Karine Ancolio

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.

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 and the Parkinson's disease-related mutant Ala53Thr-α-synuclein do not undergo proteasomal degradation in HEK293 and neuronal cells

Synucleins are neuronal proteins detectable in the neuropathological lesions of several cerebral disorders. Thus, α-synuclein immunoreactivity is found in Lewy bodies, the histopathological hallmark of sporadic Parkinson disease-affected brains. When mutated, α-synuclein seems to be responsible for some familial forms of Parkinson disease. As Lewy bodies are enriched in ubiquitinated structures and also contain proteasome-related immunoreactivity, it could be hypothesized that the proteasome contributes to the cellular degradation of α-synucleins, thereby controlling their concentration-dependent aggregation process. Here, we first demonstrate that α-synuclein is not ubiquitinated in HEK293 cells. Furthermore, by means of two specific inhibitors, we show that wild type and Ala53Thr α-synuclein do not behave as proteasome substrates in HEK293 cells and murine neurons. Our study indicates that the proteasome does not contribute to the control of cellular synucleins concentration and therefore, unlikely participates to cerebral α-synucleinopathies.

α-Secretase-Derived Product of β-Amyloid Precursor Protein Is Decreased by Presenilin 1 Mutations Linked to Familial Alzheimer's Disease

Abstract: Recent reports indicate that missense mutations on presenilin (PS) 1 are likely responsible for the main early‐onset familial forms of Alzheimers disease (FAD). Consensual data obtained through distinct histopathological, cell biology, and molecular biology approaches have led to the conclusion that these PS1 mutations clearly trigger an increased production of the 42‐amino‐acid‐long species of β‐amyloid peptide (Aβ). Here we show that overexpression of wild‐type PS1 in HK293 cells increases Aβ40 secretion. By contrast, FAD‐linked mutants of PS1 trigger increased secretion of both Aβ40 and Aβ42 but clearly favor the production of the latter species. We also demonstrate that overexpression of the wild‐type PS1 augments the α‐secretase‐derived C‐terminally truncated fragment of β‐amyloid precursor protein (APPα) recovery, whereas transfectants expressing mutated PS1 secrete drastically lower amounts of APPα when compared with cells expressing wild‐type PS1. This decrease was also observed when comparing double transfectants overexpressing wild‐type β‐amyloid precursor protein and either PS1 or its mutated congener M146V‐PS1. Altogether, our data indicate that PS mutations linked to FAD not only trigger an increased ratio of Aβ42 over total Aβ secretion but concomitantly down‐regulate the production of APPα.

Proteasome inhibitors prevent the degradation of familial Alzheimer's disease-linked presenilin 1 and potentiate A beta 42 recovery from human cells.

BackgroundSeveral lines of evidence suggest that most of the early-onset forms of familial Alzheimer’s disease (FAD) are due to inherited mutations borne by a chromosome 14–encoded protein, presenilin 1 (PS1). This is likely related to an increased production of amyloid β-peptide (A β)42, one of the main components of the extracellular deposits called senile plaques that invade human cortical areas during the disease.Materials and MethodsWe set up stably transfected HEK293 cells overexpressing wild-type (wt) and various FAD-linked mutated PS1. By Western blot analysis, we examined the influence of specific proteasome inhibitors on PS1-like immunoreactivities. Furthermore, by means of metabolic labeling and immunoprecipitation with A β40 and A β42-directed specific antibodies, we assessed the effect of the inhibitors on the production of A βs by wt and mutated PS1-expressing cells transiently transfected with βAPP751.ResultsWe show that two distinct proteasome inhibitors, Z-IE(Ot-Bu)A-Leucinal and lactacystin, increase in a time- and dose-dependent manner the immunoreactivities of both wt and mutated PS1. Furthermore, we demonstrate that PS1 is polyubiquitinated in these cells. Other inhibitors, ineffective on the proteasome, fail to protect wt and mutated PS1-like immunoreactivities. We also establish that the FAD-linked mutations of PS1 trigger a selective increased formation of Aβ42 as reflected by higher Aβ42 over total Aβ ratios when compared with wtPS1-expressing cells. Interestingly, this augmentation was further amplified by proteasome inhibitors in cells expressing mutated but not wtPS1.ConclusionAltogether, our data indicate that PS1 undergoes polyubiquitination in HEK293 cells and that the proteasome contributes to the degradation of wt and FAD-linked PS1, thereby directly influencing the Aβ production in human cells.

Effect of protein kinase A inhibitors on the production of Aβ40 and Aβ42 by human cells expressing normal and Alzheimer's disease-linked mutated βAPP and presenilin 1

We previously established that the formation of both α‐ and β/γ‐secretase‐derived products generated by human embryonic kidney 293 cells (HEK293) expressing either wild type or mutant βAPP could be stimulated by agonists of the cyclic AMP/protein kinase A pathways. This cyclic AMP‐dependent effect modulates post‐translational events since it is not prevented by actinomycin D or cycloheximide. We show here that two protein kinase A inhibitors, H89 and PKI, both trigger dose‐dependent inhibition of the basal constitutive production of Aβ40 and Aβ42 by HEK293 cells expressing wild type βAPP751. H89 also potently inhibits the total Aβ produced by the neocortical neuronal cell line TSM1. These two inhibitors also drastically reduce the recovery of Aβ40 and Aβ42 produced by HEK293 cells expressing the Swedish (Sw) βAPP and M146V‐presenilin 1 (PS1) mutations responsible for cases of the early‐onset forms of Familial Alzheimers disease (FAD). By contrast, H89 and PKI do not significantly affect the recovery of the physiological α‐secretase‐derived fragment APPα. Our study indicates that protein kinase A inhibitors selectively lower the formation of Aβ40 and Aβ42 in human cells expressing normal and mutant βAPP and PS1 without affecting the physiological α‐secretase pathway in these cells. Selective inhibitors of protein kinase A may be of therapeutic value in both sporadic and Familial Alzheimers disease, since they may decrease the production of Aβ that is thought to be responsible for the neurodegenerative process.