In the summer of 2011, we obtained the first co-localization images of succinyl-CoA synthase (a citric acid cycle enzyme) with F0–F1 ATP synthase, in an effort to demonstrate that our anti-succinyl-CoA synthase antibody was indeed staining something in the mitochondrial matrix of human brain samples (figure 1) 1. What struck me was that the F0–F1 ATP synthase staining was restricted to the neurons. ‘Keep this titbit in the back of your head,’ said Prof. Adam-Vizi, my boss at the time.
Figure 1: A1 SUCLA2 immunoreactivity in the human temporal cortex visualized by FITC-tyramide amplification immunofluorescence. Red blood cells in some capillaries are labeled because of their endogenous peroxidase activity. B1 A high-magnification confocal image demonstrates the localization of SUCLA2 within a SUCLA2-positive cell. A2 Distribution of the immunoreactivity of the mitochondrial marker F0–F1 ATP synthase subunit d in the same field as A1. B2 F0–F1 ATP synthase subunit d immunoreactivity in the same field as B1. A3 Yellow color indicates co-localization of SUCLA2 and F0–F1 ATP synthase subunit d. An almost complete absence of singly labeled structures can be observed except for the red blood cells. B3 The co-localization of SUCLA2 and F0–F1 ATP synthase subunit d is predominant even within a cell at high magnification. Scale bars 50 μm for A1–A3 and 20 μm for B1–B3. Reproduced by permission from Springer Nature.
I could stomach the idea of a truncated citric acid cycle in human brain astrocytes, but would I dare to imagine the almighty oxidative phosphorylation (OXPHOS) being absent? Back then, probably not. A few years later, we demonstrated that the α-ketoglutarate dehydrogenase complex (a critical NADH-producing dehydrogenase of the citric acid cycle) was also absent from human brain astrocytes 2. This had been previously reported in a ‘tongue-in-cheek’ manner by the group of the late John P. Blass: ‘Surprisingly, convincing [KGDHC] immunoreactivity was not found in glia’ 3.
The itch to search for OXPHOS components in human brain cells grew stronger. It took a few more years to obtain suitable human brain material. This wasn’t just because fresh human brains are hard to obtain (fixed or frozen brain samples suffer from severely decreased epitope quality, which is needed for immunohistochemistry) but also because primate brains exhibit strong autofluorescence that must be quenched, e.g., with Sudan Black B 4. After testing several antibodies, we were able to show that complex IV and cytochrome c of the mitochondrial respiratory chain are barely detectable in astrocytes . Since the antibodies luminously decorated the neurons in the same slices, there was no issue of false negativity.
Going backwards in time, delving into older literature regarding complex IV histochemical activity (a method that relies on the enzymatic, not immunological properties of the protein), I was delighted to read that Wong-Riley branded this complex as a metabolic marker for neuronal activity, especially in higher mammals 5. ‘The record is set straight,’ we thought. Astrocytes, at least those labeled with antibodies in the human brain regions that we examined, exhibit limited -if any- capacity for OXPHOS. Nicely fitting to Wong-Riley’s reports regarding an inverse relation between glial complex IV activity and animal phylogenetic hierarchy, astrocytes in rat brain exhibited positive immunoreactivity for critical OXPHOS enzymes.
But what is the significance of this finding, apart from annoying fellow researchers striving to demonstrate that human astrocyte-specific OXPHOS deficiencies underlie pathologies of the CNS? (The lack of references here is intentional). It’s a textbook definition that lack of OXPHOS leads to the formation of lactate, to regenerate NAD+ and avoid interruption of glycolysis. This fits well with the so-called Astrocyte-to-Neuron Lactate Shuttle model, which posits that in response to glutamate-mediated neuronal activity, astrocytes enhance their glycolytic flux, forming lactate, which is then shuttled to neurons 6. Although this model is not universally accepted, it has been demonstrated in the human brain in vivo 7.
Despite the absence of complex IV and cytochrome c (and other enzymes) arguing against astrocytic OXPHOS, we could not provide supportive functional evidence. Investigating cell-specific oxidative metabolism within the human brain remains unfeasible. An interesting notion, however, is that the astrocytes we observed—lacking critical OXPHOS proteins—were at rest; no one knows what they might express if stimulated. Relevant to this, it was recently shown that OXPHOS in murine microglia in vivo is essential for their response to demyelinating injury but not for survival at rest 8.
The finding that human brain astrocytes exhibit limited capacity for OXPHOS is not far-fetched: mice engineered to lack complex IV in astrocytic mitochondria in vivo were fully viable without any signs of glial or neuronal loss, even at one year of age 9. One should not forget that the pathways depicted in biochemistry textbooks are nothing more than that: paper biochemistry. In vivo, metabolism is far more dynamic—and interesting.
REFERENCES CITED
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2 Dobolyi, A. et al. Exclusive neuronal detection of KGDHC-specific subunits in the adult human brain cortex despite pancellular protein lysine succinylation. Brain Struct Funct 225, 639-667 (2020). https://doi.org:10.1007/s00429-020-02026-5
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