Folate deficiency increases the incidence of dolutegravir-associated foetal defects in a mouse pregnancy model.

BACKGROUND: Dolutegravir (DTG) is a recommended first-line regimen for all people with Human Immunodeficiency Virus (HIV) infection. Initial findings from Botswana, a country with no folate fortification program, showed an elevated prevalence of neural tube defects (NTDs) with peri-conceptional exposure to DTG. Here we explore whether a low folate diet influences the risk of DTG-associated foetal anomalies in a mouse model. METHODS: C57BL/6 mice fed a folate-deficient diet for 2 weeks, were mated and then randomly allocated to control (water), or 1xDTG (2.5 mg/kg), or 5xDTG (12.5 mg/kg) both administered orally with 50 mg/kg tenofovir disoproxil fumarate 33.3 mg/kg emtricitabine. Treatment was administered once daily from gestational day (GD) 0.5 to sacrifice (GD15.5). Foetuses were assessed for gross anomalies. Maternal and foetal folate levels were quantified. FINDINGS: 313 litters (103 control, 106 1xDTG, 104 5xDTG) were assessed. Viability, placental weight, and foetal weight did not differ between groups. NTDs were only observed in the DTG groups (litter rate: 0% control; 1.0% 1xDTG; 1.3% 5xDTG). Tail, abdominal wall, limb, craniofacial, and bleeding defects all occurred at higher rates in the DTG groups versus control. Compared with our previous findings on DTG usage in folate-replete mouse pregnancies, folate deficiency was associated with higher rates of several defects, including NTDs, but in the DTG groups only. We observed a severe left-right asymmetry phenotype that was more frequent in DTG groups than controls. INTERPRETATION: Maternal folate deficiency may increase the risk for DTG-associated foetal defects. Periconceptional folic acid supplementation could be considered for women with HIV taking DTG during pregnancy, particularly in countries lacking folate fortification programs. FUNDING: This project has been funded by Federal funds from the Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Department of Health and Human Services, under Contract No. HHSN275201800001I and award #R01HD104553. LS is supported by a Tier 1 Canada Research Chair in Maternal-Child Health and HIV. HM is supported by a Junior Investigator award from the Ontario HIV Treatment Network.

A Warburg-like metabolic program coordinates Wnt, AMPK, and mTOR signaling pathways in epileptogenesis.

Epilepsy is a complex neurological condition characterized by repeated spontaneous seizures and can be induced by initiating seizures known as status epilepticus (SE). Elaborating the critical molecular mechanisms following SE are central to understanding the establishment of chronic seizures. Here, we identify a transient program of molecular and metabolic signaling in the early epileptogenic period, centered on day five following SE in the pre-clinical kainate or pilocarpine models of temporal lobe epilepsy. Our work now elaborates a new molecular mechanism centered around Wnt signaling and a growing network comprised of metabolic reprogramming and mTOR activation. Biochemical, metabolomic, confocal microscopy and mouse genetics experiments all demonstrate coordinated activation of Wnt signaling, predominantly in neurons, and the ensuing induction of an overall aerobic glycolysis (Warburg-like phenomenon) and an altered TCA cycle in early epileptogenesis. A centerpiece of the mechanism is the regulation of pyruvate dehydrogenase (PDH) through its kinase and Wnt target genes PDK4. Intriguingly, PDH is a central gene in certain genetic epilepsies, underscoring the relevance of our elaborated mechanisms. While sharing some features with cancers, the Warburg-like metabolism in early epileptogenesis is uniquely split between neurons and astrocytes to achieve an overall novel metabolic reprogramming. This split Warburg metabolic reprogramming triggers an inhibition of AMPK and subsequent activation of mTOR, which is a signature event of epileptogenesis. Interrogation of the mechanism with the metabolic inhibitor 2-deoxyglucose surprisingly demonstrated that Wnt signaling and the resulting metabolic reprogramming lies upstream of mTOR activation in epileptogenesis. To augment the pre-clinical pilocarpine and kainate models, aspects of the proposed mechanisms were also investigated and correlated in a genetic model of constitutive Wnt signaling (deletion of the transcriptional repressor and Wnt pathway inhibitor HBP1). The results from the HBP1-/- mice provide a genetic evidence that Wnt signaling may set the threshold of acquired seizure susceptibility with a similar molecular framework. Using biochemistry and genetics, this paper outlines a new molecular framework of early epileptogenesis and advances a potential molecular platform for refining therapeutic strategies in attenuating recurrent seizures.

APC conditional knock-out mouse is a model of infantile spasms with elevated neuronal ß-catenin levels, neonatal spasms, and chronic seizures.

Infantile spasms (IS) are a catastrophic childhood epilepsy syndrome characterized by flexion-extension spasms during infancy that progress to chronic seizures and cognitive deficits in later life. The molecular causes of IS are poorly defined. Genetic screens of individuals with IS have identified multiple risk genes, several of which are predicted to alter ß-catenin pathways. However, evidence linking malfunction of ß-catenin pathways and IS is lacking. Here, we show that conditional deletion in mice of the adenomatous polyposis coli gene (APC cKO), the major negative regulator of ß-catenin, leads to excessive ß-catenin levels and multiple salient features of human IS. Compared with wild-type littermates, neonatal APC cKO mice exhibit flexion-extension motor spasms and abnormal high-amplitude electroencephalographic discharges. Additionally, the frequency of excitatory postsynaptic currents is increased in layer V pyramidal cells, the major output neurons of the cerebral cortex. At adult ages, APC cKOs display spontaneous electroclinical seizures. These data provide the first evidence that malfunctions of APC/ß-catenin pathways cause pathophysiological changes consistent with IS. Our findings demonstrate that the APC cKO is a new genetic model of IS, provide novel insights into molecular and functional alterations that can lead to IS, and suggest novel targets for therapeutic intervention.

Diphenytoin, riluzole and lidocaine: three sodium channel blockers, with different mechanisms of action, decrease hippocampal epileptiform activity.

Epilepsy is a condition affecting 1-2% of the population, characterized by the presence of spontaneous, recurrent seizures. The most common type of acquired epilepsy is temporal lobe epilepsy (TLE). Up to 30% of patients with TLE are refractory to currently available compounds, and there is an urgent need to identify novel targets for therapy. Here, we utilized the in-vitro CA3 burst preparation to examine alterations in network excitability, characterized by changes in interburst interval. Specifically, we show that bath application of three different sodium channel blockers-diphenytoin, riluzole, and lidocaine-slow spontaneous CA3 bursts. This in turn, decreased the epileptiform activity. These compounds work at different sites on voltage-gated sodium channels, but produce a similar network phenotype of decreased excitability. In the case of diphenytoin and riluzole, the change in network activity (i.e., increased interburst intervals) was persistent following drug washout. Lidocaine application, however, only increased the CA3 interburst interval when it was in the bath solution. Thus, its action was not permanent and resulted in returning CA3 bursting to baseline levels. These data demonstrate that the CA3 burst preparation provides a relatively easy and quick platform for identifying compounds that can decrease network excitability, providing the initial screen for further and more complex in-vivo, freely-behaving animal studies.

Consider muscle disease in children with elevated transaminase.

The transaminases alanine aminotransferase (ALT) and aspartate aminotransferase (AST) are markers of hepatocellular injury but are highly concentrated in muscle cells. Consequently, muscular dystrophies such as Duchenne muscular dystrophy, lead to hypertransaminasemia. Elevation in ALT and AST is most striking during the early stages of disease, before onset of or when only subtle signs of muscle disease are present. Thus, the incidental finding of elevated ALT/AST may be the presenting sign of muscle disease in many children and provides an opportunity for early diagnosis. Many physicians, however, pursue extensive workup for liver disease in children who present with the incidental finding of elevated ALT/AST. This results in delayed diagnosis and initiation of treatment and increased expense and may lead to unnecessary invasive procedures. We report 12 patients with muscle disease who presented with a variety of symptoms and were found to have an incidental finding of elevated ALT/AST. We propose a rapid screening process for evaluating children with the incidental finding of elevated ALT/AST to shorten the time to diagnosis of muscle disease.

Effects of synaptic depression and recovery on synchronous network activity.

SUMMARY: The output of an artificial neural network of spiking neurons linked by glutamatergic synapses subject to use-dependent depression was compared with physiologic data obtained from rat hippocampal area CA3 in vitro. The authors evaluated how network burst initiation and termination was affected by activity-dependent depression and recovery under a variety of experimental conditions including neuronal membrane depolarization, altered glutamate release probability, the strength of synaptic inhibition, and long-term potentiation and long-term depression of recurrent glutamatergic synapses. The results of computational experiments agreed with the in vitro data and support the idea that synaptic properties, including activity-dependent depression and recovery, play important roles in the timing and duration of spontaneous bursts of network activity. This validated network model is useful for experiments that are not feasible in vitro, and makes possible the investigation of two-dimensional aspects of burst propagation and termination.

Convulsant and anticonvulsant effects on spontaneous CA3 population bursts.

This paper analyzes the effects of a convulsant and an anticonvulsant manipulation on spontaneous bursts in CA3 pyramidal cells in the in vitro slice preparation under conditions of low (3.3 mM [K(+)](o)) and high (8.5 mM [K(+)](o)) burst probability. When burst probability was low, the anticonvulsant, pentobarbital, produced the anticipated effects: the burst duration decreased and interburst interval increased. However, when burst probability was high, both anticonvulsant and convulsant manipulations decreased the interburst interval and the burst duration. To reconcile these findings, we utilized a model in which CA3 burst duration is limited by activity-dependent depression of CA3 excitatory recurrent collateral synapses and the interburst interval is determined by the time required to recover from this depression. We defined the burst end threshold as the level of synaptic depression at which bursts terminate, and the burst start threshold as the level of synaptic depression at which burst initiation is possible. Synapses were considered to oscillate between these thresholds. When average burst duration and interburst interval data were fit using this model, the paradoxically similar effects of the convulsant and anticonvulsant manipulations could be quantitatively interpreted. The convulsant maneuver decreased both the burst start and end thresholds. The start threshold decreased more than the end threshold, so that the thresholds were closer together. This decreased the time needed to transition from one threshold to the other, i.e., the interburst interval and burst duration. The anticonvulsant manipulation primarily increased the burst end threshold. This also decreased the difference between thresholds, decreasing both interburst interval and burst duration. This model resolves the paradoxical proconvulsant effects of pentobarbital in the CA3 preparation and provides insights into the effects of anticonvulsants on epileptiform discharges in the human EEG.

Novel CLCN1 mutations with unique clinical and electrophysiological consequences.

Myotonia is a condition characterized by impaired relaxation of muscle following sudden forceful contraction. We systematically screened all 23 exons of the CLCN1 gene in 88 unrelated patients with myotonia and identified mutations in 14 patients. Six novel mutations were discovered: five were missense (S132C, L283F, T310M, F428S and T550M) found in heterozygous patients, and one was a nonsense mutation (E193X) in a homozygous patient. While five patients had a clinical diagnosis of myotonia congenita, the patient with the F428S mutation exhibited symptoms characteristic of paramyotonia congenita--a condition usually thought to be caused by mutations in the sodium channel gene SCN4A. Nevertheless, no mutations in SCN4A were identified in this patient. The functional consequences of the novel CLCN1 sequence variants were explored by recording chloride currents from human embryonic kidney cells transiently expressing homo- or heterodimeric mutant channels. The five tested mutations caused distinct functional alterations of the homodimeric human muscle chloride ion channel hClC-1. S132C and T550M conferred novel hyperpolarization-induced gating steps, L283F and T310M caused a shift of the activation curve to more positive potentials and F428S reduced the expression level of hClC-1 channels. All showed a dominant-negative effect. For S132C, L283F, T310M and T550M, heterodimeric channels consisting of one wild-type (WT) and one mutant subunit exhibited a shifted activation curve at low intracellular [Cl(-)]. WT-F428S channels displayed properties similar to WT hClC-1, but expressed at significantly lower levels. The novel mutations exhibit a broad variety of functional defects that, by distinct mechanisms, cause a significant reduction of the resting chloride conductance in muscle of heterozygous patients. Our results provide novel insights into functional alterations and clinical symptoms caused by mutations in CLCN1.