AMPK-regulated miRNA-210-3p is activated during ischaemic neuronal injury and modulates PI3K-p70S6K signalling.

Progressive neuronal injury following ischaemic stroke is associated with glutamate-induced depolarization, energetic stress and activation of AMP-activated protein kinase (AMPK). We here identify a molecular signature associated with neuronal AMPK activation, as a critical regulator of cellular response to energetic stress following ischaemia. We report a robust induction of microRNA miR-210-3p both in vitro in primary cortical neurons in response to acute AMPK activation and following ischaemic stroke in vivo. Bioinformatics and reverse phase protein array analysis of neuronal protein expression changes in vivo following administration of a miR-210-3p mimic revealed altered expression of phosphatase and tensin homolog (PTEN), 3-phosphoinositide-dependent protein kinase 1 (PDK1), ribosomal protein S6 kinase (p70S6K) and ribosomal protein S6 (RPS6) signalling in response to increasing miR-210-3p. In vivo, we observed a corresponding reduction in p70S6K activity following ischaemic stroke. Utilizing models of glutamate receptor over-activation in primary neurons, we demonstrated that induction of miR-210-3p was accompanied by sustained suppression of p70S6K activity and that this effect was reversed by miR-210-3p inhibition. Collectively, these results provide new molecular insight into the regulation of cell signalling during ischaemic injury, and suggest a novel mechanism whereby AMPK regulates miR-210-3p to control p70S6K activity in ischaemic stroke and excitotoxic injury.

Context-Specific Switch from Anti- to Pro-epileptogenic Function of the P2Y1 Receptor in Experimental Epilepsy.

Extracellular ATP activates inflammatory responses to tissue injury. It is also implicated in establishing lasting network hyperexcitability in the brain by acting upon independent receptor systems. Whereas the fast-acting P2X channels have well-established roles driving neuroinflammation and increasing hyperexcitability, the slower-acting metabotropic P2Y receptors have received much less attention. Recent studies of P2Y1 receptor function in seizures and epilepsy have produced contradictory results, suggesting that the role of this receptor during seizure pathology may be highly sensitive to context. Here, by using male mice, we demonstrate that the metabotropic P2Y1 receptor mediates either proconvulsive or anticonvulsive responses, dependent on the time point of activation in relation to the induction of status epilepticus. P2Y1 deficiency or a P2Y1 antagonist (MRS2500) administered before a chemoconvulsant, exacerbates epileptiform activity, whereas a P2Y1 agonist (MRS2365) administered at this time point is anticonvulsant. When these drugs are administered after the onset of status epilepticus, however, their effect on seizure severity is reversed, with the antagonist now anticonvulsant and the agonist proconvulsant. This result was consistent across two different mouse models of status epilepticus (intra-amygdala kainic acid and intraperitoneal pilocarpine). Pharmacologic P2Y1 blockade during status epilepticus reduces also associated brain damage, delays the development of epilepsy and, when applied during epilepsy, suppresses spontaneous seizures, in mice. Our data show a context-specific role for P2Y1 during seizure pathology and demonstrate that blocking P2Y1 after status epilepticus and during epilepsy has potent anticonvulsive effects, suggesting that P2Y1 may be a novel candidate for the treatment of drug-refractory status epilepticus and epilepsy.SIGNIFICANCE STATEMENT This is the first study to fully characterize the contribution of a metabotropic purinergic P2Y receptor during acute seizures and epilepsy. The findings suggest that targeting P2Y1 may offer a potential novel treatment strategy for drug-refractory status epilepticus and epilepsy. Our data demonstrate a context-specific role of P2Y1 activation during seizures, switching from a proconvulsive to an anticonvulsive role depending on physiopathological context. Thus, our study provides a possible explanation for seemingly conflicting results obtained between studies of different brain diseases where P2Y1 targeting has been proposed as a potential treatment strategy and highlights that the timing of pharmacological interventions is of critical importance to the understanding of how receptors contribute to the generation of seizures and the development of epilepsy.

Bok Is Not Pro-Apoptotic But Suppresses Poly ADP-Ribose Polymerase-Dependent Cell Death Pathways and Protects against Excitotoxic and Seizure-Induced Neuronal Injury.

Bok (Bcl-2-related ovarian killer) is a Bcl-2 family member that, because of its predicted structural homology to Bax and Bak, has been proposed to be a pro-apoptotic protein. In this study, we demonstrate that Bok is highly expressed in neurons of the mouse brain but that bok was not required for staurosporine-, proteasome inhibition-, or excitotoxicity-induced apoptosis of cultured cortical neurons. On the contrary, we found that bok-deficient neurons were more sensitive to oxygen/glucose deprivation-induced injury in vitro and seizure-induced neuronal injury in vivo Deletion of bok also increased staurosporine-, excitotoxicity-, and oxygen/glucose deprivation-induced cell death in bax-deficient neurons. Single-cell imaging demonstrated that bok-deficient neurons failed to maintain their neuronal Ca(2+)homeostasis in response to an excitotoxic stimulus; this was accompanied by a prolonged deregulation of mitochondrial bioenergetics.bok deficiency led to a specific reduction in neuronal Mcl-1 protein levels, and deregulation of both mitochondrial bioenergetics and Ca(2+)homeostasis was rescued by Mcl-1 overexpression. Detailed analysis of cell death pathways demonstrated the activation of poly ADP-ribose polymerase-dependent cell death in bok-deficient neurons. Collectively, our data demonstrate that Bok acts as a neuroprotective factor rather than a pro-death effector during Ca(2+)- and seizure-induced neuronal injury in vitro and in vivo SIGNIFICANCE STATEMENT: Bcl-2 proteins are essential regulators of the mitochondrial apoptosis pathway. The Bcl-2 protein Bok is highly expressed in the CNS. Because of its sequence similarity to Bax and Bak, Bok has long been considered part of the pro-apoptotic Bax-like subfamily, but no studies have yet been performed in neurons to test this hypothesis. Our study provides important new insights into the functional role of Bok during neuronal apoptosis and specifically in the setting of Ca(2+)- and seizure-mediated neuronal injury. We show that Bok controls neuronal Ca(2+)homeostasis and bioenergetics and, contrary to previous assumptions, exerts neuroprotective activities in vitro and in vivo Our results demonstrate that Bok cannot be placed unambiguously into the Bax-like Bcl-2 subfamily of pro-apoptotic proteins.

Bax regulates neuronal Ca2+ homeostasis.

Excessive Ca(2+) entry during glutamate receptor overactivation ("excitotoxicity") induces acute or delayed neuronal death. We report here that deficiency in bax exerted broad neuroprotection against excitotoxic injury and oxygen/glucose deprivation in mouse neocortical neuron cultures and reduced infarct size, necrotic injury, and cerebral edema formation after middle cerebral artery occlusion in mice. Neuronal Ca(2+) and mitochondrial membrane potential (Δψm) analysis during excitotoxic injury revealed that bax-deficient neurons showed significantly reduced Ca(2+) transients during the NMDA excitation period and did not exhibit the deregulation of Δψm that was observed in their wild-type (WT) counterparts. Reintroduction of bax or a bax mutant incapable of proapoptotic oligomerization equally restored neuronal Ca(2+) dynamics during NMDA excitation, suggesting that Bax controlled Ca(2+) signaling independently of its role in apoptosis execution. Quantitative confocal imaging of intracellular ATP or mitochondrial Ca(2+) levels using FRET-based sensors indicated that the effects of bax deficiency on Ca(2+) handling were not due to enhanced cellular bioenergetics or increased Ca(2+) uptake into mitochondria. We also observed that mitochondria isolated from WT or bax-deficient cells similarly underwent Ca(2+)-induced permeability transition. However, when Ca(2+) uptake into the sarco/endoplasmic reticulum was blocked with the Ca(2+)-ATPase inhibitor thapsigargin, bax-deficient neurons showed strongly elevated cytosolic Ca(2+) levels during NMDA excitation, suggesting that the ability of Bax to support dynamic ER Ca(2+) handling is critical for cell death signaling during periods of neuronal overexcitation.

Calpains are downstream effectors of bax-dependent excitotoxic apoptosis.

Excitotoxicity resulting from excessive Ca(2+) influx through glutamate receptors contributes to neuronal injury after stroke, trauma, and seizures. Increased cytosolic Ca(2+) levels activate a family of calcium-dependent proteases with papain-like activity, the calpains. Here we investigated the role of calpain activation during NMDA-induced excitotoxic injury in embryonic (E16-E18) murine cortical neurons that (1) underwent excitotoxic necrosis, characterized by immediate deregulation of Ca(2+) homeostasis, a persistent depolarization of mitochondrial membrane potential (Δψ(m)), and insensitivity to bax-gene deletion, (2) underwent excitotoxic apoptosis, characterized by recovery of NMDA-induced cytosolic Ca(2+) increases, sensitivity to bax gene deletion, and delayed Δψ(m) depolarization and Ca(2+) deregulation, or (3) that were tolerant to excitotoxic injury. Interestingly, treatment with the calpain inhibitor calpeptin, overexpression of the endogenous calpain inhibitor calpastatin, or gene silencing of calpain protected neurons against excitotoxic apoptosis but did not influence excitotoxic necrosis. Calpeptin failed to exert a protective effect in bax-deficient neurons but protected bid-deficient neurons similarly to wild-type cells. To identify when calpains became activated during excitotoxic apoptosis, we monitored calpain activation dynamics by time-lapse fluorescence microscopy using a calpain-sensitive Förster resonance energy transfer probe. We observed a delayed calpain activation that occurred downstream of mitochondrial engagement and directly preceded neuronal death. In contrast, we could not detect significant calpain activity during excitotoxic necrosis or in neurons that were tolerant to excitotoxic injury. Oxygen/glucose deprivation-induced injury in organotypic hippocampal slice cultures confirmed that calpains were specifically activated during bax-dependent apoptosis and in this setting function as downstream cell-death executioners.

BOK controls ER proteostasis and physiological ER stress responses in neurons.

The Bcl-2 family proteins BAK and BAX control the crucial step of pore formation in the mitochondrial outer membrane during intrinsic apoptosis. Bcl-2-related ovarian killer (BOK) is a Bcl-2 family protein with a high sequence similarity to BAK and BAX. However, intrinsic apoptosis can proceed in the absence of BOK. Unlike BAK and BAX, BOK is primarily located on the endoplasmic reticulum (ER) and Golgi membranes, suggesting a role for BOK in regulating ER homeostasis. In this study, we report that BOK is required for a full ER stress response. Employing previously characterized fluorescent protein-based ER stress reporter cell systems, we show that BOK-deficient cells have an attenuated response to ER stress in all three signaling branches of the unfolded protein response. Fluo-4-based confocal Ca2+ imaging revealed that disruption of ER proteostasis in BOK-deficient cells was not linked to altered ER Ca2+ levels. Fluorescence recovery after photobleaching (FRAP) experiments using GRP78/BiP-eGFP demonstrated that GRP78 motility was significantly lower in BOK-deficient cells. This implied that less intraluminal GRP78 was freely available and more of the ER chaperone bound to unfolded proteins. Collectively, these experiments suggest a new role for BOK in the protection of ER proteostasis and cellular responses to ER stress.

Zika virus induces FOXG1 nuclear displacement and downregulation in human neural progenitors.

Congenital alterations in the levels of the transcription factor Forkhead box g1 (FOXG1) coding gene trigger "FOXG1 syndrome," a spectrum that recapitulates birth defects found in the "congenital Zika syndrome," such as microcephaly and other neurodevelopmental conditions. Here, we report that Zika virus (ZIKV) infection alters FOXG1 nuclear localization and causes its downregulation, thus impairing expression of genes involved in cell replication and apoptosis in several cell models, including human neural progenitor cells. Growth factors, such as EGF and FGF2, and Thr271 residue located in FOXG1 AKT domain, take part in the nuclear displacement and apoptosis protection, respectively. Finally, by progressive deletion of FOXG1 sequence, we identify the C-terminus and the residues 428-481 as critical domains. Collectively, our data suggest a causal mechanism by which ZIKV affects FOXG1, its target genes, cell cycle progression, and survival of human neural progenitors, thus contributing to microcephaly.

Effect of TP53 deficiency and KRAS signaling on the bioenergetics of colon cancer cells in response to different substrates: A single cell study.

Metabolic reprogramming is a hallmark of cancer. Somatic mutations in genes involved in oncogenic signaling pathways, including KRAS and TP53, rewire the metabolic machinery in cancer cells. We here set out to determine, at the single cell level, metabolic signatures in human colon cancer cells engineered to express combinations of activating KRAS gene mutations and TP53 gene deletions. Specifically, we explored how somatic mutations in these genes and substrate availability (lactate, glucose, substrate deprivation) from the extracellular microenvironment affect bioenergetic parameters, including cellular ATP, NADH and mitochondrial membrane potential dynamics. Employing cytosolic and mitochondrial FRET-based ATP probes, fluorescent NADH sensors, and the membrane-permeant cationic fluorescent probe TMRM in HCT-116 cells as a model system, we observed that TP53 deletion and KRAS mutations drive a shift in metabolic signatures enabling lactate to become an efficient metabolite to replenish both ATP and NADH following nutrient deprivation. Intriguingly, cytosolic, mitochondrial and overall cellular ATP measurements revealed that, in WT KRAS cells, TP53 deficiency leads to an enhanced ATP production in the presence of extracellular lactate and glucose, and to the greatest increase in ATP following a starvation period. On the other hand, oncogenic KRAS in TP53-deficient cells reversed the alterations in cellular ATP levels. Moreover, cell population measurements of mitochondrial and glycolytic metabolism using a Seahorse analyzer demonstrated that WT KRAS TP53-silenced cells display an increase of the basal respiration and tightly-coupled mitochondria, in the presence of glucose as substrate, compared to TP53 competent cells. Furthermore, cells possessing oncogenic KRAS, independently of TP53 status, showed less pronounced mitochondrial membrane potential changes in response to metabolic nutrients. Furthermore, analysis of cytosolic and mitochondrial NADH levels revealed that the simultaneous presence of TP53 deletion and oncogenic KRAS showed the most pronounced alteration in cytosolic and mitochondrial NADH during metabolic stress. In conclusion, our findings demonstrate how activating KRAS mutation and loss of TP53 remodel cancer metabolism and lead to alterations in bioenergetics under metabolic stress conditions by modulating cellular ATP production, NADH oxidation, mitochondrial respiration and function.

Mitochondrial Carrier Homolog 2 Functionally Co-operates With BH3 Interacting-Domain Death Agonist in Promoting Ca2+-Induced Neuronal Injury.

The BH3 interacting-domain death agonist (BID) is a pro-apoptotic member of the Bcl-2 protein family. While proteolytic processing of BID links death receptor-induced apoptosis to the mitochondrial apoptosis pathway, we previously showed that full length BID also translocates to mitochondria during Ca2+-induced neuronal cell death. Moreover, mitochondrial carrier homolog 2 (MTCH2) was identified as a mitochondrial protein that interacts with BID during cell death. We started our studies by investigating the effect of Mtch2 silencing in a well-established model of Ca2+-induced mitochondrial permeability transition pore opening in non-neuronal HCT116 cells. We found that silencing of Mtch2 inhibited mitochondrial swelling and the associated decrease in mitochondrial energetics, suggesting a pro-death function for MTCH2 during Ca2+-induced injury. Next, we explored the role of BID and MTCH2 in mediating Ca2+-induced injury in primary cortical neurons triggered by prolonged activation of NMDA glutamate receptors. Analysis of intracellular Ca2+ transients, using time-lapse confocal microscopy, revealed that neurons lacking Bid showed markedly reduced Ca2+ levels during the NMDA excitation period. These Ca2+ transients were further decreased when Mtch2 was also silenced. Collectively, our data suggest that BID and MTCH2 functionally interact to promote Ca2+-induced neuronal injury.

Tau localises within mitochondrial sub-compartments and its caspase cleavage affects ER-mitochondria interactions and cellular Ca2+ handling.

Intracellular neurofibrillary tangles (NFT) composed by tau and extracellular amyloid beta (Aß) plaques accumulate in Alzheimer's disease (AD) and contribute to neuronal dysfunction. Mitochondrial dysfunction and neurodegeneration are increasingly considered two faces of the same coin and an early pathological event in AD. Compelling evidence indicates that tau and mitochondria are closely linked and suggests that tau-dependent modulation of mitochondrial functions might be a trigger for the neurodegeneration process; however, whether this occurs either directly or indirectly is not clear. Furthermore, whether tau influences cellular Ca2+ handling and ER-mitochondria cross-talk is yet to be explored. Here, by focusing on wt tau, either full-length (2N4R) or the caspase 3-cleaved form truncated at the C-terminus (2N4RΔC20), we examined the above-mentioned aspects. Using new genetically encoded split-GFP-based tools and organelle-targeted aequorin probes, we assessed: i) tau distribution within the mitochondrial sub-compartments; ii) the effect of tau on the short- (8-10 nm) and the long- (40-50 nm) range ER-mitochondria interactions; and iii) the effect of tau on cytosolic, ER and mitochondrial Ca2+ homeostasis. Our results indicate that a fraction of tau is found at the outer mitochondrial membrane (OMM) and within the inner mitochondrial space (IMS), suggesting a potential tau-dependent regulation of mitochondrial functions. The ER Ca2+ content and the short-range ER-mitochondria interactions were selectively affected by the expression of the caspase 3-cleaved 2N4RΔC20 tau, indicating that Ca2+ mis-handling and defects in the ER-mitochondria communications might be an important pathological event in tau-related dysfunction and thereby contributing to neurodegeneration. Finally, our data provide new insights into the molecular mechanisms underlying tauopathies.

The BAX/BAK-like protein BOK is a prognostic marker in colorectal cancer.

The intrinsic or mitochondrial apoptosis pathway is controlled by the interaction of antiapoptotic and pro-apoptotic members of the BCL-2 protein family. Activation of this death pathway plays a crucial role in cancer progression and chemotherapy responses. The BCL-2-related ovarian killer (BOK) possesses three BCL-2 homology domains and has been proposed to act in a similar pro-apoptotic pathway as the pro-apoptotic proteins BAX and BAK. In this study, we showed that stage II and III colorectal cancer patients possessed decreased levels of BOK protein in their tumours compared to matched normal tissue. BOK protein levels in tumours were also prognostic of clinical outcome but increased BOK protein levels surprisingly associated with earlier disease recurrence and reduced overall survival. We found no significant association of BOK protein tumour levels with ER stress markers GRP78 or GRP94 or with cleaved caspase-3. In contrast, BOK protein levels correlated with Calreticulin. These data indicate BOK as a prognostic marker in colorectal cancer and suggest that different activities of BOK may contribute to cancer progression and prognosis.

Control of mitochondrial physiology and cell death by the Bcl-2 family proteins Bax and Bok.

Neuronal cell death is often triggered by events that involve intracellular increases in Ca2+. Under resting conditions, the intracellular Ca2+ concentration is tightly controlled by a number of extrusion and sequestering mechanisms involving the plasma membrane, mitochondria, and ER. These mechanisms act to prevent a disruption of neuronal ion homeostasis. As these processes require ATP, excessive Ca2+ overloading may cause energy depletion, mitochondrial dysfunction, and may eventually lead to Ca2+-dependent cell death. Excessive Ca2+ entry though glutamate receptors (excitotoxicity) has been implicated in several neurologic and chronic neurodegenerative diseases, including ischemic stroke, epilepsy, and Alzheimer's disease. Recent evidence has revealed that excitotoxic cell death is regulated by the B-cell lymphoma-2 (Bcl-2) family of proteins. Bcl-2 proteins, comprising of both pro-apoptotic and anti-apoptotic members, have been shown to not only mediate the intrinsic apoptosis pathway by controlling mitochondrial outer membrane (MOM) integrity, but to also control neuronal Ca2+ homeostasis and energetics. In this review, the role of Bcl-2 family proteins in the regulation of apoptosis, their expression in the central nervous system and how they control Ca2+-dependent neuronal injury are summarized. We review the current knowledge on Bcl-2 family proteins in the regulation of mitochondrial function and bioenergetics, including the fusion and fission machinery, and their role in Ca2+ homeostasis regulation at the mitochondria and ER. Specifically, we discuss how the 'pro-apoptotic' Bcl-2 family proteins, Bax and Bok, physiologically expressed in the nervous system, regulate such 'non-apoptotic/daytime' functions.

Computational Analysis of AMPK-Mediated Neuroprotection Suggests Acute Excitotoxic Bioenergetics and Glucose Dynamics Are Regulated by a Minimal Set of Critical Reactions.

Loss of ionic homeostasis during excitotoxic stress depletes ATP levels and activates the AMP-activated protein kinase (AMPK), re-establishing energy production by increased expression of glucose transporters on the plasma membrane. Here, we develop a computational model to test whether this AMPK-mediated glucose import can rapidly restore ATP levels following a transient excitotoxic insult. We demonstrate that a highly compact model, comprising a minimal set of critical reactions, can closely resemble the rapid dynamics and cell-to-cell heterogeneity of ATP levels and AMPK activity, as confirmed by single-cell fluorescence microscopy in rat primary cerebellar neurons exposed to glutamate excitotoxicity. The model further correctly predicted an excitotoxicity-induced elevation of intracellular glucose, and well resembled the delayed recovery and cell-to-cell heterogeneity of experimentally measured glucose dynamics. The model also predicted necrotic bioenergetic collapse and altered calcium dynamics following more severe excitotoxic insults. In conclusion, our data suggest that a minimal set of critical reactions may determine the acute bioenergetic response to transient excitotoxicity and that an AMPK-mediated increase in intracellular glucose may be sufficient to rapidly recover ATP levels following an excitotoxic insult.

BID Mediates Oxygen-Glucose Deprivation-Induced Neuronal Injury in Organotypic Hippocampal Slice Cultures and Modulates Tissue Inflammation in a Transient Focal Cerebral Ischemia Model without Changing Lesion Volume.

The BH3 interacting-domain death agonist (BID) is a pro-apoptotic protein involved in death receptor-induced and mitochondria-mediated apoptosis. Recently, it has also been suggested that BID is involved in the regulation of inflammatory responses in the central nervous system. We found that BID deficiency protected organotypic hippocampal slice cultures in vitro from neuronal injury induced by oxygen-glucose deprivation. In vivo, BID-knockout (KO) mice and wild type (WT) mice were subjected to 60 min of transient middle cerebral artery occlusion (tMCAO) to induce focal cerebral ischemia, and allowed to recover for 24 h. Infarct volumes and functional outcome were assessed and the inflammatory response was evaluated using immunofluorescence, Western blotting, quantitative PCR (qPCR) and Mesoscale multiplex analysis. We observed no difference in the infarct volume or neurological outcome between BID-KO and WT mice. The inflammatory response was reduced by BID deficiency as indicated by a change in microglial/leukocyte response. In conclusion, our data suggest that BID deficiency is neuroprotective in an in vitro model and modulates the inflammatory response to focal cerebral ischemia in vivo. However, this is not translated into a robust neuroprotection in vivo.

Kidins220/ARMS interacts with Pdzrn3, a protein containing multiple binding domains.

We report the identification of a novel partner of Kidins220/ARMS (Kinase D-interacting substrate of 220 kDa/Ankyrin Repeat-rich Membrane Spanning) an adaptor of neurotrophin receptors playing crucial roles during neurogenesis. Screening a phage display library of brain cDNA products we found that D. rerio Pdzrn3, a protein containing RING-finger and PDZ-domains, interacts with Kidins220/ARMS through its first PDZ-domain. Both zebrafish proteins share high homology with the corresponding mammalian proteins and both genes are developmentally expressed in neural districts where early neurogenesis occurs. The interaction was also confirmed by biochemical assays and by co-localization at the tips of growing neurites of PC12 cells induced with nerve growth factor.

Characterization of Puma-dependent and Puma-independent neuronal cell death pathways following prolonged proteasomal inhibition.

Proteasomal stress and the accumulation of polyubiquitinated proteins are key features of numerous neurodegenerative disorders. Previously we demonstrated that stabilization of p53 and activation of its target gene, puma (p53-upregulated mediator of apoptosis), mediated proteasome inhibitor-induced apoptosis in cancer cells. Here we demonstrated that Puma also contributed to proteasome inhibitor-induced apoptosis in mouse neocortical neurons. Although protection afforded by puma gene deletion was incomplete, we found little evidence indicating contributions from other proapoptotic BH3-only proteins. Attenuation of bax expression did not further reduce Puma-independent apoptosis, suggesting that pathways other than the mitochondrial apoptosis pathway were activated. Real-time imaging experiments in wild-type and puma-deficient neurons using a fluorescence resonance energy transfer (FRET)-based caspase sensor confirmed the involvement of a second cell death pathway characterized by caspase activation prior to mitochondrial permeabilization and, more prominently, a third, caspase-independent and Puma-independent pathway characterized by rapid cell shrinkage and nuclear condensation. This pathway involved lysosomal permeabilization in the absence of autophagy activation and was sensitive to cathepsin but not autophagy inhibition. Our data demonstrate that proteasomal stress activates distinct cell death pathways in neurons, leading to both caspase-dependent and caspase-independent apoptosis, and demonstrate independent roles for Puma and lysosomal permeabilization in this model.