Wolframi sündroomi mudel hiirtel: käitumuslik, biokeemiline ja psühhofarmakoloogiline iseloomustamine

Väitekirja elektrooniline versioon ei sisalda publikatsioone.Wolframi sündroom (WS), mida kirjeldasid esmakordselt Saksa arstid Wolfram ja Wagener 1938.a, on haruldane autosomaalne retsessiivne neurodegeneratiivne häire, mida iseloomustavad varajases lapseeas algav suhkurtõbi, progresseeruv nägemisnärvi atroofia, magediabeet ja kurtus. Wolframi sünroomi põhjustavad mutatsioonid Wolframiin 1 (WFS1) geenis. Wolframiin 1 geeni on seotud hirmu ja ärevusega hiirtel ja rottidel ja inimestel on tema polümorfismid on seotud suurenenud meeleoluhäirete riskidega nagu bipolaarseste häirete psühhootilise vormi, skisofreenia, suitsidaarne käitumine ning depressioon. Käesoleval ajal ei ole veel päris selge, mis moodi WFS1 geeni puudulikkus põhjustab neuropsühhiaatriliste kõrvalekallete ilmnemist. Kuid võttes arvesse WS puhul esinevad psühhiaatrilised sümptomid ning ka Wfs1 geeni ja valgu lokalisatsiooni ajus, võib arvata, et ühetedeks põhjustajateks võivad olla kõrvalekalded GABA- ja dopamiinergilise süsteemi talitluses. Käesoleva doktoritöö keskseks eesmärgiks oli WS hiire mudeli loomine. Selleks valmistati mutantne hiireliin kustutades Wfs1 geenist 8nda eksoni, kuna WS patsientidel enamik mutatsioone asub just selles eksonis. Selline lähenemisviis aitas luua WS sarnase seisundi, et uurida sündroomi poolt esilekutsutud patoloogilisi muutusi. Katseloomade iseloomustamiseks kasutati käitumuslikke, biokeemilisi ja psühhofarmakoloogilisi uurimismeetodeid. Selgus, et Wfs1 mutantsetel hiirtel ei olnud nähtavaid sensoorseid häireid, kuid nad reageerisid rohkem stressi esilekutsuvates keskkondades (testides). Samuti suurenes neil tunduvalt enam, võrreldes metsik-tüüpi hiirtega, korikosterooni tase ringlevas veres peale füsioloogilise lahuse akuutset süstimist ning nad ei suuda kontrollida oma veresuhkru taset glükoosi tolerantsi testis. Wfs1 puudulikkusega hiirtel oli suurenenud tundlikkus GABAA retseptorite agonisti diasepaami ärevusvastase toime suhtes. Diasepaam kõrvaldas ka Wfs1 puudulikksuega hiirtele iseloomulikud häälitsemised stressogeenses situatsioonis. Neil oli vähenenud Gabra1 ja Gabra2 geenide eskpressioon oimusagaras ja otsmikukoores ehk ajustruktuurides, mis on seotud negatiivsete emotsioonide regulatsiooniga. See ekspressiooni langus on ilmselt seotud Wfs1 puudulikkusega hiirtel esineva ülitundlikkusega diasepaami ärevusvastase toime suhtes. Sarnane Gabra1 ja Gabra2 geeniekspressiooni langus esines metsik-tüüpi hiirtel peale nende eksponeerimist pluss puuris (ärevuse mudel), mis viitab, et on olemas seos GABAA retseptorite alatüüpide ekspressiooni languse ja ärevuse vahel. Lisaks kõigele muule esinesid nendel hiirtel kõrvalekalded dopamiinergilise süsteemi funktsioonis. Nende tundlikkus amfetamiini, kaudse dopamiini agonisti, motoorikat stimuleeriva toime suhtes oli vähenenud ning amfetamiini manustamine ei põhjustanud neil dopamiini ringkäiku muutusi dorsaalses ja ventraalses striatumis võrreldes metsiktüüpi pesakonnakaaslastega. Apomorfiinist tingitud motoorika stimulatsioon oli mõnevõrra tugevam mutantsetel hiirtel, kuid apomorfiin põhjustas kõikidel genotüüpidel ühesugust dopamiini ringkäiku vähenemist, mille alusel võib väita, et Wfs1 geeni puudulikkus ei mõjusta dopamiini metabolismi dopamiini retseptorite vahendusel. Seda kinni¬tavad ka geeni ekspressiooni tulemused, kus dopamiini retseptori (Drd2) ekspressioon oli ühesugune kõikide genotüüpide ventraalses striatumis. Siiski oli nii emastel kui ka isastel Wfs1 puudulikusega hiirtel vähenenud dopamiini transporteri geeni ekspressioon keskajus. See leid koos amfetamiini käitu-muslike ja biokeemiliste toimete vähenemisega viitab dopamiinergilise süsteemi funktsiooni olulisele häirumisele Wfs1 puudulikkusega hiirtel. Põhinedes glükoosi metabolismi uuringute tulemustele ja nende sarnasusele WS on selge, et Wfs1 geeni kaheksanda eksoni puudulikkusega hiir on valiidseks mudeliks WS südnroomile ja ka teistele WFS1 geenimutatsioonidest tingitud häiretele. Sellepärast võib käesolevas uuringus leitud muutusi dopamiini- ja GABA-ergilise süsteemi aktiivsuses Wfs1 puudulikkusega hiirtel laiendada WS patsientidele, et mõista nendel esinevaid Neuropsühhiaatrilisi Sümptomeid.Wolfram syndrome (WS), first described by German physicians Wolfram and Wagener in 1938, is a rare autosomal reces¬sive neurodegenerative disorder characterised by early juvenile diabetes mellitus, progressive optic nerve atrophy, diabetes insipidus and deafness. Wolfram syndrome is caused by mutations in the wolframin (WFS1) gene. In mice and rats, the wolframin gene has been associated with fear and anxiety; in humans, there is a link between WFS1 polymorphisms and increased risk for mood disorders, such as the psychotic form of bipolar disorder, schizophrenia, suicidal behaviour and depression. At present, it is not yet quite clear, how disruptions in the WFS1 gene cause neuropsychiatric deviations. However, based on the fact that WS is accompanied by psychiatric symptoms and also based on the localisation of the Wfs1 protein in the brain it can be concluded that at least partially it is caused by deviations in the functioning of the GABAergic and dopaminergic systems. The main objective of this dissertation was the creation of a WS mouse model. For that purpose, a mutant mouse line was created by deleting exon 8 in the Wfs1 gene as in WS patients most of the mutations are located in that exon. By this approach we managed to mimic WS in order to study pathological changes induced by the syndrome. To characterise the animals, behavioural, biochemical and psychopharmacological methods were used. Wfs1 mutant mice had no overt sensory deficits, however, they were more sensitive to stress-inducing environments (tests). Also, as compared to wild-type mice, they displayed a much more prominent increase in the level of corticosterone in the circulating blood in response to acute saline injection, and they were unable to keep their blood glucose level under control in the glucose tolerance test. Wfs1-deficient mice had increased sensitivity to the anxiolytic effect of GABA A recep¬tor agonist diazepam. Also, in Wfs1 mice diazepam blocked characteristic vocalisations in a stressful situation. Wfs1-deficient mice had lower expression level of Gabra1 and Gabra2 genes in the temporal and frontal lobes, brain structures involved in the regulation of negative emotions. These decreased expression levels are probably related to hypersensitivity to the anxiolytic effect of diazepam observed in Wfs1-deficient mice. In wild-type mice, a similar drop in the expression levels of Gabra1 and Gabra2 genes was evident after exposure to the plus maze (a model of anxiety), which indicates that there is a link between decreased expression levels of GABA A receptor subtypes and anxiety. Furthermore, mice lacking the wolframin protein had deviations in the function of the dopaminergic system. Their sensitivity to the motor stimulant effect of amphetamine, an indirect agonist of dopamine, had decreased and the administration of amphetamine failed to induce changes in dopamine turnover in the dorsal and ventral striatum as opposed to their wild-type littermates. Apomorphine-induced motor stimulation was somewhat stronger in mutant mice, but in all genotypes apomorphine caused a similar decrease in dopamine turnover. This enables us to conclude that Wfs1 gene deficiency has no effect on dopamine metabolism mediated by dopamine receptors. This is corroborated by the results of gene expression measurements showing that in the ventral striatum the expression level of dopamine D2 receptor (Drd2) was equal in all genotypes. However, both in male and female Wfs1-deficient mice the expression level of the dopamine transporter gene had decreased in the mesencephalon. This finding and also the decreased behavioural and biochemical effects of amphetamine are indicative of serious deviations in the function of the dopaminergic system in Wfs1-deficient mice. Based on the results of glucose metabolism studies and their similarity to WS, it is clear that the mouse, missing exon 8 of the Wfs1 gene, is a valid model of both WS syndrome and other disorders caused by mutations in the WFS1 gene. Therefore, the changes observed in the activity of the dopaminergic and GABAergic systems in Wfs1-deficient mice may also help to explain the neuropsychiatric symptoms of WS patients

Exenatide Is an Effective Antihyperglycaemic Agent in a Mouse Model of Wolfram Syndrome 1

Wolfram syndrome 1 is a very rare monogenic disease resulting in a complex of disorders including diabetes mellitus. Up to now, insulin has been used to treat these patients. Some of the monogenic forms of diabetes respond preferentially to sulphonylurea preparations. The aim of the current study was to elucidate whether exenatide, a GLP-1 receptor agonist, and glipizide, a sulphonylurea, are effective in a mouse model of Wolfram syndrome 1. Wolframin-deficient mice were used to test the effect of insulin secretagogues. Wolframin-deficient mice had nearly normal fasting glucose levels but developed hyperglycaemia after glucose challenge. Exenatide in a dose of 10 g/kg lowered the blood glucose level in both wild-type and wolframin-deficient mice when administered during a nonfasted state and during the intraperitoneal glucose tolerance test. Glipizide (0.6 or 2 mg/kg) was not able to reduce the glucose level in wolframin-deficient animals. In contrast to other groups, wolframin-deficient mice had a lower insulin-to-glucose ratio during the intraperitoneal glucose tolerance test, indicating impaired insulin secretion. Exenatide increased the insulin-to-glucose ratio irrespective of genotype, demonstrating the ability to correct the impaired insulin secretion caused by wolframin deficiency. We conclude that GLP-1 agonists may have potential in the treatment of Wolfram syndrome-related diabetes

Low cardiac content of long-chain acylcarnitines in TMLHE knockout mice prevents ischaemia-reperfusion-induced mitochondrial and cardiac damage

Copyright © 2021. Published by Elsevier Inc.Increased tissue content of long-chain acylcarnitines may induce mitochondrial and cardiac damage by stimulating ROS production. N6-trimethyllysine dioxygenase (TMLD) is the first enzyme in the carnitine/acylcarnitine biosynthesis pathway. Inactivation of the TMLHE gene (TMLHE KO) in mice is expected to limit long-chain acylcarnitine synthesis and thus induce a cardio- and mitochondria-protective phenotype. TMLHE gene deletion in male mice lowered acylcarnitine concentrations in blood and cardiac tissues by up to 85% and decreased fatty acid oxidation by 30% but did not affect muscle and heart function in mice. Metabolome profile analysis revealed increased levels of polyunsaturated fatty acids (PUFAs) and a global shift in fatty acid content from saturated to unsaturated lipids. In the risk area of ischemic hearts in TMLHE KO mouse, the OXPHOS-dependent respiration rate and OXPHOS coupling efficiency were fully preserved. Additionally, the decreased long-chain acylcarnitine synthesis rate in TMLHE KO mice prevented ischaemia-reperfusion-induced ROS production in cardiac mitochondria. This was associated with a 39% smaller infarct size in the TMLHE KO mice. The arrest of the acylcarnitine biosynthesis pathway in TMLHE KO mice prevents ischaemia-reperfusion-induced damage in cardiac mitochondria and decreases infarct size. These results confirm that the decreased accumulation of ROS-increasing fatty acid metabolism intermediates prevents mitochondrial and cardiac damage during ischaemia-reperfusion.publishersversionPeer reviewe

Globin mRNA reduction for whole-blood transcriptome sequencing

The transcriptome analysis of whole-blood RNA by sequencing holds promise for the identification and tracking of biomarkers; however, the high globin mRNA (gmRNA) content of erythrocytes hampers whole-blood and buffy coat analyses. We introduce a novel gmRNA locking assay (GlobinLock, GL) as a robust and simple gmRNA reduction tool to preserve RNA quality, save time and cost. GL consists of a pair of gmRNA-specific oligonucleotides in RNA initial denaturation buffer that is effective immediately after RNA denaturation and adds only ten minutes of incubation to the whole cDNA synthesis procedure when compared to non-blood RNA analysis. We show that GL is fully effective not only for human samples but also for mouse and rat, and so far incompletely studied cow, dog and zebrafish.Peer reviewe

Calcium Signaling and Contractility in Cardiac Myocyte of Wolframin Deficient Rats

Wolframin (Wfs1) is a membrane protein of the sarco/endoplasmic reticulum. Wfs1 mutations are responsible for the Wolfram syndrome, characterized by diabetic and neurological symptoms. Although Wfs1 is expressed in cardiac muscle, its role in this tissue is not clear. We have characterized the effect of invalidation of Wfs1 on calcium signaling-related processes in isolated ventricular myocytes of exon5-Wfs1 deficient rats (Wfs1-e5/-e5) before the onset of overt disease. Calcium transients and contraction were measured in field-stimulated isolated myocytes using confocal microscopy with calcium indicator fluo-3 AM and sarcomere length detection. Calcium currents and their calcium release-dependent inactivation were characterized in whole-cell patch-clamp experiments. At 4 months, Wfs1-e5/-e5 animals were euglycemic, and echocardiographic examination revealed fully compensated cardiac function. In field-stimulated isolated ventricular myocytes, both the amplitude and the duration of contraction of Wfs1-e5/-e5 animals were elevated relative to control Wfs1+/+ littermates. Increased contractility of myocytes resulted largely from prolonged cytosolic calcium transients. Neither the amplitude of calcium currents nor their voltage dependence of activation differed between the two groups. Calcium currents in Wfs1-e5/-e5 myocytes showed a larger extent of inactivation by short voltage prepulses applied to selectively induce calcium release-dependent inactivation of calcium current. Neither the calcium content of the sarcoplasmic reticulum, measured by application of 20 mmol/l caffeine, nor the expression of SERCA2, determined from Western blots, differed significantly in myocytes of Wfs1-e5/-e5 animals compared to control ones. These experiments point to increased duration of calcium release in ventricular myocytes of Wfs1-e5/-e5 animals. We speculate that the lack of functional wolframin might cause changes leading to upregulation of RyR2 channels resulting in prolongation of channel openings and/or a delay in termination of calcium release

Exenatide Is an Effective Antihyperglycaemic Agent in a Mouse Model of Wolfram Syndrome 1

Wolfram syndrome 1 is a very rare monogenic disease resulting in a complex of disorders including diabetes mellitus. Up to now, insulin has been used to treat these patients. Some of the monogenic forms of diabetes respond preferentially to sulphonylurea preparations. The aim of the current study was to elucidate whether exenatide, a GLP-1 receptor agonist, and glipizide, a sulphonylurea, are effective in a mouse model of Wolfram syndrome 1. Wolframin-deficient mice were used to test the effect of insulin secretagogues. Wolframin-deficient mice had nearly normal fasting glucose levels but developed hyperglycaemia after glucose challenge. Exenatide in a dose of 10 μg/kg lowered the blood glucose level in both wild-type and wolframin-deficient mice when administered during a nonfasted state and during the intraperitoneal glucose tolerance test. Glipizide (0.6 or 2 mg/kg) was not able to reduce the glucose level in wolframin-deficient animals. In contrast to other groups, wolframin-deficient mice had a lower insulin-to-glucose ratio during the intraperitoneal glucose tolerance test, indicating impaired insulin secretion. Exenatide increased the insulin-to-glucose ratio irrespective of genotype, demonstrating the ability to correct the impaired insulin secretion caused by wolframin deficiency. We conclude that GLP-1 agonists may have potential in the treatment of Wolfram syndrome-related diabetes

Mice Expressing Minimally Humanized CD81 and Occludin Genes Support Hepatitis C Virus Uptake In Vivo

Hepatitis C virus (HCV) causes chronic infections in at least 150 million individuals worldwide. HCV has a narrow host range and robustly infects only humans and chimpanzees. The underlying mechanisms for this narrow host range are incompletely understood. At the level of entry, differences in the amino acid sequences between the human and mouse orthologues of two essential host factors, the tetraspanin CD81 and the tight junction protein occludin (OCLN), explain, at least in part, HCV's limited ability to enter mouse hepatocytes. We have previously shown that adenoviral or transgenic overexpression of human CD81 and OCLN facilitates HCV uptake into mouse hepatocytes in vitro and in vivo. In efforts to refine these models, we constructed knock-in mice in which the second extracellular loops of CD81 and OCLN were replaced with the respective human sequences, which contain the determinants that are critical for HCV uptake. We demonstrate that the humanized CD81 and OCLN were expressed at physiological levels in a tissue-appropriate fashion. Mice bearing the humanized alleles formed normal tight junctions and did not exhibit any immunologic abnormalities, indicating that interactions with their physiological ligands were intact. HCV entry factor knock-in mice take up HCV with an efficiency similar to that in mice expressing HCV entry factors transgenically or adenovirally, demonstrating the utility of this model for studying HCV infection in vivo

Mice Expressing Minimally Humanized CD81 and Occludin Genes Support Hepatitis C Virus Uptake In Vivo

Hepatitis C virus (HCV) causes chronic infections in at least 150 million individuals worldwide. HCV has a narrow host range and robustly infects only humans and chimpanzees. The underlying mechanisms for this narrow host range are incompletely understood. At the level of entry, differences in the amino acid sequences between the human and mouse orthologues of two essential host factors, the tetraspanin CD81 and the tight junction protein occludin (OCLN), explain, at least in part, HCV's limited ability to enter mouse hepatocytes. We have previously shown that adenoviral or transgenic overexpression of human CD81 and OCLN facilitates HCV uptake into mouse hepatocytes in vitro and in vivo. In efforts to refine these models, we constructed knock-in mice in which the second extracellular loops of CD81 and OCLN were replaced with the respective human sequences, which contain the determinants that are critical for HCV uptake. We demonstrate that the humanized CD81 and OCLN were expressed at physiological levels in a tissue-appropriate fashion. Mice bearing the humanized alleles formed normal tight junctions and did not exhibit any immunologic abnormalities, indicating that interactions with their physiological ligands were intact. HCV entry factor knock-in mice take up HCV with an efficiency similar to that in mice expressing HCV entry factors transgenically or adenovirally, demonstrating the utility of this model for studying HCV infection in vivo. IMPORTANCE At least 150 million individuals are chronically infected with hepatitis C virus (HCV). Chronic hepatitis C can result in progressive liver disease and liver cancer. New antiviral treatments can cure HCV in the majority of patients, but a vaccine remains elusive. To gain a better understanding of the processes culminating in liver failure and cancer and to prioritize vaccine candidates more efficiently, small-animal models are needed. Here, we describe the characterization of a new mouse model in which the parts of two host factors that are essential for HCV uptake, CD81 and occludin (OCLN), which differ between mice and humans, were humanized. We demonstrate that such minimally humanized mice develop normally, express the modified genes at physiological levels, and support HCV uptake. This model is of considerable utility for studying viral entry in the three-dimensional context of the liver and to test approaches aimed at preventing HCV entry

Image_5_Treatment with the dual-incretin agonist DA-CH5 demonstrates potent therapeutic effect in a rat model of Wolfram Syndrome.jpg

AimWolfram Syndrome (WS) is a rare condition caused by mutations in Wfs1, with a poor prognosis and no cure. Mono-agonists targeting the incretin glucagon-like-peptide 1 (GLP-1) have demonstrated disease-modifying potential in pre-clinical and clinical settings. Dual agonists that target GLP-1 and glucose-dependent insulinotropic polypeptide (GIP-1) are reportedly more efficacious; hence, we evaluated the therapeutic potential of dual incretin agonism in a loss-of-function rat model of WS.MethodsEight-month-old Wfs1 knock-out (KO) and wild-type control rats were continuously treated with either the dual agonist DA-CH5 or saline for four months. Glycemic profile, visual acuity and hearing sensitivity were longitudinally monitored pre-treatment, and then at 10.5 and 12 months. Pancreata and retina were harvested for immunohistological analysis.ResultsDA-CH5 therapy reversed glucose intolerance in KO rats and provided lasting anti-diabetogenic protection. Treatment also reversed intra-islet alterations, including reduced endocrine islet area and β-cell density, indicating its regenerative potential. Although no rescue effect was noted for hearing loss, visual acuity and retinal ganglion cell density were better preserved in DA-CH5-treated rats.ConclusionWe present preclinical evidence for the pleiotropic therapeutic effects of long-term dual incretin agonist treatment; effects were seen despite treatment beginning after symptom-onset, indicating reversal of disease progression. Dual incretins represent a promising therapeutic avenue for WS patients.</p