Immunogold cytochemistry of the blood-brain barrier glucose transporter GLUT1 and endogenous albumin in the developing human brain

The blood-brain barrier (BBB) glucose transporter, GLUT1, was detected by immunogold electron microscopy on the microvascular compartment of the human foetus telencephalon at the 12th and 18th weeks of gestation. By computerized morphometry, the cellular and subcellular localization of the immunosignal for GLUT1 was quantitatively evaluated. The study showed that the glucose transporter is strongly expressed by endothelial cells while a very low signal is detected on vascular pericytes. The GLUT1 antigenic sites are preferentially associated to the ablumenal and junctional plasma membranes of the endothelial cells and tend to increase significantly with age. A parallel study carried out by the endogenous serum protein albumin demonstrated that already at the 12th week the endothelial routes are hindered to the protein as happens at the blood-endothelium interface of mature brain. The results demonstrate that in the human foetus the brain microvessels express BBB-specific functional activities early.     

The glucose transporter of the human brain and blood-brain barrier

We identified and characterized the glucose transporter in the human cerebral cortex, cerebral microvessels, and choroid plexus by specific D-glucose-displaceable [3H]cytochalasin B binding. The binding was saturable, with a dissociation constant less than 1 microM. Maximal binding capacity was approximately 7 pmol/mg protein in the cerebral cortex, approximately 42 pmol/mg protein in brain microvessels, and approximately 27 pmol/mg protein in the choroid plexus. Several hexoses displaced specific [3H]cytochalasin B binding to microvessels in a rank-order that correlated well with their known ability to cross the blood-brain barrier; the only exception was 2-deoxy-D-glucose, which had much higher affinity for the glucose transporter than the natural substrate, D-glucose. Irreversible photoaffinity labeling of the glucose transporter of microvessels with [3H]cytochalasin B, followed by solubilization and polyacrylamide gel electrophoresis, labeled a protein band with an average molecular weight of approximately 55,000. Monoclonal and polyclonal antibodies specific to the human erythrocyte glucose transporter immunocytochemically stained brain blood vessels and the few trapped erythrocytes in situ, with minimal staining of the neuropil. In the choroid plexus, blood vessels did not stain, but the epithelium reacted positively. We conclude that human brain microvessels are richly endowed with a glucose transport moiety similar in molecular weight and antigenic characteristics to that of human erythrocytes and brain microvessels of other mammalian species.     

The glucose transporter and blood-brain barrier of human brain tumors

The glucose transporter of the human brain has been localized to endothelial cells expressing the blood-brain barrier, but little is known regarding its mechanism of induction or whether its expression is exclusively linked with restricted vascular permeability. We investigated glucose transporter expression by vessels in human astrocytic tumors and pulmonary metastases to the brain using immunohistochemical techniques. Vessels in 9 of 10 low-grade astrocytomas and 8 of 10 anaplastic astrocytomas were positive for glucose transporter. Glioblastoma vessels were transporter-positive in only 2 of 10 specimens. Vessels in all three metastatic tumors were negative for the glucose transporter. The decrease in transporter expression observed in higher-grade tumors occurred independently of increases in vascular permeability. In low-grade astrocytomas and glioblastomas transporter expression and contrast enhancement were inversely related, but vessels in 6 of 9 anaplastic astrocytomas were transporter-positive despite contrast enhancement. These findings suggest that separate mechanisms induce the glucose transporter and the permeability restrictions of the human blood-brain barrier. They also have potential implications for the therapy and prognosis of astroglial neoplasms.     

Na(+)-Ca(2+) cosignaling in the stimulation of the glucose transporter GLUT1 in cultured astrocytes

Glutamate triggers an acute stimulation of the glucose transporter GLUT1 in cultured astrocytes, a phenomenon thought to facilitate energy delivery to active areas in the brain. Here we have explored the cell signaling mechanisms involved in this response. Half-stimulation of GLUT1 occurred at low micromolar glutamate, thus within the physiological range estimated in brain interstitium. The effect was mimicked by D-aspartate and inhibited by L-threo-beta-benzyloxyaspartate or Na(+) replacement with NMDG(+), showing the participation of the Na(+)-glutamate co-transporter. AMPA and the mGLURI agonist DHPG had no effect. The stimulation of GLUT1 was fully inhibited by ouabain, but independent activation of the Na(+)/K(+) ATPase pump with gramicidin did not affect glucose transport. Simultaneous with the Na(+) rise, glutamate and D-aspartate triggered a Ca(2+)signal, whose inhibition with BAPTA prevented the stimulation of GLUT1. However, an isolated Ca(2+) signal, triggered with endothelin 1, ATP or DHPG, did not affect glucose transport. The stimulation of GLUT1 could finally be mimicked by simultaneous induction of Na(+) and Ca(2+) signals. The requirement for both cations in the stimulation of the astrocytic glucose transporter, may help to explain how glucose metabolism in the brain is strongly activated by glutamate, but not by GABA or by inter-astrocytic signaling.     

Immunocytochemical distribution of the brain glucose transporter (GLUT 1) in experimental gliosis

The present study has examined the immunocytochemical expression of the blood-brain barrier glucose transporter GLUT 1 as compared with GFAP in models of experimental gliosis. In neocortical grafts, gliosis was prominent at the graft-host interface mostly associated with blood vessels. Consecutive sections examined for anti-GLUT 1 showed that the protein was distributed in nearly an identical pattern to the anti-GFAP, staining fibrillar processes and all vessels and also appeared extracellularly. In stab wounds, GLUT 1 immunoexpression was similar to GFAP reactivity and stained injured vessels and glial-like processes that were reminiscent of astrocytic end-feet. Normal glial cells and processes in unaffected neuropil, however, were never stained. This report suggests that GLUT 1 protein may be upregulated in non-endothelial components, such as reactive astroglia or possibly microglia, that are associated with injured or angiogenic vessels.     

Sympathetic modulation of the renal glucose transporter GLUT2 in diabetic rats

We have previously shown that the abolition of renal sympathetic nervous activity (RSNA) can influence cortical GLUT1 expression in diabetic rats. However, no study has examined the effects of nervous activity on expression of GLUT2, the major glucose transporter in proximal renal tubules, which participates in renal glucose handling. The aim of this study was to determine whether sympathetic activity modulates renal GLUT2 content. We studied diabetic and nondiabetic rats with normal, low, or high RSNA. The low-RSNA experiment used four groups of Wistar male rats: Wistar sham-operated, Wistar renal-denervated, Diabetic sham-operated, and Diabetic renal-denervated. The high-RSNA experiment used four groups of Wistar-Kyoto male rats: WKY (control), WKY-Diabetic, SHR (spontaneously hypertensive rats), and SHR-Diabetic. Renal denervation was confirmed by a decrease in intrarenal norepinephrine levels and sympathetic hyperactivity, by measurement of RSNA. Western blotting was used to determine the renal cortical GLUT2 protein content, and 24-h urinary sodium and glucose levels were also evaluated. Compared with controls (Wistar and WKY), diabetes increased the GLUT2 protein content in normal-RSNA Diabetics (47%) and WKY-Diabetics (83%). The renal denervation-induced decrease in RSNA reduced the GLUT2 content in both normal and diabetic rats (-21% and -15%, respectively). Compared to WKY rats, SHR presented elevated RSNA and also showed an increase in renal GLUT2 content (17%). Diabetes caused a major increase in GLUT2 protein (52%) in the SHR. These results demonstrate a direct relationship between RSNA and GLUT2 levels; they also reveal an additive effect of sympathetic hyperactivity and diabetes on GLUT2 expression, suggesting a new mechanism for modulating protein expression in renal tissue.     

The 45 kDa form of glucose transporter 1 (GLUT1) is localized in oligodendrocyte and astrocyte but not in microglia in the rat brain

We and others have previously reported that glucose transporter 1 (GLUT1)-like 45 kDa protein is localized to parenchymal cells in the brain. However, the precise cellular localization has remained unclear. In the present study, we examined the cellular localization of GLUT1 in the rat brain by double immunostaining methods and immunoelectron microscopic analysis using a rabbit antiserum specific to GLUT1. Western blot analysis of the rat brain revealed that the antiserum detected a strong band with a molecular weight of 45 kDa and a weak band of about 55 kDa, which corresponded respectively to the known molecular weights of the GLUT1 proteins in the brain parenchymal cells and the brain microvessels. Immunohistochemical staining revealed a large number of GLUT1-immunoreactive glial cells and microvessels in almost every region of the brain. Double immunofluorescence analysis demonstrated that the GLUT1-like 45 kDa protein occurred in many galactocerebroside-positive oligodendrocytes and in some glial fibrillary acidic protein (GFAP)-positive astrocytes. No GLUT1-immunoreactivity was observed in OX42-positive microglia. Immunoelectron microscopic examination confirmed that the GLUT1-immunoreactivity was mainly localized in the cytoplasm of the oligodendrocytes and astrocytes. The results indicate that the 45 kDa form of GLUT1 protein exists in the glial cells including astrocytes and oligodendrocytes.     

Spontaneous intracellular calcium oscillations in cortical astrocytes from a patient with intractable childhood epilepsy (Rasmussen's Encephalitis)

Many studies have demonstrated that astrocytes respond with fluctuations in intracellular calcium concentration ([Ca²⁺]_i) and membrane potential following the application of a number of ligands. Moreover, calcium (Ca²⁺) waves that spread through astrocytic syncitia have been described in numerous reports. We had the rare opportunity to study Ca²⁺ responses in astrocytes obtained from a patient diagnosed with Rasmussen's encephalitis, a rare form of intractable epilepsy. Using the ratiometric fluorescent indicator fura-2, we observed large spontaneous [Ca²⁺]_i oscillations. The mean time between initial rise in [Ca²⁺]_i and the return to baseline was 5.1 ± 0.19 minutes (SEM; n = 201) and [Ca²⁺]_i increased to a mean level of 271 ± 8 nM (SEM; n = 201) from a baseline of 136 ± 6 nM (SEM; n = 201). Removal of Ca²⁺ from the perfusion solution combined with the addition of the Ca²⁺ chelator EGTA (2 mM) completely but reversibly eliminated all oscillations suggesting the fluctuations were dependent on Ca²⁺ flux across the membrane. The percentage of cells undergoing spontaneous changes in [Ca²⁺]_i decreased over time in culture. At 10–11 days post-surgery, approximately 70% of the cells were exhibiting this behavior, and by day 23 transients were no longer observed. We did not observe comparable spontaneous [Ca²⁺]_i oscillations in rat cortical astrocytes. The potential that the spontaneous [Ca²⁺]_i oscillations observed may be a unique feature of epileptic tissues is discussed. GLIA 21:332–337, 1997. © 1997 Wiley-Liss, Inc.     

Role of EEG as a monitoring tool in patients with glucose transporter type I deficiency syndrome (GLUT1-DS) on ketogenic diet

Rationale

Glucose transporter type I deficiency syndrome (GLUT1-DS) is the fourth most frequent single-gene epilepsy refractory to standard antiepileptic drugs. Multiple seizure types and variable electrographic findings are reported. Ketogenic diet is expected to result in the complete resolution of the epileptiform activity.

Methods

A retrospective chart review of patients with GLUT1-DS on ketogenic diet between December 2012 and February 2022 was done. Analysis of the EEGs prior to and during the ketogenic diet was done.

Results

34 patients on ketogenic diet were reviewed. Ten had clinical diagnosis of GLUT1-DS, and seven of them had genetic confirmation. 71% were female. The average age at seizure onset was 13.85 m.o. (range: 3–60, SD ±20.52), at diagnosis was 44.57 m.o (range: 19–79), and at the onset of ketogenic diet was 46.43 m.o. (range: 20–83). 29 months (range: 13–38) delay between symptoms onset until diagnosis was noticed. At the diagnosis 100% reported seizures: 71% myoclonic, 57% generalized motor, 57% absence, 28% atonic, and 14% focal motor. Also, 71% abnormal eye movements, 57% ataxia, and 28% intolerance to fasting. 86% had normal brain MRI. 71% had abnormal EEG. All were on ketogenic diet, and four on classical (1.75:1–2.25:1 ratio). Six were clinically seizure-free after the ketogenic diet. EEG features included notch delta, focal spike and wave, and generalized spike/polyspike and wave. One patient had bilateral independent centrotemporal spikes. Spikes showed high and very high amplitude in all of them (>200 μV). The variation of the spike index decreased in three patients but increased in two.

Conclusion

Ketogenic diet is the choice treatment for patients with GLUT1-DS. Electrographic features could show worsening after initiation of the ketogenic diet even with seizure control. EEG did not prove to be a reliable tool for adjusting KD in our cohort. Centrotemporal spikes have not been reported in patients with GLUT-1 DS.     

Prenatal development of fibrous (white matter), protoplasmic (gray matter), and layer I astrocytes in the human cerebral cortex: A Golgi study

The prenatal developmental histories of layer I, fibrous (white matter), and protoplasmic (gray matter) astrocytes have been studied in the human neocortex by the rapid Golgi method. The developmental route followed by each of these astrocytes is a distinct process which evolves from a specific precursor, occurs at a different time, and is linked to a specific event. The differentiation of layer I astrocytes is linked to the neocortex external glial limiting membrane (EGLM), that of fibrous astrocytes to the early white matter vascularization and maturation, and that of protoplasmic astrocytes to the late gray matter ascending vascularization and maturation. At the start of development, three glial precursors are established in the neocortex: 1) original radial neuroectodermal cells with nuclei above the primordial plexiform layer (PPL) by losing their ependymal and retaining their pial attachments become early astrocytes of layer I and EGLM components; 2) neuroectodermal cells with nuclei below the PPL that retain their pial and ependymal attachments become type I radial glial cells which are committed to the guidance of neurons and the early EGLM maintenance; and, 3) neuroectodermal cells that lose their pial but retain their ependymal attachment are transformed into type II radial glial precursors. By progressively losing their ependymal attachment, type II radial glia precursors become freely migrating cells, establish vascular contacts, and differentiate into fibrous astrocytes (and into oligodendrocytes?) throughout the subplate, developing white matter, and paraventricular regions. After the formation of the gray matter, additional layer I astrocytes are needed for the EGLM late prenatal and postnatal maintenance because type I radial glia cells start to regress and to reabsorb their EGLM endfeet. A late ependyma-to-pia migration of glial precursors progressively repopulates layer I with additional astrocytes and establishes the ephemeral subpial granular layer (SGL) of Ranke. From the 15th week of gestation to the time of birth, late astrocytes of layer I lose their EGLM attachments, migrate freely into the maturing gray matter, establish vascular contacts, and differentiate into protoplasmic astrocytes. The protoplasmic astrocytes of the gray matter evolve from transformation of layer I astrocytes rather than from radial glia cells as is generally believed. © 1995 Wiley-Liss, Inc.     

葡萄糖载体-1在高血压性脑出血患者脑组织星形胶质细胞中的表达

目的　探讨葡萄糖载体 1(Glut 1,相对分子质量为 45 0 0 0 )在高血压性脑出血 (HIH)患者脑组织星形胶质细胞中的表达。方法　应用免疫组化方法 (Glut 1、GFAP抗体 )对 7例HIH患者和 6例非神经系统疾病死亡患者 (对照组 )的脑组织标本进行标记 ,分析脑出血后 2 4h内、72h后的星形胶质细胞中 45 0 0 0Glut 1表达的变化 ,同时采用多媒体彩色病理图文系统 (MPIAS 5 0 0 )计算星形胶质细胞中的平均灰度 (AGD)和平均光密度。结果　对照组 45 0 0 0Glut 1在星形胶质细胞中因表达较少 ,未达检测水平。 2 4h组HIH病灶周围反应性星形细胞增多、胞体增殖、肿胀 ,GFAP标记阳性 ;Glut 1在反应性星形细胞上同样呈明显阳性反应。 72h组标本显示GFAP和Glut 1强阳性反应 ,较 2 4h组表达明显增强 ,其AGD和平均吸光度均值分别为 2 0 3± 16和 0 .0 9± 0 .0 4。结论　 45 0 0 0Glut 1在HIH反应性星形胶质细胞中的表达增加 ,是一种充分保障脑组织葡萄糖能量代谢的防御机制 ;病灶周围星形胶质细胞反应性增生为血 脑屏障的破坏提供了一种代偿机制。     

Dopamine transporter mRNA is increased in the CNS of Zucker fatty (fa/fa) rats

The obese Zucker fa/fa rat is characterized by hyperinsulinemia, obesity, and altered monoamine metabolism in the central nervous system (CNS). It has been proposed that the changes in monoamine metabolism may contribute to the metabolic pathophysiology of these animals. Because it has been reported that insulin may regulate the catecholamine reuptake transporters, which terminate monoaminergic synaptic signaling, in the present study we tested whether messenger ribonucleic acid (mRNA) levels for the noradrenergic (NE) or dopaminergic (DA) transporters were altered in obese fa/fa vs. lean Fa/Fa Zucker rats. We found significantly elevated DA transporter levels in both the ventral tegmental area/substantia nigra pars compacta (VTA/SNc) and zona incerta (ZI) of obese Zucker fa/fa rats (164 ± 24% of control levels, p = .024; and 316 ± 61% of control levels, p = .019, respectively). Measurement of mRNA for tyrosine hydroxylase (TH), the rate-limiting enzyme for NE and DA synthesis revealed no effect of the fa gene in either NE or DA neurons. These findings suggest that increased DA clearance, and perhaps decreased DA signaling, may occur in the obese Zucker fa/fa rat.     

Immunocytochemical expression of the blood—brain barrier glucose transporter (GLUT-1) in neural transplants and brain wounds

The present study examined the immunocytochemical expression of the blood—brain barrier glucose transporter (GLUT-1) in a series of fetal neocortical transplants, autonomic tissue transplants, and stab wounds to the rat brain. GLUT-1 is one of a family of different glucose transporters and is found exclusively on barrier-type endothelial cells. In the brain it is responsible for the regulated facilitative diffusion of glucose acress the blood—brain barrier. This investigation is the first to determine if this important molecule is altered during the process of angiogenesis that occurs following neural transplanatation procedures of direct brain injury. Beginning in late fetal brain, e.g., E18 and continuing into maturity, GLUT-1 was strongly and exclusively expresed on normal cerebral vessels. In solid fetal central nervous system (CNS) transplants up to around 3 weeks postoperative, CLUT-1 was only weakly expressed, particularly as exemplified by colloidal gold immunostaining when compared with the host. At later times examined, up to 15 months postoperative, GLUT-1 immunoexpression was comparable with the normal adjacent brain. In autonomic tissue transplants, where the vessels do not have a blood—brain barrier, as expected, GLUT-1 was not expressed. In stab wounds, at 1 week there was extensive gliosis, and the injured vessels appeared fragmented and collapsed but still expressed GLUT-1, although to a somewhat lesser extent than normal brain. Between 3 and 6 weeks, GLUT-1 was expressed on tortuous vessels and in apparently fibrillar processes in the wound vicinity with a similar pattern to astrocyte (GFAP) reactivity. These results suggest the occurrence of a down-regulation of GLUT-1 in early transplats, perhaps related to reduced glycolytic activity or transient ischemia, or possibly due to the utilization of alternative energy sources. That GLUT-1 expression was not entirely lost in stab wounds to the mature brain suggests that the protein may be more labile in fetal or perinatal brain than in the adult and may not be affected by direct injury. Coupled with previous transplantation studies that have shown reduced neuronal glycolysis and potential barrier alteraations, the reduction of GLUT-1 activity within nearly the identical time frame could indicate a relatively early critical period in cellular metabolism following transplantation of CNS tissue. © Wiley-Liss, Inc.     

Melanin-concentrating hormone (MCH) is colocalized with α-melanocyte-stimulating hormone (α-MSH) in the rat but not in the human hypothalamus

Melanin-concentrating hormone (MCH)-containing neurons have recently been localized in the dorsolateral region of the rat hypothalamus, an area where the second α-MSH system is found which contains only α-MSH and none of the pro-opiomelanocortin (POMC)-related peptides. In order to study the morphological relationships between the MCH and α-MSH neuronal systems, we have studied the immunocytochemical localization of both MCH and α-MSH in the rat hypothalamus. The same study was also performed in the human hypothalamus where there is only one α-MSH system which contains α-MSH as well as the other POMC-related peptides (first α-MSH system). In the rat dorsolateral hypothalamus, we could demonstrate that most neuronal cell bodies stained for MCH also contained immunoreactive α-MSH. In the human hypothalamus, neuronal cell bodies stained for MCH were observed only in the periventricular area whereas cell bodies containing α-MSH were exclusively located in the infundibular (arcuate) nucleus. In the rat, immunoelectron microscopy showed labelling for MCH in the dense core vesicles of positive neurons and double-staining techniques clearly demonstrated that both immunoreactive MCH and α-MSH could be consistently detected in the same dense core vesicles. These ultrastructural studies then suggest that these two peptides should be released simultaneously from neurons located in the rat dorsolateral hypothalamus.     

The putative heme transporter HCP1 is expressed in cultured astrocytes and contributes to the uptake of hemin

Hemin, which is toxic to brain cells, has been reported to be taken up by cultured astrocytes; however, the mechanism of uptake is currently unknown. The present study investigated the mechanism of hemin uptake by rat primary astrocyte cultures. In medium containing 10% fetal calf serum, cultured astrocytes failed to accumulate significant amounts of heme‐iron, while in serum‐free medium the accumulation of heme‐iron was found to be time‐ and concentration‐dependent. After 6 h of incubation with 24 μM hemin, cells contained 36.2 ± 2.4 nmol heme‐iron/mg protein, which was 21% of the applied hemin. These results suggest that the accumulation of hemin in astrocytes does not require serum proteins such as hemopexin. A potential mechanism of hemin uptake in astrocytes involves the heme carrier protein 1 (HCP1), which is reported to mediate hemin uptake into intestinal cells. RT‐PCR analysis revealed that astrocyte cultures contained HCP1 mRNA, and immunocytochemical staining and Western blot analysis confirmed the expression of HCP1 protein in cultured astrocytes. The functionality of HCP1 in astrocytes was demonstrated by incubating cells with zinc protoporphyrin IX (ZnPPIX), which is known to be transported into cells via HCP1, and ZnPPIX autofluorescence was detected in HCP1‐positive astrocytes. In addition, ZnPPIX was found to attenuate the accumulation of heme‐iron by astrocytes. These results are the first to demonstrate that cultured astrocytes contain functional HCP1 and that this transporter contributes to hemin uptake by astrocytes. HCP1 may therefore provide a new target for reducing hemin‐related toxicity in brain cells. © 2009 Wiley‐Liss, Inc.     

Basolateral aggregated rat amyloidbeta(1-42) potentiates transmigration of primary rat monocytes through a rat blood-brain barrier

Monocytes adhere and transmigrate through a blood-brain barrier (BBB) during a normal immune patrol and after pathological events. It is well established that the transmigration of monocytes through the BBB is stimulated by soluble amyloidbeta. The aim of the present study was to explore if aggregated amyloidbeta added to the basolateral side of a BBB may modulate the adhesion and migration of primary rat monocytes through a monolayer of rat brain capillary endothelial cells (BCEC). Monocytes were freshly isolated from rat blood by negative magnetic selection and applied to the apical side of a fully confluent BCEC-monolayer with or without pre-treatment with soluble or aggregated amyloidbeta. Aggregation was performed by incubation of amyloidbeta(1-42) for 2 weeks in acidic medium at 37 degrees C. The monocytes adhered at the apical side of a BCEC-monolayer within 30-90 min (approx. 1,500 cells/well), and transmigrated to the basolateral side within 18 hours (approx. 40,000 cells/well), when stimulated with 1 ng/ml monocyte chemotactic protein-1. Soluble amyloidbeta(1-42) (100 ng/ml) significantly enhanced the adhesion and migration of monocytes after 90 min, which was modulated by antibodies against platelet-endothelial cell adhesion molecule-1, intracellular adhesion molecule-1, receptor for advanced glycosylation end products and low density lipoprotein-related protein-1 but not vascular cell adhesion molecule-1. Addition of aggregated amyloidbeta(1-42) to the basolateral side potentiated the transmigration of monocytes. In conclusion, aggregated amyloidbeta(1-42) stimulates the transmigration of monocytes through a BBB, which is of importance in Alzheimers disease.     

Canonical transient receptor potential channel 4 (TRPC4) co-localizes with the scaffolding protein ZO-1 in human fetal astrocytes in culture

Members of the mammalian transient receptor potential (TRP) family form cation-permeable channels at the plasma membrane implicated in capacitative calcium influx after activation by either second-messenger-mediated pathways or store depletion, or both. This study shows that with the use of RT-PCR, Western blotting, and immunohistochemistry, resting astrocytes express TRPC4 at the cell membrane, particularly at sites of cell-to-cell contact. By confocal imaging and immunoelectron microscopy, we detected co-localization of TRPC4 with the scaffolding protein zonula occludens 1 (ZO-1), and demonstrated that immunoprecipitation with antibodies to ZO-1 brought down TRPC4, and vice-versa. It has been proposed that the targeting of TRPC4 to the cell membrane is dependent on the interaction of the C-terminal TRL motif with PDZ domains. Using transfection of astrocytes with myc-tagged TRPC4 or TRL-motif truncated TRPC4 (deltaTRL), we found that deltaTRL localized predominantly to a juxtanuclear compartment, whereas the wild-type protein showed cell surface distribution. Deletion of the TRL motif also reduced plasma membrane expression as assessed by cell surface biotinylation experiments. Using GST fusion proteins, we found that TRPC4 interacted with the PDZ1 domain of ZO-1 and that this was also dependent on the TRL motif. Thus, our data demonstrate that the PDZ-interacting domain of TRPC4 controls its cell surface localization. These data implicate TRPC4 in the regulation of calcium homeostasis in astrocytes, particularly as part of a signaling complex that forms at junctional sites between astrocytes.     

The GABA transporter 1 (SLC6A1): a novel candidate gene for anxiety disorders

Recent evidence suggests that the GABA transporter 1 (GAT-1; SLC6A1) plays a role in the pathophysiology and treatment of anxiety disorders. In order to understand the impact of genetic variation within SLC6A1 on pathological anxiety, we performed a case–control association study with anxiety disorder patients with and without syndromal panic attacks. Using the method of sequential addition of cases, we found that polymorphisms in the 5′ flanking region of SLC6A1 are highly associated with anxiety disorders when considering the severity of syndromal panic attacks as phenotype covariate. Analysing the effect size of the association, we observed a constant increase in the odds ratio for disease susceptibility with an increase in panic severity (OR ~ 2.5 in severely affected patients). Nominally significant association effects were observed considering the entire patient sample. These data indicate a high load of genetic variance within SLC6A1 on pathological anxiety and highlight GAT-1 as a promising target for treatment of anxiety disorders with panic symptoms. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. C. K. Thoeringer and S. Ripke contributed equally to this work. Dedicated to the Special Issue “Fear, Anxiety and Anxiety Disorder”.