Erwan Paitel

Wild-type and mutated presenilins 2 trigger p53-dependent apoptosis and down-regulate presenilin 1 expression in HEK293 human cells and in murine neurons.

Presenilins 1 and 2 are two homologous proteins that, when mutated, account for most early onset Alzheimers disease. Several lines of evidence suggest that, among various functions, presenilins could modulate cell apoptotic responses. Here we establish that the overexpression of presenilin 2 (PS2) and its mutated form Asn-141-Ile-PS2 alters the viability of human embryonic kidney (HEK)293 cells as established by combined trypan blue exclusion, sodium 3′-[1-(phenylamino-carbonyl)-3,4-tetrazolium]-bis(4-methoxy-6-nitro)benzene sulfonic acid hydrate assay, and propidium iodide incorporation FACS analyses. The two parent proteins increase the acetyl-DEVD-al-sensitive caspase-3-like activity in both HEK293 cells and Telencephalon specific murine neurons, modulate Bax and bcl-2 expressions, and enhance cytochrome C translocation into the cytosol. We show that overexpression of both wild-type and mutated PS2 increases p53-like immunoreactivity and transcriptional activity. We also establish that wild-type- and mutated PS2-induced caspase activation is reduced by p53 antisense approach and by pifithrin-α, a chemical inhibitor of p53. Furthermore, mouse fibroblasts in which the PS2 gene has been knocked out exhibited strongly reduced p53-transcriptional activity. Finally, we establish that the overexpression of both wild-type and mutated PS2 is accompanied by a drastic reduction of endogenous presenilin 1 (PS1) expression. Interestingly, pifithrin-α diminished endogenous PS2 immunoreactivity, whereas the inhibitor increases PS1 expression. Altogether, our data demonstrate that wild-type and familial Alzheimers disease-linked PS2 trigger apoptosis and down-regulate PS1 expression through p53-dependent mechanisms.

α-Synuclein Lowers p53-dependent Apoptotic Response of Neuronal Cells ABOLISHMENT BY 6-HYDROXYDOPAMINE AND IMPLICATION FOR PARKINSON′S DISEASE

We have examined the influence of α-synuclein on the responsiveness of TSM1 neuronal cells to apoptotic stimulus. We show that α-synuclein drastically lowers basal and staurosporine-stimulated caspase 3 immunoreactivity and activity. This is accompanied by lower DNA fragmentation and reduced number of terminal deoxynucleotide transferase-mediated dUTP nick end labeling (TUNEL)-positive neurons. Interestingly, α-synuclein also diminishes both p53 expression and transcriptional activity. We demonstrate that the antiapoptotic phenotype displayed by α-synuclein can be fully reversed by the Parkinsons disease-associated dopamine derivative 6-hydroxydopamine. Thus, 6-hydroxydopamine fully abolishes the α-synuclein-mediated reduction of caspase 3 activity and reverses the associated decrease of p53 expression. 6-Hydroxydopamine triggers thioflavin T-positive deposits in α-synuclein, but not mock-transfected TSM1 neurons, and drastically increases α-synuclein immunoreactivity. Altogether, we suggest that α-synuclein lowers the p53-dependent caspase 3 activation of TSM1 in response to apoptotic stimuli and we propose that the natural toxin 6-hydroxydopamine abolishes this antiapoptotic phenotype by triggering α-synuclein aggregation, thereby likely contributing to Parkinsons disease neuropathology.

Phorbol ester-regulated cleavage of normal prion protein in HEK293 human cells and murine neurons.

Cellular prion protein (PrPc) undergoes a proteolytic attack at the 110/111↓112 peptide bond, whereas the PrP isoform (PrPres) that accumulates in the brain tissue in Creutzfeldt-Jakob disease reveals an alternate cleavage site at about residue 90. Interestingly, the normal processing of PrP occurs inside the 106–126 amino acid region thought to be responsible for the neurotoxicity of the pathogenic prions, whereas PrPrescleavage preserves this potentially toxic domain. Therefore, any molecular mechanisms leading to enhanced cleavage at the 110/111↓112 peptide bond could be of potential interest. We set up TSM1 neurons and HEK293 stable transfectants overexpressing the wild-type or 3F4-tagged murine PrPc, respectively. Both mock-transfected and PrPc-expressing cell lines produced an 11–12-kDa PrP fragment (referred to as N1), the immunological characterization of which strongly suggests that it corresponds to the N-terminal PrPc fragment derived from normal processing. We have established that the recovery of secreted N1 is increased by the protein kinase C agonists PDBu and PMA in a time- and dose-dependent manner in both cell lines. In contrast, secretion of N1 remains unaffected by the inactive PDBu analog αPDD and by the protein kinase A effectors dibutyryl cAMP and forskolin. Overall, our data indicate that the normal processing of PrPc is up-regulated by protein kinase C but not protein kinase A in human cells and murine neurons.

Overexpression of PrPc triggers caspase 3 activation: potentiation by proteasome inhibitors and blockade by anti-PrP antibodies.

We examined the influence of cellular prion protein (PrPc) in the control of cell death in stably transfected HEK293 cell line and in the PrPc‐inducible Rov9 cells. PrPc expression in stably transfected HEK293 human cells did not modify basal apoptotic tonus but drastically potentiated staurosporine‐stimulated cellular toxicity and DNA fragmentation as well as caspase 3‐like activity and immunoreactivity. An identical staurosporine‐induced caspase 3 activation was observed after doxycycline in the PrPc‐inducible Rov9 cell line. Interestingly, proteasome inhibitors increase PrPc‐like immunoreactivity and unmasked a basal caspase 3 activation. Conversely, we show that anti‐PrPc antibodies sequestrate PrPc at the cell surface and drastically lower PrPc‐dependent caspase activation. We suggest that intracellular PrPc could sensitize human cells to pro‐apoptotic phenotype and that blockade of PrPc internalization could be a track to prevent intracellular toxicity associated with PrPc overexpression.

Primary cultured neurons devoid of cellular prion display lower responsiveness to staurosporine through the control of p53 at both transcriptional and post-transcriptional levels.

We assessed the contribution of the cellular prion protein (PrPc) in the control of neuronal apoptosis by examining cell death in both human cells and murine primary cultured neurons. We first confirmed our previous finding that staurosporine-induced caspase activation is increased by PrPc overexpression in HEK293 cells. We show here that this phenotype is fully dependent on p53 and that the control of p53 activity by PrPc occurs at both transcriptional and post-transcriptional levels in human cells. Of most interest, we demonstrate that neuronal endogenous PrPc also controls a p53-dependent pro-apoptotic phenotype. Thus, DNA fragmentation and TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP nick end-labeling)-positive cells were lower in primary cultured neurons derived from Zrch-1 mice embryos in which PrPc has been abrogated than in wild-type neurons. PrPc knock-out neurons also displayed drastically diminished caspase-3-like activity and immunoreactivity together with reduced p53 expression and transcriptional activity, a phenotype complemented in part by PrPc transfection. Interestingly, p53 expression was also reduced in the brain of adult Prnp-/- mice. Neuronal PrPc likely controls p53 at a post-transcriptional level because the deletion of cellular prion protein is accompanied by a higher Mdm2-like immunoreactivity and reduced phosphorylated p38 MAPK expression. We therefore propose that the physiological function of endogenous cellular prion could be to regulate p53-dependent caspase-3-mediated neuronal cell death. This phenotype likely occurs through up-regulation of p53 promoter transactivation as well as downstream by controlling p53 stability via Mdm2 expression.