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BBA - Molecular Basis of Disease (v.1782, #6)

Editorial Board (pp. ii).

Therapeutic role of sirtuins in neurodegenerative disease by Tiago Fleming Outeiro; Oldriska Marques; Aleksey Kazantsev (pp. 363-369).

The sirtuins are a family of enzymes which control diverse and vital cellular functions, including metabolism and aging. Manipulations of sirtuin activities cause activation of anti-apoptotic, anti-inflammatory, anti-stress responses, and the modulation of an aggregation of proteins involved in neurodegenerative disorders. Recently, sirtuins were found to be disease-modifiers in various models of neurodegeneration. However, almost in all instances, the exact mechanisms of neuroprotection remain elusive. Nevertheless, the manipulation of sirtuin activities is appealing as a novel therapeutic strategy for the treatment of currently fatal human disorders such as Alzheimer's and Parkinson's diseases. Here, we review current data which support the putative therapeutic roles of sirtuin in aging and in neurodegenerative diseases and the feasibility of the development of sirtuin-based therapies.

Keywords: Sirtuins; Neurodegeneration; Therapeutic targets; Aging; Metabolism

Latrunculin B facilitates Shiga toxin 1 transcellular transcytosis across T84 intestinal epithelial cells by Irina Maluykova; Oksana Gutsal; Marina Laiko; Anne Kane; Mark Donowitz; Olga Kovbasnjuk (pp. 370-377).

Shiga toxins (Stx), released into the intestinal lumen by enterohemorrhagic Escherichia coli (EHEC), are major virulence factors responsible for gastrointestinal and systemic illnesses. These pathologies are believed to be due to the action of the toxins on endothelial cells, which express the Stx receptor, the glycosphingolipid Gb3. To reach the endothelial cells, Stx must translocate across the intestinal epithelial monolayer. This process is poorly understood. We investigated Stx1 movement across the intestinal epithelial T84 cell model and the role of actin turnover in this transcytosis. We showed that changes in the actin cytoskeleton due to latrunculin B, but not cytochalasin D or jasplakinolide, significantly facilitate toxin transcytosis across T84 monolayers. This trafficking is transcellular and completely inhibited by tannic acid, a cell impermeable plasma membrane fixative. This indicates that actin turnover could play an important role in Stx1 transcellular transcytosis across intestinal epithelium in vitro. Since EHEC attachment to epithelial cells causes an actin rearrangement, this finding may be highly relevant to Stx-induced disease.

Keywords: Shiga toxin; Intestinal epithelial cells; Transcytosis; Actin turnover; Latrunculin B

Five human phenylalanine hydroxylase proteins identified in mild hyperphenylalaninemia patients are disease-causing variants by Aurora Daniele; Giuseppe Cardillo; Cinzia Pennino; Maria T. Carbone; Domenico Scognamiglio; Luciana Esposito; Antonio Correra; Giuseppe Castaldo; Adriana Zagari; Francesco Salvatore (pp. 378-384).

Hyperphenylalaninemia is a group of autosomal recessive disorders caused by a wide range of phenylalanine hydroxylase (PAH) gene variants. To study the effects of mutations on PAH activity, we have reproduced five mutations (p.N223Y, p.R297L, p.F382L, p.K398N and p.Q419R) that we recently identified in a population of Southern Italy. Transient expression of mutant full-length cDNAs in human HEK293 cells yielded PAH variants whosel-phenylalanine hydroxylase activity was between 40% and 70% that of the wild-type enzyme. Moreover, Western blot analysis revealed a 50-kD monomer in all mutants thereby indicating normal synthesis of the mutant proteins. Because of the clinical mild nature of the phenotypes we performed an in vivo BH4 loading test. This was positive in all tested patients, which indicates that they are likely to respond to the coenzyme in vivo. We also analysed the environment of each mutation site in the available crystal structures of PAH by using molecular graphics tools. The structural alteration produced by each mutation was elucidated and correlated to the mutated properties of the mutant enzymes. All the data obtained demonstrate the disease-causing nature of the five novel variants.

Keywords: PKU; PKU mutations; PKU mutations functional analysis; Expression studies of PKU mutants; Structural analysis

Transport and distribution of 3-hydroxyglutaric acid before and during induced encephalopathic crises in a mouse model of glutaric aciduria type 1 by Britta Keyser; Markus Glatzel; Franziska Stellmer; Bastian Kortmann; Zoltan Lukacs; Kolker Stefan Kölker; Sven W. Sauer; Nicole Muschol; Wilhelm Herdering; Joachim Thiem; Stephen I. Goodman; David M. Koeller; Kurt Ullrich; Thomas Braulke; Muhlhausen Chris Mühlhausen (pp. 385-390).

Glutaric aciduria type 1 (GA1) is caused by the deficiency of glutaryl-CoA dehydrogenase (GCDH). Affected patients are prone to the development of encephalopathic crises during an early time window with destruction of striatal neurons and a subsequent irreversible movement disorder. 3-Hydroxyglutaric acid (3OHGA) accumulates in tissues and body fluids of GA1 patients and has been shown to mediate toxic effects on neuronal as well as endothelial cells. Injection of (³H)-labeled into 6 week-old Gcdh^−/− mice, a model of GA1, revealed a low recovery in kidney, liver, or brain tissue that did not differ from control mice. Significant amounts of 3OHGA were found to be excreted via the intestinal tract. Exposure of Gcdh^−/− mice to a high protein diet led to an encephalopathic crisis, vacuolization in the brain, and death after 4–5 days. Under these conditions, high amounts of injected³H-3OHGA were found in kidneys of Gcdh^−/− mice, whereas the radioactivity recovered in brain and blood was reduced. The data demonstrate that under conditions mimicking encephalopathic crises the blood–brain barrier appears to remain intact.

Keywords: Radiolabeled metabolite; Glutaric aciduria type 1; Mouse model; Metabolite distribution; Glutaryl-CoA dehydrogenase deficiency; Metabolic crisis

Polymorphisms in the promoter regions for human MMP-1 and MMP-13 lead to differential responses to the alpha and beta isoforms of estrogen receptor and their ligand in vitro by Yamini Achari; Ting Lu; David A. Hart (pp. 391-400).

Estrogen receptors (ER) are present in connective tissues and therefore it is possible that the loss of estrogen after menopause influences the integrity of these tissues, contributing to development of degenerative conditions such as osteoporosis and osteoarthritis in a subset of women. Aberrant expression of matrix metalloproteinases (e.g. MMP-1 and MMP-13) has been implicated in the progression of these diseases. The present study investigated potential molecular mechanisms involved in the regulation of expression of MMP-1 and MMP-13 promoter variants by ER-α and ER-β (+/−estrogen) in a transient transfection system. The results demonstrate that the activity of human MMP-1 and MMP-13 polymorphic variants is elevated in the presence of ER-α and ER-β, and the single nucleotide polymorphisms present in the promoters of MMP-1 and MMP-13 variants leads to differential activities in response to the ER isoforms. Furthermore, the influence of 17-β estradiol also varies depending upon whether the alpha or the beta isoform of ER is the modulator of these polymorphic variants. These findings support the conclusion that ER isoforms may be contributing to disease development and/or progression in genetically distinct subsets of women following menopause, and provide mechanistic insights into how such contributions are manifested.

Keywords: Polymorphism; MMP-13; ER-α; ER-β; 1G; 2G; Gene regulation

Reproductive hormones regulate the selective permeability of the blood-brain barrier by Andrea C. Wilson; Luca Clemente; Tianbing Liu; Richard L. Bowen; Sivan Vadakkadath Meethal; Craig S. Atwood / (pp. 401-407).

Reproductive hormones have been demonstrated to modulate both gap and tight junction protein expression in the ovary and other reproductive tissues, however the effects of changes in reproductive hormones on the selective permeability of the blood-brain barrier (BBB) remain unclear. Age-related declines in BBB integrity correlate with the loss of serum sex steroids and increase in gonadotropins with menopause/andropause. To examine the effect of reproductive senescence on BBB permeability and gap and tight junction protein expression/localization, female mice at 3 months of age were either sham operated (normal serum E₂ and gonadotropins), ovariectomized (low serum E₂ and high serum gonadotropins) or ovariectomized and treated with the GnRH agonist leuprolide acetate (low serum E₂ and gonadotropins). Ovariectomy induced a 2.2-fold increase in Evan's blue dye extravasation into the brain. The expression and localization of the cytoplasmic membrane-associated tight junction protein zona occludens 1 (ZO-1) in microvessels was not altered among groups indicating that the increased paracellular permeability was not due to changes in this tight junction protein. However, ovariectomy induced a redistribution of the gap junction protein connexin-43 (Cx43) such that immunoreactivity relocalized from along the extracellular microvascular endothelium to become associated with endothelial cells. An increase in Cx43 expression in the mouse brain following ovariectomy was suppressed in ovariectomized animals treated with leuprolide acetate, indicating that serum gonadotropins rather than sex steroids were modulating Cx43 expression. These results suggest that elevated serum gonadotropins following reproductive senescence may be one possible cause of the loss of selective permeability of the BBB at this time. Furthermore, these findings implicate Cx43 in mediating changes in BBB permeability, and serum gonadotropins in the cerebropathophysiology of age-related neurodegenerative diseases such as stroke and Alzheimer's disease.

Keywords: Blood-brain barrier; Selective permeability; Ovariectomy; Tight junction; Gap junction; Connexin-43; Zona occludens 1; Luteinizing hormone; Follicle-stimulating hormone; Sex steroid; Leuprolide acetate; Receptor; Stroke; Alzheimer's disease

Rescue of mutant α-galactosidase A in the endoplasmic reticulum by 1-deoxygalactonojirimycin leads to trafficking to lysosomes by Ryoji Hamanaka; Tetsuji Shinohara; Shinji Yano; Miki Nakamura; Aiko Yasuda; Shigeo Yokoyama; Jian-Qiang Fan; Kunito Kawasaki; Makoto Watanabe; Satoshi Ishii (pp. 408-413).

Active-site-specific chaperone therapy for Fabry disease is a genotype-specific therapy using a competitive inhibitor, 1-deoxygalactonojirimycin (DGJ). To elucidate the mechanism of enhancing α-galactosidase A (α-Gal A) activity by DGJ-treatment, we studied the degradation of a mutant protein and the effect of DGJ in the endoplasmic reticulum (ER). We first established an in vitro translation and translocation system using rabbit reticulocyte lysates and canine pancreas microsomal vesicles for a study on the stability of mutant α-Gal A with an amino acid substitution (R301Q) in the ER. R301Q was rapidly degraded, but no degradation of wild-type α-Gal A was observed when microsomal vesicles containing wild-type or R301Q α-Gal A were isolated and incubated. A pulse-chase experiment on R301Q-expressing TgM/KO mouse fibroblasts showed rapid degradation of R301Q, and its degradation was blocked by the addition of lactacystin, indicating that R301Q was degraded by ER-associated degradation (ERAD). Rapid degradation of R301Q was also observed in TgM/KO mouse fibroblasts treated with brefeldin A, and the amount of R301Q enzyme markedly increased by pretreatment with DGJ starting 12 h prior to addition of brefeldin A. The enhancement of α-Gal A activity and its protein level by DGJ-treatment was selectively observed in brefeldin A-treated COS-7 cells expressing R301Q but not in cells expressing the wild-type α-Gal A. Observation by immunoelectron microscopy showed that the localization of R301Q in COS-7 cells was in the lysosomes, not the ER. These data suggest that the rescue of R301Q from ERAD is a key step for normalization of intracellular trafficking of R301Q.

Keywords: Abbreviations; α-Gal A; α-galactosidase A; DGJ; 1-deoxygalactonojirimycin; ASSC; active-site-specific chaperone; ER; endoplasmic reticulum; DTT; dithiothreitol; Endo H; endoglycosidase H; ERAD; ER-associated degradation; ERT; enzyme replacement therapy; TEA; triethanolamine; RM; rough microsomes; DMEM; Dulbecco's modified Eagle's medium; FBS; fetal bovine serum; SDS-PAGE; sodium dodecyl sulfate-polyacrylamide gel electrophoresis; PBS; phosphate buffered saline; BSA; bovine serum albumin; HRP; horseradish peroxidase; EDEM; ER degradation enhancing α-mannosidase-like proteinα-galactosidase A; Active-site-specific chaperone; Fabry disease; In vitro; translation; Immunoelectron microscopy

Deletion of apolipoprotein E gene modifies the rate of depletion of alpha tocopherol (vitamin E) from mice brains by Govind T. Vatassery; Hung T. Quach; W. Ed Smith; Michael Kuskowski (pp. 414-420).

Our previous reports show that apolipoprotein E (apoE) influences the dynamics of alpha tocopherol (vitamin E) in brain. In this investigation, the patterns of depletion of alpha tocopherol from tissues of apoE deficient and wild type mice were compared after the animals were fed vitamin E deficient diets. Alpha tocopherol concentrations in specific regions of the brain and peripheral tissues at different times were determined by HPLC with electrochemical detection. ApoE deficiency significantly retarded the rate of depletion of alpha tocopherol from all regions of the brain. In addition, comparison of the rates of depletion of alpha tocopherol in both apoE deficient and wild type animals showed that cerebellum behaved differently from other areas such as cortex, hippocampus and striatum. This reinforces the uniqueness of cerebellum with regard to vitamin E biology. Patterns of depletion of tocopherol from peripheral tissues were different from brain. Serum tocopherol was higher in apoE deficient animals and remained higher than wild type during E deficiency. Depletion of liver tocopherol also tended to be unaffected by apoE deficiency. Our current and previous observations strongly suggest that apoE has an important role in modulating tocopherol concentrations in brain, probably acting in concert with other proteins as well.

Keywords: Vitamin E; Apolipoprotein E; Brain; Vitamin E deficiency; Cerebellum

Effect of lipid peroxidation products on the activity of human retinol dehydrogenase 12 (RDH12) and retinoid metabolism by Seung-Ah Lee; Olga V. Belyaeva; Natalia Y. Kedishvili (pp. 421-425).

Mutations in human Retinol Dehydrogenase 12 (RDH12) are known to cause photoreceptor cell death but the physiological function of RDH12 in photoreceptors remains poorly understood. In vitro, RDH12 recognizes both retinoids and medium-chain aldehydes as substrates. Our previous study suggested that RDH12 protects cells against toxic levels of retinaldehyde and retinoic acid [S.A. Lee, O.V. Belyaeva, I.K. Popov, N.Y. Kedishvili, Overproduction of bioactive retinoic acid in cells expressing disease-associated mutants of retinol dehydrogenase 12, J. Biol. Chem. 282 (2007) 35621–35628]. Here, we investigated whether RDH12 can also protect cells against highly reactive medium-chain aldehydes. Analysis of cell survival demonstrated that RDH12 was protective against nonanal but not against 4-hydroxynonenal. At high concentrations, nonanal inhibited the activity of RDH12 towards retinaldehyde, suggesting that nonanal was metabolized by RDH12. 4-Hydroxynonenal did not inhibit the RDH12 retinaldehyde reductase activity, but it strongly inhibited the activities of lecithin:retinol acyl transferase and aldehyde dehydrogenase, resulting in decreased levels of retinyl esters and retinoic acid and accumulation of unesterified retinol. Thus, the results of this study showed that RDH12 is more effective in protection against retinaldehyde than against medium-chain aldehydes, and that medium-chain aldehydes, especially 4-hydroxynonenal, severely disrupt cellular retinoid homeostasis. Together, these findings provide a new insight into the effects of lipid peroxidation products and the impact of oxidative stress on retinoid metabolism.

Keywords: Abbreviations; RDH12; retinol dehydrogenase 12; 4-HNE; 4-hydroxynonenal; LRAT; lecithin:retinol acyltransferase; ALDH1A1; aldehyde dehydrogenase 1 family, member A1; GSH; glutathioneRetinoic acid; Retinaldehyde; Retinol; Oxidoreductase; Nonanal; 4-hydroxynonenal

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BBA - Molecular Basis of Disease (v.1782, #6)