scholarly journals Human CPA1 mutation causes digestive enzyme misfolding and chronic pancreatitis in mice

Gut ◽  
2018 ◽  
Vol 68 (2) ◽  
pp. 301-312 ◽  
Author(s):  
Eszter Hegyi ◽  
Miklós Sahin-Tóth
Keyword(s):  
Chronic Pancreatitis ◽  
Er Stress ◽  
Digestive Enzyme ◽  
Pancreatic Disease ◽  
Mutant Mice ◽  
Inflammatory Disorder ◽  
Loss Of Function ◽  
Stress Markers ◽  
Elevated Expression

ObjectiveChronic pancreatitis is a progressive, relapsing inflammatory disorder of the pancreas, which often develops in the background of genetic susceptibility. Recently, loss-of-function mutations in CPA1, which encodes the digestive enzyme carboxypeptidase A1, were described in sporadic early onset cases and in hereditary pancreatitis. Mutation-induced misfolding of CPA1 and associated endoplasmic reticulum (ER) stress was suggested as potential disease mechanism; however, in vivo evidence has been lacking. The objective of the present study was to create a mouse model that recapitulates features of CPA1-associated chronic pancreatitis.DesignWe knocked-in the most frequently occurring p.N256K human CPA1 mutation to the mouse Cpa1 locus. Mutant mice were characterised with respect to pancreas pathology and ER stress and compared with C57BL/6N and CPA1 null control mice.ResultsIn the CPA1 N256K mutant mice, we observed hallmarks of chronic pancreatitis that included progressive acinar cell atrophy, inflammatory cell infiltration, fibrosis and acinar-ductal metaplasia. In contrast, similarly to the C57BL/6N mice, the CPA1 null control strain exhibited no signs of pancreatic disease. Mutation p.N256K induced misfolding of mouse CPA1 and resulted in elevated expression of ER stress markers Hspa5 (BiP) and Ddit3 (CHOP) both in cell culture and mutant mice.ConclusionThe results offer categorical evidence that CPA1 mutations elicit enzyme misfolding and cause chronic pancreatitis via an ER stress-related mechanism.

scholarly journals Ethanol feeding accelerates pancreatitis progression in CPA1 N256K mutant mice

2020 ◽  
Vol 318 (4) ◽  
pp. G694-G704
Author(s):  
Anna Orekhova ◽  
Andrea Geisz ◽  
Miklós Sahin-Tóth
Keyword(s):  
Endoplasmic Reticulum ◽  
Chronic Pancreatitis ◽  
Er Stress ◽  
Digestive Enzyme ◽  
Alcoholic Pancreatitis ◽  
Inflammatory Disorder ◽  
Mrna Levels ◽  
Er Chaperone ◽  
Generalized Seizures ◽  
Ethanol Feeding

Alcoholic pancreatitis is a multifactorial, progressive, inflammatory disorder of the pancreas. Alcohol initiates pancreatitis and promotes its progression in the context of genetic susceptibility and/or other environmental risk factors such as smoking. Genetic mutations can cause digestive enzyme misfolding, which induces endoplasmic reticulum (ER) stress and elicits pancreatitis. Here, we tested the hypothesis that alcohol synergizes with misfolding in promoting ER stress and thereby accelerates chronic pancreatitis progression. To this end, we fed an ethanol-containing diet to CPA1 N256K mice, which carry the human p.N256K CPA1 mutation and develop spontaneous chronic pancreatitis. Inexplicably, CPA1 N256K mice suffered generalized seizures after 2–3 wk of ethanol feeding, which resulted in high mortality and the early termination of the study. Analysis of CPA1 N256K mice euthanized after 3–3.5 wk of ethanol feeding revealed more severe chronic pancreatitis associated with significantly increased Hspa5 [ER chaperone immunoglobulin heavy chain-binding protein (BiP)] mRNA levels when compared with CPA1 N256K mice on a control liquid diet. In contrast, ethanol feeding of C57BL/6N mice for 4 wk increased Hspa5 levels to a lesser degree and caused no pancreatitis. We conclude that ethanol feeding synergizes with the misfolding CPA1 mutant in promoting ER stress and thereby accelerates progression of chronic pancreatitis in CPA1 N256K mice. NEW & NOTEWORTHY Alcoholic pancreatitis is a multifactorial, progressive, inflammatory disorder of the pancreas. This study demonstrates that alcohol synergizes with digestive enzyme misfolding in promoting endoplasmic reticulum stress and thereby accelerates progression of chronic pancreatitis.

scholarly journals FoxO suppresses endoplasmic reticulum stress to inhibit growth of Tsc1-deficient tissues under nutrient restriction

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Avantika Gupta ◽  
Hugo Stocker
Keyword(s):  
Endoplasmic Reticulum ◽  
Er Stress ◽  
Target Genes ◽  
Degradation Pathway ◽  
Nutrient Restriction ◽  
Loss Of Function ◽  
Er Stress Response ◽  
Stress Markers ◽  
Stress Response Pathway ◽  
Elevated Expression

The transcription factor FoxO has been shown to block proliferation and progression in mTORC1-driven tumorigenesis but the picture of the relevant FoxO target genes remains incomplete. Here, we employed RNA-seq profiling on single clones isolated using laser capture microdissection from Drosophila larval eye imaginal discs to identify FoxO targets that restrict the proliferation of Tsc1-deficient cells under nutrient restriction (NR). Transcriptomics analysis revealed downregulation of endoplasmic reticulum-associated protein degradation pathway components upon foxo knockdown. Induction of ER stress pharmacologically or by suppression of other ER stress response pathway components led to an enhanced overgrowth of Tsc1 knockdown tissue. Increase of ER stress in Tsc1 loss-of-function cells upon foxo knockdown was also confirmed by elevated expression levels of known ER stress markers. These results highlight the role of FoxO in limiting ER stress to regulate Tsc1 mutant overgrowth.

scholarly journals Regulation of Adipsin Expression by Endoplasmic Reticulum Stress in Adipocytes

Biomolecules ◽  
2020 ◽  
Vol 10 (2) ◽  
pp. 314
Author(s):  
Ka-Young Ryu ◽  
Eon Ju Jeon ◽  
Jaechan Leem ◽  
Jae-Hyung Park ◽  
Hochan Cho
Keyword(s):  
Endoplasmic Reticulum ◽  
Er Stress ◽  
Peroxisome Proliferator ◽  
Adipose Tissues ◽  
Peroxisome Proliferator Activated Receptor ◽  
Er Stress Response ◽  
Stress Markers ◽  
Transcriptional Reprogramming

Adpsin is an adipokine that stimulates insulin secretion from β-cells and improves glucose tolerance. Its expression has been found to be markedly reduced in obese animals. However, it remains unclear what factors lead to downregulation of adipsin in the context of obesity. Endoplasmic reticulum (ER) stress response is activated in various tissues under obesity-related conditions and can induce transcriptional reprogramming. Therefore, we aimed to investigate the relationship between adipsin expression and ER stress in adipose tissues during obesity. We observed that obese mice exhibited decreased levels of adipsin in adipose tissues and serum and increased ER stress markers in adipose tissues compared to lean mice. We also found that ER stress suppressed adipsin expression via adipocytes-intrinsic mechanisms. Moreover, the ER stress-mediated downregulation of adipsin was at least partially attributed to decreased expression of peroxisome proliferator-activated receptor γ (PPARγ), a key transcription factor in the regulation of adipocyte function. Finally, treatment with chemical chaperones recovered the ER stress-mediated downregulation of adipsin and PPARγ in vivo and in vitro. Our findings suggest that activated ER stress in adipose tissues is an important cause of the suppression of adipsin expression in the context of obesity.

scholarly journals The position of single-base deletions in the VNTR sequence of the carboxyl ester lipase (CEL) gene determines pathogenicity

2020 ◽  
Author(s):  
Anny Gravdal ◽  
Xunjun Xiao ◽  
Miriam Cnop ◽  
Khadija El Jellas ◽  
Pål R. Njølstad ◽  
...  
Keyword(s):  
Er Stress ◽  
Protein Misfolding ◽  
Digestive Enzyme ◽  
Pancreatic Disease ◽  
Variable Number ◽  
Mutational Event ◽  
Pathogenic Potential ◽  
Single Base ◽  
Exon 11 ◽  
The Unfolded Protein Response

ABSTRACTVariable number of tandem repeat (VNTR) sequences present in the genome can have functional consequences that contribute to human disease. This is the case for the CEL gene, which encodes the digestive enzyme carboxyl ester lipase. CEL has a VNTR located in exon 11, and rare single-base deletions (DELs) within this region cause MODY8, an inherited disorder characterized by exocrine pancreatic dysfunction and diabetes. Here, we have studied how the position of single-base deletions within the CEL VNTR affects the protein’s pathogenic properties. We investigated four naturally occurring CEL variants with single-base deletions in different VNTR segments (DEL1, DEL4, DEL9, DEL13), of which only DEL1 and DEL4 have been observed in MODY8 patients. When expressed in a cellular model system, only DEL1 and DEL4 exhibited significantly reduced secretion and increased intracellular aggregation compared to normal CEL. We found that all DEL variants had slightly decreased enzymatic activity and that their level of O-glycosylation was affected. Moreover, only DEL1 and DEL4 significantly increased endoplasmic reticulum (ER) stress. In conclusion, CEL single-base deletion variants have the highest pathogenic potential when the mutational event has taken place in the proximal VNTR part, resulting in the longest aberrant protein tails. Thus, DEL1 and DEL4 are pathogenic CEL variants, whereas we consider DEL13 as benign and DEL9 as likely benign. These findings have implications for our understanding of how CEL mutations cause pancreatic disease through protein misfolding and proteotoxicity, leading to ER stress and activation of the unfolded protein response.

scholarly journals MitoROS due to loss of Slc4a11 in corneal endothelial cells induces ER stress, lysosomal dysfunction and impairs autophagy

2020 ◽  
Author(s):  
Rajalekshmy Shyam ◽  
Diego G. Ogando ◽  
Moonjung Choi ◽  
Joseph A. Bonanno
Keyword(s):  
Endothelial Cells ◽  
Er Stress ◽  
Corneal Edema ◽  
Corneal Endothelial Cells ◽  
Autophagy Flux ◽  
Unfolded Protein ◽  
Stress Markers ◽  
Lysosomal Dysfunction ◽  
Mitochondrial Ros ◽  
Elevated Expression

AbstractRecent studies from Slc4a11 KO mice have identified mitochondrial dysfunction as a major contributor toward oxidative stress and cell death in Congenital Hereditary Endothelial Dystrophy. Here we asked if this stress activated autophagy in the Slc4a11 KO cell line and in KO mouse endothelial tissue. Early indicators of autophagy, phospho-mTOR and LC3-II indicated activation, however P62 was elevated suggesting an impairment of autophagy flux. The activity and the number of lysosomes, the organelle responsible for the final degradation of autophagy substrates, were found to be reduced in the KO. In addition, the expression of the master regulator of lysosomal function and biogenesis, TFEB, was significantly reduced in the KO corneal endothelia. Also, we observed increased Unfolded Protein Response, as well as elevated expression of ER stress markers, BIP and CHOP. To test if lysosomal and ER stress stems from elevated mitochondrial ROS, we treated Slc4a11 KO corneal endothelial cells with the mitochondrial ROS quencher, MitoQ. MitoQ restored lysosomal enzymes as well as TFEB, reduced ER stress, and increased autophagy flux. MitoQ injections of Slc4a11 KO mice decreased corneal edema, the major phenotype associated with CHED. We conclude that mitochondrial ROS causes ER stress and lysosomal dysfunction with impairment of autophagy in Slc4a11 KO corneal endothelium. Our study is the first to identify the presence as well as cause of lysosomal dysfunction and ER stress in an animal model of CHED, and to characterize inter-organelle relationship in a corneal cell type.

scholarly journals Jointly reduced inhibition and excitation underlies circuit-wide changes in cortical processing in Rett syndrome

2016 ◽  
Vol 113 (46) ◽  
pp. E7287-E7296 ◽  
Author(s):  
Abhishek Banerjee ◽  
Rajeev V. Rikhye ◽  
Vincent Breton-Provencher ◽  
Xin Tang ◽  
Chenchen Li ◽  
...  
Keyword(s):  
Rett Syndrome ◽  
Time Course ◽  
Pyramidal Neurons ◽  
Reversal Potential ◽  
Mutant Mice ◽  
Loss Of Function ◽  
Cortical Circuits ◽  
Recombinant Human Insulin ◽  
Cortical Pyramidal Neurons

Rett syndrome (RTT) arises from loss-of-function mutations in methyl-CpG binding protein 2 gene (Mecp2), but fundamental aspects of its physiological mechanisms are unresolved. Here, by whole-cell recording of synaptic responses in MeCP2 mutant mice in vivo, we show that visually driven excitatory and inhibitory conductances are both reduced in cortical pyramidal neurons. The excitation-to-inhibition (E/I) ratio is increased in amplitude and prolonged in time course. These changes predict circuit-wide reductions in response reliability and selectivity of pyramidal neurons to visual stimuli, as confirmed by two-photon imaging. Targeted recordings reveal that parvalbumin-expressing (PV+) interneurons in mutant mice have reduced responses. PV-specific MeCP2 deletion alone recapitulates effects of global MeCP2 deletion on cortical circuits, including reduced pyramidal neuron responses and reduced response reliability and selectivity. Furthermore, MeCP2 mutant mice show reduced expression of the cation-chloride cotransporter KCC2 (K+/Cl− exporter) and a reduced KCC2/NKCC1 (Na+/K+/Cl− importer) ratio. Perforated patch recordings demonstrate that the reversal potential for GABA is more depolarized in mutant mice, but is restored by application of the NKCC1 inhibitor bumetanide. Treatment with recombinant human insulin-like growth factor-1 restores responses of PV+ and pyramidal neurons and increases KCC2 expression to normalize the KCC2/NKCC1 ratio. Thus, loss of MeCP2 in the brain alters both excitation and inhibition in brain circuits via multiple mechanisms. Loss of MeCP2 from a specific interneuron subtype contributes crucially to the cell-specific and circuit-wide deficits of RTT. The joint restoration of inhibition and excitation in cortical circuits is pivotal for functionally correcting the disorder.

scholarly journals A genome-wide CRISPR screen identifies the glycosylation enzyme DPM1 as a modifier of DPAGT1 deficiency and ER stress

2021 ◽  
Author(s):  
Hans M. Dalton ◽  
Raghuvir Viswanatha ◽  
Ricky Brathwaite ◽  
Jae Sophia Zuno ◽  
Stephanie E. Mohr ◽  
...  
Keyword(s):  
Er Stress ◽  
Therapeutic Targets ◽  
Therapeutic Benefit ◽  
Loss Of Function ◽  
Gene Encoding ◽  
Fructose Metabolism ◽  
Genome Wide ◽  
A Genome ◽  
Crispr Screen

AbstractPartial loss-of-function mutations in glycosylation pathways underlie a set of rare diseases called Congenital Disorders of Glycosylation (CDGs). In particular, DPAGT1-CDG is caused by mutations in the gene encoding the first step in N-glycosylation, DPAGT1, and this disorder currently lacks effective therapies. To identify potential therapeutic targets for DPAGT1-CDG, we performed CRISPR knockout screens in Drosophila cells for genes associated with better survival and glycoprotein levels under DPAGT1 inhibition. We identified hundreds of candidate genes that may be of therapeutic benefit. Intriguingly, inhibition of the mannosyltransferase Dpm1, or its downstream glycosylation pathways, could rescue two in vivo models of DPAGT1 inhibition and ER stress, even though impairment of these pathways alone usually cause CDGs. While both in vivo models ostensibly cause ER stress (through DPAGT1 inhibition or a misfolded protein), we found a novel difference in fructose metabolism that may indicate glycolysis as a modulator of DPAGT1-CDG. Our results provide new therapeutic targets for DPAGT1-CDG, include the unique finding of Dpm1-related pathways rescuing DPAGT1 inhibition, and reveal a novel interaction between fructose metabolism and ER stress.

scholarly journals Selenoprotein S regulates adipogenesis through IRE1α-XBP1 pathway

2020 ◽  
Vol 244 (3) ◽  
pp. 431-443
Author(s):  
Lili Men ◽  
Junjie Yao ◽  
Shanshan Yu ◽  
Yu Li ◽  
Siyuan Cui ◽  
...  
Keyword(s):  
Er Stress ◽  
Late Phase ◽  
G1 Arrest ◽  
Mrna Levels ◽  
Protein Levels ◽  
Selenoprotein S ◽  
Stress Markers ◽  
Obese Subjects

The induction of endoplasmic reticulum (ER) stress is associated with adipogenesis, during which the inositol-requiring enzyme 1 alpha (IRE1α)-X-box-binding protein 1 (XBP1) pathway is involved. Selenoprotein S (SelS), which is an ER resident selenoprotein, is involved in ER homeostasis regulation; however, little is known about the role of SelS in regulating adipogenesis. In vivo studies showed that SelS protein levels in white adipose tissue were increased in obese subjects and high-fat diet (HFD)-fed mice. Moreover, we identified that SelS protein levels increased in the early phase of adipogenesis and then decreased in the late phase during adipogenesis. Overexpression of SelS promoted adipogenesis. Conversely, knockdown (KD) of SelS resulted in the inhibition of adipogenesis, which was related to increasing cell death, decreased mitotic clonal expansion, and cell cycle G1 arrest. In vivo studies also showed that ER stress markers (p-IRE1α/IRE1α, XBP1s, and Grp78) were significantly increased with upregulating of SelS expression in subcutaneous and visceral adipose tissues in the obese subjects and HFD-fed mice. Furthermore, in SelS KD cells, the levels of Grp78 were increased and the levels of p-IRE1α/IRE1α were unchanged , but mRNA levels of spliced XBP1 (XBP1s) produced by IRE1α-mediated splicing were decreased, suggesting a role of SelS in the modulation of IRE1α-XBP1 pathway. Moreover, inhibition of adipogenesis by SelS suppression can be rescued by overexpression of XBP1s. Thus, SelS appears to function as a novel regulator of adipogenesis through the IRE1α-XBP1 signaling pathway.

scholarly journals Novel Loss-of-Function Mutations in DNAH1 Displayed Different Phenotypic Spectrum in Humans and Mice

2021 ◽  
Vol 12 ◽  
Author(s):  
Ranjha Khan ◽  
Qumar Zaman ◽  
Jing Chen ◽  
Manan Khan ◽  
Ao Ma ◽  
...  
Keyword(s):  
Male Infertility ◽  
Mutant Mice ◽  
Pathophysiological Mechanism ◽  
Loss Of Function ◽  
Fibrous Sheath ◽  
Transmission Electron Microscopy Analysis ◽  
Central Pair ◽  
Recurrent Mutations ◽  
The Impact

Male infertility is a prevalent disorder distressing an estimated 70 million people worldwide. Despite continued progress in understanding the causes of male infertility, idiopathic sperm abnormalities such as multiple morphological abnormalities of sperm flagella (MMAF) still account for about 30% of male infertility. Recurrent mutations in DNAH1 have been reported to cause MMAF in various populations, but the underlying mechanism is still poorly explored. This study investigated the MMAF phenotype of two extended consanguineous Pakistani families without manifesting primary ciliary dyskinesia symptoms. The transmission electron microscopy analysis of cross-sections of microtubule doublets revealed a missing central singlet of microtubules and a disorganized fibrous sheath. SPAG6 staining, a marker generally used to check the integration of microtubules of central pair, further confirmed the disruption of central pair in the spermatozoa of patients. Thus, whole-exome sequencing (WES) was performed, and WES analysis identified two novel mutations in the DNAH1 gene that were recessively co-segregating with MMAF phenotype in both families. To mechanistically study the impact of identified mutation, we generated Dnah1 mice models to confirm the in vivo effects of identified mutations. Though Dnah1△iso1/△iso1 mutant mice represented MMAF phenotype, no significant defects were observed in the ultrastructure of mutant mice spermatozoa. Interestingly, we found DNAH1 isoform2 in Dnah1△iso1/△iso1 mutant mice that may be mediating the formation of normal ultrastructure in the absence of full-length protein. Altogether we are first reporting the possible explanation of inconsistency between mouse and human DNAH1 mutant phenotypes, which will pave the way for further understanding of the underlying pathophysiological mechanism of MMAF.

scholarly journals Dietary gossypol suppressed postprandial TOR signaling and elevated ER stress pathways in turbot (Scophthalmus maximus L.)

2017 ◽  
Vol 312 (1) ◽  
pp. E37-E47 ◽  
Author(s):  
Fuyun Bian ◽  
Haowen Jiang ◽  
Mingsan Man ◽  
Kangsen Mai ◽  
Huihui Zhou ◽  
...  
Keyword(s):  
Er Stress ◽  
Digestive Enzyme ◽  
Scophthalmus Maximus ◽  
Fish Growth ◽  
Tor Signaling ◽  
Body Protein ◽  
Digestive Enzyme Activities ◽  
Turbot Scophthalmus Maximus

Gossypol is known to be a polyphenolic compound toxic to animals. However, its molecular targets are far from fully characterized. To evaluate the physiological and molecular effects of gossypol, we chose turbot ( Scophthalmus maximus L.), a carnivorous fish, as our model species. Juvenile turbots (7.83 ± 0.02 g) were fed diets containing gradient levels of gossypol at 0 (G0), 600 (G1), and 1,200 (G2) mg/kg diets for 11 wk. After the feeding trial, fish growth, body protein, and fat contents were significantly reduced in the G2 group compared with those of the G0 group ( P < 0.05). Gossypol had little impact on digestive enzyme activities and intestine morphology. However, gossypol caused liver fibrosis and stimulated chemokine and proinflammatory cytokine secretions. More importantly, gossypol suppressed target of rapamycin (TOR) signaling and induced endoplasmic reticulum (ER) stress pathway in both the feeding experiment and cell cultures. Our results demonstrated that gossypol inhibited TOR signaling and elevated ER stress pathways both in vivo and in vitro, thus providing new mechanism of action of gossypol in nutritional physiology.

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