PrP mRNA and protein expression in brain and PrPc in CSF in Creutzfeldt-Jakob disease MM1 and VV2

Creutzfeldt-Jakob disease (CJD) is a heterogenic neurodegenerative disorder associated with abnormal post-translational processing of cellular prion protein (PrP^c). CJD displays distinctive clinical and pathological features which correlate with the genotype at the codon 129 (methionine or valine: M or V respectively) in the prion protein gene and with size of the protease-resistant core of the abnormal prion protein PrP^sc (type 1: 20/21 kDa and type 2: 19 kDa). MM1 and VV2 are the most common sporadic CJD (sCJD) subtypes. PrP mRNA expression levels in the frontal cortex and cerebellum are reduced in sCJD in a form subtype-dependent. Total PrP protein levels and PrP^sc levels in the frontal cortex and cerebellum accumulate differentially in sCJD MM1 and sCJD VV2 with no relation between PrP^sc deposition and spongiform degeneration and neuron loss, but with microgliosis, and IL6 and TNF-α response. In the CSF, reduced PrP^c, the only form present in this compartment, occurs in sCJD MM1 and VV2. PrP mRNA expression is also reduced in the frontal cortex in advanced stages of Alzheimer disease, Lewy body disease, progressive supranuclear palsy, and frontotemporal lobe degeneration, but PrP^c levels in brain varies from one disease to another. Reduced PrP^c levels in CSF correlate with PrP mRNA expression in brain, which in turn reflects severity of degeneration in sCJD.     

Cloning and ontogenetic expression of the uncoupling protein 1 gene <Emphasis Type="Italic">UCP1</Emphasis> in sheep

The uncoupling protein 1 (UCP1) is an indicator of brown adipocytes and is involved in the control of body temperature and regulation of energy balance. It abundantly expresses in newborns and has important functions in adults. However, little information was known on UCP1 gene expression in young and adolescent sheep. In this study, we cloned and identified the full-length DNA and cDNA sequences of the ovine UCP1 gene, which were 6659 bp and 1621 bp, respectively, and predicted the location of the gene on chromosome 17. Forty-eight animals with an equal number of males and females each for both Guangling Large Tail sheep (GLT) and Small Tail sheep Han (STH) sheep were used to study the ontogenetic expression of UCP1 mRNA in eight adipose tissues by quantitative real-time polymerase chain reaction (PCR). The results showed that the mRNA was expressed in all tissues studied and at all stages from 2 to 12 months of age. Nevertheless, the mRNA in perirenal fat was expressed significantly higher than that in other tissues and lower in superficial fat than in deep deposits. The highest expression was observed in animals at 2 months of age and then decreased gradually with age. Global expression in GLT was significantly higher than that in STH. Interactions between tissue and breed and age also influenced the mRNA expression significantly. In addition, the mRNA expression was associated with the single nucleotide polymorphism (SNP) haplotypes detected in the cDNA of the gene.     

The human uncoupling proteins 5 and 6 (UCP5/SLC25A14 and UCP6/SLC25A30) transport sulfur oxyanions,phosphate and dicarboxylates

The human genome encodes 53 members of the solute carrier family 25 (SLC25), also called the mitochondrial carrier family. In this work, two members of this family, UCP5 (BMCP1, brain mitochondrial carrier protein 1 encoded by SLC25A14) and UCP6 (KMCP1, kidney mitochondrial carrier protein 1 encoded by SLC25A30) have been thoroughly characterized biochemically. They were overexpressed in bacteria, purified and reconstituted in phospholipid vesicles. Their transport properties and kinetic parameters demonstrate that UCP5 and UCP6 transport inorganic anions (sulfate, sulfite, thiosulfate and phosphate) and, to a lesser extent, a variety of dicarboxylates (e.g. malonate, malate and citramalate) and, even more so, aspartate and (only UCP5) glutamate and tricarboxylates. Both carriers catalyzed a fast counter-exchange transport and a very low uniport of substrates. Transport was saturable and inhibited by mercurials and other mitochondrial carrier inhibitors at various degrees. The transport affinities of UCP5 and UCP6 were higher for sulfate and thiosulfate than for any other substrate, whereas the specific activity of UCP5 was much higher than that of UCP6. It is proposed that a main physiological role of UCP5 and UCP6 is to catalyze the export of sulfite and thiosulfate (the H₂S degradation products) from the mitochondria, thereby modulating the level of the important signal molecule H₂S.     

Evidence that protein kinase Calpha interacts with and regulates the glial glutamate transporter GLT-1

Many of the sodium‐dependent neurotransmitter transporters are rapidly (within minutes) regulated by protein kinase C (PKC), with changes in activity being correlated with changes in transporter trafficking to or from the plasma membrane. Our recent studies suggest that one of the classical subtypes of PKC, PKCα, may selectively mediate redistribution of the neuronal glutamate transporter, excitatory amino acid carrier (EAAC)1, and show that PKCα can be co‐immunoprecipitated with EAAC1. When the glial glutamate transporter GLT‐1a is transfected into C6 glioma cells, this transporter is internalized in response to activation of PKC, but the PKC subtype involved in this regulation is unknown. In the present study, expression of the phorbol ester‐activated subtypes of PKC was examined in C6 glioma transfected with GLT‐1. Of the classical subtypes, only PKCα was detected, and of the non‐classical subtypes, PKCδ and PKCε were detected. In this system, phorbol ester‐dependent internalization of GLT‐1 was blocked by a general inhibitor of PKCs (bisindolylmaleimide II) and by concentrations of Gö6976 that selectively block classical PKCs, but not by an inhibitor of PKCδ (rottlerin). PKCα immunoreactivity was found in GLT‐1 immunoprecipitates obtained from transfected C6 cells and from crude rat brain synaptosomes, a milieu that better mimics in vivo conditions. The amount of PKCα in both types of immunoprecipitate was modestly increased by phorbol ester, and this increase was blocked by a PKC antagonist. These studies suggest that PKCα may be required for the regulated redistribution of GLT‐1.     

High expression of SLC38A1 predicts poor prognosis in patients with de novo acute myeloid leukemia

The glutamine amino acid transporter solute carrier family 38 member 1 (SLC38A1) is associated with the occurrence and progression of solid tumors. However, it has not yet been assessed in patients with hematologic malignancy. Herein, we investigated SLC38A1 expression and explored its clinical implications in acute myeloid leukemia (AML). The results showed that patients with high SLC38A1 expression had a lower mutation rate of NPM1 gene and higher incidence of adverse-risk karyotype (p = 0.0010 and 0.0051, respectively). Patients with a high level of SLC38A1 expression presented significantly shorter overall survival in whole-cohort, chemotherapy-only, and non-inv(16) AML (p = 0.0049, 0.0247, and 0.0005 respectively). Moreover, both univariate and multivariate analyses showed that high SLC38A1 expression was an independent unfavorable prognostic biomarker for AML (p = 0.0057 and 0.0483, respectively). In summary, our study revealed SLC38A1 as a valuable prognostic and predictive marker for AML. Further, glutamine transporter SLC38A1 might serve as a potential target for the development of novel therapeutic drugs in the treatment of AML.     

Two novel mutations in the SLCO2A1 gene in a Chinese patient with primary hypertrophic osteoarthropathy

Primary hypertrophic osteoarthropathy (PHO) is a rare monogenetic disease characterized by digital clubbing, periostosis and pachydermia. Mutations in the 15-hydroxy-prostaglandin dehydrogenase (HPGD) gene and solute carrier organic anion transporter family member 2A1 (SLCO2A1) gene have been shown to be associated with PHO. Here, we described clinical characteristics in a Chinese patient with PHO, and identified two novel mutations in SLCO2A1: a heterozygous guanine-to-thymidine transition at the invariant − 1 position of the acceptor site of intron 2 (c.235-1G > T) and a heterozygous missense mutation p.Pro219Leu (c.656C > T) in exon 5.     

Frequency distribution of PRNP polymorphisms in the Pakistani population

Prion diseases are neurodegenerative conditions caused by misfolding of a normal host-encoded prion protein (PrP^C) into pathogenic scrapie prion protein (PrP^Sc). In human prion diseases, the M129V prion protein polymorphism is known to confer susceptibility to the disease, determines PrP^Sc conformation and alters clinicopathological phenotypes. To date, all clinicopathologically confirmed cases of a variant form of Cruetzfeldt-Jacob disease (vCJD) have been 129MM homozygotes. There is also predominance of 129MM homozygotes in sporadic CJD (sCJD). No information regarding prion disorders is available from Pakistan. Although only invasive procedures like brain biopsy can confirm the diagnosis of prion disorders, testing a corresponding human population for variation in the prion protein gene (PRNP) may provide some insights into the presence of these disorders in a locality. The current study therefore aimed at exploring the genetic susceptibility of Pakistani population to CJD. A total of 909 unrelated individuals including 221 hemophiliacs representing all 4 major provinces of Pakistan were screened for M129V polymorphism and insertions or deletions of octapeptide repeats (OPRIs/OPRDs) using Polymerase Chain Reaction coupled with Restriction Fragment Length Polymorphism (PCR-RFLP). Concordance of the results of some PCR-RFLP reactions was also confirmed by dideoxy automated Sanger sequencing. The frequencies of M129V alleles (129M and 129V) and genotypes (129MM, 129MV and 129VV) were found in all 909 individuals to be 0.7101, 0.2899, 0.5270, 0.3663 and 0.1067, respectively. Deletion of 1 octapeptide repeat (1-OPRD) was detected in heterozygous state in PRNP of 10 individuals and in homozygous state in 1 individual. An insertion of 3 octapeptide repeats (3-OPRI) was found in 1 individual and an insertion of 1 octapeptide repeat (1-OPRI) in two individuals. Both 3-OPRI and 1-OPRI were present in heterozygous state and were linked to 129M allele. There were no significant χ² differences between M129V allelic and genotypic frequencies of healthy individuals and hemophiliacs. However, M129V allelic and genotypic frequencies differed significantly between Pakistani population and East Asian and Western populations. Non-significant χ² differences between M129V frequencies of healthy individuals and hemophiliacs suggest that individuals manifesting single gene disorders may provide naturally randomized samples for studies aiming at surveying the genetic variation. The combined excess of 129MM and 129VV homozygosity and the presence of 3-OPRI in 1 individual imply that Pakistani population is susceptible to prion disorders. Cases of prion disorders may exist in Pakistan, albeit at lower annual prevalence than other countries where life expectancy is greater than 65 years.     

Chronic dietary vitamin A supplementation regulates obesity in an obese mutant WNIN/Ob rat model

Objective: To understand the possible role of chronic dietary high vitamin A supplementation in body weight regulation and obesity using a novel WNIN/Ob obese rat model developed at the National Centre for Laboratory Animal Sciences of National Institute of Nutrition, India. Research Methods and Procedures: Thirty‐six 7‐month‐old male rats of lean, carrier, and obese phenotypes were broadly divided into two groups; each group was subdivided into three subgroups consisting of six lean, six carrier, and six obese rats and received diets containing either 2.6 or 129 mg vitamin A/kg of diet for 2 months. Body weight gain, food intake, and weights of various organs were recorded. Adiposity index and BMI were calculated. Serum and liver retinol and brown adipose tissue (BAT)‐uncoupling protein1 (UCP1) mRNA expression levels were quantified. Results: Chronic feeding of high but non‐toxic doses of vitamin A through diet significantly reduced (P ≤ 0.05) body weight gain, adiposity index, and retroperitoneal white adipose tissue mass (without affecting food intake) in obese rats compared with their lean and carrier counterparts. In general, vitamin A treatment significantly improved hepatic retinol stores (P ≤ 0.05) in all phenotypes without affecting serum free retinol levels. However, augmented BAT‐UCP1 expression was observed only in carrier and obese rats (whose basal expression was low). Discussion: Our data suggest that chronic dietary vitamin A supplementation at high doses effectively regulates obesity in obese phenotype of the WNIN/Ob strain, possibly through up‐regulation of the BAT‐UCP1 gene and associated adipose tissue loss. However, in vitamin A‐supplemented lean and carrier rats, changes in adiposity could not be related to BAT‐UCP1 expression levels.     

Uncoupling Protein 2 (UCP2) Function in the Brain as Revealed by the Cerebral Metabolism of (1–<Superscript>13</Superscript>C)-Glucose

The mitochondrial aspartate/glutamate transporter Aralar/AGC1/Slc25a12 is critically involved in brain aspartate synthesis, and AGC1 deficiency results in a drastic fall of brain aspartate levels in humans and mice. It has recently been described that the uncoupling protein UCP2 transports four carbon metabolites including aspartate. Since UCP2 is expressed in several brain cell types and AGC1 is mainly neuronal, we set to test whether UCP2 could be a mitochondrial aspartate carrier in the brain glial compartment. The study of the cerebral metabolism of (1–¹³C)-glucose in vivo in wild type and UCP2-knockout mice showed no differences in C3 or C2 labeling of aspartate, suggesting that UCP2 does not function as a mitochondrial aspartate carrier in brain. However, surprisingly, a clear decrease (of about 30–35?%) in the fractional enrichment of glutamate, glutamine and GABA was observed in the brains of UCP2-KO mice which was not associated with differences in either glucose or lactate enrichments. The results suggest that the dilution in the labeling of glutamate and its downstream metabolites could originate from the uptake of an unlabeled substrate that could not leave the matrix via UCP2 becoming trapped in the matrix. Understanding the nature of the unlabeled substrate and its precursor(s) as alternative substrates to glucose is of interest in the context of neurological diseases associated with UCP2.     

The Escherichia coli SLC26 homologue YchM (DauA) is a C4‐dicarboxylic acid transporter

The SLC26/SulP (solute carrier/sulphate transporter) proteins are a ubiquitous superfamily of secondary anion transporters. Prior studies have focused almost exclusively on eukaryotic members and bacterial members are frequently classified as sulphate transporters based on their homology with SulP proteins from plants and fungi. In this study we have examined the function and physiological role of the Escherichia coli Slc26 homologue, YchM. We show that there is a clear YchM‐dependent growth defect when succinate is used as the sole carbon source. Using an in vivo succinate transport assay, we show that YchM is the sole aerobic succinate transporter active at acidic pH. We demonstrate that YchM can also transport other C₄‐dicarboxylic acids and that its substrate specificity differs from the well‐characterized succinate transporter, DctA. Accordingly ychM was re‐designated dauA (dicarboxylic acid uptake system A). Finally, our data suggest that DauA is a protein with transport and regulation activities. This is the first report that a SLC26/SulP protein acts as a C₄‐dicarboxylic acid transporter and an unexpected new function for a prokaryotic member of this transporter family.     

Non-Involvement of the Human Monocarboxylic Acid Transporter 1 (MCT1) in the Transport of Phenolic Acid

Phenolic acids such asp-coumaric acid and microbial metabolites of poorly absorbed polyphenols are absorbed by the monocarboxylic acid transporter (MCT)-mediated transport system which is identical to the fluorescein/H⁺ cotransport system. We focus here on the physiological impact of MCT-mediated absorption and distribution. We examined whether MCT1, the best-characterized isoform found in almost all tissues, is involved in this MCT-mediated transport system. The induction of MCT1 expression in Caco-2 cells by a treatment with sodium butyrate (NaBut) did not increase the fluorescein permeability. Moreover, the transfection of Caco-2 cells with an expression vector encoding MCT1 caused no increase in either the permeability or uptake of fluorescein. Furthermore, in the MCT1-expressing oocytes, no increase ofp-coumaric acid uptake was apparent, whereas the uptake of salicylic acid, a substrate of MCT1, nearly doubled. Our data therefore establish that MCT1 was not involved in the MCT-mediated transport of phenolic acids.     

Genetic Pathways Regulating Glutamate Levels in Retinal Müller Cells

Müller cells serve many functions including the regulation of extracellular glutamate levels. The product of two genes, Slc1a3 [aka solute carrier family 1 (glial high affinity glutamate transporter), member 3] and Glul (aka glutamine synthetase) are the primary role players that transport glutamate into the Müller cell and convert it into glutamine. In this study, we sought to identify the genetic regulation of both genes. Given their tightly coupled biological functions, we predicted that they would be similarly regulated. Using an array of 75 recombinant inbred strains of mice, we determined that Slc1a3 and Glul are differentially regulated by distinct chromosomal regions. Interestingly, despite their independent regulation, gene ontology analysis of tightly correlated genes reveals that the enriched and statistically significant molecular function categories of both directed acyclic graphs have substantial overlap, indicating that the shared functions of correlates of Slc1a3 and Glul include production and usage of ATP.     

Resistance to Diet-Induced Obesity and Associated Metabolic Perturbations in Haploinsufficient Monocarboxylate Transporter 1 Mice

The monocarboxylate transporter 1 (MCT1 or SLC16A1) is a carrier of short-chain fatty acids, ketone bodies, and lactate in several tissues. Genetically modified C57BL/6J mice were produced by targeted disruption of the mct1 gene in order to understand the role of this transporter in energy homeostasis. Null mutation was embryonically lethal, but MCT1 ^+/− mice developed normally. However, when fed high fat diet (HFD), MCT1 ^+/− mice displayed resistance to development of diet-induced obesity (24.8% lower body weight after 16 weeks of HFD), as well as less insulin resistance and no hepatic steatosis as compared to littermate MCT1 ^+/+ mice used as controls. Body composition analysis revealed that reduced weight gain in MCT1 ^+/− mice was due to decreased fat accumulation (50.0% less after 9 months of HFD) notably in liver and white adipose tissue. This phenotype was associated with reduced food intake under HFD (12.3% less over 10 weeks) and decreased intestinal energy absorption (9.6% higher stool energy content). Indirect calorimetry measurements showed ∼ 15% increase in O₂ consumption and CO₂ production during the resting phase, without any changes in physical activity. Determination of plasma concentrations for various metabolites and hormones did not reveal significant changes in lactate and ketone bodies levels between the two genotypes, but both insulin and leptin levels, which were elevated in MCT1 ^+/+ mice when fed HFD, were reduced in MCT1 ^+/− mice under HFD. Interestingly, the enhancement in expression of several genes involved in lipid metabolism in the liver of MCT1 ^+/+ mice under high fat diet was prevented in the liver of MCT1 ^+/− mice under the same diet, thus likely contributing to the observed phenotype. These findings uncover the critical role of MCT1 in the regulation of energy balance when animals are exposed to an obesogenic diet.     

The Promise of Pharmacogenomics in Reducing Toxicity During Acute Lymphoblastic Leukemia Maintenance Treatment

Pediatric acute lymphoblastic leukemia(ALL) affects a substantial number of children every year and requires a long and rigorous course of chemotherapy treatments in three stages, with the longest phase, the maintenance phase, lasting 2–3 years. While the primary drugs used in the maintenance phase, 6-mercaptopurine(6-MP) and methotrexate(MTX), are necessary for decreasing risk of relapse, they also have potentially serious toxicities, including myelosuppression, which may be life-threatening, and gastrointestinal toxicity. For both drugs, pharmacogenomic factors have been identified that could explain a large amount of the variance in toxicity between patients, and may serve as effective predictors of toxicity during the maintenance phase of ALL treatment.6-MP toxicity is associated with polymorphisms in the genes encoding thiopurine methyltransferase(TPMT), nudix hydrolase 15(NUDT15), and potentially inosine triphosphatase(ITPA), which vary between ethnic groups. Moreover, MTX toxicity is associated with polymorphisms in genes encoding solute carrier organic anion transporter family member 1B1(SLCO1B1) and dihydrofolate reductase(DHFR). Additional polymorphisms potentially associated with toxicities for MTX have also been identified, including those in the genes encoding solute carrier family 19 member 1(SLC19A1)and thymidylate synthetase(TYMS), but their contributions have not yet been well quantified. It is clear that pharmacogenomics should be incorporated as a dosage-calibrating tool in pediatric ALL treatment in order to predict and minimize the occurrence of serious toxicities for these patients.     

The glial glutamate transporter 1 (GLT1) is expressed by pancreatic beta-cells and prevents glutamate-induced beta-cell death

Glutamate is the major excitatory neurotransmitter of the central nervous system (CNS) and may induce cytotoxicity through persistent activation of glutamate receptors and oxidative stress. Its extracellular concentration is maintained at physiological concentrations by high affinity glutamate transporters of the solute carrier 1 family (SLC1). Glutamate is also present in islet of Langerhans where it is secreted by the α-cells and acts as a signaling molecule to modulate hormone secretion. Whether glutamate plays a role in islet cell viability is presently unknown. We demonstrate that chronic exposure to glutamate exerts a cytotoxic effect in clonal β-cell lines and human islet β-cells but not in α-cells. In human islets, glutamate-induced β-cell cytotoxicity was associated with increased oxidative stress and led to apoptosis and autophagy. We also provide evidence that the key regulator of extracellular islet glutamate concentration is the glial glutamate transporter 1 (GLT1). GLT1 localizes to the plasma membrane of β-cells, modulates hormone secretion, and prevents glutamate-induced cytotoxicity as shown by the fact that its down-regulation induced β-cell death, whereas GLT1 up-regulation promoted β-cell survival. In conclusion, the present study identifies GLT1 as a new player in glutamate homeostasis and signaling in the islet of Langerhans and demonstrates that β-cells critically depend on its activity to control extracellular glutamate levels and cellular integrity.     

Association of Common Polymorphisms in the Fractalkine Receptor (CX3CR1) With Obesity

The inflammatory component in obesity is now well established. The CX3CR1 gene encodes the fractalkine (CX3CL1) receptor and has two coding single‐nucleotide polymorphisms, V249I and T280M, linked to a lower risk of other inflammatory diseases such as coronary artery disease (CAD) and asthma. To determine whether CX3CR1 is associated with obesity, we genotyped the V249I and T280M polymorphisms of the CX3CR1 gene in subjects with a BMI ≥30 kg/m² and nonobese controls with a BMI <30 kg/m². Binary logistic regression analyses revealed that the 280MM genotype was associated with obesity (P = 0.022). A gender‐specific one‐way ANOVA was also conducted to investigate mean BMI and waist circumference differences between genotypes of each polymorphism. For both polymorphisms independently, women carrying two copies of the minor allele had significant higher mean waist circumference than those carrying only one copy of the minor allele (MM > TM, P = 0.031; II > VI, P = 0.013) or those who were homozygous for the major allele (MM > TT, P = 0.005; II > VV, P = 0.006). We also observed significant higher mean waist circumference in men carrying one copy of the minor allele when compared to those who were homozygous for the major allele for the T280M polymorphism (TM > TT, P = 0.029). This study suggests that CX3CR1, a biomarker of obesity in this sample, constitutes a potential target for further investigation of the role of inflammation in the expression of obesity‐related phenotypes.     

Neuronal Exosomal miRNA-dependent Translational Regulation of Astroglial Glutamate Transporter GLT1

Perisynaptic astrocytes express important glutamate transporters, especially excitatory amino acid transporter 2 (EAAT2, rodent analog GLT1) to regulate extracellular glutamate levels and modulate synaptic activation. In this study, we investigated an exciting new pathway, the exosome-mediated transfer of microRNA (in particular, miR-124a), in neuron-to-astrocyte signaling. Exosomes isolated from neuron-conditioned medium contain abundant microRNAs and small RNAs. These exosomes can be directly internalized into astrocytes and increase astrocyte miR-124a and GLT1 protein levels. Direct miR-124a transfection also significantly and selectively increases protein (but not mRNA) expression levels of GLT1 in cultured astrocytes. Consistent with our in vitro findings, intrastriatal injection of specific antisense against miR-124a into adult mice dramatically reduces GLT1 protein expression and glutamate uptake levels in striatum without reducing GLT1 mRNA levels. MiR-124a-mediated regulation of GLT1 expression appears to be indirect and is not mediated by its suppression of the putative GLT1 inhibitory ligand ephrinA3. Moreover, miR-124a is selectively reduced in the spinal cord tissue of end-stage SOD1 G93A mice, the mouse model of ALS. Subsequent exogenous delivery of miR-124a in vivo through stereotaxic injection significantly prevents further pathological loss of GLT1 proteins, as determined by GLT1 immunoreactivity in SOD1 G93A mice. Together, our study characterized a new neuron-to-astrocyte communication pathway and identified miRNAs that modulate GLT1 protein expression in astrocytes in vitro and in vivo.