Cristine Alves da Costa

Presenilin-Dependent Transcriptional Control of the Aβ-Degrading Enzyme Neprilysin by Intracellular Domains of βAPP and APLP

Amyloid beta-peptide (Abeta), which plays a central role in Alzheimers disease, is generated by presenilin-dependent gamma-secretase cleavage of beta-amyloid precursor protein (betaAPP). We report that the presenilins (PS1 and PS2) also regulate Abeta degradation. Presenilin-deficient cells fail to degrade Abeta and have drastic reductions in the transcription, expression, and activity of neprilysin, a key Abeta-degrading enzyme. Neprilysin activity and expression are also lowered by gamma-secretase inhibitors and by PS1/PS2 deficiency in mouse brain. Neprilysin activity is restored by transient expression of PS1 or PS2 and by expression of the amyloid intracellular domain (AICD), which is cogenerated with Abeta, during gamma-secretase cleavage of betaAPP. Neprilysin gene promoters are transactivated by AICDs from APP-like proteins (APP, APLP1, and APLP2), but not by Abeta or by the gamma-secretase cleavage products of Notch, N- or E- cadherins. The presenilin-dependent regulation of neprilysin, mediated by AICDs, provides a physiological means to modulate Abeta levels with varying levels of gamma-secretase activity.

Wild-type but Not Parkinson's Disease-related Ala-53 → Thr Mutant α-Synuclein Protects Neuronal Cells from Apoptotic Stimuli

Recent works suggest that α-synuclein could play a central role in Parkinsons disease (PD). Thus, two mutations were reported to be associated with rare autosomal dominant forms of the disease. We examined whether α-synuclein could modulate the caspase-mediated response and vulnerability of murine neurons in response to various apoptotic stimuli. We established TSM1 neuronal cell lines overexpressing wild-type (wt) α-synuclein or the PD-related Ala-53 → Thr mutant α-synuclein. Under basal conditions, acetyl-Asp-Glu-Val-Asp-aldehyde-sensitive caspase activity appears significantly lower in wt α-synuclein-expressing cells than in neurons expressing the mutant. Interestingly, wt α-synuclein drastically reduces the caspase activation of TSM1 neurons upon three distinct apoptotic stimuli including staurosporine, etoposide, and ceramide C2 when compared with mock-transfected cells. This inhibitory control of the caspase response triggered by apoptotic agents was abolished by the PD-related pathogenic mutation. Comparison of wild-type and mutated α-synuclein-expressing cells also indicates that the former exhibits much less vulnerability in response to staurosporine and etoposide as measured by the sodium 3′-[1-(phenylaminocarbonyl)-3,4-tetrazolium]-bis(4-methoxy-6-nitro)benzenesulfonic acid assay. Altogether, our study indicates that wild-type α-synuclein exerts an antiapoptotic effect in neurons that appears to be abolished by the Parkinsons disease-related mutation, thereby suggesting a possible mechanism underlying both sporadic and familial forms of this neurodegenerative disease.

New protease inhibitors prevent γ-secretase-mediated production of Aβ40/42 without affecting Notch cleavage

We have designed new non-peptidic potential inhibitors of γ-secretase and examined their ability to prevent production of amyloid-β 40 (Aβ40) and Aβ42 by human cells expressing wild-type and Swedish-mutant β-amyloid precursor protein (βAPP). Here we identify three such agents that markedly reduce recovery of both Aβ40 and Aβ42 produced by both cell lines, and increase that of C99 and C83, the carboxy-terminal fragments of βAPP that are derived from β-and α-secretase, respectively. Furthermore, we show that these inhibitors do not affect endoproteolysis of endogenous or overexpressed presenilins. These inhibitors are totally unable to affect the mΔEnotch-1 cleavage that leads to generation of the Notch intracellular domain (NICD). These represent the first non-peptidic inhibitors that are able to prevent γ-secretase cleavage of βAPP without affecting processing of mΔEnotch-1 or endoproteolysis of presenilins. The distinction between these two proteolytic events, which are both prevented by disruption of presenilin genes, indicates that although they are intimately linked with βAPP and Notch maturation, presenilins are probably involved in the control of maturation processes upstream of enzymes that cleave γ-secretase and Notch.

Transcriptional repression of p53 by parkin and impairment by mutations associated with autosomal recessive juvenile Parkinson's disease

Mutations of the ubiquitin ligase parkin account for most autosomal recessive forms of juvenile Parkinsons disease (AR-JP). Several studies have suggested that parkin possesses DNA-binding and transcriptional activity. We report here that parkin is a p53 transcriptional repressor. First, parkin prevented 6-hydroxydopamine-induced caspase-3 activation in a p53-dependent manner. Concomitantly, parkin reduced p53 expression and activity, an effect abrogated by familial parkin mutations known to either abolish or preserve its ligase activity. ChIP experiments indicate that overexpressed and endogenous parkin interact physically with the p53 promoter and that pathogenic mutations abolish DNA binding to and promoter transactivation of p53. Parkin lowered p53 mRNA levels and repressed p53 promoter transactivation through its Ring1 domain. Conversely, parkin depletion enhanced p53 expression and mRNA levels in fibroblasts and mouse brains, and increased cellular p53 activity and promoter transactivation in cells. Finally, familial parkin missense and deletion mutations enhanced p53 expression in human brains affected by AR-JP. This study reveals a ubiquitin ligase-independent function of parkin in the control of transcription and a functional link between parkin and p53 that is altered by AR-JP mutations.

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.

Endogenous β-amyloid production in presenilin-deficient embryonic mouse fibroblasts

Genetic and biochemical evidence have led to the suggestion that presenilins could be the long-searched-for γ-secretase, the proteolytic activity that generates the carboxy terminus of amyloid β-peptides. This activity is also thought to be responsible for the release of the Notch intracellular domain (NICD) from Notch. Here, we report the production of endogenous secreted and intracellular 40- and 42-amino-acid Aβ peptides in mouse fibroblasts deficient in presenilin 1, presenilin 2 or both. We show that the endogenous production of Aβ40 and Aβ42 was not altered by presenilin deficiency. By contrast, inactivating presenilin genes fully abolished NICD production. These data indicate that Aβ and NICD production are distinct catabolic events. Also, even though NICD formation is indeed presenilin dependent, endogenous secreted and intracellular β-amyloid peptides are still generated in absence of presenilins, indicating that there is a γ-secretase activity distinct from presenilins, at least in murine fibroblasts.

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.

The C-terminal Products of Cellular Prion Protein Processing, C1 and C2, Exert Distinct Influence on p53-dependent Staurosporine-induced Caspase-3 Activation

The cellular prion protein (PrPc) undergoes various endopro-teolytic attacks within its N-terminal domain, leading to the production of C-terminal fragments (C) tethered to the plasma membrane and soluble N-terminal peptides (N). One of these cleavages occurs at position 110/111, thereby generating C1 and N1 products. We have reported that disintegrins ADAM-10, -9, and -17 participate either directly or indirectly to this proteolytic event. An alternative proteolytic event taking place around residue 90 yields C2 and N2 fragments. The putative function of these proteolytic fragments remained to be established. We have set up two novel human embryonic kidney 293 cell lines stably overexpressing either C1 or C2. We show that C1 potentiates staurosporine-induced caspase-3 activation through a p53-dependent mechanism. Thus, C1 positively controls p53 transcription and mRNA levels and increases p53-like immunoreactivity and activity. C1-induced caspase-3 activation remained unaffected by the blockade of endocytosis in HEK 293 cells and was abolished in p53-deficient fibroblasts. Conversely, overexpression of the C2 fragment did not significantly sensitize HEK 293 cells to apoptotic stimuli and did not modify p53 mRNA levels or activity. Therefore, the nature of the proteolytic cleavage taking place on PrPc yielded C-terminal catabolites with distinct function and could be seen as a switch mechanism controlling the function of the PrPc in cell survival.

β-Synuclein Displays an Antiapoptotic p53-dependent Phenotype and Protects Neurons from 6-Hydroxydopamine-induced Caspase 3 Activation CROSS-TALK WITH α-SYNUCLEIN AND IMPLICATION FOR PARKINSON′S DISEASE

We have established stable transfectants expressing β-synuclein in TSM1 neurons. We show that in basal and staurosporine-induced conditions the number of terminal deoxynucleotidyltransferase-mediated dUTP nick end-labeling (TUNEL)-positive β-synuclein-expressing neurons was drastically lower than in mock-transfected TSM1 cells. This was accompanied by a lower DNA fragmentation as evidenced by the reduction of propidium iodide incorporation measured by fluorescence-activated cell sorter analysis. β-Synuclein strongly reduces staurosporine-induced caspase 3 activity and immunoreactivity. We establish that β-synuclein triggers a drastic reduction of p53 expression and transcriptional activity. This was accompanied by increased Mdm2 immunoreactivity while p38 expression appeared enhanced, indicating that β-synuclein-induced p53 down-regulation likely occurs at a post-transcriptional level. We showed previously that α-synuclein displays an antiapoptotic function that was abolished by the dopaminergic derived toxin 6-hydroxydopamine (6OHDA). Interestingly, β-synuclein retains its ability to protect TSM1 neurons even after 6OHDA treatment. Furthermore, β-synuclein restores the antiapoptotic function of α-synuclein in 6OHDA-treated neurons. Altogether, our data document for the first time that β-synuclein protects neurons from staurosporine and 6OHDA-stimulated caspase activation in a p53-dependent manner. Our observation that β-synuclein contributes to restoration of the α-synuclein antiapoptotic function abolished by 6OHDA may have direct implications for Parkinsons disease pathology. In this context, the cross-talk between these two parent proteins is discussed.