Neuronal autosis is Na+/K+-ATPase alpha 3-dependent and involved in hypoxic-ischemic neuronal death.

Macroautophagy (hereafter called autophagy) is an essential physiological process of degradation of organelles and long-lived proteins. The discovery of autosis, a Na+/K+-ATPase (ATP1)-dependent type of autophagic cell death with specific morphological and biochemical features, has strongly contributed to the acceptance of a pro-death role of autophagy. However, the occurrence and relevance of autosis in neurons has never been clearly investigated, whereas we previously provided evidence that autophagy mechanisms could be involved in neuronal death in different in vitro and in vivo rodent models of hypoxia-ischemia (HI) and that morphological features of autosis were observed in dying neurons following rat perinatal cerebral HI. In the present study, we demonstrated that neuronal autosis could occur in primary cortical neurons using two different stimulations enhancing autophagy flux and neuronal death: a neurotoxic concentration of Tat-BECN1 (an autophagy-inducing peptide) and a hypoxic/excitotoxic stimulus (mimicking neuronal death induced by cerebral HI). Both stimulations induce autophagic neuronal death (dependent on canonical autophagic genes and independent on apoptotic, necroptotic or ferroptotic pathways) with all morphological and biochemical (ATP1a-dependent) features of autosis. However, we demonstrated that autosis is not dependent on the ubiquitous subunit ATP1a1 in neurons, as in dividing cell types, but on the neuronal specific ATP1a3 subunit. We also provided evidence that, in different in vitro and in vivo models where autosis is induced, ATP1a3-BECN1 interaction is increased and prevented by cardiac glycosides treatment. Interestingly, an increase in ATP1a3-BECN1 interaction is also detected in dying neurons in the autoptic brains of human newborns with severe hypoxic-ischemic encephalopathy (HIE). Altogether, these results suggest that ATP1a3-BECN1-dependent autosis could play an important role in neuronal death in HI conditions, paving the way for the development of new neuroprotective strategies in hypoxic-ischemic conditions including in severe case of human HIE.

Overexpression of UCP4 in astrocytic mitochondria prevents multilevel dysfunctions in a mouse model of Alzheimer's disease.

Alzheimer's disease (AD) is becoming increasingly prevalent worldwide. It represents one of the greatest medical challenges as no pharmacologic treatments are available to prevent disease progression. Astrocytes play crucial functions within neuronal circuits by providing metabolic and functional support, regulating interstitial solute composition, and modulating synaptic transmission. In addition to these physiological functions, growing evidence points to an essential role of astrocytes in neurodegenerative diseases like AD. Early-stage AD is associated with hypometabolism and oxidative stress. Contrary to neurons that are vulnerable to oxidative stress, astrocytes are particularly resistant to mitochondrial dysfunction and are therefore more resilient cells. In our study, we leveraged astrocytic mitochondrial uncoupling and examined neuronal function in the 3xTg AD mouse model. We overexpressed the mitochondrial uncoupling protein 4 (UCP4), which has been shown to improve neuronal survival in vitro. We found that this treatment efficiently prevented alterations of hippocampal metabolite levels observed in AD mice, along with hippocampal atrophy and reduction of basal dendrite arborization of subicular neurons. This approach also averted aberrant neuronal excitability observed in AD subicular neurons and preserved episodic-like memory in AD mice assessed in a spatial recognition task. These findings show that targeting astrocytes and their mitochondria is an effective strategy to prevent the decline of neurons facing AD-related stress at the early stages of the disease.

Activation of lactate receptor HCAR1 down-modulates neuronal activity in rodent and human brain tissue.

Lactate can be used by neurons as an energy substrate to support their activity. Evidence suggests that lactate also acts on a metabotropic receptor called HCAR1, first described in the adipose tissue. Whether HCAR1 also modulates neuronal circuits remains unclear. In this study, using qRT-PCR, we show that HCAR1 is present in the human brain of epileptic patients who underwent resective surgery. In brain slices from these patients, pharmacological HCAR1 activation using a non-metabolized agonist decreased the frequency of both spontaneous neuronal Ca2+ spiking and excitatory post-synaptic currents (sEPSCs). In mouse brains, we found HCAR1 expression in different regions using a fluorescent reporter mouse line and in situ hybridization. In the dentate gyrus, HCAR1 is mainly present in mossy cells, key players in the hippocampal excitatory circuitry and known to be involved in temporal lobe epilepsy. By using whole-cell patch clamp recordings in mouse and rat slices, we found that HCAR1 activation causes a decrease in excitability, sEPSCs, and miniature EPSCs frequency of granule cells, the main output of mossy cells. Overall, we propose that lactate can be considered a neuromodulator decreasing synaptic activity in human and rodent brains, which makes HCAR1 an attractive target for the treatment of epilepsy.

Thrombolysis by PLAT/tPA increases serum free IGF1 leading to a decrease of deleterious autophagy following brain ischemia.

Cerebral ischemia is a pathology involving a cascade of cellular mechanisms, leading to the deregulation of proteostasis, including macroautophagy/autophagy, and finally to neuronal death. If it is now accepted that cerebral ischemia induces autophagy, the effect of thrombolysis/energy recovery on proteostasis remains unknown. Here, we investigated the effect of thrombolysis by PLAT/tPA (plasminogen activator, tissue) on autophagy and neuronal death. In two in vitro models of hypoxia reperfusion and an in vivo model of thromboembolic stroke with thrombolysis by PLAT/tPA, we found that ischemia enhances neuronal deleterious autophagy. Interestingly, PLAT/tPA decreases autophagy to mediate neuroprotection by modulating the PI3K-AKT-MTOR pathways both in vitro and in vivo. We identified IGF1R (insulin-like growth factor I receptor; a tyrosine kinase receptor) as the effective receptor and showed in vitro, in vivo and in human stroke patients and that PLAT/tPA is able to degrade IGFBP3 (insulin-like growth factor binding protein 3) to increase IGF1 (insulin-like growth factor 1) bioavailability and thus IGF1R activation.Abbreviations: AKT/protein kinase B: thymoma viral proto-oncogene 1; EGFR: epidermal growth factor receptor; Hx: hypoxia; IGF1: insulin-like growth factor 1; IGF1R: insulin-like growth factor I receptor; IGFBP3: insulin-like growth factor binding protein 3; Ka: Kainate; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MAPK/ERK: mitogen-activated protein kinase; MTOR: mechanistic target of rapamycin kinase; MTORC1: MTOR complex 1; OGD: oxygen and glucose deprivation; OGDreox: oxygen and glucose deprivation + reoxygentation; PepA: pepstatin A1; PI3K: phosphoinositide 3-kinase; PLAT/tPA: plasminogen activator, tissue; PPP: picropodophyllin; SCH77: SCH772984; ULK1: unc-51 like kinase 1; Wort: wortmannin.

Genetic, cellular, and structural characterization of the membrane potential-dependent cell-penetrating peptide translocation pore.

Cell-penetrating peptides (CPPs) allow intracellular delivery of bioactive cargo molecules. The mechanisms allowing CPPs to enter cells are ill-defined. Using a CRISPR/Cas9-based screening, we discovered that KCNQ5, KCNN4, and KCNK5 potassium channels positively modulate cationic CPP direct translocation into cells by decreasing the transmembrane potential (Vm). These findings provide the first unbiased genetic validation of the role of Vm in CPP translocation in cells. In silico modeling and live cell experiments indicate that CPPs, by bringing positive charges on the outer surface of the plasma membrane, decrease the Vm to very low values (-150 mV or less), a situation we have coined megapolarization that then triggers formation of water pores used by CPPs to enter cells. Megapolarization lowers the free energy barrier associated with CPP membrane translocation. Using dyes of varying dimensions in CPP co-entry experiments, the diameter of the water pores in living cells was estimated to be 2 (-5) nm, in accordance with the structural characteristics of the pores predicted by in silico modeling. Pharmacological manipulation to lower transmembrane potential boosted CPP cellular internalization in zebrafish and mouse models. Besides identifying the first proteins that regulate CPP translocation, this work characterized key mechanistic steps used by CPPs to cross cellular membranes. This opens the ground for strategies aimed at improving the ability of cells to capture CPP-linked cargos in vitro and in vivo.

Before a drug can have its desired effect, it must reach its target tissue or organ, and enter its cells. This is not easy because cells are surrounded by the plasma membrane, a fat-based barrier that separates the cell from its external environment. The plasma membrane contains proteins that act as channels, shuttling specific molecules in and out of the cell, and it also holds charge, with its inside surface being more negatively charged than its outside surface. Cell-penetrating peptides are short sequences of amino acids (the building blocks that form proteins) that carry positive charges. These positive charges allow them to cross the membrane easily, but it is not well understood how. To find out how cell-penetrating peptides cross the membrane, Trofimenko et al. attached them to dyes of different sizes. This revealed that the cell-penetrating peptides enter the cell through temporary holes called water pores, which measure about two nanometres across. The water pores form when the membrane becomes 'megapolarized', this is, when the difference in charge between the inside and the outside of the membrane becomes greater than normal. This can happen when the negative charge on the inside surface or the positive charge on the outer surface of the membrane increase. Megapolarization depends on potassium channels, which transport positive potassium ions outside the cell, making the outside of the membrane positive. When cell-penetrating peptides arrive at the outer surface of the cell near potassium channels, they make it even more positive. This increases the charge difference between the inside and the outside of the cell, allowing water pores to form. Once the peptides pass through the pores, the charge difference between the inside and the outside of the cell membrane dissipates, and the pores collapse. Drug developers are experimenting with attaching cell-penetrating peptides to drugs to help them get inside their target cells. Currently there are several experimental medications of this kind in clinical trials. Understanding how these peptides gain entry, and what size of molecule they could carry with them, provides solid ground for further drug development.

Current Evidence on Cell Death in Preterm Brain Injury in Human and Preclinical Models.

Despite tremendous advances in neonatal intensive care over the past 20 years, prematurity carries a high burden of neurological morbidity lasting lifelong. The term encephalopathy of prematurity (EoP) coined by Volpe in 2009 encompasses all aspects of the now known effects of prematurity on the immature brain, including altered and disturbed development as well as specific lesional hallmarks. Understanding the way cells are damaged is crucial to design brain protective strategies, and in this purpose, preclinical models largely contribute to improve the comprehension of the cell death mechanisms. While neuronal cell death has been deeply investigated and characterized in (hypoxic-ischemic) encephalopathy of the newborn at term, little is known about the types of cell death occurring in preterm brain injury. Three main different morphological cell death types are observed in the immature brain, specifically in models of hypoxic-ischemic encephalopathy, namely, necrotic, apoptotic, and autophagic cell death. Features of all three types may be present in the same dying neuron. In preterm brain injury, description of cell death types is sparse, and cell loss primarily concerns immature oligodendrocytes and, infrequently, neurons. In the present review, we first shortly discuss the different main severe preterm brain injury conditions that have been reported to involve cell death, including periventricular leucomalacia (PVL), diffuse white matter injury (dWMI), and intraventricular hemorrhages, as well as potentially harmful iatrogenic conditions linked to premature birth (anesthesia and caffeine therapy). Then, we present an overview of current evidence concerning cell death in both clinical human tissue data and preclinical models by focusing on studies investigating the presence of cell death allowing discriminating between the types of cell death involved. We conclude that, to improve brain protective strategies, not only apoptosis but also other cell death (such as regulated necrotic and autophagic) pathways now need to be investigated together in order to consider all cell death mechanisms involved in the pathogenesis of preterm brain damage.

Interaction between the autophagy protein Beclin 1 and Na+,K+-ATPase during starvation, exercise, and ischemia.

Autosis is a distinct form of cell death that requires both autophagy genes and the Na+,K+-ATPase pump. However, the relationship between the autophagy machinery and Na+,K+-ATPase is unknown. We explored the hypothesis that Na+,K+-ATPase interacts with the autophagy protein Beclin 1 during stress and autosis-inducing conditions. Starvation increased the Beclin 1/Na+,K+-ATPase interaction in cultured cells, and this was blocked by cardiac glycosides, inhibitors of Na+,K+-ATPase. Increases in Beclin 1/Na+,K+-ATPase interaction were also observed in tissues from starved mice, livers of patients with anorexia nervosa, brains of neonatal rats subjected to cerebral hypoxia-ischemia (HI), and kidneys of mice subjected to renal ischemia/reperfusion injury (IRI). Cardiac glycosides blocked the increased Beclin 1/Na+,K+-ATPase interaction during cerebral HI injury and renal IRI. In the mouse renal IRI model, cardiac glycosides reduced numbers of autotic cells in the kidney and improved clinical outcome. Moreover, blockade of endogenous cardiac glycosides increased Beclin 1/Na+,K+-ATPase interaction and autotic cell death in mouse hearts during exercise. Thus, Beclin 1/Na+,K+-ATPase interaction is increased in stress conditions, and cardiac glycosides decrease this interaction and autosis in both pathophysiological and physiological settings. This crosstalk between cellular machinery that generates and consumes energy during stress may represent a fundamental homeostatic mechanism.

CDK4 Regulates Lysosomal Function and mTORC1 Activation to Promote Cancer Cell Survival.

Cyclin-dependent kinase 4 (CDK4) is well-known for its role in regulating the cell cycle, however, its role in cancer metabolism, especially mTOR signaling, is undefined. In this study, we established a connection between CDK4 and lysosomes, an emerging metabolic organelle crucial for mTORC1 activation. On the one hand, CDK4 phosphorylated the tumor suppressor folliculin (FLCN), regulating mTORC1 recruitment to the lysosomal surface in response to amino acids. On the other hand, CDK4 directly regulated lysosomal function and was essential for lysosomal degradation, ultimately regulating mTORC1 activity. Pharmacologic inhibition or genetic inactivation of CDK4, other than retaining FLCN at the lysosomal surface, led to the accumulation of undigested material inside lysosomes, which impaired the autophagic flux and induced cancer cell senescence in vitro and in xenograft models. Importantly, the use of CDK4 inhibitors in therapy is known to cause senescence but not cell death. To overcome this phenomenon and based on our findings, we increased the autophagic flux in cancer cells by using an AMPK activator in combination with a CDK4 inhibitor. The cotreatment induced autophagy (AMPK activation) and impaired lysosomal function (CDK4 inhibition), resulting in cell death and tumor regression. Altogether, we uncovered a previously unknown role for CDK4 in lysosomal biology and propose a novel therapeutic strategy to target cancer cells. SIGNIFICANCE: These findings uncover a novel function of CDK4 in lysosomal biology, which promotes cancer progression by activating mTORC1; targeting this function offers a new therapeutic strategy for cancer treatment.

The Lactate Receptor HCAR1 Modulates Neuronal Network Activity through the Activation of Gα and Gßγ Subunits.

The discovery of a G-protein-coupled receptor for lactate named hydroxycarboxylic acid receptor 1 (HCAR1) in neurons has pointed to additional nonmetabolic effects of lactate for regulating neuronal network activity. In this study, we characterized the intracellular pathways engaged by HCAR1 activation, using mouse primary cortical neurons from wild-type (WT) and HCAR1 knock-out (KO) mice from both sexes. Using whole-cell patch clamp, we found that the activation of HCAR1 with 3-chloro-5-hydroxybenzoic acid (3Cl-HBA) decreased miniature EPSC frequency, increased paired-pulse ratio, decreased firing frequency, and modulated membrane intrinsic properties. Using fast calcium imaging, we show that HCAR1 agonists 3,5-dihydroxybenzoic acid, 3Cl-HBA, and lactate decreased by 40% spontaneous calcium spiking activity of primary cortical neurons from WT but not from HCAR1 KO mice. Notably, in neurons lacking HCAR1, the basal activity was increased compared with WT. HCAR1 mediates its effect in neurons through a Giα-protein. We observed that the adenylyl cyclase-cAMP-protein kinase A axis is involved in HCAR1 downmodulation of neuronal activity. We found that HCAR1 interacts with adenosine A1, GABAB, and α2A-adrenergic receptors, through a mechanism involving both its Giα and Gißγ subunits, resulting in a complex modulation of neuronal network activity. We conclude that HCAR1 activation in neurons causes a downmodulation of neuronal activity through presynaptic mechanisms and by reducing neuronal excitability. HCAR1 activation engages both Giα and Gißγ intracellular pathways to functionally interact with other Gi-coupled receptors for the fine tuning of neuronal activity.SIGNIFICANCE STATEMENT Expression of the lactate receptor hydroxycarboxylic acid receptor 1 (HCAR1) was recently described in neurons. Here, we describe the physiological role of this G-protein-coupled receptor (GPCR) and its activation in neurons, providing information on its expression and mechanism of action. We dissected out the intracellular pathway through which HCAR1 activation tunes down neuronal network activity. For the first time, we provide evidence for the functional cross talk of HCAR1 with other GPCRs, such as GABAB, adenosine A1- and α2A-adrenergic receptors. These results set HCAR1 as a new player for the regulation of neuronal network activity acting in concert with other established receptors. Thus, HCAR1 represents a novel therapeutic target for pathologies characterized by network hyperexcitability dysfunction, such as epilepsy.

Enhanced autophagy contributes to excitotoxic lesions in a rat model of preterm brain injury.

Cystic periventricular leukomalacia is commonly diagnosed in premature infants, resulting from severe hypoxic-ischemic white matter injury, and also involving some grey matter damage. Very few is known concerning the cell death pathways involved in these types of premature cerebral lesions. Excitotoxicity is a predominant mechanism of hypoxic-ischemic injury in the developing brain. Concomitantly, it has been recently shown that autophagy could be enhanced in excitotoxic conditions switching this physiological intracellular degradation system to a deleterious process. We here investigated the role of autophagy in a validated rodent model of preterm excitotoxic brain damage mimicking in some aspects cystic periventricular leukomalacia. An excitotoxic lesion affecting periventricular white and grey matter was induced by injecting ibotenate, a glutamate analogue, in the subcortical white matter (subcingulum area) of five-day old rat pups. Ibotenate enhanced autophagy in rat brain dying neurons at 24 h as shown by increased presence of autophagosomes (increased LC3-II and LC3-positive dots) and enhanced autophagic degradation (SQSTM1 reduction and increased number and size of lysosomes (LAMP1- and CATHEPSIN B-positive vesicles)). Co-injection of the pharmacological autophagy inhibitor 3-methyladenine prevented not only autophagy induction but also CASPASE-3 activation and calpain-dependent cleavage of SPECTRIN 24 h after the insult, thus providing a strong reduction of the long term brain injury (16 days after ibotenate injection) including lateral ventricle dilatation, decreases in cerebral tissue volume and in subcortical white matter thickness. The autophagy-dependent neuroprotective effect of 3-methyladenine was confirmed in primary cortical neuronal cultures using not only pharmacological but also genetic autophagy inhibition of the ibotenate-induced autophagy. Strategies inhibiting autophagy could then represent a promising neuroprotective approach in the context of severe preterm brain injuries.

Inhibition of autophagy delays motoneuron degeneration and extends lifespan in a mouse model of spinal muscular atrophy.

Spinal muscular atrophy (SMA) is a recessive autosomal neuromuscular disease, due to homozygous mutations or deletions in the telomeric survival motoneuron gene 1 (SMN1). SMA is characterized by motor impairment, muscle atrophy, and premature death following motor neuron (MN) degeneration. Emerging evidence suggests that dysregulation of autophagy contributes to MN degeneration. We here investigated the role of autophagy in the SMNdelta7 mouse model of SMA II (intermediate form of the disease) which leads to motor impairment by postnatal day 5 (P5) and to death by P13. We first showed by immunoblots that Beclin 1 and LC3-II expression levels increased in the lumbar spinal cord of the SMA pups. Electron microscopy and immunofluorescence studies confirmed that autophagic markers were enhanced in the ventral horn of SMA pups. To clarify the role of autophagy, we administered intracerebroventricularly (at P3) either an autophagy inhibitor (3-methyladenine, 3-MA), or an autophagy inducer (rapamycin) in SMA pups. Motor behavior was assessed daily with different tests: tail suspension, righting reflex, and hindlimb suspension tests. 3-MA significantly improved motor performance, extended the lifespan, and delayed MN death in lumbar spinal cord (10372.36 ± 2716 MNs) compared to control-group (5148.38 ± 94 MNs). Inhibition of autophagy by 3-MA suppressed autophagosome formation, reduced the apoptotic activation (cleaved caspase-3 and Bcl2) and the appearance of terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive neurons, underlining that apoptosis and autophagy pathways are intricately intertwined. Therefore, autophagy is likely involved in MN death in SMA II, suggesting that it might represent a promising target for delaying the progression of SMA in humans as well.

Homer1 Scaffold Proteins Govern Ca2+ Dynamics in Normal and Reactive Astrocytes.

In astrocytes, the intracellular calcium (Ca2+) signaling mediated by activation of metabotropic glutamate receptor 5 (mGlu5) is crucially involved in the modulation of many aspects of brain physiology, including gliotransmission. Here, we find that the mGlu5-mediated Ca2+ signaling leading to release of glutamate is governed by mGlu5 interaction with Homer1 scaffolding proteins. We show that the long splice variants Homer1b/c are expressed in astrocytic processes, where they cluster with mGlu5 at sites displaying intense local Ca2+ activity. We show that the structural and functional significance of the Homer1b/c-mGlu5 interaction is to relocate endoplasmic reticulum (ER) to the proximity of the plasma membrane and to optimize Ca2+ signaling and glutamate release. We also show that in reactive astrocytes the short dominant-negative splice variant Homer1a is upregulated. Homer1a, by precluding the mGlu5-ER interaction decreases the intensity of Ca2+ signaling thus limiting the intensity and the duration of glutamate release by astrocytes. Hindering upregulation of Homer1a with a local injection of short interfering RNA in vivo restores mGlu5-mediated Ca2+ signaling and glutamate release and sensitizes astrocytes to apoptosis. We propose that Homer1a may represent one of the cellular mechanisms by which inflammatory astrocytic reactions are beneficial for limiting brain injury.

The TAT-RasGAP317-326 anti-cancer peptide can kill in a caspase-, apoptosis-, and necroptosis-independent manner.

Tumor cell resistance to apoptosis, which is triggered by many anti-tumor therapies, remains a major clinical problem. Therefore, development of more efficient therapies is a priority to improve cancer prognosis. We have previously shown that a cell-permeable peptide derived from the p120 Ras GTPase-activating protein (RasGAP), called TAT-RasGAP317-326, bears anti-malignant activities in vitro and in vivo, such as inhibition of metastatic progression and tumor cell sensitization to cell death induced by various anti-cancer treatments. Recently, we discovered that this RasGAP-derived peptide possesses the ability to directly kill some cancer cells. TAT-RasGAP317-326 can cause cell death in a manner that can be either partially caspase-dependent or fully caspase-independent. Indeed, TAT-RasGAP317-326-induced toxicity was not or only partially prevented when apoptosis was inhibited. Moreover, blocking other forms of cell death, such as necroptosis, parthanatos, pyroptosis and autophagy did not hamper the killing activity of the peptide. The death induced by TAT-RasGAP317-326 can therefore proceed independently from these modes of death. Our finding has potentially interesting clinical relevance because activation of a death pathway that is distinct from apoptosis and necroptosis in tumor cells could lead to the generation of anti-cancer drugs that target pathways not yet considered for cancer treatment.

Neuroprotection by selective neuronal deletion of Atg7 in neonatal brain injury.

Perinatal asphyxia induces neuronal cell death and brain injury, and is often associated with irreversible neurological deficits in children. There is an urgent need to elucidate the neuronal death mechanisms occurring after neonatal hypoxia-ischemia (HI). We here investigated the selective neuronal deletion of the Atg7 (autophagy related 7) gene on neuronal cell death and brain injury in a mouse model of severe neonatal hypoxia-ischemia. Neuronal deletion of Atg7 prevented HI-induced autophagy, resulted in 42% decrease of tissue loss compared to wild-type mice after the insult, and reduced cell death in multiple brain regions, including apoptosis, as shown by decreased caspase-dependent and -independent cell death. Moreover, we investigated the lentiform nucleus of human newborns who died after severe perinatal asphyxia and found increased neuronal autophagy after severe hypoxic-ischemic encephalopathy compared to control uninjured brains, as indicated by the numbers of MAP1LC3B/LC3B (microtubule-associated protein 1 light chain 3)-, LAMP1 (lysosomal-associated membrane protein 1)-, and CTSD (cathepsin D)-positive cells. These findings reveal that selective neuronal deletion of Atg7 is strongly protective against neuronal death and overall brain injury occurring after HI and suggest that inhibition of HI-enhanced autophagy should be considered as a potential therapeutic target for the treatment of human newborns developing severe hypoxic-ischemic encephalopathy.

Combinative effects of ß-Lapachone and APO866 on pancreatic cancer cell death through reactive oxygen species production and PARP-1 activation.

Pancreatic cancer (PC) is one of the most lethal human malignancies and a major health problem. Patients diagnosed with PC and treated with conventional approaches have an overall 5-year survival rate of less than 5%. Novel strategies are needed to treat this disease. Herein, we propose a combinatorial strategy that targets two unrelated metabolic enzymes overexpressed in PC cells: NAD(P)H: quinone oxidoreductase-1 (NQO1) and nicotinamide phosphoribosyl transferase (NAMPT) using ß-lapachone (BL) and APO866, respectively. We show that BL tremendously enhances the antitumor activity of APO866 on various PC cell lines without affecting normal cells, in a PARP-1 dependent manner. The chemopotentiation of APO866 with BL was characterized by the following: (i) nicotinamide adenine dinucleotide (NAD) depletion; (ii) catalase (CAT) degradation; (iii) excessive H2O2 production; (iv) dramatic drop of mitochondrial membrane potential (MMP); and finally (v) autophagic-associated cell death. H2O2 production, loss of MMP and cell death (but not NAD depletion) were abrogated by exogenous supplementation with CAT or pharmacological or genetic inhibition of PARP-1. Our data demonstrates that the combination of a non-lethal dose of BL and low dose of APO866 optimizes significantly cell death on various PC lines over both compounds given separately and open new and promising combination in PC therapy.

Practical teaching of preclinical anatomy

We illustrate here why today practical teaching in preclinical anatomy is important and why the use of human cadavers is still essential for learning human anatomy by taking two examples. We explain why it is important for a student to be able to dissect and learn anatomy by exploratory anatomy. Several alternatives are discussed and modern teaching tools are illustrated with on-line and computer-based resources, anatomical models, reconstructions and radiographic images that could supplement the traditional dissection courses. Newer techniques such as anatomical body painting, projections, ultrasound or living anatomy may help in the understanding of topographical anatomy. We underline the authenticity that comes from using human tissue and consider the strengths and limitations of different teaching tools. Here we discuss also how far one should go in teaching anatomical variations in preclinical teaching. In Europe there is no consensus regarding anatomical teaching, and each institution has its own curriculum. It would be helpful to set up an anatomical data bank with images and PowerPoint slides that could be used in teaching programs. Here the Trans-European Pedagogic Anatomical Research Group (TEPARG) for Europe and the International Federation of Associations of Anatomists (IFAA) at an international level could play an essential role

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Dying neurons in thalamus of asphyxiated term newborns and rats are autophagic.

OBJECTIVE: Neonatal hypoxic-ischemic encephalopathy (HIE) still carries a high burden by its mortality and long-term neurological morbidity in survivors. Apart from hypothermia, there is no acknowledged therapy for HIE, reflecting the lack of mechanistic understanding of its pathophysiology. (Macro)autophagy, a physiological intracellular process of lysosomal degradation, has been proposed to be excessively activated in excitotoxic conditions such as HIE. The present study examines whether neuronal autophagy in the thalamus of asphyxiated human newborns or P7 rats is enhanced and related to neuronal death processes. METHODS: Neuronal autophagy and cell death were evaluated in the thalamus (frequently injured in severe HIE) of both human newborns who died after severe HIE (n = 5) and P7 hypoxic-ischemic rats (Rice-Vannuci model). Autophagic (LC3, p62), lysosomal (LAMP1, cathepsins), and cell death (TUNEL, caspase-3) markers were studied by immunohistochemistry in human and rat brain sections, and by additional methods in rats (immunoblotting, histochemistry, and electron microscopy). RESULTS: Following severe perinatal asphyxia in both humans and rats, thalamic neurons displayed up to 10-fold (p < 0.001) higher numbers of autophagosomes and lysosomes, implying an enhanced autophagic flux. The highly autophagic neurons presented strong features of apoptosis. These findings were confirmed and elucidated in more detail in rats. INTERPRETATION: These results show for the first time that autophagy is enhanced in severe HIE in dying thalamic neurons of human newborns, as in rats. Experimental neuroprotective strategies targeting autophagy could thus be a promising lead to follow for the development of future therapeutic approaches.

Mfn2 downregulation in excitotoxicity causes mitochondrial dysfunction and delayed neuronal death.

Mitochondrial fusion and fission is a dynamic process critical for the maintenance of mitochondrial function and cell viability. During excitotoxicity neuronal mitochondria are fragmented, but the mechanism underlying this process is poorly understood. Here, we show that Mfn2 is the only member of the mitochondrial fusion/fission machinery whose expression is reduced in in vitro and in vivo models of excitotoxicity. Whereas in cortical primary cultures, Drp1 recruitment to mitochondria plays a primordial role in mitochondrial fragmentation in an early phase that can be reversed once the insult has ceased, Mfn2 downregulation intervenes in a delayed mitochondrial fragmentation phase that progresses even when the insult has ceased. Downregulation of Mfn2 causes mitochondrial dysfunction, altered calcium homeostasis, and enhanced Bax translocation to mitochondria, resulting in delayed neuronal death. We found that transcription factor MEF2 regulates basal Mfn2 expression in neurons and that excitotoxicity-dependent degradation of MEF2 causes Mfn2 downregulation. Thus, Mfn2 reduction is a late event in excitotoxicity and its targeting may help to reduce excitotoxic damage and increase the currently short therapeutic window in stroke.

Aß1-42 monomers or oligomers have different effects on autophagy and apoptosis.

The role of autophagy and its relationship with apoptosis in Alzheimer disease (AD) pathogenesis is poorly understood. Disruption of autophagy leads to buildup of incompletely digested substrates, amyloid-ß (Aß) peptide accumulation in vacuoles and cell death. Aß, in turn, has been found to affect autophagy. Thus, Aß might be part of a loop in which it is both the substrate of altered autophagy and its cause. Given the relevance of different soluble forms of Aß1-42 in AD, we have investigated whether monomers and oligomers of the peptide have a differential role in causing altered autophagy and cell death. Using differentiated SK-N-BE neuroblastoma cells, we found that monomers hamper the formation of the autophagic BCL2-BECN1/Beclin 1 complex and activate the MAPK8/JNK1-MAPK9/JNK2 pathway phosphorylating BCL2. Monomers also inhibit apoptosis and allow autophagy with intracellular accumulation of autophagosomes and elevation of levels of BECN1 and LC3-II, resulting in an inhibition of substrate degradation due to an inhibitory action on lysosomal activity. Oligomers, in turn, favor the formation of the BCL2-BECN1 complex favoring apoptosis. In addition, they cause a less profound increase in BECN1 and LC3-II levels than monomers without affecting the autophagic flux. Thus, data presented in this work show a link for autophagy and apoptosis with monomers and oligomers, respectively. These studies are likely to help the design of novel disease modifying therapies.