Automated Quantification of Mitochondrial Fragmentation in an In-Vitro Parkinson’s Disease Model

AbstractNeuronal mitochondrial fragmentation is a phenotype exhibited in models of neurodegeneration such as Parkinson’s Disease. Delineating the dysfunction in mitochondrial dynamics found in diseased states can aid our understanding of underlying mechanisms for disease progression and possibly identify novel therapeutic approaches. Advances in microscopy and the availability of intuitive open-access software has accelerated the rate of image acquisition and analysis, respectively. These developments allow routine biology researchers to rapidly turn hypotheses into results. In this protocol, we describe the utilisation of cell culture techniques, high-content imaging (HCI), and subsequent open-source image analysis pipeline for the quantification of mitochondrial fragmentation in the context of an in-vitro Parkinson’s Disease model.

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Validation of an In-Vitro Parkinson’s Disease Model for the Study of Neuroprotection

Proceedings ◽

10.3390/proceedings2251559 ◽

2018 ◽

Vol 2 (25) ◽

pp. 1559 ◽

Cited By ~ 2

Author(s):

Esra Nur Yiğit ◽

Ekin Sönmez ◽

Melis Savaşan Söğüt ◽

Tunahan Çakır ◽

Işıl Aksan Kurnaz

Keyword(s):

Parkinson’S Disease ◽

Parkinson's Disease ◽

Neurological Diseases ◽

Disease Model ◽

Fold Increase ◽

Human Neuroblastoma ◽

Familial Cases ◽

Sporadic Cases ◽

Underlying Mechanisms

Parkinson’s disease (PD) is the second most common neurodegenerative disease with an estimation of 10 million people living with the disease and it is increasing in prevalence every year. Familial cases of PD are source of valuable information to determine genetical risk factors yet sporadic cases can emerge from distinct mechanisms so, identifying common pathways of familial and sporadic cases of PD may provide worthwhile insights to determine underlying mechanisms through the progression. LRRK2 mutations are the most common indicators of both sporadic and familial cases of PD and α-synuclein aggregation is one of the hallmarks of both cases of PD as well as in other synucleinopathies. As in the case of most neurological diseases, human studies addressing the molecular basis of pathology are generally restricted to post-mortem materials. For this reason, cell culture systems and animal models are widely used. There are two main approaches for modelling PD: genetically constructed PD models and neuroxin-based models. In this study, we aim to construct and compare both approaches by overexpressing wild-type (WT) and A53T mutant α-synuclein and treating cells with well-known neurotoxin 6-hydroxidopamine (6-OHDA) using dopaminergic human neuroblastoma SH-SY5Y cell line. Our findings suggest that WT or A53T α-synuclein overexpression by itself is not sufficient to cause significant toxicity in SH-SY5Y cells in the presented time scale. As expected, 6-OHDA treatment caused toxicity with an IC50 value of ~100 µM. In addition, 6-OHDA treatment causes 3- and 2.5-fold increase in SNCA and LRRK2 expression respectively.

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Automated Quantification of Mitochondrial Fragmentation in an In Vitro Parkinson's Disease Model

Current Protocols in Neuroscience ◽

10.1002/cpns.105 ◽

2020 ◽

Vol 94 (1) ◽

Author(s):

Daniel J. Rees ◽

Luke Roberts ◽

M. Carla Carisi ◽

Alwena H. Morgan ◽

M. Rowan Brown ◽

...

Keyword(s):

Parkinson’S Disease ◽

Parkinson's Disease ◽

Disease Model ◽

Mitochondrial Fragmentation ◽

Automated Quantification

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Faculty Opinions recommendation of Induction of functional dopamine neurons from human astrocytes in vitro and mouse astrocytes in a Parkinson's disease model.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.727495722.793564376 ◽

2019 ◽

Author(s):

Oliver Brüstle

Keyword(s):

Parkinson’S Disease ◽

Parkinson's Disease ◽

Disease Model ◽

Dopamine Neurons ◽

Human Astrocytes

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Cytokinin Plant Hormones Have Neuroprotective Activity in In Vitro Models of Parkinson’s Disease

Molecules ◽

10.3390/molecules26020361 ◽

2021 ◽

Vol 26 (2) ◽

pp. 361

Author(s):

Gabriel Gonzalez ◽

Jiří Grúz ◽

Cosimo Walter D’Acunto ◽

Petr Kaňovský ◽

Miroslav Strnad

Keyword(s):

Parkinson’S Disease ◽

Parkinson's Disease ◽

Cell Death ◽

Neuronal Cell Death ◽

Disease Model ◽

Neuronal Cell ◽

Zeatin Riboside ◽

Neuroprotective Activity ◽

Protective Activity

Cytokinins are adenine-based phytohormones that regulate key processes in plants, such as cell division and differentiation, root and shoot growth, apical dominance, branching, and seed germination. In preliminary studies, they have also shown protective activities against human neurodegenerative diseases. To extend knowledge of the protection (protective activity) they offer, we investigated activities of natural cytokinins against salsolinol (SAL)-induced toxicity (a Parkinson’s disease model) and glutamate (Glu)-induced death of neuron-like dopaminergic SH-SY5Y cells. We found that kinetin-3-glucoside, cis-zeatin riboside, and N6-isopentenyladenosine were active in the SAL-induced PD model. In addition, trans-, cis-zeatin, and kinetin along with the iron chelator deferoxamine (DFO) and the necroptosis inhibitor necrostatin 1 (NEC-1) significantly reduced cell death rates in the Glu-induced model. Lactate dehydrogenase assays revealed that the cytokinins provided lower neuroprotective activity than DFO and NEC-1. Moreover, they reduced apoptotic caspase-3/7 activities less strongly than DFO. However, the cytokinins had very similar effects to DFO and NEC-1 on superoxide radical production. Overall, they showed protective activity in the SAL-induced model of parkinsonian neuronal cell death and Glu-induced model of oxidative damage mainly by reduction of oxidative stress.

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Data on dose-dependent cytotoxicity of rotenone and neuroprotection conferred by Yashtimadhu (Glycyrrhiza glabra L.) in an in vitro Parkinson's disease model

Data in Brief ◽

10.1016/j.dib.2021.107535 ◽

2021 ◽

pp. 107535

Author(s):

Gayathree Karthikkeyan ◽

Ashwini Prabhu ◽

Ravishankar Pervaje ◽

Sameera Krishna Pervaje ◽

Prashant Kumar Modi ◽

...

Keyword(s):

Parkinson’S Disease ◽

Parkinson's Disease ◽

Disease Model ◽

Glycyrrhiza Glabra ◽

Glycyrrhiza Glabra L ◽

Dose Dependent

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Effects of the Toxic Metals Arsenite and Cadmium on α-Synuclein Aggregation In Vitro and in Cells

International Journal of Molecular Sciences ◽

10.3390/ijms222111455 ◽

2021 ◽

Vol 22 (21) ◽

pp. 11455

Author(s):

Emma Lorentzon ◽

Istvan Horvath ◽

Ranjeet Kumar ◽

Joana Isabel Rodrigues ◽

Markus J. Tamás ◽

...

Keyword(s):

Parkinson’S Disease ◽

Parkinson's Disease ◽

Yeast Cells ◽

Amyloid Formation ◽

Amyloid Fibers ◽

Force Microscopy ◽

Atomic Force ◽

In Vitro Characterization ◽

Underlying Mechanisms

Exposure to heavy metals, including arsenic and cadmium, is associated with neurodegenerative disorders such as Parkinson’s disease. However, the mechanistic details of how these metals contribute to pathogenesis are not well understood. To search for underlying mechanisms involving α-synuclein, the protein that forms amyloids in Parkinson’s disease, we here assessed the effects of arsenic and cadmium on α-synuclein amyloid formation in vitro and in Saccharomyces cerevisiae (budding yeast) cells. Atomic force microscopy experiments with acetylated human α-synuclein demonstrated that amyloid fibers formed in the presence of the metals have a different fiber pitch compared to those formed without metals. Both metal ions become incorporated into the amyloid fibers, and cadmium also accelerated the nucleation step in the amyloid formation process, likely via binding to intermediate species. Fluorescence microscopy analyses of yeast cells expressing fluorescently tagged α-synuclein demonstrated that arsenic and cadmium affected the distribution of α-synuclein aggregates within the cells, reduced aggregate clearance, and aggravated α-synuclein toxicity. Taken together, our in vitro data demonstrate that interactions between these two metals and α-synuclein modulate the resulting amyloid fiber structures, which, in turn, might relate to the observed effects in the yeast cells. Whilst our study advances our understanding of how these metals affect α-synuclein biophysics, further in vitro characterization as well as human cell studies are desired to fully appreciate their role in the progression of Parkinson’s disease.

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LincRNA-p21 Inhibits Cell Viability and Promotes Cell Apoptosis in Parkinson’s Disease through Activating α-Synuclein Expression

BioMed Research International ◽

10.1155/2018/8181374 ◽

2018 ◽

Vol 2018 ◽

pp. 1-10 ◽

Cited By ~ 11

Author(s):

Xiaonan Xu ◽

Chengle Zhuang ◽

Zimu Wu ◽

Hongyan Qiu ◽

Haixia Feng ◽

...

Keyword(s):

Parkinson’S Disease ◽

Parkinson's Disease ◽

Cell Apoptosis ◽

Noncoding Rna ◽

Long Intergenic Noncoding Rna ◽

Novel Target ◽

Underlying Mechanisms

Long intergenic noncoding RNA-p21 (lincRNA-p21) has been reported to be increased in Parkinson’s disease (PD). However, the function and underlying mechanisms of lincRNA-p21 remain not clear. In order to explore the role of lincRNA-p21 in PD, we used 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) to induce in vivo PD model (C57BL/6 mice) and utilized N-methyl-4-phenylpyridinium (MPP+) to create in vitro PD model (SH-SY5Y cells). Results showed that the expression level of lincRNA-p21 was increased significantly in PD models. High abundance of lincRNA-p21 inhibited viability and promoted apoptosis markedly in SH-SY5Y cells treated with MPP+. Mechanistically, further experiments demonstrated that upregulation of lincRNA-p21 could sponge miR-1277-5p and indirectly increase the expression of α-synuclein to suppress viability and activate apoptosis in SH-SY5Y cells. In short, our study illustrated that lincRNA-p21/miR-1277-5p axis regulated viability and apoptosis in SH-SY5Y cells treated with MPP+ via targeting α-synuclein. LincRNA-p21 might be a novel target for PD.

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