In Vivo Detection of Oxidized Proteins: A Practical Approach to Tissue-Derived Mitochondria - Protein Misfolding and Cellular Stress in Disease and Aging - page 247

resulting in net ROS production and oxidative modification of
proteins, lipids and DNA ( 1, 2 ). Mitochondrial dysfunction and
oxidative damage are known to be joint contributors to neurode-
generation and aging ( 3 ).
The electron transport chain is a major generator of ROS in
cells: high-throughput electron transport down the respiratory
chain inevitably results in the escape of unpaired electrons and
generation of superoxide anion (O 2 • − ), largely at complex I and
complex III ( 2, 4 ). Pharmacological inhibition of these complexes
is known to diminish ATP production, increase leakage of elec-
trons, and increase superoxide production as well as reduce ROS
removal ( 2, 5 ).
Key components in the control of assembly and functionality
of respiratory chain complexes in yeast and in mammals are ATP-
dependent metalloproteases of the AAA-superfamily (ATPases
associated with a variety of cellular activities): paraplegin and
AFG3L2 ( 6-9 ). They are ubiquitous nuclear-encoded mitochon-
drial proteins that form hetero-oligomeric paraplegin/AFG3L2
and homo-oligomeric AFG3L2 complexes in the inner mitochon-
drial membrane, named m -AAA proteases ( 10 ). Dysfunction of
m -AAA protease indeed results in impaired respiration both in
yeast and in mammals ( 6, 8, 9 ).
Mutations of paraplegin cause hereditary spastic paraplegia
(HSP), characterized by axonal degeneration of the longest motor
and sensory axons of the central nervous system ( 11 ). We previ-
ously demonstrated that HSP fibroblasts lacking the m -AAA
complex have a reduced respiratory complex I activity and
increased sensitivity to oxidant stress ( 8 ).
We have developed and characterized mutant mouse models
defective in Afg3l2 : a spontaneous mutant carrier of a missense
mutation in the core AAA domain of the protein ( Afg3l2 par/par )
and a complete knockout ( Afg3l2 Emv66/Emv66 ). Contrasting with the
mild axonal degeneration of paraplegin-deficient mouse ( 12 ),
both mouse models defective in Afg3l2 display a severe defect in
axonal development, which results in early lethality at P16. The
lack of functional AFG3L2 has deep effects on mitochondrial
morphology and metabolism. Massive accumulation of swollen
mitochondria with damaged cristae has been observed in neu-
ronal cell bodies of the central and peripheral nervous system.
Moreover, biochemical studies have revealed that enzymatic activ-
ities of respiratory chain complex I and complex III are strikingly
impaired in Afg3l2 models and result in a highly reduced ATP
production ( 9 ). We thus evaluated whether these respiratory
defects cause oxidative damage in Afg3l2 mutants measuring car-
bonyl formation, an easy detectable marker of protein oxidation
( 13 ). Carbonyl levels after reaction with dinitrophenyl hydrazine
(DNPH) are measured by Western blot on brain mitochondrial-
enriched preparations from both Afg3l2 mutant mice and controls.