Human Pathology (2013) xx, xxx–xxx

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:
Summary Immunohistochemical studies were performed on postmortem brain and spinal cord from a
Familial amyotrophic
patient with familial amyotrophic lateral sclerosis characterized by a C111Y mutation in the Cu/Zn
lateral sclerosis;
superoxide dismutase gene. Clinically, the patient presented with classical amyotrophic lateral sclerosis
Copper/zinc superoxide
and died of respiratory failure at age 53 years without ventilator dependence, 4 years after the onset.
dismutase (SOD1);
Pathologically, loss of motor neurons was more extensive than upper motor neurons. Lower motor
α-Synuclein;
neurons developed massive intracellular cytoplasmic neuronal inclusions, which were immunoreactive
Co-aggregation;
for Cu/Zn superoxide dismutase and phosphorylated α-synuclein, often colocalized. The inclusions
Immunohistochemistry
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.
© 2012 Elsevier Inc. All rights reserved.

1. Introduction ALS [2,3]. Moreover, we found that the cytoplasmic

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: oharas@cmatumoto.hosp.go.jp (S. Ohara).
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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2 Y. Takei et al.

Fig. 1 H&E stain and SOD1 immunohistochemistry of neuronal cytoplasmic inclusions. These sections were firstly stained with H&E (A-
C) 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. Electro-
myogram 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-μm-
thick sections using hematoxylin and eosin (H&E) and
Kluver-Barrera staining. Selected sections were immuno-
stained 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; Calbio-
chem, 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, Hoehen-
Yahr 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 3
4 Y. Takei et al.

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
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 immunoreac-
tive 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 immuno-
reactive 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. Immu-
noelectron 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).
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
5
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 α-
synuclein 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 α-
synuclein with SOD1 protein are speculative. However, α-
synuclein 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 α-
synuclein. 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 proper-
ties 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].
Acknowledgments
We thank Ms Mieko Chino, Department of Clinical
Laboratory, Matsumoto Medical Center, and Ms Kayo
6 Y. Takei et al.
Suzuki, Department of Analytical Science, Shinshu Univer-
sity 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.
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