Oxidative stress activates the c-Abl/p73 proapoptotic pathway in Niemann-Pick type C neurons.

Niemann-Pick type C (NPC) is a neurodegenerative disease characterized by the intralysosomal accumulation of cholesterol leading to neuronal apoptosis. We have previously reported the activation of the c-Abl/p73 proapoptotic pathway in the cerebellum of NPC mice; however, upstream signals underlying the engagement of this pathway remain unknown. Here, we investigate the possible role of oxidative stress in the activation of c-Abl/p73 using different in vitro and in vivo NPC models. Our results indicate a close temporal correlation between the appearance of nitrotyrosine (N-Tyr; a post-translational tyrosine modification caused by oxidative stress) and the activation of c-Abl/p73 in NPC models. To test the functional role of oxidative stress in NPC, we have treated NPC neurons with the antioxidant NAC and observed a dramatic decrease of c-Abl/p73 activation and a reduction in the levels of apoptosis in NPC models. In conclusion, our data suggest that oxidative stress is the main upstream stimulus activating the c-Abl/p73 pathway and neuronal apoptosis in NPC neurons.

c-Abl modulates AICD dependent cellular responses: transcriptional induction and apoptosis.

APP intracellular domain (AICD) has been proposed as a transcriptional inductor that moves to the nucleus with the adaptor protein Fe65 and regulates transcription. The two proteins, APP and Fe65, can be phosphorylated by c-Abl kinase. Neprilysin has been proposed as a target gene for AICD. We found that AICD expression is decreased by treatment with STI-571, a c-Abl inhibitor, suggesting a modulation of AICD transcription by c-Abl kinase. We observed interaction between c-Abl kinase, the AICD fragment and the Fe65 adaptor protein. In addition, STI-571 reduces apoptosis in APPSw, and the apoptotic response induced by Fe65 over-expression was inhibited by with the expression of a kinase dead (KD) c-Abl and enhanced by over-expression of WT-c-Abl. However, in the APPSw cells, the ability of the KD-c-Abl to protect against Fe65 was reduced. Finally, in APPSw clone, we detected higher trans-activation of the pro-apoptotic p73 isoform, TAp73 promoter. Our results show that c-Abl modulates AICD dependent cellular responses, transcriptional induction as well as the apoptotic response, which could participate in the onset and progression of the neurodegenerative pathology, observed in Alzheimer's disease (AD).

Imatinib therapy blocks cerebellar apoptosis and improves neurological symptoms in a mouse model of Niemann-Pick type C disease.

Niemann-Pick type C (NPC) disease is a fatal autosomal recessive disorder characterized by the accumulation of free cholesterol and glycosphingolipids in the endosomal-lysosomal system. Patients with NPC disease have markedly progressive neuronal loss, mainly of cerebellar Purkinje neurons. There is strong evidence indicating that cholesterol accumulation and trafficking defects activate apoptosis in NPC brains. The purpose of this study was to analyze the relevance of apoptosis and particularly the proapoptotic c-Abl/p73 system in cerebellar neuron degeneration in NPC disease. We used the NPC1 mouse model to evaluate c-Abl/p73 expression and activation in the cerebellum and the effect of therapy with the c-Abl-specific inhibitor imatinib. The proapoptotic c-Abl/p73 system and the p73 target genes are expressed in the cerebellums of NPC mice. Furthermore, inhibition of c-Abl with imatinib preserved Purkinje neurons and reduced general cell apoptosis in the cerebellum, improved neurological symptoms, and increased the survival of NPC mice. Moreover, this prosurvival effect correlated with reduced mRNA levels of p73 proapoptotic target genes. Our results suggest that the c-Abl/p73 pathway is involved in NPC neurodegeneration and show that treatment with c-Abl inhibitors is useful in delaying progressive neurodegeneration, supporting the use of imatinib for clinical treatment of patients with NPC disease.

c-Abl Deficiency Provides Synaptic Resiliency Against Aß-Oligomers.

Spine pathology has been implicated in the early onset of Alzheimer's disease (AD), where Aß-Oligomers (AßOs) cause synaptic dysfunction and loss. Previously, we described that pharmacological inhibition of c-Abl prevents AßOs-induced synaptic alterations. Hence, this kinase seems to be a key element in AD progression. Here, we studied the role of c-Abl on dendritic spine morphological changes induced by AßOs using c-Abl null neurons (c-Abl-KO). First, we characterized the effect of c-Abl deficiency on dendritic spine density and found that its absence increases dendritic spine density. While AßOs-treatment reduces the spine number in both wild-type (WT) and c-Abl-KO neurons, AßOs-driven spine density loss was not affected by c-Abl. We then characterized AßOs-induced morphological changes in dendritic spines of c-Abl-KO neurons. AßOs induced a decrease in the number of mushroom spines in c-Abl-KO neurons while preserving the populations of immature stubby, thin, and filopodia spines. Furthermore, synaptic contacts evaluated by PSD95/Piccolo clustering and cell viability were preserved in AßOs-exposed c-Abl-KO neurons. In conclusion, our results indicate that in the presence of AßOs c-Abl participates in synaptic contact removal, increasing susceptibility to AßOs damage. Its deficiency increases the immature spine population reducing AßOs-induced synapse elimination. Therefore, c-Abl signaling could be a relevant actor in the early stages of AD.

Physiological Control of Nitric Oxide in Neuronal BACE1 Translation by Heme-Regulated eIF2α Kinase HRI Induces Synaptogenesis.

AIMS: Hippocampus is the brain center for memory formation, a process that requires synaptogenesis. However, hippocampus is dramatically compromised in Alzheimer's disease due to the accumulation of amyloid ß-peptide, whose production is initiated by ß-site APP Cleaving Enzyme 1 (BACE1). It is known that pathological stressors activate BACE1 translation through the phosphorylation of the eukaryotic initiation factor-2α (eIF2α) by GCN2, PERK, or PKR kinases, leading to amyloidogenesis. However, BACE1 physiological regulation is still unclear. Since nitric oxide (NO) participates directly in hippocampal glutamatergic signaling, we investigated the neuronal role of the heme-regulated eukaryotic initiation factor eIF2α kinase (HRI), which can bind NO by a heme group, in BACE1 translation and its physiological consequences. RESULTS: We found that BACE1 is expressed on glutamate activation with NO being the downstream effector by triggering eIF2α phosphorylation, as it was obtained by Western blot and luciferase assay. It is due to the activation of HRI by NO as assayed by Western blot and immunofluorescence with an HRI inhibitor and HRI siRNA. BACE1 expression was early detected at synaptic spines, contributing to spine growth and consolidating the hippocampal memory as assayed with mice treated with HRI or neuronal NO synthase inhibitors. INNOVATION: We provide the first description that HRI and eIF2α are working in physiological conditions in the brain under the control of nitric oxide and glutamate signaling, and also that BACE1 has a physiological role in hippocampal function. CONCLUSION: We conclude that BACE1 translation is controlled by NO through HRI in glutamatergic hippocampal synapses, where it plays physiological functions, allowing the spine growth and memory consolidation.

EphA4 activation of c-Abl mediates synaptic loss and LTP blockade caused by amyloid-ß oligomers.

The early stages of Alzheimer's disease are characterised by impaired synaptic plasticity and synapse loss. Here, we show that amyloid-ß oligomers (AßOs) activate the c-Abl kinase in dendritic spines of cultured hippocampal neurons and that c-Abl kinase activity is required for AßOs-induced synaptic loss. We also show that the EphA4 receptor tyrosine kinase is upstream of c-Abl activation by AßOs. EphA4 tyrosine phosphorylation (activation) is increased in cultured neurons and synaptoneurosomes exposed to AßOs, and in Alzheimer-transgenic mice brain. We do not detect c-Abl activation in EphA4-knockout neurons exposed to AßOs. More interestingly, we demonstrate EphA4/c-Abl activation is a key-signalling event that mediates the synaptic damage induced by AßOs. According to this results, the EphA4 antagonistic peptide KYL and c-Abl inhibitor STI prevented i) dendritic spine reduction, ii) the blocking of LTP induction and iii) neuronal apoptosis caused by AßOs. Moreover, EphA4-/- neurons or sh-EphA4-transfected neurons showed reduced synaptotoxicity by AßOs. Our results are consistent with EphA4 being a novel receptor that mediates synaptic damage induced by AßOs. EphA4/c-Abl signalling could be a relevant pathway involved in the early cognitive decline observed in Alzheimer's disease patients.