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.

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 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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

6-hydroxydopamine-induced nuclear factor-kappa B activation in PC12 cells.

The involvement of nuclear Factor-kappa B (NF-kappa B) transcription factor in PC12 cell death triggered by the dopaminergic neurotoxin 6-hydroxydopamine (6-OHDA) was investigated. Results show that oxidative stress generated by 6-OHDA activates NF-kappa B. When the NF-kappa B activation was inhibited by parthenolide, PC12 cell death induced by 6-OHDA was significantly increased, thus suggesting an involvement of this transcription factor in a protective mechanism against 6-OHDA toxicity.

Molecular pathways involved in the neurotoxicity of 6-OHDA, dopamine and MPTP: contribution to the apoptotic theory in Parkinson's disease.

Parkinson's disease (PD) is a neurodegenerative disorder characterized by a preferential loss of the dopaminergic neurons of the substantia nigra pars compacta. Although the etiology of PD is unknown, major biochemical processes such as oxidative stress and mitochondrial inhibition are largely described. However, despite these findings, the actual therapeutics are essentially symptomatical and are not able to block the degenerative process.

A cautionary note on the use of stable transformed cells.

Gene transfection and ectopic expression is a widely used method in experimental biology. In the present report, we would like to point out that this approach may, in certain circumstances, lead to a modification of the transfected cell phenotype. Indeed, we observed that after transfection of bcl-2 gene in the neuronal PC12 cell line some of the selected clones have lost their neuronal and catecholaminergic characteristics, i.

Extracellular toxicity of 6-hydroxydopamine on PC12 cells.

6-hydroxydopamine (6-OHDA) is usually thought to cross cell membrane through dopamine uptake transporters, to inhibit mitochondrial respiration and to generate intracellular reactive oxygen species. In this study, we show that the anti-oxidants catalase, glutathione and N-acetyl-cysteine are able to reverse the toxic effects of 6-OHDA. These two latter compounds considerably slow down 6-OHDA oxidation in a cell free system suggesting a direct chemical interaction with the neurotoxin.

Triiodothyronine has diverse and multiple stimulating effects on expression of the major myelin protein genes.

If the importance of triiodothyronine (T3) on brain development including myelinogenesis has long been recognized, its mechanism of action at the gene level is still not fully elucidated. We studied the effect of T3 on the expression of myelin protein genes in aggregating brain cell cultures. T3 increases the concentrations of mRNA transcribed from the following four myelin protein genes: myelin basic protein (Mbp), myelin-associated glycoprotein (Mag), proteolipid protein (Plp), and 2',3'-cyclic nucleotide 3'-phosphodiesterase (Cnp).

Morphometric and biochemical studies in trigeminal nerve of rat after trichloroethylene or dichloroacetylene oral administration.

Trigeminal nerve impairment is one of the main features of the clinical spectrum observed after trichloroethylene (Tce) exposure. A morphometric study of teased fibres, an analysis of the fatty acid composition of total lipids of the trigeminal trunk, measurements of myelin basic protein (MBP) and of 2'-3'-cyclic nucleotide 3' phosphohydrolase (CNP) activity were used to characterize this lesion. Furthermore, the effects of Tce and of dichloroacetylene (Dca), a breakdown product supposed to be the main cause of the trigeminal neuropathy, were compared under the same experimental conditions.

A morphometric evaluation of the effects of trichloroethylene and dichloroacetylene on the rat mental nerve. Preliminary results.

Morphometric analysis was used to compare the effects of trichloroethylene (Tri) and dichloroacetylene (Dca) on the fibre parameters of the trigeminal nerve. Treated animals were clearly separated from controls according to a discriminant analysis. Furthermore, in the class of nerve fibres defined by a clustering analysis and corresponding to the largest fibres, myelin thickness was significantly decreased in the Dca group, but less so in the Tri group.

Morphometry of human nerve biopsies by means of automated cytometry: assessment with reference to ultrastructural analysis.

The morphometric analysis of myelinated fibres is a tool for diagnosis of neuropathies and for assessing nerve regeneration after reconstructive surgery. In order to obtain reliable information, sampling techniques should be avoided and the measurement of all the fibres within a nerve fascicle requiring the use of automated cytometry is necessary. We have developed a programme on the SAMBA cytological image analyser for the automated measurement of myelinated fibres.

Automated morphometric study of human peripheral nerves by image analysis.

In this paper we describe a program using the image analyzer SAMBA, which allows an automatic analysis of silver stained semithin nerve sections. The operator can interactively delimit the contour of the fascicle to be analysed by means of a digitizing tablet connected to the system which generates a mask of the region. Segmentation of the fibre images is conducted as a function of brightness threshold defined by the operator.

There is no simple adequate sampling scheme for estimating the myelinated fibre size distribution in human peripheral nerve: a statistical ultrastructural study.

Morphometric studies of peripheral nerves (PN) usually involve some sampling of the myelinated fibres (MF). In order to scrutinize the statistical properties of the sampling processes in common use and the reliability of the resulting estimates, a quantitative analysis of human superficial peroneal nerves from 8 different normal subjects was undertaken at the ultrastructural level, both in terms of MF spatial distribution and of their size distribution. This study used sampling rates involving more than 10% of the whole myelinated fibre population observed in each nerve fascicle.

A method for automatic classification of large and small myelinated fibre populations in peripheral nerves.

The statistical analysis of morphometric data collected from biopsies of human superficial peroneal nerve is complicated by the heterogeneity of the population of myelinated fibres. In order to make separate statistical analyses of the subpopulations of large and small fibres we have developed a computer program (written in PASCAL) for their automatic separation. The method is based on a dynamic centres clustering algorithm and was applied to the multifactorial space defined by the principal component analysis of the morphometric variables: axonal diameter, myelin sheath thickness, circularity index and g-ratio.

The density of myelinated fibres is related to the fascicle diameter in human superficial peroneal nerve. Statistical study of 41 normal samples.

The density of myelinated fibres in the superficial peroneal nerve was studied in 41 samples from 24 control human subjects. Photographic montages of the whole nerve fascicle were made from semithin and ultrathin transverse sections and used for a statistical analysis of sampling procedures, range of variations and relations between density and other variables. The results indicate that the spatial distribution of myelinated fibres within a nerve is often non-uniform.