Genetic assessment of familial and early-onset Parkinson's disease in a Greek population.

BACKGROUND AND PURPOSE: Although the first mutation associated with Parkinson's disease (PD) was identified several years ago in the alpha-synuclein (SNCA) gene in families of Greek and Italian ancestry, a more systematic study of this and other known PD mutations has not been performed in the Greek population. METHODS: A genetic analysis in 111 familial or sporadic with early-onset (≤50 years, EO) PD patients was performed for the presence of the A53T SNCA mutation. In separate subgroups of these patients, further mutations in the SNCA, LRRK2, Parkin, PINK1 and DJ-1 genes were searched for. Additionally, a subgroup of familial cases was analysed for mutations in the glucocerebrosidase (GBA) gene. RESULTS: In total, five patients (4.5% of our whole population) were identified with the A53T SNCA mutation, two with a heterozygote dosage mutation and one with a heterozygote point mutation in the Parkin gene, and seven patients (10.3% of our familial cohort) with GBA gene mutations. CONCLUSIONS: The A53T mutation in the SNCA gene, although uncommon, does represent a cause of PD in the Greek population, especially of familial EOPD with autosomal dominant inheritance. GBA mutations in the familial cohort tested here were as common as in a cohort of sporadic cases previously examined from the same centres. For the remainder of the genes, genetic defects that could definitively account for the disease were not identified. These results suggest that further Mendelian traits that lead to PD in the Greek population remain to be identified.

The genetics of ivermectin resistance in Caenorhabditis elegans.

The ability of organisms to evolve resistance threatens the effectiveness of every antibiotic drug. We show that in the nematode Caenorhabditis elegans, simultaneous mutation of three genes, avr-14, avr-15, and glc-1, encoding glutamate-gated chloride channel (GluCl) alpha-type subunits confers high-level resistance to the antiparasitic drug ivermectin. In contrast, mutating any two channel genes confers modest or no resistance. We propose a model in which ivermectin sensitivity in C. elegans is mediated by genes affecting parallel genetic pathways defined by the family of GluCl genes. The sensitivity of these pathways is further modulated by unc-7, unc-9, and the Dyf (dye filling defective) genes, which alter the structure of the nervous system. Our results suggest that the evolution of drug resistance can be slowed by targeting antibiotic drugs to several members of a multigene family.

Genome-wide insertional mutagenesis in human cells by the Drosophila mobile element Minos.

The development of efficient non-viral methodologies for genome-wide insertional mutagenesis and gene tagging in mammalian cells is highly desirable for functional genomic analysis. Here we describe transposon mediated mutagenesis (TRAMM), using naked DNA vectors based on the Drosophila hydei transposable element Minos. By simple transfections of plasmid Minos vectors in HeLa cells, we have achieved high frequency generation of cell lines, each containing one or more stable chromosomal integrations. The Minos-derived vectors insert in different locations in the mammalian genome. Genome-wide mutagenesis in HeLa cells was demonstrated by using a Minos transposon containing a lacZ-neo gene-trap fusion to generate a HeLa cell library of at least 10(5) transposon insertions in active genes. Multiple gene traps for six out of 12 active genes were detected in this library. Possible applications of Minos-based TRAMM in functional genomics are discussed.

Picrotoxin blockade of invertebrate glutamate-gated chloride channels: subunit dependence and evidence for binding within the pore.

Glutamate-gated chloride channels have been described in nematodes, insects, crustaceans, and mollusks. Subunits from the nematode and insect channels have been cloned and are phylogenetically related to the GABA and glycine ligand-gated chloride channels. Ligand-gated chloride channels are blocked with variable potency by the nonselective blocker picrotoxin. The first two subunits of the glutamate-gated chloride channel family, GluClalpha and GluClbeta, were cloned from the free living nematode Caenorhabditis elegans. In this study, we analyze the blockade of these novel channels by picrotoxin. In vitro synthesized GluClalpha and GluClbeta RNAs were injected individually or coinjected into Xenopus oocytes. The EC50 values for picrotoxin block of homomeric GluClalpha and GluClbeta were 59 microM and 77 nM, respectively. Picrotoxin block of homomeric GluClbeta channels was promoted during activation of membrane current with glutamate. In addition, recovery from picrotoxin block was faster during current activation by glutamate. A chimeric channel between the N-terminal extracellular domain of GluClalpha and the C-terminal membrane-spanning domain of GluClbeta localized the higher affinity picrotoxin binding site to the membrane-spanning domains of GluClbeta. A point mutation within the M2 membrane-spanning domain of GluClbeta reduced picrotoxin sensitivity >10,000-fold. We conclude that picrotoxin blocks GluCl channels by binding to a site accessible when the channel is open.

Evolutionary relationship of the ligand-gated ion channels and the avermectin-sensitive, glutamate-gated chloride channels.

Two cDNAs, GluClalpha and GluClbeta, encoding glutamate-gated chloride channel subunits that represent targets of the avermectin class of antiparasitic compounds, have recently been cloned from Caenorhabditis elegans (Cully et al., Nature, 371, 707-711, 1994). Expression studies in Xenopus oocytes showed that GluClalpha and GluClbeta have pharmacological profiles distinct from the glutamate-gated cation channels as well as the gamma-aminobutyric acid (GABA)- and glycine-gated chloride channels. Establishing the evolutionary relationship of related proteins can clarify properties and lead to predictions about their structure and function. We have cloned and determined the nucleotide sequence of the GluClalpha and GluClbeta genes. In an attempt to understand the evolutionary relationship of these channels with the members of the ligand-gated ion channel superfamily, we have performed gene structure comparisons and phylogenetic analyses of their nucleotide and predicted amino acid sequences. Gene structure comparisons reveal the presence of several intron positions that are not found in the ligand-gated ion channel superfamily, outlining their distinct evolutionary position. Phylogenetic analyses indicate that GluClalpha and GluClbeta form a monophyletic subbranch in the ligand-gated ion channel superfamily and are related to vertebrate glycine channels/receptors. Glutamate-gated chloride channels, with electrophysiological properties similar to GluClalpha and GluClbeta, have been described in insects and crustaceans, suggesting that the glutamate-gated chloride channel family may be conserved in other invertebrate species. The gene structure and phylogenetic analyses in combination with the distinct pharmacological properties demonstrate that GluClalpha and GluClbeta belong to a discrete ligand-gated ion channel family that may represent genes orthologous to the vertebrate glycine channels.

Genetic and biochemical evidence for a novel avermectin-sensitive chloride channel in Caenorhabditis elegans. Isolation and characterization.

Avermectins are a class of macrocyclic lactones that is widely used in crop protection and to treat helminth infections in man and animals. Two complementary DNAs (GluClalpha and GluClbeta) encoding chloride channels that are gated by avermectin and glutamate, respectively, were isolated from Caenorhabditis elegans. To study the role of these subunits in conferring avermectin sensitivity we isolated a mutant C. elegans strain with a Tc1 transposable element insertion that functionally inactivated the GluClalpha gene (GluClalpha::Tc1). GluClalpha::Tc1 animals exhibit a normal phenotype including typical avermectin sensitivity. Xenopus oocytes expressing GluClalpha::Tc1 strain mRNA elicited reduced amplitude avermectin and glutamate-dependent chloride currents. Avermectin binding assays in GluClalpha::Tc1 strain membranes showed the presence of high affinity binding sites, with a reduced Bmax. These experiments suggest that GluClalpha is a target for avermectin and that additional glutamate-gated and avermectin-sensitive chloride channel subunits exist in C. elegans. We isolated a cDNA (GluClalpha2) encoding a chloride channel that shares 75% amino acid identity with GluClalpha. This subunit forms homomeric channels that are gated irreversibly by avermectin and reversibly by glutamate. GluClalpha2 coassembles with GluClbeta to form heteromeric channels that are gated by both ligands. The presence of subunits related to GluClalpha may explain the low level and rarity of target site involvement in resistance to the avermectin class of compounds.

Developmental expression of a 43-kDa secreted glycoprotein from Trichinella spiralis.

Trichinella spiralis is an intracellular parasitic nematode that infects skeletal muscle cells. Infection results in loss of tissue specific characteristics and conversion of the muscle cell to a Nurse cell. The characteristic changes leading to the formation of the Nurse cell appear complete by day 12 after intramuscular infection. Proteins synthesized in the stichocytes (secretory cells) of T. spiralis and secreted in the host cell are believed to be involved in the process of Nurse cell formation. One secreted glycoprotein of 43 kDa has been considered as a candidate factor involved in Nurse cell formation. We determined the timing of synthesis and secretion of the 43-kDa glycoprotein and its temporal correlation to the changes of the infected host cell, to gain an understanding of the role of the 43-kDa glycoprotein in T. spiralis infection. We show that the 43-kDa glycoprotein is first expressed on day 11 following intramuscular infection, several days after the changes in the infected muscle cell have been initiated. Protein(s) immunologically related to the 43-kDa glycoprotein but not the 43-kDa glycoprotein itself are detected in the nuclei of mature Nurse cells. During the intramuscular stage the 43-kDa glycoprotein appears to be stored in the alpha-stichocytes of T. spiralis and appears to be secreted immediately following invasion of the intestinal columnar epithelial cells by the L1 larva. The role of the 43-kDa glycoprotein remains unknown, however, these findings argue against involvement of the 43-kDa glycoprotein in Nurse cell formation.

Trichinella pseudospiralis secretes a protein related to the Trichinella spiralis 43-kDa glycoprotein.

A 43-kDa secreted glycoprotein from the intracellular parasitic nematode Trichinella spiralis has been considered as a factor involved in the formation of the Nurse cell in infected muscle. The closely related intracellular parasitic nematode Trichinella pseudospiralis that also infects muscle cells, does not form Nurse cells and was thought not to secrete the 43-kDa glycoprotein. This implied a unique role for the 43-kDa glycoprotein in T. spiralis infection and supported the hypothesis of involvement of the 43-kDa glycoprotein in Nurse cell formation. Following cloning of a full length cDNA encoding the 43-kDa protein, antibodies were raised against several domains of the 43-kDa glycoprotein. Here we show that a protein related to the 43-kDa glycoprotein exists in T. pseudospiralis. Immunohistochemical studies reveal important similarities in the distribution of the 43-kDa glycoprotein and the related protein from T. pseudospiralis in muscle infections with either of the two parasites. The 43-kDa glycoprotein may therefore play a common role in the life cycles of these two parasites and probably is not involved in Nurse cell formation.

Molecular biology and electrophysiology of glutamate-gated chloride channels of invertebrates.

In this chapter we summarize the available data on a novel class of ligand-gated anion channels that are gated by the neurotransmitter glutamate. Glutamate is classically thought to be a stimulatory neurotransmitter, however, studies in invertebrates have proven that glutamate also functions as an inhibitory ligand. The bulk of studies conducted in vivo have been on insects and crustaceans, where glutamate was first postulated to act on H-receptors resulting in a hyperpolarizing response to glutamate. Recently, glutamate-gated chloride channels have been cloned from several nematodes and Drosophila. The pharmacology and electrophysiological properties of these channels have been studied by expression in Xenopus oocytes. Studies on the cloned channels demonstrate that the invertebrate glutamate-gated chloride channels are the H-receptors and represent important targets for the antiparasitic avermectins.

Cloning of an avermectin-sensitive glutamate-gated chloride channel from Caenorhabditis elegans.

The avermectins are a family of macrocyclic lactones used in the control of nematode and arthropod parasites. Ivermectin (22,23-dihydroavermectin B1a) is widely used as an anthelmintic in veterinary medicine and is used to treat onchocerciasis or river blindness in humans. Abamectin (avermectin B1a) is a miticide and insecticide used in crop protection. Avermectins interact with vertebrate and invertebrate GABA receptors and invertebrate glutamate-gated chloride channels. The soil nematode Caenorhabditis elegans has served as a useful model to study the mechanism of action of avermectins. A C. elegans messenger RNA expressed in Xenopus oocytes encodes an avermectin-sensitive glutamate-gated chloride channel. To elucidate the structure and properties of this channel, we used Xenopus oocytes for expression cloning of two functional complementary DNAs encoding an avermectin-sensitive glutamate-gated chloride channel. We find that the electrophysiological and structural properties of these proteins indicate that they are new members of the ligand-gated ion channel superfamily.

Analysis of a 43-kDa glycoprotein from the intracellular parasitic nematode Trichinella spiralis.

The L1 larvae of the parasitic nematode Trichinella spiralis invade skeletal muscle and initiate a process that has been interpreted to represent skeletal muscle dedifferentiation. In this process, the infected region of the muscle cell is converted into a unique structure, called the Nurse cell. The nematode T. spiralis can survive for tens of years within the cytoplasm of the Nurse cell and secretes proteins into the cytoplasm that are believed to play a role in mediating the Nurse cell formation or maintenance. We have cloned a cDNA encoding the T. spiralis-derived, 43-kDa secreted protein. Structural analysis of the predicted 344-amino acid sequence revealed an N terminally located signal peptide and a potential helix-loop-helix motif in the main body of the protein. Antibodies raised against the 43-kDa recombinant protein were used in immunocytolocalizations of T. spiralis-infected skeletal muscle sections. These antibodies strongly stained the Nurse cell nuclei and the nematode itself. Specific, though slightly weaker staining also occurred in the Nurse cell cytoplasm. In Western blots, the antibodies react with the 43-kDa protein but also detected at least two other T. spiralis-secreted proteins. DNA hybridizations reveal at least one additional 43-kDa-related sequence encoded in the T. spiralis genome. We conclude that either the 43-kDa protein and/or a closely related 43-kDa family member is secreted into the muscle and translocates to the muscle-derived nuclei. This model may provide insights into the mechanisms involved in Nurse cell formation.

High level of expression of functional human platelet alpha 2-adrenergic receptors in a stable mouse C127 cell line.

We have generated, by transfection and proper selection, a stable mouse C127 cell line which expresses the human alpha 2-adrenergic receptor gene. The size of the mRNA produced by the cloned gene is 1.8 kb. Electrophoretic analysis and autoradiography of cell membrane proteins photoaffinity labeled with p-[3H]azidoclonidine gave a broad protein band of molecular mass of approx. 64 kDa. Saturation binding with [3H]rauwolscine as ligand gave an equilibrium dissociation constant of 1.29 +/- 0.46 nM (mean +/- S.D.) and binding capacity range of 18-35 pmol/mg membrane protein, with (3-6) x 10(6) receptors per cell. Antagonist competition experiments displayed the order of potency: yohimbine greater than rauwolscine greater than phentolamine much greater than prazosin. Agonist competitions demonstrated the order of potency: p-aminoclonidine greater than (-)epinephrine much greater than (+)epinephrine much greater than (-)isoproterenol. This pharmacological profile is characteristic of the human platelet alpha 2-adrenergic receptor. The expressed receptor is able to couple to the Gi protein. Thus, when epinephrine competition for specific binding of [3H]rauwolscine was performed in the presence of 1 mM MgCl2, 1 mM Gpp[NH]p increased the Ki for epinephrine from 164 to 315 nM. Following preincubation of cultures with 1 mM isobutylmethylxanthine, 1 microM epinephrine decreased forskolin-stimulated cellular cyclic AMP accumulation by 72%. The response was biphasic, and the attenuation effect disappeared at 100 microM epinephrine. A transfected clone which did not demonstrate detectable alpha 2-adrenergic receptor mRNA displayed low levels of alpha 2-adrenergic receptor, (less than 50 fmol/mg membrane protein), similar to those found in the parent C127 cell line. In this clone, epinephrine did not attenuate but, rather, enhanced forskolin-stimulated cyclic AMP accumulation. This new C127 cell line expressing high levels of alpha 2-adrenergic receptor provides an abundant source of a single human adrenergic receptor subtype in membrane-bound conformation which is able to couple to the Gi protein and inhibit forskolin-stimulated adenylate cyclase activity. This cell line will facilitate studies of the structure: function relationship of the alpha 2-adrenergic receptor and should aid in separating the components of various signal transduction mechanisms putatively attributed to this receptor.