Weiming Xu

Mice with very low expression of the vesicular monoamine transporter 2 gene survive into adulthood: potential mouse model for parkinsonism.

ABSTRACT We have created a transgenic mouse with a hypomorphic allele of the vesicular monoamine transporter 2 (Vmat2) gene by gene targeting. These mice (KA1) have profound changes in monoamine metabolism and function and survive into adulthood. Specifically, these animals express very low levels of VMAT2, an endogenous protein which sequesters monoamines intracellularly into vesicles, a process that, in addition to being important in normal transmission, may also act to keep intracellular levels of the monoamine neurotransmitters below potentially toxic thresholds. Homozygous mice show large reductions in brain tissue monoamines, motor impairments, enhanced sensitivity to dopamine agonism, and changes in the chemical neuroanatomy of the striatum that are consistent with alterations in the balance of the striatonigral (direct) and striatopallidal (indirect) pathways. The VMAT2-deficient KA1 mice are also more vulnerable to the neurotoxic effects of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine in terms of nigral dopamine cell death. We suggest that the mice may be of value in examining, long term, the insidious damaging consequences of abnormal intracellular handling of monoamines. On the basis of our current findings, the mice are likely to prove of immediate interest to aspects of the symptomatology of parkinsonism. They may also, however, be of use in probing other aspects of monoaminergic function and dysfunction in the brain, the latter making important contributions to the pathogenesis of schizophrenia and addiction.

Localization of brain nitric oxide synthase (NOS) to human chromosome 12

Recent research has shown that nitric oxide is a novel neuronal second messenger and transmitter that may be involved in neuronal cell death and damage in neurological illness. To map the chromosomal localization of this important brain enzyme, a rat cDNA probe was prepared by RNA PCR from rat cerebellum RNA. This rat cDNA was used to isolate a human nitric oxide synthase (NOS) cDNA from a human cerebellum cDNA library. The human cDNA clone containing 1.2 kb of brain NOS cDNA was hybridized to Southern blots containing DNAs obtained from human-rodent hybrid cell line panels using EcoRI and HindIII digestion to ascertain the location of the human NOS gene. These data showed that the human brain nitric oxide synthase mapped within 12q14-qter on human chromosome 12.

Regional localization of the gene coding for human brain nitric oxide synthase (NOS1) to 12q24.2→24.31 by fluorescent in situ hybridization

The human gene for brain nitric oxide synthase (NOS1) was previously assigned to chromosome 12 by analysis of a panel of somatic cell hybrids. We report here the refinement of this localization to 12q24.2-->24.31 by fluorescent in situ hybridization.

The Affinity of a Major Ca2+ Binding Site on GRP78 Is Differentially Enhanced by ADP and ATP

GRP78 is a major protein regulated by the mammalian endoplasmic reticulum stress response, and up-regulation has been shown to be important in protecting cells from challenge with cytotoxic agents. GRP78 has ATPase activity, acts as a chaperone, and interacts specifically with other proteins, such as caspases, as part of a mechanism regulating apoptosis. GRP78 is also reported to have a possible role as a Ca2+ storage protein. In order to understand the potential biological effects of Ca2+ and ATP/ADP binding on the biology of GRP78, we have determined its ligand binding properties. We show here for the first time that GRP78 can bind Ca2+, ATP, and ADP, each with a 1:1 stoichiometry, and that the binding of cation and nucleotide is cooperative. These observations do not support the hypothesis that GRP78 is a dynamic Ca2+ storage protein. Furthermore, we demonstrate that whereas Mg2+ enhances GRP78 binding to ADP and ATP to the same extent, Ca2+ shows a differential enhancement. In the presence of Ca2+, the KD for ATP is lowered ∼11-fold, and the KD for ADP is lowered around 930-fold. The KD for Ca2+ is lowered ∼40-fold in the presence of ATP and around 880-fold with ADP. These findings may explain the biological requirement for a nucleotide exchange factor to remove ADP from GRP78. Taken together, our data suggest that the Ca2+-binding property of GRP78 may be part of a signal transduction pathway that modulates complex interactions between GRP78, ATP/ADP, secretory proteins, and caspases, and this ultimately has important consequences for cell viability.

The Ccttt polymorphism in the Nos2a gene is associated with dementia with Lewy bodies

We report the analysis of the allele distribution of a (CCTTT)n pentanucleotide repeat within the promoter region of the NOS2A gene in DNA samples from patients with autopsy confirmed Alzheimers disease (AD) and dementia with Lewy bodies (DLB) type. A significant difference was observed in the allelic distribution between the control group and the DLB group (χ2 = 15.175, df = 5; p <0.01), with an increased occurrence of the eight and nine repeat alleles, and a marked under representation of the 11 repeat allele. Genotype frequencies in the DLB group also differed significantly from controls (p <0.012). These results suggest that variations in the NOS2A gene may predispose to the development of DLB.

The molecular cloning and expression of a human synaptic vesicle amine transporter that suppresses MPP+ toxicity

A synaptic vesicle amine transporter cDNA, termed hSVAT, has been isolated by the reverse transcription and polymerase chain reaction (PCR) technique from human substantia nigra and subsequent screening of a human substantia nigra library. The hSVAT sequence obtained is highly homologous to the rat SVAT sequence (92% homology) and is essentially identical to the human sequence identified recently by Surratt and colleagues [33]. This labelled hSVAT cDNA detected a single band (approximately 5.0 kb) when used as a probe for Northern analysis of human nigral RNA extract. In situ hybridization studies using hSVAT specific antisense oligonucleotides showed a strong hybridization signal concentrated over the cells of the substantia nigra pars compacta. This cDNA sequence when expressed in chinese hamster ovary (CHO) cells conferred resistance to MPP+ the toxic metabolite of MPTP and cells containing it accumulated dopamine.

Nitric Oxide: A Novel Intercellular Messenger in the Striatum

The free radical gas nitric oxide (NO) has recently been proposed as a messenger, or novel type of neurotransmitter in the brain (Bredt et al., 1991; Snyder and Bredt, 1992). The initial evidence for a role for NO in the central nervous system came when Garthwaite et al (1988) showed that cerebellar neurones would synthesize NO in response to the excitatory neurotransmitter glutamate, an observation confirmed by Bredt and Snyder (1989) amongst others. Subsequent work by Bredt and Snyder (1990) characterized the enzyme responsible for nitric oxide production, nitric oxide synthase (NOS) from rat brain and they cloned and isolated NOS (Bredt et al., 1991). The cloning and isolation of brain NOS revealed that the enzyme was structurally related only to cytochrome P450-oxido-reductase (CP-450 OR) which like NOS has an electron-transferring/accepting carboxy-terminal sequence (Bredt et al., 1991). The ability of NOS to reduce the dye nitro-blue tetrazolium accounting for a significant amount of brain diaphorase activity and diaphorase staining reflects NOS containing neurones (Figure 1) (Hope et al., 1991). We have confirmed these observations using in situ hybridization with antisense probes for NOS and NOS specific antibodies which visualize the same population of neurones in the brain and striatum (Figure 1).