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Therapeutic Effects of Conditioned Medium of Neural Differentiated Human Bone Marrow-Derived Stem Cells on Rotenone-Induced Alpha-Synuclein Aggregation and Apoptosis.

ABSTRACT: Mesenchymal stem cells (MSCs) have been used against several diseases. Their potential mainly appears from its secreted biomolecules. Human bone marrow-derived stem cells (hBMSC) displayed neuronal functional characteristics after differentiation by basic fibroblast growth factor (bFGF) and forskolin. PD is a chronic age-related neurodegenerative disease (NDD) characterized by loss of dopaminergic neurons in the substantia nigra (SN) and abnormal accumulation of ?-synuclein (?-syn) aggregations. In this present study, we evaluated the therapeutic effects of neural differentiated hBMSC (NI-hBMSC) conditioned medium (NI-hBMSC-CM) to a rotenone- (ROT-) induced Parkinson's disease (PD) model in SH-SY5Y cells. NI-hBMSC-CM treatment (50% diluted) in the last 24?h of 48?h ROT (0.5??M) toxicity showed a significant increase in cell survival. The decreased tyrosine hydroxylase (TH) expression as a hallmark of PD was increased by NI-hBMSC-CM. The Triton X-100-soluble and Triton X-100-insoluble cell lysate fractions were used in Western blotting. The oligomeric, dimeric, and monomeric phosphorylated serine129 (p-S129) ?-syn and total monomeric ?-syn were decreased during ROT toxicity in the Triton X-100-soluble fraction. The Triton X-100-insoluble fraction revealed that ROT toxicity significantly increased the oligomeric but decreased the dimeric and monomeric p-S129 ?-syn expressions while all forms of total ?-syn were increased in SH-SY5Y cells. NI-hBMSC-CM stabilized the physiological ?-syn monomers and reduced aggregated insoluble p-S129 ?-syn against ROT. The cytoskeletal proteins, neurofilament-H (NF-H), ?3-tubulin (Tuj1), neuronal nuclei (NeuN), and synaptophysin (SYP) were significantly decreased during ROT toxicity. In addition, proapoptotic Bax was increased by ROT with decreased antiapoptotic Bcl-2 and Mcl-1 as well as proforms of caspase-9, caspase-3, caspase-7, and PARP-1. NI-hBMSC-CM ameliorated the neurotrophic protein expressions, controlled the Bax/Bcl-2 ratio, upregulated procaspases, and inactivated PARP-1. From our results, we conclude that NI-hBMSC-CM containing released biomolecules during neural differentiation employs regenerative effects on the ROT model of PD in SH-SY5Y cells.

SUBMITTER: Ramalingam M

PROVIDER: S-EPMC7847328 | biostudies-literature | 2021

REPOSITORIES: biostudies-literature

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Therapeutic Effects of Conditioned Medium of Neural Differentiated Human Bone Marrow-Derived Stem Cells on Rotenone-Induced Alpha-Synuclein Aggregation and Apoptosis.

Ramalingam Mahesh M Jang Sujeong S Jeong Han-Seong HS

Stem cells international 20210122

Mesenchymal stem cells (MSCs) have been used against several diseases. Their potential mainly appears from its secreted biomolecules. Human bone marrow-derived stem cells (hBMSC) displayed neuronal functional characteristics after differentiation by basic fibroblast growth factor (bFGF) and forskolin. PD is a chronic age-related neurodegenerative disease (NDD) characterized by loss of dopaminergic neurons in the substantia nigra (SN) and abnormal accumulation of <i>α</i>-synuclein (<i>α</i>-syn) ...[more]

PMID: 33552161

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Project description:Bone marrow-derived mesenchymal stem cells differentiate into neurons under the induction of Schwann cells. However, key microRNAs and related pathways for differentiation remain unclear. This study screened and identified differentially expressed microRNAs in bone marrow-derived mesenchymal stem cells induced by Schwann cell-conditioned medium, and explored targets and related pathways involved in their differentiation into neuronal-like cells. Primary bone marrow-derived mesenchymal stem cells were isolated from femoral and tibial bones, while primary Schwann cells were isolated from bilateral saphenous nerves. Bone marrow-derived mesenchymal stem cells were cultured in unconditioned (control group) and Schwann cell-conditioned medium (bone marrow-derived mesenchymal stem cell + Schwann cell group). Neuronal differentiation of bone marrow-derived mesenchymal stem cells induced by Schwann cell-conditioned medium was observed by time-lapse imaging. Upon induction, the morphology of bone marrow-derived mesenchymal stem cells changed into a neural shape with neurites. Results of quantitative reverse transcription-polymerase chain reaction revealed that nestin mRNA expression was upregulated from 1 to 3 days and downregulated from 3 to 7 days in the bone marrow-derived mesenchymal stem cell + Schwann cell group. Compared with the control group, microtubule-associated protein 2 mRNA expression gradually increased from 1 to 7 days in the bone marrow-derived mesenchymal stem cell + Schwann cell group. After 7 days of induction, microRNA analysis identified 83 significantly differentially expressed microRNAs between the two groups. Gene Ontology analysis indicated enrichment of microRNA target genes for neuronal projection development, regulation of axonogenesis, and positive regulation of cell proliferation. Kyoto Encyclopedia of Genes and Genomes pathway analysis demonstrated that Hippo, Wnt, transforming growth factor-beta, and Hedgehog signaling pathways were potentially associated with neural differentiation of bone marrow-derived mesenchymal stem cells. This study, which carried out successful microRNA analysis of neuronal-like cells differentiated from bone marrow-derived mesenchymal stem cells by Schwann cell induction, revealed key microRNAs and pathways involved in neural differentiation of bone marrow-derived mesenchymal stem cells. All protocols were approved by the Animal Ethics Committee of Institute of Radiation Medicine, Chinese Academy of Medical Sciences on March 12, 2017 (approval number: DWLI-20170311).

Project description:Methamphetamine (METH) is an illegal and widely abused psychoactive stimulant. METH abusers are at high risk of neurodegenerative disorders, including Parkinson's disease (PD). Previous studies have demonstrated that METH causes alpha-synuclein (?-syn) aggregation in the both laboratory animal and human. In this study, exposure to high METH doses increased the expression of ?-syn and the small ubiquitin-related modifier 1 (SUMO-1). Therefore, we hypothesized that SUMOylation of ?-syn is involved in high-dose METH-induced ?-syn aggregation. We measured the levels of ?-syn SUMOylation and these enzymes involved in the SUMOylation cycle in SH-SY5Y human neuroblastoma cells (SH-SY5Y cells), in cultures of C57 BL/6 primary mouse neurons and in brain tissues of mice exposure to METH. We also demonstrated the effect of ?-syn SUMOylation on ?-syn aggregation after METH exposure by overexpressing the key enzyme of the SUMOylation cycle or silencing SUMO-1 expression in vitro. Then, we make introduced mutations in the major SUMOylation acceptor sites of ?-syn by transfecting a lentivirus containing the sequence of WT ?-syn or K96/102R ?-syn into SH-SY5Y cells and injecting an adenovirus containing the sequence of WT ?-syn or K96/102R ?-syn into the mouse striatum. Levels of the ubiquitin-proteasome system (UPS)-related makers ubiquitin (Ub) and UbE1, as well as the autophagy-lysosome pathway (ALP)-related markers LC3, P62 and lysosomal associated membrane protein 2A (LAMP2A), were also measured in SH-SY5Y cells transfected with lentivirus and mice injected with adenovirus. The results showed that METH exposure decreases the SUMOylation level of ?-syn, although the expression of ?-syn and SUMO-1 are increased. One possible cause is the reduction of UBC9 level. The increase in ?-syn SUMOylation by UBC9 overexpression relieves METH-induced ?-syn overexpression and aggregation, whereas the decrease in ?-syn SUMOylation by SUMO-1 silencing exacerbates the same pathology. Furthermore, mutations in the major SUMOylation acceptor sites of ?-syn also aggravate ?-syn overexpression and aggregation by impairing degradation through the UPS and the ALP in vitro and in vivo. These results suggest that SUMOylation of ?-syn plays a fundamental part in ?-syn overexpression and aggregation induced by METH and could be a suitable target for the treatment of neurodegenerative diseases.

Dataset Information

Therapeutic Effects of Conditioned Medium of Neural Differentiated Human Bone Marrow-Derived Stem Cells on Rotenone-Induced Alpha-Synuclein Aggregation and Apoptosis.

Publications

Therapeutic Effects of Conditioned Medium of Neural Differentiated Human Bone Marrow-Derived Stem Cells on Rotenone-Induced Alpha-Synuclein Aggregation and Apoptosis.

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