Review 1: "SARS-CoV-2 Infection Impacts Carbon Metabolism and Depends on Glutamine for Replication in Syrian Hamster Astrocytes"

Juan de la Torre

doi:doi:10.1162/2e3983f5.ebd10c8a

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

by Lilian Gomes de Oliveira, Yan de Souza Angelo, Pedro Yamamoto, Victor Corasolla Carregari, Fernanda Crunfi, Guilherme Reis-de-Oliveira, Lícia Costa, Érica Almeida Duque, Nilton Barreto dos Santos, Glaucia Maria Almeida, Egidi Mayara Firmino, Isadora Marques Paiva, Carolina Manganeli Polonio, Nagela Ghabdan Zanluqui, Marília Garcia de Oliveira, Gustavo Gastão Davanzo, Marina Caçador Ayupe, Caio Loureiro Salgado, Antônio Francisco de Souza Filho, Marcelo Valdemir de Araújo, Taiana Tainá Silva-Pereira, Angélica Cristine de Almeida Campos, Luiz Gustavo Bentim Góes, Marielton dos Passos Cunha, Maria Regina D’Império Lima, Denise Morais Fonseca, Ana Márcia de Sá Guimarães, Paola Camargo Minoprio, Carolina Demarchi Munhoz, Cláudia Madalena Cabrera Mori, Pedro Manoel Moraes-Vieira, Thiago Mattar Cunha, Daniel Martins-de-Souza, and Jean Pierre Schatzmann Peron

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dx.doi.org

Description

ABSTRACTCoronaviruses 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.

RR:C19 Evidence Scale rating by reviewer:

Potentially informative. The main claims made are not strongly justified by the methods and data, but may yield some insight. The results and conclusions of the study may resemble those from the hypothetical ideal study, but there is substantial room for doubt. Decision-makers should consider this evidence only with a thorough understanding of its weaknesses, alongside other evidence and theory. Decision-makers should not consider this actionable unless the weaknesses are clearly understood and there is other theory and evidence to further support it.

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Review:

Acute respiratory syndrome is the hallmark feature of severe COVID-19, and therefore a main focus of research is understanding the impact of SARS-CoV-2 infection on the respiratory system. However, there is compelling evidence that SARS-CoV-2 infection can also significantly impact the normal function of other organs and systems, which has raised great interest in understanding the long-term consequences of COVID-19 beyond its effects on the respiratory system. In this regard, neurological complications of COVID-19 are frequently reported. SARS-CoV-2 RNA has been detected in autopsy brain samples and in the CSF of some patients—though different studies have reported conflicting findings. How SARS-CoV-2 enters the brain, which cell types it targets in the brain, and the mechanisms whereby SARS-CoV-2 infection can impact normal brain function are all issues that have not been solved and remain controversial. Hence the significance of the present work by Gomes de Oliveira and colleagues examining the effects of SARS-CoV-2 infection on the metabolism of hamster’s astrocytes.

The authors have provided convincing evidence that SARS-CoV-2 productively infects hamsters’ astrocytes, which is associated with very significant changes in protein expression and metabolic pathways involved in carbon metabolism, glycolysis, mitochondrial respiration, and synaptic transmission. Notably, results presented by the authors strongly support that SARS-CoV-2 multiplication in hamsters’ astrocytes does not depend on glycolysis, but rather on glutamine. Accordingly, treatment with the glutaminolysis inhibitor 6-Diazo-5-oxo-L-norleucine resulted in reduced levels of SARS-CoV-2 multiplication.

Based on their findings, the authors proposed that in COVID-19 cases, glutamine consumption due to the viral hijacking of cellular metabolism may affect glutamate/glutamine balance, and thus lead to the neurological symptoms reported in a significant number of COVID-19 cases.

The paper is well written. The experimental section of the paper was well executed and the results—which are clearly presented and nicely illustrated—support the authors’ key conclusion that SARS-CoV-2 can productively infect hamsters’ astrocytes, which is associated with disturbances in the normal astrocyte metabolism.

The main weakness of this very elegant study is the limited discussion of the authors’ findings in the context of SARS-CoV-2 infection of human brain cells. Published studies have documented that exposure of a variety of cerebral organoids (CO) to SARS-CoV-2 results in infection of different brain cell populations, but their numbers and identity have not been unequivocally established—underscoring the need for additional studies aimed at resolving these questions. Particularly relevant to the present work are studies using primary organotypic slice cultures and cortical organoids at late gliogenic stages showing that a significant number of cortical astrocytes (GFAP⁺) can be infected by SARS-CoV-2. Despite their susceptibility to SARS-COV-2, cortical astrocytes expressed undetectable levels of ACE-2 but readily detectable levels of the alternative SARS-CoV-2 receptors DPP4 and CD147. It would be interesting if the authors could provide information about results using blocking antibodies to determine the receptor used by SARS-CoV-2 to enter hamsters’ astrocytes. Another issue that needs clarification is a clear presentation of the absolute numbers (total %) of SARS-CoV-2 infected astrocytes detected in either tissue culture or in brain sections from infected hamsters. It would be important to discuss these results in the context of the apparently very low numbers of human brain cells, including astrocytes and neurons, that are detected in the different experimental systems using human cells and organoids.

Review 1: "SARS-CoV-2 Infection Impacts Carbon Metabolism and Depends on Glutamine for Replication in Syrian Hamster Astrocytes"

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