(a) The histogram shows the activity of CI measured from cerebellar homogenates, which results decreased in YG8R mice (* em P /em =0.02). Previous studies of mitochondrial pathophysiology have been performed on post-mortem tissues or fibroblasts. However, mitochondria can be affected by the procedures of tissue extraction and conservation making these studies unreliable, whereas fibroblasts are not affected in FRDA and so pathological changes in these cells may not reflect underlying disease processes. The knockout mouse model has been shown to be embryonic lethal, and this has been followed by the development of conditional knockout mouse models specific for the central nervous system or the heart.8 Although useful for understanding some features of frataxin, these models could not be studied for one of the main features of the FRDA condition, which is the slow progression.8 We used a humanised mouse model, the YG8R transgenic mouse model, which contains a human YAC with 190+90 GAA repeats on a mouse null background, that recapitulates the progressive disease phenotype shown in humans.9, 10, 11, 12 A similar approach has generated a control transgenic mouse that contains the same human YAC, but with only nine GAA repeats, called Y47R mice.13 These mouse models have been validated and extensively used in studies on FRDA.9, 14, 15 Although mitochondrial dysfunction is believed to be one of the main causes of FRDA pathology, the effect of frataxin deficiency on mitochondrial function is not yet clear. The present study sought to investigate the changes in mitochondrial physiology in FRDA-like cerebellar granule neurons and glia, by using hemizygous YG8R mice (with a defective transgene) Rabbit polyclonal to PON2 and hemizygous Y47R mice (with a normal transgene). The cerebellum is one of the most affected tissues in FRDA pathology7, 16, 17, 18 and cerebellar granule neurons have previously been shown to be lost in an inducible knockout FRDA mouse model.19 Although, patients show clear sings of cerebellar ataxia, it is not clear where the pathophysiology lies amongst the cerebellar neurons. In this work we aimed to investigate whether cerebellar granule neurons and glial cells, which are largely unexplored in FRDA, could be affected by the presence of the GAA repeat expansion and to investigate how frataxin deficiency could affect neuronal cell viability. Results YG8R cerebellar granule and glial cells show reduced frataxin levels Frataxin levels were measured in co-cultures of cerebellar granule neurons and glial cells from Y47R and YG8R mice. Using immunofluorescence, we labelled human Cinaciguat frataxin and measured the fluorescence intensity cell-by-cell, differentiating granule cells from glia with a neuronal marker (anti-MAP-2) (Physique 1A). We found that there is a significant decrease of frataxin in both cerebellar granule neurons and glial cells in the YG8R genotype, when compared to the Y47R (Physique 1B; granule cells YG8R 11.1%1.3, in YG8R cerebella compared with Y47R mice of the same Cinaciguat age. The quantification of three impartial experiments is usually represented in the histogram (Physique 1E; Y47R 1.120.24, YG8R 0.590.17; three impartial experiments in duplicates of em n /em =3 mice; *** em P /em =0.0004), normalising the hFXN signal with a mitochondrial marker, the apoptotis-inducing factor protein (AIF). Since it is known that frataxin is usually involved in the biosynthesis of haem and acts as a chaperon for ISCs,20, 21 its activity is crucial for those proteins that require ISCs to perform their functional activity,22 such as Complexes I and III of the mitochondrial electron transport chain (ETC) and aconitase.14, 22 As Complexes I and III are fundamental for the maintenance of the mitochondrial membrane potential (?m), we investigated if mitochondrial respiration could be dependent on frataxin activity and therefore be affected by the decrease of this protein. Cinaciguat YG8R cerebellar granule cells exhibit ?m.