BBA - Molecular Basis of Disease (v.1862, #7)

BMP-9 enhances fibroblast growth factor 21 expression and suppresses obesity by Sooho Kim; Senyon Choe; Dong Kun Lee (1237-1246).
Although BMP-9 has been reported to induce browning of white adipose tissues (WATs) and suppress high fat diet-induced obesity, detailed molecular mechanism needs to be further elucidated. We report here that administration of MB109, a recombinant derivative of human BMP-9, into obese mice enhanced gene expression of fibroblast growth factor 21 (FGF21), a metabolic regulator, and alleviates a spectrum of pathological symptoms due to high fat diet-induced obesity. In addition, periodical injection of MB109 (500 μg/kg/week) reduced an amount of lipid droplets in the liver, serum levels of alanine aminotransferase (ALT), and total cholesterol. These results indicate that MB109 is also effective to treat obesity-mediated non-alcoholic fatty liver disease (NAFLD).
Keywords: ALT; BMP-9; FGF21; Obesity; Stem cell; UCP1;

We investigated the effects of ketamine on both the temporal and spatial profiles of neural precursor cells located in the hippocampus, and on antidepressant-like behaviors in rats. A single dose of ketamine resulted in a significant increase in the number of 5-bromo-2-deoxyuridine-positive (BrdU+) cells in the dentate gyrus (DG) of rats at 24 h, but not at 28 days, after treatment completion. Ketamine caused antidepressant-like behaviors in the forced swim test (FST) and novelty suppressed feeding test (NSFT). Viral-mediated hippocampal knockdown of vascular endothelial growth factor (VEGF) produced depressive-like behaviors in the FST and NSFT, which were partially recovered by ketamine to the level observed in the control group. The behavioral effects of VEGF knock down were accompanied by a decrease in hippocampal neurogenesis, which was also partially recovered by ketamine.Our results suggest that basal hippocampal VEGF expression is necessary for ketamine-induced antidepressant-like behaviors in rats, but ketamine-induced VEGF expression only partially contributes to hippocampal neurogenesis and the antidepressant-like effects of ketamine.
Keywords: Rat; Depression; Ketamine; Neurogenesis; VEGF;

Loss of striatal 90-kDa ribosomal S6 kinase (Rsk) is a key factor for motor, synaptic and transcription dysfunction in Huntington's disease by Marta Anglada-Huguet; Albert Giralt; Laura Rué; Jordi Alberch; Xavier Xifró (1255-1266).
Huntington's disease (HD) is characterized by motor dysfunction due to the expression of mutant huntingtin that promotes degeneration of striatal GABAergic medium-sized spiny neurons. Here we explore the role of the 90-kDa ribosomal S6 kinase (Rsk) in the physiopathology of HD. First, we show a reduction of Rsk1 and 2 protein levels in the striatum of two HD mouse models, R6/1 and HdhQ7/Q111 knock-in mice, at ages when they suffer from motor disturbances. Interestingly, the analysis of post-mortem samples from HD patients revealed a significant reduction of both Rsk forms in the putamen and caudate, but not in the cortex. Rsk1 and 2 levels were also reduced in the striatum of BDNF heterozygous mice, and upon BDNF neutralization in striatal cultures, suggesting that striatal loss of BDNF could be involved in the decrease of Rsk levels. Finally, we injected recombinant adeno-associated-virus (AAV5)-Rsk in the striatum of R6/1 mice at the onset of motor symptoms. Four weeks later, we found higher Rsk levels in the striatum accompanied by improvements in motor coordination, enhanced expression of synaptic markers and increased expression of genes related to synaptic plasticity, such as cfos and egr1. Altogether, we identified Rsk as a key factor in striatal alterations associated with motor deficits in HD.
Keywords: Striatum; R6/1 mouse; BDNF; Huntingtin; PSD95; VGlut1;

Insulin-like growth factor 1 (IGF-1) therapy: Mitochondrial dysfunction and diseases by M.C. Sádaba; I. Martín-Estal; J.E. Puche; I. Castilla-Cortázar (1267-1278).
This review resumes the association between mitochondrial function and diseases, especially neurodegenerative diseases. Additionally, it summarizes the major role of IGF-1 as a mitochondrial protector, as studied in several experimental models (cirrhosis, aging …). The contribution of mitochondrial dysfunction to impairments in insulin metabolic signaling is also suggested by gene array analysis showing that reductions in gene expression, that regulates mitochondrial ATP production, are associated with insulin resistance and type 2 diabetes mellitus. Moreover, reductions in oxidative capacity of mitochondrial electron transport chain are manifested in obese, insulin-resistant and diabetic patients. Genetic and environmental factors, oxidative stress, and alterations in mitochondrial biogenesis can adversely affect mitochondrial function, leading to insulin resistance and several pathological conditions, such as type 2 diabetes. Finally, it remains essential to know the exact mechanisms involved in mitochondrial generation and metabolism, mitophagy, apoptosis, and oxidative stress to establish new targets in order to develop potentially effective therapies. One of the newest targets to recover mitochondrial dysfunction could be the administration of IGF-1 at low doses. In the last years, it has been observed that IGF-1 therapy has several beneficial effects: restores physiological IGF-1 levels; improves insulin resistance and lipid metabolism; exerts mitochondrial protection; and has hepatoprotective, neuroprotective, antioxidant and antifibrogenic effects. In consequence, treatment of mitochondrial dysfunctions with low doses of IGF-1 could be a powerful and useful effective therapy to restore normal mitochondrial functions.
Keywords: IGF-1; Mitochondria; Oxidative stress; Neurodegenerative diseases; Aging; Hepatic cirrhosis; Mitochondrial diseases; Free radicals;

Charcot-Marie-Tooth disease type 4C (CMT4C) is one of the commonest autosomal recessive inherited peripheral neuropathies and is associated with mutations in the Rab11 effector, SH3TC2. Disruption of the SH3TC2–Rab11 interaction is the molecular abnormality underlying this disease. However, why SH3TC2 mutations cause an isolated demyelinating neuropathy remains unanswered. Here we show that SH3TC2 is an exclusive Schwann cell protein expressed late in myelination and is downregulated following denervation suggesting a functional role in myelin sheath maintenance. We support our data with an evolutionary cell biological analysis showing that the SH3TC2 gene, and its paralogue SH3TC1, are derived from an ancestral homologue, the duplication of which occurred in the common ancestor of jawed vertebrates, coincident with the appearance of Schwann cells and peripheral axon myelination. Furthermore, we report that SH3TC2 associates with integrin-α6, suggesting that aberrant Rab11-dependent endocytic trafficking of this critical laminin receptor in myelinated Schwann cells is connected to the demyelination seen in affected nerves. Our study therefore highlights the inherent evolutionary link between SH3TC2 and peripheral nerve myelination, pointing also towards a molecular mechanism underlying the specific demyelinating neuropathy that characterizes CMT4C.
Keywords: Charcot-Marie-Tooth disease; Schwann cells; Peripheral neuropathy;

Zebrafish (Danio rerio) embryo as a platform for the identification of novel angiogenesis inhibitors of retinal vascular diseases by Sara Rezzola; Giuseppe Paganini; Francesco Semeraro; Marco Presta; Chiara Tobia (1291-1296).
Pathological angiogenesis of the retina is a main cause of blindness. Therapeutic approaches targeting vascular endothelial growth factor, a main angiogenesis inducer in retinal vascular diseases, show significant limitations. Thus, experimental models of retinal neovascularization remain crucial for investigating novel anti-angiogenic strategies and bringing them to patients. Recent observations have shown that eye neovascularization in zebrafish (Danio rerio) embryo may represent a novel target for the identification of angiogenesis inhibitors. This review highlights the use of zebrafish embryo as an innovative model system for the screening of anti-angiogenic molecules to be employed for the treatment of angiogenesis-dependent eye diseases.Display Omitted
Keywords: Angiogenesis; Angiogenesis inhibitors; Embryonic development; Vascular retina diseases; Zebrafish;

Angiotensin II type-2 receptor stimulation induces neuronal VEGF synthesis after cerebral ischemia by Laura Mateos; Maria Jose Perez-Alvarez; Francisco Wandosell (1297-1308).
Intense efforts are being undertaken to understand the pathobiology of ischemia and to develop novel and effective treatments. Angiotensin II type 2 receptor (AT2R) is related with a beneficial role in neurodegenerative disorders, including ischemia. However, the underlying molecular mechanism remains elusive. In this study, we have established that AT2R stimulation by C21 compound, a specific AT2R agonist, caused a VEGF upregulation. Using mouse primary cortical neurons exposed to oxygen–glucose deprivation (OGD), we established that this effect was mediated by a mechanism dependent of mTORC1 signaling since mTOR inhibition abolished the C21-induced VEGF upregulation. Also, we have temporally characterized the changes on VEGF levels after ischemia induction in rats using two different approaches: transient and permanent middle cerebral artery occlusion (tMCAO and pMCAO). VEGF levels were permanently augmented after reperfusion (tMCAO) whereas lower levels of VEGF were found after pMCAO, remarkably at 21 days. Therefore, C21 compound accelerated the recovery of the neurological status of pMCAO rats, reduced the ischemic damage area and abolished pMCAO-induced VEGF downregulation at 21 days. This effect of C21 compound was mainly observed in neurons of the peri-infarct area.Our results suggest that a C21-induced VEGF upregulation may be crucial after an ischemic neuronal insult in both of our experimental approaches. This upregulation was mediated by a mechanism dependent of Akt/mTOR signaling pathway, since mTOR inhibition abolished the VEGF upregulation induced by C21. Considering that VEGF is involved in regenerative processes, we propose that AT2R activation could be used as a potential pharmacological strategy after ischemic stroke.
Keywords: AT2 receptor; Ischemic stroke; MCAO; Oxygen–glucose deprivation and VEGF;

Candida albicans is an opportunist pathogen responsible for a large spectrum of infections, from superficial mycosis to the systemic disease candidiasis. Its ability to adopt various morphological forms, such as unicellular yeasts, filamentous pseudohyphae and hyphae, contributes to its ability to survive within the host. It has been suggested that the antioxidant glutathione is involved in the filamentation process. We investigated S-glutathionylation, the reversible binding of glutathione to proteins, and the functional consequences on C. albicans metabolic remodeling during the yeast-to-hyphae transition.Our work provided evidence for the specific glutathionylation of mitochondrial proteins involved in bioenergetics pathways in filamentous forms and a regulation of the main enzyme of the glyoxylate cycle, isocitrate lyase, by glutathionylation. Isocitrate lyase inactivation in the hyphal forms was reversed by glutaredoxin treatment, in agreement with a glutathionylation process, which was confirmed by proteomic data showing the binding of one glutathione molecule to the enzyme (data are available via ProteomeXchange with identifier PXD003685).We also assessed the effect of alternative carbon sources on glutathione levels and isocitrate lyase activity. Changes in nutrient availability led to morphological flexibility and were related to perturbations in glutathione levels and isocitrate lyase activity, confirming the key role of the maintenance of intracellular redox status in the adaptive metabolic strategy of the pathogen.
Keywords: Candida albicans; Filamentation; Glutathionylation; Isocitrate lyase; Mitochondria;

Mutations of the CLN3 gene lead to juvenile neuronal ceroid lipofuscinosis (JNCL), an autosomal recessive lysosomal storage disorder that causes progressive neurodegeneration in children and adolescents. There is evidence of immune system involvement in pathology that has been only minimally investigated. We characterized bone marrow stem cell-derived antigen presenting cells (APCs), peritoneal macrophages, and leukocytes from spleen and blood, harvested from the Cln3 −/− mouse model of JNCL. We detected dramatically elevated CD11c surface levels and increased total CD11c protein in Cln3 −/− cell samples compared to wild type. This phenotype was specific to APCs and also to a loss of CLN3, as surface levels did not differ from wild type in other leukocyte subtypes nor in cells from two other NCL mouse models. Subcellularly, CD11c was localized to lipid rafts, indicating that perturbation of surface levels is attributable to derangement of raft dynamics, which has previously been shown in Cln3 mutant cells. Interrogation of APC function revealed that Cln3 −/− cells have increased adhesiveness to CD11c ligands as well as an abnormal secretory pattern that closely mimics what has been previously reported for Cln3 mutant microglia. Our results show that CLN3 deficiency alters APCs, which can be a major contributor to the autoimmune response in JNCL.
Keywords: Batten disease; JNCL; CLN3; CD11c; Integrin;

Unique microbial-derived volatile organic compounds in portal venous circulation in murine non-alcoholic fatty liver disease by D.T. Reid; B. McDonald; T. Khalid; T. Vo; L.P. Schenck; M.G. Surette; P.L. Beck; R.A. Reimer; C.S. Probert; K.P. Rioux; B. Eksteen (1337-1344).
Non-alcoholic fatty liver disease is now the leading liver disease in North America. The progression of non-alcoholic fatty liver disease to the inflammatory condition, non-alcoholic steatohepatitis is complex and currently not well understood. Intestinal microbial dysbiosis has been implicated in the development of non-alcoholic fatty liver disease and progression of non-alcoholic steatohepatitis. Volatile organic compounds are byproducts of microbial metabolism in the gut that may enter portal circulation and have hepatotoxic effects contributing to the pathogenesis of non-alcoholic steatohepatitis. To test this hypothesis, we measured volatile organic compounds in cecal luminal contents and portal venous blood in a mouse model of non-alcoholic steatohepatitis.Gas chromatography–mass spectrometry analysis was conducted on cecal content and portal vein blood for volatile organic compound detection from mice fed a methionine and choline deficient diet, which induces non-alcoholic steatohepatitis. The colonic microbiome was studied by 16S rRNA gene amplification using the Illumina MiSeq platform.Sixty-eight volatile organic compounds were detected in cecal luminal content, a subset of which was also present in portal venous blood. Importantly, differences in portal venous volatile organic compounds were associated with diet-induced steatohepatitis establishing a biochemical link between gut microbiota-derived volatile organic compounds and increased susceptibility to non-alcoholic steatohepatitis.Our model creates a novel tool to further study the role of gut-derived volatile organic compounds in the pathogenesis of non-alcoholic steatohepatitis.Illustration of the impact of microbial-derived VOCs on the development of NASH. During health, VOCs produced by gut microbiota enter the healthy liver and are metabolized. At the onset of obesity, the liver begins to accumulate lipid leading to compromised metabolic function, damaged mitochondria, and reduced insulin sensitivity. Changes in gut-derived VOCs during obesity further disrupt liver function and metabolism. Presence of VOCs in the liver in combination with steatosis creates a microenvironment primed for the initiation of inflammation by release of inflammatory mediators followed by the recruitment of proinflammatory cells into the liver. This combination of detrimental cellular processes ultimately leads to the development of non-alcoholic steatohepatitis.Display Omitted
Keywords: Volatile organic compounds; Liver; Microbiome; Metabolomics;

Upregulation of the growth arrest-specific-2 in recurrent colorectal cancers, and its susceptibility to chemotherapy in a model cell system by Chi-Jung Huang; Chia-Long Lee; Shung-Haur Yang; Chih-Cheng Chien; Chi-Cheng Huang; Ruey-Neng Yang; Chun-Chao Chang (1345-1353).
Colorectal cancer (CRC) is one of the most common life-threatening malignances worldwide. CRC relapse markedly decreases the 5-year survival of patients following surgery. Aberrant expression of genes involved in pathways regulating the cell cycle, cell proliferation, or cell death are frequently reported in CRC tumorigenesis. We hypothesized that genes involved in CRC relapse might serve as prognostic indicators. We first evaluated the significance of gene sequences in the feces of patients with CRC relapse by consulting a public database. Tumorigenesis of target tissues was tested through tumor cell growth, cell cycle regulation, and chemotherapeutic efficacy. We found a highly significant correlation between CRC relapse and growth arrest-specific 2 (GAS2) gene expression. Based on cell models, the overexpressed GAS2 was associated with cellular growth rate, cell cycle regulation, and with chemotherapeutic sensitivity. Cell division was impaired by treating cells with 2-[4-(7-chloro-2-quinoxalinyloxy)phenoxy]-propionic acid (XK469), even when the cells were overexpressing GAS2. Thus, downregulation of GAS2 expression might control CRC relapse after curative resection. GAS2 could serve as a noninvasive marker from the feces of patients with prediagnosed CRC. Our findings suggest that GAS2 could have potential clinical applications for predicting early CRC relapse after radical resection, and that XK469 might impair tumor cell division by reducing GAS2 expression or blocking its cellular translocation. This will help in selecting the best therapeutic option, 5-fluorouracil in combination with XK469, for patients overexpressing GAS2 in CRC cells. Thus, GAS2 might act as a prognostic biomolecule and potential therapeutic target in patients with CRC relapse.

Ischaemic stroke, accompanied by neuroinflammation, impairs blood–brain barrier integrity through a complex mechanism involving both protein kinase C (PKC) and urokinase. Using an in vitro model of human blood–brain barrier (BBB) composed of brain microvascular endothelial cells (HBMEC) and astrocytes, this study assessed the putative roles of these elements in BBB damage evoked by enhanced availability of pro-inflammatory cytokine, TNF-α. Treatment of HBMEC with TNF-α significantly increased the mRNA and protein expressions of all plasminogen–plasmin system (PPS) components, namely tissue plasminogen activator, urokinase, urokinase plasminogen activator receptor and plasminogen activator inhibitor-1 and also the activities of urokinase, total PKC and extracellular MMP-2. Inhibition of urokinase by amiloride abated the effects of TNF-α on BBB integrity and MMP-2 activity without affecting that of total PKC. Conversely, pharmacological inhibition of conventional PKC isoforms dramatically suppressed TNF-α-induced overactivation of urokinase. Knockdown of PKC-α gene via specific siRNA in HBMEC suppressed the stimulatory effects of TNF-α on protein expression of all PPS components, MMP-2 activity, DNA fragmentation rates and pro-apoptotic caspase-3/7 activities. Establishment of co-cultures with BMEC transfected with PKC-α siRNA attenuated the disruptive effects of TNF-α on BBB integrity and function. This was partly due to elevations observed in expression of a tight junction protein, claudin-5 and partly to prevention of stress fibre formation. In conclusion, specific inhibition of PKC-α in cerebral conditions associated with exaggerated release of pro-inflammatory cytokines, notably TNF-α may be of considerable therapeutic value and help maintain endothelial cell viability, appropriate cytoskeletal structure and basement membrane.
Keywords: TNF-α; Blood–brain barrier; PKC-α; PPS components; MMP-2; Claudin-5; Apoptosis; Stress fibres;

Lipolysis and lipophagy in lipid storage myopathies by Corrado Angelini; Anna Chiara Nascimbeni; Giovanna Cenacchi; Elisabetta Tasca (1367-1373).
Triglycerides droplets are massively stored in muscle in Lipid Storage Myopathies (LSM). We studied in muscle regulators of lipophagy, the expression of the transcription factor-EB (TFEB) (a master regulator of lysosomal biogenesis), and markers of autophagy which are induced by starvation and exert a transcriptional control on lipid catabolism.We investigated the factors that regulate lipophagy in muscle biopsies from 6 patients with different types of LSM: 2 cases of riboflavin-responsive multiple acyl-CoA dehydrogenase deficiency (MADD), 1 case of primary carnitine deficiency (CD), 2 cases of neutral lipid storage myopathy (NLSD-M), 1 case of carnitine–palmitoyl-transferase-II (CPT) deficiency.Conventional morphology and electron microscopy documented the lipid accumulation and its dramatic resolution after treatment. Muscle immunofluorescence showed that while in MADD and NLSD-M there was a co-localized expression of TFEB and p62-SQSTM1 (marker of protein aggregates) in some atrophic fibers, in CD and CPT-II deficiency the reaction was almost normal. In regenerating fibers, TFEB localized in the cytoplasm (inactive form), whereas in atrophic fibers it localized in the nuclei (active form). Lipid-accumulated/atrophic fibers did not display p62-positive protein aggregates, indicating, together with the LC3-II (marker of autophagosomes) and p62-SQSTM1 analysis, that the autophagic flux is often preserved and lipophagy occurs.In atrophic and regenerating fibers of patients with NLSD-M we observed TFEB over-expression; in other conditions autophagy markers are increased, suggesting lipophagy active role on human lipid metabolism.
Keywords: Lipid storage myopathy; MADD; NLSD-M; Carnitine deficiency; TFEB;