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Research Report
Immunohistochemical localization of apoptosomerelated proteins in Lewy bodies in Parkinson's disease and dementia with Lewy bodies Yasuhiro Kawamotoa,b,n, Hidefumi Itoc, Takashi Ayakia, Ryosuke Takahashia a
Department of Neurology, Faculty of Medicine, Kyoto University, Kyoto, Japan Department of Neurology, Seijinkai Rakusaishimizu Hospital, Kyoto, Japan c Department of Neurology, Wakayama Medical University, Wakayama, Japan b
art i cle i nfo
ab st rac t
Article history:
Apoptotic stimuli induce the release of cytochrome c from the mitochondria to the cytosol,
Accepted 5 May 2014
and this released cytochrome c promotes the formation of the apoptosome, which contains cytochrome c, Apaf-1 and caspase-9, resulting in the activation of caspase-9
Keywords: Apoptosome Caspase-9 Lewy body Mitochondria Parkinson's disease
and the promotion of apoptotic cell death. To investigate the role of the apoptosome in patients with Parkinson's disease (PD), we performed immunohistochemical studies on apoptosome-related proteins in formalin-fixed, paraffin-embedded sections from 8 normal subjects, 10 patients with PD and 5 patients with dementia with Lewy bodies (DLB). Furthermore, we performed double-labeling immunohistochemistry for cleaved caspase-9 and CD68 in some sections from 8 normal subjects and 10 patients with PD. In the substantia nigra and locus ceruleus from both control and PD cases, the somata and processes of melanin-containing neurons were immunostained for cytochrome c, Apaf-1 and caspase-9. In the same areas from the PD cases, brainstem-type Lewy bodies were also immunoreactive for cytochrome c, Apaf-1 and caspase-9, and cleaved caspase-9 immunoreactivity was detected in brainstem-type Lewy bodies and CD68-immunopositive microglia. In addition to brainstem-type Lewy bodies, cortical Lewy bodies were also immunoreactive for these apoptosome-related proteins in the frontal and temporal cortices from the DLB cases. Our results suggest that apoptosome formation accompanied by caspase-9 activation may occur in the substantia nigra and locus ceruleus in brains affected by PD, and that a mitochondria-dependent apoptotic pathway may be partially associated with the pathogenesis of PD. & 2014 Published by Elsevier B.V.
Abbreviations: PD,
Parkinson's disease; CARD,
caspase recruitment domain; TUNEL,
mediated deoxyuridine triphosphate- biotin nick end labeling; DLB,
terminal deoxynucleotidyl transferase-
dementia with Lewy bodies; MPTP,
1-methyl-4-phenyl- 1,2,3,6-
tetrahydropyridine; MPPþ, 1-methyl-4-phenylpyridinium ion; H&E, hematoxylin and eosin; PBS, phosphate-buffered saline n Corresponding author at: Department of Neurology, Faculty of Medicine, Kyoto University, 54 Shogoin-Kawaharacho, Sakyoku, Kyoto 606-8507, Japan. Fax: þ81 75 751 3265. E-mail address: [email protected] (Y. Kawamoto). http://dx.doi.org/10.1016/j.brainres.2014.05.007 0006-8993/& 2014 Published by Elsevier B.V.
Please cite this article as: Kawamoto, Y., et al., Immunohistochemical localization of apoptosome-related proteins in Lewy bodies in Parkinson's disease and dementia with Lewy bodies. Brain Research (2014), http://dx.doi.org/10.1016/j. brainres.2014.05.007
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1.
Introduction
Parkinson's disease (PD) is one of the most common neurodegenerative disorders, characterized clinically by the progressive development of extrapyramidal signs consisting of a resting tremor, muscular rigidity, bradykinesia and postural instability (Hughes et al., 1992). In brains from patients with PD, neuronal loss associated with Lewy bodies is observed in several areas, including the substantia nigra and locus ceruleus (Pollanen et al., 1993). Brainstem-type Lewy bodies in the substantia nigra and locus ceruleus are eosinophilic inclusions with a hyaline core and a pale halo (Pollanen et al., 1993). Missense mutations of the α-synuclein gene (Polymeropoulos et al., 1997; Krüger et al., 1998; Zarranz et al., 2004) and multiplications of the α-synuclein locus (Singleton et al., 2003; Chartier-Harlin et al., 2004; Ibáñez et al., 2004) have been reported to be implicated in the autosomal dominant familial forms of PD. Lewy bodies are ubiquitinated (Kuzuhara et al., 1988), and α-synuclein is a main constituent of Lewy bodies (Goedert et al., 2013; Wakabayashi et al., 2013); however, the mechanism of Lewy body formation remains unclear. The mitochondria play an important role in apoptotic cell death. Upon receiving various apoptotic stimuli, cytochrome c is released from the mitochondria into the cytosol, and this released cytochrome c promotes the formation of the apoptosome, which contains cytochrome c, Apaf-1 and caspase-9 (Li et al., 1997; Adams and Cory, 2002; Riedl and Salvesen, 2007; Park, 2012). Apaf-1 is the central component of the apoptosome, and contains an N-terminal caspase recruitment domain (CARD), a nucleotide-binding domain and a C-terminal WD40 repeat domain (Adams and Cory, 2002; Riedl and Salvesen, 2007; Park, 2012). The CARD of Apaf-1
interacts with the N-terminal CARD of procaspase-9, resulting in the recruitment of procaspase-9 to the apoptosome (Adams and Cory, 2002; Riedl and Salvesen, 2007; Park, 2012). Procaspase-9 is then activated by dimerization and autocatalytic cleavage at Asp315, generating the large and small subunits (Srinivasula et al., 1998; Würstle et al., 2012). Activated caspase-9 converts procaspse-3 to the activated form of caspase-3, and promotes the downstream caspase cascade, thus leading to apoptotic cell death (Li et al., 1997; Srinivasula et al., 1998; Würstle et al., 2012). Activated caspase-3 feeds back on procaspase-9 and activates procaspase-9 by processing it at Asp330, yielding the large and small fragments, and thus amplifying the caspase cascade (Srinivasula et al., 1998; Würstle et al., 2012). DNA fragmentation associated with apoptosis was reported to be detected in pigmented neurons in the substantia nigra from patients with PD using the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling (TUNEL) method (Mochizuki et al., 1996). TUNELpositive glial cells were also shown to be localized to the substantias nigra from patients with PD (Mochizuki et al., 1996; Jellinger, 2000). Tatton and colleagues demonstrated the presence of nuclear chromatin condensation in melanized neurons in the substantia nigra from patients with PD using fluorescent nucleic acid-binding dyes such as YOYO-1 (Tatton et al., 1998; Tatton, 2000). In the present study, we performed immunohistochemical studies on apoptosomerelated proteins using autopsied brains from 8 normal subjects, 10 patients with PD and 5 patients with dementia with Lewy bodies (DLB), and found the immunohistochemical localization of apoptosome-related proteins in brainstemtype Lewy bodies in the substantia nigra and locus ceruleus and in cortical Lewy bodies in the frontal and temporal cortices.
Fig. 1 – Cytochrome c immunoreactivity in the substantia nigra from normal subjects (A: Control 2) and patients with PD (B: PD 6; C, D: PD 3). Cytochrome c immunoreactivitiy was observed in the somata and processes of melanin-containing neurons (A, B). Moderately immunopositive brainstem-type Lewy bodies were detected in some remaining neurons (B, arrow). Cytochrome c immunoreactivity was localized to the whole body (C) or the marginal zone (D) of the core of the Lewy bodies. Scale bars¼80 μm (A), 40 μm (B), and 20 μm (C and D). Please cite this article as: Kawamoto, Y., et al., Immunohistochemical localization of apoptosome-related proteins in Lewy bodies in Parkinson's disease and dementia with Lewy bodies. Brain Research (2014), http://dx.doi.org/10.1016/j. brainres.2014.05.007
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2.
Results
2.1. Cytochrome c immunoreactivity in normal and PD brains In the substantia nigra and locus ceruleus from the control cases, cytochrome c immunoreactivitiy was observed in the somata and processes of melanin-containing neurons (Fig. 1A). In the same areas from the PD cases, cytochrome c immunoreactivity was spared in the remaining neurons (Fig. 1B), and moderately immunopositive brainstem-type Lewy bodies were found in some remaining neurons (Fig. 1B). The core of the Lewy bodies was immunostained for cytochrome c (Fig. 1C), and cytochrome c immunoreactivity occasionally accumulated in the marginal zone of the core of the Lewy bodies (Fig. 1D). A semiquantitative evaluation of the substantia nigra from all 10 PD cases demonstrated that the average percentage of cytochrome c-immunoreactive Lewy bodies was 64%.
2.2.
Apaf-1 immunoreactivity in normal and PD brains
In the substantia nigra and locus ceruleus from the control cases, Apaf-1 immunoreactivitiy was observed in the somata and processes of melanin-containing neurons (Fig. 2A). In the same areas from the PD cases, Apaf-1 immunoreactivity was spared in the remaining neurons (Fig. 2B), and strongly immunopositive brainstem-type Lewy bodies were found in some remaining neurons (Fig. 2B). Apaf-1 immunoreactivity was generally localized to the halo of the Lewy bodies (Fig. 2C and D). A semiquantitative evaluation of the substantia nigra
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from all 10 PD cases demonstrated that the average percentage of Apaf-1-immunoreactive Lewy bodies was 76%.
2.3.
Caspase-9 immunoreactivity in normal and PD brains
In the substantia nigra and locus ceruleus from the control cases, caspase-9 immunoreactivity was observed in the somata and processes of melanin-containing neurons (Fig. 3A). In the same areas from the PD cases, caspase-9 immunoreactivity was spared in the remaining neurons (Fig. 3B), and intensely immunopositive brainstem-type Lewy bodies were found in some remaining neurons (Fig. 3B). Caspase- 9 immunoreactivity was generally localized to the halo of the Lewy bodies (Fig. 3C and D). A semiquantitative evaluation of the substantia nigra from all 10 PD cases demonstrated that the average percentage of caspase-9immunoreactive Lewy bodies was 80%.
2.4.
Cleaved caspase-9 immunoreactivity in PD brains
Immunohistochemical studies in the substantia nigra and locus ceruleus from the PD cases using two types of anticleaved caspase-9 antibodies showed that strongly immunopositive Lewy body-containing neurons were scattered (Fig. 4A and D), and that cleaved caspase-9 immunoreactivity was localized to the core (Fig. 4B and E) or halo (Fig. 4C and F) of brainstem-type Lewy bodies. A semiquantitative evaluation of the substantia nigra from all 10 PD cases demonstrated that the average percentages of Lewy bodies immunostained with the anti-cleaved caspase-9 antibody (h331) and the anti-cleaved caspase-9 antibody (Asp330) were 72% and 78%, respectively.
Fig. 2 – Apaf-1 immunoreactivity in the substantia nigra from normal subjects (A: Control 2) and patients with PD (B: PD 5; C: PD 3), and in the locus ceruleus from patients with PD (D: PD 6). Apaf-1 immunoreactivitiy was detected in the somata and processes of melanin-containing neurons (A and B). Strongly immunopositive brainstem- type Lewy bodies were detected in some remaining neurons (B, arrow). The brainstem- type Lewy bodies showed a ring-shaped immunostaining pattern (C and D). Scale bars¼80 μm (A), 40 μm (B), and 20 μm (C and D). Please cite this article as: Kawamoto, Y., et al., Immunohistochemical localization of apoptosome-related proteins in Lewy bodies in Parkinson's disease and dementia with Lewy bodies. Brain Research (2014), http://dx.doi.org/10.1016/j. brainres.2014.05.007
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Fig. 3 – Caspase-9 immunoreactivity in the substantia nigra from normal subjects (A: Control 4) and patients with PD (B: PD 1; C: PD 7; D PD 5). Caspase-9 immunoreactivitiy was observed in the somata and processes of melanin-containing neurons (A and B). Intensely immunopositive brainstem-type Lewy bodies were detected in some remaining neurons (B, arrow). The brainstem-type Lewy bodies showed a ring- shaped immunostaining pattern (C and D). Scale bars¼ 80 μm (A), 40 μm (B), and 20 μm (C and D).
Fig. 4 – Immunohistochemical localization of cleaved caspase-9 in the substantia nigra (A, B, D, and E) and locus ceruleus (C and F) from patients with PD (A: PD 2; B: PD 1; C: PD 3; D, F: PD 5; E: PD 6) using the anti-cleaved caspase-9 antibody (h331) (A–C) and another anti-cleaved caspase-9 antibody (Asp330) (D–F). Strongly immunopositive Lewy body-bearing neurons were scattered (A and D), and cleaved caspase-9 immunoreactivity was localized to the core (B and E) or halo (C and F) of the brainstem-type Lewy bodies. Scale bars ¼40 μm (A and D), 20 μm (B, C, E, and F).
2.5. Double-labeling immunofluorescence for cleaved caspase-9 and CD68 in normal and PD brains Midbrain sections double-immunostained with the anti-cleaved caspase-9 antibody (h331) (Fig. 5A) or another anti-cleaved caspase-9 antibody (Asp330) (Fig. 5D) plus the anti-CD68 antibody (Fig. 5B and E) showed that cleaved caspase-9
immunoreactivity was observed in CD68-immunopositive microglia in the substantia nigra from the PD cases (Fig. 5C and F). As compared with the PD cases, the activation of caspase-9 was not conspicuous in the substantia nigra from the control cases, and the average percentages of CD68-immunopositive microglia immunostained with the anti-cleaved caspase-9
Please cite this article as: Kawamoto, Y., et al., Immunohistochemical localization of apoptosome-related proteins in Lewy bodies in Parkinson's disease and dementia with Lewy bodies. Brain Research (2014), http://dx.doi.org/10.1016/j. brainres.2014.05.007
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Fig. 5 – Double-immunofluorescence staining for cleaved caspase-9 (A: h331; D: Asp331) and CD68 (B, E) in the substantia nigra from patients with PD (A–C: PD 4; D- F: PD 10). The merged images showed that the CD68-immunopositive microglia contained cleaved caspase-9 immunoreactivity (C, F). Scale bars¼ 20 μm (A–F).
Fig. 6 – Immunoreactivities of apoptosome-related proteins in the frontal cortex from patients with DLB (A–C: DLB 3; D–F: DLB 4). Cortical Lewy bodies were intensely immunostained with the anti-cytochrome c antibody (A), the anti-Apaf-1 antibody (B) anti-caspase-9 antibody (C), the anti-cleaved antibody (h331) (D) and the anti-cleaved caspase-9 antibody (Asp330) (E). No specific immunopositive staining was detected in negative control sections which were incubated with the anti-cleaved caspase-9 antibody (h331) preabsorbed with an excess amount of the blocking peptide (sc-22812P) (F). Scale bars¼ 20 μm (A–E), and 40 μm (F).
antibody (h331) and the anti-cleaved caspase-9 (Asp330) in the PD group were 94% and 96%, respectively, and those in the control group were 24% and 22%, respectively.
2.6. Immunoreactivities of apoptosome-related proteins in cortical Lewy bodies In the frontal and temporal cortices from the DLB cases, cortical Lewy bodies were intensely immunoreactive for cytochrome c (Fig. 6A), Apaf-1 (Fig. 6B), caspase-9 (Fig. 6C) and cleaved caspase-9 (Fig. 6D and E). Immunoreactivites of these apoptosome-related proteins were localized over the
entire bodies of the Lewy bodies (Fig. 6A and E). No specific immunopositive staining was detected in negative control sections which were incubated with the anti-cleaved caspase-9 (h331) antibody preabsorbed with an excess amount of the blocking peptide (sc-22812P) (Fig. 6F). The preabsorption experiments for the anti-Apaf-1 and anti-caspase-9 antibodies also resulted in no specific immunopositive staining (data not shown). A semiquantitative evaluation of the parahippocampal cortex from all 5 DLB cases demonstrated that the average percentages of Lewy bodies immunostained with the anti-cytochrome c antibody, the anti-Apaf-1 antibody, the anti-caspase-9 antibody, the anti-cleaved caspase-9
Please cite this article as: Kawamoto, Y., et al., Immunohistochemical localization of apoptosome-related proteins in Lewy bodies in Parkinson's disease and dementia with Lewy bodies. Brain Research (2014), http://dx.doi.org/10.1016/j. brainres.2014.05.007
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antibody (h331) and the anti-cleaved caspase-9 antibody (Asp330) were 60%, 72%, 74%, 66% and 72%, respectively.
2.7. Relationship between disease duration and Lewy bodies immunoreactive for apoptosome-related proteins or microglial cells immunostained for activated caspase-9
in the substantia nigra from all 10 patients with PD were summarized in Table 3. There was no significant relationship between the duration of disease and the activation of caspase-9 in microglial cells.
2.8. Activated caspase-3 immunoreactivity in PD and DLB brains
The percentages of Lewy bodies immunoreactive for cytochrome c, Apaf-1, caspase-9 and cleaved caspase-9 in the substantia nigra from all 10 patients with PD were summarized in Table 2. There was no significant relationship between the duration of disease and the proportion of Lewy bodies immunoreactive for apoptosome-related proteins. The percentages of CD68-immunopositive microglia immunostained with the anti- cleaved caspase-9 antibodies
In the substantia nigra and locus ceruleus from the PD cases, some brainstem-type Lewy bodies were strongly immunoreactive for activated caspase-3 (Fig. 7A and B), and activated caspase-3 immunoreactivity was localized to the halo (Fig. 7A) or core (Fig. 7B) of the Lewy bodies. In the frontal and temporal cortices from the DLB cases, some cortical Lewy bodies were intensely immunoreactive
Table 1 – Clinical profiles of all cases. Case Control Control Control Control Control Control Control Control PD 1 PD 2 PD 3 PD 4 PD 5 PD 6 PD 7 PD 8 PD 9 PD 10 DLB 1 DLB 2 DLB 3 DLB 4 DLB 5
1 2 3 4 5 6 7 8
Age (yr)/Sex
Diagnosis
Duration of illness (yr)/postmortem delay (h)
62/M 68/M 73/M 68/F 75/M 69/M 54/M 78/F 90/F 76/M 79/F 81/F 74/M 66/M 76/M 88/M 67/M 78/F 81/M 69/M 69/F 69/M 86/M
Pancreatic carcinoma Rheumatoid arthritis Hepatocellular carcinoma Breast cancer Pulmonary emphysema Lung cancer Pneumonia Chronic renal failure PD PD PD PD PD PD PD PD PD PD DLB DLB DLB DLB DLB
NA/3.0 NA/2.0 NA/4.5 NA/2.5 NA/2.0 NA/UD NA/2.0 NA/9.8 9/2.0 14/2.5 13/1.5 8/2.5 20/UD 10/2.3 8/1.3 10/2.1 17/UD 11/12.0 UD/9.0 9/11.5 27/1.0 2/7.5 3/5.3
PD, Parkinson's disease; DLB, dementia with Lewy bodies; M, male; F, female; NA, not applicable; and UD, undetermined.
Table 2 – Percentages of brainstem-type Lewy bodies immunoreactive for apoptosome-related proteins. Case PD PD PD PD PD PD PD PD PD PD
Cytochrome c (%)
Apaf-1 (%)
Caspase-9 (%)
Cleaved caspase-9 (h331) (%)
Cleaved caspase-9 (Asp330) (%)
66 69 71 62 60 66 58 66 68 56
78 84 69 71 76 76 68 76 80 84
84 84 72 80 84 73 84 81 77 82
77 75 62 68 78 68 74 74 69 78
84 82 68 70 80 79 84 78 75 78
1 2 3 4 5 6 7 8 9 10
PD, Parkinson's disease.
Please cite this article as: Kawamoto, Y., et al., Immunohistochemical localization of apoptosome-related proteins in Lewy bodies in Parkinson's disease and dementia with Lewy bodies. Brain Research (2014), http://dx.doi.org/10.1016/j. brainres.2014.05.007
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Table 3 – Percentages of CD68-immunopositive microglial cells immunostained for activated caspase-9. Case PD PD PD PD PD PD PD PD PD PD
Cleaved caspse-9 (h331) (%)
Cleaved caspase-9 (Asp330) (%)
94 96 92 92 96 94 92 98 92 94
97 96 95 97 96 98 94 96 98 94
1 2 3 4 5 6 7 8 9 10
PD, Parkinson's disease.
Fig. 7 – Activated caspase-3 immunoreactivity in the substantia nigra from patients with PD (A: PD 4; B: PD 2) and in the temporal cortex from patients with DLB (C: DLB 1). Strong activated caspase-3 immunoreactivity was observed in the halo (A) or core (B) of some brainstem-type Lewy bodies. Some cortical Lewy bodies were also intensely immunoreactive for activated caspase-3 (C). Scale bars¼20 μm (A–C).
for activated caspase-3 (Fig. 7C), and caspase-3 immunoreactivity was localized over the entire bodies of the Lewy bodies (Fig. 7C).
3.
Discussion
Hashimoto and colleagues reported that cytochrome c immunoreactivity was present in the peripheral portion of the central core of Lewy bodies in the substantia nigra (Hashimoto et al., 1999), In the present study, we performed immunohistochemical studies on apoptosome-related proteins using autopsied brains from patients with PD, and found that cytochrome c immunoreactivity was localized to the whole body or marginal zone of the core of Lewy bodies in the substantia nigra and locus ceruleus. This is similar to the results of the previous paper described above (Hashimoto et al., 1999). In addition to cytochrome c, we demonstrated that immunoreactivities for Apaf-1 and caspase-9 were also localized to brainstem-type Lewy bodies. Furthermore, we showed the immunoexpression of these apoptosomeproteins in cortical Lewy bodies using autopsied brains with DLB. To our best knowledge, this is the first report that documents the immunohistochemical localization of apoptosome-related proteins in both brainstem-type and cortical Lewy bodies in brains with PD and DLB. Since the discovery that MPTP (1-methyl-4-phenyl-1,2,3,6tetrahydropyridine) could cause PD-like symptoms in
humans (Langston et al., 1983), mitochondrial dysfunction in brains with PD has been attracting much attention. MPPþ (1-methyl-4-phenylpyridinium ion), a metabolite of MPTP, is selectively toxic against dopaminergic neurons (Langston et al., 1984; Markey et al., 1984). MPPþ accumulates in the mitochondria (Ramsay et al., 1986), and inhibits mitochondrial complex I activity (Mizuno et al., 1987a, 1987b). Viswanath and coworkers demonstrated that the release of cytochrome c from the mitochondria and the activation of caspase-9 occurred in the substantia nigra of MPTPtreated mice, and in MPPþ-treated cultured dopaminergic cells (Viswanath et al., 2001). In brains with PD, the activity of mitochondrial complex I was reported to be impaired (Schapira et al., 1989; Hattori et al., 1991), and activated caspase-9 immunoreactivity was shown to be localized to dopaminergic neurons in the substantia nigra from patients with PD (Viswanath et al., 2001). In the present study, we also confirmed the immunohistochemical localization of activated caspase-9 in brainstem-type Lewy bodies using two types of anti-cleaved caspase-9 antibodies. These results suggest that mitochondrial dysfunction in brains with PD, which is similar to MPTP-induced mitochondrial impairment, may promote the release of cytochrome c from the mitochondria accompanied by the activation of caspase-9. The overexpression of α-synuclein has been reported to cause mitochondrial alterations in a murine hypothalamic cell line (Hsu et al., 2000), and to induce the apoptotic cell
Please cite this article as: Kawamoto, Y., et al., Immunohistochemical localization of apoptosome-related proteins in Lewy bodies in Parkinson's disease and dementia with Lewy bodies. Brain Research (2014), http://dx.doi.org/10.1016/j. brainres.2014.05.007
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death of dopaminergic neurons in primary human mesencephalic culture (Zhou et al., 2002). The targeted overexpression of α-synuclein in the substantia nigra of rats was shown to result in the activation of caspase-9 and dopaminergic neuronal loss (Yamada et al., 2004). These in vitro and in vivo data suggest that the overexpression of α-synuclein may promote a mitochondria-dependent apoptotic pathway. In brains from patients with PD, the mitochondrial accumulation of αsynuclein was demonstrated to occur significantly in some regions, including the substantia nigra (Devi et al., 2008), and accumulated α-synuclein was shown to be associated with the inhibition of mitochondrial complex I activity (Devi et al., 2008). Pigmented neurons in the substantia nigra and locus ceruleus contain abnormal α-synuclein aggregates which eventually develop into Lewy bodies in brains affected by PD (Goedert et al., 2013; Wakabayashi et al., 2013). In the present study, we discovered the immunohistochemical localization of apoptosome-related proteins, including cleaved caspase-9, in brainstem-type Lewy bodies. These findings suggest that the abnormal accumulation of α-synuclein in brains with PD may cause mitochondrial dysfunction and promote apoptosome formation and caspase-9 activation, thus leading to the formation of Lewy bodies which contain apoptosome-related proteins. There are two main pathways of apoptosis: the extrinsic pathway associated with the formation of the death-inducing signaling complex which activates the initiator caspase-8, and the intrinsic pathway associated with the formation of the apoptosome which activates the initiator caspase-9 (Riedl and Salvesen, 2007; Park, 2012). Once activated, both initiator caspases activate the executor caspases, including caspase-3 (Srinivasula et al., 1998; Riedl and Salvesen, 2007; Würstle et al., 2012). Increased caspase-3 immunoreactivity was observed in the substantia nigra from patients with PD (Tatton, 2000), and the percentage of activated caspase-3immunopositive nigral dopaminergic neurons was shown to be increased in PD patients as compared with normal subjects (Hartmann et al., 2000). Furthermore, activated caspase-3 immunoreactivity was detected in CD68-immunopositive microglia in the ventral mesencephalon from patients with PD (Burguillos et al., 2011). In the present study, we demonstrated that cleaved caspase-9 immunoreactivity was localized to brainstem-type Lewy bodies and CD68-immunopositive microglia in the substantia nigra from patients with PD, and that activated caspase-3 immunoreactivity was also localized to brainstem-type Lewy bodies in the same areas. These results suggest that activated caspase-9 in neurons and microglia may partially contribute to the activation of caspase-3 in brains affected by PD. In conclusion, we demonstrated the immunohistochemical localization of apoptosome-related proteins in brainstem-type Lewy bodies in the substantia nigra and locus ceruleus. The activation of caspase-9 was reported to occur in brains from patients with Alzheimer's disease (Rohn et al., 2002) and amyotrophic lateral sclerosis (Inoue et al., 2003). Taken together, these data suggest that mitochondria-related apoptosis may be associated with the pathogenesis of several types of neurodegenerative diseases, including PD, and further research focused on the apoptosome may provide new treatment strategies for PD.
4.
Experimental procedures
4.1.
Tissue preparation
We studied autopsied brains from 8 control subjects without any neurological abnormalities (age range, 54–78 years; mean, 68.4 years; 6 men and 2 women), 10 patients with PD (age range, 66–90 years; mean, 77.5 years; 6 men and 4 women) and 5 patients with DLB (age range, 69–86 years; mean 74.8 years; 4 men and 1 woman). The clinical profiles from all cases are summarized in Table 1. All brains were fixed in 10% neutral formalin for about 2 weeks at room temperature. Several paraffin-embedded tissue blocks, including the frontal and temporal cortices, midbrain and upper pons, were prepared and cut into 6-μm-thick sections on a microtome. The paraffin-embedded sections were deparaffinized in xylene, followed by rehydration in a decreasing concentration of ethanol solutions. For routine pathological evaluation, deparaffinized sections from all cases were stained with the hematoxylin and eosin (H&E), Klüver–Barrera and modified Bielschowsky methods. No histological abnormalities were detected in the sections from any of the control cases. A loss of melanin-containing neurons associated with the presence of brainstem-type Lewy bodies was observed in the substantia nigra and locus ceruleus from all of the PD cases. In addition to brainstem-type Lewy bodies, many cortical Lewy bodies were observed in the cerebral cortices from all of the DLB cases.
4.2.
Primary antibodies
To examine the immunohistochemical localization of apoptosome-related proteins in both normal and diseased brains, we selected five primary antibodies. These antibodies consisted of a mouse monoclonal anti-cytochrome c antibody (BD Pharmingen, San Jose, CA, USA), a rabbit polyclonal antiApaf-1 antibody (H-324, Santa Cruz Biotechnology, Santa Cruz, CA, USA), a rabbit polyclonal anti-caspase-9 antibody (H-83, Santa Cruz Biotechnology), a goat polyclonal anticleaved caspase-9 antibody (h331, Santa Cruz Biotechnology) and a rabbit polyclonal cleaved caspase-9 antibody (Asp330, Cell Signaling Technology, Danvers, MA, USA). According to the manufacturer's instructions, the anti-cleaved caspase-9 antibody (h331) detects the small subunit of cleaved caspase-9, whereas the anti-cleaved caspase-9 antibody (Asp330) detects the large subunit of cleaved caspase-9, and both antibodies do not recognize uncleaved procaspase-9. We also used a rabbit polyclonal anti-active caspase-3 antibody (BD Pharmingen), which specifically recognizes the activated form of caspase-3.
4.3.
Immunohistochemistry
The deparaffinized sections were pretreated with 0.3% hydrogen peroxide (Santoku, Tokyo, Japan) in 0.1 M phosphatebuffered saline (PBS) for 30 min at room temperature to inhibit endogenous peroxidase activity. After washing with 0.1 M PBS, the sections were blocked with 0.1 M PBS with 3% skim milk for 2 h at room temperature. After rinsing with
Please cite this article as: Kawamoto, Y., et al., Immunohistochemical localization of apoptosome-related proteins in Lewy bodies in Parkinson's disease and dementia with Lewy bodies. Brain Research (2014), http://dx.doi.org/10.1016/j. brainres.2014.05.007
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0.1 M PBS, the anti-cytochrome c antibody (1:100), the antiApaf-1 antibody (1:50), the anti-caspase-9 antibody (1:50), the anti-cleaved caspase-9 antibody (h331; 1:100), the anticleaved caspase-9 antibody (Asp330; 1:20), or the anti-active caspase-3 antibody (1:100) diluted in 0.1 M PBS were applied onto the sections, and the sections were incubated at 4 1C overnight in a humidified chamber. After washing with 0.1 M PBS, the sections were reacted with a biotinylated antimouse, rabbit or goat IgG (Vector Laboratories, Burlingame, CA, USA) diluted in 0.1 M PBS (1:200) for 1 h at room temperature, followed by incubation with an avidin-biotinperoxidase complex kit (Vector Laboratories) diluted in 0.1 M PBS (1:400) for 1 h at room temperature. After rinsing with 0.1 M PBS and then 0.05 M Tris–HCl (pH 7.6), the sections were developed in a colorizing solution containing 0.02% diaminobenzidine tetrahydrochloride (Dojin, Kumamoto, Japan), 0.6% ammonium nickel (II) sulfate (Wako, Osaka, Japan) and 0.005% hydrogen peroxide in 0.05 M Tris–HCl (pH 7.6) for 10 min at room temperature. To confirm the specificity of the immunohistochemical reactions, the primary antibodies were omitted or replaced with normal mouse, rabbit or goat serum, and some primary antibodies, including the anti-Apaf-1 antibody, the anticaspase-9 antibody and the anti-cleaved caspase-9 antibody (h331), were preabsorbed with an excess amount of the blocking peptides (Santa Cruz Biotechnology). We detected no specific immunopositive staining in these negative control sections (Fig. 6F).
4.4. Semiquantitative assessment of immunoreactive Lewy bodies To evaluate the proportion of brainstem-type and cortical Lewy bodies which were immunoreactive for apoptosomerelated proteins, we prepared H&E-stained midbrain sections from all 10 PD cases and H&E-stained temporal sections from all 5 DLB cases. After counting the number of brainstem-type Lewy bodies in the substantia nigra and that of cortical Lewy bodies in the parahippocampal cortex, these sections were decolorized with 70% ethanol, and were then immunostained with the antibodies against apoptosome-related proteins. After counting the number of immunopositive brainstemtype and cortical Lewy bodies in the same areas, we calculated the percentage of immunolabeled brainstem-type and cortical Lewy bodies in each section, and then calculated the average percentages of brainstem-type and cortical Lewy bodies immunoreactive for cytochrome c, Apaf-1, caspase-9 and cleaved caspase-9.
4.5.
Double immunofluorescence staining
Caspase-3 has been reported to be activated in microglia in the ventral mesencephalon from patients with PD (Burguillos et al., 2011). To investigate whether caspase-9 is activated in microglia in the substantia nigra from patients with PD, we performed double-labeling immunohistochemistry using the anti-cleaved caspase-9 antibodies and a mouse monoclonal anti-CD68 antibody (Dako, Glostrup, Denmark). Midbrain sections from all of the control and PD cases were incubated with a combination of the anti-cleaved caspase-9 antibody
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(h331; 1:100) or the anti-cleaved caspase-9 antibody (Asp330; 1:20) plus the anti-CD68 antibody (1:100) in 0.1 M PBS at 4 1C overnight. After washing with 0.01 M PBS, the sections were reacted with secondary antibodies consisting of a tetramethylrhodamine-conjugated swine anti-rabbit IgG (DakoCytomation, Glostrup, Denmark) or a tetramethylrhodamine-conjugated bovine anti-goat IgG (sc-2349, Santa Cruz Biotechnology) and a fluorescein isothiocyanate- conjugate bovine anti-mouse IgG (sc-2366, Santa Cruz Biotechnology). After rinsing with 0.01 M PBS, the slides were coverslipped with Vectashield (Vector Laboratories), and were then viewed with a fluorescence microscope system (BZ-9000, Keyence, Osaka, Japan). After counting the number of CD68-immunopositive microglia in the substantia nigra from all normal and PD cases, we counted the number of CD68-immunopositive microglia immunostained with the anti-cleaved caspase-9 antibodies. We calculated the percentage of CD68-immunopositive microglia immunostained for cleaved caspase-9 in each section, and then calculated the average percentages of CD68immunopositive microglia immunostained for cleaved caspase-9 in the substantia nigra from the control and PD cases.
Acknowledgments This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan (No. 22590930).
r e f e r e nc e s
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Stenroos, T., Chandrasekharappa, S., Athanassiadou, A., Papapetropoulos, T., Johnson, W.G., Lazzarini, A.M., Duvoisin, R.C., Di Iorio, G., Golbe, L.I., Nussbaum, R.L., 1997. Mutation in the α-synuclein gene identified in families with Parkinson’s disease. Science 276, 2045–2047. Ramsay, R.R., Salach, J.I., Singer, T.P., 1986. Uptake of the neurotoxin 1-methyl-4-phenylpyridine (MPPþ) by mitochondria and its relation to the inhibition of the mitochondrial oxidation of NADþ-linked substrates by MPPþ. Biochem. Biophys. Res. Commun. 134, 743–748. Riedl, S.J., Salvesen, G.S., 2007. The apoptosome: signalling platform of cell death. Nat. Rev. Mol. Cell Biol. 8, 405–413. Rohn, T.T., Rissman, R.A., Davis, M.C., Kim, Y.E., Cotman, C.W., Head, E., 2002. Caspase-9 activation and caspase cleavage of tau in the Alzheimer’s disease brain. Neurobiol. Dis. 11, 341–354. Schapira, A.H.V., Cooper, J.M., Dexter, D., Jenner, P., Clark, J.B., Marsden, C.D., 1989. Mitochondrial complex I deficiency in Parkinson’s disease. Lancet 1, 1269. Singleton, A.B., Farrer, M., Johnson, J., Singleton, A., Hague, S., Kachergus, J., Hulihan, M., Peuralinna, T., Dutra, A., Nussbaum, R., Lincoln, S., Crawley, A., Hanson, M., Maraganore, D., Adler, C., Cookson, M.R., Muenter, M., Baptista, M., Miller, D., Blancato, J., Hardy, J., Gwinn-Hardy, K., 2003. α-Synuclein locus triplication causes Parkinson’s disease. Science 302, 841. Srinivasula, S.M., Ahmad, M., Fernandes-Alnemri, T., Alnemri, E. S., 1998. Autoactivation of procaspase-9 by Apaf-1-mediated oligomerization. Mol. Cell 1, 949–957. Tatton, N.A., Maclean-Fraser, A., Tatton, W.G., Perl, D.P., Olanow, C.W., 1998. A fluorescent double-labeling method to detect and confirm apoptotic nuclei in Parkinson’s disease. Ann. Neurol. 44, S142–S148. Tatton, N.A., 2000. Increased caspase 3 and Bax immunoreactivity accompany nuclear GAPDH translocation and neuronal apoptosis in Parkinson’s disease. Exp. Neurol. 166, 29–43. Viswanath, V., Wu, Y., Boonplueang, R., Chen, S., Stevenson, F.F., Yantiri, F., Yang, L., Beal, M.F., Andersen, J.K., 2001. Caspase-9 activation results in downstream caspase-8 activation and Bid cleavage in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridineinduced Parkinson’s disease. J. Neurosci. 21, 9519–9528. Wakabayashi, K., Tanji, K., Odagiri, S., Miki, Y., Mori, F., Takahashi, H., 2013. The Lewy body in Parkinson’s disease and related neurodegenerative disorders. Mol. Neurobiol. 47, 495–508. Wu¨rstle, M.L., Laussmann, M.A., Rehm, M., 2012. The central role of initiator caspase-9 in apoptosis signal transduction and the regulation of its activation and activity on the apoptosome. Exp. Cell Res. 318, 1213–1220. Yamada, M., Iwatsubo, T., Mizuno, Y., Mochizuki, H., 2004. Overexpression of α-synuclein in rat substantia nigra results in loss of dopaminergic neurons, phosphorylation of αsynuclein and activation of caspase-9: resemblance to pathogenetic changes in Parkinson’s disease. J. Neurochem. 91, 451–461. Zarranz, J.J., Alegre, J., Go´mez-Esteban, J.C., Lezcano, E., Ros, R., Ampuero, I., Vidal, L., Hoenicka, J., Rodriguez, O., Atare´s, B., Llorens, V., Gomez Tortosa, E., del Ser, T., Mun˜oz, D.G., de Yebenes, J.G., 2004. The new mutation, E46K, of α-synuclein causes Parkinson and Lewy body dementia. Ann. Neurol. 55, 164–173. Zhou, W., Schaack, J., Zawada, W.M., Freed, C.R., 2002. Overexpression of human α-synuclein causes dopamine neuron death in primary human mesencephalic culture. Brain Res. 926, 42–50.
Please cite this article as: Kawamoto, Y., et al., Immunohistochemical localization of apoptosome-related proteins in Lewy bodies in Parkinson's disease and dementia with Lewy bodies. Brain Research (2014), http://dx.doi.org/10.1016/j. brainres.2014.05.007
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Research Report
Immunohistochemical localization of apoptosomerelated proteins in Lewy bodies in Parkinson's disease and dementia with Lewy bodies Yasuhiro Kawamotoa,b,n, Hidefumi Itoc, Takashi Ayakia, Ryosuke Takahashia a
Department of Neurology, Faculty of Medicine, Kyoto University, Kyoto, Japan Department of Neurology, Seijinkai Rakusaishimizu Hospital, Kyoto, Japan c Department of Neurology, Wakayama Medical University, Wakayama, Japan b
art i cle i nfo
ab st rac t
Article history:
Apoptotic stimuli induce the release of cytochrome c from the mitochondria to the cytosol,
Accepted 5 May 2014
and this released cytochrome c promotes the formation of the apoptosome, which contains cytochrome c, Apaf-1 and caspase-9, resulting in the activation of caspase-9
Keywords: Apoptosome Caspase-9 Lewy body Mitochondria Parkinson's disease
and the promotion of apoptotic cell death. To investigate the role of the apoptosome in patients with Parkinson's disease (PD), we performed immunohistochemical studies on apoptosome-related proteins in formalin-fixed, paraffin-embedded sections from 8 normal subjects, 10 patients with PD and 5 patients with dementia with Lewy bodies (DLB). Furthermore, we performed double-labeling immunohistochemistry for cleaved caspase-9 and CD68 in some sections from 8 normal subjects and 10 patients with PD. In the substantia nigra and locus ceruleus from both control and PD cases, the somata and processes of melanin-containing neurons were immunostained for cytochrome c, Apaf-1 and caspase-9. In the same areas from the PD cases, brainstem-type Lewy bodies were also immunoreactive for cytochrome c, Apaf-1 and caspase-9, and cleaved caspase-9 immunoreactivity was detected in brainstem-type Lewy bodies and CD68-immunopositive microglia. In addition to brainstem-type Lewy bodies, cortical Lewy bodies were also immunoreactive for these apoptosome-related proteins in the frontal and temporal cortices from the DLB cases. Our results suggest that apoptosome formation accompanied by caspase-9 activation may occur in the substantia nigra and locus ceruleus in brains affected by PD, and that a mitochondria-dependent apoptotic pathway may be partially associated with the pathogenesis of PD. & 2014 Published by Elsevier B.V.
Abbreviations: PD,
Parkinson's disease; CARD,
caspase recruitment domain; TUNEL,
mediated deoxyuridine triphosphate- biotin nick end labeling; DLB,
terminal deoxynucleotidyl transferase-
dementia with Lewy bodies; MPTP,
1-methyl-4-phenyl- 1,2,3,6-
tetrahydropyridine; MPPþ, 1-methyl-4-phenylpyridinium ion; H&E, hematoxylin and eosin; PBS, phosphate-buffered saline n Corresponding author at: Department of Neurology, Faculty of Medicine, Kyoto University, 54 Shogoin-Kawaharacho, Sakyoku, Kyoto 606-8507, Japan. Fax: þ81 75 751 3265. E-mail address: [email protected] (Y. Kawamoto). http://dx.doi.org/10.1016/j.brainres.2014.05.007 0006-8993/& 2014 Published by Elsevier B.V.
Please cite this article as: Kawamoto, Y., et al., Immunohistochemical localization of apoptosome-related proteins in Lewy bodies in Parkinson's disease and dementia with Lewy bodies. Brain Research (2014), http://dx.doi.org/10.1016/j. brainres.2014.05.007
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1.
Introduction
Parkinson's disease (PD) is one of the most common neurodegenerative disorders, characterized clinically by the progressive development of extrapyramidal signs consisting of a resting tremor, muscular rigidity, bradykinesia and postural instability (Hughes et al., 1992). In brains from patients with PD, neuronal loss associated with Lewy bodies is observed in several areas, including the substantia nigra and locus ceruleus (Pollanen et al., 1993). Brainstem-type Lewy bodies in the substantia nigra and locus ceruleus are eosinophilic inclusions with a hyaline core and a pale halo (Pollanen et al., 1993). Missense mutations of the α-synuclein gene (Polymeropoulos et al., 1997; Krüger et al., 1998; Zarranz et al., 2004) and multiplications of the α-synuclein locus (Singleton et al., 2003; Chartier-Harlin et al., 2004; Ibáñez et al., 2004) have been reported to be implicated in the autosomal dominant familial forms of PD. Lewy bodies are ubiquitinated (Kuzuhara et al., 1988), and α-synuclein is a main constituent of Lewy bodies (Goedert et al., 2013; Wakabayashi et al., 2013); however, the mechanism of Lewy body formation remains unclear. The mitochondria play an important role in apoptotic cell death. Upon receiving various apoptotic stimuli, cytochrome c is released from the mitochondria into the cytosol, and this released cytochrome c promotes the formation of the apoptosome, which contains cytochrome c, Apaf-1 and caspase-9 (Li et al., 1997; Adams and Cory, 2002; Riedl and Salvesen, 2007; Park, 2012). Apaf-1 is the central component of the apoptosome, and contains an N-terminal caspase recruitment domain (CARD), a nucleotide-binding domain and a C-terminal WD40 repeat domain (Adams and Cory, 2002; Riedl and Salvesen, 2007; Park, 2012). The CARD of Apaf-1
interacts with the N-terminal CARD of procaspase-9, resulting in the recruitment of procaspase-9 to the apoptosome (Adams and Cory, 2002; Riedl and Salvesen, 2007; Park, 2012). Procaspase-9 is then activated by dimerization and autocatalytic cleavage at Asp315, generating the large and small subunits (Srinivasula et al., 1998; Würstle et al., 2012). Activated caspase-9 converts procaspse-3 to the activated form of caspase-3, and promotes the downstream caspase cascade, thus leading to apoptotic cell death (Li et al., 1997; Srinivasula et al., 1998; Würstle et al., 2012). Activated caspase-3 feeds back on procaspase-9 and activates procaspase-9 by processing it at Asp330, yielding the large and small fragments, and thus amplifying the caspase cascade (Srinivasula et al., 1998; Würstle et al., 2012). DNA fragmentation associated with apoptosis was reported to be detected in pigmented neurons in the substantia nigra from patients with PD using the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling (TUNEL) method (Mochizuki et al., 1996). TUNELpositive glial cells were also shown to be localized to the substantias nigra from patients with PD (Mochizuki et al., 1996; Jellinger, 2000). Tatton and colleagues demonstrated the presence of nuclear chromatin condensation in melanized neurons in the substantia nigra from patients with PD using fluorescent nucleic acid-binding dyes such as YOYO-1 (Tatton et al., 1998; Tatton, 2000). In the present study, we performed immunohistochemical studies on apoptosomerelated proteins using autopsied brains from 8 normal subjects, 10 patients with PD and 5 patients with dementia with Lewy bodies (DLB), and found the immunohistochemical localization of apoptosome-related proteins in brainstemtype Lewy bodies in the substantia nigra and locus ceruleus and in cortical Lewy bodies in the frontal and temporal cortices.
Fig. 1 – Cytochrome c immunoreactivity in the substantia nigra from normal subjects (A: Control 2) and patients with PD (B: PD 6; C, D: PD 3). Cytochrome c immunoreactivitiy was observed in the somata and processes of melanin-containing neurons (A, B). Moderately immunopositive brainstem-type Lewy bodies were detected in some remaining neurons (B, arrow). Cytochrome c immunoreactivity was localized to the whole body (C) or the marginal zone (D) of the core of the Lewy bodies. Scale bars¼80 μm (A), 40 μm (B), and 20 μm (C and D). Please cite this article as: Kawamoto, Y., et al., Immunohistochemical localization of apoptosome-related proteins in Lewy bodies in Parkinson's disease and dementia with Lewy bodies. Brain Research (2014), http://dx.doi.org/10.1016/j. brainres.2014.05.007
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2.
Results
2.1. Cytochrome c immunoreactivity in normal and PD brains In the substantia nigra and locus ceruleus from the control cases, cytochrome c immunoreactivitiy was observed in the somata and processes of melanin-containing neurons (Fig. 1A). In the same areas from the PD cases, cytochrome c immunoreactivity was spared in the remaining neurons (Fig. 1B), and moderately immunopositive brainstem-type Lewy bodies were found in some remaining neurons (Fig. 1B). The core of the Lewy bodies was immunostained for cytochrome c (Fig. 1C), and cytochrome c immunoreactivity occasionally accumulated in the marginal zone of the core of the Lewy bodies (Fig. 1D). A semiquantitative evaluation of the substantia nigra from all 10 PD cases demonstrated that the average percentage of cytochrome c-immunoreactive Lewy bodies was 64%.
2.2.
Apaf-1 immunoreactivity in normal and PD brains
In the substantia nigra and locus ceruleus from the control cases, Apaf-1 immunoreactivitiy was observed in the somata and processes of melanin-containing neurons (Fig. 2A). In the same areas from the PD cases, Apaf-1 immunoreactivity was spared in the remaining neurons (Fig. 2B), and strongly immunopositive brainstem-type Lewy bodies were found in some remaining neurons (Fig. 2B). Apaf-1 immunoreactivity was generally localized to the halo of the Lewy bodies (Fig. 2C and D). A semiquantitative evaluation of the substantia nigra
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from all 10 PD cases demonstrated that the average percentage of Apaf-1-immunoreactive Lewy bodies was 76%.
2.3.
Caspase-9 immunoreactivity in normal and PD brains
In the substantia nigra and locus ceruleus from the control cases, caspase-9 immunoreactivity was observed in the somata and processes of melanin-containing neurons (Fig. 3A). In the same areas from the PD cases, caspase-9 immunoreactivity was spared in the remaining neurons (Fig. 3B), and intensely immunopositive brainstem-type Lewy bodies were found in some remaining neurons (Fig. 3B). Caspase- 9 immunoreactivity was generally localized to the halo of the Lewy bodies (Fig. 3C and D). A semiquantitative evaluation of the substantia nigra from all 10 PD cases demonstrated that the average percentage of caspase-9immunoreactive Lewy bodies was 80%.
2.4.
Cleaved caspase-9 immunoreactivity in PD brains
Immunohistochemical studies in the substantia nigra and locus ceruleus from the PD cases using two types of anticleaved caspase-9 antibodies showed that strongly immunopositive Lewy body-containing neurons were scattered (Fig. 4A and D), and that cleaved caspase-9 immunoreactivity was localized to the core (Fig. 4B and E) or halo (Fig. 4C and F) of brainstem-type Lewy bodies. A semiquantitative evaluation of the substantia nigra from all 10 PD cases demonstrated that the average percentages of Lewy bodies immunostained with the anti-cleaved caspase-9 antibody (h331) and the anti-cleaved caspase-9 antibody (Asp330) were 72% and 78%, respectively.
Fig. 2 – Apaf-1 immunoreactivity in the substantia nigra from normal subjects (A: Control 2) and patients with PD (B: PD 5; C: PD 3), and in the locus ceruleus from patients with PD (D: PD 6). Apaf-1 immunoreactivitiy was detected in the somata and processes of melanin-containing neurons (A and B). Strongly immunopositive brainstem- type Lewy bodies were detected in some remaining neurons (B, arrow). The brainstem- type Lewy bodies showed a ring-shaped immunostaining pattern (C and D). Scale bars¼80 μm (A), 40 μm (B), and 20 μm (C and D). Please cite this article as: Kawamoto, Y., et al., Immunohistochemical localization of apoptosome-related proteins in Lewy bodies in Parkinson's disease and dementia with Lewy bodies. Brain Research (2014), http://dx.doi.org/10.1016/j. brainres.2014.05.007
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Fig. 3 – Caspase-9 immunoreactivity in the substantia nigra from normal subjects (A: Control 4) and patients with PD (B: PD 1; C: PD 7; D PD 5). Caspase-9 immunoreactivitiy was observed in the somata and processes of melanin-containing neurons (A and B). Intensely immunopositive brainstem-type Lewy bodies were detected in some remaining neurons (B, arrow). The brainstem-type Lewy bodies showed a ring- shaped immunostaining pattern (C and D). Scale bars¼ 80 μm (A), 40 μm (B), and 20 μm (C and D).
Fig. 4 – Immunohistochemical localization of cleaved caspase-9 in the substantia nigra (A, B, D, and E) and locus ceruleus (C and F) from patients with PD (A: PD 2; B: PD 1; C: PD 3; D, F: PD 5; E: PD 6) using the anti-cleaved caspase-9 antibody (h331) (A–C) and another anti-cleaved caspase-9 antibody (Asp330) (D–F). Strongly immunopositive Lewy body-bearing neurons were scattered (A and D), and cleaved caspase-9 immunoreactivity was localized to the core (B and E) or halo (C and F) of the brainstem-type Lewy bodies. Scale bars ¼40 μm (A and D), 20 μm (B, C, E, and F).
2.5. Double-labeling immunofluorescence for cleaved caspase-9 and CD68 in normal and PD brains Midbrain sections double-immunostained with the anti-cleaved caspase-9 antibody (h331) (Fig. 5A) or another anti-cleaved caspase-9 antibody (Asp330) (Fig. 5D) plus the anti-CD68 antibody (Fig. 5B and E) showed that cleaved caspase-9
immunoreactivity was observed in CD68-immunopositive microglia in the substantia nigra from the PD cases (Fig. 5C and F). As compared with the PD cases, the activation of caspase-9 was not conspicuous in the substantia nigra from the control cases, and the average percentages of CD68-immunopositive microglia immunostained with the anti-cleaved caspase-9
Please cite this article as: Kawamoto, Y., et al., Immunohistochemical localization of apoptosome-related proteins in Lewy bodies in Parkinson's disease and dementia with Lewy bodies. Brain Research (2014), http://dx.doi.org/10.1016/j. brainres.2014.05.007
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Fig. 5 – Double-immunofluorescence staining for cleaved caspase-9 (A: h331; D: Asp331) and CD68 (B, E) in the substantia nigra from patients with PD (A–C: PD 4; D- F: PD 10). The merged images showed that the CD68-immunopositive microglia contained cleaved caspase-9 immunoreactivity (C, F). Scale bars¼ 20 μm (A–F).
Fig. 6 – Immunoreactivities of apoptosome-related proteins in the frontal cortex from patients with DLB (A–C: DLB 3; D–F: DLB 4). Cortical Lewy bodies were intensely immunostained with the anti-cytochrome c antibody (A), the anti-Apaf-1 antibody (B) anti-caspase-9 antibody (C), the anti-cleaved antibody (h331) (D) and the anti-cleaved caspase-9 antibody (Asp330) (E). No specific immunopositive staining was detected in negative control sections which were incubated with the anti-cleaved caspase-9 antibody (h331) preabsorbed with an excess amount of the blocking peptide (sc-22812P) (F). Scale bars¼ 20 μm (A–E), and 40 μm (F).
antibody (h331) and the anti-cleaved caspase-9 (Asp330) in the PD group were 94% and 96%, respectively, and those in the control group were 24% and 22%, respectively.
2.6. Immunoreactivities of apoptosome-related proteins in cortical Lewy bodies In the frontal and temporal cortices from the DLB cases, cortical Lewy bodies were intensely immunoreactive for cytochrome c (Fig. 6A), Apaf-1 (Fig. 6B), caspase-9 (Fig. 6C) and cleaved caspase-9 (Fig. 6D and E). Immunoreactivites of these apoptosome-related proteins were localized over the
entire bodies of the Lewy bodies (Fig. 6A and E). No specific immunopositive staining was detected in negative control sections which were incubated with the anti-cleaved caspase-9 (h331) antibody preabsorbed with an excess amount of the blocking peptide (sc-22812P) (Fig. 6F). The preabsorption experiments for the anti-Apaf-1 and anti-caspase-9 antibodies also resulted in no specific immunopositive staining (data not shown). A semiquantitative evaluation of the parahippocampal cortex from all 5 DLB cases demonstrated that the average percentages of Lewy bodies immunostained with the anti-cytochrome c antibody, the anti-Apaf-1 antibody, the anti-caspase-9 antibody, the anti-cleaved caspase-9
Please cite this article as: Kawamoto, Y., et al., Immunohistochemical localization of apoptosome-related proteins in Lewy bodies in Parkinson's disease and dementia with Lewy bodies. Brain Research (2014), http://dx.doi.org/10.1016/j. brainres.2014.05.007
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antibody (h331) and the anti-cleaved caspase-9 antibody (Asp330) were 60%, 72%, 74%, 66% and 72%, respectively.
2.7. Relationship between disease duration and Lewy bodies immunoreactive for apoptosome-related proteins or microglial cells immunostained for activated caspase-9
in the substantia nigra from all 10 patients with PD were summarized in Table 3. There was no significant relationship between the duration of disease and the activation of caspase-9 in microglial cells.
2.8. Activated caspase-3 immunoreactivity in PD and DLB brains
The percentages of Lewy bodies immunoreactive for cytochrome c, Apaf-1, caspase-9 and cleaved caspase-9 in the substantia nigra from all 10 patients with PD were summarized in Table 2. There was no significant relationship between the duration of disease and the proportion of Lewy bodies immunoreactive for apoptosome-related proteins. The percentages of CD68-immunopositive microglia immunostained with the anti- cleaved caspase-9 antibodies
In the substantia nigra and locus ceruleus from the PD cases, some brainstem-type Lewy bodies were strongly immunoreactive for activated caspase-3 (Fig. 7A and B), and activated caspase-3 immunoreactivity was localized to the halo (Fig. 7A) or core (Fig. 7B) of the Lewy bodies. In the frontal and temporal cortices from the DLB cases, some cortical Lewy bodies were intensely immunoreactive
Table 1 – Clinical profiles of all cases. Case Control Control Control Control Control Control Control Control PD 1 PD 2 PD 3 PD 4 PD 5 PD 6 PD 7 PD 8 PD 9 PD 10 DLB 1 DLB 2 DLB 3 DLB 4 DLB 5
1 2 3 4 5 6 7 8
Age (yr)/Sex
Diagnosis
Duration of illness (yr)/postmortem delay (h)
62/M 68/M 73/M 68/F 75/M 69/M 54/M 78/F 90/F 76/M 79/F 81/F 74/M 66/M 76/M 88/M 67/M 78/F 81/M 69/M 69/F 69/M 86/M
Pancreatic carcinoma Rheumatoid arthritis Hepatocellular carcinoma Breast cancer Pulmonary emphysema Lung cancer Pneumonia Chronic renal failure PD PD PD PD PD PD PD PD PD PD DLB DLB DLB DLB DLB
NA/3.0 NA/2.0 NA/4.5 NA/2.5 NA/2.0 NA/UD NA/2.0 NA/9.8 9/2.0 14/2.5 13/1.5 8/2.5 20/UD 10/2.3 8/1.3 10/2.1 17/UD 11/12.0 UD/9.0 9/11.5 27/1.0 2/7.5 3/5.3
PD, Parkinson's disease; DLB, dementia with Lewy bodies; M, male; F, female; NA, not applicable; and UD, undetermined.
Table 2 – Percentages of brainstem-type Lewy bodies immunoreactive for apoptosome-related proteins. Case PD PD PD PD PD PD PD PD PD PD
Cytochrome c (%)
Apaf-1 (%)
Caspase-9 (%)
Cleaved caspase-9 (h331) (%)
Cleaved caspase-9 (Asp330) (%)
66 69 71 62 60 66 58 66 68 56
78 84 69 71 76 76 68 76 80 84
84 84 72 80 84 73 84 81 77 82
77 75 62 68 78 68 74 74 69 78
84 82 68 70 80 79 84 78 75 78
1 2 3 4 5 6 7 8 9 10
PD, Parkinson's disease.
Please cite this article as: Kawamoto, Y., et al., Immunohistochemical localization of apoptosome-related proteins in Lewy bodies in Parkinson's disease and dementia with Lewy bodies. Brain Research (2014), http://dx.doi.org/10.1016/j. brainres.2014.05.007
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Table 3 – Percentages of CD68-immunopositive microglial cells immunostained for activated caspase-9. Case PD PD PD PD PD PD PD PD PD PD
Cleaved caspse-9 (h331) (%)
Cleaved caspase-9 (Asp330) (%)
94 96 92 92 96 94 92 98 92 94
97 96 95 97 96 98 94 96 98 94
1 2 3 4 5 6 7 8 9 10
PD, Parkinson's disease.
Fig. 7 – Activated caspase-3 immunoreactivity in the substantia nigra from patients with PD (A: PD 4; B: PD 2) and in the temporal cortex from patients with DLB (C: DLB 1). Strong activated caspase-3 immunoreactivity was observed in the halo (A) or core (B) of some brainstem-type Lewy bodies. Some cortical Lewy bodies were also intensely immunoreactive for activated caspase-3 (C). Scale bars¼20 μm (A–C).
for activated caspase-3 (Fig. 7C), and caspase-3 immunoreactivity was localized over the entire bodies of the Lewy bodies (Fig. 7C).
3.
Discussion
Hashimoto and colleagues reported that cytochrome c immunoreactivity was present in the peripheral portion of the central core of Lewy bodies in the substantia nigra (Hashimoto et al., 1999), In the present study, we performed immunohistochemical studies on apoptosome-related proteins using autopsied brains from patients with PD, and found that cytochrome c immunoreactivity was localized to the whole body or marginal zone of the core of Lewy bodies in the substantia nigra and locus ceruleus. This is similar to the results of the previous paper described above (Hashimoto et al., 1999). In addition to cytochrome c, we demonstrated that immunoreactivities for Apaf-1 and caspase-9 were also localized to brainstem-type Lewy bodies. Furthermore, we showed the immunoexpression of these apoptosomeproteins in cortical Lewy bodies using autopsied brains with DLB. To our best knowledge, this is the first report that documents the immunohistochemical localization of apoptosome-related proteins in both brainstem-type and cortical Lewy bodies in brains with PD and DLB. Since the discovery that MPTP (1-methyl-4-phenyl-1,2,3,6tetrahydropyridine) could cause PD-like symptoms in
humans (Langston et al., 1983), mitochondrial dysfunction in brains with PD has been attracting much attention. MPPþ (1-methyl-4-phenylpyridinium ion), a metabolite of MPTP, is selectively toxic against dopaminergic neurons (Langston et al., 1984; Markey et al., 1984). MPPþ accumulates in the mitochondria (Ramsay et al., 1986), and inhibits mitochondrial complex I activity (Mizuno et al., 1987a, 1987b). Viswanath and coworkers demonstrated that the release of cytochrome c from the mitochondria and the activation of caspase-9 occurred in the substantia nigra of MPTPtreated mice, and in MPPþ-treated cultured dopaminergic cells (Viswanath et al., 2001). In brains with PD, the activity of mitochondrial complex I was reported to be impaired (Schapira et al., 1989; Hattori et al., 1991), and activated caspase-9 immunoreactivity was shown to be localized to dopaminergic neurons in the substantia nigra from patients with PD (Viswanath et al., 2001). In the present study, we also confirmed the immunohistochemical localization of activated caspase-9 in brainstem-type Lewy bodies using two types of anti-cleaved caspase-9 antibodies. These results suggest that mitochondrial dysfunction in brains with PD, which is similar to MPTP-induced mitochondrial impairment, may promote the release of cytochrome c from the mitochondria accompanied by the activation of caspase-9. The overexpression of α-synuclein has been reported to cause mitochondrial alterations in a murine hypothalamic cell line (Hsu et al., 2000), and to induce the apoptotic cell
Please cite this article as: Kawamoto, Y., et al., Immunohistochemical localization of apoptosome-related proteins in Lewy bodies in Parkinson's disease and dementia with Lewy bodies. Brain Research (2014), http://dx.doi.org/10.1016/j. brainres.2014.05.007
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death of dopaminergic neurons in primary human mesencephalic culture (Zhou et al., 2002). The targeted overexpression of α-synuclein in the substantia nigra of rats was shown to result in the activation of caspase-9 and dopaminergic neuronal loss (Yamada et al., 2004). These in vitro and in vivo data suggest that the overexpression of α-synuclein may promote a mitochondria-dependent apoptotic pathway. In brains from patients with PD, the mitochondrial accumulation of αsynuclein was demonstrated to occur significantly in some regions, including the substantia nigra (Devi et al., 2008), and accumulated α-synuclein was shown to be associated with the inhibition of mitochondrial complex I activity (Devi et al., 2008). Pigmented neurons in the substantia nigra and locus ceruleus contain abnormal α-synuclein aggregates which eventually develop into Lewy bodies in brains affected by PD (Goedert et al., 2013; Wakabayashi et al., 2013). In the present study, we discovered the immunohistochemical localization of apoptosome-related proteins, including cleaved caspase-9, in brainstem-type Lewy bodies. These findings suggest that the abnormal accumulation of α-synuclein in brains with PD may cause mitochondrial dysfunction and promote apoptosome formation and caspase-9 activation, thus leading to the formation of Lewy bodies which contain apoptosome-related proteins. There are two main pathways of apoptosis: the extrinsic pathway associated with the formation of the death-inducing signaling complex which activates the initiator caspase-8, and the intrinsic pathway associated with the formation of the apoptosome which activates the initiator caspase-9 (Riedl and Salvesen, 2007; Park, 2012). Once activated, both initiator caspases activate the executor caspases, including caspase-3 (Srinivasula et al., 1998; Riedl and Salvesen, 2007; Würstle et al., 2012). Increased caspase-3 immunoreactivity was observed in the substantia nigra from patients with PD (Tatton, 2000), and the percentage of activated caspase-3immunopositive nigral dopaminergic neurons was shown to be increased in PD patients as compared with normal subjects (Hartmann et al., 2000). Furthermore, activated caspase-3 immunoreactivity was detected in CD68-immunopositive microglia in the ventral mesencephalon from patients with PD (Burguillos et al., 2011). In the present study, we demonstrated that cleaved caspase-9 immunoreactivity was localized to brainstem-type Lewy bodies and CD68-immunopositive microglia in the substantia nigra from patients with PD, and that activated caspase-3 immunoreactivity was also localized to brainstem-type Lewy bodies in the same areas. These results suggest that activated caspase-9 in neurons and microglia may partially contribute to the activation of caspase-3 in brains affected by PD. In conclusion, we demonstrated the immunohistochemical localization of apoptosome-related proteins in brainstem-type Lewy bodies in the substantia nigra and locus ceruleus. The activation of caspase-9 was reported to occur in brains from patients with Alzheimer's disease (Rohn et al., 2002) and amyotrophic lateral sclerosis (Inoue et al., 2003). Taken together, these data suggest that mitochondria-related apoptosis may be associated with the pathogenesis of several types of neurodegenerative diseases, including PD, and further research focused on the apoptosome may provide new treatment strategies for PD.
4.
Experimental procedures
4.1.
Tissue preparation
We studied autopsied brains from 8 control subjects without any neurological abnormalities (age range, 54–78 years; mean, 68.4 years; 6 men and 2 women), 10 patients with PD (age range, 66–90 years; mean, 77.5 years; 6 men and 4 women) and 5 patients with DLB (age range, 69–86 years; mean 74.8 years; 4 men and 1 woman). The clinical profiles from all cases are summarized in Table 1. All brains were fixed in 10% neutral formalin for about 2 weeks at room temperature. Several paraffin-embedded tissue blocks, including the frontal and temporal cortices, midbrain and upper pons, were prepared and cut into 6-μm-thick sections on a microtome. The paraffin-embedded sections were deparaffinized in xylene, followed by rehydration in a decreasing concentration of ethanol solutions. For routine pathological evaluation, deparaffinized sections from all cases were stained with the hematoxylin and eosin (H&E), Klüver–Barrera and modified Bielschowsky methods. No histological abnormalities were detected in the sections from any of the control cases. A loss of melanin-containing neurons associated with the presence of brainstem-type Lewy bodies was observed in the substantia nigra and locus ceruleus from all of the PD cases. In addition to brainstem-type Lewy bodies, many cortical Lewy bodies were observed in the cerebral cortices from all of the DLB cases.
4.2.
Primary antibodies
To examine the immunohistochemical localization of apoptosome-related proteins in both normal and diseased brains, we selected five primary antibodies. These antibodies consisted of a mouse monoclonal anti-cytochrome c antibody (BD Pharmingen, San Jose, CA, USA), a rabbit polyclonal antiApaf-1 antibody (H-324, Santa Cruz Biotechnology, Santa Cruz, CA, USA), a rabbit polyclonal anti-caspase-9 antibody (H-83, Santa Cruz Biotechnology), a goat polyclonal anticleaved caspase-9 antibody (h331, Santa Cruz Biotechnology) and a rabbit polyclonal cleaved caspase-9 antibody (Asp330, Cell Signaling Technology, Danvers, MA, USA). According to the manufacturer's instructions, the anti-cleaved caspase-9 antibody (h331) detects the small subunit of cleaved caspase-9, whereas the anti-cleaved caspase-9 antibody (Asp330) detects the large subunit of cleaved caspase-9, and both antibodies do not recognize uncleaved procaspase-9. We also used a rabbit polyclonal anti-active caspase-3 antibody (BD Pharmingen), which specifically recognizes the activated form of caspase-3.
4.3.
Immunohistochemistry
The deparaffinized sections were pretreated with 0.3% hydrogen peroxide (Santoku, Tokyo, Japan) in 0.1 M phosphatebuffered saline (PBS) for 30 min at room temperature to inhibit endogenous peroxidase activity. After washing with 0.1 M PBS, the sections were blocked with 0.1 M PBS with 3% skim milk for 2 h at room temperature. After rinsing with
Please cite this article as: Kawamoto, Y., et al., Immunohistochemical localization of apoptosome-related proteins in Lewy bodies in Parkinson's disease and dementia with Lewy bodies. Brain Research (2014), http://dx.doi.org/10.1016/j. brainres.2014.05.007
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0.1 M PBS, the anti-cytochrome c antibody (1:100), the antiApaf-1 antibody (1:50), the anti-caspase-9 antibody (1:50), the anti-cleaved caspase-9 antibody (h331; 1:100), the anticleaved caspase-9 antibody (Asp330; 1:20), or the anti-active caspase-3 antibody (1:100) diluted in 0.1 M PBS were applied onto the sections, and the sections were incubated at 4 1C overnight in a humidified chamber. After washing with 0.1 M PBS, the sections were reacted with a biotinylated antimouse, rabbit or goat IgG (Vector Laboratories, Burlingame, CA, USA) diluted in 0.1 M PBS (1:200) for 1 h at room temperature, followed by incubation with an avidin-biotinperoxidase complex kit (Vector Laboratories) diluted in 0.1 M PBS (1:400) for 1 h at room temperature. After rinsing with 0.1 M PBS and then 0.05 M Tris–HCl (pH 7.6), the sections were developed in a colorizing solution containing 0.02% diaminobenzidine tetrahydrochloride (Dojin, Kumamoto, Japan), 0.6% ammonium nickel (II) sulfate (Wako, Osaka, Japan) and 0.005% hydrogen peroxide in 0.05 M Tris–HCl (pH 7.6) for 10 min at room temperature. To confirm the specificity of the immunohistochemical reactions, the primary antibodies were omitted or replaced with normal mouse, rabbit or goat serum, and some primary antibodies, including the anti-Apaf-1 antibody, the anticaspase-9 antibody and the anti-cleaved caspase-9 antibody (h331), were preabsorbed with an excess amount of the blocking peptides (Santa Cruz Biotechnology). We detected no specific immunopositive staining in these negative control sections (Fig. 6F).
4.4. Semiquantitative assessment of immunoreactive Lewy bodies To evaluate the proportion of brainstem-type and cortical Lewy bodies which were immunoreactive for apoptosomerelated proteins, we prepared H&E-stained midbrain sections from all 10 PD cases and H&E-stained temporal sections from all 5 DLB cases. After counting the number of brainstem-type Lewy bodies in the substantia nigra and that of cortical Lewy bodies in the parahippocampal cortex, these sections were decolorized with 70% ethanol, and were then immunostained with the antibodies against apoptosome-related proteins. After counting the number of immunopositive brainstemtype and cortical Lewy bodies in the same areas, we calculated the percentage of immunolabeled brainstem-type and cortical Lewy bodies in each section, and then calculated the average percentages of brainstem-type and cortical Lewy bodies immunoreactive for cytochrome c, Apaf-1, caspase-9 and cleaved caspase-9.
4.5.
Double immunofluorescence staining
Caspase-3 has been reported to be activated in microglia in the ventral mesencephalon from patients with PD (Burguillos et al., 2011). To investigate whether caspase-9 is activated in microglia in the substantia nigra from patients with PD, we performed double-labeling immunohistochemistry using the anti-cleaved caspase-9 antibodies and a mouse monoclonal anti-CD68 antibody (Dako, Glostrup, Denmark). Midbrain sections from all of the control and PD cases were incubated with a combination of the anti-cleaved caspase-9 antibody
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(h331; 1:100) or the anti-cleaved caspase-9 antibody (Asp330; 1:20) plus the anti-CD68 antibody (1:100) in 0.1 M PBS at 4 1C overnight. After washing with 0.01 M PBS, the sections were reacted with secondary antibodies consisting of a tetramethylrhodamine-conjugated swine anti-rabbit IgG (DakoCytomation, Glostrup, Denmark) or a tetramethylrhodamine-conjugated bovine anti-goat IgG (sc-2349, Santa Cruz Biotechnology) and a fluorescein isothiocyanate- conjugate bovine anti-mouse IgG (sc-2366, Santa Cruz Biotechnology). After rinsing with 0.01 M PBS, the slides were coverslipped with Vectashield (Vector Laboratories), and were then viewed with a fluorescence microscope system (BZ-9000, Keyence, Osaka, Japan). After counting the number of CD68-immunopositive microglia in the substantia nigra from all normal and PD cases, we counted the number of CD68-immunopositive microglia immunostained with the anti-cleaved caspase-9 antibodies. We calculated the percentage of CD68-immunopositive microglia immunostained for cleaved caspase-9 in each section, and then calculated the average percentages of CD68immunopositive microglia immunostained for cleaved caspase-9 in the substantia nigra from the control and PD cases.
Acknowledgments This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan (No. 22590930).
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Please cite this article as: Kawamoto, Y., et al., Immunohistochemical localization of apoptosome-related proteins in Lewy bodies in Parkinson's disease and dementia with Lewy bodies. Brain Research (2014), http://dx.doi.org/10.1016/j. brainres.2014.05.007
