Quantitative Proteomics Reveals Oxygen-Dependent Changes in Neuronal Mitochondria Affecting Function and Sensitivity to Rotenone

Mitochondria are
implicated in a variety of degenerative disorders
and aging. Mitochondria are responsive to the oxygen in their environment,
yet tissue culture is performed at atmospheric (21%) oxygen and not
at physiological (1–11%) oxygen levels found in tissues. We
employed imaging of mitochondrial probes, mass spectrometry, Western
blots, and ATP assays of the human neuroblastoma cell-line SH-SY5Y
and imaging of mitochondrial probes in human primary neurons under
standard nonphysiological oxygen conditions (atmospheric) and under
physiological oxygen levels in the nervous system to assess the impact
of oxygen on mitochondrial function. SH-SY5Y cells cultured in physiological
5% oxygen exhibited the lowest reactive oxygen species (ROS) production,
indicating that culture at 5% oxygen is favored; these results were
mimicked in primary human cells. Mass spectrometric analysis revealed
extensive mitochondrial proteomic alterations in SH-SY5Y cells based
on oxygen culture condition. Among these, the rotenone-sensitive subunit
of complex I NDUFV3 was increased in cells cultured at 5% oxygen.
Rotenone is a Parkinson’s disease-linked toxin, and correspondingly
SH-SY5Y cells cultured at 5% oxygen also exhibited over 10 times greater
sensitivity to rotenone than those cultured in atmospheric, 21%, oxygen.
Our results indicate that neuronal mitochondria are responsive to
oxygen levels and produce differential responses under different oxygen
levels.