Neuronal Endoplasmic Reticulum Research Articles

MitoTempo protects against nε-carboxymethyl lysine-induced mitochondrial dyshomeostasis and neuronal cells injury

Enhanced formation of advanced glycation end products (AGEs) is a pivotal factor in diabetes pathophysiology, increasing the risk of diabetic complications. Nε-carboxy-methyl-lysine (CML) is one of the most relevant AGEs found in several tissues including the peripheral blood of diabetic subjects. Despite recognizing diabetes as a risk factor for neurodegenerative diseases and the documented role of mitochondrial abnormalities in this connection, the impact of CML on neuronal mitochondria and its contribution to diabetes-related neurodegeneration remain uncertain. Here, we evaluated the effects of CML in differentiated SH-SY5Y human neuroblastoma cells. Due to the association between mitochondrial dysfunction and increased production of reactive oxygen species (ROS), the possible protective effects of MitoTempo, a mitochondria-targeted antioxidant, were also evaluated. Several parameters were assessed namely cells viability, mitochondrial respiration and membrane potential, ATP and ROS production, Ca2+ levels, mitochondrial biogenesis and dynamics, mito/autophagy, endoplasmic reticulum (ER) stress and amyloidogenic and synaptic integrity markers. CML caused pronounced mitochondrial defects characterized by a significant decrease in mitochondrial respiration, membrane potential, and ATP production and an increase in ROS production. An accumulation of individual mitochondria associated with disrupted mitochondrial networks was also observed. Furthermore, CML caused mitochondrial fusion and a decrease in mitochondrial mass and induced ER stress associated with altered unfolded protein response and Ca2+ dyshomeostasis. Moreover, CML increased the protein levels of β-secretase-1 and amyloid precursor protein, key proteins involved in Alzheimer's Disease pathophysiology. All these effects contributed to the decline in neuronal cells viability. Notable, MitoTempo was able to counteract most of CML-mediated mitochondrial defects and neuronal cells injury and death. Overall, these findings suggest that CML induces pronounced defects in neuronal mitochondria and ER stress, predisposing to neurodegenerative events. More, our observations suggest that MitoTempo holds therapeutic promise in mitigating CML-induced mitochondrial imbalance and neuronal damage and death.

Endoplasmic reticulum stress and autophagy in cerebral ischemia/reperfusion injury: PERK as a potential target for intervention

Abstract Several studies have shown that activation of unfolded protein response and endoplasmic reticulum (ER) stress plays a crucial role in severe cerebral ischemia/reperfusion injury. Autophagy occurs within hours after cerebral ischemia, but the relationship between ER stress and autophagy remains unclear. In this study, we established experimental models using oxygen-glucose deprivation/reoxygenation in PC12 cells and primary neurons to simulate cerebral ischemia/reperfusion injury. We found that prolongation of oxygen-glucose deprivation activated the ER stress pathway protein kinase-like endoplasmic reticulum kinase (PERK)/eukaryotic translation initiation factor 2 subunit alpha (eIF2α)-activating transcription factor 4 (ATF4)-C/EBP homologous protein (CHOP), increased neuronal apoptosis, and induced autophagy. Furthermore, inhibition of ER stress using inhibitors or by siRNA knockdown of the PERK gene significantly attenuated excessive autophagy and neuronal apoptosis, indicating an interaction between autophagy and ER stress and suggesting PERK as an essential target for regulating autophagy. Blocking autophagy with chloroquine exacerbated ER stress-induced apoptosis, indicating that normal levels of autophagy play a protective role in neuronal injury following cerebral ischemia/reperfusion injury. Findings from this study indicate that cerebral ischemia/ reperfusion injury can trigger neuronal ER stress and promote autophagy, and suggest that PERK is a possible target for inhibiting excessive autophagy in cerebral ischemia/reperfusion injury.

Exosomes derived from microglia overexpressing miR-124-3p alleviate neuronal endoplasmic reticulum stress damage after repetitive mild traumatic brain injury.

JOURNAL/nrgr/04.03/01300535-202409000-00033/figure1/v/2024-01-16T170235Z/r/image-tiff We previously reported that miR-124-3p is markedly upregulated in microglia-derived exosomes following repetitive mild traumatic brain injury. However, its impact on neuronal endoplasmic reticulum stress following repetitive mild traumatic brain injury remains unclear. In this study, we first used an HT22 scratch injury model to mimic traumatic brain injury, then co-cultured the HT22 cells with BV2 microglia expressing high levels of miR-124-3p. We found that exosomes containing high levels of miR-124-3p attenuated apoptosis and endoplasmic reticulum stress. Furthermore, luciferase reporter assay analysis confirmed that miR-124-3p bound specifically to the endoplasmic reticulum stress-related protein IRE1α, while an IRE1α functional salvage experiment confirmed that miR-124-3p targeted IRE1α and reduced its expression, thereby inhibiting endoplasmic reticulum stress in injured neurons. Finally, we delivered microglia-derived exosomes containing miR-124-3p intranasally to a mouse model of repetitive mild traumatic brain injury and found that endoplasmic reticulum stress and apoptosis levels in hippocampal neurons were significantly reduced. These findings suggest that, after repetitive mild traumatic brain injury, miR-124-3 can be transferred from microglia-derived exosomes to injured neurons, where it exerts a neuroprotective effect by inhibiting endoplasmic reticulum stress. Therefore, microglia-derived exosomes containing miR-124-3p may represent a novel therapeutic strategy for repetitive mild traumatic brain injury.

RGC-specific ATF4 and/or CHOP deletion rescues glaucomatous neurodegeneration and visual function

Endoplasmic reticulum (ER) stress has been linked with various acute and chronic neurodegenerative diseases. We previously found that optic nerve (ON) injury and diseases induce neuronal ER stress in retinal ganglion cells (RGCs). We further demonstrated that germline deletion of CHOP preserves the structure and function of both RGC somata and axons in mouse glaucoma models. Here we report that RGC-specific deletion of CHOP and/or its upstream regulator ATF4 synergistically promotes RGC and ON survival and preserves visual function in mouse ON crush and silicone oil-induced ocular hypertension (SOHU) glaucoma models. Consistently, topical application of the ATF4/CHOP chemical inhibitor ISRIB or RGC-specific CRISPR-mediated knockdown of the ATF4 downstream effector Gadd45a also delivers significant neuroprotection in the SOHU glaucoma model. These studies suggest that blocking the neuronal intrinsic ATF4/CHOP axis of ER stress is a promising neuroprotection strategy for neurodegeneration.

Neutrophil extracellular traps aggravate neuronal endoplasmic reticulum stress and apoptosis via TLR9 after traumatic brain injury

Endoplasmic reticulum (ER) stress and ER stress-mediated apoptosis play an important role during secondary brain damage after traumatic brain injury (TBI). Increased neutrophil extracellular traps (NETs) formation has been demonstrated to be associated with neurological damage after TBI. However, the correlation between ER stress and NETs remains unclear, and the specific function of NETs in neurons has not been defined. In this study, we found that the levels of NETs circulating biomarkers were remarkably elevated in the plasma of TBI patients. We then inhibited NETs formation by peptidylarginine deiminase 4 (PAD4, a critical enzyme for NETs formation) deficiency and discovered that ER stress activation and ER stress-mediated neuronal apoptosis were reduced. The degradation of NETs via DNase I showed similar outcomes. Furthermore, overexpression of PAD4 aggravated neuronal ER stress and ER stress-associated apoptosis, while TLR9 antagonist administration abrogated the damage caused by NETs. In addition to in vivo experiments, in vitro experiments revealed that treatment with a TLR9 antagonist alleviated NETs-induced ER stress and apoptosis in HT22 cells. Collectively, our results indicated that ER stress as well as the accompanying neuronal apoptosis can be ameliorated by disruption of NETs and that suppression of the TLR9-ER stress signaling pathway may contribute to positive outcomes after TBI.

Exploring the precision redox map during fasting-refeeding and satiation in C. elegans

Fasting is a popular dietary strategy because it grants numerous advantages, and redox regulation is one mechanism involved. However, the precise redox changes with respect to the redox species, organelles and tissues remain unclear, which hinders the understanding of the metabolic mechanism, and exploring the precision redox map under various dietary statuses is of great significance. Twelve redox-sensitive C. elegans strains stably expressing genetically encoded redox fluorescent probes (Hyperion sensing H2O2 and Grx1-roGFP2 sensing GSH/GSSG) in three organelles (cytoplasm, mitochondria and endoplasmic reticulum (ER)) were constructed in two tissues (body wall muscle and neurons) and were confirmed to respond to redox challenge. The H2O2 and GSSG/GSH redox changes in two tissues and three organelles were obtained by confocal microscopy during fasting, refeeding, and satiation. We found that under fasting condition, H2O2 decreased in most compartments, except for an increase in mitochondria, while GSSG/GSH increased in the cytoplasm of body muscle and the ER of neurons. After refeeding, the redox changes in H2O2 and GSSG/GSH caused by fasting were reversed in most organelles of the body wall muscle and neurons. In the satiated state, H2O2 increased markedly in the cytoplasm, mitochondria and ER of muscle and the ER of neurons, while GSSG/GSH exhibited no change in most organelles of the two tissues except for an increase in the ER of muscle. Our study systematically and precisely presents the redox characteristics under different dietary states in living animals and provides a basis for further investigating the redox mechanism in metabolism and optimizing dietary guidance.

Pharmacological Regulation of Endoplasmic Reticulum Structure and Calcium Dynamics: Importance for Neurodegenerative Diseases.

The endoplasmic reticulum (ER) is the largest organelle of the cell, composed of a continuous network of sheets and tubules, and is involved in protein, calcium (Ca2+), and lipid homeostasis. In neurons, the ER extends throughout the cell, both somal and axodendritic compartments, and is highly important for neuronal functions. A third of the proteome of a cell, secreted and membrane-bound proteins, are processed within the ER lumen and most of these proteins are vital for neuronal activity. The brain itself is high in lipid content, and many structural lipids are produced, in part, by the ER. Cholesterol and steroid synthesis are strictly regulated in the ER of the blood-brain barrier protected brain cells. The high Ca2+ level in the ER lumen and low cytosolic concentration is needed for Ca2+-based intracellular signaling, for synaptic signaling and Ca2+ waves, and for preparing proteins for correct folding in the presence of high Ca2+ concentrations to cope with the high concentrations of extracellular milieu. Particularly, ER Ca2+ is controlled in axodendritic areas for proper neurito- and synaptogenesis and synaptic plasticity and remodeling. In this review, we cover the physiologic functions of the neuronal ER and discuss it in context of common neurodegenerative diseases, focusing on pharmacological regulation of ER Ca2+ Furthermore, we postulate that heterogeneity of the ER, its protein folding capacity, and ensuring Ca2+ regulation are crucial factors for the aging and selective vulnerability of neurons in various neurodegenerative diseases. SIGNIFICANCE STATEMENT: Endoplasmic reticulum (ER) Ca2+ regulators are promising therapeutic targets for degenerative diseases for which efficacious drug therapies do not exist. The use of pharmacological probes targeting maintenance and restoration of ER Ca2+ can provide restoration of protein homeostasis (e.g., folding of complex plasma membrane signaling receptors) and slow down the degeneration process of neurons.

Neuronal Store-Operated Calcium Channels.

The endoplasmic reticulum (ER) is the major intracellular calcium (Ca2+) storage compartment in eukaryotic cells. In most instances, the mobilization of Ca2+ from this store is followed by a delayed and sustained uptake of Ca2+ through Ca2+-permeable channels of the cell surface named store-operated Ca2+ channels (SOCCs). This gives rise to a store-operated Ca2+ entry (SOCE) that has been thoroughly investigated in electrically non-excitable cells where it is the principal regulated Ca2+ entry pathway. The existence of this Ca2+ route in neurons has long been a matter of debate. However, a growing body of experimental evidence indicates that the recruitment of Ca2+ from neuronal ER Ca2+ stores generates a SOCE. The present review summarizes the main studies supporting the presence of a depletion-dependent Ca2+ entry in neurons. It also addresses the question of the molecular composition of neuronal SOCCs, their expression, pharmacological properties, as well as their physiological relevance.

The Endoplasmic Reticulum and Its Contacts: Emerging Roles in Axon Development, Neurotransmission, and Degeneration.

The neuronal endoplasmic reticulum (ER) consists of a dynamic, tubular network that extends all the way from the soma into dendrites, axons, and synapses. This morphology gives rise to an enormous membrane surface area that, through the presence of tethering proteins, lipid transfer proteins, and ion channels, plays critical roles in local calcium regulation, membrane dynamics, and the supply of ions and lipids to other organelles. Here, we summarize recent advances that highlight the various roles of the neuronal ER in axonal growth, repair, and presynaptic function. We review the variety of contact sites between the ER and other axonal organelles and describe their influence on neurodevelopment and neurotransmission.

Drosophila SPG12 ortholog, reticulon-like 1, governs presynaptic ER organization and Ca2+ dynamics.

Neuronal endoplasmic reticulum (ER) appears continuous throughout the cell. Its shape and continuity are influenced by ER-shaping proteins, mutations in which can cause distal axon degeneration in Hereditary Spastic Paraplegia (HSP). We therefore asked how loss of Rtnl1, a Drosophila ortholog of the human HSP gene RTN2 (SPG12), which encodes an ER-shaping protein, affects ER organization and the function of presynaptic terminals. Loss of Rtnl1 depleted ER membrane markers at Drosophila presynaptic motor terminals and appeared to deplete narrow tubular ER while leaving cisternae largely unaffected, thus suggesting little change in resting Ca2+ storage capacity. Nevertheless, these changes were accompanied by major reductions in activity-evoked Ca2+ fluxes in the cytosol, ER lumen, and mitochondria, as well as reduced evoked and spontaneous neurotransmission. We found that reduced STIM-mediated ER-plasma membrane contacts underlie presynaptic Ca2+ defects in Rtnl1 mutants. Our results show the importance of ER architecture in presynaptic physiology and function, which are therefore potential factors in the pathology of HSP.

Selective Serotonin Reuptake Inhibitors within Cells: Temporal Resolution in Cytoplasm, Endoplasmic Reticulum, and Membrane.

Selective serotonin reuptake inhibitors (SSRIs) are the most prescribed treatment for individuals experiencing major depressive disorder. The therapeutic mechanisms that take place before, during, or after SSRIs bind the serotonin transporter (SERT) are poorly understood, partially because no studies exist on the cellular and subcellular pharmacokinetic properties of SSRIs in living cells. We studied escitalopram and fluoxetine using new intensity-based, drug-sensing fluorescent reporters targeted to the plasma membrane, cytoplasm, or endoplasmic reticulum (ER) of cultured neurons and mammalian cell lines. We also used chemical detection of drug within cells and phospholipid membranes. The drugs attain equilibrium in neuronal cytoplasm and ER at approximately the same concentration as the externally applied solution, with time constants of a few s (escitalopram) or 200-300 s (fluoxetine). Simultaneously, the drugs accumulate within lipid membranes by ≥18-fold (escitalopram) or 180-fold (fluoxetine), and possibly by much larger factors. Both drugs leave cytoplasm, lumen, and membranes just as quickly during washout. We synthesized membrane-impermeant quaternary amine derivatives of the two SSRIs. The quaternary derivatives are substantially excluded from membrane, cytoplasm, and ER for >2.4 h. They inhibit SERT transport-associated currents sixfold or 11-fold less potently than the SSRIs (escitalopram or fluoxetine derivative, respectively), providing useful probes for distinguishing compartmentalized SSRI effects. Although our measurements are orders of magnitude faster than the therapeutic lag of SSRIs, these data suggest that SSRI-SERT interactions within organelles or membranes may play roles during either the therapeutic effects or the antidepressant discontinuation syndrome.SIGNIFICANCE STATEMENT Selective serotonin reuptake inhibitors stabilize mood in several disorders. In general, these drugs bind to SERT, which clears serotonin from CNS and peripheral tissues. SERT ligands are effective and relatively safe; primary care practitioners often prescribe them. However, they have several side effects and require 2-6 weeks of continuous administration until they act effectively. How they work remains perplexing, contrasting with earlier assumptions that the therapeutic mechanism involves SERT inhibition followed by increased extracellular serotonin levels. This study establishes that two SERT ligands, fluoxetine and escitalopram, enter neurons within minutes, while simultaneously accumulating in many membranes. Such knowledge will motivate future research, hopefully revealing where and how SERT ligands engage their therapeutic target(s).

Resveratrol Suppresses Bupivacaine-Induced Spinal Neurotoxicity in Rats by Inhibiting Endoplasmic Reticulum Stress via SIRT1 Modulation.

Bupivacaine (BUP) may cause neurotoxic effects after spinal anesthesia. Resveratrol (RSV), a natural agonist of Silent information regulator 1 (SIRT1), protects various tissues and organs from damage by regulating endoplasmic reticulum (ER) stress. The aim of this study is to explore whether RSV could alleviate the neurotoxicity induced by bupivacaine via regulating ER stress. We established a model of bupivacaine-induced spinal neurotoxicity in rats using intrathecal injection of 5% bupivacaine. The protective effect of RSV was evaluated by injecting intrathecally with 30 μg/μL RSV in total of 10 μL per day for 4 consecutive days. On day 3 after bupivacaine administration, tail-flick latency (TFL) tests and the Basso, Beattie, and Bresnahan (BBB) locomotor scores were assessed to neurological function, and the lumbar enlargement of the spinal cord was obtained. H&E and Nissl staining were used to evaluate the histomorphological changes and the number of survival neurons. TUNEL staining was conducted to determine apoptotic cells. The expression of proteins was detected by IHC, immunofluorescence, and western blot. The mRNA level of SIRT1 was determined by RT-PCR. Bupivacaine caused spinal cord neurotoxicity by inducing cell apoptosis and triggering ER stress. RSV treatment promoted the recovery of neurological dysfunction after bupivacaine administration by suppressing neuronal apoptosis and ER stress. Furthermore, RSV upregulated SIRT1 expression and inhibited PERK signaling pathway activation. In summary, resveratrol suppresses bupivacaine-induced spinal neurotoxicity in rats by inhibiting endoplasmic reticulum stress via SIRT1 modulation.

PARP16-Mediated Stabilization of Amyloid Precursor Protein mRNA Exacerbates Alzheimer's Disease Pathogenesis.

The accumulation and deposition of beta-amyloid (Aβ) are key neuropathological hallmarks of Alzheimer's disease (AD). PARP16, a Poly(ADP-ribose) polymerase, is a known tail-anchored endoplasmic reticulum (ER) transmembrane protein that transduces ER stress during pathological processes. Here, we found that PARP16 was significantly increased in the hippocampi and cortices of APPswe/PS1dE9 (APP/PS1) mice and hippocampal neuronal HT22 cells exposed to Aβ, suggesting a positive correlation between the progression of AD pathology and the overexpression of PARP16. To define the effect of PARP16 on AD progression, adeno-associated virus mediated-PARP16 knockdown was used in APP/PS1 mice to investigate the role of PARP16 in spatial memory, amyloid burden, and neuroinflammation. Knockdown of PARP16 partly attenuated impaired spatial memory, as indicated by the Morris water maze test, and decreased amyloid deposition, neuronal apoptosis, and the production of inflammatory cytokines in the brains of APP/PS1 mice. In vitro experiments demonstrated that the knockdown of PARP16 expression rescued neuronal damage and ER stress triggered by Aβ. Furthermore, we discovered that intracellular PARP16 acts as an RNA-binding protein that regulates the mRNA stability of amyloid precursor protein (APP) and protects targeted APP from degradation, thereby increasing APP levels and AD pathology. Our findings revealed an unanticipated role of PARP16 in the pathogenesis of AD, and at least in part, its association with increased APP mRNA stability.

TUG-891 inhibits neuronal endoplasmic reticulum stress and pyroptosis activation and protects neurons in a mouse model of intraventricular hemorrhage.

Pyroptosis plays an important role in hemorrhagic stroke. Excessive endoplasmic reticulum stress can cause endoplasmic reticulum dysfunction and cellular pyroptosis by regulating the nucleotide-binding oligomerization domain and leucine-rich repeat pyrin domain-containing protein 3 (NLRP3) pathway. However, the relationship between pyroptosis and endoplasmic reticulum stress after intraventricular hemorrhage is unclear. In this study, we established a mouse model of intraventricular hemorrhage and found pyroptosis and endoplasmic reticulum stress in brain tissue. Intraperitoneal injection of the selective GPR120 agonist TUG-891 inhibited endoplasmic reticulum stress, pyroptosis, and inflammation and protected neurons. The neuroprotective effect of TUG-891 appears related to inhibition of endoplasmic reticulum stress and pyroptosis activation.

AA147 ameliorates post-cardiac arrest cerebral ischemia/reperfusion injury through the co-regulation of the ATF6 and Nrf2 signaling pathways.

Ischemia/reperfusion caused by cardiac arrest (CA) disturbs endoplasmic reticulum (ER) homeostasis and redox balance in neurons. AA147, originally developed as a pharmacologic activator of the activating transcription factor 6 (ATF6), can protect multiple tissues from ischemia/reperfusion injury (IRI) by decreasing reactive oxygen species (ROS) and restoring ER function. However, it is unclear whether pharmacologic treatment of AA147 could ameliorate post-CA cerebral IRI and whether it is associated with proteostasis regulation and anti-oxidative stress mechanism. In the present study, mice were subjected to 9 min-CA surgery followed by cardiopulmonary resuscitation (CPR). AA147 or vehicle was administered 1 day before the operation and 15min after the return of spontaneous circulation. We found that AA147 restored neurological function and reduced dead neurons in mice suffering from CA. Moreover, AA147 inhibited CA/CPR-caused neuronal apoptosis and ER stress, indicated by reduced TUNEL-positive neurons, surged expression of Bcl-2/Bax, and down expression of cleaved caspase-3, caspase-12, C/EBP homologous protein (CHOP). The expression of ATF6 and its regulated gene glucose-regulated protein 78 (GRP78) increased significantly after the administration of AA147, suggesting the activation of the ATF6 pathway. In addition, AA147 also alleviated the upsurge of the ROS generation and MDA levels as well as increased SOD activity, accompanied by enhancement of the nuclear factor E2-related factor 2 (Nrf2) and its modulated heme-oxygenase-1 (HO-1) expressions. Cotreatment of AA147 with inhibitors of the ATF6 or Nrf2 significantly suppressed AA147-dependent reductions in ROS scavenging and neuronal death after CA/CPR. The results suggested that AA147 could confer neuroprotection against post-CA cerebral IRI through inhibition of oxidative stress along with ER stress-associated apoptosis, which is attributed to the coregulation of both ATF6 and Nrf2 signaling pathways activity. Our findings support the potential for AA147 as a therapeutic approach to improve post-CA brain injury.

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.