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.

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.

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.

Recent advances on alpha-synuclein cell biology: functions and dysfunctions.

Alpha-synuclein is a recently discovered protein that was first identified as the major non amyloid component of senile plaques, the cerebral lesion likely responsible for Alzheimer's disease. The role of alpha-synuclein in another brain disease namely Parkinson's disease, has been more deeply documented. It appears that alpha-synuclein fills up the intracytoplasmic inclusions called Lewy bodies that likely contribute to the etiology of Parkinson's disease. Furthermore, rare familial forms of Parkinson's disease have been shown to be linked to autosomal dominant mutations of alpha-synucleins. Is alpha-synuclein a bridge between Alzheimer's and Parkinson's diseases? Could it be seen as a common denominator for these two neurodegenerative diseases? These issues could be better addressed by further delineating the physiological function of alpha-synuclein and, as a corollary, the dysfunction taking place along with the diseases. Here, I will review the recent advances concerning the physiology of alpha-synuclein and will particularly focus on the post-traductional events leading to drastic biophysical transformations. I will describe recent works suggesting that these modifications directly modulate the normal function of alpha-synuclein, likely accounting for the dysfunction associated with Parkinson's disease and perhaps contributing to Alzheimer's pathology.

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 caspase-derived C-terminal fragment of betaAPP induces caspase-independent toxicity and triggers selective increase of Abeta42 in mammalian cells.

During its physiopathological maturation, the beta-amyloid precursor protein undergoes several distinct proteolytic events by activities called secretases. In Alzheimer's disease, the main histological hallmark called senile plaque is clearly linked to the overproduction of the amyloid peptides Abeta40 and Abeta42, two highly aggregable betaAPP-derived fragments generated by combined cleavages by beta- and gamma-secretases. Recently, an alternative hydrolytic pathway was described, involving another category of proteolytic activities called caspases, responsible for the production of a 31 amino acids betaAPP C-terminal fragment called C31. C31 was reported to lower the viability of N2a cells but the exact mechanisms mediating C31-toxicity remained to be established. Here we show that the transient transfection of pSV2 vector encoding C31 lowers by about 80% TSM1 neuronal cells viability. Arguing against a C31-stimulated apoptotic response, we demonstrate by combined enzymatic and immunological approaches that C31 expression did not modulate basal or staurosporine-induced caspase 3-like activity and pro-caspase-3 activation. Furthermore, C31 did not modify Bax and p53 expressions, poly-(ADP-ribose)-polymerase cleavage and cytochrome c translocation into the cytosol. However, we established that C31 overexpression triggers selective increase of Abeta42 but not Abeta40 production by HEK293 cells expressing wild-type betaAPP751. Altogether, our data demonstrate that C31 induces a caspase-independent toxicity in TSM1 neurons and potentiates the pathogenic betaAPP maturation pathway by increasing selectively Abeta42 species in wild type-betaAPP-expressing human cells.

New protease inhibitors prevent gamma-secretase-mediated production of Abeta40/42 without affecting Notch cleavage.

We have designed new non-peptidic potential inhibitors of gamma-secretase and examined their ability to prevent production of amyloid-beta 40 (Abeta40) and Abeta42 by human cells expressing wild-type and Swedish-mutant beta-amyloid precursor protein (betaAPP). Here we identify three such agents that markedly reduce recovery of both Abeta40 and Abeta42 produced by both cell lines, and increase that of C99 and C83, the carboxy-terminal fragments of betaAPP that are derived from beta-and alpha-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 mDeltaEnotch-1 cleavage that leads to generation of the Notch intracellular domain (NICD). These represent the first non-peptidic inhibitors that are able to prevent gamma-secretase cleavage of betaAPP without affecting processing of mDeltaEnotch-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 betaAPP and Notch maturation, presenilins are probably involved in the control of maturation processes upstream of enzymes that cleave gamma-secretase and Notch.

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.

Effect of protein kinase A inhibitors on the production of Abeta40 and Abeta42 by human cells expressing normal and Alzheimer's disease-linked mutated betaAPP and presenilin 1.

1. We previously established that the formation of both alpha- and beta/gamma-secretase-derived products generated by human embryonic kidney 293 cells (HEK293) expressing either wild type or mutant betaAPP 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. 2. We show here that two protein kinase A inhibitors, H89 and PKI, both trigger dose-dependent inhibition of the basal constitutive production of Abeta40 and Abeta42 by HEK293 cells expressing wild type betaAPP751. 3. H89 also potently inhibits the total Abeta produced by the neocortical neuronal cell line TSM1. 4. These two inhibitors also drastically reduce the recovery of Abeta40 and Abeta42 produced by HEK293 cells expressing the Swedish (Sw) betaAPP and M146V-presenilin 1 (PS1) mutations responsible for cases of the early-onset forms of Familial Alzheimer's disease (FAD). 5. By contrast, H89 and PKI do not significantly affect the recovery of the physiological alpha-secretase-derived fragment APPalpha. 6. Our study indicates that protein kinase A inhibitors selectively lower the formation of Abeta40 and Abeta42 in human cells expressing normal and mutant betaAPP and PS1 without affecting the physiological alpha-secretase pathway in these cells. Selective inhibitors of protein kinase A may be of therapeutic value in both sporadic and Familial Alzheimer's disease, since they may decrease the production of Abeta that is thought to be responsible for the neurodegenerative process.

Alzheimer's disease-linked mutation of presenilin 2 (N141I-PS2) drastically lowers APPalpha secretion: control by the proteasome.

Most of early onset familial forms of Alzheimer's disease (FAD) are due to inherited mutations located on two homologous proteins, presenilins 1 and 2 (PS1 and PS2) encoded by chromosomes 14 and 1, respectively. Here we show that the expression of wild type (wt)-PS2 in human HEK293 cells increases the production of the physiological alpha-secretase-derived product, APPalpha. By contrast, APPalpha secretion is drastically reduced in cells expressing the FAD-linked N141I-PS2. We establish that wt-PS2, N141I-PS2 and their C-terminal maturation fragment are degraded by the enzymatic multicatalytic complex, proteasome. Interestingly, two selective proteasome inhibitors, Z-IE(Ot-Bu)A-Leucinal and lactacystin potentiate the APPalpha secretion observed in wtPS2-expressing cells and further amplify the N141I-PS2-induced decrease in APPalpha production. By contrast, a series of pharmacological agents unable to affect the proteasome do not modify PS2 immunoreactivities and APPalpha recoveries. Altogether, our data indicate that: 1) wtPS2 positively modulates the alpha-secretase physiological pathway of betaAPP maturation in human cells; 2) N141I mutation on PS2 drastically lowers the secretion of APPalpha; 3) Proteasome inhibitors prevent the degradation of wtPS2, N141I-PS2 and their C-terminal maturation product. This protection against proteasomal degradation directly modulates the APPalpha secretion response elicited by wt- and FAD-linked PS2 expression in human HEK293 cells.