Prevention of ribosome collision-induced neuromuscular degeneration by SARS CoV-2–encoded Nsp1

Significance Age-related neurodegenerative disease is a looming public health crisis. Despite intensive efforts, effective disease-modifying treatment is still elusive. Development of effective therapeutic strategies applicable to a broad spectrum of neurodegenerative diseases is desirable, but little is known about the extent to which pathogenic mechanisms are shared among these diseases. We discovered that Nsp1, a viral protein encoded by SARS-CoV-2, the virus causing the ongoing COVID-19 epidemic, ameliorates neuromuscular degeneration in fly models of Alzheimer’s disease, Parkinson’s disease, and amyotrophic lateral sclerosis, which share a common mechanism—accumulation of aberrant protein species due to stalling and collision of ribosomes—which is resolved by Nsp1. These results have important implications for the understanding and treatment of neurodegenerative diseases as well as COVID-19.


Climbing activity and wing posture assays
Around 10-20 male flies were transferred to a clean plastic vial. The flies were allowed to get accustomed to the new environment for 3-4 min and subsequently measured for banginduced vertical climbing distance. The performance was scored as percentage of flies crossing the 8 cm mark within 12 seconds. Each experiment was performed ≥ 4 times. To assay wing posture, cohorts of flies raised at 25 or 29 degrees at the indicated ages were visually for straight, held-up, or droopy wing postures. The number of flies with normal (straight) or abnormal (held-up or droopy) wing postures were counted and quantified as the percentage of the total number of flies.

Aversive taste memory
Drosophila taste memory assay was performed as described previously (3). Briefly, oneweek old flies were starved for 12-18h in an empty vial on wet Kimwipe paper before test.
Flies were anesthetized on ice and fixed on a glass slide by applying nail polish to their wings. 10-15 flies were used for each set of experiment. Flies were then incubated in a humid chamber for 2 h to allow recovery from the procedure. In the pretest phase, flies were presented with 500 mM sucrose stimuli (attractive tastant) to their legs using Kimwipe wick. Flies that showed positive proboscis extension to the stimulus were used for the next phases. In the second training phase, flies were presented with 500 mM sucrose stimuli at their legs while being simultaneously punished by 10 mM quinine (aversive tastant) applied to their extended proboscis. Training was repeated 15 times for each fly.
The last phase is the test phase where the flies were given 500 mM sucrose at their legs at different time intervals (0, 5, 15, 30, 45, and 60 min), and proboscis extension was recorded. Each experiment was carried out ≥ 4 times.

Analysis of gene expression by RT-qPCR
We used TRIzol (Invitrogen) to extract mRNA from fly thorax and iscript cDNA synthesis kit (Bio-Rad) to synthesize cDNA. Real time quantitative PCR (RT-qPCR) was performed using SYBR Green. The sequences of RT-qPCR primers we used are as follows: AKT forward: GCTATGACGCCATCTGAAC

Protein extraction from cultured cells and western blotting
HeLa or U2OS cells were transfected with the respective plasmids. Cells were washed with 1X PBS 30h post transfection and lysed in lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 1% Triton X100, protease inhibitors), followed by centrifugation at 13000 rpm for 20 mins at 4 o C. Protein concentration was measured using the Bradford method. The supernatant was then mixed with 4x protein loading buffer and loaded onto either 4-12% bis-tris gels using MES as running buffer or on 16% Tricine gel and immunoblotted onto PVDF membranes. The membranes were blocked with blocking buffer (5% BSA in TBST) and incubated with following primary antibodies (Anti-Flag, Sigma-Aldrich F1804, 1:2000; were used for detection at 1:10000 dilution. Special steps were taken during SDS PAGE to better resolve the different APP.C99 species as described before (4). For quantification of western blot data, signal intensity was measured and calculated using NIH Image J.

Immunohistochemistry
Immunostaining of adult fly muscle was performed as previously described (5) Immunostaining of adult brains was performed as previously described (4)  For analysis of cGAS distribution in Nsp1 transfected cell with RQC manipulation, ZNF598 KO U2OS cells and ASCC3 KD U2OS cells were transfected with NSP1 and RFP at the ratio of 3 to 1 for two days before harvesting. All cells were harvested and fixed with 3% paraformaldehyde in PBS for 10 minutes at room temperature. This was followed by permeabilization with 0.5% Triton X-100 for 15 minutes. Cells were washed with 1 ml PBS twice and blocked with PBS containing 5% goat serum for 30 minutes at room temperature. Primary antibodies (cGAS) were diluted in blocking buffer (PBS containing 5% goat serum) and incubated in cold room overnight. Cells were washed with 1 ml PBS and incubated with secondary antibody in blocking buffer for 1 h at room temperature.

Immunostaining of cultured cells
After the final wash cells were mounted on glass slides with antifade mounting medium (Vector Laboratories, H-1700). Images were captured with Leica Confocal Microscope. and NSP1 plasmids mixed at 1:2 ratio were co-transfected for 36 hrs. Cell lysates were prepared and western blot analysis was performed. Nsp1-WT and Nsp1-KH mutant plasmids were obtained from Addgene (141255 and 164522).

Puromycin labeling of ribosome stalled newly synthesized proteins
Puromycin labeling of stalled NPCs was performed as described (4). Briefly, Hela cells were seeded on coverslips in a 6-well plate and transfected with pCAX-C99 (Addgene #30146). After 48 hr transfection, cells were treated with HHT for 5 minutes, thereafter Puromycin (50µg) and Emetine (100µg) were added and cells were incubated for 5-7 minutes. After this, cells were permeabilized by 0.02% digitonin in Permeabilization buffer (50 mM Tris-HCl, pH7.5, 5 mM MgCl2, 25 mM KCl, 355 mM cyclohexamide, 10 units RNAseOut and 0.02% digitonin) for 2 minutes. Permeabilized cells were washed twice with washing buffer (permeabilization buffer without digitonin) and fixed in 4% paraformaldehyde for 30 minutes. The Permeabilization and washing steps were performed in ice-old buffers. Cells were immunostained with the amyloid conformation-specific antibody mOC78 that recognizes aggregation-prone APP.C99 (4) and Puromycin antibody and observed under the confocal microscope.

Sucrose gradient analysis of ribosomes
For each experimental condition, one 10 cm plate of cells at around 80% confluency were first washed with ice-cold PBS and harvested by scraping. After sedimentation at 4°C at 5000 rpm for 3 min, cell pellets were resuspended in 200 μl of 1xRNC buffer containing 0.01% digitonin, 1x protease inhibitor cocktail (EDTA-free cOmplete from Roche) and 1mM DTT. After 15 min incubation on ice, cells were disrupted using a pre-chilled 26G needle appended to 1 mL syringe. Lysates were clarified by 15 min centrifugation at 15,000 g at 4°C. Lysate containing 150 μg of total RNA was loaded onto a 10%-50% analytical sucrose gradients (2 ml) and spun for 30 min at 55,000 rpm in TLS-55 rotor at 4°C using slowest acceleration and deceleration settings. Eleven fractions of 200 μl were collected 11 manually from the top of the gradient. Protein was precipitated from the solution using a final concentration of 20% Trichloroacetic acid (TCA). The resulting pellets were washed with 10% TCA, followed by 100% acetone, and dried. Samples were resuspended in Laemmli sample buffer containing β-mercaptoethanol, boiled at 95°C for 5 min, and subjected to western blot analysis.   Mitochondrial morphology was monitored with a mito-GFP reporter driven by TH-Gal4.