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Numerous studies have explored the role of cannabinoids in neurological conditions, chronic pain and neurodegenerative diseases. Restoring autophagy has been proposed as a potential target for the treatment of neurodegenerative diseases. In our study, we used a neuroblastoma cell line that overexpresses wild-type α-synuclein to investigate the effects of cannabidiol on autophagy modulation and reduction in the level of cytosolic α-synuclein. Our results demonstrated that cannabidiol enhances the accumulation of LC3-II- and GFP-LC3-positive vesicles, which indicates an increase in autophagic flux. In addition, cannabidiol-treated cells showed a reduction in cytosolic α-synuclein levels. These effects were inhibited when the cells were treated with a CB 1 receptor-selective antagonist, which indicates that the biological effects of cannabidiol are mediated via its interaction with CB 1 receptor. Additionally, we also observed that cannabinoid compounds induce autophagy and α-synuclein degradation after they interact with the CB 1 receptor. In summary, our data suggest that cannabidiol induces autophagy and reduces cytosolic α-synuclein levels. These biological effects are mediated preferentially through the interaction of cannabidiol with CB 1 receptors, and therefore, cannabinoid compounds that act selectively on this receptor could represent a new approach for autophagy modulation and degradation of protein aggregates., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Mitochondria-associated ER membranes (MAMs) are formed by close and specific components in the contact sites between the endoplasmic reticulum (ER) and mitochondria, which participate in several cell functions, including lipid metabolism, autophagy, and Ca 2+ signaling. Particularly, the presence of α-synuclein (α-syn) in MAMs was previously demonstrated, indicating a physical interaction among some proteins in this region and a potential involvement in cell dysfunctions. MAMs alterations are associated with neurodegenerative diseases such as Parkinson's disease (PD) and contribute to the pathogenesis features. Here, we investigated the effects of α-syn on MAMs and Ca 2+ transfer from the ER to mitochondria in WT- and A30P α-syn-overexpressing SH-SY5Y or HEK293 cells. We observed that α-syn potentiates the mitochondrial membrane potential (Δψ m ) loss induced by rotenone, increases mitophagy and mitochondrial Ca 2+ overload. Additionally, in α-syn-overexpressing cells, we found a reduction in ER-mitochondria contact sites through the impairment of the GRP75-IP3R interaction, however, with no alteration in VDAC1-GRP75 interaction. Consequently, after Ca 2+ release from the ER, α-syn-overexpressing cells demonstrated a reduction in Ca 2+ buffering by mitochondria, suggesting a deregulation in MAM activity. Taken together, our data highlight the importance of the α-syn/MAMs/Ca 2+ axis that potentially affects cell functions in PD., (© 2021 Wiley Periodicals LLC.)

Although the etiology of Parkinson's disease (PD) is multifactorial, it has been linked to abnormal accumulation of α-synuclein (α-syn) in dopaminergic neurons, which could lead to dysfunctions on intracellular organelles, with potential neurodegeneration. Patients with familial early-onset PD frequently present mutation in the α-syn gene (SNCA), which encodes mutant α-syn forms, such as A30P and A53T, which potentially regulate Ca 2+ unbalance. Here we investigated the effects of overexpression of wild-type α-syn (WT) and the mutant forms A30P and A53T, on modulation of lysosomal Ca 2+ stores and further autophagy activation. We found that in α-syn-overexpressing cells, there was a decrease in Ca 2+ released from endoplasmic reticulum (ER) which is related to the increase in lysosomal Ca 2+ release, coupled to lysosomal pH alkalization. Interestingly, α-syn-overexpressing cells showed lower LAMP1 levels, and a disruption of lysosomal morphology and distribution, affecting autophagy. Interestingly, all these effects were more evident with A53T mutant isoform when compared to A30P and WT α-syn types, indicating that the pathogenic phenotype for PD is potentially related to impairment of α-syn degradation. Taken together, these events directly impact PD-related dysfunctions, being considered possible molecular targets for neuroprotection.

α-Synuclein is the major component of neuronal cytoplasmic aggregates called Lewy bodies, the main pathological hallmark of Parkinson disease. Although neurons are the predominant cells expressing α-synuclein in the brain, recent studies have demonstrated that primary astrocytes in culture also express α-synuclein and regulate α-synuclein trafficking. Astrocytes have a neuroprotective role in several detrimental brain conditions; we therefore analyzed the effects of the overexpression of wild-type α-synuclein and its A30P and A53T mutants on autophagy and apoptosis. We observed that in immortalized astrocyte cell lines, overexpression of α-synuclein proteins promotes the decrease of LC3-II and the increase of p62 protein levels, suggesting the inhibition of autophagy. When these cells were treated with rotenone, there was a loss of mitochondrial membrane potential, especially in cells expressing mutant α-synuclein. The level of this decrease was related to the toxicity of the mutants because they show a more intense and sustained effect. The decrease in autophagy and the mitochondrial changes in conjunction with parkin expression levels may sensitize astrocytes to apoptosis., (© 2017 Wiley Periodicals, Inc.)

Parkinson's disease (PD) is a chronic and progressive neurodegenerative disease, characterized by loss of dopaminergic neurons of the substantia nigra (SN) and abnormal deposits of the alpha-synuclein protein (alpha-syn). Patients with PD present characteristic motor and non-motor symptom. Spheroids came as a promising alternative in the 3D culture because the scaffold-free agarose micromolded technique allows cells to produce extracellular matrix, permitting greater cell-cell interaction, recreating the microenvironment and recapitulating tissue morphogenesis. In this study, we use the SY5Y (SH-WT) cell to develop the spheroids, they express the dopaminergic machinery. As a control we will use α-syn-A53T (SH-A53T) cells, which overexpress the alpha syn. Produce the spheroids with the cell lines SH-SY5Y (SH-WT) and mutated alpha-synuclein (SH-A53T), using them as a model of Parkinson's disease. Both cells lines were grown in DMEM Medium (Dulbecco's Modified Eagle Medium) supplemented with 100U/mL of penicillin, 100 U/ mL of streptomycin and 10% fetal bovine serum (SBF) at 37°C and 5% CO 2 atmosphere. spheroids were produced with 1 × 106 cells seeded on the agarose micromolded, producing spheres with 400 µm approximately. Once a week, pictures were obtained, and the images were used for volume calculation and necrotic center evaluation. Cells viability were analyzed by Trypan Blue. Spheroids were successfully produced in both groups (SH-A53T and SH-WT). The volume on the first day was 2,9±1 × 107 (SH-WT) and 4,0±1,3 × 107 (SH-A53T). Both groups showed 95% cell viability with no changes over the time. There was significant increase in the volume of the SH-WT spheroids over time, but not in the A53T group. On day 14, SH-WT spheroids were 3X bigger than day 1 (p <0,001) and 1,5X bigger than SH-A53T (p <0,001). The necrotic center was first observed in SH-A53T group on day 7 and in SH-WT it become visible only on day 14. Here we show for the first time that cells overexpressing alpha-syn in were able to form spheroids. Using this 3D model, we identified significant changes between SH-A53T and SH-WT spheroids such as: growth pattern, necrotic center formation and volume. These differences can be explained by the aggregation potential caused by the A53T mutation. Our spheroids could be used to study the disease in another perspective opening opportunities for innovations, making a model close to what is found in vivo. [ABSTRACT FROM AUTHOR]