SARS-CoV-2 Infection Remodels the Phenotype and Promotes Angiogenesis of Primary Human Lung Endothelial Cells
Caccuri, F;Bugatti, A;Zani, A;De Palma, A;Di Silvestre, D;Manocha, E;Filippini, F;Messali, S;Chiodelli, P;Campisi, G;Fiorentini, S;Facchetti, F;Mauri, P;Caruso, A;
| DOI: 10.3390/microorganisms9071438
SARS-CoV-2-associated acute respiratory distress syndrome (ARDS) and acute lung injury are life-threatening manifestations of severe viral infection. The pathogenic mechanisms that lead to respiratory complications, such as endothelialitis, intussusceptive angiogenesis, and vascular leakage remain unclear. In this study, by using an immunofluorescence assay and in situ RNA-hybridization, we demonstrate the capability of SARS-CoV-2 to infect human primary lung microvascular endothelial cells (HL-mECs) in the absence of cytopathic effects and release of infectious particles. Preliminary data point to the role of integrins in SARS-CoV-2 entry into HL-mECs in the absence of detectable ACE2 expression. Following infection, HL-mECs were found to release a plethora of pro-inflammatory and pro-angiogenic molecules, as assessed by microarray analyses. This conditioned microenvironment stimulated HL-mECs to acquire an angiogenic phenotype. Proteome analysis confirmed a remodeling of SARS-CoV-2-infected HL-mECs to inflammatory and angiogenic responses and highlighted the expression of antiviral molecules as annexin A6 and MX1. These results support the hypothesis of a direct role of SARS-CoV-2-infected HL-mECs in sustaining vascular dysfunction during the early phases of infection. The construction of virus-host interactomes will be instrumental to identify potential therapeutic targets for COVID-19 aimed to inhibit HL-mEC-sustained inflammation and angiogenesis upon SARS-CoV-2 infection.
Virchows Archiv : an international journal of pathology
Zito Marino, F;De Cristofaro, T;Varriale, M;Zannini, G;Ronchi, A;La Mantia, E;Campobasso, CP;De Micco, F;Mascolo, P;Municinò, M;Municinò, E;Vestini, F;Pinto, O;Moccia, M;De Stefano, N;Nappi, O;Sementa, C;Zotti, G;Pianese, L;Giordano, C;Franco, R;
PMID: 35103846 | DOI: 10.1007/s00428-021-03262-8
Post-mortem examination plays a pivotal role in understanding the pathobiology of the SARS-CoV-2; thus, the optimization of virus detection on the post-mortem formalin-fixed paraffin-embedded (FFPE) tissue is needed. Different techniques are available for the identification of the SARS-CoV-2, including reverse transcription polymerase chain reaction (RT-PCR), immunohistochemistry (IHC), in situ hybridization (ISH), and electron microscopy. The main goal of this study is to compare ISH versus RT-PCR to detect SARS-CoV-2 on post-mortem lung samples of positive deceased subjects. A total of 27 samples were analyzed by RT-PCR targeting different viral RNA sequences of SARS-CoV-2, including envelope (E), nucleocapsid (N), spike (S), and open reading frame (ORF1ab) genes and ISH targeting S and Orf1ab. All 27 cases showed the N gene amplification, 22 out of 27 the E gene amplification, 26 out of 27 the S gene amplification, and only 6 the ORF1ab gene amplification. The S ISH was positive only in 12 out of 26 cases positive by RT-PCR. The S ISH positive cases with strong and diffuse staining showed a correlation with low values of the number of the amplification cycles by S RT-PCR suggesting that ISH is a sensitive assay mainly in cases carrying high levels of S RNA. In conclusion, our findings demonstrated that ISH assay has lower sensitivity to detect SARS-CoV-2 in FFPE compared to RT-PCR; however, it is able to localize the virus in the cellular context since it preserves the morphology.
Morris, C;Watkins, D;Shah, N;Pennington, T;Hens, B;Qi, G;Doud, E;Mosley, A;Atwood, B;Baucum, A;
| DOI: 10.1016/j.biopsych.2022.12.008
Background Grooming dysfunction is a hallmark of the obsessive-compulsive spectrum disorder, trichotillomania. Numerous preclinical studies have utilized SAPAP3 deficient mice for understanding the neurobiology of repetitive grooming, suggesting excessive grooming is caused by increased metabotropic glutamate receptor 5 (mGluR5) activity in striatal direct- and indirect pathway medium spiny neurons (dMSNs and iMSNs, respectively). However, MSN subtype-specific signaling mechanisms that mediate mGluR5-dependent adaptations underlying excessive grooming are not fully understood. Here, we investigate the MSN subtype-specific roles of the striatal signaling hub protein, spinophilin, in mediating repetitive motor dysfunction associated with mGluR5 function. Methods Quantitative proteomics and immunoblotting were utilized to identify how spinophilin impacts mGluR5 phosphorylation and protein interaction changes. Plasticity and repetitive motor dysfunction associated with mGluR5 action was measured using our novel conditional spinophilin mouse model that had spinophilin knocked out from striatal dMSNs or/and iMSNs. Results Loss of spinophilin only in iMSNs decreased performance of a novel motor repertoire, but loss of spinophilin in either MSN subtype abrogated striatal plasticity associated with mGluR5 function and prevented excessive grooming caused by SAPAP3 knockout mice or treatment with the mGluR5-specific positive allosteric modulator (VU0360172) without impacting locomotion-relevant behavior. Biochemically, we determined the spinophilin-mGluR5 interaction correlates with grooming behavior and loss of spinophilin shifts mGluR5 interactions from lipid-raft associated proteins toward postsynaptic density (PSD) proteins implicated in psychiatric disorders. Conclusions These results identify spinophilin as a novel striatal signaling hub molecule in MSNs that cell subtype-specifically mediates behavioral, functional, and molecular adaptations associated with repetitive motor dysfunction in psychiatric disorders.
Dowall, S;Salguero, FJ;Wiblin, N;Fotheringham, S;Hatch, G;Parks, S;Gowan, K;Harris, D;Carnell, O;Fell, R;Watson, R;Graham, V;Gooch, K;Hall, Y;Mizen, S;Hewson, R;
PMID: 34835057 | DOI: 10.3390/v13112251
The global pandemic of coronavirus disease (COVID-19) caused by infection with severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has led to an international thrust to study pathogenesis and evaluate interventions. Experimental infection of hamsters and the resulting respiratory disease is one of the preferred animal models since clinical signs of disease and virus shedding are similar to more severe cases of human COVID-19. The main route of challenge has been direct inoculation of the virus via the intranasal route. To resemble the natural infection, we designed a bespoke natural transmission cage system to assess whether recipient animals housed in physically separate adjacent cages could become infected from a challenged donor animal in a central cage, with equal airflow across the two side cages. To optimise viral shedding in the donor animals, a low and moderate challenge dose were compared after direct intranasal challenge, but similar viral shedding responses were observed and no discernible difference in kinetics. The results from our natural transmission set-up demonstrate that most recipient hamsters are infected within the system developed, with variation in the kinetics and levels of disease between individual animals. Common clinical outputs used for the assessment in directly-challenged hamsters, such as weight loss, are less obvious in hamsters who become infected from naturally acquiring the infection. The results demonstrate the utility of a natural transmission model for further work on assessing the differences between virus strains and evaluating interventions using a challenge system which more closely resembles human infection.
Matson, KJE;Russ, DE;Kathe, C;Hua, I;Maric, D;Ding, Y;Krynitsky, J;Pursley, R;Sathyamurthy, A;Squair, JW;Levi, BP;Courtine, G;Levine, AJ;
PMID: 36163250 | DOI: 10.1038/s41467-022-33184-1
After spinal cord injury, tissue distal to the lesion contains undamaged cells that could support or augment recovery. Targeting these cells requires a clearer understanding of their injury responses and capacity for repair. Here, we use single nucleus RNA sequencing to profile how each cell type in the lumbar spinal cord changes after a thoracic injury in mice. We present an atlas of these dynamic responses across dozens of cell types in the acute, subacute, and chronically injured spinal cord. Using this resource, we find rare spinal neurons that express a signature of regeneration in response to injury, including a major population that represent spinocerebellar projection neurons. We characterize these cells anatomically and observed axonal sparing, outgrowth, and remodeling in the spinal cord and cerebellum. Together, this work provides a key resource for studying cellular responses to injury and uncovers the spontaneous plasticity of spinocerebellar neurons, uncovering a potential candidate for targeted therapy.
Sci Transl Med. 2018 Oct 31;10(465).
Borel F, Gernoux G, Sun H, Stock R, Blackwood M, Brown RH Jr, Mueller C.
PMID: 30381409 | DOI: 10.1126/scitranslmed.aau6414
Amyotrophic lateral sclerosis (ALS) is a fatal neurological disease caused by degeneration of motor neurons leading to rapidly progressive paralysis. About 10% of cases are caused by gain-of-function mutations that are transmitted as dominant traits. A potential therapy for these cases is to suppress the expression of the mutant gene. Here, we investigated silencing of SOD1, a gene commonly mutated in familial ALS, using an adeno-associated virus (AAV) encoding an artificial microRNA (miRNA) that targeted SOD1 In a superoxide dismutase 1 (SOD1)-mediated mouse model of ALS, we have previously demonstrated that SOD1 silencing delayed disease onset, increased survival time, and reduced muscle loss and motor and respiratory impairments. Here, we describe the preclinical characterization of this approach in cynomolgus macaques (Macaca fascicularis) using an AAV serotype for delivery that has been shown to be safe in clinical trials. We optimized AAV delivery to the spinal cord by preimplantation of a catheter and placement of the subject with head down at 30 degrees during intrathecal infusion. We compared different promoters for the expression of artificial miRNAs directed against mutant SOD1 Results demonstrated efficient delivery and effective silencing of the SOD1 gene in motor neurons. These results support the notion that gene therapy with an artificial miRNA targeting SOD1 is safe and merits further development for the treatment of mutant SOD1-linked ALS.
Xhima K, Markham-Coultes K, Nedev H, Heinen S, Saragovi HU, Hynynen K, Aubert I
PMID: 32010781 | DOI: 10.1126/sciadv.aax6646
Free PMC Article
The degeneration of cholinergic neurons is a prominent feature of Alzheimer's disease (AD). In animal models of injury and aging, nerve growth factor (NGF) enhances cholinergic cell survival and function, contributing to improved memory. In the presence of AD pathology, however, NGF-related therapeutics have yet to fulfill their regenerative potential. We propose that stimulating the TrkA receptor, without p75NTR activation, is key for therapeutic efficacy. Supporting this hypothesis, the selective TrkA agonist D3 rescued neurotrophin signaling in TgCRND8 mice, whereas NGF, interacting with both TrkA and p75NTR, did not. D3, delivered intravenously and noninvasively to the basal forebrain using MRI-guided focused ultrasound (MRIgFUS)-mediated blood-brain barrier (BBB) permeability activated TrkA-related signaling cascades and enhanced cholinergic neurotransmission. Recent clinical trials support the safety and feasibility of MRIgFUS BBB modulation in AD patients. Neuroprotective agents targeting TrkA, combined with MRIgFUS BBB modulation, represent a promising strategy to counter neurodegeneration in AD.
Yao, Y;Barger, Z;Saffari Doost, M;Tso, CF;Darmohray, D;Silverman, D;Liu, D;Ma, C;Cetin, A;Yao, S;Zeng, H;Dan, Y;
PMID: 36170850 | DOI: 10.1016/j.neuron.2022.08.027
Sleep disturbances are strongly associated with cardiovascular diseases. Baroreflex, a basic cardiovascular regulation mechanism, is modulated by sleep-wake states. Here, we show that neurons at key stages of baroreflex pathways also promote sleep. Using activity-dependent genetic labeling, we tagged neurons in the nucleus of the solitary tract (NST) activated by blood pressure elevation and confirmed their barosensitivity with optrode recording and calcium imaging. Chemogenetic or optogenetic activation of these neurons promoted non-REM sleep in addition to decreasing blood pressure and heart rate. GABAergic neurons in the caudal ventrolateral medulla (CVLM)-a downstream target of the NST for vasomotor baroreflex-also promote non-REM sleep, partly by inhibiting the sympathoexcitatory and wake-promoting adrenergic neurons in the rostral ventrolateral medulla (RVLM). Cholinergic neurons in the nucleus ambiguous-a target of the NST for cardiac baroreflex-promoted non-REM sleep as well. Thus, key components of the cardiovascular baroreflex circuit are also integral to sleep-wake brain-state regulation.
SARS-CoV-2 infection and transmission in the North American deer mouse
Griffin, BD;Chan, M;Tailor, N;Mendoza, EJ;Leung, A;Warner, BM;Duggan, AT;Moffat, E;He, S;Garnett, L;Tran, KN;Banadyga, L;Albietz, A;Tierney, K;Audet, J;Bello, A;Vendramelli, R;Boese, AS;Fernando, L;Lindsay, LR;Jardine, CM;Wood, H;Poliquin, G;Strong, JE;Drebot, M;Safronetz, D;Embury-Hyatt, C;Kobasa, D;
PMID: 34127676 | DOI: 10.1038/s41467-021-23848-9
Widespread circulation of SARS-CoV-2 in humans raises the theoretical risk of reverse zoonosis events with wildlife, reintroductions of SARS-CoV-2 into permissive nondomesticated animals. Here we report that North American deer mice (Peromyscus maniculatus) are susceptible to SARS-CoV-2 infection following intranasal exposure to a human isolate, resulting in viral replication in the upper and lower respiratory tract with little or no signs of disease. Further, shed infectious virus is detectable in nasal washes, oropharyngeal and rectal swabs, and viral RNA is detectable in feces and occasionally urine. We further show that deer mice are capable of transmitting SARS-CoV-2 to naïve deer mice through direct contact. The extent to which these observations may translate to wild deer mouse populations remains unclear, and the risk of reverse zoonosis and/or the potential for the establishment of Peromyscus rodents as a North American reservoir for SARS-CoV-2 remains unknown.
Gastrointestinal Pathology in Samples from Coronavirus Disease 2019 (COVID-19)-Positive Patients
Archives of pathology & laboratory medicine
Westerhoff, M;Jones, D;Hrycaj, SM;Chan, MP;Pantanowitz, L;Tu, H;Choi, K;Greenson, J;Lamps, L;
PMID: 33961007 | DOI: 10.5858/arpa.2021-0137-SA
-Although primarily considered a respiratory illness, coronavirus disease 2019 (COVID-19) can cause gastrointestinal manifestations. -To evaluate histopathology and in situ hybridization for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in gastrointestinal samples from patients with recent and remote COVID-19. -Patients with positive SARS-CoV-2 nasopharyngeal tests and a gastrointestinal tissue specimen were included. SARS-CoV-2 in situ hybridization (ISH) was performed on each sample. A subset had SARS-CoV-2 next generation sequencing (NGS) performed. -Twenty-five patients met inclusion criteria. Five had positive SARS-CoV-2 nasopharyngeal tests within 7 days of their gastrointestinal procedure. Two were ulcerative colitis patients on steroid therapy who lacked typical COVID-19 symptoms. Their colectomies showed severe ulcerative colitis; one demonstrated SARS-CoV-2 by NGS but a negative ISH. Another had an ischemic colon resected as a complication of the COVID-19 course; however, both ISH and NGS were negative. A fourth had a normal-appearing terminal ileum but positive ISH and NGS. The fifth patient had ileal ulcers with SARS-CoV-2 negativity by both modalities. The remaining 20 patients had positive nasopharyngeal tests an average of 53 days prior to procedure. None of their samples demonstrated SARS-CoV-2 ISH positivity, but one was positive on NGS despite a negative nasopharyngeal test. -Gastrointestinal findings from SARS-CoV-2-infected patients ranged from normal with virus detected by ISH and NGS, to bowel ischemia secondary to systemic viral effects, without evidence of virus in the tissue. No distinct histologic finding was identified in those with gastrointestinal tissue specimens demonstrating SARS-CoV-2 positivity in this cohort.
Fluri F, Malzahn U, Homola GA, Schuhmann MK, Kleinschnitz C, Volkmann J.
PMID: 29059697 | DOI: 10.1002/ana.25086
Abstract
OBJECTIVE:
One-third of all stroke survivors are unable to walk, even after intensive physiotherapy. Thus, other concepts to restore walking are needed. Since electrical stimulation of the mesencephalic locomotor region (MLR) is known to elicit gait movements, this area might be a promising target for restorative neurostimulation in stroke patients with gait disability. The present study aims to delineate the effect of high-frequency stimulation of the MLR (MLR-HFS) on gait impairment in a rodent stroke model.
METHODS:
Male Wistar rats underwent photothrombotic stroke of the right sensorimotor cortex and chronic implantation of a stimulating electrode into the right MLR. Gait was assessed using clinical scoring of the beam walking test and videokinematic analysis (CatWalk™) at baseline and on days 3 and 4 after experimental stroke with and without MLR-HFS.
RESULTS:
Kinematic analysis revealed significant changes in several dynamic and static gait parameters resulting in overall reduced gait velocity. All rats exhibited major coordination deficits during the beam walking challenge and were unable to cross the beam. Simultaneous to the onset of MLR-HFS, a significantly higher walking speed and improvements in several dynamic gait parameters were detected by the Catwalk™-system. Rats regained the ability to cross the beam unassisted showing a reduced number of paw slips and misses.
INTERPRETATION:
MLR-HFS can improve disordered locomotor function in a rodent stroke model. It may act by shielding brainstem and spinal locomotor centers from abnormal cortical input after stroke, thus allowing for compensatory and independent action of these circuits.
Signal transduction and targeted therapy
Song, Z;Bao, L;Deng, W;Liu, J;Ren, E;Lv, Q;Liu, M;Qi, F;Chen, T;Deng, R;Li, F;Liu, Y;Wei, Q;Gao, H;Yu, P;Han, Y;Zhao, W;Zheng, J;Liang, X;Yang, F;Qin, C;
PMID: 35091528 | DOI: 10.1038/s41392-022-00891-6
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is transmitted on mink farms between minks and humans in many countries. However, the systemic pathological features of SARS-CoV-2-infected minks are mostly unknown. Here, we demonstrated that minks were largely permissive to SARS-CoV-2, characterized by severe and diffuse alveolar damage, and lasted at least 14 days post inoculation (dpi). We first reported that infected minks displayed multiple organ-system lesions accompanied by an increased inflammatory response and widespread viral distribution in the cardiovascular, hepatobiliary, urinary, endocrine, digestive, and immune systems. The viral protein partially co-localized with activated Mac-2+ macrophages throughout the body. Moreover, we first found that the alterations in lipids and metabolites were correlated with the histological lesions in infected minks, especially at 6 dpi, and were similar to that of patients with severe and fatal COVID-19. Particularly, altered metabolic pathways, abnormal digestion, and absorption of vitamins, lipids, cholesterol, steroids, amino acids, and proteins, consistent with hepatic dysfunction, highlight metabolic and immune dysregulation. Enriched kynurenine in infected minks contributed to significant activation of the kynurenine pathway and was related to macrophage activation. Melatonin, which has significant anti-inflammatory and immunomodulating effects, was significantly downregulated at 6 dpi and displayed potential as a targeted medicine. Our data first illustrate systematic analyses of infected minks to recapitulate those observations in severe and fetal COVID-19 patients, delineating a useful animal model to mimic SARS-CoV-2-induced systematic and severe pathophysiological features and provide a reliable tool for the development of effective and targeted treatment strategies, vaccine research, and potential biomarkers.
