Autosomal recessive cerebellar ataxia caused by mutations in the PEX2 gene

Objective

To expand the spectrum of genetic causes of autosomal recessive cerebellar ataxia (ARCA).

Case report

Two brothers are described who developed progressive cerebellar ataxia at 3 1/2 and 18 years, respectively. After ruling out known common genetic causes of ARCA, analysis of blood peroxisomal markers strongly suggested a peroxisomal biogenesis disorder. Sequencing of candidate PEX genes revealed a homozygous c.865_866insA mutation in the PEX2 gene leading to a frameshift 17 codons upstream of the stop codon. PEX gene mutations usually result in a severe neurological phenotype (Zellweger spectrum disorders).

Conclusions

Genetic screening of PEX2 and other PEX genes involved in peroxisomal biogenesis is warranted in children and adults with ARCA.     

Glutamate-induced swelling of cultured astrocytes is mediated by metabotropic glutamate receptor

The effects of glutamate and its agonists and antagonists on the swelling of cultured astrocytes were studied. Swelling of astrocytes was measured by [3H]-O-methyl-D-glucose uptake. Glutamate at 0.5, 1 and 10mmol/L and irons-l-aminocyclopentane-1,3-dicarboxylic acid (trans-ACPD), a metabotropic glutamate receptor (mGluR) agonist, at 1 mmol/L caused a significant increase in astrocytic volume, whereas alpha-amino-3-hydroxy-5-methyl-4-isoxazole proprionic acid (AMPA) was not effective. L-2-amino-3-phosphonopropionic acid (L-AP3), an antagonist of mGluR, blocked the astrocytic swelling induced by trans-ACPD or glutamate. In Ca2+-free condition, glutamate was no longer effective. Swelling of astrocytes induced by glutamate was not blocked by CdCl2 at 20 μmol/L, but significantly reduced by CdCl2 at 300 μmol/L and dantrolene at 30 μmol/L. These findings indicate that mGluR activation results in astrocytic swelling and both extracellular calcium and internal calcium stores play important roles in the genes     

Agonist-independent internalization of metabotropic glutamate receptor 1a is arrestin- and clathrin-dependent and is suppressed by receptor inverse agonists

Three group I mGluR antagonists CPCCOEt, LY367385 and BAY36-7620, were analyzed for their effect on cell surface expression of metabotropic glutamate receptor 1a and 1b. All three antagonists inhibited glutamate-induced internalization of mGluR1a and mGluR1b. However, when added alone, either LY367385 or BAY36-7620 increased the cell surface expression of mGluR1a but not mGluR1b. Both LY367385 and BAY36-7620 displayed inverse agonist activity as judged by their ability to inhibit basal inositol phosphate accumulation in cells expressing the constitutively active mGluR1a. Interestingly, mGluR1a but not mGluR1b was constitutively internalized in HEK293 cells and both LY367385 and BAY36-7620 inhibited the constitutive internalization of this splice variant. Furthermore, coexpression of dominant negative mutant constructs of arrestin-2 [arrestin-2-(319-418)] or Eps15 [Eps15(E Delta 95-295)] increased cell surface expression of mGluR1a and blocked constitutive receptor internalization. In the presence of these dominant negative mutants, incubation of cells with LY367385 and BAY36-7620 produced no further increase in cell surface expression of mGluR1a. Taken together, these results suggest that the constitutive activity of mGluR1a triggers the internalization of the receptor through an arrestin- and clathrin-dependent pathway, and that inverse agonists increase the cell surface expression of mGluR1a by promoting an inactive form of mGluR1a, which does not undergo constitutive internalization.     

Localization of the mGlu4a metabotropic glutamate receptor in rat cerebellar cortex

 The subcellular localization of the mGlu4a metabotropic glutamate receptor was investigated in rat cerebellum. At the light microscopical level, strong mGlu4a immunoreactivity was found in the molecular layer. A post-embedding immunogold method for electron microscopy revealed gold particles at the presynaptic sites of synapses made by parallel fiber terminals with dendritic spines of Purkinje cells. These observations support electrophysiological evidence indicating an autoreceptor function of mGlu4 receptors at these synapses. Accepted: 1 July 1997     

The metabotropic glutamate receptor mGluR5 is endocytosed by a clathrin-independent pathway

Metabotropic glutamate receptors 5 (mGluR5) are members of the growing group C G protein-coupled receptor family. Widely expressed in mammalian brain, they are involved in modulation of the glutamate transmission. By means of transfection of mGluR5 receptors in COS-7 cells and primary hippocampal neurons in culture followed by immunocytochemistry and quantitative image analysis and by a biochemical assay, we have studied the internalization of mGluR5 splice variants. mGluR5a and -5b were endocytosed in COS-7 cells as well as in axons and dendrites of cultured neurons. Endocytosis occurred even in the absence of receptor activity, because receptors mutated in the glutamate binding site were still internalized as well as receptors in which endogenous activity had been inhibited by an inverse agonist. We have measured a constitutive rate of endocytosis of 11.7%/min for mGluR5a. We report for the first time the endocytosis pathway of mGluR5. Internalization of mGluR5 is not mediated by clathrin-coated pits. Indeed, inhibition of this pathway by Eps15 dominant negative mutants did not disturb their endocytosis. However, the large GTPase dynamin 2 is implicated in the endocytosis of mGluR5 in COS-7. mGluR5 is the first shown member of the group C G-protein coupled receptor family internalized by a nonconventional pathway.     

Agonist-induced internalization of the metabotropic glutamate receptor 1a is arrestin- and dynamin-dependent

At present, little is known regarding the mechanism of metabotropic glutamate receptor (mGluR) trafficking. To facilitate this characterization we inserted a haemagglutinin (HA) epitope tag in the extracellular N-terminal domain of the rat mGluR1a. In human embryonic kidney cells (HEK293), transiently transfected with HA-mGluR1a, the epitope-tagged receptor was primarily localized to the cell surface prior to agonist stimulation. Following stimulation with glutamate (10 microM; 30 min) the HA-mGluR1a underwent internalization to endosomes. Further quantification of receptor internalization was provided by ELISA experiments which showed rapid agonist-induced internalization of the HA-mGluR1a. To determine whether agonist-induced mGluR1a internalization is an arrestin- and dynamin-dependent process, cells were cotransfected with HA-mGluR1a and either of these dynamin-K44A or arrestin-2 (319-418). Expression of either dominant negative mutant constructs with receptor strongly inhibited glutamate-induced (10 microM; 30 min) HA-mGluR1a internalization. In addition, wild-type arrestin-2-green fluorescent protein (arrestin-2-GFP) or arrestin-3-GFP underwent agonist-induced translocation from cytosol to membrane in HEK293 cells coexpressing HA-mGluR1a. Taken together our observations demonstrate that agonist-induced internalization of mGluR1a is an arrestin- and dynamin-dependent process.     

PICK1 is required for the control of synaptic transmission by the metabotropic glutamate receptor 7

Both postsynaptic density and presynaptic active zone are structural matrix containing scaffolding proteins that are involved in the organization of the synapse. Little is known about the functional role of these proteins in the signaling of presynaptic receptors. Here we show that the interaction of the presynaptic metabotropic glutamate (mGlu) receptor subtype, mGlu7a, with the postsynaptic density-95 disc-large zona occludens 1 (PDZ) domain-containing protein, PICK1, is required for specific inhibition of P/Q-type Ca(2+) channels, in cultured cerebellar granule neurons. Furthermore, we show that activation of the presynaptic mGlu7a receptor inhibits synaptic transmission and this effect also requires the presence of PICK1. These results indicate that the scaffolding protein, PICK1, plays an essential role in the control of synaptic transmission by the mGlu7a receptor complex.     

AKT2 is a downstream target of metabotropic glutamate receptor 1 (Grm1)

We reported earlier on the oncogenic properties of Grm1 by demonstrating that stable Grm1-mouse-melanocytic clones proliferate in the absence of growth supplement and anchorage in vitro. In addition, these clones also exhibit aggressive tumorigenic phenotypes in vivo with short latency in tumor formation in both immunodeficient and syngeneic mice. We also detected strong activation of AKT in allograft tumors specifically AKT2 as the predominant isoform involved. In parallel, we assessed several human melanoma biopsy samples and found again that AKT2 was the predominantly activated AKT in these human melanoma biopsies. In cultured stable Grm1-mouse-melanocytic clones, as well as an metabotropic glutamate receptor 1 (Grm1) expressing human melanoma cell line, C8161, stimulation of Grm1 by its agonist led to the activation of AKT, while preincubation with Grm1-antagonist abolished Grm1-agonist-induced AKT activation. In addition, a reduction in tumor volume of Grm1-mouse-melanocytic-allografts was detected in the presence of small interfering AKT2 RNA (siAKT2). Taken together, these results showed that, in addition to the MAPK pathway previously reported being a downstream target of stimulated Grm1, AKT2 is another downstream target in Grm1 mediated melanocyte transformation.     

Negative cooperativity of glutamate binding in the dimeric metabotropic glutamate receptor subtype 1

Metabotropic glutamate receptor (mGluR) subtype 1 is a Class III G-protein-coupled receptor that is mainly expressed on the post-synaptic membrane of neuronal cells. The receptor has a large N-terminal extracellular ligand binding domain that forms a homodimer, however, the intersubunit communication of ligand binding in the dimer remains unknown. Here, using the intrinsic tryptophan fluorescence change as a probe for ligand binding events, we examined whether allosteric properties exist in the dimeric ligand binding domain of the receptor. The indole ring of the tryptophan 110, which resides on the upper surface of the ligand binding pocket, sensed the ligand binding events. From saturation binding curves, we have determined the apparent dissociation constants (K(0.5)) of representative agonists and antagonists for this receptor (3.8, 0.46, 40, and 0.89 microm for glutamate, quisqualate, (S)-alpha-methyl-4-carboxyphenylglycine ((S)-MCPG), and (+)-2-methyl-4-carboxyphenylglycine (LY367385), respectively). Calcium ions functioned as a positive modulator for agonist but not for antagonist binding (K(0.5) values were 1.3, 0.21, 59, and 1.2 microm for glutamate, quisqualate, (S)-MCPG, and LY367385, respectively, in the presence of 2.0 mm calcium ion). Moreover, a Hill analysis of the saturation binding curves revealed the strong negative cooperativity of glutamate binding between each subunit in the dimeric ligand binding domain. As far as we know, this is the first direct evidence that the dimeric ligand binding domain of mGluR exhibits intersubunit cooperativity of ligand binding.     

Structure of the metabotropic glutamate receptor

In the twelve years since the molecular elucidation of the metabotropic glutamate receptor subtype 1, a class III family of G-protein-coupled receptors has emerged; members of this family include the calcium-sensing receptor, the GABA(B) receptor, some odorant receptors and some taste receptors. Atomic structures of the ligand-binding core of the original metabotropic glutamate receptor 1 obtained using X-ray crystallography provide a foundation for determining the initial receptor activation of this important family of G-protein-coupled receptors.     

Ligand-induced rearrangement of the dimeric metabotropic glutamate receptor 1alpha

The extracellular domain of the metabotropic glutamate receptor 1alpha (mGluR1alpha) forms a dimer and the ligand, glutamate, induces a structural rearrangement in this domain. However, the conformational change in the cytoplasmic domain, which is critical for mGluR1alpha's interaction with G proteins, remains unclear. Here we investigated the ligand-induced conformational changes in the cytoplasmic domain by fluorescence resonance energy transfer (FRET) analysis of mGluR1alpha labeled with fluorescent protein(s) under total internal reflection field microscopy. Upon ligand binding, the intersubunit FRET efficiency between the second loops increased, whereas that between first loops decreased. In contrast, the intrasubunit FRET did not change clearly. These results show that ligand binding does not change the structure of each subunit, but does change the dimeric allocation of the cytoplasmic regions, which may underlie downstream signaling.     

Pias1 interaction and sumoylation of metabotropic glutamate receptor 8

Group III presynaptic metabotropic glutamate receptors (mGluRs) play a central role in regulating presynaptic activity through G-protein effects on ion channels and signal transducing enzymes. Like all Class C G-protein-coupled receptors, mGluR8 has an extended intracellular C-terminal domain (CTD) presumed to allow for modulation of downstream signaling. In a yeast two-hybrid screen of an adult rat brain cDNA library with the CTDs of mGluR8a and 8b (mGluR8-C) as baits, we identified sumo1 and four different components of the sumoylation cascade (ube2a, Pias1, Piasgamma, Piasxbeta) as interacting proteins. Binding assays using recombinant GST fusion proteins confirmed that Pias1 interacts not only with mGluR8-C but also with all group III mGluR CTDs. Pias1 binding to mGluR8-C required a region N-terminal to a consensus sumoylation motif and was not affected by arginine substitution of the conserved lysine 882 within this motif. Co-transfection of fluorescently tagged mGluR8a-C, sumo1, and enzymes of the sumoylation cascade into HEK293 cells showed that mGluR8a-C can be sumoylated in vivo. Arginine substitution of lysine 882 within the consensus sumoylation motif, but not other conserved lysines within the CTD, abolished in vivo sumoylation. Our results are consistent with post-translational sumoylation providing a novel mechanism of group III mGluR regulation.     

Cys-140 is critical for metabotropic glutamate receptor-1 dimerization

Metabotropic glutamate receptor 1 (mGluR1) expresses at the cell surface as disulfide-linked dimers and can be reduced to monomers with sulfhydryl reagents. To identify the dimerization domain, we transiently expressed in HEK-293 cells a truncated version of mGluR1 (RhodC-R1) devoid of the extracellular domain (ECD). RhodC-R1 was a monomer in the absence or presence of the reducing agents, suggesting that dimerization occurs via the ECD. To identify cysteine residues involved in dimerization within the ECD, cysteine to serine point mutations were made at three cysteines within the amino-terminal half of the ECD. A mutation at positions Cys-67, Cys-109, and Cys-140 all resulted in significant amounts of monomers in the absence of reducing agents. The monomeric C67S and C109S mutants were not properly glycosylated, failed to reach the cell surface, and showed no glutamate response, indicating that these mutant receptors were improperly folded and/or processed and thus retained intracellularly. In contrast, the monomeric C140S mutant was properly glycosylated, processed, and expressed at the cell surface. Phosphoinositide hydrolysis assay showed that the glutamate response of the C140S mutant receptor was similar to the wild type receptor. Substitution of a cysteine for Ser-129, Lys-134, Asp-143, and Thr-146 on the C140S mutant background restored receptor dimerization. Taken together, the results suggest that Cys-140 contributes to intermolecular disulfide-linked dimerization of mGluR1.     

Membrane topology of a metabotropic glutamate receptor

The metabotropic glutamate receptors (mGluRs) have been predicted to have a classical seven transmembrane domain structure similar to that seen for members of the G-protein-coupled receptor (GPCR) superfamily. However, the mGluRs (and other members of the family C GPCRs) show no sequence homology to the rhodopsin-like GPCRs, for which this seven transmembrane domain structure has been experimentally confirmed. Furthermore, several transmembrane domain prediction algorithms suggest that the mGluRs have a topology that is distinct from these receptors. In the present study, we set out to test whether mGluR5 has seven true transmembrane domains. Using a variety of approaches in both prokaryotic and eukaryotic systems, our data provide strong support for the proposed seven transmembrane domain model of mGluR5. We propose that this membrane topology can be extended to all members of the family C GPCRs.     

Pharmacological characterization of metabotropic glutamate receptors in cultured cerebellar granule cells

A detailed pharmacological characterization of metabotropic glutamate receptors (mGluR) was performed in primary cultures of cerebellar granule cells at 6 days in vitro (DIV). The rank order of agonists induced polyphosphoinositide (PPI) hydrolysis (after correcting for the ionotropic component in the response) was as follows: in terms of efficiency, Glu>quisqualate (quis)=ibotenate (ibo)>(1_S,3_R)-1-amino-cyclopentane-1,3-dicarboxylic acid (ACPD)>-methyl-amino-l-alanine (BMAA) and in terms of potency, quis>ACPD>Glu>ibo=BMAA. Ionotropic excitatory amino acid (EAA) receptor agonists, such as -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and N-methyl-D-aspartate (NMDA) were relatively inactive (in the presence of Mg²⁺). Quis and ACPD-induced PPI hydrolysis was unaffected by ionotropic Glu receptor antagonists, but was inhibited, in part by L-2-amino-3-phosphonopropionate (AP3). In contrast, Glu-or ibo- induced PPI hydrolysis was reduced, in part, by both AP3 and NMDA receptor antagonists. Characteristic interactions involving different transmitter receptors were noted. PPI hydrolysis evoked by quis and 1_S,3_R-ACPD was not additive. In contrast, PPI hydrolysis stimulated by quis/ACPD and carbamylcholine was additive (indicating different receptors/transduction pathways). In the presence of Mg²⁺, the metabotropic response to quis/AMPA and NMDA was synergistic (this being consistent with AMPA receptor-induced depolarization activating NMDA receptor). On the other hand, in Mg²⁺-free buffer the effects of quis and NMDA, at concentrations causing maximal PPI hydrolysis, were additive (indicating that PPI hydrolysis was effected by two different mechanisms). Thus, in cerebellar granule cells EAAs elicit PPI hydrolysis by acting at two distinct receptor types: (i) metabotropic Glu receptors (mGluR), with pharmacological characteristics suggesting the expression of a unique mGluR receptor that shows certain similarities to those observed for the mGluR1 subtype (Aramori and Nakanishi, 1992) and (ii) NMDA receptors. The physiological agonist, Glu, is able to stimulate both receptor classes.Abbreviations ACPD (1_S,3_R)-1-amino-cyclopentane-1,3-dicarboxylic acid - AMPA -amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid - AP₃ L-2-amino-3-phosphono-propionate - AP₅ D-2-amino-5-phosphonopentenoate - BMAA -methyl-amino-L-alanine - DIV days in vitro - DNOX 6,7-dinitroouinoxoline-2,3-dione - EAA excitatory amino acids - Glu glutamate - InsP inositol monophosphate - mGluR metabotropic glutamate receptors - MK-801 (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]-cyclohept-5,10-imine hydrogen maleate - NMDA N-methyl-D-aspartate - PPI polyphosphoinositide - quis quisqualate     

Activation of a dimeric metabotropic glutamate receptor by intersubunit rearrangement

Although many G protein-coupled receptors (GPCRs) can form dimers, a possible role of this phenomenon in their activation remains elusive. A recent and exciting proposal is that a dynamic intersubunit interplay may contribute to GPCR activation. Here, we examined this possibility using dimeric metabotropic glutamate receptors (mGluRs). We first developed a system to perfectly control their subunit composition and show that mGluR dimers do not form larger oligomers. We then examined an mGluR dimer containing one subunit in which the extracellular agonist-binding domain was uncoupled from the G protein-activating transmembrane domain. Despite this uncoupling in one protomer, agonist stimulation resulted in symmetric activation of either transmembrane domain in the dimer with the same efficiency. This, plus other data, can only be explained by an intersubunit rearrangement as the activation mechanism. Although well established for other types of receptors such as tyrosine kinase and guanylate cyclase receptors, this is the first clear demonstration that such a mechanism may also apply to GPCRs.     

CABC1 gene mutations cause ubiquinone deficiency with cerebellar ataxia and seizures

Coenzyme Q(10) (CoQ(10)) plays a pivotal role in oxidative phosphorylation (OXPHOS) in that it distributes electrons between the various dehydrogenases and the cytochrome segments of the respiratory chain. Primary coenzyme Q(10) deficiency represents a clinically heterogeneous condition suggestive of genetic heterogeneity, and several disease genes have been previously identified. The CABC1 gene, also called COQ8 or ADCK3, is the human homolog of the yeast ABC1/COQ8 gene, one of the numerous genes involved in the ubiquinone biosynthesis pathway. The exact function of the Abc1/Coq8 protein is as yet unknown, but this protein is classified as a putative protein kinase. We report here CABC1 gene mutations in four ubiquinone-deficient patients in three distinct families. These patients presented a similar progressive neurological disorder with cerebellar atrophy and seizures. In all cases, enzymological studies pointed to ubiquinone deficiency. CoQ(10) deficiency was confirmed by decreased content of ubiquinone in muscle. Various missense mutations (R213W, G272V, G272D, and E551K) modifying highly conserved amino acids of the protein and a 1 bp frameshift insertion c.[1812_1813insG] were identified. The missense mutations were introduced into the yeast ABC1/COQ8 gene and expressed in a Saccharomyces cerevisiae strain in which the ABC1/COQ8 gene was deleted. All the missense mutations resulted in a respiratory phenotype with no or decreased growth on glycerol medium and a severe reduction in ubiquinone synthesis, demonstrating that these mutations alter the protein function.     

Proteomic analysis reveals novel binding partners of metabotropic glutamate receptor 1

Regulated trafficking of neurotransmitter receptors is critical to normal neurodevelopment and neuronal signaling. Group I mGluRs (mGluR1/5 and their splice variants) are G protein-coupled receptors enriched at excitatory synapses, where they serve to modulate glutamatergic transmission. The mGluR1 splice variants mGluR1a and mGluR1b are broadly expressed in the central nervous system and differ in their signaling and trafficking properties. Several proteins have been identified that selectively interact with mGluR1a and participate in receptor trafficking but no proteins interacting with mGluR1b have thus far been reported. We have used a proteomic strategy to isolate and identify proteins that co-purify with mGluR1b in Madin-Darby Canine Kidney (MDCK) cells, an established model system for trafficking studies. Here, we report the identification of 10 novel candidate mGluR1b-interacting proteins. Several of the identified proteins are structural components of the cell cytoskeleton, while others serve as cytoskeleton-associated adaptors and motors or endoplasmic reticulum-associated chaperones. Findings from this work will help unravel the complex cellular mechanisms underlying mGluR trafficking under physiological and pathological conditions.     

Blockade of amygdala metabotropic glutamate receptor subtype 1 impairs fear extinction

The metabotropic glutamate receptor subtype 1 (mGluR1) is thought to be crucial for several forms of memory, but its role in memory extinction has not been determined. Here, we examined a role of mGluR1 in the extinction of conditioned fear using microinjection of an mGluR1 antagonist, CPCCOEt, into the lateral amygdala (LA), a critical structure for fear conditioning and extinction. Intra-LA injection of 3 microg CPCCOEt impaired extinction that was initiated 48 h after the conditioning, but not that initiated 2h after the conditioning, indicating that the effectiveness of CPCCOEt depends upon the length of time since fear conditioning. The CPCCOEt injection failed to alter an mGluR1-like receptor (mGluR5)-dependent acquisition of fear memory, further supporting the specificity of the injected CPCCOEt on mGluR1. Together, our results suggest that amygdala mGluR1 plays a critical role in the extinction of learned fear, but not in the acquisition of fear memory.