SummaryTourette's syndrome is a common developmental neuropsychiatric disorder characterized by chronic motor and vocal tics. Despite a strong genetic contribution, inheritance is complex, and risk alleles have proven difficult to identify. Here, we describe an analysis of linkage in a two-generation pedigree leading to the identification of a rare functional mutation in the HDC gene encoding Lhistidine decarboxylase, the rate-limiting enzyme in histamine biosynthesis. Our findings, together with previously published data from model systems, point to a role for histaminergic neurotransmission in the mechanism and modulation of Tourette's syndrome and tics.Tourette's Syndrome is Characterized by Childhood Onset, Waxing and waning symptomatology, and typically, improvement in adulthood. The molecular underpinnings of the disorder remain uncertain, although multiple lines of evidence suggest involvement of dopaminergic neurotransmission and abnormalities involving cortical-striatal-thalamiccortical circuitry. 1 Current treatment focuses on tic reduction and management of prevalent coexisting conditions such as obsessive-compulsive disorder and attention deficithyperactivity disorder. However, therapeutic options have limited efficacy and may carry clinically significant side effects. Consequently, the development of new treatments based on an improved understanding of disease pathophysiology is a high priority. 2 The large genetic contribution to Tourette's syndrome is well established. 3 4,5 However, mutations are found in only a small proportion of affected persons, and the protein's normal function and the manner in which it may contribute to Tourette's syndrome are not yet well understood.In light of the probable genetic heterogeneity underlying Tourette's syndrome, we sought families in which the syndrome is transmitted in a mendelian fashion, which is rare. As has been shown for other complex disorders, gene discovery in such families may help to uncover molecular mechanisms of disease. 6 MethodsMethods are described briefly here; for complete details, see the Supplementary Appendix, available with the full text of this article at NEJM.org. All studies were approved by the Yale Institutional Review Board, and all participants provided written informed consent.A nonconsanguineous two-generation pedigree was referred to our laboratory (Fig. 1). The father and all eight offspring met the criteria for Tourette's syndrome in the Diagnostic and Statistical Manual of Mental Disorders, fourth edition, text revision (Table S1 in the Supplementary Appendix). Two children and the father also have obsessive-compulsive disorder. The mother, her parents, and her extended family are reportedly free from Tourette's syndrome, chronic tics, and obsessive-compulsive disorder.DNA samples from all family members in the two-generation pedigree were genotyped by means of Affymetrix GeneChip Human Mapping 100K Arrays and short-tandem-repeat markers that had been identified within the lod -2 linkage interval (the equivalent of a conf...

Summary Synaptogenesis is required for wiring neuronal circuits in the developing brain and continues to remodel adult networks. However, the molecules organizing synapse development and maintenance in vivo remain incompletely understood. We now demonstrate that the immunoglobulin adhesion molecule SynCAM 1 dynamically alters synapse number and plasticity. Overexpression of SynCAM 1 in transgenic mice promotes excitatory synapse number, while loss of SynCAM 1 results in fewer excitatory synapses. By turning off SynCAM 1 overexpression in transgenic brains, we show that it maintains the newly induced synapses. SynCAM 1 also functions at mature synapses to alter their plasticity by regulating long-term depression. Consistent with these effects on neuronal connectivity, SynCAM 1 expression affects spatial learning, with knock-out mice learning better. The reciprocal effects of increased SynCAM 1 expression and loss reveal that this adhesion molecule contributes to the regulation of synapse number and plasticity, and impacts how neuronal networks undergo activity-dependent changes.

cgi-58 (comparative gene identification-58) is a member of ␣/␤-hydrolase family of proteins. Mutations in CGI-58 are shown to be responsible for a rare genetic disorder known as Chanarin-Dorfman syndrome, characterized by an excessive accumulation of triacylglycerol in several tissues and ichthyosis. We have earlier reported that YLR099c encoding Ict1p in Saccharomyces cerevisiae can acylate lysophosphatidic acid to phosphatidic acid. Here we report that human CGI-58 is closely related to ICT1. To understand the biochemical function of cgi-58, the gene was overexpressed in Escherichia coli, and the purified recombinant protein was found to specifically acylate lysophosphatidic acid in an acyl-CoA-dependent manner. Overexpression of CGI-58 in S. cerevisiae showed an increase in the formation of phosphatidic acid resulting in an overall increase in the total phospholipids. However, the triacylglycerol level was found to be significantly reduced. In addition, the physiological significance of cgi-58 in mice white adipose tissue was studied. We found soluble lysophosphatidic acid acyltransferase activity in mouse white adipose tissue. Immunoblot analysis using antiIct1p antibodies followed by mass spectrometry of the immunocross-reactive protein in lipid droplets revealed its identity as cgi-58. These observations suggest the existence of an alternate cytosolic phosphatidic acid biosynthetic pathway in the white adipose tissue. Collectively, these results reveal the role of cgi-58 as an acyltransferase.

One of the major determinants of organic solvent tolerance is the increase in membrane phospholipids. Here we report for the first time that an increase in the synthesis of phosphatidic acid is responsible for enhanced phospholipid synthesis that confers tolerance to the organic solvent in Saccharomyces cerevisiae. This increase in phosphatidic acid formation is because of the induction of Ict1p, a soluble oleoyl-CoA:lysophosphatidic acid acyltransferase. YLR099C (ICT1) was reported to be maximally expressed during solvent tolerance (Miura, S., Zou, W., Ueda, M., and Tanaka, A. (2000) Appl. Environ. Microbiol. 66, 4883-4889); however, its physiological significance was not understood. In silico analysis revealed the absence of any transmembrane domain in Ict1p. Domain analysis showed that it has a hydrolase/acyltransferase domain with a distinct lipid-binding motif and a lysophospholipase domain. Analysis of ict1⌬ strain showed a drastic reduction in phosphatidic acid suggesting the role of Ict1p in phosphatidic acid biosynthesis. Overexpression of Ict1p in S. cerevisiae showed an increase in phosphatidic acid and other phospholipids on organic solvent exposure. To understand the biochemical function of Ict1p, the gene was cloned and expressed in Escherichia coli. The purified recombinant enzyme was found to specifically acylate lysophosphatidic acid. Specific activity of Ict1p was found to be higher for oleoyl-CoA as compared with palmitoyl-and stearoyl-CoAs. This study provides a mechanism for organic solvent tolerance from the point of membrane dynamics in S. cerevisiae.Tolerance to organic solvents was reported in several microorganisms, which includes Pseudomonas strains (1) and Saccharomyces cerevisiae (2). Steady accumulation of these solvents in plasma membrane resulted in the loss of structural integrity (3). Because the plasma membrane acts as a selectively permeable barrier of the cell, such a toxicity causes an impairment in the ionic and the metabolic balances, pH gradient, electrical potential, etc., leading to cell lysis.Cell surface modifications were found to be one major reason for such a tolerance. Other important factors are metabolic degradation of organic solvents, pumps responsible for extrusion (2), changes in saturated fatty acid contents, and conversion from cis to trans fatty acids (4). For example in Pseudomonas putida DOT-T1, the conversion of cis-9,10-methylene hexadecanoic acid to unsaturated cis-9-hexadecenoic acid was observed on exposure to toluene. However, the mechanism for the conversion is unknown (5). Change in the phospholipid content was shown to be an important factor in providing tolerance against the organic solvents. Elegant work on mechanisms of solvent tolerance in P. putida strain Idaho showed that the strain was able to repair the damaged membranes through efficient turnover and increased phospholipid biosynthesis. A detailed analysis of phospholipid head group turnover revealed the presence of a large amount of phosphatidylglycerol followed by phosphatidylethanol...

Human CGI-58 (for comparative gene identification-58) and YLR099c, encoding Ict1p in Saccharomyces cerevisiae, have recently been identified as acyl-CoA-dependent lysophosphatidic acid acyltransferases. Sequence database searches for CGI-58 like proteins in Arabidopsis (Arabidopsis thaliana) revealed 24 proteins with At4g24160, a member of the a/b-hydrolase family of proteins being the closest homolog. At4g24160 contains three motifs that are conserved across the plant species: a GXSXG lipase motif, a HX 4 D acyltransferase motif, and V(X) 3 HGF, a probable lipid binding motif. Dendrogram analysis of yeast ICT1, CGI-58, and At4g24160 placed these three polypeptides in the same group. Here, we describe and characterize At4g24160 as, to our knowledge, the first soluble lysophosphatidic acid acyltransferase in plants. A lipidomics approach revealed that At4g24160 has additional triacylglycerol lipase and phosphatidylcholine hydrolyzing enzymatic activities. These data establish At4g24160, a protein with a previously unknown function, as an enzyme that might play a pivotal role in maintaining the lipid homeostasis in plants by regulating both phospholipid and neutral lipid levels.

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Nicotinamide adenine dinucleotide (NAD + ) is an endogenous enzyme cofactor and cosubstrate that has effects on diverse cellular and physiologic processes, including reactive oxygen species generation, mitochondrial function, apoptosis, and axonal degeneration. A major goal is to identify the NAD + -regulated cellular pathways that may mediate these effects. Here we show that the dynamic assembly and disassembly of microtubules is markedly altered by NAD + . Furthermore, we show that the disassembly of microtubule polymers elicited by microtubule depolymerizing agents is blocked by increasing intracellular NAD + levels. We find that these effects of NAD + are mediated by the activation of the mitochondrial sirtuin sirtuin-3 (SIRT3). Overexpression of SIRT3 prevents microtubule disassembly and apoptosis elicited by antimicrotubule agents and knockdown of SIRT3 prevents the protective effects of NAD + on microtubule polymers. Taken together, these data demonstrate that NAD + and SIRT3 regulate microtubule polymerization and the efficacy of antimicrotubule agents.N icotinamide adenine dinucleotide (NAD + ) is an endogenous dinucleotide that is present in the cytosol, nucleus, and mitochondria. Athough it serves an important role as a redox cofactor in metabolism, NAD + is also a substrate for several families of enzymes, including the poly(ADP ribose) polymerases and the sirtuin deacetylase enzymes (reviewed in refs. 1 and 2). The level of intracellular NAD + is regulated by many factors, including diet and energy status (3), axonal injury (4), DNA damage (5), and certain disease states (6), suggesting that NAD + -dependent signaling is dynamically modulated in diverse contexts.NAD + -dependent signaling can be induced by treatment of cells with exogenous NAD + , which increases intracellular NAD + levels and results in diverse effects in cells and animals. These effects include enhanced oxygen consumption and ATP production (7), as well as protection from genotoxic stress and apoptosis (3). Mice treated with nicotinamide riboside, a NAD + precursor that is metabolized into NAD + , have enhanced oxidative metabolism, increased insulin sensitivity, and protection from high-fat diet-induced obesity (8). These results demonstrate that NAD + -dependent pathways can enhance metabolic function and improve a variety of disease phenotypes.An NAD + -regulated pathway also inhibits axonal degeneration elicited by axonal transection (4). Treatment of axons with 5-20 mM NAD + markedly delays the axon degenerative process (9). Additionally, animals that express the Wallerian degeneration slow (Wld S ) protein, a fusion of the NAD + biosynthetic enzyme Nicotinamide mononucleotide adenylyl transferase 1 and Ube4a, exhibit markedly delayed degeneration of the distal axonal fragment after axonal transection (10), and expression of Wld S mitigates disease phenotypes in several neurodegenerative disease models (11)(12)(13)(14). Thus, understanding the intracellular pathways regulated by NAD + may be important for understanding the pa...

The enzyme thymidylate synthase (TSase), an important chemotherapeutic drug target, catalyzes the formation of 2′-deoxythymidine-5′-monophosphate (dTMP), a precursor of one of the DNA building blocks. TSase catalyzes a multi-step mechanism that includes the abstraction of a proton from the C5 of the substrate 2′-deoxyuridine-5′-monophosphate (dUMP). Previous studies on ecTSase proposed that an active-site residue, Y94 serves the role of the general base abstracting this proton. However, since Y94 is neither very basic, nor connected to basic residues, nor located close enough to the pyrimidine proton to be abstracted, the actual identity of this base remains enigmatic. Based on crystal structures, an alternative hypothesis is that the nearest potential proton-acceptor of C5 of dUMP is a water molecule that is part of a hydrogen bond (H-bond) network comprised of several water molecules and several protein residues including H147, E58, N177, and Y94. Here, we examine the role of the residue Y94 in the proton abstraction step by removing its hydroxyl group (Y94F mutant). We investigated the effect of the mutation on the temperature dependence of intrinsic kinetic isotope effects (KIEs) and found that these KIEs are more temperature dependent than those of the wild-type enzyme (WT). These results suggest that the phenolic –OH of Y94 is a component of the transition state for the proton abstraction step. The findings further support the hypothesis that no single functional group is the general base, but a network of bases and hydroxyls (from water molecules and tyrosine) sharing H-bonds across the active site can serve the role of the general base to remove the pyrimidine proton.