Elsevier

Brain Research

Volume 1751, 15 January 2021, 147157
Brain Research

Research report
Atypical myelinogenesis and reduced axon caliber in the Scn1a variant model of Dravet syndrome: An electron microscopy pilot study of the developing and mature mouse corpus callosum

https://doi.org/10.1016/j.brainres.2020.147157Get rights and content

Highlights

  • Βefore seizure onset P16 Scn1a KO model showed atypical corpus callosum axon growth.

  • Seizure naive Scn1a KO mice, showed a trend towards smaller axon caliber.

  • Data suggests an impact of the Scn1a variant on neurodevelopment.

Abstract

Dravet Syndrome (DS) is a genetic neurodevelopmental disease. Recurrent severe seizures begin in infancy and co-morbidities follow, including developmental delay, cognitive and behavioral dysfunction. A majority of DS patients have an SCN1A heterozygous gene mutation. This mutation causes a loss-of-function in inhibitory neurons, initiating seizure onset. We have investigated whether the sodium channelopathy may result in structural changes in the DS model independent of seizures. Morphometric analyses of axons within the corpus callosum were completed at P16 and P50 in Scn1a heterozygote KO male mice and their age-matched wild-type littermates. Trainable machine learning algorithms were used to examine electron microscopy images of ~400 myelinated axons per animal, per genotype, including myelinated axon cross-section area, frequency distribution and g-ratios. Pilot data for Scn1a heterozygote KO mice demonstrate the average axon caliber was reduced in developing and adult mice. Qualitative analysis also shows micro-features marking altered myelination at P16 in the DS model, with myelin out-folding and myelin debris within phagocytic cells. The data has indicated, in the absence of behavioral seizures, factors that governed a shift toward small calibre axons at P16 have persisted in adult Scn1a heterozygote KO corpus callosum. The pilot study provides a basis for future meta-analysis that will enable robust estimates of the effects of the sodium channelopathy on axon architecture. We propose that early therapeutic strategies in DS could help minimize the effect of sodium channelopathies, beyond the impact of overt seizures, and therefore achieve better long-term treatment outcomes.

Introduction

An enduring question in clinical and basic genetic epilepsy research is whether there are structural changes in the brain antecedent to seizures (Reid et al., 2018). Structural analyses of genetic epilepsy models in seizure naïve mice suggest neurodevelopmental modifications exist including cell position and number within the cortex and hippocampus (Richards et al., 2013, Wimmer et al., 2015). The ability to identify any early neurodevelopmental changes in severe and intractable monogenic epilepsy disorders, like Dravet syndrome, would help to direct treatment strategies and their timing to achieve better therapeutic outcomes.

Dravet syndrome (DS) is a developmental and epileptic encephalopathy with disease onset in infancy. Over 85% of DS patients carry a missense mutation in the SCN1A gene, which encodes the voltage-gated sodium channel, alpha-subunit (NaV1.1). The NaV1.1 protein is mainly expressed at the axon initial segment of GABAergic interneurons, where acute loss-of-function in the inhibitory neurons is the mechanism of seizure genesis in animal models (Cheah et al., 2012, Ogiwara et al., 2007, Yu et al., 2006).

Magnetic resonance imaging (MRI) analyses of brain structure in children first diagnosed with DS suggest no evidence of abnormalities (Brunklaus et al., 2012, Lee et al., 2017). However, as severe seizures persist and comorbidities emerge, including developmental delay, cognitive dysfunction and ataxia, MRI anomalies are evident. Neuropathology ranges from subtle irregularities to more severe hippocampal sclerosis and decreased white and gray matter volumes (Catarino et al., 2011, Lee et al., 2017, Skjei et al., 2015). Limited histological studies in DS patients suggest circuit changes (Skjei et al., 2015), yet others report no clear markers of neuropathology (Catarino et al., 2011). To date, micro-circuit analysis of Scn1a heterozygote KO mice suggests altered circuitry of the cerebellum linked to motor deficits or ataxia (Kalume et al., 2007) and reduced dendritic arborization in the hippocampus, which is thought to contribute to cognitive deficits (Tsai et al., 2015). However, these results, which suggest altered micro-circuitry are confounded by the effect of recurring seizures.

In contrast to conventional structural MRI, MRI diffusion-weighted imaging (DWI) provides unique information by exploiting the interaction between diffusing water molecules and tissue microstructure. DWI has been used to compute Apparent Fiber Density (AFD) (Raffelt et al., 2012), a quantitative measure proportional to the intra-cellular volume of axons (i.e. axonal density). Unlike voxel-averaged metrics, each AFD measurement can be associated with specific populations of fibers within any voxel, even in image voxels containing multiple fiber populations, which are found in up to 90% of white matter voxels (Jeurissen et al., 2013). In a study involving whole-brain analysis of AFD in patients with Dravet syndrome (Raffelt et al., 2014), a substantial overall reduction was observed in the number of axons in many tracts throughout the white matter in Dravet syndrome compared with control, including extensive abnormalities in the corticospinal tract and mid body of the corpus callosum. The latter may reflect the mild pyramidal signs such as a crouch gait that emerge as a child with Dravet syndrome goes into puberty.

The evidence of widespread changes in white matter structure in Dravet syndrome patients, including the corpus callosum, lead us to hypothesize that the SCN1A variant could impact mammalian brain structure, not only through acute interneuron dysfunction, but enduring neurodevelopmental changes including modifying long-range connections (Flores and Mendez, 2014, Le Magueresse and Monyer, 2013). In this study, our aim was to assess neuron architecture in a DS mouse model in the developing versus the mature brain. We used electron microscopy to examine axon microstructure of the corpus callosum in Scn1a heterozygote KO (knockout); a model carrying a mutation identified in a subset of DS patients (Ogiwara et al., 2007). Our intention was to use qualitative and quantitative assessment of axon morphology done prior to the onset of overt or behavioral seizures at post-natal day 16 (P16) and in a group of P50 animals where behavioral seizures did not manifest, with each group compared to age-matched, wild type littermates.

Section snippets

Average body weight (grams) at P16 and P50

Animal weights were compared for the two age groups and were found to be similar at P16 for both genotypes. At P50 there was no significant difference in body weight between genotypes.

For the mice analyzed at P16, average body weights were (WT, 8.27 g ± 0.07, N = 2; KO, 8.35 g ± 0.01, N = 2). At P50, average body weights were not significantly different for wild type (WT) compared to Scn1a heterozygote (KO) littermates (WT, 26.65 g ± 1.02, N = 4; KO, 24.5 g ± 1.64, N = 4; p = 0.31).

SEM qualitative analysis at P16

Qualitative

Discussion

We present evidence of changes in callosal axon growth in a DS mouse model. Compared to control mice, the average cross-section axon area in adult Scn1a heterozygote KO was reduced and small caliber fibers were disproportionately higher. Preliminary analyses of the premature brain in the DS model suggest changes to axon growth and myelination may commence during early neurodevelopment. At P16, the majority of axons were unmyelinated which is consistent with myelinogenesis in developing rodent

Animals

Only male mice were used in this study and were housed in a temperature controlled, 12 h day/night light cycle environment with ad libitum access to food and water. All experiments were approved by the Florey Institute of Neuroscience and Mental Health Animal Ethics Committee (AEC# 13-097) and conducted in accordance with guidelines from National Health and Medical Research Council. Scn1a (R1407X) DS mice were maintained on 129S1/SvImJ and crossed to C57Bl/6J (N1) background for experiments.

CRediT authorship contribution statement

Kay Richards: Conceptualization, Investigation, Formal analysis, Writing - review & editing. Nikola Jancovski: Investigation, Formal analysis, Writing - review & editing. Eric Hanssen: Investigation, Writing - review & editing. Alan Connelly: Conceptualization, Writing - review & editing. Steve Petrou: Conceptualization, Writing - review & editing, Supervision.

Acknowledgements

We thank Associate Professor Finch, Deputy Director, Statistical Consulting Centre, Melbourne Statistical Consulting Platform, University of Melbourne, for assistance. We thank Prof. Kazuhiro Yamakawa (RIKEN Brain Science Institute) for donating Dravet syndrome mice. The Florey Institute of Neuroscience and Mental Health is supported by infrastructure funds from the Department of Health, State Government of Victoria.

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