Prion protein deposition and abnormal synaptic protein expression in the cerebellum in Creutzfeldt–Jakob disease

PrP and synaptic protein expression in CJD cerebellum

Pergamon

PII: S0306-4522(00)00045-2

Neuroscience Vol. 97, No. 4, pp. 715±726, 2000 715 Copyright q 2000 IBRO. Published by Elsevier Science Ltd Printed in Great Britain. All rights reserved 0306-4522/00 $20.00+0.00

www.elsevier.com/locate/neuroscience

PRION PROTEIN DEPOSITION AND ABNORMAL SYNAPTIC PROTEIN EXPRESSION IN THE CEREBELLUM IN CREUTZFELDT±JAKOB DISEASE I. FERRER,* B. PUIG, R. BLANCO and E. MARTIÂ Unidad de NeuropatologõÂa, Departamento de BiologõÂa Celular y AnatomõÂa PatoloÂgica, Universidad de Barcelona, campus de Bellvitge, 08907 Hospitalet de Llobregat, Spain

AbstractÐPrion protein (PrP C) is a cell membrane-anchored glycoprotein, which is replaced by a pathogenic protease-resistant, b-sheet-containing isoform (PrP CJD or PrP SC) in human and animal prion encephalopathies, including sporadic Creutzfeldt±Jakob disease. Cell fractionation methods show that PrP C localizes in presynaptic membrane-enriched fractions. Following infection, abnormal PrP accumulates in nerve cell processes and synaptic regions. The present study examines the possible correlation between abnormal PrP deposition and the expression of synaptic proteins controlling neurotransmission in the cerebellum of six 129 Met/Met sporadic cases of Creutzfeldt±Jakob disease. Aggregates of protease-resistant PrP-positive granules, reminiscent of cerebellar glomeruli, were found in the granular cell layer, whereas ®ne punctate PrP-immunoreactive deposits occurred in the molecular layer. Small numbers of diffuse, irregular plaque-like PrP deposits in the molecular and granular cell layers were present in every case. The somas of Purkinje cells, and stellate, basket and Golgi neurons, were not immunostained. PrP-immunoreactive ®bres were found in the album of the cerebellum and hilus of the dentate nucleus. Punctate PrP deposition decorated the neuropil of the dentate nucleus and the surface of dentate neurons. Synaptic protein expression was examined with synaptophysin, synapsin-1, synaptosomal-associated protein of 25,000 mol. wt, syntaxin-1 and Rab3a immunohistochemistry. Reduced synaptophysin, synapsin-1, synaptosomal-associated protein of 25,000 mol. wt, syntaxin-1 and Rab3a immunoreactivity was noted in the granular cell layer in every case, but reduced expression was inconstant in the molecular layer. Synaptophysin accumulated in axon torpedoes, thus indicating abnormal axon transport. Expression of synaptic proteins was relatively preserved in the dentate nucleus, although synaptophysin immunohistochemistry disclosed large coarse pericellular terminals in Creutzfeldt±Jakob disease, instead of the ®ne granular terminals in control cases, around the soma of dentate neurons. Finally, Rab3a accumulated in the cytoplasm of Purkinje cells, thus suggesting major anomalies in Rab3a transport. These observations demonstrate, for the ®rst time, abnormal expression of crucial synaptic proteins in the cerebellum of cases with Creutzfeldt±Jakob disease. However, abnormal PrP deposition is not proportional to the degree of reduction of synaptic protein expression in the different layers of the cerebellar cortex and in the dentate nucleus. Therefore, it remains to be elucidated how abnormal PrP impacts on the metabolism of proteins linked to exocytosis and neurotransmission, and how abnormal PrP deposition results in eventual synaptic loss. q 2000 IBRO. Published by Elsevier Science Ltd. Key words: Creutzfeldt±Jakob disease, PrP, synaptophysin, synapsin-1, SNAP-25, Rab3a.

Prion protein (PrP C) is a glycolipid-anchored cell membrane syalioglycoprotein with four a-helical domains and two asparagine-linked carbohydrate side-chains, and with a molecular weight of 27,000±35,000. 17 Differences in mass observed after post-translational modi®cations are due to glycosylation. 30 The function of PrP is not known. PrP-null mice grow normally, and no abnormalities in the nervous and musculoskeletal systems are detected at the age of 24 months. 10 Altered circadian rhythms and sleep have been reported in mice devoid of PrP, 66 and impaired long-term potentiation and weakened GABAA receptor-mediated fast inhibition have been observed in hippocampal slices from PrP-null mice. 14 However, normal neuronal excitability and synaptic transmission have been recorded in the CA1 region of the hippocampus 51 and cerebellar Purkinje cells 31 in PrP gene knockout mice. Prion diseases are sporadic, infectious or inherited neurodegenerative diseases in which the normal transmembrane prion protein (PrP C) is replaced by a protease-resistant,

b-sheet-containing isoform (PrP CJD or PrP SC) that is pathogenic. 17,59 Creutzfeldt±Jakob disease (CJD), Gerstmann± StraÈussler±Scheinker disease and fatal familial insomnia are the most common prion diseases in humans, whereas scrapie is the most common prion disease in sheep and goats, and bovine spongiform encephalopathy in cattle. 17 Prion diseases are transmissible. Following intracerebral inoculation, PrP SC rapidly increases at the same time as PrP C is reduced, thus suggesting that PrP C is converted to PrP SC. 17 Furthermore, PrP C is required for PrP SC propagation, as mice devoid of PrP are resistant to scrapie. 9,60 Following infection, abnormal PrP accumulates in nerve cell processes and synaptic regions. 8,15,16 Sporadic CJD is clinically manifested with mental impairment leading to rapid dementia, movement disorders including myoclonus and gait disorders, and hallucinations. Nerve cell loss, spongiform degeneration, astrocytosis and abnormal PrP deposition are typical pathological changes in CJD. 2,17,36,47,52 Electron microscopic studies have shown dilatation of the smooth endoplasmic reticulum, and dendritic and synaptic vacuolation with accumulation of loose membranes in affected neurons in human, animal and experimentally transmitted prion diseases. 1,12,46,49 In addition, examinations with the Golgi method have revealed dendritic varicosities and loss of dendritic spines. 20,34,46 Finally, reduced expression of proteins linked to exocytosis and neurotransmission has

*To whom correspondence should be addressed. Tel.: 134-93-4035808; fax: 134-93-2045065. E-mail address: [email protected] (I. Ferrer). Abbreviations: CJD, Creutzfeldt±Jakob disease; DAB, 3,3 0 -diaminobenzidine; PrP, prion protein; SNAP-25, synaptosomal-associated protein of 25,000 mol. wt. 715

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been reported in human and animal prion diseases. 22,38,44 Although the latter ®ndings indicate that PrP CJD and PrP SC accumulation results in synaptic pathology, the relationship between abnormal PrP deposition and the pathology of synapses in prion diseases is not clear. The cerebellum is relatively simple in organization, with clearly de®ned cell types, afferent and efferent pathways, and intrinsic cortical circuits. 56 Marked loss of cerebellar granular cells is a characteristic lesion in the ataxic form of CJD. 7,29,42 However, spongiform degeneration in the molecular layer, moderate loss of granular cells, focal loss of Purkinje cells, and gliosis of astrocytes and Bergmann glia are common features in classical CJD. 2,17,36 Studies with the Golgi method show loss of dendritic spines and reduction of the dendritic arbors, as well as hypertrophy and ¯attening of thick dendritic branches in Purkinje cells. 5,21 PrP diffuse plaques are commonly observed in the granular cell and molecular layers of the cerebellum in CJD. 36 Various methods of immunostaining have revealed two additional patterns. Aggregates of punctate PrP immunoreactivity, consistent with a localization in the cerebellar glomeruli, occur in the granular cell layer, whereas a ®ne punctate uniform PrP immunoreactivity may be found in the molecular layer. 17 Punctate PrP immunoreactivity is particularly notable in 129 Met/Met homozygote sporadic cases of CJD. 57 A number of proteins localized in the synaptic vesicle and presynaptic plasma membranes participate in the traf®cking, docking and fusion of the synaptic vesicle to the plasma membrane, thus facilitating synaptic transmission. 37,64,65,68 We have chosen synaptophysin, synapsin-1, synaptosomalassociated protein of 25,000 mol. wt (SNAP-25), syntaxin-1 and Rab3 as markers of synaptic protein expression. Synaptophysin is a synaptic vesicle membrane protein which interacts with other synaptic proteins and participates in exocytosis. 19,37 It is worth mentioning that mutant mice lacking synaptophysin have no detectable changes in synaptic plasticity and their synaptic transmission is normal, thus suggesting that lack of synaptophysin produces only subtle functional abnormalities or that the function of synaptophysin is redundant. 53 However, synaptophysin has been the most widely used synaptic marker in neurohistological studies. Synapsins are synaptic vesicle-associated proteins that bind to proteins of the cytoskeleton. Synapsin phosphorylation, following calcium entry in the cytosolic compartment after stimulation, liberates the synaptic vesicles from the cytoskeleton and allows them to move freely in the presynaptic terminal. 18,65,67 Mice lacking synapsin show synaptic depression, increased paired-pulse facilitation, and seizures. 48,61,62 Thus, synapsin, which controls the number of fusioncompetent vesicles, plays an important role in neurotransmitter release. SNAP-25 is a plasma membrane protein which is present in presynaptic nerve terminals, axons and growth cones. 11,33,55 Syntaxins are integral plasma membrane proteins that are implicated in the docking of synaptic vesicles at presynaptic active zones. 3,4 Clostridial neurotoxins selectively cleave different components involved in the docking and fusion of synaptic vesicles, thus producing neurotransmitter blocking. Studies with clostridial neurotoxins have demonstrated that SNAP-25 and syntaxins are essential for synaptic vesicle exocytosis. 54 Finally, Rab proteins are small GTP-binding proteins that are crucial for virtually every step in the intracellular membrane traf®c of all eukaryotes. 24,58,63 Rab3a is exclusively associated with synaptic vesicles and

dissociates from the vesicle membrane after Ca 21-dependent exocytosis. 23,25 Rab3a-de®cient synapses exhibit profound synaptic depression following repeated stimulation. 28 Therefore, Rab3 proteins are key players in the control of exocytosis. 50 The present study analyses the distribution of abnormal PrP deposition in the cerebellar cortex, white matter and dentate nucleus, and compares PrP deposition with cell damage and with modi®cations in the expression of proteins linked to exocytosis and neurotransmission in CJD. Our aim is not only to examine the spatial relationship between PrP deposition and synaptic pathology, but also to obtain information about the biochemical substrates of synaptic transmission impairment in the cerebellum in CJD. EXPERIMENTAL PROCEDURES

General aspects The brains of six patients with sporadic CJD and six age-matched controls obtained from 3 to 8 h after death were immediately ®xed by immersion in 10% buffered formalin. Samples from the cerebellum, including the cerebellar cortex, white matter and dentate nucleus, were dissected 24±48 h later, washed in phosphate-buffered saline and processed for morphological studies. All the patients (four men and two women, aged 65, 68, 72, 57, 62 and 63 years) had suffered from dementia, myoclonus, moderate or mild ataxia, and hallucinations, and had shown periodic sharp wave complexes on the electroencephalographic recording. All the patients had shown increased levels of the protein 14.3.3 in the cerebrospinal ¯uid. All the patients were homozygous methionine/methionine in codon 129 of the PrP gene (129 Met/Met). Complete neuropathological studies were carried out in CJD and controls after ®xation of the rest of the brain for about three weeks. No abnormalities, excepting small numbers of diffuse bA4-amyloid plaques throughout the neocortex and tau-immunoreactive tangles in the entorhinal cortex in two cases, were found in the control group. In each case, control and pathological samples were treated in the same way and processed in parallel to avoid differences related to tissue processing and day-to-day variations in the quality of the immunostaining. The use of human tissue was approved by the local Ethical Committee of the Hospital Princeps d'EspanyaÐ Universitat de Barcelona, following the guidelines of the Catalonia Autonomous Government. Morphological studies were carried out on paraf®n or Vibratome sections. Dewaxed paraf®n sections, 7 mm thick, were stained with haematoxylin and eosin, periodic acid±Schiff and Luxol Fast Blue± KluÈver Barrera, or processed for immunohistochemistry. Immunohistochemistry For immunohistochemistry, the sections were ®rst treated with 2% hydrogen peroxide and 10% methanol, followed by 5% normal serum for 2 h, and then incubated overnight with one of the primary antibodies. The antibody against phosphorylated neuro®laments of 200,000 mol. wt (clone RT97, Boehringer Mannheim) was used at a dilution of 1:50. The polyclonal antibody to glial ®brillary acid protein (Dako) was used at a dilution of 1:400. The rabbit PrP polyclonal antibody (kindly supplied by Prof. Diringer, Berlin) was used at a dilution of 1:500. The monoclonal antibodies to synaptophysin (Dako), synapsin-1 (Clontech) and SNAP-25 (Sternberger) were used at dilutions of 1:10, 1:100 and 1:1000, respectively. The monoclonal antibody to syntaxin (clone HPC-1, kindly supplied by T. Barnstable, New Haven) was used diluted 1:500. The rabbit polyclonal antibody to Rab3a (Calbiochem), generated by immunization with recombinant Rab3a expressed in E. coli, was used at a dilution of 1:400. Finally, the monoclonal antibody to Rab3a/b (clone CL-42.1, kindly supplied by R. Jahn, New Haven) was used at a dilution of 1:400. The sections were later incubated with speci®c biotinylated secondary immunoglobulin G antibody (Dako) diluted 1:200 and, ®nally, with the avidin±biotin±peroxidase complex (ABC kit, Vector, Vectastain) at a dilution of 1:100 for 1 h each, or with the CheMate: biotinylated secondary antibody 1 streptavidin peroxidase method (Dako). The peroxidase reaction was visualized with 0.05% 3,3 0 diaminobenzidine (DAB) and 0.01% hydrogen peroxide. Some

PrP and synaptic protein expression in CJD cerebellum

sections were counterstained with haematoxylin. Tissue sections processed for PrP were pretreated with 96% formic acid for 10 min, followed by autoclaving. PrP immunohistochemistry was also examined with another antiPrP antibody following a different procedure. Dewaxed paraf®n sections were boiled in 35% HCl for 2 min and then treated with 96% formic acid for 10 min. After blocking endogenous peroxidase with 0.03% hydrogen peroxide, the sections were incubated with the primary anti-PrP antibody used at a dilution of 1:30 in Tris buffer (pH 7.2) containing 15 mM NaN3. The PrP mouse monoclonal anti-PrP antibody (clone 3F4; Dako) is raised against the hamster scrapie strain 263K. The antibody reacts with an epitope located at amino acids 108± 111 of human and hamster PrP proteins. After washing, the sections were processed with the EnVision 1 System Peroxidase (DAB) procedure (Dako) following the instructions of the supplier. The sections were ®rst incubated with EnVision mouse labelled polymer horseradish peroxidase (peroxidase-labelled polymer conjugated to goat anti-mouse immunoglobulins in Tris±HCl buffer containing carrier protein and an antimicrobial agent) for 30 min. Then, the sections were incubated with prepared DAB 1 substrate±chromogen solution (DAB chromogen solution in buffered substrate solution, pH 7.5, containing hydrogen peroxide: 20 ml/ml for about 5 min). Incubation with Proteinase K (Dako), for 15 min diluted in 1 ml, prior to PrP immunohistochemistry was carried out in every case (control and diseased brains). Sections processed in parallel without pre-incubation with the protease served as controls for abnormal PrP deposition. The sections were lightly counterstained with haematoxylin. Vibratome sections, 25 mm thick, were processed free-¯oating for Calbindin-D28k immunohistochemistry following the avidin±biotin± peroxidase complex method. Anti-Calbindin-D28k antibodies (Sigma clone CL-300) were used at a dilution of 1:800. Double-labelling immunohistochemistry for PrP and synaptic proteins was carried out following a two-step protocol. The tissue sections were incubated with the ®rst primary antibody and the immunoreaction was visualized with 0.05% diaminobenzidine and 0.001% hydrogen peroxide. After washing, the sections were incubated with the second primary antibody and the immunoreaction was visualized with 0.01% benzidine dihydrochloride, 0.025% sodium nitroferricyanide in 0.01 M sodium phosphate buffer (pH 6) and 0.005% hydrogen peroxide. The ®rst primary antibody was recognized by a brown precipitate, whereas the second primary antibody was recognized by a granular dark blue precipitate. The speci®city of the immunoreaction was tested by omitting one or both primary antibodies and by incubating the sections with non-speci®c immunoglobulin G. RESULTS

Prion protein immunohistochemistry PrP C and PrP CJD were distinguished in tissue sections by pre-incubating the sections with Proteinase K prior to PrP immunohistochemistry. In control cases, sections processed without pre-incubation with Proteinase K showed weak PrP immunoreactivity in the granular cell layer of the cerebellum and faint diffuse immunoreactivity, which was often obscured by haematoxylin counterstaining, in the molecular layer (Fig. 1A). No Proteinase K-resistant PrP immunoreactivity was found in control cases (Fig. 1B). In contrast, strong PrP immunostaining was observed in CJD sections processed both without and with pre-incubation with Proteinase K (Fig. 1C, D). General neuropathological ®ndings Spongiform degeneration in the molecular layer, focal Purkinje cell loss, moderate loss of granule cells, and gliosis of astrocytes and Bergmann glia were observed in every case (Fig. 2A±C, Table 1). Mild loss of neurons and astrocytic gliosis were found in the dentate nucleus (Fig. 2D). Vacuolated neurons were exceptional and restricted to the dentate nucleus. Purkinje cell axon torpedoes, immunoreactive with phosphorylated neuro®lament antibodies, were present in the

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granular cell layer. No periodic acid±Schiff-positive plaques (kuru-like plaques) were observed in any case. Purkinje cells and their processes stained with antiCalbindin-D28k antibodies showed variable reduction of dendritic arbors, as well as the presence of proximal axon torpedoes and abnormal varicose or club-shaped proximal collateral axon branches surrounding the soma of Purkinje cells (Fig. 2E±G). Abnormal varicosities were also observed in Calbindin-D28k-immunoreactive ®bres in the cerebellar white matter and in terminal ®bres in the dentate nucleus (Fig. 2H). Protease-resistant prion protein immunohistochemistry in Creutzfeldt±Jakob disease Results of PrP immunohistochemistry in CJD varied depending on the antibody and the procedure used. The amount and distribution of protease-resistant PrP deposition were also dependent on individual variations (Table 1). Immunohistochemistry with the polyclonal PrP antibody disclosed diffuse irregular plaque-like PrP deposits in the molecular and granular cell layers in every case (Fig. 2I). Punctate synaptic-like PrP deposits were not manifested with the polyclonal antibody. However, immunohistochemistry with the monoclonal PrP antibody, processed following the EnVision 1 System Peroxidase procedure, revealed, in addition to diffuse plaque-like PrP deposits, punctate synaptic-like PrP deposition in the granular cell and molecular layers in every case (Fig. 3A±C). Aggregates of PrP-positive granules, reminiscent of cerebellar glomeruli, were found in the granular cell layer in the midst of the somas of granular cells. Fine punctate PrP-immunoreactive deposits occurred in the molecular layer. The somas of Purkinje cells, and stellate, basket and Golgi cells, were not stained with anti-PrP antibodies. Many ®bres in the cerebellar white matter (albus) and hilus of the dentate gyrus were decorated with anti-PrP antibodies (Fig. 3D±F, H, I). Punctate synaptic-like PrP deposition, but not diffuse plaque-like PrP deposits, was present in the neuropil of the dentate nucleus in every case (Fig. 3D, E, G±I). Punctate PrP-immunoreactive deposits decorated the surface of dentate neurons (Fig. 3G±I). In addition, granular PrP immunoprecipitates were observed in the majority of dentate neurons in two cases (Fig. 3E, F). Synaptic protein immunohistochemistry Presynaptic terminals were assessed with synaptophysin, synapsin-1, SNAP-25, syntaxin-1 and Rab3 immunohistochemistry. In control brains, aggregates of punctate synaptic protein expression, corresponding to the cerebellar glomeruli, characterized the granular cell layer. Fine uniform immunoprecipitates decorated the molecular layer. Synaptic protein expression was also observed in the neuropil of the dentate nucleus as punctate pericellular deposits. Synaptic proteins were not found in the cell bodies in control cases. Reduced synaptophysin, synapsin-1, SNAP-25, syntaxin-1 and Rab3 immunoreactivity was noted in the granular cell layer in every case. However, reduction in synaptophysin, synapsin1, SNAP-25, syntaxin-1 and Rab3 expression in the molecular layer was noted in four cases; synaptic protein expression in the molecular layer was preserved in the two remaining cases (Figs 4A, B, 5). Similar results were observed in every case with the different synaptic protein antibodies, thus indicating

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Fig. 1. PrP immunohistochemistry using the monoclonal 3F4 antibody following the EnVision 1 System Peroxidase procedure, in control (A, B) and CJD (C, D) cases. Sections A and C have been processed without Proteinase K pre-incubation, and sections B and D with pre-incubation with Proteinase K prior to PrP immunohistochemistry. Weak PrP immunoreactivity is observed in the granular cell layer in control cases. PrP immunoreactivity in controls is abolished in sections pre-incubated with Proteinase K. In contrast, strong protease-resistant PrP immunoreactivity is found in CJD. mol, p and gr, molecular, Purkinje cell and granular cell layers, respectively. Slight haematoxylin counterstaining. Magni®cation: £ 400.

non-selective reduction of synaptic protein expression. Pericellular synaptophysin-immunoreactive terminals on Purkinje cells, both perisomatic and peridendritic, were observed in the most severe case (case 5, Fig. 4C). Purkinje cell axonal torpedoes were immunostained with anti-synaptophysin antibodies (Fig. 4D). Expression of synaptic proteins was relatively

preserved in the dentate nucleus, although synaptophysin immunohistochemistry disclosed large coarse pericellular terminals in CJD, instead of the ®ne granular terminals observed in control cases, around the soma of dentate neurons (Fig. 4E, F). Finally, Rab3 immunohistochemistry disclosed Rab3 immunoreactivity in the soma of Purkinje cells in every

PrP and synaptic protein expression in CJD cerebellum

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synaptic protein expression in the different layers of the cerebellar cortex. DISCUSSION

Fig. 2. General pathological ®ndings in CJD. (A, B) Variable loss of granular cells, focal loss of Purkinje cells and spongiform degeneration in the molecular layer. (C) Reactive astrocytes in the granular cell layer and Bergmann glia in the granular cell and molecular layers, respectively. (D) Reduced numbers of neurons and reactive astrocytic gliosis in the dentate nucleus. (E) Calbindin-D28k-immunoreactive Purkinje cells showing normal dendritic arbors and proximal axons in a control case. (F) Reduced dendritic branches and axon swelling (arrowhead) in CJD. (G) Axon torpedo (arrowhead) arising from an abnormal Purkinje cell (pu), together with abnormal varicosities (arrow) just below the Purkinje cell layer. (H) Abnormal Calbindin-D28k-immunoreactive terminals (arrows) in the dentate nucleus. (I) PrP-positive plaques in the granular cell layer. mol, p and gr, molecular, Purkinje cell and granular cell layers, respectively. (A, B) Haematoxylin and eosin staining. (C, D) Glial ®brillary acidic protein (GFAP) immunohistochemistry. (E±H) Calbindin-D28k immunohistochemistry. (I) PrP immunohistochemistry using the polyclonal PrP antibody. (A±D, I) Paraf®n sections. (E±H) Vibratome sections. Magni®cations: £ 200 (A±C, E, F); £ 400 (D, G±I).

case of CJD, but, as expected, not in controls. Similar results were obtained with the monoclonal anti-Rab3a/b and with the polyclonal anti-Rab3a antibodies (Fig. 6). Double-labelling immunohistochemistry for PrP and synaptic proteins disclosed no increase in synaptic protein expression within or surrounding diffuse irregular plaquelike PrP deposits (data not shown). A semi-quantitative summary of the main neuropathological ®ndings in the six CJD cases is shown in Table 1. As seen in this table, reduced expression of synaptic proteins in the molecular layer parallels the loss of granular cells. However, no clear relationship exists between the amount and distribution of PrP deposition and the loss of granular cells, spongiform degeneration in the molecular layer and

Abnormal PrP deposition occurs in the cerebellar cortex and white matter in all patients with CJD examined here. It is worth stressing, however, that different antibodies and procedures may account for different patterns of PrP immunostaining. In the present series, the polyclonal PrP antibody recognizes diffuse plaque-like deposits, whereas the monoclonal PrP antibody, in combination with the EnVision 1 System Peroxidase procedure, recognizes, in addition, punctate synaptic-like PrP deposition. Diffuse plaque-like deposits are always encountered in the granular cell and molecular layers, as described previously in CJD. 36 Punctate, synapticlike deposits 44,45 are found in the granular cell layer as small aggregates reminiscent of cerebellar glomeruli, and in the molecular layer as ®ne immunoprecipitates. Similar patterns of PrP immunoreactivity have been reported in the cerebellar cortex in CJD. 17 Individual ®bres with focal PrP positivity, like those observed here in the cerebellar white matter and hilus of the dentate nucleus, have occasionally been reported in other brain regions in CJD. 36 As regards the dentate nucleus, punctate synaptic-like deposits are observed in all cases. Punctate perineuronal deposits, observed here around dentate neurons, have occasionally been described in other regions of the brain in CJD. 17,36,45 Finally, intracellular ®ne PrP-positive deposits are found in two cases. Taken together, these ®ndings indicate that short ®xation in buffered formalin and pre-incubation with Proteinase K, followed by PrP immunohistochemistry with the monoclonal 3F4 antibody processed following the EnVision 1 System Peroxidase procedure, provides optimal abnormal PrP immunostaining in CJD. Studies in vitro have shown that PrP C is synthesized in the endoplasmic reticulum, transferred to the Golgi complex and then transported within secretory vesicles to the external surface. PrP SC is deposited in cytoplasmic vesicles and on the cell surface. Both isoforms can be released into the extracellular space. 17,60 Studies in vivo have shown that PrP C is transported down the axons of both the central and peripheral nervous systems, 6 whereas PrP SC axon transport has been suggested in scrapie-infected rodents. 27,43 Therefore, the present results sketch a scenario of abnormal PrP traf®cking in the cerebellar pathways in CJD, which may complement previous observations of PrP metabolism in cultured cells and in scrapie-infected animals in vivo. In addition, diffuse plaque-like deposits suggest extrusion of abnormal PrP into the extracellular space. However, the chronology of the involvement of the different cerebellar pathways throughout the course of the disease still remains obscure. Although it is clear that abnormal PrP causes spongiform degeneration and cell death, 17,59 there is no direct relationship between the amount of PrP deposition and either spongiform degeneration or cell loss in the cerebellar cortex and dentate nucleus in CJD. Spongiform degeneration is invariably present in the molecular layer, whereas it is rare in the granular cell layer. Cell death mainly involves granular cells, whereas Purkinje cells are relatively spared. 17,35,36 However, the reduced dendritic arbors of Purkinje cells, as revealed previously in Golgi studies 5,21 and, herein, with CalbindinD28k immunohistochemistry, lend support to the involvement

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Table 1. Main pathological changes in the cerebellar cortex in the six cases with Creutzfeldt±Jakob disease, as shown with haematoxylin and eosin staining, protease-resistant prion protein inmunohistochemistry by using the monoclonal prion protein antibody (clone 3F4) and following the EnVision 1 System Peroxidase procedure, and synaptic protein inmunohistochemistry (synaptophysin, synapsin-1, synaptosomal-associated protein of mol. wt 25,000, syntaxin-1 and Rab3a) Case 1

2

3

4

5

6

Haematoxylin and eosin

PrP deposits

Granular cells

1

Spong mol layer

1

Granular cells

11

Spong mol layer

1

Granular cells

1

Spong mol layer

1

Granular cells

11

Spong mol layer

1

Granular cells

111

Mol spong layer

11

Granular cells

1

Spong mol layer

1

Plaque-like Synaptic-like gran cell layer Synaptic-like mol layer Plaque-like Synaptic-like gran cell layer Synaptic-like mol layer Plaque-like Synaptic-like gran cell layer Synaptic-like mol layer Plaque-like Synaptic-like gran cell layer Synaptic-like mol layer Plaque-like Synaptic-like gran cell layer Synaptic-like mol layer Plaque-like Synaptic-like gran cell layer Synaptic-like mol layer

Synaptic proteins 1 11 1 1 111 11 11 1 1 /2 1 11 1 1 11 11 1 111 11

Gran cell layer

111

Mol layer

N

Gran cell layer

111

Mol layer

1

Gran cell layer

1

Mol layer

N

Gran cell layer

11

Mol layer

1

Gran cell layer

1

Mol layer

111

Gran cell layer

11

Mol layer

N

Semi-quantitive representation of granular cell loss (granular cells), spongiform degeneration in the molecular layer (spong mol layer), plaque-like and synaptic-like PrP deposition in the granular cell and molecular layers, and reduction of synaptic protein expression in the granular cell and molecular layers (gran cell layer and mol layer, respectively). No relationship is observed between the amount of plaque-like and synaptic-like PrP deposition, and loss of granular cells and decreased synaptic protein expression in the granular cell layer. Similarly, no relationship is found between the magnitude of synaptic-like PrP deposition and synaptic protein expression in the molecular layer. However, decreased synaptic protein expression correlates with the severity of neuron loss in the granular cell layer. Spongiosis of the molecular layer: 1, moderate; 1 1 , mild; 111, severe. Loss of granular cells: 1, moderate; 11, mild; 111, severe. PrP immunoreactivity: 1, moderate; 1 1 , mild or patchy; 111, strong and generalized immunostaining. No protease-resistant PrP immunoreactivity was observed in controls. Synaptic proteins: N, no changes; 1, moderate; 11, mild; 111, severe loss of immunoreactivity when compared with age-matched controls.

of the receptive compartment of Purkinje cells in CJD. Moreover, the occurrence of axon torpedoes ®lled with phosphorylated neuro®laments and synaptophysin, the latter suggesting impaired synaptophysin transport, demonstrates the involvement of the effector compartment of Purkinje cells in this disease. Abnormal Calbindin-D28k-immunoreactive

collateral terminals in the Purkinje cell layer and dentate nucleus con®rm distant axon abnormalities of Purkinje cells. PrP is localized in synaptic boutons of the normal hamster hippocampus. 26 Synaptosomal fractionation methods have shown evidence of presynaptic localization of the PrP. 32

Fig. 3. PrP immunohistochemistry using the monoclonal 3F4 antibody following the EnVision 1 System Peroxidase procedure and Proteinase K pre-incubation. (A) Protease-resistant PrP immunoreactivity in the molecular and granular cell layers. (B, C) Aggregates of PrP granular precipitates, reminiscent of cerebellar glomeruli, in the granular cell layer and ®ne PrP deposits in the molecular layer, together with plaque-like PrP deposition of variable size (arrows) in the granular cell layer. (D, E) Synaptic-like PrP deposition in the dentate nucleus, and PrP-immunoreactive axons (arrowheads) in the cerebellar white matter: album of the cerebellum (a) and hilus of the dentate nucleus (h). PrP deposits in the hilus are labelled with white arrows. (F) Intracellular ®ne granular PrP deposition in dentate neurons (white arrows) and PrP-positive axons (arrowheads) in the neighbouring white matter. (G±I) Perisomatic PrP punctate deposits (arrows) in dentate neurons and PrP-immunoreactive axons in the dentate nucleus (arrowheads). mol, p and gr, molecular, Purkinje cell and granular cell layers, respectively; dn, dentate nucleus. Paraf®n sections (D±I): slight haematoxylin counterstaining. Magni®cations: £ 40 (A); £ 400 (B, C, F); £ 100 (D); £ 200 (E, G±I).

Fig. 3.

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Fig. 4. Synapsin-1 (A, B) and synaptophysin (C±F) immunoreactivity in the cerebellar cortex (A±D) and dentate nucleus (E, F). (A, B) Mild (A) and moderate (B) decrease of synapsin-1 immunoreactivity in the granular cell layer in CJD (cases 3 and 6, respectively). Note preserved synapsin-1 immunoreactivity in the molecular layer. (C) Decreased synaptophysin immunoreactivity in the molecular layer, with preservation of possible presynaptic terminals (arrowheads) around the cell body and primary dendrites of a Purkinje cell (case 5). (D) Increased synaptophysin immunoreactivity in axon swelling (torpedo; arrow) in the granular cell layer (case 5). (E, F) Synaptophysin immunoreactivity around the somas and main dendrites of dentate neurons in a control case (E) and in one patient with CJD (case 3). Note the coarse perineuronal immunoprecipitates in CJD. mol, p and gr, molecular, Purkinje and granular cell layers, respectively. Paraf®n sections (C±F): slight haematoxylin counterstaining. Magni®cations: £ 200 (A, B); £ 400 (C±F).

The abnormal isoform of PrP accumulates in the plasmalemma of neurites in scrapie-infected mice. 39 Furthermore, previous studies combining synaptophysin and PrP immunohistochemistry have suggested that abnormal PrP accumulates in synapses of the CNS in patients with CJD. 17,45 Combined synaptic protein and PrP immunohistochemistry, analysed here, further supports the concept that

the abnormal isoform of PrP probably accumulates in glomerular synapses, as well as in synapses of the molecular layer and perisomatic synapses of dentate nucleus neurons in CJD. There is no clear relationship between PrP deposition and synaptic protein loss in the different regions. Reduced expression of synaptophysin, synapsin-1, SNAP-25, syntaxin-1 and Rab3 is commonly found in the

Fig. 5. Haematoxylin and eosin staining (A, D, G), protease-resistant PrP deposition (B, E, H), as revealed with the monoclonal 3F4 antibody, and SNAP-25 immunohistochemistry (C, F, I) in three cases of CJD: cases 1 (A±C), 2 (D±F) and 5 (G±I), whose main neuropathological ®ndings are detailed in Table 1. Spongiform degeneration in the molecular layer, loss of granular cells, reduced synaptic protein expression and abnormal PrP deposition are variable from one case to another. mol, p and gr, molecular, Purkinje and granular cell layers, respectively. Paraf®n sections (B, C, E, F, H, I): without counterstaining. Magni®cation: £ 400.

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Fig. 6. Rab3a immunohistochemistry in the cerebellum of control (A) and CJD (B) cases. Reduced Rab3a expression is observed in the molecular layer in CJD. In addition, ®ne Rab3a immunoprecipitates decorate the cytoplasm of Purkinje cells in CJD. mol, p and gr, molecular, Purkinje and granular cell layers, respectively. Paraf®n sections: rabbit polyclonal antibody, no haematoxylin counterstaining. Magni®cation: £ 400.

granular cell layer in CJD. However, the intensity of the immunoreaction to these proteins is relatively preserved in the molecular layer and dentate nucleus. These data suggest either that glomerular synapse damage occurs at earlier stages than the damage to parallel ®bre±Purkinje cell synapses in the molecular layer, or that glomerular synapses are more vulnerable than those of the molecular layer in CJD. In fact, reduction of synaptic protein expression in the molecular layer correlates better with loss of granular cells than with the deposition of abnormal PrP in the molecular layer. In this regard, it is worth noting that loss of axon boutons is not seen until neuronal loss is already established in scrapie-infected mice 38 and in bovine spongiform encephalopathy. 41 A ®nal interesting point is the observation of Rab3a immunoreactivity in the soma of Purkinje cells in CJD. It is well established that Rab proteins cycle between GTP- and GDP-bound forms and that this cycle is paralleled by a membrane association±dissociation cycle that gears intracellular traf®cking. 24 Rab3a is associated exclusively with synaptic vesicles and it dissociates from synaptic vesicles during exocytosis; 23,25 Rab3a-de®cient synapses exhibit profound synaptic depression following repeated stimulation. 28 Therefore, Rab3a in the cytoplasm of neurons is a very abnormal situation that implies aberrant Rab3a transport, which may have serious effects on synaptic vesicle regulation. However, Rab3a in the cytoplasm of Purkinje cells in CJD does not correlate with abnormal PrP deposition in these

neurons, thus suggesting a PrP-independent mechanism of Rab3a cytoplasmic accumulation. Moreover, Rab3a in the cytoplasm of neurons is not limited to CJD. Preliminary results have shown Rab3a immunoreactivity in the somas of Purkinje cells in patients suffering from olivo-pontocerebellar atrophy and multisystemic degeneration (unpublished observations). Deposition of diffuse plaque-like PrP is not accompanied by increased or reduced expression of synaptic proteins, as revealed with double-labelling immunohistochemistry. On the one hand, the present study does not con®rm the observation of synaptic plaque-like lesions in the cerebellum of patients with CJD. 13 However, on the other hand, the present results do agree with immunohistochemical observations in the cerebral cortex in CJD in which PrP deposition, in contrast to bA4-amyloid deposition, is not associated with increased synaptophysin and SNAP-25 expression. 22 Similarly, immunoelectron microscopic studies in scrapieinfected mice have shown little or no subcellular pathology of neurites and terminals in association with extracellular PrP accumulation. 40 CONCLUSION

Taken together, these observations indicate that abnormal PrP deposition is associated with abnormal synaptic protein expression in the cerebellar cortex and dentate nucleus in CJD. However, abnormal PrP deposition is not

PrP and synaptic protein expression in CJD cerebellum

a predictor of severity, nor is it proportional to the degree of reduction of synaptic protein expression in the different layers of the cerebellar cortex and in the dentate nucleus. Therefore, these ®ndings suggest that the expression of the synaptic proteins examined is independent of PrP. It remains to be elucidated how abnormal PrP impacts on the metabolism of proteins linked to exocytosis and

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neurotransmission, and how PrP deposition results in eventual synaptic loss. AcknowledgementsÐThis work was supported by the European Commission Project Bio-4 98-6060. We thank Dr J. YaguÈe (Hospital ClõÂnico y Provincial, Barcelona) for the genetic case studies and T. Yohannan for editorial assistance.

REFERENCES

1. Beck E. and Daniel P. M. (1987) Neuropathology of transmissible spongiform encephalopathies. In Prions: Novel Infectious Pathogens Causing Scrapie and Creutzfeldt±Jakob Disease (eds Prusiner S. B. and McKinley M. P.), pp. 3105-3108. Academic, Orlando, FL. 2. Bell J. E. and Ironside J. W. (1993) Neuropathology of spongiform encephalopathies in humans. Br. med. Bull. 49, 738±777. 3. Bennett M. K., Calakos N. and Scheller R. H. (1992) Syntaxin: a synaptic protein implicated in docking of synaptic vesicles at presynaptic active zones. Science 257, 255±259. 4. Bennett M. K., GarcõÂa-ArraraÂs J. E., Elferink L. A., Peterson K., Fleming A. M., Hazuka C. D. and Scheller R. H. (1993) The syntaxin family of vesicular transport receptors. Cell 74, 863±873. 5. Berciano J., Berciano M. T., Polo J. M., Figols J., Ciudad J. and Lafarga M. (1990) Creutzfeldt±Jakob disease with severe involvement of cerebral white matter and cerebellum. Virchows Arch. A, Pathol. Anat. 417, 533±538. 6. Borchelt D. R., Koliatsos V. E., Guarnieri M., Pardo C. A., Sisodia S. S. and Price D. L. (1994) Rapid anterograde transport of the cellular prion glycoprotein in the peripheral and central nervous system. J. biol. Chem. 269, 14711±14714. 7. Brownell B. and Oppenheimer D. R. (1965) An ataxic form of subacute presenile polioencephalopathy (Creutzfeldt±Jakob disease). J. Neurol. Neurosurg. Psychiat. 28, 350±361. 8. Bruce M. E., McBride P. A. and Farquhar C. F. (1989) Precise targeting of the pathology of the sialoglycoprotein PrP and vacuolar degeneration in mouse scrapie. Neurosci. Lett. 102, 1±6. 9. BuÈeler H., Aguzzi A., Sailer A., Greiner R. A., Autenreid P., Aguet M. and Weissmann C. (1993) Mice devoid of PrP are resistant to scrapie. Cell 73, 1339±1347. 10. BuÈeler H., Fisher M., Lanfg Y., Blurthmann H., Lipp H. P., DeArmond S. J., Prusiner S. B., Aguet M. and Weissmann C. (1992) Normal development and behavior of mice lacking the neuronal surface PrP protein. Nature 365, 577±582. 11. Catsicas S., Larhammar D., Blomquist A., Sanna P. P., Milner R. J. and Wilson M. C. (1991) Expression of a conserved cell-type-speci®c protein in nerve terminals coincides with synaptogenesis. Proc. natn. Acad. Sci. U.S.A. 88, 785±789. 12. Chou S. M., Payne W. N., Gibbs C. J. and Gajdusek D. C. (1980) Transmission and scanning electron microscopy of spongiform change in Creutzfeldt± Jakob disease. Brain 103, 885±904. 13. Clinton J., Forsyth C., Royston M. C. and Roberts G. W. (1993) Synaptic degeneration is the primary neuropathological feature in prion disease: a preliminary study. NeuroReport 4, 65±68. 14. Collinge J., Whittington M. A., Sidle K. C. L., Smith J., Palmer M. S., Clarke A. R. and Jeffreys J. G. R. (1994) Prion protein is necessary for normal synaptic function. Nature 370, 295±297. 15. DeArmond S. J., Mobley W. C., De Mott D. L., Barry R. A., Beckstead J. H. and Prusiner S. B. (1987) Changes in the localization of brain prion proteins during scrapie infection. Neurology 37, 1271±1280. 16. DeArmond S. J. and Prusiner S. B. (1995) Etiology and pathogenesis of prion diseases. Am. J. Path. 146, 785±811. 17. DeArmond S. J. and Prusiner S. B. (1997) Prion diseases. In Green®eld's Neuropathology (eds Graham D. I. and Lantos P. L.), Vol. 2, pp. 122±128. Arnold, London. 18. DeCamilli P., Benfenatti P., Valtorta F. and Greengard P. (1990) The synapsins. A. Rev. cell. Biol. 6, 433±460. 19. Edelmann L., Hanson P. I., Chapman E. R. and Jahn R. (1995) Synaptobrevin binding to synaptophysin: a potential mechanism for controlling the exocytotic machine. Eur. molec. Biol. Org. J. 4, 224±231. 20. Ferrer I., Costa F. and Grau Veciana J. M. (1981) Creutzfeldt±Jakob disease: a Golgi study. Neuropath. appl. Neurobiol. 7, 237±242. 21. Ferrer I., Kulisewski J., VaÂzquez J., GonzaÂlez A. and Pineda M. (1988) Purkinje cells in degenerative diseases of the cerebellum and its connections: a Golgi study. Clin. Neuropath. 7, 237±242. 22. Ferrer I., Rivera R., Blanco R. and MartõÂ E. (1999) Expression of proteins linked to exocytosis and neurotransmission in Creutzfeldt±Jakob disease. Neurobiol. Dis. 6, 92±100. 23. Fischer von Mollard G. F., Mignery G. A., Baumert M., Perin M. S., Hanson T. J., Burger P. M., Jahn R. and SuÈdhof T. C. (1990) Rab3 is a small GTPbinding protein exclusively localized to synaptic vesicles. Proc. natn. Acad. Sci. U.S.A. 87, 1988±1992. 24. Fischer von Mollard G. F., Stahl B., Li C., SuÈdhof T. C. and Jahn R. (1994) Rab proteins in regulated exocytosis. Trends biol. Sci. 19, 164±168. 25. Fischer von Mollard G. F., SuÈdhof T. C. and Jahn R. (1991) A small GTP-binding protein dissociates from synaptic vesicles during exocytosis. Nature 349, 79±81. 26. Fournier J. G., Escaig-Haye F., Billette de Villemeur T. and Robain O. (1995) Ultrastructural localization of cellular prion protein (PrP C) in synaptic boutons of normal hamster hippocampus. C. r. hebd SeÂanc. Acad. Sci., Paris III 318, 339±344. 27. Fraser H. and Dickinson A. G. (1985) Targeting of scrapie lesions and spread of the agent via the retino-tectal projection. Brain Res. 346, 32±38. 28. Geppert M., Bolshakov A., Siegelbaum S. A., Takei K., De Camilli P., Hammer R. E. and SuÈdhof T. C. (1994) The role of Rab3a in neurotransmitter release. Nature 369, 493±497. 29. Gomori A. J., Partnow M. J., Horoupian D. S. and Hirano A. (1973) The ataxic form of Creutzfeldt±Jakob disease. Archs Neurol. 29, 318±323. 30. Haraguchi T., Fisher S., Olofson S., Endo T., Groth D. and Tarentino A., et al. (1989) Asparagine-linked glycosylation of the scrapie and cellular prion proteins. Archs Biochem. Biophys. 274, 1±13. 31. Herms J. W., Kretzchmar H. A., Tilz S. and Keller B. U. (1995) Patch-clamp analysis of synaptic transmission to cerebellar Purkinje cells of prion protein knockout mice. Eur. J. Neurosci. 12, 2508±2512. 32. Herms J. W., Tings T., Gall S., Madlung A., Giese A., Siebert H., SchuÈrmann P., Windl O., Brose N. and Kreutzschmar H. (1999) Evidence of presynaptic location and function of the prion protein. J. Neurosci. 15, 8866±8875. 33. Hess D. T., Slater T. M., Wilson M. C. and Skene J. H. P. (1992) The 25kDa synaptosomal-associated protein SNAP-25 is the major methionine-rich polypeptide in rapid axonal transport and a major substrate for palmitoylation in adult CNS. J. Neurosci. 12, 4634±4641. 34. Hogan R. N., Baringer J. R. and Prusiner S. B. (1987) Scrapie infection diminishes spines and increases varicosities of dendrites in hamsters: a quantitative Golgi study. J. Neuropath. exp. Neurol. 46, 461±473. 35. Ironside J. W. (1996) Review: Creutzfeldt±Jakob disease. Brain Path. 6, 379±388. 36. Ironside J. W. and Bell J. E. (1997) Pathology of prion diseases. In Prion Diseases (eds Collinge J. and Palmer M. S.), p. 1996. Oxford University Press, Oxford. 37. Jahn R. and SuÈdhof T. C. (1994) Synaptic vesicles and exocytosis. A. Rev. Neurosci. 17, 219±246.

726

I. Ferrer et al.

38. Jeffrey M., Fraser J. R., Halliday W. G., Fowler N., Goodsir C. M. and Brown D. A. (1995) Early unsuspected neuron and axon terminal loss in scrapieinfected mice revealed by morphometry and immunohistochemistry. Neuropath. appl. Neurobiol. 21, 41±49. 39. Jeffrey M., Goodsir C. M., Bruce M. E., McBride P. A., Scott J. R. and Halliday W. G. (1992) Infection scrapie prion protein (PrP) accumulates on neuronal plasmalemma in scrapie infected mice. Neurosci. Lett. 147, 106±109. 40. Jeffrey M., Goodsir C. M., Bruce M. E., McBride P. A. and Farquhar C. (1994) Morphogenesis of amyloid plaques in 87V murine scrapie. Neuropath. appl. Neurobiol. 20, 535±542. 41. Jeffrey M. and Halliday W. G. (1994) Numbers of neurons in vacuolated and nonvacuolated neuroanatomical nuclei in bovine spongiform encephalopathy-affected brains. J. comp. Path. 110, 287±294. 42. Jellinger K., Heiss W. D. and Deisenhammer E. (1974) The ataxic (cerebellar) form of Creutzfeldt±Jakob disease. J. Neurol. 207, 289±305. 43. Jendroska K., Heinzel F. P., Torchia M., Stowring L., Kretzschmar H. A., Kon A., Stern A., Prusiner S. B. and DeArmond S. J. (1991) Proteinase-resistant prion protein accumulation in Syrian Hamster brain correlates with regional pathology and scrapie infectivity. Neurology 41, 1482±1490. 44. Kitamoto T., Don-ura K., Muramoto T., Niyazono M. and Tateishi J. (1992) The primary structure of the prion protein in¯uences the distribution of abnormal prion protein. Am. J. Path. 141, 271±272. 45. Kitamoto T., Shin R. W., Don-ura K., Tonokane N., Miyazono N., Muramoto T. and Tateishi J. (1992) Abnormal isoform of prion protein accumulates in the synaptic structures of the central nervous system in patients with Creutzfeldt±Jakob disease. Am. J. Path. 140, 1285±1294. 46. Landis D. N. M. D., Williams R. S. and Masters C. L. (1981) Golgi and electron microscopic studies of spongiform encephalopathies. Neurology 31, 538±549. 47. Lantos P. L. (1992) From slow virus to prions: a review of transmissible spongiform encephalopathies. Histopathology 20, 1±11. 48. Li L., Chin L. S., Shupliakov O., Brodin L., Sihra T. S., Hvalby O., Jensen V., Zheng D., McNamara J. O., Greengard P. and Andersen P. (1995) Impairment of synaptic vesicle clustering and of synaptic transmission, and increased seizure propensity, in synapsin 1-de®cient mice. Proc. natn. Acad. Sci. U.S.A. 92, 9235±9239. 49. Liberski P., Yanigahira R., Asher D. M., Gibbs C. J. and Gajdusek D. C. (1990) Reevaluation of the ultrastructural pathology of experimental Creutzfeldt±Jakob disease. Brain 113, 121±137. 50. Lledo P. M., Johannes L., Vernier P., Zorec R., Darchen F., Vincent J. D., Henry J. P. and Mason W. T. (1994) Rab3 proteins: key players in the control of exocytosis. Trends Neurosci. 17, 426±432. 51. Lledo P. M., Tremblay P., DeArmond S. J., Prusiner S. B. and Nicoll R. A. (1996) Mice de®cient for prion protein exhibit normal neuronal excitability and synaptic transmission in the hippocampus. Proc. natn. Acad. Sci. U.S.A. 93, 2403±2407. 52. Masters C. L. and Gajdusek D. C. (1982) The spectrum of Creutzfeldt±Jakob disease and the virus-induced subacute spongiform encephalopathies. In Advances in Neuropathology (eds Thomas Smith W. and Cavanagh J. B.), Vol. 2, pp. 139±143. Churchill Livingstone, Edinburgh. 53. McMahon H. T., Bolshakov V. Y., Janz R., Hammer R. E., Siegelbaum S. A. and SuÈdhof T. C. (1996) Synaptophysin, a major synaptic protein, is not essential for neurotrasmitter release. Proc. natn. Acad. Sci. U.S.A. 93, 4760±4764. 54. Niemann H., Blasi J. and Jahn R. (1994) Clostridial neurotoxins: new tools for dissecting exocytosis. Trends Cell Biol. 4, 179±185. 55. Oyler G. A., Higgins G. A., Hart R. A., Battenberg E., Billingsley M., Bloom F. E. and Wilson M. C. (1989) The identi®cation of a novel synaptosomalassociated protein SNAP-25 differentially expressed by neuronal subpopulations. J. Cell Biol. 109, 3039±3052. 56. Palay S. L. and Chan-Palay V. (1974) Cerebellar Cortex: Cytology and Organization, Springer, Berlin. 57. Parchi P., Castellani R., Capellari S., Ghetti B., Young K., Chen S. G., Farlow M., Dickson D. W., Sima A. A. F., Trojanowski J. Q., Petersen R. B. and Gambetti P. (1996) Molecular basis of phenotypic variability in sporadic Creutzfeldt±Jakob disease. Ann. Neurol. 39, 767±778. 58. Pfeffer S. R. (1994) Rab GTPases: master regulators of membrane traf®cking. Curr. Opin. Cell Biol. 6, 522±526. 59. Prusiner S. B. (1997) Cell biology and transgenic models of prion diseases. In Prion Diseases (eds Collinge J. and Palmer M. S.), pp. 130±162. Oxford University Press, Oxford. 60. Prusiner S. B., Groth D., Serban A., Koehler R., Foster D., Torchia M., Burton D., Yang S. L. and DeArmond S. J. (1993) Ablation of the prion protein (PrP) gene in mice prevents scrapie and facilitates production of anti-PrP antibodies. Proc. natn. Acad. Sci. U.S.A. 90, 10608±10612. 61. Rosahl T. W., Gepert M., Spillane D., Herz J., Hammer R. E., Malenka R. C. and SuÈdhof T. C. (1993) Short-term synaptic plasticity is altered in mice lacking synapsin 1. Cell 75, 661±670. 62. Rosahl T. W., Spillane D., Missler M., Hertz J., Selig D. K., Wolff J. R., Hammer R. E., Malenka R. C. and SuÈdhof T. C. (1995) Essential functions of synapsins I and II in synaptic vesicle regulation. Nature 375, 488±493. 63. Simons K. and Zerial M. (1993) Rab proteins and the road maps for intracellular transport. Neuron 11, 789±799. 64. SoÈllner T. and Rothman J. E. (1994) Neurotransmission: harnessing fusion machinery at the synapse. Trends Neurosci. 17, 344±347. 65. SuÈdhof T. C. (1995) The synaptic vesicle cycle: a cascade of protein±protein interactions. Nature 375, 645±653. 66. Tobler I., Gaus S. E., Deboer T., Achermann P., Fisher M., RuÈlicke T., Moser M., Oesh B., McBride P. A. and Manson J. C. (1996) Altered circadian activity rhythms and sleep in mice devoid of prion protein. Nature 380, 639±642. 67. Valtorta F., Benfenatti F. and Greengard P. (1992) Structure and function of synapsins. J. Cell Biol. 267, 7195±7198. 68. Volknandt W. (1995) The synaptic vesicles and its targets. Neuroscience 64, 277±300. (Accepted 27 January 2000)