Gut Symptoms, Gut Dysbiosis and Gut-Derived Toxins in ALS.

Many pathogenetic mechanisms have been proposed for amyotrophic lateral sclerosis (ALS). Recently, there have been emerging suggestions of a possible role for the gut microbiota. Gut microbiota have a range of functions and could influence ALS by several mechanisms. Here, we review the possible role of gut-derived neurotoxins/excitotoxins. We review the evidence of gut symptoms and gut dysbiosis in ALS. We then examine a possible role for gut-derived toxins by reviewing the evidence that these molecules are toxic to the central nervous system, evidence of their association with ALS, the existence of biochemical pathways by which these molecules could be produced by the gut microbiota and existence of mechanisms of transport from the gut to the blood and brain. We then present evidence that there are increased levels of these toxins in the blood of some ALS patients. We review the effects of therapies that attempt to alter the gut microbiota or ameliorate the biochemical effects of gut toxins. It is possible that gut dysbiosis contributes to elevated levels of toxins and that these could potentially contribute to ALS pathogenesis, but more work is required.

Proteomic investigation of ALS motor cortex identifies known and novel pathogenetic mechanisms.

The key pathological feature in ALS is death of motor neurones from the brain and spinal cord, but the molecular mechanisms underlying this degeneration remain unknown. Quantifying the motor cortex proteome in autopsy brain and comparing tissues from ALS cases and non-ALS controls is critical to understanding these mechanisms. We used Sequential Window Acquisition of All Theoretical Mass Spectra (SWATH-MS) to characterize the proteomes of the motor cortex from ALS cases (n = 8) and control subjects (n = 8). A total of 1427 proteins were identified at a critical local false discovery rate < 5%; 187 of these exhibited significant expression differences between ALS cases and controls. Of these, 91 proteins were significantly upregulated and 96 proteins were significantly downregulated. Bioinformatics analysis revealed that these proteins are involved in molecular transport, protein trafficking, free radical scavenging, lipid metabolism, cell death and survival, nucleic acid metabolism, inflammatory response or amino acid metabolism and carbohydrate metabolism. Differentially expressed proteins were subjected to pathway analysis. This revealed abnormalities in pathways involving mitochondrial function, sirtuin signaling, oxidative phosphorylation, glycolysis, phagosome maturation, SNARE signaling, redox regulation and several others. Core analysis revealed mitochondrial dysfunction to be the top canonical pathway. The top-enriched networks involved JNK activation and inhibition of AKT signaling, suggesting that disruption of these signaling pathways could lead to demise of motor neurons in the ALS motor cortex.

Elevated plasma levels of D-serine in some patients with amyotrophic lateral sclerosis.

D-serine is an endogenous co-agonist with glutamate for activation of the N-methyl-D-aspartate receptor (NMDAR). D-serine exacerbates neuronal death and is elevated in the spinal cord from patients with sporadic/familial ALS. The present study was undertaken to examine whether plasma levels of D-serine of patients with ALS are different from those of healthy controls. Levels of D-serine in plasma (30 patients and 30 controls) were measured by high-performance liquid chromatography mass spectrometry. Plasma levels of D-serine in ALS patients (mean 39.27 ± 28.61 ng/ml) were significantly higher (p = 0.0293) than those of healthy control subjects (mean 21.07 ± 14.03 ng/ml) as well as previously reported values for healthy controls; ∼43% of patients had plasma D-serine levels that were 2 to 4-folds higher than those of controls. There was no association of plasma D-serine levels with disability, the duration of disease or with the age of subjects. In conclusion, we show that D-serine levels are elevated in the plasma of some ALS patients. Since D-serine serves as a co-agonist/activator of NMDAR, increases in D-serine could have a direct influence on glutamatergic neurotransmission and potentially contribute to excitotoxicity in some ALS patients.

Levels of clusterin, CD5L, ficolin-3, and gelsolin in ALS patients and controls.

In amyotrophic lateral sclerosis, there is a need for biomarkers to distinguish patients from controls, to follow disease progression and to provide information about the pathogenesis of disease. In a previous mass spectrometry study that searched for potential proteins of interest, we identified clusterin, CD5L, ficolin-3, and gelsolin as molecules that differed in abundance between ALS patients and controls, with a greater difference in patients with cognitive impairment. Here, we have measured levels of these molecules in plasma from a separate cohort of ALS patients and controls. The plasma was depleted of abundant plasma proteins. We confirmed our previous findings that levels of clusterin are decreased and ficolin-3 are increased in ALS patients compared to controls. In this study, we found that levels of CD5L were increased in patients with ALS and levels correlated with survival. We found that levels of gelsolin were modestly increased in ALS compared to controls whereas in our previous study these were decreased, especially in patients with cognitive impairment who were not included in this study. We suggest that clusterin and ficolin-3 deserve further study as potential ALS biomarkers.

Human brain neurons express a novel splice variant of excitatory amino acid transporter 5 (hEAAT5v).

Excitatory amino acid transporter 5 (EAAT5) is a protein that is known to be alternately spliced and to be abundantly expressed in the retina by populations of neurons including photoreceptors and bipolar cells. EAAT5 acts as a slow glutamate transporter and also as glutamate-gated chloride channel, the chloride conductance being large enough for EAAT5 to serve functionally as an "inhibitory" glutamate receptor. However, there has been a long-standing view that the classically spliced form of EAAT5 is not abundant or widespread in the brain and so it has not been extensively investigated in the literature. We recently identified a human-specific splicing form of EAAT5 that was not expressed by rodents but was shown to be a functional glutamate transporter. We have examined the expression of this form of EAAT5, hEAAT5v at the mRNA, and protein level in human brain, and show that populations of human cortical pyramidal neurons and cerebellar Purkinje cells show significant expression of hEAAT5v. Accordingly, we infer that EAAT5 may well be a player in modulating neuronal function in the human brain and propose that its localization in both glutamatergic and GABAergic neurons could be compatible with a role in influencing intracellular chloride and thereby neuronal parameters such as membrane potential rather than acting as a presynaptic glutamate transporter.

Elevated Levels of Homocysteinesulfinic Acid in the Plasma of Patients with Amyotrophic Lateral Sclerosis: A Potential Source of Excitotoxicity?

OBJECTIVES: Excitotoxicity is thought to be involved in the pathogenesis of amyotrophic lateral sclerosis (ALS). One possible source of excitotoxicity is the presence of sulphur amino acids (SAAs). In the brain of subjects with ALS, there are increased levels of taurine. In the metabolism of methionine to taurine, excitatory sulphur amino acids (SAAs) are formed. These could potentially contribute to excitotoxicity in ALS. The present study has examined whether plasma levels of SAAs in 38 ALS patients differ from those of 30 healthy controls. METHODS: Plasma levels of SAAs were measured by liquid chromatography mass spectrometry. RESULTS: There were no significant changes in plasma cysteic acid, cysteine sulfinic acid, and homocysteic acid in ALS patients compared to healthy subjects. Significant elevations in plasma homocysteinesulfinic acid (HCSA) levels (p < 0.0001) were observed in the ALS patients (75.91 ± 15.38 nM) compared to healthy controls (54.06 ± 8.503 nM); 50% of the ALS patients had HCSA levels that were 1.5-2-folds higher than those of controls. Plasma levels of HCSA differed significantly (p = 0.0440) between patients with bulbar onset and spinal onset (68.57 ± 14.20 vs. 79.30 ± 14.95 nM, respectively). CONCLUSION: HCSA is elevated in the blood of subjects with ALS. Since HCSA can be transported from the blood to the CNS by active transport, has neurotransmitter properties, and can activate synaptic receptors including NMDAR and metabotropic glutamate receptor, it is possible that increases in HCSA could influence glutamatergic neurotransmission and potentially contribute to excitotoxicity in some ALS patients.

Plasma from some patients with amyotrophic lateral sclerosis exhibits elevated formaldehyde levels.

INTRODUCTION: The aim of this study was to determine whether patients with amyotrophic lateral sclerosis (ALS) exhibit higher plasma levels of formaldehyde (FA) than controls, and to look for alterations in levels of FA precursors. METHODS: We studied 40 heathy controls and 50 ALS patients from the Motor Neuron Disease clinic at the Royal Brisbane & Women's Hospital. Plasma FA levels were quantified using a FA detection assay. Trimethylamine (TMA) and trimethylamine oxide (TMAO) in plasma were quantified by multiple reaction monitoring (MRM) mass spectrometry. Plasma levels in patients and controls were compared using Mann-Whitney U test and Spearman's correlation test was used to assess the correlation between levels of FA, TMA, TMAO and other variables. RESULTS: The levels of plasma FA were significantly greater in ALS subjects than controls. TMA and TMAO levels were not significantly different between healthy controls and patients, but were greater in ALS subjects with elevated FA levels than those with normal levels. Of note, levels of TMA and TMAO demonstrated a significant positive correlation with plasma FA levels (spearman's correlation coefficients of TMA with FA [r = 0.451, p = 0.010] and TMAO [r = 0.401, p = 0.023]). There was no association of FA levels with disability measured with the ALS functional rating scale, with the duration of disease or with the age of the subjects. CONCLUSION: Elevated FA is found in some patients with ALS. FA is neurotoxic and could contribute to disease pathogenesis.

Gut microbiota in ALS: possible role in pathogenesis?

Introduction: The gut microbiota has important roles in maintaining human health. The microbiota and its metabolic byproducts could play a role in the pathogenesis of neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS). Areas covered: The authors evaluate the methods of assessing the gut microbiota, and also review how the gut microbiota affects the various physiological functions of the gut. The authors then consider how gut dysbiosis could theoretically affect the pathogenesis of ALS. They present the current evidence regarding the composition of the gut microbiota in ALS and in rodent models of ALS. Finally, the authors review therapies that could improve gut dysbiosis in the context of ALS. Expert opinion: Currently reported studies suggest some instances of gut dysbiosis in ALS patients and mouse models; however, these studies are limited, and more information with well-controlled larger datasets is required to make a definitive judgment about the role of the gut microbiota in ALS pathogenesis. Overall this is an emerging field that is worthy of further investigation. The authors advocate for larger studies using modern metagenomic techniques to address the current knowledge gaps.

Mass spectrometry analysis of plasma from amyotrophic lateral sclerosis and control subjects.

Mass spectrometry was used to study blood samples from patients with amyotrophic lateral sclerosis (ALS) and healthy controls. Addenbrooke's cognitive examination-III (ACE-III) was used to test for cognitive impairment (CI). Nano liquid chromatography and time of flight mass spectrometry (MS) were performed on samples from 42 ALS patients and 18 healthy controls. SWATH™ proteomic analysis was utilized to look for differences between groups. Western blot analysis was used to study levels of 4 proteins, selected as being of possible interest in ALS, in the MS discovery cohort and a second validation group of 10 ALS patients and 10 healthy controls. INGENUITY PATHWAY ANALYSIS (IPA) was applied to the final proteomic data. Between ALS patients and controls, there were significant differences in the expression of 30 proteins. Between controls and ALS patients without CI, there were significant differences in 15 proteins. Between controls and ALS patients with CI, there were significant differences in 32 proteins. Changes in levels of gelsolin, clusterin, and CD5L were validated by using western blot analysis in the discovery cohort. Changes in the expression of gelsolin, clusterin, and ficolin 3 were replicated in a validation group. In ALS, the LXR/RXR and coagulation pathways were downregulated whereas the complement pathway was upregulated. The proteomic data were used to produce two new networks, centered on IL1 and on NFkB, which showed altered levels in ALS. This study highlights the usefulness of MS of blood samples as a tool to study ALS.

Identification and expression of a unique neonatal variant of the GABAA receptor α3 subunit.

The GABAA receptor provides the majority of inhibitory neurotransmission in the adult central nervous system but in immature brain is responsible for much of the excitatory drive, a requirement for normal brain development. It is well established that GABAA receptor subunit expression changes across the course of brain development. In the present study, we have identified a splice variant of the GABAA receptor α3 subunit which appears unique to the developing brain, referred to here as the GABAA receptor α3 subunit neonatal variant (GABAA receptor α3N). RT-PCR and sequence analysis revealed splicing of exon 8 of the α3 subunit. Western blot analysis showed expression of GABAA receptor α3N in the cortex of several neonatal species and significantly reduced expression of this splice variant in the corresponding adult brains. Expression was evident in multiple brain regions and decreased across development in the pig. Fractionation revealed differential cellular localisation in the parietal cortex, hippocampus and thalamus of the full-length GABAA receptor α3 and GABAA receptor α3N. Immunoprecipitation showed direct interaction with the GABAA receptor subunits α1 and γ2 but not with gephyrin.

Could ethanol-induced alterations in the expression of glutamate transporters in testes contribute to the effect of paternal drinking on the risk of abnormalities in the offspring?

It has been known that a preconception paternal alcoholism impacts adversely on the offspring but the mechanism of the effect is uncertain. Several findings suggest that there are signalling systems in testis that are analogous to those known to be altered by alcoholism in brain. We propose that chronic alcohol affects these systems in a manner similar to that in brain. Specifically, we hypothesise that excessive alcohol may disturb glutamatergic-like signalling in testis by increasing expression of the glutamate transporter GLAST (EAAT1). We discuss ways how to test the hypothesis as well as potential significance of some of the tests as tools in the diagnostics of chronic alcoholism.

Chloride channel ClC-5 binds to aspartyl aminopeptidase to regulate renal albumin endocytosis.

ClC-5 is a chloride/proton exchanger that plays an obligate role in albumin uptake by the renal proximal tubule. ClC-5 forms an endocytic complex with the albumin receptor megalin/cubilin. We have identified a novel ClC-5 binding partner, cytosolic aspartyl aminopeptidase (DNPEP; EC 3.4.11.21), that catalyzes the release of N-terminal aspartate/glutamate residues. The physiological role of DNPEP remains largely unresolved. Mass spectrometric analysis of proteins binding to the glutathione-S-transferase (GST)-ClC-5 C terminus identified DNPEP as an interacting partner. Coimmunoprecipitation confirmed that DNPEP and ClC-5 also associated in cells. Further experiments using purified GST-ClC-5 and His-DNPEP proteins demonstrated that the two proteins bound directly to each other. In opossum kidney (OK) cells, confocal immunofluorescence studies revealed that DNPEP colocalized with albumin-containing endocytic vesicles. Overexpression of wild-type DNPEP increased cell-surface levels of ClC-5 and albumin uptake. Analysis of DNPEP-immunoprecipitated products from rat kidney lysate identified ß-actin and tubulin, suggesting a role for DNPEP in cytoskeletal maintenance. A DNase I inhibition assay showed a significant decrease in the amount of G actin when DNPEP was overexpressed in OK cells, suggesting a role for DNPEP in stabilizing the cytoskeleton. DNPEP was not present in the urine of healthy rats; however, it was readily detected in the urine in rat models of mild and heavy proteinuria (diabetic nephropathy and anti-glomerular basement membrane disease, respectively). Urinary levels of DNPEP were found to correlate with the severity of proteinuria. Therefore, we have identified another key molecular component of the albumin endocytic machinery in the renal proximal tubule and describe a new role for DNPEP in stabilizing the actin cytoskeleton.

IL-1α reversibly inhibits skeletal muscle ryanodine receptor. a novel mechanism for critical illness myopathy?

Critical illness myopathies in patients with sepsis or sustained mechanical ventilation prolong intensive care treatment and threaten both patients and health budgets; no specific therapy is available. Underlying pathophysiological mechanisms are still patchy. We characterized IL-1α action on muscle performance in "skinned" muscle fibers using force transducers and confocal Ca(2+) fluorescence microscopy for force/Ca(2+) transients and Ca(2+) sparks. Association of IL-1α with sarcoplasmic reticulum (SR) release channel, ryanodine receptor (RyR) 1, was investigated with coimmunoprecipitation and confocal immunofluorescence colocalization. Membrane integrity was studied in single, intact fibers challenged with IL-1α. IL-1α reversibly stabilized Mg(2+) inhibition of Ca(2+) release. Low Mg(2+)-induced force and Ca(2+) transients were reversibly abolished by IL-1α. At normal Mg(2+), IL-1α reversibly increased caffeine-induced force and Ca(2+) transients. IL-1α reduced SR Ca(2+) leak via RyR1, as judged by (1) increased SR Ca(2+) retention, (2) increased IL-1α force transients being reproduced by 25 µM tetracaine, and (3) reduced Ca(2+) spark frequencies by IL-1α or tetracaine. Coimmunoprecipitation confirmed RyR1/IL-1 association. RyR1/IL-1 immunofluorescence patterns perfectly colocalized. Long-term, 8-hour IL-1α challenge of intact muscle fibers compromised membrane integrity in approximately 50% of fibers, and confirmed intracellular IL-1α deposition. IL-1α exerts a novel, specific, and reversible interaction mechanism with the skeletal muscle RyR1 macromolecular release complex without the need to act via its membrane IL-1 receptor, as IL-1R membrane expression levels were not detectable in Western blots or immunostaining of single fibers. We present a potential explanation of how the inflammatory mediator, IL-1α, may contribute to muscle weakness in critical illness.

Sgk-1 is a positive regulator of constitutive albumin uptake in renal proximal tubule cells.

BACKGROUND/AIMS: Receptor-mediated endocytosis of albumin by the renal proximal tubule requires a number of proteins including megalin/cubilin, sodium/hydrogen exchanger isoform 3 (NHE3) and ClC-5, as well as the PSD-95/Dlg/Zo-1 (PDZ) scaffold sodium/hydrogen exchanger regulatory factor 2 (NHERF2). Despite members from the AGC kinase family, v-Akt Murine Thymoma Viral Oncogene (Akt or Protein Kinase B) and Serum/Glucocorticoid regulated Kinase 1 (Sgk-1) regulating a number of essential proteins in the albumin handling pathway, their role in uptake is largely unknown. METHODS: Opossum kidney (OK) cells were exposed to Texas-Red albumin, in the presence of silencing constructs against Sgk-1, Akt and NHERF2, in addition to the NHE3 inhibitor 5-(N-ethyl-N-isopropyl)-amiloride (EIPA) and NHE3 activator dexamethasone. Target protein was also measured by Western blot analysis in OK cells following exposure to dexamethasone and albumin. RESULTS: Silencing Sgk-1 or overexpression of a dominant negative mutant (DN-Sgk-1) led to a significant reduction of albumin endocytosis compared to control. Conversely, over-expression of wildtype (WT) or constitutively active (CA) Sgk-1 significantly increased uptake. Previous reports have shown Sgk-1 can activate NHE3 through an interaction mediated by NHERF2. We found that silencing both Sgk-1 and NHERF2 demonstrated no additive effect on uptake, suggesting signaling via similar endpoints. Treatment with dexamethasone increased Sgk-1 protein levels and increased albumin endocytosis in OK cells. Interestingly, silencing Akt also lead to a reduction in albumin endocytosis, however in cells silenced for both Sgk-1 and Akt, the additive change in albumin uptake demonstrated that these proteins may act via separate pathways. CONCLUSIONS: We have characterized a Sgk-dependent pathway that regulates albumin uptake in the proximal tubule which also includes NHE3 and NHERF2. These data provide further insights into this essential tubular process.

Alternate splicing and expression of the glutamate transporter EAAT5 in the rat retina.

Excitatory amino acid transporter 5 (EAAT5) is an unusual glutamate transporter that is expressed in the retina, where it is localised to two populations of glutamatergic neurons, namely the bipolar neurons and photoreceptors. EAAT5 exhibits two distinct properties, acting both as a slow glutamate transporter and as a glutamate-gated inhibitory receptor. The latter property is attributable to a co-associated chloride conductance. EAAT5 has previously been thought to exist only as a full-length form. We now demonstrate by PCR cloning and sequencing, the presence of five novel splice variant forms of EAAT5 which skip either partial or complete exons in the rat retina. Furthermore, we demonstrate that each of these variants is expressed at the protein level as assessed by Western blotting using splice-specific antibodies that we have generated. We conclude that EAAT5 exists in multiple spliced forms, and propose, based upon retention or absence of key structural features, that these variant forms may potentially exhibit distinct properties relative to the originally described form of EAAT5.

Localisation of novel forms of glutamate transporters and the cystine-glutamate antiporter in the choroid plexus: Implications for CSF glutamate homeostasis.

The choroid plexus is a structure within each ventricle of the brain that is composed of fenestrated vessels surrounded by secretory epithelial cells. The epithelial cells are linked by tight junctions to create a permeability barrier. The epithelial cells are derived from neuroectoderm, and are thus defined by some authors as a subtype of macroglia. Glutamate is a tightly regulated substance in the CSF, as it is in the rest of the brain. In the brain macroglia express multiple sodium dependent and independent glutamate transporters and are the main regulators of extracellular glutamate. However, the identities of the transporters in the choroid plexus and their localisations have remained poorly defined. In this study we examined the expression and distribution of multiple splice variants of classical sodium-dependent glutamate transporters, as well as the cystine-glutamate antiporter, and the PDZ protein NHERF1, (which acts as a molecular anchor for proteins such as the glutamate transporter GLAST). We identified three forms of sodium-dependent transporters (GLAST1a, GLAST1c and GLT1b) that are expressed at the apical surface of the epithelial cells, a location that matches the distribution of NHERF1 and the cystine-glutamate antiporter. We propose that this coincident localisation of GLAST1a/GLAST1c/GLT1b and the cystine-glutamate antiporter would permit the cyclical trafficking of glutamate and thus optimise the accumulation of cystine for the formation of glutathione in the choroid plexus.

Differential expression of the glutamate transporter GLT-1 in pancreas.

The glutamate uptake transporter GLT-1 is best understood for its critical role in preventing brain seizures. Increasing evidence argues that GLT-1 also modulates, and is modulated by, metabolic processes that influence glucose homeostasis. To investigate further the potential role of GLT-1 in these regards, the authors examined GLT-1 expression in pancreas and found that mature multimeric GLT-1 protein is stably expressed in the pancreas of wild-type, but not GLT-1 knockout, mice. There are three primary functional carboxyl-terminus GLT-1 splice variants, called GLT-1a, b, and c. Brain and liver express all three variants; however, the pancreas expresses GLT-1a and GLT-1b but not GLT-1c. Quantitative real time-PCR further revealed that while GLT-1a is the predominant GLT-1 splice variant in brain and liver, GLT-1b is the most abundant splice variant expressed in pancreas. Confocal microscopy and immunohistochemistry showed that GLT-1a and GLT-1b are expressed in both islet ß- and α-cells. GLT-1b was also expressed in exocrine ductal domains. Finally, glutamine synthetase was coexpressed with GLT-1 in islets, which suggests that, as with liver and brain, one possible role of GLT-1 in the pancreas is to support glutamine synthesis.

A new splice variant of the glutamate-aspartate transporter: cloning and immunolocalization of GLAST1c in rat, pig and human brains.

GLAST (EAAT1) is an abundant glial glutamate transporter in the mammalian brain. It plays important roles in terminating excitatory transmission in grey matter, as well as pathophysiological roles, including protecting white matter from excitotoxic injury. In normal brain, alternative splicing of GLAST has been described: GLAST1a and GLAST1b arise from the splicing out of exons 3 and 9, respectively. This study describes the isolation of a novel cDNA clone from neonatal hypoxic pig brain, referred to as GLAST1c, where exons 5 and 6 are skipped. GLAST1c encodes a protein of 430 amino acids. RT-PCR analysis showed that GLAST1c mRNA was readily detectable in control and hypoxic pig cortex, as well as in various brain regions of rat (cortex, mid, hind and cerebellum), and human cortex, retina and optic nerve. We have raised antibodies that selectively recognize GLAST1c and demonstrate expression of this novel splice variant in astrocytes and oligodendrocytes in rat brain, pig brain and human brain, including grey and white matter. Similarly expression of GLAST1c was observed in primary astrocyte cultures and in cultured oligodendrocytes. In unstimulated astrocytes GLAST1c exhibited an intracellular peri-nuclear distribution similar to that observed when GFP-tagged GLAST1c was transfected into COS 7 cells. In astrocytes this protein rapidly redistributed to the surface upon stimulation of protein kinase with phorbol esters. We conclude that GLAST1c may represent an astrocyte and oligodendrocyte glutamate transporter, though this could not be formally validated by D-aspartate uptake studies, due to the low transfection efficiency of constructs into COS 7 cells.

Exon 4-skipping GLT-1: a new form of an abundantly expressed glutamate transporter.

GLT-1 (EAAT2) is an abundant glial glutamate transporter in the mammalian brain. It plays important roles, especially in the termination of neurotransmitter signals at excitatory synapses in grey matter. In normal brain, alternative splicing of GLT-1 has been described, where exons in the GLT-1 gene are skipped or intronic sequences spliced in to generate new sequences. This study describes the isolation of a cDNA clone encoding a new splice variant of GLT-1 where exon 4 is skipped. This novel variant was isolated by RT-PCR cloning from adult rat brain and encodes a protein of 500 amino acids (MW ~54.5 kDa). RT-PCR analysis showed that mRNA was readily detectable in various brain regions of rat, primary astrocyte cultures and in tissues such as testis, but little mRNA was detectable in retina and liver. An antibody that selectively recognizes exon-4 skipping GLT-1 revealed strong signals in Western blots and labelled grey matter astrocytes. We conclude that exon-4 skipping GLT 1 is abundantly expressed in the brain and may represent either a functional glutamate transporter or a modulator of glutamate transporter function.

Na+-H+ exchanger regulatory factor 1 (NHERF1) PDZ scaffold binds an internal binding site in the scavenger receptor megalin.

The scavenger receptor megalin binds to albumin in the microvilli of the renal proximal tubule, and transports the ligand to the intravillar cleft for processing by endocytosis. Albumin endocytosis in the proximal tubule is regulated by protein complexes containing a number of transmembrane and accessory proteins including PDZ scaffolds such as NHERF1 and NHERF2. PDZ scaffold proteins bind to class I PDZ binding motifs (S/T-X-Φ) in the extreme C-terminus of targets. Megalin contains a functional PDZ binding motif (SDV) in its distal terminus, however a potential interaction with the NHERF proteins has not been investigated. As megalin associates with NHE3 in the microvilli and NHE3 is tethered to the intravillar cleft via its interaction with NHERF1, we investigated if there is a direct interaction between megalin and NHERF1 in renal proximal tubule cells. Using confocal microscopy we determined that megalin and NHERF1 co-localise in the apical region in proximal tubule cells. Immunoprecipitation experiments performed using rat kidney lysate indicated that megalin bound NHERF1 in vivo. Using fusion proteins and peptides, we determined that PDZ2 of NHERF1 bound to megalin and that this interaction was via the C-terminus of megalin directly and in the absence of any accessory protein. We next investigated which domain in megalin was regulating this interaction. Using GST fusion proteins we determined that the loss of the most distal C-terminus of megalin containing the PDZ binding motif (SDV) did not alter its ability to bind to NHERF1. Significantly, we then identified an internal NHERF binding domain in the C-terminus of megalin. Using peptide studies we were able to demonstrate that NHERF1 bound to an internal PDZ binding motif in megalin and that a loss of a single threonine residue abolished the interaction between megalin and NHERF1. Finally, in proximal tubule cells, silencing NHERF1 increased megalin expression. Therefore, we have identified a novel protein interaction in proximal tubule cells and specifically identified a new internal PDZ binding motif in the C-terminus of megalin.