Dynamic interaction between sigma-1 receptor and Kv1.2 shapes neuronal and behavioral responses to cocaine.

The sigma-1 receptor (Sig-1R), an endoplasmic reticulum (ER) chaperone protein, is an interorganelle signaling modulator that potentially plays a role in drug-seeking behaviors. However, the brain site of action and underlying cellular mechanisms remain unidentified. We found that cocaine exposure triggers a Sig-1R-dependent upregulation of D-type K(+) current in the nucleus accumbens (NAc) that results in neuronal hypoactivity and thereby enhances behavioral cocaine response. Combining ex vivo and in vitro studies, we demonstrated that this neuroadaptation is caused by a persistent protein-protein association between Sig-1Rs and Kv1.2 channels, a phenomenon that is associated to a redistribution of both proteins from intracellular compartments to the plasma membrane. In conclusion, the dynamic Sig-1R-Kv1.2 complex represents a mechanism that shapes neuronal and behavioral response to cocaine. Functional consequences of Sig-1R binding to K(+) channels may have implications for other chronic diseases where maladaptive intrinsic plasticity and Sig-1Rs are engaged.

Benzomorphan and non-benzomorphan agonists differentially alter sigma-1 receptor quaternary structure, as does types of cellular stress.

Sigma-1 receptor (S1R) is a calcium-sensitive, ligand-operated receptor chaperone present on the endoplasmic reticulum (ER) membrane. S1R plays an important role in ER-mitochondrial inter-organelle calcium signaling and cell survival. S1R and its agonists confer resilience against various neurodegenerative diseases; however, the molecular mechanism of S1R is not yet fully understood. At resting state, S1R is either in a monomeric or oligomeric state but the ratio of these concentrations seems to change upon activation of S1R. S1R is activated by either cellular stress, such as ER-calcium depletion, or ligands. While the effect of ligands on S1R quaternary structure remains unclear, the effect of cellular stress has not been studied. In this study we utilize cellular and an in-vivo model to study changes in quaternary structure of S1R upon activation. We incubated cells with cellular stressors (H2O2 and thapsigargin) or exogenous ligands, then quantified monomeric and oligomeric forms. We observed that benzomorphan-based S1R agonists induce monomerization of S1R and decrease oligomerization, which was confirmed in the liver tissue of mice injected with (+)-Pentazocine. Antagonists block this effect but do not induce any changes when used alone. Oxidative stress (H2O2) increases the monomeric/oligomeric S1R ratio whereas ER calcium depletion (thapsigargin) has no effect. We also analyzed the oligomerization ability of various truncated S1R fragments and identified the fragments favorizing oligomerization. In this publication we demonstrate that quaternary structural changes differ according to the mechanism of S1R activation. Therefore, we offer a novel perspective on S1R activation as a nuanced phenomenon dependent on the type of stimulus.

Nox4 regulates InsP3 receptor-dependent Ca2+ release into mitochondria to promote cell survival.

Cells subjected to environmental stresses undergo regulated cell death (RCD) when homeostatic programs fail to maintain viability. A major mechanism of RCD is the excessive calcium loading of mitochondria and consequent triggering of the mitochondrial permeability transition (mPT), which is especially important in post-mitotic cells such as cardiomyocytes and neurons. Here, we show that stress-induced upregulation of the ROS-generating protein Nox4 at the ER-mitochondria contact sites (MAMs) is a pro-survival mechanism that inhibits calcium transfer through InsP3 receptors (InsP3 R). Nox4 mediates redox signaling at the MAM of stressed cells to augment Akt-dependent phosphorylation of InsP3 R, thereby inhibiting calcium flux and mPT-dependent necrosis. In hearts subjected to ischemia-reperfusion, Nox4 limits infarct size through this mechanism. These results uncover a hitherto unrecognized stress pathway, whereby a ROS-generating protein mediates pro-survival effects through spatially confined signaling at the MAM to regulate ER to mitochondria calcium flux and triggering of the mPT.

SIGMAR1 Confers Innate Resilience against Neurodegeneration.

The sigma-1 receptor (SIGMAR1) is one of a kind: a receptor chaperone protein. This 223 amino acid-long protein is enriched at the mitochondria-associated endoplasmic reticulum membrane (MAM), a specialized microdomain of the endoplasmic reticulum that is structurally and functionally connected to the mitochondria. As a receptor, SIGMAR1 binds a wide spectrum of ligands. Numerous molecules targeting SIGMAR1 are currently in pre-clinical or clinical development. Interestingly, the range of pathologies covered by these studies is broad, especially with regard to neurodegenerative disorders. Upon activation, SIGMAR1 can translocate and interact with other proteins, mostly at the MAM but also in other organelles, which allows SIGMAR1 to affect many cellular functions. During these interactions, SIGMAR1 exhibits chaperone protein behavior by participating in the folding and stabilization of its partner. In this short communication, we will shed light on how SIGMAR1 confers protection against neurodegeneration to the cells of the nervous system and why this ability makes SIGMAR1 a multifunctional therapeutic prospect.

Knocking Out Sigma-1 Receptors Reveals Diverse Health Problems.

Sigma-1 receptor (Sig-1R) is a protein present in several organs such as brain, lung, and heart. In a cell, Sig-1R is mainly located across the membranes of the endoplasmic reticulum and more specifically at the mitochondria-associated membranes. Despite numerous studies showing that Sig-1R could be targeted to rescue several cellular mechanisms in different pathological conditions, less is known about its fundamental relevance. In this review, we report results from various studies and focus on the importance of Sig-1R in physiological conditions by comparing Sig-1R KO mice to wild-type mice in order to investigate the fundamental functions of Sig-1R. We note that the Sig-1R deletion induces cognitive, psychiatric, and motor dysfunctions, but also alters metabolism of heart. Finally, taken together, observations from different experiments demonstrate that those dysfunctions are correlated to poor regulation of ER and mitochondria metabolism altered by stress, which could occur with aging.

Genetic deletion of vesicular glutamate transporter in dopamine neurons increases vulnerability to MPTP-induced neurotoxicity in mice.

A subset of midbrain dopamine (DA) neurons express vesicular glutamate transporter 2 (VgluT2), which facilitates synaptic vesicle loading of glutamate. Recent studies indicate that such expression can modulate DA-dependent reward behaviors, but little is known about functional consequences of DA neuron VgluT2 expression in neurodegenerative diseases like Parkinson's disease (PD). Here, we report that selective deletion of VgluT2 in DA neurons in conditional VgluT2-KO (VgluT2-cKO) mice abolished glutamate release from DA neurons, reduced their expression of brain-derived neurotrophic factor (BDNF) and tyrosine receptor kinase B (TrkB), and exacerbated the pathological effects of exposure to the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Furthermore, viral rescue of VgluT2 expression in DA neurons of VglutT2-cKO mice restored BDNF/TrkB expression and attenuated MPTP-induced DA neuron loss and locomotor impairment. Together, these findings indicate that VgluT2 expression in DA neurons is neuroprotective. Genetic or environmental factors causing reduced expression or function of VgluT2 in DA neurons may place some individuals at increased risk for DA neuron degeneration. Therefore, maintaining physiological expression and function of VgluT2 in DA neurons may represent a valid molecular target for the development of preventive therapeutic interventions for PD.

At the Crossing of ER Stress and MAMs: A Key Role of Sigma-1 Receptor?

Calcium exchanges and homeostasis are finely regulated between cellular organelles and in response to physiological signals. Besides ionophores, including voltage-gated Ca2+ channels, ionotropic neurotransmitter receptors, or Store-operated Ca2+ entry, activity of regulatory intracellular proteins finely tune Calcium homeostasis. One of the most intriguing, by its unique nature but also most promising by the therapeutic opportunities it bears, is the sigma-1 receptor (Sig-1R). The Sig-1R is a chaperone protein residing at mitochondria-associated endoplasmic reticulum (ER) membranes (MAMs), where it interacts with several partners involved in ER stress response, or in Ca2+ exchange between the ER and mitochondria. Small molecules have been identified that specifically and selectively activate Sig-1R (Sig-1R agonists or positive modulators) at the cellular level and that also allow effective pharmacological actions in several pre-clinical models of pathologies. The present review will summarize the recent data on the mechanism of action of Sig-1R in regulating Ca2+ exchanges and protein interactions at MAMs and the ER. As MAMs alterations and ER stress now appear as a common track in most neurodegenerative diseases, the intracellular action of Sig-1R will be discussed in the context of the recently reported efficacy of Sig-1R drugs in pathologies like Alzheimer's disease, Parkinson's disease, Huntington's disease, or amyotrophic lateral sclerosis.

The sigma-1 receptor modulates dopamine transporter conformation and cocaine binding and may thereby potentiate cocaine self-administration in rats.

The dopamine transporter (DAT) regulates dopamine (DA) neurotransmission by recapturing DA into the presynaptic terminals and is a principal target of the psychostimulant cocaine. The sigma-1 receptor (σ1R) is a molecular chaperone, and its ligands have been shown to modulate DA neuronal signaling, although their effects on DAT activity are unclear. Here, we report that the prototypical σ1R agonist (+)-pentazocine potentiated the dose response of cocaine self-administration in rats, consistent with the effects of the σR agonists PRE-084 and DTG (1,3-di-o-tolylguanidine) reported previously. These behavioral effects appeared to be correlated with functional changes of DAT. Preincubation with (+)-pentazocine or PRE-084 increased the Bmax values of [3H]WIN35428 binding to DAT in rat striatal synaptosomes and transfected cells. A specific interaction between σ1R and DAT was detected by co-immunoprecipitation and bioluminescence resonance energy transfer assays. Mutational analyses indicated that the transmembrane domain of σ1R likely mediated this interaction. Furthermore, cysteine accessibility assays showed that σ1R agonist preincubation potentiated cocaine-induced changes in DAT conformation, which were blocked by the specific σ1R antagonist CM304. Moreover, σ1R ligands had distinct effects on σ1R multimerization. CM304 increased the proportion of multimeric σ1Rs, whereas (+)-pentazocine increased monomeric σ1Rs. Together these results support the hypothesis that σ1R agonists promote dissociation of σ1R multimers into monomers, which then interact with DAT to stabilize an outward-facing DAT conformation and enhance cocaine binding. We propose that this novel molecular mechanism underlies the behavioral potentiation of cocaine self-administration by σ1R agonists in animal models.

Sigma-1 receptor regulates Tau phosphorylation and axon extension by shaping p35 turnover via myristic acid.

Dysregulation of cyclin-dependent kinase 5 (cdk5) per relative concentrations of its activators p35 and p25 is implicated in neurodegenerative diseases. P35 has a short t½ and undergoes rapid proteasomal degradation in its membrane-bound myristoylated form. P35 is converted by calpain to p25, which, along with an extended t½, promotes aberrant activation of cdk5 and causes abnormal hyperphosphorylation of tau, thus leading to the formation of neurofibrillary tangles. The sigma-1 receptor (Sig-1R) is an endoplasmic reticulum chaperone that is implicated in neuronal survival. However, the specific role of the Sig-1R in neurodegeneration is unclear. Here we found that Sig-1Rs regulate proper tau phosphorylation and axon extension by promoting p35 turnover through the receptor's interaction with myristic acid. In Sig-1R-KO neurons, a greater accumulation of p35 is seen, which results from neither elevated transcription of p35 nor disrupted calpain activity, but rather to the slower degradation of p35. In contrast, Sig-1R overexpression causes a decrease of p35. Sig-1R-KO neurons exhibit shorter axons with lower densities. Myristic acid is found here to bind Sig-1R as an agonist that causes the dissociation of Sig-1R from its cognate partner binding immunoglobulin protein. Remarkably, treatment of Sig-1R-KO neurons with exogenous myristic acid mitigates p35 accumulation, diminishes tau phosphorylation, and restores axon elongation. Our results define the involvement of Sig-1Rs in neurodegeneration and provide a mechanistic explanation that Sig-1Rs help maintain proper tau phosphorylation by potentially carrying and providing myristic acid to p35 for enhanced p35 degradation to circumvent the formation of overreactive cdk5/p25.

Sigma-1 receptor mediates cocaine-induced transcriptional regulation by recruiting chromatin-remodeling factors at the nuclear envelope.

The sigma-1 receptor (Sig-1R) chaperone at the endoplasmic reticulum (ER) plays important roles in cellular regulation. Here we found a new function of Sig-1R, in that it translocates from the ER to the nuclear envelope (NE) to recruit chromatin-remodeling molecules and regulate the gene transcription thereof. Sig-1Rs mainly reside at the ER-mitochondrion interface. However, on stimulation by agonists such as cocaine, Sig-1Rs translocate from ER to the NE, where Sig-1Rs bind NE protein emerin and recruit chromatin-remodeling molecules, including lamin A/C, barrier-to-autointegration factor (BAF), and histone deacetylase (HDAC), to form a complex with the gene repressor specific protein 3 (Sp3). Knockdown of Sig-1Rs attenuates the complex formation. Cocaine was found to suppress the gene expression of monoamine oxidase B (MAOB) in the brain of wild-type but not Sig-1R knockout mouse. A single dose of cocaine (20 mg/kg) in rats suppresses the level of MAOB at nuclear accumbens without affecting the level of dopamine transporter. Daily injections of cocaine in rats caused behavioral sensitization. Withdrawal from cocaine in cocaine-sensitized rats induced an apparent time-dependent rebound of the MAOB protein level to about 200% over control on day 14 after withdrawal. Treatment of cocaine-withdrawn rats with the MAOB inhibitor deprenyl completely alleviated the behavioral sensitization to cocaine. Our results demonstrate a role of Sig-1R in transcriptional regulation and suggest cocaine may work through this newly discovered genomic action to achieve its addictive action. Results also suggest the MAOB inhibitor deprenyl as a therapeutic agent to block certain actions of cocaine during withdrawal.

Roles of sigma-1 receptors on mitochondrial functions relevant to neurodegenerative diseases.

The sigma-1 receptor (Sig-1R) is a chaperone that resides mainly at the mitochondrion-associated endoplasmic reticulum (ER) membrane (called the MAMs) and acts as a dynamic pluripotent modulator in living systems. At the MAM, the Sig-1R is known to play a role in regulating the Ca2+ signaling between ER and mitochondria and in maintaining the structural integrity of the MAM. The MAM serves as bridges between ER and mitochondria regulating multiple functions such as Ca2+ transfer, energy exchange, lipid synthesis and transports, and protein folding that are pivotal to cell survival and defense. Recently, emerging evidences indicate that the MAM is critical in maintaining neuronal homeostasis. Thus, given the specific localization of the Sig-1R at the MAM, we highlight and propose that the direct or indirect regulations of the Sig-1R on mitochondrial functions may relate to neurodegenerative diseases including Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD) and amyotrophic lateral sclerosis (ALS). In addition, the promising use of Sig-1R ligands to rescue mitochondrial dysfunction-induced neurodegeneration is addressed.

Sigma-1 Receptors Fine-Tune the Neuronal Networks.

The endoplasmic reticular (ER) protein sigma-1 receptor (Sig-1R) has been implicated in CNS disorders including but not limited to neurodegenerative diseases, depression , amnesia, and substance abuse. Sig-1Rs are particularly enriched in the specific domain where ER membranes make contacts with the mitochondria (MAM). Within that specific domain, Sig-1Rs play significant roles governing calcium signaling and reactive oxygen species homeostasis to maintain proper neuronal functions. Studies showed that the Sig-1R is pivotal to regulate neuroplasticity and neural survival via multiple aspects of mechanism. Numerous reports have been focusing on Sig-1R's regulatory effects in ER stress, mitochondrial function, oxidative stress and protein chaperoning. In this book chapter, we will discuss the emerging role of Sig-1R in balancing the populations of neuron and glia and their implications in CNS diseases.

The non-apoptotic action of Bcl-xL: regulating Ca(2+) signaling and bioenergetics at the ER-mitochondrion interface.

Bcl-2 family proteins are known to competitively regulate Ca(2+); however, the specific inter-organelle signaling pathways and related cellular functions are not fully elucidated. In this study, a portion of Bcl-xL was detected at the ER-mitochondrion interface or MAM (mitochondria-associated ER membrane) in association with type 3 inositol 1,4,5-trisphosphate receptors (IP3R3); an association facilitated by the BH4 and transmembrane domains of Bcl-xL. Moreover, increasing Bcl-xL expression enhanced transient mitochondrial Ca(2+) levels upon ER Ca(2+) depletion induced by short-term, non-apoptotic incubation with thapsigargin (Tg), while concomitantly reducing cytosolic Ca(2+) release. These mitochondrial changes appear to be IP3R3-dependent and resulted in decreased NAD/NADH ratios and higher electron transport chain oxidase activity. Interestingly, extended Tg exposure stimulated ER stress, but not apoptosis, and further enhanced TCA cycling. Indeed, confocal analysis indicated that Bcl-xL translocated to the MAM and increased its interaction with IP3R3 following extended Tg treatment. Thus, the MAM is a critical cell-signaling junction whereby Bcl-xL dynamically interacts with IP3R3 to coordinate mitochondrial Ca(2+) transfer and alters cellular metabolism in order to increase the cells' bioenergetic capacity, particularly during periods of stress.

A role for sigma receptors in stimulant self-administration and addiction.

Sigma-1 receptors (σ1Rs) are structurally unique intracellular proteins that function as chaperones. σ1Rs translocate from the mitochondria-associated membrane to other subcellular compartments, and can influence a host of targets, including ion channels, G-protein-coupled receptors, lipids, and other signaling proteins. Drugs binding to σRs can induce or block the actions of σRs. Studies indicate that stimulant self-administration induces the reinforcing effects of σR agonists, because of dopamine transporter actions. Once established, the reinforcing effects of σR agonists are independent of dopaminergic mechanisms traditionally thought to be critical to the reinforcing effects of stimulants. Self-administered doses of σR agonists do not increase dopamine concentrations in the nucleus accumbens shell, a transmitter and brain region considered important for the reinforcing effects of abused drugs. However, self-administration of σR agonists is blocked by σR antagonists. Several effects of stimulants have been blocked by σR antagonists, including the reinforcing effects, assessed by a place-conditioning procedure. However, the self-administration of stimulants is largely unaffected by σR antagonists, indicating fundamental differences in the mechanisms underlying these two procedures used to assess the reinforcing effects. When σR antagonists are administered in combination with dopamine uptake inhibitors, an effective and specific blockade of stimulant self-administration is obtained. Actions of stimulant drugs related to their abuse induce unique changes in σR activity and the changes induced potentially create redundant and, once established, independent reinforcement pathways. Concomitant targeting of both dopaminergic pathways and σR proteins produces a selective antagonism of stimulant self-administration, suggesting new avenues for combination chemotherapies to specifically combat stimulant abuse.

Morphine drives internal ribosome entry site-mediated hnRNP K translation in neurons through opioid receptor-dependent signaling.

Heterogeneous nuclear ribonucleoprotein K (hnRNP K) binds to the promoter region of mu-opioid receptor (MOR) to regulate its transcriptional activity. How hnRNP K contributes to the analgesic effects of morphine, however, is largely unknown. We provide evidence that morphine increases hnRNP K protein expression via MOR activation in rat primary cortical neurons and HEK-293 cells expressing MORs, without increasing mRNA levels. Using the bicistronic reporter assay, we examined whether morphine-mediated accumulation of hnRNP K resulted from translational control. We identified potential internal ribosome entry site elements located in the 5' untranslated regions of hnRNP K transcripts that were regulated by morphine. This finding suggests that internal translation contributes to the morphine-induced accumulation of hnRNP K protein in regions of the central nervous system correlated with nociceptive and antinociceptive modulatory systems in mice. Finally, we found that down-regulation of hnRNP K mediated by siRNA attenuated morphine-induced hyperpolarization of membrane potential in AtT20 cells. Silencing hnRNP K expression in the spinal cord increased nociceptive sensitivity in wild-type mice, but not in MOR-knockout mice. Thus, our findings identify the role of translational control of hnRNP K in morphine-induced analgesia through activation of MOR.

Bcl-2 family in inter-organelle modulation of calcium signaling; roles in bioenergetics and cell survival.

Bcl-2 family proteins, known for their apoptosis functioning at the mitochondria, have been shown to localize to other cellular compartments to mediate calcium (Ca2+) signals. Since the proper supply of Ca2+ in cells serves as an important mechanism for cellular survival and bioenergetics, we propose an integrating role for Bcl-2 family proteins in modulating Ca2+ signaling. The endoplasmic reticulum (ER) is the main Ca2+ storage for the cell and Bcl-2 family proteins competitively regulate its Ca2+ concentration. Bcl-2 family proteins also regulate the flux of Ca2+ from the ER by physically interacting with inositol 1,4,5-trisphosphate receptors (IP3Rs) to mediate their opening. Type 1 IP3Rs reside at the bulk ER to coordinate cytosolic Ca2+ signals, while type 3 IP3Rs reside at mitochondria-associated ER membrane (MAM) to facilitate mitochondrial Ca2+ uptake. In healthy cells, mitochondrial Ca2+ drives pyruvate into the citric acid (TCA) cycle to facilitate ATP production, while a continuous accumulation of Ca2+ can trigger the release of cytochrome c, thus initiating apoptosis. Since multiple organelles and Bcl-2 family proteins are involved in Ca2+ signaling, we aim to clarify the role that Bcl-2 family proteins play in facilitating Ca2+ signaling and how mitochondrial Ca2+ is relevant in both bioenergetics and apoptosis. We also explore how these insights could be useful in controlling bioenergetics in apoptosis-resistant cell lines.

Fluvoxamine Exerts Sigma-1R to Rescue Autophagy via Pom121-Mediated Nucleocytoplasmic Transport of TFEB.

Expansion of the GGGGCC-RNA repeat is a known cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), which currently have no cure. Recent studies have indicated the activation of Sigma-1 receptor plays an important role in providing neuroprotection, especially in ALS and Alzheimer's disease. Nevertheless, the mechanisms underlying Sigma-1R activation and its effect on (G4C2)n-RNA-induced cell death remain unclear. In this study, we demonstrated that fluvoxamine is a Sigma-1R agonist that can increase chaperone activity and stabilize the protein expression of Pom121 in (G4C2)31-RNA-expressing NSC34 cells, leading to increased colocalization at the nuclear envelope. Interestingly, fluvoxamine treatment increased Pom121 protein expression without affecting transcription. In C9orf72-ALS, the nuclear translocation of TFEB autophagy factor decreased owing to nucleocytoplasmic transport defects. Our results showed that pretreatment of NSC34 cells with fluvoxamine promoted the shuttling of TFEB into the nucleus and elevated the expression of LC3-II compared to the overexpression of (G4C2)31-RNA alone. Additionally, even when used alone, fluvoxamine increases Pom121 expression and TFEB translocation. To summarize, fluvoxamine may act as a promising repurposed medicine for patients with C9orf72-ALS, as it stabilizes the nucleoporin Pom121 and promotes the translocation of TFEB in (G4C2)31-RNA-expressing NSC34 cells.

The lifetime of UDP-galactose:ceramide galactosyltransferase is controlled by a distinct endoplasmic reticulum-associated degradation (ERAD) regulated by sigma-1 receptor chaperones.

The glycosphingolipid biosynthesis is initiated by monoglycosylation of ceramides, the action of which is catalyzed either by UDP-glucose:ceramide glucosyltransferase or by UDP-galactose:ceramide galactosyltransferase (CGalT). CGalT is expressed predominantly at the endoplasmic reticulum (ER) of oligodendrocytes and is responsible for synthesizing galactosylceramides (GalCer) that play an important role in regulation of axon conductance. However, despite the importance of ceramide monoglycosylation enzymes in a spectrum of cellular functions, the mechanism that fine tunes activities of those enzymes is largely unknown. In the present study, we demonstrated that the sigma-1 receptor (Sig-1R) chaperone, the mammalian homologue of a yeast C8-C7 sterol isomerase, controls the protein level and activity of the CGalT enzyme via a distinct ER-associated degradation system involving Insig. The Sig-1R forms a complex with Insig via its transmembrane domain partly in a sterol-dependent manner and associates with CGalT at the ER. The knockdown of Sig-1Rs dramatically prolonged the lifetime of CGalT without affecting the trimming of N-linked oligosaccharides at CGalT. The increased lifetime leads to the up-regulation of CGalT protein as well as elevated enzymatic activity in CHO cells stably expressing CGalT. Knockdown of Sig-1Rs also decreased CGalT degradation endogenously expressed in D6P2T-schwannoma cells. Our data suggest that Sig-1Rs negatively regulate the activity of GalCer synthesis under physiological conditions by enhancing the degradation of CGalT through regulation of the dynamics of Insig in the lipid-activated ER-associated degradation system. The GalCer synthesis may thus be influenced by sterols at the ER.

Translational and transcriptional control of Sp1 against ischaemia through a hydrogen peroxide-activated internal ribosomal entry site pathway.

The exact mechanism underlying increases in Sp1 and the physiological consequences thereafter remains unknown. In rat primary cortical neurons, oxygen-glucose deprivation (OGD) causes an increase in H(2)O(2) as well as Sp1 in early ischaemia but apparently does not change mRNA level or Sp1 stability. We hereby identified a longer 5'-UTR in Sp1 mRNA that contains an internal ribosome entry site (IRES) that regulates rapid and efficient translation of existing mRNAs. By using polysomal fragmentation and bicistronic luciferase assays, we found that H(2)O(2) activates IRES-dependent translation. Thus, H(2)O(2) or tempol, a superoxide dismutase-mimetic, increases Sp1 levels in OGD-treated neurons. Further, early-expressed Sp1 binds to Sp1 promoter to cause a late rise in Sp1 in a feed-forward manner. Short hairpin RNA against Sp1 exacerbates OGD-induced apoptosis in primary neurons. While Sp1 levels increase in the cortex in a rat model of stroke, inhibition of Sp1 binding leads to enhanced apoptosis and cortical injury. These results demonstrate that neurons can use H(2)O(2) as a signalling molecule to quickly induce Sp1 translation through an IRES-dependent translation pathway that, in cooperation with a late rise in Sp1 via feed-forward transcriptional activation, protects neurons against ischaemic damage.

Nucleoporin POM121 signals TFEB-mediated autophagy via activation of SIGMAR1/sigma-1 receptor chaperone by pridopidine.

Macroautophagy/autophagy is an essential process for cellular survival and is implicated in many diseases. A critical step in autophagy is the transport of the transcription factor TFEB from the cytosol into the nucleus, through the nuclear pore (NP) by KPNB1/importinß1. In the C9orf72 subtype of amyotrophic lateral sclerosis-frontotemporal lobar degeneration (ALS-FTD), the hexanucleotide (G4C2)RNA expansion (HRE) disrupts the nucleocytoplasmic transport of TFEB, compromising autophagy. Here we show that a molecular chaperone, the SIGMAR1/Sigma-1 receptor (sigma non-opioid intracellular receptor 1), facilitates TFEB transport into the nucleus by chaperoning the NP protein (i.e., nucleoporin) POM121 which recruits KPNB1. In NSC34 cells, HRE reduces TFEB transport by interfering with the association between SIGMAR1 and POM121, resulting in reduced nuclear levels of TFEB, KPNB1, and the autophagy marker LC3-II. Overexpression of SIGMAR1 or POM121, or treatment with the highly selective and potent SIGMAR1 agonist pridopidine, currently in phase 2/3 clinical trials for ALS and Huntington disease, rescues all of these deficits. Our results implicate nucleoporin POM121 not merely as a structural nucleoporin, but also as a chaperone-operated signaling molecule enabling TFEB-mediated autophagy. Our data suggest the use of SIGMAR1 agonists, such as pridopidine, for therapeutic development of diseases in which autophagy is impaired.Abbreviations: ALS-FTD, amyotrophic lateral sclerosis-frontotemporal dementiaC9ALS-FTD, C9orf72 subtype of amyotrophic lateral sclerosis-frontotemporal dementiaCS, citrate synthaseER, endoplasmic reticulumGSS, glutathione synthetaseHRE, hexanucleotide repeat expansionHSPA5/BiP, heat shock protein 5LAMP1, lysosomal-associated membrane protein 1MAM, mitochondria-associated endoplasmic reticulum membraneMAP1LC3/LC3, microtubule-associated protein 1 light chain 3NP, nuclear poreNSC34, mouse motor neuron-like hybrid cell lineNUPs, nucleoporinsPOM121, nuclear pore membrane protein 121SIGMAR1/Sigma-1R, sigma non-opioid intracellular receptor 1TFEB, transcription factor EBTMEM97/Sigma-2R, transmembrane protein 97.