Check out our New Publishers' Select for Free Articles

BBA - Molecular Basis of Disease (v.1822, #11)

Editorial Board (pp. i).

Shp-2 regulates the TrkB receptor activity in the retinal ganglion cells under glaucomatous stress by Vivek K. Gupta; Yuyi You; Alexander Klistorner; Stuart L. Graham (pp. 1643-1649).

Tropomyosin‐receptor‐kinase B (TrkB receptor) activation plays an important role in the survival of retinal ganglion cells (RGCs). This study reports a novel finding that, SH2 domain-containing phosphatase-2 (Shp-2) binds to the TrkB receptor in RGCs and negatively regulates its activity under glaucomatous stress. This enhanced binding of TrkB and Shp2 is mediated through caveolin. Caveolin 1 and 3 undergo hyper-phosphorylation in RGCs under stress and bind to the Shp2 phosphatase. Shp2 undergoes activation under glaucomatous stress conditions in RGCs in vivo with a concurrent loss of TrkB activity. Inhibiting the Shp2 phosphatase restored TrkB activity in cells exposed to excitotoxic and oxidative stress. Collectively, these findings implicate a molecular basis of Shp2 mediated TrkB deactivation leading to RGC degeneration observed in glaucoma.Display Omitted► TrkB interacts with Shp2 in the retinal ganglion cells. ► TrkB–Shp2 interaction is enhanced several folds under glaucomatous stress. ► Enhanced TrkB–Shp2 interaction leads to TrkB deactivation. ► TrkB–Shp2 interaction is mediated through the protein caveolin. ► Caveolin 1 and 3 undergo hyperphosphorylation under glaucomatous stress.

Keywords: Abbreviations; RGC; retinal ganglion cell; BDNF; brain derived neurotrophic factor; Cav; caveolin; AON; antisense oligonucleotide; ON; optic nerve; IOP; intra-ocular pressureRetinal ganglion cell; Glaucoma; Optic nerve; TrkB receptor; Shp2 phosphatase; Immunoprecipitation

Chronic intermittent hypoxia induces atherosclerosis by NF-κB-dependent mechanisms by D. Song; G. Fang; S.-Z. Mao; X. Ye; G. Liu; Y. Gong; S.F. Liu (pp. 1650-1659).

Chronic intermittent hypoxia (CIH) causes atherosclerosis in mice fed a high cholesterol diet (HCD). The mechanisms by which CIH promotes atherosclerosis are incompletely understood. This study defined the mechanistic role of NF-κB pathway in CIH+HCD induced atherosclerosis. Wild type (WT) and mice deficient in the p50 subunit of NF‐κB (p50‐KO) were fed normal chow diet (ND) or HCD, and exposed to sham or CIH. Atherosclerotic lesions on the en face aortic preparation and cross‐sections of aortic root were examined. In WT mice, neither CIH nor HCD exposure alone caused, but CIH+HCD caused evident atherosclerotic lesions on both preparations after 20weeks of exposure. WT mice on ND and exposed to CIH for 35.6weeks did not develop atherosclerotic lesions. P50 gene deletion diminished CIH+HCD induced NF‐κB activation and abolished CIH+HCD induced atherosclerosis. P50 gene deletion inhibited vascular wall inflammation, reduced hepatic TNF‐α level, attenuated the elevation in serum cholesterol level and diminished macrophage foam cell formation induced by CIH+HCD exposure. These results demonstrate that inhibition of NF‐κB activation abrogates the activation of three major atherogenic mechanisms associated with an abolition of CIH+HCD induced atherosclerosis. NF‐κB may be a central common pathway through which CIH+HCD exposure activates multiple atherogenic mechanisms, leading to atherosclerosis.► Chronic intermittent hypoxia (CIH) caused atherosclerosis in mice on a high cholesterol diet. ► P50 gene deletion diminished CIH-induced NF-κB activation and atherosclerosis. ► P50 gene deletion inhibited vascular inflammation, hypercholesterolemia and foam cell formation. ► NF-κB blockade abrogated CIH activation of 3 major atherogenic mechanisms and atherosclerosis. ► NF-κB serves as a central common pathway that leads to CIH-induced atherosclerosis.

Keywords: Abbreviations; ABCA1; ATP-binding cassette transporter A1; ApoE-KO; mice lacking apoliporotein E gene; CCL2; chemokine ligand 2; CIH; chronic intermittent hypoxia; EMSA; electrophoretic mobility shift assay; FcO; ₂; fractional oxygen concentration; HCD; high cholesterol diet; HDL; high density lipoprotein; HMGCR; hydroxy-3-methyl-glutaryl-CoA reductase; IL6; interleukin-6; iNOS; inducible nitric oxide synthase; LDL; low density lipoprotein; LDLr; LDL receptor; ND; normal chow diet; ORO; Oil Red O; OSA; obstructive sleep apnea; p50-KO; mice deficient in NF-κB p50 gene; SR-B1; scavenger receptor class B1; TRAF3; TNF receptor-associated factor 3Atherosclerosis; Cardiovascular risk factor; Intermittent hypoxia; Nuclear factor kappa B; Obstructive sleep apnea

Downregulation of A₁ and A_2B adenosine receptors in human trisomy 21 mesenchymal cells from first-trimester chorionic villi by Stefania Gessi; Stefania Merighi; Angela Stefanelli; Prisco Mirandola; Alessandra Bonfatti; Sergio Fini; Alberto Sensi; Roberto Marci; Katia Varani; Pier Andrea Borea; Fortunato Vesce (pp. 1660-1670).

Human reproduction is complex and prone to failure. Though causes of miscarriage remain unclear, adenosine, a proangiogenic nucleoside, may help determine pregnancy outcome. Although adenosine receptor (AR) expression has been characterized in euploid pregnancies, no information is available for aneuploidies, which, as prone to spontaneous abortion (SA), are a potential model for shedding light on the mechanism regulating this event. AR expression was investigated in 71 first-trimester chorionic villi (CV) samples and cultured mesenchymal cells (MC) from euploid and TR21 pregnancies, one of the most frequent autosomal aneuploidy, with a view to elucidating their potential role in the modulation of vascular endothelial growth factor (VEGF) and nitric oxide (NO). Compared to euploid cells, reduced A₁ and A_2B expression was revealed in TR21 CV and MCs. The non-selective adenosine agonist 5′- N-ethylcarboxamidoadenosine (NECA) increased NO, by activating, predominantly, A₁AR and A_2AAR through a molecular pathway involving hypoxia-inducible-factor-1 (HIF-1α), and increased VEGF, mainly through A_2B. In conclusion the adenosine transduction cascade appears to be disturbed in TR21 through reduced expression of A_2B and A₁ARs. These anomalies may be implicated in complications such as fetal growth restriction, malformation and/or SA, well known features of aneuploid pregnancies. Therefore A₁ and A_2BARs could be potential biomarkers able to provide an early indication of SA risk and their stimulation may turn out to improve fetoplacental perfusion by increasing NO and VEGF.► Adenosine receptors (ARs) have never been characterized in aneuploid pregnancies. ► Here ARs were investigated in euploid and trisomy 21 chorionic villi and mesenchymal cells. ► Reduced A₁ and A_2B expression was revealed in aneuploid chorionic villi and mesenchymal cells. ► A₁ and A_2BARs increasing NO and VEGF secretion may improve fetoplacental perfusion.

Keywords: Abbreviations; CV; chorionic villi; DPCPX; 1,3-dipropyl-8-cyclopentyl-xanthine; MC; mesenchymal cells; MRE 2029-F20; N; -benzo[1,3]dioxol-5-yl-2-[5-(1,3-dipropyl-2,6-dioxo-2,3,6,7-tetrahydro-1; H-purin-8-yl)-1-methyl-1; H-pyrazol-3-yl-oxy]-acetamide]; MRE 3008-F20; 5-; N-; (4-methoxyphenylcarbamoyl)-amino-8-propyl-2-(2-furyl)-pyrazolo[4,3e]-,2,4triazolo[1,5c]pyrimidine; NECA; 5′-; N; -Ethylcarboxamidoadenosine; PSB36; 1-Butyl-8-(hexahydro-2,5-methanopentalen-3a(1; H; )-yl)-3,7-dihydro-3-(3-hydroxypropyl)-1; H; -purine-2,6-dione; PSB603; 8-[4-[4-(4-Chlorophenzyl)piperazide-1-sulfonyl)phenyl]]-1-propylxanthine; SCH442416; 2-(2-Furanyl)-7-[3-(4-methoxyphenyl)propyl]-7; H; -pyrazolo[4,3-; e; ][1,2,4]triazolo[1,5-; c; ]pyrimidin-5-amine; ZM; 241385 (4-(2-[7-amino-2-(2-furyl)-[1,2,4]triazolo-[2,32][1,3,6]triazinyl-amino]ethyl)-phenol)Adenosine receptors; Trisomy 21; Mesenchymal stem cells; Angiogenesis

Na⁺ and K⁺ ion imbalances in Alzheimer's disease by Victor M. Vitvitsky; Sanjay K. Garg; Richard F. Keep; Roger L. Albin; Ruma Banerjee (pp. 1671-1681).

Alzheimer's disease (AD) is associated with impaired glutamate clearance and depressed Na⁺/K⁺ ATPase levels in AD brain that might lead to a cellular ion imbalance. To test this hypothesis, [Na⁺] and [K⁺] were analyzed in postmortem brain samples of 12 normal and 16 AD individuals, and in cerebrospinal fluid (CSF) from AD patients and matched controls. Statistically significant increases in [Na⁺] in frontal (25%) and parietal cortex (20%) and in cerebellar [K⁺] (15%) were observed in AD samples compared to controls. CSF from AD patients and matched controls exhibited no differences, suggesting that tissue ion imbalances reflected changes in the intracellular compartment. Differences in cation concentrations between normal and AD brain samples were modeled by a 2-fold increase in intracellular [Na⁺] and an 8–15% increase in intracellular [K⁺]. Since amyloid beta peptide (Aβ) is an important contributor to AD brain pathology, we assessed how Aβ affects ion homeostasis in primary murine astrocytes, the most abundant cells in brain tissue. We demonstrate that treatment of astrocytes with the Aβ 25–35 peptide increases intracellular levels of Na⁺ (~2–3-fold) and K⁺ (~1.5-fold), which were associated with reduced levels of Na⁺/K⁺ ATPase and the Na⁺-dependent glutamate transporters, GLAST and GLT-1. Similar increases in astrocytic Na⁺ and K⁺ levels were also caused by Aβ 1–40, but not by Aβ 1–42 treatment. Our study suggests a previously unrecognized impairment in AD brain cell ion homeostasis that might be triggered by Aβ and could significantly affect electrophysiological activity of brain cells, contributing to the pathophysiology of AD.► Intracellular [Na⁺] and [K⁺] levels are increased in brain regions of Alzheimer's disease patients. ► Similar ion increases were observed ex vivo in murine astrocytes treated with amyloid β. ► This ion imbalance might contribute to the pathophysiology of Alzheimer's disease.

Keywords: Abbreviations; AD; Alzheimer's disease; Aβ; amyloid β peptide; CAβ42; levels of Aβ 1–42 peptide in CSF; Cb; cerebellum; CDR; clinical dementia rating; CSF; cerebrospinal fluid; FC; frontal cortex; MRI; magnetic resonance imaging; PC; parietal cortex; PIB; results of [; ¹¹; C]-Pittsburgh B compound positron emission tomography imaging; Ptau181; CSF phospho-tau 181 levels; Tau; CSF tau levelsAlzheimer's disease; Amyloid β; Astrocyte; Brain; Sodium; Potassium

Alkaptonuria is a novel human secondary amyloidogenic disease by Lia Millucci; Adriano Spreafico; Laura Tinti; Daniela Braconi; Lorenzo Ghezzi; Eugenio Paccagnini; Giulia Bernardini; Loredana Amato; Marcella Laschi; Enrico Selvi; Mauro Galeazzi; Alessandro Mannoni; Maurizio Benucci; Pietro Lupetti; Federico Chellini; Maurizio Orlandini; Annalisa Santucci (pp. 1682-1691).

Alkaptonuria (AKU) is an ultra-rare disease developed from the lack of homogentisic acid oxidase activity, causing homogentisic acid (HGA) accumulation that produces a HGA-melanin ochronotic pigment, of unknown composition. There is no therapy for AKU. Our aim was to verify if AKU implied a secondary amyloidosis. Congo Red, Thioflavin-T staining and TEM were performed to assess amyloid presence in AKU specimens (cartilage, synovia, periumbelical fat, salivary gland) and in HGA-treated human chondrocytes and cartilage. SAA and SAP deposition was examined using immunofluorescence and their levels were evaluated in the patients' plasma by ELISA. 2D electrophoresis was undertaken in AKU cells to evaluate the levels of proteins involved in amyloidogenesis. AKU osteoarticular tissues contained SAA-amyloid in 7/7 patients. Ochronotic pigment and amyloid co-localized in AKU osteoarticular tissues. SAA and SAP composition of the deposits assessed secondary type of amyloidosis. High levels of SAA and SAP were found in AKU patients' plasma. Systemic amyloidosis was assessed by Congo Red staining of patients' abdominal fat and salivary gland. AKU is the second pathology after Parkinson's disease where amyloid is associated with a form of melanin. Aberrant expression of proteins involved in amyloidogenesis has been found in AKU cells. Our findings on alkaptonuria as a novel type II AA amyloidosis open new important perspectives for its therapy, since methotrexate treatment proved to significantly reduce in vitro HGA-induced A-amyloid aggregates.► Alkaptonuria (no therapy) is a novel type II secondary amyloidosis. ► Ochronotic melanin-based pigment and SAA/SAP amyloid co-localize in Alkaptonuria. ► High levels of SAA and SAP were found in AKU patients' plasma. ► Systemic amyloidosis was assessed in patients' abdominal fat and salivary gland. ► Methotrexate reduces (- 97%) HGA-induced A-amyloid aggregates.

Keywords: Ochronosis; Amyloidosis; Homogentisic acid

MiR-26a enhances metastasis potential of lung cancer cells via AKT pathway by targeting PTEN by Boning Liu; Xiang Wu; Bin Liu; Changli Wang; Yunde Liu; Qinghua Zhou; Ke Xu (pp. 1692-1704).

Lung cancer is the leading cause of cancer related death, 90% of lung cancer patients die of metastasis. Many microRNAs (miRNAs) are deregulated in cancer. They are involved in tumorigenesis and function as oncogenes or tumor suppressor genes. Recent studies show that miRNAs may be responsible for tumor metastasis. Several functional studies show that miR-26a plays an important role in carcinogenesis; however, none of these studies is related to tumor metastasis. In the present study, we investigated the effect of miR-26a on metastasis potential of lung cancer cells. Our data showed that miR-26a expression level was higher in lymph node metastasis tumor tissues than in primary tumor tissues. Ectopic expression of miR-26a dramatically enhanced lung cancer cell migration and invasion abilities. Metastasis-related genes matrix metallopeptidase 2 (MMP-2), vascular endothelial growth factor (VEGF), Twist and β-catenin were upregulated. Phosphatase and tensin homolog (PTEN) was a direct target of miR-26a. Further mechanistic study revealed that miR-26a increased AKT phosphorylation and nuclear factor kappa B (NFκB) transcriptional activation. Our study demonstrated that miR-26a enhanced lung cancer cell metastasis potential via modulation of metastasis-related gene expression, and activation of AKT pathway by PTEN suppression, suggesting that miR-26a might be a potential therapeutic candidate in patients with metastatic lung cancer.► miR-26a level is higher in metastasis tumor tissues than in primary tumor tissues. ► miR-26a enhances lung cancer cell migration and invasion. ► miR-26a modulates metastasis-related gene expression. ► miR-26a activates AKT pathway by PTEN suppression.

Keywords: MiR-26a; PTEN; Lung cancer; Metastasis; AKT

GCK-MODY diabetes associated with protein misfolding, cellular self-association and degradation by Maria Negahdar; Ingvild Aukrust; Bente B. Johansson; Janne Molnes; Anders Molven; Franz M. Matschinsky; Sovik Oddmund Søvik; Rohit N. Kulkarni; Torgeir Flatmark; Njolstad Pål Rasmus Njølstad; Bjorkhaug Lise Bjørkhaug (pp. 1705-1715).

GCK-MODY, dominantly inherited mild fasting hyperglycemia, has been associated with >600 different mutations in the glucokinase (GK)-encoding gene ( GCK). When expressed as recombinant pancreatic proteins, some mutations result in enzymes with normal/near-normal catalytic properties. The molecular mechanism(s) of GCK-MODY due to these mutations has remained elusive. Here, we aimed to explore the molecular mechanisms for two such catalytically ‘normal’ GCK mutations (S263P and G264S) in the F260-L270 loop of GK. When stably overexpressed in HEK293 cells and MIN6 β-cells, the S263P- and G264S-encoded mutations generated misfolded proteins with an increased rate of degradation (S263P>G264S) by the protein quality control machinery, and a propensity to self-associate (G264S>S263P) and form dimers (SDS resistant) and aggregates (partly Triton X-100 insoluble), as determined by pulse-chase experiments and subcellular fractionation. Thus, the GCK-MODY mutations S263P and G264S lead to protein misfolding causing destabilization, cellular dimerization/aggregation and enhanced rate of degradation. In silico predicted conformational changes of the F260-L270 loop structure are considered to mediate the dimerization of both mutant proteins by a domain swapping mechanism. Thus, similar properties may represent the molecular mechanisms for additional unexplained GCK-MODY mutations, and may also contribute to the disease mechanism in other previously characterized GCK-MODY inactivating mutations.► GCK-MODY mutations S263P and G264S cause reduced GK catalytic activity (S263PG264S). ► The mutant proteins self-associate (G264S>S263P) and form homodimers/aggregates. ► Prevention of self-association may represent new therapeutic approach.

Keywords: Abbreviations; Ab; antibody; GK; glucokinase; GKRP; glucokinase regulatory protein; Glc; α-; d; -glucose; HEK293; human embryonic kidney 293 cells; hGK; human glucokinase; MIN6; mouse insulinoma cell; MODY; maturity-onset diabetes of the young; PDB; protein data bank; PNS; post‐nuclear supernatant; RRL; rabbit reticulocyte lysate; TEV protease; tobacco etch virus protease; UPS; ubiquitin–proteasome systemGCK-MODY; Catalytic activity; Protein misfolding; Self-association; Dimerization; Degradation

The role of AMPK/mTOR/S6K1 signaling axis in mediating the physiological process of exercise-induced insulin sensitization in skeletal muscle of C57BL/6 mice by Xiaolei Liu; Hairui Yuan; Yanmei Niu; Wenyan Niu; Li Fu (pp. 1716-1726).

The crosstalk between mTORC1/S6K1 signaling and AMPK is emerging as a powerful and highly regulated way to gauge cellular energy and nutrient content. The aim of the current study was to determine the mechanism by which exercise training reverses lipid-induced insulin resistance and the role of AMPK/mTOR/S6K1 signaling axis in mediating this response in skeletal muscle. Our results showed that high-fat feeding resulted in decreased glucose tolerance, which was associated with decreased Akt expression and increased intramuscular triglyceride deposition in the skeletal muscle of C57BL/6 mice. Impairments in lipid metabolism were accompanied by increased total protein and phosphorylation of S6K1, SREBP-1c cleavage, and decreased AMPK phosphorylation. Exercise training reversed these impairments, resulting in improved serum lipid profiles and glucose tolerance. C2C12 myotubes were exposed to palmitate, resulting in an increased insulin-dependent Akt Ser473 phosphorylation, associated with a significant increase in the level of phosphorylation of S6K1 on T389. All these changes were reversed by activation of AMPK. Consistent with this, inhibition of AMPK by compound C induced an enhanced phosphorylation of both S6K1 and Akt, and silencing of S6K1 with siRNA showed no effect on Akt phosphorylation in both the absence and presence of palmitate cultured myotubes. In addition, compound C led to an elevated SREBP-1c cleavage but was blocked by S6K1 siRNA. In summary, exercise training inhibits SREBP-1c cleavage through AMPK/mTOR/S6K1 signaling, resulting in decreased intramyocellular lipid accumulation. Our results provide new insights into the mechanism by which AMPK/mTOR/S6K1 signaling axis mediates the physiological process of exercise-induced insulin sensitization.Display Omitted► Exercise training improved glucose tolerance in insulin-resistant mice. ► Exercise training induced an elevated AMPK and decreased mTOR/S6K1 activity. ► Activation of AMPK inhibited Akt and S6K1 phosphorylation in C2C12 cells. ► S6K1 is responsible for the inhibition of SREBP1 by AMPK in C2C12 myotubes. ► Our data suggest that exercise training inhibits SREBP-1 through AMPK/mTOR pathway.

Keywords: Abbreviations; 4E-BP1; Initiation factor 4E-binding protein 1; ACC; Acetyl-CoA carboxylase; Akt; Protein kinase B; AMPK; AMP-activated protein kinase; CPT1; Carnitine palmitoyltransferase I; DAG; Diacylglycerol; FAT/CD36; Fatty acid translocase; LCFA; Long-chain fatty acids; mTOR; Mammalian target of rapamycin; NEFA; Non-esterified fatty acids; S6K1; p70 ribosomal protein S6 kinase 1; SREBP-1c; Sterol regulatory element binding protein 1c; WAT; White adipose tissueAerobic exercise; Insulin resistance; High-fat diet; Insulin signaling; Lipogenesis

RAPADILINO RECQL4 mutant protein lacks helicase and ATPase activity by Deborah L. Croteau; Marie L. Rossi; Jennifer Ross; Lale Dawut; Christopher Dunn; Tomasz Kulikowicz; Vilhelm A. Bohr (pp. 1727-1734).

The RecQ family of helicases has been shown to play an important role in maintaining genomic stability. In humans, this family has five members and mutations in three of these helicases, BLM, WRN and RECQL4, are associated with disease. Alterations in RECQL4 are associated with three diseases, Rothmund–Thomson syndrome, Baller–Gerold syndrome, and RAPADILINO syndrome. One of the more common mutations found in RECQL4 is the RAPADILINO mutation, c.1390+2delT which is a splice-site mutation leading to an in-frame skipping of exon 7 resulting in 44 amino acids being deleted from the protein (p.Ala420–Ala463del). In order to characterize the RAPADILINO RECQL4 mutant protein, it was expressed in bacteria and purified using an established protocol. Strand annealing, helicase, and ATPase assays were conducted to characterize the protein's activities relative to WT RECQL4. Here we show that strand annealing activity in the absence of ATP is unchanged from that of WT RECQL4. However, the RAPADILINO protein variant lacks helicase and ssDNA-stimulated ATPase activity. These observations help explain the underlying molecular etiology of the disease and our findings provide insight into the genotype and phenotype association among RECQL4 syndromes.Display Omitted► RAPADILINO syndrome is associated with mutations in RECQL4. ► We characterize RAPADILINO RECQL4's catalytic activities relative to WT RECQL4. ► We show that RAPADILINO RECQL4 lacks helicase and ssDNA-dependent ATPase activity. ► RAPADILINO and RTS patients may develop cancer due to loss of helicase activity.

Keywords: RecQ helicase; RECQL4; RAPADILINO; Rothmund–Thomson syndrome; ATPase

Preventing the calorie restriction-induced increase in insulin-stimulated Akt2 phosphorylation eliminates calorie restriction's effect on glucose uptake in skeletal muscle by Naveen Sharma; Edward B. Arias; Donel A. Sequea; Gregory D. Cartee (pp. 1735-1740).

Calorie restriction (CR; ~60% of ad libitum, AL, consumption) improves insulin-stimulated glucose uptake in skeletal muscle. The precise cellular mechanism for this healthful outcome is unknown, but it is accompanied by enhanced insulin-stimulated activation of Akt. Previous research using Akt2-null mice demonstrated that Akt2 is essential for the full CR-effect on insulin-stimulated glucose uptake by muscle. However, because Akt2-null mice were completely deficient in Akt2 in every cell throughout life, it would be valuable to assess the efficacy of transient, muscle-specific Akt inhibition for attenuation of CR-effects on glucose uptake. Accordingly, we used a selective Akt inhibitor (MK-2206) to eliminate the CR-induced elevation in insulin-stimulated Akt2 phosphorylation and determined the effects on Akt substrates and glucose uptake. We incubated isolated epitrochlearis muscles from 9-month-old AL and CR (~60–65% of AL intake for 6months) rats with or without MK-2206 and measured insulin-stimulated (1.2nM) glucose uptake and phosphorylation of the insulin receptor (Tyr1162/1163), pan-Akt (Thr308 and Ser473), Akt2 (Thr308 and Ser473), AS160/TBC1D4 (Thr642), and Filamin C (Ser2213). Incubation of isolated skeletal muscles with a dose of a selective Akt inhibitor that eliminated the CR-induced increases in Akt2 phosphorylation prevented CR's effects on insulin-stimulated glucose uptake, pAS160^Thr642 and pFilamin C^Ser2213 without altering pIR^Tyr1162/1163. These data provide compelling new evidence linking the CR-induced increase in insulin-stimulated Akt2 phosphorylation to CR's effects on insulin-mediated phosphorylation of Akt substrates and glucose uptake in skeletal muscle.► Greater insulin sensitivity leads to improved health with calorie restriction (CR). ► Enhanced insulin effects on Akt2 and glucose uptake in muscle are hallmarks of CR. ► Akt inhibitor MK-2206 eliminated these CR effects in isolated rat skeletal muscle. ► MK-2206 also eliminated CR effects on Akt2 substrates in insulin-stimulated muscle. ► CR effect on Akt2 in insulin-activated muscle is likely a key for health benefits.

Keywords: Insulin sensitivity; Insulin resistance; Glucose transport; Caloric restriction; GLUT4

Crosstalk between the ubiquitin–proteasome system and autophagy in a human cellular model of Alzheimer's disease by Valentina Cecarini; Laura Bonfili; Massimiliano Cuccioloni; Matteo Mozzicafreddo; Giacomo Rossi; Laura Buizza; Daniela Uberti; Mauro Angeletti; Anna Maria Eleuteri (pp. 1741-1751).

Alzheimer's disease is the most common progressive neurodegenerative disorder characterized by the abnormal deposition of amyloid plaques, likely as a consequence of an incorrect processing of the amyloid-β precursor protein (AβPP). Dysfunctions in both the ubiquitin–proteasome system and autophagy have also been observed. Recently, an extensive cross-talk between these two degradation pathways has emerged, but the exact implicated processes are yet to be clarified. In this work, we gained insight into such interplay by analyzing human SH-SY5Y neuroblastoma cells stably transfected either with wild-type AβPP gene or 717 valine-to-glycine AβPP-mutated gene. The over-expression of the AβPP mutant isoform correlates with an increase in oxidative stress and a remodeled pattern of protein degradation, with both marked inhibition of proteasome activities and impairment in the autophagic flux. To compensate for this altered scenario, cells try to promote the autophagy activation in a HDAC6-dependent manner. The treatment with amyloid-β₄₂ oligomers further compromises proteasome activity and also contributes to the inhibition of cathepsin-mediated proteolysis, finally favoring the neuronal degeneration and suggesting the existence of an Aβ₄₂ threshold level beyond which proteasome-dependent proteolysis becomes definitely dysfunctional.► Mutated AβPP increases oxidative stress and remodels the proteolysis in an AD model. ► Alterations in the autophagic flux are evident at the step of autophagosome formation. ► Cells try to counteract the altered scenario activating the HDAC6-dependent autophagy. ► Existence of an Aβ₄₂ level beyond which proteolysis becomes definitely dysfunctional

Keywords: Abbreviations; AβPP; amyloid-β precursor protein; Aβ; ₄₀; amyloid-β (1–40); Aβ; ₄₂; amyloid-β (1–42); UPS; ubiquitin–proteasome system; HDAC6; histone deacetylase 6; ChT-L; chymotrypsin-like; T-L; trypsin-like; PGPH; peptidylglutamyl-peptide hydrolyzing; AP-N; aminopeptidase N; BrAAP; branched-chain amino acids preferring; CM-H; ₂; DCFDA; 5-(and-6) chloromethyl-2′,7′ dichlorodihydrofluorescein diacetate acetyl esterProteasome; Autophagy; Amyloid; Alzheimer's disease; HDAC6

Functional characterization of nine novel naturally occurring human melanocortin-3 receptor mutations by Fan Yang; Ya-Xiong Tao (pp. 1752-1761).

The melanocortin-3 receptor (MC3R) is a member of family A rhodopsin-like G protein-coupled receptors. Mouse genetic studies suggested that MC3R and the related MC4R are non-redundant regulators of energy homeostasis. Lack of Mc3r leads to higher feed efficiency and fat mass. However, until now only a few MC3R mutations have been identified in humans and the role of MC3R in the pathogenesis of obesity was unclear. In the present study, we performed detailed functional studies on nine naturally occurring MC3R mutations recently reported. We found that all nine mutants had decreased cell surface expression. A260V, M275T, and L297V had decreased total expression whereas the other six mutants had normal total expression. Mutants S69C and T280S exhibited significant defects in ligand binding and signaling. The dramatic defects of T280S might be partially caused by decreased cell surface expression. In addition, we found mutants M134I and M275T had decreased maximal binding but displayed similar signaling properties as wild-type MC3R. All the other mutants had normal binding and signaling activities. Co-expression studies showed that all mutants except L297V did not affect wild-type MC3R signaling. Multiple mutations at T280 demonstrated the necessity of Thr for cell surface expression, ligand binding, and signaling. In summary, we provided detailed data of these novel human MC3R mutations leading to a better understanding of structure–function relationship of MC3R and the role of MC3R mutation in obesity.► MC3R mutations predispose humans to obesity. ► T280 is important for cell surface expression, ligand binding, and signaling. ► Decreased cell surface expression is common among missense MC3R mutations.

Keywords: Abbreviations; GPCR; G protein-coupled receptor; hMC3R; human melanocortin-3 receptor; MC4R; melanocortin-4 receptor; MSH; melanocyte stimulating hormone; POMC; proopiomelanocortin; WT; wild-typeMelanocortin-3 receptor; Naturally occurring mutations; Obesity; Binding; Signaling; Cell surface expression

DHA induces apoptosis by altering the expression and cellular location of GRP78 in colon cancer cell lines by Elena Fasano; Simona Serini; Elisabetta Piccioni; Amelia Toesca; Giovanni Monego; Achille R. Cittadini; Franco O. Ranelletti; Gabriella Calviello (pp. 1762-1772).

n−3 polyunsaturated fatty acids exert growth-inhibitory and pro-apoptotic effects in colon cancer cells. We hypothesized that the anti-apoptotic glucose related protein of 78kDa (GRP78), originally described as a component of the unfolded protein response in endoplasmic reticulum (ER), could be a molecular target for docosahexaenoic acid (DHA) in these cells. GRP78 total and surface overexpression was previously associated with a poor prognosis in several cancers, whereas its down-regulation with decreased cancer growth in animal models. DHA treatment induced apoptosis in three colon cancer cell lines (HT-29, HCT116 and SW480), and inhibited their total and surface GRP78 expression. The cell ability to undergo DHA-induced apoptosis was inversely related to their level of GRP78 expression. The transfection of the low GRP78-expressing SW480 cells with GRP78-GFP cDNA significantly induced cell growth and inhibited the DHA-driven apoptosis, thus supporting the essential role of GRP78 in DHA pro-apoptotic effect. We suggest that pERK1/2 could be the first upstream target for DHA, and demonstrate that, downstream of GRP78, DHA may exert its proapoptotic role by augmenting the expression of the ER resident factors ERdj5 and inhibiting the phosphorylation of PKR-like ER kinase (PERK), known to be both physically associated with GRP78, and by activating caspase-4. Overall, the regulation of cellular GRP78 expression and location is suggested as a possible route through which DHA can exert pro-apoptotic and antitumoral effects in colon cancer cells.► Colon cancer cell ability to undergo apoptosis is related to basal GRP78 expression. ► DHA is able to induce apoptosis in colon cancer cells by inhibiting GRP78 total and surface expression. ► Downstream of GRP78, DHA increases ERdj5 expression and inhibits PERK phosphorylation. ► DHA-induced apoptosis in colon cancer cells is accompanied by caspase-4 activation.

Keywords: DHA; Colon cancer cell; Apoptosis; GRP78; ERK1/2

Altered transcription factor trafficking in oxidatively-stressed neuronal cells by Vivek P. Patel; Donald B. DeFranco; Charleen T. Chu (pp. 1773-1782).

Age-related neurodegenerative diseases are associated with alterations in gene expression in affected neurons. One of the mechanisms that could account for this is altered subcellular localization of transcription factors, which has been observed in human post-mortem brains of each of the major neurodegenerative diseases, including Parkinson's disease (PD). The specific mechanisms are yet to be elucidated; however a potential mechanism involves alterations in nuclear transport. In this study, we examined the nucleocytoplasmic trafficking of select transcription factors in response to a PD-relevant oxidative injury, 6-hydroxydopamine (6OHDA). Utilizing a well-established model of ligand-regulated nucleocytoplasmic shuttling, the glucocorticoid receptor, we found that 6OHDA selectively impaired nuclear import through an oxidative mechanism without affecting nuclear export or nuclear retention. Interestingly, impaired nuclear import was selective as Nrf2 (nuclear factor E2-related factor 2) nuclear localization remained intact in 6OHDA-treated cells. Thus, oxidative stress specifically impacts the subcellular localization of some but not all transcription factors, which is consistent with observations in post-mortem PD brains. Our data further implicate a role for altered microtubule dependent trafficking in the differential effects of 6OHDA on transcription factor import. Oxidative disruption of microtubule-dependent nuclear transport may contribute to selective declines in transcriptional responses of aging or diseased dopaminergic cells.► 6OHDA-induced oxidative stress impairs nuclear import of transcription factors. ► 6OHDA does not affect nuclear export or nuclear retention. ► Effect is selective as consistent with observations in post-mortem PD brains. ► 6OHDA alters the state of tubulin polymerization. ► Results support a role for altered microtubule dependent trafficking.

Keywords: Abbreviations; 6OHDA; 6-Hydroxydopamine; AD; Alzheimer's disease; ALS; amyotrophic lateral sclerosis; ATF2; activating transcription factor 2; Bcl2; B-cell lymphoma 2; BDNF; brain-derived neurotrophic factor; Cort; cortisol; CREB; cAMP response element binding protein; Dex; dexamethasone; DMEM; Dulbecco's Modified Eagle's Medium; DPBS; Dulbecco's Phosphate-Buffered Saline; ERK; extracellular signal-regulated kinase; GFP; green fluorescent protein; GR; glucocorticoid receptor; HD; Huntington's disease; Keap1; kelch-like ECH-associated protein 1; LDH; lactate dehydrogenase; LRRK2; leucine-rich repeat kinase 2; MPTP; 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine; MT; microtubule; PD; Parkinson's disease; Nrf2; nuclear factor E2-related factor 2; Nurr1; nuclear receptor related 1 protein; PINK1; PTEN-induced putative kinase 1; ROS; reactive oxygen species; TDP-43; TAR DNA-binding protein 43Parkinson's disease; Nuclear trafficking; 6-Hydroxydopamine; Oxidative stress; Microtubules

Intravitreal injection or topical eye-drop application of a μ-calpain C2L domain peptide protects against photoreceptor cell death in Royal College of Surgeons' rats, a model of retinitis pigmentosa by Taku Ozaki; Mitsuru Nakazawa; Tetsuro Yamashita; Hiroyuki Sorimachi; Shoji Hata; Hiroshi Tomita; Hitomi Isago; Ayaka Baba; Sei-ichi Ishiguro (pp. 1783-1795).

Mitochondrial μ-calpain initiates apoptosis-inducing factor (AIF)-dependent apoptosis in retinal photoreceptor degeneration. Mitochondrial μ-calpain inhibitors may represent therapeutic targets for the disease. Therefore, we sought to identify inhibitors of mitochondrial calpains and determine their effects in Royal College of Surgeons' (RCS) rats, an animal model of retinitis pigmentosa (RP). We synthesized 20-mer peptides of the C2-like (C2L) domain of μ-calpain. Two μ-calpain peptides N2 and N9 inhibited mitochondrial μ-calpain activity (IC₅₀; 892 and 498nM, respectively), but not other proteases. Western blotting showed that 50μM of both μ-calpain peptides caused specific degradation of mitochondrial μ-calpain. Three-dimensional structure of calpains suggested that the peptides N2 and N9 corresponded to the regions forming salt bridges between the protease core domain 2 and the C2L domain. We determined the inhibitory regions of μ-calpain peptides N2 and N9 using 10-mers, and one peptide, N2-10-2, inhibited the activity of mitochondrial μ-calpain (IC₅₀; 112nM). We next conjugated the peptide N2-10-2 to the C-terminal of HIV-1 tat (HIV), a cell-penetrating peptide. Using isolated rat liver mitochondria, 50μM HIV-conjugated μ-calpain N2-10-2 peptide (HIV-Nμ, IC₅₀; 285nM) significantly inhibited AIF truncation. The intravitreal injection of 20mM HIV-Nμ also prevented retinal photoreceptor apoptosis determined by TUNEL staining, and preserved retinal function assessed by electroretinography in RCS rats. Topical application of 40mM HIV-Nμ also prevented apoptosis of retinal photoreceptors in RCS rats. Our results demonstrate that HIV-Nμ, a peptide inhibitor of mitochondrial μ-calpain, offers a new modality for treating RP.► We identified the specific peptide inhibitor of mitochondrial μ-calpain. ► We intravitreally injected the peptide into an animal model of retinitis pigmentosa. ► The peptide prevented retinal photoreceptor apoptosis and preserved retinal function. ► Eye drops containing the peptide also inhibited photoreceptor apoptosis. ► The peptide offers a new modality for treating retinitis pigmentosa.

Keywords: Calpain; Mitochondria; Peptide; Apoptosis-inducing factor; Retinal photoreceptor degeneration; Retinitis pigmentosa

Metformin affects the circadian clock and metabolic rhythms in a tissue-specific manner by Maayan Barnea; Liyan Haviv; Roee Gutman; Nava Chapnik; Zecharia Madar; Oren Froy (pp. 1796-1806).

Metformin is a commonly-used treatment for type 2 diabetes, whose mechanism of action has been linked, in part, to activation of AMP-activated protein kinase (AMPK). However, little is known regarding its effect on circadian rhythms. Our aim was to evaluate the effect of metformin administration on metabolism, locomotor activity and circadian rhythms. We tested the effect of metformin treatment in the liver and muscle of young lean, healthy mice, as obesity and diabetes disrupt circadian rhythms. Metformin led to increased leptin and decreased glucagon levels. The effect of metformin on liver and muscle metabolism was similar leading to AMPK activation either by liver kinase B1 (LKB1) and/or other kinases in the muscle. AMPK activation resulted in the inhibition of acetyl CoA carboxylase (ACC), the rate limiting enzyme in fatty acid synthesis. Metformin also led to the activation of liver casein kinase I α (CKIα) and muscle CKIε, known modulators of the positive loop of the circadian clock. This effect was mainly of phase advances in the liver and phase delays in the muscle in clock and metabolic genes and/or protein expression. In conclusion, our results demonstrate the differential effects of metformin in the liver and muscle and the critical role the circadian clock has in orchestrating metabolic processes.Display Omitted► Metformin leads to increased leptin and decreased glucagon levels. ► The metabolic effect of metformin on liver and muscle metabolism is similar. ► Metformin leads to circadian phase advances in the liver but phase delays in the muscle.

Keywords: Metformin; Circadian rhythm; Clock; Metabolism; AMPK

NMR-based metabolomics study of canine bladder cancer by Jian Zhang; Siwei Wei; Lingyan Liu; G.A. Nagana Gowda; Patty Bonney; Jane Stewart; Deborah W. Knapp; Daniel Raftery (pp. 1807-1814).

Bladder cancer is one of the leading lethal cancers worldwide. With the high risk of recurrence for bladder cancer following the initial diagnoses, lifelong monitoring of patients is necessary. The lack of adequate sensitivity and specificity of current noninvasive monitoring approaches including urine cytology, other urine tests, and imaging, underlines the importance of studies that focus on the detection of more reliable biomarkers for this cancer. The emerging area of metabolomics, which deals with the analysis of a large number of small molecules in a single step, promises immense potential for discovering metabolite markers for screening and monitoring treatment response and recurrence in patients with bladder cancer. Since naturally-occurring canine transitional cell carcinoma of the urinary bladder is very similar to human invasive bladder cancer, spontaneous canine transitional cell carcinoma has been applied as a relevant animal model of human invasive transitional cell carcinoma. In this study, we have focused on profiling the metabolites in urine from dogs with transitional cell carcinoma and healthy control dogs combining nuclear magnetic resonance spectroscopy and statistical analysis methods.¹H NMR-based metabolite profiling analysis was shown to be an effective approach for differentiating samples from dogs with transitional cell carcinoma and healthy controls based on a partial least square-discriminant analysis of the NMR spectra. In addition, there were significant differences in the levels of six individual metabolites between samples from dogs with transitional cell carcinoma and the control group based on the Student's t-test. These metabolites were selected to build a separate partial least square‐discriminant analysis model that was then used to test the classification accuracy. The result showed good classification between transitional cell carcinoma and control groups with the area under the receiver operating characteristic curve of 0.85. The sensitivity and specificity of the model were 86% and 78%, respectively. These results suggest that urine metabolic profiling may have potential for early detection of bladder cancer and of bladder cancer recurrence following treatment, and may enhance our understanding of the mechanisms involved.► NMR based metabolomics of urine differentiates dogs with bladder cancer from healthy controls. ► Six candidate metabolite markers had good discriminatory ability and low p-values. ► A statistical model using these markers had good sensitivity and selectivity to detect the cancer. ► Energy metabolism changes were dominant in canine bladder cancer biochemistry.

Keywords: Bladder cancer; Metabolomics; Biomarker; Nuclear magnetic resonance spectroscopy; Transitional cell carcinoma; Dog

Activation of SIRT1 protects pancreatic β-cells against palmitate-induced dysfunction by Ling Wu; Libin Zhou; Yan Lu; Juan Zhang; Fangfang Jian; Yun Liu; Fengying Li; Wenyi Li; Xiao Wang; Guo Li (pp. 1815-1825).

Sirtuin 1 (SIRT1), a nicotinamide adenosine dinucleotide-dependent histone deacetylase, is an important regulator of energy homeostasis in response to nutrient availability. In pancreatic β-cells, SIRT1 has been shown to up-regulate insulin secretion in response to glucose stimulation. However, the potential roles of SIRT1 in islet β-cells against lipotoxicity remain poorly understood. Here, we demonstrated that SIRT1 mRNA and protein expressions were markedly reduced in the islets isolated from rats infused with 20% Intralipid for 24h. Long-term exposure to 0.4mmol/L palmitate also decreased SIRT1 expression in cultured INS-1 cells and isolated rat islets, which was prevented by 10μmol/L resveratrol, a SIRT1 agonist. In addition, resveratrol improved glucose-stimulated insulin secretion decreased by palmitate, which was abrogated by EX527, a specific SIRT1 inhibitor. Furthermore, inhibition of SIRT1 activity by EX527 or a knockdown of SIRT1 suppressed insulin promoter activity, along with decreased insulin, v‐maf musculoaponeurotic fibrosarcoma oncogene homolog A (MafA), and NK6 homeodomain 1 (NKX6.1) mRNA expressions. Activation of SIRT1 by resveratrol or overexpression of SIRT1 antagonized palmitate-inhibited insulin transcriptional activity. SIRT1 overexpression exerted an additive effect on pancreatic and duodenal homeobox 1 (PDX1)-stimulated insulin promoter activity and abolished forkhead box O1 protein (FOXO1)-decreased insulin transcriptional activity. Resveratrol reversed FOXO1 nuclear translocation induced by palmitate. Our findings indicate that SIRT1 protects against palmitate-induced β-cell dysfunction.► Fatty acids decrease SIRT1 expression in β cells. ► Resveratrol ameliorates palmitate-suppressed insulin secretion. ► Knockdown or inhibition of SIRT1 suppresses insulin transcription. ► SIRT1 activation ameliorates palmitate-inhibited insulin promoter activity.

Keywords: Abbreviations; FFA; free fatty acid; PDX1; pancreatic and duodenal homeobox 1; GSIS; glucose-stimulated insulin secretion; JNK; Jun N-terminal kinase; SIRT1; sirtuin 1; PGC-1; peroxisome proliferator-activated receptor gamma coactivator 1; NeuroD; neurogenic differentiation; NKX6.1; NK6 homeodomain 1; MafA; v-maf musculoaponeurotic fibrosarcoma oncogene homolog A; AMPK; AMP-activated protein kinaseSIRT1; Palmitate; β-cell; Lipotoxicity; Insulin transcription

Autophagy-dependent and -independent involvement of AMP-activated protein kinase in 6-hydroxydopamine toxicity to SH-SY5Y neuroblastoma cells by Katarina Arsikin; Tamara Kravic-Stevovic; Maja Jovanovic; Biljana Ristic; Gordana Tovilovic; Nevena Zogovic; Vladimir Bumbasirevic; Vladimir Trajkovic; Ljubica Harhaji-Trajkovic (pp. 1826-1836).

The role of the main intracellular energy sensor adenosine monophosphate (AMP)-activated protein kinase (AMPK) in the induction of autophagic response and cell death was investigated in SH-SY5Y human neuroblastoma cells exposed to the dopaminergic neurotoxin 6-hydroxydopamine (6-OHDA). The induction of autophagy in SH-SY5Y cells was demonstrated by acridine orange staining of intracellular acidic vesicles, the presence of autophagosome- and autophagolysosome-like vesicles confirmed by transmission electron microscopy, as well as by microtubule‐associated protein 1 light‐chain 3 (LC3) conversion and p62 degradation detected by immunoblotting. 6-OHDA induced phosphorylation of AMPK and its target Raptor, followed by the dephosphorylation of the major autophagy inhibitor mammalian target of rapamycin (mTOR) and its substrate p70S6 kinase (S6K). 6-OHDA treatment failed to suppress mTOR/S6K phosphorylation and to increase LC3 conversion, p62 degradation and cytoplasmatic acidification in neuroblastoma cells in which AMPK expression was downregulated by RNA interference. Transfection of SH-SY5Y cells with AMPK or LC3β shRNA, as well as treatment with pharmacological autophagy inhibitors suppressed, while mTOR inhibitor rapamycin potentiated 6-OHDA-induced oxidative stress and apoptotic cell death. 6-OHDA induced phosphorylation of p38 mitogen-activated protein (MAP) kinase in an AMPK-dependent manner, and pharmacological inhibition of p38 MAP kinase reduced neurotoxicity, but not AMPK activation and autophagy triggered by 6-OHDA. Finally, the antioxidant N-acetyl cysteine antagonized 6-OHDA-induced activation of AMPK, p38 and autophagy. These data suggest that oxidative stress-mediated AMPK/mTOR-dependent autophagy and AMPK/p38-dependent apoptosis could be valid therapeutic targets for neuroprotection.Display Omitted► Pro-oxidant neurotoxin 6-OHDA induces autophagy in SH-SY5Y neuroblastoma cells. ► Energy sensor AMPK-dependent mTOR inhibition mediates autophagy induction by 6-OHDA. ► AMPK/mTOR-dependent autophagy is involved in 6-OHDA toxicity to SH-SY5Y cells. ► AMPK-induced p38 activity mediates 6-OHDA neurotoxicity independently of autophagy. ► AMPK controls 6-OHDA neurotoxicity through autophagy- and p38-dependent mechanisms.

Keywords: Abbreviations; 6-OHDA; 6-hydroxydopamine; AMPK; AMP-activated protein kinase; DHE; dihydroethidium; DHR; dihydrorhodamine; ERK; extracellular signal-regulated kinase; FACS; fluorescence-activated cell sorting; FITC; fluorescein isothiocyanate; LC3; microtubule-associated protein 1 light-chain 3; LDH; lactate dehydrogenase; MAPK; mitogen-activated protein kinase; mTOR; mammalian target of rapamycin; MTT; 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; PD; Parkinson's disease; PI; propidium iodide; ROS; reactive oxygen species; S6K; p70S6 kinase; shRNA; short hairpin RNA; TEM; transmission electron microscopy6-Hydroxydopamine; Neurotoxicity; AMPK; Autophagy; p38; Oxidative stress

Sections

Personal tools

BBA - Molecular Basis of Disease (v.1822, #11)

Sections

Personal tools

Local resources

BBA - Molecular Basis of Disease (v.1822, #11)