Neuronal Endoplasmic Reticulum Research Articles

Spermidine inhibits high glucose-induced endoplasmic reticulum stress in HT22 cells by upregulation of growth differentiation factor 11.

Hyperglycemia-induced neuronal endoplasmic reticulum (ER) stress is particularly important for the pathogenesis of diabetic encephalopathy. Spermidine (Spd) has neuroprotection in several nervous system diseases. Our current study to explore the potential protective role of Spd in hyperglycemia-induced neuronal ER stress and the underlying mechanisms. HT22 cells were treated with high glucose (HG) to establish an in-vitro model of hyperglycemia toxicity. The HT22 cells' activity was tested by cell counting kit-8 assay. RNA interference technology was used to silence the expression of growth differentiation factor 11 (GDF11) in HT22 cells. The GDF11 expression levels of mRNA were assessed using reverse transcription-PCR (RT-PCR). Western blotting analysis was applied to evaluate the expressions of GRP78 and cleaved caspase-12. Spd markedly abolished HG-exerted decline in cell viability as well as upregulations of GRP78 and cleaved caspase-12 in HT22 cells, indicating the protection of Spd against HG-induced neurotoxicity and ER stress. Furthermore, we showed that Spd upregulated the expression of GDF11 in HG-exposed HT22 cells. While, silenced GDF11 expression by RNA interference reversed the protective effects of Spd on HG-elicited neurotoxicity and ER stress in HT22 cells. These results indicated that Spd prevents HG-induced neurotoxicity and ER stress through upregulation of GDF11. Our findings identify Spd as a potential treatment for diabetic encephalopathy as well as ER stress-related neurologic diseases.

1, 25-(OH)2D3 protects against ER stress and miRNA dysregulation in Mus musculus neurons.

The pathophysiology of neurodegenerative diseases (NDDs) is closely associated with cellular oxidative stress which can result in the accumulation of toxic proteins in the endoplasmic reticulum (ER) leading to ER stress and subsequent unfolded protein response (UPR) signaling, a mechanism that aggravate these disorders. Vitamin D has been suggested to have important neuroprotective role and its administration has been shown to reduce neuronal injury, neurotoxicity and oxidative stress in various animal systems. The current study was undertaken to examine the effect of vitamin D3 on UPR in ER stress induced Mus musculus neuronal cells. Mus musculus cortical and hippocampal primary neuronal cultures were pretreated with 1,25-dihydroxyvitamin D3 (1, 25-(OH)2D3), the active form of vitamin D, followed by ER stress induction with a chemical ER stress inducer thapsigargin and with an advanced glycated protein, AGE-BSA. The UPR genes and related microRNAs (miRNA) expressions were analyzed mainly using real-time PCR. The experiment resulted in the suppression of ER stress marker BiP and UPR pathway genes such as Perk, Ire1α, Chop and Puma which mediate cellular apoptosis indicating the protective effect of 1, 25-(OH)2D3 against neuronal ER stress. Further studies into the molecular aspects showed that ER stress mediated down-regulated expression of microRNAs (miRNAs) such as mmu-miR-24, 27b, 124, 224, 290, 351 and 488 which are known to regulate the UPR pathway genes were also reduced with vitamin pretreatment, of which the miRNAs miR-24 and 27b which shares the same cluster are potentially involved in various human diseases. This study emphasizes the therapeutic role of vitamin D in reducing neuronal ER stress and the need for maintaining sufficient amount of this vitamin in our diet.

New insights into the regulation of synaptic transmission and plasticity by the endoplasmic reticulum and its membrane contacts

Mammalian neurons are highly compartmentalized yet very large cells. To provide each compartment with its distinct properties, metabolic homeostasis and molecular composition need to be precisely coordinated in a compartment-specific manner. Despite the importance of the endoplasmic reticulum (ER) as a platform for various biochemical reactions, such as protein synthesis, protein trafficking, and intracellular calcium control, the contribution of the ER to neuronal compartment-specific functions and plasticity remains elusive. Recent advances in the development of live imaging and serial scanning electron microscopy (sSEM) analysis have revealed that the neuronal ER is a highly dynamic organelle with compartment-specific structures. sSEM studies also revealed that the ER forms contacts with other membranes, such as the mitochondria and plasma membrane, although little is known about the functions of these ER-membrane contacts. In this review, we discuss the mechanisms and physiological roles of the ER structure and ER-mitochondria contacts in synaptic transmission and plasticity, thereby highlighting a potential link between organelle ultrastructure and neuronal functions.

Microglia are involved in phagocytosis and extracellular digestion during Zika virus encephalitis in young adult immunodeficient mice

BackgroundZika virus (ZIKV) has been associated with several neurological complications in adult patients.MethodsWe used a mouse model deficient in TRIF and IPS-1 adaptor proteins, which are involved in type I interferon production, to study the role of microglia during brain infection by ZIKV. Young adult mice were infected intravenously with the contemporary ZIKV strain PRVABC59 (1 × 105 PFUs/100 µL).ResultsInfected mice did not present overt clinical signs of the disease nor body weight loss compared with noninfected animals. However, mice exhibited a viremia and a brain viral load that were maximal (1.3 × 105 genome copies/mL and 9.8 × 107 genome copies/g of brain) on days 3 and 7 post-infection (p.i.), respectively. Immunohistochemistry analysis showed that ZIKV antigens were distributed in several regions of the brain, especially the dorsal hippocampus. The number of Iba1+/TMEM119+ microglia remained similar in infected versus noninfected mice, but their cell body and arborization areas significantly increased in the stratum radiatum and stratum lacunosum-moleculare layers of the dorsal hippocampus cornu ammoni (CA)1, indicating a reactive state. Ultrastructural analyses also revealed that microglia displayed increased phagocytic activities and extracellular digestion of degraded elements during infection. Mice pharmacologically depleted in microglia with PLX5622 presented a higher brain viral load compared to untreated group (2.8 × 1010versus 8.5 × 108 genome copies/g of brain on day 10 p.i.) as well as an increased number of ZIKV antigens labeled with immunogold in the cytoplasm and endoplasmic reticulum of neurons and astrocytes indicating an enhanced viral replication. Furthermore, endosomes of astrocytes contained nanogold particles together with digested materials, suggesting a compensatory phagocytic activity upon microglial depletion.ConclusionsThese results indicate that microglia are involved in the control of ZIKV replication and/or its elimination in the brain. After depletion of microglia, the removal of ZIKV-infected cells by phagocytosis could be partly compensated by astrocytes.

Altered neuronal physiology, development, and function associated with a common chromosome 15 duplication involving CHRNA7

BackgroundCopy number variants (CNVs) linked to genes involved in nervous system development or function are often associated with neuropsychiatric disease. While CNVs involving deletions generally cause severe and highly penetrant patient phenotypes, CNVs leading to duplications tend instead to exhibit widely variable and less penetrant phenotypic expressivity among affected individuals. CNVs located on chromosome 15q13.3 affecting the alpha-7 nicotinic acetylcholine receptor subunit (CHRNA7) gene contribute to multiple neuropsychiatric disorders with highly variable penetrance. However, the basis of such differential penetrance remains uncharacterized. Here, we generated induced pluripotent stem cell (iPSC) models from first-degree relatives with a 15q13.3 duplication and analyzed their cellular phenotypes to uncover a basis for the dissimilar phenotypic expressivity.ResultsThe first-degree relatives studied included a boy with autism and emotional dysregulation (the affected proband-AP) and his clinically unaffected mother (UM), with comparison to unrelated control models lacking this duplication. Potential contributors to neuropsychiatric impairment were modeled in iPSC-derived cortical excitatory and inhibitory neurons. The AP-derived model uniquely exhibited disruptions of cellular physiology and neurodevelopment not observed in either the UM or unrelated controls. These included enhanced neural progenitor proliferation but impaired neuronal differentiation, maturation, and migration, and increased endoplasmic reticulum (ER) stress. Both the neuronal migration deficit and elevated ER stress could be selectively rescued by different pharmacologic agents. Neuronal gene expression was also dysregulated in the AP, including reduced expression of genes related to behavior, psychological disorders, neuritogenesis, neuronal migration, and Wnt, axonal guidance, and GABA receptor signaling. The UM model instead exhibited upregulated expression of genes in many of these same pathways, suggesting that molecular compensation could have contributed to the lack of neurodevelopmental phenotypes in this model. However, both AP- and UM-derived neurons exhibited shared alterations of neuronal function, including increased action potential firing and elevated cholinergic activity, consistent with increased homomeric CHRNA7 channel activity.ConclusionsThese data define both diagnosis-associated cellular phenotypes and shared functional anomalies related to CHRNA7 duplication that may contribute to variable phenotypic penetrance in individuals with 15q13.3 duplication. The capacity for pharmacological agents to rescue some neurodevelopmental anomalies associated with diagnosis suggests avenues for intervention for carriers of this duplication and other CNVs that cause related disorders.

Morphological Heterogeneity of the Endoplasmic Reticulum within Neurons and Its Implications in Neurodegeneration.

The endoplasmic reticulum (ER) is a multipurpose organelle comprising dynamic structural subdomains, such as ER sheets and tubules, serving to maintain protein, calcium, and lipid homeostasis. In neurons, the single ER is compartmentalized with a careful segregation of the structural subdomains in somatic and neurite (axodendritic) regions. The distribution and arrangement of these ER subdomains varies between different neuronal types. Mutations in ER membrane shaping proteins and morphological changes in the ER are associated with various neurodegenerative diseases implying significance of ER morphology in maintaining neuronal integrity. Specific neurons, such as the highly arborized dopaminergic neurons, are prone to stress and neurodegeneration. Differences in morphology and functionality of ER between the neurons may account for their varied sensitivity to stress and neurodegenerative changes. In this review, we explore the neuronal ER and discuss its distinct morphological attributes and specific functions. We hypothesize that morphological heterogeneity of the ER in neurons is an important factor that accounts for their selective susceptibility to neurodegeneration.

LncRNA Xist regulates sevoflurane-induced social and emotional impairment by modulating miR-98-5p/EDEM1 signaling axis in neonatal mice

Long non-coding RNA (lncRNA) X-inactive specific transcript (Xist) is involved in apoptosis and inflammatory injury. This study aimed to assess the role of lncRNA Xist in sevoflurane-induced social and emotional impairment and neuronal apoptosis in neonatal mice and hippocampal neuronal cells. The performance in social and emotional tests and the expression levels of lncRNA Xist and microRNA (miR)-98-5p after sevoflurane exposure were measured. Moreover, the effects of suppression of lncRNA Xist on neuronal apoptosis and endoplasmic reticulum (ER) stress were determined. Subsequently, the association among lncRNA Xist, miR-98-5p, and ER degradation-enhancing α-mannosidase-like 1 protein (EDEM1) was explored. Our results showed that lncRNA Xist increased, miR-98-5p decreased, and social and emotional impairment appeared after sevoflurane exposure. Furthermore, suppression of lncRNA Xist improved sevoflurane-induced social and emotional impairment and reduced sevoflurane-induced neuronal apoptosis and ER stress in vivo and in vitro. Moreover, lncRNA Xist negatively regulated miR-98-5p expression, and it contributed to sevoflurane-induced neuronal apoptosis and ER stress by sponging miR-98-5p. Additionally, EDEM1 was identified as a target of miR-98-5p. Our findings revealed that the knockdown of lncRNA Xist ameliorates sevoflurane-induced social and emotional impairment through inhibiting neuronal apoptosis and ER stress by targeting the miR-98-5p/EDEM1 axis.

LncRNA MALAT1 aggravates oxygen\u2010glucose deprivation/reoxygenation-induced neuronal endoplasmic reticulum stress and apoptosis via the miR-195a-5p/HMGA1 axis

BackgroundThis study aimed to investigate the potential role and molecular mechanism of lncRNA metastasis associated lung adenocarcinoma transcript 1 (MALAT1) in cerebral ischemia/reperfusion injury.ResultsUsing an oxygen-glucose deprivation/reoxygenation (OGD/R) cell model, we determined that the expression of MALAT1 was significantly increased during OGD/R. MALAT1 knockdown reversed OGD/R-induced apoptosis and ER stress. Mechanistically, MALAT1 promoted OGD/R-induced neuronal injury through sponging miR-195a-5p to upregulating high mobility group AT-hook1 (HMGA1).ConclusionsCollectively, these data demonstrate the mechanism underlying the invovlvement of MALAT1 in cerebral ischemia/reperfusion injury, thus providing translational evidence that MALAT1 may serve as a novel biomarker and therapeutic target for ischemic stroke.

MANF is neuroprotective against ethanol-induced neurodegeneration through ameliorating ER stress

Fetal alcohol spectrum disorders (FASD) are a spectrum of developmental disorders caused by prenatal alcohol exposure. Neuronal loss or neurodegeneration in the central nervous system (CNS) is one of the most devastating features in FASD. It is imperative to delineate the underlying mechanisms to facilitate the treatment of FASD. Endoplasmic reticulum (ER) stress is a hallmark and an underlying mechanism of many neurodegenerative diseases, including ethanol-induced neurodegeneration. Mesencephalic astrocyte-derived neurotrophic factor (MANF) responds to ER stress and has been identified as a protein upregulated in response to ethanol exposure during the brain development. To investigate the role of MANF in ethanol-induced neurodegeneration and its association with ER stress regulation, we established a CNS-specific Manf knockout mouse model and examined the effects of MANF deficiency on ethanol-induced neuronal apoptosis and ER stress using a third-trimester equivalent mouse model. We found MANF deficiency exacerbated ethanol-induced neuronal apoptosis and ER stress and that blocking ER stress abrogated the harmful effects of MANF deficiency on ethanol-induced neuronal apoptosis. Moreover, using an animal model of ER-stress-induced neurodegeneration, we demonstrated that MANF deficiency potentiated tunicamycin (TM)-induced ER stress and neurodegeneration. A whole transcriptome RNA sequencing also supported the functionality of MANF in ER stress modulation and revealed targets that may mediate the ER stress-buffering capacity of MANF. Collectively, these results suggest that MANF is a neurotrophic factor that can protect neurons against ethanol-induced neurodegeneration by ameliorating ER stress.

Divergent Nodes of Non-autonomous UPRER Signaling through Serotonergic and Dopaminergic Neurons

In multicellular organisms, neurons integrate a diverse array of external cues to affect downstream changes in organismal health. Specifically, activation of the endoplasmic reticulum (ER) unfolded protein response (UPRER) in neurons increases lifespan by preventing age-onset loss of ER proteostasis and driving lipid depletion in a cell non-autonomous manner. The mechanism of this communication is dependent on the release of small clear vesicles from neurons. We find dopaminergic neurons are necessary and sufficient for activation of cell non-autonomous UPRER to drive lipid depletion in peripheral tissues, whereas serotonergic neurons are sufficient to drive protein homeostasis in peripheral tissues. These signaling modalities are unique and independent and together coordinate the beneficial effects of neuronal cell non-autonomous ER stress signaling upon health and longevity.

Divergent Nodes of Non-Autonomous UPR <sup>ER</sup> Signaling Through Serotonergic and Dopaminergic Neurons

In multicellular organisms, neurons integrate a diverse array of external cues to affect downstream changes in organismal health. Specifically, activation of the endoplasmic reticulum (ER) unfolded protein response (UPRER) in neurons increases lifespan by preventing age-onset loss of ER proteostasis and driving lipid depletion in a cell non-autonomous manner. The mechanism of this communication is dependent on release of small clear vesicles from neurons. We find dopaminergic neurons are necessary and sufficient for activation of cell non-autonomous UPRER to drive lipid depletion in peripheral tissues, while serotonergic neurons are necessary and sufficient to drive protein homeostasis in peripheral tissues. These signaling modalities are unique and independent, and together coordinate the benefits of neuronal cell non-autonomous ER stress signaling upon health and longevity.

Synthesis, biological activities, and SAR studies of novel 1-(2-chloro-4,5-difluorophenyl)-1H-pyrazole derivatives

In order to search for the new ryanodine receptor (RyR) regulator, a series of 35 novel fluoro-substituted compounds introduced 1-(2-chloro-4,5-difluorophenyl)-1H-pyrazole moiety containing modified pyrazole heterocycle were designed and synthesized. Then, they were tested for the insecticidal activities against Mythimna separata and Plutella xylostella in our greenhouse. After a systematic biological screening, it was found out that IVc showed 50% larvicidal activities against Mythimna separata at 0.1 mg L−1, equivalent to that of chlorantraniliprole (36%, 0.1 mg L−1). The activity of IVc against Plutella xylostella was 90% at 10−5 mg L−1, whereas the chlorantraniliprole was 70% at the same concentration. Then, insect electrophysiology experiments were conducted to study the pattern of action of IVc and IVe. It was confirmed by the experimental results that both compounds could lead to the release of calcium from the endoplasmic reticulum of neurons as classical anthranilic diamide insecticides.

Dysregulation of miRNA and its reversal with l-ascorbic acid during AGE-BSA induced ER stress in Mus musculus neuronal cells

Loss of function and progressive death of neurons, the pathological mechanism of neurodegenerative diseases (NDDs) can occur as a result of apoptotic unfolded protein response (UPR) signaling induced by endoplasmic reticulum (ER) stress resulting from misfolded protein accumulation in ER lumen. Non-enzymatic glycation of proteins have been implicated in the pathophysiology of NDDs and are known to cause cellular oxidative stress leading to ER stress mediated neuronal apoptosis. MicroRNAs (miRNAs), post transcriptional regulators of gene expression are identified as regulators of UPR and the altered brain miRNA profiles in NDDs suggest the involvement of miRNA dysregulation in neuronal death. l-ascorbic acid, a micro nutrient and an important neuroprotectant has been suggested to be a modulator of gene expression. However there are few reports on miRNAs targeting the genes involved in neuronal apoptotic UPR signaling as well as l-ascorbic acid mediated regulation of neuronal UPR pathway and miRNA expression. Here, we identified a statistically significant downregulation of 7 miRNAs such as mmu-miR-24, 27b, 124, 224, 290, 351 and 488 and inverse expression relationship with their UPR related targets such as Perk, Ire1α, Chop and Puma during advanced glycated BSA (AGE-BSA) induced ER stress in Mus musculus cortical and hippocampal neurons. We also observed that a pretreatment with l-ascorbic acid alleviated AGE-BSA induced neuronal ER stress as well as the downregulation of the 7 potential miRNAs.

Embelin as Lead Compound for New Neuroserpin Polymerization Inhibitors.

Familial encephalopathy with neuroserpin inclusion bodies (FENIB) is a severe and lethal neurodegenerative disease. Upon specific point mutations in the SERPINI1gene-coding for the human protein neuroserpin (NS) the resulting pathologic NS variants polymerize and accumulate within the endoplasmic reticulum of neurons in the central nervous system. To date, embelin (EMB) is the only known inhibitor of NS polymerization in vitro. This molecule is capable of preventing NS polymerization and dissolving preformed polymers. Here, we show that lowering EMB concentration results in increasing size of NS oligomers in vitro. Moreover, we observe that in cells expressing NS, the polymerization of G392E NS is reduced, but this effect is mediated by an increased proteasomal degradation rather than polymerization impairment. For these reasons we designed a systematic chemical evolution of the EMB scaffold aimed to improve its anti-polymerization properties. The effect of EMB analogs against NS polymerization was assessed in vitro. None of the EMB analogs displayed an anti-polymerization activity better than the one reported for EMB, indicating that the EMB–NS interaction surface is very specific and highly optimized. Thus, our results indicate that EMB is, to date, still the best candidate for developing a treatment against NS polymerization.

Glycosylation Tunes Neuroserpin Physiological and Pathological Properties.

Neuroserpin (NS) is a member of the serine protease inhibitors superfamily. Specific point mutations are responsible for its accumulation in the endoplasmic reticulum of neurons that leads to a pathological condition named familial encephalopathy with neuroserpin inclusion bodies (FENIB). Wild-type NS presents two N-glycosylation chains and does not form polymers in vivo, while non-glycosylated NS causes aberrant polymer accumulation in cell models. To date, all in vitro studies have been conducted on bacterially expressed NS, de facto neglecting the role of glycosylation in the biochemical properties of NS. Here, we report the expression and purification of human glycosylated NS (gNS) using a novel eukaryotic expression system, LEXSY. Our results confirm the correct N-glycosylation of wild-type gNS. The fold and stability of gNS are not altered compared to bacterially expressed NS, as demonstrated by the circular dichroism and intrinsic tryptophan fluorescence assays. Intriguingly, gNS displays a remarkably reduced polymerisation propensity compared to non-glycosylated NS, in keeping with what was previously observed for wild-type NS in vivo and in cell models. Thus, our results support the relevance of gNS as a new in vitro tool to study the molecular bases of FENIB.

MANF Ablation Causes Prolonged Activation of the UPR without Neurodegeneration in the Mouse Midbrain Dopamine System.

Mesencephalic astrocyte-derived neurotrophic factor (MANF) is an endoplasmic reticulum (ER) localized protein that regulates ER homeostasis and unfolded protein response (UPR). The biology of endogenous MANF in the mammalian brain is unknown and therefore we studied the brain phenotype of MANF-deficient female and male mice at different ages focusing on the midbrain dopamine system and cortical neurons. We show that a lack of MANF from the brain led to the chronic activation of UPR by upregulation of the endoribonuclease activity of the inositol-requiring enzyme 1α (IRE1α) pathway. Furthermore, in the aged MANF-deficient mouse brain in addition the protein kinase-like ER kinase (PERK) and activating transcription factor 6 (ATF6) branches of the UPR pathways were activated. Neuronal loss in neurodegenerative diseases has been associated with chronic ER stress. In our mouse model, increased UPR activation did not lead to neuronal cell loss in the substantia nigra (SN), decrease of striatal dopamine or behavioral changes of MANF-deficient mice. However, cortical neurons lacking MANF were more vulnerable to chemical induction of additional ER stress in vitro We conclude that embryonic neuronal deletion of MANF does not cause the loss of midbrain dopamine neurons in mice. However, endogenous MANF is needed for maintenance of neuronal ER homeostasis both in vivo and in vitro.

Neuronal ER–plasma membrane junctions organized by Kv2–VAP pairing recruit Nir proteins and affect phosphoinositide homeostasis

The association of plasma membrane (PM)-localized voltage-gated potassium (Kv2) channels with endoplasmic reticulum (ER)-localized vesicle-associated membrane protein-associated proteins VAPA and VAPB defines ER-PM junctions in mammalian brain neurons. Here, we used proteomics to identify proteins associated with Kv2/VAP-containing ER-PM junctions. We found that the VAP-interacting membrane-associated phosphatidylinositol (PtdIns) transfer proteins PYK2 N-terminal domain-interacting receptor 2 (Nir2) and Nir3 specifically associate with Kv2.1 complexes. When coexpressed with Kv2.1 and VAPA in HEK293T cells, Nir2 colocalized with cell-surface-conducting and -nonconducting Kv2.1 isoforms. This was enhanced by muscarinic-mediated PtdIns(4,5)P2 hydrolysis, leading to dynamic recruitment of Nir2 to Kv2.1 clusters. In cultured rat hippocampal neurons, exogenously expressed Nir2 did not strongly colocalize with Kv2.1, unless exogenous VAPA was also expressed, supporting the notion that VAPA mediates the spatial association of Kv2.1 and Nir2. Immunolabeling signals of endogenous Kv2.1, Nir2, and VAP puncta were spatially correlated in cultured neurons. Fluorescence-recovery-after-photobleaching experiments revealed that Kv2.1, VAPA, and Nir2 have comparable turnover rates at ER-PM junctions, suggesting that they form complexes at these sites. Exogenous Kv2.1 expression in HEK293T cells resulted in significant differences in the kinetics of PtdIns(4,5)P2 recovery following repetitive muscarinic stimulation, with no apparent impact on resting PtdIns(4,5)P2 or PtdIns(4)P levels. Finally, the brains of Kv2.1-knockout mice had altered composition of PtdIns lipids, suggesting a crucial role for native Kv2.1-containing ER-PM junctions in regulating PtdIns lipid metabolism in brain neurons. These results suggest that ER-PM junctions formed by Kv2 channel-VAP pairing regulate PtdIns lipid homeostasis via VAP-associated PtdIns transfer proteins.

Methylmercury exposure induces ROS/Akt inactivation-triggered endoplasmic reticulum stress-regulated neuronal cell apoptosis

Epidemiological studies have positively linked mercury exposure and neurodegenerative diseases (ND). Methylmercury (MeHg), an organic form of mercury, is a ubiquitous and potent environmental neurotoxicant that easily crosses the blood-brain barrier and causes irreversible injury to the central nervous system (CNS). However, the molecular mechanisms underlying MeHg-induced neurotoxicity remain unclear. Here, the present study found that Neuro-2a cells underwent apoptosis in response to MeHg (1–5 μM), which was accompanied by increased phosphatidylserine (PS) exposure on the outer cellular membrane leaflets, caspase-3 activity, and the activation of caspase cascades and poly (ADP-ribose) polymerase (PARP). Exposure of Neuro-2a cells to MeHg also triggered endoplasmic reticulum (ER) stress, which was identified via several key molecules (including: glucose-regulated protein (GRP)78, GRP94, C/EBP homologous protein (CHOP) X-box binding protein(XBP)-1, protein kinase R-like ER kinase (PERK), eukaryotic initiation factor 2α (eIF2α), inositol-requiring enzyme(IRE)-1, activation transcription factor(AFT)4, and ATF6. Transfection with GRP78-, GRP94-, CHOP-, and XBP-1-specific small interfering (si)RNA significantly suppressed the expression of these proteins, and attenuated cytotoxicity and caspase-12, -7, and -3 activation in MeHg-exposed cells. Furthermore, MeHg dramatically decreased Akt phosphorylation, and the overexpression of activation of Akt1 (myr-Akt1) could significantly prevent MeHg-induced Akt inactivation, as well as apoptotic and ER stress-related signals. Pretreatment with the antioxidant N-acetylcysteine (NAC) effectively prevented MeHg-induced neuronal cell reactive oxygen species (ROS) generation, apoptotic and ER stress-related signals, and Akt inactivation. Collectively, these results indicate that MeHg exerts its cytotoxicity in neurons by inducing ROS-mediated Akt inactivation up-regulated ER stress, which induces apoptosis and ultimately leads to cell death.

24(S)-Hydroxycholesterol induces ER dysfunction-mediated unconventional cell death

Endoplasmic reticulum (ER) stress induced by disruption of protein folding activates the unfolded protein response (UPR), which while generally pro-survival in effect can also induce cell death under severe ER stress. 24(S)-hydroxycholesterol (24S-OHC), which is enzymatically produced in the ER of neurons, plays an important role in maintaining brain cholesterol homeostasis but also shows neurotoxicity when subjected to esterification by acyl-CoA:cholesterol acyltransferase 1 (ACAT1) in the ER. In this study, we demonstrated that the accumulation of 24S-OHC esters in human neuroblastoma SH-SY5Y cells evoked the UPR with substantially no pro-survival adaptive response but with significant activation of pro-death UPR signaling via regulated IRE1-dependent decay (RIDD). We further found that accumulation of 24S-OHC esters caused disruption of ER membrane integrity and release of ER luminal proteins into cytosol. We also found that de novo synthesis of global proteins was robustly suppressed in 24S-OHC-treated cells. Collectively, these results show that ER dysfunction and the accompanying RIDD-mediated pro-death UPR signaling and global protein synthesis inhibition are responsible for 24S-OHC ester-induced unconventional cell death.

Nickel Enhances Zinc-Induced Neuronal Cell Death by Priming the Endoplasmic Reticulum Stress Response.

Trace metals such as zinc (Zn), copper (Cu), and nickel (Ni) play important roles in various physiological functions such as immunity, cell division, and protein synthesis in a wide variety of species. However, excessive amounts of these trace metals cause disorders in various tissues of the central nervous system, respiratory system, and other vital organs. Our previous analysis focusing on neurotoxicity resulting from interactions between Zn and Cu revealed that Cu2+ markedly enhances Zn2+-induced neuronal cell death by activating oxidative stress and the endoplasmic reticulum (ER) stress response. However, neurotoxicity arising from interactions between zinc and metals other than copper has not been examined. Thus, in the current study, we examined the effect of Ni2+ on Zn2+-induced neurotoxicity. Initially, we found that nontoxic concentrations (0–60 μM) of Ni2+ enhance Zn2+-induced neurotoxicity in an immortalized hypothalamic neuronal cell line (GT1-7) in a dose-dependent manner. Next, we analyzed the mechanism enhancing neuronal cell death, focusing on the ER stress response. Our results revealed that Ni2+ treatment significantly primed the Zn2+-induced ER stress response, especially expression of the CCAAT-enhancer-binding protein homologous protein (CHOP). Finally, we examined the effect of carnosine (an endogenous peptide) on Ni2+/Zn2+-induced neurotoxicity and found that carnosine attenuated Ni2+/Zn2+-induced neuronal cell death and ER stress occurring before cell death. Based on our results, Ni2+ treatment significantly enhances Zn2+-induced neuronal cell death by priming the ER stress response. Thus, compounds that decrease the ER stress response, such as carnosine, may be beneficial for neurological diseases.