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Exp Neurobiol 2018; 27(6): 550-563
Published online December 12, 2018
© The Korean Society for Brain and Neural Sciences
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: firstname.lastname@example.org
Seung Hyun Kim, TEL: 82-2-2290-8371, FAX: 82-2-2296-8370, e-mail: email@example.com
Seungbok Lee, TEL: 82-2-880-2330, FAX: 82-2-762-2583, e-mail: firstname.lastname@example.org
† These authors contributed equally to this work.
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
Keywords: Amyotrophic lateral sclerosis, Whole exome sequencing,
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 .
Impaired mitochondrial trafficking in motor neurons is a well-established phenomenon in ALS pathophysiology . 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 . 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
In this study, we employed a trio-WES approach to identify pathogenic variants in a sALS patient and detected a
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 (http://evs.gs.washington.edu/EVS/), the 1000 Genomes Project (http://www.1000genomes.org/), and gnomAD (http://gnomad.broadinstitute.org/). All amino acid-altering
Full-length cDNAs for
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  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).
Flies were maintained at 25℃ on standard food. Transgenic
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
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
A climbing test was used to assess the locomotor function of adult flies as previously described . 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.
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 . 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).
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 . 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.
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).
Comparisons were made by one-way ANOVA analysis with a post-hoc Turkey test. Data are presented as mean±SEM.
While performing WES on sALS trios, we identified a 28-year-old female patient carrying a
Disruptions in microtubule network assembly have been proposed as a critical component of ALS pathogenesis . 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 ), and tyrosinated α-tubulin (detecting both free tubulin and newly formed microtubules ). 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.
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) , which is the major deacetylase of α-tubulin . 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.
To validate the effects of the RAPGEF2-E1357K mutant on microtubule maintenance and mitochondrial distribution in motor axons, we generated
We have recently shown that loss of the
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 (
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) . 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.
Mitochondrial dysfunction is intimately linked to apoptotic cell death, which involves the mitochondrial recruitment of the proapoptotic regulator BAX from the cytosol . 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
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 . 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
Consistent with our previous findings , knockdown of a
In conclusion, we identified a