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α-Synuclein coaggregation in familial amyotrophic lateral sclerosis with SOD1 gene mutation
Human Pathology (2013) 44, 1171–1176
www.elsevier.com/locate/humpath
Case study
α-Synuclein coaggregation in familial amyotrophic lateral sclerosis with SOD1 gene mutation Yo-ichi Takei MD a , Kenya Oguchi MD a , Hiroshi Koshihara MD a , Akiyo Hineno MD b , Akinori Nakamura MD b , Shinji Ohara MD a,⁎ a
Department of Neurology, Matsumoto Medical Center, Chushin-Matsumoto Hospital, Kotobuski 811, Matsumoto, 399–0021 Japan b Department of Medicine (Neurology and Rheumatology), Shinshu University School of Medicine, Asahi 3-1-1, 390–8621, Japan Received 19 September 2012; revised 21 October 2012; accepted 31 October 2012
Keywords: Familial amyotrophic lateral sclerosis; Copper/zinc superoxide dismutase (SOD1); α-Synuclein; Co-aggregation; Immunohistochemistry
Summary Immunohistochemical studies were performed on postmortem brain and spinal cord from a patient with familial amyotrophic lateral sclerosis characterized by a C111Y mutation in the Cu/Zn superoxide dismutase gene. Clinically, the patient presented with classical amyotrophic lateral sclerosis and died of respiratory failure at age 53 years without ventilator dependence, 4 years after the onset. Pathologically, loss of motor neurons was more extensive than upper motor neurons. Lower motor neurons developed massive intracellular cytoplasmic neuronal inclusions, which were immunoreactive for Cu/Zn superoxide dismutase and phosphorylated α-synuclein, often colocalized. The inclusions were TAR DNA-binding protein 43 negative. The clinicopathologic significance of coaggregation of α-synuclein and Cu/Zn superoxide dismutase protein, a novel finding in neurodegenerative disorders, needs further investigation. © 2013 Elsevier Inc. All rights reserved.
1. Introduction Approximately 10% of amyotrophic lateral sclerosis (ALS) cases are familial, 20% of which are caused by a mutation in the gene encoding Cu/Zn superoxide dismutase (SOD1). Misfolding and aggregation of SOD1 are related to a gain of toxic function in SOD1-related ALS [1]. We report the autopsy findings of a case with familial motor neuron disease associated with a SOD1 C111Y mutation. Unlike previous reports of patients with a SOD1 C111Y mutation, there was no immunoreactivity for 43-kd TAR DNA-binding protein (TDP-43), which is commonly found in cytoplasmic aggregates in sporadic ALS and SOD1-unrelated familial ⁎ Corresponding author. E-mail address: [email protected] (S. Ohara). 0046-8177/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.humpath.2012.10.024
ALS [2,3]. Moreover, we found that the cytoplasmic aggregates in the present case were often immunoreactive for α-synuclein, which was colocalized with SOD1 protein, despite the absence of clinical and pathologic evidence of concurrent Parkinson disease.
2. Case report The detailed genetic and clinical features of the family, showing marked intrafamilial phenotypic variations, have been previously reported [4]. The patient noticed weakness of his right hand muscle at age 49 years and, a year later, experienced similar changes in his left hand. Neurologic findings included fasciculation of bilateral arm muscles,
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Fig. 1 H&E stain and SOD1 immunohistochemistry of neuronal cytoplasmic inclusions. These sections were firstly stained with H&E (AC) and destained and processed for SOD1 immunohistochemistry (D-F). The fibrillar inclusions show asteroid-like (A and D) or brushed-bar (B and E) appearance and often found multiple within a cell (C and F). SOD1 immunohistochemistry could reveal intracellular and extracellular fibrils (arrowheads) not well identified with conventional H&E. Bar, 20 μm.
atrophy of dorsal interosseus muscles, increased deep tendon reflexes, and bilaterally positive Babinski signs. Electromyogram revealed neurogenic patterns involving his leg muscles. He gradually developed bulbar symptoms but continued to walk until 1 year before his death. He died of respiratory failure after 4 years of illness.
3. Materials and methods The brain and spinal cord were fixed in 10% buffered formalin, and multiple tissue blocks were embedded in paraffin. Histologic examination was performed on 4-μmthick sections using hematoxylin and eosin (H&E) and Kluver-Barrera staining. Selected sections were immunostained by the streptavidin-biotin method and double immunofluorescence with the following primary antibodies: mouse monoclonal antibodies against human SOD1 (0.5 μg/
mL; MBL, Aich, Japan), phosphorylated α-synuclein (1:5000; WAKO, Osaka, Japan; 1:5000 LB509; Santa Cruz Biotech, Santa Cruz, CA), phosphorylated TDP-43 (1:5000 pS409/410; CosmoBio, Tokyo, Japan), a sheep polyclonal antibody against human SOD1 (1:100; Calbiochem, San Diego, CA), and a rabbit polyclonal antibody against TDP-43 (1:3000; Protein Tech Group, Chicago, IL). Reaction products were visualized with diaminobenzidine (DAB). For double immunofluorescence, deparaffinized sections were incubated with Sudan Black B to suppress autofluorescence, and fluorescein isothiocyanate (FITC)- or Cy3-labeled secondary antibodies were applied (Jackson Labs, Pittsburgh, PA) and were mounted in 50% glycerol in phosphate-buffered saline. Sections of midbrain from a case of classical Parkinson disease (65-year-old man, HoehenYahr Scale, stage II) harboring numerous Lewy bodies were used as positive control. To examine ultrastructural features of α-synuclein immunoreactive inclusions, areas of paraffin sections
Fig. 2 Immunostaining for SOD1 (A, C, E, G, and H) and phosphorylated α-synuclein (B, D, F, I, and J) examined on serial sections. A and B, Nucleus prehypoglossi. The intracytoplasmic inclusions (arrowhead) are immunoreactive for both SOD1 and α-synuclein. A neuron (arrow) undergoing neuronophagia contains 2 cytoplasmic inclusions, which are positive for SOD1 but negative for α-synuclein. Bar, 20 μm. C and D, Cervical anterior horn. Fibrillar aggregates in 2 neurons (indicated by arrow and arrowhead) are immunoreactive both for α-synuclein and SOD1. Bar, 50 μm. E and F, Lumbar anterior horn. SOD1 and α-synuclein are colocalized in one neuron (thin arrow), but not another (thick arrow). Bar, 100 μm. G-J, Enlarged photos of the area indicated by the arrows. The neuronal surface containing SOD1-positive inclusions (H) is covered by α-synuclein–positive cell processes (arrowheads in J). Bar, 20 μm.
α-Synuclein coaggregation with SOD1 mutation in ALS
1173
1174 immunostained by α-synuclein antibody were processed for conventional electron microscopy.
4. Results The postfixed brain weighed 1440 g with a normal external appearance. Microscopic examination revealed moderate loss of neurons with reactive astrocytosis involving the anterior horn of the spinal cord, most prominently in the lumbar segments. The cytoplasm of remaining anterior horn cells frequently contained coarse fibrils or bundles, often forming asteroid-like shapes (Fig. 1). There were no Bunina bodies. Myelin pallor was slight in the lateral corticospinal tracts but was not evident in the posterior columns. The Clarke column and posterior horn neurons were well preserved with occasional inclusions. Onuf nucleus in the
Y. Takei et al. sacral segment, which is related to bladder and rectal function, revealed intracytoplasmic inclusions in preserved neurons. Although relatively rare, inclusions were also found in glial cells. In the brainstem, the hypoglossal and ambiguus nuclei and reticular formation revealed mild neuronal loss with many cytoplasmic inclusions. In the precentral gyrus, there was a mild loss of Betz neurons. Immunohistochemically, the inclusions were immunoreactive for SOD1 but did not stain with antibodies to TDP-43. On the other hand, many inclusions were also positive for phosphorylated α-synuclein. Immunohistochemistry of serial sections and using double immunofluorescence established that SOD1 and phosphorylated α-synuclein were often colocalized (Figs. 2 and 3). In addition, in several anterior horn neurons with SOD1-positive inclusions, the motoneuron cell surface was covered with punctate α-synuclein immunoreactive structures (Fig. 2J), likely corresponding to
Fig. 3 Double immunofluorescence immunohistochemistry. The section of medulla was double labeled for SOD1 (green) and phosphorylated α-synuclein (red). In the neurons of reticular formation, colocalization of SOD1 and α-synuclein is evident in the inclusions as well as intracytoplasmic fibrils. There are α-synuclein–positive and SOD1-negative neuritic processes (arrowheads). Bar, 50 μm.
α-Synuclein coaggregation with SOD1 mutation in ALS
Fig. 4 α-Synuclein immunoelectron microscopy of an inclusion in the medullary reticular formation. A, Electron-dense immune reaction products are located on filaments of various sizes at the periphery of the inclusion. Bar, 5 μm. B, High-power view of the area indicated with arrow in A. Bar, 1 μm.
presynaptic axon terminals of axosomatic synapses. Neurons of the dorsal vagal nucleus, locus ceruleus, and substantia nigra were well preserved without Lewy bodies or Lewy neurites, confirmed by the absence of α-synuclein immunoreactivity. Ultrastructurally, inclusions were composed of radially oriented bundles of 15- to 20-nm diameter fibrils. Immunoelectron microscopy revealed that the filaments in the periphery of the inclusions were coated with α-synuclein immunoreactive product, with little immunoreactivity in the center of the inclusions (Fig. 4).
1175 possible that SOD1-related ALS and sporadic preclinical Parkinson disease may have coexisted in our patient. However, this is unlikely because there was no neuronal loss nor α-synuclein-positive inclusions in the substantia nigra and locus ceruleus and in the dorsal vagal nucleus of medulla, which are commonly affected sites in the early stage of Parkinson disease [5,6]. Sporadic or familial cases of motor neuron disease with parkinsonism have been occasionally reported, suggesting a possible common pathogenic mechanism [7,8]. Doherty et al immunohistochemically studied the spinal cords of 27 patients with motor neuron disease including 1 case of SOD1-related ALS and found a significant increase of αsynuclein staining in the anterior horn, especially involving axonal spheroids [9]. Noda et al [10] reported similar pathologic findings to our case involving an autopsy case of a 78-year-old patient clinically diagnosed with pure autonomic failure. That case showed that, in addition to the severe loss of sympathetic neurons in the intermediolateral column, there was moderate loss of neurons in the anterior horn of the spinal cord and hypoglossal and facial nuclei as well as in the medullary reticular formation, all of which were associated with the appearance of intracytoplasmic Lewy body–like hyaline inclusions. Clarke column nuclei and posterior columns were also degenerated, thus sharing the characteristic pathologic features of SOD1-related ALS. Importantly, there were neither Lewy bodies nor neuronal loss in the substantia nigra [10]. The underlying mechanisms of coaggregation of αsynuclein with SOD1 protein are speculative. However, αsynuclein is prone to aggregate by various posttranslational modifications, which favor protein-protein interaction [11]. It is possible that a “cross seeding phenomenon,” in which aggregated protein can act as a heterogeneous template inducing the misfolding and aggregation of other dissimilar proteins [12], may also apply to mutant SOD1 protein and αsynuclein. Because α-synuclein is known to be localized to the presynaptic terminal [13], interaction of mutant SOD1 protein and α-synuclein across axosomatic synapses may have occurred, triggering α-synuclein aggregation. Alternatively, it is also possible that mutant SOD1 protein and its aggregates provide a pro-oxidative cellular environment, which may impair normal α-synuclein metabolism, leading to aggregation. Wild-type SOD1 may acquire binding and toxic properties of mutant SOD1 protein through oxidative damage [14]. Therefore, α-synuclein coaggregation may also occur in sporadic ALS and in other neurodegenerative diseases in which SOD1 protein is considered a major target of oxidative damage such as Alzheimer and Parkinson diseases [15].
5. Discussion The striking finding in our patient is that phosphorylated α-synuclein was present in aggregates often colocalized with SOD1. Because α-synuclein is a major component of Lewy bodies, a pathologic hallmark of Parkinson disease, it is
Acknowledgments We thank Ms Mieko Chino, Department of Clinical Laboratory, Matsumoto Medical Center, and Ms Kayo
1176 Suzuki, Department of Analytical Science, Shinshu University School of Medicine, for their expert technical assistance. We also thank Prof Hitoshi Takahashi, Department of Neuropathology, Niigata Brain Research Institute, Niigata; Dr Hisae Sumi, Department of Neurology, Osaka University School of Medicine, for helpful discussion; and Prof Robert E. Schmidt, Department of Neuropathology, Washington University School of Medicine, St Louis, MO, for reviewing the manuscript. This study was supported by Grants-in-Aid from the Ministry of Health, Labor and Welfare, Japan.
References [1] Kerman A, Liu HN, Croul S, et al. Amyotrophic lateral sclerosis is a non-amyloid disease in which extensive misfolding of SOD1 is unique to the familial form. Acta Neuropathol 2010;119:335-44. [2] Sumi H, Kato S, Mochimaru Y, et al. Nuclear TAR DNA binding protein 43 expression in spinal cord neurons correlates with the clinical course in amyotrophic lateral sclerosis. J Neuropathol Exp Neurol 2009;68:37-47. [3] Okamoto Y, Ihara M, Urushitani M, et al. An autopsy case of SOD1related ALS with TDP-43 positive inclusions. Neurology 2011;77: 1993-5. [4] Nakamura A, Hineno A, Yoshida K, et al. Marked intrafamilial phenotypic variation in a family with SOD1 C111Y mutation. Amyotroph Lateral Scler 2012;13:479-86.
Y. Takei et al. [5] Braak H, Del Tredici K, Rub U, et al. Staging of brain pathology related to sporadic Parkinson's disease. Neurobiol Aging 2003;24: 197-211. [6] Kingsbury AE, Bandopadhyay R, Silveira-Moriyama L, et al. Brain stem pathology in Parkinson's disease: an evaluation of the Braak staging model. Mov Disord 2010;25:2508-15. [7] Delisle MB, Gorce P, Hirsch E, et al. Motor neuron disease, parkinsonism and dementia. Report of a case with diffuse Lewy body–like intracytoplasmic inclusions. Acta Neuropathol 1987;75:104-8. [8] Al Qureshi, Wilmot G, Dihenia B, et al. Motor neuron disease with Parkisonism. Arch Neurol 1996;53:987-91. [9] Doherty MJ, Bird T, Leverenz JB. Alpha-synuclein in motor neuron disease: an immunohistologic study. Acta Neuropathol 2004;107: 169-75. [10] Noda K, Katayama S, Watanabe C, et al. Pure autonomic failure with motor neuron disease: report of a clinical study and postmortem examination of a patient. J Neurol Neurosurg Psychiatry 1996;60: 351-2. [11] Breydo L, Wu JW, Uversky VN. α-synuclein misfolding and Parkinson's disease. Biochim Biophys Acta 2012;1822:261-85. [12] Polymenidou M, Cleaveland DW. The seeds of neurodegeneration: prion-like spreading in ALS. Cell 2011;147:498-508. [13] Schulz-Schaeffer WJ. The synaptic pathology of alpha-synuclein aggregation in dementia with Lewy bodies, Parkinson's disease and Parkinson's disease dementia. Acta Neuropathol 2010;120:131-43. [14] Ezzi SA, Urushitani M, Julien J-P. Wild-type superoxide dismutase acquires binding and toxic properties of ALS-linked mutant forms through oxidation. J Neurochem 2007;102:170-8. [15] Choi J, Rees HD, Weintraub ST, et al. Oxidative modifications and aggregation of Cu, Zn-Superoxide dismutase associated with Alzheimer and Parkinson diseases. J Biol Chem 2005;280:11648-55.
www.elsevier.com/locate/humpath
Case study
α-Synuclein coaggregation in familial amyotrophic lateral sclerosis with SOD1 gene mutation Yo-ichi Takei MD a , Kenya Oguchi MD a , Hiroshi Koshihara MD a , Akiyo Hineno MD b , Akinori Nakamura MD b , Shinji Ohara MD a,⁎ a
Department of Neurology, Matsumoto Medical Center, Chushin-Matsumoto Hospital, Kotobuski 811, Matsumoto, 399–0021 Japan b Department of Medicine (Neurology and Rheumatology), Shinshu University School of Medicine, Asahi 3-1-1, 390–8621, Japan Received 19 September 2012; revised 21 October 2012; accepted 31 October 2012
Keywords: Familial amyotrophic lateral sclerosis; Copper/zinc superoxide dismutase (SOD1); α-Synuclein; Co-aggregation; Immunohistochemistry
Summary Immunohistochemical studies were performed on postmortem brain and spinal cord from a patient with familial amyotrophic lateral sclerosis characterized by a C111Y mutation in the Cu/Zn superoxide dismutase gene. Clinically, the patient presented with classical amyotrophic lateral sclerosis and died of respiratory failure at age 53 years without ventilator dependence, 4 years after the onset. Pathologically, loss of motor neurons was more extensive than upper motor neurons. Lower motor neurons developed massive intracellular cytoplasmic neuronal inclusions, which were immunoreactive for Cu/Zn superoxide dismutase and phosphorylated α-synuclein, often colocalized. The inclusions were TAR DNA-binding protein 43 negative. The clinicopathologic significance of coaggregation of α-synuclein and Cu/Zn superoxide dismutase protein, a novel finding in neurodegenerative disorders, needs further investigation. © 2013 Elsevier Inc. All rights reserved.
1. Introduction Approximately 10% of amyotrophic lateral sclerosis (ALS) cases are familial, 20% of which are caused by a mutation in the gene encoding Cu/Zn superoxide dismutase (SOD1). Misfolding and aggregation of SOD1 are related to a gain of toxic function in SOD1-related ALS [1]. We report the autopsy findings of a case with familial motor neuron disease associated with a SOD1 C111Y mutation. Unlike previous reports of patients with a SOD1 C111Y mutation, there was no immunoreactivity for 43-kd TAR DNA-binding protein (TDP-43), which is commonly found in cytoplasmic aggregates in sporadic ALS and SOD1-unrelated familial ⁎ Corresponding author. E-mail address: [email protected] (S. Ohara). 0046-8177/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.humpath.2012.10.024
ALS [2,3]. Moreover, we found that the cytoplasmic aggregates in the present case were often immunoreactive for α-synuclein, which was colocalized with SOD1 protein, despite the absence of clinical and pathologic evidence of concurrent Parkinson disease.
2. Case report The detailed genetic and clinical features of the family, showing marked intrafamilial phenotypic variations, have been previously reported [4]. The patient noticed weakness of his right hand muscle at age 49 years and, a year later, experienced similar changes in his left hand. Neurologic findings included fasciculation of bilateral arm muscles,
1172
Y. Takei et al.
Fig. 1 H&E stain and SOD1 immunohistochemistry of neuronal cytoplasmic inclusions. These sections were firstly stained with H&E (AC) and destained and processed for SOD1 immunohistochemistry (D-F). The fibrillar inclusions show asteroid-like (A and D) or brushed-bar (B and E) appearance and often found multiple within a cell (C and F). SOD1 immunohistochemistry could reveal intracellular and extracellular fibrils (arrowheads) not well identified with conventional H&E. Bar, 20 μm.
atrophy of dorsal interosseus muscles, increased deep tendon reflexes, and bilaterally positive Babinski signs. Electromyogram revealed neurogenic patterns involving his leg muscles. He gradually developed bulbar symptoms but continued to walk until 1 year before his death. He died of respiratory failure after 4 years of illness.
3. Materials and methods The brain and spinal cord were fixed in 10% buffered formalin, and multiple tissue blocks were embedded in paraffin. Histologic examination was performed on 4-μmthick sections using hematoxylin and eosin (H&E) and Kluver-Barrera staining. Selected sections were immunostained by the streptavidin-biotin method and double immunofluorescence with the following primary antibodies: mouse monoclonal antibodies against human SOD1 (0.5 μg/
mL; MBL, Aich, Japan), phosphorylated α-synuclein (1:5000; WAKO, Osaka, Japan; 1:5000 LB509; Santa Cruz Biotech, Santa Cruz, CA), phosphorylated TDP-43 (1:5000 pS409/410; CosmoBio, Tokyo, Japan), a sheep polyclonal antibody against human SOD1 (1:100; Calbiochem, San Diego, CA), and a rabbit polyclonal antibody against TDP-43 (1:3000; Protein Tech Group, Chicago, IL). Reaction products were visualized with diaminobenzidine (DAB). For double immunofluorescence, deparaffinized sections were incubated with Sudan Black B to suppress autofluorescence, and fluorescein isothiocyanate (FITC)- or Cy3-labeled secondary antibodies were applied (Jackson Labs, Pittsburgh, PA) and were mounted in 50% glycerol in phosphate-buffered saline. Sections of midbrain from a case of classical Parkinson disease (65-year-old man, HoehenYahr Scale, stage II) harboring numerous Lewy bodies were used as positive control. To examine ultrastructural features of α-synuclein immunoreactive inclusions, areas of paraffin sections
Fig. 2 Immunostaining for SOD1 (A, C, E, G, and H) and phosphorylated α-synuclein (B, D, F, I, and J) examined on serial sections. A and B, Nucleus prehypoglossi. The intracytoplasmic inclusions (arrowhead) are immunoreactive for both SOD1 and α-synuclein. A neuron (arrow) undergoing neuronophagia contains 2 cytoplasmic inclusions, which are positive for SOD1 but negative for α-synuclein. Bar, 20 μm. C and D, Cervical anterior horn. Fibrillar aggregates in 2 neurons (indicated by arrow and arrowhead) are immunoreactive both for α-synuclein and SOD1. Bar, 50 μm. E and F, Lumbar anterior horn. SOD1 and α-synuclein are colocalized in one neuron (thin arrow), but not another (thick arrow). Bar, 100 μm. G-J, Enlarged photos of the area indicated by the arrows. The neuronal surface containing SOD1-positive inclusions (H) is covered by α-synuclein–positive cell processes (arrowheads in J). Bar, 20 μm.
α-Synuclein coaggregation with SOD1 mutation in ALS
1173
1174 immunostained by α-synuclein antibody were processed for conventional electron microscopy.
4. Results The postfixed brain weighed 1440 g with a normal external appearance. Microscopic examination revealed moderate loss of neurons with reactive astrocytosis involving the anterior horn of the spinal cord, most prominently in the lumbar segments. The cytoplasm of remaining anterior horn cells frequently contained coarse fibrils or bundles, often forming asteroid-like shapes (Fig. 1). There were no Bunina bodies. Myelin pallor was slight in the lateral corticospinal tracts but was not evident in the posterior columns. The Clarke column and posterior horn neurons were well preserved with occasional inclusions. Onuf nucleus in the
Y. Takei et al. sacral segment, which is related to bladder and rectal function, revealed intracytoplasmic inclusions in preserved neurons. Although relatively rare, inclusions were also found in glial cells. In the brainstem, the hypoglossal and ambiguus nuclei and reticular formation revealed mild neuronal loss with many cytoplasmic inclusions. In the precentral gyrus, there was a mild loss of Betz neurons. Immunohistochemically, the inclusions were immunoreactive for SOD1 but did not stain with antibodies to TDP-43. On the other hand, many inclusions were also positive for phosphorylated α-synuclein. Immunohistochemistry of serial sections and using double immunofluorescence established that SOD1 and phosphorylated α-synuclein were often colocalized (Figs. 2 and 3). In addition, in several anterior horn neurons with SOD1-positive inclusions, the motoneuron cell surface was covered with punctate α-synuclein immunoreactive structures (Fig. 2J), likely corresponding to
Fig. 3 Double immunofluorescence immunohistochemistry. The section of medulla was double labeled for SOD1 (green) and phosphorylated α-synuclein (red). In the neurons of reticular formation, colocalization of SOD1 and α-synuclein is evident in the inclusions as well as intracytoplasmic fibrils. There are α-synuclein–positive and SOD1-negative neuritic processes (arrowheads). Bar, 50 μm.
α-Synuclein coaggregation with SOD1 mutation in ALS
Fig. 4 α-Synuclein immunoelectron microscopy of an inclusion in the medullary reticular formation. A, Electron-dense immune reaction products are located on filaments of various sizes at the periphery of the inclusion. Bar, 5 μm. B, High-power view of the area indicated with arrow in A. Bar, 1 μm.
presynaptic axon terminals of axosomatic synapses. Neurons of the dorsal vagal nucleus, locus ceruleus, and substantia nigra were well preserved without Lewy bodies or Lewy neurites, confirmed by the absence of α-synuclein immunoreactivity. Ultrastructurally, inclusions were composed of radially oriented bundles of 15- to 20-nm diameter fibrils. Immunoelectron microscopy revealed that the filaments in the periphery of the inclusions were coated with α-synuclein immunoreactive product, with little immunoreactivity in the center of the inclusions (Fig. 4).
1175 possible that SOD1-related ALS and sporadic preclinical Parkinson disease may have coexisted in our patient. However, this is unlikely because there was no neuronal loss nor α-synuclein-positive inclusions in the substantia nigra and locus ceruleus and in the dorsal vagal nucleus of medulla, which are commonly affected sites in the early stage of Parkinson disease [5,6]. Sporadic or familial cases of motor neuron disease with parkinsonism have been occasionally reported, suggesting a possible common pathogenic mechanism [7,8]. Doherty et al immunohistochemically studied the spinal cords of 27 patients with motor neuron disease including 1 case of SOD1-related ALS and found a significant increase of αsynuclein staining in the anterior horn, especially involving axonal spheroids [9]. Noda et al [10] reported similar pathologic findings to our case involving an autopsy case of a 78-year-old patient clinically diagnosed with pure autonomic failure. That case showed that, in addition to the severe loss of sympathetic neurons in the intermediolateral column, there was moderate loss of neurons in the anterior horn of the spinal cord and hypoglossal and facial nuclei as well as in the medullary reticular formation, all of which were associated with the appearance of intracytoplasmic Lewy body–like hyaline inclusions. Clarke column nuclei and posterior columns were also degenerated, thus sharing the characteristic pathologic features of SOD1-related ALS. Importantly, there were neither Lewy bodies nor neuronal loss in the substantia nigra [10]. The underlying mechanisms of coaggregation of αsynuclein with SOD1 protein are speculative. However, αsynuclein is prone to aggregate by various posttranslational modifications, which favor protein-protein interaction [11]. It is possible that a “cross seeding phenomenon,” in which aggregated protein can act as a heterogeneous template inducing the misfolding and aggregation of other dissimilar proteins [12], may also apply to mutant SOD1 protein and αsynuclein. Because α-synuclein is known to be localized to the presynaptic terminal [13], interaction of mutant SOD1 protein and α-synuclein across axosomatic synapses may have occurred, triggering α-synuclein aggregation. Alternatively, it is also possible that mutant SOD1 protein and its aggregates provide a pro-oxidative cellular environment, which may impair normal α-synuclein metabolism, leading to aggregation. Wild-type SOD1 may acquire binding and toxic properties of mutant SOD1 protein through oxidative damage [14]. Therefore, α-synuclein coaggregation may also occur in sporadic ALS and in other neurodegenerative diseases in which SOD1 protein is considered a major target of oxidative damage such as Alzheimer and Parkinson diseases [15].
5. Discussion The striking finding in our patient is that phosphorylated α-synuclein was present in aggregates often colocalized with SOD1. Because α-synuclein is a major component of Lewy bodies, a pathologic hallmark of Parkinson disease, it is
Acknowledgments We thank Ms Mieko Chino, Department of Clinical Laboratory, Matsumoto Medical Center, and Ms Kayo
1176 Suzuki, Department of Analytical Science, Shinshu University School of Medicine, for their expert technical assistance. We also thank Prof Hitoshi Takahashi, Department of Neuropathology, Niigata Brain Research Institute, Niigata; Dr Hisae Sumi, Department of Neurology, Osaka University School of Medicine, for helpful discussion; and Prof Robert E. Schmidt, Department of Neuropathology, Washington University School of Medicine, St Louis, MO, for reviewing the manuscript. This study was supported by Grants-in-Aid from the Ministry of Health, Labor and Welfare, Japan.
References [1] Kerman A, Liu HN, Croul S, et al. Amyotrophic lateral sclerosis is a non-amyloid disease in which extensive misfolding of SOD1 is unique to the familial form. Acta Neuropathol 2010;119:335-44. [2] Sumi H, Kato S, Mochimaru Y, et al. Nuclear TAR DNA binding protein 43 expression in spinal cord neurons correlates with the clinical course in amyotrophic lateral sclerosis. J Neuropathol Exp Neurol 2009;68:37-47. [3] Okamoto Y, Ihara M, Urushitani M, et al. An autopsy case of SOD1related ALS with TDP-43 positive inclusions. Neurology 2011;77: 1993-5. [4] Nakamura A, Hineno A, Yoshida K, et al. Marked intrafamilial phenotypic variation in a family with SOD1 C111Y mutation. Amyotroph Lateral Scler 2012;13:479-86.
Y. Takei et al. [5] Braak H, Del Tredici K, Rub U, et al. Staging of brain pathology related to sporadic Parkinson's disease. Neurobiol Aging 2003;24: 197-211. [6] Kingsbury AE, Bandopadhyay R, Silveira-Moriyama L, et al. Brain stem pathology in Parkinson's disease: an evaluation of the Braak staging model. Mov Disord 2010;25:2508-15. [7] Delisle MB, Gorce P, Hirsch E, et al. Motor neuron disease, parkinsonism and dementia. Report of a case with diffuse Lewy body–like intracytoplasmic inclusions. Acta Neuropathol 1987;75:104-8. [8] Al Qureshi, Wilmot G, Dihenia B, et al. Motor neuron disease with Parkisonism. Arch Neurol 1996;53:987-91. [9] Doherty MJ, Bird T, Leverenz JB. Alpha-synuclein in motor neuron disease: an immunohistologic study. Acta Neuropathol 2004;107: 169-75. [10] Noda K, Katayama S, Watanabe C, et al. Pure autonomic failure with motor neuron disease: report of a clinical study and postmortem examination of a patient. J Neurol Neurosurg Psychiatry 1996;60: 351-2. [11] Breydo L, Wu JW, Uversky VN. α-synuclein misfolding and Parkinson's disease. Biochim Biophys Acta 2012;1822:261-85. [12] Polymenidou M, Cleaveland DW. The seeds of neurodegeneration: prion-like spreading in ALS. Cell 2011;147:498-508. [13] Schulz-Schaeffer WJ. The synaptic pathology of alpha-synuclein aggregation in dementia with Lewy bodies, Parkinson's disease and Parkinson's disease dementia. Acta Neuropathol 2010;120:131-43. [14] Ezzi SA, Urushitani M, Julien J-P. Wild-type superoxide dismutase acquires binding and toxic properties of ALS-linked mutant forms through oxidation. J Neurochem 2007;102:170-8. [15] Choi J, Rees HD, Weintraub ST, et al. Oxidative modifications and aggregation of Cu, Zn-Superoxide dismutase associated with Alzheimer and Parkinson diseases. J Biol Chem 2005;280:11648-55.