Phylogenesis of Brain-Derived Neurotrophic Factor (BDNF)‏ In

Gene 450 (2010) 85–93 Contents lists available at ScienceDirect Gene journal homepage: www.elsevier.com/locate/gene Phylogenesis of brain-derived neurotrophic factor (BDNF) in vertebrates Gianluca Tettamanti, Anna Giulia Cattaneo, Rosalba Gornati, Magda de Eguileor, Giovanni Bernardini, Giorgio Binelli ⁎ Dipartimento di Biotecnologie e Scienze Molecolari, Università degli Studi dell'Insubria, Varese, Italy article info abstract Article history: Brain-derived neurotrophic factor (BDNF) belongs to neurotrophin family, a class of molecules playing key Received 12 June 2009 roles in neuronal development, survival and regeneration, neurite growth and plasticity: memory processes Received in revised form 7 July 2009 are mainly affected, and mutations of the human BDNF gene are associated to cognitive and behavioural Accepted 8 July 2009 disturbances. Available online 29 October 2009 All neurotrophins contain a highly conserved C-terminal domain and bind to the same receptor family. Both Received by M. Di Giulio correct folding and post-translational processing of the entire preproprotein are pivotal for sorting to the extracellular space, dimerization and receptor binding. Keywords: Evolutionary studies conducted so far demonstrate that a single ancestor gene underwent two independent BDNF duplication events at an early stage of vertebrate evolution, leading to the formation of the current Neurotrophins neurotrophins. However, works focusing on BDNF evolution are scarce and fragmentary, mainly in lower Molecular phylogenetics vertebrates. Structure prediction In this work, we report cloning of eight DNA sequences from amphibians and teleosts, and analysis of the entire coding regions (cDNA sequences) of BDNF from 35 organisms, from teleosts to mammals. A phylogenetic tree was constructed and the analysis of non-synonymous–synonymous substitution rates performed for the different branches. Our results suggest that natural selection is acting on mammals, separating them from other classes. Since preproprotein cleavage and 3D structure of mature protein are important for functional activity of BDNF, we also propose a de novo prediction of the 3D structure of translates in at least one species for each class, in order to get hints about the functional constraints of the protein. © 2009 Elsevier B.V. All rights reserved. 1. Introduction discovery of nerve growth factor (NGF) in the 1950s (Aloe, 2004), the family has progressively grown and now includes NGF, brain-derived Neurotrophin factors regulate neuronal differentiation and play neurotrophic factor (BDNF), neurotrophin-3 (NT-3), neurotrophin-4/ key roles in neuronal survival, neurite growth and plasticity. Since the 5 (NT-4/5), neurotrophin 6 (NT-6) and neurotrophin 7 (NT-7) (Barde et al., 1982; Götz et al., 1994; Lai et al., 1998; Leibrock et al., 1989; “ Abbreviations: ASPM, asp (abnormal spindle) homolog microcephaly associated Levi-Montalcini, 1987). While all neurotrophins bind to the com- (Drosophila); BDNF, brain-derived neurotrophic factor; cDNA, DNA complementary to mon” p75 receptor, the binding to three distinct Trk tyrosine kinase RNA; DIANNA, DiAminoacid Neural Network Application; dN, non-synonymous receptors mediates the specificity in the activity of these neurotrophic differences; DNT1, Drosophila neurotrophin 1; dNTPs, deoxyribonucleoside triphos- polypeptides (Chao, 2003; Reichardt, 2006). phate; dS, synonymous differences; DTT, dithiothreitol; ML, maximum likelihood; BDNF acts on neurons of the central and peripheral nervous NCBI, National Center for Biotechnology Information; NGF, nerve growth factor; NT, neurotrophin; PAML, phylogenetic analyses with maximum likelihood; PCR, PHYLIP: system (Levi-Montalcini and Calissano, 1979; Leibrock et al., 1989; phylogeny inferring program; PDB, Protein Data Bank; PSIPRED, Protein Structure Purvues et al., 2001); it also rescues many sensory neurons from Prediction Server; RNase, ribonuclease; Trk, tyrosine kinase receptors; STAN, STructure naturally occurring cell death (Hofer and Barde, 1988) and has been ANalysis server; UPGMA, unweighted pair group method with arithmetic mean; UCSF, shown to be involved in the regulation of vascular development in University of California San Francisco; USF, Uppsala Software Factory; ω, dN/dS rate. ⁎ Corresponding author. Dipartimento di Biotecnologie e Scienze Molecolari, ischemic tissues (Donovan et al., 2000; Kermani and Hempstead, Università degli Studi dell'Insubria, Via J-H Dunant 3, 21100 Varese, Italy. Tel.: +39 2007). Two main mutations in the BDNF protein are associated with 0332 421533; fax: +39 0332 421500. diseases in humans: the most common is V66M, found in association E-mail addresses: [email protected] (G. Tettamanti), with memory loss and hippocampus hyperactivation (Egan et al., [email protected] (A.G. Cattaneo), [email protected] 2003); the other one is L2I, which is associated with the congenital (R. Gornati), [email protected] (M. de Eguileor), fi [email protected] (G. Bernardini), [email protected] central hypoventilation syndrome, rstly described by Weese-Mayer (G. Binelli). et al. (2002). 0378-1119/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.gene.2009.07.023 86 G. Tettamanti et al. / Gene 450 (2010) 85–93 The BDNF protein contains a domain shared by all neurotrophins 2.2. RNA extraction, mRNA retrotranscription and PCR characterized by six strictly conserved cysteine residues, fundamental for the correct folding of the molecule. This feature, together with the Total RNA was extracted from liver or brain with TRIzol Reagent high percentage of amino acid identity shown by vertebrate BDNF, (Life Technologies) from 100 mg of frozen tissue according to the indicates that this factor reached an optimally functioning structure manufacturer's instructions. The first-strand cDNA was synthesized very early in vertebrate evolution, thus hindering further variations using 4 μg of total RNA, 50 pmol dT16 primer, 1 μl dNTPs mix 10 mM, in (Götz et al., 1992), due to an increased selective pressure on the a volume of 12 μl. The mix was heated at 65 °C for 15 min, chilled on coding region. ice and then 4 μl 5× retrotranscription buffer, 2 μl 0.1 M DTT, 1 μl In the past years several phylogenetic analyses of neurotrophin RNaseOUT™ and 200 U M-MLV retrotranscriptase (Invitrogen) were and Trk receptor gene families have been accomplished and the added to a final volume of 20 μl. After incubation at 37 °C for 50 min, majority of them have been devoted to the search of differences able the reaction was stopped at 75 °C for 15 min. The generated cDNA was to explain how the specificity in the activity of the different neuro- diluted to 20 μl with H2O and stored at −20 °C. trophins is achieved. Evolutionary studies on neurotrophins con- PCR amplification of BDNF cDNA was run using 2 μM solution of ducted so far demonstrate that two independent duplication events specific primers (Table 1), 5 μl of the first-strand cDNA synthesis mix, of a single ancestor gene occurred at an early stage of vertebrate 1 U of GoTaq DNA polymerase (Promega) in its own buffer, 0.2 mM evolution, leading to the formation of the current neurotrophins dNTPs mix. For each sample a set of PCR was run without retro- (Hallböök, 1999). The recent discovery of a putative neurotrophin-Trk transcription product. The efficiency of the retrotranscription reaction system after Strongylocentrotus purpuratus genome analysis (Burke et was evaluated by amplification of β-actin with appropriate primers al., 2006) and the characterization of Trk homologs in Invertebrates (Table 1). such as Lymnea stagnalis (Van Kesteren et al., 1998) and amphioxus (Benito-Gutiérrez et al., 2005) led to the hypothesis that the neuro- 2.3. Cloning and sequencing trophin/Trk signalling system could have been present in the common ancestor of protostomes and deuterostomes (Hallböök The PCR products were resolved by a 1% agarose gel and visualized et al., 2006); thus, the neurotrophin/Trk signalling system would by ethidium bromide staining. The amplified bands were extracted not be a vertebrate innovation, as partially confirmed by the structural from the gel using the Wizard SV Gel and PCR Clean-Up System and functional investigation on DNT1, a neurotrophin present in (Promega, Madison, WI), cloned in pGEM-T Easy vector (Promega) Drosophila melanogaster (Zhu et al., 2008). following the commercial kit instructions and the standard laboratory In this context, works focusing on BDNF evolution are scarce and procedures (Sambrook et al., 1989) and successively sequenced. The fragmentary (Leaché and McGuire, 2006; Kullander et al., 1997; Götz obtained sequences were deposited in the GenBank database. A list of et al., 1992; Van der Meijden et al., 2007) and, with the exception of all sequences used in this work together with their accession numbers Danio rerio (Hashimoto and Heinrich, 1997; Heinrich and Lum, is reported in Table 2. 2000; Heinrich and Pagtakhan, 2004; Lum et al., 2001), molecular data on BDNF in lower vertebrates are still lacking. Besides the phylo- 2.4. Sequence analysis genetic work of Heinrich and Lum (2000), only recently a consider- able number or BDNF sequences of a wide array of organisms were Sequence analysis was focused on two main aspects. collected and analyzed (Lanave et al., 2007). The main aim of this work is to study BDNF phylogenesis in 2.4.1. Phylogenesis vertebrates, devoting particular attention to the

Load more