CON LAS HIPÓTESIS

Initial experiments were designed to identify cytochrome C release from the mitochondria to the cytosol. In order to confirm that SK-N-SH (ABAD-EGFP) cells undergo apoptosis when incubated with Aβ42, subcellular fractionation was carried out on the cells after incubation for 24hr with 2µM Aβ42 peptide. For comparison and as a positive control, SK-N-SH (ABAD-EGFP) cells were also incubated with actinomycin D which is an antineoplastic antibiotic and inhibits cell proliferation by binding to double stranded DNA, inhibiting RNA synthesis and thus transcription (Sobell, 1985). It is a known inducer of apoptosis in tumour cells (Kleeff et al., 2000).

The live imaging studies (section 3.3.2) appeared to show GFP release into the cytosol after a 4hr incubation with 2µM Aβ42 (Figure 3.14). Therefore it was proposed that ABAD-EGFP may be released rapidly from the mitochondria into the cytosol in a similar manner to cytochrome C and this could be another initial marker for apoptosis.

The experiment was designed to isolate the mitochondria from the cells by cellular fractionation after the addition of Aβ, and identify whether cytochrome C and/or ABAD were released into the cytosol by western immunoblotting. The SK-N-SH (ABAD-EGFP) cells were fractionated using a mitochondrial fractionation kit (Active Motif), which is designed to provide an optimized protocol to eliminate cross-contamination and produce high yields of properly

segregated mitochondrial and cytosolic fractions. The kit provides gentle lysis buffers and requires only a pestle homogeniser and the use of a benchtop centrifuge. The detailed protocol is found in section 2.4.2. The isolated fractions were analysed by western blot analysis using 0.5µg ml-1 anti-cytochrome C primary antibody (Santa Cruz mouse anti-cytochrome C) and 0.5µg ml-1 anti- mouse HRP (horseradish peroxidase) secondary antibody (Figure 3.16).

Fig. 3.16. Western blot of fractionated SK-N-SH (ABAD-EGFP) cells incubated for 24hr with 100µM actinomycin D or 2µM Aβ42, using anti-cytochrome C antibody. Lanes 1,

2, 3: mitochondrial fractions. Lanes 5, 6, 7: cytosolic fractions. Lanes 1&5: no toxins (negative control). Lanes 2&6: actinomycin D incubation. Lanes 3&7: Aβ incubation. Lane 4: empty lane. Lane 9: molecular weight markers. Lane 10: positive control for cytochrome C. This shows that cytochrome C is present in the mitochondria but only present in the cytosol after addition of the toxins, actinomycin D and Aβ (lanes 6&7), indicating that both toxins cause apoptosis after 24hr incubation with SK-N-SH (ABAD-

In order to identify if ABAD-EGFP is released from the mitochondria into the cytosol in a similar manner to cytochrome C, a repeat experiment was carried out. The SK-N-SH (ABAD-EGFP) cells were incubated with Aβ42 overnight for 16hr. The cells were fractionated as before. The isolated fractions were analysed by western blot using 0.5µg ml-1 anti-ABAD antibody (kindly donated by SD Yan, Columbia University NY, USA) (Figure 3.17).

Fig. 3.17. Western blot of fractionated SK-N-SH (ABAD-EGFP) cells incubated for 16hr, using 0.5µg ml-1 anti-ABAD antibody. Lane 1, mitochondrial fraction, lane 2,

cytosolic fraction and lane 3, positive control for ABAD. This shows that ABAD-EGFP is not released into the cytosol when SK-N-SH (ABAD-EGFP) cells are incubated with 2µM Aβ42 for 16hr.

These western blot analysis results did not appear to match the live imaging results. To verify if ABAD-EGFP release into the cytosol is time dependent, a further experiment was carried out where SK-N-SH (ABAD-EGFP) cells were incubated with 2µM Aβ42 for up to 48hr. Samples were taken and fractionated at 18hr, 26hr and 48hr after incubation with Aβ (Figure 3.18).

Fig. 3.18. Western blot of fractionated SK-N-SH (ABAD-EGFP) cells incubated for up to 48hr with 2µM Aβ42. Western blot analysis using anti-ABAD antibody. Lanes (a), (c),

(e) and (h) mitochondrial fractions and lanes (b), (d), (f) and (i) cytosolic fractions (green arrows). Lanes (a) and (b) cells with no Aβ incubation. Lanes (c) and (d) cells after 48hr incubation with Aβ. Lanes (e) and (f) cells after 26hr incubation with Aβ. Lane (g) Positive control for ABAD. Lanes (h) and (i) cells after 18hr incubation with Aβ. This shows that when SK-N-SH (ABAD-EGFP) cells are incubated with 2µM Aβ42for 48h,

These results may indicate that ABAD is released from the mitochondria late in the death of a cell, or these results may be due to the poor sensitivity of the antibodies used that may only detect high concentrations of the proteins in the cellular lysate. The live imaging experiments performed previously appeared to show ABAD-EGFP beginning to be released into the cytosol after 4hr incubation with 2µM Aβ42. This may indicate that ABAD-EGFP is released slowly from the mitochondria over a period of hours. This is supported by the western blots after 24hr and 48hr incubation with the Aβ42 peptide. It is therefore difficult to make a direct comparison of the results here, using these different techniques. Similarly the MTT assays may not be sensitive enough and the results could be masked by the cells dividing.

However, all the western blots show that Aβ42 peptide induces apoptosis in SK- N-SH (ABAD-EGFP) cells, shown by cytochrome C release from the mitochondria into the cytosol within 16hr. ABAD is seen in the cytosol after 48hr incubation with the Aβ peptide. From these results in SK-N-SH cells, ABAD does not appear to be mimicking the rapid release of cytochrome C into the cytosol and it is therefore not an early marker for apoptosis.

3.4.3 Investigation of apoptotic pathways induced in SK-N-SH (ABAD-