Amyotrophic lateral sclerosis (ALS) is an incurable neurodegenerative disease that primarily affects motoneurons in the brain and spinal cord. Dominant mutations in superoxide dismutase-1 (SOD1) cause a familial form of ALS. Mutant SOD1-damaged glial cells contribute to ALS pathogenesis by releasing neurotoxic factors, but the mechanistic basis of the motoneuron-specific elimination is poorly understood. Here, we describe a motoneuron-selective death pathway triggered by activation of lymphotoxin-b receptor (LT-bR) by LIGHT, and operating by a novel signaling scheme. We show that astrocytes expressing mutant SOD1 mediate the selective death of motoneurons through the proinflammatory cytokine interferon-c (IFNc), which activates the LIGHT-LT-bR death pathway. The expression of LIGHT and LT-bR by motoneurons in vivo correlates with the preferential expression of IFNc by motoneurons and astrocytes at disease onset and symptomatic stage in ALS mice. Importantly, the genetic ablation of Light in an ALS mouse model retards progression, but not onset, of the disease and increases lifespan. We propose that IFNc contributes to a cross-talk between motoneurons and astrocytes causing the selective loss of some motoneurons following activation of the LIGHT-induced death pathway.

Cellular responses to protein misfolding are thought to play key roles in triggering neurodegeneration. In the mutant superoxide dismutase (mSOD1) model of amyotrophic lateral sclerosis (ALS), subsets of motoneurons are selectively vulnerable to degeneration. Fast fatigable motoneurons selectively activate an endoplasmic reticulum (ER) stress response that drives their early degeneration while a subset of mSOD1 motoneurons show exacerbated sensitivity to activation of the motoneuron-specific Fas/NO pathway. However, the links between the two mechanisms and the molecular basis of their cellular specificity remained unclear. We show that Fas activation leads, specifically in mSOD1 motoneurons, to reductions in levels of calreticulin (CRT), a calcium-binding ER chaperone. Decreased expression of CRT is both necessary and sufficient to trigger SOD1 G93A motoneuron death through the Fas/NO pathway. In SOD1 G93Amice in vivo, reductions in CRT precede muscle denervation and are restricted to vulnerable motor pools. In vitro, both reduced CRT and Fas activation trigger an ER stress response that is restricted to, and required for death of, vulnerable SOD1 G93A motoneurons. Our data reveal CRT as a critical link between a motoneuron-specific death pathway and the ER stress response and point to a role of CRT levels in modulating motoneuron vulnerability to ALS.

The MET tyrosine kinase, the receptor of hepatocyte growth factor-scatter factor (HGF/SF), is known to be essential for normal development and cell survival. We report that stress stimuli induce the caspase-mediated cleavage of MET in physiological cellular targets, such as epithelial cells, embryonic hepatocytes, and cortical neurons. Cleavage occurs at aspartic residue 1000 within the SVD site of the juxtamembrane region, independently of the crucial docking tyrosine residues Y1001 or Y1347 and Y1354. This cleavage generates an intracellular 40-kDa MET fragment containing the kinase domain. The p40 MET fragment itself causes apoptosis of MDCK epithelial cells and embryonic cortical neurons, whereas its kinase-dead version is impaired in proapoptotic activity. Finally, HGF/SF treatment does not favor MET cleavage and apoptosis, confirming the known survival role of ligand-activated MET. Our results show that stress stimuli convert the MET survival receptor into a proapoptotic factor.

Coordination of cell death and survival is crucial during embryogenesis and adulthood, and alteration of this balance can result in degeneration or cancer. Growth factor receptors such as Met can activate phosphatidyl-inositol-3Ј kinase (PI3K), a major intracellular mediator of growth and survival. PI3K can then antagonize p53-triggered cell death, but the underlying mechanisms are not fully understood. We used genetic and pharmacological approaches to uncover Met-triggered signaling pathways that regulate hepatocyte survival during embryogenesis. Here, we show that PI3K acts via mTOR (Frap1) to regulate p53 activity both in vitro and in vivo. mTOR inhibits p53 by promoting the translation of Mdm2, a negative regulator of p53. We also demonstrate that the PI3K effector Akt is required for Met-triggered Mdm2 upregulation, in addition to being necessary for the nuclear translocation of Mdm2. Inhibition of either mTOR or Mdm2 is sufficient to block cell survival induced by Hgf-Met in vitro. Moreover, in vivo inhibition of mTOR downregulates Mdm2 protein levels and induces p53-dependent apoptosis. Our studies identify a novel mechanism for Met-triggered cell survival during embryogenesis, involving translational regulation of Mdm2 by mTOR. Moreover, they reinforce mTOR as a potential drug target in cancer.

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J. Neurochem. (2010) 114, 795–809. Abstract A dominant mutation in the gene coding for the vesicle‐associated membrane protein‐associated protein B (VAPB) was associated with amyotrophic lateral sclerosis, a fatal paralytic disorder characterized by the selective loss of motoneurons in the brain and spinal cord. Adeno‐associated viral vectors that we show to transduce up to 90% of motoneurons in vitro were used to model VAPB‐associated neurodegenerative process. We observed that Adeno‐associated viral‐mediated over‐expression of both wild‐type and mutated form of human VAPB selectively induces death of primary motoneurons, albeit with different kinetics. We provide evidence that ER stress and impaired homeostatic regulation of calcium (Ca2+) are implicated in the death process. Finally, we found that completion of the motoneuron death program triggered by the over‐expression of wild‐type and mutant VAPB implicates calpains, caspase 12 and 3. Our viral‐based in vitro model, which recapitulates the selective vulnerability of motoneurons to the presence of mutant VAPB and also to VAPB gene dosage effect, identifies aberrant Ca2+ signals and ER‐derived death pathways as important events in the motoneuron degenerative process.

Several neurological disorders manifest symptoms that result from the degeneration and death of specific neurons. p53 is an important modulator of cell death, and its inhibition could be a therapeutic approach to several neuropathologies. Here, we report the design, synthesis, and biological evaluation of novel p53 inhibitors based on the imino-tetrahydrobenzothiazole scaffold. By performing studies on their mechanism of action, we find that cyclic analogue 4b and its open precursor 2b are more potent than pifithrin-alpha (PFT-alpha), which is known to block p53 pro-apoptotic activity in vitro and in vivo without acting on other pro-apoptotic pathways. Using spectroscopic methods, we also demonstrate that open form 2b is more stable than 4b in biological media. Compound 2b is converted into its corresponding active cyclic form through an intramolecular dehydration process and was found two log values more active in vivo than PFT-alpha. Thus, 2b can be considered as a new prodrug prototype that prevents in vivo p53-triggered cell death in several neuropathologies and possibly reduces cancer therapy side effects.