TY - JOUR
T1 - BNIP3L-mediated mitophagy is required for mitochondrial remodeling during the differentiation of optic nerve oligodendrocytes
AU - Yazdankhah, Meysam
AU - Ghosh, Sayan
AU - Shang, Peng
AU - Stepicheva, Nadezda
AU - Hose, Stacey
AU - Liu, Haitao
AU - Chamling, Xitiz
AU - Tian, Shenghe
AU - Sullivan, Mara L.G.
AU - Calderon, Michael Joseph
AU - Fitting, Christopher S.
AU - Weiss, Joseph
AU - Jayagopal, Ashwath
AU - Handa, James T.
AU - Sahel, José Alain
AU - Zigler, J. Samuel
AU - Kinchington, Paul R.
AU - Zack, Donald J.
AU - Sinha, Debasish
N1 - Funding Information:
This work was supported by the Eunice Kennedy Shriver National Institute of Child Health and Human Development [NIH R21HD059008-01]; Knights Templar Eye Foundation; National Eye Institute [1K99EY029011]; National Eye Institute [P30EY001765]; National Institutes of Health [NIH P30 EY08098]; Jennifer Salvitti Davis Chair in Ophthalmology, University of Pittsburgh; Research to Prevent Blindness [to UMPC Department of Ophthalmology]; University of Pittsburgh [Start-up funds]. We thank Drs. Simon Watkins (Director of the Center for Biologic Imaging and Vice-Chair, Department of Cell Biology, University of Pittsburgh School of Medicine, USA) and Shomi Bhattacharya (UCL Institute of Ophthalmology, UK) for critical reading and insightful discussions. We would like to thank Dr. Toren Finkel, Director of UPMC Aging Institute, for kindly providing the mt-Keima plasmid. We would also like to thank the Imaging Core Facility, Department of Pediatrics, University of Pittsburgh School of Medicine for their help with the confocal microscopy studies. This study was funded by the Knights Templar Eye Foundation (award to MY), NIH R21HD059008-01 (to DS), University of Pittsburgh start-up funds (to DS), Jennifer Salvitti Davis Chair in Ophthalmology (to DS), NIH P30 EY08098 (to Department of Ophthalmology Molecular Biology Core), Research to Prevent Blindness, NY (to Department of Ophthalmology, UPMC), 1K99EY029011 (to XC) and P30EY001765 (to DJZ).
Funding Information:
We thank Drs. Simon Watkins (Director of the Center for Biologic Imaging and Vice-Chair, Department of Cell Biology, University of Pittsburgh School of Medicine, USA) and Shomi Bhattacharya (UCL Institute of Ophthalmology, UK) for critical reading and insightful discussions. We would like to thank Dr. Toren Finkel, Director of UPMC Aging Institute, for kindly providing the mt-Keima plasmid. We would also like to thank the Imaging Core Facility, Department of Pediatrics, University of Pittsburgh School of Medicine for their help with the confocal microscopy studies. This study was funded by the Knights Templar Eye Foundation (award to MY), NIH R21HD059008-01 (to DS), University of Pittsburgh start-up funds (to DS), Jennifer Salvitti Davis Chair in Ophthalmology (to DS), NIH P30 EY08098 (to Department of Ophthalmology Molecular Biology Core), Research to Prevent Blindness, NY (to Department of Ophthalmology, UPMC), 1K99EY029011 (to XC) and P30EY001765 (to DJZ).
Publisher Copyright:
© 2021 Informa UK Limited, trading as Taylor & Francis Group.
PY - 2021
Y1 - 2021
N2 - Retinal ganglion cell axons are heavily myelinated (98%) and myelin damage in the optic nerve (ON) severely affects vision. Understanding the molecular mechanism of oligodendrocyte progenitor cell (OPC) differentiation into mature oligodendrocytes will be essential for developing new therapeutic approaches for ON demyelinating diseases. To this end, we developed a new method for isolation and culture of ON-derived oligodendrocyte lineage cells and used it to study OPC differentiation. A critical aspect of cellular differentiation is macroautophagy/autophagy, a catabolic process that allows for cell remodeling by degradation of excess or damaged cellular molecules and organelles. Knockdown of ATG9A and BECN1 (pro-autophagic proteins involved in the early stages of autophagosome formation) led to a significant reduction in proliferation and survival of OPCs. We also found that autophagy flux (a measure of autophagic degradation activity) is significantly increased during progression of oligodendrocyte differentiation. Additionally, we demonstrate a significant change in mitochondrial dynamics during oligodendrocyte differentiation, which is associated with a significant increase in programmed mitophagy (selective autophagic clearance of mitochondria). This process is mediated by the mitophagy receptor BNIP3L (BCL2/adenovirus E1B interacting protein 3-like). BNIP3L-mediated mitophagy plays a crucial role in the regulation of mitochondrial network formation, mitochondrial function and the viability of newly differentiated oligodendrocytes. Our studies provide novel evidence that proper mitochondrial dynamics is required for establishment of functional mitochondria in mature oligodendrocytes. These findings are significant because targeting BNIP3L-mediated programmed mitophagy may provide a novel therapeutic approach for stimulating myelin repair in ON demyelinating diseases. Abbreviations: A2B5: a surface antigen of oligodendrocytes precursor cells, A2B5 clone 105; ACTB: actin, beta; APC: an antibody to label mature oligodendrocytes, anti-adenomatous polyposis coli clone CC1; ATG5: autophagy related 5; ATG7: autophagy related 7; ATG9A: autophagy related 9A; AU: arbitrary units; BafA1: bafilomycin A1; BCL2: B cell leukemia/lymphoma 2; BECN1: beclin 1, autophagy related; BNIP3: BCL2/adenovirus E1B interacting protein 3; BNIP3L/NIX: BCL2/adenovirus E1B interacting protein 3-like; CASP3: caspase 3; CNP: 2′,3′-cyclic nucleotide 3′-phosphodiesterase; Ctl: control; COX8: cytochrome c oxidase subunit; CSPG4/NG2: chondroitin sulfate proteoglycan 4; DAPI: 4′6-diamino-2-phenylindole; DNM1L: dynamin 1-like; EGFP: enhanced green fluorescent protein; FACS: fluorescence-activated cell sorting; FIS1: fission, mitochondrial 1; FUNDC1: FUN14 domain containing 1; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GFAP: glial fibrillary growth factor; GFP: green fluorescent protein; HsESC: human embryonic stem cell; IEM: immunoelectron microscopy; LAMP1: lysosomal-associated membrane protein 1; LC3B: microtubule-associated protein 1 light chain 3; MBP: myelin basic protein; MFN2: mitofusin 2; Mito-Keima: mitochondria-targeted monomeric keima-red; Mito-GFP: mitochondria-green fluorescent protein; Mito-RFP: mitochondria-red fluorescent protein; MitoSOX: red mitochondrial superoxide probe; MKI67: antigen identified by monoclonal antibody Ki 67; MMP: mitochondrial membrane potential; O4: oligodendrocyte marker O4; OLIG2: oligodendrocyte transcription factor 2; ON: optic nerve; OPA1: OPA1, mitochondrial dynamin like GTPase; OPC: oligodendrocyte progenitor cell; PDL: poly-D-lysine; PINK1: PTEN induced putative kinase 1; PRKN/Parkin: parkin RBR E3 ubiquitin protein ligase; RFP: red fluorescent protein; RGC: retinal ganglion cell; ROS: reactive oxygen species; RT-PCR: real time polymerase chain reaction; SEM: standard error of the mean; SOD2: superoxide dismutase 2, mitochondrial; SQSTM1/p62: sequestosome 1; TEM: transmission electron microscopy; TMRM: tetramethylrhodamine methyl ester; TOMM20: translocase of outer mitochondrial membrane 20; TUBB: tubulin, beta; TUBB3: tubulin, beta 3 class III.
AB - Retinal ganglion cell axons are heavily myelinated (98%) and myelin damage in the optic nerve (ON) severely affects vision. Understanding the molecular mechanism of oligodendrocyte progenitor cell (OPC) differentiation into mature oligodendrocytes will be essential for developing new therapeutic approaches for ON demyelinating diseases. To this end, we developed a new method for isolation and culture of ON-derived oligodendrocyte lineage cells and used it to study OPC differentiation. A critical aspect of cellular differentiation is macroautophagy/autophagy, a catabolic process that allows for cell remodeling by degradation of excess or damaged cellular molecules and organelles. Knockdown of ATG9A and BECN1 (pro-autophagic proteins involved in the early stages of autophagosome formation) led to a significant reduction in proliferation and survival of OPCs. We also found that autophagy flux (a measure of autophagic degradation activity) is significantly increased during progression of oligodendrocyte differentiation. Additionally, we demonstrate a significant change in mitochondrial dynamics during oligodendrocyte differentiation, which is associated with a significant increase in programmed mitophagy (selective autophagic clearance of mitochondria). This process is mediated by the mitophagy receptor BNIP3L (BCL2/adenovirus E1B interacting protein 3-like). BNIP3L-mediated mitophagy plays a crucial role in the regulation of mitochondrial network formation, mitochondrial function and the viability of newly differentiated oligodendrocytes. Our studies provide novel evidence that proper mitochondrial dynamics is required for establishment of functional mitochondria in mature oligodendrocytes. These findings are significant because targeting BNIP3L-mediated programmed mitophagy may provide a novel therapeutic approach for stimulating myelin repair in ON demyelinating diseases. Abbreviations: A2B5: a surface antigen of oligodendrocytes precursor cells, A2B5 clone 105; ACTB: actin, beta; APC: an antibody to label mature oligodendrocytes, anti-adenomatous polyposis coli clone CC1; ATG5: autophagy related 5; ATG7: autophagy related 7; ATG9A: autophagy related 9A; AU: arbitrary units; BafA1: bafilomycin A1; BCL2: B cell leukemia/lymphoma 2; BECN1: beclin 1, autophagy related; BNIP3: BCL2/adenovirus E1B interacting protein 3; BNIP3L/NIX: BCL2/adenovirus E1B interacting protein 3-like; CASP3: caspase 3; CNP: 2′,3′-cyclic nucleotide 3′-phosphodiesterase; Ctl: control; COX8: cytochrome c oxidase subunit; CSPG4/NG2: chondroitin sulfate proteoglycan 4; DAPI: 4′6-diamino-2-phenylindole; DNM1L: dynamin 1-like; EGFP: enhanced green fluorescent protein; FACS: fluorescence-activated cell sorting; FIS1: fission, mitochondrial 1; FUNDC1: FUN14 domain containing 1; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GFAP: glial fibrillary growth factor; GFP: green fluorescent protein; HsESC: human embryonic stem cell; IEM: immunoelectron microscopy; LAMP1: lysosomal-associated membrane protein 1; LC3B: microtubule-associated protein 1 light chain 3; MBP: myelin basic protein; MFN2: mitofusin 2; Mito-Keima: mitochondria-targeted monomeric keima-red; Mito-GFP: mitochondria-green fluorescent protein; Mito-RFP: mitochondria-red fluorescent protein; MitoSOX: red mitochondrial superoxide probe; MKI67: antigen identified by monoclonal antibody Ki 67; MMP: mitochondrial membrane potential; O4: oligodendrocyte marker O4; OLIG2: oligodendrocyte transcription factor 2; ON: optic nerve; OPA1: OPA1, mitochondrial dynamin like GTPase; OPC: oligodendrocyte progenitor cell; PDL: poly-D-lysine; PINK1: PTEN induced putative kinase 1; PRKN/Parkin: parkin RBR E3 ubiquitin protein ligase; RFP: red fluorescent protein; RGC: retinal ganglion cell; ROS: reactive oxygen species; RT-PCR: real time polymerase chain reaction; SEM: standard error of the mean; SOD2: superoxide dismutase 2, mitochondrial; SQSTM1/p62: sequestosome 1; TEM: transmission electron microscopy; TMRM: tetramethylrhodamine methyl ester; TOMM20: translocase of outer mitochondrial membrane 20; TUBB: tubulin, beta; TUBB3: tubulin, beta 3 class III.
KW - ATG9A
KW - autophagy
KW - autophagy flux
KW - co-culture
KW - demyelinating diseases
KW - glial cells
KW - mitochondrial dynamics
KW - myelin
KW - oligodendrocyte lineage cells
KW - retinal ganglion cell axons
UR - http://www.scopus.com/inward/record.url?scp=85099648035&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=85099648035&partnerID=8YFLogxK
U2 - 10.1080/15548627.2020.1871204
DO - 10.1080/15548627.2020.1871204
M3 - Article
C2 - 33404293
AN - SCOPUS:85099648035
SN - 1554-8627
VL - 17
SP - 3140
EP - 3159
JO - Autophagy
JF - Autophagy
IS - 10
ER -