Knockout Animal Models Research Articles

275 Permanent Alteration of Abcc6 With In Vivo CRISPR-Cas9 Genome Editing

Pseudoxanthoma elasticum (PXE), a heritable ectopic mineralization disorder, is caused by mutations in the Abcc6 gene primarily expressed in the liver and the kidneys. The fundamental question on the pathogenesis of PXE, whether lack of Abcc6 expression in liver or kidney is the primary site of molecular pathology in peripheral tissues, has not been addressed. Previous studies have used a systemic knockout to construct mice or zebrafish model, these animal models do not simulate the onset of PXE very well. Specific knock-out of the Abcc6 gene in the liver may better mimic the onset of PXE. Therefore, it is imperative to construct an animal model that specifically knocks out the ABCC6 gene in the liver. The goal of this study was to construct a liver-specific knockout animal model of Abcc6 gene, aiming to better simulate the pathogenesis of PXE and provide new ideas for its pathogenesis research and treatment. Our previous study found a new PXE site in Chinese, so we designed sgRNA at the same sites in mice and constructed a CRISPR/Cas9 plasmid co-expressing lentivirus. Then we used tail vein injection to achieve knock-out Abcc6 gene in the liver. We found that 1 month of administration of the virus, the mutagenesis of Abcc6 in the liver was found. We also found that 6 months of administration of the virus, pyrophosphate (PPi) level was reduced in the knock-out mice and depicted ectopic mineralization in the dermal sheath of vibrissae in muzzle skin. Gene editing with the CRISPR/Cas9 system disrupts the Abcc6 gene in vivo with high efficiency and reduces PPi levels in mice, and depicts ectopic mineralization in the skin. Specific knock-out of the Abcc6 gene in the liver may better mimic the onset of PXE. This approach may have therapeutic potential for the prevention of PXE in humans.

Characterization and Proteomic-Transcriptomic Investigation of Monocarboxylate Transporter 6 Knockout Mice: Evidence of a Potential Role in Glucose and Lipid Metabolism.

Monocarboxylate transporter 6 [(MCT6), SLC16A5] is an orphan transporter with no known endogenous substrates or physiological role. Previous in vitro and in vivo experiments investigated MCT6 substrate/inhibitor specificity in Xenopus laevis oocytes; however, these data remain limited. Transcriptomic changes in the livers of mice undergoing different dieting schemes have suggested that Mct6 plays a role in glucose and lipid metabolism. The objectives of this study were 1) to develop a novel knockout (KO) mouse model (Mct6-/-) using CRISPR/Cas9 technology, 2) to characterize the KO animal model by examining physiological and biochemical parameters, and 3) to understand the physiological role of MCT6 in vivo through global proteomic and liver transcriptomic profiling. mRNA tissue analysis demonstrated knockout of Mct6, which showed greater than 90% knockdown of Mct6 (Slc16a5) gene expression in all major tissues analyzed when normalized to Mct6+/+ mice. Proteomic analyses identified greater than 4000 unique proteins in kidney, liver, and colon tissues, among which 51, 38, and 241 proteins were significantly altered, respectively (for each tissue), between Mct6+/+ and Mct6-/- mice. Additionally, Mct6-/- mice demonstrated significant changes in 199 genes in the liver compared with Mct6+/+ mice. In silico biological pathway analyses revealed significant changes in proteins and genes involved in glucose and lipid metabolism-associated pathways. This study is the first to provide evidence for an association of Mct6 in the regulation of glucose and lipid metabolism. SIGNIFICANCE STATEMENT: This paper focuses on elucidating the innate biological role of an orphan transporter in vivo, which has not been investigated thus far. Using efficient and high-throughput technologies, such as CRISPR/Cas9 gene editing, liquid chromatography-tandem mass spectrometry-based proteomic and RNA-sequencing transcriptomic analyses, our laboratory provides the first existence and characterization of a Mct6 knockout mouse model. The evidence gathered in this paper, as well as other laboratories, support the importance of MCT6 in regulating a variety of glucose and lipid metabolic pathways, which may indicate its significance in metabolic diseases.

Abstract LB-112: Unraveling biology and identifying targets with functional genomics approaches using lentiarray crispr libraries

Abstract The ability of CRISPR/Cas9, to efficiently and precisely edit a cell’s DNA and introduce a complete genetic knockout, while minimizing off-target effects, offers an improved approach to target identification. Identifying and validating targets that underlie disease mechanisms still remains a significant challenge in the drug discovery and development process. The transcription factor NF-κB is a crucial player in many aspects of cancer initiation and progression. Here we demonstrate a knock-out screening approach that utilizes an arrayed CRISPR library to interrogate the impact of individual gene knock-outs on the NF-κB pathway in a cervical carcinoma cell line as measured by a functional cell-based assay. We describe the library design concepts, the assay development, screening results and validation of specific identified hits. We tested the approach using a library that targets the human kinome and developed a loss-of-function assay using CellSensor™ NF-κB-bla ME-180 cell line, which stably expresses Cas9. The CellSensor™ reporter assay enables easy identification of genomic targets associated with the NF-κB pathway. We elucidate the key factors in developing a robust assay including both transduction and assay optimization to achieve the highest levels of transduction efficiency and assay window. Using the optimized parameters, we screened the LentiArray CRISPR human kinome library that targets >800 kinases and demonstrate how we followed-up on and validated a subset of the identified hits. The hits were further validated by confirming nucleotide(s) deletion/ insertion in target DNA and additional screening using target specific siRNA and chemical compounds. The ability to scale this approach through the generation of genome wide CRISPR libraries, coupled with the use of lentiviral delivery methods enables high-throughput (HTP) loss-of-function screens to be performed rapidly and identify genes whose activity is important for the specific endpoint being measured. These screenings can provide a wealth of data on the normal functioning of a target and in turn, should yield better validated targets for progression into full drug discovery. Ultimately, the hits from the HTP screenings can be followed up by using CRISPR technology to generate animal knockout models that would support translating the screen to the pre-clinical trials. This in turn could provide better correlation to the clinical setting and thereby reduce candidate drug attrition. Citation Format: Chetana M. Revankar, Julia Braun, Jian-Ping Yang, Justin Wetter, Tufan Gokirmak, Namritha Ravinder, Jon Chesnut, David Piper. Unraveling biology and identifying targets with functional genomics approaches using lentiarray crispr libraries [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-112.

Carnosine Essential For Cardiac Function. A Study With Knockout Rats For The Carnosine Synthase Gene

Carnosine is present in high concentrations in heart, where it appears to increase the sensitivity of the contractile apparatus to Ca2+. However, it is currently unknown whether this role is relevant to the cardiac physiology. PURPOSE: To evaluate the impact of the lack of carnosine on myocardial contractile function in rats knockout (KO) for the CARNS1 gene (carnosine synthase1). METHODS: We developed the first KO animal model for the CARNS1 gene through CRISPR-Cas9 technology. Male wild-type (WT) and KO rats (4 months-old) were used. In vivo cardiac function was assessed by echocardiography (ECO) and cardiac electrical activity by electrocardiography (ELECTRO). Cardiomyocyte contractile function was assessed in isolated cardiomyocytes by measuring cardiomyocyte and sarcomere contractility analysis, along with the determination of Ca2+ transient. Unpaired t-tests were used to compare variables between WT and KO. The study was approved by the Ethics Committee on the Use of Animals of USP. RESULTS: ECO (WT:n=4; KO:n=6) showed that KO rats presented a higher systolic diameter (WT:0.008±0.001mm/g; KO:0.011±0.001mm/g; p=0.002), lower Fraction of Left Ventricular Ejection (WT:80.26±6.86%; KO:69.86±4.67%; p=0.01) and lower Fraction of Left Ventricular Shortening (WT:51.12±7.39%; KO:40.88±3.92%; p=0.01), characterizing systolic dysfunction. ELECTRO (WT: n=8; KO:n=8) showed higher T wave amplitude in the KO group (WT:0.06±0.02mV; KO:0.13±0.03mV; p=0.005), indicating a disturbance in the Ca2+ channels. In vitro contractility data (WT: n=3; KO:n=3) showed that sarcomere shortening is reduced in the KO (WT:11.75±4.20%; KO:9.28±3.71%) (WT:6.81±3.15s; KO:4.33±1.87s ; p<0.0001), and the time to reach 50% of maximal shortening is longer in the KO (WT:0.04±0.01s; KO:0.05±0.01s; p<0.0001). Ca2+ transient analysis showed lower amplitude of Ca2+ in the KO group (WT:0.20±0.10; KO:0.16±0.06 F340/380 ratio; p=0.001) and longer time for to reach 50% of the Ca2+ decay rate (WT:0.21±0.04s; KO:0.24±0.03s; p=0.001). CONCLUSION: Absence of carnosine resulted in systolic dysfunction associated with Ca2+ transient changes in cardiac muscle. This is the first evidence to demonstrate in vivo, ex vivo and in vitro that carnosine is essential for normal cardiac function. Supported by FAPESP 2014/11948-8 and CAPES

Matrix metalloproteinases and inhibitors in dentistry.

Matrix metalloproteinase (MMP) expression has been associated with tissue development, invasive cancer cell behavior, and inflammation. The associations of increased expression of MMPs with diseases have led to intensive research activities to develop MMP inhibitors. Here, the questions are addressed which associations between increased levels of any MMP with dental diseases may be cause or consequence, whether MMP levels may be of diagnostic value and whether and which MMP inhibitors need further investigations for use in dental diseases. To study the role of MMPs and to discriminate between cause or consequence, the literature about measurements of MMPs and about the use of inhibitory drugs and genetic knockout animal models in dentistry was compared. The only FDA-approved treatment with MMP inhibitors is tetracyclines for periodontitis, whereas a diagnostic test for activated MMP-8 in oral fluids is valued in practical periodontology. The MMP literature in dentistry is artificially skewed to the gelatinases MMP-2 and MMP-9 and to enamelysin, alias MMP-20. The basis for this observation is, respectively, the widely used and sensitive technique of gelatin zymography and enamel proteins as substrates of MMP-20. Studies on additional MMPs are gaining interest in dentistry and MMP inhibitors may provide new applications. In addition, drugs with proven effects for the treatment of dental diseases may be found to act through MMP inhibition. In conclusion, research on MMPs and inhibitors may provide practical applications beyond diagnosis and treatment of periodontitis and will be, directly or indirectly, beneficial for patients with dental or periodontal diseases.

MON-100 Unique miRNA Signature in HIV Lipodystrophy with Reduced Adipose Dicer Expression

Suppression of Dicer, an endoribonuclease that regulates microRNAs(miRNA), has evolved as a viral mechanism to enhance host HIV infectivity and may have unintended metabolic consequences. Animal knockout models of adipose-specific dicer(ADicer) acquire lipodystrophy accompanied by severe metabolic abnormalities. Data show adipose is a source of exosomal miRNAs, functioning as adipokines influencing metabolic homeostasis. We hypothesized a unique miRNA profile among individuals well-phenotyped for HIV lipodystrophy and reduced ADicer expression. We evaluated >1000 miRNAs from exosomes derived from sera among the 27 male individuals [9 HIV lipodystrophy(HIV/lipo), 9 HIV without lipodystrophy(HIV/non-lipo), 9 non-HIV] whom we previously showed variations in ADicer: most suppressed among HIV lipo, followed by HIV non-lipo and non-HIV (2.49[0.02,4.88] vs. 11.20[4.83,21.45] vs. 17.69[10.72,47.91],P=.002). To estimate miRNA abundance, data was normalized using the geomean of expressed Ct values. A false discovery rate analysis(FDR) was applied. We investigated whether significant exosomal miRNAs demonstrated a similar pattern in corresponding subcutaneous adipose tissue(SAT) samples. Using target prediction databases (TargetScan, miRDB, Diana), we identified genes related to fat biology (target score ≥85%) with clinical relevance to lipodystrophic phenotypes and further evaluated these in the adipose depot. HIV/lipo individuals(age 56±3years, BMI 30±1kg/m2, duration HIV 24±2years, duration ART 20±2years, CD4+ count 482±90cells/μl, undetectable VL 67%) were similar to HIV/non-lipo(age 52±3 years, BMI 30±1 kg/m2) and non-HIV(age 55±3years, BMI 30±1kg/m2) individuals. Reduced ADicer expression was significantly related to reduced CD4+ count(r=0.55,P=.02), duration ART use(r=-0.70,P=.001) and tended to be related to duration HIV(r=-0.44,P=.07) and reduced CD8+ count(r=0.42,P=.08). Applying FDR, we detected exosomal miRNA-20a-3p(P=.0026), 324-5p(P=.0059), and 186-5p(P=.0977) were expressed differentially in HIV/lipo vs. non-HIV and 324-5p(P=.0348) in HIV/lipo vs. HIV/non-lipo. We further confirmed that miRNA-20a-3p was significantly and differentially expressed in the SAT, expressed highest in HIV lipo followed by HIV non-lipo and non-HIV [9.73±12.38 vs. 2.24±1.84 vs. 1.02±0.19, P<0.05] in a pattern similar to the exosomal profiles. Relevant miRNA and their target genes which tended to be differentially expressed in the SAT of HIV lipo vs. HIV non-lipo include: 20a-3p (TDG, P=0.07) and 186-5p (PDE10A, PID1; P<0.05). Altered ADicer expression and specific exosomal miRNAs may affect gene expression of regulators important to fat biology and metabolic homeostasis in HIV. In particular, miRNA-20a-3p may function as an adipokine demonstrating similar expression profiles in exosomes and adipose tissue of individuals with HIV lipodystrophy.

Implications of Genetic Manipulation of Caveolin‐1 Protein Expression in a Mouse Model of Marfan Syndrome‐Associated Aortic Aneurysm

Marfan Syndrome (MFS), a connective tissue disorder, resulting from mutations in the Fibrillin‐1 gene, is associated with several clinical manifestations with the most life‐threatening being aortic aneurysm, dissection, and rupture. The mechanism underlying MFS pathogenesis seems to result from crosstalk between the Angiotensin‐II (AngII) pathway and over activation of transforming growth factor‐beta (TGF‐b) signaling. Studies show that Losartan, an angiotensin II receptor type I (ATRI) blocker, can block progression of aortic aneurysm in mice, partially due to its inhibitory effects on TGF‐b signaling. It has been established that caveolin‐1 (Cav1), a coat protein of caveolae that is highly expressed in endothelial and smooth muscle, regulates AngII and TGF‐β signaling pathways, through its interactions with ATR1 and TGF‐β receptors. Interestingly, Cav1 knockout (Cav1KO) animal models illustrated increased elastin synthesis and nitric oxide (NO) production. Our previous study in the MFS mouse model reported reduced NO production in the aortic wall, proposing a potential link between Cav‐1 activation and aneurysm progression in MFS mice. Hence, in this study, we aimed to investigate the effects of genetic manipulation of Cav1 expression on the progression of aortic aneurysm in a well‐established mouse model of MFS‐associated aortic aneurysm, by generating MFS mice lacking Cav1 expression (MFS/Cav1KO).In vivo analysis of biophysical properties in 3‐ and 6‐ month old wild type, MFS, Cav1KO, and MFS/Cav1KO mice was performed using Vevo 2100 high‐resolution ultrasound imaging system (FUJIFILM VisualSonics). Aortic diameters at the aortic annulus, sinus of Valsalva, and sinotubular junction were significantly increased in MFS and MFS/CAV1KO mice as compared to control and Cav1KO groups at 3, and 6 months in systole and diastole. Pulse wave velocity (PWV), a reliable indicator of aortic wall stiffness, was significantly increased in MFS as compared to control and Cav1KO mice, with no significant changes observed between MFS and MFS/Cav1KO groups at 3 months of age. Interestingly, at 6 months of age, MFS/Cav1KO mice presented higher values for PWV as compared to MFS groups, indicating that deletion of Cav1 gene in MFS mice may have detrimental effects on aortic wall structure as MFS mice age.Cardiac parameters were also measured. Our data showed that at 3 months of age, a marked increase in heart rate, ejection fraction, and fractional shortening were detectable in MFS mice as compared to control subjects. Measurements of cardiac output and stroke volume revealed no significant differences among experimental groups. Interestingly, left ventricular mass was significantly greater in MFS/CAV1KO compared to wild type and MFS mice, suggesting that Cav1 plays a protective role in maintaining normal cardiac structure in MFS mice. At this point, our collected data suggest that Cav1 may have some protective effects on aortic and cardiac structure and function during the development of aortic aneurysm in the mouse model of MFS.Support or Funding InformationThis study was funded by The Marfan Foundation (M.E.), and a Midwestern University Graduate Fund (T.C.).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Neurodevelopmental Disorders: Functional Role of Ambra1 in Autism and Schizophrenia.

The activating molecule in Beclin-1-regulated autophagy (Ambra1) is a highly intrinsically disordered protein best known for its role as a mediator in autophagy, by favoring the formation of autophagosomes. Additional studies have revealed that Ambra1 is able to coordinate cell responses to stress conditions such as starvation, and it actively participates in cell proliferation, cytoskeletal modification, apoptosis, mitochondria removal, and cell cycle downregulation. All these functions highlight the importance of Ambra1 in crucial physiological events, including metabolism, cell death, and cell division. Importantly, Ambra1 is also crucial for proper embryonic development, and its complete absence in knock-out animal models leads to severe brain morphology defects. In line with this, it has recently been implicated in neurodevelopmental disorders affecting humans, particularly autism spectrum disorders and schizophrenia. Here, we discuss the recent links between Ambra1 and neurodevelopment, particularly focusing on its role during the maturation of hippocampal parvalbumin interneurons and its importance for maintaining a proper excitation/inhibition balance in the brain.

Interleukin-2 and the Septohippocampal System: An Update on Intrinsic Actions and Autoimmune Processes Relevant to Neuropsychiatric Disorders.

Recent research suggests that lymphocytes can secrete classic neuropeptides, whereas peripheral immunization may signal hypothalamic neuronal cells. These results have led to more analysis of the function of cytokines as modulators of the peripheral and central nervous systems. In the past, the role of brain cytokines was thought to be a mere redundancy of their activity within the peripheral immune system. Nevertheless, it is currently appreciated that central nervous system (CNS) cytokines have selective effects on neuronal cells. Furthermore, recent research has revealed the involvement of various cytokines in the pathophysiologic processes of neurologic and neuropsychiatric diseases. Yet, despite a plethora of published literature, most of this clinical knowledge remains correlative, and much of the basic research has understandably relied on in vitro experimental designs. However, animal knockout models have provided valuable insight into the complex biology of cytokines, mainly of interleukin-2 (IL-2). Indeed, research has tried to unveil the effects of IL-2 on the septohippocampal system and its associated pathways that regulate learning, memory, and other processes. In this chapter, we provide a comprehensive summary of the studies investigating the role of intrinsic and extrinsic IL-2 in the CNS, particularly at the level of the septohippocampal system. We also discuss the function of other cytokines in this system and propose possible clinical correlates.

Gene Knockdown in Zebrafish (Danio rerio) as a Tool to Model Photoreceptor Diseases.

Disturbances in the general mRNA metabolism have been recognized as a major defect in a growing number of hereditary human diseases. One prominent example of this disease group is retinitis pigmentosa (RP), characterized by selective loss of photoreceptor cells. RP can be caused by dominant mutations in key factors of the pre-mRNA processing spliceosome. In these cases, the complex events leading to the RP phenotype can only insufficiently be analyzed in animal knockout models due to the essential functions of splice factors. Furthermore knockout animals frequently miss the specific phenotypes caused by knockdown of the respective genes. Here we introduce the zebrafish Danio rerio as a valuable vertebrate model system to study RP and related diseases in knockdown case scenarios.

Hydrogen sulfide and hepatic lipid metabolism - a critical pairing for liver health.

Hydrogen sulfide (H2 S) is the most recently recognized gasotransmitter, influencing a wide range of physiological processes. As a critical regulator of metabolism, H2 S has been suggested to be involved in the pathology of many diseases, particularly obesity, diabetes and cardiovascular disorders. Its involvement in liver health has been brought to light more recently, particularly through knockout animal models, which show severe hepatic lipid accumulation upon ablation of H2 S metabolic pathways. A complex relationship between H2 S and lipid metabolism in the liver is emerging, which has significant implications for liver disease establishment and/or progression, regardless of the disease-causing agent. In this review, we discuss the critical importance of H2 S in hepatic lipid metabolism. We then describe the animal models so far related with H2 S and lipid-associated liver disease, as well as H2 S-based treatments available. Finally, we highlight important considerations for future studies and identify areas in which much still remains to be determined. LINKED ARTICLES: This article is part of a themed section on Hydrogen Sulfide in Biology & Medicine. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v177.4/issuetoc.

Research progress on composite animal models of inflammatory bowel disease based on gene knockout

Establishing a suitable animal model is important for studying the mechanism of inflammatory bowel disease (IBD) and exploring new therapeutic approaches. Although a large number of IBD single gene knockout animal models have been established, single knockout of certain genes associated with human IBD susceptibility does not manifest symptoms of IBD or manifest extremely milder symptoms, while composite animal models based on other modeling factors can better simulate the clinical features of IBD. This article mainly introduces three novel composite animal models and elaborates the possible pathogenesis of each composite model:animal models established by gene double knockout have more obvious and earlier symptoms than single-knockout models; single gene knockout model with Helicobacter infection can help to study the role of microbial infections in the pathogenesis of IBD; on the basis of gene knockout, specific deletion of certain immune cells can be used to study the role of the immune cells in the development of IBD. Among the above composite animal models, Muc2/IL-10 double knockout mice may be important animal models for IBD study.

Genetic Alterations in Oxidant and Anti-Oxidant Enzymes in the Vascular System.

Cardiovascular diseases (CVD) are one of the prime causes of mortality worldwide. Experimental animal models have become a valuable tool to investigate and further advance our knowledge on etiology, pathophysiology and intervention. They also provide a great opportunity to understand the contribution of different genes and effector molecules in the pathogenesis and development of diseases at the sub-cellular levels. High levels of reactive oxygen species (ROS) have been associated with the progression of CVD such as ischemic heart disease (IHD), myocardial infarction, hypertension, atherosclerosis, aortic aneurysm, aortic dissection and others. On the contrary, low levels of antioxidants were associated with exacerbated cardiovascular event. Major focus of this review is on vascular pathogenesis that leads to CVD, with special emphasis on the roles of oxidant/antioxidant enzymes in health and disease progression in vascular cells including vascular endothelium. The major oxidant enzymes that have been implicated with the progression of CVD include NADPH Oxidase, nitric oxide synthase, monoamine oxidase, and xanthine oxidoreductase. The major antioxidant enzymes that have been attributed to normalizing the levels of oxidative stress include superoxide dismutases, catalase and glutathione peroxidases (GPx), and thioredoxin. Cardiovascular phenotypes of major oxidants and antioxidants knockout and transgenic animal models are discussed here.

Construction of EZH2 Knockout Animal Model by CRISPR/Cas9 Technology

背景与目的已有的研究证明CRISPR/Cas9（Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated 9）系统是一种能够在哺乳动物细胞中高效操作的新型基因编辑技术，应用人工设计的向导RNA（single-guide RNA, sgRNA）介导外源表达的Cas9蛋白与靶点DNA特异性结合以实现对基因组DNA的切割，被切割后的基因组DNA通过非同源重组或同源重组的方式进行修复，从而实现基因的敲除、或者外源基因的敲入等目标。本研究的目的是应用CRISPR/Cas9技术构建小鼠肺部EZH2基因敲除的动物模型。方法针对EZH2基因的编码区，设计两个靶向EZH2基因Exon3和Exon4的sgRNA，通过慢病毒包装、感染细胞、SURVEYOR assay等一系列体外实验，验证所设计的sgRNA的有效性。应用支气管插管的方式把慢病毒灌注到小鼠肺部，利用免疫组化方法和qRT-PCR进行检测。结果NIH-3T3细胞的体外实验结果验证了实验所设计的sgEZH2能够有效地在体外细胞系中介导Cas9切割靶DNA；小鼠支气管插管实验及肺部组织免疫组化和qRT-PCR方法检测EZH2基因敲除的效率，发现实验组小鼠肺部组织EZH2表达明显降低。结论本研究成功设计了两条能够敲除EZH2功能的sgRNA，并应用CRISPR/Cas9技术成功建立了肺部EZH2基因敲除的小鼠模型，为研究EZH2的功能和作用机制提供了有效的动物模型。

A Potential Compensatory Role of Panx3 in the VNO of a Panx1 Knock Out Mouse Model.

Pannexins (Panx) are integral membrane proteins, with Panx1 being the best-characterized member of the protein family. Panx1 is implicated in sensory processing, and knockout (KO) animal models have become the primary tool to investigate the role(s) of Panx1 in sensory systems. Extending previous work from our group on primary olfaction, the expression patterns of Panxs in the vomeronasal organ (VNO), an auxiliary olfactory sense organ with a role in reproduction and social behavior, were compared. Using qRT-PCR and Immunohistochemistry (IHC), we confirmed the loss of Panx1, found similar Panx2 expression levels in both models, and a significant upregulation of Panx3 in mice with a global ablation of Panx1. Specifically, Panx3 showed upregulated expression in nerve fibers of the non-sensory epithelial layer in juvenile and adult KO mice and in the sensory layer of adults, which overlaps with Panx1 expression areas in WT populations. Since both social behavior and evoked ATP release in the VNO was not compromised in KO animals, we hypothesized that Panx3 could compensate for the loss of Panx1. This led us to compare Panx1 and Panx3 channels in vitro, demonstrating similar dye uptake and ATP release properties. Outcomes of this study strongly suggest that Panx3 may functionally compensate for the loss of Panx1 in the VNO of the olfactory system, ensuring sustained chemosensory processing. This finding extends previous reports on the upregulation of Panx3 in arterial walls and the skin of Panx1 KO mice, suggesting that roles of Panx1 warrant uncharacterized safeguarding mechanisms involving Panx3.

MALAT1: An Epigenetic Regulator of Inflammation in Diabetic Retinopathy

Despite possessing limited protein-coding potential, long non-coding RNAs (lncRNAs) have been implicated in a myriad of pathologic conditions. Most well documented in cancer, one prominent intergenic lncRNA known as MALAT1 is notorious for its role in impacting epigenetic mechanisms. In this study, we established a novel epigenetic paradigm for MALAT in diabetic retinopathy (DR) by employing siRNA-mediated MALAT1 knockdown in human retinal endothelial cells (HRECs), a Malat1 knockout animal model, vitreous humor from diabetic patients, pharmacological inhibitors for histone and DNA methylation, RNA immunoprecipitation, western blotting, and a unique DNA methylation array to determine glucose-related alterations in MALAT1. Our findings indicated that MALAT1 is capable of impacting the expressions of inflammatory transcripts through its association with components of the PRC2 complex in diabetes. Furthermore, the vitreous humors from diabetic patients revealed increased expressions of MALAT1, TNF-α, and IL-6. Intriguingly, our DNA methylation array demonstrated that transient high glucose exposure in HRECs does not contribute to significant methylation alterations at CpG sites across the MALAT1 gene. However, global inhibition of DNA methyltransferases induced significant increases in MALAT1 and associated inflammatory transcripts in HRECs. Our findings collectively demonstrate the importance of MALAT1 in inflammation and epigenetic regulation in DR.

Randomised controlled trial of simvastatin treatment for autism in young children with neurofibromatosis type 1 (SANTA).

BackgroundNeurofibromatosis 1 (NF1) is a monogenic model for syndromic autism. Statins rescue the social and cognitive phenotype in animal knockout models, but translational trials with subjects > 8 years using cognition/behaviour outcomes have shown mixed results. This trial breaks new ground by studying statin effects for the first time in younger children with NF1 and co-morbid autism and by using multiparametric imaging outcomes.MethodsA single-site triple-blind RCT of simvastatin vs. placebo was done. Assessment (baseline and 12-week endpoint) included peripheral MAPK assay, awake magnetic resonance imaging spectroscopy (MRS; GABA and glutamate+glutamine (Glx)), arterial spin labelling (ASL), apparent diffusion coefficient (ADC), resting state functional MRI, and autism behavioural outcomes (Aberrant Behaviour Checklist and Clinical Global Impression).ResultsThirty subjects had a mean age of 8.1 years (SD 1.8). Simvastatin was well tolerated. The amount of imaging data varied by test. Simvastatin treatment was associated with (i) increased frontal white matter MRS GABA (t(12) = − 2.12, p = .055), GABA/Glx ratio (t(12) = − 2.78, p = .016), and reduced grey nuclei Glx (ANCOVA p < 0.05, Mann-Whitney p < 0.01); (ii) increased ASL perfusion in ventral diencephalon (Mann-Whitney p < 0.01); and (iii) decreased ADC in cingulate gyrus (Mann-Whitney p < 0.01). Machine-learning classification of imaging outcomes achieved 79% (p < .05) accuracy differentiating groups at endpoint against chance level (64%, p = 0.25) at baseline. Three of 12 (25%) simvastatin cases compared to none in placebo met ‘clinical responder’ criteria for behavioural outcome.ConclusionsWe show feasibility of peripheral MAPK assay and autism symptom measurement, but the study was not powered to test effectiveness. Multiparametric imaging suggests possible simvastatin effects in brain areas previously associated with NF1 pathophysiology and the social brain network.Trial registrationEU Clinical Trial Register (EudraCT) 2012-005742-38 (www.clinicaltrialsregister.eu)

Conditional ablation of the RFX4 isoform 1 transcription factor: Allele dosage effects on brain phenotype.

Regulatory factor X4 (RFX4) isoform 1 is a recently discovered isoform of the winged helix transcription factor RFX4, which can bind to X-box consensus sequences that are enriched in the promoters of cilia-related genes. Early insertional mutagenesis studies in mice first identified this isoform, and demonstrated that it was crucial for mouse brain development. RFX4 isoform 1 is the only RFX4 isoform significantly expressed in the mouse fetal and adult brain. In this study, we evaluated conditional knock-out (KO) mice in which one or two floxed alleles of Rfx4 were deleted early in development through the use of a Sox2-Cre transgene. Heterozygous deletion of Rfx4 resulted in severe, non-communicating congenital hydrocephalus associated with hypoplasia of the subcommissural organ. Homozygous deletion of Rfx4 resulted in formation of a single ventricle in the forebrain, and severe dorsoventral patterning defects in the telencephalon and midbrain at embryonic day 12.5, a collection of phenotypes that resembled human holoprosencephaly. No anatomical abnormalities were noted outside the brain in either case. At the molecular level, transcripts encoded by the cilia-related gene Foxj1 were significantly decreased, and Foxj1 was identified as a direct gene target of RFX4 isoform 1. The phenotypes were similar to those observed in the previous Rfx4 insertional mutagenesis studies. Thus, we provide a novel conditional KO animal model in which to investigate the downstream genes directly and/or indirectly regulated by RFX4 isoform 1. This model could provide new insights into the pathogenesis of obstructive hydrocephalus and holoprosencephaly in humans, both relatively common and disabling birth defects.

The role of toll-like receptors in diabetic kidney disease.

Involved in innate immunity, toll-like receptors (TLRs) recognize pathogenic and endogenous ligands. Ligand binding initiates an inflammatory cascade which if sustained leads to fibrosis. This review summarizes the role of TLRs in diabetic kidney disease (DKD) with particular emphasis on TLR2 and TLR4. Collectively, preclinical evidence to date supports the causative role of TLR2 and TLR4 in both type I and type II DKD. The relative importance of each is still unclear. In experimental models, there are increased TLR2 and TLR4 ligands, expression and signalling. Functional studies using inhibitors or knockout animal models confirm causality. Clinical evidence also supports increased ligands and TLR2 and TLR4 expression in diabetes however there are no clinical studies examining whether interruption of these pathways confer renoprotection. Preclinical evidence to date supports the role of TLR2 and TLR4 in DKD. It will be useful to examine the value of interrupting these signalling pathways in clinical trials.

Methods to Investigate the Molecular Basis of Progranulin Actions on Brain and Behavior In Vivo Using Knockout Mice.

Currently one of the few molecules that equally excites a neuroscientist, a cancer biologist, an immunologist, and a developmental biologist is progranulin (GRN/Grn)-a pluripotent growth factor that plays key roles in cell survival, proliferation, development, tissue regeneration, inflammation, wound healing, and angiogenesis. However, the molecular pathways associated with GRN signaling involved in these varied physiological processes are not understood. Gene inactivation has been considered as one of the best methods to delineate the biological role of a protein, and gene targeting is a direct means to disrupt a gene's open reading frame and block its expression, for instance, in a mouse. Such a gene knockout animal model also served as an in vivo disease model where loss of gene or its function is thought to be the primary disease mechanism, as is the case with progranulin loss of function in frontotemporal lobar degeneration (FTLD). It is estimated that up to half of the cases of familial, dominant FTLD might be due to GRN haploinsufficiency. To understand the molecular pathways associated with GRN loss, constitutive and conditional progranulin knockout (Grn-/-) mice have also been constructed in several laboratories, including ours. These mice show several disease-characteristic features and suggest that continued studies on the Grn-/- mice would be instructive in the understanding of complex GRN biology in health and disease.