Hexb enzyme deficiency leads to lysosomal abnormalities in radial glia and microglia in zebrafish brain development

Kuil, Laura E.; Martí, Anna López; Mascaro, Ana Carreras; Bosch, Jeroen C. van den; Berg, Paul van den; Linde, Herma C. van der; Schoonderwoerd, Kees; Ruijter, George J. G.; Ham, Tjakko J. van

doi:10.1002/glia.23641

Cited by 33 publications

(62 citation statements)

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“…Finally, to model the consequences of the lack of UGP2 in vivo , we generated zebrafish mutants for both ugp2a and ugp2b , the zebrafish homologs of UGP2 , using CRISPR-Cas9 injections in fertilized oocytes in a background of a radial glia/neural stem cell reporter 76 . Double homozygous mutant lines having frameshift deletions for both genes confirmed by Sanger sequencing could be generated but the only viable combination, obtained with ugp2a loss, created a novel ATG in exon 2 of ugp2b, leading to a hypomorphic allele ( Figure 6A ).…”

Section: Resultsmentioning

confidence: 99%

“…For live imaging, the medium was changed at 1 dpf to E3 + 0.003% 1-phenyl 2-thiourea (PTU) to prevent pigmentation. Ugp2a and ugp2b were targeted by Cas9/gRNA RNP-complex as we did before 76 . Briefly, fertilized oocytes from a tgBAC(slc1a2b:Citrine)re01tg reporter line 76 maintained on an TL background strain were obtained, and injected with Cas9 protein and crRNA and tracrRNA synthesized by IDT (Alt-R CRISPR-Cas9 System), targeting the open reading frame of zebrafish ugp2a and ugp2b .…”

Section: Methodsmentioning

confidence: 99%

“…Ugp2a and ugp2b were targeted by Cas9/gRNA RNP-complex as we did before 76 . Briefly, fertilized oocytes from a tgBAC(slc1a2b:Citrine)re01tg reporter line 76 maintained on an TL background strain were obtained, and injected with Cas9 protein and crRNA and tracrRNA synthesized by IDT (Alt-R CRISPR-Cas9 System), targeting the open reading frame of zebrafish ugp2a and ugp2b . DNA was extracted from fin clips and used for genotyping using primers flanking the gRNA location ( Supplementary Table 8 ) followed by sequencing.…”

Section: Methodsmentioning

confidence: 99%

“…In this study, we describe two new mutant fish lines containing deletions in ugp2a ( ugp2a Δ/Δ ) and ugp2b ( ugp2b Δ/Δ ): ugp2a re08/re08 containing a 37 bp deletion in exon 2 and ugp2b re09/re09 containing a 5 bp deletion in exon 2. Intravital imaging, and analysis of eye movement, was performed as previously described 76 . Briefly, zebrafish larvae anesthetized in tricaine were mounted in low melting point agarose containing tricaine and imaged using a Leica SP5 intravital imaging setup with a 20×/1.0 NA water-dipping lens.…”

Section: Methodsmentioning

confidence: 99%

“…Briefly, zebrafish larvae anesthetized in tricaine were mounted in low melting point agarose containing tricaine and imaged using a Leica SP5 intravital imaging setup with a 20×/1.0 NA water-dipping lens. To assess the locomotor activity of zebrafish larvae from 3 to 5 dpf, locomotor activity assays were performed using an infrared camera system (DanioVision™ Observation chamber, Noldus) and using EthoVision® XT software (Noldus) as described 76 . Briefly, control ( n = 24) and ugp2a Δ/Δ ; ugp2b Δ/Δ ( n = 24) zebrafish larvae, in 48 well plates, were subjected to gradually increasing (to bright light) and decreasing light conditions (darkness) as in Kuil et al 76 .…”

Section: Methodsmentioning

confidence: 99%

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Loss of UGP2 in brain leads to a severe epileptic encephalopathy, emphasizing that bi-allelic isoform specific start-loss mutations of essential genes can cause genetic diseases

Perenthaler

Nikoncuk

Yousefi

et al. 2019

Preprint

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2 52 Running title: Loss of UGP2 causes a severe epileptic encephalopathy 53 54 55 Abstract: 60 Developmental and/or epileptic encephalopathies (DEEs) are a group of devastating genetic 61 disorders, resulting in early onset, therapy resistant seizures and developmental delay. Here we 62 report on 12 individuals from 10 families presenting with a severe form of intractable epilepsy, 63 severe developmental delay, progressive microcephaly and visual disturbance. Whole exome 64 sequencing identified a recurrent, homozygous variant (chr2:64083454A>G) in the essential UDP-65 glucose pyrophosphorylase (UGP2) gene in all probands. This rare variant results in a tolerable 66Met12Val missense change of the longer UGP2 protein isoform but causes a disruption of the start 67 codon of the shorter isoform. We show that the absence of the shorter isoform leads to a reduction 68 of functional UGP2 enzyme in brain cell types, leading to altered glycogen metabolism, upregulated 69unfolded protein response and premature neuronal differentiation, as modelled during pluripotent 70 stem cell differentiation in vitro. In contrast, the complete lack of all UGP2 isoforms leads to 71 differentiation defects in multiple lineages in human cells. Reduced expression of Ugp2a/Ugp2b in 72 vivo in zebrafish mimics visual disturbance and mutant animals show a behavioral phenotype. Our 73 study identifies a recurrent start codon mutation in UGP2 as a cause of a novel autosomal recessive 74 DEE. Importantly, it also shows that isoform specific start-loss mutations causing expression loss of a 75 tissue relevant isoform of an essential protein can cause a genetic disease, even when an organism-76 wide protein absence is incompatible with life. We provide additional examples where a similar 77 disease mechanism applies. 78 79 80 81 82 83 84 85 86 87 3 Introduction: 88 Developmental and/or epileptic encephalopathies (DEEs) are a heterogeneous group of genetic 89 disorders, characterized by severe epileptic seizures in combination with developmental delay or 90 regression 1 . Genes involved in multiple pathophysiological pathways have been implicated in DEEs, 91 including synaptic impairment, ion channel alterations, transporter defects and metabolic processes 92 such as disorders of glycosylation 2 . Mostly, dominant acting, de novo mutations have been identified 93 in children suffering from DEEs 3 , and only a limited number of genes with a recessive mode of 94 inheritance are known so far, with a higher occurrence rate in consanguineous populations 4 . A recent 95 cohort study on DEEs employing whole exome sequencing (WES) and copy-number analysis, 96however, found that up to 38% of diagnosed cases might be caused by recessive genes, indicating 97 that the importance of this mode of inheritance in DEEs has been underestimated 5 . 98

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%