Parallel Metabolomics and Lipidomics of a PSMA/GCPII Deficient Mouse Model Reveal Alteration of NAAG Levels and Brain Lipid Composition.

Glutamate carboxypeptidase II (GCPII, also known as PSMA or FOLH1) is responsible for the cleavage of N-acetyl-aspartyl-glutamate (NAAG) to N-acetyl-aspartate and glutamate in the central nervous system and facilitates the intestinal absorption of folate by processing dietary folyl-poly-γ-glutamate in the small intestine. The physiological function of GCPII in other organs like kidneys is still not known. GCPII inhibitors are neuroprotective in various conditions (e.g., ischemic brain injury) in vivo; however, their utilization as potential drug candidates has not been investigated in regard to not yet known GCPII activities. To explore the GCPII role and possible side effects of GCPII inhibitors, we performed parallel metabolomic and lipidomic analysis of the cerebrospinal fluid (CSF), urine, plasma, and brain tissue of mice with varying degrees of GCPII deficiency (fully deficient in Folh1, -/-; one allele deficient in Folh1, +/-; and wild type, +/+). Multivariate analysis of metabolites showed no significant differences between wild-type and GCPII-deficient mice (except for NAAG), although changes were observed between the sex and age. NAAG levels were statistically significantly increased in the CSF, urine, and plasma of GCPII-deficient mice. However, no difference in NAAG concentrations was found in the whole brain lysate likely because GCPII, as an extracellular enzyme, can affect only extracellular and not intracellular NAAG concentrations. Regarding the lipidome, the most pronounced genotype-linked changes were found in the brain tissue. In brains of GCPII-deficient mice, we observed statistically significant enrichment in phosphatidylcholine-based lipids and reduction of sphingolipids and phosphatidylethanolamine plasmalogens. We hypothesize that the alteration of the NAA-NAAG axis by absent GCPII activity affected myelin composition. In summary, the absence of GCPII and thus similarly its inhibition do not have detrimental effects on metabolism, with just minor changes in the brain lipidome.

Iridium-based Polymeric Multifunctional Imaging Tools for Biochemistry.

Herein, we report the development of a macromolecular multifunctional imaging tool for biological investigations, which is comprised of an N-(2-hydroxypropyl)methacrylamide backbone, iridium-based luminescent probe, glutamate carboxypeptidase II (GCPII) targeting ligand, and biotin affinity tag. The iridium luminophore is a tris-cyclometalated complex based on [Ir(ppy)3] with one of its 2-phenylpyridine ligands functionalized to allow conjugation. Synthesized macromolecular probes differed in the structure of the polymer and content of the iridium complex. The applicability of the developed imaging tools has been tested in flow cytometry (FACS) based assay, laser confocal microscopy, and fluorescence lifetime imaging microscopy (FLIM). The FACS analysis has shown that the targeted iBodies containing the iridium luminophore exhibit selective labelling of GCPII expressing cells. This observation was also confirmed in the imaging experiments with laser confocal microscopy. The FLIM experiment has shown that the iBodies with the iridium label exhibit a lifetime greater than 100 ns, which distinguishes them from typically used systems labelled with organic fluorophores exhibiting short fluorescence lifetimes. The results of this investigation indicate that the system exhibits interesting properties, which supports the development of additional biological tools utilizing the key components (iridium complexes, iBody concept), primarily focusing on the longer lifetime of the iridium emitter.