PMCCPMCCPMCC

Search tips
Search criteria 

Advanced

 
Logo of actahistAuthor GuidelinesHomepageActa Histochemica et Cytochemica
 
Acta Histochem Cytochem. 2010 May 1; 43(2): 69–75.
Published online 2010 April 21. doi:  10.1267/ahc.10005
PMCID: PMC2875863

Involvement of 4-hydroxy-2-nonenal Accumulation in Multiple System Atrophy

Abstract

Recent studies have suggested implications for α-synuclein cytotoxicity in the pathomechanism of multiple system atrophy (MSA). Given in vitro evidence that α-synuclein generates oxidative stress, it is proposed that lipid peroxidation may be accelerated in MSA. To address this issue, we performed an immunohistochemical analysis of protein-bound 4-hydroxy-2-nonenal (P-HNE) in sections of archival, formalin-fixed, paraffin-embedded pontine materials of eight sporadic MSA patients and eight age-matched control subjects. In the MSA cases, P-HNE immunoreactivity was localized in all of the neuronal cytoplasmic inclusions and glial cytoplasmic inclusions, both of them identified with α-synuclein and ubiquitin. It was also detectable in reactive astrocytes and phagocytic microglia but undetectable in activated microglia. By contrast, P-HNE immunoreactivity in the control cases was only very weak or not at all in the parenchyma including neurons and glia. The present results provide in vivo evidence that HNE participates in α-synuclein-induced cytotoxicity and neuroinflammation in MSA.

Keywords: alpha-synuclein, 4-hydeoxy-2-nonenal, immunohistochemistry, multiple system atrophy, oxidative stress

I. Introduction

Multiple system atrophy (MSA) is a neurodegenerative disease characterized by selective involvement of the pontocerebellar, striatonigral, and autonomic systems, corresponding to the classical clinical entities such as olivopontocerebellar atrophy (OPCA), striatonigral degeneration (SND) and Shy-Drager syndrome (SDS), respectively [41]. Compelling evidence identifies α-synuclein cytotoxicity in the pathological processes of α-synucleinopathies including neurodegenerative disorders such as MSA as well as Parkinson’s disease [9, 10, 39, 42]. Several studies focused on implications for aggregation and ubiquitination of α-synuclein molecules [21, 26] in neuronal cytoplasmic inclusions (NCIs) and (oligodendro)glial cytoplasmic inclusions (GCIs), both of which are known to be pathognomonic hallmarks of MSA and appear in the affected systems [40, 41].

Recent investigations have documented the occurrence of oxidative stress in human MSA and its animal models [8, 18, 33] as well as in vitro evidence that α-synuclein molecules generate oxidative stress [34]. It is well known that oxidative stress gives rise to the formation of oxidative modification products derived from nucleic acids, proteins and lipids [11]. A previous study demonstrated oxidative injury evidenced by the presence of 4-hydroxy-2-nonenal (HNE) in Purkinje cells in the cerebellum of patients with OPCA [43]. HNE, a member of 4-hydroxyalkenals, is a well characterized lipid peroxidation product derived from n-6 polyunsaturated fatty acids (PUFAs), including linoleic and γ-linoleinic acids; it attacks and modifies protein amino acid residues to form its protein-bound forms such as “hemiacetal adduct” with histidine, cysteine or lysine, “pyrrole adduct” with lysine, and “fluorescent crosslinker” with lysine (Fig. 1) [38]. These processes give rise to stress response such as expression of proinflammatory gene products including monocyte chemoattractant protein-1 (MCP-1) and cyclooxygenase-2 (COX-2) in macrophages [20, 25]. Given such conditions, it is plausible that HNE triggers neuroinflammation in MSA. However, the relationship between α-synuclein aggregation, lipid peroxidation, and glial inflammatory response in MSA has not been fully understood. To address this issue, we investigated and compared immunohistochemical localizations of protein-bound HNE (P-HNE), α-synuclein, ubiquitin and glial cell markers in MSA pons, which is essentially involved in this disease.

Fig. 1
Schematic diagram showing nonenzymatic processes of n-6 polyunsaturated fatty acid (PUFA) peroxidation and subsequent protein carbonylation to form several protein adducts of 4-hydroxy-2-nonenal (HNE). His, Cys, and Lys indicate protein amino acid residues ...

II. Materials and Methods

Subjects

This study was performed after obtaining written consent from the family member of each case, in accordance with the Declaration of Helsinki, and carried out on archival, formalin (10%)-fixed, paraffin-embedded brain stem pontine materials of eight sporadic MSA patients (age: 55–68 yr; sex: 4 males and 4 females) and eight age-matched control subjects (age: 52–68; sex: 6 males and 2 females). There was no significant difference in age at death between the control and MSA groups (P>0.8 by unpaired Student’s t-test). The clinical features of the cases examined are summarized in Table 1. Multiple 6-µm-thick sections of each pons were used for histopathological staining with hematoxylin-eosin (H&E) and Klüver-Barrera (K&B) and for immunohistochemical staining.

Table 1
Clinical features of examined cases

Antibodies

The primary antibodies employed in immunohistochemistry were mouse monoclonal IgG1 against P-HNE (Clone HNEJ2; NOF, Tokyo, Japan) at a concentration of 0.1 µg/mL, rabbit polyclonal IgG against α-synuclein (Cat. No. 18671; IBL, Fujioka, Japan) at a concentration of 1 µg/mL, rabbit polyclonal IgG against ubiquitin (Cat. No. Z458; Dako, Glostrup, Denmark) at a dilution of 1:1,000, rabbit polyclonal IgG against glial fibrillary acidic protein (GFAP) (Cat. No. GTX16997; GeneTex; Irvine, CA, USA) at a dilution of 1:1,000, and rabbit polyclonal IgG against glucose transporter-5 (GLUT5) (Cat. No. 18905; IBL) at a concentration of 0.5 µg/mL. Of these antigens, GFAP and GLUT5 were used as histological markers of astrocytes and microglia, respectively. As described in the data sheet, the antibody HNEJ2 specifically recognizes hemiacetal adduct with histidine among the protein-bound forms of HNE [37].

Immunohistochemical staining

Sections were deparaffinized, rehydrated, quenched for 10 min at room temperature with 3% hydrogen peroxide, rinsed in phosphate-buffered saline, pH 7.6 (PBS), pretreated for 20 min at room temperature with 3% bovine serum albumin (BSA) in PBS, and then incubated overnight at 4°C with the primary antibodies. Antibody binding was visualized by the polymer-immunocomplex (PIC) method using the appropriate EnVision system (Dako). 3,3'-Diaminobenzidine tetrahydrochloride (DAB) was the chromogen, and hematoxylin, the counterstain. Sections processed with omission of the primary antibodies or incubated with nonimmune serum derived from the same animal species as those producing the antibodies gave negative reaction controls. The specificity of the anti-P-HNE antibody on sections was comfirmed by preimmunoabsorption test; sections were incubated with the antibody preincubated with 1 mg/mL BSA-HNE conjugates in PBS.

Immunohistochemical evaluation

Immunohistochemical localization of P-HNE was verified by comparison with consecutive sections stained with H&E and immunostained with the antibodies to α-synuclein, ubiquitin, GFAP, and GLUT5, and it was also identified by the double-immunolabeled staining method as described previously [32]. Sections were incubated with one antibody, and immunoreaction was detected by the PIC method using DAB as the chromogen. After taking microphotographs, sections were processed by eluting the immune complex by microwaving (400 W, 95°C, 10 min) in citrate buffer, pH 6.0, and these sections were subsequently incubated with another antibody. Immunoreaction was detected by the PIC method using NiCl2/DAB as the chromogen. Immunohistochemical localizations of the examined substances on NiCl2/DAB-colored sections were compared with the initially taken microphotographs at the corresponding regions of DAB-colored sections.

III. Results

Histopathological observations

Histopathological examination revealed that the MSA pons showed the characteristic features of this disease as described previously [41], whereas the control pons displayed no significant abnormality. In brief, at a low magnification of K&B-stained sections, the MSA patients showed prominent atrophy of pontine base and myelin pallor of transverse fibers (Fig. 2b), while the control subjects seemed to be morphologically intact (Fig. 2a). At a higher magnification of H&E-stained sections, the MSA cases exhibited atrophy and reduced number of pontine neurons, reactive gliosis, and appearance of NCIs and GCIs (Fig. (Fig.2d),2d), but by contrast the control cases did not show any histological alteration (Fig. 2c).

Fig. 2
Semimacroscopic findings of Klüver-Barrera-stained pontine sections (a, b) and microscopic findings of pontine nucleus sections stained with hematoxylin-eosin (c, d) and immunostained for protein-bound 4-hydroxy-2-nonenal (e, f) in control subjects ...

Immunohistochemical observations

Immunoreaction products were invisible on sections processed for negative reaction control (Fig. 2g) and preimmunoabsorption test of the anti-P-HNE antibody (Fig. (Fig.2h).2h). P-HNE immunoreactivity was only very weak or not at all in the control cases (Fig. 2e). In the MSA cases, the immunoreactivity was mainly distributed in the pontine base and distinct in the abnormal structures NCIs and GCIs, as well as the cytoplasm of all of the normal-looking and degenerative pontine neurons and reactive glia, and it was more intense in these abnormal structures (Fig. 2f). As described in the literature [7], microglial morphology in pathological conditions was defined as the following: activated microglia were characterized by thin perikarya and double-branching, arborized, thick cell processes, and phagocytic microglia were characterized by hypertrophic perikarya. Moreover, activated microglia were scattered in the MSA lesions (Fig. 3h), while phagocytic microglia were aggregated to constitute so-called glial nodules (Fig. 3j). P-HNE immunoreactivity in the MSA pons was colocalized with all of the NCIs and GCIs immunoreactive for α-synuclein (Fig. 3a, b) and ubiquitin (Fig. 3c, d), and it was also detectable in almost all of the GFAP-immunoreactive reactive astrocytes in the lesions (Fig. 3e, f) and GLUT5-immunoreactive “aggregated” phagocytic microglia (Fig. 3i, j) but not in any of the GLUT5-immunoreactive “scattered” activated microglia (Fig. 3g, h). No distinct α-synuclein immunoreactivity was detectable in GFAP-immunoreactive astrocytes (Fig. 4a, b) or GLUT5-immunoreactive microglia (Fig. (Fig.4c,4c, d) in the MSA cases. There was no significant difference in these immunohistochemical results among the MSA cases.

Fig. 3
Immunohistochemical comparison of protein-bound 4-hydroxy-2-nonenal (a, c, e, g, i) with α-synuclein (b), ubiquitin (d), glial fibrillary acidic protein (f) and glucose transporter-5 (h, j) in pontine sections from multiple system atrophy patients. ...
Fig. 4
Double immunohistochemical staining of α-synuclein (blue in a; brown in b) with glial fibrillary acidic protein-immunoreactive reactive astrocytes (brown in a) and glucose transporter-5-immunoreactive phagocytic microglia (blue in b) in pontine ...

IV. Discussion

In the present study, noteworthy was the coexistence of P-HNE accumulates with α-synuclein- and ubiquitin-containing NCIs and GCIs in the MSA pons. These observations suggest a close link between α-synuclein, ubiquitin, and HNE in the pathomechanism of MSA. A number of investigations have indicated that oligomers of α-synuclein molecules generate reactive oxygen species (ROS) such as hydrogen peroxide and hydroxyl radical [35], which are responsible for lipid peroxidation [11, 38]. The representative lipid peroxidation product HNE modifies and crosslinks amino acid residues of α-synuclein to promote molecular aggregation of this protein [28]. HNE also inhibits normal proteasomal and lysosomal functions but not ubiquitination in neurons [4, 5, 15, 17], impairs glutamate and glucose transport in neurons [16, 27], and induces programmed cell death of neurons and oligodendrocytic progenitors [6, 19, 22]; these mechanisms are mediated by HNE-driven oxidative stress. Thus, our results suggest a critical role for HNE, as an α-synuclein-induced lipid peroxidation product, in the processes of neuronal and oligodendroglial degeneration in MSA, including ROS-induced cell damage, inhibition of the proteolytic mechanisms, and formation of the NCIs and GCIs.

Another interesting finding in this study was the disclosure of P-HNE accumulates in pontine base astrocytes and microglia in the MSA cases. Given that α-synuclein immunoreactivity was localized in NCIs and GCIs, it is likely that HNE primarily occurs in lesional neurons and oligodendroglia in the processes of α-synuclein aggregation and diffuses into the surrounding parenchyma. The finding that α-synuclein immunoreactivity was undetectable in astrocytes and microglia suggests that these cells digested this protein after uptake. The lack of P-HNE immunoreactivity in activated microglia may result from evidence that these cells promptly scavenge HNE via their strong antioxidative defense system using a large amount of intra-cellular glutathione produced by cystine that is taken up from the extracellular space in return for HNE-induced glutamate release [1]. The fact that P-HNE accumulates were detectable in reactive astrocytes and phagocytic microglia points to the possibility that these glial cells may phagocytose extracellular P-HNE or undergo intracellular protein adduct formation of HNE diffused from the extracellular space. This discrepancy of P-HNE immunostaining between phagocytic and activated forms of microglia may reflect a difference in property of these cells [7].

Glial activation has been observed in the brain lesions of MSA patients and α-synuclein-overexpressing mice as an animal model of this disease [13, 34]. A number of studies have demonstrated that HNE impairs glutamate uptake by astrocytes and microglia and promotes glutamate release from these glial cells [13, 36], leading to exposure of neurons and oligodendrocytes to an excess of extracellular glutamate. It has been shown that an imbalance of glial glutamate release and uptake interacts with neuroinflammation [36]. Activated microglia modulate expression of astroglial enzymes responsible for oxidative and inflammatory stimuli [30]. Furthermore, several studies have focused on the role of α-synuclein in lipid peroxidation metabolism and downstream inflammatory response in the brain [10]. In fact, upregulation of proinflammatory gene products has been shown in reactive astrocytes and activated microglia in neurodegenerative diseases such as Alzheimer’s disease, Parkinson’s disease, and amyotrophic lateral sclerosis [12, 23, 29], all of which are associated with increased tissue HNE levels [14, 24, 31]. These observations could indicate that glial cells in MSA lesions participate in local inflammation in response to oxidative stress.

Taken together, we demonstrated the accumulation of P-HNE in NCIs, GCIs, reactive astrocytes, and phagocytic microglia, but not activated microglia in MSA pontine lesions. Our results suggest the involvement of HNE-mediated cytotoxic effects on pontine neurons and oligodendroglia in association with the formation of NCIs and GCIs, as well as inflammatory response and glutamate toxicity by astrocytes and microglia. Elucidating the mechanism by which α-synuclein-induced HNE production is regulated in the central nervous system will contribute to a better understanding of MSA and establishment of therapeutic strategies for this disease.

V. Acknowledgements

The authors wish to thank N. Sakayori and M. Karita for technical assistance.

VI. References

1. Barger S. W., Goodwin M. E., Porter M. M., Beggs M. L. Glutamate release from activated microglia requires the oxidative burst and lipid peroxidation. J. Neurochem. 2007;101:1205–1213. [PMC free article] [PubMed]
2. Blanc E. M., Bruce-Keller A. J., Mattson M. P. Astrocytic gap junctional communication decreases neuronal vulnerability to oxidative stress-induced disruption of Ca2+ homeostasis and cell death. J. Neurochem. 1998;70:958–970. [PubMed]
3. Blanc E. M., Keller J. N., Fernandez S., Mattson M. P. 4-Hydroxynonenal, a lipid peroxidation product, impairs glutamate transport in astrocytes. Glia. 1998;22:149–160. [PubMed]
4. Dasuri K., Nguyen A., Zhang L., Ferdenandez-Kim O. S., Bruce-Keller A. J., Blalock B. A., Cabo R. D., Keller J. N. Comparison of rat liver and brain proteasomes for oxidative stress-induced inactivation: influence of ageing and dietary restriction. Free Radic. Res. 2009;43:28–36. [PMC free article] [PubMed]
5. Ferrington D. A., Kapphahn R. J. Catalytic site-specific inhibition of the 20S proteasome by 4-hydroxynonenal. FEBS Lett. 2004;578:217–223. [PubMed]
6. Gard A. L., Solodushko V. G., Waeg G., Majic T. 4-Hydroxynonenal, a lipid peroxidation byproduct, is cytotoxic for oligodendrocyte progenitors and inhibits their responsiveness to PDGF. Microsc. Res. Tech. 2001;52:709–718. [PubMed]
7. Garden G. A., Möller T. Microglia biology in health and disease. J. Neuroimmune Pharmacol. 2006;1:127–137. [PubMed]
8. Giasson B. I., Duda J. E., Murray I. V., Chen Q., Souza J. M., Hurtig H. I., Ischiropoulos H., Trojanowski J. Q., Lee V. M. Oxidative damage linked to neurodegeneration by selective α-synuclein nitration in synucleinopathy lesions. Science. 2000;290:985–989. [PubMed]
9. Goedert M. Alpha-synuclein and neurodegenerative diseases. Nat. Rev. Neurosci. 2001;2:492–501. [PubMed]
10. Golovko M. Y., Barceló-Coblijn G., Castagnet P. I., Austin S., Combs C. K., Murphy E. J. The role of α-synuclein in brain lipid metabolism: a downstream impact on brain inflammatory response. Mol. Cell. Biochem. 2009;326:55–66. [PubMed]
11. Halliwell B. Oxidative stress and neurodegeneration: where are we now? J. Neurochem. 2006;97:1634–1658. [PubMed]
12. Hirsch E. C., Hunot S. Neuroinflammation in Parkinson’s disease: a target for neuroprotection? Lancet Neurol. 2009;8:382–397. [PubMed]
13. Ishizawa K., Komori T., Sasaki S., Arai N., Mizutani T., Hirose T. Microglial activation parallels system degeneration in multiple system atrophy. J. Neuropathol. Exp. Neurol. 2004;63:43–52. [PubMed]
14. Jenner P. Oxidative stress in Parkinson’s disease. Ann. Neurol. 2003;53 (suppl. 3):S26–S38. [PubMed]
15. Kaemmerer E., Schutt F., Krohne T. U., Holz F. G., Kopitz J. Effects of lipid peroxidation-related protein modifications on RPE lysosomal functions and POS phagocytosis. Invest. Ophthalmol. Vis. Sci. 2007;48:1342–1347. [PubMed]
16. Keller J. N., Mark R. J., Bruce A. J., Blanc E., Rothstein J. D., Uchida K., Waeg G., Mattson M. P. 4-Hydroxynonenal, an aldehydic product of membrane lipid peroxidation, impairs glutamate transport and mitochondrial function in synaptosomes. Neuroscience. 1997;80:685–696. [PubMed]
17. Keller J. N., Huang F. F., Zhu H., Yu J., Ho Y.-S., Kindy M. S. Oxidative stress-associated impairment of proteasome activity during ischemia-reperfusion injury. J. Cereb. Blood Flow Metab. 2000;20:1467–1473. [PubMed]
18. Kikuchi A., Takeda A., Onodera H., Kimpara T., Hisanaga K., Sato N., Nunomura A., Castellani R. J., Perry G., Smith M. A., Itoyama Y. Systemic increase of oxidative nucleic acid damage in Parkinson’s disease and multiple system atrophy. Neurobiol. Dis. 2002;9:244–248. [PubMed]
19. Kruman I., Bruce-Keller A. J., Bredesen D., Waeg G., Mattson M. P. Evidence that 4-hydroxynonenal mediates oxidative-stress-induced neuronal apoptosis. J. Neurosci. 1997;17:5089–5100. [PubMed]
20. Kumagai T., Matsukawa N., Kaneko Y., Kusumi Y., Mitsumata M., Uchida K. A lipid peroxidation-derived inflammatory mediator. Identification of 4-hydroxy-2-nonenal as a potential inducer of cyclooxygenase-2 in macrophages. J. Biol. Chem. 2004;279:48389–48396. [PubMed]
21. Lee J. T., Wheeler T. C., Chin L.-C. Ubiquitination of α-synuclein by Siah-1 promotes α-synuclein aggregation and apoptotic cell death. Hum. Mol. Genet. 2008;17:906–917. [PubMed]
22. Malecki A., Garrido R., Mattson M. P., Hennig B., Toborek M. 4-Hydroxynonenal induces oxidative stress and death of cultured spinal cord neurons. J. Neurochem. 2000;74:2278–2287. [PubMed]
23. McGeer P. L., McGeer E. G. Inflammatory processes in amyotrophic lateral sclerosis. Muscle Nerve. 2002;26:459–470. [PubMed]
24. Montine T. J., Morrow J. D. Fatty acid oxidation in the pathogenesis of Alzheimer’s disease. Am. J. Pathol. 2005;166:1283–1289. [PubMed]
25. Nitti M., Domenicotti C., d’Abramo C., Assereto S., Cottalasso D., Melloni E., Poli G., Biasi F., Marinari U. M., Pronzato M. A. Activation of PKC-β isoforms mediates HNE-induced MCP-1 release by macrophages. Biochem. Biophys. Res. Commun. 2002;294:547–552. [PubMed]
26. Nonaka T., Iwatsubo T., Hasegawa M. Ubiquitination of α-synuclein. Biochemistry. 2005;44:361–368. [PubMed]
27. Pedersen W. A., Cashman N. R., Mattson M. P. The lipid peroxidation product 4-hydroxynonenal impairs glutamate and glucose transport and choline acetyltransferase activity in NSC-19 motor neuron cells. Exp. Neurol. 1999;155:1–10. [PubMed]
28. Quin Z., Hu D., Han S., Reaney S. H., Di Monte D. A., Fink A. L. Effect of 4-hydroxy-2-nonenal modification on α-synuclein aggregation. J. Biol. Chem. 2007;282:5862–5870. [PubMed]
29. Rogers J. The Inflammatory response in Alzheimer’s disease. J. Periodontol. 2008;79:1535–1543. [PubMed]
30. Röhl C., Armbrust E., Kolbe K., Maser E., Venz S., Gülden M. Activated microglia modulate astroglial enzymes involved in oxidative stress and inflammatory stress and increase the resistance of astrocytes to oxidative stress in vitro. Glia. 2008;56:1114–1126. [PubMed]
31. Shibata N., Nagai R., Uchida K., Horiuchi S., Yamada S., Hirano A., Kawaguchi M., Yamamoto T., Sasaki S., Kobayashi M. Morphological evidence for lipid peroxidation and protein glycoxidation in spinal cords from sporadic amyotrophic lateral sclerosis patients. Brain Res. 2001;917:97–104. [PubMed]
32. Shibata N., Kawaguchi M., Uchida K., Kakita A., Takahashi H., Nakano R., Fujimura H., Sakoda S., Ihara Y., Nobukuni K., Takehisa Y., Kuroda S., Kokubo Y., Kuzuhara S., Honma T., Mochizuki Y., Mizutani T., Yamada S., Toi S., Sasaki S., Iwata M., Hirano A., Yamamoto T., Kato Y., Sawada T., Kobayashi M. Protein-bound crotonaldehyde accumulates in the spinal cord of superoxide dismutase-1 mutation-associated familial amyotrophic lateral sclerosis and its transgenic mouse model. Neuropathology. 2007;27:49–61. [PubMed]
33. Stefanova N., Reindle M., Neumann M., Haass C., Poewe W., Kahle P. J., Wenning G. K. Oxidative stress in transgenic mice with oligodendroglial α-synuclein overexpression replicates the characteristic neuropathology of multiple system atrophy. Am. J. Pathol. 2005;166:869–876. [PubMed]
34. Stefanova N., Reindl M., Neumann M., Kahle P. J., Poewe W., Wenning G. K. Microglial activation mediates neurodegeneration related to oligodendrolial α-synucleinopathy: implications for multiple system atrophy. Movement Dis. 2007;22:2196–2203. [PubMed]
35. Tabner B. J., Turnbull S., El-Agnaf O. M. A., Allsop D. Formation of hydrogen peroxide and hydroxyl radicals from Aβ and α-synuclein as a possible mechanism of cell death in Alzheimer’s disease and Parkinson’s disease. Free Radic. Biol. Med. 2002;32:1076–1083. [PubMed]
36. Tilleux S., Hermans E. Neuroinflammation and regulation of glial glutamate uptake in neurological disorders. J. Neurosci. Res. 2007;85:2059–2070. [PubMed]
37. Toyokuni S., Miyake N., Hiai H., Hagiwara M., Kawakishi S., Osawa T., Uchida K. The monoclonal antibody specific for the 4-hydroxy-2-nonenal histidine adduct. FEBS Lett. 1995;359:189–191. [PubMed]
38. Uchida K. Role of reactive aldehydes in cardiovascular diseases. Free Radic. Biol. Med. 2000;28:1685–1696. [PubMed]
39. Uversky V. N. Neuropathology, biochemistry, and biophysics of α-synuclein aggregation. J. Neurochem. 2007;103:17–37. [PubMed]
40. Wakabayashi K., Yoshimoto M., Tsuji S., Takahashi H. α-Synuclein immunoreactivity in glial cytoplasmic inclusions in multiple system atrophy. Neurosci. Lett. 1998;249:180–182. [PubMed]
41. Wenning G. K., Colosimo C., Geser F., Poewe W. Multiple system atrophy. Lancet Neurol. 2004;3:93–103. [PubMed]
42. Wenning G. K., Stefanova N., Jellinger K. A., Poewe W., Schlossmacher M. G. Multiple system atrophy: a primary oligodendrogliopathy. Ann. Neurol. 2008;64:239–246. [PubMed]
43. Yamashita T., Ando Y., Obayashi K., Terazaki H., Sakashita N., Uchida K., Ohama E., Ando M., Uchino M. Oxidative injury is present in Purkinje cells in patients with olivopontocerebellar atrophy. J. Neurol. Sci. 2000;175:107–110. [PubMed]

Articles from Acta Histochemica et Cytochemica are provided here courtesy of Japan Society of Histochemistry and Cytochemistry