Int J Stem Cells.  2019 Mar;12(1):114-124. 10.15283/ijsc18110.

CRISPR/Cas9 Edited sRAGE-MSCs Protect Neuronal Death in Parkinson's Disease Model

• Lee J
• Bayarsaikhan D
• Arivazhagan R
• Park H
• Lim B
• Gwak P
• Jeong GB
• Lee J
• Byun K
• Lee B

• KMID: 2447230
• DOI: http://doi.org/10.15283/ijsc18110

Abstract

BACKGROUND AND OBJECTIVES
Parkinson's disease (PD) is a fatal and progressive degenerative disease of the nervous system. Until recently, its promising treatment and underlying mechanisms for neuronal death are poorly understood. This study was investigated to identify the molecular mechanism of neuronal death in the substantia nigra and corpus striatum of PD.
METHODS
The soluble RAGE (sRAGE) secreting Umbilical Cord Blood"”derived Mesenchymal Stem Cell (UCB-MSC) was generated by gene editing method using clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9 (CRISPR/Cas9). These cells were transplanted into Corpus Striatum of rotenone-induced PD animal models then behavioral test, morphological analysis, and immunohistochemical experiments were performed to determine the neuronal cell death and recovery of movement.
RESULTS
The neuronal cell death in Corpus Striatum and Substantia Nigra was dramatically reduced and the movement was improved after sRAGE secreting UCB-MSC treatment in PD mice by inhibition of RAGE in neuronal cells.
CONCLUSIONS
We suggest that sRAGE secreting UCB-MSC based therapeutic approach could be a potential treatment strategy for neurodegenerative disease including PD.

Keyword

Parkinson's disease; CRISPR/Cas9; sRAGE secreting UCB-MSC; Microglia; AGE-albumin

MeSH Terms

Animals
Behavior Rating Scale
Cell Death
Corpus Striatum
Mesenchymal Stromal Cells
Methods
Mice
Microglia
Models, Animal
Nervous System
Neurodegenerative Diseases
Neurons*
Parkinson Disease*
Rage
Substantia Nigra
Umbilical Cord

Figure

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  Fig. 1 Distribution of co-localized AGE-Albumin in activated microglial cells in the striatum of mouse PD model. (A) Triple immunostaining of AGE (blue), Albumin (green), and Iba1 (red, activated microglial cell marker) in the striatum of control and mouse PD model (n=3). Merged image shows that AGE, ALB, and Iba1 were co-localized mostly in striatum area of mouse PD brain. The fluorescence expression level (B) and co-localization coefficient analyzed by densitometric analysis software using Zen software. Scale bar=50 μm. ***p<0.001.

• 

  Fig. 2 Generation and characterization of sRAGE secreting UCB-MSC. (A) The illustration picture represents the gene information of pZDonor-AAVS1 puromycin vector. Each arrow describes certain gene. (B) The illustration of the sRAGE insertion coding sequence. (C) Genome integration was confirmed by Junction PCR with genomic DNAs of UCB-MSCs which were transfected with mock, GFP and sRAGE containing pZDonor-AAVS1 plasmids. (D) Immunoblot analysis of supernatant and extract from UCB-MSC cells transfected with mock (lane 1) and FLAG-tagged sRAGE in pZDonor-AAVS1 vector (lane 2). β-actin loaded as a positive control. The secretion of human sRAGE levels (E) was confirmed with ELISA. ***p<0.001.

• 

  Fig. 3 Protective effect of sRAGE secreting UCB-MSC on AGE-albumin induced neuronal cell death by decreasing RAGE level. (A) RAGE expression is shown in double-labeled confocal images RAGE (red) and DAPI (blue) using neuronal (SHSY-5Y) cell before and after exposing AGE-albumin or co-treated with AGE-albumin and sRAGE secreting UCB-MSC conditioned medium. Neuronal death was evaluated by double staining TUNEL (red) and DAPI (blue). Scale bar=50 μm. (B) Cell activity and viability were determined using the MTS assay. (C) Immunoblot analysis of neuronal cell lysates after AGE-albumin or AGE-albumin with sRAGE secreting UCB-MSC conditioned medium co-treatment. (D~G) Densitometry analyses of MAPK proteins were evaluated using the Image-J software. Each experiment was performed in triplicated and repeated three times. *p<0.05.

• 

  Fig. 4 Protection against neuronal death by sRAGE secreting UCB-MSC in a PD model. (A) Cresyl violet staining of control, PD, and sRAGE secreting UCB-MSC treated PD mouse brains showing the population changes of neuronal cells in control (n=3), rotenone-treated (n=3), rotenone with sRAGE secreting UCB-MSC treated groups(n=3). (B) The neuronal population was reduced in rotenone-treated animals, however, the population increased in rotenone with sRAGE secreting UCB-MSC treated group. Scale bar=200 μm. (C) Double immunostaining of RAGE (red) or NEUN (green) in control, rotenone-treated, rotenone with sRAGE secreting UCB-MSC treated groups. Double labeling increased in rotenone-treated animals, however, the labeling decreased in rotenone with sRAGE secreting UCB-MSC treated group. (D) Neuronal death was evaluated by triple staining NEUN (green), TUNEL (red) and DAPI (blue). The TUNEL signal increased in rotenone treated animals but it decreased in rotenone with sRAGE secreting UCB-MSC treated group. Scale bar=50 μm. *p<0.05.

• 

  Fig. 5 A proposed model of AGE-albumin mediated neuronal cell death and its protection in PD. The schematic diagram illustrates the synthesis in microglial cells and extracellular secretion of AGE-albumin, which induces neuronal cell death and ultimately contributes to neurodegeneration. AGE-albumin synthesis and secretion in microglial cells are increased in PD models. sRAGE secretion from sRAGE UCB-MSC can protect AGE-albumin induced neuronal death in PD. Taken together, AGE-albumin promotes the death of primary neuronal cells and sRAGEMSC treatment can protect the pathobiology of PD.

Reference

References

1. Walker Z, Possin KL, Boeve BF, Aarsland D. Lewy body dementias. Lancet. 2015; 386:1683–1697. DOI: 10.1016/S0140-6736(15)00462-6. PMID: 26595642. PMCID: 5792067.

2. Castellani R, Smith MA, Richey PL, Perry G. Glycoxidation and oxidative stress in Parkinson disease and diffuse Lewy body disease. Brain Res. 1996; 737:195–200. DOI: 10.1016/0006-8993(96)00729-9. PMID: 8930366.

3. Münch G, Lüth HJ, Wong A, Arendt T, Hirsch E, Ravid R, Riederer P. Crosslinking of alpha-synuclein by advanced glycation endproducts--an early pathophysiological step in Lewy body formation? J Chem Neuroanat. 2000; 20:253–257. DOI: 10.1016/S0891-0618(00)00096-X.

4. Cepeda IL, Flores J, Cornfeldt ML, O’Kusky JR, Doudet DJ. Human retinal pigment epithelial cell implants ameliorate motor deficits in two rat models of Parkinson disease. J Neuropathol Exp Neurol. 2007; 66:576–584. DOI: 10.1097/nen.0b013e318093e521. PMID: 17620983.

5. Przedborski S. Pathogenesis of nigral cell death in Parkinson’s disease. Parkinsonism Relat Disord. 2005; 11(Suppl 1):S3–7. DOI: 10.1016/j.parkreldis.2004.10.012. PMID: 15885625.

6. Chen Z. Cell Therapy for Parkinson’s disease: new hope from reprogramming technologies. Aging Dis. 2015; 6:499–503. DOI: 10.14336/AD.2014.1201. PMID: 26618051. PMCID: 4657821.

7. Fu MH, Li CL, Lin HL, Chen PC, Calkins MJ, Chang YF, Cheng PH, Yang SH. Stem cell transplantation therapy in Parkinson’s disease. Springerplus. 2015; 4:597. DOI: 10.1186/s40064-015-1400-1. PMID: 26543732. PMCID: 4628010.

8. Lindvall O. Treatment of Parkinson’s disease using cell transplantation. Philos Trans R Soc Lond B Biol Sci. 2015; 370:20140370. DOI: 10.1098/rstb.2014.0370. PMID: 26416681. PMCID: 4633999.

9. Przedborski S. Inflammation and Parkinson’s disease pathogenesis. Mov Disord. 2010; 25(Suppl 1):S55–57. DOI: 10.1002/mds.22638. PMID: 20187228.

10. Damien P, Allan DS. Regenerative therapy and immune modulation using umbilical cord blood-derived cells. Biol Blood Marrow Transplant. 2015; 21:1545–1554. DOI: 10.1016/j.bbmt.2015.05.022. PMID: 26079441.

11. Bayarsaikhan E, Bayarsaikhan D, Lee J, Son M, Oh S, Moon J, Park HJ2, Roshini A2, Kim SU4, Song BJ5, Jo SM6, Byun K3, Lee B3. Microglial AGE-albumin is critical for neuronal death in Parkinson’s disease: a possible implication for theranostics. Int J Nanomedicine. 2016; 10(Spec Iss):281–292. DOI: 10.2147/IJN.S95077. PMID: 27601894. PMCID: 5003553.

12. Kalea AZ, Schmidt AM, Hudson BI. Alternative splicing of RAGE: roles in biology and disease. Front Biosci (Landmark Ed). 2011; 16:2756–2770. DOI: 10.2741/3884.

13. Son M, Kang WC, Oh S, Bayarsaikhan D, Ahn H, Lee J, Park H, Lee S, Choi J, Lee HS, Yang PC, Byun K, Lee B. Advanced glycation end-product (AGE)-albumin from activated macrophage is critical in human mesenchymal stem cells survival and post-ischemic reperfusion injury. Sci Rep. 2017; 7:11593. DOI: 10.1038/s41598-017-11773-1. PMID: 28912521. PMCID: 5599509.

14. Son M, Oh S, Park H, Ahn H, Choi J, Kim H, Lee HS, Lee S, Park HJ, Kim SU, Lee B, Byun K. Protection against RAGE-mediated neuronal cell death by sRAGE-secreting human mesenchymal stem cells in 5xFAD transgenic mouse model. Brain Behav Immun. 2017; 66:347–358. DOI: 10.1016/j.bbi.2017.07.158. PMID: 28760504.

15. Byun K, Yoo Y, Son M, Lee J, Jeong GB, Park YM, Salekdeh GH, Lee B. Advanced glycation end-products produced systemically and by macrophages: a common contributor to inflammation and degenerative diseases. Pharmacol Ther. 2017; 177:44–55. DOI: 10.1016/j.pharmthera.2017.02.030. PMID: 28223234.

16. Schmidt AM, Yan SD, Yan SF, Stern DM. The multiligand receptor RAGE as a progression factor amplifying immune and inflammatory responses. J Clin Invest. 2001; 108:949–955. DOI: 10.1172/JCI200114002. PMID: 11581294. PMCID: 200958.

17. Bucciarelli LG, Wendt T, Rong L, Lalla E, Hofmann MA, Goova MT, Taguchi A, Yan SF, Yan SD, Stern DM, Schmidt AM. RAGE is a multiligand receptor of the immunoglobulin superfamily: implications for homeostasis and chronic disease. Cell Mol Life Sci. 2002; 59:1117–1128. DOI: 10.1007/s00018-002-8491-x. PMID: 12222959.

18. Byun K, Bayarsaikhan E, Kim D, Kim CY, Mook-Jung I, Paek SH, Kim SU, Yamamoto T, Won MH, Song BJ, Park YM, Lee B. Induction of neuronal death by microglial AGE-albumin: implications for Alzheimer’s disease. PLoS One. 2012; 7:e37917. DOI: 10.1371/journal.pone.0037917. PMID: 22662249. PMCID: 3360664.

19. Byun K, Bayarsaikhan E, Kim D, Son M, Hong J, Jeong GB, Paek SH, Won MH, Lee B. Activated microglial cells synthesize and secrete AGE-albumin. Anat Cell Biol. 2012; 45:47–52. DOI: 10.5115/acb.2012.45.1.47. PMID: 22536551. PMCID: 3328740.

20. Wang H, La Russa M, Qi LS. CRISPR/Cas9 in genome editing and beyond. Annu Rev Biochem. 2016; 85:227–264. DOI: 10.1146/annurev-biochem-060815-014607. PMID: 27145843.

21. Chang CY, Ting HC, Su HL, Jeng JR. Combining induced pluripotent stem cells and genome editing technologies for clinical applications. Cell Transplant. 2018; 27:379–392. DOI: 10.1177/0963689718754560. PMID: 29806481. PMCID: 6038034.

22. Komor AC, Badran AH, Liu DR. CRISPR-based technologies for the manipulation of eukaryotic genomes. Cell. 2017; 168:20–36. DOI: 10.1016/j.cell.2016.10.044. PMCID: 5235943.

23. Hendriks WT, Warren CR, Cowan CA. Genome editing in human pluripotent stem cells: approaches, pitfalls, and solutions. Cell Stem Cell. 2016; 18:53–65. DOI: 10.1016/j.stem.2015.12.002. PMID: 26748756. PMCID: 4709030.

24. Ceasar SA, Rajan V, Prykhozhij SV, Berman JN, Ignacimuthu S. Insert, remove or replace: a highly advanced genome editing system using CRISPR/Cas9. Biochim Biophys Acta. 2016; 1863:2333–2344. DOI: 10.1016/j.bbamcr.2016.06.009. PMID: 27350235.

25. Gaj T, Gersbach CA, Barbas CF 3rd. ZFN, TALEN, and CRISPR/Cas-based methods for genome engineering. Trends Biotechnol. 2013; 31:397–405. DOI: 10.1016/j.tibtech.2013.04.004. PMID: 23664777. PMCID: 3694601.

26. Tycko J, Myer VE, Hsu PD. Methods for optimizing CRISPR-Cas9 genome editing specificity. Mol Cell. 2016; 63:355–370. DOI: 10.1016/j.molcel.2016.07.004. PMID: 27494557. PMCID: 4976696.

27. Chandrasegaran S. Recent advances in the use of ZFNmediated gene editing for human gene therapy. Cell Gene Ther Insights. 2017; 3:33–41. DOI: 10.18609/cgti.2017.005. PMID: 29270315. PMCID: 5736148.

28. Renaud JB, Boix C, Charpentier M, De Cian A, Cochennec J, Duvernois-Berthet E, Perrouault L, Tesson L, Edouard J, Thinard R, Cherifi Y, Menoret S, Fontanière S, de Crozé N, Fraichard A, Sohm F, Anegon I, Concordet JP, Giovannangeli C. Improved genome editing efficiency and flexibility using modified oligonucleotides with TALEN and CRISPR-Cas9 nucleases. Cell Rep. 2016; 14:2263–2272. DOI: 10.1016/j.celrep.2016.02.018. PMID: 26923600.

29. Luo Y, Rao M, Zou J. Generation of GFP reporter human induced pluripotent stem cells using AAVS1 safe harbor transcription activator-like effector nuclease. Curr Protoc Stem Cell Biol. 2014; 29:5.A.7.1–18. DOI: 10.1002/9780470151808.sc05a07s29.

30. Münch G, Westcott B, Menini T, Gugliucci A. Advanced glycation endproducts and their pathogenic roles in neurological disorders. Amino Acids. 2012; 42:1221–1236. DOI: 10.1007/s00726-010-0777-y. PMID: 20949363.

31. Perry VH, Nicoll JA, Holmes C. Microglia in neurodegenerative disease. Nat Rev Neurol. 2010; 6:193–201. DOI: 10.1038/nrneurol.2010.17. PMID: 20234358.

32. Vasan S, Foiles PG, Founds HW. Therapeutic potential of AGE inhibitors and breakers of AGE protein cross-links. Expert Opin Investig Drugs. 2001; 10:1977–1987. DOI: 10.1517/13543784.10.11.1977. PMID: 11772301.

33. Walker DG, Lue LF. Investigations with cultured human microglia on pathogenic mechanisms of Alzheimer’s disease and other neurodegenerative diseases. J Neurosci Res. 2005; 81:412–425. DOI: 10.1002/jnr.20484. PMID: 15957156.