We show that neuronal MRC deficiency affecting all respiratory complexes occurs in the brain of patients with sCJD. This deficiency shows a strong association with the severity of pathological markers of disease and has a predilection for the MM1 molecular disease subtype. In spite of the pronounced MRC loss, the outer mitochondrial membrane marker VDAC1 remains intact, indicating that the total neuronal mitochondrial mass remains unchanged. The finding of deficient MRC in sCJD corroborate the work of Ansoleaga et al., who reported deficiency of multiple MRC complexes in the frontal cortex of MM1 type sCJD [11].
Neuronal MRC deficiency in sCJD shows a strong correlation with the severity of the neurodegenerative changes. It has a clear regional predilection for the severely affected temporal cortex, whereas the relatively spared CA4/CA3 hippocampal regions show only a mild decrease in MRC staining. Moreover, the extent of MRC deficiency in the temporal cortex shows a positive correlation with the severity of neuropathological changes including gliosis, vacuolation and PrPsc load. We believe that the lack of association between MRC deficiency and severity of neuropathology in the CA4/CA3 region may reflect insufficient statistical power due to the substantially milder MRC involvement in that area.
The severity of MRC deficiency is associated with the molecular subtype of sCJD. MRC deficiency is significantly more pronounced in neurons of individuals with MM1 type sCJD compared to VV2 type sCJD. In fact, with the exception of complex V, MRC deficiency in VV2 type sCJD was not statistically significant compared to controls, although a trend was seen for higher numbers of MRC-deficient neurons. Regression analysis shows that the difference between molecular subtype of sCJD is partly confounded by the more severe pathological changes in MM1 molecular phenotype. Spongiform changes are more pronounced in the neocortex of MM1 type sCJD compared to VV2, where the basal ganglia and thalamus are predominantly affected [19]. However, as disease subtype remains a significant predictor of MRC deficiency in our model, we cannot exclude the possibility of additional factors rendering MM1 neurons more susceptible to MRC impairment.
Our findings suggest that MRC deficiency is an integral part of sCJD pathology and plays an important role in the pathogenesis of the disorder. While MRC deficiency correlates strongly with the severity of disease specific pathological markers, the fact that it is also present in morphologically normal neurons of the mildly affected CA4/CA3 region suggests it is not a terminal phenomenon in dying neuronal populations. The mechanisms underlying MRC loss in sCJD remain unknown. Since mitochondrial mass is unchanged, it is possible MRC is depleted via an active downregulation or increased degradation of the peptide subunits of the respiratory complexes. mtDNA encodes subunits of complexes I, III, IV and V and quantitative and/or qualitative mtDNA defects are a common cause of MRC deficiency [20]. However, mtDNA defects should not affect complex II, which is entirely encoded in the nucleus, and very rarely cause a uniform loss of the remaining respiratory complexes. Therefore, the MRC deficiency of sCJD does not fit the profile of pure mtDNA defects.
The observed pan-respiratory complex deficiency without a change in the mitochondrial outer membrane marker VDAC1 may reflect damage to the inner mitochondrial membrane. This is in line with previous reports of abnormal mitochondrial cristae architecture in prion infected rodents [8, 21, 22]. Interestingly, while PrPc has been shown to localize mostly on the plasma membrane, it has been suggested that it may also be present in the inner mitochondrial membrane of healthy mice [23]. Furthermore, dysmorphic mitochondria with abnormal cristae morphology have been observed in PRNP knockout mice [24]. It is therefore possible that PrPsc aggregation injures the inner mitochondrial membrane, both directly and via loss of its physiological counterpart, PrPc.
Irrespective of the cause, deficiency of the entire MRC is expected to compromise neuronal metabolism causing ATP depletion and a shift toward increased glycolysis [25]. Neurons are highly dependent on oxidative phosphorylation and use most of their ATP to maintain their membrane potential. This is achieved via the action of the sodium-potassium ATPase, which maintains the intra- and extracellular concentrations of sodium and potassium ions against their electrochemical gradients and, by extension, regulates intracellular water balance [26]. Pump failure due to ATP deficiency causes a shift of water from the extracellular to the intracellular compartment (i.e. cytotoxic edema), leading to disruption of essential cellular processes and ultimately cell death. In fact, diffusion weighted imaging (DWI) of the brain typically shows evidence of restricted water diffusion in affected areas of the sCJD brain [27], suggesting that cytotoxic edema is indeed an important mechanism underlying neuronal death in sCJD. Based on our findings, we propose that cytotoxic changes in the sCJD brain are caused by ATP depletion due to neuronal MRC deficiency. Similar signs of restricted water diffusion on MRI are seen in other disorders involving neuronal energy failure such as mitochondrial disease [28,29,30], hypoxia/ischemia [31], hypoglycaemia [32, 33] and carbon monoxide intoxication [34].
Another consequence of neuronal MRC impairment in sCJD would be a shift toward glycolytic metabolism, resulting in higher production of lactate. Elevated concentration of cerebrospinal fluid (CSF) lactate has indeed been reported in patients with CJD, corroborating this hypothesis [35]. In addition to decreased ATP production, a glycolytic shift could render neurons more susceptible to oxidative damage. Normally, neurons can consume glucose through the pentose phosphate pathway, which helps to regenerate reduced glutathione, an important component against oxidative stress. Up-regulation of glycolysis may therefore cause increased levels of oxidized glutathione, in turn, increased susceptibility to the formation of reactive oxygen species in neurons [36]. Oxidative damage has indeed been implicated in cell models of prion disease [37,38,39].
While cause and effect cannot be confidently discerned by this type of study, our findings strongly suggest that MRC loss in sCJD is deleterious and actively involved in the neurodegenerative process. Interestingly, the pattern of MRC deficiency in sCJD is distinct from that of other neurodegenerative proteinopathies. In Parkinson’s disease, MRC deficiency is selective for complex I (and to a much lesser degree complex IV) and does not correlate with the severity of neurodegeneration. In fact, unlike sCJD where the load of PrPsc correlates with MRC deficiency, in PD there is an inverse relationship between complex I loss and Lewy pathology leading to the hypothesis that it may be a partly protective event [17, 40]. It is conceivable that downregulation of complex I levels without a decrease in the remaining respiratory complexes, as observed in PD, serves to limit reactive oxygen species (ROS) production and oxidative damage without a major compromise in energy transduction. In contrast, the global MRC loss seen in sCJD is highly likely to cause ATP depletion.
In conclusion, we show that mitochondrial dysfunction is an important mechanism underlying neuronal injury and neurodegeneration in sCJD. These findings provide an explanation for the clinical observations of restricted MRI diffusion and elevated CSF lactate in patients. Our findings suggest that mitochondria should be the focus of further study in sCJD and should be assessed as potential therapeutic targets for this incurable and devastating disorder.