The ion pump Na⁺,K⁺-ATPase is a critical determinant of neuronal excitability; however, its role in the etiology of diseases of the central nervous system (CNS) is largely unknown. We describe here the molecular phenotype of a Trp931Arg mutation of the Na⁺,K⁺-ATPase catalytic α1 subunit in an infant diagnosed with therapy-resistant lethal epilepsy. In addition to the pathological CNS phenotype, we also detected renal wasting of Mg²⁺. We found that membrane expression of the mutant α1 protein was low, and ion pumping activity was lost. Arginine insertion into membrane proteins can generate water-filled pores in the plasma membrane, and our molecular dynamic (MD) simulations of the principle states of Na⁺,K⁺-ATPase transport demonstrated massive water inflow into mutant α1 and destabilization of the ion-binding sites. MD simulations also indicated that a water pathway was created between the mutant arginine residue and the cytoplasm, and analysis of oocytes expressing mutant α1 detected a nonspecific cation current. Finally, neurons expressing mutant α1 were observed to be depolarized compared with neurons expressing wild-type protein, compatible with a lowered threshold for epileptic seizures. The results imply that Na⁺,K⁺-ATPase should be considered a neuronal locus minoris resistentia in diseases associated with epilepsy and with loss of plasma membrane integrity.

In children, tick-borne encephalitis and neuroborreliosis are common infections affecting the central nervous system. As inflammatory pathways including cytokine expression are activated in these children and appear to be of importance for outcome, we hypothesized that induction of the kynurenine pathway may be part of the pathophysiological mechanism. Inflammatory biomarkers were analyzed in cerebrospinal fluid from 22 children with tick-borne encephalitis (TBE), 34 children with neuroborreliosis (NB) and 6 children with no central nervous system infection. Cerebrospinal fluid levels of kynurenine and kynurenic acid were increased in children with neuroborreliosis compared to the comparison group. A correlation was seen between expression of several cerebrospinal fluid cytokines and levels of kynurenine and kynurenic acid in children with neuroborreliosis but not in children with tick-borne encephalitis. These findings demonstrate a strong induction of the kynurenine pathway in children with neuroborreliosis which differs from that seen in children with tick-borne encephalitis. The importance of brain kynurenic acid (KYNA) in both immune modulation and neurotransmission raises the possibility that abnormal levels of the compound in neuroborreliosis might be of importance for the pathophysiology of the disease. Drugs targeting the enzymes of this pathway may open the venue for novel therapeutic interventions.

The pyruvate dehydrogenase complex (PDC) is responsible for the conversion of pyruvate into acetyl-CoA, which is used for energy conversion in cells. PDC activity is regulated by phosphorylation via kinases and phosphatases (PDK/PDP). Variants in all subunits of the PDC and in PDK3 have been reported, with varying phenotypes including lactic acidosis, neurodevelopmental delay, peripheral neuropathy, or seizures. Here, we report a de novo heterozygous missense variant in PDK1 (c.1139G > A; p.G380D) in a girl with developmental delay and early onset severe epilepsy. To investigate the role of PDK1^G380D in energy metabolism and neuronal development, we used a zebrafish model. In zebrafish embryos we show a reduced number of cells with mitochondria with membrane potential, reduced movements, and a delay in neuronal development. Furthermore, we observe a reduction in the phosphorylation of PDH-E1α by PDK^G380D, which suggests a disruption in the regulation of PDC activity. Finally, in patient fibroblasts, a mild reduction in the ratio of phosphorylated PDH over total PDH-E1α was detected. In summary, our findings support the notion that this aberrant PDK1 activity is the cause of clinical symptoms in the patient.

Pyruvate dehydrogenase complex deficiency (PDCD) is a defect of aerobic carbohydrate metabolism that causes neurological disorders with varying degrees of severity. We report the clinical, biochemical, and molecular findings in patients with primary and secondary PDCD caused by novel atypical genetic variants. Whole-genome sequencing (WGS) identified the synonymous variants c.447AG, p.(Lys149=) and c.570CT, p.(Cys190=) in pyruvate dehydrogenase E1 subunit alpha 1 (PDHA1), the deep intronic variants c.1023+2267GA and c.1023+2302AG in pyruvate dehydrogenase complex component X (PDHX), and c.185+15054GA in thiamine pyrophosphokinase (TPK1). Analysis by Sanger and RNA sequencing of cDNA from patient blood and/or cultured fibroblasts showed that the synonymous variants in PDHA1 lead to aberrant splicing and skipping of exons 5 and 5-6 in one of the patients and transcripts lacking exon 6 in the other. The deep intronic variants in PDHX and TPK1 lead to insertion of intronic sequence in the corresponding transcripts. The splice defects in PDHA1 were more pronounced in cultured fibroblasts than in blood. Our findings expand the spectrum of pathogenic variants causing PDCD and highlight the importance of atypical variants leading to aberrant splicing. The severity of the splice defects and resulting biochemical dysfunction varied between tissues, stressing the importance of performing biochemical and transcript analysis in affected tissues. The two males with hemizygous synonymous PDHA1 variants have a mild phenotype and higher PDH enzyme activity than expected, which is consistent with aberrant but leaky splicing with a proportion of the transcripts remaining correctly spliced.

PPFIBP1 encodes for the liprin-β1 protein which has been shown to play a role in neuronal outgrowth and synapse formation in Drosophila melanogaster . By exome sequencing, we detected nine ultra-rare homozygous loss-of-function variants in 14 individuals from 10 unrelated families. The individuals presented with moderate to profound developmental delay, often refractory early-onset epilepsy and progressive microcephaly. Further common clinical findings included muscular hypertonia, spasticity, failure to thrive and short stature, feeding difficulties, impaired hearing and vision, and congenital heart defects. Neuroimaging revealed abnormalities of brain morphology with leukoencephalopathy, cortical abnormalities, and intracranial periventricular calcifications as major features. In a fetus with intracranial calcifications, we identified a rare homozygous missense variant that by structural analysis was predicted to disturb the topology of the SAM-domain region that is essential for protein-protein interaction. For further insight in the effects of PPFIBP1 loss-of-function, we performed automated behavioural phenotyping of a Caenorhabditis elegans PPFIBP1/hlb-1 knockout model which revealed defects in spontaneous and light-induced behaviour and confirmed resistance to the acetylcholinesterase inhibitor aldicarb suggesting a defect in the neuronal presynaptic zone. In conclusion, we present bi-allelic loss-of-function variants in PPFIBP1 as a novel cause of an autosomal recessive neurodevelopmental disorder.

Objective: The aim of this study was to characterize intellectual and motor function, neurological features including epilepsy, treatment response, and adaptive behavior in patients with pyruvate dehydrogenase complex deficiency (PDCD) in Sweden. Methods: Forty‐two individuals with genetically confirmed PDCD (86% PDHA1 ‐related disease) were identified from a nationwide epidemiological study and were included in this cross‐sectional study comprising systematic neurological evaluations ( n = 41) and caregiver interviews assessing adaptive behavior ( n = 35). Results: Intellectual disability was detected in 33/42 (79%) individuals, while 31/35 (89%) demonstrated significant impairments in adaptive functioning. Although 27/39 (69%) were ambulatory, only 8/39 (21%) demonstrated age‐appropriate walking ability. Clinical signs of polyneuropathy were observed in 24/41 (59%), bulbar symptoms in 22/41 (54%), spasticity in 19/41 (46%), ataxia in 13/41 (32%), and dystonia in 9/41 (22%). Lifetime epilepsy was present in 16/41 (39%) of individuals. Ketogenic diet treatment, administered to 30 individuals, was effective in both prenatal‐ and postnatal‐onset disease. Seizure frequency decreased in individuals with epilepsy (8/9; 89%) and relapses of dystonia, ataxia, exercise intolerance, and lactic acidosis were prevented in all affected individuals (15/15). Improvements in communication and motor function were also noted. Discussion: Intellectual disability and deficits in adaptive behavior are frequent in PDCD, although cognitive outcomes are more heterogeneous among individuals with postnatal onset. Prenatal onset and epilepsy are associated with severe‐profound intellectual disability. Although most are ambulatory, motor deficits are frequent. A ketogenic diet treatment is a safe and effective therapeutic option, contributing to both seizure control and remission of neurological deterioration relapses.