SARS-CoV-2 Infection Impacts Carbon Metabolism and Depends on Glutamine for Replication in Syrian Hamster Astrocytes

Abstract

Coronaviruses belong to a well-known family of enveloped RNA viruses and are the causative agent of the common cold. Although the seasonal coronaviruses do not pose a threat to human life, three members of this family, i.e., SARS-CoV, MERS-CoV and recently, SARS-CoV2, may cause severe acute respiratory syndrome and lead to death. Unfortunately, COVID-19 has already caused more than 4.4 million deaths worldwide. Although much is better understood about the immunopathogenesis of the lung disease, important information about systemic disease is still missing, mainly concerning neurological parameters. In this context, we sought to evaluate immunometabolic changes using in vitro and in vivo models of hamsters infected with SARS-CoV-2. Here we show that, besides infecting hamster’s astrocytes, SARS-CoV-2 induces changes in protein expression and metabolic pathways involved in carbon metabolism, glycolysis, mitochondrial respiration, and synaptic transmission. Interestingly, many of the differentially expressed proteins are concurrent with proteins that correlate with neurological diseases, such as Parkinsons’s disease, multiple sclerosis, amyotrophic lateral sclerosis, and Huntington’s disease. Metabolic analysis by high resolution real-time respirometry evidenced hyperactivation of glycolysis and mitochondrial respiration. Further metabolomics analysis confirmed the consumption of many metabolites, including glucose, pyruvate, glutamine, and alpha ketoglutarate. Interestingly, we observed that glutamine was significantly reduced in infected cultures, and the blockade of mitochondrial glutaminolysis significantly reduced viral replication and pro-inflammatory response. SARS-CoV-2 was confirmed in vivo as hippocampus, cortex, and olfactory bulb of intranasally infected hamsters were positive for viral genome several days post-infection. Altogether, our data reveals important changes in overall protein expression, mostly of those related to carbon metabolism and energy generation, causing an imbalance in important metabolic molecules and neurotransmitters. This may suggest that some of the neurological features observed during COVID-19, as memory and cognitive impairment, may rely on altered energetic profile of brain cells, as well as an unbalanced glutamine/glutamate levels, whose importance for adequate brain function is unquestionable.

ScreenIT

Nov 19, 2021

SciScore for 10.1101/2021.10.23.465567: (What is this?)

Please note, not all rigor criteria are appropriate for all manuscripts.

Table 1: Rigor

Ethics	IACUC: The protocols were approved by the Ethics Committee for Animal Research of University of Sao Paulo (CEUA n° 7971160320 / 3147240820) and all efforts were made to minimize animal suffering.
Sex as a biological variable	Brain samples from SARS-CoV-2-infected and uninfected hamsters from two unrelated experiments were used herein (one performed with 15–16-week-old females and another with 18-week-old males).
Randomization	Quantification was made using ImageJ software (NIH), where 5 to 10 randomly chosen visual fields per coverslip from three independent cultures were analyzed.
Blinding	not detected.
Power Analysis	not detected.
Cell Line Authentication	not detected.

Table 2: Resources

Antibodies
Sente…

SciScore for 10.1101/2021.10.23.465567: (What is this?)

Please note, not all rigor criteria are appropriate for all manuscripts.

Table 1: Rigor

Ethics	IACUC: The protocols were approved by the Ethics Committee for Animal Research of University of Sao Paulo (CEUA n° 7971160320 / 3147240820) and all efforts were made to minimize animal suffering.
Sex as a biological variable	Brain samples from SARS-CoV-2-infected and uninfected hamsters from two unrelated experiments were used herein (one performed with 15–16-week-old females and another with 18-week-old males).
Randomization	Quantification was made using ImageJ software (NIH), where 5 to 10 randomly chosen visual fields per coverslip from three independent cultures were analyzed.
Blinding	not detected.
Power Analysis	not detected.
Cell Line Authentication	not detected.

Table 2: Resources

Antibodies
Sentences	Resources
After antigen retrieval (Tris/EDTA-Tween-20 buffer -10mM Tris, 1mM EDTA, 0.05% Tween 20, pH 9 - for 30 minutes at 95°C, followed by 20 minutes at room temperature), cells were permeabilized with 0.5% Triton-X100 in PBS for 10 minutes, blocked with blocking solution (5% donkey serum + 0.05% Triton X-100 in PBS) (Sigma-Aldrich) for 2 hours at room temperature and incubated with primary antibodies: GFAP (1:1000, Sigma-Aldrich), IBA1 (1:500, Abcam), and MAP2 (1:500, Sigma-Aldrich) diluted in blocking solution, overnight at 4°C.	GFAP suggested: None IBA1 suggested: (ChromoTek Cat# smsG1Cys2-1, RRID:AB_2864263) MAP2 suggested: None
Experimental Models: Cell Lines
Sentences	Resources
The inoculum was then added to the flask containing Vero cells and maintained for 1 hour at 37°C, following addition of fresh DMEM low glucose media supplemented with 2% FBS and 1% penicillin/streptomycin.	Vero suggested: None
This virus was obtained from nasopharyngeal swabs from the first patient (HIAE01) to be diagnosed with COVID-19 in Brazil, isolated in Vero ATCC CCL-81 cells and quantified by using the Median Tissue Culture Infectious Dose (TCID50) assay.	Vero ATCC CCL-81 suggested: None
Plaque-forming unit assay: For virus titration, Vero CCL81 cells were seeded in 24 well plates one day before the infection for adhesion.	Vero CCL81 suggested: None
Software and Algorithms
Sentences	Resources
Label free quantitative analysis was obtained using the relative abundance intensity integrated in Progenesis software, using all peptides identified for normalization.	Progenesis suggested: (Progenesis QI, RRID:SCR_018923)
Generated images were loaded into Fiji software for further analysis.	Fiji suggested: (Fiji, RRID:SCR_002285)
Mitochondrial analysis: For measurements of mitochondrial superoxide, the cells were stained with LIVE/DEAD™ Fixable Green (L34970; ThermoFisher®, 15 min) and MitoSOX™ Red mitochondrial superoxide indicator (M36008; ThermoFisher®; 2.5uM, 10 min).	ThermoFisher® suggested: (ThermoFisher; SL 8; Centrifuge, RRID:SCR_020809)
Next, cells were analyzed using a Accuri C6 Plus (BD Biosciences®) cytometer and data analyzed using FlowJo X software.	BD Biosciences® suggested: (BD Biosciences, RRID:SCR_013311) FlowJo suggested: (FlowJo, RRID:SCR_008520)
Quantification was made using ImageJ software (NIH), where 5 to 10 randomly chosen visual fields per coverslip from three independent cultures were analyzed.	ImageJ suggested: (ImageJ, RRID:SCR_003070)
Single Nuclei Transcriptomic Profile Analysis: Raw snRNA-seq data were obtained from frozen medial frontal cortex tissue from six post- mortem control and seven COVID-19 patients assessed herein were obtained from the dataset GSE159812 on NCBI GeneExpression Omnibus public databank.	NCBI GeneExpression Omnibus suggested: None
(DEGs) corresponding to the Differentially Expressed Proteins (DEPs) from hamster brain proteomics and concordant fold-change were subjected to Gene Ontology analysis, performed through online database PANTHER using PANTHER Pathways dataset34.	PANTHER suggested: (PANTHER, RRID:SCR_004869)
Statistical analysis: Data was plotted and analyzed using the GraphPad Prism 8.0 software (GraphPad Software®, San Diego, CA).	GraphPad suggested: (GraphPad Prism, RRID:SCR_002798)

Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.

Results from TrialIdentifier: No clinical trial numbers were referenced.

Results from Barzooka: We found bar graphs of continuous data. We recommend replacing bar graphs with more informative graphics, as many different datasets can lead to the same bar graph. The actual data may suggest different conclusions from the summary statistics. For more information, please see Weissgerber et al (2015).

Results from JetFighter: Please consider improving the rainbow (“jet”) colormap(s) used on page 41. At least one figure is not accessible to readers with colorblindness and/or is not true to the data, i.e. not perceptually uniform.

Results from rtransparent:

Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
No protocol registration statement was detected.

Results from scite Reference Check: We found no unreliable references.

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SARS-CoV-2 Infection Impacts Carbon Metabolism and Depends on Glutamine for Replication in Syrian Hamster Astrocytes

Abstract

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