Expression of Wild-Type Rp1 Protein in Rp1 Knock-in Mice Rescues the Retinal Degeneration Phenotype

Mutations in the retinitis pigmentosa 1 (RP1) gene are a common cause of autosomal dominant retinitis pigmentosa (adRP), and have also been found to cause autosomal recessive RP (arRP) in a few families. The 33 dominant mutations and 6 recessive RP1 mutations identified to date are all nonsense or frameshift mutations, and almost exclusively (38 out of 39) are located in the 4(th) and final exon of RP1. To better understand the underlying disease mechanisms of and help develop therapeutic strategies for RP1 disease, we performed a series of human genetic and animal studies using gene targeted and transgenic mice. Here we report that a frameshift mutation in the 3(rd) exon of RP1 (c.686delC; p.P229QfsX35) found in a patient with recessive RP1 disease causes RP in the homozygous state, whereas the heterozygous carriers are unaffected, confirming that haploinsufficiency is not the causative mechanism for RP1 disease. We then generated Rp1 knock-in mice with a nonsense Q662X mutation in exon 4, as well as Rp1 transgenic mice carrying a wild-type BAC Rp1 transgene. The Rp1-Q662X allele produces a truncated Rp1 protein, and homozygous Rp1-Q662X mice experience a progressive photoreceptor degeneration characterized disorganization of photoreceptor outer segments. This phenotype could be prevented by expression of a normal amount of Rp1 protein from the BAC transgene without removal of the mutant Rp1-Q662X protein. Over-expression of Rp1 protein in additional BAC Rp1 transgenic lines resulted in retinal degeneration. These findings suggest that the truncated Rp1-Q662X protein does not exert a toxic gain-of-function effect. These results also imply that in principle gene augmentation therapy could be beneficial for both recessive and dominant RP1 patients, but the levels of RP1 protein delivered for therapy will have to be carefully controlled.     

Transgenic Mice Overexpressing Serum Retinol-Binding Protein Develop Progressive Retinal Degeneration through a Retinoid-Independent Mechanism

Serum retinol-binding protein 4 (RBP4) is the sole specific transport protein for retinol in the blood, but it is also an adipokine with retinol-independent, proinflammatory activity associated with obesity, insulin resistance, type 2 diabetes, and cardiovascular disease. Moreover, two separate studies reported that patients with proliferative diabetic retinopathy have increased serum RBP4 levels compared to patients with mild or no retinopathy, yet the effect of increased levels of RBP4 on the retina has not been studied. Here we show that transgenic mice overexpressing RBP4 (RBP4-Tg mice) develop progressive retinal degeneration, characterized by photoreceptor ribbon synapse deficiency and subsequent bipolar cell loss. Ocular retinoid and bisretinoid levels are normal in RBP4-Tg mice, demonstrating that a retinoid-independent mechanism underlies retinal degeneration. Increased expression of pro-interleukin-18 (pro-IL-18) mRNA and activated IL-18 protein and early-onset microglia activation in the retina suggest that retinal degeneration is driven by a proinflammatory mechanism. Neither chronic systemic metabolic disease nor other retinal insults are required for RBP4 elevation to promote retinal neurodegeneration, since RBP4-Tg mice do not have coincident retinal vascular pathology, obesity, dyslipidemia, or hyperglycemia. These findings suggest that elevation of serum RBP4 levels could be a risk factor for retinal damage and vision loss in nondiabetic as well as diabetic patients.     

莱茵衣藻LZTFL1蛋白的多克隆抗体制备及应用

目的:亮氨酸拉链转录因子1(LZTFL1)是一种与纤毛信号转导相关的蛋白质,关于其如何在信号转导中发挥作用以及作用机制目前尚不清楚。莱茵衣藻(Chlamydomonas reinhardtii)是研究纤毛信号传导的模式生物,而目前对莱茵衣藻LZTFL1蛋白的研究甚少,尚未有相应的检测抗体,因此制备LZTFL1多克隆抗体用于后续实验研究。方法:采用RT-PCR技术从C.reinhardtii CC125中提取总RNA,扩增981bp的目的基因Lztfl1,插入到p ET-28a(+)原核表达载体,成功构建p ET-28a(+)-Lztfl1重组质粒,转入E.coli BL21(DE3)经IPTG诱导后成功表达6×His-LZTFL1融合蛋白,融合蛋白经亲和纯化后免疫新西兰大白兔制备多克隆抗体,最后采用间接ELISA法测得抗血清效价达到1∶512 000,经Western blot对C.reinhardtii CC125检测具有较高特异性。结果:首次实现了莱茵衣藻LZTFL1蛋白的原核表达,制备出一支兔抗莱茵衣藻LZTFL1蛋白的多克隆抗体,为后续研究LZTFL1蛋白在莱茵衣藻中的结构功能及在纤毛信号转导中的相互作用奠定了基础。     

LINE-1 Mediated Insertion into Poc1a (Protein of Centriole 1 A) Causes Growth Insufficiency and Male Infertility in Mice

Skeletal dysplasias are a common, genetically heterogeneous cause of short stature that can result from disruptions in many cellular processes. We report the identification of the lesion responsible for skeletal dysplasia and male infertility in the spontaneous, recessive mouse mutant chagun. We determined that Poc1a, encoding protein of the centriole 1a, is disrupted by the insertion of a processed Cenpw cDNA, which is flanked by target site duplications, suggestive of a LINE-1 retrotransposon-mediated event. Mutant fibroblasts have impaired cilia formation and multipolar spindles. Male infertility is caused by defective spermatogenesis early in meiosis and progressive germ cell loss. Spermatogonial stem cell transplantation studies revealed that Poc1a is essential for normal function of both Sertoli cells and germ cells. The proliferative zone of the growth plate is small and disorganized because chondrocytes fail to re-align after cell division and undergo increased apoptosis. Poc1a and several other genes associated with centrosome function can affect the skeleton and lead to skeletal dysplasias and primordial dwarfisms. This mouse mutant reveals how centrosome dysfunction contributes to defects in skeletal growth and male infertility.     

Depletion of Selenoprotein GPx4 in Spermatocytes Causes Male Infertility in Mice

Phospholipid hydroperoxide glutathione peroxidase (GPx4) is an intracellular antioxidant enzyme that directly reduces peroxidized phospholipids. GPx4 is strongly expressed in the mitochondria of testis and spermatozoa. We previously found a significant decrease in the expression of GPx4 in spermatozoa from 30% of infertile human males diagnosed with oligoasthenozoospermia (Imai, H., Suzuki, K., Ishizaka, K., Ichinose, S., Oshima, H., Okayasu, I., Emoto, K., Umeda, M., and Nakagawa, Y. (2001) Biol. Reprod. 64, 674–683). To clarify whether defective GPx4 in spermatocytes causes male infertility, we established spermatocyte-specific GPx4 knock-out mice using a Cre-loxP system. All the spermatocyte-specific GPx4 knock-out male mice were found to be infertile despite normal plug formation after mating and displayed a significant decrease in the number of spermatozoa. Isolated epididymal GPx4-null spermatozoa could not fertilize oocytes in vitro. These spermatozoa showed significant reductions of forward motility and the mitochondrial membrane potential. These impairments were accompanied by the structural abnormality, such as a hairpin-like flagella bend at the midpiece and swelling of mitochondria in the spermatozoa. These results demonstrate that the depletion of GPx4 in spermatocytes causes severe abnormalities in spermatozoa. This may be one of the causes of male infertility in mice and humans.     

Deficiency of Angulin-2/ILDR1, a Tricellular Tight Junction-Associated Membrane Protein,Causes Deafness with Cochlear Hair Cell Degeneration in Mice

Tricellular tight junctions seal the extracellular spaces of tricellular contacts, where the vertices of three epithelial cells meet, and are required for the establishment of a strong barrier function of the epithelial cellular sheet. Angulins and tricellulin are known as specific protein components of tricellular tight junctions, where angulins recruit tricellulin. Mutations in the genes encoding angulin-2/ILDR1 and tricellulin have been reported to cause human hereditary deafness DFNB42 and DFNB49, respectively. To investigate the pathogenesis of DFNB42, we analyzed mice with a targeted disruption of Ildr1, which encodes angulin-2/ILDR1. Ildr1 null mice exhibited profound deafness. Hair cells in the cochlea of Ildr1 null mice develop normally, but begin to degenerate by two weeks after birth. Tricellulin localization at tricellular contacts of the organ of Corti in the cochlea was retained in Ildr1 null mice, but its distribution along the depth of tricellular contacts was affected. Interestingly, compensatory tricellular contact localization of angulin-1/LSR was observed in the organ of Corti in Ildr1 null mice although it was hardly detected in the organ of Corti in wild-type mice. The onset of hair cell degeneration in Ildr1 null mice was earlier than that in the reported Tric mutant mice, which mimic one of the tricellulin mutations in DFNB49 deafness. These results indicate that the angulin-2/ILDR1 deficiency causes the postnatal degenerative loss of hair cells in the cochlea, leading to human deafness DFNB42. Our data also suggest that angulin family proteins have distinct functions in addition to their common roles of tricellulin recruitment and that the function of angulin-2/ILDR1 for hearing cannot be substituted by angulin-1/LSR.     

Deletion of Aldose Reductase from Mice Inhibits Diabetes-Induced Retinal Capillary Degeneration and Superoxide Generation

Purpose

Pharmacologic inhibition of aldose reductase (AR) previously has been studied with respect to diabetic retinopathy with mixed results. Since drugs can have off-target effects, we studied the effects of AR deletion on the development and molecular abnormalities that contribute to diabetic retinopathy. Since recent data suggests an important role for leukocytes in the development of the retinopathy, we determined also if AR in leukocytes contributes to leukocyte-mediated death of retinal endothelial cells in diabetes.

Methods

Wild-type (WT; C57BL/6J) and AR deficient (AR^−/−) mice were made diabetic with streptozotocin. Mice were sacrificed at 2 and 10 months of diabetes to evaluate retinal vascular histopathology, to quantify retinal superoxide production and biochemical and physiological abnormalities in the retina, and to assess the number of retinal endothelial cells killed by blood leukocytes in a co-culture system.

Results

Diabetes in WT mice developed the expected degeneration of retinal capillaries, and increased generation of superoxide by the retina. Leukocytes from diabetic WT mice also killed more retinal endothelial cells than did leukocytes from nondiabetic animals (p<0.0001). Deletion of AR largely (P<0.05) inhibited the diabetes-induced degeneration of retinal capillaries, as well as the increase in superoxide production by retina. AR-deficiency significantly inhibited the diabetes-induced increase in expression of inducible nitric oxide synthase (iNOS) in retina, but had no significant effect on expression of intercellular adhesion molecule-1 (ICAM-1), phosphorylated p38 MAPK, or killing of retinal endothelial cells by leukocytes.

Conclusions

AR contributes to the degeneration of retinal capillaries in diabetic mice. Deletion of the enzyme inhibits the diabetes-induced increase in expression of iNOS and of superoxide production, but does not correct a variety of other pro-inflammatory abnormalities associated with the development of diabetic retinopathy.     

莱茵衣藻LZTFL1蛋白的多克隆抗体制备及应用

目的:亮氨酸拉链转录因子1(LZTFL1)是一种与纤毛信号转导相关的蛋白质,关于其如何在信号转导中发挥作用以及作用机制目前尚不清楚。莱茵衣藻(Chlamydomonas reinhardtii)是研究纤毛信号传导的模式生物,而目前对莱茵衣藻LZTFL1蛋白的研究甚少,尚未有相应的检测抗体,因此制备LZTFL1多克隆抗体用于后续实验研究。方法:采用RT-PCR技术从C.reinhardtii CC125中提取总RNA,扩增981bp的目的基因Lztfl1,插入到p ET-28a(+)原核表达载体,成功构建p ET-28a(+)-Lztfl1重组质粒,转入E.coli BL21(DE3)经IPTG诱导后成功表达6×His-LZTFL1融合蛋白,融合蛋白经亲和纯化后免疫新西兰大白兔制备多克隆抗体,最后采用间接ELISA法测得抗血清效价达到1∶512 000,经Western blot对C.reinhardtii CC125检测具有较高特异性。结果:首次实现了莱茵衣藻LZTFL1蛋白的原核表达,制备出一支兔抗莱茵衣藻LZTFL1蛋白的多克隆抗体,为后续研究LZTFL1蛋白在莱茵衣藻中的结构功能及在纤毛信号转导中的相互作用奠定了基础。     

Optic Nerve Compression and Retinal Degeneration in Tcirg1 Mutant Mice Lacking the Vacuolar-Type H+-ATPase a3 Subunit

Background

Vacuolar-type proton transporting ATPase (V-ATPase) is involved in the proper development of visual function. Mutations in the Tcirg1 (also known as Atp6V0a3) locus, which encodes the a3 subunit of V-ATPase, cause severe autosomal recessive osteopetrosis (ARO) in humans. ARO is often associated with impaired vision most likely because of nerve compression at the optic canal. We examined the ocular phenotype of mice deficient in Tcirg1 function.

Methodology/Principal Findings

X-ray microtomography showed narrowed foramina in the skull, suggesting that optic nerve compression occurred in the a3-deficient (Tcirg1 ^−/−) mice. The retina of the mutant mice had normal architecture, but the number of apoptotic cells was increased at 2–3 wks after birth. In the ocular system, the a3 subunit accumulated in the choriocapillary meshwork in uveal tissues. Two other subunit isoforms a1 and a2 accumulated in the retinal photoreceptor layer. We found that the a4 subunit, whose expression has previously been shown to be restricted to several transporting epithelia, was enriched in pigmented epithelial cells of the retina and ciliary bodies. The expression of a4 in the uveal tissue was below the level of detection in wild-type mice, but it was increased in the mutant choriocapillary meshwork, suggesting that compensation may have occurred among the a subunit isoforms in the mutant tissues.

Conclusions

Our findings suggest that a similar etiology of visual impairment is involved in both humans and mice; thus, a3-deficient mice may provide a suitable model for clinical and diagnostic purposes in cases of ARO.     

CYCLIC-NUCLEOTIDE PHOSPHODIESTERASE : An Early Defect in Inherited Retinal Degeneration of C3H Mice

Cyclic nucleotides have been implicated in the differentiation and function of the vertebrate retina. In the normal retina of DBA mice, the specific activity of cyclic-nucleotide phosphodiesterase (PDE), with cyclic-AMP as the substrate (cAMP-PDE), increases eightfold between the 6th and 20th postnatal day. Kinetic analysis of retinae from newborn mice reveals a PDE with a single Michaelis constant (K_m) value for cyclic-AMP (low K_m-PDE). After the 6th postnatal day, a second PDE with a high K_m for cyclic-AMP (high K_m-PDE) can be demonstrated. The appearance and increasing activity of the high K_m-PDE coincides with the differentiation and growth of photoreceptor outer segments. Additionally, the high K_m-PDE is shown by microchemical techniques to be concentrated in the photoreceptor cell layer and the low K_m-PDE within the inner layers of the normal retina. In C3H mice afflicted with an inherited degeneration of the photoreceptor layer, the postnatal increase in the specific activity of cAMP-PDE is substantially lower than in the normal retina. The postnatal increase in the specific activity of cAMP-PDE in two regions of the brain of C3H mice is the same as in the normal strain. A deficiency in high K_m-PDE activity in the C3H retina is evident on the 7th postnatal day, when the activity of low K_m-PDE, photoreceptor morphology, and rhodopsin content of these retina are essentially normal. In the adult C3H retina, the PDE activity with cyclic-GMP and cyclic-UMP as substrates is significantly below that of the normal retina. These data indicate that an alteration in cyclic-AMP metabolism occurs before photoreceptor cell degeneration in the retinae of C3H mice.     

Involvement of Endoplasmic Reticulum Stress in TULP1 Induced Retinal Degeneration

Inherited retinal disorders (IRDs) result in severe visual impairments in children and adults. A challenge in the field of retinal degenerations is identifying mechanisms of photoreceptor cell death related to specific genetic mutations. Mutations in the gene TULP1 have been associated with two forms of IRDs, early-onset retinitis pigmentosa (RP) and Leber congenital amaurosis (LCA). TULP1 is a cytoplasmic, membrane-associated protein shown to be involved in transportation of newly synthesized proteins destined for the outer segment compartment of photoreceptor cells; however, how mutant TULP1 causes cell death is not understood. In this study, we provide evidence that common missense mutations in TULP1 express as misfolded protein products that accumulate within the endoplasmic reticulum (ER) causing prolonged ER stress. In an effort to maintain protein homeostasis, photoreceptor cells then activate the unfolded protein response (UPR) complex. Our results indicate that the two major apoptotic arms of the UPR pathway, PERK and IRE1, are activated. Additionally, we show that retinas expressing mutant TULP1 significantly upregulate the expression of CHOP, a UPR signaling protein promoting apoptosis, and undergo photoreceptor cell death. Our study demonstrates that the ER-UPR, a known mechanism of apoptosis secondary to an overwhelming accumulation of misfolded protein, is involved in photoreceptor degeneration caused by missense mutations in TULP1. These observations suggest that modulating the UPR pathways might be a strategy for therapeutic intervention.     

Loss of the Metalloprotease ADAM9 Leads to Cone-Rod Dystrophy in Humans and Retinal Degeneration in Mice

Cone-rod dystrophy (CRD) is an inherited progressive retinal dystrophy affecting the function of cone and rod photoreceptors. By autozygosity mapping, we identified null mutations in the ADAM metallopeptidase domain 9 (ADAM9) gene in four consanguineous families with recessively inherited early-onset CRD. We also found reduced photoreceptor responses in Adam9 knockout mice, previously reported to be asymptomatic. In 12-month-old knockout mice, photoreceptors appear normal, but the apical processes of the retinal pigment epithelium (RPE) cells are disorganized and contact between photoreceptor outer segments (POSs) and the RPE apical surface is compromised. In 20-month-old mice, there is clear evidence of progressive retinal degeneration with disorganized POS and thinning of the outer nuclear layer (ONL) in addition to the anomaly at the POS-RPE junction. RPE basal deposits and macrophages were also apparent in older mice. These findings therefore not only identify ADAM9 as a CRD gene but also identify a form of pathology wherein retinal disease first manifests at the POS-RPE junction.     

Depletion and Reconstitution of Macrophages in Mice

Macrophages are critical players in the innate immune response to infectious challenge or injury, initiating the innate immune response and directing the acquired immune response. Macrophage dysfunction can lead to an inability to mount an appropriate immune response and as such, has been implicated in many disease processes, including inflammatory bowel diseases. Macrophages display polarized phenotypes that are broadly divided into two categories. Classically activated macrophages, activated by stimulation with IFNγ or LPS, play an essential role in response to bacterial challenge whereas alternatively activated macrophages, activated by IL-4 or IL-13, participate in debris scavenging and tissue remodeling and have been implicated in the resolution phase of inflammation. During an inflammatory response in vivo, macrophages are found amid a complex mixture of infiltrating immune cells and may participate by exacerbating or resolving inflammation. To define the role of macrophages in situ in a whole animal model, it is necessary to examine the effect of depleting macrophages from the complex environment. To ask questions about the role of macrophage phenotype in situ, phenotypically defined polarized macrophages can be derived ex vivo, from bone marrow aspirates and added back to mice, with or without prior depletion of macrophages. In the protocol presented here clodronate-containing liposomes, versus PBS injected controls, were used to deplete colonic macrophages during dextran sodium sulfate (DSS)-induced colitis in mice. In addition, polarized macrophages were derived ex vivo and transferred to mice by intravenous injection. A caveat to this approach is that clodronate-containing liposomes deplete all professional phagocytes, including both dendritic cells and macrophages so to ensure the effect observed by depletion is macrophage-specific, reconstitution of phenotype by adoptive transfer of macrophages is necessary. Systemic macrophage depletion in mice can also be achieved by backcrossing mice onto a CD11b-DTR background, which is an excellent complementary approach. The advantage of clodronate-containing liposome-mediated depletion is that it does not require the time and expense involved in backcrossing mice and it can be used in mice regardless of the background of the mice (C57BL/6, BALB/c, or mixed background).     

Expression of Lysostaphin in Milk of Transgenic Mice Affects the Growth of Neonates

As an initial step towards enhancing mastitis resistance in dairy animals, we generated BLG-Lys transgenic mice that secrete lysostaphin, a potent antistaphylococcal protein, in their milk. In the current study, we continue our assessment of lysostaphin as a suitable antimicrobial protein for mastitis resistance and have investigated mammary gland development and function in three lines of transgenic mice. As the lines were propagated, there was a tendency for fewer BLG-Lys litters to survive to weaning (51% as compared to 90% for nontransgenic lines, p = 0.080). Nontransgenic pups fostered on dams from these three lines exhibited diminished growth rates during the first week of lactation. Rates of gain became comparable to pups on nontransgenic dams at later time points. Initial slow growth also resulted in decreased weaning weights for pups nursed by transgenic dams (15.35±0.27 g) when compared to pups delivered and nursed by nontransgenic dams (18.61 ± 0.61 g; p < 0.001), but the effect was temporary, as similar weights were attained by adulthood. Milk yield at peak lactation was not different between BLG-Lys (0.79 ± 0.33 g) and nontransgenic (0.91 ± 0.38 g; p = 0.166) dams. Histological examination of the transgenic mammary glands during gestation revealed no differences when compared to control glands; however, at early lactational stages, the BLG-Lys glands exhibited less alveolar area than control glands and a delay in lobulo-alveolar maturation. The results clearly demonstrate reduced growth of neonates on BLG-Lys dams; whether the poor pup performance can be attributed to delayed mammary development or the gland development merely reflects reduced suckling stimuli from the pups remains to be determined.Authors Abhijit Mitra and Kathleen S. Hruska contributed equally to this work     

Spy1 Protein Mediates Phosphorylation and Degradation of SCG10 Protein in Axonal Degeneration

Axon loss is a destructive consequence of a wide range of neurological diseases without a clearly defined mechanism. Recent data demonstrate that SCG10 is a novel axonal maintenance factor and that rapid SCG10 loss after injury requires JNK activity; how JNK induces degradation of SCG10 is not well known. Here we showed that SCG10 was a binding partner of Spy1, a Speedy/RINGO family protein, which participated in cellular response to sciatic nerve injury. During the early stage of axonal injury, Spy1 expression was inversely correlated with SCG10. Spy1 mediated SCG10 phosphorylation and degradation partly in a JNK-dependent manner. Inhibition of Spy1 attenuated SCG10 phosphorylation and delayed injury-induced axonal degeneration. Taken together, these data suggest that Spy1 is an important regulator of SCG10 and can be targeted in future axo-protective therapeutics.     

Depletion of Cutaneous Macrophages and Dendritic Cells Promotes Growth of Basal Cell Carcinoma in Mice

Basal cell carcinoma (BCC) belongs to the group of non-melanoma skin tumors and is the most common tumor in the western world. BCC arises due to mutations in the tumor suppressor gene Patched1 (Ptch). Analysis of the conditional Ptch knockout mouse model for BCC reveals that macrophages and dendritic cells (DC) of the skin play an important role in BCC growth restraining processes. This is based on the observation that a clodronate-liposome mediated depletion of these cells in the tumor-bearing skin results in significant BCC enlargement. The depletion of these cells does not modulate Ki67 or K10 expression, but is accompanied by a decrease in collagen-producing cells in the tumor stroma. Together, the data suggest that cutaneous macrophages and DC in the tumor microenvironment exert an antitumor effect on BCC.