Expression and Localisation of AIF

5.2.3.2 Expression and Localisation of AIF

The activation of mitochondrial driven apoptosis is dependent on the influx of cell death proteins from within the mitochondria into the cytosol. Upon exposure to toxins such as damaged DNA or ROS, PARP-1 receptors are activated leading to the subsequent activation of Bcl pathways which in turn results in the permeabilisation of the outer mitochondrial membrane (154). This permeabilisation leads to the cleavage of the apoptosis inducing factor (AIF) to its death-inducing active form into the cytosol, which then translocates to the nucleus where it leads to DNA degradation (110). Furthermore, the breakdown of the mitochondrial membrane allows the release of cytochrome-C. Free cytochrome C within the cytosol of the cell is then able to induce further cell death pathways through activation of caspase-3 dependent apoptosis ((106)).

To explore this, fresh murine hearts were homogenized and mitochondria were separated to look at the differential distribution of AIF and cytochrome C. In addition to the whole cell expression of AIF protein, cells were fractionated into the cytosol and mitochondria portions in order to investigate alterations of AIF compartmentalisation following TH.

Western blotting analysis demonstrated that the protein expression levels of AIF were comparable in both sham and trauma haemorrhage animals with mean densities of 0.20 vs. 0.17 (p=0.50).

To further elucidate the distribution of proteins in the different sub cellular fractions obtained from heart tissue of TH models, the subcellular fractionation procedures are followed and differential distribution of specific proteins of interest, between the cytosol and mitochondria were investigated. Heart tissue was homogenized and then processed by differential centrifugation; at low speed to remove large organelles and debris and using a specific mitochondrial extraction buffer (see methods section 2.4.3) by high-speed centrifugation to isolate the mitochondria. The fractionated samples were run through the gels and probed for AIF and positive controls were used to confirm mitochondrial separation- MTCO2 as a mitochondrial marker and GAPDH as a cytosolic marker. The change in distribution of AIF in the mitochondria versus the cytosol was assessed. The subcellular fractionation procedure removes the nuclei from the supernatant and therefore, further analysis was done to compare the mitochondrial expression of AIF against the total expression.

Figure 5.12: AIF expression as demonstrated by western blot analysis. Mean optical density for sham group was 0.20 vs 0.17 for the TH group

In comparison to the sham animals, the TH animals show a reduction in AIF distribution in the mitochondria to supernatant ratio. The mean ratio in the sham models is 1.5 in comparison to 0.4 in the TH models, however this is not statistically significant. This is suggestive that there is an increase in release of AIF in to the supernatant where the trauma becomes lethal in these mice. When the ratios are studied as the distribution between the mitochondria and total cell content that include the nuclei, the difference in AIF expression becomes statistically different.

The mean ratio in the sham models is 0.6 when compared to 0.3 in the TH models

(p=0.04). This is suggestive of mitochondrial AIF leaching into cytosol and potentially its translocation to the nucleus.

Figure 5.13: Comparison of mitochondrial vs Supernatant AIF compartmentalisation in sham and trauma haemorrhage murine hearts as demonstrated by western blots. The mean ratio of mitochondrial AIF (M AIF) to supernatant AIF (S AIF) is 1.5 in sham group compared to 0.4 in TH group, p=0.16.

Figure 5.14: Comparison of AIF expression in whole tissue and in the mitochondria of sham and trauma haemorrhage murine hearts as demonstrated by western blots.

The ratio of total AIF (T AIF) to mitochondrial AIF (M AIF) in sham operated group is 0.6 compared to 0.3 in TH animals, p=0.04.

Immunofluorescent microscopy was used to visualise the localisation of the cell death associated protein AIF with concurrent staining of a mitochondrial marker, MTCO2. The nucleus was directly stained with DAPI (blue). MTCO2 was a primary mouse antibody, that was further incubated with an anti-mouse secondary labeled

with AF 594 (red) and AIF was an anti-rabbit primary antibody that was further labeled with an anti-rabbit secondary antibody with AF 488 label (green).

AIF expression was examined through immunohistochemistry. In contrast to the expression measured by western blot, this demonstrated an increase in AIF expression in the TH models. The mean immunofluorescence exhibited by AIF in the TH models was double that of the sham animals 13 vs. 6 (p0.001).

Figure 5.15: AIF expression as measured by immunofluorescence in paraffin sections. The mean fluorescence was calculated to be 13 in sham animals vs. 6 in the TH animals, p=0.001

On IHC analysis the nuclear localisation of AIF was more apparent in the resuscitated TH models when compared to the Sham models (Figure 5.16). The image-j software was utilised to account for the degree of co-localisation of the proteins. Mitochondrial localisation was calculated to be 0.05 in the TH models compared to 0.18 in the sham, however this was not statistically significant.

Whereas, the difference in nuclear localization was statistically different with a mean of 0 in the sham and 0.06 in the TH models.

Figu.re 5.16: Sections from sham-operated animals, DAPI (blue), AIF (green), MTCO2 (red) and combined images

Figure 5.17: Paraffin embedded sections from TH animals, DAPI (blue), AIF (green), MTCO2 (red) and combined images. In the combined images, localisation of AIF in the nucleus can be seen

Figure 5.18: Mitochondrial Localisation of AIF as calculated by Image-J in sham animals 0.18 in comparison to 0.05 in TH animals, p=0.25

Figure 5.19: Nuclear Localisation of AIF as calculated by Image-J in sham animals 0 in comparison to 0.06 in TH animals, p=0.04