Delivery of an anti-transthyretin Nanobody to the brain through intranasal administration reveals transthyretin expression and secretion by motor neurons

Transthyretin (TTR) is a transport protein of retinol and thyroxine in serum and CSF, which is mainly secreted by liver and choroid plexus, and in smaller amounts in other cells throughout the body. The exact role of TTR and its specific expression in Central Nervous System (CNS) remains understudied. We investigated TTR expression and metabolism in CNS, through the intranasal and intracerebroventricular delivery of a specific anti-TTR Nanobody to the brain, unveiling Nanobody pharmacokinetics to the CNS. In TTR deficient mice, we observed that anti-TTR Nanobody was successfully distributed throughout all brain areas, and also reaching the spinal cord. In wild-type mice, a similar distribution pattern was observed. However, in areas known to be rich in TTR, reduced levels of Nanobody were found, suggesting potential targetmediated effects. Indeed, in wild-type mice, the anti-TTR Nanobody was specifically internalized in a receptor-mediated process, by neuronal-like cells, which were identified as motor neurons. Whereas in KO TTR mice Nanobody was internalized by all cells, for late lysosomal degradation. Moreover, we demonstrate that in vivo motor neurons also actively synthesize TTR. Finally, in vitro cultured primary motor neurons were also found to synthesize and secrete TTR into culture media. Thus, through a novel intranasal CNS distribution study with an anti-TTR Nanobody, we disclose a new cell type capable of synthesizing TTR, which might be important for the understanding of the physiological role of TTR, as well as in pathological conditions where TTR levels are altered in CSF, such as amyotrophic lateral sclerosisThis work was supported by FEDER funds through the Operational Competitiveness Programme – COMPETE, by national funding from the Portuguese Foundation for Science and Technology (FCT) under the project PEst-C/SAU/LA0001/2011, QREN Brainiac 13141 and a post-doctoral fellowship (SFRH/BPD/84178/2012) to João Gomes. The authors acknowledge Paul Moreira for support in recombinant protein production and Paula Gon©calves, for tissue processing. The authors of the manuscript have the following potential competing interest: IC, HS, PV and, AW (left in 04/2012) were employees of ABLYNX at the time of work development. The other authors have no conflict of interests. The anti-TTR Nanobody discovery and characterization, as well as the quantification of Nanobodies in brain samples was performed by ABLYNX, whereas Nanobody delivery, tissue imaging, biochemistry processing as well as cellular cultures and data processing was performed by IBMC