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BBA - Molecular Basis of Disease (v.1802, #9)

Editorial Board (pp. i).

Molecular characterization of dopamine-derived quinones reactivity toward NADH and glutathione: Implications for mitochondrial dysfunction in Parkinson disease by Marco Bisaglia; Maria Eugenia Soriano; Irene Arduini; Stefano Mammi; Luigi Bubacco (pp. 699-706).

Oxidative stress and mitochondrial dysfunction, especially at the level of complex I of the electronic transport chain, have been proposed to be involved in the pathogenesis of Parkinson disease (PD). A plausible source of oxidative stress in nigral dopaminergic neurons is the redox reactions that specifically involve dopamine (DA) and produce various toxic molecules, i.e., free radicals and quinone species (DAQ). It has been shown that DA oxidation products can induce various forms of mitochondrial dysfunction, such as mitochondrial swelling and decreased electron transport chain activity. In the present work, we analyzed the potentially toxic effects of DAQ on mitochondria and, specifically, on the NADH and GSH pools. Our results demonstrate that the generation of DAQ in isolated respiring mitochondria triggers the opening of the permeability transition pore most probably by inducing oxidation of NADH, while GSH levels are not affected. We then characterized in vitro, by UV and NMR spectroscopy, the reactivity of different DA-derived quinones, i.e., dopamine-o-quinone (DQ), aminochrome (AC) and indole-quinone (IQ), toward NADH and GSH. Our results indicate a very diverse reactivity for the different DAQ studied that may contribute to unravel the complex molecular mechanisms underlying oxidative stress and mitochondria dysfunction in the context of PD.► Evaluation of the toxic effects of dopamine quinones (DAQ) on mitochondria. ► A very diverse reactivity was observed for the different DAQ investigated. ► DAQ triggers the opening of the permeability transition pore. ► DAQ induce oxidation of NADH, while GSH levels are not affected.

Keywords: Dopamine quinone; Glutathione; Mitochondria; NADH; NMR

Effects of ALS-related SOD1 mutants on dynein- and KIF5-mediated retrograde and anterograde axonal transport by Ping Shi; Strom Anna-Lena Ström; Jozsef Gal; Haining Zhu (pp. 707-716).

Transport of material and signals between extensive neuronal processes and the cell body is essential to neuronal physiology and survival. Slowing of axonal transport has been shown to occur before the onset of symptoms in amyotrophic lateral sclerosis (ALS). We have previously shown that several familial ALS-linked copper–zinc superoxide dismutase (SOD1) mutants (A4V, G85R, and G93A) interacted and colocalized with the retrograde dynein–dynactin motor complex in cultured cells and affected tissues of ALS mice. We also found that the interaction between mutant SOD1 and the dynein motor played a critical role in the formation of large inclusions containing mutant SOD1. In this study, we showed that, in contrast to the dynein situation, mutant SOD1 did not interact with anterograde transport motors of the kinesin-1 family (KIF5A, B and C). Using dynein and kinesin accumulation at the sciatic nerve ligation sites as a surrogate measurement of axonal transport, we also showed that dynein mediated retrograde transport was slower in G93A than in WT mice at an early presymptomatic stage. While no decrease in KIF5A-mediated anterograde transport was detected, the slowing of anterograde transport of dynein heavy chain as a cargo was observed in the presymptomatic G93A mice. The results from this study along with other recently published work support that mutant SOD1 might only interact with and interfere with some kinesin members, which, in turn, could result in the impairment of a selective subset of cargos. Although it remains to be further investigated how mutant SOD1 affects different axonal transport motor proteins and various cargos, it is evident that mutant SOD1 can induce defects in axonal transport, which, subsequently, contribute to the propagation of toxic effects and ultimately motor neuron death in ALS.

Keywords: Abbreviations; ALS; amyotrophic lateral sclerosis; SOD1; copper–zinc superoxide dismutase; FBS; fetal bovine serum; PBS; phosphate buffered saline; GST; glutathione; S; -transferase; WT; wild type; DHC; dynein heavy chain; DIC; dynein intermediate chain; KHC; kinesin heavy chain; KLC; kinesin light chainAmyotrophic lateral sclerosis; Copper–zinc superoxide dismutase; Axonal transport; Kinesin; Dynein

Celiac anti-tissue transglutaminase antibodies interfere with the uptake of alpha gliadin peptide 31–43 but not of peptide 57–68 by epithelial cells by Ivana Caputo; Maria Vittoria Barone; Marilena Lepretti; Stefania Martucciello; Ivan Nista; Riccardo Troncone; Salvatore Auricchio; Daniele Sblattero; Carla Esposito (pp. 717-727).

Celiac disease is characterized by the secretion of IgA-class autoantibodies that target tissue transglutaminase (tTG). It is now recognized that anti-tTG antibodies are functional and not mere bystanders in the pathogenesis of celiac disease. Here we report that interaction between anti-tTG antibodies and extracellular membrane-bound tTG inhibits peptide 31–43 (but not peptide 57–68) uptake by cells, thereby impairing the ability of p31–43 to drive Caco-2 cells into S-phase. This effect did not involve tTG catalytic activity. Because anti-tTG antibodies interfered with epidermal growth factor endocytosis, we assume that they exert their effect by reducing peptide 31–43 endocytosis. Our results suggest that cell-surface tTG plays a hitherto unknown role in the regulation of gliadin peptide uptake and endocytosis.

Keywords: Abbreviations; tTG; tissue transglutaminase; CD; celiac disease; p31–43; peptide 31–43; p57–68; peptide 57–68; PBS; phosphate-buffered saline; PFA; paraformaldehyde; BrdU; bromodeoxyuridine; EGF; epidermal growth factor; ERK; extracellular signal-regulated kinase; FBS; fetal bovine serum; M-β-CD; methyl-β-cyclodextrin; MDC; monodansylcadaverineTransglutaminase; Anti-transglutaminase antibody; Gliadin peptide; Celiac disease; Endocytosis

Carnitine palmitoyltransferase 2: New insights on the substrate specificity and implications for acylcarnitine profiling by Sara Violante; Lodewijk IJlst; Henk van Lenthe; Isabel Tavares de Almeida; Ronald J. Wanders; Fátima V. Ventura (pp. 728-732).

Over the last years acylcarnitines have emerged as important biomarkers for the diagnosis of mitochondrial fatty acid β-oxidation (mFAO) and branched-chain amino acid oxidation disorders assuming they reflect the potentially toxic acyl-CoA species, accumulating intramitochondrially upstream of the enzyme block. However, the origin of these intermediates still remains poorly understood. A possibility exists that carnitine palmitoyltransferase 2 (CPT2), member of the carnitine shuttle, is involved in the intramitochondrial synthesis of acylcarnitines from accumulated acyl-CoA metabolites. To address this issue, the substrate specificity profile of CPT2 was herein investigated. Saccharomyces cerevisiae homogenates expressing human CPT2 were incubated with saturated and unsaturated C2–C26 acyl-CoAs and branched-chain amino acid oxidation intermediates. The produced acylcarnitines were quantified by ESI-MS/MS. We show that CPT2 is active with medium (C8–C12) and long-chain (C14–C18) acyl-CoA esters, whereas virtually no activity was found with short- and very long-chain acyl-CoAs or with branched-chain amino acid oxidation intermediates. Trans-2-enoyl-CoA intermediates were also found to be poor substrates for CPT2. Inhibition studies performed revealed that trans-2-C16:1-CoA may act as a competitive inhibitor of CPT2 ( K_i of 18.8μM). The results obtained clearly demonstrate that CPT2 is able to reverse its physiological mechanism for medium and long-chain acyl-CoAs contributing to the abnormal acylcarnitines profiles characteristic of most mFAO disorders. The finding that trans-2-enoyl-CoAs are poorly handled by CPT2 may explain the absence of trans-2-enoyl-carnitines in the profiles of mitochondrial trifunctional protein deficient patients, the only defect where they accumulate, and the discrepancy between the clinical features of this and other long-chain mFAO disorders such as very long-chain acyl-CoA dehydrogenase deficiency.

Keywords: Abbreviations; mFAO; mitochondrial fatty acid β-oxidation; CPT1; carnitine palmitoyltransferase 1; CPT2; carnitine palmitoyltransferase 2; CACT; carnitine/acylcarnitine translocase; MTP; mitochondrial trifunctional protein; LCHAD; long-chain 3-hydroxyacyl-CoA dehydrogenase; VLCAD; very long-chain acyl-CoA dehydrogenase; DMN; dimethylnonanoate; DMH; dimethylheptanoate; Prist-CoA; Pristanoyl-CoAAcylcarnitine; Carnitine palmitoyltransferase 2 (CPT2); Long-chain acyl-CoA; Fatty acid oxidation (FAO); Mitochondrial Trifunctional Protein (MTP)

Activation of ASK1, downstream MAPKK and MAPK isoforms during cardiac ischaemia by Stephen J. Harding; Gareth J. Browne; Bryan W. Miller; Sally A. Prigent; Martin Dickens (pp. 733-740).

p38 MAPK is activated potently during cardiac ischaemia, although the precise mechanism by which it is activated is unclear. We used the isolated perfused rat heart to investigate the signalling pathways activated upstream of p38 during global cardiac ischaemia. Ischaemia strongly activated p38α but not the JNK pathway. The MAPKKs, MKK3, MKK4 and MKK6 have previously been identified as potential upstream activators of p38; however, in the ischaemic perfused heart, we saw activation of MKK3 and MKK6 but not MKK4. MKK3 and MKK6 showed different temporal patterns of activity, indicating distinct modes of activation and physiological function. Consistent with a lack of JNK activation, we saw no activation of MKK4 or MKK7 at any time point during ischaemia. A lack of MKK4 activation indicates, at least in the ischaemic heart, that MKK4 is not a physiologically relevant activator of p38. The MAPKKK, ASK1, was strongly activated late during ischaemia, with a similar time course to that of MKK6 and in ischaemic neonatal cardiac myocytes ASK1 expression preferentially activated MKK6 rather than MKK3. These observations suggest that during ischaemia ASK1 is coupled to p38 activation primarily via MKK6. Potent activation of ASK1 during ischaemia without JNK activation shows that during cardiac ischaemia, ASK1 preferentially activates the p38 pathway. These results demonstrate a specificity of responses seldom seen in previous studies and illustrate the benefits of using direct assays in intact tissues responding to physiologically relevant stimuli to unravel the complexities of MAPK signalling.

Keywords: Abbreviations; ASK1; apoptosis-sensitive kinase; MAPK; mitogen-activated protein kinase; MAPKK/MKK; MAPK kinase; MAPKKK; MAPKK kinase; JNK; c-Jun N-terminal kinase; ERK; extracellularly regulated kinase; SAPK; stress-activated protein kinase; MEKK; MAPK/ERK kinase; MLK; mixed lineage kinase; TAK; TGF β-activated kinase; GCK; germinal centre kinase; Tpl-2; tumour progression locus-2; PAK; p21-activated protein kinase; TAO Kinase; thousand and one kinase; DTT; dithiothreitol; BSA; bovine serum albumin; GST; glutathione S-transferaseIschaemia; Heart; ASK1; JNK; p38; MAPK

Plasma globotriaosylsphingosine: Diagnostic value and relation to clinical manifestations of Fabry disease by S.M. Rombach; N. Dekker; M.G. Bouwman; G.E. Linthorst; A.H. Zwinderman; F.A. Wijburg; S. Kuiper; M.A. vd Bergh Weerman; J.E.M. Groener; B.J. Poorthuis; C.E.M. Hollak; J.M.F.G. Aerts (pp. 741-748).

Fabry disease is an X-linked lysosomal storage disorder due to deficiency of alpha-Galactosidase A, causing accumulation of globotriaosylceramide and elevated plasma globotriaosylsphingosine (lysoGb3). The diagnostic value and clinical relevance of plasma lysoGb3 concentration was investigated. All male and adult female patients with classical Fabry disease could be discerned by an elevated plasma lysoGb3. In young pre-symptomatic Fabry heterozygotes, lysoGb3 levels can be normal. Individuals carrying the R112H and P60L mutations, without classical Fabry symptoms, showed no elevated plasma lysoGb3. Multiple regression analysis showed that there is no correlation of plasma lysoGb3 concentration with total disease severity score in Fabry males. However, plasma lysoGb3 concentration did correlate with white matter lesions (odds ratio: 6.1 per 100 nM lysoGb3 increase (95% CI: 1.4–25.9, p=0.015). In females, plasma lysoGb3 concentration correlated with overall disease severity. Furthermore, plasma lysoGb3 level was related to left ventricular mass (19.5±5.5g increase per 10nM lysoGb3 increase; p=0.001). In addition, it was assessed whether lifetime exposure to lysoGb3 correlates with disease manifestations. Male Fabry patients with a high lysoGb3 exposure (>10,000U), were moderately or severely affected, only one mildly. Female patients with a low exposure (<1000U) were asymptomatic or mildly affected. A large proportion of the females with an exposure >1000U showed disease complications. Plasma lysoGb3 is useful for the diagnosis of Fabry disease. LysoGb3 is an independent risk factor for development of cerebrovascular white matter lesions in male patients and left ventricular hypertrophy in females. Disease severity correlates with exposure to plasma lysoGb3.

Keywords: Fabry disease; Lysoglycolipids; Pathogenesis

DNA-binding and transcriptional activities of human HSF4 containing mutations that associate with congenital and age-related cataracts by Yasuaki Enoki; Yuka Mukoda; Chie Furutani; Hiroshi Sakurai (pp. 749-753).

Heat shock transcription factor HSF4 is necessary for ocular lens development and fiber cell differentiation. Mutations of the human HSF4 gene have been implicated in congenital and age-related cataracts. Here, we show that HSF4 activates transcription of genes encoding crystallins and beaded filament structural proteins in lens epithelial cells. Five missense mutations that have been associated with congenital cataract inhibited DNA-binding of HSF4, which demonstrates the relationship between HSF4 mutations, loss of lens protein gene expression, and cataractogenesis. However, two missense mutations that have been associated with age-related cataract did not or only slightly alter HSF4 activity, implying that other genetic and environmental factors affect the functions of these mutant proteins.

Keywords: HSF4; Congenital cataract; Age-related cataract; Crystallin; Beaded filament structural protein

The levels of both lipid rafts and raft-located acetylcholinesterase dimers increase in muscle of mice with muscular dystrophy by merosin deficiency by María Teresa Moral-Naranjo; María Fernanda Montenegro; Munoz-Delgado Encarnación Muñoz-Delgado; Francisco J. Campoy; Cecilio J. Vidal (pp. 754-764).

Wild type and dystrophic (merosin-deficient) Lama2dy mice muscles were compared for their density of lipid rafts. The 5-fold higher level of caveolin-3 and the 2-3 times higher level of ecto-5’-nucleotidase activity in raft preparations (Triton X-100-resistant membranes) of dystrophic muscle supported expansion of caveolar and non-caveolar lipid rafts. The presence in rafts of glycosylphosphatidylinositol (GPI)-linked acetylcholinesterase (AChE) dimers, which did not arise from erythrocyte or nerve, not only revealed for the first time the capacity of the myofibre for translating the AChE-H mRNA but also an unrecognized pathway for targeting AChE-H to specialized membrane domains of the sarcolemma. Rafts of dystrophic muscle contained a 5-fold higher AChE activity/mg protein. RT-PCR for 3’-alternative mRNAs of AChE revealed AChE-T mRNA prevailing over AChE-R and AChE-H mRNAs in wild type mouse muscle. It also displayed principal 5’-alternative AChE mRNAs with exons E1c and E1e (the latter coding for N-terminally extended subunits) and fewer with E1d, E1a and E1b. The levels of AChE and butyrylcholinesterase mRNAs were unaffected by dystrophy. Finally, the decreased level of proline-rich membrane anchor (PRiMA) mRNA in Lama2dy muscle provided for a rational explanation to the loss of PRiMA-bearing AChE tetramers in dystrophic muscle.

Keywords: Caveolae; Cholinesterase; Ecto-5’-nucleotidase (CD73); Muscular dystrophy

Caenorhabditis elegans, a pluricellular model organism to screen new genes involved in mitochondrial genome maintenance by Matthew Glover Addo; Raynald Cossard; Damien Pichard; Kwasi Obiri-Danso; Rotig Agnès Rötig; Agnès Delahodde (pp. 765-773).

The inheritance of functional mitochondria depends on faithful replication and transmission of mitochondrial DNA (mtDNA). A large and heterogeneous group of human disorders is associated with mitochondrial genome quantitative and qualitative anomalies. Several nuclear genes have been shown to account for these severe OXPHOS disorders. However, in several cases, the disease-causing mutations still remain unknown. Caenorhabditis elegans has been largely used for studying various biological functions because this multicellular organism has short life cycle and is easy to grow in the laboratory. Mitochondrial functions are relatively well conserved between human and C. elegans, and heteroplasmy exists in this organism as in human. C. elegans therefore represents a useful tool for studying mtDNA maintenance. Suppression by RNA interference of genes involved in mtDNA replication such as polg-1, encoding the mitochondrial DNA polymerase, results in reduced mtDNA copy number but in a normal phenotype of the F1 worms. By combining RNAi of genes involved in mtDNA maintenance and EtBr exposure, we were able to reveal a strong and specific phenotype (developmental larval arrest) associated to a severe decrease of mtDNA copy number. Moreover, we tested and validated the screen efficiency for human orthologous genes encoding mitochondrial nucleoid proteins. This allowed us to identify several genes that seem to be closely related to mtDNA maintenance in C. elegans.This work reports a first step in the further development of a large-scale screening in C. elegans that should allow to identify new genes of mtDNA maintenance whose human orthologs will obviously constitute new candidate genes for patients with quantitative or qualitative mtDNA anomalies.

Keywords: Mitochondria; mtDNA maintenance; Nematode/; Caenorhabditis elegans

Analysis of TSC1 truncations defines regions involved in TSC1 stability, aggregation and interaction by Marianne Hoogeveen-Westerveld; Carla Exalto; Anneke Maat-Kievit; Ans van den Ouweland; Dicky Halley; Mark Nellist (pp. 774-781).

Tuberous sclerosis complex (TSC) is an autosomal dominant disorder characterised by the development of hamartomas in a variety of organs and tissues. The disease is caused by mutations in either the TSC1 gene on chromosome 9q34, or the TSC2 gene on chromosome 16p13.3. The TSC1 and TSC2 gene products, TSC1 and TSC2, interact to form a protein complex that inhibits signal transduction to the downstream effectors of the target of rapamycin complex 1 (TORC1). Here we investigate TSC1 structure and function by analysing a series of truncated TSC1 proteins. We identify specific regions of the protein that are important for TSC1 stability, localisation, interactions and function.

Keywords: Abbreviations; TSC; tuberous sclerosis complex; RHEB; ras homolog expressed in brain; TORC1; target of rapamycin complex 1; S6K; p70 S6 kinase; GAP; GTPase activating proteinTuberous sclerosis complex; TSC1; TSC2; TORC1

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BBA - Molecular Basis of Disease (v.1802, #9)