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Original Article

Exp Neurobiol 2018; 27(6): 550-563

Published online December 12, 2018

© The Korean Society for Brain and Neural Sciences

A De NovoRAPGEF2 Variant Identified in a Sporadic Amyotrophic Lateral Sclerosis Patient Impairs Microtubule Stability and Axonal Mitochondria Distribution

Keunjung Heo1,†, Su Min Lim2,†, Minyeop Nahm2,†, Young-Eun Kim3, Ki-Wook Oh2, Hwan Tae Park4, Chang-Seok Ki5*, Seung Hyun Kim2*, and Seungbok Lee1*

1Department of Brain and Cognitive Sciences and Dental Research Institute, Seoul National University, Seoul 08826, Korea.

2Department of Neurology, College of Medicine, Hanyang University, Seoul 04763, Korea.

3Department of Laboratory Medicine, College of Medicine, Hanyang University, Seoul 04763, Korea.

4Department of Molecular Neuroscience, College of Medicine, Dong-A University, Busan 49201, Korea.

5Green Cross Genome Corporation, Yongin 16924, Korea.

Correspondence to: *To whom correspondence should be addressed.
Chang-Seok Ki, TEL: 82-31-260-0601, FAX: 82-31-260-9087, e-mail:
Seung Hyun Kim, TEL: 82-2-2290-8371, FAX: 82-2-2296-8370, e-mail:
Seungbok Lee, TEL: 82-2-880-2330, FAX: 82-2-762-2583, e-mail:
These authors contributed equally to this work.

Received: October 31, 2018; Revised: November 24, 2018; Accepted: November 26, 2018

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that is frequently linked to microtubule abnormalities and mitochondrial trafficking defects. Whole exome sequencing (WES) of patient-parent trios has proven to be an efficient strategy for identifying rare de novo genetic variants responsible for sporadic ALS (sALS). Using a trio-WES approach, we identified a de novoRAPGEF2 variant (c.4069G>A, p.E1357K) in a patient with early-onset sALS. To assess the pathogenic effects of this variant, we have used patient-derived skin fibroblasts and motor neuron-specific overexpression of the RAPGEF2-E1357K mutant protein in Drosophila. Patient fibroblasts display reduced microtubule stability and defective microtubule network morphology. The intracellular distribution, ultrastructure, and function of mitochondria are also impaired in patient cells. Overexpression of the RAPGEF2 mutant in Drosophila motor neurons reduces the stability of axonal microtubules and disrupts the distribution of mitochondria to distal axons and neuromuscular junction (NMJ) synapses. We also show that the recruitment of the pro-apoptotic protein BCL2-associated X (BAX) to mitochondria is significantly increased in patient fibroblasts compared with control cells. Finally, increasing microtubule stability through pharmacological inhibition of histone deacetylase 6 (HDAC6) rescues defects in the intracellular distribution of mitochondria and BAX. Overall, our data suggest that the RAPGEF2 variant identified in this study can drive ALS-related pathogenic effects through microtubule dysregulation.

Keywords: Amyotrophic lateral sclerosis, Whole exome sequencing, RAPGEF2, Missense mutation, Microtubules, Mitochondria

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease that affects both upper and lower motor neurons, leading to muscle weakness and atrophy followed by paralysis [1,2]. ALS is usually fatal due to respiratory failure within 5 years after symptom onset and represents the most common form of adult-onset motor neuron diseases with an incidence of 2 per 100,000. Approximately 10% of cases show familial inheritance, while the remaining majority of cases occur sporadically. Over the last two decades, substantial progress has been made in understanding of the genetic landscape of familial ALS (fALS). To date, two-thirds of fALS are associated with mutations in any of more than 25 genes [3,4,5], encoding proteins involved in protein homeostasis, RNA metabolism, vesicular trafficking, and cytoskeletal organization. Despite this progress in identifying fALS-associated genes, the genetic etiology of sporadic ALS (sALS) remains largely unknown [3].

Impaired mitochondrial trafficking in motor neurons is a well-established phenomenon in ALS pathophysiology [6]. Electron microscopic studies of post-mortem ALS cases demonstrated remarkable accumulation of mitochondria in the somata and proximal axons of motor axons in the spinal cord [7]. Consistently, abnormal clustering of mitochondria in proximal axons of motor neurons was also observed in transgenic mice and rats expressing the ALS mutant SOD1-G93A [8,9]. Similar defects in mitochondrial distribution were also observed in motor neurons from transgenic mice expressing ALS-associated TDP-43 mutants [10,11]. However, the underlying mechanisms of mitochondrial trafficking defects in ALS remain to be fully understood.

Whole exome sequencing (WES) has significantly contributed to our knowledge of ALS genetics. First, WES studies on ALS families identified novel pathogenic variants in known fALS genes, including SOD1, SPG11, and UBQLN2 [12,13,14]. Second, the WES analysis of ALS families or in large ALS cohorts discovered new fALS genes such as VCP, PFN1, hnRNPA1, NEK1, and TBK1 [15,16,17,18,19]. Finally, a previous WES-based study on family trios (the affected person and his/her parents) identified a de novo variant in the new sALS gene SS18L1 (also known as CREST) that was subsequently found to be associated with two fALS cases [20,21], demonstrating the power of WES to identify new ALS-associated genes.

In this study, we employed a trio-WES approach to identify pathogenic variants in a sALS patient and detected a de novo missense variant (c.4069G>A, p.E1357K) in the RAPGEF2 gene. Our data obtained from patient-derived fibroblasts and a Drosophila model expressing the mutant protein suggests that the RAPGEF2 variant identified in this study impairs mitochondrial distribution and morphology through microtubule dysregulation, which has been increasingly recognized as a core component of ALS pathogenesis [22].

Subjects, exome sequencing, and genetic variant analysis

The 28-year-old female patient and her healthy parents provided written informed consent as approved by the Institutional Review Boards of Hanyang University Hospital (Seoul, Korea). Genomic DNA was isolated from peripheral blood leukocytes using the Wizard Genomic DNA Purification kit (Promega, Madison, WI, USA). The exomes of subjects were captured using the Agilent SureSelect all Exon 50Mb kit (Agilent, Santa Clara, CA, USA) and sequenced on an Illumina NextSeq500 machine (paired-end and 100-bp reads) (Illumina, San Diego, CA, USA). Reads were mapped to a custom GRCh37/hg19 build using the Burrows-Wheeler Aligner (BWA). Annotation was performed using an in-house custom-made script. We selected rare variants with allele frequency less than 0.01 identified in the NHLBI Exome Sequencing Project (, the 1000 Genomes Project (, and gnomAD ( All amino acid-altering de novo variants with filtering criteria were validated by Sanger sequencing on DNA samples from the family trio using primers designed by the authors.

Molecular biology and cell culture

Full-length cDNAs for RAPGEF2 and Bcl-2-associated X protein (BAX) were PCR-amplified from total human cDNAs and cloned into the pEGFP-C1 vector (Clontech, Mountain View, CA, USA) to generate pEGFP-RAPGEF2 and pEGFP-BAX. The E1357K mutation was introduced into pEGFP-RAPGEF2 by PCR-mediated mutagenesis to produce pEGFP-RAPGEF2-E1357K.

Primary human fibroblasts were established from punch biopsies on the forearm skin of the patient and a 43-year-old male control as described previously [23] and maintained in Dulbecco's modified Eagle's medium (DMEM) containing 20% heat-inactivated (30 min, 55℃) fetal bovine serum (FBS), 1% non-essential amino acids, and antibiotics. Passage-matched control and patient fibroblasts (prior to passage 10) were used in each experiment. For inhibition of HDAC6, human skin fibroblasts were treated with 1 µM tubastatin A (Sigma-Aldrich, St. Louis, MI, USA) overnight at 37℃. Human HeLa cells were maintained in DMEM supplemented with 10% heat-inactivated FBS and transfected using FuGENE HD transfection reagent (Promega, Madison, WI, USA).

Fly strains

Flies were maintained at 25℃ on standard food. Transgenic D42-GAL4 and D42-GAL4, UAS-mito-HA-GFP flies were obtained from the Bloomington Stock Center (Bloomington, IN, USA). The UAS-HA-RAPGEF2-WT and UAS-HA-RAPGEF2-E1357K transgenes were produced in the w1118 background by standard germline transformation. The RNAi line PDZ-GEFKK102612 (referred to here as to UAS-gef26RNAi) was obtained from the Vienna Drosophila Resources Center (Vienna, Austria).

Immunostaining, imaging, and quantification

Cultured cells were fixed with 4% formaldehyde in PBS for 20 min at room temperature, permeabilized with 0.2% Triton X-100 in PBS for 10 min, and blocked with 1% BSA in PBS for 1 h. Samples were then incubated with primary antibodies for 1 h and sequentially incubated with fluorescently labeled secondary antibodies for 30 min at room temperature. Wandering third-instar Drosophila larvae were dissected in Ca2+-free HL3 saline, fixed with 4% formaldehyde in PBS for 20 min, and immunostained as previously described [24]. The following primary antibodies were used in this study: anti-mitochondria (1:1000; Millipore, Burlington, MA, USA), anti-acetylated α-tubulin (1:500; Sigma-Aldrich), anti-tyrosinated α-tubulin (1:500; Millipore, Bedford, MA, USA), anti-α-tubulin (1:500; Sigma-Aldrich), and anti-Futsch (1:50; DSHB, Iowa City, IA, USA). The FITC-, Cy3-, and Cy5-conjugated secondary antibodies were obtained from Jackson ImmunoResearch (1:200; West Grove, PA, USA).

Fluorescent images were acquired with an LSM 700 laser-scanning confocal microscope using a C Apo 40x W or Plan Apo 63x 1.4 NA objective (Carl Zeiss, Jena, Germany). The length of Mito-RFP-labeled mitochondria in fibroblasts was determined using ImageJ. For quantification of the number and area of mitochondria in Drosophila motor neurons, we acquired confocal images of A5 segment in dissected third instar larvae labeled with anti-GFP (for Mito-GFP) and anti-HRP antibodies. The number and area of Mito-GFP puncta normalized to the respective area were also determined using ImageJ as previously described [25].

Adult behavioral analysis

A climbing test was used to assess the locomotor function of adult flies as previously described [24]. Briefly, 45 adult flies aged for 20 days were transferred into a glass graduated cylinder. Following 5-min acclimation, flies were gently tapped to the bottom and the distance climbed by individual flies in a 30 s period was measured.

Western blot analysis

Primary human skin fibroblasts were homogenized in ice-cold lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% Triton X-100, and protease inhibitors) and subjected to western blotting as previously described [23]. For some experiments, we separated the mitochondrial and cytosolic fractions from fibroblast lysates using the BioVision Mitochondria/Cytosol Fractionation kit (BioVision, Milpitas, CA, USA). The following primary antibodies were used: anti-acetylated α-tubulin (1:1000; Sigma-Aldrich), anti-tyrosinated α-tubulin (1:1000; Millipore), anti-α-tubulin (1:1000; Sigma-Aldrich), anti-BAX (1:1000; BD, Franklin Lakes, NJ, USA), and anti-GAPDH (1:1000; Santa Cruz Biotechnology, Dallas, TX, USA).

Analysis of mitochondrial membrane potential

The mitochondrial membrane potential was assessed in live primary fibroblasts using 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide (JC-1; Sigma-Aldrich) as described [26]. Briefly, fibroblasts were washed and incubated with 5 µg/ml JC-1 dye for 20 min at 37℃. The cells were then rinsed with culture medium, and their images were obtained using the Applied Precision DeltaVision fluorescence microscopy system (GE Healthcare, Chicago, IL, USA). JC-1 accumulates as red fluorescent aggregates within polarized mitochondria but does as green fluorescent monomers within less polarized mitochondria.

Electron microscopy

Primary human skin fibroblasts were fixed in PBS containing 4% paraformaldehyde and 2.5% glutaraldehyde for 24 h and rinsed in PBS. The samples were then subjected to 70-nm sectioning after gradual dehydration in ethanol solutions and propylene oxide (Acros Organics, Morris Plains, NJ, USA), and stained with epoxy resins using standard procedures. Images were acquired with a Hitachi electron microscope (Hitachi, Tokyo, Japan) equipped with a ES500W digital camera (GATAN, Pleasanton, CA, USA).

Statistical analysis

Comparisons were made by one-way ANOVA analysis with a post-hoc Turkey test. Data are presented as mean±SEM.

Identification of a de novo variant in the RAPGEF2 gene in a sALS patient

While performing WES on sALS trios, we identified a 28-year-old female patient carrying a de novo variant in the RAPGEF2 gene (c.4069G>A, p.E1357K) (Fig. 1A). The de novo occurrence of this variant in the patient was confirmed by Sanger sequencing (Fig. 1B). In addition, we proved no additional pathogenic variants in the most common fALS genes including C9orf72, SOD1, FUS, TARDBP, and TBK1. She developed progressive weakness first in the lower limbs and then in the upper limbs from 27 years of age. The neurologic examination at our clinic showed clinical signs of upper motor neuron involvement, mild hand wasting, and fasciculation in the lower limbs. Needle electromyography (EMG) showed widespread active denervation potentials with fasciculation in both upper and lower limbs. In addition, we observed long-duration, large-amplitude polyphasic potentials during volitional contraction, suggesting that there is chronic re-innervation. Based on the revised El Escorial criteria [27], she was diagnosed with clinically probable ALS.

The RAPGEF2 gene encodes a Rap1-specific guanine nucleotide exchange factor (GEF) that harbors an N-terminal cyclic nucleotide monophosphate-binding (cNMP) domain, a Ras-GEFN domain, a PSD-95/Dlg/ZO-1 (PDZ) domain, a Ras association (RA) domain, and a RasGEF domain, which is followed by two C-terminal low-complexity domains (LCDs). Previous studies have shown that ALS-causing mutations are frequently found in the LCD domains of RNA-binding proteins, including TDP-43, FUS, hnRNP A1, and TIA1 [28,29,30]. The RAPGEF2-E1357 residue is highly conserved among species ranging from Drosophila to vertebrates and locates within the second LCD domain (Fig. 1C).

Reduction of the stable microtubule network in patient-derived skin fibroblasts carrying the E1357K variant of RAPGEF2

Disruptions in microtubule network assembly have been proposed as a critical component of ALS pathogenesis [22]. We therefore asked whether the E1357K variant of RAPGEF2 affects microtubule dynamics or organization. To address this, we visualized microtubule networks in control- and patient-derived skin fibroblasts using antibodies against α-tubulin (detecting both free α-tubulin and microtubules), acetylated α-tubulin (detecting long-lived, stable microtubules [31]), and tyrosinated α-tubulin (detecting both free tubulin and newly formed microtubules [32]). In control cells, all of these tubulin antibodies revealed the typical microtubule pattern of an aster-like distribution extending toward the cell periphery (Fig. 2A). The levels and distribution of α-tubulin and tyrosinated α-tubulin remained unchanged in patient fibroblasts (Fig. 2A). In sharp contrast, anti-acetylated α-tubulin signals were weaker in patient fibroblasts than in control fibroblasts (Fig. 2A). In addition, acetylated α-tubulin networks were restricted only in the perinuclear region. We confirmed the selective reduction of acetylated α-tubulin in patient cells by western blotting (Fig. 2B~D). These results suggest that the E1357K variant of RAPGEF2 affects the stability of the microtubule network.

To corroborate the above conclusion, we analyzed the effect of the RAPGEF2-E1357K variant on the level of α-tubulin acetylation in HeLa cells. Levels of anti-acetylated α-tubulin signal were significantly decreased in RAPGEF2-E1357K-transfected cells compared with untransfected control cells (Fig. 2E), confirming a deleterious effect of the RAPGEF2 variant on microtubule stability.

Abnormal mitochondrial distribution in patient fibroblasts

Since appropriate mitochondrial distribution critically depends on microtubule-based transport, we investigated whether alteration of the microtubule network in patient-derived fibroblasts is paralleled with abnormalities in mitochondrial morphology and distribution. Control and patient fibroblasts were transfected with a mitochondrial matrix-localized RFP (Mito-RFP) reporter construct. In control cells, Mito-RFP signals largely appeared as tubular networks extending throughout the cytoplasm (Fig. 3A). In contrast, the Mito-RFP-labeled mitochondria networks in patient-derived cells were fragmented and more restricted around the perinuclear area (Fig. 3A). To quantify mitochondrial fragmentation, we measured mitochondrial length. Patient fibroblasts showed a reduction of about 88% in average mitochondrial length compared with control cells (Fig. 3B). However, the size of cells was comparable between both genotypes (Fig. 3C).

Next, we investigated whether the decrease in the stability of microtubules is a causative mechanism of abnormal mitochondrial distribution in patient-derived fibroblasts. Acetylation of lysine 40 in α-tubulin, an indication of microtubule stabilization, is increased by inhibiting the catalytic activity of histone deacetylase 6 (HDAC6) [33], which is the major deacetylase of α-tubulin [34]. We examined the effect of an HDAC6-selective inhibitor (tubastatin A) on mitochondrial distribution in patient fibroblasts. Treatment of fibroblasts with 1 µM tubastatin A restored abnormalities in acetylated α-tubulin distribution and mitochondrial distribution and length (Fig. 3A~C). These results suggest that impaired microtubule stability is a causative mechanism accounting for abnormal mitochondrial distribution in patient-derived fibroblasts expressing the RAPGEF2-E1357K variant.

Transgenic expression of the RAPGEF2-E1357K variant in Drosophila motor neurons impairs microtubule stability and mitochondria distribution along axons

To validate the effects of the RAPGEF2-E1357K mutant on microtubule maintenance and mitochondrial distribution in motor axons, we generated Drosophila carrying UAS transgenes of HA-tagged wild-type RAPGEF2 (UAS-HA-RAPGEF2-WT) and its E1357K mutant (UAS-HA-RAPGEF2-E1357K). We targeted expression of these transgenes in larval motor neurons using the D42-GAL4 driver. First, we performed confocal analysis on third instar larvae with antibodies against acetylated α-tubulin and Futsch, a microtubule-associated protein stabilizing microtubules [35]. Both acetylated α-tubulin and Futsch staining in motor axons and neuromuscular junction (NMJ) synapses were not altered by HA-RAPGEF2-WT overexpression (Fig. 4A~H). However, levels of both markers were significantly reduced in motor axons and NMJ synapses in larvae overexpressing HA-RAPGEF2-E1357K (Fig. 4A~H). These results suggest that expression of the RAPGEF2-E1357K mutation deleteriously impacts microtubule stability through a toxic gain-of-function mechanism rather than a simple dose-dependent mechanism. Next, we compared the distribution of Mito-GFP-labeled mitochondria in the soma and at the terminals of motor neurons in control larvae and larvae expressing HA-RAPGEF2-WT or HA-RAPGEF2-E1357K. The levels and distribution of Mito-GFP in the soma of motor neurons were not significantly different between the three genotypes (Fig. 5A). However, mitochondria number and area in motor axons were significantly reduced in HA-RAPGEF2-E1357K-expressing larvae compared with control or HA-RAPGEF2-WT-expressing larvae (Fig. 5B~E). We also observed a similar reduction in the area of mitochondria in NMJ synapses of HA-RAPGEF2-E1357K-expressing larvae (Fig. 5C and F). The aggregated distribution of mitochondria precluded comparison of mitochondria number in NMJ synapses.

We have recently shown that loss of the Drosophila RAPGEF2 homolog (Gef26) causes an increase of axonal Futsch staining [24]. To confirm this and to preclude the possibility that the E1357K mutation exerts its deleterious effect on mitochondria distribution through a loss-of-function mechanism, we depleted expression of Gef26 in motor neurons using a transgenic RNA interference (RNAi) approach. We first confirmed an increase of both acetylated α-tubulin and Futsch staining in motor axons and NMJ synapses in Gef26-knockdowned larvae (Fig. 4A~H). We found no obvious changes in the levels and distribution of mitochondria in the soma and at the terminals of motor neurons (Fig. 5A~F). These results support the notion that the RAPGEF2-E1357K mutation deleteriously impact mitochondria distribution through a toxic gain-of-function mechanism.

To test if expression of the RAPGEF2-E1357K mutant induces motor dysfunction, we performed climbing assays on adult flies at 20 days of age. Compared with transgenic controls (D42-GAL4/+), flies expressing UAS-HA-RAPGEF2-WT in motor neurons under the control of D42-GAL4(D42-GAL4/UAS-HA-RAPGEF2-WT) displayed normal climbing ability (D42-GAL4/+: 20.56±0.22 cm; D42-GAL4/UAS-HA-RAPGEF2-WT: 20.04±0.29 cm; p>0.05; Fig. 5G and H). However, age-matched flies expressing the RAPGEF2-E1357K variant made relatively short climbs (16.98±0.47 cm; p<0.01 from D42-GAL4/+; Fig. 5G and H), demonstrating the toxic effect of the human RAPGEF2-E1357K variant in Drosophila motor neurons.

Defective mitochondrial ultrastructure and function in patient fibroblasts

To investigate the effects of the RAPGEF2-E1357K variant on mitochondrial structures, we performed transmission electron microscopy (EM) for control and patient fibroblasts. Control mitochondria showed a typical crista structure with electron-dense deposits in the matrix (Fig. 6A). In contrast, patient fibroblasts displayed swollen and vacuolated mitochondria without lamella cristae and electron-dense deposits (Fig. 6A).

To investigate whether the ultrastructural mitochondrial abnormalities in patient fibroblasts are paralleled with mitochondrial dysfunction, we assessed mitochondrial membrane potential using JC-1, a cationic lipophilic dye. The JC-1 dye accumulates as red-fluorescent J-aggregates within mitochondria at high membrane potentials (energized mitochondria), while it accumulates as green fluorescent monomers within mitochondria at low membrane potentials (deenergized mitochondria) [36]. In live control fibroblasts loaded with JC-1, we observed strong red fluorescent signals but not green fluorescent signals (Fig. 6B), suggesting the majority of mitochondria are functional. In contrast, both red and green fluorescent signals were prominent in JC-1-loaded patient fibroblasts (Fig. 6B). These results suggest that mitochondria activity is lower in patient fibroblasts than in control fibroblasts.

Abnormal translocation of BAX to mitochondria in patient fibroblasts is rescued by pharmacological inhibition of HDAC6

Mitochondrial dysfunction is intimately linked to apoptotic cell death, which involves the mitochondrial recruitment of the proapoptotic regulator BAX from the cytosol [37]. We therefore examined whether mitochondrial dysfunction in patient-derived skin fibroblasts is paralleled with abnormal accumulation of BAX on the mitochondria. To this end, we transfected control and patient fibroblasts with a GFP-BAX construct and stained them with anti-GFP and anti-mitochondria. In control fibroblasts, BAX showed a diffuse cytoplasmic distribution with a minimal overlap with mitochondria (Fig. 7A). Notably, we observed a prominent overlap between BAX signals and mitochondria in patient fibroblasts (Fig. 7A). As an additional approach, we separated cytosolic and mitochondrial fractions from lysates of control and patient fibroblasts and performed Western blot analysis of these fractions using anti-BAX. This experiment demonstrated that BAX was more recruited to the mitochondria in patient fibroblasts than in control fibroblasts (Fig. 7B and C). In a control experiment, we confirmed that total BAX levels were comparable between control and patient derived fibroblasts (data not shown). Finally, we found that the abnormal accumulation of BAX at the mitochondria was rescued by inhibiting HDAC6 with 1 µM tubastatin A (Fig. 7A~C). The same dose of tubastatin A had no effect on the BAX distribution in control-derived fibroblasts (Fig. 7A~C).

In this study, we report the identification of a de novo variant of the RAPGEF2 gene (c.4069G>A, p.E1357K) in a patient with sALS through a trio-based WES approach. To assess the pathogenic potential of the RAPGEF2-E1357K variant, we have used skin fibroblasts derived from the patient and a Drosophila model carrying a RAPGEF2-E1357K transgene. In this way, we have investigated the effects of the RAPGEF2 variant on the microtubule cytoskeleton and the mitochondria, two cellular components that are frequently affected in ALS [22,38]. The patient fibroblasts exhibit reduction of stable microtubules as well as abnormal distribution and fragmentation of the mitochondrial network. Overexpression of the RAPGEF2-E1357K variant in Drosophila motor neurons impairs microtubule stability and mitochondrial distribution along axons and at NMJ presynaptic terminals. Aside from mitochondrial network abnormalities, disruption of mitochondrial structure including swelling, vacuolization, and loss of cristae have been also observed in various in vitro and in vivo models of ALS [38]. Our EM analysis reveals that patient fibroblasts carrying the RAPGEF-E1357K mutation also contain swollen and vacuolated mitochondria without cristae. Finally, patient fibroblasts show accumulation of nonfunctional mitochondria and induction of BAX recruitment to mitochondria, which is a key cellular event of mitochondria-dependent apoptosis [37]. Taken together, our results indicate that the de novo RAPGEF2-E1357K variant identified in our patient exerts deleterious effects on cells, which may be related to the pathogenesis of ALS.

Microtubules are dynamic polymers that undergo polymerization and depolymerization of α- and β-tubulin heterodimer. While developing neurons keep microtubules in a highly dynamic state during process outgrowth, mature neurons progressively increase microtubule stability to maintain many aspects of cellular functions [39,40]. This change in microtubule stability is associated with chemical modifications of microtubules including acetylation [39]. Microtubule acetylation and deacetylation mainly occur at the conserved lysine 40 residue of α-tubulin by α-tubulin acetyltransferase 1 (αTAT1) and histone deacetylase 6 (HDAC6), respectively [34,41]. Interestingly, several studies have demonstrated HDAC6-mediated modulation of ALS pathogenesis. For example, removal of the HDAC6 gene is shown to delay disease progression in the SOD1-G93A mouse model of ALS [42]. More recently, it has been reported that pharmacological inhibition of HDAC6 rescues axonal transport defects in FUS-ALS patient-derived motor neurons [43]. Here we show that pharmacological inhibition of HDAC6 is able to rescue both morphological mitochondria defects and abnormal accumulation of BAX in the mitochondria in patient fibroblasts carrying the RAPGEF2-E1357K variant. Thus, this study supports the notion that dysregulation of microtubule stability could act as a primary driver for ALS pathophysiology.

Consistent with our previous findings [24], knockdown of a Drosophila RAPGEF2 homolog (Gef26) increases the levels of acetylated α-tubulin and Futsch/MAP1B in motor axons. These phenotypes are the opposites of those induced by overexpression of the human RAFGEF2-E1357K variant in Drosophila motor neurons, precluding the possibility that the RAPGEF2 mutant exerts a deleterious effect on microtubule stability through a dominant negative mechanism. Moreover, overexpression of wild-type human RAPGEF2 in Drosophila motor neurons has no effect on axonal microtubule stability, further suggesting that the RAPGEF2-E1357K mutant impairs microtubule stability through a toxic gain-of-function mechanism rather than a simple dose-dependent mechanism.

In conclusion, we identified a de novoRAPGEF2 variant in a sALS patient, which causes dysregulation of microtubule stability as well as of the distribution, structure, and function of mitochondria. Since these defects are becoming increasingly recognized as core components of ALS pathogenesis [22,38], the current study warrants re-sequencing this gene in larger ALS cohorts to validate the pathogenicity of mutations in RAPGEF2.

Fig. 1. Detection of a RAPGEF2de novo variant (p.E1357K) in a sALS trio. (A) Family pedigree. Square indicates male; circle, female; and solid, affected. (B) Confirmation of the p.E1357K variant in RAPGEF2 by Sanger sequencing. (C) Domain architecture of the human RAPGEF2 protein along with the position of the identified substitution. Conserved domains are identified by SMART (Simple Modular Architecture Research Tool, and PONDR (Predictor of Natural Disordered Regions, The asterisk indicates the position of the substitution p.E1357K within the second LCD domain of RAPGEF2. Note that the amino acid E1357 is highly conserved among species. cNMP, cyclic nucleotide monophosphate-binding; Ras-GEFN, the N-terminal domain of guanine nucleotide exchange factor for Ras-like GTPases; PDZ, PSD-95/Dlg/ZO-1 binding domain; RA, Ras/Rap association; RasGEF, guanine nucleotide exchange factor for Ras-like GTPases; LCD, low-complexity domain.
Fig. 2. ALS patient-derived fibroblasts display reduced levels of acetylated α-tubulin and disrupted microtubule network. (A) Confocal images of control-derived fibroblasts and ALS patient-derived fibroblasts (E1357K) labeled with antibodies against acetylated α-tubulin (Ac-tub), tyrosinated α-tubulin (Tyr-tub), or α-tubulin (Tub) and DAPI. Scale bar, 5 µm. (B) Western blot analysis of lysates from control and patient fibroblasts using anti-acetylated α-tubulin, anti-tyrosinated α-tubulin, anti-α-tubulin, and anti-GAPDH antibodies. (C and D) Quantitative analysis by densitometric measurements (n=3). The intensities of Ac-tub and Tyr-tub were normalized to that of Tub. Data are presented as mean±SEM. *p<0.001. (E) Confocal images of HeLa cells transiently expressing wild-type GFP-RAPGEF2 (WT) or GFP-RAPGEF2-E1357K (E1357K) labeled with anti-GFP and anti-Actub antibodies. Note that the microtubule network is disrupted in GFP-RAPGEF2-E1357K-expressing cells but not in GFP-RAPGEF2-expressing cells. Scale bar, 5 µm.
Fig. 3. Pharmacological inhibition of HDAC6 rescues abnormalities in the mitochondrial network in ALS patient-derived fibroblasts carrying the RAPGEF2-E1357K variant. (A) Confocal images of Mito-RFP-expressing control and patient (E1357K) fibroblasts treated with DMSO or 1 µM tubastatin A (Tub A). Cells were labeled with anti-acetylated α-tubulin (Ac-tub) and DAPI. Bottom panels show higher magnification views of the regions marked by boxes. Scale bar, 5 µm. (B and C) Quantification of mitochondria length and cell size (n=7 for DMSO-treated control fibroblasts; n=6 for DMSO-treated patient fibroblasts (E1357K); n=11 for Tub A-treated control fibroblasts; n=11 for Tub A-treated patient fibroblasts (E1357K). Data are presented as mean±SEM. Comparisons are made with DMSO-treated control fibroblasts (*p<0.001).
Fig. 4. Overexpression of the human RAPGEF2-E1357K variant in Drosophila motor neurons impairs microtubule stability in axons and at NMJ terminals. (A~D) Confocal images of motor axons (A and B) and NMJ terminals (C and D) of abdominal segment 5 in third instar larvae doubly labeled with anti-acetylated α-tubulin (Ac-tub) or anti-Futsch and anti-HRP antibodies. Genotypes include D42-GAL4/+ (control), D42-GAL4/UASHA-RAPGEF2-WT(HA-RAPGEF2-WT), UAS-HA-RAPGEF2-E1357K/+; D42-GAL4/+ (HA-RAPGEF2-E1357K), and D42-GAL4/UAS-gef26RNAi(Gef26 KD). Scale bars, 5 µm. (E and F) Quantification of the ratios of mean Ac-tub to HRP fluorescence intensities in axons (E) and at NMJ terminals (F). (G and H) Quantification of the ratios of mean Futsch to HRP fluorescence intensities in axons (G) and at NMJ terminals (H). Data are presented as mean±SEM. All comparisons are made with the D42-GAL4/+ control (*p<0.001).
Fig. 5. Overexpression of the human RAPGEF2-E1357K variant in Drosophila motor neurons disrupts the distribution of mitochondria to axons and NMJ terminals. (A~C) Confocal images of ventral nerve cord (VNC, A), axon (B), and NMJ 6/7 (C) in third instar larvae stained with anti-GFP alone (A) or together with anti-HRP (B and C). Axons and NMJs in abdominal segment 5 were analyzed. Genotypes include D42-GAL4,UAS-mito-GFP/+ (control), D42-GAL4,UAS-mito-GFP/UAS-HA-RAPGEF2-WT(HA-RAPGEF2-WT), UAS-HA-RAPGEF2-E1357K/+; D42-GAL4,UAS-mito-GFP/+ (HA-RAPGEF2-E1357K), and D42-GAL4,UAS-mito-GFP/UAS-gef26RNAi(Gef26 KD). Scale bars, 5 µm. (D~F) Quantification of the number (D) and area (E and F) of Mito-GFP-positive puncta in axons (D and E) and at NMJ terminals (F). Values were normalized to the respective area of axon or NMJ terminals. (G) Distribution of the distance climbed by 20-day-old D42-GAL4/+ (control), D42-GAL4/UAS-HA-RAPGEF2-WT (HA-RAPGEF2-WT), and UAS-HA-RAPGEF2-E1357K/+; D42-GAL4/+ (HA-RAPGEF2-E1357K) flies over 30 s. (H) Quantification of average climbing distance. Data are presented as mean±SEM. All comparisons are made with the D42-GAL4,UAS-mito-GFP/+ control (*p<0.001; **p<0.01).
Fig. 6. Abnormalities in mitochondrial ultrastructure and function in ALS patient-derived fibroblasts. (A) Transmission electron micrographs of cross-sectioned control and patient fibroblasts. N indicates the nucleus, and arrowheads indicate mitochondria. Note that patient fibroblasts show enlarged mitochondria without cristae structures. Scale bar, 0.2 µm. (B) Fluorescent images of JC-1-loaded control and patient (E1357K) fibroblasts. Note abnormal accumulation of green-fluorescent JC-1 monomers in the mitochondria of patient but not control cells. Scale bar, 15 µm.
Fig. 7. Pharmacological inhibition of HDAC6 restores abnormal accumulation of BAX in the mitochondria in ALS patient-derived fibroblasts. (A) Confocal images of control and patient (E1357K) fibroblasts expressing GFP-BAX stained with anti-mitochondria antibody and DAPI. Cells were treated with DMSO or 1 µM tubastatin A (Tub A) prior to immunostaining. Scale bar, 5 µm. (B) Western blot analysis of mitochondrial and cytosolic fractions from DMSO- or Tub A-treated control and patient fibroblasts using anti-BAX and anti-α-tubulin antibodies. (C) Quantitative analysis of densitometric measurements (n=3). The band intensities of BAX in each fraction were normalized to the sum of mitochondrial and cytosolic BAX intensities. Data are presented as mean±SEM. Comparisons are made with the DMSO-treated control (*p<0.001).
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