BBA - Molecular Basis of Disease (v.1802, #7-8)

CK2 and GSK3 phosphorylation on S29 controls wild-type ATXN3 nuclear uptake by V. Pastori; E. Sangalli; P. Coccetti; C. Pozzi; S. Nonnis; G. Tedeschi; P. Fusi (583-592).
In the present work we show that murine ATXN3 (ATXN3Q6) nuclear uptake is promoted by phosphorylation on serine 29, a highly conserved residue inside the Josephin domain. Both casein kinase 2 (CK2) and glycogen synthase kinase 3 (GSK3) are able to carry out phosphorylation on this residue. S29 phosphorylation was initially assessed in vitro on purified ATXN3Q6, and subsequently confirmed in transfected COS-7 cells, by MS analysis. Site-directed mutagenesis of S29 to an alanine was shown to strongly reduce nuclear uptake, in COS-7 transiently transfected cells overexpressing ATXN3Q6, while substitution with phospho-mimic aspartic acid restored the wild-type phenotype. Finally, treatment with CK2 and GSK3 inhibitors prevented S29 phosphorylation and strongly inhibited nuclear uptake, showing that both kinases are involved in ATXN3Q6 subcellular sorting. Although other authors have previously addressed this issue, we show for the first time that ATXN3 is phosphorylated inside the Josephin domain and that S29 phosphorylation is involved in nuclear uptake of ATXN3.
Keywords: ATXN3; Phosphorylation; CK2; GSK3; Subcellular localization; Mass spectrometry;

Ablation of LKB1 in the heart leads to energy deprivation and impaired cardiac function by Niels Jessen; Ho-Jin Koh; Clifford D. Folmes; Cory Wagg; Nobuharu Fujii; Bo Løfgren; Cordula M. Wolf; Charles I. Berul; Michael F. Hirshman; Gary D. Lopaschuk; Laurie J. Goodyear (593-600).
Energy deprivation in the myocardium is associated with impaired heart function and increased morbidity. LKB1 is a kinase that is required for activation of AMP-activated protein kinase (AMPK) as well as 13 AMPK-related protein kinases. AMPK stimulates ATP production during ischemia and prevents post-ischemic dysfunction. We used the Cre–Lox system to generate mice where LKB1 was selectively knocked out in cardiomyocytes and muscle cells (LKB1-KO) to assess the role of LKB1 on cardiac function in these mice.Heart rates of LKB1-KO mice were reduced and ventricle diameter was increased. Ex vivo, cardiac function was impaired during aerobic perfusion of isolated working hearts, and recovery of function after ischemia was reduced. Although oxidative metabolism and mitochondrial function were normal, the AMP/ATP ratio was increased in LKB1-KO hearts. This was associated with a complete ablation of AMPKα2 activity, and a stimulation of signaling through the mammalian target of rapamycin. Our results establish a critical role for LKB1 for normal cardiac function under both aerobic conditions and during recovery after ischemia. Ablation of LKB1 leads to a decreased cardiac efficiency despite normal mitochondrial oxidative metabolism.
Keywords: Cardiac function; LKB1; AMP-activated protein kinase; mTOR;

Factors that affect postnatal bone growth retardation in the twitcher murine model of Krabbe disease by Miguel Agustin Contreras; William Louis Ries; Srinivasan Shanmugarajan; Gonzalo Arboleda; Inderjit Singh; Avtar Kaur Singh (601-608).
Krabbe disease is an inherited lysosomal disorder in which galactosylsphingosine (psychosine) accumulates mainly in the central nervous system. To gain insight into the possible mechanism(s) that may be participating in the inhibition of the postnatal somatic growth described in the animal model of this disease (twitcher mouse, twi), we studied their femora. This study reports that twi femora are smaller than of those of wild type (wt), and present with abnormality of marrow cellularity, bone deposition (osteoblastic function), and osteoclastic activity. Furthermore, lipidomic analysis indicates altered sphingolipid homeostasis, but without significant changes in the levels of sphingolipid-derived intermediates of cell death (ceramide) or the levels of the osteoclast–osteoblast coupling factor (sphingosine-1-phosphate). However, there was significant accumulation of psychosine in the femora of adult twi animals as compared to wt, without induction of tumor necrosis factor-alpha or interleukin-6. Analysis of insulin-like growth factor-1 (IGF-1) plasma levels, a liver secreted hormone known to play a role in bone growth, indicated a drastic reduction in twi animals when compared to wt. To identify the cause of the decrease, we examined the IGF-1 mRNA expression and protein levels in the liver. The results indicated a significant reduction of IGF-1 mRNA as well as protein levels in the liver from twi as compared to wt littermates. Our data suggest that a combination of endogenous (psychosine) and endocrine (IGF-1) factors play a role in the inhibition of postnatal bone growth in twi mice; and further suggest that derangements of liver function may be contributing, at least in part, to this alteration.
Keywords: Galactosylsphingosine; Insulin-like growth factor-1; Krabbe disease; Lysosomal disorders; Osteopenia; Psychosine; Twitcher mice;

Fibroblast growth factor-2 (FGF2)-mediated signaling plays an important role in fiber cell differentiation in eye lens. We had previously shown that kynurenine (KYN) produced from the overexpression of indoleamine 2,3-dioxygenase (IDO) causes defects in the differentiation of fiber cells, induces fiber cell apoptosis and cataract formation in the mouse lens, and leads to cell cycle arrest in cultured mouse lens epithelial cells (mLEC). In this study, we demonstrate that exogenous KYN reduces FGF2-mediated expression of α-, β-, and γ-crystallin and MIP26 in mLEC. We show that endogenously produced KYN in mLEC of IDO transgenic animals causes similar defects in FGF2-induced protein expression and that a competitive inhibitor of IDO prevents such defects. Our data also show that KYN inhibits FGF2-induced Akt and ERK1/2 phosphorylation in mLEC, which are required for crystallin and MIP26 expression in the lens. KYN does not inhibit FGF2 binding to cells but inhibit phosphorylation of FGFR1in mLEC. Together our data suggest that KYN might inhibit FGF2-mediated fiber cell differentiation by preventing expression of crystallins and MIP26. Our studies provide a novel mechanism by which KYN can exert deleterious effects in cells.
Keywords: Lens epithelial cell; Kynurenine; FGF-2; FGFR1; Crystallin; MIP26;

The small heat shock protein αA-crystallin is expressed in pancreas and acts as a negative regulator of carcinogenesis by Mi Deng; Pei-Chao Chen; Sisi Xie; Junqiong Zhao; Lili Gong; Jinping Liu; Lan Zhang; Shuming Sun; Jiao Liu; Haili Ma; Surinder K. Batra; David Wan-Cheng Li (621-631).
The small heat shock protein αA-crystallin is a structural protein in the ocular lens. In addition, recent studies have also revealed that it is a molecular chaperone, an autokinase and a strong anti-apoptotic regulator. Besides its lenticular distribution, a previous study demonstrates that a detectable level of αA-crystallin is found in other tissues including thymus and spleen. In the present study, we have re-examined the distribution of αA-crystallin in various normal human and mouse tissues and found that the normal pancreas expresses a moderate level of αA-crystallin. Moreover, αA-crystallin is found significantly downregulated in 60 cases of pancreatic carcinoma of different types than it is in 11 normal human pancreas samples. In addition, we demonstrate that αA-crystallin can enhance the activity of the activating protein-1 (AP-1) through modulating the function of the MAP kinase, and also upregulates components of TGFβ pathway. Finally, expression of αA-crystallin in a pancreatic cancer cell line, MiaPaCa, results in retarded cell migration. Together, these results suggest that αA-crystallin seems to negatively regulate pancreatic carcinogenesis.
Keywords: Small heat shock protein; αA; Pancreas; AP-1; Smad2/3/5; TGFβ; Pancreatic cancer; Cell migration; Lens;

Pharmacological inhibition of c-Jun N-terminal kinase signaling prevents cardiomyopathy caused by mutation in LMNA gene by Wei Wu; Jian Shan; Gisèle Bonne; Howard J. Worman; Antoine Muchir (632-638).
Mutations in LMNA, which encodes A-type nuclear lamins, cause disorders of striated muscle that have as a common feature dilated cardiomyopathy. We have demonstrated an abnormal activation of both the extracellular signal-regulated kinase (ERK) and the c-Jun N-terminal kinase (JNK) branches of the mitogen-activated protein kinase signaling cascade in hearts from Lmna H222P/H222P mice that develop dilated cardiomyopathy. We previously showed that pharmacological inhibition of cardiac ERK signaling in these mice delayed the development of left ventricle dilatation and deterioration in ejection fraction. In the present study, we treated Lmna H222P/H222P mice with SP600125, an inhibitor of JNK signalling. Systemic treatment with SP600125 inhibited JNK phosphorylation, with no detectable effect on ERK. It also blocked increased expression of RNAs encoding natriuretic peptide precursors and proteins involved in the architecture of the sarcomere that occurred in placebo-treated mice. Furthermore, treatment with SP600125 significantly delayed the development of left ventricular dilatation and prevented decreases in cardiac ejection fraction and fibrosis. These results demonstrate a role for JNK activation in the development of cardiomyopathy caused by LMNA mutations. They further provide proof-of-principle for JNK inhibition as a novel therapeutic option to prevent or delay the cardiomyopathy in humans with mutations in LMNA.
Keywords: Cardiomyopathy; Lamin; MAP kinase; JNK; Emery–Dreifuss muscular dystrophy;

Dihydropyrimidinase deficiency: Phenotype, genotype and structural consequences in 17 patients by André B.P. van Kuilenburg; Doreen Dobritzsch; Judith Meijer; Rutger Meinsma; Jean-François Benoist; Birgit Assmann; Susanne Schubert; Georg F. Hoffmann; Marinus Duran; Maaike C. de Vries; Gerd Kurlemann; François J.M. Eyskens; Lawrence Greed; Jörn Oliver Sass; K. Otfried Schwab; Adrian C. Sewell; John Walter; Andreas Hahn; Lida Zoetekouw; Antonia Ribes; Suzanne Lind; Raoul C.M. Hennekam (639-648).
Dihydropyrimidinase (DHP) is the second enzyme of the pyrimidine degradation pathway and catalyses the ring opening of 5,6-dihydrouracil and 5,6-dihydrothymine. To date, only 11 individuals have been reported suffering from a complete DHP deficiency. Here, we report on the clinical, biochemical and molecular findings of 17 newly identified DHP deficient patients as well as the analysis of the mutations in a three-dimensional framework. Patients presented mainly with neurological and gastrointestinal abnormalities and markedly elevated levels of 5,6-dihydrouracil and 5,6-dihydrothymine in plasma, cerebrospinal fluid and urine. Analysis of DPYS, encoding DHP, showed nine missense mutations, two nonsense mutations, two deletions and one splice-site mutation. Seventy-one percent of the mutations were located at exons 5–8, representing 41% of the coding sequence. Heterologous expression of 11 mutant enzymes in Escherichia coli showed that all but two missense mutations yielded mutant DHP proteins without significant activity. Only DHP enzymes containing the mutations p.R302Q and p.T343A possessed a residual activity of 3.9% and 49%, respectively. The crystal structure of human DHP indicated that the point mutations p.R490C, p.R302Q and p.V364M affect the oligomerization of the enzyme. In contrast, p.M70T, p.D81G, p.L337P and p.T343A affect regions near the di-zinc centre and the substrate binding site. The p.S379R and p.L7V mutations were likely to cause structural destabilization and protein misfolding. Four mutations were identified in multiple unrelated DHP patients, indicating that DHP deficiency may be more common than anticipated.
Keywords: Dihydropyrimidinase; DPYS; Neurological and gastrointestinal abnormalities; Crystal structure; Functional and structural protein analysis;

Inactivation of IL-6 and soluble IL-6 receptor by neutrophil derived serine proteases in cystic fibrosis by Eamon P. McGreal; Philip L. Davies; Wendy Powell; Stefan Rose-John; O. Bradley Spiller; Iolo Doull; Simon A. Jones; Sailesh Kotecha (649-658).
The ability of IL-6 to signal via both membrane bound and soluble receptors is thought to explain the capacity of this cytokine to act in both the initiation and resolution of acute inflammatory responses. In cystic fibrosis (CF), poorly resolved neutrophillic inflammation of the lungs is a primary cause of morbidity and mortality. Expression of IL-6 has been reported to be low in CF lung secretions, despite ongoing inflammation, but the status of soluble IL-6 receptor (sIL-6R) in these patients is unknown. We hypothesised that sIL-6R may be an important potentiator of IL-6 activity in CF associated lung disease. IL-6, sIL-6R and sgp130 (a natural antagonist of responses mediated by the sIL-6R) were analysed by ELISA and Western blot in bronchoalveolar lavage fluid (BALF) from 28 paediatric CF patients and nine non-CF controls. Total cell counts in CF were four fold higher compared to controls (median: 1.4 × 106 cells/ml v. 0.35 × 106 cells/ml in controls) (p  < 0.001) and the infiltrate was dominated by neutrophils which were elevated by 89 fold (0.62 × 106 cells/ml v. 0.007 × 106 cells/ml in controls) (p  < 0.001). Other markers of inflammation such as IL-8 and MCP-1 were elevated 17.5 and 3.8 fold respectively (IL-8; median: 1122 pg/ml v. 64 pg/ml in controls, p  < 0.01 and MCP-1; median: 692 pg/ml v. 182 pg/ml in controls, p  < 0.05). IL-6, although present in 23/32 CF BALF specimens compared to 1/9 controls (p  < 0.01), was weakly expressed (median: 50 pg/ml). Expression of sIL-6R and sgp130 in CF was no different to control patients. We tested whether weak expression of all three molecules was due to degradation by CF BALF. Degradative activity was observed in association with BALF elastase activity and could be specifically blocked by serine protease inhibitors. Degradation of sIL-6R by purified serine proteases (elastase, cathepsin G and proteinase 3) was also observed leading to a loss of trans-signalling activity. Interestingly, sIL-6R was protected from proteolysis by interaction with IL-6. Our data identify and define a novel protease mediated deficiency of IL-6 signalling in the CF lung.
Keywords: Cystic fibrosis; IL-6; Cytokine; Inflammation; Protease; Neutrophil;

Muscle degeneration in neuraminidase 1-deficient mice results from infiltration of the muscle fibers by expanded connective tissue by Edmar Zanoteli; Diantha van de Vlekkert; Erik J. Bonten; Huimin Hu; Linda Mann; Elida M. Gomero; A. John Harris; Giulio Ghersi; Alessandra d'Azzo (659-672).
Neuraminidase 1 (NEU1) regulates the catabolism of sialoglycoconjugates in lysosomes. Congenital NEU1 deficiency in children is the basis of sialidosis, a severe neurosomatic disorder in which patients experience a broad spectrum of clinical manifestations varying in the age of onset and severity. Osteoskeletal deformities and muscle hypotonia have been described in patients with sialidosis. Here we present the first comprehensive analysis of the skeletal muscle pathology associated with loss of Neu1 function in mice. In this animal model, skeletal muscles showed an expansion of the epimysial and perimysial spaces, associated with proliferation of fibroblast-like cells and abnormal deposition of collagens. Muscle fibers located adjacent to the expanded connective tissue underwent extensive invagination of their sarcolemma, which resulted in the infiltration of the fibers by fibroblast-like cells and extracellular matrix, and in their progressive cytosolic fragmentation. Both the expanded connective tissue and the juxtaposed infiltrated muscle fibers were strongly positive for lysosomal markers and displayed increased proteolytic activity of lysosomal cathepsins and metalloproteinases. These combined features could lead to abnormal remodeling of the extracellular matrix that could be responsible for sarcolemmal invagination and progressive muscle fiber degeneration, ultimately resulting in an overt atrophic phenotype. This unique pattern of muscle damage, which has never been described in any myopathy, might explain the neuromuscular manifestations reported in patients with the type II severe form of sialidosis. More broadly, these findings point to a potential role of NEU1 in cell proliferation and extracellular matrix remodeling.
Keywords: NEU1; Sialidosis; Metalloproteinase; Muscle biopsy; Lysosome; ECM;

Reduced creatine kinase as a central and peripheral biomarker in Huntington's disease by Jinho Kim; Daniel J. Amante; Jennifer P. Moody; Christina K. Edgerly; Olivia L. Bordiuk; Karen Smith; Samantha A. Matson; Wayne R. Matson; Clemens R. Scherzer; H. Diana Rosas; Steven M. Hersch; Robert J. Ferrante (673-681).
A major goal of current clinical research in Huntington's disease (HD) has been to identify preclinical and manifest disease biomarkers, as these may improve both diagnosis and the power for therapeutic trials. Although the underlying biochemical alterations and the mechanisms of neuronal degeneration remain unknown, energy metabolism defects in HD have been chronicled for many years. We report that the brain isoenzyme of creatine kinase (CK-BB), an enzyme important in buffering energy stores, was significantly reduced in presymptomatic and manifest disease in brain and blood buffy coat specimens in HD mice and HD patients. Brain CK-BB levels were significantly reduced in R6/2 mice by ∼ 18% to ∼ 68% from 21 to 91 days of age, while blood CK-BB levels were decreased by ∼ 14% to ∼ 44% during the same disease duration. Similar findings in CK-BB levels were observed in the 140 CAG mice from 4 to 12 months of age, but not at the earliest time point, 2 months of age. Consistent with the HD mice, there was a grade-dependent loss of brain CK-BB that worsened with disease severity in HD patients from ∼ 28% to ∼ 63%, as compared to non-diseased control patients. In addition, CK-BB blood buffy coat levels were significantly reduced in both premanifest and symptomatic HD patients by ∼ 23% and ∼ 39%, respectively. The correlation of CK-BB as a disease biomarker in both CNS and peripheral tissues from HD mice and HD patients may provide a powerful means to assess disease progression and to predict the potential magnitude of therapeutic benefit in this disorder.
Keywords: Biomarker; Creatine kinase; Huntington's disease; Energetic defects; Blood buffy coat;

Niemann–Pick type C cells show cholesterol dependent decrease of APP expression at the cell surface and its increased processing through the β-secretase pathway by Martina Malnar; Marko Kosicek; Stefan Mitterreiter; Damir Omerbasic; Stefan F. Lichtenthaler; Alison Goate; Silva Hecimovic (682-691).
The link between cholesterol and Alzheimer's disease has recently been revealed in Niemann–Pick type C disease. We found that NPC1 / cells show decreased expression of APP at the cell surface and increased processing of APP through the β-secretase pathway resulting in increased C99, sAPPβ and intracellular Aβ40 levels. This effect is dependent on increased cholesterol levels, since cholesterol depletion reversed cell surface APP expression and lowered Aβ/C99 levels in NPC1 / cells to the levels observed in wt cells. Finding that overexpression of C99, a direct γ-secretase substrate, does not lead to increased intracellular Aβ levels in NPC1 / cells vs. CHOwt suggests that the effect on intracellular Aβ upon cholesterol accumulation in NPC1 / cells is not due to increased APP cleavage by γ-secretase. Our results indicate that cholesterol may modulate APP processing indirectly by modulating APP expression at the cell surface and thus its cleavage by β-secretase.
Keywords: Alzheimer's disease; Amyloid-β; APP; Cholesterol; β-secretase; NPC1;

Analysis of the functional consequences of lethal mutations in mitochondrial translational elongation factors by Kenta Akama; Brooke E. Christian; Christie N. Jones; Takuya Ueda; Nono Takeuchi; Linda L. Spremulli (692-698).
Mammalian mitochondria synthesize a set of thirteen proteins that are essential for energy generation via oxidative phosphorylation. The genes for all of the factors required for synthesis of the mitochondrially encoded proteins are located in the nuclear genome. A number of disease-causing mutations have been identified in these genes. In this manuscript, we have elucidated the mechanisms of translational failure for two disease states characterized by lethal mutations in mitochondrial elongation factor Ts (EF-Tsmt) and elongation factor Tu (EF-Tumt). EF-Tumt delivers the aminoacyl-tRNA (aa-tRNA) to the ribosome during the elongation phase of protein synthesis. EF-Tsmt regenerates EF-Tumt:GTP from EF-Tumt:GDP. A mutation of EF-Tsmt (R325W) leads to a two-fold reduction in its ability to stimulate the activity of EF-Tumt in poly(U)-directed polypeptide chain elongation. This loss of activity is caused by a significant reduction in the ability of EF-Tsmt R325W to bind EF-Tumt, leading to a defect in nucleotide exchange. A mutation of Arg336 to Gln in EF-Tumt causes infantile encephalopathy caused by defects in mitochondrial translation. EF-Tumt R336Q is as active as the wild-type protein in polymerization using Escherichia coli 70S ribosomes and E. coli [14C]Phe-tRNA but is inactive in polymerization with mitochondrial [14C]Phe-tRNA and mitochondrial 55S ribosomes. The R336Q mutation causes a two-fold decrease in ternary complex formation with E. coli aa-tRNA but completely inactivates EF-Tumt for binding to mitochondrial aa-tRNA. Clearly the R336Q mutation in EF-Tumt has a far more drastic effect on its interaction with mitochondrial aa-tRNAs than bacterial aa-tRNAs.
Keywords: EF-Tu; EF-Ts; Translation; Elongation; Mitochondria; Disease;