Glioma tumor grade correlates with parkin depletion in mutant p53-linked tumors and results from loss of function of p53 transcriptional activity.

Gliomas represent the most frequent form of primary brain tumors in adults, the prognosis of which remains extremely poor. Inactivating mutations on the tumor suppressor TP53 were proposed as a key etiological trigger of glioma development. p53 has been recently identified as a transcriptional target of parkin. Interestingly, somatic mutations on parkin have also been linked to glioma genesis. We examined the possibility that a disruption of a functional interaction between p53 and parkin could contribute to glioma development in samples devoid of somatic parkin mutations or genetic allele deletion. We show here that parkin levels inversely correlate to brain tumor grade and p53 levels in oligodendrogliomas, mixed gliomas and glioblastomas. We demonstrate that p53 levels negatively and positively correlate to bax and Bcl2 respectively, underlying a loss of p53 transcriptional activity in all types of glial tumors. Using various cell models lacking p53 or harboring either transcriptionally inactive or dominant negative p53, as well as in p53 knockout mice brain, we establish that p53 controls parkin promoter transactivation, mRNA and protein levels. Furthermore, we document an increase of parkin expression in mice brain after p53-bearing viral infection. Finally, both cancer-related p53 inactivating mutations and deletion of a consensus p53 binding sequence located on parkin promoter abolish p53-mediated control of parkin transcription, demonstrating that p53 regulates parkin transcription via its DNA binding properties. In conclusion, our work delineates a functional interplay between mutated p53 and parkin in glioma genesis that is disrupted by cancer-linked pathogenic mutations. It also allows envisioning parkin as a novel biomarker of glioma biopsies enabling to follow the progression of this type of cancers.

Interplay between parkin and p53 governs a physiological homeostasis that is disrupted in Parkinson's disease and cerebral cancer.

Parkin is responsible for most autosomal juvenile recessive cases of Parkinson's disease (PD). Besides its well-characterized function as ubiquitin ligase, we previously established that parkin could repress p53 at the transcriptional level. Interestingly, p53 was recently shown to upregulate parkin, suggesting a feedback loop by which parkin and p53 interplay, thereby contributing to their physiological homeostasis. This equilibrium is disrupted in both PD and cerebral cancer. Thus, when parkin is mutated in PD, its transcriptional ability to repress p53 is abolished. Therefore, p53 elevation could likely contribute to the exacerbated cell death observed in PD-affected brains. Inversely, in brain-associated tumors linked to p53 mutations, the transcriptional control of parkin is reduced, and thereby, parkin expression is lowered. The reduction in parkin level could, in turn, contribute to an increase in the levels of transcriptionally inactive p53 that could explain, at least in part, the defect in cellular apoptotic commitment observed in cerebral cancer. Here, we discuss in detail the various studies demonstrating the importance of the functional interplay between parkin and p53 and its impairment by pathogenic mutations likely contributing to the etiology of PD and gliomas.

The caspase 6 derived N-terminal fragment of DJ-1 promotes apoptosis via increased ROS production.

In pathological conditions, the amount of DJ-1 determines whether a cell can survive or engage a cell death program. This is exemplified in epithelial cancers, in which DJ-1 expression is increased, while autosomal recessive early onset Parkinson's disease mutations of DJ-1 generally lead to decreased stability and expression of the protein. We have shown previously that DJ-1 is cleaved by caspase-6 during induction of apoptosis. We demonstrate here that the N-terminal cleaved fragment of DJ-1 (DJ-1 Nt) is specifically expressed in the nucleus and promotes apoptosis in SH-SY5Y neuroblastoma cell lines. In addition, overexpression of DJ-1 Nt in different cell lines leads to a loss of clonogenic potential and sensitizes to staurosporin and 1-methyl-4-phenylpyridinium (MPP+)-mediated caspase activation and apoptosis. Importantly, inhibition of endogenous DJ-1 expression with sh-RNA or DJ-1 deficiency mimics the effect of DJ-1 Nt on cell growth and apoptosis. Moreover, overexpression of DJ-1 Nt increases reactive oxygen species (ROS) production, and sensitizes to MPP+-mediated apoptosis and DJ-1 oxidation. Finally, specific exclusion of DJ-1 Nt from the nucleus abrogates its pro-apoptotic effect. Taken together, our findings identify an original pathway by which generation of a nuclear fragment of DJ-1 through caspase 6-mediated cleavage induces ROS-dependent amplification of apoptosis.

α-Secretase-derived cleavage of cellular prion yields biologically active catabolites with distinct functions.

The cellular prion protein (PrP(c)) undergoes α-secretase-derived processing by disintegrins. This cleavage occurs within the 106-126 putative toxic domain of PrP(c), yielding two complementary N- and C-terminal fragments referred to as N1 and C1, respectively. Here we review our recent data showing that these two PrP(c)-derived products harbor distinct p53-dependent functions. Thus, C1 potentiates staurosporine (STS)-induced caspase-3 activation by upregulating p53 transcription, mRNA levels and activity. Conversely, N1 is protective both in vitro and in vivo. Thus, N1 inhibits STS-induced caspase-3 activation by downregulating p53 in various cell systems and protects rat retinal ganglion cells from hypoxia-induced apoptosis. Furthermore, N1 protects cells against C1-induced toxicity. Therefore, our data show that disintegrin-associated processing of PrP(c) gives rise to two fragments that display opposite effects on p53-dependent cell death and that can functionally interact.

Cellular prion and its catabolites in the brain: production and function.

During the last thirty years, part of the scientific community focused on the mechanisms by which a naturally occurring protein called cellular prion (PrP(c)) converts into a protease-resistant isoform (PrP(sc)) responsible for fatal Transmissible Spongiform Encephalopathies (TSE). Concomitantly, the physiology of PrP(c) has also been studied. PrP(c) undergoes proteolytic attacks leading to both membrane-attached and secreted fragments, the nature of which differs in normal and TSE-affected human brains. Does proteolysis of PrP(c) correspond to an inactivating mechanism impairing the biological function of the protein, or alternatively, does it represent a maturation process allowing the produced fragments to trigger their own physiological function? Here we review the mechanisms involved in the production of PrP(c) catabolites and we focus on the function of PrP(c) and its derived fragments in the cell death/ survival regulation in the nervous system.

Parkin: much more than a simple ubiquitin ligase.

Parkin is mainly a cytosolic protein involved in a subset of Parkinson's disease (PD) cases referred to as autosomal juvenile recessive forms of PD. Most studies have established as a dogma that parkin function could be resumed as an ubiquitin ligase activity. Accordingly, several cellular functions ascribed to parkin derive from its ability to ubiquitinate a series of proteins, thereby rendering them prone to proteasomal degradation. Several lines of data indicated that parkin could display antiapoptotic properties and we demonstrated that indeed, parkin could downregulate the p53-dependent pathway. However, we showed that such function remained independent of parkin's ability to act as an ubiquitin ligase. Thus, we established that parkin repressed p53 transcription by physically interacting with its promoter. Here, we describe this novel parkin-associated transcription factor function and we speculate on putative additional transcriptional targets.

p53, a pivotal effector of a functional cross-talk linking presenilins and Pen-2.

The γ-secretase is a multiprotein complex responsible for the ultimate cut yielding amyloid-ß peptides and their N-terminal truncated species. This complex is composed of at least four distinct entities, namely presenilin-1 (PS1) or PS2, anterior pharynx defective-1, presenilin enhancer-2 (Pen-2) and nicastrin. Very few studies examined the transcriptional regulation of this complex, and more precisely, whether some of the members functionally interact. Here, we summarize our previous data documenting the fact that Pen-2 controls cell death in a p53-dependent manner and our recent demonstration of a pivotal role of p53 as a regulator of Pen-2 transcription. As PS trigger amyloid precursor protein intracellular domain-dependent regulation of p53, our studies delineate a feedback control mechanism by which PS and Pen-2 functionally interact in a p53-dependent manner.

Loss of function of DJ-1 triggered by Parkinson's disease-associated mutation is due to proteolytic resistance to caspase-6.

DJ-1 was recently identified as a gene product responsible for a subset of familial Parkinson's disease (PD). The mechanisms by which mutations in DJ-1 alter its function and account for PD-related pathology remained largely unknown. We show that DJ-1 is processed by caspase-6 and that the caspase-6-derived C-terminal fragment of DJ-1 fully accounts for associated p53-dependent cell death. In line with the above data, we show that a recently described early-onset PD-associated mutation (D149A) renders DJ-1 resistant to caspase-6 proteolysis and abolishes its protective phenotype. Unlike the D149A mutation, the L166P mutation that prevents DJ-1 dimerization does not impair its proteolysis by caspase-6 although it also abolishes DJ-1 antiapoptotic function. Therefore, we show here that DJ-1 loss of function could be due to impaired caspase-6 proteolysis and we document the fact that various DJ-1 mutations could lead to PD pathology through distinct molecular mechanisms.

Neprilysin activity and expression are controlled by nicastrin.

We recently demonstrated that the presenilin-dependent gamma-secretase complex regulates the expression and activity of neprilysin, one of the main enzymes that degrade the amyloid beta-peptide (Abeta) which accumulates in Alzheimer's disease. Here, we examined the influence of endogenous nicastrin (NCT), a member of the gamma-secretase complex, on neprilysin physiology. We show that nicastrin deficiency drastically lowers neprilysin expression, membrane-bound activity and mRNA levels, but it did not modulate the expression of two other putative Abeta-cleaving enzymes, endothelin-converting enzyme and insulin-degrading enzyme. Furthermore, we show that nicastrin restores neprilysin activity and expression in nicastrin-deficient, but not presenilin-deficient fibroblasts, indicating that the control of neprilysin necessitates the complete gamma-secretase complex harbouring its four reported components. Finally, we show that NCT expression peaked 24 h after NCT cDNA transfection of wild-type and NCT-/- fibroblasts, while neprilysin expression drastically increased only after 36 h and was maximal at 48 h. This delayed effect on neprilysin expression correlates well with our demonstration of an indirect gamma-secretase-dependent modulation of neprilysin at its transcriptional level.

JLK isocoumarin inhibitors: selective gamma-secretase inhibitors that do not interfere with notch pathway in vitro or in vivo.

gamma-Secretase activity is involved in the generation of Abeta and therefore likely contributes to the pathology of Alzheimer's disease. Blocking this activity was seen as a major therapeutic target to slow down or arrest Abeta-related AD progression. This strategy seemed more doubtful when it was established that gamma-secretase also targets other substrates including Notch, a particularly important transmembrane protein involved in vital functions, at both embryonic and adulthood stages. We have described previously new non-peptidic inhibitors able to selectively inhibit Abeta cellular production in vitro without altering Notch pathway. We show here that in vivo, these inhibitors do not alter the Notch pathway responsible for somitogenesis in the zebrafish embryo. In addition, we document further the selectivity of JLK inhibitors by showing that, unlike other described gamma-secretase inhibitors, these agents do not affect E-cadherin processing. Finally, we establish that JLKs do not inhibit beta-site APP cleaving enzymes (BACE) 1 and BACE2, alpha-secretase, the proteasome, and GSK3beta kinase. Altogether, JLK inhibitors are the sole agents to date that are able to prevent Abeta production without triggering unwanted cleavages of other proteins.

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.

The C-terminal fragment of the Alzheimer's disease amyloid protein precursor is degraded by a proteasome-dependent mechanism distinct from gamma-secretase.

The beta-amyloid protein (Abeta) is derived by proteolytic processing of the amyloid protein precursor (APP). Cleavage of APP by beta-secretase generates a C-terminal fragment (APP-CTFbeta), which is subsequently cleaved by gamma-secretase to produce Abeta. The aim of this study was to examine the cleavage of APP-CTFbeta by gamma-secretase in primary cortical neurons from transgenic mice engineered to express the human APP-CTFbeta sequence. Neurons were prepared from transgenic mouse cortex and proteins labelled by incubation with [35S]methionine and [35S]cysteine. Labelled APP-CTFbeta and Abeta were then immunoprecipitated with a monoclonal antibody (WO2) specific for the transgene sequences. Approximately 30% of the human APP-CTFbeta (hAPP-CTFbeta) was converted to human Abeta (hAbeta), which was rapidly secreted. The remaining 70% of the hAPP-CTFbeta was degraded by an alternative pathway. The cleavage of hAPP-CTFbeta to produce hAbeta was inhibited by specific gamma-secretase inhibitors. However, treatment with proteasome inhibitors caused an increase in both hAPP-CTFbeta and hAbeta levels, suggesting that the alternative pathway was proteasome-dependent. A preparation of recombinant 20S proteasome was found to cleave a recombinant cytoplasmic domain fragment of APP (APPcyt) directly. The study suggests that in primary cortical neurons, APP-CTFbeta is degraded by two distinct pathways, one involving gamma-secretase, which produces Abeta, and a second major pathway involving direct cleavage of APP-CTFbeta within the cytoplasmic domain by the proteasome. These results raise the possibility that defective proteasome function could lead to an increase in Abeta production in the AD brain.

The disintegrins ADAM10 and TACE contribute to the constitutive and phorbol ester-regulated normal cleavage of the cellular prion protein.

We showed previously that PrPc undergoes constitutive and phorbol ester-regulated cleavage inside the 106-126 toxic domain of the protein, leading to the production of a fragment referred to as N1. Here we show by a pharmacological approach that o-phenanthroline, a general zinc-metalloprotease inhibitors, as well as BB3103 and TAPI, the inhibitors of metalloenzymes ADAM10 (A disintegrin and metalloprotease); and TACE, tumor necrosis factor alpha-converting enzyme; ADAM17), respectively, drastically reduce N1 formation. We set up stable human embryonic kidney 293 transfectants overexpressing human ADAM10 and TACE, and we demonstrate that ADAM10 contributes to constitutive N1 production whereas TACE mainly participates in regulated N1 formation. Furthermore, constitutive N1 secretion is drastically reduced in fibroblasts deficient for ADAM10 whereas phorbol 12,13-dibutyrate-regulated N1 production is fully abolished in TACE-deficient cells. Altogether, our data demonstrate for the first time that disintegrins could participate in the catabolism of glycosyl phosphoinositide-anchored proteins such as PrPc. Second, our study identifies ADAM10 and ADAM17 as the protease candidates responsible for normal cleavage of PrPc. Therefore, these disintegrins could be seen as putative cellular targets of a therapeutic strategy aimed at increasing normal PrPc breakdown and thereby depleting cells of the putative 106-126 "toxic" domain of PrPc.

Involvement of cytosolic prolyl endopeptidase in degradation of p40-phox splice variant protein in myeloid cells.

Our previous studies indicated that an alternatively spliced variant mRNA of p40-phox, a cytosolic component of NADPH oxidase, is expressed but its protein is hardly detected in myeloid cells such as promyelocytic HL-60 cells and neutrophils. Here, we have examined the stability of p40-phox variant protein in undifferentiated HL-60 cells. When in vitro-translated proteins were incubated with subcellular fractions of HL-60 cells, p40-phox variant protein but not native p40-phox was degraded by the cytosol and granule fractions. The degradation of variant protein by the granule fraction was observed using sonicated but not intact granules, suggesting that the variant protein is unlikely to be degraded by the granules in intact cells. To identify the enzyme(s) involved, we examined the effects of various enzyme inhibitors on the degradation of variant protein by the cytosol fraction. Degradation was completely inhibited by proline-specific serine protease (prolyl endopeptidase) inhibitors but not by proteasome, calpain, and metalloprotease inhibitors. Furthermore, the variant protein was degraded by a purified prolyl endopeptidase, and the degradation was protected by treating HL-60 cells with a cell-permeable inhibitor (S17092-1) for prolyl endopeptidase. These observations suggest that a cytosolic prolyl endopeptidase is involved in the degradation of p40-phox variant protein in myeloid cells.

Constitutive alpha-secretase cleavage of the beta-amyloid precursor protein in the furin-deficient LoVo cell line: involvement of the pro-hormone convertase 7 and the disintegrin metalloprotease ADAM10.

The beta-amyloid precursor protein (betaAPP) undergoes a physiological cleavage triggered by one or several proteolytic activities referred to as alpha-secretases, leading to the secretion of sAPPalpha. Several lines of evidence indicate that the alpha-secretase cleavage is a highly regulated process. Thus, besides constitutive production of sAPPalpha, several studies have reported on protein kinase C-regulated sAPPalpha secretion. Studies aimed at identifying alpha-secretase(s) candidates suggest the involvement of enzymes belonging to the pro-hormone convertases and disintegrin families. The delineation of respective contributions of proteolytic activities in constitutive and regulated sAPPalpha secretion is rendered difficult by the fact that the overall regulated response always includes the basal constitutive counterpart that cannot be selectively abolished. Here we report on the fact that the furin-deficient LoVo cells are devoid of regulated PKC-dependent sAPPalpha secretion and therefore represent an interesting model to study exclusively the constitutive sAPPalpha secretion. We show here, by a pharmacological approach using selective inhibitors, that pro-hormone convertases and proteases of the ADAM (disintegrin metalloproteases) family participate in the production/secretion of sAPPalphas in LoVo cells. Transfection analysis allowed us to further establish that the pro-hormone convertase 7 and ADAM10 but not ADAM17 (TACE, tumour necrosis factor alpha-converting enzyme) likely contribute to constitutive sAPPalpha secretion by LoVo cells.

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

Cellular prion protein (PrP(c)) undergoes a proteolytic attack at the 110/111 downward arrow112 peptide bond, whereas the PrP isoform (PrP(res)) 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 PrP(res) cleavage preserves this potentially toxic domain. Therefore, any molecular mechanisms leading to enhanced cleavage at the 110/111 downward arrow112 peptide bond could be of potential interest. We set up TSM1 neurons and HEK293 stable transfectants overexpressing the wild-type or 3F4-tagged murine PrP(c), respectively. Both mock-transfected and PrP(c)-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 PrP(c) 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 alphaPDD and by the protein kinase A effectors dibutyryl cAMP and forskolin. Overall, our data indicate that the normal processing of PrP(c) is up-regulated by protein kinase C but not protein kinase A in human cells and murine neurons.

Role of the proteasome in Alzheimer's disease.

The proteasome is a multicatalytic complex involved in the degradation of polyubiquitinated proteins. Here we review the clues of a possible involvement of the proteasome in Alzheimer's disease neuropathology. Thus, we discuss the fact that the proteasome modulates the intracellular concentrations of presenilins 1 and 2. These two proteins, when mutated, appear responsible for most of early onset forms of Alzheimer's disease and this is thought to be due to the exacerbation of the pathogenic pathway of the maturation of the beta-amyloid precursor protein. Controlling presenilins concentrations could have drastic repercussions on cell physiology as suggested by the fact that proteasome inhibitors drastically potentiate the 'normal' or 'pathogenic' presenilins phenotype related with betaAPP processing. The possibility of considering the proteasome as a potential target for therapeutic intervention in Alzheimer's disease is discussed.

Laminar specific loss of isocortical presenilin 1 immunoreactivity in Alzheimer's disease. Correlations with the amyloid load and the density of tau-positive neurofibrillary tangles.

Presenilin 1 has been shown to be mutated in a high proportion of cases of familial Alzheimer's disease. Immunoreactive epitopes of the protein have been found mainly in neurones devoid of neurofibrillary tangles - an observation that has led to the conclusion that presenilin 1 could have a protective role. In this study, the relationship between deposits of Abeta peptide (both the 40 and 42 isoforms), tau positive neurofibrillary tangles and presenilin 1-positive neuronal profiles were analysed in three cases of presenilin 1 mutation, four cases of sporadic Alzheimer's disease and five controls. Immunohistochemistry was performed in a sample from the supramarginal gyrus. The proportion of volume occupied by the Abeta1-40 and Abeta1-42 deposits (amyloid load) was evaluated by a point-counting technique. Tau-positive neurofibrillary tangles, and presenilin 1-positive neuronal profiles were directly counted. The location of the lesions in the thickness of the cortex was recorded. The density of PS1-positive neuronal profiles in Alzheimer's disease cases was lower than in the controls. The deficit was significant only in the upper layers of the cortex. The density of presenilin 1 neuronal profiles was negatively correlated with Abeta1-40 and Abeta1-42 loads, and with the density of tau-positive neurofibrillary tangles. Multivariate analysis showed that the Abeta1-42 load was the best determinant of the decrease in presenilin 1-positive neuronal profiles. Presenilin 1-positive neurones appear to be lost rather than protected in the course of Alzheimer disease.

Alpha-synuclein and the Parkinson's disease-related mutant Ala53Thr-alpha-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, alpha-synuclein immunoreactivity is found in Lewy bodies, the histopathological hallmark of sporadic Parkinson disease-affected brains. When mutated, alpha-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 alpha-synucleins, thereby controlling their concentration-dependent aggregation process. Here, we first demonstrate that alpha-synuclein is not ubiquitinated in HEK293 cells. Furthermore, by means of two specific inhibitors, we show that wild type and Ala53Thr alpha-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 alpha-synucleinopathies.

Wild-type but not Parkinson's disease-related ala-53 --> Thr mutant alpha -synuclein protects neuronal cells from apoptotic stimuli.

Recent works suggest that alpha-synuclein could play a central role in Parkinson's disease (PD). Thus, two mutations were reported to be associated with rare autosomal dominant forms of the disease. We examined whether alpha-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) alpha-synuclein or the PD-related Ala-53 --> Thr mutant alpha-synuclein. Under basal conditions, acetyl-Asp-Glu-Val-Asp-aldehyde-sensitive caspase activity appears significantly lower in wt alpha-synuclein-expressing cells than in neurons expressing the mutant. Interestingly, wt alpha-synuclein drastically reduces the caspase activation of TSM1 neurons upon three distinct apoptotic stimuli including staurosporine, etoposide, and ceramide C(2) 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 alpha-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 alpha-synuclein exerts an antiapoptotic effect in neurons that appears to be abolished by the Parkinson's disease-related mutation, thereby suggesting a possible mechanism underlying both sporadic and familial forms of this neurodegenerative disease.