Genetics
Abstract
The retinal pigment epithelium (RPE) provides vital metabolic support for retinal photoreceptor cells and also is an important player in numerous retinal diseases. Gene manipulation in mice using the Cre-LoxP system is an invaluable tool for studying the genetic basis of these retinal diseases. However, existing RPE-targeted Cre mouse lines have critical limitations that restrict their reliability for studies of disease pathogenesis and treatment, including mosaic Cre expression, inducer-independent activity, off-target Cre expression, and intrinsic toxicity. Here, we report the generation and characterization of a knock-in mouse line in which a P2A-CreERT2 coding sequence is fused with the native RPE-specific 65 kDa protein (Rpe65) gene for co-translational expression of CreERT2. Cre+/- mice were able to recombine a stringent Cre reporter allele with >99% efficiency and absolute RPE specificity upon tamoxifen induction at both post-natal days (PD) 21 and 50. Tamoxifen-independent Cre activity was negligible at PD64. Moreover, tamoxifen-treated Cre+/- mice displayed no signs of structural or functional retinal pathology up to 4 months of age. Despite weak RPE65 expression from the knock-in allele, visual cycle function was normal in Cre+/- mice. These data indicate that Rpe65CreERT2 mice are well-suited for studies of gene function and pathophysiology in the RPE.
Authors
Elliot H. Choi, Susie Suh, David E. Einstein, Henri Leinonen, Zhiqian Dong, Sriganesh Ramachandra Rao, Steven J. Fliesler, Seth Blackshaw, Minzhong Yu, Neal S. Peachey, Krzysztof Palczewski, Philip D. Kiser
Abstract
One of the most common malignancies affecting adults with Neurofibromatosis type 1 (NF1) is the malignant peripheral nerve sheath tumor (MPNST), an aggressive and often fatal sarcoma which commonly arises from benign plexiform neurofibromas. Despite advances in our understanding of MPNST pathobiology, there are few effective therapeutic options, and no investigational agents have proven success in clinical trials. To further understand the genomic heterogeneity of MPNST, and to generate a preclinical platform that encompasses this heterogeneity, we developed a collection of NF1-MPNST patient-derived xenografts (PDX). These PDX were compared to the primary tumors from which they were derived using copy number analysis, whole-exome and RNA sequencing. We identified chromosome 8 gain as a recurrent genomic event in MPNST and validated its occurrence by FISH in the PDX and parental tumors, in a validation cohort, and by single cell sequencing in the PDX. Finally, we show that chromosome 8 gain is associated with inferior overall survival in soft tissue sarcomas. Taken together, these data suggest that chromosome 8 gain is a critical event in MPNST pathogenesis, and may account for the aggressive nature and poor outcomes in this sarcoma subtype.
Authors
Carina A. Dehner, Chang In Moon, Xiyuan Zhang, Zhaohe Zhou, Christopher A. Miller, Hua Xu, Xiaodan Wan, Kuangying Yang, R. Jay Mashl, Sara J.C. Gosline, Yuxi Wang, Xiaochun Zhang, Abigail Godec, Paul A. Jones, Sonika Dahiya, Himanshi Bhatia, Tina Primeau, Shunqiang Li, Kai Pollard, Fausto J. Rodriguez, Li Ding, Christine A. Pratilas, Jack F. Shern, Angela C. Hirbe
Abstract
DNA methylation (DNAm) has been shown to play a role in mediating food allergy, however, the mechanism by which it does so is poorly understood. In this study, we used targeted NextGen bisulfite sequencing to evaluate DNAm levels in 125 targeted highly informative genomic regions containing 602 CpG sites on 70 immune-related genes to understand whether DNAm can differentiate peanut allergy (PA) vs non-allergy (NA). We found PA-associated DNAm signatures associated with 12 genes (7 novel to food allergy, 3 associated with Th1/Th2, and 2 associated with innate immunity) as well as DNAm signature combinations with superior diagnostic potential compared to serum peanut specific-IgE for PA vs. NA. Further, we found that following peanut protein stimulation, peripheral blood mononuclear cell (PBMCs) from PA participants showed increased production of cognate cytokines compared to NA participants. The varying responses between PA and NA participants may be associated with the interaction between the modification of DNAm and the interference of environment. Using Euclidean distance analysis, we found that the distances of methylation profile comprising 12 DNAm signatures between PA and NA pairs in monozygotic (MZ) twins were smaller than that in randomly paired genetically unrelated individuals, suggesting that PA related DNAm signatures may be associated with genetic factors.
Authors
Xiaoying Zhou, Xiaorui Han, Shu-Chen Lyu, Bryan J. Bunning, Laurie Kost, Iris Chang, Shu Cao, Vanitha Sampath, Kari C. Nadeau
Abstract
To unequivocally address their unresolved intimate structures in blood, we scrutinized the size distribution of circulating cell-free DNA (cfDNA) using whole genome sequencing (WGS) from both double- and single-strand DNA library preparations (DSP and SSP), as well as using Q-PCR. The size profile in healthy individuals was remarkably homogenous when using either DSP sequencing (DSP-S) or SSP sequencing (SSP-S). Our findings also confirmed that cfDNA size profile shows a characteristic nucleosome fragmentation pattern. Overall, our data indicate that the proportion of cfDNA inserted in mono-nucleosomes, di-nucleosomes and chromatin of higher molecular size (>1,000bp) can be estimated as 67.5-80%, 9.4-11.5% and 8.5-21.0%, respectively. Thus, our data on WGS (N=7) and Q-PCR (N=116 taken together suggests that only a minor proportion of cfDNA is bigger than that existing in mono-nucleosome or transcription factor complexes circulating in blood. Although DNA on single chromatosomes or mono-nucleosomes is detectable, our data revealed that cfDNA is highly nicked (97-98%) on those structures, which appear to be subjected to continuous nuclease activity in the bloodstream. Fragments analysis allows the distinction of cfDNA of different origins: first, cfDNA size profile analysis may be useful in cfDNA extract quality control; second, subtle but reliable differences between healthy metastatic colorectal cancer (mCRC) patients and healthy individuals vary with the proportion of malignant cell-derived cfDNA in plasma extracts, pointing to a higher degree of cfDNA fragmentation and nuclease activity in samples with high malignant cell cfDNA content. Size profile analysis, or ‘fragmentomics’, has shown significant potential to improve diagnostics and cancer screening.
Authors
Cynthia Sanchez, Benoit Roch, Thilbault Mazard, Philippe Blache, Zahra Al Amir Dache, Brice Pastor, Ekaterina Pisareva, Rita Tanos, Alain R. Thierry
Modulation of ATXN1 S776 phosphorylation reveals the importance of allele-specific targeting in SCA1
Abstract
Spinocerebellar ataxia type 1 (SCA1) is an adult-onset neurodegenerative disorder characterized by motor incoordination, mild cognitive decline, respiratory dysfunction, and early lethality. It is caused by the expansion of the polyglutamine (polyQ) tract in Ataxin-1 (ATXN1), which stabilizes the protein, leading to its toxic accumulation in neurons. Previously, we showed that serine 776 (S776) phosphorylation is critical for ATXN1 stability and contributes to its toxicity in cerebellar Purkinje cells. Still, the therapeutic potential of disrupting S776 phosphorylation on noncerebellar SCA1 phenotypes remains unstudied. Here, we report that abolishing S776 phosphorylation specifically on the polyQ-expanded ATXN1 of SCA1-knockin mice reduces ATXN1 throughout the brain and not only rescues the cerebellar motor incoordination but also improves respiratory function and extends survival while not affecting the hippocampal learning and memory deficits. As therapeutic approaches are likely to decrease S776 phosphorylation on polyQ-expanded and WT ATXN1, we further disrupted S776 phosphorylation on both alleles and observed an attenuated rescue, demonstrating a potential protective role of WT allele. This study not only highlights the role of S776 phosphorylation to regulate ATXN1 levels throughout the brain but also suggests distinct brain region–specific disease mechanisms and demonstrates the importance of developing allele-specific therapies for maximal benefits in SCA1.
Authors
Larissa Nitschke, Stephanie L. Coffin, Eder Xhako, Dany B. El-Najjar, James P. Orengo, Elizabeth Alcala, Yanwan Dai, Ying-Wooi Wan, Zhandong Liu, Harry T. Orr, Huda Y. Zoghbi
Mutations of MAP1B encoding a microtubule-associated phosphoprotein cause sensorineural hearing loss
Abstract
The pathophysiology underlying spiral ganglion cell defect–induced deafness remains elusive. Using the whole exome sequencing approach, in combination with functional assays and a mouse disease model, we identified the potentially novel deafness-causative MAP1B gene encoding a highly conserved microtubule-associated protein. Three novel heterozygous MAP1B mutations (c.4198A>G, p.1400S>G; c.2768T>C, p.923I>T; c.5512T>C, p.1838F>L) were cosegregated with autosomal dominant inheritance of nonsyndromic sensorineural hearing loss in 3 unrelated Chinese families. Here, we show that MAP1B is highly expressed in the spiral ganglion neurons in the mouse cochlea. Using otic sensory neuron–like cells, generated by pluripotent stem cells from patients carrying the MAP1B mutation and control subject, we demonstrated that the p.1400S>G mutation caused the reduced levels and deficient phosphorylation of MAP1B, which are involved in the microtubule stability and dynamics. Strikingly, otic sensory neuron–like cells exhibited disturbed dynamics of microtubules, axonal elongation, and defects in electrophysiological properties. Dysfunctions of these derived otic sensory neuron–like cells were rescued by genetically correcting MAP1B mutation using CRISPR/Cas9 technology. Involvement of MAP1B in hearing was confirmed by audiometric evaluation of Map1b heterozygous KO mice. These mutant mice displayed late-onset progressive sensorineural hearing loss that was more pronounced in the high frequencies. The spiral ganglion neurons isolated from Map1b mutant mice exhibited the deficient phosphorylation and disturbed dynamics of microtubules. Map1b deficiency yielded defects in the morphology and electrophysiology of spiral ganglion neurons, but it did not affect the morphologies of cochlea in mice. Therefore, our data demonstrate that dysfunctions of spiral ganglion neurons induced by MAP1B deficiency caused hearing loss.
Authors
Limei Cui, Jing Zheng, Qiong Zhao, Jia-Rong Chen, Hanqing Liu, Guanghua Peng, Yue Wu, Chao Chen, Qiufen He, Haosong Shi, Shankai Yin, Rick A. Friedman, Ye Chen, Min-Xin Guan
Abstract
Mutations of CNTNAP1 were associated with myelination disorders, suggesting the role of CNTNAP1 in myelination processes. Whether CNTNAP1 may have a role in early cortical neuronal development is largely unknown. In this study, we identified 4 compound heterozygous mutations of CNTNAP1 in 2 Chinese families. Using mouse models, we found that CNTNAP1 is highly expressed in neurons and is located predominantly in MAP2+ neurons during the early developmental stage. Importantly, Cntnap1 deficiency results in aberrant dendritic growth and spine development in vitro and in vivo, and it delayed migration of cortical neurons during early development. Finally, we found that the number of parvalbumin+ neurons in the cortex and hippocampus of Cntnap1–/– mice is strikingly increased by P15, suggesting that excitation/inhibition balance is impaired. Together, this evidence elucidates a critical function of CNTNAP1 in cortical development, providing insights underlying molecular and circuit mechanisms of CNTNAP1-related disease.
Authors
Wanxing Li, Lin Yang, Chuanqing Tang, Kaiyi Liu, Yulan Lu, Huijun Wang, Kai Yan, Zilong Qiu, Wenhao Zhou
Abstract
Complex I (also known as NADH-ubiquinone oxidoreductase) deficiency is the most frequent mitochondrial disorder present in childhood. NADH-ubiquinone oxidoreductase iron-sulfur protein 3 (NDUFS3) is a catalytic subunit of the mitochondrial complex I; NDUFS3 is conserved from bacteria and essential for complex I function. Mutations affecting complex I, including in the Ndufs3 gene, cause fatal neurodegenerative diseases, such as Leigh syndrome. No treatment is available for these conditions. We developed and performed a detailed molecular characterization of a neuron-specific Ndufs3 conditional KO mouse model. We showed that deletion of Ndufs3 in forebrain neurons reduced complex I activity, altered brain energy metabolism, and increased locomotor activity with impaired motor coordination, balance, and stereotyped behavior. Metabolomics analyses showed an increase of glycolysis intermediates, suggesting an adaptive response to the complex I defect. Administration of metformin to these mice delayed the onset of the neurological symptoms but not of neuronal loss. This improvement was likely related to enhancement of glucose uptake and utilization, which are known effects of metformin in the brain. Despite reports that metformin inhibits complex I activity, our findings did not show worsening a complex I defect nor increases in lactic acid, suggesting that metformin should be further evaluated for use in patients with mitochondrial encephalopathies.
Authors
Susana Peralta, Milena Pinto, Tania Arguello, Sofia Garcia, Francisca Diaz, Carlos T. Moraes
Abstract
Phenylalanine hydroxylase–deficient (PAH-deficient) phenylketonuria (PKU) results in systemic hyperphenylalaninemia, leading to neurotoxicity with severe developmental disabilities. Dietary phenylalanine (Phe) restriction prevents the most deleterious effects of hyperphenylalaninemia, but adherence to diet is poor in adult and adolescent patients, resulting in characteristic neurobehavioral phenotypes. Thus, an urgent need exists for new treatments. Additionally, rodent models of PKU do not adequately reflect neurocognitive phenotypes, and thus there is a need for improved animal models. To this end, we have developed PAH-null pigs. After selection of optimal CRISPR/Cas9 genome-editing reagents by using an in vitro cell model, zygote injection of 2 sgRNAs and Cas9 mRNA demonstrated deletions in preimplantation embryos, with embryo transfer to a surrogate leading to 2 founder animals. One pig was heterozygous for a PAH exon 6 deletion allele, while the other was compound heterozygous for deletions of exon 6 and of exons 6–7. The affected pig exhibited hyperphenylalaninemia (2000–5000 μM) that was treatable by dietary Phe restriction, consistent with classical PKU, along with juvenile growth retardation, hypopigmentation, ventriculomegaly, and decreased brain gray matter volume. In conclusion, we have established a large-animal preclinical model of PKU to investigate pathophysiology and to assess new therapeutic interventions.
Authors
Erik A. Koppes, Bethany K. Redel, Marie A. Johnson, Kristen J. Skvorak, Lina Ghaloul-Gonzalez, Megan E. Yates, Dale W. Lewis, Susanne M. Gollin, Yijen L. Wu, Shawn E. Christ, Martine Yerle, Angela Leshinski, Lee D. Spate, Joshua A. Benne, Stephanie L. Murphy, Melissa S. Samuel, Eric M. Walters, Sarah A. Hansen, Kevin D. Wells, Uta Lichter-Konecki, Robert A. Wagner, Joseph T. Newsome, Steven F. Dobrowolski, Jerry Vockley, Randall S. Prather, Robert D. Nicholls
Abstract
Somatic KRAS mutations are highly prevalent in many human cancers. In addition, a distinct spectrum of germline KRAS mutations cause developmental disorders called RASopathies. The mutant proteins encoded by these germline KRAS mutations are less biochemically and functionally activated than the mutant proteins found in cancer. We generated mice harboring conditional KrasLSL-P34R and KrasLSL-T58I “knock in” alleles and characterized the consequences of each mutation in vivo. Embryonic expression of KrasT58I resulted in craniofacial abnormalities reminiscent of RASopathy disorders, and these mice also exhibited hyperplastic growth of multiple organs, modest alterations in cardiac valvulogenesis, myocardial hypertrophy, and myeloproliferation. By contrast, embryonic KrasP34R expression resulted in early perinatal lethality from respiratory failure due to defective lung sacculation, which was associated with aberrant ERK activity in lung epithelial cells. Somatic Mx1-Cre-mediated activation in the hematopoietic compartment showed that KrasP34R and KrasT58I expression had distinct signaling effects despite causing a similar spectrum of hematologic diseases. These novel mouse strains are robust models for investigating the consequences of endogenous hyperactive K-Ras signaling in different developing and adult tissues, for comparing how oncogenic and germline K-Ras proteins perturb signaling networks and cell fate decisions, and for performing preclinical therapeutic trials.
Authors
Jasmine C. Wong, Pedro A. Perez-Mancera, Tannie Q. Huang, Jangkyung Kim, Joaquim Grego-Bessa, Maria del pilar Alzamora, Scott C. Kogan, Amnon Sharir, Susan H. Keefe, Carolina E. Morales, Denny Schanze, Pau Castel, Kentaro Hirose, Guo N. Huang, Martin Zenker, Dean Sheppard, Ophir Klein, David Tuveson, Benjamin S. Braun, Kevin Shannon
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