Familial Dysautonomia (FD) Human Embryonic Stem Cell Derived PNS Neurons Reveal that Synaptic Vesicular and Neuronal Transport Genes Are Directly or Indirectly Affected by IKBKAP Downregulation.

Sharon Lefler,Tikva Turetsky,Nibal Bashir,Benjamim E Reubinoff,Channa Maayan,Malkiel A Cohen,Aviel Even,Miguel Weil,Anastasya Birger,Sharona Even-Ram,Gal Kantor,Aharon Razin,David Cheishvili,Einat Aizenman,Yaniv Gil,Junming Yue

doi:10.1371/journal.pone.0138807

Sharon Lefler, Tikva Turetsky + Show 14 more

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https://doi.org/10.1371/journal.pone.0138807

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Abstract

A splicing mutation in the IKBKAP gene causes Familial Dysautonomia (FD), affecting the IKAP protein expression levels and proper development and function of the peripheral nervous system (PNS). Here we found new molecular insights for the IKAP role and the impact of the FD mutation in the human PNS lineage by using a novel and unique human embryonic stem cell (hESC) line homozygous to the FD mutation originated by pre implantation genetic diagnosis (PGD) analysis. We found that IKBKAP downregulation during PNS differentiation affects normal migration in FD-hESC derived neural crest cells (NCC) while at later stages the PNS neurons show reduced intracellular colocalization between vesicular proteins and IKAP. Comparative wide transcriptome analysis of FD and WT hESC-derived neurons together with the analysis of human brains from FD and WT 12 weeks old embryos and experimental validation of the results confirmed that synaptic vesicular and neuronal transport genes are directly or indirectly affected by IKBKAP downregulation in FD neurons. Moreover we show that kinetin (a drug that corrects IKBKAP alternative splicing) promotes the recovery of IKAP expression and these IKAP functional associated genes identified in the study. Altogether, these results support the view that IKAP might be a vesicular like protein that might be involved in neuronal transport in hESC derived PNS neurons. This function seems to be mostly affected in FD-hESC derived PNS neurons probably reflecting some PNS neuronal dysfunction observed in FD.

Highlights

Familial Dysautonomia (FD; Riley-Day syndrome, hereditary sensory and autonomic neuropathy type III) is an autosomal recessive congenital neuropathy that affects the peripheral nervous system (PNS) [1, 2]
Our previous study suggests that IKAP/human elongator protein-1 (hELP1) expression is much higher in peripheral neurons derived from human embryonic stem cells compared to primary fibroblasts [22], and fibroblasts do not exhibit the same ratio of IKBKAP exon 20 including: exon 20 skipping transcripts as observed in nervous system-derived tissues of FD patients [5]
We characterized the differentiation of the FD human embryonic stem cells (hESC) into homogenous neural crest cells (NCC) population, following an established protocol [38] as depicted in S4 Fig. While in these experiments we could not observe any differences in the efficiency of FD cells to differentiate to NCC, we have noticed that the delamination from plated human neural precursor clusters and migration of FD-NCC was reduced in comparison to wild type (WT)-NCC (Fig 1A)

Summary

Introduction

Familial Dysautonomia (FD; Riley-Day syndrome, hereditary sensory and autonomic neuropathy type III) is an autosomal recessive congenital neuropathy that affects the PNS [1, 2]. Our previous study suggests that IKAP/hELP1 expression is much higher in peripheral neurons derived from human embryonic stem cells (hESC) compared to primary fibroblasts [22], and fibroblasts do not exhibit the same ratio of IKBKAP exon 20 including: exon 20 skipping transcripts as observed in nervous system-derived tissues of FD patients [5]. This finding narrows the understanding of disease mechanisms in FD down to human cellular models where neural cells can be generated. These two FD stem cell models were used for validating the potency of therapeutic agents such as kinetin, a cytokinin that has been shown to increase IKBKAP mRNA and protein expression in FD cell lines and in vivo models [25,26,27] as well as in leukocytes of healthy carriers of the FD mutation [28]

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