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Parkinson’s disease (PD) is a neurodegenerative disease characterized by the loss of dopaminergic neurons in the substantia nigra resulting in severe and progressive motor impairments. However, the mechanisms underlying this neuronal loss remain largely unknown. Oxidative stress and ER stress have been implicated in PD and these factors are known to activate the integrated stress response (ISR). Activating transcription factor 4 (ATF4), a key mediator of the ISR, and has been reported to induce the expression of genes involved in cellular homeostasis. However, during prolonged activation ATF4 can also induce the expression of pro-death target genes. Therefore, in the present study, we investigated the role of ATF4 in neuronal cell death in models of PD. We demonstrate that PD neurotoxins (MPP+ and 6-OHDA) and α-synuclein aggregation induced by pre-formed human alpha-synuclein fibrils (PFFs) cause sustained upregulation of ATF4 expression in mouse cortical and mesencephalic dopaminergic neurons. Furthermore, we demonstrate that PD neurotoxins induce the expression of the pro-apoptotic factors Chop, Trb3, and Puma in dopaminergic neurons in an ATF4-dependent manner. Importantly, we have determined that PD neurotoxin and α-synuclein PFF induced neuronal death is attenuated in ATF4-deficient dopaminergic neurons. Furthermore, ectopic expression of ATF4 but not transcriptionally defective ATF4ΔRK restores sensitivity of ATF4-deficient neurons to PD neurotoxins. Finally, we demonstrate that the eIF2α kinase inhibitor C16 suppresses MPP+ and 6-OHDA induced ATF4 activation and protects against PD neurotoxin induced dopaminergic neuronal death. Taken together these results indicate that ATF4 promotes dopaminergic cell death induced by PD neurotoxins and pathogenic α-synuclein aggregates and highlight the ISR factor ATF4 as a potential therapeutic target in PD.

In response to micro-environmental cues such as microbial infections or T-helper 1 and 2 (TH1 and TH2) cytokines, macrophages (Mϕs) develop into M1- or M2-like phenotypes. Phenotypic polarization/activation of Mϕs are also essentially regulated by autocrine signals. Type-A γ-aminobutyric acid receptor (GABAAR)-mediated autocrine signaling is critical for phenotypic differentiation and transformation of various cell types. The present study explored whether GABAAR signaling regulates lung Mϕ (LMϕ) phenotypic activation under M1/TH1 and M2/TH2 environments. Results showed that GABAAR subunits were expressed by primary LMϕ of mice and the mouse Mϕ cell line RAW264.7. The expression levels of GABAAR subunits in mouse LMϕs and RAW264.7 cells decreased or increased concurrently with classical (M1) or alternative (M2) activation, respectively. Moreover, activation or blockade of GABAARs distinctively influenced the phenotypic characteristics of Mϕ. These results suggested that microenvironments leading to LMϕ phenotypic polarization concurrently modulates autocrine GABA signaling and its role in Mϕ activation.

Parkinson’s Disease (PD) is characterized by the loss of dopaminergic neurons in the substantia nigra resulting in severe motor impairments. However, the mechanisms underlying this neuronal loss remain largely unknown. Activating Transcription Factor 4 (ATF4), a key mediator of the Integrated Stress Response (ISR), is a transcription factor that during prolonged activation can induce the expression of pro-apoptotic target genes. Oxidative stress and ER stress have been implicated in PD and these factors are known to activate the ISR. In this study, we have determined, that both PD neurotoxins (MPP+ and 6-OHDA) and α-synuclein aggregation induced by pre-formed human alpha-synuclein fibrils (PFFs) cause sustained upregulation of ATF4 expression in mouse primary cortical and mesencephalic neurons. Furthermore, we demonstrate that PD neurotoxins induce the expression of the pro-apoptotic factors Chop, Trb3 and Puma in an ATF4-dependent manner. Importantly, using neurons derived from ATF4 +/+ and ATF4 -/- mice, we demonstrate that ATF4 promotes neuronal apoptosis and dopaminergic cell loss in cellular models of PD. Finally, we demonstrate that the eIF2α kinase inhibitor C16 suppresses MPP+ and 6-OHDA induced ATF4 activation and protects against PD neurotoxin induced neuronal death. Taken together these results indicate that ATF4 is a key regulator of dopaminergic cell death induced by PD neurotoxins and pathogenic α-synuclein aggregates and highlight the ISR as a potential therapeutic target in PD.

Activation of Group I metabotropic glutamate receptors (mGluRs) activates signaling cascades, resulting in calcium release from intracellular stores, ERK1/2 activation, and long term changes in synaptic activity that are implicated in learning, memory, and neurodegenerative diseases. As such, elucidating the molecular mechanisms underlying Group I mGluR signaling is important for understanding physiological responses initiated by the activation of these receptors. In the current study, we identify the multifunctional scaffolding protein spinophilin as a novel Group I mGluR-interacting protein. We demonstrate that spinophilin interacts with the C-terminal tail and second intracellular loop of Group I mGluRs. Furthermore, we show that interaction of spinophilin with Group I mGluRs attenuates receptor endocytosis and phosphorylation of ERK1/2, an effect that is dependent upon the interaction of spinophilin with the C-terminal PDZ binding motif encoded by Group I mGluRs. Spinophilin knock-out results in enhanced mGluR5 endocytosis as well as increased ERK1/2, AKT, and Ca(2+) signaling in primary cortical neurons. In addition, the loss of spinophilin expression results in impaired mGluR5-stimulated LTD. Our results indicate that spinophilin plays an important role in regulating the activity of Group I mGluRs as well as their influence on synaptic activity.

How the brain’s antioxidant defenses adapt to changing demand is incompletely understood. Here we show that synaptic activity is coupled, via the NMDA receptor (NMDAR), to control of the glutathione antioxidant system. This tunes antioxidant capacity to reflect the elevated needs of an active neuron, guards against future increased demand and maintains redox balance in the brain. This control is mediated via a programme of gene expression changes that boosts the synthesis, recycling and utilization of glutathione, facilitating ROS detoxification and preventing Puma-dependent neuronal apoptosis. Of particular importance to the developing brain is the direct NMDAR-dependent transcriptional control of glutathione biosynthesis, disruption of which can lead to degeneration. Notably, these activity-dependent cell-autonomous mechanisms were found to cooperate with non-cell-autonomous Nrf2-driven support from astrocytes to maintain neuronal GSH levels in the face of oxidative insults. Thus, developmental NMDAR hypofunction and glutathione system deficits, separately implicated in several neurodevelopmental disorders, are mechanistically linked., How the brain’s antioxidant defenses adapt to changing demand is not well understood. Here the authors demonstrate that synaptic activity is coupled to transcriptional control of the glutathione antioxidant system via NMDA receptors, enabling neurons to tune their antioxidant defenses.

A novel role for ATF3 is identified in acinar-to–duct cell metaplasia during pancreatic injury. ATF3 targets transcriptional regulators that affect acinar cell fate and reduces the severity of pancreatic injury. However, by doing so, ATF3 may also increase the susceptibility for progression to pancreatic adenocarcinoma., Pancreatitis is a debilitating disease of the exocrine pancreas that, under chronic conditions, is a major susceptibility factor for pancreatic ductal adenocarcinoma (PDAC). Although down-regulation of genes that promote the mature acinar cell fate is required to reduce injury associated with pancreatitis, the factors that promote this repression are unknown. Activating transcription factor 3 (ATF3) is a key mediator of the unfolded protein response, a pathway rapidly activated during pancreatic insult. Using chromatin immunoprecipitation followed by next-generation sequencing, we show that ATF3 is bound to the transcriptional regulatory regions of >30% of differentially expressed genes during the initiation of pancreatitis. Of importance, ATF3-dependent regulation of these genes was observed only upon induction of pancreatitis, with pathways involved in inflammation, acinar cell differentiation, and cell junctions being specifically targeted. Characterizing expression of transcription factors that affect acinar cell differentiation suggested that acinar cells lacking ATF3 maintain a mature cell phenotype during pancreatitis, a finding supported by maintenance of junctional proteins and polarity markers. As a result, Atf3–/– pancreatic tissue displayed increased tissue damage and inflammatory cell infiltration at early time points during injury but, at later time points, showed reduced acinar-to–duct cell metaplasia. Thus our results reveal a critical role for ATF3 as a key regulator of the acinar cell transcriptional response during injury and may provide a link between chronic pancreatitis and PDAC.

Although Numb exhibits its tumor-suppressive function in breast cancer in part by binding to and stabilizing p53, it is unknown how the Numb-p53 interaction is regulated in cells. We found that Numb is methylated in its phosphotyrosine-binding (PTB) domain by the lysine methyltransferase Set8. Moreover, methylation uncouples Numb from p53, resulting in increased p53 ubiquitination and degradation. While Numb promotes apoptosis in a p53-dependent manner, the apoptotic function is abolished when Numb is methylated by Set8 or the Lys methylation sites in Numb are mutated. Conversely, the Numb-p53 interaction and Numb-mediated apoptosis are significantly enhanced by depletion of Set8 from cancer cells or by treating the cells with doxorubicin, a chemotherapeutic drug that causes a reduction in the mRNA and protein levels of Set8. Our work identifies the Set8-Numb-p53 signaling axis as an important regulatory pathway for apoptosis and suggests a therapeutic strategy by targeting Numb methylation.

Stimulation of Group I metabotropic glutamate receptors (mGluR1 and mGluR5) leads to activation of a wide variety of signalling pathways. mGluRs couple to Gα(q/)₁₁ proteins, activating phospholipase Cβ1 resulting in both diacylglycerol and inositol-1,4,5-triphosphate formation followed by the activation of protein kinase C. In addition, mGluR activation can lead to modulation of a number of ion channels, such as different types of calcium and potassium channels. Group I mGluRs can also activate other downstream protein kinases, such as ERK1/2 and AKT, which are implicated in cellular growth, differentiation, and survival. Moreover, Group I mGluRs interact with a variety of different proteins that are important for the regulation of synaptic signalling, such as Homer and PDZ domain containing proteins, such as Tamalin. A role for mGluR1/5 in a number of disease states has also been proposed. As mGluR1/5 signal transduction is complex and involves multiple partners, a better understanding of alterations in mGluR signalling in brain disorders will be required in order to discern the molecular and cellular basis of these pathologies. This review will highlight recent findings concerning mGluR signaling alterations in brain pathologies, such as stroke, fragile X syndrome, Alzheimer's disease, Parkinson's disease, Huntington's disease, epilepsy, and drug addiction.

Developmental and pathological death of neurons requires activation of a defined pathway of cell cycle proteins. However, it is unclear how this pathway is regulated and whether it is relevantin vivo. A screen for transcripts robustly induced in cultured neurons by DNA damage identified Sertad1, a Cdk4 (cyclin-dependent kinase 4) activator. Sertad1 is also induced in neurons by nerve growth factor (NGF) deprivation and Aβ (β-amyloid). RNA interference-mediated downregulation of Sertad1 protects neurons in all three death models. Studies of NGF withdrawal indicate that Sertad1 is required to initiate the apoptotic cell cycle pathway since its knockdown blocks subsequent pathway events. Finally, we find that Sertad1 expression is required for developmental neuronal death in the cerebral cortex. Sertad1 thus appears to be essential for neuron death in trophic support deprivationin vitroandin vivoand in models of DNA damage and Alzheimer's disease. It may therefore be a suitable target for therapeutic intervention.

Huntington's disease (HD) is an autosomal-dominant neurodegenerative disorder caused by a polyglutamine expansion in the huntingtin protein (Htt). Group I metabotropic glutamate receptors (mGluRs) are coupled to Gαqand play an important role in neuronal survival. We have previously demonstrated that mGluRs interact with Htt. Here we used striatal neuronal primary cultures and acute striatal slices to demonstrate that mGluR-mediated signaling pathways are altered in a presymptomatic mouse model of HD (HdhQ111/Q111), as compared to those of control mice (HdhQ20/Q20). mGluR1/5-mediated inositol phosphate (InsP) formation is desensitized in striatal slices fromHdhQ111/Q111mice and this desensitization is PKC-mediated. Despite of decreased InsP formation, (S)-3,5-dihydroxylphenylglycine (DHPG)-mediated Ca2+release is higher inHdhQ111/Q111than inHdhQ20/Q20neurons. Furthermore, mGluR1/5-stimulated AKT and extracellular signal-regulated kinase (ERK) activation is altered inHdhQ111/Q111mice. Basal AKT activation is higher inHdhQ111/Q111neurons and this increase is mGluR5 dependent. Moreover, mGluR5 activation leads to higher levels of ERK activation inHdhQ111/Q111than inHdhQ20/Q20striatum. PKC inhibition not only bringsHdhQ111/Q111DHPG-stimulated InsP formation toHdhQ20/Q20levels, but also causes an increase in neuronal cell death inHdhQ111/Q111neurons. However, PKC inhibition does not modify neuronal cell death inHdhQ20/Q20neurons, suggesting that PKC-mediated desensitization of mGluR1/5 inHdhQ111/Q111mice might be protective in HD. Together, these data indicate that group I mGluR-mediated signaling pathways are altered in HD and that these cell signaling adaptations could be important for striatal neurons survival.

The angiotensin II type 1 receptor (AT(1)R) plays an important role in cardiovascular function and as such represents a primary target for therapeutic intervention. The AT(1)R has traditionally been considered to be coupled to the activation of phospholipase C (PLC) beta via its association with G alpha(q/11), leading to increases in intracellular inositol phosphate (IP) and release of calcium from intracellular stores. In the present study, we investigated whether the small GTPase RalA contributed to the regulation of AT(1)R endocytosis and signaling. We find that neither RalA nor RalB is required for the endocytosis of the AT(1)R, but that RalA expression is required for AT(1)R-stimulated IP formation but not 5-HT(2A) receptor-mediated IP formation. AT(1)R-activated IP formation is lost in the absence of Ral guanine nucleotide dissociation stimulator (RalGDS), and requires the beta-arrestin-dependent plasma membrane translocation of RalGDS. G alpha(q/11) small interfering RNA (siRNA) treatment also significantly attenuates both AT(1)R- and 5-HT(2A) receptor-stimulated IP formation after 30 min of agonist stimulation. PLC-delta1 has been reported to be activated by RalA, and we show that AT(1)R-stimulated IP formation is attenuated after PLC-delta 1 siRNA treatment. Taken together, our results provide evidence for a G protein-coupled recepto-activated and RalGDS/Ral-mediated mechanism for PLC-delta 1 stimulation.

DNA damage is an important initiator of neuronal apoptosis and activates signaling events not yet fully defined. Using the camptothecin-induced DNA damage model in neurons, we previously showed that cyclin D1-associated cell cycle cyclin-dependent kinases (Cdks) (Cdk4/6) and p53 activation are two major events leading to activation of the mitochondrial apoptotic pathway. With gene array analyses, we detected upregulation of Cited2, a CBP (cAMP response element-binding protein-binding protein)/p300 interacting transactivator, in response to DNA damage. This upregulation was confirmed by reverse transcription-PCR and Western blot. CITED2 was functionally important because CITED2 overexpression promotes death, whereas CITED2 deficiency protects. Cited2 upregulation is upstream of the mitochondrial death pathway (BAX, Apaf1, or cytochromecrelease) and appears to be independent of p53. However, inhibition of the Cdk4 blocked Cited2 induction. The Cited2 prodeath mechanism does not involve Bmi-1 or p53. Instead, Cited2 activates peroxisome proliferator-activated receptor-γ (PPARγ), an activity that we demonstrate is critical for DNA damage-induced death. These results define a novel neuronal prodeath pathway in which Cdk4-mediated regulation of Cited2 activates PPARγ and consequently caspase.

Oxidative stress has been implicated as a key trigger of neuronal apoptosis in stroke and neurodegenerative conditions such as Alzheimer's disease, Parkinson's disease and amyotrophic lateral sclerosis. The Bcl-2 homology 3 (BH3)-only subfamily of Bcl-2 genes consists of multiple members that can be activated in a cell-type- and stimulus-specific manner to promote cell death. In the present study, we demonstrate that, in cortical neurons, oxidative stress induces the expression of the BH3-only members Bim, Noxa, and Puma. Importantly, we have determined thatPuma−/− neurons, but notBim−/− orNoxa−/− neurons, are remarkably resistant to the induction of apoptosis by multiple oxidative stressors. Furthermore, we have determined that Bcl-2-associated X protein (Bax) is also required for oxidative stress induced cell death and that Puma plays a dominant role in regulating Bax activation. Specifically, we have established that the induction of Puma, but not Bim or Noxa, is necessary and sufficient to induce a conformational change in Bax to its active state, its translocation to the mitochondria and mitochondrial membrane permeabilization. Finally, we demonstrate that whereas both Puma and BimELcan bind to the antiapoptotic family member Bcl-XL, only Puma was found to associate with Bax. This suggests that in addition to neutralizing antiapoptotic members, Puma may play a dominant role by complexing with Bax and directly promoting its activation. Overall, we have identified Puma as a dominant regulator of oxidative stress induced Bax activation and neuronal apoptosis, and suggest that Puma may be an effective therapeutic target for the treatment of a number of neurodegenerative conditions.

Extending the productive lifespan of human cells could have major implications for diseases of aging, such as atherosclerosis. We identified a relationship between aging of human vascular smooth muscle cells (SMCs) and nicotinamide phosphoribosyltransferase (Nampt/PBEF/Visfatin), the rate-limiting enzyme for NAD+ salvage from nicotinamide. Replicative senescence of SMCs was preceded by a marked decline in the expression and activity of Nampt. Furthermore, reducing Nampt activity with the antagonist FK866 induced premature senescence in SMCs, assessed by serial quantification of the proportion of cells with senescence-associated beta-galactosidase activity. In contrast, introducing the Nampt gene into aging human SMCs delayed senescence and substantially lengthened cell lifespan, together with enhanced resistance to oxidative stress. Nampt-mediated SMC lifespan extension was associated with increased activity of the NAD+-dependent longevity enzyme SIRT1 and was abrogated in Nampt-overexpressing cells transduced with a dominant-negative form of SIRT1 (H363Y). Nampt overexpression also reduced the fraction of p53 that was acetylated on lysine 382, a target of SIRT1, suppressed an age-related increase in p53 expression, and increased the rate of p53 degradation. Moreover, add-back of p53 with recombinant adenovirus blocked the anti-aging effects of Nampt. These data indicate that Nampt is a longevity protein that can add stress-resistant life to human SMCs by optimizing SIRT1-mediated p53 degradation.

Alzheimer’s disease (AD) is characterized by the deposition of Beta-Amyloid (Aβ) peptides in the brain. Aβ peptides are generated by cleavage of the Amyloid Precursor Protein (APP) by the β − and γ − secretase enzymes. Although this process is tightly linked to the internalization of cell surface APP, the compartments responsible are not well defined. We have found that APP can be rapidly internalized from the cell surface to lysosomes, bypassing early and late endosomes. Here we show by confocal microscopy and electron microscopy that this pathway is mediated by macropinocytosis. APP internalization is enhanced by antibody binding/crosslinking of APP suggesting that APP may function as a receptor. Furthermore, a dominant negative mutant of Arf6 blocks direct transport of APP to lysosomes, but does not affect classical endocytosis to endosomes. Arf6 expression increases through the hippocampus with the development of Alzheimer’s disease, being expressed mostly in the CA1 and CA2 regions in normal individuals but spreading through the CA3 and CA4 regions in individuals with pathologically diagnosed AD. Disruption of lysosomal transport of APP reduces both Aβ40 and Aβ42 production by more than 30 %. Our findings suggest that the lysosome is an important site for Aβ production and that altering APP trafficking represents a viable strategy to reduce Aβ production. Electronic supplementary material The online version of this article (doi:10.1186/s13041-015-0129-7) contains supplementary material, which is available to authorized users.

Huntington disease is caused by a polyglutamine expansion in the huntingtin protein (Htt) and is associated with excitotoxic death of striatal neurons. Group I metabotropic glutamate receptors (mGluRs) that are coupled to inositol 1,4,5-triphosphate formation and the release of intracellular Ca(2+) stores play an important role in regulating neuronal function. We show here that mGluRs interact with the Htt-binding protein optineurin that is also linked to normal pressure open angled glaucoma and, when expressed in HEK 293 cells, optineurin functions to antagonize agonist-stimulated mGluR1a signaling. We find that Htt is co-precipitated with mGluR1a and that mutant Htt functions to facilitate optineurin-mediated attenuation of mGluR1a signaling. In striatal cell lines derived from Htt(Q111/Q111) mutant knock-in mice mGluR5-stimulated inositol phosphate formation is also severely impaired when compared with striatal cells derived from Htt(Q7/Q7) knock-in mice. In addition, we show that a missense single nucleotide polymorphism optineurin H486R variant previously identified to be associated with glaucoma is selectively impaired in mutant Htt binding. Although optineurin H486R retains the capacity to bind to mGluR1a, optineurin H486R-dependent attenuation of mGluR1a signaling is not enhanced by the expression of mutant Htt. Because G protein-coupled receptor kinase 2 (GRK2) protein expression is relatively low in striatal tissue, we propose that optineurin may substitute for GRK2 in the striatum to mediate mGluR desensitization. Taken together, these studies identify a novel mechanism for mGluR desensitization and an additional biochemical link between altered glutamate receptor signaling and Huntington disease.

Previous studies have shown that DNA damage-evoked death of primary cortical neurons occurs in a p53 and cyclin-dependent kinase-dependent (CDK) manner. The manner by which these signals modulate death is unclear. Nuclear factor-kappaB (NF-kappaB) is a group of transcription factors that potentially interact with these pathways. Presently, we show that NF-kappaB is activated shortly after induction of DNA damage in a manner independent of the classic IkappaB kinase (IKK) activation pathway, CDKs, ATM, and p53. Acute inhibition of NF-kappaB via expression of a stable IkappaB mutant, downregulation of the p65 NF-kappaB subunit by RNA interference (RNAi), or pharmacological NF-kappaB inhibitors significantly protected against DNA damage-induced neuronal death. NF-kappaB inhibition also reduced p53 transcripts and p53 activity as measured by the p53-inducible messages, Puma and Noxa, implicating the p53 tumor suppressor in the mechanism of NF-kappaB-mediated neuronal death. Importantly, p53 expression still induces death in the presence of NF-kappaB inhibition, indicating that p53 acts downstream of NF-kappaB. Interestingly, neurons cultured from p65 or p50 NF-kappaB-deficient mice were not resistant to death and did not show diminished p53 activity, suggesting compensatory processes attributable to germline deficiencies, which allow p53 activation still to occur. In contrast to acute NF-kappaB inhibition, prolonged NF-kappaB inhibition caused neuronal death in the absence of DNA damage. These results uniquely define a signaling paradigm by which NF-kappaB serves both an acute p53-dependent pro-apoptotic function in the presence of DNA damage and an anti-apoptotic function in untreated normal neurons.

Sustained cellular function and viability of high-energy demanding post-mitotic cells rely on the continuous supply of ATP. The utilization of mitochondrial oxidative phosphorylation for efficient ATP generation is a function of oxygen levels. As such, oxygen deprivation, in physiological or pathological settings, has profound effects on cell metabolism and survival. Here we show that mild extracellular acidosis, a physiological consequence of anaerobic metabolism, can reprogramme the mitochondrial metabolic pathway to preserve efficient ATP production regardless of oxygen levels. Acidosis initiates a rapid and reversible homeostatic programme that restructures mitochondria, by regulating mitochondrial dynamics and cristae architecture, to reconfigure mitochondrial efficiency, maintain mitochondrial function and cell survival. Preventing mitochondrial remodelling results in mitochondrial dysfunction, fragmentation and cell death. Our findings challenge the notion that oxygen availability is a key limiting factor in oxidative metabolism and brings forth the concept that mitochondrial morphology can dictate the bioenergetic status of post-mitotic cells., In hypoxic conditions, cells depend on anaerobic respiration, which results in extracellular acidosis. Khacho et al. find that acidosis serves a protective function, enhancing mitochondrial respiratory capacity and sustaining ATP synthesis despite limited oxygen availability, by both promoting mitochondrial fusion and inhibiting fission.

Huntington's disease (HD) is an autosomal-dominant neurodegenerative disorder caused by a polyglutamine expansion in the amino-terminal region of the huntingtin protein, which promotes progressive neuronal cell loss, neurological symptoms and death. In the present study, we show that blockade of mGluR5 with MTEP promotes increased locomotor activity in both control (Hdh(Q20/Q20)) and mutant HD (Hdh(Q111/Q111)) mice. Although acute injection of MTEP increases locomotor activity in both control and mutant HD mice, locomotor activity is increased in only control mice, not mutant HD mice, following the genetic deletion of mGluR5. Interestingly, treatment of mGluR5 knockout mice with either D1 or D2 dopamine antagonists eliminates the increased locomotor activity of mGluR5 knockout mice. Amphetamine treatment increases locomotor activity in control mice, but not mGluR5 null mutant HD mice. However, the loss of mGluR5 expression improves rotarod performance and decreases the number of huntingtin intranuclear inclusions in mutant HD mice. These adaptations may be due to mutant huntingtin-dependent alterations in gene expression, as microarray studies have identified several genes that are altered in mutant, but not wild-type HD mice lacking mGluR5 expression. qPCR experiments confirm that the mRNA transcript levels of dynein heavy chain, dynactin 3 and dynein light chain-6 are altered following the genetic deletion of mGluR5 in mutant HD mice, as compared with wild-type mutant HD mice. Thus, our data suggest that mutant huntingtin protein and mGluR5 exhibit a functional interaction that may be important for HD-mediated alterations in locomotor behavior and the development of intranuclear inclusions.

Recombinant adenovirus vectors have provided a major advance in gene delivery systems for post-mitotic neurons. However, the use of these first generation vectors has been limited due to the onset of virally mediated effects on cellular function and viability. In the present study we have used primary cultures of cerebellar granule neurons to examine the efficacy and cytotoxic effects of a helper-dependent adenovirus vector (hdAd) in comparison with a first generation vector. Our results demonstrate that the hdAd system provides equally efficient infectivity with significantly reduced toxicity in comparison to first generation vectors. Neurons transduced with a high titre of a first generation vector exhibited a time-dependent shut down in global protein synthesis and impaired physiological function as demonstrated by a loss of glutamate receptor responsiveness. This was followed by an increase in the fraction of TUNEL-positive cells and a loss of neuronal survival. In contrast, hdAds could be used at titres that transduce >85% of neurons with little cytotoxic effect: cellular glutamate receptor responses and rates of protein synthesis were indistinguishable from uninfected controls. Furthermore, cell viability was not significantly affected for at least 7 days after infection. At excessive viral titres, however, infection with hdAd did cause moderate but significant changes in cell function and viability in primary neuronal cultures. Thus, while these vectors are remarkably improved over first generation vectors, these also have limitations with respect to viral effects on cellular function and viability. Gene Therapy (2000) 7, 1200-1209.

p53 is a pivotal molecule regulating the death of neurons both after acute injury and during development. The molecular mechanisms by which p53 induces apoptosis in neuronal cells, however, are not well understood. We have shown previously that adenovirus-mediated p53 gene delivery to neurons was sufficient to induce apoptosis. In the present study we have examined the molecular mechanism by which p53 evokes neuronal cell death. Adenovirus-mediated delivery of p53 to cerebellar granule neurons resulted in caspase-3 (CPP32) activation followed by terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling (TUNEL) staining and loss of viability as determined by an MTT survival assay. To determine whether Bax is essential for caspase-3 activation, p53 was expressed in Bax-deficient cells. Bax null neurons did not exhibit caspase-3 activation in response to p53 and were protected from apoptosis. To determine whether Bax-dependent caspase-3 activation was required in p53-mediated neuronal cell death, caspase-3-deficient neurons were examined. Our results indicate that caspase-3-deficient neurons exhibit a remarkable delay in apoptosis and a dramatic decrease in TUNEL-positive cells. These studies demonstrate that p53-induced cell death in postmitotic neurons involves a Bax-dependent caspase-3 activation, suggesting that these molecules are important determinants in neuronal cell death after injury.

Neuroinflammation is a common feature of acute neurological conditions such as stroke and spinal cord injury, as well as neurodegenerative conditions such as Parkinson's disease, Alzheimer's disease, and amyotrophic lateral sclerosis. Previous studies have demonstrated that acute neuroinflammation can adversely affect the survival of neural precursor cells (NPCs) and thereby limit the capacity for regeneration and repair. However, the mechanisms by which neuroinflammatory processes induce NPC death remain unclear. Microglia are key mediators of neuroinflammation and when activated to induce a pro-inflammatory state produce a number of factors that could affect NPC survival. Importantly, in the present study we demonstrate that tumor necrosis factor a (TNFα) produced by lipopolysaccharide-activated microglia is necessary and sufficient to trigger apoptosis in mouse NPCs in vitro. Furthermore, we demonstrate that microglia-derived TNFα induces NPC apoptosis via a mitochondrial pathway regulated by the Bcl-2 family protein Bax. BH3-only proteins are known to play a key role in regulating Bax activation and we demonstrate that microglia-derived TNFα induces the expression of the BH3-only family member Puma in NPCs via an NF-kB-dependent mechanism. Specifically, we show that NF-kB is activated in NPCs treated with conditioned media from activated microglia and that Puma induction and NPC apoptosis is blocked by the NF-kB inhibitor BAY-117082. Importantly, we have determined that NPC apoptosis induced by activated microglia-derived TNFα is attenuated in Puma-deficient NPCs, indicating that Puma induction is required for NPC death. Consistent with this, we demonstrate that Puma-deficient NPCs exhibit anB13-fold increase in survival as compared with wild-type NPCs following transplantation into the inflammatory environment of the injured spinal cord in vivo. In summary, we have identified a key signaling pathway that regulates neuroinflammation induced apoptosis in NPCs in vitro and in vivo that could be targeted to promote regeneration and repair in diverse neurological conditions. © 2013 Macmillan Publishers Limited.

An increasing body of evidence points to a key role of endoplasmic reticulum (ER) stress in acute and chronic neurodegenerative conditions. Extensive ER stress can trigger neuronal apoptosis, but the signaling pathways that regulate this cell death remain unclear. In the present study, we demonstrate that PUMA, a Bcl-2 homology 3 (BH3)-only member of the Bcl-2 family, is transcriptionally activated in cortical neurons by ER stress and is essential for ER-stress-induced cell death. PUMA is known to be a key transcriptional target of p53, but we have found that ER stress triggers PUMA induction and cell death through a p53-independent mechanism mediated by the ER-stress-inducible transcription factor ATF4 (activating transcription factor 4). Specifically, we demonstrate that ectopic expression of ATF4 sensitizes mouse cortical neurons to ER-stress-induced apoptosis and that ATF4-deficient neurons exhibit markedly reduced levels of PUMA expression and cell death. However, chromatin immunoprecipitation experiments suggest that ATF4 does not directly regulate the PUMA promoter. Rather, we found that ATF4 induces expression of the transcription factor CHOP (C/EBP homologous protein) and that CHOP in turn activates PUMA induction. Specifically, we demonstrate that CHOP binds to the PUMA promoter during ER stress and that CHOP knockdown attenuates PUMA induction and neuronal apoptosis. In summary, we have identified a key signaling pathway in ER-stress-induced neuronal death involving ATF4–CHOP-mediated transactivation of the proapoptotic Bcl-2 family member PUMA. We propose that this pathway may be an important therapeutic target relevant to a number of neurodegenerative conditions.

Background A central feature of Alzheimer's disease is the cleavage of the amyloid precursor protein (APP) to form beta-amyloid peptide (Aβ) by the β-secretase and γ-secretase enzymes. Although this has been shown to occur after endocytosis of APP from the cell surface, the exact compartments of APP processing are not well defined. We have previously demonstrated that APP and γ-secretase proteins and activity are highly enriched in purified rat liver lysosomes. In order to examine the lysosomal distribution and trafficking of APP in cultured cells, we generated constructs containing APP fused to a C-terminal fluorescent protein tag and N-terminal HA-epitope tag. These were co-transfected with a panel of fluorescent-protein tagged compartment markers. Results Here we demonstrate using laser-scanning confocal microscopy that although APP is present throughout the endosomal/lysosomal system in transfected Cos7 and neuronal SN56 cell lines as well as in immunostained cultured mouse neurons, it is enriched in the lysosome. We also show that the Swedish and London mutations reduce the amount of APP in the lysosome. Surprisingly, in addition to its expected trafficking from the cell surface to the early and then late endosomes, we find that cell-surface labelled APP is transported rapidly and directly from the cell surface to lysosomes in both Cos7 and SN56 cells. This rapid transit to the lysosome is blocked by the presence of either the London or Swedish mutations. Conclusions These results demonstrate the presence of a novel, rapid and specific transport pathway from the cell surface to the lysosomes. This suggests that regulation of lysosomal traffic could regulate APP processing and that the lysosome could play a central role in the pathophysiology of Alzheimer's disease.

DNA damage is a critical component of neuronal death underlying neurodegenerative diseases and injury. Neuronal death evoked by DNA damage is characterized by inappropriate activation of multiple cell cycle components. However, the mechanism regulating this activation is not fully understood. We demonstrated previously that the cell division cycle (Cdc) 25A phosphatase mediates the activation of cyclin-dependent kinases and neuronal death evoked by the DNA damaging agent camptothecin. We also showed that Cdc25A activation is blocked by constitutive checkpoint kinase 1 activity under basal conditions in neurons. Presently, we report that an additional factor is central to regulation of Cdc25A phosphatase in neuronal death. In a gene array screen, we first identified Pim-1 as a potential factor up-regulated following DNA damage. We confirmed the up-regulation of Pim-1 transcript, protein and kinase activity following DNA damage. This induction of Pim-1 is regulated by the nuclear factor kappa beta (NF-kappaB) pathway as Pim-1 expression and activity are significantly blocked by siRNA-mediated knockdown of NF-kappaB or NF-kappaB pharmacological inhibitors. Importantly, Pim-1 activity is critical for neuronal death in this paradigm and its deficiency blocks camptothecin-mediated neuronal death. It does so by activating Cdc25A with consequent activation of cyclin D1-associated kinases. Taken together, our results demonstrate that Pim-1 kinase plays a central role in DNA damage-evoked neuronal death by regulating aberrant neuronal cell cycle activation.

Neurogenesis persists in the adult brain and can contribute to learning and memory processes and potentially to regeneration and repair of the affected nervous system. Deregulated neurogenesis has been observed in neuropathological conditions including neurodegenerative diseases, trauma and stroke. However, the survival of neural precursor cells (NPCs) and newly born neurons is adversely affected by the inflammatory environment that arises as a result of microglial activation associated with injury or disease processes. In the present study, we have investigated the mechanisms by which microglia affect NPC proliferation and survival. Importantly, we demonstrate that interleukin-1β (IL-1β) produced by lipopolysaccharide/interferon-γ-activated microglia is necessary to induce cell cycle arrest and apoptosis in NPCs in vitro. Mechanistically, we show that IL-1β activates the tumor suppressor p53 through an oxidative stress-dependent mechanism resulting in p53-mediated induction of the cyclin-dependent kinase inhibitor p21 and the proapoptotic Bcl-2 (B-cell lymphoma-2) family members Puma (p53-upregulated modulator of apoptosis) and Noxa. Furthermore, we demonstrate that cell cycle arrest and apoptosis induced by recombinant IL-1β or activated microglia is attenuated in p53-deficient NPCs. Finally, we have determined that IL-1β induces NPC death via the p53-dependent induction of Puma leading to the activation of a Bax (Bcl-2-associated X protein)-mediated mitochondrial apoptotic pathway. In summary, we have elucidated a novel role for p53 in the regulation of NPC proliferation and survival during neuroinflammatory conditions that could be targeted to promote neurogenesis and repair in a number of neurological conditions.

Mitochondria release proteins that propagate both caspase-dependent and caspase-independent cell death pathways. AIF (apoptosis-inducing factor) is an important caspase-independent death regulator in multiple neuronal injury pathways. Presently, there is considerable controversy as to whether AIF is neuroprotective or proapoptotic in neuronal injury, such as oxidative stress or excitotoxicity. To evaluate the role of AIF in BAX-dependent (DNA damage induced) and BAX-independent (excitotoxic) neuronal death, we used Harlequin (Hq) mice, which are hypomorphic for AIF. Neurons carrying double mutations forHq/Apaf1-/-(apoptosis proteases-activating factor) are impaired in both caspase-dependent and AIF-mediated mitochondrial cell death pathways. These mutant cells exhibit extended neuroprotection against DNA damage, as well as glutamate-induced excitotoxicity. Specifically, AIF is involved in NMDA- and kainic acid- but not AMPA-induced excitotoxicity.In vivoexcitotoxic studies using kainic acid-induced seizure showed thatHqmice had significantly less hippocampal damage than wild-type littermates. Our results demonstrate an important role for AIF in both BAX-dependent and BAX-independent mechanisms of neuronal injury.

Thep53tumor suppressor gene has been implicated in the regulation of apoptosis in a number of different neuronal death paradigms. Because of the importance of p53 in neuronal injury, we questioned the mechanism underlying p53-mediated apoptosis in neurons. Using adenoviral-mediated gene delivery, reconstitution experiments, and mice carrying a knock-in mutation in the endogenousp53gene, we show that the transactivation function of p53 is essential to induce neuronal cell death. Although p53 possesses two transactivation domains that can activate p53 targets independently, we demonstrate that the first activation domain (ADI) is required to drive apoptosis after neuronal injury. Furthermore, the BH3-only proteins Noxa and PUMA exhibit differential regulation by the two transactivation domains. Here, we show that Noxa can be induced by either activation domain, whereas PUMA induction requires both activation domains to be intact. Unlike Noxa, the upregulation of PUMA alone is sufficient to induce neuronal cell death. We demonstrate, therefore, that the first transactivation domain of p53 is indispensable for the induction of neuronal cell death.

The p53 tumor suppressor gene is believed to play an important role in neuronal cell death in acute neurological disease and in neurodegeneration. The p53 signaling cascade is complex, and the mechanism by which p53 induces apoptosis is cell type-dependent. Using DNA microarray analysis, we have found a striking induction of the proapoptotic gene, SIVA. SIVA is a proapoptotic protein containing a death domain and interacts with members of the tumor necrosis factor receptor family as well as anti-apoptotic Bcl-2 family proteins. SIVA is induced following direct p53 gene delivery, treatment with a DNA-damaging agent camptothecin, and stroke injury in vivo. SIVA up-regulation is sufficient to initiate the apoptotic cascade in neurons. Through isolation and analysis of the SIVA promoter, we have identified response elements for both p53 and E2F1. Like p53, E2F1 is another tumor suppressor gene involved in the regulation of apoptosis, including neuronal injury models. We have identified E2F consensus sites in the promoter region, whereas p53 recognition sequences were found in intron1. Sequence analysis has shown that these consensus sites are also conserved between mouse and human SIVA genes. Electrophoretic mobility shift assays reveal that both transcription factors are capable of binding to putative consensus sites, and luciferase reporter assays reveal that E2F1 and p53 can activate transcription from the SIVA promoter. Here, we report that the proapoptotic gene, SIVA, which functions in a broad spectrum of cell types, is a direct transcriptional target for both tumor suppressors, p53 and E2F1.

The major challenge in treating cancer is that many tumor cells carry mutations in key apoptotic genes such as p53, Bcl family proteins or those affecting caspase signaling. Such defects render treatment with traditional chemotherapeutic agents ineffective. Many studies have demonstrated the importance of caspase-independent cell death pathways in injury, degenerative diseases and tumor tissue. It is now recognized that in addition to their critical role in the production of cellular energy, mitochondria are also the source of key proapoptotic molecules involved in caspase activation. More recently, it has been discovered that in response to apoptotic stimuli, mitochondria can also release caspase-independent cell death effectors such as AIF and Endonuclease G. In this review, we examine the role of Bcl family proteins and poly(ADP-ribose) polymerase-1 signaling in the regulation of these apoptotic pathways and address the ongoing controversies in this field. Continued study of the mechanisms of apoptosis including caspase-independent death processes are likely to reveal novel therapeutic targets for the treatment of diverse human pathologies including cancer, neurodegenerative diseases and acute injuries such as stroke or myocardial infarction.

Caspase-independent death mechanisms have been shown to execute apoptosis in many types of neuronal injury. P53 has been identified as a key regulator of neuronal cell death after acute injury such as DNA damage, ischemia, and excitotoxicity. Here, we demonstrate that p53 can induce neuronal cell death via a caspase-mediated process activated by apoptotic activating factor-1 (Apaf1) and via a delayed onset caspase-independent mechanism. In contrast to wild-type cells, Apaf1-deficient neurons exhibit delayed DNA fragmentation and only peripheral chromatin condensation. More importantly, we demonstrate that apoptosis-inducing factor (AIF) is an important factor involved in the regulation of this caspase-independent neuronal cell death. Immunofluorescence studies demonstrate that AIF is released from the mitochondria by a mechanism distinct from that of cytochrome-c in neurons undergoing p53-mediated cell death. The Bcl-2 family regulates this release of AIF and subsequent caspase-independent cell death. In addition, we show that enforced expression of AIF can induce neuronal cell death in a Bax- and caspase-independent manner. Microinjection of neutralizing antibodies against AIF significantly decreased injury-induced neuronal cell death in Apaf1-deficient neurons, indicating its importance in caspase-independent apoptosis. Taken together, our results suggest that AIF may be an important therapeutic target for the treatment of neuronal injury.

p53 is a transcriptional activator which has been implicated as a key regulator of neuronal cell death after acute injury. We have shown previously that p53-mediated neuronal cell death involves a Bax-dependent activation of caspase 3; however, the transcriptional targets involved in the regulation of this process have not been identified. In the present study, we demonstrate that p53 directly upregulates Apaf1 transcription as a critical step in the induction of neuronal cell death. Using DNA microarray analysis of total RNA isolated from neurons undergoing p53-induced apoptosis a 5–6-fold upregulation of Apaf1 mRNA was detected. Induction of neuronal cell death by camptothecin, a DNA-damaging agent that functions through a p53-dependent mechanism, resulted in increased Apaf1 mRNA in p53-positive, but not p53-deficient neurons. In both in vitro and in vivo neuronal cell death processes of p53-induced cell death, Apaf1 protein levels were increased. We addressed whether p53 directly regulates Apaf1 transcription via the two p53 consensus binding sites in the Apaf1 promoter. Electrophoretic mobility shift assays demonstrated p53–DNA binding activity at both p53 consensus binding sequences in extracts obtained from neurons undergoing p53-induced cell death, but not in healthy control cultures or when p53 or the p53 binding sites were inactivated by mutation. In transient transfections in a neuronal cell line with p53 and Apaf1 promoter–luciferase constructs, p53 directly activated the Apaf1 promoter via both p53 sites. The importance of Apaf1 as a p53 target gene in neuronal cell death was evaluated by examining p53-induced apoptotic pathways in primary cultures of Apaf1-deficient neurons. Neurons treated with camptothecin were significantly protected in the absence of Apaf1 relative to those derived from wild-type littermates. Together, these results demonstrate that Apaf1 is a key transcriptional target for p53 that plays a pivotal role in the regulation of apoptosis after neuronal injury.

Growing evidence suggests that certain cell cycle regulators also mediate neuronal death. Of relevance, cyclin D1-associated kinase activity is increased and the retinoblastoma protein (Rb), a substrate of the cyclin D1-Cdk4/6 complex, is phosphorylated during K(+) deprivation-evoked death of cerebellar granule neurons (CGNs). Cyclin-dependent kinase (CDK) inhibitors block this death, suggesting a requirement for the cyclin D1/Cdk4/6-Rb pathway. However, the downstream target(s) of this pathway are not well defined. The transcription factor E2F-1 is regulated by Rb and is reported to evoke death in proliferating cells when overexpressed. Accordingly, we examined whether E2F-1 was sufficient to evoke death of CGNs and whether it was required for death evoked by low K(+). We show that adenovirus-mediated expression of E2F-1 in CGNs results in apoptotic death, which is independent of p53, dependent upon Bax, and associated with caspase 3-like activity. In addition, we demonstrate that levels of E2F-1 mRNA and protein increase during K(+) deprivation-evoked death. The increase in E2F-1 protein is blocked by the CDK inhibitor flavopiridol. Finally, E2F-1-deficient neurons are modestly resistant to death induced by low K(+). These results indicate that E2F-1 expression is sufficient to promote neuronal apoptosis and that endogenous E2F-1 modulates the death of CGNs evoked by low K(+).

Background Group I metabotropic glutamate receptors (mGluR) are coupled via Gαq/11 to the activation of phospholipase Cβ, which hydrolyzes membrane phospholipids to form inositol 1,4,5 trisphosphate and diacylglycerol. In addition to functioning as neurotransmitter receptors to modulate synaptic activity, pathological mGluR5 signaling has been implicated in a number of disease processes including Fragile X, amyotrophic lateral sclerosis, multiple sclerosis, Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, epilepsy, and drug addiction. The expression of mGluR5 in astrocytes has been shown to be increased in several acute and chronic neurodegenerative conditions, but little is known about the functional relevance of mGluR5 up-regulation in astrocytes following injury. Results In the current study, we investigated primary mouse cortical astrocyte cell death in response to oxygen glucose deprivation (OGD) and found that OGD induced both necrotic and apoptotic cell death of astrocytes. OGD resulted in an increase in astrocytic mGluR5 protein expression, inositol phosphate formation and extracellular regulated kinase (ERK1/2) phosphorylation, but only inositol phosphate formation was blocked with the mGluR5 selective antagonist MPEP. Cortical astrocytes derived from mGluR5 knockout mice exhibited resistance to OGD-stimulated apoptosis, but a lack of mGluR5 expression did not confer protection against necrotic cell death. The antagonism of the inositol 1,4,5 trisphosphate receptor also reduced apoptotic cell death in wild-type astrocytes, but did not provide any additional protection to astrocytes derived from mGluR5 null mice. Moreover, the disruption of Homer protein interactions with mGluR5 also reduced astrocyte apoptosis. Conclusion Taken together these observations indicated that mGluR5 up-regulation contributed selectively to the apoptosis of astrocytes via the activation of phospholipase C and the release of calcium from intracellular stores as well as via the association with Homer proteins.

The AKT, GSK3 and JNK family kinases have been implicated in neuronal apoptosis associated with neuronal development and several neurodegenerative conditions. However, the mechanisms by which these kinase pathways regulate apoptosis remain unclear. In this study we have investigated the role of these kinases in neuronal cell death using an established model of trophic factor deprivation induced apoptosis in cerebellar granule neurons. BCL-2 family proteins are known to be central regulators of apoptosis and we have determined that the pro-apoptotic family member Puma is transcriptionally up-regulated in trophic factor deprived neurons and that Puma induction is required for apoptosis in vitro and in vivo. Importantly, we demonstrate that Puma induction is dependent on both JNK activation and AKT inactivation. AKT is known to regulate a number of downstream pathways, however we have determined that PI3K-AKT inactivation induces Puma expression through a GSK3β-dependent mechanism. Finally we demonstrate that the JNK and AKT/GSK3β pathways converge to regulate FoxO3a-mediated transcriptional activation of Puma. In summary we have identified a novel and critical link between the AKT, GSK3β and JNK kinases and the regulation of Puma induction and suggest that this may be pivotal to the regulation of neuronal apoptosis in neurodegenerative conditions.

Background Alzheimer’s disease (AD) pathology occurs in part as the result of excessive production of β-amyloid (Aβ). Metabotropic glutamate receptor 5 (mGluR5) is now considered a receptor for Aβ and consequently contributes to pathogenic Aβ signaling in AD. Results Genetic deletion of mGluR5 rescues the spatial learning deficits observed in APPswe/PS1ΔE9 AD mice. Moreover, both Aβ oligomer formation and Aβ plaque number are reduced in APPswe/PS1ΔE9 mice lacking mGluR5 expression. In addition to the observed increase in Aβ oligomers and plaques in APPswe/PS1ΔE9 mice, we found that both mTOR phosphorylation and fragile X mental retardation protein (FMRP) expression were increased in these mice. Genetic deletion of mGluR5 reduced Aβ oligomers, plaques, mTOR phosphorylation and FMRP expression in APPswe/PS1ΔE9 mice. Conclusions Thus, we propose that Aβ activation of mGluR5 appears to initiate a positive feedback loop resulting in increased Aβ formation and AD pathology in APPswe/PS1ΔE9 mice via mechanism that is regulated by FMRP.