Effect of Cell Genome Editing on the Outcome of Neurotransplantation in Experimental Parkinsonism

The aim of the study was to evaluate the effect of genome editing on the outcome of experimental transplantation of neuronal precursors obtained from a patient with the hereditary form of Parkinson’s disease.

Materials and Methods. Parkinsonian syndrome was modeled in Wistar rats (n=24) by unilateral administration (into the substantia nigra) of the neurotoxin 6-hydroxydopamine, that selectively destroy dopaminergic neurons. The neural progenitors were differentiated from induced pluripotent stem cells (iPSCs) derived from a patient with the PARK8 form of Parkinson’s disease carrying the G2019S mutation in the LRRK2 gene. In two series of experiments, the cells containing the mutated gene and the isogenic “normalized” cells (that underwent genome editing using the artificial endonuclease system CRISP/Cas9) were used for neurotransplantation. The neuronal precursors were transplanted via a unilateral injection into the brain striatum of rats. We then monitored changes in the behavior of experimental animals using the open field test and the passive avoidance response (PAR) test. The data was processed using the Statistica 7.0 software with the single-factor analysis of variance (ANOVA).

Results. The administration of neuronal precursors into the rat brain striatum led to a gradual restoration of motor activity in the experimental animals of both groups, i.e., those transplanted with either mutant or “normalized” cells. In the PAR test, the rats transplanted with mutant cells failed to reproduce the conditioned responses, whereas the rats transplanted with “normalized” cells were able to reproduce the avoidance responses in the way similar to that in intact rats. The latent period before entering the dark box differed between the two groups of animals with a high degree of statistical significance.

Conclusion. The study confirms the possibility of correcting motor and cognitive impairments in experimental parkinsonian rats by replenishing the pool of dopaminergic neurons with neuronal precursors produced from iPSCs derived from somatic cells (fibroblasts). Using genome editing to correct the causal mutation in iPSCs before transplantation significantly improves the treatment results. This study, therefore, provides the rationale for further development of this promising technique and its eventual use in patients with genetically determined forms of Parkinson’s disease.

1. Illarioshkin S.N. Modern view on etiology of Parkinson’s disease. Nevrologicheskiy zhurnal 2015; 20(4): 4–13.
2. Ekstrapiramidnye rasstroystva [Extrapyramidal disorders]. Pod red. Shtoka V.N., Ivanovoy-Smolenskoy I.A., Levina O.S. [Shtok V.N., Ivanova-Smolenskaya I.A., Levin O.S. (editors)]. Moscow: MEDpress-inform; 2002.
3. Wirdefeldt K., Adami H.-O., Cole P., Trichopoulos D., Mandel J. Epidemiology and etiology of Parkinson’s disease: a review of the evidence. Eur J Epidemiol 2011; 26(S1): 1–58, https://doi.org/10.1007/s10654-011-9581-6.
4. Airavaara M., Voutilainen M.H., Wang Y., Hoffer B. Neurorestoration. Parkinsonism Relat Disord 2012; 18: S143–S146, https://doi.org/10.1016/s1353-8020(11)70045-1.
5. Korecka J.A., Verhaagen J., Hol E.M. Cell-replacement and gene-therapy strategies for Parkinson’s and Alzheimer’s disease. Regen Med 2007; 2(4): 425–446, https://doi.org/10.2217/17460751.2.4.425.
6. Langston J.W. The promise of stem cells in Parkinson disease. J Clin Invest 2005; 115(1): 23–25, https://doi.org/10.1172/jci24012.
7. Lindvall O. Developing dopaminergic cell therapy for Parkinson’s disease — give up or move forward? Mov Disord 2013; 28(3): 268–273, https://doi.org/10.1002/mds.25378.
8. Lindvall O., Kokaia Z., Martinez-Serrano A. Stem cell therapy for human neurodegenerative disorders — how to make it work. Nat Med 2004; 10(7): S42–S50, https://doi.org/10.1038/nm1064.
9. Lebedeva О.S., Lagarkova М.А., Illarioshkin S.N., Khaspekov L.G., Grivennikov I.А. Induced pluripotent stem cells: new possibilities in neurobiology and neurotransplantaion. Annaly klinicheskoy i eksperimental’noy nevrologii 2011; 5(4): 37–45.
10. Gao A., Peng Y., Deng Y., Qing H. Potential therapeutic applications of differentiated induced pluripotent stem cells (iPSCs) in the treatment of neurodegenerative diseases. Neuroscience 2013; 228: 47–59, https://doi.org/10.1016/j.neuroscience.2012.09.076.
11. Hwang D.Y., Kim D.S., Kim D.W. Human ES and iPS cells as cell sources for the treatment of Parkinson’s disease: current state and problems. J Cell Biochem 2010; 109(2): 292–301, https://doi.org/10.1002/jcb.22411.
12. Nishimura K., Takahashi J. Therapeutic application of stem cell technology toward the treatment of Parkinson’s disease. Biol Pharm Bull 2013; 36(2): 171–175, https://doi.org/10.1248/bpb.b12-00929.
13. Takahashi K., Tanabe K., Ohnuki M., Narita M., Ichisaka T., Tomoda K., Yamanaka S. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 2007; 131(5): 861–872, https://doi.org/10.1016/j.cell.2007.11.019.
14. Lebedeva O.S., Lagar’kova M.A., Kiselev S.L., Mukhina I.V., Vedunova M.V., Usova O.V., Stavrovskaya A.V., Yamshchikova N.G., Fedotova E.Y., Grivennikov I.A., Khaspekov L.G., Illarioshkin S.N. The morphofunctional properties of induced pluripotent stem cells derived from human skin fibroblasts and differentiated to dopaminergic neurons. Neurochemical Journal 2013; 7(3): 207–214, https://doi.org/10.1134/s1819712413030082.
15. Stavrovskaya A.V., Voronkov D.N., Yamshchikova N.G., Ol’shanskiy A.S., Khudoerkov R.M., Khaspekov L.G., Illarioshkin S.N. Morphochemical evaluation of neurotransplantation outcomes in experimental Parkinsonism. Annaly klinicheskoy i eksperimental’noy nevrologii 2015; 9(2): 28–32.
16. Paxinos G., Watson C. The rat brain in stereotaxic coordinates. Academic Press; 1998.
17. Vetchinova A.S., Novosadova E.V., Abramycheva N.Yu., Grivennikov I.A., Illarioshkin S.N. PARK8 form of Parkinson’s disease: induced pluripotent stem cells-based model and genome editing of LRRK2 mutation. Asimmetriya 2016; 10(4): 90–96.
18. Miroshnichenko E.V., Stavrovskaya A.V., Shugalev N.P., Lenard L., Hartmann G. Emotional state variations in rats during recall of passive avoidance reactions after neurotensin administration into nucleus accumbens of brain. Zhurnal vysshey nervnoy deyatel’nosti im. I.P. Pavlova 2010; 60(6): 704–711.
19. Shugalev N.P., Stavrovskaya A.V., Yamshikova N.G., Olshansky A.S., Kashirina E.A. Neurotensin influence on painful stress afteractions in rats with neurotoxic damages of serotoninergic structures of brain substantia nigra. Zhurnal vysshey nervnoy deyatel’nosti im. I.P. Pavlova 2013; 63(3): 384–394, https://doi.org/10.7868/s004446771303012x.
20. Shugalev N.P., Stavrovskaya A.V., Yamshikova N.G., Olshansky A.S., Miroshnichenko E.V. Reproduction of passive avoidance reactions after neurotensin microinjection into nucleus accumbens of rat brain against the background of reserpine action. Zhurnal vysshey nervnoy deyatel’nosti im. I.P. Pavlova 2012; 62(3): 357–363.
21. Stein M.B., Heuser I.J., Juncos J.L., Uhde T.W. Anxiety disorders in patients with Parkinson’s disease. Am J Psychiatry 1990; 147(2): 217–220, https://doi.org/10.1176/ajp.147.2.217.
22. Uc E.Y., Tippin J., Chou K.L., Erickson B.A., Doerschug K.C., Jimmeh Fletcher D.M. Non-motor symptoms in Parkinson’s disease. European Neurological Review 2012; 7(1): 35–40, https://doi.org/10.17925/enr.2012.07.01.35.
23. Vendrova M.I., Golubev V.L., Sadekov R.A., Vein A.M. Motor, cognitive and affective disorders in Parkinson’s disease. Zhurnal nevrologii i psikhiatrii imeni S.S. Korsakova 2002; 102(3): 13–17. Chaudhuri K.R., Healy D.G., Schapira A.H. Non-motor symptoms of Parkinson’s disease: diagnosis and management. Lancet Neurol 2006; 5(3): 235–245, https://doi.org/10.1016/s1474-4422(06)70373-8.
24. Chaudhuri K.R., Healy D.G., Schapira A.H. Non-motor symptoms of Parkinson’s disease: diagnosis and management. Lancet Neurol 2006; 5(3): 235–245, https://doi.org/10.1016/s1474-4422(06)70373-8 .
25. Yakhno N.N. Cognitive impairment in neurological clinical practice. Nevrologicheskiy zhurnal 2006; 11(S1): 4–12.
26. Alves C.S., Andreatini R., da Cunha C., Tufik S., Vital M.A.B. Phosphatidylserine reverses reserpine-induced amnesia. Eur J Pharmacol 2000; 404(1–2): 161–167, https://doi.org/10.1016/s0014-2999(00)00607-5.
27. Dubrovina N.I., Popova E.V., Ilyuchenok R.Yu. A сompensating-reactivating effect of quinpirole on the extinction of conditional habit and amnesia in mice with alternative behavioral stereotypes. Eksperimental’naya i klinicheskaya farmakologiya 2001; 64(3): 13–16.
28. Molodtsova G.F. Metabolism and receptor binding of serotonin in brain structures during performance of a conditioned passive avoidance response. Neurosci Behav Physiol 2005; 35(7): 685–692, https://doi.org/10.1007/s11055-005-0111-4.
29. Goldman-Rakic P.S. The cortical dopamine system: role in memory and cognition. Adv Pharmacol 1997; 42: 707–711, https://doi.org/10.1016/s1054-3589(08)60846-7.
30. Cabib S., Puglisi-Allegra S. Opposite responses of mesolimbic dopamine system to controllable and uncontrollable aversive experiences. J Neurosci 1994; 14(5 Pt 2): 3333–3340.

Stavrovskaya A.V., Novosadova E.V., Olshansky A.S., Yamshchikova N.G., Gushchina A.S., Arsenyeva E.L., Grivennikov I.A., Illarioshkin S.N. Effect of Cell Genome Editing on the Outcome of Neurotransplantation in Experimental Parkinsonism. Sovremennye tehnologii v medicine 2017; 9(4): 7, https://doi.org/10.17691/stm2017.9.4.01