使用高分辨率呼吸测量法分析 果蝇黑腹 果蝇 PINK1B9-Null 突变体的线粒体功能
Summary
在这里，我们提出了一种高分辨率呼吸测量方案，用于分析 PINK1B9-null 突变果蝇的生物能量学。该方法使用底物-解偶联剂-抑制剂滴定 （SUIT） 方案。
Abstract
神经退行性疾病，包括帕金森病 （PD），和细胞紊乱，如癌症，是一些破坏能量代谢并损害线粒体功能的疾病。线粒体是控制能量代谢和细胞存活和死亡的细胞过程的细胞器。出于这个原因，评估线粒体功能的方法可以为病理和生理过程中的细胞状况提供重要的见解。在这方面，高分辨率呼吸测量 （HRR） 方案允许评估整个线粒体呼吸链功能或特定线粒体复合物的活性。此外，研究线粒体生理学和生物能量学需要遗传和实验上可处理的模型，例如 果蝇黑腹果蝇。
该模型具有多种优点，例如与人体生理学的相似性、生命周期快、易于维护、成本效益高、高通量能力以及将伦理问题降至最低。这些属性共同确立了它作为解剖复杂细胞过程的宝贵工具。本工作解释了如何使用 果蝇黑腹 果蝇 PINK1B9-null 突变体分析线粒体功能。 pink1 基因负责编码 PTEN 诱导的推定激酶 1，通过一个被认为是线粒体自噬的过程，这对于从线粒体网络中去除功能失调的线粒体至关重要。该基因的突变与常染色体隐性遗传性早发性家族性 PD 有关。该模型可用于研究与帕金森病病理生理学有关的线粒体功能障碍。
Introduction
线粒体是控制重要功能的细胞器，包括细胞凋亡调节、钙稳态和参与生物合成途径。通过拥有自主的遗传物质，它们能够为细胞维持和修复过程做出贡献。它们的结构包含电子传递链和氧化磷酸化，这两者都对细胞能量至关重要 1,2,3。特别是，能量控制是通过氧化磷酸化 （OXPHOS） 产生三磷酸腺苷 （ATP） 来实现的2。能量代谢紊乱伴线粒体功能受损发生在细胞存活和死亡中 4,5，通常与多种人类病理学（如癌症）和神经退行性疾病（如帕金森病 （PD）3,6）有关。
帕金森病是一种慢性、进行性和神经系统疾病。这种疾病的主要原因是脑细胞的死亡，特别是在黑质中，黑质负责产生控制运动的神经递质多巴胺 6,7,8。最早将帕金森综合征与线粒体功能障碍联系起来的观察是在 1988 年，在使用抑制呼吸链复合物 I9 的毒素的实验模型中。
目前，有几种方法可以评估线粒体功能障碍 10,11,12,1 3;然而，与传统方法相比，高分辨率呼吸测量法 （HRR） 具有卓越的灵敏度和优势13,14。例如，HRR方案允许评估整个线粒体呼吸链功能或特定线粒体复合物的活性14,15。线粒体功能障碍可以在完整细胞、分离的线粒体甚至体外10、11、13、14 中进行评估。
线粒体功能障碍与许多病理和生理过程密切相关。因此，使用遗传和实验上可处理的模型系统研究线粒体生理学和生物能量学非常重要。在这方面，对果蝇果蝇（Drosophila melanogaster）的研究有几个优点。该模型与人类共享基本的细胞特征和过程，包括使用 DNA 作为遗传物质、共同细胞器以及参与发育、免疫和细胞信号传导的保守分子通路。此外，果蝇具有生命周期快、易于维护、成本低、通量高、伦理问题少等特点，是解剖复杂细胞过程的宝贵工具16,17,18,19,20。
此外，PTEN诱导的推定激酶1（pink1）基因的同源物在黑腹果蝇中表达。它通过线粒体自噬过程在去除受损线粒体方面起着至关重要的作用8.在人类中，该基因的突变使个体易患与线粒体功能障碍相关的常染色体隐性遗传家族性 PD 8,21,22,23。因此，果蝇是一种强大的动物模型，用于研究帕金森病的病理生理学和筛选以线粒体功能障碍和生物能量学为重点的候选药物。因此，本工作解释了如何使用 HRR 技术在 OROBOROS 中使用底物-解偶联剂-抑制剂-滴定 （SUIT） 方案分析黑腹果蝇 PD 模型中的线粒体功能。
Protocol
我们使用了来自布卢明顿果蝇种群中心（ID号：34749）的菌株w1118（白色）和w[*] Pink1[B9]/FM7i，P{w[+mC]=ActGFP}JMR3（称为Pink1B9）（FlyBase ID：FBgn0029891）。在这项研究中，将雄性黑腹果蝇PINK1B9-null突变体与来自w1118菌株的雄性黑腹果蝇进行比较，后者用作对照组（遗传背景）。其他参数必须与呼吸测量实验同时进行分析，以确保果蝇具有?…
Representative Results
在这里，我们发现，与对照果蝇相比，PINK1 B9 无效果蝇在 PINK1B9 无效果蝇中，OXPHOS CI （P = 0.0341） 和 OXPHOS CI&II （P = 0.0392） 状态下的 O2 通量降低（图 4）。这一结果在我们小组29,30 的先前研究结果中也观察到。 CI 和 CII 是电子传输系统 （ETS） 的关键组成部分，其中 CI 负?…
Discussion
HRR 是一种强大的技术，用于研究 黑腹果蝇 和其他生物体的线粒体呼吸和能量代谢。它提供了对线粒体功能的详细和定量评估，使研究人员能够深入了解细胞的生物能量学。此处介绍的方案描述了使用SUIT协议在 黑腹果蝇中评估线粒体呼吸链功能和特定线粒体复合物的活性。SUIT方案涉及系统地操纵各种底物、解偶联剂和抑制剂，以检查线粒体呼吸的不同方面。
?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
作者感谢巴西机构 Coordenação de Aperfeiçoamento de Pesquisa Pessoal de Nível Superior （CAPES EPIDEMIAS 09 #88887.505377/2020）。P.M. （#88887.512821/2020-00） 和 TD （#88887.512883/2020-00） 是研究奖学金获得者。
Materials
| ADP | Sigma-Aldrich | A5285 | Adenosine 5′-diphosphate sodium sal (CAS number 72696-48-1); ≥95%; molecular weight = 501.31 g/mol. |
| Ágar | Kasv | K25-1800 | For bacteriologal use |
| Antimycin-A | Sigma-Aldrich | A8674 | Antimycin A from Streptomyces sp. (CAS number 1397-94-0); molecular weight 540 g/mol; |
| Bovine Serum Albumin (BSA) | Sigma-Aldrich | A7030 | Bovine Serum Albumin (CAS number 9048-46-8); pH 7,0 ≥ 98% |
| Datlab software | Oroboros Instruments, Innsbruck, Austria | 20700 | Software for data acquisition and analysis |
| Digitonin | Sigma-Aldrich | D 5628 | CAS number 11024-24-1 |
| Distilled water | |||
| Drosophila melanogaster strain w[*] Pink1[B9]/FM7i, P{w[+mC]=ActGFP}JMR3 | Obtained from Bloomington Drosophila stock center | ||
| Drosophila melanogaster strain w1118 | Obtained from the Federal University of Santa Maria | ||
| EGTA | Sigma-Aldrich | E8145 | Ethylene glycol-bis(2-aminoethylether)-N,N,N',N'-tetraacetic acid (CAS number 13638-13-3); ≥97%; molecular weight =468.28 g/mol |
| FCCP | Sigma-Aldrich | C2920 | Carbonyl cyanide 4- (trifluoromethoxy)phenylhydrazone (CAS number 370-86-5); ≥98% (TLC), powder |
| GraphPad Prism version 8.0.1. | Software for data acquisition and analysis | ||
| Hepes | Sigma-Aldrich | H4034 | 4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid (CAS number 7365-45-9); ≥99,5% (titration), cell cultured tested; molecular weight = 238.30 g/mol |
| High-resolution respirometer Oxygraph O2K | Oroboros Instruments, Innsbruck, Austria | 10022-02 | Startup O2K respirometer kit |
| KH2PO4 | Sigma-Aldrich | P5379 | Monopotassium phosphate (CAS number 7778-77-0); Reagente Plus, molecular weigt = 136.09 g/mol |
| KOH | Sigma-Aldrich | 211473 | Potassium hydroxide (CAS number 1310-58-3); ACS reagent, ≥85%, pellets |
| Malate | Sigma-Aldrich | M1296 | Malonic acid (CAS number 141-82-2); 99%, molecular weight = 104.06 g/mol). A solution is pH adjusted to approximately 7.0. |
| Malic acid | Sigma-Aldrich | M1000 | (S)-(−)-2-Hydroxysuccinic acid (CAS number 97-67-6); ≥95% ; molecular weight = 134.09 g/mol |
| MES | Sigma-Aldrich | M3671 | 2-(N-Morpholino)ethanesulfonic acid (CAS number 4432-31-9); ≥99% (titration); molecular weight = 195.24 g/mol |
| MgCl2 | Sigma-Aldrich | M8266 | Magnesium chloride (CAS number 7786-30-3); anhydrous, ≥98%, molecular weight = 95.21 g/mol |
| Microcentrifuge tubes | Eppendorf | ||
| O2K-Titration Set | Oroboros Instruments, Innsbruck, Austria | 20820-03 | Hamilton syringes with different volumes |
| Oligomycin | Sigma-Aldrich | O 4876 | Oligomycin from Streptomyces diastatochromogenes (CAS number 1404-19-9); ≥90% total oligomycins basis (HPLC) |
| Pistil to homogenization | |||
| Proline | Sigma-Aldrich | P0380 | L-Proline (CAS number 147-85-3); powder; 99%; molecular weight = 115.13 g/mol |
| Pyruvate | Sigma-Aldrich | P2256 | Sodium pyruvate (CAS number 113-24-6), ≥99%; molecular weight = 110.04 g/mol |
| Rotenone | Sigma-Aldrich | R8875 | Rotetone (CAS number 83-79-4); ≥95%, molecular weight 394.42 g/ mol |
| Succinate | Sigma-Aldrich | S 2378 | Sodium succinate dibasic hexahydrate (CAS number 6106-21-4); ≥99% |
| Sucrose | Merck | 107,651,000 | Sucrose for microbiology use (CAS number 57-50-1) |
| Taurine | Sigma-Aldrich | T0625 | CAS number 107-35-7 |
References
- Brand, M. D., Orr, A. L., Perevoshchikova, I. V., Quinlan, C. L. The role mitochondrial function and cellular bioenergetics in ageing and disease. Br J Dermatol. 169, 1-8 (2013).
- Ramaccini, D., et al. Mitochondrial function and dysfunction in dilated cardiomyopathy. Front Cell Dev Biol. 8, 624216 (2021).
- Zhunina, O. A., et al. The role of mitochondrial dysfunction in vascular disease, tumorigenesis, and diabetes. Front Mol Biosci. 8, 671908 (2021).
- Prasun, P. Mitochondrial dysfunction in metabolic syndrome. Biochim Biophys Acta. 1866 (10), 165838 (2020).
- Bhatti, J. S., Bhatti, G. K., Reddy, P. H. Mitochondrial dysfunction and oxidative stress in metabolic disorders – A Step towards mitochondria based therapeutic strategies. Biochim Biophys Acta. 1863 (5), 1066-1077 (2017).
- Perier, C., Vila, M. Mitochondrial biology and Parkinson’s disease. Cold Spring Harb Perspect Med. 2 (2), 009332 (2012).
- DeMaagd, G., Philip, A. Parkinson’s disease and its management. Pharmacy and Therapeutics. 40 (8), 504-532 (2015).
- Miyazaki, N., et al. PINK1-dependent and Parkin-independent mitophagy is involved in reprogramming of glycometabolism in pancreatic cancer cells. Biochem Biophys Res Commun. 625, 167-173 (2022).
- Kopin, I. J., Markey, S. P. MPTP toxicity: implications for research in Parkinson’s disease. Annual Review of Neuroscience. 11, 81-96 (1988).
- Connolly, N. M. C., et al. Guidelines on experimental methods to assess mitochondrial dysfunction in cellular models of neurodegenerative diseases. Cell Death Differ. 25 (3), 542-572 (2018).
- Brand, M. D., Nicholls, D. G. Assessing mitochondrial dysfunction in cells. Biochem J. 435, 297-312 (2011).
- Haynes, C. M., Fiorese, C. J., Lin, Y. -. F. Evaluating and responding to mitochondrial dysfunction: The UPRmt and beyond. Trends Cell Mol Biol. 23 (7), 311-318 (2013).
- Long, Q., Huang, L., Huang, K., Yang, Q. Assessing mitochondrial bioenergetics in isolated mitochondria from mouse heart tissues using oroboros 2k-oxygraph. Methods Mol Biol. 1966, 237-246 (2019).
- Scheiber, D., et al. High-resolution respirometry in human endomyocardial biopsies shows reduced ventricular oxidative capacity related to heart failure. Exp Mol Med. 51 (2), 1-10 (2019).
- Krumschnabel, G., Eigentler, A., Fasching, M., Gnaiger, E. Chapter nine – use of safranin for the assessment of mitochondrial membrane potential by high-resolution respirometry and fluorometry. Methods Enzymol. 542, 163-181 (2014).
- Demir, E. The potential use of Drosophila as an in vivo model organism for COVID-19-related research: a review. Turk J Biol. 45 (4), 559-569 (2021).
- Hales, K. G., Korey, C. A., Larracuente, A. M., Roberts, D. M. Genetics on the fly: a primer on the Drosophila model system. 遗传学. 201 (3), 815-842 (2015).
- McBride, S. M. J., Holloway, S. L., Jongens, T. A. Using Drosophila as a tool to identify pharmacological therapies for fragile X syndrome. Drug Discov Today Technol Technologies. 10 (1), e129-e136 (2013).
- Tennessen, J. M., Barry, W. E., Cox, J., Thummel, C. S. Methods for studying metabolism in Drosophila. Methods (San Diego, Calif). 68 (1), 105-115 (2014).
- Bar, S., Prasad, M., Datta, R. Neuromuscular degeneration and locomotor deficit in a Drosophila model of mucopolysaccharidosis VII is attenuated by treatment with resveratrol). Dis Model Mech. 11 (11), (2018).
- Imai, Y. PINK1-Parkin signaling in Parkinson’s disease: Lessons from Drosophila. Neurosci Res. 159, 40-46 (2020).
- Julienne, H., Buhl, E., Leslie, D. S., Hodge, J. J. L. Drosophila PINK1 and parkin loss-of-function mutants display a range of non-motor Parkinson’s disease phenotypes. Neurobiol Dis. 104, 15-23 (2017).
- Biswas, S., Bagchi, A. Analysis of the structural dynamics of the mutations in the kinase domain of PINK1 protein associated with Parkinson’s disease. Gene. 857, 147183 (2023).
- Koh, H., Chung, J. PINK1 and Parkin to control mitochondria remodeling. Anat Cell Biol. 43 (3), 179-184 (2010).
- Zhu, M., Li, X., Tian, X., Wu, C. Mask loss-of-function rescues mitochondrial impairment and muscle degeneration of Drosophila pink1 and parkin mutants. Hum Mol Genet. 24 (11), 3272-3285 (2015).
- Park, J., et al. Mitochondrial dysfunction in Drosophila PINK1 mutants is complemented by parkin. Nature. 441 (7097), 1157-1161 (2006).
- Dytham, C. . Choosing and using statistics: a biologist’s guide. , (2011).
- Bonora, M., et al. ATP synthesis and storage. Purinergic Signal. 8 (3), 343-357 (2012).
- Gonçalves, D. F., et al. Caffeine improves mitochondrial function in PINK1B9-null mutant Drosophila melanogaster. J Bioenerg Biomembr. 55 (1), 1-13 (2023).
- Gonçalves, D. F., et al. Mitochondrial function and cellular energy maintenance during aging in a Drosophila melanogaster model of Parkinson disease. Mitochondrion. 65, 166-175 (2022).
- Janowska, J. I., et al. Mitochondrial respiratory chain complex I dysfunction induced by N-methyl carbamate ex vivo can be alleviated with a cell-permeable succinate prodrug. Toxicol In Vitro. 65, 104794 (2020).
- Ngo, D. T. M., et al. Oxidative modifications of mitochondrial complex II are associated with insulin resistance of visceral fat in obesity. Physiol Endocrinol Metab. 316 (2), E168-E177 (2019).
- Stroud, D. A., et al. Accessory subunits are integral for assembly and function of human mitochondrial complex I. Nature. 538 (7623), 123-126 (2016).
- Senyilmaz, D., et al. Regulation of mitochondrial morphology and function by stearoylation of TFR1. Nature. 525 (7567), 124-128 (2015).
- Poddighe, S., et al. Mucuna pruriens (Velvet bean) rescues motor, olfactory, mitochondrial and synaptic impairment in PINK1B9 Drosophila melanogaster genetic model of Parkinson’s disease. PloS One. 9 (10), e110802 (2014).
- Pesta, D., Gnaiger, E. High-resolution respirometry: OXPHOS protocols for human cells and permeabilized fibers from small biopsies of human muscle. Methods Mol Biol (Clifton, N.J). 810, 25-58 (2012).
- Winer, L. S. P., Wu, M. Rapid analysis of glycolytic and oxidative substrate flux of cancer cells in a microplate. PloS One. 9 (10), e109916 (2014).
- Plitzko, B., Loesgen, S. Measurement of oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) in culture cells for assessment of the energy metabolism. Bio Protoc. 8 (10), e2850 (2018).
- Wu, M., et al. Multiparameter metabolic analysis reveals a close link between attenuated mitochondrial bioenergetic function and enhanced glycolysis dependency in human tumor cells. Am J Physiol. Cell Physiol. 292 (1), C125-C136 (2007).
- Divakaruni, A. S., Jastroch, M. A practical guide for the analysis, standardization and interpretation of oxygen consumption measurements. Nat Metab. 4 (8), 978-994 (2022).
- Bingol, B., Sheng, M. Mechanisms of mitophagy: PINK1, Parkin, USP30 and beyond. Free Radic Biol Med. 100, 210-222 (2016).
Cite This Article
Michelotti, P., Duarte, T., Dalla Corte, C. L. Analyzing Mitochondrial Function in a Drosophila melanogaster PINK1B9-Null Mutant Using High-resolution Respirometry. J. Vis. Exp. (201), e65664, doi:10.3791/65664 (2023).