A new method for quantifying mitochondrial axonal transport

Abstract

Axonal transport of mitochondria is critical for neuronal survival and function. Automatically quantifying and analyzing mitochondrial movement in a large quantity remain challenging. Here, we report an efficient method for imaging and quantifying axonal mitochondrial transport using microfluidic-chamber-cultured neurons together with a newly developed analysis package named “MitoQuant”. This tool-kit consists of an automated program for tracking mitochondrial movement inside live neuronal axons and a transient-velocity analysis program for analyzing dynamic movement patterns of mitochondria. Using this method, we examined axonal mitochondrial movement both in cultured mammalian neurons and in motor neuron axons of Drosophila in vivo. In 3 different paradigms (temperature changes, drug treatment and genetic manipulation) that affect mitochondria, we have shown that this new method is highly efficient and sensitive for detecting changes in mitochondrial movement. The method significantly enhanced our ability to quantitatively analyze axonal mitochondrial movement and allowed us to detect dynamic changes in axonal mitochondrial transport that were not detected by traditional kymographic analyses.

Keywords

mitochondrial transport / image processing and analysis / FUS proteinopathy and mitochondrial transport defect

References

[1] Ashrafi G, Schlehe JS, LaVoie MJ, Schwarz TL (2014) Mitophagy of damaged mitochondria occurs locally in distal neuronal axons and requires PINK1 and Parkin. J Cell Biol 206:655–670

[2] Borland MK, Trimmer PA, Rubinstein JD, Keeney PM, Mohanakumar K, Liu L, Bennett JP Jr (2008) Chronic, low-dose rotenone reproduces Lewy neurites found in early stages of Parkinson’s disease, reduces mitochondrial movement and slowly kills differentiated SH-SY5Y neural cells. Mol Neurodegener 3:21

[3] Bros H, Hauser A, Paul F, Niesner R, Infante-Duarte C (2015) Assessing mitochondrial movement within neurons: manual versus automated tracking methods. Traffic 16(8):906–917

[4] Buades A, Coll B, Morel JM (2005) A non-local algorithm for image denoising. Comput Vis Pattern Recognit 2:60–65

[5] Chan DC (2006) Mitochondrial fusion and fission in mammals. Annu Rev Cell Dev Biol 22:79–99

[6] Chang DT, Honick AS, Reynolds IJ (2006) Mitochondrial trafficking to synapses in cultured primary cortical neurons. J Neurosci 26:7035–7045

[7] Chen Y, Yang M, Deng J, Chen X, Ye Y, Zhu L, Liu J, Ye H, Shen Y, Li Y, Rao EJ, Fushimi K, Zhou X, Bigio EH, Mesulam M, Xu Q, Wu JY (2011) Expression of human FUS protein in Drosophila leads to progressive neurodegeneration. Protein Cell 2(6):477–486

[8] Cui B, Wu C, Chen L, Ramirez A, Bearer EL, Li WP, Mobley WC, Chu S (2007) One at a time, live tracking of NGF axonal transport using quantum dots. Proc Natl Acad Sci USA 104:13666–13671

[9] Deng J, Yang M, Chen Y, Chen X, Liu J, Sun S, Cheng H, Li Y, Bigio EH, Mesulam M (2015) FUS interacts with HSP60 to promote mitochondrial damage. PLoS Genet 11:e1005357

[10] Devine MJ, Birsa N, Kittler JT (2015) Miro sculpts mitochondrial dynamics in neuronal health and disease. Neurobiol Dis S0969–9961(15):30110–30118

[11] Fukami JI, Yamamoto I, Casida JE (1967) Metabolism of rotenone in vitro by tissue homogenates from mammals and insects. Science 155(3763):713–716

[12] Greenamyre JT, Betarbet R, Sherer TB (2003) The rotenone model of Parkinson’s disease: genes, environment and mitochondria. Parkinsonism Relat Disord 9(Suppl 2):S59–S64

[13] Guo W, Chen Y, Zhou X, Kar A, Ray P, Chen X, Rao EJ, Yang M, Ye H, Zhu L, Liu J, Xu M, Yang Y, Wang C, Zhang D, Bigio EH, Mesulam M, Shen Y, Xu Q, Fushimi K, Wu JY (2011) An ALS-associated mutation affecting TDP-43 enhances protein aggregation, fibril formation and neurotoxicity. Nat Struct Mol Biol 18(7):822–830

[14] Hollenbeck M, Grabensee B (1993) Hemolytic-uremic syndrome and thrombotic thrombocytopenic purpura in the adult. Deutsche medizinische Wochenschrift 118:69–75

[15] Hollenbeck PJ, Saxton WM (2005) The axonal transport of mitochondria. J Cell Sci 118:5411–5419

[16] Huang C, Zhou H, Tong J, Chen H, Liu YJ (2011) FUS transgenic rats develop the phenotypes of amyotrophic lateral sclerosis and frontotemporal lobar degeneration. PLoS Genet 7: e1002011

[17] Kalman RE (1960) A new approach to linear filtering and prediction problems. J Basic Eng 82:35

[18] Kang JS, Tian JH, Pan PY, Zald P, Li C, Deng C, Sheng ZH (2008) Docking of axonal mitochondria by syntaphilin controls their mobility and affects short-term facilitation. Cell 132:137–148

[19] Kann O, Kovacs R (2007) Mitochondria and neuronal activity. Am J Physiol Cell Physiol 292:C641–C657

[20] Lanson NA Jr, Maltare A, King H, Smith R, Kim JH (2011) A Drosophila model of FUS-related neurodegeneration reveals genetic interaction between FUS and TDP-43. Hum Mol Genet 20:2510–2523

[21] Li Y, Yang M, Huang Z, Chen X, Maloney MT, Zhu L, Liu J, Yang Y, Du S, Jiang X, Wu JY (2014) AxonQuant: a microfluidic chamber culture-coupled algorithm that allows high-throughput quantification of axonal damage. Neurosignals 22(1):14–29

[22] Lin MY, Sheng ZH (2015) Regulation of mitochondrial transport in neurons. Exp Cell Res 334:35–44

[23] Liu S, Sawada T, Lee S, Yu W, Silverio G, Alapatt P, Millan I, Shen A, Saxton W, Kanao T (2012) Parkinson’s disease-associated kinase PINK1 regulates Miro protein level and axonal transport of mitochondria. PLoS Genet 8:e1002537

[24] Mackenzie IR, Munoz DG, Kusaka H, Yokota O, Ishihara K (2011) Distinct pathological subtypes of FTLD-FUS. Acta Neuropathol 121:207–218

[25] Mattson MP, Gleichmann M, Cheng A (2008) Mitochondria in neuroplasticity and neurological disorders. Neuron 60:748–766

[26] Miller KE, Sheetz MP (2004) Axonal mitochondrial transport and potential are correlated. J Cell Sci 117(13):2791–2804

[27] Morris RL, Hollenbeck PJ (1995) Axonal transport of mitochondria along microtubules and F-actin in living vertebrate neurons. J Cell Biol 131:1315–1326

[28] Muller MJ, Klumpp S, Lipowsky R (2008) Tug-of-war as a cooperative mechanism for bidirectional cargo transport by molecular motors. Proc Natl Acad Sci USA 105:4609–4614

[29] Muller MJ, Klumpp S, Lipowsky R (2010) Bidirectional transport by molecular motors: enhanced processivity and response to external forces. Biophys J 98:2610–2618

[30] Overly CC, Hollenbeck PJ (1996) Dynamic organization of endocytic pathways in axons of cultured sympathetic neurons. J Neurosci 16:6056–6064

[31] Overly CC, Rieff HI, Hollenbeck PJ (1996) Organelle motility and metabolism in axons vs dendrites of cultured hippocampal neurons. J Cell Sci 109(Pt 5):971–980

[32] Radad K, Rausch WD, Gille G (2006) Rotenone induces cell death in primary dopaminergic culture by increasing ROS production and inhibiting mitochondrial respiration. Neurochem Int 49:379–386

[33] Reddy PH (2011) Abnormal tau, mitochondrial dysfunction, impaired axonal transport of mitochondria, and synaptic deprivation in Alzheimer’s disease. Brain Res 1415:136–148

[34] Reddy PH, Shirendeb UP (2012) Mutant huntingtin, abnormal mitochondrial dynamics, defective axonal transport of mitochondria, and selective synaptic degeneration in Huntington’s disease. Biochim Biophy Acta 1822:101–110

[35] Reeve AK, Krishnan KJ, Turnbull D (2008) Mitochondrial DNA mutations in disease, aging, and neurodegeneration. Ann N Y Acad Sci 1147:21–29

[36] Rizzuto R, Pinton P, Carrington W, Fay FS, Fogarty KE, Lifshitz LM, Tuft RA, Pozzan T (1998) Close contacts with the endoplasmic reticulum as determinants of mitochondrial Ca2+ responses. Science 280:1763–1766

[37] Sabatelli M, Moncada A, Conte A, Lattante S, Marangi G (2013) Mutations in the 3’ untranslated region of FUS causing FUS overexpression are associated with amyotrophic lateral sclerosis. Hum Mol Genet 22:4748–4755

[38] Salinas S, Bilsland LG, Schiavo G (2008) Molecular landmarks along the axonal route: axonal transport in health and disease. Curr Opin Cell Biol 20:445–453

[39] Sanders LH, Greenamyre JT (2013) Oxidative damage to macromolecules in human Parkinson disease and the rotenone model. Free Radic Biol Med 62:111–120

[40] Sheng ZH, Cai Q (2012) Mitochondrial transport in neurons: impact on synaptic homeostasis and neurodegeneration. Nat Rev Neurosci 13:77–93

[41] Singh RP, Thangaraj K, Kale RK (2014) Mitochondria in health and disease. Mitochondrion 16:1

[42] Srivastava P, Panda D (2007) Rotenone inhibits mammalian cell proliferation by inhibiting microtubule assembly through tubulin binding. FEBS J 274:4788–4801

[43] Tourtellotte WG (2015) Axon transport and target tissue innervation hemostasis: relevant perspectives on the etiopathogenesis of familial dysautonomia. Am J Pathol S0002–9440(15):00644–00646

[44] Wallace DC (2013) A mitochondrial bioenergetic etiology of disease. J Clin Invest 123(4):1405–1412

[45] Wang X, Schwarz TL (2009a) Chapter 18 imaging axonal transport of Mitochondria. Methods Enzymol 457:319–333

[46] Wang X, Schwarz TL (2009b) The mechanism of Ca2+ -dependent regulation of kinesin-mediated mitochondrial motility. Cell 136:163–174

[47] Wang X, Su B, Lee HG, Li X, Perry G, Smith MA, Zhu X (2009) Impaired balance of mitochondrial fission and fusion in Alzheimer’s disease. J Neurosci 29:9090–9103

[48] Wang X, Winter D, Ashrafi G, Schlehe J, Wong YL, Selkoe D, Rice S, Steen J, LaVoie MJ, Schwarz TL (2011) PINK1 and parkin target miro for phosphorylation and degradation to arrest mitochondrial motility. Cell 147:893–906

[49] Yadav A, Agarwal S, Tiwari SK, Chaturvedi RK (2014) Mitochondria: prospective targets for neuroprotection in Parkinson’s disease. Curr Pharm Des 20:5558–5573

[50] Yang L, Parton R, Ball G, Qiu Z, Greenaway AH, Davis I, Lu W (2010) An adaptive non-local means filter for denoising live-cell images and improving particle detection. J Struct Biol 172:233–243

[51] Yang L, Qiu Z, Greenaway AH, Lu W (2012) A new framework for particle detection in low-SNR fluorescence live-cell images and its application for improved particle tracking. IEEE Trans Biomed Eng 7:2040–2050

[52] Zhang K, Osakada Y, Vrljic M, Chen L, Mudrakola HV, Cui B (2010) Single-molecule imaging of NGF axonal transport in microfluidic devices. Lab Chip 10:2566–2573

[53] Zhang K, Osakada Y, Xie W, Cui B (2011) Automated image analysis for tracking cargo transport in axons. Microsc Res Tech 74:605–613

RIGHTS & PERMISSIONS

2016 The Author(s) 2016. This article is published with open access at Springerlink.com and journal.hep.com.cn