Speciation Genetics in Drosophila: Insights from Male and Female Transcriptomes
Population genetic models predict that the X chromosome will evolve at a faster rate than autosomes (i.e., faster-X evolution). The basis for faster-X evolution is that recessive genes located on the X chromosome can be exposed in males while these genes are hidden if located on the autosomes. Evaluations of protein-coding sequences have indeed shown mixed results with some studies showing an excess of divergence on the X chromosome compared to autosomes, particularly in genes expressed higher in males than in females (i.e., male-biased genes). In addition, whole-genome analyses of gene expression in Drosophila yakuba and D. santomea males indicate that X-linked genes are more differentially expressed between species (i.e., faster-X evolution of gene expression) than autosomal genes. This trend is once again strongest for male-biased genes. However, these studies utilized only males and were therefore limited in their expression profiles. Here, we investigate the whole-genome profiles of D. yakuba and D. santomea males and females to determine the relative rates of evolution for all gene classes. In addition, we explore the implications of sex chromosome inheritance on autosomal gene expression in the hybrids of these species.
THE ROLE OF ENDOGENOUS MICROBIOTA IN SEIZURE-LIKE BEHAVIOR OF SHUDDERER, A DROSOPHILA VOLTAGE-GATED SODIUM CHANNEL MUTANT
It is widely recognized that mutations in genes encoding voltage-gated sodium (Nav) channels contribute to the etiology underlying various seizure disorders. Shudderer (Shu), a gain-of-function mutant for the Drosophila Nav channel gene, exhibits neuronal hyperexcitability and seizure-like behavioral defects, including spontaneous jerking and heat-induced convulsion. Results of microarray analyses indicated that Shu mutants have an increased innate immune response and reduced insulin signaling. Because the endogenous gut microbiota has a substantial impact on the host immune system and insulin-mediated metabolic regulation, we hypothesized that the microbiome plays a role in Shu phenotypes. Intriguingly, removing the gut microbiota of Shu and wild-type (WT) flies using antibiotic-containing or sterile food significantly suppressed Shu behavioral phenotypes while having no effect on WT behavior. Culturing homogenized guts dissected from Shu and WT flies on MRS agar revealed drastic differences in the number of culturable bacteria. Further, we performed high-throughput sequencing of the bacterial 16S ribosomal RNA gene which revealed species-specific differences in the gut microbiome of Shu and WT flies. Because neurological disorders and changes in the microbiota can individually increase oxidative stress in an organism, we examined the antioxidant response in the guts of Shu and WT flies using a GFP reporter, (GST-D-GFP). GST-D-GFP expression was altered in Shu and WT flies fed antibiotics, suggesting a potential involvement of oxidative stress in the antibiotic-dependent rescue of seizure-like behavior. Additional experiments will determine ROS levels in Shu and WT flies and are expected to yield a better understanding of the role of the microbiome in the context of seizure disorders.
Mutation in regulatory protease, CAPN5, results in eye disease
Uveitis (intraocular inflammation) causes significant visual morbidity and blindness in all ages, yet it has proven refractive to current therapies, underscoring the critical need for new clinical approaches. Nevertheless, a major barrier to developing new medicines has been the lack of any specific molecular cause for the disease. To gain insight into the molecular mechanisms driving uveitis, we sought the disease-associated gene in kindreds with a Mendelian form—Autosomal Dominant Neovascular Inflammatory Vitreoretinopathy (ADNIV). Our studies showed the underlying cause of ADNIV was mutations in CAPN5 (calpain-5), making CAPN5 the first nonsyndromic uveitis gene discovered. This discovery provides an unprecedented starting point for investigating the substantial gap in our understanding of the molecular basis of ocular inflammation.
CAPN5 is a calcium-activated, intracellular protease expressed at photoreceptor synapses. Similar to the function of other calpains, we believe that proteolysis by CAPN5 is a tightly controlled, post-translational regulatory mechanism that deploys protein fragments with altered activity. Although it is a member of a relatively large, well-studied family, little is known regarding the CAPN5 natural substrates. One exception to this is CAPN5 autoproteolysis, which uncovers one natural substrate of CAPN5: CAPN5 itself. We aim to identify the sequences in CAPN5 that are the targets of autoproteolysis and also to determine the CAPN5 cleavage site in a substrate we recently identified, platelet-derived growth factor B (PDGFB). PDGFB was previously identified as a protein cleaved into an active angiogenic form by an unknown protease, and PDGFB is active in a number of similar eye diseases, including AMD, PVR and diabetic retinopathy. The insight gained from these studies may eventually be used to design therapies for a variety of previously untreatable eye diseases.
Functional Characterization of Epilepsy Related Genes in Zebrafish
TD Fuller1,2, TA Westfall1, DC Slusarski1
1Department of Biology, University of Iowa, Iowa City, IA 52242, 2Interdisciplinary Graduate Program in Genetics
Statement of Purpose: Epilepsy is a chronic condition of recurrent seizures which affects approximately one percent of the general population. Though several causative genes have already been identified, these account for only a small percentage of all the genetically caused cases of epilepsy. Further, even when genes are identified, we lack tractable animal models to rapidly translate these findings into mechanistic insights and ultimately new anti-epileptic therapies. For this reason, the zebrafish is increasingly being used as a model of epilepsy due to its high genetic and physiologic homology to humans and its seizure-like behavior in response to various pharmacological and genetic manipulations. Using High throughput sequencing, a significant number of gene variants are being identified, yet their role in the disease state remain unknown. My project utilizes the zebrafish and focuses on characterizing the functional role of 15 genes in the NIH Undiagnosed Diseases Program for which mutations have been associated with epilepsy, and for which zebrafish orthologues have been identified. Methods: I isolated the zebrafish orthologues and characterized gene expression patterns by RTPCR and whole mount in situ hybridization techniques. We previously demonstrated that knockdown of Prickle (PK), a gene associated with human epilepsy, sensitizes zebrafish to seizure-inducing drugs through the use of larval motility assays. To facilitate high-throughput in vivo screens, I adapted this approach and developed a code to rapidly and efficiently analyze the generated data sets. This allowed for the characterization of the 15 candidate genes in the context of seizure sensitization. Results: Of the fifteen candidates, I found five: syne1b, sms, ccdc89, wscd1, and nid2a, to result in seizure sensitization when knocked down in the zebrafish. I show that each of these genes is expressed in specific regions in the brain during critical times of neuronal development. Further, I find genes expressed in the retina result in axon defects when knocked down.
The Conserved and Unique Determinants of Crossover Events
Meiotic recombination promotes genetic diversity and allows for the continuous adaptation of natural populations to ever changing environments. Mechanistically, recombination relies heavily on protein-protein and protein-DNA interactions. The goal of my project is to find out how genetic incompatibilities through failed interactions disrupt the tight control of recombination localization across the genome in interspecific hybrids. To this end, I am currently generating high-resolution recombination maps in two closely related species of fruit flies, Drosophila yakuba and D. santomea, and their hybrids. Our findings will have a positive impact in our understanding of the role of protein interactions in recombination control, will illuminate the pervasiveness of sexual reproduction and recombination in nature, and will enable the development of new theoretical models on the maintenance of genetic variation.
Genetic therapeutic strategies for the Bardet-Biedl Syndrome (BBS) M390R mutation
Bardet-Beidl Syndrome (BBS) is a pleiotropic ciliopathy that causes a variety of phenotypes in humans and animal models, including blindness and male infertility. BBS is considered a model disease for ciliopathies in general, which affect approximately 1 in 1000 people worldwide. There are no current efficient non-surgical treatments for ciliopathies, illustrating a critical need for new therapeutic strategies. The most common cause of BBS is the M390R mutation in Bardet-Biedl Syndrome 1 (BBS1) gene, an essential component of the BBsome that is required for basal body function and primary ciliogenesis. This mutation leads to photoreceptor degeneration and dysfunctional flagella in sperm. My objective is to use gene replacement and editing techniques to restore male fertility and prevent retinal degeneration in a Bbs1M390R/M390R mouse model. I hypothesize that these phenotypes are amenable to correction by gene therapy and gene correction. Here, I will test this hypothesis by using BBS1 gene therapy and correction as a strategy to preserve photoreceptors in a mouse model. First, I will elucidate the mechanism by which BBS1 overexpression in the retina causes toxicity. I hypothesize that overexpression of BBS1 leads to homodimerization and subsequent inability to incorporate into the BBsome, thereby affecting ciliary function. I will deliver adeno-associated viruses (AAVs) expressing BBS1 driven by the BBS1 promoter to the Bbs1M390R/M390R mice postnatally, and subsequently test photoreceptor preservation by electroretinogram (ERG) and immunohistology. I will also use CRISPR/Cas9 mediated homologous recombination to correct the M390R mutation in vitro and in vivo. In addition to Cas9, I will employ Cpf1, a Cas9-like class 2 CRISPR endonuclease, to simultaneously knock out dysfunctional BBS1 and introduce functional BBS1 cDNA driven by the BBS1 promoter. By correcting the germline in vivo, all downstream progeny should effectively be corrected. While most studies using gene editing techniques are completed ex vivo, my approach will show the level of correction that can occur in vivo using various novel methods. These techniques may be applied to other degenerative genetic diseases.
LINCing prostaglandin signaling and the regulation of collective cell migration.
Emily Toombs, Tina Tootle
Collective cell migration – the coordinated movement of tightly or loosely associated cells – is important for development and tumor invasion. Many signals are involved in collective cell migration including mechanotransduction, or the transfer of physical force into electrical or chemical signals. While there are many ways in which cells can respond to force, a key mechanism important for cellular migration is the direct connection of the cytoskeleton to the nucleoskeleton via the Linker of Nucleoskeleton and Cytoskeleton (LINC) complex. Alterations in the LINC complex affect nuclear shape, nuclear position, and transcription; however, not much is known about its regulation. Another mechanism regulating cell migration is prostaglandin (PG) signaling. PGs are short-range lipid signals that are implicated in many processes, but, at the same time, little is known about their downstream targets. Our lab has shown that PGs have a large affect on actin remodeling via regulating actin binding proteins. Importantly, actin binding proteins play key roles in mechanotransduction; however, PG signaling has not been previously implicated in regulating mechanotransduction. Using the robust genetic model system of Drosophila, and the well characterized process of oogenesis or follicle development, we present the first evidence that PGs regulate the LINC Complex during the collective and invasive cell migration of the border cells. We hypothesize that PG signaling regulates perinuclear Fascin to control LINC complex function. This hypothesis is based on our prior studies that revealed PG signaling regulates Fascin, an actin bundling protein widely implicated in cell migration. This regulation occurs, in part, by PGs modulating the localization of Fascin, including Fascin’s new perinuclear localization. In both PG synthesis and LINC complex mutant follicles Fascin’s localization to the nuclear periphery is lost. Furthermore, our collaborators have found that in cancer cells, Fascin interacts directly with the LINC Complex. Here we present that loss of PGs or the LINC complex results in delayed and aberrant border cell migration; importantly, Fascin is highly expressed in the border cells. We have identified several tools and approaches that will allow us to quantitatively assess the connection between PG signaling and the LINC complex, as well as the role of Fascin in LINC complex regulation. This research is expected to provide the mechanistic insight into how PGs regulate cellular migration by controlling actin binding proteins to modulate the LINC complex, and, therefore, affect mechanotransduction. These findings will improve our understanding of the functions of PGs, Fascin, and the LINC complex both developmentally and during tumor progression.
Defining the Mediator CDK8 Module in Cardiac Stress
The Mediator complex plays key roles in regulating the transcription of nearly all RNA PolII transcribed genes. Mediator is comprised of four modules: the head, middle, tail (collectively known as the core Mediator), and the CDK8 module which is known to transiently interact with the core Mediator. Classically, it was believed that the composition of Mediator was largely invariant and that the CDK8 module was largely repressive in function. However, recent evidence suggests that during development and cellular differentiation, the composition of core Mediator and CDK8-Mediator changes. This compositional change may account for the large scale transcriptional reprogramming observed in differentiated cells. One component of the CDK8 module is Med13. Cardiac Med13 has been previously shown to regulate cardiac and whole body metabolism, with cardiac expression of Med13 being inversely proportional to susceptibility to metabolic syndromes. In murine cardiomyocytes, the expression of most CDK8 module proteins (including MED13) is decreased following birth. However, recent findings suggest that in cardiomyocytes isolated from mice exposed to cardiac stress, the expression of the CDK8 module proteins is increased. This change in expression may result in a change in composition of CDK8-Mediator, resulting in a modified transcriptome in the cardiomyocytes exposed to cardiac stress. This research aims to define the composition of the CDK8 module as well as components of core Mediator during disease progression and in CDK8 submodule mutants as a means of elucidating the etiology of cardiovascular disease.
Ana Castro’s Abstract
The role of the anti-sigma factor RsiV in lysozyme sensing and stress response
A Castro1, Jessica Hastie1, Craig Ellermeier1
Microbiology Department, College of Medicine, University of Iowa
Bacteria respond to changes in their environment by altering gene expression. Understanding the mechanism by which information is transmitted from outside the cells across the membrane is critical, as it will provide insight into how cells regulate resistance mechanisms. The goal is to gain insight into stress response mechanisms that may contribute to microbial survival in rapidly changing conditions. Extra cytoplasmic function (ECF) s factors are a subset of s factors that allow many organisms to sense and respond to changes in the environment. Most ECF s factors are held in an inactive state by an anti-s factor that prevents the ECF s from interacting with RNA polymerase. Certain envelope stresses in bacteria include the regulated intramembrane proteolysis (RIP), a mechanisms which results in the sequential cleavage of membrane-bound proteins, including s factors. In Bacillus subtilis, the ECF s factor, sV, belongs to the ECF30 subfamily of ECF s factors, members which are primarily found in firmicutes.sV is activated specifically by lysozyme, a critical component of the host innate immune system. sV induces resistance to lysozyme in organisms such as Clostridium difficile. In the absence of lysozyme sV is inhibited by the anti-s factor RsiV. In response to lysozyme, RsiV is cleaved by signal peptidase at site-1 leading to the release and activation of sV. We have shown that RIP dependent mechanism of sV activation is dependent on the anti-s, RsiV, binding to the inducing signal, lysozyme. My project involves using B. subtilis as a model to determine RsiV factors that allow signal peptidase cleavage in the presence of lysozyme. We will determine how σV is important for pathogenesis and virulence in B. subtilis. The objective of this project is to determine the mechanisms by which RsiV binding to lysozyme allow for signal peptidase cleavage at site-1 in the extracellular domain.
Joseph Giacalone’s Abstract
Disease modeling using patient-specific photoreceptor precursor cells
Induced pluripotent stem cell (iPSC)-derived photoreceptor precursor cells can be used to study biological processes and have the potential to restore vision to patients with retinal degenerative diseases like retinitis pigmentosa. Biopsies were obtained from patients with inherited retinal degeneration and fibroblast lines were established. Patient-specific iPSCs were then generated, clonally expanded and validated. Post-mitotic photoreceptor precursor cells were generated using a stepwise 3D differentiation protocol. This method will serve as a platform for studying RPGR-associated XLRP.
Matt Strub’s Abstract
Genomic Signature-Based Approaches to Drug Repositioning for ΔF508-CFTR Rescue
Cystic fibrosis (CF) is a lethal autosomal recessive disease caused by mutations in the CF transmembrane conductance regulator (CFTR) gene. The most common CFTR mutation, termed ΔF508, is a 3 base deletion resulting in the loss of a phenylalanine residue. This causes protein misfolding, resulting in proteosomal degradation. However, if CFTR-ΔF508 is allowed to traffick to the cell membrane, anion channel function may be partially restored. The McCray Lab previously reported that transfection with a miR-138 mimic or siRNA knockdown of SIN3A in primary cultures of CF airway epithelia increases CFTR mRNA and protein levels, and partially restores cAMP-stimulated Cl– conductance. The Connectivity Map (CMAP) is a catalog of gene expression profiles from cultured human cells treated with a variety of bioactive chemical compounds and has pattern-matching software to mine data. A CMAP query using previously generated gene expression signatures identified 27 candidate small molecules that mimicked miR-138 and DsiRNA SIN3A treatments. Our lab reported the identification of 4 small molecules that partially restored CFTR-ΔF508 function in primary CF airway epithelia, highlighting the utility of a genomic signature approach in drug discovery. Currently, the NIH is greatly expanding the CMAP dataset into the Library of Integrated Network-based Cellular Signatures (LINCS). Here we query the LINCS database using a meta-analysis of rescue signatures to identify candidate compounds for rescue of CFTR-ΔF508 function.
In collaboration with the Broad Institute, we used previously generated gene sets to iteratively query the Library of Integrated Network-based Cellular Signatures (LINCS). 125 candidate small molecules were selected for further testing. Functional screens performed in CFBE (ΔF508/ΔF508) cells identified 7/125 compounds that partially rescued ΔF508 function, as assessed by cAMP-activated Cl– conductance. Additional experiments performed to assess their activity in primary human CF epithelial cells confirmed the ability of these seven compounds to partially rescue ΔF508 function. Interestingly, some of these compounds showed significant cooperativity when administered with C18. We then obtained 70 congeners, derivatives, or related compounds of the seven validated LINCS hits and identified 18/70 compounds that increased cAMP-activated chloride conductance by at least 50% compared to a DMSO control. We also tested compounds in the presence of C18 and identified four compounds that increased conductance by at least 40% compared to a C18 control.
We recently used a meta-analytic approach to analyze multiple rescue signatures and relevant pathways to create a prioritized candidate drug list for functional screens. Transcriptomic profiles of RNAi (siRNA knockdown of SIN3A, SYVN1, and NEDD8, and miR-138 overexpression) and temperature (27°C for 24 hours, 40°C for 24 hours, and 27°C for 24 hours followed by 40°C for 24 hours) treatments were used to represent a rescue signature. Profiles of primary cells (both human and pig) from CF and healthy donors were used to represent a disease signature. The rescue and disease signatures were then analyzed together to create representative lists of up- and down-regulated genes. Meanwhile, gene sets were extracted from relevant curated pathways related to CFTR trafficking (from Thomson Reuters MetaCore). Gene sets from both strategies were then scored against all drug profiles contained in the CMAP and LINCS databases. The rescue signature- and disease signature-based scores were combined to form an overall score that was used to identify and prioritize candidate molecules. We have tested 115 compounds from the resulting meta-analysis-based candidate drug list and 36 compounds have been shown to increase cAMP-activated chloride conductance by at least 30% compared to DMSO. Additionally, 21 compounds in the presence of C18 increased conductance by at least 30% compared to a C18 control. Further analysis of preliminary hits will include validation in primary cells. We also have the ability to test the most efficacious drugs in our ΔF508/ΔF508 porcine model. Lastly, we are performing chemogenomic enrichment analysis using the results of all tested compounds to elucidate possible classification, structural, or transcriptomic similarities between efficacious drugs. Such analysis may help us to identify and prioritize additional candidates.
Tricia Braun’s Abstract
Genome-wide DNA methylation analysis of glucocorticoid treatment in human Blood and saliva and the correlation of DNA methylation between peripheral tissues and brain
Patricia Braun1, Yasunori Nagahama2, Marie Hafner1, Lauren O’Sullivan1, Melissa McKane1, Tanner D. Gardiner1, Andrew Grossbach2, Matthew A Howard III2, Hiroto Kawasaki2, James B. Potash1, Gen Shinozaki1
1Department of Psychiatry, University of Iowa Carver College of Medicine
2Department of Neurosurgery, University of Iowa Carver College of Medicine
Glucocorticoids help regulate the stress response, and an imbalance of glucocorticoids has been implicated in depression. Within mouse models, candidate genes have been shown to be differentially methylated in response to glucocorticoid treatment. Using the Infinium HumanMethylation 450K Array, which covers over 450,000 CpGs, we performed preliminary studies on genome-wide DNA methylation (DNAm) changes that occur within saliva samples from 10 subjects and blood samples from 6 subjects before and after treatment with dexamethasone, a corticosteroid, in the context of neurosurgery. Within saliva samples, average DNAm differences of >10% were observed for 84 CpGs. One CpG in a long intergenic non-coding RNA (LINC00871) attained near genome-wide level of significance (average DNAm: pre-dexamethasone 49%, post-dexamethasone 38%; p=5×10-7). Within blood samples, over 200 CpGs had >20% difference; however, none were statistically significant. These findings provide initial evidence for an influence of glucocorticoids on DNAm within humans. To understand the relevance DNAm in saliva and blood to brain DNAm, we also examined the correlation of DNAm between peripheral tissues and brain tissues. This is an essential issue for not only our analysis, but for the field more broadly. We obtained saliva, blood, and live brain tissue samples from 13 patients undergoing neurosurgery and analyzed genome-wide DNAm with the 450K Array. Blood and saliva showed a high degree of correlation for DNAm (r2=0.97), and saliva DNAm was revealed to be more similar to brain DNAm (r2=0.84) than blood (r2=0.81; p<1×10-4). As we have ongoing access to samples from neurosurgery patients, we will expand these studies to understand the extent to which peripheral tissues can be used as surrogate tissues for DNAm in the brain. Furthermore, we will collect samples from oral surgery patients given a higher dosage of glucocorticoids to more fully ascertain the global effects of glucocorticoids on DNAm.
Jessica Ponce’s Abstract
Stephanie Haase’s Abstract
The Social Activities Committee would like to remind you all that the second Genetics Winter Party is THIS FRIDAY, December 11 from 7-10p.m. at the North Ridge Pavilion in Coralville (2250 Holiday Rd.). This will be a BYOB (beer and wine only) and potluck-style event, so we request but do not require that people bring an appetizer or dessert option to share. Additionally, this will be an ugly sweater party, so we would love to see everyone’s most garish, gaudy, and hideous holiday wear. Prizes will be awarded for the ugliest sweaters!
The entrance to the North Ridge Pavilion can be difficult to see in the dark. If you are driving in from the East, you will see a blue street sign on the right side of the road for the pavilion and the turn will be on your left. There is a sign for the pavilion next to a row of pine trees. If you are driving in from the West, you will see a blue street sign on the right side of the road for the pavilion and it will be the next right turn.
Please feel free to contact anyone on the Social Activities Committee with any questions.
We look forward to seeing you there!
The Genetics Social Activities Committee would like to congratulate Dr. Fengxiao Bu on the successful defense of his thesis and completion of his Doctorate!
Bu is from the Smith lab in the Otolaryngology department. His thesis seminar, entitled “Exploring the Genetics of a Complex Disease – Atypical Hemolytic Uremic Syndrome”, was on Friday, December 4th, 2015. Bu has accepted a postdoctoral appointment here at the University of Iowa to continue his research, and we expect that he will continue to excel.
We asked Bu a few questions about his experience in the Genetics program here at Iowa:
Q: What is your favorite thing that you have experienced here at Iowa?
A: The best thing is that I had a great advisors and met so many awesome people here.
Q: What was your favorite class to take?
Q: What was your favorite class to TA?
A: Bioinformatics techniques.
Q: Do you have any advice about preparing for defense?
A: Better to start early. I would say 6 months is a good number for prepare everything.
Again, congratulations Bu and good luck on your future research!
A Targeted Sequencing Study of Glutamatergic Candidate Genes in Attempted Suicide
Suicidal behavior has been shown to have a heritable component that is partly driven by psychiatric disorders. However, there is also an independent factor contributing to the heritability of suicidality. We previously conducted a whole exome sequencing study of bipolar suicide attempters and bipolar non-attempters to assess this independent factor. This whole exome study implicated glutamatergic neurotransmission in attempted suicide, as did our genome-wide association study (GWAS) of the attempted suicide phenotype. In the current study, we have conducted a targeted next-generation sequencing study of the glutamatergic N-methyl-D-aspartate (NMDA) receptor, neurexin, and neuroligin gene families in 476 bipolar suicide attempters and 473 bipolar non-attempters. The goal of this study was to gather sequence information from coding and regulatory regions of these glutamatergic genes to identify variants associated with attempted suicide. We identified 186 coding variants and 4,298 regulatory variants predicted to be functional in these genes. No individual variants were overrepresented in cases or controls to a degree that was statistically significant after correction for multiple testing. Additionally, none of the gene-level results were statistically significant following correction. While this study provides no direct support for a role of the examined glutamatergic candidate genes, further sequencing in expanded gene sets will be required to understand the role of glutamatergic signaling in the risk for suicidal behavior.
Soluble CR1 Gene therapy rescues renal phenotypes in a murine model of C3G
Xue Xiao1,2, Yuzhou Zhang1, Janice Staber3, Sanjeev Sethi5, Paul B. McCray, Jr.2,3,
Carla M. Nester1,3,4, Richard JH Smith1,2,3,4
1Molecular Otolaryngology and Renal Research Laboratories, Caver College of Medicine, University of Iowa, Iowa City, Iowa, USA; 2Interdepartmental PhD Program in Genetics, Carver College of Medicine, University of Iowa, Iowa City, Iowa, USA; 3, 4Departments of Pediatrics and Internal Medicine, Carver College of Medicine, University of Iowa, Iowa City, Iowa, USA; 5Division of Anatomic Pathology, Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, Minnesota, USA
C3 glomerulopathy (C3G) encompasses two prototypical diseases, dense deposit disease (DDD) and C3 glomerulonephritis (C3GN). Both diseases are characterized by fluid-phase dysregulation of the alternative pathway (AP) of complement that leads to C3 deposition in the renal glomerulus. Unknown disease triggers and lacks of specific treatments lead to end-stage renal failure in 50% of patients. Soluble complement receptor 1 (sCR1) is a soluble form of a membrane bound regulator of complement. Short-term studies show that sCR1 is capable of restoring complement control in a murine model of C3G, the Cfh-/-/huCR1-Tg mouse. However, within days of terminating treatment, complement dysregulation is again evident. In this study, we sought to determine whether continuous presence of sCR1 could provide long-term complement control in the C3G murine model. Using the piggyBac transposon system coupled with hydrodynamic tail vein injection, we delivered a construct of sCR1 (LHR A-C) to the C3G murine model to provide constitutive sCR1 expression in mouse circulation. Animals were followed for 6 months. sCR1 expression was detected by real time PCR and ELISA in mouse liver and circulation respectively in 6 months. C3 levels approximately doubled and clearance of glomerular C3 and C3 fragments deposition was documented by immunofluorescence. Electronic microscopy showed a reduction in dense deposits in injected as compared to control animals. There were no changes by light microscopy. Renal function improvements had been revealed from stabilized 24-hr urine albumin creatinine ratio. In this study, we proved that long-term expression of sCR1 could rescue the renal phenotype in C3G mice and may be a viable treatment for patients with this disease.