Document Type : Original Research


1 Faculty of Life Sciences, Shahid-Beheshti University, Tehran, Iran

2 Institute for Cognitive and Brain Science, Shahid Beheshti University, Tehran, Iran

3 Department o Physiology, School of Medicine, Zanjan University of Medical Sciences, Iran

4 Cancer Gene Therapy Research Center, Zanjan University of Medical Sciences, Zanjan, Iran

5 Department of Medical Nanotechnology, School of Medicine, Zanjan University of Medical Sciences, Zanjan, Iran


Background & Objective: Parkinson's disease (PD) is a progressive neurodegenerative disorder in which the cause is attributed to the alpha-synuclein (α-Syn) accumulation due to the decreased rate of autophagy. Due to the many advantages, mesenchymal stem cells (MSCs), such as the secretion of neurotrophic factors, have been proposed for PD cell therapy. The present study, in continuation of the previous study, aimed to investigate the therapeutic effect of human-derived Conjunctival MSCs (CJ-MSCs) on the clearance of α-Syn by the microRNA-149(miR-149)/Akt/mTOR/ pathway.
Methods: Stereotaxic 6-hydroxy dopamine (6-OHDA) was injected directly into the medial forebrain bundle (MFB) to induce Parkinson's disease. An apomorphine-induced rotation test was used to confirm the model establishment. CJ-MSCs were encapsulated in alginate microgel using a microfluidic system. The green fluorescent protein (GFP) labeled CJ-MSCs were encapsulated, and free cells were transplanted into the rats' right striatum. Behavioral and molecular analyses evaluated the potency of CJ-MSCs (encapsulated and free cells) in PD rats. Real-Time Quantitative Reverse Transcription PCR (qRT-PCR) was performed to investigate the expression of the miR-149-5p, Akt, mTOR, and α-Syn.
Results: Our obtained results indicated that transplantation of CJ-MSCs leads to a decrease in the number of rotations while raising the balance and motor abilities. The gene expression evaluation showed a significant reduction in Akt, mTOR, and α-Syn mRNA levels and a significant increase in the level of miR-149-5p compared to the control group.  
Conclusion: It seems that CJ-MSCs can promote the degradation of intracellular α-Syn by miR-149-5p/Akt/mTOR pathway and improve rats' motor functions.


Main Subjects

  1. Obeso JA, Stamelou M, Goetz CG, Poewe W, Lang AE, Weintraub D, et al. Past, present, and future of Parkinson's disease: a special essay on the 200th anniversary of the shaking palsy. Mov Disord. 2017;32(9):1264-310. [DOI:10.1002/mds.27115] [PMID] [PMCID]
  2. Armstrong MJ, Okun MS. Diagnosis and Treatment of Parkinson Disease A Review. JAMA. 2020;323(6):548-560. [DOI:10.1001/jama.2019.22360] [PMID]
  3. Bridi, JC, Hirth F. Mechanisms of α-synuclein induced synaptopathy in Parkinson's disease. Front. Neurosci. 2018;12:80. [PMID] [PMCID] [DOI:10.3389/fnins.2018.00080]
  4. Ganapathy K, Datta I, Sowmithra S, Joshi P. Influence of 6-Hydroxydopamin Toxicity on α-Synuclein Phosphorylation, Resting Vesicle Expression, and Vesicular Dopamine Release. Cell Biothec. 2016;9999:1-18. [DOI:10.1002/jcb.25570] [PMID]
  5. Wu KP, Weinstock DS, Narayanan C, Levy RM, Baum J. Structural reorganization of alpha-synuclein at low pH observed by NMR and REMD simulations. J Mol Biol. 2009;391(4): 784-96. [DOI:10.1016/j.jmb.2009.06.063] [PMID] [PMCID]
  6. Klein AD, Mazzulli JR. Is Parkinson's disease a lysosomal disorder? Brain. 2018;141(8);2255-62. [DOI:10.1093/brain/awy147] [PMID] [PMCID]
  7. Scrivo A, Bourdenx M, Pampliega O, Cuervo AM. Selective autophagy as a potential therapeutic target for neurodegenerative disorders. Lancet. Neurol.2018;17(9):802-15. [DOI:10.1016/S1474-4422(18)30238-2]
  8. Maiese K, Chong ZZ, Shang YC, Wang S. mTOR: on target for novel therapeutic strategies in the nervous system. Trends. Mol. Med. 2016; 19(1):51-60. [PMID] [PMCID] [DOI:10.1016/j.molmed.2012.11.001]
  9. Sardi SP, Cedarbaum JM, Brundin P. Targeted therapies for Parkinson's disease: from genetics to the clinic. Mov. Disord. 2018;33(5):684-96. [DOI:10.1002/mds.27414] [PMID] [PMCID]
  10. Ghosh A, Tyson T, George S, Hildebrandt EN, Steiner JA, Madaj Z, et al. Mitochondrial pyruvate carrier regulates autophagy, infammation, and neurodegeneration in experimental models of Parkinson's disease. Sci. Transl. Med. 2016;8(368):368-74. [DOI:10.1126/scitranslmed.aag2210] [PMCID]
  11. Jankovic J. Pathogenesis-targeted therapeutic strategies in Parkinson's disease. Mov Disord. 2019;34(1):41-4. [DOI:10.1002/mds.27534] [PMID]
  12. Marei HE, El‐Gamal A, Althani A, Afifi N, Abd‐Elmaksoud A, Farag A, et al. Cholinergic and dopaminergic neuronal differentiation of human adipose tissue derived mesenchymal stem cells. Cell. Physiol. 2018;233(2):936-45. [DOI:10.1002/jcp.25937] [PMID]
  13. Di-Benedetto A, Posa F, Carbone C, Cantore S, Brunetti G, Centonze M, et al. NURR1 downregulation favors osteoblastic differentiation of MSCs. Stem Cell Int. 2017; 2017:6975251. [DOI:10.1155/2017/7617048] [PMID] [PMCID]
  14. Schwerk A, Altschüler J, Roch M, Gossen M, Winter C, Berg J, et al. Human adipose-derived mesenchymal stromal cells increase endogenous neurogenesis in the rat subventricular zone acutely after 6-hydroxydopamine lesioning. Cytotherapy. 2015;17(2), 199-214. [DOI:10.1016/j.jcyt.2014.09.005] [PMID]
  15. Marote A, Teixeira FG, Medens-Pinheiro B, Salgado AJ. MSCs-Derived Exosomes: Cell-Secreted Nanovesicles withRegenerative Potential. Front Pharmacol. 2016;7:231. [PMCID]  [DOI:10.3389/fphar.2016.00231] [PMID]
  16. Ghasemloo E, Oryan S, Reza M, Hossein B, Mehdi M, Eskandari . The neuroprotective efect of MicroRNA-149-5p and coenzymeQ10 by reducing levels of infammatory cytokines and metalloproteinases following focal brain ischemia in rats. Brain Research Bulletin. 2021a; 169205-213. [DOI:10.1016/j.brainresbull.2021.01.013] [PMID]
  17. Ghasemloo E, Mostafavi H, Hosseini M, Forouzandeh M, Eskandari M, Mousavi SS . Neuroprotective efects of coenzyme Q10 in Parkinson's model via a novel Q10/miR‑149‑5p/MMPs pathway. Met Brain Dis. 2021b; 36:2089-2100. [DOI:10.1007/s11011-021-00795-4] [PMID]
  18. Zhang Y, Guo X, Xiong L, Yu L, Guo Q, Li Z, Li B, Lin N. Comprehensive analysis of microRNA-regulated protein interaction network reveals the tumor suppressive role of microRNA-149 in human hepatocellular carcinoma via targeting AKT-mTOR pathway. Molecular Cancer. 2014;13:253-268. [DOI:10.1186/1476-4598-13-253] [PMID] [PMCID]
  19. Wei ZG, Wu RL, Lavker RM, Sun TT. In vitro growth and differentiation of rabbit bulbar, fornix, and palpebral conjunctival epithelia. Implications on conjunctival epithelial transdifferentiation and stem cells. Invest Ophthalmol Vis Sci. 1993;34(5):1814-28.
  20. Harun MHN, Sepian SN, Chua KH, Ropilah AR, Abd-Ghafar N, Che-Hamzah J, et al. Human forniceal region is the stem cell-rich zone of the conjunctival epithelium. Hum Cell. 2013;26(1): 35-40. [DOI:10.1007/s13577-011-0025-0] [PMID]
  21. Nadri S, Soleimani M, Mobarra Z, Amini S. Expression of dopamine-associated genes on conjunctiva stromal-derived human mesenchymal stem cells. Biochem. Biophys. Res. Commun. 2008a;377(2):423-8. [DOI:10.1016/j.bbrc.2008.09.148] [PMID]
  22. Tan WH, Takeuchi S. Monodisperse alginate hydrogel microbeads for cell encapsulation. Adv. Mater. 2007;19(18):2696-2701. [DOI:10.1002/adma.200700433]
  23. Yao R, Zhang R, Lin F, Luan J. Injectable cell/hydrogel microspheres induce the formation of fat lobulelike microtissues and vascularized adipose tissue regeneration. Biofabrication. 2012;4(2):Article ID045003. [DOI:10.1088/1758-5082/4/4/045003] [PMID]
  24. Bozza A, Coates EE, Incitti T, Ferlin KM, Messina A, Menna E, et al. Neural differentiation of pluripotent cells in 3D alginate-based cultures. Biomaterials. 2014;35(16):4636-45. [DOI:10.1016/j.biomaterials.2014.02.039] [PMID]
  25. Forouzandeh M, Bigdeli MR, Mostafavi H, Nadri S, Eskandari M. Therapeutic potentials of human microfluidic encapsulated conjunctival mesenchymal stem cells on the rat model of Parkinson's disease. Exp Mol Pathol. 2021;123: 104723. [DOI:10.1016/j.yexmp.2021.104703] [PMID]
  26. Raza C, Anjum R, Shakeel N. Parkinson's disease: mechanisms, translational models and management strategies. Life Sci. 2019;226:77-90. [DOI:10.1016/j.lfs.2019.03.057] [PMID]
  27. Nadri S, Soleimani M, Kiani J, Atashi A, Izadpanah R. Multipotent mesenchymal stem cells from adult human eye con junctiva stromal cells. Differentiation. 2008b;76(3):223-231. [DOI:10.1111/j.1432-0436.2007.00216.x] [PMID]
  28. Mostafavi H, Ghasemifard L, Rostami A, Alipour A, Nadri S. Trabecular meshwork mesenchymal stem cell transplantation improve motor symptoms of parkinsonian rat model. Biologicals. 2019;61:61-7. [DOI:10.1016/j.biologicals.2019.06.006] [PMID]
  29. Venkatesh K, Sen D. Mesenchymal stem cells as a source of dopaminergic neurons: a potential cell based therapy for Parkinson's disease. Curr Stem Cell Res Ther. 2017;12(4):326-47. [PMID] [DOI:10.2174/1574888X12666161114122059]
  30. Teixeira FG, Carvalho MM, Souza N, Salgado AJ. Mesenchymal stem cells secretome: a new paradigm for central nervous system regeneration? Cell Mol Life Sci. 2017;70(20): 3871-82. [DOI:10.1007/s00018-013-1290-8] [PMID]
  31. Blaber SP, Vebster RA, Hill CJ, Breen EJ, Kuah D, Vesey G, et al. Analysis of in vitro secretion profiles from adipose-derived cell populations. Transl Med. 2012;0:Article number:172. [DOI:10.1186/1479-5876-10-172] [PMID] [PMCID]
  32. Perez RG, Waymire JC, Lin E, Liu JJ, Guo F, Zigmond MJ. A role for alpha-synuclein in the regulation of dopamine biosynthesis. Neurosci. 2002;22:3090-3099. [PMCID] [DOI:10.1523/JNEUROSCI.22-08-03090.2002]
  33. Gao N, Li YH, Li X, Yu S, Fu GL, Chen B. Effect of alphasynuclein on the promoter activity of tyrosine hydroxylase gene. Neurosci. Bulletin. 2007;23(1):53-57. [DOI:10.1007/s12264-007-0008-z] [PMID] [PMCID]
  34. Luk KC, Kehm V, Carroll J, Zhang B, O'Brien P, Trojanowski JQ, et al. Pathologicalα-Synuclein transmission initiates Parkinson-like neurodegeneration in non-transgenic mice. Science. 2012;338(6109):949-953. [PMID] [PMCID] [DOI:10.1126/science.1227157]
  35. Bockaert J, Marin P. mTOR in brain physiology and pathologies. Physiol Rev. 2015;95(4): 1157-87. [DOI:10.1152/physrev.00038.2014] [PMID]
  36. Dijkstra A, Ingrassia A, Menezes RX, Kesteren RE, Rozemuller AJM, Heutink P, et al. Evidence for immune response, axonal dysfunction and reduced endocytosis in the substantia nigra in early stage Parkinson's disease. PLoS ONE. 2015;10(6): e0128651, e0128651. [PMID] [PMCID] [DOI:10.1371/journal.pone.0128651]
  37. Wang Y, Ding L, Wang X, Zhang J, Han W, Feng L, et al. Pterostilbene simultaneously induces apoptosis, cell cycle arrest and cyto-protective autophagy in breast cancer cells. American. Translational. Res. 2012;4(1):44-51.
  38. Sheng YL, Chen X, Hou XO, Yuan X, Yuan BS, Yuan YQ, Zhan QL, Cao X, Liu CF, Luo WF, Hu LF. Urate promotes SNCA/α-synuclein clearance via regulating mTOR dependent macroautophagy. Exp Neurol . 2017;297:138-147. [DOI:10.1016/j.expneurol.2017.08.007] [PMID]
  39. Chen LL, Song JX, Lu JH, Yuan ZW, Liu LF, Kumar Durairajan SS, Li M. Corynoxine, a Natural Autophagy Enhancer, Promotes the Clearance of Alpha-Synuclein via Akt/mTOR Pathway. J Neuroimmune Pharmacol. 2014;12: 125-138. [DOI:10.1007/s11481-014-9528-2] [PMID]
  40. Decressac M, Mattsson B, Weikop P, Lundblad M, Jakobsson JA, Bjorklund A. TFEB-mediated autophagy rescues midbrain dopamine neurons froma-synuclein toxicity. Proceedings of the National Aca of Sci of USA. 2013;110(19): E1817-26. [DOI:10.1073/pnas.1305623110] [PMID] [PMCID]
  41. Subramaniam S, Napolitano F, Mealar R, Kim S, Errico F, Barrow R, et al. a striatal-enriched small G protein, mediates mTOR signaling and L-DOPA-induced dyskinesia. Nature. Neurosci. 2012;15:191-3. [DOI:10.1038/nn.2994] [PMID] [PMCID]
  42. Ebrahim N, Ahmad IA, Hussein NI, Dessoukey AA, Farid AS, et al. Mesenchymal Stem Cell-Derived Exosomes Ameliorated Diabetic Nephropathy by Autophagy Induction through the mTOR Signaling Pathway. Cells. 2018;7(12):1-25. [DOI:10.3390/cells7120226] [PMID] [PMCID]
  43. Liu L, Jin X, Hu CF, Li R, Zhou Z, Shen CX. Exosomes Derived from Mesenchymal Stem Cells Rescue Myocardial Ischaemia/Reperfusion Injury by Inducing Cardiomyocyte Autophagy Via AMPK and Akt Pathways. Cell Physiol Biochem. 2018;43:52-68. [DOI:10.1159/000480317] [PMID]
  44. Zonneveld MI, Keulers TGH, Rouschop KMA. Extracellular vesicles as transmitters of hypoxia tolerance in solid cancers. Cancers (Basel). 2019;11:15. [DOI:10.3390/cancers11020154] [PMID] [PMCID]
  45. Xing H, Tan J, Miao Y, Lv Y, Zhang Q. Crosstalk between exosomes and autophagy: A review of molecular mechanisms and therapies. J Cell Mol Med. 2021;25:2297-2308. [DOI:10.1111/jcmm.16276] [PMID] [PMCID]
  46. Park HJ, Shin JY, Kim HN, Oh SH, Lee PH. Neuroprotective effects of mesenchymal stem cells through autophagy modulation in a parkinsonian model. Neurobiol Aging. 2014; 35(8):1920-28. [PMID] [DOI:10.1016/j.neurobiolaging.2014.01.028]
  47. Lee KY, Mooney DJ. Alginate: properties and biomedical applications. Prog Polym Sci. 2012;37(1):106-26. [PMID] [PMCID] [DOI:10.1016/j.progpolymsci.2011.06.003]
  48. Hashemi M, Kalalinia F. Application of encapsulation technology in stem cell therapy. Life Sci. 2015;143:139-146. [DOI:10.1016/j.lfs.2015.11.007] [PMID]
  49. Moriarty N, Pandit A, Dowd E. Encapsulation of primary dopaminergic neurons in a GDNF loaded collagen hydrogel increases their survival, re-innervation and function after intra-striatal transplantation. Sci Rep. 2017;7:16033. [PMCID] [DOI:10.1038/s41598-017-15970-w] [PMID]
  50. Adill MM, Vazin T, Ananthanaryana B, Rodrigues GMC, Rao AT, Kulkarni RU, et al. Engineered hydrogels increase the post-transplantation survival of encapsulated hESC-derived midbrain dopaminergic neurons. Biomaterials. 2017;136:1-11. [PMID]  [DOI:10.1016/j.biomaterials.2017.05.008]