Michael J. Lumb

Delivery of GABAARs to Synapses Is Mediated by HAP1-KIF5 and Disrupted by Mutant Huntingtin

The density of GABA(A) receptors (GABA(A)Rs) at synapses regulates brain excitability, and altered inhibition may contribute to Huntingtons disease, which is caused by a polyglutamine repeat in the protein huntingtin. However, the machinery that delivers GABA(A)Rs to synapses is unknown. We demonstrate that GABA(A)Rs are trafficked to synapses by the kinesin family motor protein 5 (KIF5). We identify the adaptor linking the receptors to KIF5 as the huntingtin-associated protein 1 (HAP1). Disrupting the HAP1-KIF5 complex decreases synaptic GABA(A)R number and reduces the amplitude of inhibitory postsynaptic currents. When huntingtin is mutated, as in Huntingtons disease, GABA(A)R transport and inhibitory synaptic currents are reduced. Thus, HAP1-KIF5-dependent GABA(A)R trafficking is a fundamental mechanism controlling the strength of synaptic inhibition in the brain. Its disruption by mutant huntingtin may explain some of the defects in brain information processing occurring in Huntingtons disease and provides a molecular target for therapeutic approaches.

Functional Synergism between the Most Common Polymorphism in Human Alanine:Glyoxylate Aminotransferase and Four of the Most Common Disease-causing Mutations

The autosomal recessive disorder primary hyperoxaluria type 1 (PH1) is caused by a deficiency of the liver-specific pyridoxal-phosphate-dependent enzyme alanine:glyoxylate aminotransferase (AGT). Numerous mutations and polymorphisms in the gene encoding AGT have been identified, but in only a few cases has the causal relationship between genotype and phenotype actually been demonstrated. In this study, we have determined the effects of the most common naturally occurring amino acid substitutions (both normal polymorphisms and disease-causing mutations) on the properties, especially specific catalytic activity, of purified recombinant AGT. The results presented in this paper show the following: 1) normal human His-tagged AGT can be expressed at high levels in Escherichia coli and purified in a correctly folded, dimerized and catalytically active state; 2) presence of the common P11L polymorphism decreases the specific activity of purified recombinant AGT by a factor of three; 3) AGTs containing four of the most common PH1-specific mutations (G41R, F152I, G170R, and I244T) are all soluble and catalytically active in the absence of the P11L polymorphism, but in its presence all lead to protein destabilization and aggregation into inclusion bodies; 4) naturally occurring and artificial amino acid substitutions that lead to peroxisome-to-mitochondrion AGT mistargeting in mammalian cells also lead to destabilization and aggregation in E. coli; and 5) the PH1-specific G82E mutation abolishes AGT catalytic activity by interfering with cofactor binding, as does the artificial K209R mutation at the putative site of cofactor Shiff base formation. These results are discussed in the light of the high allelic frequency (∼20%) of the P11L polymorphism and its importance in determining the phenotypic manifestations of mutations in PH1.

Ubiquitin-dependent lysosomal targeting of GABA A receptors regulates neuronal inhibition

The strength of synaptic inhibition depends partly on the number of GABAA receptors (GABAARs) found at synaptic sites. The trafficking of GABAARs within the endocytic pathway is a key determinant of surface GABAAR number and is altered in neuropathologies, such as cerebral ischemia. However, the molecular mechanisms and signaling pathways that regulate this trafficking are poorly understood. Here, we report the subunit specific lysosomal targeting of synaptic GABAARs. We demonstrate that the targeting of synaptic GABAARs into the degradation pathway is facilitated by ubiquitination of a motif within the intracellular domain of the γ2 subunit. Blockade of lysosomal activity or disruption of the trafficking of ubiquitinated cargo to lysosomes specifically increases the efficacy of synaptic inhibition without altering excitatory currents. Moreover, mutation of the ubiquitination site within the γ2 subunit retards the lysosomal targeting of GABAARs and is sufficient to block the loss of synaptic GABAARs after anoxic insult. Together, our results establish a previously unknown mechanism for influencing inhibitory transmission under normal and pathological conditions.

An intronic duplication in the alanine: glyoxylate aminotransferase gene facilitates identification of mutations in compound heterozygote patients with primary hyperoxaluria type 1

SummaryWe report here the identification of a duplication within the first intron of the gene encoding human alanine:glyoxylate aminotransferase (AGT); this duplication is closely linked to two point mutations associated with peroxisome-to-mitochondrion mistargeting of AGT in primary hyperoxaluria type 1 (PH1) patients. Polymerase chain reaction amplification of regions of the AGT gene including the insertion site from individuals heterozygous for this duplication, produces allele-specific fragments of different sizes. We have taken advantage of this to identify a nonsense mutation within a non-expressed allele of a compound heterozygote PH1 patient with mitochondrial AGT.

Benzodiazepines Modulate GABAA Receptors by Regulating the Preactivation Step after GABA Binding

GABAA receptors (GABAARs) composed of αβγ subunits are allosterically modulated by the benzodiazepines (BDZs). Agonists at the BDZ binding site potentiate submaximal GABA responses by increasing the apparent affinity of GABAARs for GABA. Although BDZs were initially thought to affect the binding of GABA agonists, recent studies suggest an effect on receptor gating; however, the involvement of preactivation steps in the modulation by BDZs has not been considered. Consequently, we examined whether BDZ agonists could exert their modulatory effect by displacing the equilibrium between resting and preactivated states of recombinant α1β2γ2 GABAARs expressed in Xenopus oocytes. For GABA and the partial agonists 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol and piperidine-4-sulfonic acid, we examined BDZ modulation using a simple three-step model incorporating agonist binding, receptor preactivation, and channel opening. The model accounted for diazepam modulation simply by increasing the preactivation constant by approximately fourfold. To assess whether BDZs preferentially affected a specific GABA binding site, pentameric concatamers were used. This demonstrated that single GABA-binding site mutant receptors were equally sensitive to modulation by BDZs compared with wild-type counterparts. Overall, our results suggest that BDZs affect the preactivation step to cause a global conformational rearrangement of GABAARs, thereby modulating receptor function.

Correction of an enzyme trafficking defect in hereditary kidney stone disease in vitro

In normal human hepatocytes, the intermediary-metabolic enzyme alanine:glyoxylate aminotransferase (AGT) is located within the peroxisomes. However, in approx. one-third of patients suffering from the hereditary kidney stone disease primary hyperoxaluria type 1, AGT is mistargeted to the mitochondria. AGT mistargeting results from the synergistic interaction between a common P11L (Pro11-->Leu) polymorphism and a disease-specific G170R mutation. The polymorphism generates a functionally weak mitochondrial targeting sequence, the efficiency of which is increased by the mutation. The two substitutions together, but not in isolation, inhibit AGT dimerization, highlighting the different structural requirements of the peroxisomal and mitochondrial protein-import machineries. In the present study, we show that treatments known to increase the stability of proteins non-specifically (i.e. lowering the temperature from 37 to 30 degrees C or by the addition of glycerol) completely normalize the intracellular targeting of mutant AGT expressed in transfected COS cells. On the other hand, treatments known to decrease protein stability (e.g. increasing the temperature from 37 to 42 degrees C) exacerbate the targeting defect. Neither of the treatments affects the relative efficiencies of the peroxisomal and mitochondrial protein-import pathways intrinsically. Results are discussed in the light of the known structural requirements of the two protein trafficking pathways and the formulation of possible treatment strategies for primary hyperoxaluria type 1.

Alanine:glyoxylate aminotransferase peroxisome-to-mitochondrion mistargeting in human hereditary kidney stone disease.

The pyridoxal-phosphate (PLP)-dependent enzyme alanine:glyoxylate aminotransferase (AGT) is mistargeted from peroxisomes to mitochondria in patients with the hereditary kidney stone disease primary hyperoxaluria type 1 (PH1) due to the synergistic interaction between a common Pro(11)Leu polymorphism and a PH1-specific Gly(170)Arg mutation. The kinetic partitioning of newly synthesised AGT between peroxisomes and mitochondria is determined by the combined effects of (1) the generation of cryptic mitochondrial targeting information, and (2) the inhibition of AGT dimerization. The crystal structure of AGT has recently been solved, allowing the effects of the various polymorphisms and mutations to be rationalised in terms of AGTs three-dimensional conformation. Procedures that increase dimer stability and/or increase the rate of dimer formation have potential in the formulation of novel strategies to treat this otherwise intractable life-threatening disease.

Molecular characterization and clinical use of a polymorphic tandem repeat in an intron of the human alanine:glyoxylate aminotransferase gene.

The autosomal recessive disease primary hyperoxaluria type 1 (PH1) is caused by a deficiency of the liver-specific peroxisomal enzyme alanine:glyoxylate aminotransferase (AGT). This paper concerns the identification, characterization and clinical use of an unusual discretely polymorphic tandem repeat sequence in the fourth intron of the human AGT gene (gene locus designation AGXT). In a random Caucasian population, three alleles could be clearly recognized that consisted of either 12 (type III), 17 (type 11) or ∼38 (type I) tandemly repeated copies of a highly conserved 29/32-bp sequence with frequencies of 33%, 7% and 60%, respectively. In a random Japanese population, the allelic frequencies were markedly different (i.e. 31%, 45% and 19%, respectively). In addition, a fourth allele was identified, consisting of ∼32 repeats (type IV), with an allelic frequency of ∼5% in Japanese. The repetitive sequence was similar to previously identified mammalian sequences with homology to the Epstein-Barr virus IR3 repetitive element involving a 12/15-bp region GCA(GGN)GGAGGAGGG within the repeat unit. This IR3-like sequence was interspersed with a 17-bp sequence with no similarity to any currently known repetitive element. The type I and type III alleles were judged to be equivalent to a previously identifiedTagI polymorphism. Two polymorphisms previously shown to be associated with the peroxisome-to-mitochondrion mistargeting of AGT in PHI (a C154→T point substitution in exon 1 and a 74-bp duplication in intron 1) were found to segregate exclusively with the type I intron 4 polymorphism in Caucasians, but not in Japanese. The polymorphic nature of the intron 4 tandem repeats makes them of potential use in the prenatal diagnosis of PH1, especially when coupled with the exon 1 C154→T substitution or intron 1 duplication polymorphisms. A PH1 family, in which a fetus had been predicted previously to be either normal or a carrier by AGT enzymic analysis of a fetal liver biopsy, but who had been shown to be only partially informative with respect to the C154→T/intron 1 polymorphisms, was analysed retrospectively. The family was completely informative for the intron 4 tandem repeat polymorphism and the carrier status of the fetus was confirmed.

Postsynaptic GABAB Receptor Activity Regulates Excitatory Neuronal Architecture and Spatial Memory

Cognitive dysfunction is a common symptom in many neuropsychiatric disorders and directly correlates with poor patient outcomes. The majority of prolonged inhibitory signaling in the brain is mediated via GABAB receptors (GABABRs), but the molecular function of these receptors in cognition is ill defined. To explore the significance of GABABRs in neuronal activity and cognition, we created mice with enhanced postsynaptic GABABR signaling by mutating the serine 783 in receptor R2 subunit (S783A), which decreased GABABR degradation. Enhanced GABABR activity reduced the expression of immediate-early gene-encoded protein Arc/Arg3.1, effectors that are critical for long-lasting memory. Intriguingly, S783A mice exhibited increased numbers of excitatory synapses and surface AMPA receptors, effects that are consistent with decreased Arc/Arg3.1 expression. These deficits in Arc/Arg3.1 and neuronal morphology lead to a deficit in spatial memory consolidation. Collectively our results suggest a novel and unappreciated role for GABABR activity in determining excitatory neuronal architecture and spatial memory via their ability to regulate Arc/Arg3.1.

Proteomic Characterization of Inhibitory Synapses Using a Novel pHluorin-tagged γ-Aminobutyric Acid Receptor, Type A (GABAA), α2 Subunit Knock-in Mouse

The accumulation of γ-aminobutyric acid receptors (GABAARs) at the appropriate postsynaptic sites is critical for determining the efficacy of fast inhibitory neurotransmission. Although we know that the majority of synaptic GABAAR subtypes are assembled from α1–3, β, and γ2 subunits, our understanding of how neurons facilitate their targeting to and stabilization at inhibitory synapses is rudimentary. To address these issues, we have created knock-in mice in which the pH-sensitive green fluorescent protein (GFP) and the Myc epitope were introduced to the extracellular domain of the mature receptor α2 subunit (pHα2). Using immunoaffinity purification and mass spectroscopy, we identified a stable complex of 174 proteins that were associated with pHα2, including other GABAAR subunits, and previously identified receptor-associated proteins such as gephyrin and collybistin. 149 of these proteins were novel GABAAR binding partners and included G-protein-coupled receptors and ion channel subunits, proteins that regulate trafficking and degradation, regulators of protein phosphorylation, GTPases, and a number of proteins that regulate their activity. Notably, members of the postsynaptic density family of proteins that are critical components of excitatory synapses were not associated with GABAARs. Crucially, we demonstrated for a subset of these novel proteins (including cullin1, ephexin, potassium channel tetramerization domain containing protein 12, mitofusin2, metabotropic glutamate receptor 5, p21-activated kinase 7, and Ras-related protein 5A) bind directly to the intracellular domains of GABAARs, validating our proteomic analysis. Thus, our experiments illustrate the complexity of the GABAAR proteome and enhance our understanding of the mechanisms neurons use to construct inhibitory synapses.