Imatinib mesylate (IM), a potent inhibitor of the BCR/ABL tyrosine kinase, has become standard first-line therapy for patients with chronic myeloid leukemia (CML), but the frequency of resistance increases in advancing stages of disease. Elimination of BCR/ABL-dependent intracellular signals triggers apoptosis, but it is unclear whether this activates additional cell survival and/or death pathways. We have shown here that IM induces autophagy in CML blast crisis cell lines, CML primary cells, and p210 BCR/ABL -expressing myeloid precursor cells. IM-induced autophagy did not involve c-Abl or Bcl-2 activity but was associated with ER stress and was suppressed by depletion of intracellular Ca 2+ , suggesting it is mechanistically nonoverlapping with IM-induced apoptosis. We further demonstrated that suppression of autophagy using either pharmacological inhibitors or RNA interference of essential autophagy genes enhanced cell death induced by IM in cell lines and primary CML cells. Critically, the combination of a tyrosine kinase inhibitor (TKI), i.e., IM, nilotinib, or dasatinib, with inhibitors of autophagy resulted in near complete elimination of phenotypically and functionally defined CML stem cells. Together, these findings suggest that autophagy inhibitors may enhance the therapeutic effects of TKIs in the treatment of CML.

In brain ischemia, gating of postsynaptic glutamate receptors and other membrane channels triggers intracellular Ca2+ overload and cell death. In excitotoxic settings, the initial Ca2+ influx through glutamate receptors is followed by a second uncontrolled Ca2+ increase that leads to neuronal demise. Here we report that the major plasma membrane Ca2+ extruding system, the Na+/Ca2+ exchanger (NCX), is cleaved during brain ischemia and in neurons undergoing excitotoxicity. Inhibition of Ca2+-activated proteases (calpains) by overexpressing their endogenous inhibitor protein, calpastatin or the expression of an NCX isoform not cleaved by calpains, prevented Ca2+ overload and rescued neurons from excitotoxic death. Conversely, down-regulation of NCX by siRNA compromised neuronal Ca2+ handling, transforming the Ca2+ transient elicited by non-excitotoxic glutamate concentrations into a lethal Ca2+overload. Thus, proteolytic inactivation of NCX-driven neuronal Ca2+ extrusion is responsible for the delayed excitotoxic Ca2+ deregulation and neuronal death.

SummaryA minority of individuals experiencing traumatic events develop anxiety disorders. The reason for the lack of correspondence between the prevalence of exposure to psychological trauma and the development of anxiety is unknown. Extracellular proteolysis contributes to fear-associated responses by facilitating neuronal plasticity at the neuron-matrix interface1-4. Here we show that the serine protease neuropsin is critical for stress-related plasticity in the amygdala by regulating the dynamics of EphB2/NMDA receptor interaction, the expression of Fkbp5 and anxiety-like behaviour. Stress results in neuropsin-dependent cleavage of EphB2 in the amygdala causing dissociation of EphB2 from the NR1-subunit of NMDA receptor and promoting membrane turnover of EphB2 receptors. Dynamic EphB2/NR1 interaction enhances NMDA receptor current, induces the Fkbp5 gene expression and enhances behavioural signatures of anxiety. Upon stress, neuropsin-deficient mice do not show EphB2 cleavage and its dissociation from NR1 resulting in a static EphB2/NR1 interaction, attenuated induction of the Fkbp5 gene and low anxiety. The behavioural response to stress can be restored by intra-amygdala injection of neuropsin into neuropsin-deficient mice and disrupted by the injection of either anti-EphB2 antibodies or silencing the Fkbp5 gene in the amygdala of wild-type animals. Our findings establish a novel neuronal pathway linking stress-induced proteolysis of EphB2 in the amygdala to anxiety.

The p53 family member TAp73 is a transcription factor that plays a key role in many biological processes, including neuronal development. In particular, we have shown that p73 drives the expression of miR-34a, but not miR-34b and c, in mouse cortical neurons. miR-34a in turn modulates the expression of synaptic targets including synaptotagmin-1 and syntaxin-1A. Here we show that this axis is retained in mouse ES cells committed to differentiate toward a neurological phenotype. Moreover, overexpression of miR-34a alters hippocampal spinal morphology, and results in electrophysiological changes consistent with a reduction in spinal function. Therefore, the TAp73/miR-34a axis has functional relevance in primary neurons. These data reinforce a role for miR-34a in neuronal development.cell death | synaptogenesis | neuronal differentiation | hippocampus M icro-RNAs (miRs) are one family of a number of small noncoding regulatory RNAs (1). They are initially transcribed as pri-miRs, which are processed by a nuclear RNase III enzyme to form stem-loop structured premiRs. The premiRs are transported to the cytosol, where another RNase III cleaves off double-stranded portions of the hairpin to generate a short-lived dsRNA of approximately 20 to 25 nt. This duplex becomes unwound, and one strand (forming the mature miR) becomes incorporated into miR-protein complexes. The mature miR within the miR-protein complex recognizes complementary sites in the 3′ UTR of target genes, resulting in translational inhibition or destabilization of the target mRNAs and down-regulation of the encoded protein. During development, a number of miRs show distinct expression patterns during maturation of the CNS (2). For example, microarray miR profiling of embryonic, early postnatal, and adult brain revealed differential changes in nine miRNAs, including miR-9 and -124, and the levels of both these miRs increase markedly during the transition from neuronal precursors to mature neurons. miR-124 has also been implicated in the differentiation of neuroblastoma cells induced by retinoic acid (3).p73 is a member of the p53 family. Two distinct promoters transcribe different isoforms containing-TAp73-or lackingΔNp73-the aminoterminal transactivation domain (4); furthermore, extensive alternative 3′-splicing produces additional isoforms (5, 6). Trp73-KO mice have significant developmental abnormalities of the central nervous system, including congenital hydrocephalus, hippocampal dysgenesis, and defects of pheromone detection (7). Isoform-selective KOs have shown both a distinct neuronal phenotype and altered tumor susceptibility (8, 9). p53 can regulate several miRs (10). Indeed, the miR-34 family (miR-34a-c) is a p53 target (11-13), which can mimic several p53 effects in a cell type-specific manner. miR-34a is ubiquitous with the highest expression in mouse brain, and overexpression of miR-34a in neuroblastoma cell lines modulates neuronal-specific genes (14), whereas miR-34b and c are mainly expressed in the lung (15). Less information is available ...

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