- Email: [email protected]
Deficient RNA-editing enzyme ADAR2 in an amyotrophic lateral sclerosis patient with a FUSP525L mutation
Journal of Clinical Neuroscience xxx (2016) xxx–xxx
Contents lists available at ScienceDirect
Journal of Clinical Neuroscience journal homepage: www.elsevier.com/locate/jocn
Case report
Deficient RNA-editing enzyme ADAR2 in an amyotrophic lateral sclerosis patient with a FUSP525L mutation Hitoshi Aizawa a,⇑, Takuto Hideyama b, Takenari Yamashita c, Takashi Kimura d, Naoki Suzuki e, Masashi Aoki e, Shin Kwak c,f a
Department of Neurology, Tokyo Medical University, 6-7-1 Nishishinjuku, Shinjuku-ku, Tokyo 160-0023, Japan Department of Neurology, Tokyo Teishin Hospital, Tokyo 102-0071, Japan Division of Clinical Biotechnology, Center for Disease Biology and Integrative Medicine, Graduate School of Medicine, The University of Tokyo, Bunkyo-ku, Tokyo 113-0033, Japan d Department of Neurology, Asahikawa Medical Center, National Hospital Organization, Asahikawa 070-8644, Japan e Department of Neurology, Tohoku University School of Medicine, Sendai, Miyagi 980-8577, Japan f Clinical Research Center of Medicine, International University of Health and Welfare, Minato-ku, Tokyo 108-8329, Japan b c
a r t i c l e
i n f o
Article history: Received 9 October 2015 Accepted 12 December 2015 Available online xxxx Keywords: Adenosine deaminase acting on RNA 2 Amyotrophic lateral sclerosis Fused in sarcoma
a b s t r a c t Mutations in the fused in sarcoma (FUS) gene can cause amyotrophic lateral sclerosis (ALS), and FUS gene mutations have been reported in sporadic ALS patients with basophilic cytoplasmic inclusions. Deficiency of adenosine deaminase acting on RNA 2 (ADAR2), an enzyme that specifically catalyzes GluA2 Q/R siteediting, has been reported in considerable proportions of spinal motor neurons of the majority of sporadic ALS patients. We describe the relationship between GluA2 Q/R site-editing efficiency and FUS-positive inclusions in a patient with FUSP525L. A 24-year-old woman with ALS presented with basophilic cytoplasmic inclusions, significantly reduced GluA2 Q/R site-editing efficiency in the spinal motor neurons, and markedly decreased ADAR2 mRNA levels. Neuropathologic examination showed that not all spinal motor neurons expressed ADAR2 and revealed FUS-positive cytoplasmic inclusions in motor neurons irrespective of ADAR2 immunoreactivity. There were no phosphorylated transactive response (TAR) DNA-binding protein 43 kDa (TDP-43)-positive inclusions, indicating that there was no tight correlation between ADAR2 deficiency and TDP-43 deposition. ADAR2 deficiency can occur in ALS patients with a FUSP525L mutation and is unrelated to the presence of FUS-positive inclusions. FUS-associated ALS may share neurodegenerative characteristics with classical sporadic ALS. Ó 2016 Elsevier Ltd. All rights reserved.
1. Introduction Motor neurons exhibiting transactive response (TAR) DNA-binding protein 43 kDa (TDP-43) pathology in sporadic amyotrophic lateral sclerosis (ALS) patients are always devoid of immunoreactivity for adenosine deaminase acting on RNA 2 (ADAR2) [1]. ADAR2 catalyzes adenosine-to-inosine conversion at specific positions in pre-mRNAs, including the glutamine/arginine (Q/R) site of GluA2 pre-mRNA, and ADAR2 downregulation results in Q/R site-unedited abnormal GluA2 expression in the motor neurons of sporadic ALS patients [2,3]. A sporadic juvenile-onset ALS patient with basophilic cytoplasmic inclusions [4] was recently shown to carry a fused in sarcoma (FUS)P525L mutation. We
⇑ Corresponding author. Tel.: +81 3 3342 6111; fax: +81 3 3342 6272. E-mail address: [email protected] (H. Aizawa).
investigated FUS pathology by analyzing ADAR2 gene expression in motor neurons to determine whether ALS with FUS mutations is characterized by the death cascade observed in sporadic ALS. 2. Case presentation This case was reported previously as sporadic juvenile-onset ALS with basophilic inclusions [4]. In brief, a 24-year-old female without neurological family history presented with progressive muscle atrophy and right upper extremity weakness, which rapidly extended to the left upper extremity and eventually to the neck and lower extremities in 5 months. Neurologic examination revealed involvement of multiple lower motor neuron systems. She died 7 months after symptom onset. Written informed consent for neuropathologic and genetic analysis was obtained from her relatives. This patient was recently shown to carry a FUSP525L mutation using standard protocols.
http://dx.doi.org/10.1016/j.jocn.2015.12.039 0967-5868/Ó 2016 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Aizawa H et al. Deficient RNA-editing enzyme ADAR2 in an amyotrophic lateral sclerosis patient with a FUSP525L mutation. J Clin Neurosci (2016), http://dx.doi.org/10.1016/j.jocn.2015.12.039
2
Case report / Journal of Clinical Neuroscience xxx (2016) xxx–xxx
Fig. 2. Association between adenosine deaminase acting on RNA 2 (ADAR2) and basophilic inclusions (BIs). (A) Immunohistochemical analysis of autopsy specimens. (a) ADAR2-negative neuron without BIs, (b) ADAR2-positive neuron without BIs, (c) ADAR2-negative neuron with a BI (arrow), and (d) ADAR2-positive neuron with a BI (arrow). Scale bars represent 20 lm. (B) Percent distribution of spinal motor neurons with ADAR2 immunoreactivity and/or BIs.
Fig. 1. Histopathologic analysis of autopsy specimens. (A) Basophilic inclusion (BI) (arrow) in the cytoplasm of a spinal motor neuron stained with hematoxylin and eosin (400). (B) BI (arrow) showing fused in sarcoma (FUS) immunoreactivity (400). (C) Transactive response (TAR) DNA-binding protein 43 kDa (TDP-43) is positive in the nucleus, but is not found in the BI (arrow) (400). (D) BI (arrow) negative for phosphorylated TDP-43 (400). Scale bars represent 20 lm.
2.1. Histologic and immunohistochemical analysis Microscopic examination revealed cytoplasmic basophilic inclusions in the spinal motor neurons (Fig. 1A), which were immunoreactive for FUS (Fig. 1B). TDP-43 was positive in the nuclei of motor neurons with cytoplasmic basophilic inclusions (Fig. 1C), but there were no phosphorylated TDP-43-immunoreactive inclusions (Fig. 1D), indicating that TDP-43 was not mislocalized. 2.2. Quantification of motor neurons in the spinal cord ADAR2-positive and -negative spinal motor neurons were noted in the anterior horn. (Fig. 2A) Twenty-two sections of thoracolumbar spinal cord were immunostained with ADAR2 antibody. In total, 52 motor neurons were detected in the anterior horns. ADAR2-positive and -negative neurons with nucleoli in the anterior horns were counted in each section. There were ADAR2-negative neurons without basophilic inclusions (Fig. 2A-a), ADAR2-positive neurons without basophilic inclusions (Fig. 2A-b), ADAR2-negative neurons with basophilic inclusions (Fig. 2A-c), and ADAR2-positive neurons with basophilic inclusions (Fig. 2A-d). The graph in Figure 2B shows that ADAR2 immunoreactivity was independent of the presence of basophilic cytoplasmic inclusions. 3. Discussion Some spinal motor neurons lacked ADAR2 immunoreactivity, an observation consistent with both our previous reports on sporadic ALS patients [1] and the finding that editing efficiency at the GluA2 Q/R site and ADAR2 mRNA levels were decreased in motor neurons [3]. ADAR2-positive and -negative motor neurons may represent normal and decreased editing at the GluA2 Q/R site, respectively. GluA2 Q/R site editing occurs with 100% efficiency in normal human motor neurons but is highly variable among
individual motor neurons in this case and all sporadic ALS cases examined to our knowledge [2]. Given that ADAR2 downregulation to below the level necessary to edit the Q/R site of all GluA2 mRNAs is the cause of motor neuron death in conditional ADAR2-knockout mice [5], these results indicate that ALS due to a FUSP525L mutation is closely related to ADAR2 deficiency, as well as sporadic ALS. Independence of ADAR2 immunoreactivity from the presence of cytoplasmic basophilic inclusions suggests that basophilic inclusion formation itself is not mechanistically associated with ADAR2 downregulation. This finding is in marked contrast with sporadic ALS, where TDP-43 pathology is closely associated with the loss of ADAR2 immunoreactivity [1,6], representing a molecular link between ADAR2 downregulation and TDP-43 pathology [7]. Many different FUS mutations have been reported to be associated with ALS, indicating that ADAR2 activity should be examined in ALS patients carrying other FUS mutations to determine whether ADAR2 downregulation universally occurs in association with ALS-linked FUS mutations. The present results suggest ADAR2 downregulation as a mechanism underlying FUS-associated ALS, although the possibility that mutant FUS enhances motor neuron death by other mechanisms cannot be ruled out. Elucidation of the mechanism underlying ADAR2 downregulation in FUSP525L and possibly other FUS mutations may improve our understanding of ALS pathophysiology. 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. Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jocn.2015.12.039. References [1] Aizawa H, Sawada J, Hideyama T, et al. TDP-43 pathology in sporadic ALS occurs in motor neurons lacking the RNA editing enzyme ADAR2. Acta Neuropathol 2010;120:75–84. [2] Kawahara Y, Ito K, Sun H, et al. Glutamate receptors: RNA editing and death of motor neurons. Nature 2004;427:801. [3] Hideyama T, Yamashita T, Aizawa H, et al. Profound downregulation of the RNA editing enzyme ADAR2 in ALS spinal motor neurons. Neurobiol Dis 2012;45:1121–8. [4] Aizawa H, Kimura T, Hashimoto K, et al. Basophilic cytoplasmic inclusions in a case of sporadic juvenile amyotrophic lateral sclerosis. J Neurol Sci 2000;176:109–13.
Please cite this article in press as: Aizawa H et al. Deficient RNA-editing enzyme ADAR2 in an amyotrophic lateral sclerosis patient with a FUSP525L mutation. J Clin Neurosci (2016), http://dx.doi.org/10.1016/j.jocn.2015.12.039
Case report / Journal of Clinical Neuroscience xxx (2016) xxx–xxx [5] Hideyama T, Yamashita T, Suzuki T, et al. Induced loss of ADAR2 engenders slow death of motor neurons from Q/R site-unedited GluR2. J Neurosci 2010;30:11917–25. [6] Hideyama T, Teramoto S, Hachiga K, et al. Co-occurrence of TDP-43 mislocalization with reduced activity of an RNA editing enzyme, ADAR2, in aged mouse motor neurons. PLoS ONE 2012;7:e43469.
3
[7] Yamashita T, Hideyama T, Hachiga K, et al. A role for calpain-dependent cleavage of TDP-43 in amyotrophic lateral sclerosis pathology. Nat Commun 2012;3:1307.
Please cite this article in press as: Aizawa H et al. Deficient RNA-editing enzyme ADAR2 in an amyotrophic lateral sclerosis patient with a FUSP525L mutation. J Clin Neurosci (2016), http://dx.doi.org/10.1016/j.jocn.2015.12.039
Contents lists available at ScienceDirect
Journal of Clinical Neuroscience journal homepage: www.elsevier.com/locate/jocn
Case report
Deficient RNA-editing enzyme ADAR2 in an amyotrophic lateral sclerosis patient with a FUSP525L mutation Hitoshi Aizawa a,⇑, Takuto Hideyama b, Takenari Yamashita c, Takashi Kimura d, Naoki Suzuki e, Masashi Aoki e, Shin Kwak c,f a
Department of Neurology, Tokyo Medical University, 6-7-1 Nishishinjuku, Shinjuku-ku, Tokyo 160-0023, Japan Department of Neurology, Tokyo Teishin Hospital, Tokyo 102-0071, Japan Division of Clinical Biotechnology, Center for Disease Biology and Integrative Medicine, Graduate School of Medicine, The University of Tokyo, Bunkyo-ku, Tokyo 113-0033, Japan d Department of Neurology, Asahikawa Medical Center, National Hospital Organization, Asahikawa 070-8644, Japan e Department of Neurology, Tohoku University School of Medicine, Sendai, Miyagi 980-8577, Japan f Clinical Research Center of Medicine, International University of Health and Welfare, Minato-ku, Tokyo 108-8329, Japan b c
a r t i c l e
i n f o
Article history: Received 9 October 2015 Accepted 12 December 2015 Available online xxxx Keywords: Adenosine deaminase acting on RNA 2 Amyotrophic lateral sclerosis Fused in sarcoma
a b s t r a c t Mutations in the fused in sarcoma (FUS) gene can cause amyotrophic lateral sclerosis (ALS), and FUS gene mutations have been reported in sporadic ALS patients with basophilic cytoplasmic inclusions. Deficiency of adenosine deaminase acting on RNA 2 (ADAR2), an enzyme that specifically catalyzes GluA2 Q/R siteediting, has been reported in considerable proportions of spinal motor neurons of the majority of sporadic ALS patients. We describe the relationship between GluA2 Q/R site-editing efficiency and FUS-positive inclusions in a patient with FUSP525L. A 24-year-old woman with ALS presented with basophilic cytoplasmic inclusions, significantly reduced GluA2 Q/R site-editing efficiency in the spinal motor neurons, and markedly decreased ADAR2 mRNA levels. Neuropathologic examination showed that not all spinal motor neurons expressed ADAR2 and revealed FUS-positive cytoplasmic inclusions in motor neurons irrespective of ADAR2 immunoreactivity. There were no phosphorylated transactive response (TAR) DNA-binding protein 43 kDa (TDP-43)-positive inclusions, indicating that there was no tight correlation between ADAR2 deficiency and TDP-43 deposition. ADAR2 deficiency can occur in ALS patients with a FUSP525L mutation and is unrelated to the presence of FUS-positive inclusions. FUS-associated ALS may share neurodegenerative characteristics with classical sporadic ALS. Ó 2016 Elsevier Ltd. All rights reserved.
1. Introduction Motor neurons exhibiting transactive response (TAR) DNA-binding protein 43 kDa (TDP-43) pathology in sporadic amyotrophic lateral sclerosis (ALS) patients are always devoid of immunoreactivity for adenosine deaminase acting on RNA 2 (ADAR2) [1]. ADAR2 catalyzes adenosine-to-inosine conversion at specific positions in pre-mRNAs, including the glutamine/arginine (Q/R) site of GluA2 pre-mRNA, and ADAR2 downregulation results in Q/R site-unedited abnormal GluA2 expression in the motor neurons of sporadic ALS patients [2,3]. A sporadic juvenile-onset ALS patient with basophilic cytoplasmic inclusions [4] was recently shown to carry a fused in sarcoma (FUS)P525L mutation. We
⇑ Corresponding author. Tel.: +81 3 3342 6111; fax: +81 3 3342 6272. E-mail address: [email protected] (H. Aizawa).
investigated FUS pathology by analyzing ADAR2 gene expression in motor neurons to determine whether ALS with FUS mutations is characterized by the death cascade observed in sporadic ALS. 2. Case presentation This case was reported previously as sporadic juvenile-onset ALS with basophilic inclusions [4]. In brief, a 24-year-old female without neurological family history presented with progressive muscle atrophy and right upper extremity weakness, which rapidly extended to the left upper extremity and eventually to the neck and lower extremities in 5 months. Neurologic examination revealed involvement of multiple lower motor neuron systems. She died 7 months after symptom onset. Written informed consent for neuropathologic and genetic analysis was obtained from her relatives. This patient was recently shown to carry a FUSP525L mutation using standard protocols.
http://dx.doi.org/10.1016/j.jocn.2015.12.039 0967-5868/Ó 2016 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Aizawa H et al. Deficient RNA-editing enzyme ADAR2 in an amyotrophic lateral sclerosis patient with a FUSP525L mutation. J Clin Neurosci (2016), http://dx.doi.org/10.1016/j.jocn.2015.12.039
2
Case report / Journal of Clinical Neuroscience xxx (2016) xxx–xxx
Fig. 2. Association between adenosine deaminase acting on RNA 2 (ADAR2) and basophilic inclusions (BIs). (A) Immunohistochemical analysis of autopsy specimens. (a) ADAR2-negative neuron without BIs, (b) ADAR2-positive neuron without BIs, (c) ADAR2-negative neuron with a BI (arrow), and (d) ADAR2-positive neuron with a BI (arrow). Scale bars represent 20 lm. (B) Percent distribution of spinal motor neurons with ADAR2 immunoreactivity and/or BIs.
Fig. 1. Histopathologic analysis of autopsy specimens. (A) Basophilic inclusion (BI) (arrow) in the cytoplasm of a spinal motor neuron stained with hematoxylin and eosin (400). (B) BI (arrow) showing fused in sarcoma (FUS) immunoreactivity (400). (C) Transactive response (TAR) DNA-binding protein 43 kDa (TDP-43) is positive in the nucleus, but is not found in the BI (arrow) (400). (D) BI (arrow) negative for phosphorylated TDP-43 (400). Scale bars represent 20 lm.
2.1. Histologic and immunohistochemical analysis Microscopic examination revealed cytoplasmic basophilic inclusions in the spinal motor neurons (Fig. 1A), which were immunoreactive for FUS (Fig. 1B). TDP-43 was positive in the nuclei of motor neurons with cytoplasmic basophilic inclusions (Fig. 1C), but there were no phosphorylated TDP-43-immunoreactive inclusions (Fig. 1D), indicating that TDP-43 was not mislocalized. 2.2. Quantification of motor neurons in the spinal cord ADAR2-positive and -negative spinal motor neurons were noted in the anterior horn. (Fig. 2A) Twenty-two sections of thoracolumbar spinal cord were immunostained with ADAR2 antibody. In total, 52 motor neurons were detected in the anterior horns. ADAR2-positive and -negative neurons with nucleoli in the anterior horns were counted in each section. There were ADAR2-negative neurons without basophilic inclusions (Fig. 2A-a), ADAR2-positive neurons without basophilic inclusions (Fig. 2A-b), ADAR2-negative neurons with basophilic inclusions (Fig. 2A-c), and ADAR2-positive neurons with basophilic inclusions (Fig. 2A-d). The graph in Figure 2B shows that ADAR2 immunoreactivity was independent of the presence of basophilic cytoplasmic inclusions. 3. Discussion Some spinal motor neurons lacked ADAR2 immunoreactivity, an observation consistent with both our previous reports on sporadic ALS patients [1] and the finding that editing efficiency at the GluA2 Q/R site and ADAR2 mRNA levels were decreased in motor neurons [3]. ADAR2-positive and -negative motor neurons may represent normal and decreased editing at the GluA2 Q/R site, respectively. GluA2 Q/R site editing occurs with 100% efficiency in normal human motor neurons but is highly variable among
individual motor neurons in this case and all sporadic ALS cases examined to our knowledge [2]. Given that ADAR2 downregulation to below the level necessary to edit the Q/R site of all GluA2 mRNAs is the cause of motor neuron death in conditional ADAR2-knockout mice [5], these results indicate that ALS due to a FUSP525L mutation is closely related to ADAR2 deficiency, as well as sporadic ALS. Independence of ADAR2 immunoreactivity from the presence of cytoplasmic basophilic inclusions suggests that basophilic inclusion formation itself is not mechanistically associated with ADAR2 downregulation. This finding is in marked contrast with sporadic ALS, where TDP-43 pathology is closely associated with the loss of ADAR2 immunoreactivity [1,6], representing a molecular link between ADAR2 downregulation and TDP-43 pathology [7]. Many different FUS mutations have been reported to be associated with ALS, indicating that ADAR2 activity should be examined in ALS patients carrying other FUS mutations to determine whether ADAR2 downregulation universally occurs in association with ALS-linked FUS mutations. The present results suggest ADAR2 downregulation as a mechanism underlying FUS-associated ALS, although the possibility that mutant FUS enhances motor neuron death by other mechanisms cannot be ruled out. Elucidation of the mechanism underlying ADAR2 downregulation in FUSP525L and possibly other FUS mutations may improve our understanding of ALS pathophysiology. 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. Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jocn.2015.12.039. References [1] Aizawa H, Sawada J, Hideyama T, et al. TDP-43 pathology in sporadic ALS occurs in motor neurons lacking the RNA editing enzyme ADAR2. Acta Neuropathol 2010;120:75–84. [2] Kawahara Y, Ito K, Sun H, et al. Glutamate receptors: RNA editing and death of motor neurons. Nature 2004;427:801. [3] Hideyama T, Yamashita T, Aizawa H, et al. Profound downregulation of the RNA editing enzyme ADAR2 in ALS spinal motor neurons. Neurobiol Dis 2012;45:1121–8. [4] Aizawa H, Kimura T, Hashimoto K, et al. Basophilic cytoplasmic inclusions in a case of sporadic juvenile amyotrophic lateral sclerosis. J Neurol Sci 2000;176:109–13.
Please cite this article in press as: Aizawa H et al. Deficient RNA-editing enzyme ADAR2 in an amyotrophic lateral sclerosis patient with a FUSP525L mutation. J Clin Neurosci (2016), http://dx.doi.org/10.1016/j.jocn.2015.12.039
Case report / Journal of Clinical Neuroscience xxx (2016) xxx–xxx [5] Hideyama T, Yamashita T, Suzuki T, et al. Induced loss of ADAR2 engenders slow death of motor neurons from Q/R site-unedited GluR2. J Neurosci 2010;30:11917–25. [6] Hideyama T, Teramoto S, Hachiga K, et al. Co-occurrence of TDP-43 mislocalization with reduced activity of an RNA editing enzyme, ADAR2, in aged mouse motor neurons. PLoS ONE 2012;7:e43469.
3
[7] Yamashita T, Hideyama T, Hachiga K, et al. A role for calpain-dependent cleavage of TDP-43 in amyotrophic lateral sclerosis pathology. Nat Commun 2012;3:1307.
Please cite this article in press as: Aizawa H et al. Deficient RNA-editing enzyme ADAR2 in an amyotrophic lateral sclerosis patient with a FUSP525L mutation. J Clin Neurosci (2016), http://dx.doi.org/10.1016/j.jocn.2015.12.039