Laboratory Studies
Hypomyelinating leukodystrophy-associated missense mutant of FAM126A/hyccin/DRCTNNB1A aggregates in the endoplasmic reticulum

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Abstract

Hypomyelinating leukodystrophies (HLD) are hereditary central nervous system diseases in which the myelin sheath does not form properly. The disease prototype is the X-linked recessive Pelizaeus-Merzbacher disease (also now known as HLD1), which is caused by the mutation, multiplication, or deletion of the plp1 gene. PLP1 missense mutations lead to protein aggregation and accumulation in subcellular compartments such as the endoplasmic reticulum (ER). The gene responsible for an autosomal recessive Pelizaeus-Merzbacher-like disease called HLD5 is named fam126a (also known as hyccin or drctnnb1a). While the gene mutations often cause FAM126A protein deficiency, one known missense mutation, Leu-53-to-Pro (L53P), allows some protein to be produced. Here, we show that the L53P mutant aggregates in cells, accumulating primarily in the ER. This is in contrast to the wild type FAM126A, which distributes throughout the cytoplasm. In addition, the L53P mutant expression promotes the activities of kinases involved in unfolded protein response. These results suggest that a disease-associated FAM126A missense mutation causes protein accumulation in subcellular compartments, possibly to mediate a disease-associated phenotype, which is similar to what is seen with PLP1.

Introduction

The myelin sheath is a unique multi-layer structure that acts as an insulator between surrounding axons [1]. The myelin sheath is derived from myelin-forming glial cells, which are called oligodendrocytes in the central nervous system (CNS) and Schwann cells in the peripheral nervous system. Over time, the myelin sheaths grow to more than one hundred times larger than the collective surface area of the premyelinating oligodendrocyte or Schwann cell plasma membranes [1].

Pelizaeus-Merzbacher disease is the prototypic hereditary hypomyelinating leukodystrophy (HLD) in oligodendrocytes [1], [2], [3], [4], [5], [6], [7], [8]. In this disease (known as HLD1), missense PLP1 mutations generally cause protein aggregation, which results in accumulation of mutant PLP1 proteins in some subcellular compartments and triggers an unfolded protein response (UPR) [1], [2], [3], [4], [5], [6], [7], [8]. Recent studies have identified other genes responsible for HLD (HLD2, HLD4, and HLD5) [9]. HLD5 (Mendelian Inheritance in Man number 610532) is a recessive disorder that causes hypomyelination and cataracts [10], [11], [12]. In most cases, HLD5 is caused by premature stop signal causing mutations in the fam126a (also known as hyccin or drctnnb1a) gene and FAM126A protein deficiency leads to neurological phenotypes such as intellectual disabilities and walking difficulties [10], [11], [12]. One unique fam126a gene mutation, which results in Leu-53-to-Pro (L53P), allows the FAM126A protein to be produced [10]. The aim of this study was to investigate whether this unique missense mutation changes the biochemical properties of FAM126A. We show, to our knowledge, for the first time, that the L53P mutation causes protein aggregation, similar to the missense mutations of PLP1. The protein accumulation primarily occurs in the endoplasmic reticulum (ER), stimulating UPR-responsible kinase activities.

Section snippets

Antibodies

The following antibodies were purchased: anti-calnexin (ER membrane marker protein) from Enzo Life Sciences (Farmingdale, NY, USA); anti-Golgi matrix protein (GM) 130 from BD Biosciences Pharmingen (Franklin Lake, NJ, USA); anti-Lamp1 (lysosome marker protein) from Abcam (Cambridge, UK); anti-FLAG from Sigma-Aldrich (St. Louis, MO, USA); anti-eukaryotic translation initiation factor 2α subunit (eIF2α), anti-(pSer51) eIF2α (antibody specifically recognizing eIF2α phosphorylation through UPR),

Expression of FAM126A L53P mutant in Cos-7 cells

The purpose of this study was to examine the effect of fam126a gene missense mutation on FAM126A protein properties. To investigate whether the L53P mutation of FAM126A protein has an effect on cellular localization, we transfected EGFP-tagged wild type or the L53P mutant construct into Cos-7 cells. Cos cell lines are widely utilized to determine the subcellular localization of various proteins, since they have large cell bodies [7], [8], [15]. We first inserted EGFP-tag into the N-terminal

Discussion

While recent studies of hereditary hypomyelinating diseases in the CNS identify a number of genes other than plp1 as disease-responsible genes with various mutations, comparatively less is known about the effect of gene mutations on the biochemical properties of their products [9]. In HLD5, fam126a mutations mostly result in a deficiency of FAM126A protein expression. It is thus likely that loss of function of FAM126A is often responsible for pathogenesis of HLD5 [9], [10], [11], [12].

Conflicts of Interest/Disclosures

The authors declare that they have no financial or other conflicts of interest in relation to this research and its publication.

Acknowledgements

We thank Drs. J.R. Chan, N. Kitamura, and E.M. Shooter for helpful discussions. This work was supported by Grants-in-Aid for Scientific Research from the Japanese Ministry of Education, Culture, Sports, Science, and Technology (MEXT) and the Japanese Ministry of Health, Labour, and Welfare (MHLW). This work was also partially supported by grants from the Kanehara Foundation, the Kowa Foundation, the Mochida Foundation, the Naito Foundation, and the Takeda Foundation.

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