Mutations in PYCR2, Encoding Pyrroline-5-Carboxylate Reductase 2, Cause Microcephaly and Hypomyelination

Despite recent advances in understanding the genetic bases of microcephaly, a large number of cases of microcephaly remain unexplained, suggesting that many microcephaly syndromes and associated genes have yet to be identified. Here, we report mutations in PYCR2, which encodes an enzyme in the proline biosynthesis pathway, as the cause of a unique syndrome characterized by postnatal microcephaly, hypomyelination, and reduced cerebral white-matter volume. Linkage mapping and whole-exome sequencing identified homozygous mutations (c.355C>T [p.Arg119Cys] and c.751C>T [p.Arg251Cys]) in PYCR2 in the affected individuals of two consanguineous families. A lymphoblastoid cell line from one affected individual showed a strong reduction in the amount of PYCR2. When mutant cDNAs were transfected into HEK293FT cells, both variant proteins retained normal mitochondrial localization but had lower amounts than the wild-type protein, suggesting that the variant proteins were less stable. A PYCR2-deficient HEK293FT cell line generated by genome editing with the clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 system showed that PYCR2 loss of function led to decreased mitochondrial membrane potential and increased susceptibility to apoptosis under oxidative stress. Morpholino-based knockdown of a zebrafish PYCR2 ortholog, pycr1b, recapitulated the human microcephaly phenotype, which was rescued by wild-type human PYCR2 mRNA, but not by mutant mRNAs, further supporting the pathogenicity of the identified variants. Hypomyelination and the absence of lax, wrinkly skin distinguishes this condition from that caused by previously reported mutations in the gene encoding PYCR2’s isozyme, PYCR1, suggesting a unique and indispensable role for PYCR2 in the human CNS during development

Microarray Noninvasive Neuronal Seizure Recordings from Intact Larval Zebrafish.

Zebrafish epilepsy models are emerging tools in experimental epilepsy. Zebrafish larvae, in particular, are advantageous because they can be easily genetically altered and used for developmental and drug studies since agents applied to the bath penetrate the organism easily. Methods for electrophysiological recordings in zebrafish are new and evolving. We present a novel multi-electrode array method to non-invasively record electrical activity from up to 61 locations of an intact larval zebrafish head. This method enables transcranial noninvasive recording of extracellular field potentials (which include multi-unit activity and EEG) to identify epileptic seizures. To record from the brains of zebrafish larvae, the dorsum of the head of an intact larva was secured onto a multi-electrode array. We recorded from individual electrodes for at least three hours and quantified neuronal firing frequency, spike patterns (continuous or bursting), and synchrony of neuronal firing. Following 15 mM potassium chloride- or pentylenetetrazole-infusion into the bath, spike and burst rate increased significantly. Additionally, synchrony of neuronal firing across channels, a hallmark of epileptic seizures, also increased. Notably, the fish survived the experiment. This non-invasive method complements present invasive zebrafish neurophysiological techniques: it affords the advantages of high spatial and temporal resolution, a capacity to measure multiregional activity and neuronal synchrony in seizures, and fish survival for future experiments, such as studies of epileptogenesis and development

Correction: Microarray Noninvasive Neuronal Seizure Recordings from Intact Larval Zebrafish.

[This corrects the article DOI: 10.1371/journal.pone.0156498.]

Zebrafish models of candidate human epilepsy-associated genes provide evidence of hyperexcitability

Summary: Hundreds of novel candidate human epilepsy-associated genes have been identified thanks to advancements in next-generation sequencing and large genome-wide association studies, but establishing genetic etiology requires functional validation. We generated a list of >2,200 candidate epilepsy-associated genes, of which 48 were developed into stable loss-of-function (LOF) zebrafish models. Of those 48, evidence of seizure-like behavior was present in 5 (arfgef1, kcnd2, kcnv1, ubr5, and wnt8b). Further characterization provided evidence for epileptiform activity via electrophysiology in kcnd2 and wnt8b mutants. Additionally, arfgef1 and wnt8b mutants showed a decrease in the number of inhibitory interneurons in the optic tectum of larval animals. Further, RNA sequencing (RNA-seq) revealed convergent transcriptional abnormalities between mutant lines, consistent with their developmental defects and hyperexcitable phenotypes. These zebrafish models provide strongest experimental evidence supporting the role of ARFGEF1, KCND2, and WNT8B in human epilepsy and further demonstrate the utility of this model system for evaluating candidate human epilepsy genes

Provoked seizures in 5-second raw data traces of action potentials.

<p><b>a:</b> Baseline activity before PTZ addition. <b>b:</b> A typical seizure starting with a prolonged action potential burst 1–2 minutes after PTZ application. <b>c:</b> Seizures continue as short paroxysmal action potential bursts. PTZ addition leads to a seizure firing pattern. Markings denote sustained (> 2 sec; b) versus short (< 500 ms; c) bursting.</p

Confirmation of cell stability.

<p>Top panels in (<b>a)</b> show representative single cell recordings before and after KCl addition, top panels in (<b>b</b>) before and after PTZ addition. Bottom panels show averages of baseline and KCl recordings (<b>a</b>) and of baseline and PTZ recordings (<b>b</b>). Dashed lines indicate the width of the averaged action potential at half maximum of the amplitude and they approximate 1.3 ms in both recordings. Action potentials are stable over time as indicated by the unchanged shape of the traces.</p

Increase in active electrode firing fraction and spatial coherence of electrical activity after drug addition.

<p><b>a:</b> Area under the curve (AUC), a numerical integration, of the total firing fraction between baseline and post-drug periods shows significant increase (** p < 0.01, *** p < 0.001) after KCl (p = 0.0009) and PTZ (p = 0.002) addition relative to baseline. Note the similarity in the firing fraction across time when no drug (sham) was added to the bath (p = 0.86). <b>b:</b> Area under the curve of spatial coherence, a measure derived by comparing the correlation of coefficient of a central pixel with respect to its nearest-matching neighbor along time, also did not differ between baseline and follow-up in the sham condition (p = 0.19). Notably, more channels fired simultaneously indicating a significant increase (* p < 0.05, ** p < 0.01) in spatial coherence after KCl (p = 0.002) and PTZ (p = 0.02) administration.</p

Addition of TTX to seizing larvae abolishes spiking.

<p>Spike rate plotted as a function of time (one channel shown). Recordings are made before and after PTZ addition. The addition of 0.4 mM TTX at 3900 s abolished firing. These experiments demonstrate a high likelihood that recorded signals arise from neurons firing action potentials (in contrast to myogenic potentials arising from tail-movements).</p

Tools and steps for larva-mounting and timeline of recording.

<p><b>a:</b> Electrode chamber used for recordings (scale: 10 mm). <b>b:</b> Nylon mesh and slice anchor used to hold larva in place (scale: 5 mm). <b>c:</b> Placement of larva onto its dorsal side into the chamber with a drop of water (scale: 300 μm). <b>d:</b> Securing larva with nylon mesh (scale: 300 μm); microelectrode array is highlighted with red dots for enhanced visibility in this photo and the next. <b>e:</b> Larva positioned with head onto the microelectrode array (scale 300 μm). <b>f:</b> Timeline for experiments—each starting with a 15 min acclimation period, a 30 min control recording, and a 30 min recording time after drug application.</p