Mitochondrial abnormalities in PrP-null mice and Mecp2-null mice

Abstract

The transmissible spongiform encephalopathies (TSE's), also known as prion diseases, are believed to arise following the conversion ofthe endogenous cellular prion protein, PrPc, to a pathogenic isoform, PrPSc. Although the physiological function(s) ofPrPc remain uncertain, recent reports have demonstrated increased levels of oxidative stress markers and mitochondrial abnormalities in PrP-null mice. This thesis will demonstrate further evidence of significant mitochondrial abnormality in PrP-null mice. In addition the opportunity arose to study mitochondrial function in Mecp2-null mice, a model of the neurodevelopmental disorder Rett syndrome. A mitochondrial abnormality has long been postulated as a contributing factor in Rett Syndrome and altered transcription levels of mitochondrial respiratory Complex subunits have recently been identified in the Mecp2-n\iW model. This thesis will confirm and describe significant mitochondrial pathology in this model.The phenotype associated with the PrP-null mouse has proved to be subtle, the animals exhibiting minor behavioral modification and controversial electrophysiological differences. There is also a growing body of evidence implicating the involvement of oxidative stress in both TSE's and the PrP-null model. Recent studies ofPrP-null mice have uncovered alterations in mitochondrial morphology, MnSOD levels and some weak evidence of altered transcription levels of some subunits of respiratory chain Complexes. Additionally electrophysiological studies of hippocampal slices, which synaptically express PrPc at high levels, have recently shown a reduction in post-tetanic potentiation, which is also suggestive of altered mitochondrial function in the brain. Experiments were therefore conducted to augment this body of evidence in PrP-null mice. The sensitivity ofhippocampal slices to experimental tissue culture and the oxidative nature of the culture environment together with basic measures of synaptic efficacy were carried out to investigate the possibility of an oxidative stress-related phenotype for the PrP-null mouse. Evidence of a mitochondrial abnormality in PrP-null mice was sought by extensive assessment of the morphological and functional characteristics of isolated brain mitochondria.Study of isolated mitochondrial suspensions by electron microscopy revealed that PrPnull mitochondria were larger and, when cristae density was quantitatively measured using a novel technique, to have a reduced cristae density when compared to controls.Measurement of oxygen consumption by the mitochondrial electron transport chain of PrP-null mice was carried out using mitochondrial and submitochondrial particle suspensions in a Clark-type Oxygen Electrode. A significant increase in respiratory capacity was detected in PrP-null mitochondria when metabolising Complex I substrates, but not when electrons entered downstream of Complex I. This implicates Complex I as a site for pathological change. Analysis of mitochondrial coupling indicated no difference between the genotypes, suggesting that the permeability of the inner mitochondrial membrane to protons was unchanged.Assessment of superoxide production by the respiratory chain using tempone-H spintrapping and electron paramagnetic resonance (EPR) spectroscopy proved impossible using intact mitochondria due to interference by endogenous antioxidant systems. However the data suggested that tempone spin-trapping and EPR may yield novel methods for the assay of mitochondrial antioxidants. The mitochondrial antioxidant effect was circumvented by the use of submitochondrial particles (SMP) which lacked the interfering antioxidant system. EPR experiments demonstrated maximal superoxide production by Complex I was significantly increased by around 40% in PrP-null SMP suspensions whilst Complex III superoxide production was unchanged.As recent studies ofthe Mecp2-null mouse model of Rett syndrome detected increased transcription of Uqcrcl (which encodes a core subunit of Complex III), mitochondrial morphology and respiration were studied in Mecp2-null mice. Whilst electron microscopy did not reveal any gross alterations in mitochondrial size or cristae density, respiration measurements revealed a severe phenotype in symptomatic, but not presymptomatic, Mecp2-null mice. Mitochondrial coupling was considerably reduced in isolated brain mitochondria from Mecp2-nu\\ mice indicating reduced mitochondrial efficiency. This effect was accompanied by an increase in respiratory capacity through Complex HI. To determine ifthe overexpression of Uqcrcl was causative in the production of the observed increase in respiratory capacity, the respiration rates ofN2A cells overexpressing Uqcrcl were measured. As transfected N2A cells showed significantly increased respiration rates through Complex IE, the up-regulation of Uqcrcl may be causative in producing the respiratory capacity increase observed in the animal model.These results enhance the evidence for dysfunctional mitochondria in both PrP-null and Mecp2-nu\\ mouse models and are discussed in relation to other investigations of prion disease, Rett syndrome and other models of neurodegenerative disease.