TFG binds LC3C to regulate ULK1 localization and autophagosome formation.

The early secretory pathway and autophagy are two essential and evolutionarily conserved endomembrane processes that are finely interlinked. Although growing evidence suggests that intracellular trafficking is important for autophagosome biogenesis, the molecular regulatory network involved is still not fully defined. In this study, we demonstrate a crucial effect of the COPII vesicle-related protein TFG (Trk-fused gene) on ULK1 puncta number and localization during autophagy induction. This, in turn, affects formation of the isolation membrane, as well as the correct dynamics of association between LC3B and early ATG proteins, leading to the proper formation of both omegasomes and autophagosomes. Consistently, fibroblasts derived from a hereditary spastic paraparesis (HSP) patient carrying mutated TFG (R106C) show defects in both autophagy and ULK1 puncta accumulation. In addition, we demonstrate that TFG activity in autophagy depends on its interaction with the ATG8 protein LC3C through a canonical LIR motif, thereby favouring LC3C-ULK1 binding. Altogether, our results uncover a link between TFG and autophagy and identify TFG as a molecular scaffold linking the early secretion pathway to autophagy.

AMBRA1 phosphorylation by CDK1 and PLK1 regulates mitotic spindle orientation.

AMBRA1 is a crucial factor for nervous system development, and its function has been mainly associated with autophagy. It has been also linked to cell proliferation control, through its ability to regulate c-Myc and D-type cyclins protein levels, thus regulating G1-S transition. However, it remains still unknown whether AMBRA1 is differentially regulated during the cell cycle, and if this pro-autophagy protein exerts a direct role in controlling mitosis too. Here we show that AMBRA1 is phosphorylated during mitosis on multiple sites by CDK1 and PLK1, two mitotic kinases. Moreover, we demonstrate that AMBRA1 phosphorylation at mitosis is required for a proper spindle function and orientation, driven by NUMA1 protein. Indeed, we show that the localization and/or dynamics of NUMA1 are strictly dependent on AMBRA1 presence, phosphorylation and binding ability. Since spindle orientation is critical for tissue morphogenesis and differentiation, our findings could account for an additional role of AMBRA1 in development and cancer ontogenesis.

Targeting cancer stem cells in medulloblastoma by inhibiting AMBRA1 dual function in autophagy and STAT3 signalling.

Medulloblastoma (MB) is a childhood malignant brain tumour comprising four main subgroups characterized by different genetic alterations and rate of mortality. Among MB subgroups, patients with enhanced levels of the c-MYC oncogene (MBGroup3) have the poorest prognosis. Here we identify a previously unrecognized role of the pro-autophagy factor AMBRA1 in regulating MB. We demonstrate that AMBRA1 expression depends on c-MYC levels and correlates with Group 3 patient poor prognosis; also, knockdown of AMBRA1 reduces MB stem potential, growth and migration of MBGroup3 stem cells. At a molecular level, AMBRA1 mediates these effects by suppressing SOCS3, an inhibitor of STAT3 activation. Importantly, pharmacological inhibition of autophagy profoundly affects both stem and invasion potential of MBGroup3 stem cells, and a combined anti-autophagy and anti-STAT3 approach impacts the MBGroup3 outcome. Taken together, our data support the c-MYC/AMBRA1/STAT3 axis as a strong oncogenic signalling pathway with significance for both patient stratification strategies and targeted treatments of MBGroup3.

Neuroprotective coordination of cell mitophagy by the ATPase Inhibitory Factor 1.

The mitochondrial ATPase Inhibitory Factor 1 (hereafter referred to as IF1) blocks the reversal of the F1Fo-ATPsynthase to prevent detrimental consumption of cellular ATP and associated demise. Herein, we infer further its molecular physiology by assessing its protective function in neurons during conditions of challenged homeostatic respiration. By adopting in vitro and in vivo protocols of hypoxia/ischemia and re-oxygenation, we show that a shift in the IF1:F1Fo-ATPsynthase expression ratio occurs in neurons. This increased IF1 level is essential to induce accumulation of the PTEN-induced putative kinase 1 (PINK-1) and recruitment of the mitophagic ubiquitin ligase PARK-2 to promote autophagic "control" of the mitochondrial population. In IF1 overexpressing neurons ATP depletion is reduced during hypoxia/ischemia and the mitochondrial membrane potential (ΔYm) resilient to re-oxygenation as well as resistant to electrogenic, Ca(2+) dependent depolarization. These data suggest that in mammalian neurons mitochondria adapt to respiratory stress by upregulating IF1, which exerts a protective role by coordinating pro-survival cell mitophagy and bioenergetics resilience.

Reduction of the ATPase inhibitory factor 1 (IF1) leads to visual impairment in vertebrates.

In vertebrates, mitochondria are tightly preserved energy producing organelles, which sustain nervous system development and function. The understanding of proteins that regulate their homoeostasis in complex animals is therefore critical and doing so via means of systemic analysis pivotal to inform pathophysiological conditions associated with mitochondrial deficiency. With the goal to decipher the role of the ATPase inhibitory factor 1 (IF1) in brain development, we employed the zebrafish as elected model reporting that the Atpif1a-/- zebrafish mutant, pinotage (pnt tq209 ), which lacks one of the two IF1 paralogous, exhibits visual impairment alongside increased apoptotic bodies and neuroinflammation in both brain and retina. This associates with increased processing of the dynamin-like GTPase optic atrophy 1 (OPA1), whose ablation is a direct cause of inherited optic atrophy. Defects in vision associated with the processing of OPA1 are specular in Atpif1-/- mice thus confirming a regulatory axis, which interlinks IF1 and OPA1 in the definition of mitochondrial fitness and specialised brain functions. This study unveils a functional relay between IF1 and OPA1 in central nervous system besides representing an example of how the zebrafish model could be harnessed to infer the activity of mitochondrial proteins during development.

Distinct Mechanisms of Pathogenic DJ-1 Mutations in Mitochondrial Quality Control.

The deglycase and chaperone protein DJ-1 is pivotal for cellular oxidative stress responses and mitochondrial quality control. Mutations in PARK7, encoding DJ-1, are associated with early-onset familial Parkinson's disease and lead to pathological oxidative stress and/or disrupted protein degradation by the proteasome. The aim of this study was to gain insights into the pathogenic mechanisms of selected DJ-1 missense mutations, by characterizing protein-protein interactions, core parameters of mitochondrial function, quality control regulation via autophagy, and cellular death following dopamine accumulation. We report that the DJ-1M26I mutant influences DJ-1 interactions with SUMO-1, in turn enhancing removal of mitochondria and conferring increased cellular susceptibility to dopamine toxicity. By contrast, the DJ-1D149A mutant does not influence mitophagy, but instead impairs Ca2+ dynamics and free radical homeostasis by disrupting DJ-1 interactions with a mitochondrial accessory protein known as DJ-1-binding protein (DJBP/EFCAB6). Thus, individual DJ-1 mutations have different effects on mitochondrial function and quality control, implying mutation-specific pathomechanisms converging on impaired mitochondrial homeostasis.

A role for TSPO in mitochondrial Ca2+ homeostasis and redox stress signaling.

The 18 kDa translocator protein TSPO localizes on the outer mitochondrial membrane (OMM). Systematically overexpressed at sites of neuroinflammation it is adopted as a biomarker of brain conditions. TSPO inhibits the autophagic removal of mitochondria by limiting PARK2-mediated mitochondrial ubiquitination via a peri-organelle accumulation of reactive oxygen species (ROS). Here we describe that TSPO deregulates mitochondrial Ca2+ signaling leading to a parallel increase in the cytosolic Ca2+ pools that activate the Ca2+-dependent NADPH oxidase (NOX) thereby increasing ROS. The inhibition of mitochondrial Ca2+ uptake by TSPO is a consequence of the phosphorylation of the voltage-dependent anion channel (VDAC1) by the protein kinase A (PKA), which is recruited to the mitochondria, in complex with the Acyl-CoA binding domain containing 3 (ACBD3). Notably, the neurotransmitter glutamate, which contributes neuronal toxicity in age-dependent conditions, triggers this TSPO-dependent mechanism of cell signaling leading to cellular demise. TSPO is therefore proposed as a novel OMM-based pathway to control intracellular Ca2+ dynamics and redox transients in neuronal cytotoxicity.

Inhibition of hippocampal plasticity in rats performing contrafreeloading for water under repeated administrations of pramipexole.

RATIONALE: Compulsive symptoms develop in patients exposed to pramipexole (PPX), a dopaminergic agonist with high selectivity for the D3 receptor. Consistently, we demonstrated that PPX produces an exaggerated increase in contrafreeloading (CFL) for water, a repetitive and highly inflexible behavior that models core aspects of compulsive disorders. OBJECTIVES: Given the role of the hippocampus in behavioral flexibility, motivational control, and visuospatial working memory, we investigated the role of hippocampus in the expression of PPX-induced CFL. To this aim, rats were subjected to CFL under chronic PPX, and then examined for the electrophysiological, structural, and molecular properties of their hippocampus. METHODS: We measured long-term potentiation (LTP) at CA1 Schaffer collaterals, dendritic spine density in CA1 pyramidal neurons, and then glutamate release and expression of pre and postsynaptic proteins in hippocampal synaptosomes. The effects of PPX on hippocampal-dependent working memory were assessed through the novel object recognition (NOR) test. RESULTS: We found that PPX-treated rats showing CFL exhibited a significant decrease in hippocampal LTP and failed to exhibit the expected increase in hippocampal spine density. Glutamate release and PSD-95 expression were decreased, while pSYN expression was increased in hippocampal synaptosomes of PPX-treated rats showing CFL. Despite a general impairment of hippocampal synaptic function, working memory was unaffected by PPX treatment. CONCLUSIONS: Our findings demonstrate that chronic PPX affects synaptic function in the hippocampus, an area that is critically involved in the expression of flexible, goal-centered behaviors. We suggest that the hippocampus is a promising target in the pharmacotherapy of compulsive disorders.

Presynaptic c-Jun N-terminal Kinase 2 regulates NMDA receptor-dependent glutamate release.

Activation of c-Jun N-terminal kinase (JNK) signaling pathway is a critical step for neuronal death occurring in several neurological conditions. JNKs can be activated via receptor tyrosine kinases, cytokine receptors, G-protein coupled receptors and ligand-gated ion channels, including the NMDA glutamate receptors. While JNK has been generally associated with postsynaptic NMDA receptors, its presynaptic role remains largely unexplored. Here, by means of biochemical, morphological and functional approaches, we demonstrate that JNK and its scaffold protein JIP1 are also expressed at the presynaptic level and that the NMDA-evoked glutamate release is controlled by presynaptic JNK-JIP1 interaction. Moreover, using knockout mice for single JNK isoforms, we proved that JNK2 is the essential isoform in mediating this presynaptic event. Overall the present findings unveil a novel JNK2 localization and function, which is likely to play a role in different physiological and pathological conditions.

Age-related changes of protein SUMOylation balance in the AßPP Tg2576 mouse model of Alzheimer's disease.

Alzheimer's disease (AD) is a complex disorder that affects the central nervous system causing a severe neurodegeneration. This pathology affects an increasing number of people worldwide due to the overall aging of the human population. In recent years SUMO protein modification has emerged as a possible cellular mechanism involved in AD. Some of the proteins engaged in the physiopathological process of AD, like BACE1, GSK3-ß tau, AßPP, and JNK, are in fact subject to protein SUMO modifications or interactions. Here, we have investigated the SUMO/deSUMOylation balance and SUMO-related proteins during the onset and progression of the pathology in the Tg2576 mouse model of AD. We examined four age-stages (1.5, 3, 6, 17 months old) and observed shows an increase in SUMO-1 protein conjugation at 3 and 6 months in transgenic mice with respect to WT in both cortex and hippocampus. Interestingly this is paralleled by increased expression levels of Ubc9 and SENP1 in both brain regions. At 6 months of age also the SUMO-1 mRNA resulted augmented. SUMO-2-ylation was surprisingly decreased in old transgenic mice and was unaltered in the other time windows. The fact that alterations in SUMO/deSUMOylation equilibrium occur from the early phases of AD suggests that global posttranslational modifications may play an important role in the mechanisms underlying disease pathogenesis, thus providing potential targets for pharmacological interventions.

Key role of mitochondria in Alzheimer's disease synaptic dysfunction.

Neuronal transmission and functional synapses require mitochondria, which are mainly involved in the generation of energy (ATP and NAD(+)), regulation of cell signaling and calcium homeostasis. Particularly intriguing is emerging data suggesting the relationship between mitochondria and neurotrophic factors that can act at the synaptic level promoting neuronal transmission and plasticity. On the other hand, disturbances in mitochondrial functions might contribute to impaired synaptic transmission and neuronal degeneration in Alzheimer's Disease and other chronic and acute neurodegenerative disorders. Here, we review the molecular mediators controling mitochondrial function and their impact on synaptic dysfunction associated with the pathogenesis of Alzheimer's Disease.

Age-related changes of hippocampal synaptic plasticity in AßPP-null mice are restored by NGF through p75NTR.

Amyloid-ß protein precursor (AßPP) is a ubiquitous protein found in all cell types, suggesting basic and yet important roles, which still remain to be fully elucidated. Loss of function of AßPP has been linked to abnormal neuronal morphology and synaptic function within the hippocampus and alterations in spatial learning, suggesting a neurotrophic role for this protein. Besides AßPP, nerve growth factor (NGF) and other neurotrophins have also been shown to finely modulate neuronal excitability, synaptic plasticity, and cognitive functions. In addition, recent data support the hypothesis of a functional interconnection between AßPP and NGF pathway. Here, we demonstrated that loss of AßPP function, leading to progressive decrease of choline acetyltransferase expression in the septum, correlates with age-related impairment of long-term potentiation (LTP) in the dentate gyrus. We next addressed whether impaired hippocampal plasticity in AßPP-null mice can be restored upon NGF treatment. Notably, NGF, as well as Pro-NGF, can fully revert LTP deficits in AßPP-null mice through p75NTR and JNK pathway activation. Overall the present study may unveil a new mechanism by which, in the absence of AßPP, NGF treatment may preferentially direct p75-neurotrophin-dependent JNK activation toward regeneration and plasticity in functionally relevant brain circuits.