Functions of LKB1 in neural crest development: The story unfolds.

Neural crest cells (NCCs) are highly motile, multipotent, embryonic cells that delaminate from the dorsal edges of the neural tube. NCCs follow stereotypical long-range migratory pathways to reach target organs during development, where they give rise to multiple derivatives. The identification of reservoirs of neural crest stem cells that persist to adulthood has recently aroused renewed interest in the biology of NCCs. In this context, several recent studies have demonstrated the essential role of the metabolic kinase LKB1 in NCC establishment. This review surveys how LKB1 governs the formation and maintenance of several neural crest derivatives, including facial bones, melanocytes, Schwann cells, and the enteric nervous system. We also detail the underlying molecular mechanisms that involve downstream effectors of LKB1, in particular the contribution of the AMPK-mTOR signaling pathway to both polarity and metabolic processes. Collectively, these recent discoveries open promising perspectives for new therapeutic applications for the treatment of neural crest disorders.

LKB1 specifies neural crest cell fates through pyruvate-alanine cycling.

Metabolic processes underlying the development of the neural crest, an embryonic population of multipotent migratory cells, are poorly understood. Here, we report that conditional ablation of the Lkb1 tumor suppressor kinase in mouse neural crest stem cells led to intestinal pseudo-obstruction and hind limb paralysis. This phenotype originated from a postnatal degeneration of the enteric nervous ganglia and from a defective differentiation of Schwann cells. Metabolomic profiling revealed that pyruvate-alanine conversion is enhanced in the absence of Lkb1. Mechanistically, inhibition of alanine transaminases restored glial differentiation in an mTOR-dependent manner, while increased alanine level directly inhibited the glial commitment of neural crest cells. Treatment with the metabolic modulator AICAR suppressed mTOR signaling and prevented Schwann cell and enteric defects of Lkb1 mutant mice. These data uncover a link between pyruvate-alanine cycling and the specification of glial cell fate with potential implications in the understanding of the molecular pathogenesis of neural crest diseases.

LKB1 signaling in cephalic neural crest cells is essential for vertebrate head development.

Head development in vertebrates proceeds through a series of elaborate patterning mechanisms and cell-cell interactions involving cephalic neural crest cells (CNCC). These cells undergo extensive migration along stereotypical paths after their separation from the dorsal margins of the neural tube and they give rise to most of the craniofacial skeleton. Here, we report that the silencing of the LKB1 tumor suppressor affects the delamination of pre-migratory CNCC from the neural primordium as well as their polarization and survival, thus resulting in severe facial and brain defects. We further show that LKB1-mediated effects on the development of CNCC involve the sequential activation of the AMP-activated protein kinase (AMPK), the Rho-dependent kinase (ROCK) and the actin-based motor protein myosin II. Collectively, these results establish that the complex morphogenetic processes governing head formation critically depends on the activation of the LKB1 signaling network in CNCC.

Alix is involved in caspase 9 activation during calcium-induced apoptosis.

The cytoplasmic protein Alix/AIP1 (ALG-2 interacting protein X) is involved in cell death through mechanisms which remain unclear but require its binding partner ALG-2 (apoptosis-linked gene-2). The latter was defined as a regulator of calcium-induced apoptosis following endoplasmic reticulum (ER) stress. We show here that Alix is also a critical component of caspase 9 activation and apoptosis triggered by calcium. Indeed, expression of Alix dominant-negative mutants or downregulation of Alix afford significant protection against cytosolic calcium elevation following thapsigargin (Tg) treatment. The function of Alix in this paradigm requires its interaction with ALG-2. In addition, we demonstrate that caspase 9 activation is necessary for apoptosis induced by Tg and that this activation is impaired by knocking down Alix. Altogether, our findings identify, for the first time, Alix as a crucial mediator of Ca(2+) induced caspase 9 activation.

Alix and ALG-2 make a link between endosomes and neuronal death.

Alix [ALG-2 (apoptosis-linked gene 2)-interacting protein X] is a ubiquitinous adaptor protein first described for its capacity to bind to the calcium-binding protein, ALG-2. Alix regulates neuronal death in ways involving interactions with ALG-2 and with proteins of the ESCRT (endosomal sorting complex required for transport). Even though all Alix interactors characterized to date are involved in endosomal trafficking, the genuine function of the protein in this process remains unclear. We have demonstrated recently that Alix and ALG-2 form in the presence of calcium, a complex with apical caspases and with the endocytosed death receptor TNFR1 (tumour necrosis factor alpha receptor 1), thus suggesting a molecular coupling between endosomes and the cell death machinery.

Alix and ALG-2 are involved in tumor necrosis factor receptor 1-induced cell death.

Alix/AIP1 regulates cell death in a way involving interactions with the calcium-binding protein ALG-2 and with proteins of ESCRT (endosomal sorting complex required for transport). Using mass spectrometry we identified caspase-8 among proteins co-immunoprecipitating with Alix in dying neurons. We next demonstrated that Alix and ALG-2 interact with pro-caspase-8 and that Alix forms a complex with the TNFalpha receptor-1 (TNF-R1), depending on its capacity to bind ESCRT proteins. Thus, Alix and ALG-2 may allow the recruitment of pro-caspase-8 onto endosomes containing TNF-R1, a step thought to be necessary for activation of the apical caspase. In line with this, expression of Alix deleted of its ALG-2-binding site (AlixDeltaALG-2) significantly reduced TNF-R1-induced cell death, without affecting endocytosis of the receptor. In a more physiological setting, we found that programmed cell death of motoneurons, which can be inhibited by AlixDeltaALG-2, is regulated by TNF-R1. Taken together, these results highlight Alix and ALG-2 as new actors of the TNF-R1 pathway.

Alix differs from ESCRT proteins in the control of autophagy.

Alix/AIP1 is a cytosolic protein that regulates cell death through mechanisms that remain unclear. Alix binds to two protein members of the so-called Endosomal Sorting Complex Required for Transport (ESCRT), which facilitates membrane fission events during multivesicular endosome formation, enveloped virus budding and cytokinesis. Alix itself has been suggested to participate in these cellular events and is thus often considered to function in the ESCRT pathway. ESCRT proteins were recently implicated in autophagy, a process involved in bulk degradation of cytoplasmic constituents in lysosomes, which can also participate in cell death. In this study, we shown that, unlike ESCRT proteins, Alix is not involved in autophagy. These results strongly suggest that the capacity of several mutants of Alix to block both caspase-dependent and independent cell death does not relate to their capacity to modulate autophagy. Furthermore, they reinforce the conclusion of other studies demonstrating that the role of Alix is different from that of classical ESCRT proteins.

Survival response-linked Pyk2 activation during potassium depletion-induced apoptosis of cerebellar granule neurons.

Numerous extracellular stimuli trigger trans-autophosphorylation at Tyr402 of Pyk2, inducing its activation. Pyk2 is a key mediator of several signaling pathways and has been implicated in apoptosis induced by specific stress signals. We investigated whether Pyk2 participates in cerebellar granule neuron (CGN) apoptosis induced by the suppression of membrane depolarization. We demonstrate that shifting CGN cultures from 25 mM to 5 mM KCl-containing medium induces an early, transient 70% increase in phosphorylated Tyr402 and Tyr580 Pyk2 levels that is triggered by Ca(2+) released from intracellular stores and mediated by calmodulin (CaM). Overexpression of Pyk2 increases CGN survival after 24 h by 70% compared to the control, thus suggesting that Pyk2 is involved in an anti-apoptotic response to K+ lowering. Furthermore, we show that CGN grown in K25 medium exhibit detectable CaM-dependent Pyk2 activity. When silencing Pyk2 activity by expressing a dominant-negative form, only 40% of the transfected neurons were alive 24 h after transfection when compared to the control. Overall, the present findings demonstrate for the first time that Pyk2 is a critical mediator of CGN survival.

Increased Alix (apoptosis-linked gene-2 interacting protein X) immunoreactivity in the degenerating striatum of rats chronically treated by 3-nitropropionic acid.

Chronic intoxication by 3-nitropropionic acid in the Lewis rat reproduces many features reminiscent of Huntington's disease including behavioural alterations and cortico-striatal degeneration. In particular, in this model, striatal degeneration is accompanied by calpain activation as found in the human disease. The present study was undertaken to determine whether the expression of Alix (apoptosis linked gene-2 interacting protein), a widespread protein involved in neuronal death, would be modified in the striatum and cortex of 3NP-treated rats. The results clearly show that Alix immunoreactivity is increased in the neuronal cell bodies of the lateral striatum, where degeneration is massive. The medial striatum and the cortex that lack neurodegeneration remain only weakly labelled. This is further evidence suggesting an involvement of Alix in the events driving neuronal death.

Alix, a protein regulating endosomal trafficking, is involved in neuronal death.

Alix/AIP1 is a cytoplasmic protein, which was first characterized as an interactor of ALG-2, a calcium-binding protein necessary for cell death. Alix has also recently been defined as a regulator of the endo-lysosomal system. Here we have used post-mitotic cerebellar neurons to test Alix function in caspase-dependent and -independent cell death. Indeed, these neurons survived when cultured in 25 mm potassium-containing medium but underwent apoptosis soon after the extracellular potassium was lowered to 5 mm. In agreement with other studies, we show that caspases are activated after K+ deprivation, but that inhibition of these proteases, using the pancaspase inhibitor boc-aspartyl(OMe)-fluoromethylketone, has no effect on cell survival. Transfection experiments demonstrated that Alix overexpression is sufficient to induce caspase activation, whereas overexpression of its C-terminal half, Alix-CT, blocks caspase activation and cell death after K+ deprivation. We also define a 12-amino acid PXY repeat of the C-terminal proline-rich domain necessary for binding ALG-2. Deletion of this domain in Alix or in Alix-CT abolished the effects of the overexpressed proteins on neuronal survival, demonstrating that the ALG-2-binding region is crucial for the death-modulating function of Alix. Overall, these findings define the Alix/ALG-2 complex as a regulator of cell death controlling both caspase-dependent and -independent pathways. They also suggest a molecular link between the endo-lysosomal system and the effectors of the cell death machinery.

Alix (ALG-2-interacting protein X), a protein involved in apoptosis, binds to endophilins and induces cytoplasmic vacuolization.

ALG-2-interacting protein X (Alix), also known as AIP1, is a cytoplasmic protein ubiquitously expressed and concentrated in phagosomes and exosomes. Alix may regulate apoptosis since it binds apoptosis-linked gene 2 (ALG-2), a Ca2+-binding protein necessary for cell death, and also overexpression of its C-terminal half (Alix-CT) blocks death induced by several stimuli. This part of Alix contains a long proline-rich domain containing several potential SH3-binding sites. Using Alix as bait in a yeast two-hybrid system to screen a mouse brain library, we have found that SH3p4, SH3p8, and SH3p13, collectively known as endophilins, bind to Alix. Co-immunoprecipitations and overlay experiments allowed us to demonstrate that endophilins bind to Alix-CT through an SH3/proline-rich domain interaction. We have narrowed the region of Alix interacting with endophilins down to 14 amino acids containing a PXRPPPP consensus sequence, also present in synaptojanin and germinal center kinase-like kinase, allowing their interaction to endophilins. We further show that overexpression of Alix-CT, which blocks cell death, leads to cytoplasmic vacuolization into tubulo-vesicular structures delineated by Alix-CT. This vacuolization phenomenon is greatly enhanced upon co-expression with endophilins and may be part of the protecting mechanism afforded by Alix-CT.