Cyclic GMP-AMP synthase promotes the inflammatory and autophagy responses in Huntington disease

Significance Huntington disease (HD) is a genetic disorder caused by glutamine-expansion in the huntingtin (mHTT) protein, which affects motor, psychiatric, and cognitive function, but the mechanisms remain unclear. mHTT is known to induce DNA damage and affect autophagy, both associated with inflammatory responses, but what mediates all these were unknown. Here we report that cGAS, a DNA damage sensor, is highly upregulated in the striatum of a mouse model and HD human patient’s tissue. We found ribosomes, which make proteins, are robustly accumulated on the cGAS mRNA in HD cells. cGAS depletion decreases—and cGAS expression increases—both inflammatory and autophagy responses in HD striatal cells. Thus, cGAS is a therapeutic target for HD. Blocking cGAS will prevent/slow down HD symptoms.


Generation of cGAS depleted striatal cells
First, we transfected the striatal neuronal cells (control, HD-het and HD-homo) with cGAS CRISPR/Cas9 plasmid (sc-437363) or CRISPR/Cas9 control plasmid (sc-418922) in 10cm dish. After 48 h we sorted the cells based on GFP fluorescence and re-cultured them. We passaged them 2-3 time and prepared lysate to confirm the cGAS depletion by Western blotting using cGAS antibody. Exogenous pTRIP-SFFV-EGFP-FLAG-cGAS (GFP-cGAS) was transfected into the cGASD-HD-homo cells and cGASD-control cells to further investigate the role of cGAS in autophagy and inflammatory responses in HD.

Western blotting
To check the protein expression level striatal neuronal cells (control and HD) were plated in 6 well plate (2x10 5 cells per well). After 24 hours, cells were washed in PBS and lysed in buffer containing 1% Triton X-100 in 50 mM Tris-HCl, pH 7.4, 150 mM NaCl and 1x protease inhibitor cocktail (Roche, Sigma) and 1x phosphatase inhibitor (PhosStop, Roche, Sigma), sonicated for 3 x 5 sec at 20% amplitude, and cleared by centrifugation for 10 min at 11,000g at 4°C . Protein concentration was determined with a bicinchoninic acid (BCA) protein assay reagent (Pierce). Equal amounts of protein (30-40 µg) were loaded and were separated by electrophoresis in 4 to 12% Bis-Tris Gel (Thermo Fisher Scientific), transferred to polyvinylidene difluoride membranes, and probed with the indicated antibodies. HRP-conjugated secondary antibodies (Jackson ImmunoResearch Inc.) were probed to detect bound primary IgG with a chemiluminescence imager (Alpha Innotech) using enhanced chemiluminescence from WesternBright Quantum (Advansta) (SI ref. 4). The band intensities were quantified with ImageJ software (NIH).
Phosphorylated proteins were then normalized against the total protein levels (normalized to actin or GAPDH). Mice striatal tissues were homogenized in radioimmunoprecipitation assay (RIPA) buffer [50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1.0% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS,] with 1x complete protease inhibitor cocktail (Roche, Sigma) and 1x phosphatase inhibitor (PhosStop, Roche, Sigma), followed by a brief sonication for 2 x 5 sec at 20% amplitude and cleared by centrifugation for 10 min at 11,000g at 4°C. Protein estimation was done using a BCA method and proceeded to Western Blotting as mentioned above. Immunoprecipitation from HD-homo cells was carried out using HTT antibody as described before (SI ref. 5). Human tissue was homogenized in lysis buffer [50 mM tris (pH 7.4), 150 mM NaCl, 10% glycerol, and 1.0% Triton X-100] with protease and phosphatase inhibitors, followed by a brief sonication for 6 s at 20% amplitude and cleared by centrifugation for 10 min at 11,000g at 4°C. Protein estimation was done using a BCA method and proceeded to Western Blotting as mentioned above.

Real-time PCR
RNA was extracted from the fractionated samples following lysis in Trizol reagent. 250 ng RNA was used to prepare cDNA using Takara

Nuclear/cytoplasmic and Subcellular Fractionation
For subcellular fractionation, HD-homo cells were trypsinized and lysed in buffer A of mitochondria isolation buffer (Thermo Fisher Scientific 89874) and kept on ice for 2 minutes. Buffer C was added to each sample and mixed by inverting 5 times. The homogenates were centrifuged at low speed (700g) for 10 minutes to separate nuclei and cell debris. The supernatants were immediately loaded on top of 10-50% sucrose gradients and centrifuged at 40000 RPM (SW41Ti rotor) at 4°C for 2 hours. The gradients were fractionated manually (10 x 1 ml fractions). Using methanol/chloroform, total protein of each fraction was precipitated. The protein pellets were resuspended in 2x LDS/bmercaptoethanol lysis buffer and proceeded to Western Blotting as mentioned above.

Staining with DAPI
Striatal neuronal cells (Control and HD-het, HD-homo) plated in glass bottom dishes or hESC-derived striatal neurons on coverslips were washed in D-PBS and fixed for 10 min in 4% PFA (Electron Microscopy Sciences). The cells were washed 3 times with D-PBS and incubated with DAPI (4′,6-diamidino-2-phenylindole, Sigma) for 10 min.

Image processing and micronuclei quantification
For control and HD-homo cells, fluorescent confocal images were taken in Zeiss LSM 880 microscope using 63X oil immersion Plan-apochromat objective (1.4 NA). Excitation was via a 405 nm diode-pumped solid-state laser. Pinholes were set so that the section thickness was equal for all channels and ≤ 1 AU. Images were acquired with an optimal Z-step of 0.27 µm covering the whole cellular volume. Processing was performed with Zen software black/blue edition 2012. HD-het cells were imaged using Leica DM5500B microscope (100x objective). For micronuclei quantification, we counted the cells with small DAPI positive puncta separated from nucleus.