A mitochondrial location for haemoglobins--dynamic distribution in ageing and Parkinson's disease

Abstract

Haemoglobins are iron-containing proteins that transport oxygen in the blood of most vertebrates. The mitochondrion is the cellular organelle which consumes oxygen in order to synthesise ATP. Mitochondrial dysfunction is implicated in neurodegeneration and ageing. We find that α and β haemoglobin (Hba and Hbb) proteins are altered in their distribution in mitochondrial fractions from degenerating brain. We demonstrate that both Hba and Hbb are co-localised with the mitochondrion in mammalian brain. The precise localisation of the Hbs is within the inner membrane space and associated with inner mitochondrial membrane. Relative mitochondrial to cytoplasmic ratios of Hba and Hbb show changing distributions of these proteins during the process of neurodegeneration in the pcd(5j) mouse brain. A significant difference in mitochondrial Hba and Hbb content in the mitochondrial fraction is seen at 31 days after birth, this corresponds to a stage when dynamic neuronal loss is measured to be greatest in the Purkinje Cell Degeneration mouse. We also report changes in mitochondrial Hba and Hbb levels in ageing brain and muscle. Significant differences in mitochondrial Hba and Hbb can be seen when comparing aged brain to muscle, suggesting tissue specific functions of these proteins in the mitochondrion. In muscle there are significant differences between Hba levels in old and young mitochondria. To understand whether the changes detected in mitochondrial Hbs are of clinical significance, we examined Parkinson's disease brain, immunohistochemistry studies suggest that cell bodies in the substantia nigra accumulate mitochondrial Hb. However, western blotting of mitochondrial fractions from PD and control brains indicates significantly less Hb in PD brain mitochondria. One explanation could be a specific loss of cells containing mitochondria loaded with Hb proteins. Our study opens the door to an examination of the role of Hb function, within the context of the mitochondrion-in health and disease.

Keywords: Ageing; Haemoglobin; Mitochondria; Neurodegeneration; Parkinson's Disease.

Fig. 1

HbA and HbB colocalise with mitochondria in control human frontal lobe. Confocal images. Mitochondria are visualised with the COXIV antibody (red) and HbA and HbB (green) are overlaid to provide colocalisation information. Sections from human frontal lobe and substantia nigra were each investigated for both haemoglobins. A. HbA and COXIV colocalise in many regions of the frontal lobe, it appears that there are areas which are specific for HbA where mitochondria are not present. B. HbB and COXIV also overlap in expression in the frontal lobe, though again there are areas of HbB expression where mitochondria are not detected. C. In the substantia nigra HbA and COXIV expression also overlap, a close look suggests some mitochondria do not express Hba and there are areas of diffuse HbA expression that do not correspond to mitochondrial staining. D. Again in the substantia nigra there is considerable overlap of HbB and COXIV localisation, though there are distinct regions where either one or the other protein is expressed or colocalisation doesn't appear to be uniform.

Fig. 2

HbA and HbB are localised within the mitochondrion—to the inner membrane and inter-membrane space. A. HbA is localised (white arrows) to the mitochondrion and can be observed in the inter-membrane space and within cristae structures. A characteristic rosette pattern of gold particles is seen when immuno electron microscopy of HbA is performed. Immuno-electron microscopy was performed on control human brain tissues using 6 nm immunogold. B. Secondary antibody no primary antibody control-conjugated immunogold. C. HbA and HbB are co-isolated with inner membrane and inter-membrane space sub-fractions from human mitochondrial preparations. Smac/Diablo confirms isolation of inter-membrane space fractions. The inner membrane enrichment is confirmed by NDUFS3 reactivity, faint bands in this lane suggest that Hbs also associate with the mitochondrial inner membrane. Abbreviations used: mito fr.—whole mitochondrial fraction before sub-fractionation, IMS—inter-membrane space, OM—outer membrane, IM—inner membrane space.

Fig. 3

Comparison of mitochondrial Hba and Hbb in pcd^5j during neurodegeneration and in normal, ageing mouse. A Hba and B Hbb relative quantities are shown in pcd^5j (ataxic) and control mouse neuronal mitochondria at intervals from weaning to maturity. Both Hba and Hbb are reduced relatively in mitochondria in mouse brain undergoing a neurodegenerative process. The difference between ataxic and control samples reaches significance in the brains taken at 31 days. At this time point both Hba and Hbb are significantly reduced in ataxic mitochondria when compared with quantities in the cytoplasm. A + B *p < 0.05 (2-way anova) n = 3. C. Western blot example of Hbs variation through the process of degeneration. Each sample lane is first analysed, for normalisation, to determine total mitochondrial content ratio with respect to cytoplasmic contamination (as described for D and E below). Hbs are then compared with the normalised (compared to cytoplamic) VDAC/porin quantification in each case. D. Hba and E. Hbb were measured in mitochondrial and cytoplasmic fractions prepared from 8 week and 18 month old mouse brain and skeletal muscle. The ratio of Hba and Hbb in the mitochondrial fraction (normalised to porin) compared with the cytoplasm (normalised to hsp-90) is plotted. Significant differences between mitochondrial and cytoplasmic ratios of both Hbs were found between the two tissue types, brain and muscle. Hba shows a significant increase when measured in old muscle mitochondria compared with young muscle mitochondria. Data shown are mean ± SEM of 3 experiments *p < 0.05 (one-tailed t-test).

Fig. 4

A. HbA colocalises with mitochondria in human brain regions frontal lobe and substantia nigra (HbA-green, COXIV-red, DAPI stain—blue). Control human frontal lobe is compared with Alzheimer's (AD) frontal lobe. There is a suggestion that in AD frontal lobe there is less diffuse mitochondrial staining than in controls with many more frequent clumps of mitochondrial and HbA colocalisation. In control substantia nigra large cell bodies show both separate staining and also colocalisation of HbA and COXIV. PD substantia nigra cell bodies also show overlap of these proteins, with perhaps a greater distribution of colocalisation both intra and extracellularly. B HbA mitochondrial to cytoplasmic ratio is reduced in PD brain *p = 0.065 (1 tail t-test) n = 4, compared with controls. There is no significant difference in AD mitochondrial/cytoplasmic ratios of HbA when compared with controls. C. HbB is significantly reduced in PD mitochondria compared with cytoplasmic HbB **p = 0.038 (1 tail t-test) n = 4. Again ratios in AD brain fractions do not show a significant change when compared with controls. Abbreviations: FL—frontal lobe, SN—subtantia nigra, AD—Alzheimer's Disease, PD—Parkinson's Disease.