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BBA - Molecular Basis of Disease (v.1832, #4)

Editorial Board (pp. i).

Enhanced parkin levels favor ER-mitochondria crosstalk and guarantee Ca2⁺ transfer to sustain cell bioenergetics by Cali Tito Calì; Denis Ottolini; Alessandro Negro; Marisa Brini (pp. 495-508).

Loss-of-function mutations in PINK1 or parkin genes are associated with juvenile-onset autosomal recessive forms of Parkinson disease. Numerous studies have established that PINK1 and parkin participate in a common mitochondrial-quality control pathway, promoting the selective degradation of dysfunctional mitochondria by mitophagy. Upregulation of parkin mRNA and protein levels has been proposed as protective mechanism against mitochondrial and endoplasmic reticulum (ER) stress. To better understand how parkin could exert protective function we considered the possibility that it could modulate the ER–mitochondria inter-organelles cross talk. To verify this hypothesis we investigated the effects of parkin overexpression on ER–mitochondria crosstalk with respect to the regulation of two key cellular parameters: Ca2⁺ homeostasis and ATP production. Our results indicate that parkin overexpression in model cells physically and functionally enhanced ER–mitochondria coupling, favored Ca2⁺ transfer from the ER to the mitochondria following cells stimulation with an 1,4,5 inositol trisphosphate (InsP₃) generating agonist and increased the agonist-induced ATP production. The overexpression of a parkin mutant lacking the first 79 residues (ΔUbl) failed to enhance the mitochondrial Ca2⁺ transients, thus highlighting the importance of the N-terminal ubiquitin like domain for the observed phenotype. siRNA-mediated parkin silencing caused mitochondrial fragmentation, impaired mitochondrial Ca2⁺ handling and reduced the ER–mitochondria tethering. These data support a novel role for parkin in the regulation of mitochondrial homeostasis, Ca2⁺ signaling and energy metabolism under physiological conditions.► Parkin overexpression augments mitochondrial Ca2⁺ transients and ATP production ► Parkin overexpression enhances endoplasmic reticulum-mitochondria interactions ► Enhanced levels of parkin sustain mitochondrial function and bioenergetics ► ER–mitochondria communication represents a key determinant in PD pathogenesis

Keywords: Parkin; Mitochondria; Endoplasmic reticulum; Ca; 2; ⁺; homeostasis; ATP production; Parkinson disease

Long-term dietary effects on substrate selection and muscle fiber type in very-long-chain acyl-CoA dehydrogenase deficient (VLCAD^−/−) mice by Sara Tucci; Sonja Pearson; Diran Herebian; Ute Spiekerkoetter (pp. 509-516).

Dietary fat restriction and increased carbohydrate intake are part of treatment in very-long-chain acyl-CoA dehydrogenase (VLCAD)-deficiency, the most common defect of long-chain fatty acid oxidation. The long-term impact of these interventions is unknown. We characterized here the effects of a fat-reduced, carbohydrate-enriched diet and an increased fat intake on energy metabolism in a mouse model of VLCAD-deficiency.Wild-type and VLCAD^−/− mice were fed one year either with a normal (5.1%), a high fat (10.6%) or a low-fat, carbohydrate-enriched (2.6%) diet. Dietary effects on genes involved in lipogenesis, energy homeostasis and substrate selection were quantified by real-time-PCR. Acylcarnitines as sign of impaired energy production were determined in dried blood spots and tissues. White skeletal muscle was analyzed for muscle fiber type as well as for glycogen and triglyceride content.Both dietary modifications induced enhanced triacylglyceride accumulation in skeletal muscle and inhibition of glucose oxidation. This was accompanied by an up-regulation of genes coding for oxidative muscle fiber type I and a marked accumulation of acylcarnitines, especially prominent in the heart (164±2.8 in VLCAD^−/− vs. 82.3±2.1 in WT μmol/mg) under a low-fat, carbohydrate-enriched diet.We demonstrate here that both dietary interventions with respect to the fat content of the diet reverse endogenous compensatory mechanisms in muscle that have evolved in VLCAD^−/− mice resulting in pronounced energy deficiency. In particular, the low-fat carbohydrate-enriched diet was not effective in the long term. Further experiments are necessary to define the optimal energy provision for fatty acid oxidation defects.► A fat-reduced, carbohydrate-enriched diet is part of the treatment of VLCAD-deficiency. ► Organs of long-term treated VLCAD^−/− mice showed evidence of energy deficiency. ► Compensatory mechanisms in VLCAD^−/− mice are reversed. ► Especially the muscular phenotype is worsened. ► Long-term dietary modifications are not efficient in the treatment of VLCAD-deficiency.

Keywords: VLCAD-deficient mouse; Dietary intervention; Muscle fibers type

Cleavage of extracellular matrix in periodontitis: Gingipains differentially affect cell adhesion activities of fibronectin and tenascin-C by Sabrina Ruggiero; Raluca Cosgarea; Jan Potempa; Barbara Potempa; Sigrun Eick; Matthias Chiquet (pp. 517-526).

Gingipains are cysteine proteases that represent major virulence factors of the periodontopathogenic bacterium Porphyromonas gingivalis. Gingipains are reported to degrade extracellular matrix (ECM) of periodontal tissues, leading to tissue destruction and apoptosis. The exact mechanism is not known, however. Fibronectin and tenascin-C are pericellular ECM glycoproteins present in periodontal tissues. Whereas fibronectin mediates fibroblast adhesion, tenascin-C binds to fibronectin and inhibits its cell-spreading activity. Using purified proteins in vitro, we asked whether fibronectin and tenascin-C are cleaved by gingipains at clinically relevant concentrations, and how fragmentation by the bacterial proteases affects their biological activity in cell adhesion. Fibronectin was cleaved into distinct fragments by all three gingipains; however, only arginine-specific HRgpA and RgpB but not lysine-specific Kgp destroyed its cell-spreading activity. This result was confirmed with recombinant cell-binding domain of fibronectin. Of the two major tenascin-C splice variants, the large but not the small was a substrate for gingipains, indicating that cleavage occurred primarily in the alternatively spliced domain. Surprisingly, cleavage of large tenascin-C variant by all three gingipains generated fragments with increased anti-adhesive activity towards intact fibronectin. Fibronectin and tenascin-C fragments were detected in gingival crevicular fluid of a subset of periodontitis patients. We conclude that cleavage by gingipains directly affects the biological activity of both fibronectin and tenascin-C in a manner that might lead to increased cell detachment and loss during periodontal disease.► Bacterial proteases degrade extracellular matrix proteins during periodontitis. ► Only arginine-specific gingipains destroy the cell adhesion activity of fibronectin. ► All gingipains produce tenascin-C fragments with increased anti-adhesive activity. ► Fibronectin and tenascin-C fragments are found in certain periodontitis patients. ► By disturbing adhesion, gingipains might induce cell loss in periodontitis.

Keywords: Abbreviations; HRgpA; high molecular mass arginine-specific gingipain A; RgpB; arginine-specific gingipain B; Kgp; lysine-specific gingipain; GCF; gingival crevicular fluid; ECM; extracellular matrix; FN; fibronectin; FNIII; fibronectin type III domain; TNC; tenascin-C; RGD; arginine-glycine-aspartic acid; GST; glutathione-S-transferase; PBS; phosphate buffered saline; DMEM; Dulbecco's modified Eagle medium; SDS-PAGE; sodium dodecyl sulfate-polyacrylamide gel electrophoresis; mAb; monoclonal antibodyGingipain; Extracellular matrix; Fibronectin; Tenascin-C; Cell adhesion; Periodontitis

Crosstalk between diabetes and brain: Glucagon-like peptide-1 mimetics as a promising therapy against neurodegeneration by A.I. Duarte; E. Candeias; S.C. Correia; R.X. Santos; C. Carvalho; S. Cardoso; Placido A. Plácido; M.S. Santos; C.R. Oliveira; P.I. Moreira (pp. 527-541).

According to World Health Organization estimates, type 2 diabetes (T2D) is an epidemic (particularly in under development countries) and a socio-economic challenge. This is even more relevant since increasing evidence points T2D as a risk factor for Alzheimer's disease (AD), supporting the hypothesis that AD is a “type 3 diabetes” or “brain insulin resistant state”. Despite the limited knowledge on the molecular mechanisms and the etiological complexity of both pathologies, evidence suggests that neurodegeneration/death underlying cognitive dysfunction (and ultimately dementia) upon long-term T2D may arise from a complex interplay between T2D and brain aging. Additionally, decreased brain insulin levels/signaling and glucose metabolism in both pathologies further suggests that an effective treatment strategy for one disorder may be also beneficial in the other. In this regard, one such promising strategy is a novel successful anti-T2D class of drugs, the glucagon-like peptide-1 (GLP-1) mimetics (e.g. exendin-4 or liraglutide), whose potential neuroprotective effects have been increasingly shown in the last years. In fact, several studies showed that, besides improving peripheral (and probably brain) insulin signaling, GLP-1 analogs minimize cell loss and possibly rescue cognitive decline in models of AD, Parkinson's (PD) or Huntington's disease. Interestingly, exendin-4 is undergoing clinical trials to test its potential as an anti-PD therapy. Herewith, we aim to integrate the available data on the metabolic and neuroprotective effects of GLP-1 mimetics in the central nervous system (CNS) with the complex crosstalk between T2D-AD, as well as their potential therapeutic value against T2D-associated cognitive dysfunction.► A complex interaction between brain aging and type 2 diabetes may lead to dementia. ► A successful therapy against type 2 diabetes may be also successful against dementia. ► GLP-1 mimetics are novel successful insulinotropic drugs. ► Besides their peripheral effects, GLP-1 mimetics have neuroprotective effects. ► GLP-1 analogs may be a potential therapy against diabetes-associated dementia.

Keywords: Alzheimer's disease; Brain; Diabetes; Glucagon-like peptide-1 mimetics; Insulin signaling; Neuroprotection

Epigallocatechin-3-gallate prevents oxidative phosphorylation deficit and promotes mitochondrial biogenesis in human cells from subjects with Down's syndrome by Daniela Valenti; Domenico De Rasmo; Anna Signorile; Leonardo Rossi; Lidia de Bari; Iris Scala; Barbara Granese; Sergio Papa; Rosa Anna Vacca (pp. 542-552).

A critical role for mitochondrial dysfunction has been proposed in the pathogenesis of Down's syndrome (DS), a human multifactorial disorder caused by trisomy of chromosome 21, associated with mental retardation and early neurodegeneration. Previous studies from our group demonstrated in DS cells a decreased capacity of the mitochondrial ATP production system and overproduction of reactive oxygen species (ROS) in mitochondria. In this study we have tested the potential of epigallocatechin-3-gallate (EGCG) – a natural polyphenol component of green tea – to counteract the mitochondrial energy deficit found in DS cells. We found that EGCG, incubated with cultured lymphoblasts and fibroblasts from DS subjects, rescued mitochondrial complex I and ATP synthase catalytic activities, restored oxidative phosphorylation efficiency and counteracted oxidative stress. These effects were associated with EGCG-induced promotion of PKA activity, related to increased cellular levels of cAMP and PKA-dependent phosphorylation of the NDUFS4 subunit of complex I. In addition, EGCG strongly promoted mitochondrial biogenesis in DS cells, as associated with increase in Sirt1-dependent PGC-1α deacetylation, NRF-1 and T-FAM protein levels and mitochondrial DNA content.In conclusion, this study shows that EGCG is a promoting effector of oxidative phosphorylation and mitochondrial biogenesis in DS cells, acting through modulation of the cAMP/PKA- and sirtuin-dependent pathways. EGCG treatment promises thus to be a therapeutic approach to counteract mitochondrial energy deficit and oxidative stress in DS.► The role of EGCG to counteract the mitochondrial energy deficit in human DS cells was studied. ► EGCG stimulates complex I activity through activation of cAMP-PKA signaling pathway. ► EGCG promotes mitochondrial biogenesis through Sirt1-mediated PGC1α activation. ► This study could provide a useful background for the development of novel drug therapies for DS.

Keywords: Abbreviations; CREB; cAMP response element-binding; DCF; dichlorofluorescein; DS; Down's syndrome; EGCG; epigallocatechin-3-gallate; H; ₂; O; ₂; hydrogen peroxide; NRF-1; nuclear respiratory factor 1; OXPHOS; mitochondrial oxidative phosphorylation; PGC-1α; peroxisome proliferator-activated receptor-γ coactivator; ROS; reactive oxygen species; Sirt1; sirtuin 1; T-FAM; mitochondrial transcription factor ADown syndrome; EGCG; Mitochondria; Sirt1; PGC-1α; cAMP/PKA pathway

Loss of connexin43 expression in Ewing's sarcoma cells favors the development of the primary tumor and the associated bone osteolysis by Julie Talbot; Brion Régis Brion; Picarda Gaëlle Picarda; Amiaud Jérome Amiaud; Julie Chesneau; Gwenola Bougras; Verena Stresing; Franck Tirode; Dominique Heymann; Redini Françoise Redini; Franck Verrecchia (pp. 553-564).

Ewing's sarcoma (ES) is a primary bone tumor characterized by a chromosomic translocation between the EWS gene and a member of the ETS gene family, mainly FLI1, which leads to an aberrant transcription factor EWS–FLI1 that promotes tumorigenicity. Gap junctions are intercellular channels composed of transmembrane proteins (connexin: Cx), that allow direct intercellular communication between adjacent cells. Numerous studies have shown that tumorigenesis may be associated with a loss of gap junctional intercellular communication (GJIC).Loss of Cx43 expression was observed at the protein and mRNA levels in ES cell lines compared to those measured in human mesenchymal stem cells. A673 ES cells stably transfected with an shRNA targeting EWS–FLI1 showed an increase in Cx43 expression (at the mRNA, protein and transcriptional levels) and GJIC. In an osteolytic murine model of ES, the overexpression of Cx43 in ES cells dramatically reduced tumor growth, leading to a significant increase in animal survival. In vitro assays showed that Cx43 overexpression increases the p27 level with an associated marked decrease of Rb phosphorylation, consistent with the observed blockade of the cell cycle in G0/G1 phase. In addition, the bone microarchitectural parameters, assessed by micro-CT analysis, showed an increased bone volume when Cx43 expression was enhanced. Histological analysis demonstrated that the overexpression of Cx43 in ES tumor cells inhibits osteoclast activity and therefore bone resorption.Our study demonstrated that the loss of Cx43 expression in ES cells plays a crucial role in the development of the primary tumor and the associated bone osteolysis.► A loss of Cx43 expression was observed in human ES cell lines. ► An shRNA targeting EWS–FLI1 leads to an increase of Cx43 expression in ES cell lines. ► Cx43 overexpression in ES cells reduces the in vivo growth of primary tumor. ► An increased bone volume is observed when Cx43 expression was enhanced in tumor cells. ► Cx43 overexpression in ES tumor cells inhibits osteoclast activity.

Keywords: Connexin43; Ewing's sarcoma; EWS–FLI1; Osteolysis; Tumor growth

Impairment of the proteasome is crucial for glucose-induced lifespan reduction in the mev-1 mutant of Caenorhabditis elegans by Elena Fitzenberger; Michael Boll; Uwe Wenzel (pp. 565-573).

Hyperglycemia is a hallmark of diabetes that is associated with diabetic complications and a reduction of lifespan. Using the mev-1 mutant of the nematode Caenorhabditis elegans we here tried to identify molecular mechanisms underlying the lifespan reducing effects of glucose. The lowest glucose concentration tested (10mM) caused a significant lifespan reduction at 37°C and was used to assess effects on mitochondrial efficiency, formation of protein carbonyls and levels of methylglyoxal, a precursor of advanced glycation end products (AGEs). RNA-interference (RNAi) served the identification of targets for glucose-induced damage. Levels of protein carbonyls and AGEs remained unaffected by 10mM glucose. Levels of reactive oxygen species inside mitochondria were increased but their scavenging by ascorbic acid did not influence lifespan reduction by glucose. Mitochondrial efficiency was reduced by glucose as concluded from a lowered P/O-ratio. A reduced lifespan of mev-1 that was unaffected by the addition of glucose resulted from RNAi of key players of mitochondrial unfolded protein response. Besides increased accumulation of misfolded proteins, reduced proteasomal degradation caused the same phenotype as was evidenced by RNAi for UBQ-1 or UBA-1. Accumulation of functionally impaired proteins, e.g. in mitochondria, underlies the lifespan reducing effects of glucose. Our study provides evidence for a crucial importance of the proteostasis network for lifespan regulation which is impaired by glucose.► Glucose reduces mitochondrial efficiency. ► Glucose impairs the unfolded protein response and proteasomal degradation. ► Blocking autophagosomal degradation triggers proteasomal degradation. ► Activation of the proteasome prevents lifespan reduction by glucose.

Keywords: Abbreviations; AA; ascorbic acid; ABU; activated in blocked unfolded protein response; AGEs; advanced glycation end products; ER; endoplasmic reticulum; MG; methylglyoxal; NGM; nematode growth medium; PVDF; polyvinylidene difluoride; QPCR; quantitative polymerase chain reaction; RNAi; RNA interference; ROS; reactive oxygen species; SDS-PAGE; sodium dodecyl polyacrylamide gel electrophoresis; UPR; ^mt; unfolded protein response of mitochondria; UPR; ^ER; unfolded protein response of the endoplasmic reticulumGlucose-induced toxicity; Aging; Mitochondrial efficiency; Unfolded protein response; Proteasome

Mitochondrial aldehyde dehydrogenase obliterates endoplasmic reticulum stress-induced cardiac contractile dysfunction via correction of autophagy by Bingfang Zhang; Yingmei Zhang; Karissa H. La Cour; Kacy L. Richmond; Xiao-Ming Wang; Jun Ren (pp. 574-584).

ER stress triggers myocardial contractile dysfunction while effective therapeutic regimen is still lacking. Mitochondrial aldehyde dehydrogenase (ALDH2), an essential mitochondrial enzyme governing mitochondrial and cardiac function, displays distinct beneficial effect on the heart. This study was designed to evaluate the effect of ALDH2 on ER stress-induced cardiac anomalies and the underlying mechanism involved with a special focus on autophagy. WT and ALDH2 transgenic mice were subjected to the ER stress inducer thapsigargin (1mg/kg, i.p., 48h). Echocardiographic, cardiomyocyte contractile and intracellular Ca2⁺ properties as well as myocardial histology, autophagy and autophagy regulatory proteins were evaluated. ER stress led to compromised echocardiographic indices (elevated LVESD, reduced fractional shortening and cardiac output), cardiomyocyte contractile and intracellular Ca2⁺ properties and cell survival, associated with upregulated autophagy, dampened phosphorylation of Akt and its downstream signal molecules TSC2 and mTOR, the effects of which were alleviated or mitigated by ALDH2. Thapsigargin promoted ER stress proteins Gadd153 and GRP78 without altering cardiomyocyte size and interstitial fibrosis, the effects of which were unaffected by ALDH2. Treatment with thapsigargin in vitro mimicked in vivo ER stress-induced cardiomyocyte contractile anomalies including depressed peak shortening and maximal velocity of shortening/relengthening as well as prolonged relengthening duration, the effect of which was abrogated by the autophagy inhibitor 3-methyladenine and the ALDH2 activator Alda-1. Interestingly, Alda-1-induced beneficial effect against ER stress was obliterated by autophagy inducer rapamycin, Akt inhibitor AktI and mTOR inhibitor RAD001. These data suggest a beneficial role of ALDH2 against ER stress-induced cardiac anomalies possibly through autophagy reduction.► We examine the effect of ALDH2 in ER stress-induced cardiomyopathy. ► ALDH2 overexpression alleviates ER stress-induced cardiac mechanical defect. ► ALDH2 inhibits autophagy and restores phosphorylation of Akt and mTOR.

Keywords: ER stress; ALDH2; Autophagy; Akt; mTOR; Cardiomyocyte mechanics

XLID CUL4B mutants are defective in promoting TSC2 degradation and positively regulating mTOR signaling in neocortical neurons by Hung-Li Wang; Ning-Chun Chang; Yi-Hsin Weng; Tu-Hsueh Yeh (pp. 585-593).

Truncating or missense mutation of cullin 4B (CUL4B) is one of the most prevalent causes underlying X-linked intellectual disability (XLID). CUL4B-RING E3 ubiquitin ligase promotes ubiquitination and degradation of various proteins. Consistent with previous studies, overexpression of wild-type CUL4B in 293 cells enhanced ubiquitylation and degradation of TSC2 or cyclin E. The present study shows that XLID mutant (R388X), (R572C) or (V745A) CULB failed to promote ubiquitination and degradation of TSC2 or cyclin E. Adenoviruses-mediated expression of wild-type CUL4B decreased protein level of TSC2 or cyclin E in cultured neocortical neurons of frontal lobe. Furthermore, shRNA-mediated CUL4B knockdown caused an upregulation of TSC2 or cyclin E. XLID mutant (R388X), (R572C) or (V745A) CUL4B did not downregulate protein expression of TSC2 or cyclin E in neocortical neurons. By promoting TSC2 degradation, CUL4B could positively regulate mTOR activity in neocortical neurons of frontal cortex. Consistent with this hypothesis, CUL4B knockdown-induced upregulation of TSC2 in neocortical neurons resulted in a decreased protein level of active phospho-mTOR^Ser2448 and a reduced expression of active phospho-p70S6K^Thr389 and phospho-4E-BP1^Thr37/46, two main substrates of mTOR-mediated phosphorylation. Wild-type CUL4B also increased protein level of active phospho-mTOR^Ser2448, phospho-p70S6K^Thr389 or phospho-4E-BP1^Thr37/46. XLID CUL4B mutants did not affect protein level of active phospho-mTOR^Ser2448, phospho-p70S6K^Thr389 or phospho-4E-BP1^Thr37/46. Our results suggest that XLID CUL4B mutants are defective in promoting TSC2 degradation and positively regulating mTOR signaling in neocortical neurons.► Wild-type CUL4B promotes the ubiquitylation and degradation of TSC2. ► XLID CULB mutants are defective in promoting the degradation of TSC2. ► CUL4B knockdown-induced increase in TSC2 augments mTOR activity in cortical neurons. ► CUL4B positively regulates mTOR signaling in neocortical neurons of frontal cortex. ► XLID CUL4B mutants fail to positively modulate mTOR activity in neocortical neurons.

Keywords: X-linked intellectual disability; Cullin 4B; TSC2; Cyclin E; mTOR; Neocortical neurons

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