To identify susceptibility variants for Parkinson's disease (PD), we performed a genome-wide association study (GWAS) and two replication studies in a total of 2,011 cases and 18,381 controls from Japan. We identified a new susceptibility locus on 1q32 (P = 1.52 x 10(-12)) and designated this as PARK16, and we also identified BST1 on 4p15 as a second new risk locus (P = 3.94 x 10(-9)). We also detected strong associations at SNCA on 4q22 (P = 7.35 x 10(-17)) and LRRK2 on 12q12 (P = 2.72 x 10(-8)), both of which are implicated in autosomal dominant forms of parkinsonism. By comparing results of a GWAS performed on individuals of European ancestry, we identified PARK16, SNCA and LRRK2 as shared risk loci for PD and BST1 and MAPT as loci showing population differences. Our results identify two new PD susceptibility loci, show involvement of autosomal dominant parkinsonism loci in typical PD and suggest that population differences contribute to genetic heterogeneity in PD.

Neural progenitor cells, including neural stem cells, are a potential expandable source of graft material for transplantation aimed at repairing the damaged CNS. Here we present the first evidence that in vitro-expanded fetusderived neurosphere cells were able to generate neurons in vivo and improve motor function upon transplantation into an adult rat spinal-cord-contusion injury model. As the source of graft material, we used a neural stem cell-enriched population that was derived from rat embryonic spinal cord (E14.5) and expanded in vitro by neurosphere formation. Nine days after contusion injury, these neurosphere cells were transplanted into adult rat spinal cord at the injury site. Histological analysis 5 weeks after the transplantation showed that mitotic neurogenesis occurred from the transplanted donor progenitor cells within the adult rat spinal cord, a nonneurogenic region; that these donor-derived neurons extended their processes into the host tissues; and that the neurites formed synaptic structures. Furthermore, analysis of motor behavior using a skilled reaching task indicated that the treated rats showed functional recovery. These results indicate that in vitro-expanded neurosphere cells derived from the fetal spinal cord are a potential source for transplantable material for treatment of spinal cord injury.

Apo E is a 34-kDa plasma protein important for the metabolism of plasma lipoproteins (1). Like other apolipoproteins, apo E contains multiple 22-amino acid repeats that form amphipathic helices, enabling it to associate with the surface of plasma lipoproteins. Apo E also contains a stretch of basic residues (136-150) that is important for high-affinity binding to the LDL receptor and subsequent endocytosis of the associated lipoprotein particle (2). In addition, apo E mediates lipoprotein interactions with LDL receptor-related protein (LRP) (3), the VLDL receptor (4), other lipoprotein receptors (5), endothelial heparin sulfate (6), and plasma lipases (7,8). The phenotype of severe hyperlipidemia and spontaneous development of atherosclerosis in mice lacking apo E clearly demonstrates the central role of apo E in mammalian lipid metabolism (9, 10).In humans, the APOE gene is polymorphic and has 3 alleles: APOE*2, APOE*3, and APOE*4. These alleles have frequencies of 7%, 77%, and 15%, respectively, in the general population (11). The APOE*3 allele codes for cysteine at position 112 and for arginine at 158. The APOE*2 allele codes cysteines at both positions, whereas the APOE*4 allele codes for arginines at both positions. Various population-based studies have suggested that the different APOE alleles have distinct influences on lipid metabolism in humans. Possession of at least 1 copy of the APOE*2 allele has been associated with higher plasma apo E (12) and lower plasma cholesterol, LDL cholesterol, and apo B levels (11) when compared with APOE*3 homozygotes. The APOE*2 allele is also associated with lower risk of coronary artery disease (13), except in 5-10% of APOE*2 homozygotes who develop type III hyperlipoproteinemia and premature atherosclerosis (14). On the other hand, the presence of at least 1 APOE*4 allele is associated with lower plasma apo E (12) and increased plasma cholesterol, LDL cholesterol, and apo B levels (11), and a greater risk of coronary artery disease (13), when compared with APOE*3 homozygotes. Davignon et al. (11) estimate that the apo E polymorphism accounts for 2.8% of the variation of risk for atherosclerosis, which is a large contribution for a single locus in this complex, polygenic disease.The availability of a well-defined model system should benefit studies of the role of the human apo E polymorphism in lipid metabolism and atherosclerosis. To develop such a model, we have used gene targeting to replace the murine Apoe gene with the 3 human APOE alleles. These mice retain the murine Apoe regulatory sequences and solely produce human apo E proteins with different We have generated mice expressing the human apo E4 isoform in place of the endogenous murine apo E protein and have compared them with mice expressing the human apo E3 isoform. Plasma lipid and apolipoprotein levels in the mice expressing only the apo E4 isoform (4/4) did not differ significantly from those in mice with the apo E3 isoform (3/3) on chow and were equally elevated in response to increased lipid and choles...

Eight cases of intraspinal cysts communicating with the intervertebral disc presenting symptoms identical to those of disc herniation are presented. Because all cysts were connected to the corresponding disc and the development of the cyst was assumed to be related to underlying disc injury, it is proposed to name this clinical entity discal cyst.

In the retina, the glutamate transporter GLAST is expressed in Müller cells, whereas the glutamate transporter GLT-1 is found only in cones and various types of bipolar cells. To investigate the functional role of this differential distribution of glutamate transporters, we have analyzed GLAST and GLT-1 mutant mice. In GLAST-deficient mice, the electroretinogram b-wave and oscillatory potentials are reduced and retinal damage after ischemia is exacerbated, whereas GLT-1-deficient mice show almost normal electroretinograms and mild increased retinal damage after ischemia. These results demonstrate that GLAST is required for normal signal transmission between photoreceptors and bipolar cells and that both GLAST and GLT-1 play a neuroprotective role during ischemia in the retina.L-Glutamate is the major excitatory neurotransmitter in the mammalian retina (1). High-affinity glutamate transporters are believed to be essential for terminating synaptic transmission as well as for keeping the extracellular glutamate concentration below neurotoxic levels (1, 2). Five subtypes of glutamate transporter (GLAST, GLT-1, EAAC1, EAAT4, and EAAT5) (3-8) have been cloned, but the contributions of individual transporter subtypes to retinal function are poorly understood. Studies have been hampered by the lack of subtype-selective glutamate transporter drugs. As an alternative approach, we have analyzed GLAST-and GLT-1-deficient mice (9, 10). Our results demonstrate that GLAST is required in retinal signal transmission at the level of the photoreceptor and bipolar cell and that GLAST and GLT-1 are crucial for the protection of retinal cells from glutamate neurotoxicity. MATERIALS AND METHODSImmunohistochemistry. Mice were anesthetized with diethyl ether and perfused transcardially with saline, followed by 4% paraformaldehyde in 0.1 M sodium phosphate buffer containing 0.5% picric acid at room temperature. Eyes were removed and postfixed overnight in the same fixative, and 7-m-thick paraffin or frozen sections were cut and mounted onto gelatin-and poly-L[D]-lysine-coated slides. The sections were incubated overnight with an affinity-purified rabbit polyclonal antibody against the carboxyl-terminal sequence of the mouse GLAST (1.0 g͞ml) (KKPYQLIAQDNEPEKPVAD-SETKM) (11, 12), an affinity-purified rabbit polyclonal antibody against the rat GLT-1 (0.2 g͞ml) [anti-B12; gift from N. C. Danbolt] (13), or a mouse monoclonal antibody against glutamate synthetase (GS) (2.0 g͞ml) (Chemicon) at room temperature. The sections were then incubated with biotinylated goat anti-rabbit IgG (Nichirei, Tokyo) for GLAST and GLT-1 or biotinylated rabbit anti-mouse IgG (Nichirei) for GS for 1 hr, followed by further incubation with streptavidin-Texas red (NEN) for 30 min at room temperature. Sections were examined by a confocal laser scanning microscope (Molecular Dynamics).Electroretinograms (ERGs). Mice (9-11 weeks old) were anesthetized by intraperitoneal injection of a mixture of xylazine (10 mg͞kg) and ketamine (25 mg͞kg). The pupils were dilat...

Trehalose is potentially a useful cryo-or anhydroprotectant molecule for cells and biomolecules such as proteins and nucleotides. A major obstacle to application is that cellular membranes are impermeable to trehalose. In this study, we isolated and characterized the functions of a facilitated trehalose transporter [trehalose transporter 1 (TRET1)] from an anhydrobiotic insect, Polypedilum vanderplanki. Tret1 cDNA encodes a 504-aa protein with 12 predicted transmembrane structures. Tret1 expression was induced by either desiccation or salinity stress. Expression was predominant in the fat body and occurred concomitantly with the accumulation of trehalose, indicating that TRET1 is involved in transporting trehalose synthesized in the fat body into the hemolymph. Functional expression of TRET1 in Xenopus oocytes showed that transport activity was stereochemically specific for trehalose and independent of extracellular pH (between 4.0 and 9.0) and electrochemical membrane potential. These results indicate that TRET1 is a trehalose-specific facilitated transporter and that the direction of transport is reversible depending on the concentration gradient of trehalose. The extraordinarily high values for apparent K m (>100 mM) and Vmax (>500 pmol/min per oocyte) for trehalose both indicate that TRET1 is a high-capacity transporter of trehalose. Furthermore, TRET1 was found to function in mammalian cells, suggesting that it confers trehalose permeability on cells, including those of vertebrates as well as insects. These characteristic features imply that TRET1 in combination with trehalose has high potential for basic and practical applications in vivo.anhydrobiosis ͉ insect ͉ trehalose transport ͉ Polypedilum vanderplanki dessication-inducible gene

l-Serine is synthesized from glycolytic intermediate 3-phosphoglycerate and is an indispensable precursor for the synthesis of proteins, membrane lipids, nucleotides, and neuroactive amino acids d-serine and glycine. We have recently shown that l-serine and its interconvertible glycine act as Bergmann glia-derived trophic factors for cerebellar Purkinje cells. To investigate whether such a metabolic neuron-glial relationship is fundamental to the developing and adult brain, we examined by in situ hybridization and immunohistochemistry the cellular expression of 3-phosphoglycerate dehydrogenase (3PGDH), the initial step enzyme for de novo l-serine biosynthesis in animal cells. At early stages when the neural wall consists exclusively of the ventricular zone, neuroepithelial stem cells expressed 3PGDH strongly and homogeneously. Thereafter, 3PGDH expression was downregulated and eventually disappeared in neuronal populations, whereas its high expression was transmitted to the radial glia and later to astrocytes in the gray and white matters. In addition, 3PGDH was highly expressed throughout development in the olfactory ensheathing glia, a specialized supporting cell that thoroughly ensheathes olfactory nerves. These results establish a fundamental link of the radial glia/astrocyte lineage and olfactory ensheathing glia to l-serine biosynthesis in the brain. We discuss this finding in the context of the hypothesis that 3PGDH expression in these glia cells contributes to energy metabolism in differentiating and differentiated neurons and other glia cells, which are known to be vulnerable to energy loss.

Anhydrobiosis is an extremely dehydrated state in which organisms show no detectable metabolism but retain the ability to revive after rehydration. Thus far, two hypotheses have been proposed to explain how cells are protected during dehydration: (i) water replacement by compatible solutes and (ii) vitrification. The present study provides direct physiological and physicochemical evidence for these hypotheses in an African chironomid, Polypedilum vanderplanki, which is the largest multicellular animal capable of anhydrobiosis. Differential scanning calorimetry measurements and Fourier-transform infrared (FTIR) analyses indicated that the anhydrobiotic larvae were in a glassy state up to as high as 65°C. Changing from the glassy to the rubbery state by either heating or allowing slight moisture uptake greatly decreased the survival rate of dehydrated larvae. In addition, FTIR spectra showed that sugars formed hydrogen bonds with phospholipids and that membranes remained in the liquid-crystalline state in the anhydrobiotic larvae. These results indicate that larvae of P. vanderplanki survive extreme dehydration by replacing the normal intracellular medium with a biological glass. When entering anhydrobiosis, P. vanderplanki accumulated nonreducing disaccharide trehalose that was uniformly distributed throughout the dehydrated body by FTIR microscopic mapping image. Therefore, we assume that trehalose plays important roles in water replacement and intracellular glass formation, although other compounds are surely involved in these phenomena.trehalose ͉ water replacement ͉ Fourier-transform infrared microspectroscopy ͉ biological glass ͉ cryptobiosis