Challenges in glioblastoma research: focus on the tumor microenvironment.

Glioblastoma (GBM) is the most deadly type of malignant brain tumor, despite extensive molecular analyses of GBM cells. In recent years, the tumor microenvironment (TME) has been recognized as an important player and therapeutic target in GBM. However, there is a need for a full and integrated understanding of the different cellular and molecular components involved in the GBM TME and their interactions for the development of more efficient therapies. In this review, we provide a comprehensive report of the GBM TME, which assembles the contributions of physicians and translational researchers working on brain tumor pathology and therapy in France. We propose a holistic view of the subject by delineating the specific features of the GBM TME at the cellular, molecular, and therapeutic levels.

Parkin differently regulates presenilin-1 and presenilin-2 functions by direct control of their promoter transcription.

We previously established that besides its canonical function as E3-ubiquitin ligase, parkin also behaves as a transcriptional repressor of p53. Here we show that parkin differently modulates presenilin-1 and presenilin-2 expression and functions at transcriptional level. Thus, parkin enhances/reduces the protein expression, promoter activity and mRNA levels of presenilin-1 and presenilin-2, respectively, in cells and in vivo. This parkin-associated function is independent of its ubiquitin-ligase activity and remains unrelated to its capacity to repress p53. Accordingly, physical interaction of endogenous or overexpressed parkin with presenilins promoters is demonstrated by chromatin immunoprecipitation assays (ChIP). Furthermore, we identify a consensus sequence, the deletion of which abolishes parkin-dependent modulation of presenilins-1/2 and p53 promoter activities. Interestingly, electrophoretic mobility shift assays (EMSA) revealed a physical interaction between this consensus sequence and wild-type but not mutated parkin. Finally, we demonstrate that the RING1-IBR-RING2 domain of parkin harbors parkin's potential to modulate presenilins promoters. This transcriptional control impacts on presenilins-associated phenotypes, since parkin increases presenilin-1-associated γ-secretase activity and reduces presenilin-2-linked caspase-3 activation. Overall, our data delineate a promoter responsive element targeted by parkin that drives differential regulation of presenilin-1 and presenilin-2 transcription with functional consequences for γ-secretase activity and cell death.

Periphilin is a novel interactor of synphilin-1, a protein implicated in Parkinson's disease.

Parkinson's disease (PD) is a common neurodegenerative disorder characterized by the loss of dopaminergic neurons and the presence of Lewy bodies. Alpha-synuclein and its interactor synphilin-1 are major components of these inclusions. Rare mutations in the alpha-synuclein and synphilin-1 genes have been implicated in the pathogenesis of PD; however, the normal function of these proteins is far from being completely elucidated. We, thus, searched for novel synphilin-1-interacting proteins and deciphered periphilin as new interactor. Periphilin isoforms are involved in multiple cellular functions in vivo, and the protein is broadly expressed during embryogenesis and in the adult brain. We show that periphilin displays an overlapping expression pattern with synphilin-1 in cellular and animal models and in Lewy bodies of PD patients. Functional studies demonstrate that periphilin, as previously shown for synphilin-1, displays an antiapoptotic function by reducing caspase-3 activity. Searching for mutations in the periphilin gene, we detected a K69E substitution in two patients of a PD family. Taken together, these findings support for the first time an involvement of periphilin in PD.

p53-dependent control of transactivation of the Pen2 promoter by presenilins.

The senile plaques found in the brains of patients with Alzheimer's disease are mainly due to the accumulation of amyloid beta-peptides (A beta) that are liberated by gamma-secretase, a high molecular weight complex including presenilins, PEN-2, APH-1 and nicastrin. The depletion of each of these proteins disrupts the complex assembly into a functional protease. Here, we describe another level of regulation of this multimeric protease. The depletion of both presenilins drastically reduces Pen2 mRNA levels and its promoter transactivation. Furthermore, overexpression of presenilin-1 lowers Pen2 promoter transactivation, a phenotype abolished by a double mutation known to prevent presenilin-dependent gamma-secretase activity. PEN-2 expression is decreased by depletion of beta-amyloid precursor protein (APP) and increased by the APP intracellular domain (AICD). We show that AICD and APP complement for Pen2 mRNA levels in APP/APLP1-2 knockout fibroblasts. Interestingly, overexpression of presenilin-2 greatly increases Pen2 promoter transactivation. The opposite effect triggered by both presenilins was reminiscent of our previous study, which showed that these two proteins elicit antagonistic effects on p53. Therefore, we examined the contribution of p53 on Pen2 transcription. Pen2 promoter transactivation, and Pen2 mRNA and protein levels were drastically reduced in p53(-/-) fibroblasts. Furthermore, PEN-2 expression could be rescued by p53 complementation in p53- and APP-deficient cells. Interestingly, PEN-2 expression was also reduced in p53-deficient mouse brain. Overall, our study describes a p53-dependent regulation of PEN-2 expression by other members of the gamma-secretase complex, namely presenilins.

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 Parkinson's 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.

DJ-1: a newcomer in Parkinson's disease pathology.

Parkinson's disease (PD) is a movement disorder of high prevalence in the elderly. It is characterized by a loss of dopaminergic neurons and the presence of intracytoplasmic inclusions named Lewy bodies. To date six familial PD-associated proteins have been identified so far. Some of them are implicated in the development of either autosomal dominant (alpha-synuclein and LRRK2 (leucine-rich repeat kinase 2/dardarin) or early-onset recessive (parkin, DJ-1, PINK1 (PTEN-induced kinase-1) and ATP13A2) PD forms. A number of genetic studies have shown that 50% of the recessive forms are linked to mutations on parkin gene, followed by PINK1 (8-15%) and DJ-1 (1%). The purpose of this review is to provide an overview of the emerging data on the cellular and molecular biology of DJ-1. DJ-1 is a ubiquitous protein that was first described as an oncogene. Nevertheless, after its association to monogenic PD a number considerable data aiming at understanding its implication in the physiopathology of PD was produced. This review will describe the main advances concerning the function of DJ-1. A considerable progress that was only possible due to a better understanding of DJ-1 structure, genetics, distribution and development of in vivo models. All these points along with the description of recent data showing the interaction of DJ-1 with other PD-associated proteins will be given.

The gamma/epsilon-secretase-derived APP intracellular domain fragments regulate p53.

Amyloid beta-peptide (Abeta), which plays a central role in Alzheimer Disease, is generated by presenilin-dependent and presenilin-independent gamma-secretase cleavages of beta-amyloid precursor protein (betaAPP). We report that the presenilins (PS1 and PS2) also regulate p53-associated cell death. Thus, we established that PS deficiency, catalytically inactive PS mutants, gamma-secretase inhibitors and betaAPP or APLP2 depletion reduced the expression and activity of p53, and lowered the transactivation of its promoter and mRNA levels. p53 expression was also reduced in the brains or betaAPP-deficient mice or in brains where both PS had been invalidated by double conditional knock out. AICDC59 and AICDC50, the gamma- and epsilon-secretase-derived C-terminal fragments of betaAPP, respectively, trigger the activation of caspase-3, p53-dependent cell death, and increase p53 activity and mRNA. Finally, HEK293 cells expressing PS1 harboring familial AD (FAD) mutations or FAD-affected brains, all display enhanced p53 activity and p53 expression. Our studies demonstrate that AICDs control p53 at a transcriptional level, in vitro and in vivo and unravel a still unknown function for presenilins.

p53-Dependent Aph-1 and Pen-2 anti-apoptotic phenotype requires the integrity of the gamma-secretase complex but is independent of its activity.

The presenilin-dependent gamma-secretase activity, which is responsible for the generation of amyloid beta-peptide, is a high molecular weight complex composed of at least four components, namely, presenilin-1 (or presenilin-2), nicastrin, Aph-1, and Pen-2. Previous data indicated that presenilins, which are thought to harbor the catalytic core of the complex, also control p53-dependent cell death. Whether the other components of the gamma-secretase complex could also modulate the cell death process in mammalian neurons remained to be established. Here, we examined the putative contribution of Aph-1 and Pen-2 in the control of apoptosis in TSM1 cells from a neuronal origin. We show by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling and DNA fragmentation analyses that the overexpression of Aph-1a, Aph-1b, or Pen-2 drastically lowered staurosporine-induced cellular toxicity. In support of an apoptosis rather than necrosis process, Aph-1 and Pen-2 also lower staurosporine- and etoposide-induced caspase-3 expression and diminished caspase-3 activity and poly(ADP-ribose) polymerase inactivation. The Aph-1 and Pen-2 anti-apoptotic phenotype was associated with a drastic reduction of p53 expression and activity and lowered p53 mRNA transcription. Furthermore, the Aph-1- and Pen-2-associated reduction of staurosporine-induced caspase-3 activation was fully abolished by p53 deficiency. Conversely, Aph-1a, Aph-1b, and Pen-2 gene inactivation increases both caspase-3 activity and p53 mRNA levels. Finally, we show that Aph-1 and Pen-2 did not trigger an anti-apoptotic response in cells devoid of presenilins or nicastrin, whereas the protective response was still observed in fibroblasts devoid of beta-amyloid precursor protein and amyloid precursor protein like-protein 2. Furthermore, Aph-1- and Pen-2-associated protection against staurosporine-induced caspase-3 activation was not affected by the gamma-secretase inhibitors N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester and difluoromethylketone. Altogether, our study indicates that Aph-1 and Pen-2 trigger an anti-apoptotic response by lowering p53-dependent control of caspase-3. Our work also demonstrates that this phenotype is strictly dependent on the molecular integrity of the gamma-secretase complex but remains independent of the gamma-secretase catalytic activity.

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 (PrP(c)) 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 PrP(c) yielded C-terminal catabolites with distinct function and could be seen as a switch mechanism controlling the function of the PrP(c) in cell survival.

Caspase-3-derived C-terminal product of synphilin-1 displays antiapoptotic function via modulation of the p53-dependent cell death pathway.

Parkinson disease is the second most frequent neurodegenerative disorder after Alzheimer disease. A subset of genetic forms of Parkinson disease has been attributed to alpha-synuclein, a synaptic protein with remarkable chaperone properties. Synphilin-1 is a cytoplasmic protein that has been identified as a partner of alpha-synuclein (Engelender, S., Kaminsky, Z., Guo, X., Sharp, A. H., Amaravi, R. K., Kleiderlein, J. J., Margolis, R. L., Troncoso, J. C., Lanahan, A. A., Worley, P. F., Dawson, V. L., Dawson, T. M., and Ross, C. A. (1999) Nat. Gen. 22, 110-114), but its function remains totally unknown. We show here for the first time that synphilin-1 displays an antiapoptotic function in the control of cell death. We have established transient and stable transfectants overexpressing wild-type synphilin-1 in human embryonic kidney 293 cells, telecephalon-specific murine 1 neurons, and SH-SY5Y neuroblastoma cells, and we show that both cell systems display lower responsiveness to staurosporine and 6-hydroxydopamine. Thus, synphilin-1 reduces procaspase-3 hydrolysis and thereby caspase-3 activity and decreases poly(ADP-ribose) polymerase cleavage, two main indicators of apoptotic cell death. Furthermore, we establish that synphilin-1 drastically reduces p53 transcriptional activity and expression and lowers p53 promoter transactivation and mRNA levels. Interestingly, we demonstrate that synphilin-1 catabolism is enhanced by staurosporine and blocked by caspase-3 inhibitors. Accordingly, we show by transcription/translation assay that recombinant caspase-3 and, to a lesser extent, caspase-6 but not caspase-7 hydrolyze synphilin-1. Furthermore, we demonstrate that mutated synphilin-1, in which a consensus caspase-3 target sequence has been disrupted, resists proteolysis by cellular and recombinant caspases and displays drastically reduced antiapoptotic phenotype. We further show that the caspase-3-derived C-terminal fragment of synphilin-1 was probably responsible for the antiapoptotic phenotype elicited by the parent wild-type protein. Altogether, our study is the first demonstration that synphilin-1 harbors a protective function that is controlled by the C-terminal fragment generated by its proteolysis by caspase-3.

Recent insights on the pro-apoptotic phenotype elicited by presenilin 2 and its caspase and presenilinase-derived fragments.

Programmed cell death (PCD) also called apoptosis, is a normal and genetically controlled event that could play, when mis-regulated, a pivotal role in the development of several neurodegenerative disorders such as Parkinson's disease. Sporadic Alzheimer's disease is one of the most prominent age-related syndromes whose etiology, although still unknown, could be related to biochemical or environmental causes. A few cases of Alzheimer's disease are likely of genetic origin and linked to mutations on the genes coding for the amyloid precursor protein (betaAPP) and presenilins 1 and 2. Although still discussed, the hypothesis of an implication of apoptotic cell death in Alzheimer's disease neuropathology has been recently supported by a growing body of biochemical evidences. Thus, the implication of presenilins in apoptotic processes in vitro has been well documented but the mechanisms underlying this function are still a matter of intense research. The aim of this review is to focus on the mechanisms by which presenilin 2 affects the programmed cell death with special emphasis on the role of the proteolytically derived presenilin fragments generated by both presenilinase- and caspases. The distinct apoptotic phenotypes elicited by the two parent proteins presenilins 1 and 2 and their functional cross talk will be briefly discussed.

Beta-synuclein displays an antiapoptotic p53-dependent phenotype and protects neurons from 6-hydroxydopamine-induced caspase 3 activation: cross-talk with alpha-synuclein and implication for Parkinson's disease.

We have established stable transfectants expressing beta-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 beta-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. beta-Synuclein strongly reduces staurosporine-induced caspase 3 activity and immunoreactivity. We establish that beta-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 beta-synuclein-induced p53 down-regulation likely occurs at a post-transcriptional level. We showed previously that alpha-synuclein displays an antiapoptotic function that was abolished by the dopaminergic derived toxin 6-hydroxydopamine (6OHDA). Interestingly, beta-synuclein retains its ability to protect TSM1 neurons even after 6OHDA treatment. Furthermore, beta-synuclein restores the antiapoptotic function of alpha-synuclein in 6OHDA-treated neurons. Altogether, our data document for the first time that beta-synuclein protects neurons from staurosporine and 6OHDA-stimulated caspase activation in a p53-dependent manner. Our observation that beta-synuclein contributes to restoration of the alpha-synuclein antiapoptotic function abolished by 6OHDA may have direct implications for Parkinson's disease pathology. In this context, the cross-talk between these two parent proteins is discussed.