alterado para: “MicroRNAs-enriched exosomes as a new therapy for Parkinson’s disease” MicroRNAs-enriched exosomes as a new therapy for brain repair in stroke

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MicroRNAs-enriched exosomes as a new therapy for Parkinson’s disease

Publication . Esteves, Marta Raquel Carrola; Bernardino, Liliana Inácio; Ferreira, Lino Silva

Parkinson’s disease (PD), a progressive and chronic neurological disorder characterized by the selective degeneration of the nigrostriatal dopaminergic pathway, has a huge socioeconomic impact in modern society. Alterations in alpha (α)-synuclein protein expression and aggregation have been regarded as a primary cause of dopaminergic neurons death in the substantia nigra (SN) pars compacta. So far, there are no treatments that halt or reverse the progression of PD. Recent evidence showed that microRNAs (miRNA), small non-coding RNAs that negatively regulate gene expression, are dysregulated in PD patients. In particular, miR-124 levels were found decreased in plasma and postmortem brain parenchyma of PD patients. Thus, miR-124 has become a relevant molecular therapeutic target for PD. Increasing intracellular levels of miR-124 enhances neurogenesis and neuroprotection in PD models. However, the effect of miR- 124 on α-synuclein dynamics has not yet been reported. One of the main goals of this thesis was to evaluate the role of miR-124-3p in the expression and aggregation of α-synuclein protein using the rat model of PD based on the acute administration of paraquat (PQ). The first research work showed that intranigral administration of miR-124-3p significantly reduces the protein levels of α- synuclein and α-synuclein phosphorylated at serine 129 (present in large amounts in Lewy bodies) in the SN of rats exposed to PQ. Moreover, the protein levels of NADPH oxidase 1 (Nox1), responsible for the oxidative stress production and eventually involved in the development of α-synuclein toxicity, and its activator GTPase Rac1, decreased in SN after miR-124-3p administration in PQ-treated rats. Additionally, the reduced levels of Pitx3 in the SN caused by the administration of PQ were found to increase after the administration of miR-124-3p. This work demonstrates for the first time the role of miR- 124-3p in the regulation of α-synuclein toxicity, possibly through the modulation of the Nox1/Rac1 signaling pathway and in the regulation of Pitx3 expression important for the survival and maintenance of dopaminergic neurons. The efficient delivery of miRNA at the intracellular and intracerebral levels has several limitations since they are highly susceptible to degradation by nucleases and are difficult to take up by cells due to their negative charge. Recently, several approaches capable of guaranteeing an efficient delivery of miRNA have been developed. In particular, small extracellular vesicles (sEV), also referred to as exosomes, have been highlighted as efficient delivery systems for miRNA due to their intrinsic ability to interact with cells and tissues. The second main goal of this thesis was to use sEV isolated from human umbilical cord blood-derived mononuclear cells as a biological vehicle of miR-124 and to evaluate the efficiency of these sEV transfected with miR-124-3p (miR-124-3p sEV) in inducing neurogenesis and neuroprotection in 6-hydroxydopamine (6-OHDA) PD model. The second research work showed that in vitro, sEV efficiently deliver miR-124- 3p to subventricular zone (SVZ) neural stem cells (NSC) and to N27 dopaminergic cells. Treatment of NSC with miR-124-3p sEV induced neuronal differentiation of SVZ NSC under physiological conditions and protected N27 dopaminergic cells against 6-OHDAinduced toxicity. In vivo, sEV intracerebroventricularly injected in mice were detected in SVZ lining the lateral ventricles and in striatum and midbrain sections, the brain regions most affected by the disease. Although miR-124-3p sEV did not increase the number of new neurons in the 6-OHDA-lesioned striatum, the formulation protected dopaminergic neurons in the SN and striatal fibers, which fully counteracted motor behavior symptoms in mice administered with 6-OHDA. In conclusion, the results obtained during this thesis provide new evidence on the role of miR-124-3p in α-synuclein protein pathology and evidence supporting the application of sEV as biologic delivery agents for miR-124-3p to promote neuroprotection of dopaminergic neurons. In short, the enrichment of sEV with miR-124-3p may configure a promising therapeutic strategy able to halt or slow-down dopaminergic neuronal death in PD.

2023-07-05Doctoral thesis

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