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

Affiliations
  • 1Center for Genomics and Proteomics & Stem Cell Core Facility, Lee Gil Ya Cancer and Diabetes Institute, Gachon University, Incheon, Korea. bhlee@gachon.ac.kr khbyun1@gachon.ac.kr
  • 2Department of Anatomy & Cell Biology, Graduate School of Medicine, Gachon University, Incheon, Korea.
  • 3Department of Aquatic Life Medicine, Pukyong National University, Busan, Korea.

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

  • 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

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