Julien Puyal

A long-standing controversy is whether autophagy is a bona fide cause of mammalian cell death. We used a cell-penetrating autophagy-inducing peptide, Tat-Beclin 1, derived from the autophagy protein Beclin 1, to investigate whether high levels of autophagy result in cell death by autophagy. Here we show that Tat-Beclin 1 induces dose-dependent death that is blocked by pharmacological or genetic inhibition of autophagy, but not of apoptosis or necroptosis. This death, termed "autosis," has unique morphological features, including increased autophagosomes/autolysosomes and nuclear convolution at early stages, and focal swelling of the perinuclear space at late stages. We also observed autotic death in cells during stress conditions, including in a subpopulation of nutrient-starved cells in vitro and in hippocampal neurons of neonatal rats subjected to cerebral hypoxia-ischemia in vivo. A chemical screen of ~5,000 known bioactive compounds revealed that cardiac glycosides, antagonists of Na(+),K(+)-ATPase, inhibit autotic cell death in vitro and in vivo. Furthermore, genetic knockdown of the Na(+),K(+)-ATPase α1 subunit blocks peptide and starvation-induced autosis in vitro. Thus, we have identified a unique form of autophagy-dependent cell death, a Food and Drug Administration-approved class of compounds that inhibit such death, and a crucial role for Na(+),K(+)-ATPase in its regulation. These findings have implications for understanding how cells die during certain stress conditions and how such cell death might be prevented.

To evaluate the contributions of autophagic, necrotic, and apoptotic cell death mechanisms after neonatal cerebral ischemia and hence define the most appropriate neuroprotective approach for postischemic therapy.Rats were exposed to transient focal cerebral ischemia on postnatal day 12. Some rats were treated by postischemic administration of pan-caspase or autophagy inhibitors. The ischemic brain tissue was studied histologically, biochemically, and ultrastructurally for autophagic, apoptotic, and necrotic markers.Lysosomal and autophagic activities were increased in neurons in the ischemic area from 6 to 24 hours postinjury, as shown by immunohistochemistry against lysosomal-associated membrane protein 1 and cathepsin D, by acid phosphatase histochemistry, by increased expression of autophagosome-specific LC3-II and by punctate LC3 staining. Electron microscopy confirmed the presence of large autolysosomes and putative autophagosomes in neurons. The increases in lysosomal activity and autophagosome formation together demonstrate increased autophagy, which occurred mainly in the border of the lesion, suggesting its involvement in delayed cell death. We also provide evidence for necrosis near the center of the lesion and apoptotic-like cell death in its border, but in nonautophagic cells. Postischemic intracerebroventricular injections of autophagy inhibitor 3-methyladenine strongly reduced the lesion volume (by 46%) even when given >4 hours after the beginning of the ischemia, whereas pan-caspase inhibitors, carbobenzoxy-valyl-alanyl-aspartyl(OMe)-fluoromethylketone and quinoline-val-asp(OMe)-Ch2-O-phenoxy, provided no protection.The prominence of autophagic neuronal death in the ischemic penumbra and the neuroprotective efficacy of postischemic autophagy inhibition indicate that autophagy should be a primary target in the treatment of neonatal cerebral ischemia.

Accumulating evidence suggests that changes in the metabolic signature of astrocytes underlie their response to neuroinflammation, but how proinflammatory stimuli induce these changes is poorly understood. By monitoring astrocytes following acute cortical injury, we identified a differential and region-specific remodeling of their mitochondrial network: while astrocytes within the penumbra of the lesion undergo mitochondrial elongation, those located in the core—the area invaded by proinflammatory cells—experience transient mitochondrial fragmentation. In brain slices, proinflammatory stimuli reproduced localized changes in mitochondrial dynamics, favoring fission over fusion. This effect was triggered by Drp1 phosphorylation and ultimately resulted in reduced respiratory capacity. Furthermore, maintenance of the mitochondrial architecture critically depended on the induction of autophagy. Deletion of Atg7, required for autophagosome formation, prevented the reestablishment of tubular mitochondria, leading to marked reactive oxygen species accumulation and cell death. Thus, our data reveal autophagy to be essential for regenerating astrocyte mitochondrial networks during inflammation.

There is currently no approved neuroprotective pharmacotherapy for acute conditions such as stroke and cerebral asphyxia. One of the reasons for this may be the multiplicity of cell death mechanisms, because inhibition of a particular mechanism leaves the brain vulnerable to alternative ones. It is therefore essential to understand the different cell death mechanisms and their interactions. We here review the multiple signaling pathways underlying each of the three main morphological types of cell death – apoptosis, autophagic cell death and necrosis – emphasizing their importance in the neuronal death that occurs during cerebral ischemia and hypoxia-ischemia, and we analyze the interactions between the different mechanisms. Finally, we discuss the implications of the multiplicity of cell death mechanisms for the design of neuroprotective strategies.

Lactate is increasingly described as an energy substrate of the brain. Beside this still debated metabolic role, lactate may have other effects on brain cells. Here, we describe lactate as a neuromodulator, able to influence the activity of cortical neurons. Neuronal excitability of mouse primary neurons was monitored by calcium imaging. When applied in conjunction with glucose, lactate induced a decrease in the spontaneous calcium spiking frequency of neurons. The effect was reversible and concentration dependent (IC50 ∼4.2 mM). To test whether lactate effects are dependent on energy metabolism, we applied the closely related substrate pyruvate (5 mM) or switched to different glucose concentrations (0.5 or 10 mM). None of these conditions reproduced the effect of lactate. Recently, a Gi protein-coupled receptor for lactate called HCA1 has been introduced. To test if this receptor is implicated in the observed lactate sensitivity, we incubated cells with pertussis toxin (PTX) an inhibitor of Gi-protein. PTX prevented the decrease of neuronal activity by L-lactate. Moreover 3,5-dyhydroxybenzoic acid, a specific agonist of the HCA1 receptor, mimicked the action of lactate. This study indicates that lactate operates a negative feedback on neuronal activity by a receptor-mediated mechanism, independent from its intracellular metabolism.

Neuronal autophagy is enhanced in many neurological conditions, such as cerebral ischemia and traumatic brain injury, but its role in associated neuronal death is controversial, especially under conditions of apoptosis. We therefore investigated the role of autophagy in the apoptosis of primary cortical neurons treated with the widely used and potent pro-apoptotic agent, staurosporine (STS). Even before apoptosis, STS enhanced autophagic flux, as shown by increases in autophagosomal (LC3-II level, LC3 punctate labeling) and lysosomal (cathepsin D, LAMP1, acid phosphatase, β-hexasominidase) markers. Inhibition of autophagy by 3-methyladenine, or by lentivirally-delivered shRNAs against Atg5 and Atg7, strongly reduced the STS-induced activation of caspase-3 and nuclear translocation of AIF, and gave partial protection against neuronal death. Pan-caspase inhibition with Q-VD-OPH likewise protected partially against neuronal death, but failed to affect autophagy. Combined inhibition of both autophagy and caspases gave strong synergistic neuroprotection. The autophagy contributing to apoptosis was Beclin 1-independent, as shown by the fact that Beclin 1 knockdown failed to reduce it but efficiently reduced rapamycin-induced autophagy. Moreover the Beclin 1 knockdown sensitized neurons to STS-induced apoptosis, indicating a cytoprotective role of Beclin 1 in cortical neurons. Caspase-3 activation and pyknosis induced by two other pro-apoptotic stimuli, MK801 and etoposide, were likewise found to be associated with Beclin 1-independent autophagy and reduced by the knockdown of Atg7 but not Beclin 1. In conclusion, Beclin 1-independent autophagy is an important contributor to both the caspase-dependent and -independent components of neuronal apoptosis and may be considered as an important therapeutic target in neural conditions involving apoptosis.

Glial cells are increasingly recognized as active players that profoundly influence neuronal synaptic transmission by specialized signaling pathways. In particular, astrocytes have been shown recently to release small molecules, such as the amino acids l-glutamate and d-serine as "gliotransmitters," which directly control the efficacy of adjacent synapses. However, it is still controversial whether gliotransmitters are released from a cytosolic pool or by Ca(2+)-dependent exocytosis from secretory vesicles, i.e., by a mechanism similar to the release of synaptic vesicles in synapses. Here we report that rat cortical astrocytes contain storage vesicles that display morphological and biochemical features similar to neuronal synaptic vesicles. These vesicles share some, but not all, membrane proteins with synaptic vesicles, including the SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) synaptobrevin 2, and contain both l-glutamate and d-serine. Furthermore, they show uptake of l-glutamate and d-serine that is driven by a proton electrochemical gradient. d-Serine uptake is associated with vesicle acidification and is dependent on chloride. Whereas l-serine is not transported, serine racemase, the synthesizing enzyme for d-serine, is anchored to the membrane of the vesicles, allowing local generation of d-serine. Finally, we reveal a previously unexpected mutual vesicular synergy between d-serine and l-glutamate filling in glia vesicles. We conclude that astrocytes contain vesicles capable of storing and releasing d-serine, l-glutamate, and most likely other neuromodulators in an activity-dependent manner.

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.

The multiplicity of cell death mechanisms induced by neonatal hypoxia-ischemia makes neuroprotective treatment against neonatal asphyxia more difficult to achieve. Whereas the roles of apoptosis and necrosis in such conditions have been studied intensively, the implication of autophagic cell death has only recently been considered. Here, we used the most clinically relevant rodent model of perinatal asphyxia to investigate the involvement of autophagy in hypoxic-ischemic brain injury. Seven-day-old rats underwent permanent ligation of the right common carotid artery, followed by 2 hours of hypoxia. This condition not only increased autophagosomal abundance (increase in microtubule-associated protein 1 light chain 3-11 level and punctuate labeling) but also lysosomal activities (cathepsin D, acid phosphatase, and β-N-acetylhexosaminidase) in cortical and hippocampal CA3-damaged neurons at 6 and 24 hours, demonstrating an increase in the autophagic flux. In the cortex, this enhanced autophagy may be related to apoptosis since some neurons presenting a high level of autophagy also expressed apoptotic features, including cleaved caspase-3. On the other hand, enhanced autophagy in CA3 was associated with a more purely autophagic cell death phenotype. In striking contrast to CA3 neurons, those in CA1 presented only a minimal increase in autophagy but strong apoptotic characteristics. These results suggest a role of enhanced autophagy in delayed neuronal death after severe hypoxia-ischemia that is differentially linked to apoptosis according to the cerebral region.

Neuronal autophagy is increased in numerous excitotoxic conditions including neonatal cerebral hypoxia-ischemia (HI). However, the role of this HI-induced autophagy remains unclear. To clarify this role we established an in vitro model of excitotoxicity combining kainate treatment (Ka, 30 µM) with hypoxia (Hx, 6% oxygen) in primary neuron cultures. KaHx rapidly induced excitotoxic death that was completely prevented by MK801 or EGTA. KaHx also stimulated neuronal autophagic flux as shown by a rise in autophagosome number (increased levels of LC3-II and punctate LC3 labeling) accompanied by increases in lysosomal abundance and activity (increased SQSTM1/p62 degradation, and increased LC3-II levels in the presence of lysosomal inhibitors) and fusion (shown using an RFP-GFP-LC3 reporter). To determine the role of the enhanced autophagy we applied either pharmacological autophagy inhibitors (3-methyladenine or pepstatinA/E64) or lentiviral vectors delivering shRNAs targeting Becn1 or Atg7. Both strategies reduced KaHx-induced neuronal death. A prodeath role of autophagy was also confirmed by the enhanced toxicity of KaHx in cultures overexpressing BECN1 or ATG7. Finally, in vivo inhibition of autophagy by intrastriatal injection of a lentiviral vector expressing a Becn1-targeting shRNA increased the volume of intact striatum in a rat model of severe neonatal cerebral HI. These results clearly show a death-mediating role of autophagy in hypoxic-excitotoxic conditions and suggest that inhibition of autophagy should be considered as a neuroprotective strategy in HI brain injuries.

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 Giβγ 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 Giβγ 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.

The term autophagic cell death (ACD) initially referred to cell death with greatly enhanced autophagy, but is increasingly used to imply a death-mediating role of autophagy, as shown by a protective effect of autophagy inhibition. In addition, many authors require that autophagic cell death must not involve apoptosis or necrosis. Adopting these new and restrictive criteria, and emphasizing their own failure to protect human osteosarcoma cells by autophagy inhibition, the authors of a recent Editor's Corner article in this journal argued for the extreme rarity or nonexistence of autophagic cell death. We here maintain that, even with the more stringent recent criteria, autophagic cell death exists in several situations, some of which were ignored by the Editor's Corner authors. We reject their additional criterion that the autophagy in ACD must be the agent of ultimate cell dismantlement. And we argue that rapidly dividing mammalian cells such as cancer cells are not the most likely situation for finding pure ACD.

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.

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.

Neuronal nitric oxide synthase (nNOS) and p38MAPK are strongly implicated in excitotoxicity, a mechanism common to many neurodegenerative conditions, but the intermediary mechanism is unclear. NOS1AP is encoded by a gene recently associated with sudden cardiac death, diabetes-associated complications, and schizophrenia (Arking et al., 2006; Becker et al., 2008; Brzustowicz, 2008; Lehtinen et al., 2008). Here we find it interacts with p38MAPK-activating kinase MKK3. Excitotoxic stimulus induces recruitment of NOS1AP to nNOS in rat cortical neuron culture. Excitotoxic activation of p38MAPK and subsequent neuronal death are reduced by competing with the nNOS:NOS1AP interaction and by knockdown with NOS1AP-targeting siRNAs. We designed a cell-permeable peptide that competes for the unique PDZ domain of nNOS that interacts with NOS1AP. This peptide inhibits NMDA-induced recruitment of NOS1AP to nNOS and in vivo in rat, doubles surviving tissue in a severe model of neonatal hypoxia-ischemia, a major cause of neonatal death and pediatric disability. The highly unusual sequence specificity of the nNOS:NOS1AP interaction and involvement in excitotoxic signaling may provide future opportunities for generation of neuroprotectants with high specificity.

Autophagy is a cellular mechanism for degrading proteins and organelles. It was first described as a physiological process essential for cellular health and survival, and this is its role in most cells. However, it can also be a mediator of cell death, either by the triggering of apoptosis or by an independent "autophagic" cell death mechanism. This duality is important in the central nervous system, where the activation of autophagy has recently been shown to be protective in certain chronic neurodegenerative diseases but deleterious in acute neural disorders such as stroke and hypoxic/ischemic injury. The authors here discuss these distinct roles of autophagy in the nervous system with a focus on the role of autophagy in mediating neuronal death. The development of new therapeutic strategies based on the manipulation of autophagy will need to take into account these opposing roles of autophagy.

Neonatal hypoxic-ischemic encephalopathy is a critical cerebral event occurring around birth with high mortality and neurological morbidity associated with long-term invalidating sequelae. In view of the great clinical importance of this condition and the lack of very efficacious neuroprotective strategies, it is urgent to better understand the different cell death mechanisms involved with the ultimate aim of developing new therapeutic approaches. The morphological features of three different cell death types can be observed in models of perinatal cerebral hypoxia-ischemia: necrotic, apoptotic and autophagic cell death. They may be combined in the same dying neuron. In the present review, we discuss the different cell death mechanisms involved in neonatal cerebral hypoxia-ischemia with a special focus on how autophagy may be involved in neuronal death, based: (1) on experimental models of perinatal hypoxia-ischemia and stroke, and (2) on the brains of human neonates who suffered from neonatal hypoxia-ischemia.

BAK1, pro-apoptotic protein; BCL2, B-cell lymphoma 2 protein; BECN1, Beclin 1; CASP3, caspase 3; CTSD, cathepsin D; CYCS, cytochrome c; LAMP1, lysosomal-associated membrane protein 1; LC3, microtubule-associated protein 1 light chain 3; LDH, lactate dehydrogenase; MAPK8/JNK1, mitogen-activated protein kinase 8; MAPK9/JNK2, mitogen-activated protein kinase 9; SQSTM1/p62,

Neurodegeneration in mitochondrial disorders is considered irreversible because of limited metabolic plasticity in neurons, yet the cell-autonomous implications of mitochondrial dysfunction for neuronal metabolism in vivo are poorly understood. Here, we profiled the cell-specific proteome of Purkinje neurons undergoing progressive OXPHOS deficiency caused by disrupted mitochondrial fusion dynamics. We found that mitochondrial dysfunction triggers a profound rewiring of the proteomic landscape, culminating in the sequential activation of precise metabolic programs preceding cell death. Unexpectedly, we identified a marked induction of pyruvate carboxylase (PCx) and other anaplerotic enzymes involved in replenishing tricarboxylic acid cycle intermediates. Suppression of PCx aggravated oxidative stress and neurodegeneration, showing that anaplerosis is protective in OXPHOS-deficient neurons. Restoration of mitochondrial fusion in end-stage degenerating neurons fully reversed these metabolic hallmarks, thereby preventing cell death. Our findings identify a previously unappreciated pathway conferring resilience to mitochondrial dysfunction and show that neurodegeneration can be reversed even at advanced disease stages.

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.

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.

Cell death due to cerebral ischemia has been attributed to necrosis and apoptosis, but autophagic mechanisms have recently been implicated as well. Using rats exposed to neonatal focal cerebral ischemia, we have shown that lysosomal and autophagic activities are increased in ischemic neurons, and have obtained strong neuroprotection by post-ischemic inhibition of autophagy.

Endoplasmic reticulum (ER) homeostasis alteration contributes to pancreatic β-cell dysfunction and death and favors the development of diabetes. In this study, we demonstrate that HDLs protect β-cells against ER stress induced by thapsigargin, cyclopiazonic acid, palmitate, insulin overexpression, and high glucose concentrations. ER stress marker induction and ER morphology disruption mediated by these stimuli were inhibited by HDLs. Using a temperature-sensitive viral glycoprotein folding mutant, we show that HDLs correct impaired protein trafficking and folding induced by thapsigargin and palmitate. The ability of HDLs to protect β-cells against ER stress was inhibited by brefeldin A, an ER to Golgi trafficking blocker. These results indicate that HDLs restore ER homeostasis in response to ER stress, which is required for their ability to promote β-cell survival. This study identifies a cellular mechanism mediating the beneficial effect of HDLs on β-cells against ER stress-inducing factors.

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. Ann Neurol 2014;76:695–711

Abstract 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.

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.

Despite tremendous advances in neonatal intensive care, prematurity nowadays carries a high burden of neurological morbidity lasting lifelong. Preterm brain injury, covered by the term encephalopathy of prematurity (EoP), associates white matter injury and inflammation, but more and more evidence implicates also gray matter damage and excitotoxicity. 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 EoP. 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 concern primarily immature oligodendrocytes and infrequently neurons. In the present review, we first shortly discuss the different main preterm brain injury conditions including periventricular leucomalacia (PVL), diffuse white matter injury (dWMI) and intraventricular hemorrhages as well as potentially harmful iatrogenic conditions linked to premature birth. Then, we present an overview of current evidences concerning cell death in both clinical human tissue data and preclinical models. We conclude that, to improve brain protective strategies, not only apoptosis but also other cell death (such as regulated necrotic and autophagic) pathways have now to be investigated together in order to consider all cell death mechanisms involved in the pathogenesis of preterm brain damage.

In insulin-secreting cells, activation of the c-Jun NH2-terminal kinase (JNK) pathway triggers apoptosis. Whereas JNK1 and JNK2 are ubiquitously produced, JNK3 has been described exclusively in neurons. This report aims to characterise the expression and role in apoptosis of the three JNK isoforms in insulin-secreting cells exposed to cytokines. Sections of human and mouse pancreases were used for immunohistochemistry studies with isoform-specific anti-JNK antibodies. Human, pig, mouse and rat pancreatic islets were isolated by enzymatic digestion and RNA or protein extracts were prepared. RNA and protein levels were determined by quantitative RT-PCR and western blotting respectively, using JNK-isoform-specific primers and isoform-specific antibodies; activities of the three JNK isoforms were determined by kinase assays following quantitative immunoprecipitation/depletion of JNK3. JNK silencing was performed with small interfering RNAs and apoptotic rates were determined in INS-1E cells by scoring cells displaying pycnotic nuclei. JNK3 and JNK2 mRNAs are the predominant isoforms expressed in human pancreatic islets. JNK3 is nuclear while JNK2 is also cytoplasmic. In INS-1E cells, JNK3 knockdown increases c-Jun levels and caspase-3 cleavage and sensitises cells to cytokine-induced apoptosis; in contrast, JNK1 or JNK2 knockdown is protective. In insulin-secreting cells, JNK3 plays an active role in preserving pancreatic beta cell mass from cytokine attacks. The specific localisation of JNK3 in the nucleus, its recruitment by cytokines, and its effects on key transcription factors such as c-Jun, indicate that JNK3 is certainly an important player in the transcriptional control of genes expressed in insulin-secreting cells.

Clathrin-dependent endocytosis is mediated by a tightly regulated network of molecular interactions that provides essential protein-protein and protein-lipid binding activities. Here we report the hydrolysis of the alpha- and beta2-subunits of the tetrameric adaptor protein complex 2 by calpain. Calcium-dependent alpha- and beta2-adaptin hydrolysis was observed in several rat tissues, including brain and primary neuronal cultures. Neuronal alpha- and beta2-adaptin cleavage was inducible by glutamate stimulation and was accompanied by the decreased endocytosis of transferrin. Heterologous expression of truncated forms of the beta2-adaptin subunit significantly decreased the membrane recruitment of clathrin and inhibited clathrin-mediated receptor endocytosis. Moreover, the presence of truncated beta2-adaptin sensitized neurons to glutamate receptor-mediated excitotoxicity. Proteolysis of alpha- and beta2-adaptins, as well as the accessory clathrin adaptors epsin 1, adaptor protein 180, and the clathrin assembly lymphoid myeloid leukemia protein, was detected in brain tissues after experimentally induced ischemia and in cases of human Alzheimer disease. The present study further clarifies the central role of calpain in regulating clathrin-dependent endocytosis and provides evidence for a novel mechanism through which calpain activation may promote neurodegeneration: the sensitization of cells to glutamate-mediated excitotoxicity via the decreased internalization of surface receptors.

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.

Abstract The patterns of development of the vestibular nuclei (VN) and their main connections involving glutamate neurotransmission offer a good model for studying the function of the glial‐derived neuromodulator D ‐serine in synaptic plasticity. In this study we show that D ‐serine is present in the VN and we analyzed its distribution and the levels of expression of serine racemase and D ‐amino acid oxidase ( D ‐AAO) at different stages of postnatal (P) development. From birth to P21, high levels of D ‐serine were detected in glial cells and processes in all parts of the VN. This period corresponded to high expression of serine racemase and low expression of D ‐AAO. On the other hand, in the mature VN D ‐serine displayed very low levels and was mainly localized in neuronal cell bodies and dendrites. This drop of D ‐serine in adult stages corresponded to an increasing expression of D ‐AAO at mature stages. High levels of glial D ‐serine during the first 3 weeks of postnatal development correspond to an intense period of plasticity and synaptogenesis and maturation of VN afferents, suggesting that D ‐serine could be involved in these phenomena. These results demonstrate for the first time that changes in D ‐serine levels and distribution occur during postnatal development in the central nervous system. The strong decrease of D ‐serine levels and the glial‐to‐neuronal switch suggests that D ‐serine may have distinct functional roles depending on the developmental stage of the vestibular network. J. Comp. Neurol. 497:610–621, 2006. © 2006 Wiley‐Liss, Inc.

Glucose is an important metabolic substrate of the retina and diabetic patients have to maintain a strict normoglycemia to avoid diabetes secondary effects, including cardiovascular disease, nephropathy, neuropathy and retinopathy. Others and we recently demonstrated the potential role of hypoglycemia in diabetic retinopathy. We showed acute hypoglycemia to induce retinal cell death both in vivo during an hyperinsulinemic/hypoglycemic clamp and in vitro in 661W photoreceptor cells cultured at low glucose concentration. In the present study, we showed low glucose to induce a decrease of BCL2 and BCL-XL anti-apoptotic proteins expression, leading to an increase of free pro-apoptotic BAX. In parallel, we showed that, in retinal cells, low glucose-induced apoptosis is involved in the process of autophagosomes formation through the AMPK/RAPTOR/mTOR pathway. Moreover, the decrease of LAMP2a expression led to a defect in the autophagosome/lysosome fusion process. Specific inhibition of autophagy, either by 3-methyladenine or by down-regulation of ATG5 or ATG7 proteins expression, increased caspase 3 activation and 661W cell death. We show that low glucose modifies the delicate equilibrium between apoptosis and autophagy. Cells struggled against low nutrient condition-induced apoptosis by starting an autophagic process, which led to cell death when inhibited. We conclude that autophagy defect is associated with low glucose-induced 661W cells death that could play a role in diabetic retinopathy. These results could modify the way of addressing negative effects of hypoglycemia. Short-term modulation of autophagy could be envisioned to treat diabetic patients in order to avoid secondary complications of the disease.

APO866, an inhibitor of NAD biosynthesis, exhibits potent antitumor properties in various malignancies. Recently, it has been shown that APO866 induces apoptosis and autophagy in human hematological cancer cells, but the role of autophagy in APO866-induced cell death remains unclear. Here, we report studies on the molecular mechanisms underlying APO866-induced cell death with emphasis on autophagy. Treatment of leukemia and lymphoma cells with APO866 induced both autophagy, as evidenced by an increase in autophagosome formation and in SQSTM1/p62 degradation, but also increased caspase activation as revealed by CASP3/caspase 3 cleavage. As an underlying mechanism, APO866-mediated autophagy was found to deplete CAT/catalase, a reactive oxygen species (ROS) scavenger, thus promoting ROS production and cell death. Inhibition of autophagy by ATG5 or ATG7 silencing prevented CAT degradation, ROS production, caspase activation, and APO866-induced cell death. Finally, supplementation with exogenous CAT also abolished APO866 cytotoxic activity. Altogether, our results indicated that autophagy is essential for APO866 cytotoxic activity on cells from hematological malignancies and also indicate an autophagy-dependent CAT degradation, a novel mechanism for APO866-mediated cell killing. Autophagy-modulating approaches could be a new way to enhance the antitumor activity of APO866 and related agents.

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.

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.

D-JNKI1, a cell-permeable peptide inhibitor of the c-Jun N-terminal kinase (JNK) pathway, has been shown to be a powerful neuroprotective agent after focal cerebral ischemia in adult mice and young rats. We have investigated the potential neuroprotective effect of D-JNKI1 and the involvement of the JNK pathway in a neonatal rat model of cerebral hypoxia-ischemia (HI). Seven-day-old rats underwent a permanent ligation of the right common carotid artery followed by 2 h of hypoxia (8% oxygen). Treatment with D-JNKI1 (0.3 mg/kg intraperitoneally) significantly reduced early calpain activation, late caspase 3 activation and, in the thalamus, autophagosome formation, indicating an involvement of JNK in different types of cell death: necrotic, apoptotic, and autophagic. However, the size of the lesion was unchanged. Further analysis showed that neonatal HI induced an immediate decrease in JNK phosphorylation (reflecting mainly JNK1 phosphorylation) followed by a slow progressive increase (including JNK3 phosphorylation 54 kDa), whereas c-jun and c-fos expression were both strongly activated immediately after HI. In conclusion, unlike in adult ischemic models, JNK is only moderately activated after severe cerebral HI in neonatal rats and the observed positive effects of D-JNKI1 are insufficient to give neuroprotection. Thus, for perinatal asphyxia, D-JNKI1 can only be considered in association with other therapies.

The effects of high frequency stimulation (HFS) of the primary vestibular afferents on synaptic transmission in the ventral part of the medial vestibular nuclei (vMVN) were studied during postnatal development and compared with the changes in the expression of the group I metabotropic glutamate receptor (mGluR) subtypes, mGluR1 and mGluR5. During the first stages of development, HFS always induced a mGluR5- and GABAA-dependent long-term depression (LTD) which did not require NMDA receptor and mGluR1 activation. The probability of inducing LTD decreased progressively throughout the development and it was zero at about the end of the second postnatal week. Conversely, long-term potentiation (LTP) appeared at the beginning of the second week and its occurrence increased to reach the adult value at the end of the third week. Of interest, the sudden change in the LTP frequency occurred at the time of eye opening, about the end of the second postnatal week. LTP depended on NMDA receptor and mGluR1 activation. In parallel with the modifications in synaptic plasticity, we observed that the expression patterns and localizations of mGluR5 and mGluR1 in the medial vestibular nuclei (MVN) changed during postnatal development. At the earlier stages the mGluR1 expression was minimal, then increased progressively. In contrast, mGluR5 expression was initially high, then decreased. While mGluR1 was exclusively localized in neuronal compartments and concentrated at the postsynaptic sites at all stages observed, mGluR5 was found mainly in neuronal compartments at immature stages, then preferentially in glial compartments at mature stages. These results provide the first evidence for a progressive change from LTD to LTP accompanied by a distinct maturation expression of mGluR1 and mGluR5 during the development of the MVN.

Recent studies showed that endocytosis is enhanced in neurons exposed to an excitototoxic stimulus. We here confirm and analyze this new phenomenon using dissociated cortical neuronal cultures. NMDA-induced uptake (FITC-dextran or FITC or horseradish peroxidase) occurs in these cultures and is due to endocytosis, not to cell entry through damaged membranes; it requires an excitotoxic dose of NMDA and is dependent on extracellular calcium, but occurs early, while the neuron is still intact and viable. It involves two components, NMDA-induced and constitutive, with different characteristics. Neither component involves specific binding of the endocytosed molecules to a saturable receptor. Strikingly, molecules internalized by the NMDA-induced component are targeted to neuronal nuclei. This component, but not the constitutive one, is blocked by a c-Jun N-terminal protein kinase inhibitor. In conclusion, an excitotoxic dose of NMDA triggers c-Jun N-terminal protein kinase-dependent endocytosis in cortical neuronal cultures, providing an in vitro model of the excitotoxicity-induced endocytosis reported in intact tissues.

Macroautophagy (hereafter called autophagy) is an essential physiological process of degradation of organelles and long-lived proteins. The discovery of autosis, a Na

Aims. Pulmonary arterial hypertension (PAH) is a life-threatening disease that still lacks a direct therapeutic approach targeted to the molecular defects associated with the disease. In this study, we focused on the control of the sodium/hydrogen exchange, which is at the root of impaired regulation of intracellular acidity, as well as of the sodium and calcium intracellular overload. We tested the hypothesis that inhibiting the sodium/hydrogen exchanger isoform 1 (NHE-1) with rimeporide enables the recovery of the pulmonary and right ventricular dysfunction in the Sugen5416/hypoxia rat preclinical model of PAH. Methods and Results. We studied 44 rats divided into two broad groups, control, and Sugen5416/hypoxia. After verifying the insurgence of PAH in the Sugen5416/hypoxia group by transthoracic echocardiography and pulse-wave Doppler analysis, two subgroups were assigned to treatment with either 100 mg/kg/day rimeporide or placebo in drinking water for three weeks. The functional, morphological (fibrosis and hypertrophy) and biochemical (inflammation, signalling pathways) myocardial and pulmonary dysfunctions caused by PAH can be at least partially reverted by treatment with rimeporide. Interestingly, the most striking effects of rimeporide were observed in the right ventricle. Rimeporide was able to improve the hemodynamic variables in the pulmonary circulation and the right ventricle, to decrease right ventricle hypertrophy, pulmonary vascular remodelling, inflammation, and fibrosis. No effect of rimeporide is detected in control rats. We also showed that the protective effect of rimeporide was accompanied by a decrease of the p-Akt/Akt ratio and a stimulation of the autophagy flux mainly in the right ventricle. Conclusion. By specifically inhibiting NHE-1, rimeporide at the selected dosage revealed remarkable anti-PAH effects by preventing functional, morphological, and biochemical deleterious effects of PAH on right ventricle and lung. Rimeporide has to be considered as a potential treatment for PAH.

Targeting neuroprotectants specifically to the cells that need them is a major goal in biomedical research. Many peptidic protectants contain an active sequence linked to a carrier such as the transactivator of transcription (TAT) transduction sequence, and here we test the hypothesis that TAT-linked peptides are selectively endocytosed into neurons stressed by excitotoxicity and focal cerebral ischemia.In vivo experiments involved intracerebroventricular injection of TAT peptides or conventional tracers (peroxidase, fluorescein isothiocyanate-dextran) in young rats exposed to occlusion of the middle cerebral artery at postnatal day 12. Cellular mechanisms of uptake were analyzed in dissociated cortical neuronal cultures.In both models, all tracers were taken up selectively into stressed neurons by endocytosis. In the in vivo model, this was neuron specific and limited to the ischemic area, where the neurons displayed enhanced immunolabeling for early endosomal antigen-1 and clathrin. The highly efficient uptake of TAT peptides occurred by the same selective mechanism as for conventional tracers. All tracers were targeted to the nucleus and cytoplasm of neurons that appeared viable, although ultimately destined to die. In dissociated cortical neuronal cultures, an excitotoxic dose of N-methyl-D-aspartate induced a similar endocytosis. It was 100 times more efficient with TAT peptides than with dextran, because the former bound to heparan sulfate proteoglycans at the cell surface, but it depended on dynamin and clathrin in both cases.Excitotoxicity-induced endocytosis is the main entry route for protective TAT peptides and targets selectively the neurons that need to be protected.

Excitotoxicity and cerebral ischemia induce strong endocytosis in neurons, and we here investigate its functional role in neuroprotection by a functional transactivator of transcription (TAT)-peptide, the c-Jun N-terminal kinase (JNK) inhibitor D-JNKI1, against NMDA-excitotoxicity in vitro and neonatal ischemic stroke in P12 Sprague-Dawley rats. In both situations, the neuroprotective efficacy of D-JNKI1 was confirmed, but excessively high doses were counterproductive. Importantly, the induced endocytosis was necessary for neuroprotection, which required that the TAT-peptide be administered at a time when induced endocytosis was occurring. Uptake by other routes failed to protect, and even promoted cell death at high doses. Blocking the induced endocytosis of D-JNKI1 with heparin or with an excess of D-TAT-peptide eliminated the neuroprotection. We conclude that excitotoxicity-induced endocytosis is a basic property of stressed neurons that can target neuroprotective TAT-peptides into the neurons that need protection. Furthermore, it is the main mediator of neuroprotection by D-JNKI1. This may explain promising reports of strong neuroprotection by TAT-peptides without apparent side effects, and warns that the timing of peptide administration is crucial.

HDLs protect pancreatic beta cells against apoptosis induced by several endoplasmic reticulum (ER) stressors, including thapsigargin, cyclopiazonic acid, palmitate and insulin over-expression. This protection is mediated by the capacity of HDLs to maintain proper ER morphology and ER functions such as protein folding and trafficking. Here, we identified a distinct mode of protection exerted by HDLs in beta cells challenged with tunicamycin (TM), a protein glycosylation inhibitor inducing ER stress. HDLs were found to inhibit apoptosis induced by TM in the MIN6 insulinoma cell line and this correlated with the maintenance of a normal ER morphology. Surprisingly however, this protective response was neither associated with a significant ER stress reduction, nor with restoration of protein folding and trafficking in the ER. These data indicate that HDLs can use at least two mechanisms to protect beta cells against ER stressors. One that relies on the maintenance of ER function and one that operates independently of ER function modulation. The capacity of HDLs to activate several anti-apoptotic pathways in beta cells may explain their ability to efficiently protect these cells against a variety of insults.

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 β-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.

We investigated the distribution of the glutamate receptor subunits, α-amino-3-hydroxy-5-methyl-4 isoazolepropionic acid (AMPA) GluR2 and GluR2/R3, and N-methyl-d-aspartate (NMDA) NR1, and the timing of their appearance during early development of the mouse vestibular and spiral ganglia. NMDA NR1 was the first to be expressed, in the statoacoustic ganglion neurons on E11. GluR2/R3 immunoreactivity was detected in these neurons on E12. This signal probably corresponded exclusively to GluR3, as no signal was obtained for GluR2 alone at this stage. The appearance of these proteins began much earlier than previously reported. GluR2 staining was observed later, on E14 in the vestibular neurons and on E17 in the spiral neurons. The sequence in which these three glutamate receptors appeared suggested possible differences in their roles in the establishment of neuronal circuitry in the inner ear sensory epithelia. The production of NR1 and GluR2/R3 began during the early period of neuron growth and fasciculation. GluR2 appeared later and its expression paralleled synaptogenesis in the vestibular sensory epithelia and in the organ of Corti.

Abstract We investigated whether three calcium‐binding proteins, calretinin, parvalbumin, and calbindin, could identify specific aspects of the postnatal development of the rat lateral (LVN) and medial (MVN) vestibular nuclei and their vestibular and cerebellar connections. Calretinin levels in the vestibular nuclei, increased significantly between birth and postnatal day (P) 45. In situ hybridization and immunocytochemical staining showed that calretinin‐immunoreactive neurons were mostly located in the parvocellular MVN at birth and that somatic and dendritic growth occurred between birth and P14. During the first week, parvalbumin‐immunoreactive fibers and endings were confined to specific areas, i.e., the ventral LVN and magnocellular MVN, and identified exclusively the maturation of the vestibular afferents. Calbindin was located within the dorsal LVN and the parvocellular MVN and identified the first arrival of the corticocerebellar afferents. From the second week, in addition to labeling vestibular afferents in their specific target areas, parvalbumin was also found colocalized with calbindin in mature Purkinje cell afferents. Thus, the specific spatiotemporal distribution of parvalbumin and calbindin could correspond to two successive phases of synaptic remodeling involving integration of the vestibular sensory messages and their cerebellar control. On the basis of the sequence of distribution patterns of these proteins during the development of the vestibular nuclei, calretinin is an effective marker for neuronal development of the parvocellular MVN, parvalbumin is a specific marker identifying maturation of the vestibular afferents and endings, and calbindin is a marker of the first appearance and development of Purkinje cell afferents. J. Comp. Neurol. 451:374–391, 2002. © 2002 Wiley‐Liss, Inc.

Autophagy is a cellular mechanism for degrading proteins and organelles. It was first described as a physiological process essential for maintaining homeostasis and cell survival, but understanding its role in conditions of stress has been complicated by the recognition of a new type of cell death ("type 2") characterized by deleterious autophagic activity. This paradox is important in the central nervous system where the activation of autophagy seems to be protective in certain neurodegenerative diseases but deleterious in cerebral ischemia. The development of new therapeutic strategies based on the manipulation of autophagy will need to take into account these opposing roles of autophagy.

Using immunocytochemistry and multiunit recording of afferent activity of the whole vestibular nerve, we investigated the role of metabotropic glutamate receptors (mGluR) in the afferent neurotransmission in the frog semicircular canals (SCC). Group I (mGluR1α) and group II (mGluR2/3) mGluR immunoreactivities were distributed to the vestibular ganglion neurons, and this can be attributed to a postsynaptic locus of metabotropic regulation of rapid excitatory transmission. The effects of group I/II mGluR agonist (1S,3R)-1-aminocyclopentane-trans-1,3-dicarboxylic acid (ACPD) and antagonist (R,S)-α-methyl-4-carboxyphenylglycine (MCPG) on resting and chemically induced afferent activity were studied. ACPD (10–100 μM) enhanced the resting discharge frequency. MCPG (5–100 μM) led to a concentration-dependent decrease of both resting activity and ACPD-induced responses. If the discharge frequency had previously been restored by l-glutamate (l-Glu) in high-Mg2+ solution, ACPD elicited a transient increase in the firing rate in the afferent nerve suggesting that ACPD acts on postsynaptic receptors. The l-Glu agonists, α-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) and N-methyl-d-aspartate (NMDA), were tested during application of ACPD. AMPA- and NMDA-induced responses were higher in the presence than absence of ACPD, implicating mGluR in the modulation of ionotropic glutamate receptors. These results indicate that activation of mGluR potentiates AMPA and NMDA responses through a postsynaptic interaction. We conclude that ACPD may exert modulating postsynaptic effects on vestibular afferents and that this process is activity-dependent.

Abstract Cell-penetrating peptides (CPPs) allow intracellular delivery of cargo molecules. They provide efficient methodology to transfer bioactive molecules in cells, in particular in conditions when transcription or translation of cargo-encoding sequences is not desirable or achievable. 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 (V m ). 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 V m 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 sizes, we assessed the diameter of the water pores in living cells and found that they readily accommodated the passage of 2 nm-wide molecules, in accordance with the structural characteristics of the pores predicted by in silico modeling. Pharmacological manipulation to lower transmembrane potential boosted CPPs 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 membrane. This opens the ground for strategies aimed at improving the ability of cells to capture CPP-linked cargos in vitro and in vivo .

19 NO UV EL LE S M AG AZ IN E les parenchymateuses (probablement hepatiques) et non par les cellules hematopoietiques (probablement dendritiques). Ces donnees permettent de reconstituer le scenario suivant : apres inoculation dermique au cours d’une piqure de moustique, les sporozoites migreraient dans le ganglion lymphatique de drainage, ou ils seraient presentes par des cellules dendritiques a des lymphocytes T CD8+ naifs, permettant leur activation. Ces cellules activees migreraient alors dans la circulation et le foie ou elles seraient alors capables de reconnaitre des antigenes parasitaires presentes directement par l’hepatocyte, et non plus par une cellule presentatrice d’antigene professionnelle. Bien que la reponse immunologique induite ne soit pas complete, elle pourrait permettre de contenir l’infection parasitaire [2]. Ces donnees nouvelles vont certainement avoir des consequences determinantes sur la facon de concevoir la vaccination antipalustre. Elles constituent un argument supplementaire pour une approche vaccinale fondee sur l’inoculation intradermique de parasites entiers ou tout du moins d’antigenes presentes a la fois sur le sporozoite et sur le foie infecte. ‡ Lymph nodes: first barrier against Plasmodium

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.

<p>Supplementary Methods.</p>

<div>Abstract<p>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 <i>in vitro</i> 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.</p>Significance:<p>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.</p></div>

<p>Supplementary Tables. Supplementary Table 1. Target sequences for guide RNA used to create KO stable cell lines with CRISP/Cas9 technology, Supplementary Table 2. Primers used for qPCR Analysis of Transcription Factor EB (TFEB) regulated genes (human), Supplementary Table 3. Primers used for qPCR Analysis of Senescence genes (human), Supplementary Table 4. Primers used for qPCR Analysis of housekeeping genes (human), Supplementary Table 5. Primers used for FLCN mutagenesis</p>

<p>Supplementary Tables. Supplementary Table 1. Target sequences for guide RNA used to create KO stable cell lines with CRISP/Cas9 technology, Supplementary Table 2. Primers used for qPCR Analysis of Transcription Factor EB (TFEB) regulated genes (human), Supplementary Table 3. Primers used for qPCR Analysis of Senescence genes (human), Supplementary Table 4. Primers used for qPCR Analysis of housekeeping genes (human), Supplementary Table 5. Primers used for FLCN mutagenesis</p>

<p>Supplementary Figures and Supplementary Figure legends. Supplementary Fig. 1.- CDK4 inhibition or depletion decreases mTORC1 activity in several cancer cell lines, Supplementary Fig. 2.- CDK4 effects on mTORC1 are α-ketoglutarate and E2F1-independent, Supplementary Fig. 3.- CDK4 phosphorylates FLCN, Supplementary Fig. 4.- CDK4 regulates mTORC1 activity through phosphorylating FLCN, Supplementary Fig. 5.- CDK4 inhibition or depletion increases lysosomal mass, Supplementary Fig. 6.- Hematoxylin and eosin staining and gene expression analysis of tumors from mice treated with or without LY2835219, Supplementary Fig. 7.- In vitro cell death analysis and in vivo electron microscopy analysis of tumors treated with DMSO, LY2835219, A769662 and the combination of A769662 with LY2835219</p>

<p>Supplementary Figures and Supplementary Figure legends. Supplementary Fig. 1.- CDK4 inhibition or depletion decreases mTORC1 activity in several cancer cell lines, Supplementary Fig. 2.- CDK4 effects on mTORC1 are α-ketoglutarate and E2F1-independent, Supplementary Fig. 3.- CDK4 phosphorylates FLCN, Supplementary Fig. 4.- CDK4 regulates mTORC1 activity through phosphorylating FLCN, Supplementary Fig. 5.- CDK4 inhibition or depletion increases lysosomal mass, Supplementary Fig. 6.- Hematoxylin and eosin staining and gene expression analysis of tumors from mice treated with or without LY2835219, Supplementary Fig. 7.- In vitro cell death analysis and in vivo electron microscopy analysis of tumors treated with DMSO, LY2835219, A769662 and the combination of A769662 with LY2835219</p>

<div>Abstract<p>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 <i>in vitro</i> 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.</p>Significance:<p>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.</p></div>

<p>Supplementary Methods.</p>

Abstract 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 dose 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.

The postnatal developmental expression and the distribution of the glutamate transporters (GLAST, GLT-1 and EAAC1) were analyzed in rat vestibular nuclei (VN), at birth and during the following 4 weeks. Analyses were performed using reverse transcriptase-polymerase chain reaction and immunoblotting of GLAST, GLT-1 and EAAC1 mRNA and protein during the postnatal development of the VN neurons and their afferent connections. We also studied the distribution of each glutamate transporter in the medial and lateral VN by use of immunocytochemistry and confocal microscopy. GLAST, GLT-1 and EAAC1 mRNA and protein were present in the VN at each developmental stage. GLAST was highly expressed mainly in glia from birth to the adult stage, its distribution pattern was heterogeneous depending on the region of the medial and lateral VN. GLT-1 expression increased dramatically during the second and third postnatal weeks. At least during the first postnatal week, GLT-1 was expressed in the soma of neurons. EAAC1 was detected in neurons and decreased from the third week. These temporal and regional patterns of GLAST, GLT-1 and EAAC1 suggest that they play different roles in the maturation of glutamatergic synaptic transmission in the medial and lateral VN during postnatal development.

Perinatal asphyxia remains one of the challenging conditions in neonatology, with high prevalence, high mortality and neurological morbidity and no pharmacological neuroprotective strategy. Three main types of neuronal cell death have been described: apoptosis, necrosis and more recently, autophagic cell death, which is a physiological process of degradation, but excessive autophagy may be involved in cell death. Recent studies from others as well as from our own group showed that inhibition of autophagy is neuroprotective in neonatal models of cerebral ischemia in animals. We showed in a model of perinatal asphyxia in rats an increased neuronal autophagic flux linked to cell death. Following our observations in animals, our objectives for the present study were: 8 newborns at term who died in the Division of Neonatology from 2004-2008 were selected retrospectively and anonymously on the criteria: asphyctic event, Sarnat III, death and accorded autopsy. In histological sections from the frontal cortex, hippocampus, basal ganglia and thalamus, necrosis, apoptosis, but also autophagy were present, with a distinct distribution pattern. The different markers of autophagosomes, lysosomes and apoptosis were tested by immunohistochemistry, showing an increase of autophagic flux in areas affected by asphyxia. These observations lead to a new understanding in the mechanisms of neuronal cell death following perinatal asphyxia.

Introduction: Various studies from hypoxic-ischemic animals haveinvestigated neuroprotection by targeting necrosis and apoptosis with inconclusive results. Three types of cell death have been described: apoptosis, necrosis and more recently, autophagic cell death. While autophagy is a physiological process of degradation of cellular components, excessive autophagy may be involved in cell death. Recent studies showed that inhibition of autophagy is neuroprotective in rodent neonatal models of cerebral ischemia. Furthermore, neonatal hypoxia-ischemia strongly increased neuronal autophagic flux which is linked to cell death in a rat model of perinatal asphyxia. Following our observations in animals, the aim of the present study was to characterize the different neuronal death phenotypes and to clarify whether autophagic cell death could be also involved in neuronal death in the human newborns after perinatal asphyxia. Methods: we selected retrospectively and anonymously all newborns who died in our unit of neonatology between 2004 and 2009, with the following criteria: gestational age >36 weeks, diagnosis of perinatal asphyxia (Apgar <5 at 5 minutes, arterial pH <7.0 at 1 hour of life and encephalopathy Sarnat III) and performed autopsy. The brain of 6 cases in asphyxia group and 6 control cases matching gestational age who died of pulmonary or other malformations were selected. On histological sections of thalamus, frontal cortex and hippocampus, different markers of apoptosis (caspase 3, TUNEL), autophagosomes (LC3-II) and lysosomes (LAMP1, Cathepsin D) were tested by immunohistochemistry. Results: Preliminary studies on markers of apoptosis (TUNEL, caspase 3) and of autophagy (Cathepsin D, LC3II, LAMP1) showed an expected increase of apoptosis, but also an increase of neuronal autophagic flux in the selected areas. The distribution seems to be region specific. Conclusion: This is the first time that autophagic flux linked with cell death is shown in brain of human babies, in association with hypoxicischemic encephalopathy. This work leads to a better understanding of the mechanisms associated with neuronal death following perinatal asphyxia and determines whether autophagy could be a promising therapeutic target.

Background Moderate autophagy occurs in healthy cells to maintain cellular homeostasis, but excessive autophagy has been involved in cell death mechanisms. Enhanced autophagy has been shown to occur in different excitotoxicity-related conditions, but its function remains unclear. Objectives The present study investigated the effect of cerebral hypoxia-ischemia on autophagy and autophagic cell death in a neonatal rodent model. Methods P7 rats underwent permanent carotid occlusion followed by 2 h of hypoxia (8%) with subsequent cortical, striatal, hippocampal and thalamic lesions. Using acid phosphatase histochemistry, immunocytochemistry and Western blots against lysosomal and autophagic markers, autophagy was studied and confirmed by electron microscopy. Results There was a significant increase in LC3-II expression in all four studied areas at 6 and 24 h after HI, mainly in neurons. Lysosomal activities were also increased following HI. Many neurons were strongly labeled for acid phophatase, and cathepsin B and D. These increases were confirmed using enzymatic assays and the presence of both autophagosomes and autolysosomes was detected using colocalization studies in the same neuronal cells. Electron microscopy confirmed the presence of numerous autophagosomes in the cytoplasm of dying neurons in the lesion (at 6 h) and also autolysosomes (at 24 h). Preliminary in vitro results using siRNA for Beclin 1 (Atg 8) was protective in a serum deprivation model. Conclusions HI strongly enhanced autophagy by the presence of increased numbers of autophagosomes and autolysosomes mainly in neuronal cells and suggests that autophagic cell death mediates neuronal cell death in hypoxia-ischemia, opening new neuroprotective approaches.

Summary Cell-penetrating peptides (CPPs) allow intracellular delivery of cargo molecules. CPPs provide efficient methodology to transfer bioactive molecules in cells, in particular in conditions when transcription or translation of cargo-encoding sequences is not desirable or achievable. The mechanisms allowing CPPs to enter cells are ill-defined and controversial. This work identifies potassium channels as key regulators of cationic CPP translocation. Using a CRISPR/Cas9-based screening, we discovered that KCNQ5, KCNN4, and KCNK5 positively modulate CPP cellular direct translocation by reducing transmembrane potential (Vm). Cationic CPPs further decrease the Vm to megapolarization values (about −150 mV) leading to the formation of ∼2 nm-wide water pores used by CPPs to access the cell’s cytoplasm. Pharmacological manipulation to lower transmembrane potential boosted CPPs cellular uptake in zebrafish and mouse models. Besides identifying the first genes that regulate CPP translocation, this work characterizes key mechanistic steps used by CPPs to cross cellular membrane. This opens the ground for pharmacological strategies augmenting the susceptibility of cells to capture CPP-linked cargos in vitro and in vivo.

Endocytosis and autophagy are known to be enhanced in some cases of neuronal death, and there is evidence that they can be directly involved in the death mechanism. We here report that both these trafficking events are enhanced by cerebral ischemia in neonatal (P12) rats, in which the main (cortical) branch of the middle cerebral artery was occluded by cauterization in association with clamping of the common carotid artery on the same side for 1.5 h. We also analyze the endocytic phenomenon pharmacologically in dissociated neuronal cultures exposed to NMDA. The enhanced endocytosis was shown in rats that received an intracerebroventricular injection of 4.4 kD FITC-dextran at the time of carotid unclamping, and were sacrificed 24 h later. There was strong neuronal endocytosis restricted to the ischemic zone, which was specifically labelled by immunocytochemistry for early endosomal antigen 1 (EEA1). In dissociated neuronal cultures a similar NMDA-induced endocytosis of FITC-dextran was observed; it increased with the dose of NMDA and the duration of NMDA treatment, and with a toxic dose the strongest endocytosis was observed microscopically to be in neurons stained with propidium iodide (a marker for dying or dead cells). However, to study specifically the endocytotic mechanism without the complication of cell death, we determined a combination of NMDA concentration and timing that enhanced endocytosis but with minimal cell death, as judged by LDH release, and measured the endocytosis in cellular extracts by fluorescence spectrometry. This revealed two components of dextran internalisation: they were both confirmed to be endocytic by the fact that they did not occur at 4°C, but they had completely different pharmacological profiles. One occurred constitutively (even in the absence of NMDA) and was blocked by inhibitors of classical fluid-phase dynamin-independent endocytosis (e.g. by wortmannin). The other component was induced by NMDA, insensitive to fluid-phase inhibitors, but sensitive to inhibitors of dynamin-mediated endocytosis such as 0.4 M sucrose and also to the JNK pathway inhibitor D-JNKI1, a powerful neuroprotectant. The latter inhibitors had little effect on the constitutive component of endocytosis. Although most dynamin-mediated endocytosis is receptor-mediated, competition experiments showed that the NMDA-induced endocytosis did not depend on a receptor specific for dextran. Enhanced lysosomal activity, presumably autophagy, was shown in neurones within the ischemic region (again at 24 h after carotid unclamping) by acid phosphatase histochemistry and by immunohistochemistry for lysosome-associated membrane protein (LAMP)-1. Cathepsin D was also increased in the ischemic region, but appeared to be in glia, not in neurons. The importance of these results is twofold.

Article Figures and data Abstract Editor's evaluation eLife digest Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract 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. Editor's evaluation Although the role of membrane potential in cell-penetrating peptide (CPP) translocation has been consistently described in artificial systems, this multi scale study combining cell biology, genetics and in silico approaches further extends this topic to a live cell context where it shows that internalization stops when the membrane polarization is decreased by the removal of potassium channels. It proposes an original mechanism of CPP translocation based on transient water pore formation, which should be of interest for biophysicists, cell biologists and for applications such as drug deliver. https://doi.org/10.7554/eLife.69832.sa0 Decision letter Reviews on Sciety eLife's review process eLife digest 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. Introduction Cell-penetrating peptides (CPPs) are short non-toxic sequences of 5–30 amino acids present in proteins able to cross membranes such as homeoproteins and some viral components. CPPs can also be used to deliver bioactive cargos (siRNAs, DNA, polypeptides, liposomes, nanoparticles, and others) in cells for therapeutic or experimental purposes (Bechara and Sagan, 2013; Futaki et al., 2013; Guidotti et al., 2017; Illien et al., 2016; Jones and Sayers, 2012; Koren and Torchilin, 2012; Madani et al., 2011; Mueller et al., 2008; Ruseska and Zimmer, 2020; Trabulo et al., 2010; Vasconcelos et al., 2013). Even though they differ in their origin (Frankel and Pabo, 1988; Green and Loewenstein, 1988; Joliot et al., 1991; Oehlke et al., 1998) and physico-chemical properties, the majority of CPPs carry positive charges in their sequence (Bechara and Sagan, 2013; Guidotti et al., 2017; Jones and Sayers, 2012; Madani et al., 2011). Polyarginine (e.g. R9), HIV-1 TAT47-57, and Penetratin (Antennapedia43-58) are among the most used and studied CPPs. The mode of CPP cellular entry is still debated and no proteins have been identified that regulate this process. CPP entry starts after the initial electrostatic interactions between the positively charged CPP and the negatively charged components of the cell membrane (Bechara and Sagan, 2013; Futaki et al., 2013; Guidotti et al., 2017; Jones and Sayers, 2012; Koren and Torchilin, 2012; Madani et al., 2011; Ruseska and Zimmer, 2020; Trabulo et al., 2010; Vasconcelos et al., 2013). Interaction with acid sphingomyelinase (Verdurmen et al., 2010) and glycosaminoglycans (Amand et al., 2012; Bechara et al., 2013; Butterfield et al., 2010; Fuchs and Raines, 2004; Futaki and Nakase, 2017; Ghibaudi et al., 2005; Gonçalves et al., 2005; Hakansson and Caffrey, 2003; Rullo et al., 2011; Rusnati et al., 1999; Ziegler, 2008; Ziegler and Seelig, 2004; Ziegler and Seelig, 2011), local membrane deformation (Hirose et al., 2012), as well as calcium fluxes (Melikov et al., 2015) have been suggested to play a role in CPP internalization. CPPs enter cells through a combination of two non-mutually exclusive mechanisms (Illien et al., 2016; Bechara et al., 2013): endocytosis and direct translocation (Bechara and Sagan, 2013; Futaki et al., 2013; Guidotti et al., 2017; Jones and Sayers, 2012; Koren and Torchilin, 2012; Madani et al., 2011; Ruseska and Zimmer, 2020; Trabulo et al., 2010; Vasconcelos et al., 2013). The nature of these entry mechanisms is debated and not fully understood at the molecular level. The vesicular internalization of CPPs has been suggested to occur through clathrin-dependent endocytosis, macropinocytosis, and caveolin-1-mediated endocytosis (Bechara and Sagan, 2013; Futaki et al., 2013; Guidotti et al., 2017; Jones and Sayers, 2012; Koren and Torchilin, 2012; Madani et al., 2011; Trabulo et al., 2010). However, recent data indicate that CPP endocytosis proceeds via a newly discovered pathway that is Rab14-dependent but Rab5- and Rab7-independent (Trofimenko et al., 2021). When CPPs are endocytosed, access to the cytosol requires that the CPPs break out of endosomes through a poorly understood process called endosomal escape. Direct translocation allows the CPPs to access the cytosol through their ability to cross the plasma membrane. There is currently no unifying model to explain mechanistically how direct translocation proceeds and no genes have yet been identified to modulate the manner by which CPPs cross cellular membranes. Direct translocation across the plasma membrane often seemed to originate from specific areas of the cells, suggesting discrete structures on the plasma membrane involved in CPP entry (Allolio et al., 2018; Duchardt et al., 2007; Hirose et al., 2012; Wallbrecher et al., 2017; Ziegler et al., 2005). There is a general consensus though that an adequate plasma membrane potential (Vm) is required for direct translocation to occur based on live cell experiments (Rothbard et al., 2004; Wallbrecher et al., 2017; Zhang et al., 2009), as well as in silico studies (Gao et al., 2019; Lin and Alexander-Katz, 2013; Moghal et al., 2020; Via et al., 2018). Electrophysiological and pharmacological Vm modulations have revealed that depolarization blocks CPP internalization (Rothbard et al., 2004; Zhang et al., 2009) and hyperpolarization improves the internalization of cationic CPPs (Chaloin et al., 1998; Henriques et al., 2005; Moghal et al., 2020; Rothbard et al., 2004; Wallbrecher et al., 2017). By itself, a sufficiently low Vm (i.e. hyperpolarization) appears to trigger CPP direct translocation in live cells (Rothbard et al., 2004; Wallbrecher et al., 2017; Zhang et al., 2009). In silico modeling has provided evidence that membrane hyperpolarization leads to the formation of transient water pores, allowing CPP translocation into cells (Gao et al., 2019; Herce and Garcia, 2007; Herce et al., 2009; Lin and Alexander-Katz, 2013; Via et al., 2018), but the free energy landscape governing CPP translocation has not been determined. Moreover, the nature and the structural characteristics of the pores used by CPPs to cross the plasma membrane have not been investigated in live cells. Here, we provide the first genetic evidence that validates the importance of Vm for CPP direct translocation and we characterize the diameter of the water pores used by CPPs to enter live cells. We also determined the role of the Vm in modulating the free energy barrier associated with membrane translocation and the impact of the Vm on CPP translocation kinetics. Results Modes of TAT-RasGAP317-326 cellular entry In the present work, we have used TAT-RasGAP317-326 as a model compound to investigate the molecular basis of CPP cellular internalization. This peptide is made up of the TAT48-57 CPP and a 10 amino acid sequence derived from the SH3 domain of p120 RasGAP (Michod et al., 2004). TAT-RasGAP317-326 sensitizes cancer cells to chemo-, radio-, and photodynamic therapies (Chevalier et al., 2015; Michod et al., 2009; Pittet et al., 2007; Tsoutsou et al., 2017) and prevents cell migration and invasion (Barras et al., 2014). This peptide also exhibits antimicrobial activity (Heulot et al., 2017; Georgieva et al., 2021; Heinonen et al., 2021). Some cancer cell lines, such as Raji (Burkitt’s lymphoma), SKW6.4 (transformed B-lymphocytes), and HeLa (cervix carcinoma), are directly killed by this peptide (Heulot et al., 2016). The manner by which TAT-RasGAP317-326 kills cells has recently been uncovered (Serulla et al., 2020). The peptide first accesses the cell’s cytosol by direct translocation through the plasma membrane. It then binds to specific phospholipids, such as phosphatidylserine and phosphatidylinositol-bisphosphate that are enriched in the inner leaflet of the plasma membrane. This binding allows the peptide to disrupt the cell’s membrane causing its death by necrosis. Most CPPs can enter cells by direct translocation and by endocytosis (Bechara and Sagan, 2013; Futaki et al., 2013; Guidotti et al., 2017; Illien et al., 2016; Jones and Sayers, 2012; Koren and Torchilin, 2012; Madani et al., 2011; Mueller et al., 2008; Ruseska and Zimmer, 2020; Trabulo et al., 2010; Vasconcelos et al., 2013). This is also the case for TAT-RasGAP317-326 (Figure 1A–B and Videos 1–3). Two types of staining were observed in cells incubated with this peptide: (i) vesicular only and (ii) vesicular and cytosolic (Figure 1A and Figure 1—figure supplement 1A). When the peptide cytosolic signal was strong, it masked the vesicular staining (Figure 1A). In our experimental settings, the cytosolic acquisition of TAT-RasGAP317-326 occurred only through direct translocation and not through endosomal escape (Figure 1—figure supplement 2, Video 4) and was not due to phototoxicity (Figure 1—figure supplement 3) as can occur in some settings (Dixit and Cyr, 2003; Ha and Tinnefeld, 2012; Levitus and Ranjit, 2011; Zheng et al., 2014). Figure 1 with 4 supplements see all Download asset Open asset TAT-RaGAP317-326 cellular entry modes. (A) Depiction of the different modes of cell-penetrating peptide (CPP) entry into cells. Confocal microscopy was performed on the indicated cell lines incubated for 1 hr with 40 μM FITC-TAT-RasGAP317-326 in RPMI, 10% fetal bovine serum (FBS). Cells were washed with PBS prior to visualization. Vesicular staining is indicative of CPP endocytosis while diffuse cytosolic staining is a consequence of CPP direct translocation into cells. Scale bar: 10 μm. (B) Quantitation of the different modes of CPP entry as a function of time (FITC-TAT-RasGAP317-326 continually present in the media) using the experimental conditions presented in panel A. Types of staining were visually quantitated as indicated in Figure 1—figure supplement 1A (n = 157 cells per condition). There was no indication of fluorescence quenching, due to endosomal acidification, preventing the detection of CPP-containing endosomes (in at least during the first hour of CPP exposure) (Figure 1—figure supplement 1B). TAT-RasGAP317-326 enters cells via endocytosis and direct translocation, but only direct translocation mediates its biological activity and leads to cell death (Figure 1—figure supplement 4). Results correspond to the average of three independent experiments. Video 1 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg TAT-RasGAP317-326 internalization in Raji cells over a 16-hr period. Representative confocal time-lapse recording of wild-type Raji cells incubated with 5 μM TAT-RasGAP317-326 for 16 hr in RPMI in the absence of serum. For the first 30 min of the recording, images were taken every 30 s, then until the end of the recording, images were taken every 5 min. Peptide was present in the media throughout the recording. Yellow and pink arrows indicate cells taking up the peptide by direct translocation and by endocytosis, respectively. Cyan arrows point toward labeled endosomes and green asterisks to dead cells. Scale bar: 20 μm. Time is displayed in hours:minutes. Video 2 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg Early peptide entry in wild-type Raji cells. Time-lapse recording of Raji cells incubated with 40 μM TAT-RasGAP317-326 for 30 min in RPMI, 10% fetal bovine serum (FBS). Peptide was present in the media throughout the recording and images were taken for 30 min at 10 s intervals. Scale bar: 10 μm. Time is displayed in minutes:seconds. Video 3 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg Early peptide entry in wild-type HeLa cells. Time-lapse recording of HeLa cells incubated with 80 μM FITC-TAT-RasGAP317-326 in RPMI, 10% fetal bovine serum (FBS). Yellow and pink arrows indicate cells experiencing direct translocation and endocytosis, respectively. Images were taken for 30 min at 10 s intervals. Scale bar: 20 μm. Time is displayed in minutes:seconds. Video 4 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg Distinction between endosomal escape and direct translocation. Wild-type HeLa cells were pre-incubated with 80 μM FITC-TAT-RasGAP317-326 for 30 min in RPMI, 10% fetal bovine serum (FBS) and then imaged every 5 min for 4 hr at 37°C, 5% CO2. Video on the left was recorded in the continuous presence of the peptide. Video on the right was recorded after the peptide was washed out three times with RPMI, 10% FBS. Scale bar: 10 μm. Time is displayed in hours:minutes. Identification of potassium channels as mediators of TAT cargo direct translocation into cells As TAT-RasGAP317-326 needs to translocate through the plasma membrane to reach the cytosol, a prerequisite for the peptide to kill cells (Serulla et al., 2020), we used the killing ability of the peptide in a CRISPR/Cas9 screen to identify genes involved in CPP direct translocation in two different cell lines (Raji and SKW6.4 cells) (Figure 2—figure supplement 1A). The top candidate genes identified through this approach were specific potassium channels or genes coding for proteins known to regulate such channels indirectly (e.g. PIP5K1A; Suh and Hille, 2008; Figure 2A and Figure 2—figure supplement 1B). KCNQ5, identified in Raji cells, is a voltage-dependent potassium channel. KCNN4 and KCNK5, identified in SKW6.4 cells, are calcium-activated channels and belong to the two-pore (voltage-independent) potassium channel family (Shieh et al., 2000), respectively. Figure 2 with 4 supplements see all Download asset Open asset Identification of potassium channels as mediators of direct translocation of cell-penetrating peptides (CPPs) into cells. (A) Identification of genes implicated in TAT-RasGAP317-326 internalization in Raji and SKW6.4 cells. The graphs depict the p-value (calculated using the MAGeCK procedure; see Materials and methods) for the difference in sgRNA expression between peptide-treated and control cells for the ~20,000 genes targeted by the CRISPR/Cas9 library. (B) Quantitation of TAT-RasGAP317-326 entry (top) and induced death (bottom) in wild-type (WT) and knock-out (KO) cells. The WT and the corresponding potassium channel KO versions of the indicated cell lines were pretreated or not for 30 min with 10 μM XE-991 or with TRAM-34 and then incubated (still in the presence of the inhibitors when initially added) with or without 40 μM (Raji and SKW6.4 cells) or 80 μM (HeLa cells) TAT-RasGAP317-326. Internalization was recorded after 1 hr and cell death after 16 hr (Raji and SKW6.4) or 24 hr (HeLa). Results correspond to the average of three independent experiments. TAT-RasGAP317-326 concentrations and time of incubation used were adjusted so that the CPP induced similar cell death (between 60% and 90%) in the WT versions of the different cell lines. (C) Quantitation of the modalities of TAT-RasGAP317-326 entry in WT and KCNQ5 KO Raji cells. Cells were incubated with FITC-TAT-RasGAP317-326 for various periods of time and peptide staining was visually quantitated on confocal images (n = 165 cells for each time-point). The high percentage of cells with vesicular staining in the KO cells results from the absence of strong diffuse staining masking endosomes. The results correspond to the average of three experiments. These potassium channels were pharmacologically or genetically inactivated (Figure 2B and Figure 2—figure supplement 2A-C) to validate their involvement in the direct translocation of TAT-RasGAP317-326 through the plasma membrane and the resulting death induction. The KCNQ family inhibitor, XE-991 (Schroeder et al., 2000), as well as KCNQ5 genetic invalidation (Figure 2—figure supplement 2A), fully blocked peptide internalization in Raji cells and protected them from the killing activity of the peptide (Figure 2B and Figure 2—figure supplement 2D). SKW6.4 cells individually lacking KCNN4 or KCNK5 (Figure 2—figure supplement 2B), or SKW6.4 cells treated with TRAM-34, a KCNN4 inhibitor (Wulff et al., 2001; Wulff et al., 2000), were impaired in their ability to take up the peptide and were partially protected against its cytotoxic activity (Figure 2B and Figure 2—figure supplement 2D). Inhibition of KCNN4 activity with TRAM-34 in KCNK5 knock-out cells did not further protect the cells against TAT-RasGAP317-326-induced death. In HeLa cells, TRAM-34, but not XE-991, inhibited TAT-RasGAP317-326 internalization and subsequent death (Figure 2B). Thus, in HeLa cells, KCNN4 channels regulate the membrane translocation of the peptide. This was confirmed by knocking out KCNN4 in these cells (Figure 2B and Figure 2—figure supplement 2C). Resistance to TAT-RasGAP317-326-induced death in KCNQ5 knock-out Raji cells and KCNN4 knock-out SKW6.4 or HeLa cells was restored through ectopic expression of the corresponding FLAG- or V5-tagged channels (Figure 2—figure supplement 2E-F), ruling out off-target effects. We next determined whether vesicular internalization or direct translocation were affected in cells with impaired potassium channel activities. Compared to their respective wild-type controls, the percentage of cells with diffuse cytosolic location of FITC-TAT-RasGAP317-326 was drastically diminished in cells lacking one of the CRISPR/Cas9 screen-identified potassium channels in the respective cell lines (Figure 2C and Figure 2—figure supplement 3A-B). This was mirrored by an increase in the percentage of knock-out cells with vesicular staining. The invalidation of potassium channels did not affect transferrin or dextran internalization into cells (Figure 2—figure supplement 3C) or the infectivity of vesicular stomatitis virus (Torriani et al., 2019), substantiating the non-involvement of these channels in endocytic pathways. One possibility to explain the above-mentioned results is that the absence of potassium channels reduces peptide binding to cells, thereby hampering subsequent peptide cellular uptake. At a 20 μM concentration, TAT-RasGAP317-326 is readily taken up by wild-type Raji cells but not by KCNQ5 knock-out cells. At this concentration, peptide binding was slightly lower in knock-out than in wild-type cells (Figure 2—figure supplement 3D, upper graph). However, augmenting the peptide concentrations in the extracellular medium of KCNQ5 knock-out cells to reach surface binding signals equivalent or higher than what was obtained in wild-type cells still did not result in peptide cellular internalization unless ≥80 μM of the peptides were used and even in this case, the uptake remained inefficient (Figure 2—figure supplement 3D). Difference in peptide binding is therefore not the cause of the inability of potassium channel knock-out cells to take up TAT-RasGAP317-326. We then assessed whether the role of potassium channels in cellular internalization also applied to TAT cargos other than RasGAP317-326. TAT-PNA is an oligonucleotide covalently bound to TAT, which can correct a splicing mutation within the luciferase-coding sequence (Abes et al., 2007; Kang et al., 1998). This can only occur if TAT-PNA reaches the cytosol. The luciferase activity triggered by TAT-PNA was diminished in the presence of potassium channel inhibitors and in potassium channel knock-out cell lines (Figure 2—figure supplement 4A). Cytosolic access of TAT-Cre, which can recombine a loxP-RFP-STOP-loxP-GFP (D’Astolfo et al., 2015; Wadia et al., 2004) gene construct, was then assessed. Switch from red to green fluorescence occurs only when TAT-Cre reaches the nucleus. This took place in wild-type Raji cells but not in the KCNQ5 knock-out cells (Figure 2—figure supplement 4B). We finally tested a clinical phase III therapeutic D-JNKI1 compound (Guidotti et al., 2017; Vasconcelos et al., 2013) used in the context of hearing loss and intraocular inflammation. The internalization of this peptide was completely blocked in Raji cells lacking KCNQ5 (Figure 2—figure supplement 4C, left). D-JNKI1 internalization was also diminished in SKW6.4 cells lacking KCNN4 and KCNK5 channels, as well as in HeLa cells lacking the KCNN4 potassium channel (Figure 2—figure supplement 4C, middle and right panels). These data demonstrate that the absence of specific potassium channels diminishes or even blocks the entry of various TAT-bound cargos. Potassium channels maintain plasma membrane polarization that is required for cationic CPP entry into cells Potassium is the main ion involved in setting the plasma membrane potential (Vm). The potassium channels identified in the CRISPR/Cas9 screen may therefore participate in the establishment of an adequate Vm permissive for CPP direct translocation (Chaloin et al., 1998; Henriques et al., 2005; Moghal et al., 2020; Rothbard et al., 2004; Wallbrecher et al., 2017; Zhang et al., 2009). Figure 3A (left graph) shows that genetic disruption or pharmacological inhibition of KCNQ5 in Raji cells led to an increase in their Vm (from –26 to –15 mV, validated with electrophysiological recordings; see Figure 3—figure supplement 1A). Surprisingly, such minimal increase in Vm in the KCNQ5 knock-out Raji cells practically abolished CPP internalization (Figure 3B, left graph), indicating that above a certain threshold, the Vm is no longer permissive for CPP direct translocation. In SKW6.4 and HeLa cells, Vm measurement was much more variable than in Raji cells. Nevertheless, a trend of increased Vm was observed when KCNN4 or KCNK5 were invalidated genetically or pharmacologically (Figure 3A, middle and right graphs) and this was accompanied by reduced peptide uptake (Figure 3B, middle and right graphs). As the CRISPR/Cas9 screens performed in various cell lines identified a variety of potassium channels required for efficient CPP internalization, we conclude that it is the Vm maintenance activity of these channels that is important for CPP direct translocation and not some specific features of the channels. Figure 3 with 5 supplements see all Download asset Open asset Potassium channels maintain plasma membrane polarization that is required for cell-penetrating peptide (CPP) entry into cells. (A) Assessment of the resting plasma membrane potential in the indicated wild-type cell lines and the corresponding potassium channel knock-out (KO) clones in the presence or in the absence 10 μM XE-991 or TRAM-34. The gray and white zones correspond to non-treated cells and inhibitor-treated cells, respectively. NT, not treated. The p-values correspond to the assessment of the significance of the differences with the control wild-type condition using ANOVA multiple comparison analysis with Dunnett’s correction. Each dot in a given condition represents an independent experiment. (B) Effect of cellular depolarization (left of the gray zone) and hyperpolarization (right of the gray zone) on peptide internalization in the absence of serum. The indicated cell lines and the corresponding channel KO clones were pretreated or not with depolarization agents (2 μg/ml gramicidin for 5 min or high extracellular potassium buffer for 30 min) or with hyperpolarization inducer (10 μM valinomycin), followed by the addition of TAT-RasGAP317-326 for 1 hr. Alternatively, hyperpolarization was achieved by ectopic expression of the KCNJ2 potassium channel. Membrane potential and peptide internalization were then determined. Membrane potential was measured in the presence of DiBac4(3) by flow cytometry. Peptide internalization was measured by flow cytometry in the presence of 0.2% trypan blue. The p-values correspond to the assessment of the significance of the differences with the control wild-type condition using ANOVA multiple comparison analysis with Dunnett’s correction. Each dot in a given condition represents an independent experiment. Treatment with valinomycin was used in the absence of serum as the latter is expected to interfere with the drug (Rimmele and Chatton, 2014). As shown in Figure 3—figure supplement 5A, removing serum from the culture medium sensitized cells to TAT-RasGAP317-326 and consequently, the CPP concentration had to be adapted accordingly (Figure 3—figure supplement 5B). Serum withdrawal does not affect the Vm (Figure 3—figure supplement 5C). (C) Quantitation of cytosolic CPP signal (top) and the number of endocytic vesicles per cell (bottom) in wild-type HeLa cells (n = 158 cells) incubated for 1 hr with 10 μM FITC-CPP in control, depolarizing (2 μg/ml gramicidin), or hyperpolarizing (10 μM valinomycin) conditions in the absence of serum based on confocal microscopy images (Figure 3—figure supplement 2D). Comparison between different conditions to non-treated control was done using ANOVA test with Dunnett’s correction for multiple comparison. The number of endocytic vesicles per cell was quantitated based on confocal images. Statistical comparison was done using t-tests. Quantitation of vesicles was not performed in hyperpolarizing conditions due to masking from strong cytosolic signal. (D) Modulation of the Vm membrane potential by varying extracellular potassium concentrations. Assessment of membrane potential changes in Raji cells incubated in RPMI medium containing the indicated concentrations of potassium chloride (isotonicity was maintained by adapting the sodium chloride concentrations; see Materials and methods). Membrane potential was measured with DiBac4(3). The results correspond to the median of six independent experiments. (E) Internalization of various CPPs in the presence of different concentrations of potassium chloride in the media. Data for a given experiment are linked with thin b