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, 52 (1), 359-371

Activation of p53 in Down Syndrome and in the Ts65Dn Mouse Brain Is Associated With a Pro-Apoptotic Phenotype

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Activation of p53 in Down Syndrome and in the Ts65Dn Mouse Brain Is Associated With a Pro-Apoptotic Phenotype

Antonella Tramutola et al. J Alzheimers Dis.

Abstract

Down syndrome (DS) is the most common genetic cause of intellectual disability, resulting from trisomy of chromosome 21. The main feature of DS neuropathology includes early onset of Alzheimer's disease (AD), with deposition of senile plaques and tangles. We hypothesized that apoptosis may be activated in the presence of AD neuropathology in DS, thus we measured proteins associated with upstream and downstream pathways of p53 in the frontal cortex from DS cases with and without AD pathology and from Ts65Dn mice, at different ages. We observed increased acetylation and phosphorylation of p53, coupled to reduced MDM2/p53 complex level and lower levels of SIRT1. Activation of p53 was associated with a number of targets (BAX, PARP1, caspase-3, p21, heat shock proteins, and PGC1α) that were modulated in both DS and DS/AD compared with age-matched controls. In particular, the most relevant changes (increased p-p53 and acetyl-p53 and reduced formation of MDM2/p53 complex) were found to be modified only in the presence of AD pathology in DS. In addition, a similar pattern of alterations in the p53 pathway was found in Ts65Dn mice. These results suggest that p53 may integrate different signals, which can result in a pro-apoptotic-phenotype contributing to AD neuropathology in people with DS.

Keywords: Apoptosis; Ts65Dn mouse model; caspase; p53; sirtuins; trisomy 21.

Figures

Figure 1
Figure 1. p53 protein levels and post-translational modifications
p53 phosphorylation at Ser-20 (1)(ph.), acetylation at Lys382 (K382) (2)(ac.) and total expression (exp.) levels (3)were measured by Western Blot in the frontal cortex of controls and DS cases (Panel A) and in the frontal cortex of Ts65Dn mice at 6 and 12 months of age (T6, T12) compared to age-matched euploid animals (E6, E12 -Panel B). Densitometric values shown in the bargraph are the mean of 8 samples per group normalized to total protein load and are given as percentage of control (E6 mice), set as 100%. On the top a representative blot image with protein bands is shown (*p < 0.05).
Figure 2
Figure 2. Upstream regulation of p53
Sirtuins: SIRT1 (1) and SIRT2 (2) levels were measured by Western Blot in the frontal cortex of controls and DS cases (Panel A) and in the frontal cortex of euploid and Ts65Dn mice at 6 and 12 months of age (Panel B). Densitometric values shown in the bargraph are the mean of 8 samples per group normalized per total protein load and are given as percentage of control, set as 100%. On the top a representative blot image with protein bands is shown (*p < 0.05).
Figure 3
Figure 3. p53-MDM2 complex
Levels of p53-MDM2 complex were measured by immunoprecipitation/Western Blot in the frontal cortex of controls and DS cases (panel A) and in the frontal cortex of euploid and Ts65Dn mice at 6 and 12 months of age (Panel B). Densitometric values shown in the bargraph are the mean of 8 samples per group and are given as percentage of control, set as 100%. On the top a representative blot image with protein bands is shown (*p < 0.05).
Figure 4
Figure 4. Bax, PARP1 and caspase-3 down stream targets of p53
Proteins associated with the apoptotic pathway including bax (1), PARP1 (2) and Caspase-3 (3) levels were measured by Western Blot in the frontal cortex of controls and DS cases (Panel A) and in the frontal cortex of Ts65Dn mice at 6 and 12 months of age compared to age-matched euploid animals (Panel B). Densitometric values shown in the bargraph are the mean of 8 samples per group normalized per total protein load and are given as percentage of control, set as 100%. On the top a representative blot image with protein bands is shown (*p < 0.05).
Figure 5
Figure 5. Heat shock protein in response to p53 activation
Two heat shock proteins Hsp70 (1) and Hsp27 (2) were measured by Western Blot in the frontal cortex of controls and DS cases (Panel A) and in the frontal cortex of Ts65Dn mice at 6 and 12 months of age compared to age-matched euploid animals (Panel B). Densitometric values shown in the bargraph are the mean of 8 samples per group normalized per total protein load and are given as percentage of control, set as 100%. On the top a representative blot image with protein bands is shown (*p < 0.05).
Figure 6
Figure 6. PGC1α, a down stream target of p53
PGC1α levels were measured by Western Blot in the frontal cortex of controls and DS cases (Panel A) and in the frontal cortex of Ts65Dn mice at 6 and 12 months of age compared to age-matched euploid animals (Panel B). Densitometric values shown in the bargraph are the mean of 8 samples per group normalized per total protein load and are given as percentage of control, set as 100%. On the top a representative blot image with protein bands is shown (*p < 0.05).
Figure 7
Figure 7. Putative scenario of p53 network in Down Syndrome
Accumulation and transcriptional activation of p53 occurs in response to a wide variety of insults such as DNA damage, oxidative stress, excitotoxicity, etc. p53 undergoes PTMs that critically control its stability and function, including phosphorylation, acetylation, ubiquitination, oxidation and others. Cellular stress affects the interaction between p53 and MDM2, an ubiquitin ligase responsible for p53 degradation. In parallel, transcriptional activity of p53 requires acetylation and/or phosphorylation that may promote cell death pathway through the activation of downstream targets (Bax, Puma, PARP1, Caspase 3, PGC-1α and others). HSPs (27 and 70) act as pro-survival signals by interacting with pro-apoptotic mediators and stabilizing p53 inactive form (p53/MDM2 complex).

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