The Parkinson’s disease-associated gene ITPKB protects against α-synuclein aggregation by regulating ER-to-mitochondria calcium release

Significance Parkinson’s disease (PD) is the second most prevalent neurodegenerative disease of aging, affecting approximately 10 million patients worldwide with no approved therapies to modify progression of disease. Further understanding of the cellular mechanisms contributing to the development of PD is necessary to discover therapies. Here, we characterize the role of a recently identified GWAS hit for sporadic PD, ITPKB, in the aggregation of α-synuclein, the primary pathological feature of disease. These results identify inhibition of inositol-1,4,5,-triphosphate (IP3)-mediated ER-to-mitochondria calcium release as a potential therapeutic approach for reducing neuropathology in PD.


Immunocytochemistry
For PFF experiments, MCNs were rinsed in 1x PBS and fixed in 4% PFA/PBS for 15 min at RT. Fixed cells were washed 3x in PBS, 3 min each followed by immunocytochemistry (ICC) or storage at 4°C in 0.01% sodium azide/PBS until day of ICC. Fixed cells were permeabilized in 0.1% Triton X-100/PBS for 20 min followed by blocking in 0.05% Triton X-100/PBS (PBS-T) supplemented with 5% normal goat serum (NGS) for 1 h. Cells were then incubated overnight with primary antibodies diluted in 2% BSA/PBS-T at 4°C, as described below. The next day, cells were washed 3x in PBS-T, 15 min each followed by incubation with goat anti-mouse IgG, anti-rabbit IgG, and/or anti-Chicken IgY secondary antibodies conjugated with Alexa Fluor fluorescent dyes (Invitrogen, Cat# A32728, A-11036, A32933) for 2 h at RT protected from light by aluminum foil. All secondary antibodies were diluted 1:700 in 5% NGS/PBS-T block buffer supplemented with 0.5 µg/ml DAPI (Invitrogen, Cat# 62248). After secondary antibody labeling, cells were washed 3x, 15 min each in PBS-T and stored in 0.01% sodium azide/PBS at 4°C until imaging and analysis by confocal microscopy. . After 1 h, cells were gently rinsed 3x with pre-warmed Dulbecco's PBS (DPBS) and immediately imaged at 40x magnification at 37°C using the Perkin Elmer Opera Phenix high-content confocal imager. After imaging, cells were then lysed in 1x Passive Lysis buffer (Promega, Cat# E1941) to be compatible with subsequent analysis by Cell Titer Glo (Promega, Cat# G7571) luminescence assays, which were performed according to manufacturer's instructions.

Image Analysis
Live-cell and fixed-cell ICC experiments were imaged using the Perkin Elmer Opera Phenix high-content confocal imager. Images were captured using Harmony software and transferred to the Columbus image analysis platform for subsequent analysis by batch processing. All statistical analysis was performed using Graphpad Prism software. Whenever possible, the figure legends in this manuscript provide the exact p  values, sample size, statistical test and post-hoc correction used, if applicable, and the number of biological  replicate experiments performed; individual data points and/or box-and whisker plots are shown for all figures.

Agilent Seahorse Mito Stress Test Assay
For Seahorse experiments, primary neurons were isolated as described above, and plated at a density of 20,000 cells per well in 96-well XF Cell Culture Microplates pre-coated with 100 µg/ml Poly-D-Lysine (Millipore, Cat# A-003-E) and 50 µg/ml Laminin (Invitrogen, Cat# 23017-015) overnight. Mitochondrial oxygen consumption was measured according to the instructions of the XF Cell Mito Stress Test Kit (Agilent Technologies, Cat# 103015-100). On the day of the assay (DIV14-DIV16), cells were washed 2x with and incubated in Assay Medium (10mM pyruvate, 10mM glucose in Agilent Seahorse XF DMEM Medium pH 7.4, Cat# 103575-100) in a 37°C non-CO2 incubator for 1 h. Oxygen consumption rates (OCRs) were measured with an XF96 extracellular flux analyzer (Agilent Seahorse) at baseline, after addition of 1 µM oligomycin to evaluate respiration associated with cellular ATP production, after addition of 1.5 µM Carbonyl cyanide-4 (trifluoromethoxy) phenylhydrazone (FCCP) to evaluate uncoupled respiration, and after addition of 0.5 µM rotenone/antimycin A to measure non-mitochondrial respiration. Upon completion of analysis, cells were fixed in 2% paraformaldehyde for 20 min at room temperature, washed 2x with PBS and nuclei were stained using NucBlue (Invitrogen, Cat# R37606). The number of cells per well were imaged using the Permin Elmer Opera Phenix high-content automated imager, as described above (Opera Phenix, Perkin Elmer). Raw OCR values were then normalized to the number of cells per well.

Supporting Information Appendix -Materials and Methods (pertaining to Figures S1 to S17)
Generation of human iPSC derived neurons Human iPSCs were obtained from a patient with a SNCA gene triplication, as described previously (Zambon et al. 2019, PMID: 30753527). iPSCs were thawed in mTESR Plus basal media (StemCell Technologies, Cat# 05826) supplemented with 1x mTESR Plus Supplement (StemCell Technologies, Cat# 05827) plus 10 µM Rock inhibitor Y-27632 (Sigma, Cat# Y0503), and seeded in 6 well plates pre-coated with hESCqualified Matrigel (Corning, Cat# 354277). Cells were maintained in mTESR Complete media. When iPSCs reached ~75% confluence, cells were passaged 1:10 into a new Matrigel-coated 6 well plate. On the next day (Day 1), cells were fed by complete media exchange with fresh mTESR Complete media. On Day 2, cells were transduced with 20 µl NGN2 LV plus 0.5 µl Polybrene. On Day 3, cells were fed by complete media exchange with fresh mTESR Complete media. On Day 4-6, NGN2 LV transduced iPSCs were expanded and frozen in mTESR complete media supplemented with 10% DMSO. For differentiation into neural progenitor cells (NPCs), NGN2 LV transduced iPSCs were thawed as described above. On the next day (Day 1), the media was changed to N2B27 media Cat# 2837], and 10 ng/ml GDNF [R&D Systems, Cat# 212-GD-050]). Wells were replenished with 50% fresh NGN2 Induction media on DIV 2, 4, and 7, at which point doxycycline was removed from the media. On DIV 10, cells were fed with 50% fresh media and pre-treated for 1 h with 0, 10, or 100 nM GNF362 followed by addition of 5 µg/ml mPFFs for 11 days. On DIV 21, cells were fixed for immunocytochemistry as described below.
Cloning, Expression and Purification of Murine α-Synuclein Monomer Wild type full length murine αSyn with an N-terminal 6xHis tag followed by an eXact tag (Bio-Rad; Profinity eXact Fusion-Tag System) was cloned, expressed in E. coli and purified as previously described (Weihofen et al 2019). Cell pellets were suspended in lysis buffer and lysed by a microfluidizer at 12,000 psi on ice. The lysate was clarified by centrifugation and the 6xHis-eXact-murine αSyn was captured by batch binding to Ni-NTA resin (Qiagen, Superflow). The protein-bound resin was packed into a column, washed with Ni wash buffer containing 20 mM imidazole, 0.5 mM dithiothreitol and protease inhibitors, and eluted with 250 mM imidazole in Ni wash buffer. The eluate was loaded onto a Profinity eXact affinity column (Bio-Rad) equilibrated with 100 mM Na2HPO4, pH 7.2, 500 mM sodium acetate, and washed with the same buffer until a stable baseline was obtained. Cleavage of the eXact tag was triggered by washing with 100 mM Na2HPO4, pH 8, 100 mM NaF, leading to release of the untagged protein from the column. Pooled peak fractions were further purified by size exclusion chromatography (SEC). The final purified protein was concentrated to 10 mg/mL in 10 mM Tris-HCl, pH 7.6, 50 mM NaCl, filtered through a 0.1 µm filter, and 0.2 mL aliquots were stored at -80°C. The purified αSyn was >95% pure by SDS-PAGE and had a calculated molecular weight of 14 kDa by SEC-multiangle light scattering. The purified αSyn had a hydrodynamic radius of 3.5 nm with no detectable large particles by dynamic light scattering.
Preparation of murine α-synuclein preformed fibrils Purified full length murine α-syn (0.2 mL) was thawed in a 37°C water bath. The protein was centrifuged with a Beckman tabletop high speed centrifuge at 600,000 g for 30 min at 4°C. The 200 µL clarified α-syn monomer solution was transferred to a 1.7 mL Eppendorf tube and fibrillar α-syn formed by continuous shaking at 1100 rpm in an Eppendorf Thermomixer at 37°C for 5 days.

Antibodies
The following antibodies were used in conjunction with either Western blot, ELISA, or immunocytochemistry analysis as described below. eQTL analysis The MetaBrain eQTL summary statistics were derived from public and protected RNA-seq and DNA genotyping available to the scientific community and Biogen, and consists of the GTEx, AMP-AD, BrainSeq, PsychENCODE, NABEC, and TargetALS cohorts. Additionally, public RNA-seq data for brain was obtained from the European Nucleotide Archive (ENA), following genotyping on RNA-seq reads. The data was harmonized using standard alignment using STAR to GRCh38, and FeatureCounts was used to quantify genes using GENCODE v24. QC and harmonization was performed using a standardized pipeline (3,4). eQTL analysis focused on RNA-seq samples from cortex tissues and was limited to individuals of European descent. The eQTLs were computed and normalized using the QTL pipeline described in Westra et al. Postnatal AAV injections Adeno-associated viruses (AAVs) were injected into mice on postnatal day 0 (P0). Pups were anesthetized on ice and were then injected intracerebroventricularly (i.c.v.) with 4 µl virus (1E11 GCs) mixed with fast green dye (<1% total). Mice were then warmed on a heating pad until recovered and monitored postsurgery according to IACUC approved guidelines. P0 injected pups were then injected with saline (PBS) or murine α-syn PFFs at 2 months of age (see below).
Stereotaxic injections At 2 months of age, animals were anesthetized with 1.5% isoflurane and stereotaxically injected into the right hemisphere with recombinant murine α-syn PFFs (2 µl of 5 mg/ml sonicated fibrils). Control animals received sterile PBS (vehicle). A 26-gauge single needle insertion (co-ordinates: AP 0.2mm, ML -2.0mm, DV -2.6mm) into the right forebrain was used to target the inoculum to the dorsal neostriatum. Injections were performed at a rate of 0.25 µl/min with the needle in place for > 5 min at each target. Animals were monitored regularly following recovery from surgery according to IACUC approved guidelines, and sacrificed 30 days post-injection as described below.
Biochemical fractionation for in vivo study For PFF injected mice, animals were anesthetized by CO2 inhalation followed by cardiac perfusion with icecold PBS according to Biogen IACUC guidelines. Brains were removed from the skull and the cerebral cortex, hippocampus, midbrain, and striatum tissues were dissected and immediately frozen by submersion in liquid nitrogen. Tissues were stored at -80°C until day of homogenization. Frozen cortices (ipsilateral hemisphere to PFF injection site) were homogenized in 8 volumes per weight (v/w) high-salt (HS) buffer (50 mM HEPES KOH pH 7.6, 750 mM NaCl, 5 mM EDTA) supplemented with Halt protease/phosphatase inhibitor cocktail with EDTA (Pierce, Cat# 78442) using the Qiagen TissueLyser II homogenization apparatus (Cat# 85300). 600 µl total HS lysate per sample was centrifuged at 20,000 g for 45 min at 4°C. The supernatant ('HS fraction') was collected and then the pellet was washed by resuspension in HS buffer followed by centrifugation at 20,000 g for 45 min at 4°C. Washed supernatants were discarded. The pellets were then resuspended in 300 µl 1% Triton X-100 (TX) buffer (50 mM HEPES KOH pH 7.6, 750 mM NaCl, 5 mM EDTA, 1% Triton X-100 plus protease/phosphatase inhibitors), vortexed, and incubated on ice for 15 min prior to centrifugation at 20,000 g for 45 min at 4°C. The supernatant ('TX fraction') was collected and then the pellet was washed by resuspension in TX buffer followed by centrifugation at 20,000 g for 45 min at 4°C. Washed supernatants were discarded. The pellets were then resuspended in 100 µl SDS buffer (50 mM HEPES pH 7.6, 2% SDS plus protease/phosphatase inhibitors), vortexed, and incubated at RT for 30 min followed by centrifugation at 20,000 g for 45 min at 25°C. The supernatant ('SDS fraction') was collected. Protein concentration in each fraction was determined by BCA assay and analyzed by Western blotting as described above.
Imaging and cell quantification Methodologies for imaging and counting the number of transduced cells, glial cell numbers, a P129+ cells in the cortex were adapted from those previously reported (6). In brief, a total of two sections per animal were used to quantify the percentage of transduced cells in the cortex, the number of glial cells in the cortex, and the percentage of P129+ cells in the cortex. Images were acquired on a TissueGnostics TissueFAXS SL automated slide scanner, equipped with a Zeiss AxioImager Z2 microscope, Crestoptics X-light V2 spinning disc confocal head, Lumencore Spectra 3 light source, and Hamamatsu ORCA Flash 4.0 V2+ camera. Quantitative analysis was performed on three and four-labeled fluorescent images generated by 20x confocal montage imaging of an entire sagittal mouse brain section compiled from individual images acquired using a Zeiss 20x/0.8NA Plan-Apochromat lens. The cortex was delineated as an active ROI based on neuroanatomical landmarks and with reference to the sagittal atlas of the mouse brain (Allen Brain Atlas). All slides were scanned under the same conditions for magnification, exposure time, lamp intensity and camera gain. For quantitative assessment, NeuN +, IBA1 +, GFAP + and P129+ cells from the selected ROIs were automatically detected using a custom automated image analysis software created at Biogen, and all images were batch analyzed together. Once two sections of each brain were counted, NeuN +, IBA1 +, GFAP + and P129+ cells were normalized to the area of the ROI (um2) or to the total number of DAPI+ nuclei within the ROI. Representative 20x confocal montage images were generated for each treatment group using a Zeiss AxioImager Z2 microscope and a Zeiss 20x/0.8NA Plan-Apochromat lens.   Figure S2. EM characterization of murine α-synuclein preformed fibrils. A-B. Wild type full length murine α-Syn was cloned, expressed in E. coli, and purified as previously described (Weihofen et al. 2019) prior to aggregation into preformed fibrils (PFFs) by continuous shaking at 1100 rpm in an Eppendorf Thermomixer at 37°C for 5 days (please refer to Materials and Methods for a complete description of the PFF preparation protocol). PFFs were analyzed by electron microscopy (EM) prior to (A) and immediately after (B) sonication using a Q Sonica water bath (1 seconds on, 1 seconds off for 1 minutes at Amplitude 50). Lower panels depict high magnification images of representative fibrils from A (left) and B (right). Scale bar = 200 nm. C. Diagram of experimental design for in vitro α-syn preformed fibril (PFF) model in primary mouse cortical neurons. Lentiviruses (LVs) were added on day in vitro (DIV) 5 at a multiplicity of infection (MOI) equal to 3 prior to treatment with 2 µg/ml murine α-syn monomer or sonicated PFFs on DIV 9. For GNF362 treated cells, neurons were pretreated with GNF362 or vehicle (DMSO) for 1 h prior to addition of PFFs with GNF362 co-treatment until DIV 20. D-E. Number (D) and total fluorescence intensity (E) of pS129 α-syn inclusions in cultures treated with α-syn monomer or PFFs. Note that α-syn monomer treatment did not induce pS129 α-syn pathology. ***p<0.0001 by 1-Way ANOVA with Tukey's post-hoc test; n=4-6 wells per group. Table S1. MetaBrain eQTL analysis for rs4653767-T/C allele. Meta eQTL analysis of rs4653767-C compared to rs4653767-T; Z-score = -0.2010, P value = 0.8405, FDR = 1.