Biochemical Fractionation of Human α-Synuclein in a <i>Drosophila</i> Model of Synucleinopathies
Synucleinopathies are a group of central nervous system pathologies that are characterized by the intracellular accumulation of misfolded and aggregated α-synuclein in proteinaceous depositions known as Lewy Bodies (LBs). The transition of α-synuclein from its physiological to pathological form has...
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MDPI AG
2024-03-01
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| Series: | International Journal of Molecular Sciences |
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| Online Access: | https://www.mdpi.com/1422-0067/25/7/3643 |
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| author | Khondamir Imomnazarov Joshua Lopez-Scarim Ila Bagheri Valerie Joers Malú Gámez Tansey Alfonso Martín-Peña |
| author_facet | Khondamir Imomnazarov Joshua Lopez-Scarim Ila Bagheri Valerie Joers Malú Gámez Tansey Alfonso Martín-Peña |
| author_sort | Khondamir Imomnazarov |
| collection | DOAJ |
| description | Synucleinopathies are a group of central nervous system pathologies that are characterized by the intracellular accumulation of misfolded and aggregated α-synuclein in proteinaceous depositions known as Lewy Bodies (LBs). The transition of α-synuclein from its physiological to pathological form has been associated with several post-translational modifications such as phosphorylation and an increasing degree of insolubility, which also correlate with disease progression in post-mortem specimens from human patients. Neuronal expression of α-synuclein in model organisms, including <i>Drosophila melanogaster</i>, has been a typical approach employed to study its physiological effects. Biochemical analysis of α-synuclein solubility via high-speed ultracentrifugation with buffers of increasing detergent strength offers a potent method for identification of α-synuclein biochemical properties and the associated pathology stage. Unfortunately, the development of a robust and reproducible method for the evaluation of human α-synuclein solubility isolated from <i>Drosophila</i> tissues has remained elusive. Here, we tested different detergents for their ability to solubilize human α-synuclein carrying the pathological mutation A53T from the brains of aged flies. We also assessed the effect of sonication on the solubility of human α-synuclein and optimized a protocol to discriminate the relative amounts of soluble/insoluble human α-synuclein from dopaminergic neurons of the <i>Drosophila</i> brain. Our data established that, using a 5% SDS buffer, the three-step protocol separates cytosolic soluble, detergent-soluble and insoluble proteins in three sequential fractions according to their chemical properties. This protocol shows that sonication breaks down α-synuclein insoluble complexes from the fly brain, making them soluble in the SDS buffer and thus enriching the detergent-soluble fraction of the protocol. |
| first_indexed | 2024-04-24T10:43:14Z |
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| issn | 1661-6596 1422-0067 |
| language | English |
| last_indexed | 2024-04-24T10:43:14Z |
| publishDate | 2024-03-01 |
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| record_format | Article |
| series | International Journal of Molecular Sciences |
| spelling | doaj.art-4c907d634087406aad1aa247a634c3432024-04-12T13:19:20ZengMDPI AGInternational Journal of Molecular Sciences1661-65961422-00672024-03-01257364310.3390/ijms25073643Biochemical Fractionation of Human α-Synuclein in a <i>Drosophila</i> Model of SynucleinopathiesKhondamir Imomnazarov0Joshua Lopez-Scarim1Ila Bagheri2Valerie Joers3Malú Gámez Tansey4Alfonso Martín-Peña5Center for Translational Research in Neurodegenerative Disease, Department of Neuroscience, McKnight Brain Institute, University of Florida, Gainesville, FL 32610, USACenter for Translational Research in Neurodegenerative Disease, Department of Neuroscience, McKnight Brain Institute, University of Florida, Gainesville, FL 32610, USACenter for Translational Research in Neurodegenerative Disease, Department of Neuroscience, McKnight Brain Institute, University of Florida, Gainesville, FL 32610, USACenter for Translational Research in Neurodegenerative Disease, Department of Neuroscience, McKnight Brain Institute, University of Florida, Gainesville, FL 32610, USACenter for Translational Research in Neurodegenerative Disease, Department of Neuroscience, McKnight Brain Institute, University of Florida, Gainesville, FL 32610, USACenter for Translational Research in Neurodegenerative Disease, Department of Neuroscience, McKnight Brain Institute, University of Florida, Gainesville, FL 32610, USASynucleinopathies are a group of central nervous system pathologies that are characterized by the intracellular accumulation of misfolded and aggregated α-synuclein in proteinaceous depositions known as Lewy Bodies (LBs). The transition of α-synuclein from its physiological to pathological form has been associated with several post-translational modifications such as phosphorylation and an increasing degree of insolubility, which also correlate with disease progression in post-mortem specimens from human patients. Neuronal expression of α-synuclein in model organisms, including <i>Drosophila melanogaster</i>, has been a typical approach employed to study its physiological effects. Biochemical analysis of α-synuclein solubility via high-speed ultracentrifugation with buffers of increasing detergent strength offers a potent method for identification of α-synuclein biochemical properties and the associated pathology stage. Unfortunately, the development of a robust and reproducible method for the evaluation of human α-synuclein solubility isolated from <i>Drosophila</i> tissues has remained elusive. Here, we tested different detergents for their ability to solubilize human α-synuclein carrying the pathological mutation A53T from the brains of aged flies. We also assessed the effect of sonication on the solubility of human α-synuclein and optimized a protocol to discriminate the relative amounts of soluble/insoluble human α-synuclein from dopaminergic neurons of the <i>Drosophila</i> brain. Our data established that, using a 5% SDS buffer, the three-step protocol separates cytosolic soluble, detergent-soluble and insoluble proteins in three sequential fractions according to their chemical properties. This protocol shows that sonication breaks down α-synuclein insoluble complexes from the fly brain, making them soluble in the SDS buffer and thus enriching the detergent-soluble fraction of the protocol.https://www.mdpi.com/1422-0067/25/7/3643α-synuclein<i>Drosophila</i>synucleinopathyParkinson’s diseaseSDSchemical fractionation |
| spellingShingle | Khondamir Imomnazarov Joshua Lopez-Scarim Ila Bagheri Valerie Joers Malú Gámez Tansey Alfonso Martín-Peña Biochemical Fractionation of Human α-Synuclein in a <i>Drosophila</i> Model of Synucleinopathies International Journal of Molecular Sciences α-synuclein <i>Drosophila</i> synucleinopathy Parkinson’s disease SDS chemical fractionation |
| title | Biochemical Fractionation of Human α-Synuclein in a <i>Drosophila</i> Model of Synucleinopathies |
| title_full | Biochemical Fractionation of Human α-Synuclein in a <i>Drosophila</i> Model of Synucleinopathies |
| title_fullStr | Biochemical Fractionation of Human α-Synuclein in a <i>Drosophila</i> Model of Synucleinopathies |
| title_full_unstemmed | Biochemical Fractionation of Human α-Synuclein in a <i>Drosophila</i> Model of Synucleinopathies |
| title_short | Biochemical Fractionation of Human α-Synuclein in a <i>Drosophila</i> Model of Synucleinopathies |
| title_sort | biochemical fractionation of human α synuclein in a i drosophila i model of synucleinopathies |
| topic | α-synuclein <i>Drosophila</i> synucleinopathy Parkinson’s disease SDS chemical fractionation |
| url | https://www.mdpi.com/1422-0067/25/7/3643 |
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