Abstract
Impaired neuronal proteostasis is a salient feature of many neurodegenerative diseases, highlighting alterations in the function of the endoplasmic reticulum (ER). We previously reported that targeting the transcription factor XBP1, a key mediator of the ER stress response, delays disease progression and reduces protein aggregation in various models of neurodegeneration. To identify disease modifier genes that may explain the neuroprotective effects of XBP1 deficiency, we performed gene expression profiling of brain cortex and striatum of these animals and uncovered insulin-like growth factor 2 (Igf2) as the major upregulated gene. Here, we studied the impact of IGF2 signaling on protein aggregation in models of Huntington’s disease (HD) as proof of concept. Cell culture studies revealed that IGF2 treatment decreases the load of intracellular aggregates of mutant huntingtin and a polyglutamine peptide. These results were validated using induced pluripotent stem cells (iPSC)-derived medium spiny neurons from HD patients and spinocerebellar ataxia cases. The reduction in the levels of mutant huntingtin was associated with a decrease in the half-life of the intracellular protein. The decrease in the levels of abnormal protein aggregation triggered by IGF2 was independent of the activity of autophagy and the proteasome pathways, the two main routes for mutant huntingtin clearance. Conversely, IGF2 signaling enhanced the secretion of soluble mutant huntingtin species through exosomes and microvesicles involving changes in actin dynamics. Administration of IGF2 into the brain of HD mice using gene therapy led to a significant decrease in the levels of mutant huntingtin in three different animal models. Moreover, analysis of human postmortem brain tissue and blood samples from HD patients showed a reduction in IGF2 level. This study identifies IGF2 as a relevant factor deregulated in HD, operating as a disease modifier that buffers the accumulation of abnormal protein species.
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References
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Acknowledgements
We are grateful to Dr. Oliver Bracko for providing IGF2 overexpression constructs. We thank the Harvard Brain Tissue Resource Center for providing HD post-mortem samples. We also thank Drs. Dimitri Krainc and Katarina Trajkovic for feedback on protocols to detect mHtt secretion. We also thank Dr. Cristina Alberini for feedback and providing tools to study IGF2 signaling. We thank Marioly Müller and Dr. Josefina Barrera and her team for their kind help taking the blood samples. We thank Dr. Felipe Oyarzun and Rodrigo Sierpe for the kindly help with the use of the Nanosight NS300. We specially acknowledge Carolina Jerez, Claudia Rivera, Valentina Castillo and Constanza Gonzalez for their technical assistance.
Funding
This work was directly funded by ANID/FONDAP program 15150012 (C.H. and R.L.V.), Millennium Institute P09-015-F (C.H. and R.L.V.), CONICYT-Brazil 441921/2016–7, FONDEF ID16I10223, FONDEF D11E1007 and FONDECYT 1180186 (C.H.). We also thank FONDECYT 3150097 (P.G-H.), FONDECYT 1191003 (R.L.V.), FONDECYT 1150069 (H.G.R.), CONICYT Ph.D. fellowship 21160843 (P.T-E.), CSC and the Postgraduate Student Research and Innovation Project of Jiangsu Province KYLX15_0558 (D.W.), the Hersenstichting and NWO-ALW (S.B.), NIH R01 NS100529 (L.M.E.) and NIH NS092829 (R.L.W.). ISF Legacy Heritage Fund (2394/17) (G.Z.L.)
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PG-H, PT-E, CH and RLV designed the study; PG-H and PT-E designed, performed and analyzed most of the in vitro and in vivo experiments; DW and SB designed, performed and analyzed the pulse-chase experiments; CS and FC analyzed the NTA experiments and assisted with the extracellular vesicle experiments; PC-C provided the information and blood samples from Chilean HD patients; DRH and CG-B designed and performed the experiments of actin remodeling; PT, KAL and BJG performed the microarray analysis using the brains samples from the XBP1cko mice; LP and RLW performed the quantitative proteomics in Neuro2a cells; CS and HGR performed the Ingenuity pathway analysis; SPS produced the AAV2-IGF2 and AAV2-Mock; GZL, NS and MS performed the experiments in HEK293 cells using the full-lenghtHttQ103-myc plasmid; SN and LME designed, performed and analyzed the experiments using MSNs derived from HD patients. AT designed, performed and analyzed the experiments using neurons derived from SCA3 patients.
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The authors declare no conflict of interest in this study. AAV-IGF2 for gene therapy and its use in misfolded protein diseases as Huntington’s disease (Chile no. 201603282 and PCT. provisional application no. PCT/CL2017/000040).
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MTA may be required for access to AAV-IGF2 and some vectors. All data are available upon request from C.H.
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Supplementary file1 (AVI 1209 kb) Supplementary video 1. Neuro2a treated with mock medium
Supplementary file2 (AVI 1561 kb) Supplementary video 2. Neuro2a treated with IGF2-enriched medium
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Supplementary file3 (TIF 1831 kb) Supplementary fig. 1 XBP1 deficiency does not alter Igf1 mRNA levels. a Igf2 mRNA levels were measured by real-time PCR in cDNA generated from striatal tissue of 9-month-old XBP1cKO-YAC128 mice. Data represent the average and SEM of the analysis of three animals per group. b Igf1 mRNA levels were measured by real-time PCR in cDNA generated from striatal tissue of 9-month-old XBP1cKO-YAC128 mice. Data represent the average and SEM of five animals. Statistically significant differences detected by one-tailed unpaired t-test (**: p < 0.01)
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Supplementary file4 (TIF 2666 kb) Supplementary fig. 2 IGF2 expression reduces polyQ79 levels. a PolyQ79-EGFP monomers from Fig. 2B were analyzed in whole cell extracts by western blot analysis using an anti-GFP antibody (left panel). PolyQ79-EGFP monomer levels after 24 and 48 h of expression were quantified and normalized to Hsp90 levels (middle and right panel, respectively) b Neuro2a cells were co-transfected with a polyQ79-EGFP expression vector and IGF2 plasmid or empty vector (Mock). 24 h later, cells were collected in Trizol and Gfp mRNA levels were measured by semi-quantitative PCR. Values represent the mean and SEM of at least three independent experiments. c Quantification of filter trap experiment from Fig. 2c (time point 48 h)
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Supplementary file5 (TIF 7168 kb) Supplementary fig. 3 IGF2 does not inhibit global protein synthesis or degradation. a HEK293T cells were co-transfected with GFP-mHttQ43 expression vector with IGF2 plasmid (right, upper panel) or empty vector (left, upper panel) and then pulse labeled with 35S for indicated time points. Autoradiography (AR) represents total proteins that were synthesized in the time of harvesting cells. All data were normalized to time point 0 h (lower panel). b HEK293T cells were co-transfected with GFP-mHttQ43 expression vector with IGF2 plasmid (right, upper panel) or empty vector (Mock) (left, upper panel) and then pulse labeled with 35S for indicated time points to follow the decay of the labeled protein while chasing with unlabeled precursor. Autoradiography (AR) represents total extracts in each time (upper panel). All data were normalized to time point 1 h (lower panel). c HEK293T cells were transfected with GFP-mHttQ43 and IGF2 expression vectors. Pulse was performed 24 h after transfection. Cells were treated with 30 μM chloroquine (CQ) or 1 µM bortezomib (Bort) at the beginning of the chasing for additional 21 h (upper panel). Ubiquitin, p62 and LC3 were measured as control of the autophagy inhibition
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Supplementary file6 (TIF 16029 kb) Supplementary fig. 4 Proteasome, autophagy or conventional secretion are not involved in the reduction in polyQ79 levels after IGF2 expression (control experiments). a Neuro2a cells were co-transfected with expression vectors for polyQ79-EGFP and IGF2 or empty vector (Mock). After 8 h, cells were treated with 1 μM MG132 for additional 16 h. PolyQ79-EGFP inclusions were visualized by fluorescence microscopy (upper panel) and polyQ79-EGFP aggregation levels measured by filter trap (lower panel). Image cropped from the same membrane and film. b Filter trap assay was performed to detect polyQ79-EGFP aggregates using the same cells extracts of Fig. 4e using an anti-GFP antibody (upper panel) and quantified (lower panel). Image cropped from the same membrane and film. c Neuro2a cells were transfected with IGF2 for 8 h and then treated with 1 μM lactacystin (Lact) or 1 μM MG132 for additional 16 h. Ubiquitin accumulation was measured by western blot to confirm the activity of the inhibitors. Hsp90 expression was measured as loading control. d Neuro2a cells where co-transfected with IGF2 plasmid or empty vector (Mock) in the presence or absence of polyQ79-EGFP expression vector for 8 h, and then treated with 30 μM chloroquine (CQ) for additional 16 h. p62 levels were monitored by western blot using anti-p62 antibody. Hsp90 expression was monitored as loading control (upper panel). p62 levels were quantified and normalized to Hsp90 (lower panel). e Neuro2a cells were co-transfected with polyQ79-EGFP and IGF2 expression vectors or empty vector (Mock) for 8 h and then treated with 30 μM CQ for additional 16 h. p62 and LC3 levels were measured by western blot as control for autophagy inhibitors in experiment shown in Fig. 4h. Hsp90 levels were determined as loading control. f HEK293T cells were co-transfected with polyQ79-EGFP and IGF2 expression vectors or empty vector (Mock). After 8 h, cells were treated with 30 μM chloroquine (CQ) for additional 16 h. PolyQ79-EGFP aggregation was analyzed in whole cells extracts by western blot using anti-GFP antibody. Hsp90 expression was monitored as loading control (left panel). PolyQ79-EGFP HMW species levels were quantified and normalized to Hsp90 levels (right panel). g Filter trap assay was performed using the same cells extracts analyzed in (f), and polyQ79-EGFP HMW species were detected using anti-GFP antibody (upper panel) and quantified (lower panel). h HEK293T cells were transiently transfected with IGF2 expressing vector (IGF2) or empty plasmid (Mock) for 8 h and then treated with 30 μM CQ for additional 16 h. p62 and LC3 levels were measured by western blot as control for autophagy inhibitors in experiment shown in Fig. 4f and Fig. 4g. Hsp90 levels were determined as loading control. i, j Neuro2a cells were co-transfected with polyQ79-EGFP and IGF2 or empty vector (Mock) for 8 h and then treated with 200 nM bafilomycin A1 (Baf) or 250 mM 3-methyladenine (3-MA) for additional 16 h. PolyQ79-EGFP HMW species were analyzed in cell lysates using western blot with an anti-GFP antibody. Hsp90 or GAPDH expressions were monitored as loading control. In all quantifications, average and SEM of at least three independent experiments are shown. Statistically significant differences detected by two-tailed unpaired t-test (**: p < 0.01; *: p < 0.05). k HEK293 cells were co-transfected with FL-Htt103Q and IGF2 plasmid or empty vector (Mock). 24 h later, culture media was collected and loaded for dot blot detection of FL-Htt103Q using anti-Htt antibody. l Neuro2a cells were co-transfected with a polyQ79-EGFP expression vector and IGF2 plasmid or empty vector (Mock). 48 h later, cell death was measured by FACS after propidium iodide staining. m NanoSight profile of exosomes purified form IGF2 expressing cells. Isolated exosomes from Neuro2a cells co-transfected with polyQ79-EGFP and IGF2 plasmid or empty vector (Mock) were analyzed by NanoSight nanotracking analysis and plotted by size to confirm proper purification method. n Western blot analysis of enriched fractions of microvesicles (MV) and exosomes using the markers Alix and CD63
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Supplementary file7 (TIF 3537 kb) Supplementary fig. 5 Gene therapy to deliver IGF2 into the brain. a Neuro2a cells were co-transfected with expression vectors for mHtt588Q95-RFP and IGF2 plasmid or empty vector (Mock) using an AAV back bound. After 24 h, mHttQ95-RFP inclusions were visualized by fluorescence microscopy (left panel). Protein aggregation was analyzed in whole cells extracts by western blot (right panel). Hsp90 was measured as loading control. b Igf2-HA expression was confirmed by conventional PCR in cDNA obtained from dissected striatum of animals co-injected with AAV mHtt588Q95-RFP and AAV-IGF2 or AAV-Mock showed in Fig. 8a. c Igf2-HA expression was confirmed by conventional PCR in cDNA obtained from dissected striatum of YAC128 animals injected with AAV-IGF2 or AAV-Mock intra-cerebroventricular at neonatal stage P1-P2 shown in Fig. 8c. d Disease progression was monitored once every two weeks, using the rotarod test. YAC128 and littermate control mice were injected with AAVs as indicated in Fig. 8c. Motor performance was monitored once every two weeks from 2-month to 8-month old. The analysis shows the average of the group at each time point
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García-Huerta, P., Troncoso-Escudero, P., Wu, D. et al. Insulin-like growth factor 2 (IGF2) protects against Huntington’s disease through the extracellular disposal of protein aggregates. Acta Neuropathol 140, 737–764 (2020). https://doi.org/10.1007/s00401-020-02183-1
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DOI: https://doi.org/10.1007/s00401-020-02183-1