Wojciech Kedzierski

Transgenic mice expressing variants of complement factor H develop AMD-like retinal findings.

PURPOSE Complement factor H (Cfh) is a key regulator of the alternative complement pathway. A Cfh variant (Y402H) increases the risk for AMD. The purpose of this study was to develop a pathophysiologically relevant animal model of AMD based on this genetic risk factor. METHODS The authors generated chimeric Cfh transgenic mouse lines using two constructs consisting of the human CFH sequence for SCR6-8 (with either 402Y or 402H), flanked by the mouse sequence for SCR1-5 and SCR9-20. They tested the expression of the transgenic mRNA and protein molecules and examined the mice at 12 to 14 months of age for clinical and histologic retinal changes. RESULTS Nuclease protection assay and qRT-PCR analysis demonstrated transgenic mRNA expression in the liver and in the posterior segment of the eye. Western blot analysis showed that the transgenic proteins are present in the circulation at levels comparable to those of mouse Cfh. The chimeric proteins were found to be functional, as demonstrated by their ability to restore physiological serum levels of complement component C3 in Cfh KO mice. Clinical examination showed subretinal drusen-like deposits. Histology demonstrated an accumulation of subretinal cells that stained with a macrophage/microglia marker. Basal laminar deposits, long-spaced collagen, and increased numbers of lipofuscin granules were seen on electron microscopy. Immunohistochemistry showed a thicker sub-RPE band of C3d staining. CONCLUSIONS Chimeric Cfh proteins led to AMD-like characteristics in mice. This may represent a good model for studying the role of complement and other components of the immune system in early AMD.

Deficiency of rds/peripherin causes photoreceptor death in mouse models of digenic and dominant retinitis pigmentosa

Retinitis pigmentosa (RP) is a group of inherited blinding diseases caused by mutations in multiple genes including RDS. RDS encodes rds/peripherin (rds), a 36-kDa glycoprotein in the rims of rod and cone outer-segment (OS) discs. Rom1 is related to rds with similar membrane topology and the identical distribution in OS. In contrast to RDS, no mutations in ROM1 alone have been associated with retinal disease. However, an unusual digenic form of RP has been described. Affected individuals in several families were doubly heterozygous for a mutation in RDS causing a leucine 185 to proline substitution in rds (L185P) and a null mutation in ROM1. Neither mutation alone caused clinical abnormalities. Here, we generated transgenic/knockout mice that duplicate the amino acid substitutions and predicted levels of rds and rom1 in patients with RDS-mediated digenic and dominant RP. Photoreceptor degeneration in the mouse model of digenic RP was faster than in the wild-type and monogenic controls by histological, electroretinographic, and biochemical analysis. We observed a positive correlation between the rate of photoreceptor loss and the extent of OS disorganization in mice of several genotypes. Photoreceptor degeneration in RDS-mediated RP appears to be caused by a simple deficiency of rds and rom1. The critical threshold for the combined abundance of rds and rom1 is ≈60% of wild type. Below this value, the extent of OS disorganization results in clinically significant photoreceptor degeneration.

A Stargardt disease-3 mutation in the mouse Elovl4 gene causes retinal deficiency of C32-C36 acyl phosphatidylcholines.

Stargardt disease‐3 (STGD3) is a juvenile dominant macular degeneration caused by mutations in elongase of very long chain fatty acid‐4. All identified mutations produce a truncated protein which lacks a motif for protein retention in endoplasmic reticulum, the site of fatty acid synthesis. In these studies of Stgd3‐knockin mice carrying a human pathogenic mutation, we examined two potential pathogenic mechanisms: truncated protein‐induced cellular stress and lipid product deficiency. Analysis of mutant retinas detected no cellular stress but demonstrated selective deficiency of C32–C36 acyl phosphatidylcholines. We conclude that this deficit leads to the human STGD3 pathology.

Regulator of G Protein Signaling (RGS16) Inhibits Hepatic Fatty Acid Oxidation in a Carbohydrate Response Element-binding Protein (ChREBP)-dependent Manner

G protein-coupled receptor (GPCR) pathways control glucose and fatty acid metabolism and the onset of obesity and diabetes. Regulators of G protein signaling (RGS) are GTPase-activating proteins (GAPs) for Gi and Gq α-subunits that control the intensity and duration of GPCR signaling. Herein we determined the role of Rgs16 in GPCR regulation of liver metabolism. Rgs16 is expressed during the last few hours of the daily fast in periportal hepatocytes, the oxygen-rich zone of the liver where lipolysis and gluconeogenesis predominate. Rgs16 knock-out mice had elevated expression of fatty acid oxidation genes in liver, higher rates of fatty acid oxidation in liver extracts, and higher plasma β-ketone levels compared with wild type mice. By contrast, transgenic mice that overexpressed RGS16 protein specifically in liver exhibited reciprocal phenotypes as well as low blood glucose levels compared with wild type littermates and fatty liver after overnight fasting. The transcription factor carbohydrate response element-binding protein (ChREBP), which induces fatty acid synthesis genes in response to high carbohydrate feeding, was unexpectedly required during fasting for maximal Rgs16 transcription in liver and in cultured primary hepatocytes during gluconeogenesis. Thus, RGS16 provides a signaling mechanism for glucose production to inhibit GPCR-stimulated fatty acid oxidation in hepatocytes.

Analysis of the rds/peripherin·rom1 Complex in Transgenic Photoreceptors That Express a Chimeric Protein

Mice homozygous for the retinal degeneration slow (rds) mutation completely lack photoreceptor outer segments. The rds gene encodes rds/peripherin (rds), a membrane glycoprotein in the rims of rod and cone outer segment discs. rds is present as a complex with the related protein, rom1. Here, we generated transgenic mice that express a chimeric protein (rom/D2) containing the intradiscal D2 loop of rds in the context of rom1. rom/D2 was N-glycosylated, formed covalent homodimers, and interacted non-covalently with itself, rds, and rom1. The rds·rom/D2 interaction was significantly more stable than the non-covalent interaction between rds and rom1 by detergent/urea titration. Analysis of mice expressing rom/D2 revealed that rds is 2.5-fold more abundant than rom1, interacts non-covalently with itself and rom1 via the D2 loop, and forms a high order complex that may extend the entire circumference of the disc. Expression of rom/D2 fully rescued the ultrastructural phenotype inrds+/− mutant mice, but it had no effect on the phenotype in rds−/− mutants. Together, these observations explain the striking differences in null phenotypes and frequencies of disease-causing mutations between the RDS andROM1 genes.

Transgenic analysis of rds/peripherin N-glycosylation: effect on dimerization, interaction with rom1, and rescue of the rds null phenotype.

Abstract : Rds/peripherin is an integral membrane glycoprotein that is present in the rims of photoreceptor outer segment disks. In mammals, it is thought to stabilize the disk rim through heterophilic interactions with the related nonglycosylated protein rom1. Glycosylation of rds/peripherin at asparagine 229 is widely conserved in vertebrates. In this study, we investigated the role of rds/peripherin N‐glycosylation. We generated transgenic mice that expressed only S231A‐substituted rds/peripherin in their retinas. This protein was not glycosylated but formed covalent dimers with itself and with glycosylated rds/peripherin. Nonglycosylated rds/peripherin also interacted noncovalently with rom1 homodimers to form a heterooligomeric complex. The glycosylated rds/peripherin ·· rom1 complex bound to concanavalin A‐Sepharose, suggesting that the glycan is not directly involved in the interaction between these proteins. In double transgenic mice expressing normal and S231A‐substituted rds/peripherin, the mRNA‐to‐protein ratios were similar for both transgenes, indicating no effect of N‐glycosylation on rds/peripherin stability. Finally, expression of nonglycosylated rds/peripherin in transgenic mice rescued the phenotype of outer segment nondevelopment in retinal degeneration slow (rds‐/‐) null mutants. These observations indicate that N‐glycosylation of rds/peripherin is not required for its normal processing, stability, or in vivo function.

Epidermal expression of an Elovl4 transgene rescues neonatal lethality of homozygous Stargardt disease-3 mice.

Elongase of very long chain fatty acids-4 (ELOVL4) is the only mammalian enzyme known to synthesize C28-C36 fatty acids. In humans, ELOVL4 mutations cause Stargardt disease-3 (STGD3), a juvenile dominant macular degeneration. Heterozygous Stgd3 mice that carry a pathogenic mutation in the mouse Elovl4 gene demonstrate reduced levels of retinal C28-C36 acyl phosphatidylcholines (PC) and epidermal C28-C36 acylceramides. Homozygous Stgd3 mice die shortly after birth with signs of disrupted skin barrier function. In this study, we report generation of transgenic (Tg) mice with targeted Elovl4 expression driven by an epidermal-specific involucrin promoter. In homozygous Stgd3 mice, this transgene reinstates both epidermal Elovl4 expression and synthesis of two missing epidermal lipid groups: C28-C36 acylceramides and (O-linoleoyl)-omega-hydroxy C28-C36 fatty acids. Transgene expression also restores skin barrier function and rescues the neonatal lethality of homozygous Stgd3 mice. These studies establish the critical requirement for epidermal C28-C36 fatty acid synthesis for animal viability. In addition to the skin, Elovl4 is also expressed in other tissues, including the retina, brain, and testes. Thus, these mice will facilitate future studies to define the roles of C28-C36 fatty acids in the Elovl4-expressing tissues.

Polyunsaturated very-long-chain C28–C36 fatty acids and retinal physiology

Recent studies have established that retinal health depends on the presence of polyunsaturated C28–C36 fatty acids, in addition to docosahexaenoic acid (DHA, C22:6n-3). Initially characterised 20 years ago, these C28–C36 fatty acids are found as sn-1 acyl components of retinal phosphatidylcholines (PCs), which have DHA in the sn-2 position. This unique PC species is found in both rod- and cone-dominant retinas, mainly in the photoreceptor outer segments where the majority of phototransduction reactions take place. In bovine photoreceptor outer segments, this species is a significant component of lipid membranes. Its C28–C36 fatty acids account for 10 mol % of total PC fatty acids. Polyunsaturated C28–C36 fatty acids are synthesised in the retina, in contrast to eicosapentaenoic acid (EPA, C20:5n-3) and DHA which in humans are predominantly of dietary origin. Synthesis of C28–C36 fatty acids appears to be exclusively catalysed by elongase of very-long-chain fatty acids-4 (Elovl4). Mutations in Elovl4 cause Stargardt disease-3, a juvenile autosomal dominant macular degeneration. A mouse genetic model of the disease carries a human pathogenic 5 bp deletion in the mouse Elovl4 gene. It demonstrates early selective deficiency of retinal C28–C36 acyl PCs, followed later by reduced electroretinographic signals and increased accumulation of toxic N-retinylidene-N-retinylethanolamine (A2E).

Expression of Tyrosine Hydroxylase Gene in Cultured Hypothalamic Cells: Roles of Protein Kinase A and C

Abstract: In hypothalamic cells cultured in serum‐free medium, the quantity of tyrosine hydroxylase mRNA increases after treatment with an activator of the protein kinase A pathway (8‐bromoadenosine cyclic AMP, 3‐isobutyl‐1‐methylxanthine, or forskolin) or an activator of protein kinase C (12‐O‐tetradecanoylphorbol 13‐acetate or sn‐1,2‐diacylglycerol). The tyrosine hydroxylase mRNA level decreases in the cells after inhibition of protein kinase C with calphostin C or after depletion of protein kinase C by extended phorbol ester treatment. These data suggest that both protein kinase pathways regulate tyrosine hydroxylase gene expression in hypothalamic cells. As simultaneous activation of both pathways has less than an additive effect on the tyrosine hydroxylase mRNA level, they appear to be interrelated. Compared with the rapid and dramatic increase of the tyrosine hydroxylase mRNA level in pheochromocytoma cells, activation of the protein kinase A or protein kinase C pathway in the cultured hypothalamic cells induces slow changes of a small magnitude in the amount of tyrosine hydroxylase mRNA. The slow regulation of tyrosine hydroxylase gene expression in hypothalamic dopaminergic neurons corresponds to the relatively high stability of tyrosine hydroxylase mRNA (half‐life = 14 ± 1 h) in these cells.

Mass and activity of tyrosine hydroxylase in tuberoinfundibular dopaminergic neurons of the aged brain. Control by prolactin and ovarian hormones

The roles of prolactin (PRL) and the ovarian hormones, estradiol and progesterone, in the control of tuberoinfundibular dopaminergic neurons of aged female rats were investigated. The in situ molar activity of tyrosine hydroxylase (TH) in neurites of these neurons was assayed by measuring the rate of accumulation of L-dihydroxyphenylalanine in the median eminence following the administration of a L-dihydroxiphenylalanine decarboxylase inhibitor. The TH mass was measured by an immunoblot assay using rat TH as the standard. Pituitary implants in aged ovariectomized animals resulted in a significant increase in the median eminence of both the mass and in situ molar activity of TH. When circulating PRL of aged rats was neutralized by administration of antiserum against rat PRL, the activity of TH was reduced significantly compared to that of animals treated with preimmune serum. In aged ovariectomized rats treated with both estradiol and progesterone, the in situ molar activity of TH increased significantly compared to animals treated only with the solvent vehicle, estradiol, or progesterone. The stimulatory effect of estradiol and progesterone appeared to be mediated through a mechanism that did not involve PRL, since neutralization of circulating PRL failed to prevent an increase in TH activity in estradiol-progesterone-treated animals. None of these treatments affected the in situ activity of TH in the superior cervical ganglion. We conclude that PRL as well as combined estradiol-progesterone treatment have important roles in the maintenance of TH activity in aged tuberoinfundibular dopaminergic neurons.