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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online September 14, 2005 as doi:10.1096/fj.05-3839fje. |
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,1
* INSERM U 573, Centre Paul Broca, Paris, France; and
CNRS, UMR 8542, ENS, Paris, France
1 Correspondence: INSERM U 573, 2 ter rue d’Alésia, Paris 75014, France. E-mail: bernadette.allinquant{at}broca.inserm.fr
SPECIFIC AIMS
We previously demonstrated that intraneuronal accumulation of the juxtamembranar cytoplasmic domain (Jcasp) of amyloid precursor protein (APP) extending until the caspase cleavage site (aa649-664) induces apoptosis in vitro and in vivo. This effect seems highly specific since it is lost when tyrosine (Y653) is replaced by an aspartate. This domain is unmasked in the brains of Alzheimer’s disease (AD) patients, but also in cellular models like Aß neuroblastoma cell death or human apoptotic neurons, suggesting a role in the development of the disease. The aim of this study was to understand the first mechanisms involved in the neuronal cell death induced by this APP domain. We focused our interest on partners attached early to the toxic APP peptide and able to induce downstream apoptosis.
PRINCIPAL FINDINGS
1. SET (TAF1ß, I2PP2A) binds to Jcasp sequence ex vivo and in vitro
The biotinylated Jcasp peptide, its mutant with a Y->D change in position 653 (J(Y>D)casp) and a more distal H domain were internalized into cultured embryonic cortical neurons through the help of a cell permeable peptide (penetratin). Internalized peptides remain intact for at least 3 h (Fig. 1
A). Jcasp is proapototic whereas the J(Y->D)casp, and the H domain have no effect over background (Fig. 1B
). Large-scale neuronal cultures were treated for 3 h by the three biotinylated peptides for ex vivo pulldown experiment and the silver-stained pattern of retrieved proteins shows different proteins attached to the internalized peptides. In this report we have focused our interest on the 39 kDa protein because of its preferential binding to Jcasp (Fig. 1C
).
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We identified this 39 kDa band by mass spectrometry and MS/MS sequencing as the SET protein also called template activating factor (TAF1ß) or phosphatase 2A inhibitor2 (I2PP2A). An antibody raised against a SET peptide recognizes a band at 39 kDa in total neuronal extract (Fig. 1D
). We repeated the experiment of Fig. 1C
, with more stringent conditions and used the antibody to reveal SET on a Western blot. This experiment confirms that Jcasp specifically binds to SET ex vivo and that the affinity of SET for Jcasp is reduced in the Y->D variant (Fig. 1E
).
Internalized Jcasp has access mainly to the cytoplasm but can reach the nucleus. In primary neurons, SET is present in the nucleus with limited staining of cytoplasm and neurites but redistributes between the cytoplasm and the nucleus in the presence of Jcasp 3 h after internalization, suggesting that SET can interact with Jcasp in both compartments.
A recombinant His-SET protein binds Jcasp in vitro and a longer version of the Jcasp sequence extending until the gamma-secretase site and also able to induce neuronal cell death, but not Myc nor to the full-length APP C-terminal domain which does not induce cell death in our conditions. Biacore analyses show that this interaction is 100-fold lower for the J(Y->D)casp.
2. SET (TAF1ß, I2PP2A) is an actor of Jcasp-induced cell death and a positive regulator of apoptosis
The specific interaction between Jcasp and SET suggested that SET could be in the pathway of Jcasp proapoptotic activity. To investigate this possibility, we designed a down-regulation strategy and tested its influence on Jcasp-induced cell death (Fig. 2
A–D).
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Neuron morphology indicates a clear survival effect of the antisense oligonucleotides directed against SET mRNA (Fig. 2E-G
). This effect is on Jcasp-induced cell death (Fig. 2G
); it is not seen in basal conditions or with the sense oligonucleotides and is still present after 72 h (Fig. 2I
).
Cell death induced by a longer version of Jcasp extending until the gamma-secretase site (Gcasp) is also antagonized by the SET antisense oligonucleotides (Fig. 2H
).
Conversely, overexpression of SET in primary neurons was investigated. A first approach was the use of a recombinant Penetratin-SET protein. The internalization of Penetratin-SET within primary neurons induces cell death in a dose-dependent manner while Penetratin alone has no effect. To confirm this observation, a plasmid encoding SET bearing a N-terminal myc tag was expressed in primary neurons, transfected EGFP serving as a control. After 24 h all SET expressing cells were dead and only cells expressing EGFP could be observed. Taken together, SET gain and loss of function experiments demonstrate that SET is a positive regulator of neuronal death.
CONCLUSIONS AND SIGNIFICANCE
We demonstrate here that SET plays an important role in the regulation of cell death induced by a proapoptotic domain of the APP cytoplasmic tail (Jcasp). SET, also called Template-Activating Factor 1ß (TAF 1ß), is a multifunctional protein. In an AD context, SET represses the expression of hop1, the mammalian presenilin2 homologue in C. elegans and acts as an activator of the Fe 65-mediated transactivation after the transfer of APP cytoplasmic domain to the nucleus. SET also enhances the activity of cdk5/p35 through its association with p35, a kinase involved in cell cycle and in tau phosphorylation. Finally, SET is a potent inhibitor of protein phosphatase 2A (I2PP2A), a phosphatase involved in cell cycle progression and in tau dephosphorylation.
SET interacts with the Jcasp, both ex vivo (within primary neurons in culture) and in vitro. As the binding of SET for the nonproapoptotic J(Y->D)casp variant of Jcasp is of lower affinity, the high affinity SET/Jcasp interaction correlates with the ability of Jcasp to induce cell death. Down-regulation of SET expression by primary neurons confirms a role for SET in Jcasp induced apoptosis. Cell death induced by a longer peptide including the Jcasp sequence and extending to the gamma secretase site is also antagonized by SET down-regulation. This suggests that in basal conditions unmasking Jcasp sequence drives SET proapoptotic activity. However, overexpressing SET induces neuronal apoptosis independently of Jcasp internalization.
SET has been involved in the model of granzyme A-dependent cell death where it plays a protective role. This discrepancy might be a reflection of the different cell types (hematopoietic cells vs. neurons) and death mechanisms involved. Granzyme A cell death, as opposed to Jcasp-induced apoptosis, is caspase independent.
SET activity might be related to its PP2A inhibition activity. Calyculin A, a selective inhibitor of PP2A and PP1 is neurotoxic to SH-SY5Y neuroblastoma cells, a good model of primary neurons. However, the mechanism of cell death induction, downstream of Jcasp/SET interaction, is not known and its understanding will require further investigations of cell modifications in conditions of SET gain and loss of function.
A link between SET and neuronal loss, associated with AD, is suggested by SET up-regulation in the hippocampus of AD patients, in correlation with neurofibrillar tangles and, negatively, with Mini-Mental Status Exam. In this context, it is noteworthy that SET regulates the activity of the phosphoseryl/phosphothreonyl protein phosphatase PP2A, which is compromised in AD brain, a possible cause of abnormal tau hyperphosphorylation. Finally, caspase 3 activation and the accumulation of caspase-cleaved APP fragments precede mature neurofibrillary tangles in AD. The putative role of SET in linking these two events requires further investigations. In conclusion, our findings suggest a new function of SET as a regulator of apoptosis induced by the accumulation of an unmasked APP cytoplasmic domain in AD after caspase activation (Fig. 3
). The specificity of this apoptotic pathway might help to better understand some aspects of the disease.
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FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-3839fje;
This article has been cited by other articles:
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  V. Simon, J. Guidry, T. W. Gettys, A. B. Tobin, and S. M. Lanier The Proto-oncogene SET Interacts with Muscarinic Receptors and Attenuates Receptor Signaling J. Biol. Chem., December 29, 2006; 281(52): 40310 - 40320. [Abstract] [Full Text] [PDF]
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