The present study demonstrates, for the first time, the evidence of intrinsic sex differences in mitochondrial biogenesis in hypoxic-ischemic neuronal injury using segregated XY and XX CGNs. First, measurement of the relative amount of mtDNA during OGD and Reox showed significant increase in mtDNA content in XY neurons, which either remained unchanged or reduced below the control levels in XX neurons under identical conditions. Secondly, sex differences in the activation of the nuclear-encoded regulatory program for mitochondrial biogenesis including the PGC-1α co-activator, the NRF-1 transcription factor and the mitochondrial transcription factor TFAM. Thirdly, balanced increase of both the fusion and fission genes transcription, increase in donut formation, and enhanced recovery of ΔΨm and ATP levels in XY neurons at the OGD/Reox periods. On the contrary, fusion and fission genes transcription was imbalanced in XX neurons with simultaneous decrease in ΔΨm and ATP levels, thus promoting (apoptosis process) cell death following OGD/Reox. Our findings clearly show intrinsic sex differences in mitochondrial biogenesis and shed new light on sex-specific changes in mitochondrial transcription factors involved in this process, which could aid sex-specific mitochondrial adaptation, functional recovery and neuronal survival after OGD.
Numerous studies have shown that mitochondrial dysfunction plays a key role in the pathophysiology of many neurological diseases [9, 36]. Conditions that hinder mitochondrial performance such as hypoxic-ischemia place the brain at risk for compromised energy production and secondary injury . Thus one option to minimize the damage attributable to lost energy is to increase the number of mitochondria themselves. Previously, we have reported sex differences in the process of initiating mitochondria-mediated cell death between male and female neurons during the OGD/Reox . We found the apoptotic pathology is mediated by at least two signaling cascades activated in XY and XX neurons following OGD/Reox. The caspase-dependent intrinsic mitochondria-mediated mechanisms were more pronounced in XX neurons and contributed to higher degree of neuronal death, whereas, the extrinsic caspase-independent pathway involves poly(ADP)ribose polymerase-1 (PARP-1) activation and apoptosis-inducing factor (AIF) release at a much earlier time than in XX neurons that play an important role in mediating XY neuronal death during the OGD/Reox . Thus specific inhibition of these pathways may improve brain outcomes from hypoxic-ischemic brain injury in male vs. female neurons. To further address this sex-difference in neuronal death, we show that neuronal cells respond to OGD/Reox by activating critical nuclear and mitochondrial factors in a sex-specific way. These responses are accompanied by increase in mitochondrial DNA transcription and mtDNA content, transcription factors and proteins expression followed by structural evidence of mitochondrial donut formation. Cerebral hypoxia-ischemia has been shown to cause mitochondrial swelling , rupture of mitochondrial membrane with resultant release of mtDNA and subsequent endonuclease digestion , which could account for the decreased mtDNA observed in XX neurons. Furthermore, oxidative stress is also responsible for mtDNA damage, which is more susceptible to damage than nuclear DNA . Thus, this decrease in mtDNA content in XX cells is consistent with our previous findings of more cell death in XX CGNs during the Reox phase . This is further supported by enhanced ΔΨm loss and higher levels of ATP depletion in XX neurons upon exposure to OGD. In contrast, XY CGNs with greater recovery of ΔΨm and ATP levels during the OGD/Reox suggests that XY neurons have more potential for preserving the mitochondrial integrity and function.
A highly novel aspect of the present work is the linkage of sex specificity with mitochondrial biogenesis. Histological evidence of mitochondrial biogenesis was found after transient global ischemia in adult rats . We found enhanced expression of PGC-1α, Tfam and NRF-1 mRNA, and PGC-1α and TFAM protein levels in XY neurons in comparison to XX neurons. It is known that the transcriptional activity of NRF-1 is enhanced by the PGC-1α coactivator in the process of mitochondrial biogenesis [3, 40], and the expression of TFAM is, at least partially, under the control of NRF-1 [18, 41]. Thus, up-regulation of the PGC-1α co-activator in XY neurons coordinates gene activation and facilitates mitochondrial biogenesis in XY neurons, but not in XX neurons during the OGD/Reox periods. Furthermore, the nuclear transcriptional program activated by OGD/Reox includes increase in TFAM expression in XY neurons, which possibly contributes to the increase in mtDNA content [3, 42]. Our findings suggest effective nuclear-mitochondrial communication in a sex-specific way.
The up-regulation of HSP60, observed in XY neurons, is another response that occurs after many stressors, and is indicative of mitochondrial biogenesis [18, 43]. HSP60 is involved in stabilizing both newly synthesize proteins and mtDNA and discrete protein-DNA complexes critical for the regulation of mtDNA transmission and biogenesis of new mitochondria . Thus, the higher levels of HSP60 observed in XY neurons suggest enhanced mitochondrial biogenesis in XY neurons compared to XX neurons. In addition, HSP60 and COXIV are markers for the presence of mitochondria; their increased protein levels may be an integral part of the mechanism involved in mitochondrial biogenesis in surviving XY neurons after OGD.
The morphology of mitochondria including the expression of fusion and fission genes are indicators of mitochondrial vitality, and that fusion and fission processes are linked to cell viability and apoptosis . Furthermore, ATP is required to support the production of GTP which is needed for both outer (Mfn 1 and 2) and inner (Opa 1) membrane fusion proteins . Thus, the observed differences in sex-specific cell death during the OGD/Reox could be correlated with fusion/fission genes transcription. Chen et al., (2003) using knock-out mice of either Mfn-1 or Mfn-2 have demonstrated the essential role of the mitochondrial fusion/fission machinery and cell viability . We found that XY neurons showed a mainly balanced increase of fusion gene Mfn-1 and fission gene Fis-1 genes, supporting the increased viability of XY neurons during the Reox period . On the contrary, XX neurons showed an imbalance in fusion and fission genes expression under similar conditions, thus promoting apoptotic processes in XX neurons . Mitochondrial shape is largely determined by a balance between fusion-fission events, and this equilibrium maintains steady state mitochondrial morphology, mtDNA and metabolic mixing, bioenergetics functionality and organelle number [20, 46]. Mitochondrial donut formation have been documented in both primary cells and several cell lines, and have some advantages over the linear rod shaped structure to quickly regain ΔΨm loss . We found higher number of donut-shaped mitochondria in XY neurons than in XX neurons during the Reox after OGD. This could aid mitochondrial functional recovery and increased viability observed in XY neurons, whereas, ATP depletion and failure to restore ΔΨm loss in XX neurons affected the fusion process and enhanced vulnerability in XX neurons. Thus, reshaping of mitochondria to donuts might be a component of a protective mechanism that helps to preserve the organelles under the conditions of metabolic stress during the OGD/Reox periods. Together, our findings suggest an intrinsic sex-specific mitochondrial protective mechanism that helps to preserve organelles under hypoxic-ischemic stress conditions.