Retinal aging and inherited neurodegenerative diseases

视网膜老化和遗传性神经退行性疾病

• 批准号: 8737647
• 负责人: ANAND SWAROOP
• 金额: $ 240.88万
• 依托单位: NATIONAL EYE INSTITUTE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8737647
• 关键词: Affect; Age related macular degeneration; Age-Months; Aging; Alleles; Animal Model; Biogenesis; Biological; Blindness; C3 gene; Cells; Centrioles; ChIP-seq; Choroid; Cilia; Clinical; Code; Collaborations; Complement; Complex; Data; Data Analyses; Defect; Development; Disease; Down-Regulation; Embryo; Environmental Risk Factor; Epigenetic Process; Family; Family member; Fibroblasts; Frameshift Mutation; Functional disorder; Gene Expression; Genes; Genetic; Genetic Variation; Genotype; Genus Hippocampus; Homeostasis; Human; Human Genetics; Human Pathology; Hydrocephalus; Immune; Individual; Inherited; Investigation; Joubert syndrome; Knowledge; Lead; Leber' s amaurosis; Link; Longevity; Macular degeneration; Maintenance; Massachusetts; Measurement; Membrane; Michigan; Microglia; Mitochondria; Modeling; Molecular; Molecular Profiling; Morphogenesis; Mothers; Mus; Mutation; Neuraxis; Neurodegenerative Disorders; Nuclear; Opsin; Oxygen Consumption; Pathogenesis; Pathology; Pathway interactions; Patients; Penetrance; Pennsylvania; Phenotype; Photoreceptors; Pluripotent Stem Cells; RNA; Regulation; Research; Residual state; Respiratory Chain; Retina; Retinal; Retinal Cone; Retinal Degeneration; Retinal Diseases; Retinitis Pigmentosa; Rhodopsin; Role; Scientist; Signal Transduction; Somatic Cell; Sorting - Cell Movement; Symptoms; Synapses; Testing; Therapeutic; Transgenes; Universities; Variant; age related; age related neurodegeneration; angiogenesis; appendage; base; ciliopathy; cilium biogenesis; design; exome sequencing; genetic variant; genome-wide; human disease; immune function; in vitro Assay; in vitro Model; induced pluripotent stem cell; insight; interest; kinetosome; male; mouse model; neurotrophic factor; normal aging; novel; photoreceptor degeneration; pleiotropism; pluripotency; postnatal; retinal rods; screening; senescence; therapeutic target; transcription factor; transcriptome sequencing

Background Identification of genetic defects and elucidation of underlying pathways of photoreceptor degeneration constitute an essential step in developing targeted therapeutic strategies for retinal neurodegenerative diseases. N-NRL collaborates with numerous clinicians and scientists to identify genes associated with retinitis pigmentosa (RP), Leber congenital amaurosis (LCA), and syndromic diseases involving photoreceptor dysfunction. Genetics of human retinal diseases Retinal diseases often display genetic and clinical variability ascribable to combinations of allelic differences, modifier loci, epigenetic and environmental factors. A modifier gene affects penetrance, expressivity as well as pleiotropy. We are exploring the effect of modifiers in LCA, which often manifests unexplained genetic and clinical variability even in related individuals. Combined with global retinal RNA-Seq and ChIP-Seq data we can apply additional filtering to evaluate candidate genetic variants. We aim to define a systematic genome-wide approach for searching rare alleles that may contribute to LCA pathogenesis and phenotypic variability. RP defines a large group of inherited retinal disorders, characterized by progressive photoreceptor degeneration and vision loss. A large proportion of RP patients is isolates or simplex, with no known affected family members. RP2 and RPGR genes have been identified as the primary causative genes in X-linked RP (XLRP), as well as in X-linked cone dystrophy (CD) and cone-rod dystrophy (CRD). In collaboration with groups at University of Michigan and University of Pennsylvania, we have identified pathogenic mutations in RPGR and RP2 genes in 15% of simplex male patients screened and proposed RPGR as a first tier gene for screening isolated males with retinal degeneration (1). CD and CRD are clinically and genetically heterogeneous and display various modes of Mendelian inheritance. Mutations in several genes have been identified, however the genetic cause is still unknown for several cases. Upon whole exome sequencing of 27 individuals from eight unrelated Israeli families (in collaboration with Hebrew University), we have identified rare variants in GUCY2D, CDHR1, C8orf37 and CACNA1F, previously associated with CD/CRD. In another autosomal recessive family we have identified a potential causative variant in a gene linked to a syndromic disorder involving loss of vision. We are pursuing exome sequencing of additional Israeli families and investigation of the biological effect(s) of novel gene variants on disease pathology. Animal models of human retinal disease Mutations in RP2 gene are associated with 10-15% of XLRP. In collaboration with colleagues at University of Massachusetts, we investigated the pathogenesis of retinal degeneration in Rp2-/- mice. Despite no overt signs of degeneration, photopic and scotopic ERG declined gradually from one month of age in Rp2-/- retina. Slow, progressive degeneration of photoreceptor membrane discs was associated with mislocalization of cone opsins to the nuclear and synaptic layers, and to reduced rhodopsin content in outer segments (OS), suggesting that RP2 contributes to maintenance of photoreceptor function and that cone opsin mislocalization represents an early step in RP2-associated XLRP (3). Cep290 is a common cause of vision loss in LCA and is involved in several syndromic ciliopathies. Cep290 is essential for photoreceptor ciliogenesis and OS formation. Since the phenotype of the hypomorphic mouse model Cep290rd16 is relatively mild compared to Cep290-associated human pathology, we generated Cep290-/- mice. Most Cep290-/- mice die around weaaning from hydrocephalus. However, tthose that survive have a normal lifespan and develop rapid retinal degeneration starting at postnatal day (P)12. The effects on OS morphogenesis are more pronounced than in Cep290rd16 retinas, supporting a critical role of CEP290 in photoreceptor cilia development. CC2D2A is associated with Meckel (MKS) and Joubert syndrome ciliopathies. The Cc2d2a-/- mouse we generated is embryonic lethal and recapitulates the clinical symptoms of MKS. Embryonic fibroblasts (MEF) from Cc2d2a-/- mice presented defect in cilia basal body in the mother centriole subdistal appendages (SDA), where Cc2d2a is localized. The cilia biogenesis defect was rescued by Cc2d2a transgene. Mutations in transcription factor CRX lead to autosomal dominant LCA. The mechanism of CRX-LCA is not understood. We have identified a mouse with a 1-bp frameshift deletion in Crx coding sequence, similar to many human LCA-causing dominant CRX mutations (CrxRip). Unlike Crx-/- mouse retina, the dominant Crx c.763del1 mutation in CrxRip results in congenital blindness with complete loss of ERG, yet photoreceptors do not degenerate. Dominant CRX frameshift mutations associated with LCA mimic the CrxRip phenotype and can be rescued by Crx. RNA-Seq profiling revealed progressive and complete loss of rod differentiation factor Nrl in CrxRip, while residual Nrl remains in Crx-/- retina. Moreover, Nrl partially restored the rod phenotype in CrxRip/+ mice. Our study provides the mechanism of congenital blindness caused by dominant CRX mutations and should assist in therapeutic design. Altered homeostasis in the aging retina Molecular pathways leading to rod dysfunction in aging are not delineated. By microarray and RNA-Seq profiling of aging mouse rods (flow sorted from Nrl-GFP mice), we have identified changes in gene expression in mitochondrial and proteasomal genes. Although down-regulation of individual genes of the respiratory chain complexes is minimal, the combined effect could be significant in photoreceptors. To test this hypothesis, we have adapted an in vitro assay for measurement of oxygen consumption rate (as an indicator of mitochondrial function) in freshly isolated retina punches using the Seahorse, HF24 Analyzer. The analysis of data from aging and caloric-restricted mice is in progress. To gain additional insights, we have collected human retina, RPE and choroid at different ages and from normal and disease individuals to correlate genotype with aging-associated expression changes using RNA-Seq. Microglia (MG), the resident immune cells of the central nervous system, are thought to contribute to the pathogenesis of age-related neurodegeneration. We collaborated with W. Wong, NEI, to compare gene expression profiles of aging mouse MG. Molecular pathways involving immune function and regulation, angiogenesis, and neurotrophin signaling showed age-related changes. In particular, expression of complement genes, C3 and CFB, previously associated with age-related macular degeneration (AMD), increased with aging, suggesting that senescent MG may contribute to complement dysregulation during disease pathogenesis. The age-related gene expression changes we detected appear to alter MG constitutive support functions and regulation of their activation status (4). In vitro models of human retinal disease Reprogramming of somatic cells to pluripotency allows de novo differentiation to photorecetors and modeling of retinal disease in a dish using the patients' own cells to study disease pathogenesis and discover new molecules for therapy. We are focusing on patients with mutations in CEP290 that present LCA or Joubert syndrome phenotypes. In parallel, we have generated induced pluripotent stem cells (iPSCs) from the Cep290rd16 mouse and controls to complement our human studies using a mouse model of which we have extensive knowledge. Significance We have identified and characterized a few disease genes. Research on animal models has highlighted an interesting convergence on biogenesis and transport functions associated with primary cilia and revealed novel pathways contributing to photoreceptor homeostasis.

背景 鉴定遗传缺陷和光感受器变性的潜在途径是开发视网膜神经退行性疾病的靶向治疗策略的重要步骤。 N-NRL与众多临床医生和科学家合作，以鉴定与视网膜炎色素炎（RP），Leber先天性症（LCA）（LCA）和综合疾病有关的基因，涉及光感受器功能障碍。 人类视网膜疾病的遗传学 视网膜疾病经常显示出可归因于等位基因差异，修饰基因座，表观遗传和环境因素的组合的遗传和临床变异性。修饰符基因会影响外观，表现性和多效性。我们正在探索修饰符在LCA中的影响，LCA在相关个体中即使在相关个体中也经常表现出无法解释的遗传和临床变异性。结合全局视网膜RNA-SEQ和CHIP-SEQ数据，我们可以应用其他过滤以评估候选遗传变异。 我们旨在定义一种全系统的基因组方法，用于搜索可能有助于LCA发病机理和表型变异性的稀有等位基因。 RP定义了一大批遗传性视网膜疾病，其特征是进行性光感受器变性和视力丧失。 RP患者很大一部分是分离株或单纯株，没有已知的受影响的家庭成员。 RP2和RPGR基因已被鉴定为X连锁RP（XLRP）以及X连锁的锥体营养不良（CD）和锥体rod Droptrophy（CRD）中的主要因果基因。与密歇根大学和宾夕法尼亚大学的小组合作，我们在RPGR和RP2基因中确定了15％的单纯形男性患者的RPGR和RP2基因的致病突变，并提议RPGR作为首个筛查具有视视性变性的分离型男性的第一个层基因（1）。 CD和CRD在临床和遗传上是异质的，并显示了Mendelian遗传的各种模式。已经鉴定出几种基因的突变，但是在几种情况下，遗传原因仍然未知。在对来自八个无关以色列家庭（与希伯来大学合作的）的27个个体进行了整个外显子组测序后，我们已经确定了以前与CD/CRD相关的GUCY2D，CDHR1，C8ORF37和CACNA1F中的稀有变体。在另一个常染色体隐性家族中，我们已经确定了与涉及视力丧失的综合症疾病有关的基因中的潜在原因变异。我们正在追求其他以色列家庭的外显子组测序，并研究了新基因变体对疾病病理学的生物学作用。 人类视网膜疾病的动物模型 RP2基因的突变与XLRP的10-15％有关。与马萨诸塞大学的同事合作，我们研究了RP2 - / - 小鼠视网膜变性的发病机理。尽管没有明显的退化迹象，但在RP2 - / - 视网膜中，光波器和Scotopic ERG逐渐下降。 Slow, progressive degeneration of photoreceptor membrane discs was associated with mislocalization of cone opsins to the nuclear and synaptic layers, and to reduced rhodopsin content in outer segments (OS), suggesting that RP2 contributes to maintenance of photoreceptor function and that cone opsin mislocalization represents an early step in RP2-associated XLRP (3). CEP290是LCA视力丧失的常见原因，并且参与了几种综合征纤毛病。 CEP290对于光感受器纤毛发生和OS形成至关重要。由于与CEP290相关的人类病理相比，由于型小鼠模型CEP290RD16的表型相对温和，因此我们产生了CEP290 - / - 小鼠。大多数CEP290 - / - 小鼠死于脑积水周围。然而，生存的tthose具有正常的寿命，并从产后（p）12开始发展快速的视网膜变性。 与CEP290RD16视网膜相比，对OS形态发生的影响更为明显，这支持CEP290在光感受器纤毛发育中的关键作用。 CC2D2A与Meckel（MKS）和Joubert综合征纤毛病有关。我们生成的CC2D2A - / - 小鼠是胚胎致死的，并概括了MK的临床症状。来自CC2D2A-/ - 小鼠的胚胎成纤维细胞（MEF）在母亲Centriole subdistal Appendage（SDA）中出现缺陷，其中CC2D2A被定位。 CC2D2A转基因挽救了纤毛生物发生缺陷。 转录因子CRX中的突变导致常染色体显性LCA。 CRX-LCA的机制尚不清楚。我们已经在CRX编码序列中鉴定出具有1 bp移码缺失的小鼠，类似于许多引起人类LCA的主要CRX突变（CRXRIP）。与CRX - / - 小鼠视网膜不同，CRXRIP中的主要CRX C.763DEL1突变导致先天性失明，而ERG完全丢失，但感光体并未退化。与LCA模拟CRXRIP表型相关的主要CRX移码突变，可以通过CRX救出。 RNA-seq分析表明，CRXRIP中杆分化因子NRL的渐进性和完全丢失，而残留的NRL仍保留在CRX - / - 视网膜中。此外，NRL在CRXRIP/+小鼠中部分恢复了杆表型。我们的研究提供了由主要CRX突变引起的先天性失明机制，应有助于治疗设计。 改变了视网膜老化的体内平衡 未划定导致衰老中杆功能障碍的分子途径。通过衰老小鼠棒的微阵列和RNA-seq分析（从NRL-GFP小鼠分类），我们已经确定了线粒体和蛋白酶体基因中基因表达的变化。尽管呼吸链复合物的单个基因的下调很小，但合并作用在感光体中可能很重要。为了检验这一假设，我们已经改编了一种体外测定法，以使用Seahorse HF24分析仪在新鲜分离的视网膜打孔器中测量氧消耗率（作为线粒体功能的指标）。对衰老和热量限制小鼠的数据的分析正在进行中。为了获得更多的见解，我们在不同年龄以及从正常和疾病个体的人群中收集了人类视网膜，RPE和脉络膜，以将基因型与使用RNA-Seq相关的基因型与衰老相关的表达变化相关。 小胶质细胞（MG）是中枢神经系统的驻留免疫细胞，被认为有助于与年龄相关的神经变性的发病机理。我们与Nei的W. Wong合作，比较了老化小鼠Mg的基因表达谱。涉及免疫功能和调节，血管生成和神经营养蛋白信号传导的分子途径显示与年龄相关的变化。特别是，以前与年龄相关的黄斑变性（AMD）相关的补体基因C3和CFB的表达随着衰老而增加，这表明衰老MG可能在疾病发病机理期间有助于补体失调。我们检测到的与年龄相关的基因表达变化似乎改变了MG组成型支持函数及其激活状态的调节（4）。 人类视网膜疾病的体外模型 将体细胞重新编程为多能性允许从从患者自己的细胞研究疾病发病机理并发现新的治疗分子的菜肴中对光子分离和视网膜疾病进行建模。我们专注于在CEP290中出现LCA或Joubert综合征表型的突变患者。同时，我们从CEP290RD16小鼠中生成了诱导的多能干细胞（IPSC），并使用了我们具有广泛知识的小鼠模型来补充人类研究。 意义 我们已经确定并表征了一些疾病基因。动物模型的研究强调了与原发性纤毛相关的生物发生和运输功能的有趣融合，并揭示了有助于感光体稳态的新型途径。