Alpha-synuclein-immunoreactive deposits in human and animal prion ...

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clein-immunoreactive deposits in the central nervous sys- tem of various prion diseases (sporadic, iatrogenic and new variant Creutzfeldt-Jakob diseases, and ...
Acta Neuropathol (2002) 103 : 516–520 DOI 10.1007/s00401-001-0499-z

R E G U L A R PA P E R

S. Haïk · N. Privat · K. T. Adjou · V. Sazdovitch · D. Dormont · C. Duyckaerts · J. J. Hauw

Alpha-synuclein-immunoreactive deposits in human and animal prion diseases

Received: 19 July 2001 / Revised, accepted: 6 November 2001 / Published online: 5 February 2002 © Springer-Verlag 2002

Abstract Prion related disorders are associated with the accumulation of a misfolded isoform (PrPsc) of the hostencoded prion protein, PrP. There is strong evidence for the involvement of unidentified co-factors in the PrP to PrPsc conversion process. In this study, we show α-synuclein-immunoreactive deposits in the central nervous system of various prion diseases (sporadic, iatrogenic and new variant Creutzfeldt-Jakob diseases, and experimental scrapie of hamsters). α-Synuclein accumulated close to PrPsc deposits but we did not observe strict colocalization of prion protein and α-synuclein immunoreactivities particularly in PrPsc plaques. α-Synuclein is thought to be a key player in some neurodegenerative disorders, is able to interact with amyloid structures and has known chaperone-like activities. Our results, in various prion diseases, suggest a role for α-synuclein in regulating PrPsc formation.

conversion can be mediated by cofactors [13, 17]. α-Synuclein is the major component of Lewy bodies [15]. This presynaptic protein [11] can self-aggregate and form amyloid-like filaments [5]. In addition, α-synuclein can bind amyloid structures in vitro [19], has been observed in huntingtin polyglutamine aggregate disorders [4], and can induce neuronal death [8, 20]. We report that α-synuclein-immunoreactive deposits can be observed in three varieties of Creutzfeldt-Jakob disease (sporadic, iatrogenic and new variant CJD) and in brains of Syrian hamsters inoculated with the scrapie strain 263K. α-Synuclein-immunoreactive deposits and deposits of PrPsc did not colocalize. These results, found in an archetype of protein misfolding-induced encephalopathy, suggest that this chaperone-like protein [14] may be involved in the abnormal process leading to protein accumulation and neurodegeneration in prion diseases.

Keywords Creutzfeldt-Jakob disease · Prion · Aggregation · Chaperone · Neurodegeneration

Material and methods Patients

Introduction Prion diseases, the most frequent variety of which in human is Creutzfeldt-Jakob disease (CJD), are characterized by post-translational accumulation of the host-encoded prion protein PrP in an amyloid misfolded isoform (PrPsc). Several lines of evidence indicate that the PrP to PrPsc

We studied eight cases of definite sporadic CJD [3], one case of iatrogenic CJD secondary to contaminated growth hormone treatment and one case of new variant CJD with well-documented clinical data and PrP genotype. The patients’ families gave informed consent for the genetic and post-mortem studies. Controls were nine normal brains from patients who had died of non-neurological diseases. Hamsters

S. Haïk (✉) · N. Privat · V. Sazdovitch · C. Duyckaerts · J.J. Hauw Raymond Escourolle Neuropathology Laboratory, Association Claude Bernard, INSERM U 360, Pitié-Salpêtrière Hospital, 47 Bd. de l’Hôpital, 75013 Paris, France e-mail: [email protected], Tel.: +33-1-42161881, Fax: +33-1-44239828 K.T. Adjou · D. Dormont Service de Neurovirologie, CEA, DSV/DRM, CRSSA, Fontenay aux Roses, France

Five female golden Syrian hamsters (Centre d’Elevage René Janvier, Le Genest-St-Isle, France) were inoculated intracerebrally with the 263K scrapie agent. The titer of the stock brain homogenate was 2.2×1011 50% lethal doses/g; 50 µl of 1% w/v dilution were injected into the right cerebral hemisphere of each animal. Control animals were four non-inoculated hamsters and four hamsters inoculated with a brain homogenate obtained from a normal animal. All efforts were made to minimize the number of animals.

517 Histopathology and immunohistochemistry Human brains A standard neuropathological study of human brains was performed after fixation in 10% formalin for at least 2 months. We sampled isocortical and allocortical areas, basal ganglia, cerebellum and brain stem from 1-cm-thick coronal sections. Specimens were embedded in paraffin (after formic acid treatment for all CJD cases and four control cases) as previously described [9]. Hamster brains Animals were killed 75 days after inoculation at the clinical phase of the disease. Hamster brains were removed and pretreated by formic acid treatment as previously described [1]. Antibodies Immunohistochemistry of α-synuclein was performed using a monoclonal antibody (1:1,500, IgG1 Kappa, epitope 115–122; Zymed, San Francisco, Calif.) and a rabbit polyclonal antibody (1:3,200, epitope 111–131; Chemicon, Temecula, Calif.). For negative controls, we used a monoclonal antibody directed against Aβ-amyloid peptide (IgG1 kappa; Dako, Trappes, France) and a rabbit polyclonal antibody directed against Tau protein (rabbit polyclonal; Dako). For hamster brain analysis, we used the rabbit polyclonal anti α-synuclein antibody (1/3,200; Chemicon); the rabbit polyclonal antibody directed against Tau protein and the IgG1 Kappa directed against human α-synuclein were used as negative controls. The antibodies directed against α-synuclein used in this study specifically recognize Lewy bodies and neurites in dementia with Lewy bodies and Parkinson’s disease. Immunohistochemistry of PrP was performed using 3F4 mouse anti-human PrP monoclonal antibody (Senetek Maryland Height, Mo.). For some double immunostaining, mouse monoclonal antibodies directed against GFAP (Dako), CD68 (Dako), and SNAP25 (Sternberger, Lutherville, Md.), and polyclonal anti-synaptophysin (Dako) and antiubiquitin ((Dako) were used. Immunohistochemistry PrP immunohistochemistry was performed on 7-µm-thick tissue sections from cerebellum and frontal cortex specimens using different pretreatment stages of deparaffined sections by microwave irradiation (1,000 W for 10 min, three times), formic acid treatment and enzymatic digestion (proteinase K diluted 1:200 for 8 min at 24°C) as previously described [9]. The following varieties of PrP deposits were distinguished: vacuolar (surrounding the vacuoles of spongiform changes), synaptic (diffuse staining), granular (small deposits less than 5 µm wide), focal (large, 5- to 50-µmwide, non-amyloid round deposits) and kuru-type amyloid plaques (10- to 50-µm-wide deposits which were also identified on Congo red sections) [9]. For α-synuclein immunochemistry, the pretreatment was microwave irradiation only. A standard streptavidin-biotin peroxidase kit (Dako) was used for single immunolabeling. Double labeling of PrP and α-synuclein was performed using a mouse monoclonal anti-PrP antibody (3F4) and a rabbit polyclonal antibody (Chemicon). In the streptavidin-biotin peroxidase system,

Fig. 1A–E α-Synuclein deposits in the CNS of different varieties of CJD. α-Synuclein immunohistochemistry in the cerebellum of a sporadic case of CJD (A), an iatrogenic case of CJD (B), a case of new variant CJD (C), a control case (D) and in the frontal cortex of a sporadic case of CJD (E). Granular deposits in the neuropil of the molecular layer (A–C), within (arrow) and on the edge of (small arrow) morula-type spongiosis of the deep cortical layers (E) (CJD Creutzfeldt-Jakob disease). Bar 33 µm

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Fig. 2A–D α-Synuclein deposits accumulate in hamster brains inoculated by a strain of scrapie (263K). Immunohistochemistry of α-synuclein in the thalamus (A, C) and hippocampus (B, D) of a 263K-inoculated hamster at the terminal stage of the disease (A, B) and of a normal control (C, D). Bar 33 µm

the α-synuclein labeling was first performed and revealed using a diaminobenzidine (DAB) substrate, followed by PrP labeling using a DAB-nickel substrate. For immunofluorescence detection, the two primary antibodies were used together and revealed using species-specific secondary antibodies. Confocal microscopic analyses were performed using a Leica TCS 4D (Krypton/Argon laser) with sequential acquisition for the two fluorochromes and a confocal depth of 0.6–1 µm. Excitation laser beams of 488 and 568 nm were used. The digital images were acquired using eight averaged line scans at 1,024×1,024 pixel resolution and Scanware software (Leica, Heidelberg, Germany).

Results and discussion α-Synuclein-immunoreactive granular deposits (1–6 µm in diameter) were found in 7 of the 10 studied cases of CJD. These granular deposits were always associated with prominent PrP accumulation: they were mostly seen in the cerebellar cortex (Fig. 1A–C), where PrPsc is easily detected, and were only present in cases with disease duration of more than 1 year. They were not found in the other three patients who died 3, 4, or 8 months, respectively, after the inaugural symptoms. This could be due to a lack of α-synuclein immunodetection under a critical threshold of accumulation. The deposits were numerous in new variant CJD and growth hormone-induced CJD, in

which the PrP load is usually high [2, 18]. In sporadic CJD, they were generally more sparse, and were mainly observed in the cerebellum in three cases. α-Synucleinimmunoreactive deposits were distinctly seen in the neuropil of the cerebellar molecular layer. The perikaryon of Purkinje cells was free of α-synuclein accumulation. In two other cases with no or mild cerebellar pathology, α-synuclein deposits were seen exclusively in the isocortex (Fig. 1E) around areas of spongiosis. Abundant deposits of α-synuclein were also found in the thalamus and hippocampus of scrapie-infected golden Syrian hamster (Fig. 2). Sparse deposits were seen in the frontal cortex and the cerebellum. Accumulation of α-synuclein was thus prominent in the brain areas where deposits of PrPsc principally occurred. In contrast, brains of human and hamster controls harbored neither α-synuclein nor PrP-immunoreactive deposits in addition to the fine punctuate pattern usually observed in areas rich in synapses of normal brains. Therefore, we observed α-synuclein-immunoreactive deposits in the central nervous system of different species with various prion diseases. This indicates that perturbation of the α-synuclein metabolism is a common mechanism involved in these disorders. The pathological cascade in prion diseases is not fully understood. The accumulation of α-synuclein close to PrPsc deposits suggests an interaction between the metabolism of PrP and α-synuclein. Since α-synuclein and its peptide byproducts have been shown to be neurotoxic [8, 20], α-synuclein accumulation may be an as yet unexpected factor for prion-induced neurodegeneration. The α-synuclein immunostain-

519 Fig. 3 Double labeling of α-synuclein and PrP (A–D), α-synuclein and ubiquitin (E, F) and α-synuclein and synaptophysin (G) in the cerebellum (molecular layer) of sporadic CJD. A–C DAB substrate for α-synuclein (brown) and DAB-nickel substrate for PrP (dark brown) were used. D Confocal microscopy using green labeling (FITC) for PrP and red labeling (Cy3) for α-synuclein. α-Synuclein immunolabeling was found either isolated (arrow) or in areas of PrPsc granular deposits (double arrow) or close to PrPsc plaques (arrowhead). E, F Confocal microscopy using green labeling (FITC) for α-synuclein and red labeling (Cy3) for ubiquitin in two different areas. G Confocal microscopy using green labeling (FITC) for α-synuclein and red labeling (Cy3) for synaptophysin (PrP prion protein, PrPsc misfolded isoform of PrP, DAB diaminobenzidine, FITC fluorescein isothiocyanate, Cy3 cyanin 3). Bar 15 µm

ing may also correspond to the dystrophic neurites that have been observed around PrPsc plaques and that are positive for ubiquitin immunostaining [10, 16]. Therefore, we performed double staining of α-synuclein and ubiqui-

tin (Fig. 3). We observed that α-synuclein staining exhibits a distinct pattern. We did not observe any colocalization of α-synuclein immunostaining and other neural cell markers [GFAP, CD68, SNAP25 (data not shown)

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and synaptophysin (Fig. 3)]. Thus, although the precise location at the cellular level of α-synuclein accumulation remains to be determined by electron microscopy, our results suggest that α-synuclein deposits cannot be explained only by a disturbance in the presynaptic structure. Double labeling of PrP and α-synuclein in CJD (Fig. 3) and scrapie hamsters (data not shown) indicated that these protein deposits did not regularly colocalize. α-Synuclein was seen as discrete deposits that sometimes grouped together close to granular, focal or plaque-type PrP deposits. Interestingly, no examples of colocalization of PrP and α-synuclein immunoreactivities were found in the plaques of PrP (Fig. 3). The underlying pathogenic event in prion diseases is a conformational modification of the normal host-encoded PrP from a soluble form to the pathogenic form that is aggregated and rich in β-sheets [13]. There is increasing evidence that various chaperones can mediate the PrP to PrPsc conversion [7]. Moreover, data from transgenic mice models suggest the requirement of an accessory molecule in the conversion process [17], which may be a cellular chaperone [13]. α-Synuclein has been shown to bind Aβ and to regulate its formation [19]. Moreover, α-synuclein also exhibits chaperone-like activity and inhibits the aggregation of denatured proteins [14]. We cannot exclude that α-synuclein might similarly regulate the conversion of PrP into its amyloid isoform, in which case the absence of colocalization of α-synuclein and PrPsc amyloid deposits would suggest a role of α-synuclein in a negative modulation of PrP aggregation process. In Alzheimer’s disease, the presence of α-synuclein in amyloid plaques is still a debated question [6]. However, deposits of α-synuclein, the major component of Lewy bodies, have been observed in other neurodegenerative disorders, including Huntington’s disease [4]. α-Synuclein deposits are thus seen in disorders associated with amyloid or aggregated proteins [12]. Our observation of α-synuclein deposits in prion diseases, which represent an archetype of protein conformation-induced encephalopathies [13], suggests that α-synuclein may be involved in the abnormal post-translational process leading to misfolding of proteins and neurodegeneration. Acknowledgements We are grateful to the members of the Epidemiological Network for Creutzfeldt-Jakob survey in France and to C. Nzé and F. Fierville for providing technical assistance. This study was supported in part by the “Programme de Recherche sur les ESST et les Prions”, EU Concerted Action on Human TSE (Biomed 2, contracts QLK2-CT-2000-00837 and BMH4-CT986003) and PHRC (grant no. AOM 96-117).

References 1. Adjou T, Privat N, Demart S, Deslys J, Seman M, Hauw JJ, Dormont D (2000) MS-8209, an amphotericin B analogue, delays the appearance of spongiosis, astrogliosis and PrPres accumulation in the brain of scrapie-infected hamsters. J Comp Pathol 122:3–8 2. Billette de Villemeur T, Gelot A, Deslys JP, Dormont D, Duyckaerts C, Jardin L, Denni J, Robain O (1994) Iatrogenic Creutzfeldt-Jakob disease in three growth hormone recipients: a neuropathological study. Neuropathol Appl Neurobiol 20: 111–117

3. Budka H, Aguzzi A, Brown P, Brucher JM, Bugiani O, Gullotta F, Haltia M, Hauw JJ, Ironside JW, Jellinger K, Kretzschmar HA, Lantos PL, Masullo C, Schlote W, Tateishi J, Weller RO (1995) Neuropathological diagnostic criteria for Creutzfeldt-Jakob disease (CJD) and other human spongiform encephalopathies (Prion diseases). Brain Pathol 5:459–466 4. Charles V, Mezey E, Reddy PH, Dehejia A, Young TA, Polymeropoulos MH, Brownstein MJ, Tagle DA (2000) Alphasynuclein immunoreactivity of huntingtin polyglutamine aggregates in striatum and cortex of Huntington’s disease patients and transgenic mouse models. Neurosci Lett 289:29–32 5. Conway KA, Harper JD, Lansbury PT Jr (2000) Fibrils formed in vitro from alpha-synuclein and two mutant forms linked to Parkinson’s disease are typical amyloid. Biochemistry 39:2552– 2563 6. Culvenor JG, McLean CA, Cutt S, Campbell BC, Maher F, Jakala P, Hartmann T, Beyreuther K, Masters CL, Li QX (1999) Non-Aβ component of Alzheimer’s disease amyloid (NAC) revisited NAC and alpha-synuclein are not associated with Aβ amyloid. Am J Pathol 155:1173–1181 7. Debburman SK, Raymond GJ, Caughey B, Lindquist S (1997) Chaperone-supervised conversion of prion protein to its protease-resistant form. Proc Natl Acad Sci USA 94:13938–13943 8. Forloni G, Bertani I, Calella AM, Thaler F, Invernizzi R (2000) Alpha-synuclein and Parkinson’s disease: selective neurodegenerative effect of alpha-synuclein fragment on dopaminergic neurons in vitro and in vivo. Ann Neurol 47:632–640 9. Hauw JJ, Sazdovitch V, Laplanche JL, Peoc’h K, Kopp N, Kemeny J, Privat N, Delasnerie-Laupretre N, Brandel JP, Deslys JP, Dormont D, Alperovitch A (2000) Neuropathologic variants of sporadic Creutzfeldt-Jakob disease and codon 129 of PrP gene. Neurology 54:1641–1646 10. Ironside JW, Mccardle L, Hayward PA, Bell JE (1993) Ubiquitin immunocytochemistry in human spongiform encephalopathies. Neuropathol Appl Neurobiol 19:134–140 11. Iwai A, Masliah E, Yoshimoto M, Ge N, Flanagan L, Silva HA de, Kittel A, Saitoh T (1995) The precursor protein of non-Aβ component of Alzheimer’s disease amyloid is a presynaptic protein of the central nervous system. Neuron 14:467–475 12. McGowan DP, Roon-Mom W van, Holloway H, Bates GP, Mangiarini L, Cooper GJ, Faull RL, Snell RG (2000) Amyloidlike inclusions in Huntington’s disease. Neuroscience 100: 677–680 13. Prusiner SB (1998) Prions. Proc Natl Acad Sci USA 95: 13363–13383 14. Souza JM, Giasson BI, Lee VM, Ischiropoulos H (2000) Chaperone-like activity of synucleins. FEBS Lett 474:116–119 15. Spillantini MG, Schmidt ML, Lee VM, Trojanowski JQ, Jakes R, Goedert M (1997) Alpha-synuclein in Lewy bodies. Nature 388:839–840 16. Suenaga T, Hirano A, Llena J.F, Ksiezak-Reding H, Yen S-H, Dickson DW (1990) Ubiquitin immunoreactivity in Kuru plaques in Creutzfeldt-Jakob Disease. Ann Neurol 28:174–177 17. Telling GC, Scott M, Mastrianni J, Gabizon R, Torchia M, Cohen FE, DeArmond SJ, Prusiner SB (1995) Prion propagation in mice expressing human and chimeric PrP transgenes implicates the interaction of cellular PrP with another protein. Cell 83:79–90 18. Will RG, Ironside JW, Zeidler M, Cousens SN, Estibeiro K, Alperovitch A, Poser S, Pocchiari M, Hofman A, Smith PG (1996) A new variant of Creutfeldt-Jakob disease in the UK. Lancet 347:921–925 19. Yoshimoto M, Iwai A, Kang D, Otero DA, Xia Y, Saitoh T (1995) NACP, the precursor protein of the non-amyloid beta/A4 protein (Aβ) component of Alzheimer disease amyloid, binds Aβ and stimulates Aβ aggregation. Proc Natl Acad Sci USA 92:9141–9145 20. Zhou W, Hurlbert MS, Schaack J, Prasad KN, Freed CR (2000) Overexpression of human alpha-synuclein causes dopamine neuron death in rat primary culture and immortalized mesencephalon-derived cells. Brain Res 866:33–43