Data on the effects of Charcot-Marie-Tooth disease type 2N-associated AARS missense mutation (Arg329-to-His) on the cell biological properties

Charcot-Marie-Tooth (CMT) diseases are genetic neuropathies in the peripheral nervous system (PNS). Type 1 CMT diseases are neuropathies in Schwann cells, PNS myelinating glial cells, whereas type 2 CMT diseases are axonal neuropathies. In addition, there are other types of categories in CMT diseases. CMT diseases are associated with approximately 100 responsible genes. Taiwanese mutation (Asn71-to-Tyr) of alanyl-tRNA synthetase (AARS) in type 2N CMT disease has been reported to have several pathological effects on properties of AARS proteins themselves [1]. Also, some mutations in other responsible genes affect cell biological properties of their gene products [2,3]. Herein we provide the data regarding the effects of another type 2N CMT disease-associated AARS mutation (Arg329-to-His) in French family on the cellular properties.


Data
The data shared in this article provide immunofluorescent and microscopic analyses of type 2N CMT disease-associated AARS mutant proteins (Arg329-to-His) for AARS protein localization and cellular differentiation. This position of the mutation in French family [1e4] is different from the Asn71-to-Tyr mutation in Taiwanese family [5]. Fig. 1 describes cytoplasmic localization of GFP-tagged wild type AARS proteins and intracellular punctate localization of GFP-tagged AARS mutant proteins in COS-7 cells. In Figs. 2e4, GFP-tagged AARS mutant proteins are co-stained with antibodies against antigens of the endoplasmic reticulum (ER), Golgi body, and lysosome, respectively. Mutant proteins are partially co-localized with Golgi and lysosomal antigens (Figs. 3 and 4). Additionally, parental neuronal N1E-115 cell line exhibits differentiated phenotypes with long processes whereas cells stably harboring mutant constructs exhibit decreased differentiated ones (Fig. 5).

Value of the data
This data set is of value to the scientific community to need the information for the effects of various types of Charcot-Marie-Tooth disease-associated mutations on cell biological changes. The data can provide the method to examine changes of the properties of gene products by Charcot-Marie-Tooth diseaseassociated mutations.
The data allow us to promote how various Charcot-Marie-Tooth disease type 2-associated mutations have similar effects in vitro.
template, using the site-directed mutagenesis kit (TOYOBO Life Science, Osaka, Japan), according to the manufacturer's instructions. All DNA sequences were confirmed by sequencing (Fasmac, Kanagawa, Japan).

Cell culture, differentiation, and transfection
African green monkey epithelial-like COS-7cells (Human Health Science Research Resources Bank, Osaka, Japan) and mouse neuroblastoma N1E-115 cells (kindly provided by Dr. Daisuke Shiokawa, Tokyo Science University, Chiba, Japan) were cultured on 3.5-cm cell culture dishes (Greiner, Ober€ osterreich, Germany) with or without a coverslip in DMEM (Nakalai Tesque, Kyoto, Japan) containing 10% heat-inactivated FBS (Thermo Fisher Scientific, Waltham, MA, USA) and PenStrep (Thermo Fisher Scientific) in 5% CO 2 at 37 C. Cells were transfected with the plasmids using the ScreenFect A or ScreenFect A Plus transfection kit (FujiFilm, Tokyo, Japan), according to the manufacturers' instruction. The medium was replaced 4h after transfection. Transfected cells were used for experiments 48h after transfection. To induce differentiation of N1E-115 cells, cells were cultured in DMEM containing PenStrep in 5% CO 2 at 37 C for 5 days. Cells harboring processes longer than one-cell-body length were estimated as differentiated cells [1].

Stable clone isolation
For isolation of N1E-115 cells stably harboring AARS (Arg329-to-His), cells were transfected with pEGFP-N3-AARS (Arg329-to-His). Growth medium containing 500 mg/ml G418 (Nacalai Tesque) was changed every 2 or 3 days, according to the manufacturer's instructions. After 14 days, G418-resistant colonies were collected and compared with phenotypes of their control parental cells.

Immunofluorescence
Cells on a coverslip were fixed with 4% paraformaldehyde (Nacalai Tesque) or 100% cold methanol (Nacalai Tesque). Cells were blocked with the Blocking One reagent (Nacalai Tesque) and incubated  [6]. The fluorescent TIFF images were collected with a microscope system equipped with a laser-scanning Fluoview apparatus (Olympus, Tokyo, Japan) using Fluoview software (Olympus). Their resulting colored images were analyzed in the line plot analysis mode using the Image J software (URL: https://imagej.nih.gov/).