This study established, for the first time, increased mitochondrial biogenesis after neonatal H-I brain injury. Measurement of the relative amount of brain mtDNA up to 24 hours after H-I showed an increase in cortical mtDNA content. Comparable to the increase in mtDNA, a temporal increase in the number of mitochondria, the expression of two mitochondrial proteins, HSP60 and COXIV, and citrate synthase activity was found. Moreover, increased expression of NRF-1 and TFAM were also detected. These results suggest that rapidly increased mitochondrial biogenesis is an inducible response by the neonatal brain after H-I injury and constitutes a novel component of the endogenous repair mechanisms of the brain.
Numerous studies support the hypothesis that disruption of mitochondrial function plays a central role in the pathophysiology of many neurological diseases.20
Conditions or events that specifically hinder mitochondrial performance, such as H-I–induced cerebral damage, place the brain at risk for compromised energy production and thus secondary injury. An obvious strategy to help minimize damage attributable to lost energy resources is to increase the number of mitochondria themselves. The evidence for this occurring in cerebral H-I models is not clear and has been examined in only a few studies using adult ischemic models. For example, increased mitochondrial elongation, a well documented step in the process of mitochondrial biogenesis, was observed in the CA1 region of the hippocampus after transient global ischemia in adults.9,11
Histological evidence of mitochondrial biogenesis was also found after transient global ischemia in adult rats.11
In transient focal ischemia, 30 minutes of ischemia induced a reduction in mtDNA content; however, mtDNA was restored to nearly preischemic levels 24 hours later.13
This transient loss might be explained by the hypothesis that in adult mice mtDNA deletions or additions are more likely to occur than in young mice to produce decreased viable numbers of mitochondria in the adult.21,22
Our data using the neonatal H-I model lends credence to this possibility.
Other dynamic changes besides mitochondrial senescence, however, contribute to loss of mitochondria after H-I.5
One of the major factors that are upregulated after hypoxic insults is hypoxia-inducible factor 1 (HIF-1).23
While HIF-1 can enhance the ability of tissue to survive reduced oxygen levels, one of the byproducts of this process is the inhibition of mitochondrial biogenesis via loss of c-myc upregulation by PGC-1.24
We in fact found that mRNA and protein levels of PGC-1 did not increase after H-I, indicating that the maximum potential for mitochondrial biogenesis may not have been achieved in our study. Thus, at least one acute response, the upregulation of HIF-1 that is protective against H-I, may be maladaptive in the long term overriding any beneficial mechanisms that are attempting to increase mitochondrial biogenesis.
The upregulation of HSP60 is another response that occurs after many stressors, and is indicative of mitochondrial biogenesis.25
The majority of constitutively expressed HSP60 is in the mitochondria and is involved in stabilizing both newly synthesized proteins and mtDNA, the latter via mitochondrial nucleoids, discrete protein-DNA complexes critical for the regulation of mtDNA transmission and biogenesis of new mitochondria.26
In our H-I model, we did find increased levels of this mitochondrial-enriched protein in surviving neurons, as would be predicted after enhanced biogenesis. In addition to HSP60 being a marker for the presence of mitochondria, it may also be an integral part of the mechanism involved in mitochondrial biogenesis after H-I.
Recent studies have shown that exogenously supplied factors may be able to drive mitochondrial biogenesis in addition to or by augmenting endogenous signaling responses. One such compound is resveratrol, a polyphenol that can activate AMP-activated kinase and that also induces mitochondrial biogenesis in neurons.27
Because resveratrol is also protective in several cerebral ischemic models including neonatal ischemia,28
this raises the possibility that mitochondrial biogenesis may be one of its neuroprotective mechanisms. The involvement of the AMP-kinase cascade is also worth investigating in future studies of neonatal H-I because it plays a key role in sensing and transduction of cellular energy levels.29
Future studies also should include examining exogenously supplied compounds capable of inducing or aiding mitochondrial biogenesis to limit or even contribute to neonatal brain repair.
The functional significance of mitochondrial biogenesis is unknown. Increased mitochondrial mass would clearly improve the overall oxidative function and energy state of the H-I brain. This may be an endogenous neuroprotective response against H-I injury. Evidence for such a role cannot be made until the H-I–induced signaling mechanisms controlling neuronal mitochondrial biogenesis have been elucidated. Further studies to identify the specific signaling pathways will help directly address this issue and determine whether these signaling pathways can be enhanced to ameliorate brain damage due to perinatal H-I.