Deletion of the γ-Aminobutyric Acid Transporter 2 (GAT2 and SLC6A13) Gene in Mice Leads to Changes in Liver and Brain Taurine Contents

The GABA transporters (GAT1, GAT2, GAT3, and BGT1) have mostly been discussed in relation to their potential roles in controlling the action of transmitter GABA in the nervous system. We have generated the first mice lacking the GAT2 (slc6a13) gene. Deletion of GAT2 (both mRNA and protein) neither affected growth, fertility, nor life span under nonchallenging rearing conditions. Immunocytochemistry showed that the GAT2 protein was predominantly expressed in the plasma membranes of periportal hepatocytes and in the basolateral membranes of proximal tubules in the renal cortex. This was validated by processing tissue from wild-type and knockout mice in parallel. Deletion of GAT2 reduced liver taurine levels by 50%, without affecting the expression of the taurine transporter TAUT. These results suggest an important role for GAT2 in taurine uptake from portal blood into liver. In support of this notion, GAT2-transfected HEK293 cells transported [3H]taurine. Furthermore, most of the uptake of [3H]GABA by cultured rat hepatocytes was due to GAT2, and this uptake was inhibited by taurine. GAT2 was not detected in brain parenchyma proper, excluding a role in GABA inactivation. It was, however, expressed in the leptomeninges and in a subpopulation of brain blood vessels. Deletion of GAT2 increased brain taurine levels by 20%, suggesting a taurine-exporting role for GAT2 in the brain.

Download Full-text

The betaine-GABA transporter (BGT1, slc6a12) is predominantly expressed in the liver and at lower levels in the kidneys and at the brain surface

AJP Renal Physiology ◽

10.1152/ajprenal.00464.2011 ◽

2012 ◽

Vol 302 (3) ◽

pp. F316-F328 ◽

Cited By ~ 79

Author(s):

Y. Zhou ◽

S. Holmseth ◽

R. Hua ◽

A. C. Lehre ◽

A. M. Olofsson ◽

...

Keyword(s):

Plasma Membranes ◽

Renal Medulla ◽

Brain Parenchyma ◽

Ependymal Cells ◽

Main Role ◽

Mrna Levels ◽

Physiological Importance ◽

Brain Surface ◽

Negative Controls ◽

The Brain

The Na+- and Cl−-dependent GABA-betaine transporter (BGT1) has received attention mostly as a protector against osmolarity changes in the kidney and as a potential controller of the neurotransmitter GABA in the brain. Nevertheless, the cellular distribution of BGT1, and its physiological importance, is not fully understood. Here we have quantified mRNA levels using TaqMan real-time PCR, produced a number of BGT1 antibodies, and used these to study BGT1 distribution in mice. BGT1 (protein and mRNA) is predominantly expressed in the liver (sinusoidal hepatocyte plasma membranes) and not in the endothelium. BGT1 is also present in the renal medulla, where it localizes to the basolateral membranes of collecting ducts (particularly at the papilla tip) and the thick ascending limbs of Henle. There is some BGT1 in the leptomeninges, but brain parenchyma, brain blood vessels, ependymal cells, the renal cortex, and the intestine are virtually BGT1 deficient in 1- to 3-mo-old mice. Labeling specificity was assured by processing tissue from BGT1-deficient littermates in parallel as negative controls. Addition of 2.5% sodium chloride to the drinking water for 48 h induced a two- to threefold upregulation of BGT1, tonicity-responsive enhancer binding protein, and sodium- myo-inositol cotransporter 1 (slc5a3) in the renal medulla, but not in the brain and barely in the liver. BGT1-deficient and wild-type mice appeared to tolerate the salt treatment equally well, possibly because betaine is one of several osmolytes. In conclusion, this study suggests that BGT1 plays its main role in the liver, thereby complementing other betaine-transporting carrier proteins (e.g., slc6a20) that are predominantly expressed in the small intestine or kidney rather than the liver.

Download Full-text

Distribution of Suramin, an Antitrypanosomal Drug, across the Blood-Brain and Blood-Cerebrospinal Fluid Interfaces in Wild-Type and P-Glycoprotein Transporter-Deficient Mice

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00372-07 ◽

2007 ◽

Vol 51 (9) ◽

pp. 3136-3146 ◽

Cited By ~ 25

Author(s):

Lisa Sanderson ◽

Adil Khan ◽

Sarah Thomas

Keyword(s):

Choroid Plexus ◽

Brain Perfusion ◽

Brain Parenchyma ◽

Sleeping Sickness ◽

New Drugs ◽

Wild Type ◽

P Glycoprotein ◽

Blood Brain ◽

Targeted Mutation ◽

The Brain

ABSTRACT Although 60 million people are exposed to human African trypanosomiasis, drug companies have not been interested in developing new drugs due to the lack of financial reward. No new drugs will be available for several years. A clearer understanding of the distribution of existing drugs into the brains of sleeping sickness patients is needed if we are to use the treatments that are available more safely and effectively. This proposal addresses this issue by using established animal models. Using in situ brain perfusion and isolated incubated choroid plexus techniques, we investigated the distribution of [3H]suramin into the central nervous systems (CNSs) of male BALB/c, FVB (wild-type), and P-glycoprotein-deficient (Mdr1a/Mdr1b-targeted mutation) mice. There was no difference in the [3H]suramin distributions between the three strains of mice. [3H]suramin had a distribution similar to that of the vascular marker, [14C]sucrose, into the regions of the brain parenchyma that have a blood-brain barrier. However, the association of [3H]suramin with the circumventricular organ samples, including the choroid plexus, was higher than that of [14C]sucrose. The association of [3H]suramin with the choroid plexus was also sensitive to phenylarsine oxide, an inhibitor of endocytosis. The distribution of [3H]suramin to the brain was not affected by the presence of other antitrypanosomal drugs or the P-glycoprotein efflux transporter. Overall, the results confirm that [3H]suramin would be unlikely to treat the second or CNS stage of sleeping sickness.

Download Full-text

Comparison of receptor-mediated endocytosis kinetics between wild-type t-PA and recombinant pamiteplase in isolated rat hepatocytes and liver cell plasma membranes

Xenobiotica ◽

10.1080/00498250050078002 ◽

2000 ◽

Vol 30 (7) ◽

pp. 693-705 ◽

Cited By ~ 1

Author(s):

K. Oikawa ◽

T. Watanabe ◽

S. Higuchi

Keyword(s):

Liver Cell ◽

Plasma Membranes ◽

Rat Hepatocytes ◽

Wild Type ◽

Isolated Rat Hepatocytes ◽

Receptor Mediated Endocytosis ◽

Cell Plasma ◽

Isolated Rat

Download Full-text

Brain Acetyl-CoA Production and Phosphorylation of Cytoskeletal Proteins Are Targets of CYP46A1 Activity Modulation and Altered Sterol Flux

Neurotherapeutics ◽

10.1007/s13311-021-01079-6 ◽

2021 ◽

Author(s):

Natalia Mast ◽

Alexey M. Petrov ◽

Erin Prendergast ◽

Ilya Bederman ◽

Irina A. Pikuleva

Keyword(s):

Cell Biology ◽

Plasma Membranes ◽

Cytoskeletal Proteins ◽

Brain Diseases ◽

Wild Type ◽

Acetyl Coa ◽

Model Of Alzheimer’S Disease ◽

5Xfad Mice ◽

And Control ◽

The Brain

AbstractCholesterol and 24-hydroxycholesterol are the most abundant brain sterols and represent the substrate and product, respectively, of cytochrome P450 46A1 (CYP46A1), a CNS-specific enzyme. CYP46A1 controls cholesterol elimination and turnover in the brain, the two processes that determine the rate of brain sterol flux through the plasma membranes and thereby the properties of these membranes. Brain sterol flux is decreased in Cyp46a1−/− mice compared to wild-type mice and increased in 5XFAD mice (a model of Alzheimer’s disease) when they are treated with a small dose of efavirenz, a CYP46A1 activator. Herein, we first assessed the brain proteome (synaptosomal fractions) and phospho-proteome (synaptosomal fractions and brain homogenates) of efavirenz-treated and control 5XFAD mice. Then, based on the pattern of protein abundance change, we conducted acetyl-CoA measurements (brain homogenates and mitochondria) and metabolic profiling (brain homogenates). The phospho-proteomics datasets were used for comparative analyses with the datasets obtained by us previously on mice with the same changes (efavirenz-treated and control 5XFAD mice from a different treatment paradigm) or with changes in the opposite direction (Cyp46a1−/− vs wild-type mice) in brain sterol flux. We found that CYP46A1 activity or the rate of brain sterol flux affects acetyl-CoA-related metabolic pathways as well as phosphorylation of cytoskeletal and other proteins. Knowledge of the key roles of acetyl-CoA and cytoskeletal phosphorylation in cell biology expands our understanding of the significance of CYP46A1-mediated cholesterol 24-hydroxylation in the brain and provides an additional explanation for why CYP46A1 activity modulations are beneficial in mouse models of different brain diseases.

Download Full-text

Is FAM19A5 an adipokine? Peripheral FAM19A5 in wild-type, FAM19A5 knock-out, and LacZ knock-in mice

10.1101/2020.02.19.955351 ◽

2020 ◽

Author(s):

Hoyun Kwak ◽

Eun-Ho Cho ◽

Eun Bee Cho ◽

Yoo-Na Lee ◽

Anu Shahapal ◽

...

Keyword(s):

Matrix Protein ◽

Hek293 Cells ◽

Extracellular Matrix Protein ◽

Dependent Manner ◽

Wild Type ◽

Adipose Tissues ◽

Peripheral Tissues ◽

Knock Out ◽

Protein Levels ◽

The Brain

AbstractFAM19A5 (also called TAFA5) is a novel secretory protein that is primarily expressed in the brain. However, a recent study reported that FAM19A5 is an adipocyte-derived adipokine that regulates vascular smooth muscle function. Furthermore, genome-wide association study (GWAS) and RNA-seq analyses revealed that the FAM19A5 was associated with a variety of diseases and tumorigenesis in peripheral tissues. We investigated FAM19A5 transcript and protein levels in the peripheral tissues, including adipose tissues from wild-type, FAM19A5 knock-out, and LacZ knock-in mice. In general, total FAM19A5 transcript levels in the central and peripheral nervous systems were higher than levels in any of the peripheral tissues including adipose tissues. Brain tissues expressed similar levels of the FAM19A5 transcript isoforms 1 and 2, whereas expression in the peripheral tissues predominantly expressed isoform 2. In the peripheral tissues, but not the brain, FAM19A5 protein levels in adipose and reproductive tissues were below detectable limits for analysis by Western blot. Additionally, we found that FAM19A5 protein did not interact with the S1PR2 receptor for G-protein-mediated signal transduction, β-arrestin recruitment, and ligand-mediated internalization. Instead, FAM19A5 was internalized into HEK293 cells in an extracellular matrix protein-dependent manner. Taken together, the present study determined basal levels of FAM19A5 transcripts and proteins in peripheral tissues, which provides compelling evidence to further investigate the function of FAM19A5 in peripheral tissues under pathological conditions, including metabolic diseases and/or tumorigenesis.

Download Full-text

Membrane structure in mammalian astrocytes: a review of freeze-fracture studies on adult, developing, reactive and cultured astrocytes

Journal of Experimental Biology ◽

10.1242/jeb.95.1.35 ◽

1981 ◽

Vol 95 (1) ◽

pp. 35-48

Author(s):

D. M. Landis ◽

T. S. Reese

Keyword(s):

Cerebrospinal Fluid ◽

Membrane Structure ◽

Plasma Membranes ◽

Freeze Fracture ◽

Brain Parenchyma ◽

Specific Pattern ◽

Cultured Astrocytes ◽

Fluid Compartments ◽

Polygonal Particle ◽

The Brain

The application of freeze-fracture techniques to studies of brain structure has led to the recognition of two unsuspected specializations of membrane structure, each distributed in a specific pattern across the surface of astrocytes. ‘Assemblies’ (aggregates of uniform, small particles packed in orthogonal array into rectangular or square aggregates) are found to characterize astrocytic plasma membranes apposed to blood vessels or to the cerebrospinal fluid at the surface of the brain. These particle aggregates are much less densely packed in astrocytic processes in brain parenchyma. Assemblies are not fixation artifacts, have been shown to extend to the true outer surface of the membrane, are remarkably labile in the setting of anoxia, and are at least in part protein. The function of assemblies is unknown, but their positioning suggests that they may have a role in the transport of some material into or out of the blood and cerebrospinal fluid compartments. A second specialization of intramembrane particle distribution, the polygonal particle junction, links astrocytic processes at the surface of the brain, and also links proximal, large caliber astrocytic processes in brain parenchyma. The function of this membrane specialization also is unknown, but it may subserve a mechanical role.

Download Full-text

Distribution of Glycylsarcosine and Cefadroxil among Cerebrospinal Fluid, Choroid Plexus, and Brain Parenchyma after Intracerebroventricular Injection is Markedly Different between Wild-Type and Pept2 Null Mice

Journal of Cerebral Blood Flow & Metabolism ◽

10.1038/jcbfm.2010.84 ◽

2010 ◽

Vol 31 (1) ◽

pp. 250-261 ◽

Cited By ~ 22

Author(s):

David E Smith ◽

Yongjun Hu ◽

Hong Shen ◽

Tavarekere N Nagaraja ◽

Joseph D Fenstermacher ◽

...

Keyword(s):

Cerebrospinal Fluid ◽

Choroid Plexus ◽

Interstitial Fluid ◽

Brain Parenchyma ◽

Wild Type ◽

Septal Region ◽

Biodistribution Studies ◽

Choroid Plexuses ◽

Clearance Kinetics ◽

The Brain

The purpose of this study was to define the cerebrospinal fluid (CSF) clearance kinetics, choroid plexus uptake, and parenchymal penetration of PEPT2 substrates in different regions of the brain after intracerebroventricular administration. To accomplish these objectives, we performed biodistribution studies using [14C]glycylsarcosine (GlySar) and [3H]cefadroxil, along with quantitative autoradiography of [14C]GlySar, in wild-type and Pept2 null mice. We found that PEPT2 deletion markedly reduced the uptake of GlySar and cefadroxil in choroid plexuses at 60 mins by 94% and 82% ( P<0.001), respectively, and lowered their CSF clearances by about fourfold. Autoradiography showed that GlySar concentrations in the lateral, third, and fourth ventricle choroid plexuses were higher in wild-type as compared with Pept2 null mice ( P<0.01). Uptake of GlySar by the ependymal–subependymal layer and septal region was higher in wild-type than in null mice, but the half-distance of penetration into parenchyma was significantly less in wild-type mice. The latter is probably because of the clearance of GlySar from interstitial fluid by brain cells expressing PEPT2, which stops further penetration. These studies show that PEPT2 knockout can significantly modify the spatial distribution of GlySar and cefadroxil (and presumably other peptides/mimetics and peptide-like drugs) in brain.

Download Full-text