Mutant LRRK2 Elicits Calcium Imbalance and Depletion of Dendritic Mitochondria in Neurons
2012; Elsevier BV; Volume: 182; Issue: 2 Linguagem: Inglês
10.1016/j.ajpath.2012.10.027
ISSN1525-2191
AutoresSalvatore J. Cherra, Erin Steer, Aaron M. Gusdon, Kirill Kiselyov, Charleen T. Chu,
Tópico(s)Genetic Neurodegenerative Diseases
ResumoMutations in the leucine-rich repeat kinase 2 (LRRK2) have been associated with familial and sporadic cases of Parkinson disease. Mutant LRRK2 causes in vitro and in vivo neurite shortening, mediated in part by autophagy, and a parkinsonian phenotype in transgenic mice; however, the underlying mechanisms remain unclear. Because mitochondrial content/function is essential for dendritic morphogenesis and maintenance, we investigated whether mutant LRRK2 affects mitochondrial homeostasis in neurons. Mouse cortical neurons expressing either LRRK2 G2019S or R1441C mutations exhibited autophagic degradation of mitochondria and dendrite shortening. In addition, mutant LRRK2 altered the ability of the neurons to buffer intracellular calcium levels. Either calcium chelators or inhibitors of voltage-gated L-type calcium channels prevented mitochondrial degradation and dendrite shortening. These data suggest that mutant LRRK2 causes a deficit in calcium homeostasis, leading to enhanced mitophagy and dendrite shortening. Mutations in the leucine-rich repeat kinase 2 (LRRK2) have been associated with familial and sporadic cases of Parkinson disease. Mutant LRRK2 causes in vitro and in vivo neurite shortening, mediated in part by autophagy, and a parkinsonian phenotype in transgenic mice; however, the underlying mechanisms remain unclear. Because mitochondrial content/function is essential for dendritic morphogenesis and maintenance, we investigated whether mutant LRRK2 affects mitochondrial homeostasis in neurons. Mouse cortical neurons expressing either LRRK2 G2019S or R1441C mutations exhibited autophagic degradation of mitochondria and dendrite shortening. In addition, mutant LRRK2 altered the ability of the neurons to buffer intracellular calcium levels. Either calcium chelators or inhibitors of voltage-gated L-type calcium channels prevented mitochondrial degradation and dendrite shortening. These data suggest that mutant LRRK2 causes a deficit in calcium homeostasis, leading to enhanced mitophagy and dendrite shortening. CME Accreditation Statement: This activity ("ASIP 2013 AJP CME Program in Pathogenesis") has been planned and implemented in accordance with the Essential Areas and policies of the Accreditation Council for Continuing Medical Education (ACCME) through the joint sponsorship of the American Society for Clinical Pathology (ASCP) and the American Society for Investigative Pathology (ASIP). ASCP is accredited by the ACCME to provide continuing medical education for physicians.The ASCP designates this journal-based CME activity ("ASIP 2013 AJP CME Program in Pathogenesis") for a maximum of 48 AMA PRA Category 1 Credit(s)™. Physicians should only claim credit commensurate with the extent of their participation in the activity.CME Disclosures: The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose. CME Accreditation Statement: This activity ("ASIP 2013 AJP CME Program in Pathogenesis") has been planned and implemented in accordance with the Essential Areas and policies of the Accreditation Council for Continuing Medical Education (ACCME) through the joint sponsorship of the American Society for Clinical Pathology (ASCP) and the American Society for Investigative Pathology (ASIP). ASCP is accredited by the ACCME to provide continuing medical education for physicians. The ASCP designates this journal-based CME activity ("ASIP 2013 AJP CME Program in Pathogenesis") for a maximum of 48 AMA PRA Category 1 Credit(s)™. Physicians should only claim credit commensurate with the extent of their participation in the activity. CME Disclosures: The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose. Parkinson disease (PD) is a progressive neurodegenerative disease that affects both subcortical and cortical brain regions, leading to deficits in motor control and cognitive decline. Although most cases of PD are sporadic, familial mutations account for nearly 10% of patients with PD.1Toulouse A. Sullivan A.M. Progress in Parkinson's disease: where do we stand?.Prog Neurobiol. 2008; 85: 376-392Crossref PubMed Scopus (153) Google Scholar Interestingly, mutations in the leucine-rich repeat kinase 2 (LRRK2) have been identified in approximately 5% of familial PD cases and 1% of sporadic PD cases.2Brice A. Genetics of Parkinson's disease: lRRK2 on the rise.Brain. 2005; 128: 2760-2762Crossref PubMed Scopus (74) Google Scholar Although the underlying pathogenesis remains poorly understood, changes in autophagy regulation and mitochondrial homeostasis are emerging as common pathways in toxin and genetic models of PD.3Schon E.A. Przedborski S. Mitochondria: the next (neurode)generation.Neuron. 2011; 70: 1033-1053Abstract Full Text Full Text PDF PubMed Scopus (442) Google Scholar, 4Gusdon A.M. Zhu J. Van Houten B. Chu C.T. ATP13A2 regulates mitochondrial bioenergetics through macroautophagy.Neurobiol Dis. 2011; 45: 962-972Crossref PubMed Scopus (129) Google Scholar Previous studies have highlighted the importance of mitochondrial quality control in PD models using mitochondrial neurotoxins or proteins encoded by recessive PD-associated genes (Parkin, DJ-1, and PINK1) that traffic to the mitochondria.5Betarbet R. Sherer T.B. MacKenzie G. Garcia-Osuna M. Panov A.V. Greenamyre J.T. Chronic systemic pesticide exposure reproduces features of Parkinson's disease.Nat Neurosci. 2000; 3: 1301-1306Crossref PubMed Scopus (3037) Google Scholar, 6Narendra D. Tanaka A. Suen D.F. Youle R.J. Parkin is recruited selectively to impaired mitochondria and promotes their autophagy.J Cell Biol. 2008; 183: 795-803Crossref PubMed Scopus (2984) Google Scholar, 7Poole A.C. Thomas R.E. Andrews L.A. McBride H.M. Whitworth A.J. Pallanck L.J. The PINK1/Parkin pathway regulates mitochondrial morphology.Proc Natl Acad Sci U S A. 2008; 105: 1638-1643Crossref PubMed Scopus (739) Google Scholar, 8Ved R. Saha S. Westlund B. Perier C. Burnam L. Sluder A. Hoener M. Rodrigues C.M. Alfonso A. Steer C. Liu L. Przedborski S. Wolozin B. Similar patterns of mitochondrial vulnerability and rescue induced by genetic modification of alpha-synuclein, parkin, and DJ-1 in Caenorhabditis elegans.J Biol Chem. 2005; 280: 42655-42668Abstract Full Text Full Text PDF PubMed Scopus (228) Google Scholar Although these mitochondrial toxins and mutant proteins perturb mitochondrial respiration, degradation, and calcium buffering, less is known about how nonmitochondrially targeted PD gene products, such as LRRK2 or α-synuclein, affect mitochondrial homeostasis.9Chu C.T. Diversity in the regulation of autophagy and mitophagy: lessons from Parkinson's disease.Parkinsons Dis. 2011; 2011: 789431Crossref PubMed Scopus (34) Google Scholar, 10Lin M.T. Beal M.F. Mitochondrial dysfunction and oxidative stress in neurodegenerative diseases.Nature. 2006; 443: 787-795Crossref PubMed Scopus (4835) Google Scholar, 11Mattson M.P. Calcium and neurodegeneration.Aging Cell. 2007; 6: 337-350Crossref PubMed Scopus (597) Google Scholar Recent studies suggest that autophagy regulation may be a point of convergence among the Parkin, PINK1, DJ-1, α-synuclein, and LRRK2 pathways.6Narendra D. Tanaka A. Suen D.F. Youle R.J. Parkin is recruited selectively to impaired mitochondria and promotes their autophagy.J Cell Biol. 2008; 183: 795-803Crossref PubMed Scopus (2984) Google Scholar, 12Plowey E.D. Cherra 3rd, S.J. Liu Y.J. Chu C.T. Role of autophagy in G2019S-LRRK2-associated neurite shortening in differentiated SH-SY5Y cells.J Neurochem. 2008; 105: 1048-1056Crossref PubMed Scopus (435) Google Scholar, 13Dagda R.K. Cherra 3rd, S.J. Kulich S.M. Tandon A. Park D. Chu C.T. Loss of pink1 function promotes mitophagy through effects on oxidative stress and mitochondrial fission.J Biol Chem. 2009; 284: 13843-13855Abstract Full Text Full Text PDF PubMed Scopus (781) Google Scholar, 14Geisler S. Holmstrom K.M. Skujat D. Fiesel F.C. Rothfuss O.C. Kahle P.J. Springer W. PINK1/Parkin-mediated mitophagy is dependent on VDAC1 and p62/SQSTM1.Nat Cell Biol. 2010; 12: 119-131Crossref PubMed Scopus (2119) Google Scholar, 15Cuervo A.M. Stefanis L. Fredenburg R. Lansbury P.T. Sulzer D. Impaired degradation of mutant alpha-synuclein by chaperone-mediated autophagy.Science. 2004; 305: 1292-1295Crossref PubMed Scopus (1614) Google Scholar Autophagy is a highly conserved, cytoplasmic recycling pathway that engulfs proteins, aggregates, and organelles in autophagosomes and traffics them to the lysosome for degradation.16Klionsky D.J. Emr S.D. Autophagy as a regulated pathway of cellular degradation.Science. 2000; 290: 1717-1721Crossref PubMed Scopus (3048) Google Scholar Autophagic turnover of mitochondria and aggregate-prone proteins is one way to maintain healthy, functioning neurons; however, excessive degradation may have deleterious effects leading to neurodegeneration.17Cherra 3rd, S.J. Chu C.T. Autophagy in neuroprotection and neurodegeneration: a question of balance.Future Neurol. 2008; 3: 309-323Crossref PubMed Scopus (160) Google Scholar Therefore, PD-associated genes may modulate mitochondrial homeostasis by directly regulating mitochondrial function or by affecting mitochondrial degradation.4Gusdon A.M. Zhu J. Van Houten B. Chu C.T. ATP13A2 regulates mitochondrial bioenergetics through macroautophagy.Neurobiol Dis. 2011; 45: 962-972Crossref PubMed Scopus (129) Google Scholar Although LRRK2 function remains unknown, previous studies indicate a role in regulating neurite morphological characteristics, cytoskeletal dynamics, or the endosomal-autophagic system.12Plowey E.D. Cherra 3rd, S.J. Liu Y.J. Chu C.T. Role of autophagy in G2019S-LRRK2-associated neurite shortening in differentiated SH-SY5Y cells.J Neurochem. 2008; 105: 1048-1056Crossref PubMed Scopus (435) Google Scholar, 18Alegre-Abarrategui J. Christian H. Lufino M.M. Mutihac R. Venda L.L. Ansorge O. Wade-Martins R. LRRK2 regulates autophagic activity and localizes to specific membrane microdomains in a novel human genomic reporter cellular model.Hum Mol Genet. 2009; 18: 4022-4034Crossref PubMed Scopus (368) Google Scholar, 19Cherra 3rd, S.J. Kulich S.M. Uechi G. Balasubramani M. Mountzouris J. Day B.W. Chu C.T. Regulation of the autophagy protein LC3 by phosphorylation.J Cell Biol. 2010; 190: 533-539Crossref PubMed Scopus (259) Google Scholar, 20Gillardon F. Leucine-rich repeat kinase 2 phosphorylates brain tubulin-beta isoforms and modulates microtubule stability: a point of convergence in parkinsonian neurodegeneration?.J Neurochem. 2009; 110: 1514-1522Crossref PubMed Scopus (186) Google Scholar, 21Jaleel M. Nichols R.J. Deak M. Campbell D.G. Gillardon F. Knebel A. Alessi D.R. LRRK2 phosphorylates moesin at threonine-558: characterization of how Parkinson's disease mutants affect kinase activity.Biochem J. 2007; 405: 307-317Crossref PubMed Scopus (429) Google Scholar, 22Li Y. Liu W. Oo T.F. Wang L. Tang Y. Jackson-Lewis V. Zhou C. Geghman K. Bogdanov M. Przedborski S. Beal M.F. Burke R.E. Li C. Mutant LRRK2(R1441G) BAC transgenic mice recapitulate cardinal features of Parkinson's disease.Nat Neurosci. 2009; 12: 826-828Crossref PubMed Scopus (421) Google Scholar, 23MacLeod D. Dowman J. Hammond R. Leete T. Inoue K. Abeliovich A. The familial parkinsonism gene LRRK2 regulates neurite process morphology.Neuron. 2006; 52: 587-593Abstract Full Text Full Text PDF PubMed Scopus (481) Google Scholar, 24Parisiadou L. Xie C. Cho H.J. Lin X. Gu X.L. Long C.X. Lobbestael E. Baekelandt V. Taymans J.M. Sun L. Cai H. Phosphorylation of ezrin/radixin/moesin proteins by LRRK2 promotes the rearrangement of actin cytoskeleton in neuronal morphogenesis.J Neurosci. 2009; 29: 13971-13980Crossref PubMed Scopus (246) Google Scholar, 25Ramonet D. Daher J.P. Lin B.M. Stafa K. Kim J. Banerjee R. Westerlund M. Pletnikova O. Glauser L. Yang L. Liu Y. Swing D.A. Beal M.F. Troncoso J.C. McCaffery J.M. Jenkins N.A. Copeland N.G. Galter D. Thomas B. Lee M.K. Dawson T.M. Dawson V.L. Moore D.J. Dopaminergic neuronal loss: reduced neurite complexity and autophagic abnormalities in transgenic mice expressing G2019S mutant LRRK2.PLoS One. 2011; 6: e18568Crossref PubMed Scopus (311) Google Scholar PD-associated mutations in the GTPase domain (R1441C) and the kinase domain (G2019S) have previously been shown to cause neurite shortening and autophagy induction.12Plowey E.D. Cherra 3rd, S.J. Liu Y.J. Chu C.T. Role of autophagy in G2019S-LRRK2-associated neurite shortening in differentiated SH-SY5Y cells.J Neurochem. 2008; 105: 1048-1056Crossref PubMed Scopus (435) Google Scholar, 19Cherra 3rd, S.J. Kulich S.M. Uechi G. Balasubramani M. Mountzouris J. Day B.W. Chu C.T. Regulation of the autophagy protein LC3 by phosphorylation.J Cell Biol. 2010; 190: 533-539Crossref PubMed Scopus (259) Google Scholar, 22Li Y. Liu W. Oo T.F. Wang L. Tang Y. Jackson-Lewis V. Zhou C. Geghman K. Bogdanov M. Przedborski S. Beal M.F. Burke R.E. Li C. Mutant LRRK2(R1441G) BAC transgenic mice recapitulate cardinal features of Parkinson's disease.Nat Neurosci. 2009; 12: 826-828Crossref PubMed Scopus (421) Google Scholar, 23MacLeod D. Dowman J. Hammond R. Leete T. Inoue K. Abeliovich A. The familial parkinsonism gene LRRK2 regulates neurite process morphology.Neuron. 2006; 52: 587-593Abstract Full Text Full Text PDF PubMed Scopus (481) Google Scholar, 25Ramonet D. Daher J.P. Lin B.M. Stafa K. Kim J. Banerjee R. Westerlund M. Pletnikova O. Glauser L. Yang L. Liu Y. Swing D.A. Beal M.F. Troncoso J.C. McCaffery J.M. Jenkins N.A. Copeland N.G. Galter D. Thomas B. Lee M.K. Dawson T.M. Dawson V.L. Moore D.J. Dopaminergic neuronal loss: reduced neurite complexity and autophagic abnormalities in transgenic mice expressing G2019S mutant LRRK2.PLoS One. 2011; 6: e18568Crossref PubMed Scopus (311) Google Scholar LRRK2 may also play roles in synaptic transmission26Piccoli G. Condliffe S.B. Bauer M. Giesert F. Boldt K. De Astis S. Meixner A. Sarioglu H. Vogt-Weisenhorn D.M. Wurst W. Gloeckner C.J. Matteoli M. Sala C. Ueffing M. LRRK2 controls synaptic vesicle storage and mobilization within the recycling pool.J Neurosci. 2011; 31: 2225-2237Crossref PubMed Scopus (212) Google Scholar, 27Shin N. Jeong H. Kwon J. Heo H.Y. Kwon J.J. Yun H.J. Kim C.H. Han B.S. Tong Y. Shen J. Hatano T. Hattori N. Kim K.S. Chang S. Seol W. LRRK2 regulates synaptic vesicle endocytosis.Exp Cell Res. 2008; 314: 2055-2065Crossref PubMed Scopus (287) Google Scholar, 28Li X. Patel J.C. Wang J. Avshalumov M.V. Nicholson C. Buxbaum J.D. Elder G.A. Rice M.E. Yue Z. Enhanced striatal dopamine transmission and motor performance with LRRK2 overexpression in mice is eliminated by familial Parkinson's disease mutation G2019S.J Neurosci. 2010; 30: 1788-1797Crossref PubMed Scopus (264) Google Scholar and protein translation.29Gehrke S. Imai Y. Sokol N. Lu B. Pathogenic LRRK2 negatively regulates microRNA-mediated translational repression.Nature. 2010; 466: 637-641Crossref PubMed Scopus (324) Google Scholar, 30Imai Y. Gehrke S. Wang H.Q. Takahashi R. Hasegawa K. Oota E. Lu B. Phosphorylation of 4E-BP by LRRK2 affects the maintenance of dopaminergic neurons in Drosophila.EMBO J. 2008; 27: 2432-2443Crossref PubMed Scopus (365) Google Scholar A recent study of skin fibroblasts from patients bearing the G2019S mutation showed altered mitochondrial function.31Mortiboys H. Johansen K.K. Aasly J.O. Bandmann O. Mitochondrial impairment in patients with Parkinson disease with the G2019S mutation in LRRK2.Neurology. 2010; 75: 2017-2020Crossref PubMed Scopus (233) Google Scholar Because LRRK2 partially associates with mitochondria18Alegre-Abarrategui J. Christian H. Lufino M.M. Mutihac R. Venda L.L. Ansorge O. Wade-Martins R. LRRK2 regulates autophagic activity and localizes to specific membrane microdomains in a novel human genomic reporter cellular model.Hum Mol Genet. 2009; 18: 4022-4034Crossref PubMed Scopus (368) Google Scholar, 32Biskup S. Moore D.J. Celsi F. Higashi S. West A.B. Andrabi S.A. Kurkinen K. Yu S.W. Savitt J.M. Waldvogel H.J. Faull R.L. Emson P.C. Torp R. Ottersen O.P. Dawson T.M. Dawson V.L. Localization of LRRK2 to membranous and vesicular structures in mammalian brain.Ann Neurol. 2006; 60: 557-569Crossref PubMed Scopus (425) Google Scholar and interacts with PD-associated proteins that regulate mitochondrial stress,33Venderova K. Kabbach G. Abdel-Messih E. Zhang Y. Parks R.J. Imai Y. Gehrke S. Ngsee J. Lavoie M.J. Slack R.S. Rao Y. Zhang Z. Lu B. Haque M.E. Park D.S. Leucine-rich repeat kinase 2 interacts with Parkin, DJ-1 and PINK-1 in a Drosophila melanogaster model of Parkinson's disease.Hum Mol Genet. 2009; 18: 4390-4404Crossref PubMed Scopus (145) Google Scholar we investigated mechanisms by which mutations in LRRK2 affect mitochondrial homeostasis in neurons. All experiments were performed in accordance with research protocols approved by the University of Pittsburgh (Pittsburgh, PA) Institutional Animal Care and Use Committee. Mouse E15 cortical neurons were maintained in Neurobasal, supplemented with 2% B27 and 2 mmol/L Glutamax (all from Invitrogen, Carlsbad, CA). The cortical neurons analyzed were pyramidal neurons based on soma size (212 ± 6 μm2) and dendrite morphological characteristics.34Ballesteros-Yáñez I. Benavides-Piccione R. Bourgeois J.P. Changeux J.P. DeFelipe J. Alterations of cortical pyramidal neurons in mice lacking high-affinity nicotinic receptors.Proc Natl Acad Sci U S A. 2010; 107: 11567-11572Crossref PubMed Scopus (74) Google Scholar, 35Schubert D. Kötter R. Luhmann H.J. Staiger J.F. Morphology, electrophysiology and functional input connectivity of pyramidal neurons characterizes a genuine layer va in the primary somatosensory cortex.Cereb Cortex. 2006; 16: 223-236Crossref PubMed Scopus (114) Google Scholar For mitochondrial content and dendrite length measurements, neurons were transfected with the indicated plasmids at 7 days in vitro (DIV). Neurons were then fixed with 4% paraformaldehyde 5 days after transfection (12 DIV) for analysis of mitochondrial content and autophagy and 14 days after transfection (21 DIV) for dendrite length. Neurons were stained with antibodies to microtubule associated protein-2 (MAP2) (Millipore, Billerica, MA) to identify dendrites and with anti-green fluorescent protein (GFP; Invitrogen) to identify transfected neurons for dendrite length analysis. Axons were defined as MAP2-negative processes that exhibited known morphological characteristics (thin and uniform diameter and length of more than two high-power fields) that distinguish axons from dendrites.36Kang J.S. Tian J.H. Pan P.Y. Zald P. Li C. Deng C. Sheng Z.H. Docking of axonal mitochondria by syntaphilin controls their mobility and affects short-term facilitation.Cell. 2008; 132: 137-148Abstract Full Text Full Text PDF PubMed Scopus (431) Google Scholar ImageJ software version 1.42q (NIH) was used to measure the mitochondrial content in primary dendrites, axons, and soma; the primary dendrite area/neuron; and the summated dendrite length/neuron, as illustrated in Supplemental Figure S1. Mitochondrial content was calculated from raw images as follows: area of cytochrome c oxidase subunit 8 targeting sequence (COX8)–GFP–labeled mitochondria/area for each compartment. For mitochondrial content and autophagy analysis, neurons were treated with 1 nmol/L bafilomycin A (Merck KGaA, Darmstadt, Germany), 2 μmol/L 1,2-bis (aminophenoxy) ethane-N,N,N′,N′-tetraacetic acid acetoxymethyl ester (BAPTA-AM; Invitrogen), 2 mmol/L EGTA (Sigma-Aldrich Corp, St Louis, MO), 1 μmol/L nitrendipine (Tocris Bioscience, Ellisville, MO), 1 μmol/L NiCl2 (Sigma-Aldrich Corp), 50 nmol/L ω-agatoxin (Tocris Bioscience), or 100 nmol/L w-conotoxin (Tocris Bioscience) 3 days after transfection and analyzed 2 days later. For dendrite length analysis, neurons were treated as indicated at 10 days after transfection and analyzed 4 days later. SH-SY5Y and HEK293 cells were cultured in Dulbecco's modified Eagle's medium (BioWhittaker, Walkersville, MD) with 10% fetal bovine serum (BioWhittaker), 2 mmol/L l-glutamine (BioWhittaker), and 10 μmol/L retinoic acid (Sigma-Aldrich Corp) for 3 days before transfection. For neurite length, autophagosome quantification, and mitochondrial content in SH-SY5Y cells, the cells were cotransfected with GFP and LRRK2 [wild type (WT), mutants, or vector] at 2 days before imaging. For BAPTA-AM treatments, cells were incubated in BAPTA-AM supplemented media for 1 day before imaging. The mitochondria were identified in GFP-positive cells by staining for endogenous TOM20 (Santa Cruz Biotechnology, Santa Cruz, CA). Autophagy was inhibited using RNA interference (RNAi) against human ATG7 (Thermo Scientific, Rockford, IL), as previously described.12Plowey E.D. Cherra 3rd, S.J. Liu Y.J. Chu C.T. Role of autophagy in G2019S-LRRK2-associated neurite shortening in differentiated SH-SY5Y cells.J Neurochem. 2008; 105: 1048-1056Crossref PubMed Scopus (435) Google Scholar Cortical neurons were transfected at 7 DIV and imaged at 2, 3, 4, or 5 days after transfection. Neurons were placed in warm Dulbecco's PBS, and mitochondrial movement in the proximal dendrites (up to 75 μm from the soma) was imaged every 5 seconds for a total of 5 minutes37De Vos K.J. Sheetz M.P. Visualization and quantification of mitochondrial dynamics in living animal cells.Methods Cell Biol. 2007; 80: 627-682Crossref PubMed Scopus (74) Google Scholar on a 37°C microscope stage using an inverted epifluorescence microscope (Olympus IX71) and Microsuite Basic Edition version 2.3 (build 1121) imaging software (Olympus America, Center Valley, PA). The net mitochondrial movement was tracked and quantified using ImageJ software version 1.46r (NIH, Bethesda, MD), with the MTrackJ plug-in. Mitochondrial movement was defined as a >2-μm change in position within the imaging period. The percentages of mitochondria moving in the anterograde and retrograde directions were quantified for each neuron and expressed as a ratio. The velocities of individual mitochondria were quantified at 3 and 5 days after transfection. GFP–light chain 3 (LC3)–labeled autophagosomes were identified as bright puncta (>1.5 SDs higher than the mean cytoplasmic fluorescence), ranging from 0.5 to 1.0 μm in diameter. The puncta were counted in each cellular compartment for primary neurons. Cortical neurons were transfected at 7 DIV. Neurons were stained with 50 nmol/L tetramethylrhodamine methyl ester (TMRM; Invitrogen) and imaged in Krebs-Ringer buffer with 12.5 nmol/L TMRM on day 3 after transfection, using a Nikon A1 confocal microscope (Melville, NY) with <1% laser power at 37°C. TMRM equilibrates across the plasma and mitochondrial membranes in a nernstian manner; thus, the resulting whole cell fluorescence after TMRM loading reflects the potentials of both membranes.38Ward M.W. Rego A.C. Frenguelli B.G. Nicholls D.G. Mitochondrial membrane potential and glutamate excitotoxicity in cultured cerebellar granule cells.J Neurosci. 2000; 20: 7208-7219Crossref PubMed Google Scholar Mitochondrial membrane potential (Ψm) was calculated as the ratio of mitochondrial/cytosolic TMRM staining intensity, as previously described.39Lemasters J.J. Ramshesh V.K. Imaging of mitochondrial polarization and depolarization with cationic fluorophores.Methods Cell Biol. 2007; 80: 283-295Crossref PubMed Scopus (96) Google Scholar This ratio accounts for variations in cellular polarization that affect the uptake of TMRM, thus allowing for the comparison of ratios between mitochondrial and cytosolic TMRM staining across multiple cells. The staining intensity of proximal dendrites (<75 μm from the soma) was measured in the cytosol and mitochondria using ImageJ software. Several mitochondrial regions of interest were selected in the dendrites of each cell and divided by adjacent cytosolic control regions to obtain the nernstian quotient. The average was taken as the relative mitochondrial polarization of dendritic mitochondria. Cortical neurons or SH-SY5Y cells were lysed in 25 mmol/L HEPES (pH 7.5), 150 mmol/L NaCl, 5 mmol/L EDTA, 10% glycerol, and 1% Triton X-100. Proteins were separated by SDS-PAGE and transferred to a polyvinylidene difluoride membrane. Blots were probed with antibodies against ATG7 (Rockland Inc., Gilbersville, PA), glyceraldehyde-3-phosphate dehydrogenase (Abcam, Cambridge, MA), Lamin A/C (Cell Signaling Technology, Danvers, MA), LC3 (Nanotools, Teningen, Germany), and LRRK2 (C41-2; Michael J. Fox Foundation, New York City, NY). Cortical neurons were incubated with 5 mmol/L Fura2-AM (Invitrogen) in normal buffer: 10 mmol/L HEPES (pH 7.6), 140 mmol/L NaCl, 5 mmol/L KCl, 1 mmol/L CaCl2, 1 mmol/L MgCl2, and 10 mmol/L glucose at 37°C for 1 hour. Cells were washed in normal buffer for 10 minutes, and then imaged at 25°C; 50 mmol/L KCl in normal buffer was added to determine the peak amplitude of calcium influx. Changes in cytoplasmic calcium were followed by measuring the Fura2 340nm/380nm fluorescence ratio in regions of interest containing one cell. The signal deflection from the baseline (ΔF) was normalized to the mean baseline value (F), which was established prior to stimulating the cells. Calcium buffering capacity was defined as the difference in Fura2-AM intensity between peak amplitude and the plateau phase during KCl stimulation. To study calcium clearance, intracellular calcium was allowed to reach the plateau phase in the presence of 50 mmol/L KCl in normal buffer; Fura2-AM intensity was then measured on washout with normal buffer. The curve between the Fura2-AM staining at the plateau phase with 50 mmol/L KCl and at baseline after KCl washout was fit by a mono-exponential equation, which was used to calculate the time constant (τ). In some experiments, the calcium clearance was measured in the presence of 10 μmol/L carbonyl cyanide 3-chlorophenylhydrazone (CCCP; Sigma-Aldrich Corp). All graphical data are compiled from multiple independent experiments. One-way analysis of variance was used to compare the treatment groups, followed by post hoc t-tests with Bonferroni corrections for multiple comparisons. The sample size for all graphical data was calculated to provide a statistical power of 0.75. A corrected P value of <0.05 was considered statistically significant. To determine whether LRRK2 mutants affected mitochondrial homeostasis, we measured the percent mitochondrial content [100 × (mitochondrial area/cytoplasmic area)] in the axons, dendrites, and soma of cortical neurons co-expressing COX8-GFP with LRRK2-WT, LRRK2-G2019S, or LRRK2-R1441C (Figure 1A and Supplemental Figure S1). Neurons were counterstained for MAP2 to distinguish dendrites from axons (Figure 1A and Supplemental Figure S2A). We found that LRRK2 PD-associated mutants, but not WT LRRK2, caused significant reductions in mitochondrial content in dendrites, but not axons, at 5 days after transfection (Figure 1, B and E, and Supplemental Figure S2A). The LRRK2-G2019S mutant also caused a significant decrease in somatic mitochondrial content that was not observed with the LRRK2-R1441C mutant (Figure 1D and Supplemental Figure S2B), although the LRRK2 plasmids resulted in equivalent protein expression (Supplemental Figure S2C). There were no morphological changes to dendrites at this time point (Figure 1C), indicating that the decrease in mitochondria density preceded subsequent reductions in dendritic area observed at 14 days after transfection.23MacLeod D. Dowman J. Hammond R. Leete T. Inoue K. Abeliovich A. The familial parkinsonism gene LRRK2 regulates neurite process morphology.Neuron. 2006; 52: 587-593Abstract Full Text Full Text PDF PubMed Scopus (481) Google Scholar The kinase-deficient LRRK2 K1906M mutant had no effect on dendritic mitochondrial content (Supplemental Figure S3A). Because decreased dendritic mitochondrial content could underlie the dendrite/neurite retraction observed in culture and mouse models of LRRK2-related PD,19Cherra 3rd, S.J. Kulich S.M. Uechi G. Balasubramani M. Mountzouris J. Day B.W. Chu C.T. Regulation of the autophagy protein LC3 by phosphorylation.J Cell Biol. 2010; 190: 533-539Crossref PubMed Scopus (259) Google Scholar, 22Li Y. Liu W. Oo T.F. Wang L. Tang Y. Jackson-Lewis V. Zhou C. Geghman K. Bogdanov M. Przedborski S. Beal M.F. Burke R.E. Li C. Mutant LRRK2(R1441G) BAC transgenic mice recapitulate cardinal features of Parkinson's disease.Nat Neurosci. 2009; 12: 826-828Crossref PubMed Scopus (421) Google Scholar, 23MacLeod D. Dowman J. Hammond R. Leete T. Inoue K. Abeliovich A. The familial parkinsonism gene LRRK2 regulates neurite process morphology.Neuron. 2006; 52: 587-593Abstract Full Text Full Text PDF PubMed Scopus (481) Google Scholar, 25Ramonet D. Daher J.P. Lin B.M. Stafa K. Kim J. Banerjee R. Westerlund M. Pletnikova O. Glauser L. Yang L. Liu Y. Swing D.A. Beal M.F. Troncoso J.C. McCaffery J.M. Jenkins N.A. Copeland N.G. Galter D. Thomas B. Lee M.K. Dawson T.M. Dawson V.L. Moore D.J. Dopaminergic neuronal loss: reduced neurite complexity and autophagic abnormalities in transgenic mice expressing G2019S mutant LRRK2.PLoS One. 2011; 6: e18568Crossref PubMed Scopus (311) Google Scholar we further investigated causes of this decrease in mitochondrial content. Mitochondrial trafficking is one process that could regulate the quantity of mitochondria by delivering or removing mitochondria from the dendrites. To determine whether LRRK2 mutants affected mitochondrial trafficking, we measured anterograde (away from the soma) and retrograde (toward the soma) movement of COX8-GFP–labeled mitochondria in dendrites at 2 to 5 days after transfection. The percentage of mobile mitochondria was not significantly changed between treatment conditions (eg, at 5 days, vector = 15.64 ± 0.42, WT = 15.71 ± 0.11, G2019S = 17.52 ± 1.41, and R1441C =
Referência(s)