Investigation of the Ub G76V -GFP mouse model

As expected, no correlation was found between the steady-state level of ubiquitinated proteins and all three 20S proteasome activities in the WT mice. Only in the KO mice, the steady-state levels of ubiquitinated proteins correlated inversely with the chymotrypsin-like and the trypsin-like activity, i.e. the steady-state levels of ubiquitinated proteins were low, when the two activities were high or vice versa. No correlation was found in the KI mice concerning these parameters. Between the steady-state levels of ubiquitinated proteins and the caspase-like activity, no correlation was observed in both KI and KO mice.

The expected 400 bp-fragment for the transgene was only amplified in the transgenic Ub^G76V-GFP mice.

Fibroblasts were isolated from subcutis biopsies of an adult Ub^G76V-GFP mouse, propagated for several weeks, plated on Lab-Tek™ chamber slides and treated with different proteasome inhibitors for 15 h. The reversible proteasome inhibitor MG132 was used in a concentration of 0.1 µM, 1 µM or 10 µM, the reversible proteasome inhibitor MG262 in a concentration of 1 nM, 10 nM, 100 nM or 1 µM and the irreversible proteasome inhibitor epoxomicin in a concentration of 5 nM, 50 nM, 500 nM or 5 µM. Afterwards, the GFP fluorescence was analyzed by confocal microscopy (Fig. 3.36).

Figure 3.35: Genotyping of the Ub^G76V-GFP mouse model by PCR. PCR was performed on genomic tail DNA from transgenic Ub^G76V-GFP (UG) and WT mice using transgene-specific primers.

MW stands for the 100-bp molecular weight marker.

Figure 3.36: Direct fluorescence micrographs of treated fibroblasts from an adult Ub^G76V-GFP mouse. Fibroblasts were untreated (control) or treated with the proteasome inhibitors MG132, MG262 or epoxomicin (epo) in the indicated concentrations. Confocal images were recorded after 15 h

Control

Epo 5 nM MG262 1 nM

MG132 100 nM

Epo 50 nM MG262 10 nM

MG132 1000 nM

Epo 500 nM MG262 100 nM

Epo 5000 nM MG262 1000 nM

MG132 10000 nM Control

Epo 5 nM Epo 5 nM MG262 1 nM MG262 1 nM

MG132 100 nM

Epo 50 nM Epo 50 nM MG262 10 nM MG262 10 nM

MG132 1000 nM

Epo 500 nM Epo 500 nM MG262 100 nM MG262 100 nM

Epo 5000 nM Epo 5000 nM MG262 1000 nM

MG132 10000 nM MW WT UG UG UG

400 bp

MW WT UG UG UG 400 bp

Without proteasome inhibition, no GFP-positive fibroblasts could be found, which proved that the reporter was rapidly degraded by the UPS under normal conditions. In contrast, after proteasome inhibition an increase of GFP-positive fibroblasts was observed in a concentration-dependent manner. This was found with all 3 inhibitors;

the inhibition by MG262 and epoxomicin was more potent than by MG132.

To analyze whether proteasome inhibition leads to the same effect in cardiomyocytes, cardiomyocytes were isolated from adult Ub^G76V-GFP mice, plated on laminin-coated Lab-Tek™ chamber slides and treated with 10 µM MG132, 100 nM MG262 or 500 nM epoxomicin for 20 h. Afterwards, GFP-positive, rod-shaped cardiomyocytes were counted under the confocal microscope (Fig. 3.37).

GFP-positive cells were found in the untreated cardiomyocytes, and the number of GFP-positive cardiomyocytes did not increase after UPS inhibition. This suggests that the adult cardiomyocytes were exposed to stress during isolation and responded with a stress-induced GFP accumulation already visible without proteasome inhibition.

Finally, 9 µmol/kg MG262, which is a high dose (Lindsten et al., 2003), was injected intraperitoneally (i.p.) into an adult Ub^G76V-GFP mouse. As a control, a 60% DMSO-NaCl-solution (vehicle) was i.p. injected into a second Ub^G76V-GFP mouse. Both mice were killed 24 h after injection, and the liver and heart were examined by direct

Control MG132 10 µM MG262 100 nM Epo 500 nM Control MG132 10 µM MG262 100 nM Epo 500 nM

Figure 3.37: Direct fluorescence micrographs of treated cardiomyocytes from adult Ub^G76V-GFP mice. Cardiomyocytes were untreated (control) or treated with the proteasome inhibitors MG132, MG262 or epoxomicin (epo) in the indicated concentrations. Confocal images were recorded after 20 h treatment with a Zeiss LSM 5 Image sytem using a Zeiss Axiovert 200 M microscope.

No GFP-fluorescence was detected in the control liver and heart, which indicated that the reporter was rapidly degraded by the UPS. After proteasome inhibition by MG262, a GFP-fluorescence was obtained in the liver, but not in the heart. Moreover, proteins were extracted from the heart and liver of these treated mice and analyzed by Western blot using an antibody directed against ubiquitin (Fig. 3.39).

First, the Western blot analysis showed that the steady-state level of ubiquitinated proteins was much higher in the liver than in the heart under normal conditions.

Figure 3.38: Direct fluorescence micrographs of tissues from treated adult Ub^G76V-GFP mice.

MG262 (9 µmol/kg) or vehicle only (60% DMSO-NaCl-solution; control) were injected i.p. into adult Ub^G76V-GFP mice. Direct fluorescence micrographs were taken from both liver and heart 24 h after injection.

Figure 3.39: Ubiquitination in treated adult Ub^G76V-GFP mice. Proteins were extracted from both heart and liver of adult Ub^G76V-GFP mice treated i.p. with MG262 (9 µ mol/kg) or vehicle only (60% DMSO-NaCl-solution). The blot was stained with an antibody directed against ubiquitin.

+ MG262

Heart Liver

Control

+ MG262

Heart Liver

Control

MG262 i.p. (µmol/kg): 0 9 0 9 Heart Liver

Ubiquitinated proteins

MG262 i.p. (µmol/kg): 0 9 0 9 Heart Liver

Ubiquitinated proteins

after treatment. This suggests that the concentration of MG262 that reached the heart after i.p. injection was insufficient to inhibit the proteasome. This would explain why no GFP fluorescence was visualized by fluorescence microscopy in the heart of the MG262-treated Ub^G76V-GFP mouse. In contrast, a strong increase in the steady-state level of ubiquitinated proteins was revealed in the liver of the MG262-treated mouse compared to the control, which was in line with the strong GFP-accumulation examined by the fluorescence microscopy.