Поражения в мозге
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chuka_lisИсследователи из Балтимора обнаружили, что у некоторых людей коронавирус поражает мозг по типу болезни Альцгеймера.
Считается, что коронавирус SARS-CoV-2 может проникать в нейроны. У многих заболевших ковидом возникают неврологические расстройства, включая и острые, а у трети выживших после тяжелой болезни имеются неврологические проявления нарушений работы мозга. При этом не до конца ясно, каковы фенотипы инфицированных нейронов, и куда проникает вирус.
Авторы показали в свое работе, что коронавирус проник в ткани мозга (кора мозга: энторинальная кора, нижняя лобная извилина и дорсолатеральная префронтальная кора), имеющие отношения к когнитивным функциям, у пяти ковидных пациентов (с болезнью Альцгеймера, аутизмом, лобно-височной деменцией или без основного заболевания), заражая нейроны и другие клетки коры головного мозга. В контрольных образцах (19, по возрасту- основной болезни мозга, если была, но без ковида) ничего подобного не обнаружено.
Согласно их данным, коронавирус индуцирует или усиливает невропатию, подобную болезни Альцгеймера, с проявлениями агрегации бета-амилоида и образованием бляшек, тау-патией, нейровоспалением (повышенная продукция интерлейкинов 6 и 1-бета) и гибелью клеток (разными путями: некроптозис, ферроптозис и изнашивание). Коронавирус SARS-CoV-2, так же, заражает в культуре только зрелые нейроны, полученные из индуцируемых плюрипотентных стволовых клеток здоровых людей и людей с болезнью Альцгеймера, через свой рецептор ACE2 и облегчающий проникновение нейропилин-1. Он запускает генные программы, подобные работающим в клетках при болезни Альцгеймера, в здоровых нейронах после заражения, и усугубляет невропатию болезни Альцгеймера, если она есть. Так что при ковиде такая же почти "генная подпись", как и при болези Альцгеймера (с инфекционной подоплекой): идет подавление активности трех основных генов - GJA8, CryAA2 и PSG6 в первичных нейронах человека, что ведет к проявлению фенотипа болезни (накопление патологически уложенного белка).
Because ACE2, the SARS-CoV-2 cellular receptor, and NRP1, a facilitator of SARS-CoV-2 entry, are expressed in CNS neurons, we hypothesized that SARS-CoV-2 can infect neural cells in the brain, especially under conditions such as Alzheimer’s disease and autism with BBB compromise. SARS-CoV- 2 has been observed in cells of olfactory bulbs. We focused on cognitive centers of the brain. The SARS CoV-2 spike protein and nucleocapsid protein were detected in cells of the inferior frontal cortexes of five COVID-19 cases: two autism cases, one Alzheimer’s case, one frontotemporal dementia (FTD) case and one case without any underlying health conditions (apparently healthy case) (Fig. 1, Fig. S1). In contrast, there was no positive staining of spike protein or nucleocapsid protein in the cortexes of the non-COVID- 19 autism brains (Fig. 1A, C, Fig. S1A). Spike protein- and nucleocapsid protein-positive viral particles were robustly present in the cytoplasm and cellular projections of cortical cells (Fig. 1E, F). RNAscope in situ hybridization detected the abundant presence of SARS-CoV-2 genomic RNA in cortical cells of the five COVID-19 cases (Fig. 1G, H; Fig. S1F). PCR analysis using the CDC method 26 confirmed the presence of SARS-CoV-2 RNA in the three cases that had frozen brain tissues (Fig. 1I). In the 31-year123 old COVID-19 autism case, the amounts of SARS-CoV-2 RNA in the inferior frontal cortex and the dorsolateral prefrontal cortex were comparable to those in the lungs (Fig. 1I). In the COVID-19 FTD case and the COVID-19 apparently healthy case, SARS-CoV-2 RNA was also detected, whereas there was no SARS-CoV-2 RNA in the age-matched non-COVID-19 FTD individual or in the age-matched non- COVID-19 apparently healthy individual (Fig. 1I). Additional analyses showed that cells of the three cortical regions, the entorhinal cortex, the inferior frontal cortex and the dorsolateral prefrontal cortex, in the two COVID-19 autism cases and the COVID-19 Alzheimer’s case possessed abundant spike protein staining signals (Fig. 1J, K, L). The numbers of SARS-CoV-2 spike protein-positive cells in these 131 three regions were different in these three COVID-19 cases (Fig. 1J, K, L). These findings demonstrated that SARS-CoV-2 was neurotropic in this cohort of COVID-19 patients (Table S1). SARS-CoV-2 infects CNS cells expressing ACE2 and NRP1 and NRP1. Indeed, the SARS-CoV-2 spike protein staining signal was colocalized with both ACE2 and NRP1 signals (Fig. 2A, B), indicating that SARS-CoV-2 infects cortical cells using these proteins. ACE2 was colocalized with the neuron marker neurofilament light chain (NFL) (Fig. 2C, D), the pan-neuron marker class III beta-tubulin (Tuj1) (Fig. 2E), the oligodendrocyte marker 2',3'-cyclic nucleotide-3'- phosphodiesterase (CNPase) (Fig. 2F), and the microglia marker ionized calcium-binding adapter molecule 1 (Iba1) (Fig. 2G). Quantitative analysis of double ACE2 and cell marker positive cells indicated that all neurons expressed ACE2 (Fig. 2H). Next, we determined which cell types in the cortexes were infected by SARS-CoV-2. We used dual immunohistological labeling with antibodies against cell type144 specific markers and the SARS-CoV-2 spike protein. Both the mature neuron marker neuronal nuclear protein (NeuN) and the pan-neuron marker class III beta-tubulin (Tuj1) were colocalized with the spike protein in cells of the inferior frontal cortex (Fig. 2I, J). In the same region, both GABAergic inhibitory neurons (GAD65-positive) and glutamatergic excitatory neurons (glutamine synthetase-positive) contained SARS-CoV-2 spike protein staining signals (Fig. 2K, L). The SARS-CoV-2 spike protein staining signal was also present in Iba1-positive microglia and CNPase-positive oligodendrocytes in the same region (Fig. 2M, N). However, there was no detectable SARS-CoV-2 spike protein staining signal in glial fibrillary acidic protein (GFAP)-positive astrocytes (Fig. 2O). Quantification of double SARS152 CoV-2 spike protein- and different cell type marker-positive cells indicated that spike protein was present in all neurons (Fig. 2P). Thus, SARS-CoV-2 infects various CNS cell types. Because infected peripheral immune cells may enter the brain, T and B cells 154 were examined in the inferior frontal cortex. There were no T, B cell, or macrophage marker staining signals in this region of the COVID-19 autism cases (Fig. S2). However, a significant number of these immune cells were detected in the COVID-19 Alzheimer’s brain (Fig. S2). SARS-CoV-2 causes cell death via multiple pathways To determine whether SARS-CoV-2-infected cells in the cortical regions undergo cell death, we used immunohistological dual labeling with antibodies against the spike protein and cleaved caspase-3. There was no detectable cleaved caspase 3 staining signal in cells of the inferior frontal cortexes of non-COVID- 19 autism controls (Fig. 3A). Over 90% of cleaved caspase 3-positive cells were SARS-CoV-2 spike protein positive in the cortexes of two COVID-19 autism cases (Fig. 3A). In the COVID-19 Alzheimer’s disease case, over 20% of cleaved caspase 3-positive cells were SARS-CoV-2 spike protein positive (Fig. 3B). Cleaved caspase 3 was induced in two COVID-19 autism cases and the COVID-19 case without underlying health conditions and was enhanced in the COVID-19 Alzheimer’s case and the COVID-19 FTD case (Fig. S3A, B, C). To further determine the type of programmed cell death, we examined the presence of necroptotic, ferroptotic, and senescent cells. The cell necroptosis markers phospho-MLKL (mixed lineage kinase domain-like) and phospho-RIPK3 coexisted in spike protein-positive cells (Fig. 3C, Fig. S3D). Similarly, the two cell ferroptosis markers TfR1 (transferrin receptor) and ASCL4 (long-chain fatty acyl-CoA synthetase 4) were expressed in cells with positive spike protein signals (Fig. 3D, Fig. S3E). Because the cytokine associated with SARS-CoV-2 may trigger the cellular senescence program, we detected the senescence marker DPP4 (dipeptidyl-peptidase 4) in a subset of spike protein-positive cells (Fig. 3E). There were negligible double spike protein- and TfR1- or p-RIPK3-positive cells in the COVID-19 Alzheimer’s case (Fig. 3D, Fig. S3D). These findings suggest that SARS-CoV-2 leads to cell death and senescence through multiple pathways. SARS-CoV-2 induces neuroinflammation It has been suggested that SARS-CoV-2 infection leads to neuroinflammation. However, direct evidence on the link between SARS-CoV-2 and neuroinflammation is still lacking. The protein expression of two cytokines, IL-1 and IL-6 (interleukin 6), was significantly increased in the cortical cells of COVID-19 autism patients compared to the cortical cells of non-COVID-19 autism patients (Fig. S4A, B, C), providing the direct evidence of neuroinflammation induced by SARS-CoV-2. SARS-CoV-2 induces Alzheimer’s-like phenotype development and exacerbation Cellular A (amyloid beta) aggregates were observed in the cortexes of the two COVID-19 autism cases (Fig. 4A) and the one COVID-19 case without underlying health conditions (Fig. 4B), whereas it was not present in the cortexes of age-matched non-COVID-19 autism cases and an apparently healthy individual (Fig. 4A, B). Extracellular A plaques were present in one of the COVID-19 autism cases (Fig. 4A). Immunofluorescence analysis with thioflavin-T confirmed that the cytoplasmic deposition of A in the COVID-19 autism cases consisted of aggregated A (Fig. 4A). There was more A plaque deposition per measured area in the cortexes of the COVID-19 Alzheimer’s and FTD brains than in the cortexes of age-matched non-COVID-19 Alzheimer’s and FTD brains (Fig. 4C, D). One of the neuropathological hallmarks of Alzheimer’s disease is the development of intracellular neurofibrillary tangles (NFTs) composed of hyperphosphorylated Tau (microtubule-associated protein tau). p-Tau-containing NFTs are associated with neuronal dysfunction, cognitive deficits and neuronal death 27,28. p-Tau-containing NFTs were present in the inferior cortexes of the two COVID-19 autism cases (Fig. 4E). Cellular p-Tau deposition was induced in these two COVID-19 autism cases and the COVID-19 case without underlying health conditions (Fig. 4E, F), whereas there were no signals or negligible p-Tau staining signals in the cortexes of non-COVID-19 control brains (Fig. 4E, F). There were significantly higher numbers of Pick bodies in the COVID-19 FTD case than in age-matched non-COVID-19 FTD cases (Fig. 4G, H). Thus, SARS-CoV-2 infection is linked to Alzheimer’s neuropathology. SARS-CoV-2 infects iPSC-derived mature neurons Based on the above findings in neurons of COVID-19 patients’ brain cortexes, we propose that SARS204 CoV-2 can effectively infect neurons. To establish an in vitro platform to study this process, we obtained iPSCs derived from age-matched healthy individuals and Alzheimer’s patients and differentiated it into neurons, followed by SARS-CoV-2 infection. SARS-CoV-2-GFP (in which GFP replaced the viral open reading frame ORF7a 29) at a multiplicity of infection (MOI) of 0.1 or 0.2 did not infect any cells at iPSC neuron differentiation day 35 (Fig. 5A, Fig. S5A) and some of these cells expressed the pan-neuron marker Tuj1 but did not express the mature neuron marker NeuN (Fig. 5B, Fig. S5B). At iPSC differentiation day 50, SARS-CoV-2-GFP at an MOI of 0.05, 0.1 or 0.2 effectively infected cells differentiated from iPSCs from healthy individuals and Alzheimer’s patients (Fig. 5C, Fig. S5C, D), and SARS-CoV-2-GFP could be detected in cell culture media 72 hours post SARS-CoV-2-GFP infection (Fig. 5D), indicating that the virus not only infects cells but also replicates intracellularly. SARS-CoV-2-infected cells were essentially all Tuj1-positive cells at iPSC differentiation day 50 (Fig. 5E), and no GFAT-positive astrocytes were detected in mock- and SARS-CoV-2-infected cells (Fig. 5F). At iPSC differentiation day 35, there was no expression of ACE2 or NRP1 proteins (Fig. 5G, H). At iPSC differentiation day 50, robust ACE2 and NRP1 protein expression existed in Tuj1-positive neurons (Fig. 5I, J). Over 40% of Tuj1-positive neurons were ACE2- and/or NRP1-positive (Fig. 5I, J). Subsequent experiments were conducted on iPSC differentiation day 50. These results suggest that SARS-CoV-2 infects mature neurons via ACE2 with the facilitation of NRP1. SARS-CoV-2 induces Alzheimer’s phenotypes in iPSC-derived cells Because SARS-CoV-2 induces A cellular aggregates and extracellular plaques, p-Tau cellular deposition and NFTs and neuroinflammation in COVID-19 patients, we hypothesized that SARS-CoV-2 infection can turn neurons derived from iPSCs from healthy individuals into Alzheimer’s-phenotype neurons. Neurons differentiated from iPSCs of healthy individuals and Alzheimer’s patients were infected with wild-type SARS-CoV-2 (WA-1 strain 30) at an MOI of 0.1 for 48 hours (Fig. 6, Fig. S6). SARS-CoV- 2 induced cellular A aggregates in healthy neurons and increased cellular A aggregates in Alzheimer’s neurons (Fig. 6A). Cellular p-Tau deposition was induced in healthy neurons after 72 hours of SARS229 CoV-2 infection (Fig. 6B), and the virus further increased cellular p-Tau deposition in Alzheimer’s neurons (Fig. 6B). Compared to their mock-infected counterparts, both healthy neurons and Alzheimer’s neurons had higher levels of major inflammatory cytokines including IL-1, IL-6, IFN and TNF after SARS CoV-2 infection (Fig. 6C). Among the critical Alzheimer’s mediators, amyloid precursor protein (APP), enzyme -secretase 1 (BACE1), and presenilin 1/2 (PSEN1/2), SARS-CoV-2 significantly increased BACE1 expression in healthy neurons and Alzheimer’s neurons but did not affect the expression of the other Alzheimer’s mediators (Fig. 6D, E). Likewise, SARS-CoV-2 significantly increased the number of cleaved caspase 3-positive cells differentiated from iPSCs of healthy individuals and Alzheimer’s patients (Fig. 6F). Thus, SARS-CoV-2 triggers an Alzheimer’s-like cellular program in neurons derived from iPSCs of healthy individuals and enhances Alzheimer’s phenotypes in cells derived from Alzheimer’s iPSCs. Alzheimer’s infectious etiology genes identified via SARS-CoV-2 Over 95% of Alzheimer’s cases are sporadic and their causes are still unclear. Studies of DNA viruses have shown that Alzheimer’s etiology has an infectious component 31,32. Based on the above observation that SARS-CoV-2 induces Alzheimer’s phenotypes in neurons derived from iPSCs of healthy non- Alzheimer’s individuals, we aimed to utilize SARS-CoV-2 infection to reveal genes responsible for the Alzheimer’s infectious etiology. The transcriptomes of neurons differentiated from iPSCs of healthy individuals and Alzheimer’s patients were determined by RNA sequencing (Fig. 7A-E). Under mock infection conditions, 553 genes were significantly upregulated, while 71 genes were significantly downregulated, in Alzheimer’s neurons compared to neurons from iPSCs of healthy individuals (designated healthy neurons) (Fig. 7A). SARS-CoV-2 upregulated 75 genes and downregulated 19 genes in healthy neurons (Fig. 7B). To extract the genes responsible for Alzheimer’s infectious etiology, 24 overlapping genes were identified between the Alzheimer’s neuron-mock-infected group and the healthy neuron-SARS-CoV-2-infected group (Fig. 7D). Pathway analysis revealed that the changes in these 24 genes activated infection pathways elicited by bacteria and viruses (Fig. 7D). Compared to healthy neurons without viral infection, Alzheimer’s neurons infected with SARS-CoV-2 had 517 upregulated genes and 256 downregulated genes (Fig. 7C). This number of downregulated genes (256) was higher than the number of downregulated genes in Alzheimer’s neurons (71) (Fig. 7A, C). In Alzheimer’s neurons, SARS-CoV-2 further increased the expression of 25 upregulated genes and decreased the expression of 34 downregulated genes by several-fold (Fig. S6A), indicating that the virus deteriorates Alzheimer’s conditions, and pathway analysis pointed to the further activation of neuroinflammation and other processes in Alzheimer’s neurons (Fig. S6B).
Считается, что коронавирус SARS-CoV-2 может проникать в нейроны. У многих заболевших ковидом возникают неврологические расстройства, включая и острые, а у трети выживших после тяжелой болезни имеются неврологические проявления нарушений работы мозга. При этом не до конца ясно, каковы фенотипы инфицированных нейронов, и куда проникает вирус.
Авторы показали в свое работе, что коронавирус проник в ткани мозга (кора мозга: энторинальная кора, нижняя лобная извилина и дорсолатеральная префронтальная кора), имеющие отношения к когнитивным функциям, у пяти ковидных пациентов (с болезнью Альцгеймера, аутизмом, лобно-височной деменцией или без основного заболевания), заражая нейроны и другие клетки коры головного мозга. В контрольных образцах (19, по возрасту- основной болезни мозга, если была, но без ковида) ничего подобного не обнаружено.
Согласно их данным, коронавирус индуцирует или усиливает невропатию, подобную болезни Альцгеймера, с проявлениями агрегации бета-амилоида и образованием бляшек, тау-патией, нейровоспалением (повышенная продукция интерлейкинов 6 и 1-бета) и гибелью клеток (разными путями: некроптозис, ферроптозис и изнашивание). Коронавирус SARS-CoV-2, так же, заражает в культуре только зрелые нейроны, полученные из индуцируемых плюрипотентных стволовых клеток здоровых людей и людей с болезнью Альцгеймера, через свой рецептор ACE2 и облегчающий проникновение нейропилин-1. Он запускает генные программы, подобные работающим в клетках при болезни Альцгеймера, в здоровых нейронах после заражения, и усугубляет невропатию болезни Альцгеймера, если она есть. Так что при ковиде такая же почти "генная подпись", как и при болези Альцгеймера (с инфекционной подоплекой): идет подавление активности трех основных генов - GJA8, CryAA2 и PSG6 в первичных нейронах человека, что ведет к проявлению фенотипа болезни (накопление патологически уложенного белка).
Because ACE2, the SARS-CoV-2 cellular receptor, and NRP1, a facilitator of SARS-CoV-2 entry, are expressed in CNS neurons, we hypothesized that SARS-CoV-2 can infect neural cells in the brain, especially under conditions such as Alzheimer’s disease and autism with BBB compromise. SARS-CoV- 2 has been observed in cells of olfactory bulbs. We focused on cognitive centers of the brain. The SARS CoV-2 spike protein and nucleocapsid protein were detected in cells of the inferior frontal cortexes of five COVID-19 cases: two autism cases, one Alzheimer’s case, one frontotemporal dementia (FTD) case and one case without any underlying health conditions (apparently healthy case) (Fig. 1, Fig. S1). In contrast, there was no positive staining of spike protein or nucleocapsid protein in the cortexes of the non-COVID- 19 autism brains (Fig. 1A, C, Fig. S1A). Spike protein- and nucleocapsid protein-positive viral particles were robustly present in the cytoplasm and cellular projections of cortical cells (Fig. 1E, F). RNAscope in situ hybridization detected the abundant presence of SARS-CoV-2 genomic RNA in cortical cells of the five COVID-19 cases (Fig. 1G, H; Fig. S1F). PCR analysis using the CDC method 26 confirmed the presence of SARS-CoV-2 RNA in the three cases that had frozen brain tissues (Fig. 1I). In the 31-year123 old COVID-19 autism case, the amounts of SARS-CoV-2 RNA in the inferior frontal cortex and the dorsolateral prefrontal cortex were comparable to those in the lungs (Fig. 1I). In the COVID-19 FTD case and the COVID-19 apparently healthy case, SARS-CoV-2 RNA was also detected, whereas there was no SARS-CoV-2 RNA in the age-matched non-COVID-19 FTD individual or in the age-matched non- COVID-19 apparently healthy individual (Fig. 1I). Additional analyses showed that cells of the three cortical regions, the entorhinal cortex, the inferior frontal cortex and the dorsolateral prefrontal cortex, in the two COVID-19 autism cases and the COVID-19 Alzheimer’s case possessed abundant spike protein staining signals (Fig. 1J, K, L). The numbers of SARS-CoV-2 spike protein-positive cells in these 131 three regions were different in these three COVID-19 cases (Fig. 1J, K, L). These findings demonstrated that SARS-CoV-2 was neurotropic in this cohort of COVID-19 patients (Table S1). SARS-CoV-2 infects CNS cells expressing ACE2 and NRP1 and NRP1. Indeed, the SARS-CoV-2 spike protein staining signal was colocalized with both ACE2 and NRP1 signals (Fig. 2A, B), indicating that SARS-CoV-2 infects cortical cells using these proteins. ACE2 was colocalized with the neuron marker neurofilament light chain (NFL) (Fig. 2C, D), the pan-neuron marker class III beta-tubulin (Tuj1) (Fig. 2E), the oligodendrocyte marker 2',3'-cyclic nucleotide-3'- phosphodiesterase (CNPase) (Fig. 2F), and the microglia marker ionized calcium-binding adapter molecule 1 (Iba1) (Fig. 2G). Quantitative analysis of double ACE2 and cell marker positive cells indicated that all neurons expressed ACE2 (Fig. 2H). Next, we determined which cell types in the cortexes were infected by SARS-CoV-2. We used dual immunohistological labeling with antibodies against cell type144 specific markers and the SARS-CoV-2 spike protein. Both the mature neuron marker neuronal nuclear protein (NeuN) and the pan-neuron marker class III beta-tubulin (Tuj1) were colocalized with the spike protein in cells of the inferior frontal cortex (Fig. 2I, J). In the same region, both GABAergic inhibitory neurons (GAD65-positive) and glutamatergic excitatory neurons (glutamine synthetase-positive) contained SARS-CoV-2 spike protein staining signals (Fig. 2K, L). The SARS-CoV-2 spike protein staining signal was also present in Iba1-positive microglia and CNPase-positive oligodendrocytes in the same region (Fig. 2M, N). However, there was no detectable SARS-CoV-2 spike protein staining signal in glial fibrillary acidic protein (GFAP)-positive astrocytes (Fig. 2O). Quantification of double SARS152 CoV-2 spike protein- and different cell type marker-positive cells indicated that spike protein was present in all neurons (Fig. 2P). Thus, SARS-CoV-2 infects various CNS cell types. Because infected peripheral immune cells may enter the brain, T and B cells 154 were examined in the inferior frontal cortex. There were no T, B cell, or macrophage marker staining signals in this region of the COVID-19 autism cases (Fig. S2). However, a significant number of these immune cells were detected in the COVID-19 Alzheimer’s brain (Fig. S2). SARS-CoV-2 causes cell death via multiple pathways To determine whether SARS-CoV-2-infected cells in the cortical regions undergo cell death, we used immunohistological dual labeling with antibodies against the spike protein and cleaved caspase-3. There was no detectable cleaved caspase 3 staining signal in cells of the inferior frontal cortexes of non-COVID- 19 autism controls (Fig. 3A). Over 90% of cleaved caspase 3-positive cells were SARS-CoV-2 spike protein positive in the cortexes of two COVID-19 autism cases (Fig. 3A). In the COVID-19 Alzheimer’s disease case, over 20% of cleaved caspase 3-positive cells were SARS-CoV-2 spike protein positive (Fig. 3B). Cleaved caspase 3 was induced in two COVID-19 autism cases and the COVID-19 case without underlying health conditions and was enhanced in the COVID-19 Alzheimer’s case and the COVID-19 FTD case (Fig. S3A, B, C). To further determine the type of programmed cell death, we examined the presence of necroptotic, ferroptotic, and senescent cells. The cell necroptosis markers phospho-MLKL (mixed lineage kinase domain-like) and phospho-RIPK3 coexisted in spike protein-positive cells (Fig. 3C, Fig. S3D). Similarly, the two cell ferroptosis markers TfR1 (transferrin receptor) and ASCL4 (long-chain fatty acyl-CoA synthetase 4) were expressed in cells with positive spike protein signals (Fig. 3D, Fig. S3E). Because the cytokine associated with SARS-CoV-2 may trigger the cellular senescence program, we detected the senescence marker DPP4 (dipeptidyl-peptidase 4) in a subset of spike protein-positive cells (Fig. 3E). There were negligible double spike protein- and TfR1- or p-RIPK3-positive cells in the COVID-19 Alzheimer’s case (Fig. 3D, Fig. S3D). These findings suggest that SARS-CoV-2 leads to cell death and senescence through multiple pathways. SARS-CoV-2 induces neuroinflammation It has been suggested that SARS-CoV-2 infection leads to neuroinflammation. However, direct evidence on the link between SARS-CoV-2 and neuroinflammation is still lacking. The protein expression of two cytokines, IL-1 and IL-6 (interleukin 6), was significantly increased in the cortical cells of COVID-19 autism patients compared to the cortical cells of non-COVID-19 autism patients (Fig. S4A, B, C), providing the direct evidence of neuroinflammation induced by SARS-CoV-2. SARS-CoV-2 induces Alzheimer’s-like phenotype development and exacerbation Cellular A (amyloid beta) aggregates were observed in the cortexes of the two COVID-19 autism cases (Fig. 4A) and the one COVID-19 case without underlying health conditions (Fig. 4B), whereas it was not present in the cortexes of age-matched non-COVID-19 autism cases and an apparently healthy individual (Fig. 4A, B). Extracellular A plaques were present in one of the COVID-19 autism cases (Fig. 4A). Immunofluorescence analysis with thioflavin-T confirmed that the cytoplasmic deposition of A in the COVID-19 autism cases consisted of aggregated A (Fig. 4A). There was more A plaque deposition per measured area in the cortexes of the COVID-19 Alzheimer’s and FTD brains than in the cortexes of age-matched non-COVID-19 Alzheimer’s and FTD brains (Fig. 4C, D). One of the neuropathological hallmarks of Alzheimer’s disease is the development of intracellular neurofibrillary tangles (NFTs) composed of hyperphosphorylated Tau (microtubule-associated protein tau). p-Tau-containing NFTs are associated with neuronal dysfunction, cognitive deficits and neuronal death 27,28. p-Tau-containing NFTs were present in the inferior cortexes of the two COVID-19 autism cases (Fig. 4E). Cellular p-Tau deposition was induced in these two COVID-19 autism cases and the COVID-19 case without underlying health conditions (Fig. 4E, F), whereas there were no signals or negligible p-Tau staining signals in the cortexes of non-COVID-19 control brains (Fig. 4E, F). There were significantly higher numbers of Pick bodies in the COVID-19 FTD case than in age-matched non-COVID-19 FTD cases (Fig. 4G, H). Thus, SARS-CoV-2 infection is linked to Alzheimer’s neuropathology. SARS-CoV-2 infects iPSC-derived mature neurons Based on the above findings in neurons of COVID-19 patients’ brain cortexes, we propose that SARS204 CoV-2 can effectively infect neurons. To establish an in vitro platform to study this process, we obtained iPSCs derived from age-matched healthy individuals and Alzheimer’s patients and differentiated it into neurons, followed by SARS-CoV-2 infection. SARS-CoV-2-GFP (in which GFP replaced the viral open reading frame ORF7a 29) at a multiplicity of infection (MOI) of 0.1 or 0.2 did not infect any cells at iPSC neuron differentiation day 35 (Fig. 5A, Fig. S5A) and some of these cells expressed the pan-neuron marker Tuj1 but did not express the mature neuron marker NeuN (Fig. 5B, Fig. S5B). At iPSC differentiation day 50, SARS-CoV-2-GFP at an MOI of 0.05, 0.1 or 0.2 effectively infected cells differentiated from iPSCs from healthy individuals and Alzheimer’s patients (Fig. 5C, Fig. S5C, D), and SARS-CoV-2-GFP could be detected in cell culture media 72 hours post SARS-CoV-2-GFP infection (Fig. 5D), indicating that the virus not only infects cells but also replicates intracellularly. SARS-CoV-2-infected cells were essentially all Tuj1-positive cells at iPSC differentiation day 50 (Fig. 5E), and no GFAT-positive astrocytes were detected in mock- and SARS-CoV-2-infected cells (Fig. 5F). At iPSC differentiation day 35, there was no expression of ACE2 or NRP1 proteins (Fig. 5G, H). At iPSC differentiation day 50, robust ACE2 and NRP1 protein expression existed in Tuj1-positive neurons (Fig. 5I, J). Over 40% of Tuj1-positive neurons were ACE2- and/or NRP1-positive (Fig. 5I, J). Subsequent experiments were conducted on iPSC differentiation day 50. These results suggest that SARS-CoV-2 infects mature neurons via ACE2 with the facilitation of NRP1. SARS-CoV-2 induces Alzheimer’s phenotypes in iPSC-derived cells Because SARS-CoV-2 induces A cellular aggregates and extracellular plaques, p-Tau cellular deposition and NFTs and neuroinflammation in COVID-19 patients, we hypothesized that SARS-CoV-2 infection can turn neurons derived from iPSCs from healthy individuals into Alzheimer’s-phenotype neurons. Neurons differentiated from iPSCs of healthy individuals and Alzheimer’s patients were infected with wild-type SARS-CoV-2 (WA-1 strain 30) at an MOI of 0.1 for 48 hours (Fig. 6, Fig. S6). SARS-CoV- 2 induced cellular A aggregates in healthy neurons and increased cellular A aggregates in Alzheimer’s neurons (Fig. 6A). Cellular p-Tau deposition was induced in healthy neurons after 72 hours of SARS229 CoV-2 infection (Fig. 6B), and the virus further increased cellular p-Tau deposition in Alzheimer’s neurons (Fig. 6B). Compared to their mock-infected counterparts, both healthy neurons and Alzheimer’s neurons had higher levels of major inflammatory cytokines including IL-1, IL-6, IFN and TNF after SARS CoV-2 infection (Fig. 6C). Among the critical Alzheimer’s mediators, amyloid precursor protein (APP), enzyme -secretase 1 (BACE1), and presenilin 1/2 (PSEN1/2), SARS-CoV-2 significantly increased BACE1 expression in healthy neurons and Alzheimer’s neurons but did not affect the expression of the other Alzheimer’s mediators (Fig. 6D, E). Likewise, SARS-CoV-2 significantly increased the number of cleaved caspase 3-positive cells differentiated from iPSCs of healthy individuals and Alzheimer’s patients (Fig. 6F). Thus, SARS-CoV-2 triggers an Alzheimer’s-like cellular program in neurons derived from iPSCs of healthy individuals and enhances Alzheimer’s phenotypes in cells derived from Alzheimer’s iPSCs. Alzheimer’s infectious etiology genes identified via SARS-CoV-2 Over 95% of Alzheimer’s cases are sporadic and their causes are still unclear. Studies of DNA viruses have shown that Alzheimer’s etiology has an infectious component 31,32. Based on the above observation that SARS-CoV-2 induces Alzheimer’s phenotypes in neurons derived from iPSCs of healthy non- Alzheimer’s individuals, we aimed to utilize SARS-CoV-2 infection to reveal genes responsible for the Alzheimer’s infectious etiology. The transcriptomes of neurons differentiated from iPSCs of healthy individuals and Alzheimer’s patients were determined by RNA sequencing (Fig. 7A-E). Under mock infection conditions, 553 genes were significantly upregulated, while 71 genes were significantly downregulated, in Alzheimer’s neurons compared to neurons from iPSCs of healthy individuals (designated healthy neurons) (Fig. 7A). SARS-CoV-2 upregulated 75 genes and downregulated 19 genes in healthy neurons (Fig. 7B). To extract the genes responsible for Alzheimer’s infectious etiology, 24 overlapping genes were identified between the Alzheimer’s neuron-mock-infected group and the healthy neuron-SARS-CoV-2-infected group (Fig. 7D). Pathway analysis revealed that the changes in these 24 genes activated infection pathways elicited by bacteria and viruses (Fig. 7D). Compared to healthy neurons without viral infection, Alzheimer’s neurons infected with SARS-CoV-2 had 517 upregulated genes and 256 downregulated genes (Fig. 7C). This number of downregulated genes (256) was higher than the number of downregulated genes in Alzheimer’s neurons (71) (Fig. 7A, C). In Alzheimer’s neurons, SARS-CoV-2 further increased the expression of 25 upregulated genes and decreased the expression of 34 downregulated genes by several-fold (Fig. S6A), indicating that the virus deteriorates Alzheimer’s conditions, and pathway analysis pointed to the further activation of neuroinflammation and other processes in Alzheimer’s neurons (Fig. S6B).
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