beta-Trace protein in human cerebrospinal fluid: a diagnostic marker for N-glycosylation defects in brain.

As carbohydrate-deficient glycoprotein syndromes (CDGS) are multisystemic disorders with impaired central nervous function in nearly all cases, we tested isoforms of beta-trace protein (beta TP), a 'brain-type' glycosylated protein in cerebrospinal fluid (CSF) of nine patients with the characteristic CDGS type I pattern of serum transferrin. Whereas the serum transferrin pattern did not discriminate between the various subtypes of CDGS type I (CDGS type Ia, type Ic, and patients with unknown defect), beta TP isoforms of CDGS type Ia patients differed from that of the other CDGS type I patients. The percentage of abnormal beta TP isoforms correlated with the severity of the neurological symptoms. Furthermore, two patients are described, who illustrate that abnormal protein N-glycosylation can occur restricted to either the 'peripheral' serum or the central nervous system compartment. This is the first report presenting evidence for an N-glycosylation defect restricted to the brain. Testing beta TP isoforms is a useful tool to detect protein N-glycosylation disorders in the central nervous system.     

A calcium-inhibited Drosophila adenylyl cyclase

Mammals possess a family of transmembrane, G-protein-responsive adenylyl cyclase isoforms (tmACs) encoded by distinct genes differing in their patterns of expression and modes of biochemical regulation. Our previous work confirmed that Drosophila melanogaster also possesses a family of tmAC isoforms defining the fly as a suitable genetic model for discerning mammalian tmAC function. We now describe a Drosophila tmAC, DAC39E, which employs a novel means for regulating its expression; differential exon utilization results in a developmental switch in DAC39E protein. DAC39E protein sequence is most closely related to mammalian type III AC, and it is predominantly expressed in the central nervous system (CNS) and olfactory organs, suggesting a role in processing sensory signaling inputs. DAC39E catalytic activity is inhibited by micromolar concentrations of calcium; therefore, DAC39E is oppositely regulated by calcium compared to the only other tmAC shown to be expressed in the Drosophila CNS, Rutabaga AC. The presence of both positively and negatively regulated tmACs suggests a complex mode of cross-talk between cAMP and calcium signal transduction pathways in the fly CNS.     

Functional expression of metabotropic GABAB receptors in primary cultures of astrocytes from rat cerebral cortex

GABA(B) receptor subunits are widely expressed on neurons throughout the central nervous system (CNS), at both pre- and postsynaptic sites, where they mediate the late and slow component of the inhibitory response to the major inhibitory neurotransmitter GABA. Recently, GABA(B) receptors have been reported to be expressed in astrocytes and microglia in the rat CNS by immunocytochemistry. However, there are few reports available for the functional characterization of GABA(B) receptors on astrocytes. In the present study, we therefore investigated the functional expression and characteristics of GABA(B) receptors in primary cultures of astrocytes from rat cerebral cortex. In the presence of 10 microM GTP, forskolin concentration-dependently increased adenylylcyclase (AC) activity in membranes prepared from rat astrocytes. The selective GABA(B) agonist (R)-baclofen concentration-dependently reduced forskolin-stimulated AC activity in the presence of 10 microM GTP. This effect was reversed by the selective GABA(B) antagonists, CGP-55845 and CGP-54626, and was completely abolished by treatment of astrocytic membranes with pertussis toxin. In addition, RT-PCR, Western blotting, and immunocytochemistry clearly showed that metabotropic GABA(B) receptor isoforms (GABA(B)R1 and GABA(B)R2) are expressed in rat cerebrocortical astrocytes. Taken collectively, these results demonstrate that functionally active metabotropic GABA(B) receptors are expressed in rat cerebrocortical astrocytes.     

Dynamic expression of alternate splice forms of D-cbl during embryogenesis

The Cbl family of proteins act as E3 ubiquitin-protein ligases and have been associated with the down regulation of a variety of receptor tyrosine kinases. Cbl proteins associate with many different cell signalling molecules suggesting that they may have functions outside of the RING finger-mediated ubiquitin ligase activity. The Drosophila melanogaster cbl gene (D-cbl) encodes two splice forms (Oncogene 19 (2000) 3299). Here we report on the differential expression of these isoforms during Drosophila embryogenesis. Both isoforms are maternally expressed but the long isoform of D-cbl is also transiently expressed in the invaginating mesoderm and later is specifically expressed in neurons of the central nervous system (CNS). Cbl protein is shown to be localised to axons of the longitudinal connectives and commissures in the central nervous system.     

Different isoforms of the non-integrin laminin receptor are present in mouse brain and bind PrP

The prion protein (PrP) plays a central role in prion diseases, and identifying its cellular receptor appears to be of crucial interest. We previously showed in the yeast two-hybrid system that PrP interacts with the 37 kDa precursor (LRP) of the high affinity 67 kDa laminin receptor (LR), which acts as the cellular receptor of PrP in cellular models. However, among the various isoforms of the receptor that have been identified so far, those which are present in the central nervous system and which bind PrP are still unknown. In this study, we have purified mouse brain fractions enriched in the laminin receptor and have performed overlay assays in order to identify those isoforms that interact with the prion protein. We demonstrate (i) the presence, in mouse brain, of several isoforms of the LRP/LR corresponding to different maturation states of the receptor (44, 60, 67 and 220 kDa) and (ii) the binding of all of these isoforms to PrP. Our data strongly support a physiological role of the laminin receptor/PrP interaction in the brain and highlight its relevance for transmissible spongiform encephalopathies.     

Locustatachykinin isoforms in the locust: distribution and quantification in the central nervous system and action on the oviduct muscle.

The presence of locustatachykinin (LomTK)-like immunoreactivity is demonstrated in the central nervous system (CNS) of Locusta migratoria with the use of a polyclonal antiserum raised against LomTK1. By developing a radioimmunoassay with the same antiserum, we have demonstrated picomolar amounts of LomTK-like material in the tissues of the central nervous system. In contrast, only femptomolar amounts of LomTK-like material are associated with the oviduct tissue. The relative amounts of the different LomTK isoforms in the brain and the abdominal ganglionic chain were examined by separating the native peptides on high-performance liquid chromatography and comparing their retention times to synthetic LomTK standards. The amounts of the different isoforms of LomTK differed between and within the two regions of the central nervous system. However, the ratios of the different isoform amounts were similar between the two regions. The myostimulatory activities of LomTKs 1 to 4 were characterized by using the locust oviduct bioassay. LomTKs 1, 2, and 3 appeared to be more efficacious than LomTK4.     

Functional studies and distribution define a family of transmembrane AMPA receptor regulatory proteins

Functional expression of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors in cerebellar granule cells requires stargazin, a member of a large family of four-pass transmembrane proteins. Here, we define a family of transmembrane AMPA receptor regulatory proteins (TARPs), which comprise stargazin, gamma-3, gamma-4, and gamma-8, but not related proteins, that mediate surface expression of AMPA receptors. TARPs exhibit discrete and complementary patterns of expression in both neurons and glia in the developing and mature central nervous system. In brain regions that express multiple isoforms, such as cerebral cortex, TARP-AMPA receptor complexes are strictly segregated, suggesting distinct roles for TARP isoforms. TARPs interact with AMPA receptors at the postsynaptic density, and surface expression of mature AMPA receptors requires a TARP. These studies indicate a general role for TARPs in controlling synaptic AMPA receptors throughout the central nervous system.     

Functional Differences between Ultrabithorax Protein Isoforms in Drosophila Melanogaster: Evidence from Elimination, Substitution and Ectopic Expression of Specific Isoforms

The homeotic selector gene Ultrabithorax (Ubx) specifies regional identities in multiple tissues within the thorax and abdomen of Drosophila melanogaster. Ubx encodes a family of six developmentally specific homeodomain protein isoforms translated from alternatively spliced mRNAs. The mutant allele Ubx(195) contains a stop codon in exon mII, one of three differential elements, and consequently produces functional UBX protein only from mRNAs of type IVa and IVb, which are expressed mainly in the central nervous system. Although it retains activity for other processes, Ubx(195) behaves like a null allele with respect to development of the peripheral nervous system, indicating that UBX-IVa and IVb alone do not contribute detectable Ubx function for this tissue. The mutant allele Ubx(MX17) contains an inversion of exon mII. We find that this allele only produces mRNAs of type IVa, but the expression pattern of the resulting UBX-IVa protein is indistinguishable from that of total UBX protein expression in wild-type embryos. The phenotype of homozygous Ubx(MX17) embryos indicates that UBX-IVa cannot substitute functionally for other isoforms to promote normal development of the peripheral nervous system. This functional limitation is confirmed by a detailed analysis of the peripheral nervous system in embryos that express specific UBX isoforms ectopically under control of a heat shock promoter. Additional observations suggest that UBX isoforms also differ in their ability to function in other tissues.     

hnRNP A1 and secondary structure coordinate alternative splicing of Mag

Myelin-associated glycoprotein (MAG) is a major component of myelin in the vertebrate central nervous system. MAG is present in the periaxonal region of the myelin structure, where it interacts with neuronal proteins to inhibit axon outgrowth and protect neurons from degeneration. Two alternatively spliced isoforms of Mag mRNA have been identified. The mRNA encoding the shorter isoform, known as S-MAG, contains a termination codon in exon 12, while the mRNA encoding the longer isoform, known as L-MAG, skips exon 12 and produces a protein with a longer C-terminal region. L-MAG is required in the central nervous system. How inclusion of Mag exon 12 is regulated is not clear. In a previous study, we showed that heteronuclear ribonucleoprotein A1 (hnRNP A1) contributes to Mag exon 12 skipping. Here, we show that hnRNP A1 interacts with an element that overlaps the 5′ splice site of Mag exon 12. The element has a reduced ability to interact with the U1 snRNP compared with a mutant that improves the splice site consensus. An evolutionarily conserved secondary structure is present surrounding the element. The structure modulates interaction with both hnRNP A1 and U1. Analysis of splice isoforms produced from a series of reporter constructs demonstrates that the hnRNP A1-binding site and the secondary structure both contribute to exclusion of Mag exon 12.     

Genetic evidence for adenylyl cyclase 1 as a target for preventing neuronal excitotoxicity mediated by N-methyl-D-aspartate receptors

The excessive activation of N-methyl-D-aspartate (NMDA) receptors by glutamate results in neuronal excitotoxicity. cAMP is a key second messenger and contributes to NMDA receptor-dependent synaptic plasticity. Adenylyl cyclases 1 (AC1) and 8 (AC8) are the two major calcium-stimulated ACs in the central nervous system. Previous studies demonstrate AC1 and AC8 play important roles in synaptic plasticity, memory, and persistent pain. However, little is known about the possible roles of these two ACs in glutamate-induced neuronal excitotoxicity. Here, we report that genetic deletion of AC1 significantly attenuated neuronal death induced by glutamate in primary cultures of cortical neurons, whereas AC8 deletion did not produce a significant effect. AC1, but not AC8, contributes to intracellular cAMP production following NMDA receptor activation by glutamate in cultured cortical neurons. AC1 is involved in the dynamic modulation of cAMP-response element-binding protein activity in neuronal excitotoxicity. To explore the possible roles of AC1 in cell death in vivo, we studied neuronal excitotoxicity induced by an intracortical injection of NMDA. Cortical lesions induced by NMDA were significantly reduced in AC1 but not in AC8 knock-out mice. Our findings provide direct evidence that AC1 plays an important role in neuronal excitotoxicity and may serve as a therapeutic target for preventing excitotoxicity in stroke and neurodegenerative diseases.     

Mapping of antigenic epitopes on the alpha 1 subunit of the inhibitory glycine receptor.

The inhibitory glycine receptor (GlyR) is a ligand-gated chloride channel protein that occurs in developmentally regulated isoforms in the vertebrate central nervous system. Monoclonal antibodies (mAbs) against the GlyR distinguish neonatal and adult GlyR proteins by identifying distinct alpha subunit variants within these receptor isoforms. Here, bacterially expressed fusion proteins of the rat GlyR alpha 1 subunit were used to localize the major antigenic epitopes of this protein within its N-terminal 105 amino acids. Synthetic peptides allowed further fine mapping of two mAb binding domains. MAb 2b, specific for the adult alpha 1 subunit, bound to a peptide corresponding to amino acids 1-10, whereas mAb 4a, which recognizes both neonatal and adult GlyR isoforms, reacted with a peptide representing residues 96-105 of the alpha 1 polypeptide. These data define unique and common antigenic epitopes on GlyR alpha subunit variants.     

Insulin-Regulated Adenylyl Cyclase Signaling System in Cell Culture of Mouse Fibroblasts of L Line

In terms of the subsequent study of the earlier discovered adenylyl cyclase signaling mechanism of insulin action [1, 2], we studied participation in it of various isoforms of protein kinase C. As object of study, a culture of mouse fibroblast cells of the L line (LSM subline) was chosen. It has been shown that insulin and a non-hydrolyzed analog of GTP, Gpp[NH]p, stimulate the adenylyl cyclase (AC) activity in these cells both individually and in combination. Activators of phorbol-sensitive isoforms of protein kinase C, diacylglycerol and phorbol ester (phorbol-12-myristate-13-acetate) at their concentrations of 1–100 nM stimulate basal activity of AC. In their presence, a significant decrease of stimulating effects of insulin and Gpp[NH]p or their complete disappearance are observed. Calphostin C (1–100 nM), an inhibitor of both phorbol-sensitive and atypical, phorbol-insensitive isoforms of protein kinase C, somewhat increased the basal AC activity. However, the stimulating effects of insulin and Gpp[NH]p in the presence of calphostin C decreased markedly. On the whole, the obtained data allow us to suggest participation of various isoforms of protein kinase C (sensitive and insensitive to the phorbol esters) in regulation of the process of the insulin signal transduction in mouse fibroblasts through the adenylyl cyclase signaling mechanism. Thus, mechanisms of functioning of the insulin-regulated AC-system in fibroblasts, representatives of connective tissue cells, are similar to those that we described earlier in muscle tissues of vertebrate and invertebrate animals. Taken together, these data indicate the absence of tissue- and species-specificity in functioning of the insulin-regulated AC system and its wide spread in tissues of different animals.     

Neuronal regulation of otolith growth and kinetotic behaviour

Inner ear stones (otoliths) of larval cichlid fish were labelled with the calcium-tracer alizarin-complexone (AC) before animals were subjected to hypergravity (hg; 3 g). After the experiment, the otoliths' area between the two AC-labellings was measured. Growth of hg-otoliths was significantly slowed down as compared to 1 g-control specimens. In the course of a second experiment, the vestibular nerve was unilaterally transacted in neonate swordtail fish which were subsequently incubated in AC. Incorporation of AC was considerably lower in the otoliths of the transacted side. The results strongly suggest that otolith growth is continuously regulated in dependence of the environmental gravity vector. Since the otolithic calcium incorporation ceased on the transected head sides, it is concluded that the regulation of otolith growth is based on the central nervous efferent vestibular system.     

Sulfogalactosylceramides in motor and psycho-cognitive adult metachromatic leukodystrophy: relations between clinical, biochemical analysis and molecular aspects

Metachromatic leukodystrophy (MLD) is a human autosomal recessive lysosomal neurodegenerative disorder that results from the accumulation of sulfatides in the central and peripheral nervous system. It is due to the enzyme deficiency of the sulfatide sulfatase i.e. arylsulfatase A (ASA). During adolescence and/or adulthood, there are 2 clinical presentations. It may be that of a degenerative disease of the central nervous system with mainly spastic manifestations or a spino-cerebellar ataxia, or that of a psychosis. As several lines of evidence indicate that the psychotic form of MLD could be a model of psychosis, we decided to do a pluridisciplinary study on 11 psycho-cognitive cases involving mental and psychiatric testing, in comparison with 5 adult motor cases, a biochemical study with enzyme assays and quantitative mass spectrometry of urinary sulfatides, so as to determine whether there were biochemical particularities related to the psychotic forms. For quantitative mass spectrometry (MS), a non physiological sulfatide with C17:0 fatty acid was synthesized. The major sulfatide isoforms were present in the 2 clinical forms with the following fatty acids and sphingoid bases: C22:1/d18:1, and /or C22:0/d18:2 (m/z 862.5), C22:0 (OH)/d18:1 (m/z 878,5), C24:0/d18:1 and / or C24:0/C23:1(OH)/d18:2 (m/z 890,3), C24:0 (OH)/d18:1(m/z 906.5). We had shown previously that there were different ASA mutations in the psychiatric adult form (heterozygous I179S) versus the adult motor form (homozygous P426L). We show here that there were no relations with the level of ASA and with the mass spectrometric study of the sulfatide isoforms which were identical in the 2 clinical forms.     

Regulation of adenylyl cyclase in the central nervous system

Adenylyl cyclases (ACs) are a family of enzymes that synthesize one of the major second messengers, cAMP, upon stimulation. Since the report of the first adenylyl cyclase (AC) gene in 1989, tremendous efforts have been devoted to identifying and characterizing more AC isozymes. In the past decade, significant knowledge regarding the basic structure, tissue distribution, and regulation of AC isozymes has been accumulated. Because members of the AC superfamily are tightly controlled by various signals, one of the most important impacts of these AC isozymes is their contribution to the complexity and fine-tuning of cellular signalling, especially in the central nervous system (CNS) where multiple signals constantly occur. This review focuses on recent progress toward understanding the physiological roles of ACs in the CNS.     

The A-kinase Anchoring Protein Yotiao Facilitates Complex Formation between Adenylyl Cyclase Type 9 and the IKs Potassium Channel in Heart

The scaffolding protein Yotiao is a member of a large family of protein A-kinase anchoring proteins with important roles in the organization of spatial and temporal signaling. In heart, Yotiao directly associates with the slow outward potassium ion current (I(Ks)) and recruits both PKA and PP1 to regulate I(Ks) phosphorylation and gating. Human mutations that disrupt I(Ks)-Yotiao interaction result in reduced PKA-dependent phosphorylation of the I(Ks) subunit KCNQ1 and inhibition of sympathetic stimulation of I(Ks), which can give rise to long-QT syndrome. We have previously identified a subset of adenylyl cyclase (AC) isoforms that interact with Yotiao, including AC1-3 and AC9, but surprisingly, this group did not include the major cardiac isoforms AC5 and AC6. We now show that either AC2 or AC9 can associate with KCNQ1 in a complex mediated by Yotiao. In transgenic mouse heart expressing KCNQ1-KCNE1, AC activity was specifically associated with the I(Ks)-Yotiao complex and could be disrupted by addition of the AC9 N terminus. A survey of all AC isoforms by RT-PCR indicated expression of AC4-6 and AC9 in adult mouse cardiac myocytes. Of these, the only Yotiao-interacting isoform was AC9. Furthermore, the endogenous I(Ks)-Yotiao complex from guinea pig also contained AC9. Finally, AC9 association with the KCNQ1-Yotiao complex sensitized PKA phosphorylation of KCNQ1 to β-adrenergic stimulation. Thus, in heart, Yotiao brings together PKA, PP1, PDE4D3, AC9, and the I(Ks) channel to achieve localized temporal regulation of β-adrenergic stimulation.     

GABAB receptor complex as a potential target for tumor therapy

γ-Aminobutyric acid (GABA) is the main inhibitory neurotransmitter in the vertebrate central nervous system. Metabotropic GABA(B) receptors are heterodimeric G-protein-coupled receptors (GPCRs) consisting of GABA(B1) and GABA(B2) subunits. The intracellular C-terminal domains of GABA(B) receptors are involved in heterodimerization, oligomerization, and association with other proteins, which results in a large receptor complex. Multiple splice variants of the GABA(B1) subunit have been identified in which GABA(B1a) and GABA(B1b) are the most abundant isoforms in the nervous system. Isoforms GABA(B1c) through GABA(B1n) are minor isoforms and are detectable only at mRNA levels. Some of the minor isoforms have been detected in peripheral tissues and encode putative soluble proteins with C-terminal truncations. Interestingly, increased expression of GABA(B) receptors has been detected in several human cancer cells and tissues. Moreover, GABA(B) receptor agonist baclofen inhibited tumor growth in rat models. GABA(B) receptor activation not only induces suppressing the proliferation and migration of various human tumor cells but also results in inactivation of CREB (cAMP-responsive element binding protein) and ERK in tumor cells. Their structural complexity makes it possible to disrupt the functions of GABA(B) receptors in various ways, raising GABA(B) receptor diversity as a potential therapeutic target in some human cancers.