SARS-CoV-2-encoded protein ameliorates neuromuscular degeneration in fly models of neurodegenerative diseases

In a recent study published in PNAS, researchers explored the in vivo efficacy of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-encoded proteins against neuromuscular (NM) degeneration associated with ribosome stalling.

Study: Prevention of ribosome collision-induced neuromuscular degeneration by SARS CoV-2–encoded Nsp1. Image Credit: Kateryna Kon/Shutterstock
Study: Prevention of ribosome collision-induced neuromuscular degeneration by SARS CoV-2–encoded Nsp1. Image Credit: Kateryna Kon/Shutterstock

Background

Age-associated neurodegeneration is a public health crisis that requires the development of targeted, effective, and broad disease-altering therapeutic agents; however, data on the extent to which pathophysiological mechanisms are shared by the diverse neurodegenerative diseases are limited. The control of translation machinery is essential for proteostasis maintenance during aging and to combat viral infections.

About the study

In the present study, researchers explored the neuroprotective effects of SARS-CoV-2-encoded proteins in vivo using Drosophila species.

For Drosophila genetic analysis, fly culture and crosses were performed, and the newly synthesized Drosophila larvae proteins were puromycin (Pum)-labeled, following which fly lifespan was analyzed. HeLa, plate transfluor cell line (U2OS), and human embryonic kidney (HEK)293T cells were used for cell culture experiments, and drug treatment analysis was performed wherein HeLa cells were treated with the drugs such as cycloheximide, homoharringtonine (HHT), Pum and emetine.

Further, translation stalling reporter assays were performed. To evaluate the potential effectiveness of SARS-CoV-2 proteins on NM function, 12 SARS-CoV-2 proteins likely to be involved in intracellular host-virus interactions were expressed, including non-structural proteins (Nsp)-1,2,3,6, open reading frames (Orf)-3a,3b,6,7a,7b,8, 9b, and 10. Wing posture and locomotor activity assays were performed to evaluate indirect flight musculature integrity and muscle function, respectively.

The team investigated whether SARS-CoV-2 protein expression can exacerbate compromised NM functioning among neurodegenerative disease patients. Western blot analysis and immunostaining were performed to confirm SAR-CoV-2 protein expression. Next, Nsp1 effects on Alzheimer’s disease (AD)-associated phenotypes were investigated in the context of full-length-amyloid precursor protein (FL-APP) and on APP- or APP.C99-induced memory and learning defects.

For the behavioral assays, transgenic flies coexpressing β-secretase and FL-APP in neurons were used, and BACE1 (beta-secretase 1) was used for facilitating APP.C99 production from FL-APP. Further, the underlying mechanisms of Nsp1 activity against APP.C99-induced pathologies were explored, and Nsp1 effects on FL-APP were assessed. Protein synthesis in Drosophila tissues, based on Pum incorporation into nascent peptide chains (NPCs), was measured to explore Nsp1 effects on global translation.

Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis was performed to assess messenger ribonucleic acid (mRNA) levels. Mammalian cells were used to investigate Nsp1-mediated stalled translation regulation mechanisms for which stalled NPCs were Pum-labeled and treated by Pum-emetine combinations Nsp1 effects on ribosome stalling were assessed by green fluorescent protein (GFP)-P2A-K20-P2A-red fluorescent protein (RFP) reporter assays and by evaluating Nsp1 effects on 40S subunit Rps3 (ribosomal protein s3) ribosome collision indicator.

The team explored the potential relevance of ribosome collision-induced Cyclic GMP-AMP synthase and stimulator of interferon genes (cGAS/STING) signaling among APP.C99 cells NM toxicity. In addition, published literature on Nsp1 and RQC-associated factor interactions was searched to understand Nsp1 ribosome stalling regulation mechanisms. Sucrose gradient ribosomal analysis was performed to examine activating signal cointegrator 1 complex subunit 3 (ASCC3) and endothelial differentiation-related factor 1 (EDF1) distributions.

Results

Nsp1 overexpression effectively rescued behavioral phenotypes and NM degeneration among Drosophila species models of Parkinson’s disease, amyotrophic-type lateral sclerosis, and AD. The mechanism of aberrant protein accumulation due to translatory ribosome collision and stalling (and resultant proteostasis failure) was shared by the three diseases.

Nsp1 demonstrated a multipronged action in resolving collided ribosomes, aborting stalled translations, and removing faulty translationary products causative of the modeled diseases, at least partially through ABCE1, ribosome-related quality-control factors, AKT signaling, and autophagy. Nsp1 effects were exquisitely specific, and it rescued AD-associated proteostasis failure and NM degeneration in APP C-terminal fragment (APP.C99) transgenic flies.

Nsp1 promoted the removal of aberrant APP.C99 species resulting from inadequate ribosome-associated protein quality control (RQC) of ribosome stalling. Nsp1 did not cause a global shutdown of protein synthesis or turnover of APP.C99 mRNA in Drosophila. Nsp1 promoted collided ribosome resolution and inhibited the cGAS/STING pathway. ABCE1 (ATP-binding cassette sub-family E member 1) mediated Nsp1 effects on stalled APP.C99 translation.

Nsp1 effect on APP/APP.C99 expression was unlikely through mRNA degradation or global translation inhibition. For Nsp1, no effect was observed on the RFP/GFP ratio expressed by the non-stalling reporter, GFP-P2A-K0-RFP, for Nsp1; however, using the GFP-P2A-Flag-K20- P2A-mKate2 translation stall reporter, Nsp1 lowered the ration of mKate2/GFP.

In coronavirus disease 2019 (COVID-19), Nsp1 probably promotes stalled ribosome disassembly on SARS-CoV-2 RNA to prevent aberrant viral protein accumulation or for recycling ribosomes that are stalled on host mRNA to increase their availability for SARS-CoV-2 translation. Nsp1-induced reduction of total APP.C99 and FL-APP levels was consistent with Nsp1’s abortive discontinuation modes on the stalled ribosomal molecules.

Conclusion

Overall, the study findings showed amelioration of NM degeneration by Nsp1 in several neurodegenerative disease models and provided novel insights into the biochemical functions of Nsp1 in manipulating the host translation machinery. The findings uncovered a novel mechanism of Nsp1 in manipulating host translation to combat age-associated neurogenerative diseases.

Journal reference:
Pooja Toshniwal Paharia

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Pooja Toshniwal Paharia

Dr. based clinical-radiological diagnosis and management of oral lesions and conditions and associated maxillofacial disorders.

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