Mitochondrial matrix pH acidifies during anoxia and is maintained by the F₁F_o‐ATPase in anoxia‐tolerant painted turtle cortical neurons

Abstract

The western painted turtle (Chrysemys picta bellii) can survive extended periods of anoxia via a series of mechanisms that serve to reduce its energetic needs. Central to these mechanisms is the response of mitochondria, which depolarize in response to anoxia in turtle pyramidal neurons due to an influx of K+. It is currently unknown how mitochondrial matrix pH is affected by this response and we hypothesized that matrix pH acidifies during anoxia due to increased K+/H+ exchanger activity. Inhibition of K+/H+ exchange via quinine led to a collapse of mitochondrial membrane potential (Ψm) during oxygenated conditions in turtle cortical neurons, as indicated by rhodamine‐123 fluorescence, and this occurred twice as quickly during anoxia which indicates an elevation in K+ conductance. Mitochondrial matrix pH acidified during anoxia, as indicated by SNARF‐1 fluorescence imaged via confocal microscopy, and further acidification occurred during anoxia when the F1Fo‐ATPase was inhibited with oligomycin‐A, indicating that ΔpH collapse is prevented during anoxic conditions. Collectively, these results indicate that the mitochondrial proton electrochemical gradient is actively preserved during anoxia to prevent a collapse of Ψm and ΔpH.

Migrasomes: From Biogenesis, Release, Uptake, Rupture to Homeostasis and Diseases

“…By genetically manipulating the expression of TSPAN4, TSPAN9, dynein, KIF5B and integrin, and by blocking migration using the myosin II inhibitor “blebbistatin” in L929 cells, Jiao et al found that blocking mitocytosis causes loss of MMP and reduction of spare respiration capacity in cells that are not exposed to mitochondrial stressors, while enhanced mitocytosis improves the loss of MMP and preserves the spare respiration capacity in cells with or without CCCP treatment [ 6 ]. Besides CCCP, other mitochondrial stressors, such as deferiprone (a typical iron chelator [ 54 , 55 ]), antimycin A (a complex III inhibitor [ 56 ]), and oligomycin (a selective F1FO-ATPase inhibitor [ 57 , 58 ]), and starvation can also induce mitocytosis in L929 cells [ 6 ]. CCCP treatment also induces mitocytosis in mouse bone marrow-derived macrophages, human pancreatic cancer cells (MIACaPa-2 [ 59 ]), and human umbilical vein ECs [ 6 ].…”

Parkinson’s Disease–Associated LRRK2 Interferes with Astrocyte-Mediated Alpha-Synuclein Clearance

“…Indeed, the inflection point of free and conjugated SNARF curves are 7.826 (R = 0.9956) and 7.606 (R = 0.9918), respectively (Supplementary Figure 5). SNARF is particularly suitable to sense pH that ranges between 6 and 9 (Supplementary Figure 5) [51,52] allowing to monitor α-syn PFFs trafficking through the endo-lysosomal pathway. We treated cells with 0.5 μM SNARF-labeled-α-syn PFFs and captured images after 24 h using live cell confocal microscopy at the two relevant emission ranges (Fig.…”

Cytoskeletal Arrest: An Anoxia Tolerance Mechanism

“…If MMP in turtle cells were to depolarize completely in the absence of oxygen-dependent proton pumping, the organelle would not be able to maintain ion homeostasis, triggering cytochrome C release and pro-apoptotic signalling [ 240 ]; therefore, an alternate source of proton pumping must be mobilized in order to maintain anoxic MMP. In turtles, anoxic proton pumping is widely regarded to occur through reversal of ATP synthase and at the expense of ATP [ 161 , 194 , 239 , 241 , 242 , 243 , 244 ], though this has not yet been demonstrated in non-excitable turtle cells such as hepatocytes. This reversal makes the mitochondria an energetic “liability” during anoxia when ATP supply is limiting [ 245 ].…”

“…In anoxia-intolerant mammals, hypoxia induces mitochondrial fission and mitophagy [ 252 , 253 ], and these processes may be active during anoxia in turtles in order to reduce ATP demand for mitochondrial maintenance by reversal of ATP synthase [ 161 , 194 , 239 , 241 , 242 , 243 , 244 ]. In cold- and anoxia-intolerant mammals, MMP depolarization inhibits mitochondrial fusion [ 253 , 254 , 255 ] and thus favours fission.…”