Foxo3a induces motoneuron death through the Fas pathway in cooperation with JNK

BACKGROUND: Programmed cell death of motoneurons in the developing spinal cord is thought to be regulated through the availability of target-derived neurotrophic factors. When deprived of trophic support, embryonic spinal motoneurons in vitro over-express FasL, a ligand activating a Fas-mediated death pathway. How trophic factors regulate the expression of FasL is presently unclear, but two regulators of FasL, FOXO3a (FKHRL1) and JNK have been described to play a role in other cell types. Thus, their potential function in motoneurons was investigated in this study. RESULTS: We show here that as a result of removal of neurotrophic factors and the consequent reduction in signalling through the PI3K/Akt pathway, Foxo3a translocates from the cytoplasm to the nucleus where it triggers cell death. Death is reduced in Fas and FasL mutant motoneurons and in the presence of JNK inhibitors indicating that a significant part of it requires activation of the Fas/FasL pathway through JNK. CONCLUSIONS: Therefore, in motoneurons as in other cell types, FOXO transcriptional regulators provide an important link between other signalling pathways and the cell death machinery

Optimised and Rapid Pre-clinical Screening in the SOD1G93A Transgenic Mouse Model of Amyotrophic Lateral Sclerosis (ALS)

The human SOD1G93A transgenic mouse has been used extensively since its development in 1994 as a model for amyotrophic lateral sclerosis (ALS). In that time, a great many insights into the toxicity of mutant SOD1 have been gained using this and other mutant SOD transgenic mouse models. They all demonstrate a selective toxicity towards motor neurons and in some cases features of the pathology seen in the human disease. These models have two major drawbacks. Firstly the generation of robust preclinical data in these models has been highlighted as an area for concern. Secondly, the amount of time required for a single preclinical experiment in these models (3–4 months) is a hurdle to the development of new therapies. We have developed an inbred C57BL/6 mouse line from the original mixed background (SJLxC57BL/6) SOD1G93A transgenic line and show here that the disease course is remarkably consistent and much less prone to background noise, enabling reduced numbers of mice for testing of therapeutics. Secondly we have identified very early readouts showing a large decline in motor function compared to normal mice. This loss of motor function has allowed us to develop an early, sensitive and rapid screening protocol for the initial phases of denervation of muscle fibers, observed in this model. We describe multiple, quantitative readouts of motor function that can be used to interrogate this early mechanism. Such an approach will increase throughput for reduced costs, whilst reducing the severity of the experimental procedures involved

Implication de la Calréticuline et de CRMP4 dans la dégénérescence des motoneurones dans la Sclérose Latérale Amyotrophique

La Sclérose Latérale Amyotrophique se caractérise par la perte sélective de motoneurones (MNs) du cortex, du tronc cérébral et de la moelle épinière. Les souris surexprimant le gène humain muté codant pour la superoxide dismutase 1 (mSOD1) constitue un bon modèle d'étude. Les MNs mSOD1 présentent une hypersensibilité à la mort après activation du récepteur Fas et la production d'oxyde nitrique (NO). Notre étude protéomique a identifié deux effecteurs du NO, la calréticuline (CRT) et CRMP4. CRT est une protéine chaperonne de stockage du calcium dans le réticulum endoplasmique. Nous montrons que, in vivo, CRT diminue de moitié dans une sous population de MNs mSOD1 dits vulnérables, car dégénérant les premiers. Sa diminution est nécessaire et suffisante pour induire la mort des MNs mSOD1 en activant le stress du RE. CRMP4 est une protéine de régulation de la croissance axonale, qui augmente in vivo dans les MNs mSOD1 à un stade pré-symptomatique. Sa surexpression est suffisante pour induire une dénervation périphérique et la dégénérescence de MNs mSOD1. Nos résultats mettent en évidence CRT et CRMP4 comme étant deux cibles thérapeutiques potentielles dans la SLA.Amyotrophic Lateral Sclerosis (ALS) is characterized by the selective degeneration of upper and lower motoneurons (MNs). The most common familial form and best characterized mouse model of ALS is linked to mutations in the gene coding for the superoxide dismutase 1 (mSOD1). MNs expressing mSOD1 show an increased sensitivity to the death induced by Fas/NO activation. Our proteomic study identified two downstream effectors of NO, Calreticulin (CRT) and CRMP4. CRT is a chaperone-calcium-binding protein of the endoplasmic reticulum, which is decreased two-fold in vivo, in an early degenerating MNs sub-population, named vulnerable. The decrease in CRT expression is both necessary and sufficient to kill mSOD1 MNs through ER stress activation. CRMP4 is a neurite outgrowth regulator which expression is increased in vivo in mSOD1 MNs at a presymptomatic stage. CRMP4 overexpression is sufficient to induce peripheral denervation and, through a dying-back effect, to kill mSOD1 MNs. Our results point out CRT and CRMP-4 as two potential therapeutic targets for ALS.AIX-MARSEILLE2-Bib.electronique (130559901) / SudocSudocFranceF

Etude fonctionnelle chez le rat de la protéine SMN (Survival Motor Neuron), produit du gène candidat pour les amyotrophies spinales infantiles

AIX-MARSEILLE2-BU Sci.Luminy (130552106) / SudocSudocFranceF

Mort développementale et pathologique du motoneurone spinal (Implication du récepteur de mort Fas (CD95))

AIX-MARSEILLE2-BU Sci.Luminy (130552106) / SudocSudocFranceF

Motoneurons were electroporated with HA-wt-Foxo3a and cultured for 2 d with NTFs in the presence or not of a JNK inhibitor, L-JNKl1 (1 μM)

<p><b>Copyright information:</b></p><p>Taken from "Foxo3a induces motoneuron death through the Fas pathway in cooperation with JNK"</p><p>BMC Neuroscience 2004;5():48-48.</p><p>Published online 29 Nov 2004</p><p>PMCID:PMC538283.</p><p>Copyright © 2004 Barthélémy et al; licensee BioMed Central Ltd.</p> Blocking the JNK pathway in motoneuron reduces the killing effect of TM-Foxo3a by about 20 %. Results are mean ± SD of 6 wells in 3 independent experiments. The 100 % corresponds to the number of motoneurons electroporated with HA-wt-Foxo3a and untreated. Differences with the TM-Foxo3a effect in L-JNKl1-treated compared with control neurons were found significant using Student's t-test (p < 0.001)