH</span></p> </td> <td> <p><span>PANANTICOVID_I_Auto_user_S5-00513-163-30pM_24_IgM_VH_414__REANALYSIS_488_rawtf.fa</span></p> </td> </tr> <tr> <td> <p><span>lib 1 IgG_IgA VH</span></p> </td> <td> <p><span>PANANTICOVID_I_Auto_user_S5-00513-162-30pM_24_IgG_IgA_VH_407_469_rawtf.fa</span></p> </td> </tr> <tr> <td> <p><span>lib 2 IgM VH</span></p> </td> <td> <p><span>PANANTICOVID_I_Auto_user_S5-00513-157-30pM_pz26_VH_IgM_404_REANALYSIS_460_rawtf.fa</span></p> </td> </tr> <tr> <td> <p><span>lib 2 IgG_IgA VH</span></p> </td> <td> <p><span>PANANTICOVID_I_Auto_user_S5-00513-158-30pM_pz26_VH_IgG_IgA_403_458_rawtf.fa</span></p> </td> </tr> <tr> <td> <p><span>lib 3 IgM VH</span></p> </td> <td> <p><span>PANANTICOVID_I_Auto_user_S5-00513-161-30pM_27_IgM_VH_408_465_rawtf.fa</span></p> </td> </tr> <tr> <td> <p><span>lib 3 IgG_IgA VH</span></p> </td> <td> <p><span>PANANTICOVID_I_Auto_user_S5-00513-164-30pM_27_IgG_IgA_VH_413_482_rawtf.fa</span></p> </td> </tr> <tr> <td> <p><span>lib 4 IgM VH</span></p> </td> <td> <p><span>PANANTICOVID_II_Auto_user_S5-00513-159-30pM_pz39_IgM_VH_406_461_rawtf.fa</span></p> <p><span>PANANTICOVID_I_Auto_user_S5-00513-159-30pM_pz39_IgM_VH_406_REANALYSIS_477_rawtf.fa</span></p> </td> </tr> <tr> <td> <p><span>lib 4 IgG_IgA VH</span></p> </td> <td> <p><span>PANANTICOVID_I_Auto_user_S5-00513-160-30pM_pz39_IgG_IgA_VH_405_463_rawtf.fa</span></p> </td> </tr> <tr> <td> <p><span>lib 5 IgM VH</span></p> </td> <td> <p><span>PANANTICOVID_II_Auto_user_S5-00513-166-30pM_pz42_VH_IgM_415_486_rawtf.fa</span></p> </td> </tr> <tr> <td> <p><span>lib 5 IgG_IgA VH</span></p> </td> <td> <p><span>PANANTICOVID_I_Auto_user_S5-00513-167-30pM_42_IgG_IgA_VH_418_489_rawtf.fa</span></p> </td> </tr> <tr> <td> <p><span>lib 6 IgM VH</span></p> </td> <td> <p><span>PANANTICOVID_I_Auto_user_S5-00513-168-30pM_44_IgM_VH_417_491_rawtf.fa</span></p> </td> </tr> <tr> <td> <p><span>lib 6 IgG_IgA<span>  </span>VH</span></p> </td> <td> <p><span>PANANTICOVID_I_Auto_user_S5-00513-169-30pM_pz44_IgG_IgA_VH_420_493_rawtf.fa</span></p> </td> </tr> </tbody> </table&gt

Selection and characterization of human scFvs targeting the SARS-CoV-2 nucleocapsid protein isolated from antibody libraries of COVID-19 patients

Abstract In 2019, the novel SARS-CoV-2 coronavirus emerged in China, causing the pneumonia named COVID-19. At the beginning, all research efforts were focused on the spike (S) glycoprotein. However, it became evident that the nucleocapsid (N) protein is pivotal in viral replication, genome packaging and evasion of the immune system, is highly immunogenic, which makes it another compelling target for antibody development alongside the spike protein. This study focused on the construction of single chain fragments variable (scFvs) libraries from SARS-CoV-2-infected patients to establish a valuable, immortalized and extensive antibodies source. We used the Intracellular Antibody Capture Technology to select a panel of scFvs against the SARS-CoV-2 N protein. The whole panel of scFv was expressed and characterized both as intrabodies and recombinant proteins. ScFvs were then divided into 2 subgroups: those that exhibited high binding activity to N protein when expressed in yeast or in mammalian cells as intrabodies, and those purified as recombinant proteins, displaying affinity for recombinant N protein in the nanomolar range. This panel of scFvs against the N protein represents a novel platform for research and potential diagnostic applications