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Pyridoxine-dependent epilepsy (PDE-ALDH7A1), also known as antiquitin deficiency, is an inborn error of lysine metabolism that presents with refractory epilepsy in newborns. Bi-allelic ALDH7A1 variants lead to deficiency of α-aminoadipic semialdehyde dehydrogenase, resulting in accumulation of piperideine-6-carboxylate (P6C), and secondary deficiency of the important co-factor pyridoxal-5’-phosphate (PLP, active vitamin B6) through its complexation with P6C. Vitamin B6 supplementation resolves epilepsy in patients, but despite this treatment, intellectual disability may occur. Early diagnosis and treatment, preferably based on newborn screening, potentially optimize long-term clinical outcome. However, the currently known diagnostic PDE-ALDH7A1 biomarkers are incompatible with newborn screening procedures. Using a combination of the innovative analytical methods untargeted metabolomics and infrared ion spectroscopy, we have been able to discover novel biomarkers for PDE-ALDH7A1: 2S,6S-and 2S,6R-oxopropylpiperidine-2-carboxylic acid (2-OPP) and 6-oxopiperidine-2-carboxylic acid (6-oxoPIP). We demonstrate the applicability of 2-OPP as a PDE-ALDH7A1 biomarker in newborn screening. Additionally, we show that 2-OPP accumulates in brain tissue of patients and Aldh7a1 knock-out mice, and induces epilepsy-like behavior in a zebrafish model system. We speculate that 2-OPP may contribute to ongoing neurotoxicity, also in treated PDE-ALDH7A1 patients. As 2-OPP formation appears to increase upon ketosis, we emphasize the importance of avoiding catabolism in PDE-ALDH7A1 patients.

The unfolded protein response (UPR) is one of the major cell‐autonomous proteostatic stress responses. The UPR has been implicated in the pathogenesis of neurodegenerative diseases and is therefore actively investigated as therapeutic target. In this respect, cell non‐autonomous effects of the UPR including the reported cell‐to‐cell transmission of UPR activity may be highly important. A pharmaca‐based UPR induction was employed to generate conditioned media (CM) from CM‐donating neuronal (‘donor’) cells (SK‐N‐SH and primary mouse neurons). As previously reported, upon subsequent transfer of CM to naive neuronal ‘acceptor’ cells, we confirmed UPR target mRNA and protein expression by qPCR and automated microscopy. However, UPR target gene expression was also induced in the absence of donor cells, indicating carry‐over of pharmaca. Genetic induction of single pathways of the UPR in donor cells did not result in UPR transmission to acceptor cells. Moreover, no transmission was detected upon full UPR activation by nutrient deprivation or inducible expression of the heavy chain of immunoglobulin M in donor HeLa cells. In addition, in direct co‐culture of donor cells expressing the immunoglobulin M heavy chain and fluorescent UPR reporter acceptor HeLa cells, UPR transmission was not observed. In conclusion, carry‐over of pharmaca is a major confounding factor in pharmaca‐based UPR transmission protocols that are therefore unsuitable to study cell‐to‐cell UPR transmission. In addition, the absence of UPR transmission in non‐pharmaca‐based models of UPR activation indicates that cell‐to‐cell UPR transmission does not occur in cell culture., The unfolded protein response (UPR) is a major proteostatic stress response that was reported to be transmitted from cell‐to‐cell. Pharmacological induction of the UPR in ‘donor’ cells results in UPR activation in ‘acceptor’ cells via transfer of conditioned media. However, we show that carry‐over of pharmaca is a major confounding factor in this UPR transmission paradigm. Therefore, we employed multiple non‐pharmacological UPR transmission assays. Our data demonstrate that cell‐to‐cell transmission of UPR activity does not occur in cell culture. Open Science: This manuscript was awarded with the Open Materials Badge For more information see: https://cos.io/our-services/open-science-badges/