Role of Bone Morphogenetic Proteins for Catecholaminergic Neurons in Vivo : Use of the Tyrosine Hydroxylase Locus for Cell-Specific inactivation of Signal Transduction

Abstract: Members of the Transforming Growth factor-β (TGF-β) superfamily and its subclass Bone Morphogenetic Proteins (BMP) play important roles for nervous system development. In order to study the BMP role for catecholaminergic neurons in vivo, we generated three knock-in mice, expressing the transgenes specifically in the targeting cells. Two genetic modifications result in expression of dominant negative (dn) BMP receptors (BMPRII and ALK2). The tissue-specific expression was achieved by the transgene insertion into 3’- untranslated region of the endogenous gene for tyrosine hydroxylase (TH), the first enzyme in catecholamine biosynthesis. An Internal Ribosome Entry site (IRES) preceded inserted cDNAs, allowing for functional bicistronic mRNA production. While almost no defects in Th-IRES-dnALK2, the Th-IRES-dnBMPRII mouse demonstrated declined levels of catecholamines, including dopamine in the striatum. Losses of midbrain dopaminergic neurons (MDN) might cause the effect. Additionally, intermediate lines of these mice, preserving a neo-cassette, oriented opposite to the locus transcription, demonstrate dramatic decrease of catecholamine level, hence, represent models for rare catecholamine-deficiency diseases, including L-DOPA-responsive dystonia.The third mouse, expressing in the same way Cre-recombinase (Th-IRES-Cre), represents a tool for catecholaminergic cell-limited deletion of any gene, which has to be flanked by loxP sites. Besides TH-positive areas, unexpected sites of Cre-recombination were identified, indicating regions of transient TH expression. Surprising recombination in oocytes opens a possibility to use our mouse as a general Cre-deletor.Using TH-IRES-Cre mouse we generated tissue-specific knockout mice for two BMP signal transducers: Smad1 and Smad4 (also crucial for TGF-β). While no phenotype in Smad1 knockout, TH-IRES-Cre/Smad4 mouse revealed several defects including decreased level of striatal dopamine. These results demonstrate a positive role of BMPs for MDN fate in vivo. Generated mice represent a tool-box for comprehensive study of the BMP function in catecholaminergic neurons. This study is of potential interest for understanding some aspects of Parkinson’s disease.