[show abstract][hide abstract] ABSTRACT: Several human small heat shock proteins (sHsps) are phosphorylated oligomeric chaperones that enhance stress resistance. They are characterized by their ability to interact and form polydispersed hetero-oligomeric complexes. We have analyzed the cellular consequences of the stable expression of either wild type HspB5 or its cataracts and myopathies inducing R120G mutant in growing and oxidative stress treated HeLa cells that originally express only HspB1. Here, we describe that wild type and mutant HspB5 induce drastic and opposite effects on cell morphology and oxidative stress resistance. The cellular distribution and phosphorylation of these polypeptides as well as the oligomerization profile of the resulting hetero-oligomeric complexes formed by HspB1 with the two types of exogenous polypeptides revealed the dominant effects induced by HspB5 polypeptides towards HspB1. The R120G mutation enhanced the native size and salt resistance of HspB1-HspB5 complex. However, in oxidative conditions the interaction between HspB1 and mutant HspB5 was drastically modified resulting in the aggregation of both partners. The mutation also induced the redistribution of HspB1 phosphorylated at serine 15, originally observed at the level of the small oligomers that do not interact with wild type HspB5, to the large oligomeric complex formed with mutant HspB5. This phosphorylation stabilized the interaction of HspB1 with mutant HspB5. A dominant negative effect towards HspB1 appears therefore as an important event in the cellular sensitivity to oxidative stress mediated by mutated HspB5 expression. These observations provide novel data that describe how a mutated sHsp can alter the protective activity of another member of this family of chaperones.
PLoS ONE 01/2013; 8(8):e70545. · 3.73 Impact Factor
[show abstract][hide abstract] ABSTRACT: The multifunctional protein netrin-1 was initially discovered as the main attractive cue for commissural axon guidance by acting through its receptor DCC. Recently, we have shown that netrin-1 also interacts with the orphan transmembrane receptor amyloid precursor protein (APP). APP is cleaved by proteases, generating amyloid-β peptide, the main component of the amyloid plaques that are associated with Alzheimer disease. Our previous work demonstrated that via its interaction with APP, netrin-1 is a negative regulator of amyloid-β production in adult brain, but the biological relevance of APP/netrin-1 interaction under non-pathological conditions was unknown. We show here that during commissural axon navigation, APP, expressed at the growth cone, is part of the DCC receptor complex mediating netrin-1-dependent axon guidance. APP interacts with DCC in the presence of netrin-1 and enhances netrin-1-mediated DCC intracellular signaling, such as MAPK activation. Inactivation of APP in mice is associated with reduced commissural axon outgrowth. Thus, APP functionally acts as a co-receptor for DCC to mediate axon guidance.
Journal of Biological Chemistry 07/2012; 287(35):30014-23. · 4.65 Impact Factor
[show abstract][hide abstract] ABSTRACT: Sonic hedgehog (Shh) and its main receptor, Patched (Ptc), are implicated in both neural development and tumorigenesis. Besides its classic morphogenic activity, Shh is also a survival factor. Along this line, Ptc has been shown to function as a dependence receptor; it induces apoptosis in the absence of Shh, whereas its pro-apoptotic activity is blocked in the presence of Shh. Here we show that, in the absence of its ligand, Ptc interacts with the adaptor protein DRAL (downregulated in rhabdomyosarcoma LIM-domain protein; also known as FHL2). DRAL is required for the pro-apoptotic activity of Ptc both in immortalized cells and during neural tube development in chick embryos. We demonstrate that, in the absence of Shh, Ptc recruits a protein complex that includes DRAL, one of the caspase recruitment (CARD)-domain containing proteins TUCAN (family member, 8) or NALP1 (NLR family, pyrin domain containing 1) and apical caspase-9. Ptc triggers caspase-9 activation and enhances cell death through a caspase-9-dependent mechanism. Thus, we propose that in the absence of its ligand Shh the dependence receptor Ptc serves as the anchor for a caspase-activating complex that includes DRAL, and caspase-9.
[show abstract][hide abstract] ABSTRACT: The enteric nervous system (ENS) is derived from vagal and sacral neural crest cells (NCC) that delaminate from the neural tube and undergo extensive migration and proliferation in order to colonize the entire length of the gut and differentiate into many millions of neurons and glial cells. Although apoptotic programmed cell death is an essential physiological process during development of the majority of the vertebrate nervous system, apoptosis within early ENS development has not been comprehensively investigated. The aim of this study was to determine the presence and extent of apoptosis within the vagal NCC population that gives rise to most of the ENS in the chick embryo. We demonstrated that apoptotic cells, as shown by terminal deoxynucleotidyl transferase biotin-dUTP nick end labelling and active caspase-3 immunoreactivity, are present within an electroporated green fluorescent protein (GFP) and human natural killer-1 (HNK-1) immunopositive NCC population migrating from the vagal region of the neural tube to the developing foregut. Inhibition of caspase activity in vagal NCC, by electroporation with a dominant-negative form of caspase-9, increased the number of vagal NCC available for ENS formation, as shown by 3-dimensional reconstruction of serial GFP or HNK-1 labelled sections, and resulted in hyperganglionosis within the proximal foregut, as shown by NADPH-diaphorase whole gut staining. These findings suggest that apoptotic cell death may be a normal process within the precursor pool of pre-enteric NCC that migrates to the gut, and as such it may play a role in the control of ENS formation.
Neurogastroenterology and Motility 05/2009; 21(7):768-e49. · 2.94 Impact Factor
[show abstract][hide abstract] ABSTRACT: The beta-amyloid precursor protein (APP) is an orphan transmembrane receptor whose physiological role is largely unknown. APP is cleaved by proteases generating amyloid-beta (Abeta) peptide, the main component of the amyloid plaques that are associated with Alzheimer's disease. Here, we show that APP binds netrin-1, a multifunctional guidance and trophic factor. Netrin-1 binding modulates APP signaling triggering APP intracellular domain (AICD)-dependent gene transcription. Furthermore, netrin-1 binding suppresses Abeta peptide production in brain slices from Alzheimer model transgenic mice. In this mouse model, decreased netrin-1 expression is associated with increased Abeta concentration, thus supporting netrin-1 as a key regulator of Abeta production. Finally, we show that netrin-1 brain administration in Alzheimer model transgenic mice may be associated with an amelioration of the Alzheimer's phenotype.
Cell death and differentiation 02/2009; 16(5):655-63. · 8.24 Impact Factor
[show abstract][hide abstract] ABSTRACT: DCC (Deleted in Colorectal Cancer) is a putative tumor suppressor whose expression is lost in numerous cancers and whose tumor suppressor activity appears to be dependent on its ability to trigger apoptosis when disengaged by its ligand netrin-1. In this sense, netrin-1 is a survival factor that controls tumorigenesis. However, netrin-1 is also the prototypical axon guidance cue and has been shown to orient many neurons or axons, especially commissural axons, during spinal cord development. Here we show that netrin-1 is not only an attractive cue for developing commissural axons but also promotes their survival. In primary neuronal culture, in mice or in chick embryos, netrin-1 inhibits the proapoptotic activity of DCC in developing commissural neurons. Thus, adequate commissural neurons navigation requires both the attractive activity of netrin-1 and the anti-apoptotic function of this cue.
Proceedings of the National Academy of Sciences 10/2008; 105(38):14465-70. · 9.74 Impact Factor
[show abstract][hide abstract] ABSTRACT: DCC (deleted in colorectal cancer) is a putative tumor suppressor gene whose expression is lost in numerous cancers. DCC also encodes the main receptor for the neuronal navigation cue netrin-1. It has been shown that DCC belongs to the so-called family of dependence receptors. Such receptors induce apoptosis when their ligand is absent, thus conferring a state of cellular dependence on ligand availability. We recently proposed that DCC is a tumor suppressor because it induces the death of tumor cells that grow in settings of ligand unavailability. Moreover, it seems that the DCC/netrin-1 pair may also regulate neuron survival during nervous system development. However, the mechanisms by which DCC triggers cell death are still unknown. We show here that the localization of DCC to lipid rafts is a prerequisite for its proapoptotic activity, both in immortalized cells and in primary neurons. The presence of DCC in lipid rafts probably allows the formation of an adequate submembrane complex, because the interaction of caspase-9 with DCC is inhibited by the disorganization of lipid rafts. Thus, dependence receptors may require lipid raft localization for cell death signaling.
Proceedings of the National Academy of Sciences 03/2006; 103(11):4128-33. · 9.74 Impact Factor
[show abstract][hide abstract] ABSTRACT: During development, axons migrate long distances in responses to attractive or repulsive signals that are detected by their growth cones. One of these signals is mediated by netrin-1, a diffusible laminin-related molecule that both attracts and repels growth cones via interaction with its receptor DCC (deleted in colorectal cancer). Here we show that DCC in both commissural neurons and immortalized cells, is partially associated with cholesterol- and sphingolipid-enriched membrane domains named lipid rafts. This localization of DCC in lipid rafts is mediated by the palmitoylation within its transmembrane region. Moreover, this raft localization of DCC is required for netrin-1-induced DCC-dependent ERK activation, and netrin-1-mediated axon outgrowth requires lipid raft integrity. Thus, the presence of axon guidance-related receptors in lipid rafts appears to be a crucial pre-requisite for growth cone response to chemo-attractive or repulsive cues.
[show abstract][hide abstract] ABSTRACT: Neuronal growth cones are guided to their targets by attractive and repulsive guidance cues. In mammals, netrin-1 is a bifunctional cue, attracting some axons and repelling others. Deleted in colorectal cancer (Dcc) is a receptor for netrin-1 that mediates its chemoattractive effect on commissural axons, but the signalling mechanisms that transduce this effect are poorly understood. Here we show that Dcc activates mitogen-activated protein kinase (MAPK) signalling, by means of extracellular signal-regulated kinase (ERK)-1 and -2, on netrin-1 binding in both transfected cells and commissural neurons. This activation is associated with recruitment of ERK-1/2 to a Dcc receptor complex. Inhibition of ERK-1/2 antagonizes netrin-dependent axon outgrowth and orientation. Thus, activation of MAPK signalling through Dcc contributes to netrin signalling in axon growth and guidance.
[show abstract][hide abstract] ABSTRACT: The expression of DCC (deleted in colorectal cancer) is often markedly reduced in colorectal and other cancers. However, the rarity of point mutations identified in DCC coding sequences and the lack of a tumor predisposition phenotype in DCC hemizygous mice have raised questions about its role as a tumor suppressor. DCC also mediates axon guidance and functions as a dependence receptor; such receptors create cellular states of dependence on their respective ligands by inducing apoptosis when unoccupied by ligand. We now show that DCC drives cell death independently of both the mitochondria-dependent pathway and the death receptor/caspase-8 pathway. Moreover, we demonstrate that DCC interacts with both caspase-3 and caspase-9 and drives the activation of caspase-3 through caspase-9 without a requirement for cytochrome c or Apaf-1. Hence, DCC defines an additional pathway for the apoptosome-independent caspase activation.
Proceedings of the National Academy of Sciences 04/2001; 98(6):3416-21. · 9.74 Impact Factor
[show abstract][hide abstract] ABSTRACT: The RET (rearranged during transfection) proto-oncogene encodes a tyrosine kinase receptor involved in both multiple endocrine neoplasia type 2 (MEN 2), an inherited cancer syndrome, and Hirschsprung disease (HSCR), a developmental defect of enteric neurons. We report here that the expression of RET receptor induces apoptosis. This pro-apoptotic effect of RET is inhibited in the presence of its ligand glial cell line-derived neurotrophic factor (GDNF). Furthermore, we present evidence that RET induces apoptosis via its own cleavage by caspases, a phenomenon allowing the liberation/exposure of a pro-apoptotic domain of RET. In addition, we report that Hirschsprung-associated RET mutations impair GDNF control of RET pro-apoptotic activity. These results indicate that HSCR may result from apoptosis of RET-expressing enteric neuroblasts.
The EMBO Journal 09/2000; 19(15):4056-63. · 9.82 Impact Factor