Glutaric aciduria type I (GA-I, OMIM # 231670) is an inborn error of metabolism caused by a deficiency of glutaryl-CoA dehydrogenase (GCDH). Patients develop acute encephalopathic crises (AEC) with striatal injury most often triggered by catabolic stress. The pathophysiology of GA-I, particularly in brain, is still not fully understood. We generated the first knock-in rat model for GA-I by introduction of the mutation p.R411W, the rat sequence homologue of the most common Caucasian mutation p.R402W, into the Gcdh gene of Sprague Dawley rats by CRISPR/Cas9 technology. Homozygous Gcdhki/ki rats revealed a high excretor phenotype, but did not present any signs of AEC under normal diet (ND). Exposure to a high lysine diet (HLD, 4.7%) after weaning resulted in clinical and biochemical signs of AEC. A significant increase of plasmatic ammonium concentrations was found in Gcdhki/ki rats under HLD, accompanied by a decrease of urea concentrations and a concomitant increase of arginine excretion. This might indicate an inhibition of the urea cycle. Gcdhki/ki rats exposed to HLD showed highly diminished food intake resulting in severely decreased weight gain and moderate reduction of body mass index (BMI). This constellation suggests a loss of appetite. Under HLD, pipecolic acid increased significantly in cerebral and extra-cerebral liquids and tissues of Gcdhki/ki rats, but not in WT rats. It seems that Gcdhki/ki rats under HLD activate the pipecolate pathway for lysine degradation. Gcdhki/ki rat brains revealed depletion of free carnitine, microglial activation, astroglyosis, astrocytic death by apoptosis, increased vacuole numbers, impaired OXPHOS activities and neuronal damage. Under HLD, Gcdhki/ki rats showed imbalance of intra- and extracellular creatine concentrations and indirect signs of an intracerebral ammonium accumulation. We successfully created the first rat model for GA-I. Characterization of this Gcdhki/ki strain confirmed that it is a suitable model not only for the study of pathophysiological processes, but also for the development of new therapeutic interventions. We further brought up interesting new insights into the pathophysiology of GA-I in brain and periphery.

Methylmalonic aciduria (MMA) is an inborn error of metabolism resulting in loss of function of the enzyme methylmalonyl-CoA mutase (MMUT). Despite acute and persistent neurological symptoms, the pathogenesis of MMA in the central nervous system is poorly understood, which has contributed to a dearth of effective brain specific treatments. Here we utilised patient-derived induced pluripotent stem cells and in vitro differentiation to generate a human neuronal model of MMA. We reveal strong evidence of mitochondrial dysfunction caused by deficiency of MMUT in patient neurons. By employing patch-clamp electrophysiology, targeted metabolomics, and bulk transcriptomics, we expose an altered state of excitability, which is exacerbated by application of dimethyl-2-oxoglutarate, and we suggest may be connected to metabolic rewiring. Our work provides first evidence of mitochondrial driven neuronal dysfunction in MMA, which through our comprehensive characterisation of this paradigmatic model, enables first steps to identifying effective therapies.

Deficiency of antiquitin (α‐aminoadipic semialdehyde dehydrogenase), an enzyme involved in lysine degradation and encoded by ALDH7A1, is the major cause of vitamin B₆‐dependent epilepsy (PDE‐ALDH7A1). Despite seizure control with high dose pyridoxine (PN), developmental delay still occurs in approximately 70% of patients. We aimed to investigate metabolic perturbations due to possible previously unidentified roles of antiquitin, which may contribute to developmental delay, as well as metabolic effects of high dose pyridoxine supplementation reflecting the high doses used for seizure control in patients with PDE‐ALDH7A1. Untargeted metabolomics by high resolution mass spectrometry (HRMS) was used to analyze plasma of patients with PDE‐ALDH7A1 and two independently generated lines of cultured ReNcell CX human neuronal progenitor cells (NPCs) with CRISPR/Cas mediated antiquitin deficiency. Accumulation of lysine pathway metabolites in antiquitin‐deficient NPCs and western‐blot analysis confirmed knockdown of ALDH7A1. Metabolomics analysis of antiquitin‐deficient NPCs in conditions of lysine restriction and PN supplementation identified changes in metabolites related to the transmethylation and transsulfuration pathways and osmolytes, indicating a possible unrecognized role of antiquitin outside the lysine degradation pathway. Analysis of plasma samples of PN treated patients with PDE‐ALDH7A1 and antiquitin‐deficient NPCs cultured in conditions comparable to the patient plasma samples demonstrated perturbation of metabolites of the gamma‐glutamyl cycle, suggesting potential oxidative stress‐related effects in PN‐treated patients with PDE‐ALDH7A1. We postulate that a model of human NPCs with CRISPR/Cas mediated antiquitin deficiency is well suited to characterize previously unreported roles of antiquitin, relevant to this most prevalent form of pyridoxine‐dependent epilepsy.

Citrullinemia type I (CTLN1) is a rare autosomal recessive disorder caused by mutations in the gene encoding argininosuccinate synthetase 1 (ASS1) that catalyzes the third step of the urea cycle. CTLN1 patients suffer from impaired elimination of nitrogen, which leads to neurotoxic levels of circulating ammonia and urea cycle byproducts that may cause severe metabolic encephalopathy, death or irreversible brain damage. Standard of care (SOC) of CTLN1 consists of daily nitrogen-scavenger administration, but patients remain at risk of life-threatening decompensations. We evaluated the therapeutic efficacy of a recombinant adeno-associated viral vector carrying the ASS1 gene under the control of a liver-specific promoter (VTX-804). When administered to three-week-old CTLN1 mice, all the animals receiving VTX-804 in combination with SOC gained body weight normally, presented with a normalization of ammonia and reduction of citrulline levels in circulation, and 100% survived for 7 months. Similar to what has been observed in CTLN1 patients, CTLN1 mice showed several behavioral abnormalities such as anxiety, reduced welfare and impairment of innate behavior. Importantly, all clinical alterations were notably improved after treatment with VTX-804. This study demonstrates the potential of VTX-804 gene therapy for future clinical translation to CTLN1 patients.

Cotranslational protein folding is a vectorial process, and for membrane proteins, N-terminal helical segments are the first that become available for membrane insertion. While structures of many G-protein coupled receptors (GPCRs) in various states have been determined, the details of their folding pathways are largely unknown. The seven transmembrane (TM) helices of GPCRs often contain polar residues within the hydrophobic core, and some of the helices in isolation are predicted to be only marginally stable in a membrane environment. Here we review our efforts to describe how marginally hydrophobic TM helices of GPCRs integrate into the membrane in the absence of all compensating interhelical contacts, ideally capturing early biogenesis events. To this end, we use truncated GPCRs, here referred to as fragments. We present data from the human Y4 and the yeast Ste2p receptors in detergent micelles derived from solution NMR techniques. We find that the secondary structure in the fragments is similar to corresponding parts of the entire receptors. However, uncompensated polar or charged residues destabilize the helices, and prevent proper integration into the lipid bilayer, in agreement with the biophysical scales from Wimley and White for the partitioning of amino acids into the membrane-interior. We observe that the stability and integration of single TM helices is improved by adding neighboring helices. We describe a topology study, in which all possible forms of the Y4 receptor were made so that the entire receptor is truncated from the N-terminus by one TM helix at a time. We discover that proteins with an increasing number of helices assume a more defined topology. In a parallel study, we focused on the role of extracellular loops in ligand recognition. We demonstrate that transferring all loops of the human Y1 receptor onto the E. coli outer membrane protein OmpA in a suitable topology results in a chimeric receptor that displays, albeit reduced, affinity and specificity for the cognate ligand. Our data indicate that not all TM helices will spontaneously insert into the helix, and we suggest that at least for some GPCRs, N-terminal segments might remain associated with the translocon until their interacting partners are biosynthesized.