Endoplasmic Reticulum Chaperone Research Articles

Vx-809, a CFTR Corrector, Acts through a General Mechanism of Protein Folding and on the Inflammatory Process

Correct protein folding is the basis of cellular well-being; thus, accumulation of misfolded proteins within the endoplasmic reticulum (ER) leads to an imbalance of homeostasis that causes stress to the ER. Various studies have shown that protein misfolding is a significant factor in the etiology of many human diseases, including cancer, diabetes, and cystic fibrosis. Misfolded protein accumulation in the ER triggers a sophisticated signal transduction pathway, the unfolded protein response (UPR), which is controlled by three proteins, resident in ER: IRE1α, PERK, and ATF6. Briefly, when ER stress is irreversible, IRE1α induces the activation of pro-inflammatory proteins; PERK phosphorylates eIF2α which induces ATF4 transcription, while ATF6 activates genes encoding ER chaperones. Reticular stress causes an alteration of the calcium homeostasis, which is released from the ER and taken up by the mitochondria, leading to an increase in the oxygen radical species production, and consequently, to oxidative stress. Accumulation of intracellular calcium, in combination with lethal ROS levels, has been associated with an increase of pro-inflammatory protein expression and the initiation of the inflammatory process. Lumacaftor (Vx-809) is a common corrector used in cystic fibrosis treatment which enhances the folding of mutated F508del-CFTR, one of the most prevalent impaired proteins underlying the disease, promoting a higher localization of the mutant protein on the cell membrane. Here, we demonstrate that this drug reduces the ER stress and, consequently, the inflammation that is caused by such events. Thus, this molecule is a promising drug to treat several pathologies that present an etiopathogenesis due to the accumulation of protein aggregates that lead to chronic reticular stress.

Scaffold Hopping and Screening for Potent Small Molecule Agonists for GRP94: Implications to Alleviate ER Stress-Associated Pathogenesis.

Disparity in the activity of Endoplasmic reticulum (ER) leads to degenerative diseases, mainly associated with protein misfolding and aggregation leading to cellular dysfunction and damage, ultimately contributing to ER stress. ER stress activates the complex network of Unfolded Protein Response (UPR) signaling pathways mediated by transmembrane proteins IRE1, ATF6, and PERK. In addition to UPR, many ER chaperones have evolved to optimize the output of properly foldedsecretory and membrane proteins. Glucose-regulated protein 94 (GRP94), an ER chaperone of heat shock protein HSP90 family, directs protein folding through interaction with other components of the ER protein folding machinery and assists in ER-associated degradation (ERAD). Activation of GRP94 would increase the efficacy of protein folding machinery and regulate the UPR pathway toward homeostasis. The present study aims to screen for novel agonists for GRP94 based on Core hopping, pharmacophore hypothesis, 3D-QSAR, and virtual screening with small-molecule compound libraries in order to improve the efficiency of native protein folding by enhancing GRP94 chaperone activity, therefore to reduce protein misfolding and aggregation. In this study, we have employed the strategy of small molecule-dependent ER programming to enhance the chaperone activity of GRP94 through scaffold hopping-based screening approach to identify specific GRP94 agonists. New scaffolds generated by altering the cores of NECA, the known GRP94 agonist, were validated by employing pharmacophore hypothesis testing, 3D-QSAR modeling, and molecular dynamics simulations. This facilitated the identification of small molecules to improve the efficiency of native protein folding by enhancing GRP94 activity. High-throughput virtual screening of the selected pharmacophore hypothesis against Selleckchem and ZINC databases retrieved a total of 2,27,081 compounds. Further analysis on docking and ADMET properties revealed Epimedin A, Narcissoside, Eriocitrin 1,2,3,4,6-O-Pentagalloylglucose, Secoisolariciresinol diglucoside, ZINC92952357, ZINC67650204, and ZINC72457930 as potential lead molecules. The stability and interaction of these small molecules were far better than the known agonist, NECA indicating their efficacy in selectively alleviating ER stress-associated pathogenesis. These results substantiate the fact that small molecule-dependent ER reprogramming would activate the ER chaperones and therefore reduce the protein misfolding as well as aggregation associated with ER stress in order to restore cellular homeostasis.

PDIA3 Expression Is Altered in the Limbic Brain Regions of Triple-Transgenic Mouse Model of Alzheimer's Disease.

In the present study, we used a mouse model of Alzheimer's disease (AD) (3×Tg-AD mice) to longitudinally analyse the expression level of PDIA3, a protein disulfide isomerase and endoplasmic reticulum (ER) chaperone, in selected brain limbic areas strongly affected by AD-pathology (amygdala, entorhinal cortex, dorsal and ventral hippocampus). Our results suggest that, while in Non-Tg mice PDIA3 levels gradually reduce with aging in all brain regions analyzed, 3×Tg-AD mice showed an age-dependent increase in PDIA3 levels in the amygdala, entorhinal cortex, and ventral hippocampus. A significant reduction of PDIA3 was observed in 3×Tg-AD mice already at 6 months of age, as compared to age-matched Non-Tg mice. A comparative immunohistochemistry analysis performed on 3×Tg-AD mice at 6 (mild AD-like pathology) and 18 (severe AD-like pathology) months of age showed a direct correlation between the cellular level of Aβ and PDIA3 proteins in all the brain regions analysed, even if with different magnitudes. Additionally, an immunohistochemistry analysis showed the presence of PDIA3 in all post-mitotic neurons and astrocytes. Overall, altered PDIA3 levels appear to be age- and/or pathology-dependent, corroborating the ER chaperone's involvement in AD pathology, and supporting the PDIA3 protein as a potential novel therapeutic target for the treatment of AD.

Antibodies to calnexin and mutated calreticulin are common in human sera

Purpose of the studyCalreticulin is an endoplasmic reticulum chaperone protein, which is involved in protein folding and in peptide loading of major histocompatibility complex class I molecules together with its homolog calnexin. Mutated calreticulin is associated with a group of hemopoietic disorders, especially myeloproliferative neoplasms. Currently only the cellular immune response to mutated calreticulin has been described, although preliminary findings have indicated that antibodies to mutated calreticulin are not specific for myeloproliferative disorders. These findings have prompted us to characterize the humoral immune response to mutated calreticulin and its chaperone homologue calnexin. Patients and methodsWe analyzed sera from myeloproliferative neoplasm patients, healthy donors and relapsing-remitting multiple sclerosis patients for the occurrence of autoantibodies to wild type and mutated calreticulin forms and to calnexin by enzyme-linked immunosorbent assay. ResultsAntibodies to mutated calreticulin and calnexin were present at similar levels in serum samples of myeloproliferative neoplasm and multiple sclerosis patients as well as healthy donors. Moreover, a high correlation between antibodies to mutated calreticulin and calnexin was seen for all patient and control groups. Epitope binding studies indicated that cross-reactive antibodies bound to a three-dimensional epitope encompassing a short linear sequence in the C-terminal of mutated calreticulin and calnexin. ConclusionCollectively, these findings indicate that calreticulin mutations may be common and not necessarily lead to onset of myeloproliferative neoplasm, possibly due to elimination of cells with mutations. This, in turn, may suggest that additional molecular changes may be required for development of myeloproliferative neoplasm.

Infancy-onset diabetes caused by de-regulated AMPylation of the human endoplasmic reticulum chaperone BiP.

Dysfunction of the endoplasmic reticulum (ER) in insulin-producing beta cells results in cell loss and diabetes mellitus. Here we report on five individuals from three different consanguineous families with infancy-onset diabetes mellitus and severe neurodevelopmental delay caused by a homozygous p.(Arg371Ser) mutation in FICD. The FICD gene encodes a bifunctional Fic domain-containing enzyme that regulates the ER Hsp70 chaperone, BiP, via catalysis of two antagonistic reactions: inhibitory AMPylation and stimulatory deAMPylation of BiP. Arg371 is a conserved residue in the Fic domain active site. The FICDR371S mutation partially compromises BiP AMPylation in vitro but eliminates all detectable deAMPylation activity. Overexpression of FICDR371S or knock-in of the mutation at the FICD locus of stressed CHO cells results in inappropriately elevated levels of AMPylated BiP and compromised secretion. These findings, guided by human genetics, highlight the destructive consequences of de-regulated BiP AMPylation and raise the prospect of tuning FICD's antagonistic activities towards therapeutic ends.

Comparative transcriptomic analysis revealed changes in multiple signaling pathways involved in protein degradation in the digestive gland of Mytilus coruscus during high-temperatures

As a result of global warming, the Mytilus coruscus living attached in the intertidal zone experience extreme and fluctuating changes in temperature, and extreme temperature changes are causing mass mortality of intertidal species. This study explores the transcriptional response of M. coruscus at different temperatures (18 °C, 26 °C, and 33 °C) and different times (0, 12, and 24 h) of action by analyzing the potential temperature of the intertidal zone. In response to high temperatures, several signaling pathways in M. coruscus, ribosome, endocytosis, endoplasmic reticulum stress, protein degradation, and lysosomes, interact to counter the adverse effects of high temperatures on protein homeostasis. Increased expression of key genes, including heat shock proteins (Hsp70, Hsp20, and Hsp110), Lysosome-associated membrane glycoprotein (LAMP), endoplasmic reticulum chaperone (BiP), and baculoviral IAP repeat-containing protein 7 (BIRC7), may further mitigate the effects of heat stress and delay mortality in M. coruscus. These results reveal changes in multiple signaling pathways involved in protein degradation during high-temperature stress, which will contribute to our overall understanding of the molecular mechanisms underlying the response of M. coruscus to high-temperature stress.

Calnexin, More Than Just a Molecular Chaperone.

Calnexin is a type I integral endoplasmic reticulum (ER) membrane protein with an N-terminal domain that resides in the lumen of the ER and a C-terminal domain that extends into the cytosol. Calnexin is commonly referred to as a molecular chaperone involved in the folding and quality control of membrane-associated and secreted proteins, a function that is attributed to its ER- localized domain with a structure that bears a strong resemblance to another luminal ER chaperone and Ca2+-binding protein known as calreticulin. Studies have discovered that the cytosolic C-terminal domain of calnexin undergoes distinct post-translational modifications and interacts with a variety of proteins. Here, we discuss recent findings and hypothesize that the post-translational modifications of the calnexin C-terminal domain and its interaction with specific cytosolic proteins play a role in coordinating ER functions with events taking place in the cytosol and other cellular compartments.

Leukocyte surface expression of the endoplasmic reticulum chaperone GRP78 is increased in severe COVID-19.

Hyperinflammation present in individuals with severe COVID-19 has been associated with an exacerbated cytokine production and hyperactivated immune cells. Endoplasmic reticulum stress leading to the unfolded protein response has been recently reported as an active player in inducing inflammatory responses. Once unfolded protein response is activated, GRP78, an endoplasmic reticulum-resident chaperone, is translocated to the cell surface (sGRP78), where it is considered a cell stress marker; however, its presence has not been evaluated in immune cells during disease. Here we assessed the presence of sGRP78 on different cell subsets in blood samples from severe or convalescent COVID-19 patients. The frequency of CD45+sGRP78+ cells was higher in patients with the disease compared to convalescent patients. The latter showed similar frequencies to healthy controls. In patients with COVID-19, the lymphoid compartment showed the highest presence of sGRP78+ cells versus the myeloid compartment. CCL2, TNF-α, C-reactive protein, and international normalized ratio measurements showed a positive correlation with the frequency of CD45+sGRP78+ cells. Finally, gene expression microarray data showed that activated T and B cells increased the expression of GRP78, and peripheral blood mononuclear cells from healthy donors acquired sGRP78 upon activation with ionomycin and PMA. Thus, our data highlight the association of sGRP78 on immune cells in patients with severe COVID-19.

Proteoform Identification in 2D Electrophoresis Maps by Using Isoelectric Point Prediction

The possibility of identifying specific protein proteoforms with post-translational modifications (PTM) by analyzing two-dimensional (2D) gel electrophoresis maps and using the prediction of the isoelectric point of proteins (pI) has been investigated. The pI values were predicted using the pIPredict 3 program, supporting a wide range of chemical and post-translational modifications. Eleven 11 proteins (albumin, alpha-1-microglobulin, annexin A2, apolipoprotein E, gastric triacylglycerol lipase, mitochondrial isocitrate dehydrogenase, clusterin, plasmin, prothrombin, endoplasmic reticulum chaperone, S-adenosylmethionine synthase type 1) identified on six 2D electrophoresis maps were used as examples. Various options for selecting hypotheses are considered. These take into consideration the following available information about a particular protein: possible modification sites, processing features, variability of the amino acid composition. The obtained results indicate that the use of predicting the pI value for proteins with hypothetical PTMs can form a set of hypotheses about specific proteoform occurrence on 2D electrophoresis maps.

Induction of ER stress-mediated apoptosis through SOD1 upregulation by deficiency of CHI3L1 inhibits lung metastasis.

Chitinase-3-like protein 1 (CHI3L1), which is secreted by immune and inflammatory cells, is associated with several inflammatory diseases. However, the basic cellular pathophysiological functions of CHI3L1 are not well characterized. To investigate the novel pathophysiological function of CHI3L1, we performed LC-MS/MS analysis of cells transfected with Myc-vector and Myc-CHI3L1. We analyzed the changes in the protein distribution in Myc-CHI3L1 transfected-cells, and identified 451 differentially expressed proteins (DEPs) compared with Myc-vector-transfected-cells. The biological function of the 451 DEPs was analyzed and it was found that the proteins with endoplasmic reticulum (ER)-associated function were much more highly expressed in CHI3L1-overexpressing cells. We then compared and analyzed the effect of CHI3L1 on the ER chaperon levels in normal lung cells and cancer cells. We identified that CHI3L1 is localized in the ER. In normal cells, the depletion of CHI3L1 did not induce ER stress. However, the depletion of CHI3L1 induces ER stress and eventually activates the unfolded protein response, especially the activation of Protein kinase R-like endoplasmic reticulum kinase (PERK), which regulates protein synthesis in cancer cells. CHI3L1 may not affect ER stress owing to the lack of misfolded proteins in normal cells, but instead activate ER stress as a defense mechanism only in cancer cells. Under ER stress conditions induced by the application of thapsigargin, the depletion of CHI3L1 induces ER stress through the upregulation of PERK and PERK downstream factors (eIF2α and ATF4) in both normal and cancer cells. However, these signaling activations occur more often in cancer cells than in normal cells. The expression of Grp78 and PERK in the tissues of patients with lung cancer was higher compared with healthy tissues. It is well known that ER stress-mediated PERK-eIF2α-ATF4 signaling activation causes apoptotic cell death. ER stress-mediated apoptosis induced by the depletion of CHI3L1 occurs in cancer cells, but rarely occurs in normal cells. Consistent with results from the in vitro model, ER stress-mediated apoptosis was greatly increased during tumor growth and in the lung metastatic tissue of CHI3L1-knockout (KO) mice. The analysis of "big data" identified superoxide dismutase-1 (SOD1) as a novel target of CHI3L1 and interacted with CHI3L1. The depletion of CHI3L1 increased SOD1 expression, resulting in ER stress. Furthermore, the depletion of SOD1 reduced the expression of ER chaperones and ER-mediated apoptotic marker proteins, as well as apoptotic cell death induced by the depletion of CHI3L1 in in vivo and in vitro models. These results suggest that the depletion of CHI3L1 increases ER stress-mediated apoptotic cell death through SOD1 expression, and subsequently inhibits lung metastasis.

Response to endoplasmic reticulum stress in arginine vasopressin neurons.

Arginine vasopressin (AVP) is an antidiuretic hormone synthesized principally in the hypothalamic supraoptic and paraventricular nuclei. The immunoglobulin heavy chain binding protein (BiP), one of the most abundant endoplasmic reticulum (ER) chaperones, is highly expressed in AVP neurons, even under basal conditions. Moreover, its expression is upregulated in proportion to the increase in AVP expression under dehydration. These data suggest that AVP neurons are constantly exposed to ER stress. BiP knockdown in AVP neurons induces ER stress and autophagy, resulting in AVP neuronal loss, indicating that BiP is pivotal in maintaining the AVP neuron system. Furthermore, inhibition of autophagy after BiP knockdown exacerbates AVP neuronal loss, suggesting that autophagy induced under ER stress is a protective cellular mechanism by which AVP neurons cope with ER stress. Familial neurohypophysial diabetes insipidus (FNDI) is an autosomal dominant disorder caused by mutations in the AVP gene. It is characterized by delayed-onset progressive polyuria and eventual AVP neuronal loss. In AVP neurons of FNDI model mice, mutant protein aggregates are confined to a specific compartment of the ER, called the ER-associated compartment (ERAC). The formation of ERACs contributes to maintaining the function of the remaining intact ER, and mutant protein aggregates in ERACs undergo autophagic-lysosomal degradation without isolation or translocation from the ER, representing a novel protein degradation system in the ER.

Genotype-phenotype correlation and adaptive proteome reorganization in Marinesco-Sjögren syndrome

Marinesco-Sjögren Syndrome (MSS) causes cerebellar ataxia, myopathy and congenital cataracts in people carrying SIL1 mutations. SIL1 is an ATP exchange factor for BiP, the major endoplasmic reticulum (ER) chaperone involved in protein folding. SIL1 loss influences BiP activity, leading to ER stress and the activation of unfolded protein response (UPR). Purkinje cells and skeletal muscle fibers are the most sensitive cells to prolonged pathologic UPR, but adverse effects are detectable in other cell types. Currently a clear genotype-phenotype correlation is missing, due to the variable symptomatology and to the discovery of new SIL1 variants. We decided to focus our attention on two recent works providing different strategies to shed light on the pathophysiology of MSS. In the first one several cellular biomarkers have been evaluated to distinguish between malignant and benign SIL1 mutations. The other study proposed a proteomic approach to clarify adaptative mechanisms of MSS fibroblasts in response to SIL1 loss. Further investigations are needed to better understand the pathogenesis of MSS and to simplify the diagnosis in patients.

Overexpression of PvBiP2 improved biomass yield and cadmium tolerance in switchgrass (Panicum virgatum L.)

Switchgrass (Panicum virgatum L.), the prime bioenergy feedstock crop, is one ideal candidate for phytoremediation of cadmium (Cd). The absorption of Cd imposes severe endoplasmic reticulum (ER)-stress in plants. ER chaperone binding proteins (BiPs) are important modulators in ER-stress responses. The objective of this study was to characterize one Cd-responsive BiP gene, PvBiP2, in switchgrass for its roles in Cd tolerance and plant growth. PvBiP2 was up-regulated by Cd and the ER-stress inducer, dithiothreitol (DTT) and could be trans-activated by one Cd-responsive heat shock transcription factor PvHsfA4. Overexpression of PvBiP2 in switchgrass significantly increased its plant growth with higher height, stem diameter, leaf width, internode length, and tiller numbers than those of the wildtype (WT) plants under non-stress conditions. After 30 days of Cd treatment, the PvBiP2 over-expression transgenic lines showed 40–45% higher dry biomass accumulation with net photosynthesis rate (Pn), but lower electrolyte leakage (EL), malondialdehyde (MDA), and glutathione (GSH) levels than WT. Moreover, over-expressing PvBiP2 led to ∼90–140% Cd accumulation in plants but 46–57% lower Cd translocation rates to shoots. Together, the PvHSFA4-PvBiP2 module acted as positive regulators in plant Cd tolerance, and over-expressing PvBiP2 promoted plant vegetative growth as well as Cd tolerance making it an ideal molecular target for genetic improvement in switchgrass in the future.

Knockdown of endoplasmic reticulum chaperone BiP leads to the death of parvocellular AVP/CRH neurons in mice.

Arginine vasopressin (AVP) is expressed in both magnocellular (magnAVP) and parvocellular AVP (parvAVP) neurons of the paraventricular nucleus, and AVP colocalizes with corticotropin-releasing hormone (CRH) only in the parvocellular neurons. The immunoglobulin heavy chain binding protein (BiP) is a major endoplasmic reticulum (ER) chaperone which regulates the unfolded protein response under ER stress. We previously demonstrated that knockdown of BiP in magnAVP neurons exacerbated ER stress, which resulted in the autophagy-associated cell death of magnAVP neurons. Using the same approach, in the present study we examined the role of BiP in mouse parvAVP/CRH neurons. Our data demonstrate that BiP is expressed in mouse parvAVP/CRH neurons under nonstress conditions and is upregulated in proportion to the increase in CRH expression after adrenalectomy. For BiP knockdown in parvAVP/CRH neurons, we utilized a viral approach in combination with shRNA interference. Knockdown of BiP expression induced ER stress in parvAVP/CRH neurons, as reflected by the expression of C/EBP homologous protein. Furthermore, BiP knockdown led to the loss of parvAVP/CRH neurons after 4 weeks. In summary, our results demonstrate that BiP plays a pivotal role in parvAVP/CRH neurons, which function as neuroendocrine cells producing a large number of secretory proteins.

Endoplasmic Reticulum Stress Signaling and Neuronal Cell Death

Besides protein processing, the endoplasmic reticulum (ER) has several other functions such as lipid synthesis, the transfer of molecules to other cellular compartments, and the regulation of Ca2+ homeostasis. Before leaving the organelle, proteins must be folded and post-translationally modified. Protein folding and revision require molecular chaperones and a favorable ER environment. When in stressful situations, ER luminal conditions or chaperone capacity are altered, and the cell activates signaling cascades to restore a favorable folding environment triggering the so-called unfolded protein response (UPR) that can lead to autophagy to preserve cell integrity. However, when the UPR is disrupted or insufficient, cell death occurs. This review examines the links between UPR signaling, cell-protective responses, and death following ER stress with a particular focus on those mechanisms that operate in neurons.

Cannabidiol Protects Striatal Neurons by Attenuating Endoplasmic Reticulum Stress.

Introduction: The aggregation of misfolded proteins in the endoplasmic reticulum (ER) is a pathological trait shared by many neurodegenerative disorders. This aggregation leads to the persistent activation of the unfolded protein response (UPR) and ultimately apoptosis as a result of ER stress. Cannabidiol (CBD) has been demonstrated to be neuroprotective in various cellular and animal models of neurodegeneration, which has been attributed to its antioxidant and anti-inflammatory properties. However, little is known about the role of CBD in the context of protein folding and ER stress. The purpose of this study was to investigate whether CBD is neuroprotective against an in vitro model of ER stress. Materials and Methods: Using different exposure models, mouse striatal STHdhQ7/Q7 cells were exposed to either the ER stress inducer thapsigargin (TG) and/or CBD. Cell viabilities assays were used to investigate the effect of CBD pre-treatment, co-treatment, and post-treatment on TG-induced cell death. Real-time quantitative polymerase chain reaction was used to measure changes in ER stress regulators and UPR genes such as glucose-regulated protein-78 (GRP78), mesencephalic astrocyte-derived neurotrophic factor (MANF), B cell lymphoma 2 (BCL-2), BCL-2 interacting mediator of cell death (BIM), and caspase-12. Results: Cell viability increased significantly when cells were pre-treated with CBD before TG exposure. An increase in the gene expression of pro-survival ER chaperone GRP78 and ER-resident neurotrophic factor MANF coincided with this effect and decreased ER-mediated pro-apoptotic markers such as BIM, and caspase-12 was observed. Conclusions: These data suggest that CBD pre-treatment is neuroprotective against TG-induced cell death. Understanding the role of ER stress in CBD-driven neuroprotection provides insight into the therapeutic potential of CBD and the role of ER dysfunction in neurodegenerative disorders.

Oxytocin action on components of endoplasmic reticulum in hippocampal neuronal cells

Despite the known importance of the endoplasmic reticulum (ER) in protein synthesis and vesicular transport, it is not clear whether neuropeptide and neuromodulator oxytocin can directly affect components of the ER in neuronal cells. Therefore, in the present study, we hypothesize that incubation of hippocampal neuronal cells in a presence of oxytocin 1) plays a role in the regulation of the expression of selected ER chaperone components and molecules involved in unfolded protein response pathway 2) affects distribution of the intracellular fluorescence signal highly selective for the ER. We found that oxytocin (1 μM) after 60 min significantly decreased the gene expression of oxidoreductase Ero1β, chaperone glucose-regulated proteins (Grp) 78 and Grp94. A significant decrease in GRP78 protein levels in response to oxytocin treatment occurred after 30, 60 and 120 min. We also observed a time-dependent increase in calreticulin protein levels with a statistically significant increase observed after 360 min. We found that the dynamics of the ER network changes significantly within 2 h of incubation under the influence of oxytocin. In conclusion we have shown that ER chaperones, oxidoreductases and trafficking molecules in neuronal cells are changing in response to oxytocin treatment in a short-term scenario potentially relevant for growth of dendrites and axons.

Sigma-1 receptor agonist PRE-084 confers protection against TAR DNA-binding protein-43 toxicity through NRF2 signalling

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease affecting upper and lower motor neurons. As a consequence, ALS patients display a locomotor disorder related to muscle weakness and progressive paralysis. Pathological mechanisms that participate in ALS involve deficient unfolded protein response, mitochondrial dysfunction and oxidative stress, among others. Finding a therapeutic target to break the vicious circle is particularly challenging. Sigma-1 receptor (S1R) is an endoplasmic reticulum (ER) chaperone that may be one of those targets. We here address and decipher the efficiency of S1R activation on a key ALS gene, TDP43, in zebrafish vertebrate model. While expression of mutant TDP43 (TDP43G348C) led to locomotor defects, treatment with the reference S1R agonist PRE-084 rescued motor performances in a zebrafish model. Treatment with the agonist ameliorated maximal mitochondrial respiration in the TDP43 context. We observed that TDP43G348C exacerbated ER stress induced by tunicamycin, resulting in increased levels of ER stress chaperone BiP and pro-apoptotic factor CHOP. Importantly, PRE-084 treatment in the same condition further heightened BiP levels but also EIF2α/ATF4 and NRF2 signalling cascades, both known to promote antioxidant protection during ER stress. Moreover, we showed that increasing NRF2 levels directly or by sulforaphane treatment rescued locomotor defects of TDP43G348C zebrafish. For the first time, we here provide the proof of concept that PRE-084 prevents mutant TDP43 toxicity by boosting ER stress response and antioxidant cascade through NRF2 signalling.

The regulatory role of endoplasmic reticulum chaperone proteins in neurodevelopment.

The endoplasmic reticulum (ER) is the largest tubular reticular organelle spanning the cell. As the main site of protein synthesis, Ca2+ homeostasis maintenance and lipid metabolism, the ER plays a variety of essential roles in eukaryotic cells, with ER molecular chaperones participate in all these processes. In recent years, it has been reported that the abnormal expression of ER chaperones often leads to a variety of neurodevelopmental disorders (NDDs), including abnormal neuronal migration, neuronal morphogenesis, and synaptic function. Neuronal development is a complex and precisely regulated process. Currently, the mechanism by which neural development is regulated at the ER level remains under investigation. Therefore, in this work, we reviewed the recent advances in the roles of ER chaperones in neural development and developmental disorders caused by the deficiency of these molecular chaperones.

Synthesis and Pharmacological Evaluation of Novel Triazole-Pyrimidine Hybrids as Potential Neuroprotective and Anti-neuroinflammatory Agents.

Neuroprotection is a precise target for the treatment of neurodegenerative diseases, ischemic stroke, and traumatic brain injury. Pyrimidine and its derivatives have been proven to use antiviral, anticancer, antioxidant, and antimicrobial activity prompting us to study the neuroprotection and anti-inflammatory activity of the triazole-pyrimidine hybrid on human microglia and neuronal cell model. A series of novel triazole-pyrimidine-based compounds were designed, synthesized and characterized by mass spectra, 1HNMR, 13CNMR, and a single X-Ray diffraction analysis. Further, the neuroprotective, anti-neuroinflammatory activity was evaluated by cell viability assay (MTT), Elisa, qRT-PCR, western blotting, and molecular docking. The molecular results revealed that triazole-pyrimidine hybrid compounds have promising neuroprotective and anti-inflammatory properties. Among the14synthesized compounds,ZA3-ZA5, ZB2-ZB6,and intermediateS5showed significantanti-neuroinflammatory propertiesthrough inhibition ofnitric oxide (NO)and tumor necrosis factor-α (TNF-α) production in LPS-stimulated human microglia cells. From 14 compounds, six (ZA2toZA6and intermediateS5)exhibited promising neuroprotective activity by reduced expression ofthe endoplasmic reticulum (ER) chaperone, BIP, and apoptosis marker cleaved caspase-3 in human neuronalcells.Also,a molecular docking studyshowed thatlead compounds havefavorable interactionwithactive residues ofATF4 and NF-kB proteins. The possible mechanism of action was observed through the inhibition of ER stress, apoptosis, andthe NF-kB inflammatory pathway. Thus, our study strongly indicates that the novel scaffolds of triazole-pyrimidine-based compounds can potentially be developed as neuroprotective and anti-neuroinflammatory agents.