Inhibition of bioenergetics provides novel insights into recruitment of PINK1-dependent neuronal mitophagy

Yea Seul Shin, James G. Ryall, Joanne M. Britto, Chew L. Lau, Rodney J. Devenish, Phillip Nagley, Philip M. Beart

Research output: Contribution to journalArticleResearchpeer-review

Abstract

Contributions of damaged mitochondria to neuropathologies have stimulated interest in mitophagy. We investigated triggers of neuronal mitophagy by disruption of mitochondrial energy metabolism in primary neurons. Mitophagy was examined in cultured murine cerebellar granule cells after inhibition of mitochondrial respiratory chain by drugs rotenone, 3-nitropropionic acid, antimycin A, and potassium cyanide, targeting complexes I, II, III, and IV, respectively. Inhibitor concentrations producing slow cellular demise were determined from analyses of cellular viability, morphology of neuritic damage, plasma membrane permeability, and oxidative phosphorylation. Live cell imaging of dissipation of mitochondrial membrane potential (ΔΨ m ) by drugs targeting mitochondrial complexes was referenced to complete depolarization by carbonyl cyanide m-chlorophenyl hydrazone. While inhibition of complexes I, III and IV effected rapid dissipation of ΔΨ m , inhibition of complex II using 3-nitropropionic acid led to minimal depolarization of mitochondria. Nonetheless, all respiratory chain inhibitors triggered mitophagy as indicated by increased aggregation of mitochondrially localized PINK1. Mitophagy was further analyzed using a dual fluorescent protein biosensor reporting mitochondrial relocation to acidic lysosomal environment. Significant acidification of mitochondria was observed in neurons treated with rotenone or 3-nitropropionic acid, revealing mitophagy at distal processes. Neurons treated with antimycin A or cyanide failed to show mitochondrial acidification. Minor dissipation of ΔΨ m by 3-nitropropionic acid coupled with vigorous triggering of mitophagy suggested depolarization of mitochondria is not a necessary condition to trigger mitophagy. Moreover, weak elicitation of mitophagy by antimycin A, subsequent to loss of ΔΨ m , suggested that mitochondrial depolarization is not a sufficient condition for triggering robust neuronal mitophagy. Our findings provide new insight into complexities of mitophagic clearance of neuronal mitochondria. (Figure presented.).

Original languageEnglish
Pages (from-to)269-283
Number of pages15
JournalJournal of Neurochemistry
Volume149
Issue number2
DOIs
Publication statusPublished - Apr 2019

Keywords

  • bioenergetics
  • depolarization
  • lysosome
  • mitophagy
  • neuron
  • PINK1

Cite this

@article{db180f68a8944e00b85b009a46c0e19e,
title = "Inhibition of bioenergetics provides novel insights into recruitment of PINK1-dependent neuronal mitophagy",
abstract = "Contributions of damaged mitochondria to neuropathologies have stimulated interest in mitophagy. We investigated triggers of neuronal mitophagy by disruption of mitochondrial energy metabolism in primary neurons. Mitophagy was examined in cultured murine cerebellar granule cells after inhibition of mitochondrial respiratory chain by drugs rotenone, 3-nitropropionic acid, antimycin A, and potassium cyanide, targeting complexes I, II, III, and IV, respectively. Inhibitor concentrations producing slow cellular demise were determined from analyses of cellular viability, morphology of neuritic damage, plasma membrane permeability, and oxidative phosphorylation. Live cell imaging of dissipation of mitochondrial membrane potential (ΔΨ m ) by drugs targeting mitochondrial complexes was referenced to complete depolarization by carbonyl cyanide m-chlorophenyl hydrazone. While inhibition of complexes I, III and IV effected rapid dissipation of ΔΨ m , inhibition of complex II using 3-nitropropionic acid led to minimal depolarization of mitochondria. Nonetheless, all respiratory chain inhibitors triggered mitophagy as indicated by increased aggregation of mitochondrially localized PINK1. Mitophagy was further analyzed using a dual fluorescent protein biosensor reporting mitochondrial relocation to acidic lysosomal environment. Significant acidification of mitochondria was observed in neurons treated with rotenone or 3-nitropropionic acid, revealing mitophagy at distal processes. Neurons treated with antimycin A or cyanide failed to show mitochondrial acidification. Minor dissipation of ΔΨ m by 3-nitropropionic acid coupled with vigorous triggering of mitophagy suggested depolarization of mitochondria is not a necessary condition to trigger mitophagy. Moreover, weak elicitation of mitophagy by antimycin A, subsequent to loss of ΔΨ m , suggested that mitochondrial depolarization is not a sufficient condition for triggering robust neuronal mitophagy. Our findings provide new insight into complexities of mitophagic clearance of neuronal mitochondria. (Figure presented.).",
keywords = "bioenergetics, depolarization, lysosome, mitophagy, neuron, PINK1",
author = "Shin, {Yea Seul} and Ryall, {James G.} and Britto, {Joanne M.} and Lau, {Chew L.} and Devenish, {Rodney J.} and Phillip Nagley and Beart, {Philip M.}",
year = "2019",
month = "4",
doi = "10.1111/jnc.14667",
language = "English",
volume = "149",
pages = "269--283",
journal = "Journal of Neurochemistry",
issn = "0022-3042",
publisher = "Wiley-Blackwell",
number = "2",

}

Inhibition of bioenergetics provides novel insights into recruitment of PINK1-dependent neuronal mitophagy. / Shin, Yea Seul; Ryall, James G.; Britto, Joanne M.; Lau, Chew L.; Devenish, Rodney J.; Nagley, Phillip; Beart, Philip M.

In: Journal of Neurochemistry, Vol. 149, No. 2, 04.2019, p. 269-283.

Research output: Contribution to journalArticleResearchpeer-review

TY - JOUR

T1 - Inhibition of bioenergetics provides novel insights into recruitment of PINK1-dependent neuronal mitophagy

AU - Shin, Yea Seul

AU - Ryall, James G.

AU - Britto, Joanne M.

AU - Lau, Chew L.

AU - Devenish, Rodney J.

AU - Nagley, Phillip

AU - Beart, Philip M.

PY - 2019/4

Y1 - 2019/4

N2 - Contributions of damaged mitochondria to neuropathologies have stimulated interest in mitophagy. We investigated triggers of neuronal mitophagy by disruption of mitochondrial energy metabolism in primary neurons. Mitophagy was examined in cultured murine cerebellar granule cells after inhibition of mitochondrial respiratory chain by drugs rotenone, 3-nitropropionic acid, antimycin A, and potassium cyanide, targeting complexes I, II, III, and IV, respectively. Inhibitor concentrations producing slow cellular demise were determined from analyses of cellular viability, morphology of neuritic damage, plasma membrane permeability, and oxidative phosphorylation. Live cell imaging of dissipation of mitochondrial membrane potential (ΔΨ m ) by drugs targeting mitochondrial complexes was referenced to complete depolarization by carbonyl cyanide m-chlorophenyl hydrazone. While inhibition of complexes I, III and IV effected rapid dissipation of ΔΨ m , inhibition of complex II using 3-nitropropionic acid led to minimal depolarization of mitochondria. Nonetheless, all respiratory chain inhibitors triggered mitophagy as indicated by increased aggregation of mitochondrially localized PINK1. Mitophagy was further analyzed using a dual fluorescent protein biosensor reporting mitochondrial relocation to acidic lysosomal environment. Significant acidification of mitochondria was observed in neurons treated with rotenone or 3-nitropropionic acid, revealing mitophagy at distal processes. Neurons treated with antimycin A or cyanide failed to show mitochondrial acidification. Minor dissipation of ΔΨ m by 3-nitropropionic acid coupled with vigorous triggering of mitophagy suggested depolarization of mitochondria is not a necessary condition to trigger mitophagy. Moreover, weak elicitation of mitophagy by antimycin A, subsequent to loss of ΔΨ m , suggested that mitochondrial depolarization is not a sufficient condition for triggering robust neuronal mitophagy. Our findings provide new insight into complexities of mitophagic clearance of neuronal mitochondria. (Figure presented.).

AB - Contributions of damaged mitochondria to neuropathologies have stimulated interest in mitophagy. We investigated triggers of neuronal mitophagy by disruption of mitochondrial energy metabolism in primary neurons. Mitophagy was examined in cultured murine cerebellar granule cells after inhibition of mitochondrial respiratory chain by drugs rotenone, 3-nitropropionic acid, antimycin A, and potassium cyanide, targeting complexes I, II, III, and IV, respectively. Inhibitor concentrations producing slow cellular demise were determined from analyses of cellular viability, morphology of neuritic damage, plasma membrane permeability, and oxidative phosphorylation. Live cell imaging of dissipation of mitochondrial membrane potential (ΔΨ m ) by drugs targeting mitochondrial complexes was referenced to complete depolarization by carbonyl cyanide m-chlorophenyl hydrazone. While inhibition of complexes I, III and IV effected rapid dissipation of ΔΨ m , inhibition of complex II using 3-nitropropionic acid led to minimal depolarization of mitochondria. Nonetheless, all respiratory chain inhibitors triggered mitophagy as indicated by increased aggregation of mitochondrially localized PINK1. Mitophagy was further analyzed using a dual fluorescent protein biosensor reporting mitochondrial relocation to acidic lysosomal environment. Significant acidification of mitochondria was observed in neurons treated with rotenone or 3-nitropropionic acid, revealing mitophagy at distal processes. Neurons treated with antimycin A or cyanide failed to show mitochondrial acidification. Minor dissipation of ΔΨ m by 3-nitropropionic acid coupled with vigorous triggering of mitophagy suggested depolarization of mitochondria is not a necessary condition to trigger mitophagy. Moreover, weak elicitation of mitophagy by antimycin A, subsequent to loss of ΔΨ m , suggested that mitochondrial depolarization is not a sufficient condition for triggering robust neuronal mitophagy. Our findings provide new insight into complexities of mitophagic clearance of neuronal mitochondria. (Figure presented.).

KW - bioenergetics

KW - depolarization

KW - lysosome

KW - mitophagy

KW - neuron

KW - PINK1

UR - http://www.scopus.com/inward/record.url?scp=85061435372&partnerID=8YFLogxK

U2 - 10.1111/jnc.14667

DO - 10.1111/jnc.14667

M3 - Article

VL - 149

SP - 269

EP - 283

JO - Journal of Neurochemistry

JF - Journal of Neurochemistry

SN - 0022-3042

IS - 2

ER -