Endoplasmic Reticulum Storage Research Articles

Sestrin2 drives ER-phagy in response to protein misfolding

Protein biogenesis within the endoplasmic reticulum (ER) is crucial for organismal function. Errors during protein folding necessitate the removal of faulty products. ER-associated protein degradation and ER-phagy target misfolded proteins for proteasomal and lysosomal degradation. The mechanisms initiating ER-phagy in response to ER proteostasis defects are not well understood. By studying mouse primary cells and patient samples as a model of ER storage disorders (ERSDs), we show that accumulation of faulty products within the ER triggers a response involving SESTRIN2, a nutrient sensor controlling mTORC1 signaling. SESTRIN2 induction by XBP1 inhibits mTORC1's phosphorylation of TFEB/TFE3, allowing these transcription factors toenter the nucleus and upregulate the ER-phagy receptor FAM134B along with lysosomal genes. This response promotes ER-phagy of misfolded proteins via FAM134B-Calnexin complex. Pharmacological induction of FAM134B improves clearance of misfolded proteins in ERSDs. Our study identifies the interplay between nutrient signaling and ER quality control, suggesting therapeutic strategies for ERSDs.

ER procollagen storage defect without coupled unfolded protein response drives precocious arthritis.

Collagenopathies are a group of clinically diverse disorders caused by defects in collagen folding and secretion. For example, mutations in the gene encoding collagen type-II, the primary collagen in cartilage, can lead to diverse chondrodysplasias. One example is the Gly1170Ser substitution in procollagen-II, which causes precocious osteoarthritis. Here, we biochemically and mechanistically characterize an induced pluripotent stem cell-based cartilage model of this disease, including both hetero- and homozygous genotypes. We show that Gly1170Ser procollagen-II is notably slow to fold and secrete. Instead, procollagen-II accumulates intracellularly, consistent with an endoplasmic reticulum (ER) storage disorder. Likely owing to the unique features of the collagen triple helix, this accumulation is not recognized by the unfolded protein response. Gly1170Ser procollagen-II interacts to a greater extent than wild-type with specific ER proteostasis network components, consistent with its slow folding. These findings provide mechanistic elucidation into the etiology of this disease. Moreover, the easily expandable cartilage model will enable rapid testing of therapeutic strategies to restore proteostasis in the collagenopathies.

Characterization of PDGF-Induced Subcellular Calcium Regulation through Calcium Channels in Airway Smooth Muscle Cells by FRET Biosensors

The homeostasis of cellular calcium is fundamental for many physiological processes, while the calcium levels remain inhomogeneous within cells. During the onset of asthma, epithelial and inflammatory cells secrete platelet-derived growth factor (PDGF), inducing the proliferation and migration of airway smooth muscle (ASM) to the epidermal layer, narrowing the airway. The regulation of ASM cells by PDGF is closely related to the conduction of calcium signals. In this work, we generated subcellular-targeted FRET biosensors to investigate calcium regulation in the different compartments of ASM cells. A PDGF-induced cytoplasmic calcium [Ca2+]C increase was attributed to both extracellular calcium influx and endoplasmic reticulum (ER) calcium [Ca2+]ER release, which was partially regulated by the PLC-IP3R pathway. Interestingly, the removal of the extracellular calcium influx led to inhibited ER calcium release, likely through inhibitory effects on the calcium-dependent activation of the ER ryanodine receptor. The inhibition of the L-type calcium channel on the plasma membrane or the SERCA pump on the ER resulted in both reduced [Ca2+]C and [Ca2+]ER from PDGF stimulation, while IP3R channel inhibition led to reduced [Ca2+]C only. The inhibited SERCA pump caused an immediate [Ca2+]C increase and [Ca2+]ER decrease, indicating active calcium exchange between the cytosol and ER storage in resting cells. PDGF-induced calcium at the outer mitochondrial membrane sub-region showed a similar regulatory response to cytosolic calcium, not influenced by the inhibition of the mitochondrial calcium uniporter channel. Therefore, our work identifies calcium flow pathways among the extracellular medium, cell cytosol, and ER via regulatory calcium channels. Specifically, extracellular calcium flow has an essential function in fully activating ER calcium release.

Protein quality control and aggregation in the endoplasmic reticulum: From basic to bedside.

Endoplasmic reticulum (ER) is the largest membrane-bound compartment in all cells and functions as a key regulator in protein biosynthesis, lipid metabolism, and calcium balance. Mammalian endoplasmic reticulum has evolved with an orchestrated protein quality control system to handle defective proteins and ensure endoplasmic reticulum homeostasis. Nevertheless, the accumulation and aggregation of misfolded proteins in the endoplasmic reticulum may occur during pathological conditions. The inability of endoplasmic reticulum quality control system to clear faulty proteins and aggregates from the endoplasmic reticulum results in the development of many human disorders. The efforts to comprehensively understand endoplasmic reticulum quality control network and protein aggregation will benefit the diagnostics and therapeutics of endoplasmic reticulum storage diseases. Herein, we overview recent advances in mammalian endoplasmic reticulum protein quality control system, describe protein phase transition model, and summarize the approaches to monitor protein aggregation. Moreover, we discuss the therapeutic applications of enhancing endoplasmic reticulum protein quality control pathways in endoplasmic reticulum storage diseases.

A lower motor neuron disease in takahē (Porphyrio hochstetteri) is an endoplasmic reticulum storage disease

Aims: To investigate the pathogenesis of a disease in takahē with intracytoplasmic inclusion bodies in lower motor neurons. Methods: Four cases aged between 5 and 12 years, from three different wildlife sanctuaries in New Zealand were examined. Of these, only one had signs of spinal dysfunction in the form of paresis. Stained paraffin sections of tissues were examined by light microscopy and immunostained sections of the ventral horn of the spinal cord by confocal microscopy. Epoxy resin sections of the bird with spinal dysfunction were examined by electron microscopy. Results: Two types of inclusion bodies were noted, but only in motor neurons of the ventral spinal cord and brain stem. These were large globoid eosinophilic bodies up to 5 µm in diameter, and yellow/brown granular inclusions mostly at the pole of the cell. The globoid bodies stained with Luxol fast blue but not with periodic acid Schiff (PAS), or Sudan black. The granular inclusions stained with Luxol fast blue, PAS and Sudan black. Both bodies were slightly autofluorescent. On electron microscopy the globoid bodies had an even electron-dense texture and were bound by a membrane. Beneath the membrane were large numbers of small intraluminal vesicles. The smaller granular bodies were more heterogeneous, irregularly rounded and membrane-bound accumulations of granular electron-dense material, often with electron-lucent vacuoles. Others were more vesicular but contained varying amounts of electron-dense material. The large globoid bodies did not immunostain for lysosomal markers lysosomal associated protein 1 (LAMP1) or cathepsin D so they were not lysosomal. The small granular bodies stained for cathepsin D by a chromogenic method. A kindred matrix analysis showed two cases to be as closely related as first cousins, and another case was almost as closely related to one of them, but the fourth bird was unrelated to any other. Conclusions: It was concluded that this was an endoplasmic reticulum storage disease due to a specific protein misfolding within endoplasmic reticulum. It was rationalised that the two types of inclusions reflected the same aetiology, but that misfolded protein in the smaller granular bodies had entered the lysosomal system via endoplasmic reticulum autophagy. Although the cause was unclear, it most likely had a genetic aetiology or predisposition and, as such, has clinical relevance.

Modulation of calcium signaling by nanosecond electric pulses and cell death through apoptosis in A549 lung cancerous cells

Calcium ion (Ca2+), which serves as one of the important signaling agents, is imperative to control normal cell function. The altered Ca2+ signaling is known to play a crucial role in the resistance of cancerous cells against apoptosis. Herein, nanosecond pulsed electric field (nsPEF) was applied to non-small lung cancerous cells A549, and Ca2+ mobilization along with mitochondria-mediated apoptotic cell death that accompanies morphological changes was investigated. In the presence of nsPEF, intracellular mobilization of Ca2+ ions through the efflux from endoplasmic reticulum storage was dependent on the pulse-width of the applied field. The field-induced Ca2+ mobilization has shown to correlate with the superoxide anion (O2−) generation, which probably occurred in mitochondria utilizing the nicotinamide adenine dinucleotide (NADH), as supported by the field-induced change in autofluorescence intensity and lifetime of NADH. Mitochondrial dysfunction and O2− generation, which promoted apoptotic cell death, were also induced by nsPEF with strong correlation with intracellular Ca2+ ions.

Maintaining the thyroid gland in mutant thyroglobulin–induced hypothyroidism requires thyroid cell proliferation that must continue in adulthood

Congenital hypothyroidism with biallelic thyroglobulin (Tg protein, encoded by the TG gene) mutation is an endoplasmic reticulum (ER) storage disease. Many patients (and animal models) grow an enlarged thyroid (goiter), yet some do not. In adulthood, hypothyroid TGcog/cog mice (bearing a Tg-L2263P mutation) exhibit a large goiter, whereas adult WIC rats bearing the TGrdw/rdw mutation (Tg-G2298R) exhibit a hypoplastic thyroid. Homozygous TG mutation has been linked to thyroid cell death, and cytotoxicity of the Tg-G2298R protein was previously thought to explain the lack of goiter in WIC-TGrdw/rdw rats. However, recent studies revealed that TGcog/cog mice also exhibit widespread ER stress–mediated thyrocyte death, yet under continuous feedback stimulation, thyroid cells proliferate in excess of their demise. Here, to examine the relative proteotoxicity of the Tg-G2298R protein, we have used CRISPR–CRISPR-associated protein 9 technology to generate homozygous TGrdw/rdw knock-in mice in a strain background identical to that of TGcog/cog mice. TGrdw/rdw mice exhibit similar phenotypes of defective Tg protein folding, thyroid histological abnormalities, hypothyroidism, and growth retardation. TGrdw/rdw mice do not show evidence of greater ER stress response or stress-mediated cell death than TGcog/cog mice, and both mouse models exhibit sustained thyrocyte proliferation, with comparable goiter growth. In contrast, in WIC-TGrdw/rdw rats, as a function of aging, the thyrocyte proliferation rate declines precipitously. We conclude that the mutant Tg-G2298R protein is not intrinsically more proteotoxic than Tg-L2263P; rather, aging-dependent difference in maintenance of cell proliferation is the limiting factor, which accounts for the absence of goiter in adult WIC-TGrdw/rdw rats.

Protein Aggregation in the ER: Calm behind the Storm.

As one of the largest organelles in eukaryotic cells, the endoplasmic reticulum (ER) plays a vital role in the synthesis, folding, and assembly of secretory and membrane proteins. To maintain its homeostasis, the ER is equipped with an elaborate network of protein folding chaperones and multiple quality control pathways whose cooperative actions safeguard the fidelity of protein biogenesis. However, due to genetic abnormalities, the error-prone nature of protein folding and assembly, and/or defects or limited capacities of the protein quality control systems, nascent proteins may become misfolded and fail to exit the ER. If not cleared efficiently, the progressive accumulation of misfolded proteins within the ER may result in the formation of toxic protein aggregates, leading to the so-called “ER storage diseases”. In this review, we first summarize our current understanding of the protein folding and quality control networks in the ER, including chaperones, unfolded protein response (UPR), ER-associated protein degradation (ERAD), and ER-selective autophagy (ER-phagy). We then survey recent research progress on a few ER storage diseases, with a focus on the role of ER quality control in the disease etiology, followed by a discussion on outstanding questions and emerging concepts in the field.

Protein Misfolding and Aggregation: The Relatedness between Parkinson's Disease and Hepatic Endoplasmic Reticulum Storage Disorders.

Dysfunction of cellular homeostasis can lead to misfolding of proteins thus acquiring conformations prone to polymerization into pathological aggregates. This process is associated with several disorders, including neurodegenerative diseases, such as Parkinson’s disease (PD), and endoplasmic reticulum storage disorders (ERSDs), like alpha-1-antitrypsin deficiency (AATD) and hereditary hypofibrinogenemia with hepatic storage (HHHS). Given the shared pathophysiological mechanisms involved in such conditions, it is necessary to deepen our understanding of the basic principles of misfolding and aggregation akin to these diseases which, although heterogeneous in symptomatology, present similarities that could lead to potential mutual treatments. Here, we review: (i) the pathological bases leading to misfolding and aggregation of proteins involved in PD, AATD, and HHHS: alpha-synuclein, alpha-1-antitrypsin, and fibrinogen, respectively, (ii) the evidence linking each protein aggregation to the stress mechanisms occurring in the endoplasmic reticulum (ER) of each pathology, (iii) a comparison of the mechanisms related to dysfunction of proteostasis and regulation of homeostasis between the diseases (such as the unfolded protein response and/or autophagy), (iv) and clinical perspectives regarding possible common treatments focused on improving the defensive responses to protein aggregation for diseases as different as PD, and ERSDs.

The Recruitment-Secretory Block ("R-SB") Phenomenon and Endoplasmic Reticulum Storage Diseases.

In this article, we review the biological and clinical implication of the Recruitment-Secretory Block (“R-SB”) phenomenon. The phenomenon refers to the reaction of the liver with regard to protein secretion in conditions of clinical stimulation. Our basic knowledge of the process is due to the experimental work in animal models. Under basal conditions, the protein synthesis is mainly carried out by periportal (zone 1) hepatocytes that are considered the “professional” synthesizing protein cells. Under stimulation, midlobular and centrolobular (zones 2 and 3) hepatocytes, are progressively recruited according to lobular gradients and contribute to the increase of synthesis and secretion. The block of secretion, operated by exogenous agents, causes intracellular retention of all secretory proteins. The Pi MZ phenotype of Alpha-1-antitrypsin deficiency (AATD) has turned out to be the key for in vivo studies of the reaction of the liver, as synthesis and block of secretion are concomitant. Indeed, the M fraction of AAT is stimulated for synthesis and regularly exported while the Z fraction is mostly retained within the cell. For that reason, the phenomenon has been designated “Recruitment-Secretory Block” (“R-SB”). The “R-SB” phenomenon explains why: (a) the MZ individuals can correct the serum deficiency; (b) the resulting immonohistochemical and electron microscopic (EM) patterns are very peculiar and specific for the diagnosis of the Z mutation in tissue sections in the absence of genotyping; (c) the term carrier is no longer applicable for the heterozygous condition as all Pi MZ individuals undergo storage and the storage predisposes to liver damage. The storage represents the true elementary lesion and consequently reflects the phenotype-genotype correlation; (d) the site and function of the extrahepatic AAT and the relationship between intra and extracellular AAT; (e) last but not least, the concept of Endoplasmic Reticulum Storage Disease (ERSD) and of a new disease, hereditary hypofibrinogenemia with hepatic storage (HHHS). In the light of the emerging phenomenon, described in vitro, namely that M and Z AAT can form heteropolymers within hepatocytes as well as in circulation, we have reviewed the whole clinical and experimental material collected during forty years, in order to evaluate to what extent the polymerization phenomenon occurs in vivo. The paper summarizes similarities and differences between AAT and Fibrinogen as well as between the related diseases, AATD and HHHS. Indeed, fibrinogen gamma chain mutations undergo an aggregation process within the RER of hepatocytes similar to AATD. In addition, this work has clarified the intriguing phenomenon underlying a new syndrome, hereditary hypofibrinogenemia and hypo-APO-B-lipoproteinemia with hepatic storage of fibrinogen and APO-B lipoproteins. It is hoped that these studies could contribute to future research and select strategies aimed to simultaneously correct the hepatocytic storage, thus preventing the liver damage and the plasma deficiency of the two proteins.

225-LB: Targeting ß-Cell Endoplasmic Reticulum (ER) TALK-1 Channels to Restore Ca2+ Homeostasis and Reduce ER Stress in Diabetes

Perturbations in Ca2+ homeostasis contribute to β-cell failure in diabetes, limiting insulin secretion and exacerbating hyperglycemia. TALK-1 is the most islet-restricted and most abundantly expressed K+ channel of the β-cell. ER TALK-1 channels provide K+ countercurrent for [Ca2+]ER release, thereby tuning [Ca2+]ER storage and release. During β-cell Ca2+ influx, TALK-1-enhanced [Ca2+]ER release leads to activation of the hyperpolarizing Ca2+ activated K+ channels, thus increasing the silent phase duration between Ca2+ oscillations. A gain-of-function mutation in TALK-1(L114P) causes MODY, by disrupting cytoplasmic and ER Ca2+ handling. However, the ability to probe ER TALK-1 channels as a therapeutic target to normalize β-cell Ca2+ handling and reduce ER stress in diabetes has not been determined. Here, using a high throughput fluorescence-based thallium (TI+) flux assay (z’ =0.64) in a TALK-1 overexpression cell line, we identified 80 TALK-1 selective inhibitors from a small molecule library of 50,000 compounds. 4 potent inhibitors that also inhibited the ER membrane TALK-1 channels were identified by monitoring cyclopiazonic acid-induced [Ca2+]ER release (e.g. K2P16-i7(IC50=5.3μM); 63.36% [Ca2+]ER recovery, p-value=0.02). We also tested these inhibitors for their efficacy in controlling mouse islet [Ca2+]ER storage as well as cytosolic Ca2+ oscillations. TALK-1 inhibitors significantly increased islet [Ca2+]ER storage and Ca2+ oscillation frequency (e.g.K2P16-i7; [Ca2+]ER AUC=60.9a.u., peaks/min=0.45 vs. DMSO; [Ca2+]ER AUC=49.7a.u., peaks/min= 0.17, p-value<0.05). Importantly, the inhibitors did not alter [Ca2+]ER and glucose-stimulated Ca2+ oscillation frequency in TALK-1 KO islets. In summary, we have identified the first ER TALK-1 selective inhibitors that can not only be utilized to assess the role of TALK-1 in human β-cell Ca2+ handling and GSIS, but also potentially reduce ER dysfunction during diabetes pathogenesis. Disclosure A. Y. Nakhe: None. P. Dadi: None. D. Westover: None. E. Days: None. J. Denton: None. D. Jacobson: None.

Hepatic and Extrahepatic Sources and Manifestations in Endoplasmic Reticulum Storage Diseases.

Alpha-1-antitrypsin (AAT) and fibrinogen are secretory acute phase reactant proteins. Circulating AAT and fibrinogen are synthesized exclusively in the liver. Mutations in the encoding genes result in conformational abnormalities of the two molecules that aggregate within the rough endoplasmic reticulum (RER) instead of being regularly exported. That results in AAT-deficiency (AATD) and in hereditary hypofibrinogenemia with hepatic storage (HHHS). The association of plasma deficiency and liver storage identifies a new group of pathologies: endoplasmic reticulum storage disease (ERSD).

The Discovery of Endoplasmic Reticulum Storage Disease. The Connection between an H&E Slide and the Brain.

The revolutionary evolution in science and technology over the last few decades has made it possible to face more adequately three main challenges of modern medicine: changes in old diseases, the appearance of new diseases, and diseases that are unknown (mostly genetic), despite research efforts. In this paper we review the road travelled by pathologists in search of a method based upon the use of routine instruments and techniques which once were available for research only. The application to tissue studies of techniques from immunology, molecular biology, and genetics has allowed dynamic interpretations of biological phenomena with special regard to gene regulation and expression. That implies stepwise investigations, including light microscopy, immunohistochemistry, in situ hybridization, electron microscopy, molecular histopathology, protein crystallography, and gene sequencing, in order to progress from suggestive features detectable in routinely stained preparations to more characteristic, specific, and finally, pathognomonic features. Hematoxylin and Eosin (H&E)-stained preparations and appropriate immunohistochemical stains have enabled the recognition of phenotypic changes which may reflect genotypic alterations. That has been the case with hepatocytic inclusions detected in H&E-stained preparations, which appeared to correspond to secretory proteins that, due to genetic mutations, were retained within the rough endoplasmic reticulum (RER) and were deficient in plasma. The identification of this phenomenon affecting the molecules alpha-1-antitrypsin and fibrinogen has led to the discovery of a new field of cell organelle pathology, endoplasmic reticulum storage disease(s) (ERSD). Over fifty years, pathologists have wandered through a dark forest of complicated molecules with strange conformations, and by detailed observations in simple histopathological sections, accompanied by a growing background of molecular techniques and revelations, have been able to recognize and identify arrays of grotesque polypeptide arrangements.

The loss-of-function PCSK9Q152H variant increases ER chaperones GRP78 and GRP94 and protects against liver injury.

Individuals harboring the loss-of-function (LOF) proprotein convertase subtilisin/kexin type 9 Gln152His variation (PCSK9Q152H) have low circulating low-density lipoprotein cholesterol levels and are therefore protected against cardiovascular disease (CVD). This uncleavable form of proPCSK9, however, is retained in the endoplasmic reticulum (ER) of liver hepatocytes, where it would be expected to contribute to ER storage disease (ERSD), a heritable condition known to cause systemic ER stress and liver injury. Here, we examined liver function in members of several French-Canadian families known to carry the PCSK9Q152H variation. We report that PCSK9Q152H carriers exhibited marked hypocholesterolemia and normal liver function despite their lifelong state of ER PCSK9 retention. Mechanistically, hepatic overexpression of PCSK9Q152H using adeno-associated viruses in male mice greatly increased the stability of key ER stress-response chaperones in liver hepatocytes and unexpectedly protected against ER stress and liver injury rather than inducing them. Our findings show that ER retention of PCSK9 not only reduced CVD risk in patients but may also protect against ERSD and other ER stress-driven conditions of the liver. In summary, we have uncovered a cochaperone function for PCSK9Q152H that explains its hepatoprotective effects and generated a translational mouse model for further mechanistic insights into this clinically relevant LOF PCSK9 variant.

Abstract 17415: Mitochondrial Ca2+/Calmodulin-Dependent Kinase II Inhibition Changes the Calcium Homeostasis in the Endothelium and Decreases the Vascular Relaxation in Mesenteric Arteries

Introduction: The Ca2+/Calmodulin-dependent Kinase II (CaMKII) is present in mitochondria and cytosol. In mitochondria, it regulates the mitochondrial Ca 2+ uptake via the mitochondrial Ca2+ uniporter. Since endothelial nitric oxide synthase activity is regulated by intracellular [Ca2+], we hypothesized that it affects cytosolic Ca2+, NO production and ACh-dependent vasodilation. Hypothesis: Inhibition of mitochondrial CaMKII in endothelium increases the cytosolic [Ca2+], and decreases vasorelaxation by Acetylcholine. Methods: CaMKII in mitochondria was inhibited through expression of the mitochondria-targeted CaMKII inhibitor peptide (mito-CaMKIIN) in a novel transgenic mouse model (endo-mtCaMKIIN) in endothelial cells only or delivered by adenoviral transduction (Ad-mtCaMKIIN) in human Aortic Endothelium cells (HAEC). In HAEC, cytosolic Ca2+ levels (by FURA-2 AM), eNOS activation and NOx levels were measured. Results: The basal Ca2+ levels were higher in the cytosol of mitoCaMKIIN cells (1.08 ± 0.02 Fura-2 ratio normalized by control, p<0.05). Thapsigargin-induced ER Ca 2+ release was significantly higher with mitoCaMKIIN (AUC 0.252 ± 0.027 versus 0.112 ± 0.01275, p<0.05), whereas cytosolic Ca 2+ levels after ACh were reduced (AUC 0.191 ± 0.025 versus 0.435 ± 0.054). Higher levels of phosphorylation of eNOS at Ser1177 and Thr495 sites were seen at baseline. The concentration-response curve of vascular relaxation to acetylcholine and SNP shifted to the right (p<0.05) in mesenteric resistance artery of mitoCaMKIIN mice. Conclusions: The inhibition of mitochondrial CaMKII in the endothelium increases the cytosolic levels, endoplasmic reticulum storage of calcium and eNOS phosphorylation. However, there are lower calcium release and lower sensitivity to acetylcholine and SNP.

Intraventricular Hemorrhage Induces Rapid Calcium Signaling and Transcriptional Changes in the Developing Choroid Plexus

Abstract INTRODUCTION Intraventricular hemorrhage (IVH) leads to acute hydrocephalus in a subset of preterm infants by unclear mechanisms that potentially include increased cerebrospinal fluid (CSF) production by the choroid plexus (ChP), based on recent work in adult rats (Karimy et al., 2017). The earliest physiologic changes in ChP epithelial cells likely involve calcium signaling, which is a known cellular messenger in other secretory epithelial cell populations. The following experiments utilize rapid calcium imaging to evaluate how calcium signaling is regulated in embryonic ChP explants, and additionally determine specific transcriptional alterations in Vivo. METHODS 1) Calcium imaging of embryonic explants: Lateral ventricle ChP is dissected at embryonic day 14.5 (E14.5) from a conditional transgenic mouse line (FoxJ1-Cre; Gcamp6f). The ChP explants are kept viable with artificial CSF, exposed to focal blood injections, and imaged for calcium responses using epifluorescence and two-photon imaging. 2) Transcriptional analysis in a model of preterm IVH: Quantitative RT-PCR is performed on acutely dissected ChP at 24 and 48 hr, following in utero injection of age-matched blood in the lateral ventricles of individual embryos (E14.5 pregnant CD1 mice). RESULTS Upon exposure to age-matched E14.5 plasma, nearly all ChP epithelial cells rapidly release highly concentrated intracellular calcium followed by a mosaic pattern of recurrent calcium release. Using different conditions of calcium blockade, we identify endoplasmic reticulum storage as the primary source of intracellular calcium release. Expression of NKCC1 (the predominant apical ion co-transporter in the ChP) rises by 24 hr, suggesting increased ChP ion permeability, consistent with prior reports of a CSF hypersecretion phenotype. CONCLUSION IVH causes immediate intracellular calcium release by the ChP epithelia, followed by increased NKCC1 transcription within 24 hr. Together, these data suggest a rapid and dynamic embryonic ChP responsiveness to hemorrhage and contributes to our understanding of IVH-induced hydrocephalus pathophysiology.

40-OR: The Role of TALK-1 K+ Channels in Pancreatic ß-Cell Insulin Secretion, Mitochondrial Function, and the ER Stress Response

During Glucose-Stimulated Insulin Secretion (GSIS), β-cells exhibit oscillations in cytosolic Ca2+ ([Ca2+]c) and endoplasmic reticulum (ER) Ca2+ ([Ca2+]ER), which are regulated by the two-pore domain K+ channel, TALK-1. TALK-1 is the most abundant β-cell K+ channel and it controls Ca2+ homeostasis by modulating K+ permeability at both the plasma and ER membrane. However, how TALK-1 modulates ER stress and β-cell failure during the pathogenesis of diabetes has not been determined. To understand how TALK-1 controls β-cell function under diabetic conditions, we assessed how a mutation in TALK-1 (ND) which causes neonatal diabetes, impacts TALK-1 channel activity and [Ca2+]ER handling. While plasmallemal TALK-1 activity was inhibited with the ND mutation, [Ca2+]ER storage was significantly reduced by the ND mutation compared to control TALK-1. This suggests that TALK-1 ND enhances TALK-1 activity on the ER membrane leading to reduced [Ca2+]ER storage. Reducing [Ca2+]ER enhances ER stress, which would be expected to cause β-cell dysfunction. Indeed, TALK-1 ND significantly reduces β-cell insulin secretion compared to control. To further investigate the role of TALK-1 we developed a β-cell-specific TALK-1 KO mouse model (β-TALK1-KO). On a high fat diet, β-TALK1-KO mice show improved glucose tolerance compared to controls. Mitochondrial calcium ([Ca2+]mito) was also examined because it is modulated by [Ca2+]ER; we found that β-TALK1-KO mice have increased [Ca2+]mito. As reactive oxygen species (ROS) production is a byproduct of mitochondrial respiration, we investigated if TALK-1 affects ROS production. TALK1 KO islets were found to have reduced ROS production compared to wild type islets. Together, these data indicate that increased TALK-1 channel activity reduces [Ca2+]ER, [Ca2+]mito, and increases ROS; suggesting that TALK-1 plays an important role in modulating the β-cell responses to stress associated with diabetic conditions. Disclosure S.M. Graff: None. P. Dadi: None. C.E. Ibsen: None. M. Dickerson: None. K.L. Jordan: None. D. Jacobson: None.

Cytokine-mediated changes in K+ channel activity promotes an adaptive Ca2+ response that sustains \u03b2-cell insulin secretion during inflammation

Cytokines present during low-grade inflammation contribute to β-cell dysfunction and diabetes. Cytokine signaling disrupts β-cell glucose-stimulated Ca2+ influx (GSCI) and endoplasmic reticulum (ER) Ca2+ ([Ca2+]ER) handling, leading to diminished glucose-stimulated insulin secretion (GSIS). However, cytokine-mediated changes in ion channel activity that alter β-cell Ca2+ handling remain unknown. Here we investigated the role of K+ currents in cytokine-mediated β-cell dysfunction. Kslow currents, which control the termination of intracellular Ca2+ ([Ca2+]i) oscillations, were reduced following cytokine exposure. As a consequence, [Ca2+]i and electrical oscillations were accelerated. Cytokine exposure also increased basal islet [Ca2+]i and decreased GSCI. The effect of cytokines on TALK-1 K+ currents were also examined as TALK-1 mediates Kslow by facilitating [Ca2+]ER release. Cytokine exposure decreased KCNK16 transcript abundance and associated TALK-1 protein expression, increasing [Ca2+]ER storage while maintaining 2nd phase GSCI and GSIS. This adaptive Ca2+ response was absent in TALK-1 KO islets, which exhibited decreased 2nd phase GSCI and diminished GSIS. These findings suggest that Kslow and TALK-1 currents play important roles in altered β-cell Ca2+ handling and electrical activity during low-grade inflammation. These results also reveal that a cytokine-mediated reduction in TALK-1 serves an acute protective role in β-cells by facilitating increased Ca2+ content to maintain GSIS.

Calneuron 1 Increased Ca2+ in the Endoplasmic Reticulum and Aldosterone Production in Aldosterone-Producing Adenoma.

Aldosterone production is initiated by angiotensin II stimulation and activation of intracellular Ca2+ signaling. In aldosterone-producing adenoma (APA) cells, the activation of intracellular Ca2+ signaling is independent of the renin-angiotensin-aldosterone systems. The purpose of our study was to clarify molecular mechanisms of aldosterone production related to Ca2+ signaling. Transcriptome analysis revealed that the CALN1 gene encoding calneuron 1 had the strongest correlation with CYP11B2 (aldosterone synthase) among genes encoding Ca2+-binding proteins in APA. CALN1 modulation and synthetic or fluorescent compounds were used for functional studies in human adrenocortical carcinoma (HAC15) cells. CALN1 expression was 4.4-fold higher in APAs than nonfunctioning adrenocortical adenomas. CALN1 expression colocalized with CYP11B2 expression as investigated using immunohistochemistry in APA and zona glomerulosa of male rats fed by a low-salt diet. CALN1 expression was detected in the endoplasmic reticulum (ER) by using GFP-fused CALN1, CellLight ER-RFP, and the corresponding antibodies. CALN1-overexpressing HAC15 cells showed increased Ca2+ in the ER and cytosol fluorescence-based studies. Aldosterone production was potentiated in HAC15 cells by CALN1 expression, and dose-responsive inhibition with TMB-8 showed that CALN1-mediated Ca2+ storage in ER involved sarcoendoplasmic reticulum calcium transport ATPase. The silencing of CALN1 decreased Ca2+ in ER, and abrogated angiotensin II- or KCNJ5 T158A-mediated aldosterone production in HAC15 cells. Increased CALN1 expression in APA was associated with elevated Ca2+ storage in ER and aldosterone overproduction. Suppression of CALN1 expression prevented angiotensin II- or KCNJ5 T158A-mediated aldosterone production in HAC15 cells, suggesting that CALN1 is a potential therapeutic target for excess aldosterone production.

α1-antitrypsin Deficiency: A Misfolded Secretory Protein Variant with Unique Effects on the Endoplasmic Reticulum.

In the classical form of α1-antitrypsin deficiency (ATD) a point mutation leads to accumulation of a misfolded secretory glycoprotein in the endoplasmic reticulum (ER) of liver cells and so ATD has come to be considered a prototypical ER storage disease. It is associated with two major types of clinical disorders, chronic obstructive pulmonary disease (COPD) by loss-of-function mechanisms and hepatic cirrhosis and carcinogenesis by gain-of-function mechanisms. The lung disease predominantly results from proteolytic damage to the pulmonary connective tissue matrix because of reduced levels of protease inhibitor activity of α1-anitrypsin (AT) in the circulating blood and body fluids. Cigarette smoking is a powerful disease-promoting modifier but other modifiers are known to exist because variation in the lung disease phenotype is still found in smoking and non-smoking homozygotes. The liver disease is highly likely to be caused by the proteotoxic effects of intracellular misfolded protein accumulation and a high degree of variation in the hepatic phenotype among affected homozygotes has been hypothetically attributed to genetic and environmental modifiers that alter proteostasis responses. Liver biopsies of homozygotes show intrahepatocytic inclusions with dilation and expansion of the ER and recent studies of iPS-derived hepatocyte-like cells from individuals with ATD indicate that the changes in the ER directly vary with the hepatic phenotype i.e there is much lesser alteration in the ER in cells derived from homozygotes that do not have clinically significant liver disease. From a signaling perspective, studies in mammalian cell line and animal models expressing the classical α1-antitrypsin Z variant (ATZ) have found that ER signaling is perturbed in a relatively unique way with powerful activation of autophagy and the NFκB pathway but relatively limited, if any, UPR signaling. It is still not known how much these unique structural and functional changes and the variation among affected homozygotes relate to the tendency of this variant to polymerize and aggregate and/or to the repertoire of proteostasis mechanisms that are activated.