Characterization of LRRTM and NGR gene families: Expression and functions

1 REVIEW OF THE LITERATURE.1 1.1 Identifying protein-coding genes in the post-genomic era . 1 1.2 The zeitgeist of ’omics’ vs. bottom-up approaches in biology. 2 1.3 How to elucidate what a gene does: from gene to phenotype or vice versa? 3 1.4 The leucine-rich repeat protein superfamily. 4 1.4.1 Structure of the LRR domain . 4 1.4.2 Prevalence of the LRR domains in the proteome. 5 1.4.3 On the evolution of genes encoding proteins with signal peptide and LRR domain . 6 1.5 Neuronal LRR membrane proteins . 6 1.5. 1 Structural features of neuronal LRR membrane proteins. 7 1.5. 2 Emerging common themes in neuronal LRR membrane protein biology. 9 1.5. 3 Example of neuronal LRR membrane proteins: NGL family. 10 1.5. 4 Example of neuronal LRR membrane proteins: SALM family . 10 1.5. 5 Example of neuronal LRR membrane proteins: Slitrk family . 11 1.5. 6 Example of neuronal LRR membrane proteins: AMIGO family. 11 1.6 Nogo-A and Nogo-66 Receptor in axonal regeneration. 12 1.6. 1 Myelin inhibitors of neurite growth . 12 1.6. 2 Identification of Nogo-A . 12 1.6. 3 Nogo-66 receptor (NgR1) and its ligands . 13 1.6. 4 Therapeutic potential of perturbing Nogo-A and NgR1 function. 14 1.7 Genetic basis of left-right brain asymmetry. 14 1.7. 1 Co-evolution of handedness and brain asymmetry . 15 1.7. 2 Genes and genomic regions implicated in handedness. 15 1.8 Schizophrenia. 16 1.8. 1 Schizophrenia genetics. 16 1.8. 2 Schizophrenia and handedness . 16 1.8. 3 Animal models in schizophrenia research . 16 2 AIMS OF THIS STUDY.18 3 MATERIALS AND METHODS . 19 3.1 Bioinformatics. 19 3.2 Recombinant DNA techniques . 19 3.3 RT-PCR expression analysis . 19 3.4 In situ hybridization . 20 3.5 Cell culture . 20 3.6 Neuronal cell culture . 20 3.7 Recombinant protein production and affinity purification . 21 3.8 Immunocytochemistry and light and confocal microscopy. 21 3.9 Cell-based ligand binding assays. 21 3.10 Generation of knock out mice . 21 3.10. 1 Targeting constructs . 21 3.10. 2 Embryonic stem cell targeting. 21 3.10. 3 Mouse breeding schemes. 21 3.10. 4 Removal of the neo-cassette . 22 3.11 SNP genotyping and human genetics. 22 4 RESULTS AND DISCUSSION . 23 4.1 Characterization of LRRTM and NGRL gene families . 23 4.1.1 The emergence of LRRTM and NGR gene families. 23 4.2 Analysis of the primary, secondary and tertiary structure of the proteins encoded by LRRTM and NGR gene families. 24 4.2. 1 Analysis of the cytoplasmic segment of LRRTMs . 26 4.3 Localization of LRRTM and NGR family mRNAs and proteins. 26 4.3.1 mRNA expression pattern of mouse and human LRRTMs and NGRs . 26 4.3.2 LRRTMs and NGRs are expressed neuronally . 27 4.3.3 Subcellular localization of LRRTM1 and NgR family members. 27 4.4 Mapping the NgR family interactome. 27 4.4.1 Interactions between multiple reticulons and NgR1 . 27 4.4.2 MAG is a high affinity ligand for NgR2. 28 4.4.3 Characterization of OMgp-MAG interaction. 28 4.5 Mechanistic insight into how NgR1 interacts with multiple ligands . 29 4.5. 1 Multiple sites in Nogo-A interact with NgR1 . 29 4.5. 2 Myelin ligands’ binding to NgR1 requires overlapping but partially separate residues 29 4.6 Lrrtm1 and Lrrtm3 knock out mice as tools to pioneer in vivo studies on neuronal LRR membrane proteins . 30 4.6. 1 Lrrtm1 and Lrrtm3 genes can be efficiently targeted . 31 4.6. 2 tau-GFP and tau-LacZ reporter expression. 32 4.6. 3 On the phenotype of Lrrtm3 knock out mice. 32 4.7 Association of LRRTM1 locus to schizophrenia and handedness. 32 4.7. 1 No association of LRRTM1 gene 5 ́ region to schizophrenia in Han Chinese families33 5 CONCLUDING REMARKS . 36 6 FUTURE DIRECTIONS . 36 REFERENCES . 38 LIST OF ORIGINAL PUBLICATIONS This thesis is based on the following original articles, which are referred to in the text by their Roman numerals (I-IV): I Laurén J., Airaksinen M.S., Saarma M. , and Timmusk T. : A novel gene family encoding leucine-rich repeat transmembrane proteins differentially expressed in the nervous system. Genomics, 2003, 81(4): 411-421 II Laurén J., Airaksinen M.S., Saarma M. , and Timmusk T. : Two Novel Mammalian Nogo Receptor Homologs Differentially Expressed in the Central and Peripheral Nervous System. Molecular and Cellular Neuroscience, 2003, 24(3): 581-594 III Laurén J., Hu F. , Chin J. Liao J., Airaksinen M.S., and Strittmatter S.M. : Characterization of Myelin Ligand Complexes with Neuronal Nogo-66 Receptor Family Members. Journal of Biological Chemistry, 2007, 282(8): 5715-5725 IV Francks C. , Maegawa S. *, Laurén J.*, Abrahams B. , Velayos-Baeza A. , Medland S.E. , Colella S. , Groszer M. , McAuley E.Z. , Caffrey T.M, Timmusk T. , Pruunsild P., Koppel I. , Lind P.A. , Matsumoto-Itaba N. , Nicod J., Xiong L. , Joober R. , Enard W. , Krinsky B. , Nanba E. , Richardson A.J., Riley B.P, Martin N.G. , Strittmatter S.M. , Möller H-J., Rujescu D. , Clair D. , Muglia P., Roos J.L. , Fisher S.E. , Wade-Martins R. , Rouleau G.A. , Stein J.F. , Karayiorgou M. , Geschwind D.H. , Ragoussis J., Kendler K.S. , Airaksinen M.S., Oshimura M. , DeLisi L.E. , and Monaco A.P.: LRRTM1 on chromosome 2p12 is a maternally suppressed gene that is associated paternally with handedness and schizophrenia. Molecular Psychiatry, 2007 (in press)