Regional Differences in Penetration of the Protein Stabilizer Trimethoprim (TMP) in the Rat Central Nervous System.

Serena Di Palma,Martin E. Schwab,Shane A. Liddelow,Susanne Neumann,Alice C. Mosberger,Benjamin V. Ineichen,Endre Laczko

doi:10.3389/fnmol.2020.00167

Abstract

Regulating gene expression at the protein level is becoming increasingly important for answering basic questions in neurobiology. Several techniques using destabilizing domains (DD) on transgenes, which can be activated or deactivated by specific drugs, have been developed to achieve this goal. A DD from bacterial dihydrofolate reductase bound and stabilized by trimethoprim (TMP) represents such a tool. To control transgenic protein levels in the brain, the DD-regulating drugs need to have sufficient penetration into the central nervous system (CNS). Yet, very limited information is available on TMP pharmacokinetics in the CNS following systemic injection. Here, we performed a pharmacokinetic study on the penetration of TMP into different CNS compartments in the rat. We used mass spectrometry to measure TMP concentrations in serum, cerebrospinal fluid (CSF) and tissue samples of different CNS regions upon intraperitoneal TMP injection. We show that TMP quickly (within 10 min) penetrates from serum to CSF through the blood-CSF barrier. TMP also shows quick penetration into brain tissue but concentrations were an order of magnitude lower compared to serum or CSF. TMP concentration in spinal cord was lower than in any other analyzed CNS area. Nevertheless, effective levels of TMP to stabilize DDs can be reached in the CNS with half-lives around 2 h. These data show that TMP has good and fast penetration properties into the CNS and is therefore a valuable ligand for precisely controlling protein expression in the CNS in rodents.

Highlights

Temporal and spatial regulation of genes and proteins are key methods in modern biology and neuroscience
A recent promising technique to regulate protein levels in central nervous system (CNS) tissue in vivo uses a destabilizing domain fused to a protein of interest which leads to degradation of this protein unless it is bound by TMP (Cho et al, 2013)
We provide a detailed pharmacokinetic study describing the concentration of TMP in serum, cerebrospinal fluid (CSF), and different CNS regions in rats after a high intraperitoneal TMP dose

Summary

Introduction

Temporal and spatial regulation of genes and proteins are key methods in modern biology and neuroscience. A destabilizing domain (DD) modified from FK506- and rapamycin-binding protein (FKBP) fused to a protein of interest has been shown to lead to degradation by the proteasome, unless bound by the ligand Shield-1 (Banaszynski and Wandless, 2006; Banaszynski et al, 2006, 2008) To optimize this tool for use in the brain and spinal cord, a DD from bacterial dihydrofolate reductase (DHFR) which is bound and stabilized by the CNS permeable antibiotic trimethoprin (TMP, Tu et al, 1989) was developed. When fused to YFP and virally expressed in rat striatum, YFP protein levels can be controlled by TMP administered in drinking water (Iwamoto et al, 2010; Tai et al, 2012) This ligand-induced rescue from degradation has been successfully achieved for a secreted protein (GDNF) (Quintino et al, 2013) and for Crerecombinase in mice (Sando et al, 2013), and more recently with a constitutively active form of Akt kinase (Park et al, 2018). Sando et al (2013) expressed DD-Cre under control of a synapsin promoter and showed fast induction of the transgene with TMP after intraperitoneal administration in mice, suggesting that systemic TMP reaches the brain within minutes to hours

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