p53 upregulated mediator of apoptosis (Puma) deficiency increases survival of adult neural stem cells generated physiologically in the hippocampus, but does not protect stem cells generated in surplus after an excitotoxic lesion

Objectives: Neurogenesis occurs in the mammalian brain throughout adulthood and increases in response to metabolic, toxic or traumatic insults. To remove potentially superfluous or unwanted neural stem cells/neuronal progenitors, their rate of proliferation and differentiation is fine-tuned against their rate of apoptosis. Apoptosis requires the transcriptional and posttranslational activation of Bcl-2-homolgy domain 3 (BH3)-only proteins. Previously, we demonstrated that the BH3-only protein p53-upregulated mediator of apoptosis (Puma) controls the physiological rate of apoptosis of neural precursor cells in the adult mouse hippocampus. Puma's role in controlling a lesion-induced increase in neural stem cells is currently not known.Methods: We employed a model of local, N-methyl-D-asparte (NMDA)-induced excitotoxic injury to the CA1 hippocampal subfield and immunofluorescence labelling to produce increased neural stem cell proliferation/ neurogenesis in the dentate gyrus at two survival times following the excitotoxic lesion.Results: Deletion of puma failed to rescue any NMDA-induced increase in adult born cells as assessed by BrdU or Doublecortin labelling in the long-term. No difference in the proportion of BrdU/NeuN-positive cells comparing the different genotypes and treatments suggested that the phenotypic fate of the cells was preserved regardless of the genotype and the treatment.Conclusions: While neurogenesis is up-regulated in puma-deficient animals following NMDA-induced excitotoxicity to the hippocampal CA1 subfield, puma deficiency could not protect this surplus of newly generated cells from apoptotic cell death.</p

Additional file 1: of Increased A20-E3 ubiquitin ligase interactions in bid-deficient glia attenuate TLR3- and TLR4-induced inflammation

Figure S1. Increased A20-TRAF3, A20-Peli1, and TRAF6-Peli1 interactions in TLR3- and TLR4-stimulated bid-deficient glia and macrophages, compared with wt. (A) wt and bid −/− mixed glia were transfected with Ubiquitin-HA and stimulated for 1 h with LPS (100 ng/ml) 20 h following transfection. Co-immunoprecipitation of anti-TRAF6 was carried out for each sample, and TRAF6-A20 interactions were determined by Western blot using an anti-A20 antibody. TRAF6-A20 interactions were quantified using optical density (n = 1 experiment). (B) wt and bid-deficient mixed glia were transfected with TRAF6-FLAG and stimulated for 1 h with LPS (100 ng/ml) 20 h post transfection. The cells were lysed in RIPA buffer, and anti-FLAG was immunoprecipitated from each of the samples. Peli1 was detected by Western blot, indicating the interaction between TRAF6-FLAG and Peli1 (n = 1 experiment). (C) wt and bid-deficient macrophages were stimulated with PolyI:C (100 ng/ml) or LPS (100 ng/ml) for 1 h and lysed in RIPA buffer. Anti-Peli1 was co-immunoprecipitated from each sample, and Peli1-A20 interactions were determined by Western blot using an anti-A20 antibody (n = 1 experiment). (TIFF 1770 kb

AMPK preferentially depresses retrograde transport of axonal mitochondria during localised nutrient deprivation

Mitochondrial clusters are found at regions of high energy demand, allowing cells to meet local metabolic requirements while maintaining neuronal homeostasis. AMP-activated protein kinase (AMPK), a key energy stress sensor, responds to increases in AMP/ATP ratio by activating multiple signalling cascades to overcome the energetic deficiency. In many neurological conditions, the distal axon experiences energetic stress independent of the soma. Here, we used microfluidic devices to physically isolate these two neuronal structures and to investigate whether localised AMPK signalling influenced axonal mitochondrial transport. Nucleofection of primary cortical neurons, derived from E16 mouse embryos (both sexes), with mito-GFP allowed monitoring of the transport dynamics of mitochondria within the axon, by confocal microscopy.Pharmacological activation of AMPK at the distal axon (0.1 mM AICAR) induced a depression of the mean frequency, velocity and distance of retrograde mitochondrial transport in the adjacent axon. Anterograde mitochondrial transport was less sensitive to local AMPK stimulus, with the imbalance of bi-directional mitochondrial transport resulting in accumulation of mitochondria at the region of energetic stress signal. Mitochondria in the axon-rich white matter of the brain rely heavily on lactate as a substrate for ATP synthesis. Interestingly, localised inhibition of lactate uptake (10 nM AR-C155858) reduced mitochondrial transport in the adjacent axon in all parameters measured, similar to that observed by AICAR treatment. Co-addition of compound C restored all parameters measured to baseline levels, confirming the involvement of AMPK. This study highlights a role of AMPK signalling in the depression of axonal mitochondrial mobility during localised energetic stress.</p

AMP-activated protein kinase (AMPK)-induced preconditioning in primary cortical neurons involves activation of MCL-1.

Neuronal preconditioning is a phenomenon where a previous exposure to a sub-lethal stress stimulus increases the resistance of neurons towards a second, normally lethal stress stimulus. Activation of the energy stress sensor, AMP-activated protein kinase (AMPK) has been shown to contribute to the protective effects of ischaemic and mitochondrial uncoupling-induced preconditioning in neurons, however, the molecular basis of AMPK-mediated preconditioning has been less well characterized. We investigated the effect of AMPK preconditioning using 5-aminoimidazole-4-carboxamide riboside (AICAR) in a model of NMDA-mediated excitotoxic injury in primary mouse cortical neurons. Activation of AMPK with low concentrations of AICAR (0.1 mM for 2 h) induced a transient increase in AMPK phosphorylation, protecting neurons against NMDA-induced excitotoxicity. Analysing potential targets of AMPK activation, demonstrated a marked increase in mRNA expression and protein levels of the anti-apoptotic BCL-2 family protein myeloid cell leukaemia sequence 1 (MCL-1) in AICAR-preconditioned neurons. Interestingly, over-expression of MCL-1 protected neurons against NMDA-induced excitotoxicity while MCL-1 gene silencing abolished the effect of AICAR preconditioning. Monitored intracellular Ca²⁺ levels during NMDA excitation revealed that MCL-1 over-expressing neurons exhibited improved bioenergetics and markedly reduced Ca²⁺ elevations, suggesting a potential mechanism through which MCL-1 confers neuroprotection. This study identifies MCL-1 as a key effector of AMPK-induced preconditioning in neurons.</p

A constitutively-active IKK-complex at the axon initial segment.

BACKGROUND: Previous studies provided evidence for an accumulation of IκB-kinase (IKK) α/β at the axon initial segment (AIS), a neuronal compartment defined by ankyrin-G expression. Here we explored whether the presence of the IKK-complex at the AIS was associated with the activation of IKK signaling at this site. METHODS AND RESULTS: Proximity-ligation assays (PLAs) using pan-IKKα/β, phospho-IKKα/β-specific as well as ankyrin-G specific antibodies validated their binding to proximal epitopes in the AIS, while antibodies to other phosphorylated signaling proteins showed no preference for the AIS. Small-hairpin mediated silencing of IKKβ significantly reduced anti-phospho-IKKα/β-immunoreactivities in the AIS. ank3 gene-deficient cerebellar Purkinje cells also exhibited no phosphorylated IKKα/β at the proximal region of their axons. Transient ankyrin-G overexpression in PC12 cells augmented NF-κB transactivation in an ankyrin-G death-domain dependent manner. Finally, small molecule inhibitors of IKK-activity, including Aspirin, inhibited the accumulation of activated IKK proteins in the AIS. CONCLUSION: Our data suggest the existence of a constitutively-active IKK signaling complex in the AIS.</p