Anti-prion Protein Antibodies Research Articles

A high-content neuron imaging assay demonstrates inhibition of prion disease-associated neurotoxicity by an anti-prion protein antibody

There is an urgent need to develop disease-modifying therapies to treat neurodegenerative diseases which pose increasing challenges to global healthcare systems. Prion diseases, although rare, provide a paradigm to study neurodegenerative dementias as similar disease mechanisms involving propagation and spread of multichain assemblies of misfolded protein (“prion-like” mechanisms) are increasingly recognised in the commoner conditions such as Alzheimer’s disease. However, studies of prion disease pathogenesis in mouse models showed that prion propagation and neurotoxicity can be mechanistically uncoupled and in vitro assays confirmed that highly purified prions are indeed not directly neurotoxic. To aid development of prion disease therapeutics we have therefore developed a cell-based assay for the specific neurotoxicity seen in prion diseases rather than to simply assess inhibition of prion propagation. We applied this assay to examine an anti-prion protein mouse monoclonal antibody (ICSM18) known to potently cure prion-infected cells and to delay onset of prion disease in prion-infected mice. We demonstrate that whilst ICSM18 itself lacks inherent neurotoxicity in this assay, it potently blocks prion disease-associated neurotoxicity.

Intermolecular crosslinking of abnormal prion protein is efficiently induced by a primuline-sensitized photoreaction

In prion diseases, infectious pathogenic particles that are composed of abnormal prion proteins (PrPSc) accumulate in the brain. PrPSc is biochemically characterized by its protease-resistance core (PrPres), but its structural features have not been fully elucidated. Here, we report that primuline, a fluorescent dye with photosensitization activity, dramatically enhances UV-irradiation-induced SDS-resistant PrPSc/res oligomer formation that can be detected by immunoblot analysis of prion-infected materials. This oligomer formation occurs specifically with PrPSc/res but not with normal prion protein, and it was demonstrated using purified PrPSc/res as well as unpurified materials. The oligomer formation proceeded in both primuline-dose- and UV irradiation time-dependent manners. Treatment with urea or formic acid did not break oligomers into monomers. Neither did the presence of aromatic amino acids modify oligomer formation. Analysis with a panel of anti-prion protein antibodies showed that the antibodies against the N-terminal region of PrPres were less reactive in the dimer than the monomer. These findings suggest that the primuline-sensitized photoreaction enhances intermolecular crosslinking of PrPSc/res molecules at a hydrophobic area of the N-terminal region of PrPres. In the screening of other compounds, photoreactive compounds such as luciferin exhibited a similar but lower activity with respect to oligomer formation than primuline. The enhanced photoreaction with these compounds will be useful for evaluating the structural features of PrPSc/res, especially the interactions between PrPSc/res molecules.

Anti-prion Protein Antibody 6D11 Restores Cellular Proteostasis of Prion Protein Through Disrupting Recycling Propagation of PrPSc and Targeting PrPSc for Lysosomal Degradation.

PrPSc is an infectious and disease-specific conformer of the prion protein, which accumulation in the CNS underlies the pathology of prion diseases. PrPSc replicates by binding to the cellular conformer of the prion protein (PrPC) expressed by host cells and rendering its secondary structure a likeness of itself. PrPC is a plasma membrane anchored protein, which constitutively recirculates between the cell surface and the endocytic compartment. Since PrPSc engages PrPC along this trafficking pathway, its replication process is often referred to as "recycling propagation." Certain monoclonal antibodies (mAbs) directed against prion protein can abrogate the presence of PrPSc from prion-infected cells. However, the precise mechanism(s) underlying their therapeutic propensities remains obscure. Using N2A murine neuroblastoma cell line stably infected with 22L mouse-adapted scrapie strain (N2A/22L), we investigated here the modus operandi of the 6D11 clone, which was raised against the PrPSc conformer and has been shown to permanently clear prion-infected cells from PrPSc presence. We determined that 6D11 mAb engages and sequesters PrPC and PrPSc at the cell surface. PrPC/6D11 and PrPSc/6D11 complexes are then endocytosed from the plasma membrane and are directed to lysosomes, therefore precluding recirculation of nascent PrPSc back to the cell surface. Targeting PrPSc by 6D11 mAb to the lysosomal compartment facilitates its proteolysis and eventually shifts the balance between PrPSc formation and degradation. Ongoing translation of PrPC allows maintaining the steady-state level of prion protein within the cells, which was not depleted under 6D11 mAb treatment. Our findings demonstrate that through disrupting recycling propagation of PrPSc and promoting its degradation, 6D11 mAb restores cellular proteostasis of prion protein.

Establishment of a simple cell-based ELISA for the direct detection of abnormal isoform of prion protein from prion-infected cells without cell lysis and proteinase K treatment

ABSTRACTPrion-infected cells have been used for analyzing the effect of compounds on the formation of abnormal isoform of prion protein (PrPSc). PrPSc is usually detected using anti-prion protein (PrP) antibodies after the removal of the cellular isoform of prion protein (PrPC) by proteinase K (PK) treatment. However, it is expected that the PK-sensitive PrPSc (PrPSc-sen), which possesses higher infectivity and conversion activity than the PK-resistant PrPSc (PrPSc-res), is also digested through PK treatment. To overcome this problem, we established a novel cell-based ELISA in which PrPSc can be directly detected from cells persistently infected with prions using anti-PrP monoclonal antibody (mAb) 132 that recognizes epitope consisting of mouse PrP amino acids 119–127. The novel cell-based ELISA could distinguish prion-infected cells from prion-uninfected cells without cell lysis and PK treatment. MAb 132 could detect both PrPSc-sen and PrPSc-res even if all PrPSc molecules were not detected. The analytical dynamic range for PrPSc detection was approximately 1 log. The coefficient of variation and signal-to-background ratio were 7%–11% and 2.5–3.3, respectively, demonstrating the reproducibility of this assay. The addition of a cytotoxicity assay immediately before PrPSc detection did not affect the following PrPSc detection. Thus, all the procedures including cell culture, cytotoxicity assay, and PrPSc detection were completed in the same plate. The simplicity and non-requirement for cell lysis or PK treatment are advantages for the high throughput screening of anti-prion compounds.

Coexistence of two forms of disease-associated prion protein in extracerebral tissues of cattle infected with H-type bovine spongiform encephalopathy.

H-type bovine spongiform encephalopathy (H-BSE) is an atypical form of BSE in aged cattle. H-BSE is characterized by the presence of two proteinase K-resistant forms of disease-associated prion protein (PrPSc), identified as PrPSc #1 and PrPSc #2, in the brain. To investigate the coexistence of different PrPSc forms in the extracerebral tissues of cattle experimentally infected with H-BSE, immunohistochemical and molecular analyses were performed by using N-terminal-, core-region- and C-terminal-specific anti-prion protein antibodies. Our results demonstrated that two distinct forms of PrPSc coexisted in the various extracerebral tissues.

Tumor necrosis factor-α reduces beta-amyloid accumulation primarily by lowering cellular prion protein levels in a brain endothelial cell line

Disruption of beta-amyloid (Aβ) transport across the blood–brain barrier is thought to cause Aβ accumulation in the brain, thus leading to the development of Alzheimer’s disease (AD). As AD patients show increased serum tumor necrosis factor-α (TNFα) levels, we examined the effect of TNFα on the function and expression of Aβ transport-related proteins including cellular prion protein (PrPC) in the mouse brain microvascular endothelial cell line MBEC4. TNFα decreased PrPC levels and intracellular radiolabeled Aβ. Similarly, anti-prion protein antibody also decreased radiolabeled Aβ. These results suggest that TNFα lowers PrPC levels, which in turn, reduces Aβ in the brain endothelium.

Peripheral administration of a humanized anti-PrP antibody blocks Alzheimer's disease Aβ synaptotoxicity.

Alzheimer's disease (AD) is associated with pathological assembly states of amyloid-β protein (Aβ). Aβ-related synaptotoxicity can be blocked by anti-prion protein (PrP) antibodies, potentially allowing therapeutic targeting of this aspect of AD neuropathogenesis. Here, we show that intravascular administration of a high-affinity humanized anti-PrP antibody to rats can prevent the plasticity-disrupting effects induced by exposure to soluble AD brain extract. These results provide an in vivo proof of principle for such a therapeutic strategy.

Abstract 5207: Targeting prion protein as a potential oncogene in pancreatic cancer.

Abstract Background: Human prion protein (PrP) has been found to be up-regulated in gastric, breast, pancreatic cancers and its expression in cancer cells contributes to cancer progression and resistance to various cancer therapies. Current literature suggests that overexpression of PrP induces inherent resistance to cancer therapeutics, while repression of PrP induces sensitization to cancer therapeutics. Our previous studies using expression microarray have revealed that PrP is not detected in normal human pancreatic ductal cells but is expressed in human pancreatic ductal adenocarcinoma (PDAC) cell lines and 40% of pancreatic cancer specimens. Further, expression of PrP confers a worse prognosis in PDAC patients. The poor prognosis of pancreatic cancer demonstrates needs for better target and drug development. Our recent studies also found that PrP is universally expressed in fetal pancreatic precursor ductal cells, and co-expresses with pancreatic stem cell markers, such as Pdx1 and Notch1, suggesting that PrP is a marker of pancreatic stem cells and may cross-talk with Notch1 during development and pancreatic tumorigenesis. However, the role for PrP in the establishment or progression of cancer is unknown, and whether PrP cross-talks with oncogenes such as Notch in these processes need to be elucidated. Methods: We applied immunoprecipitation (Abcam, Anti-Prion protein antibody 8H4) and western blotting to investigate the binding partners for PrP. Knockdown and over-expression of PRNP gene were carried out on various pancreatic cell lines, followed by in vitro proliferation assay and apoptotic activity analysis of these cells. Results: Pancreatic cancer cell lines express different levels of PrP, e.g. BxPC3 and Pan02.03 have higher PrP expression when compared to Capan-1. Western blot analysis revealed that PrP can bind with Notch1 protein in both BxPC-3 and CAPAN-1 cell lines. Moreover, down-regulation of PrP led to decreased cell proliferation and accelerated apoptosis, accompanied by down-regulation of cleaved/activated Notch1 (ICN1) and Notch2 (ICN2) expression in BxPC3 cells, but had lesser effect on CAPAN-1 cells. In comparison, over-expression of PrP significantly increased the proliferation rate of CAPAN-1 cells and dramatically upregulated the expression of ICN1. In conjunction with our previous findings that PrP exists as a pro-prion that binds filamin A in pancreatic cancer cells, these findings suggest that PrP may mediate pancreatic cancer cell progression through regulation of Notch activity and other cell mechanic and signaling integrators. This hypothesis is being further tested by xenograft analysis. Elucidating the mechanisms for PrP mediated Notch activation in PDAC will further our long-term goal of understanding the role of oncogenic PrP in conferring pancreatic cancer aggressiveness, and potentially will lead to novel therapeutic options for treatment of pancreatic cancer. Citation Format: Yiwei Wang, Lan Zhou, Wei Xin. Targeting prion protein as a potential oncogene in pancreatic cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5207. doi:10.1158/1538-7445.AM2013-5207

Therapeutic effect of peripheral administration of an anti-prion protein antibody on mice infected with prions

Therapeutic effect of peripheral administration of an anti-prion protein antibody on mice infected with prions

Neuroanatomical Distribution of Disease-Associated Prion Protein in Cases of Bovine Spongiform Encephalopathy Detected by Fallen Stock Surveillance in Japan

Bovine spongiform encephalopathy (BSE) is a fatal neurodegenerative disorder of cattle characterized by accumulation of the disease-associated prion protein (PrP(Sc)) in the central nervous system (CNS). The immunohistochemical patterns and distribution of PrP(Sc) were investigated in the CNS, brains, and spinal cords of 7 naturally occurring BSE cases confirmed by the fallen stock surveillance program in Japan. No animals showed characteristic clinical signs of the disease. Coronal slices of 14 different brain areas in each case were immunohistochemically analyzed using an anti-prion protein antibody. Immunolabeled PrP(Sc) deposition was widely observed throughout each brain and spinal cord. Intense PrP(Sc) deposition was greater in the thalamus, brainstem, and spinal cord of the gray matter than in the neocortices. The topographical distribution pattern and severity of PrP(Sc) accumulation were mapped and plotted as immunohistochemical profiles of the different brain areas along the caudal-rostral axis of the brain. The distribution pattern and severity of the immunolabeled PrP(Sc) in the CNS were almost the same among the 7 cases analyzed, suggesting that the naturally occurring cases in this study were at the preclinical stage of the disease. Immunohistochemical mapping of the PrP(Sc) deposits will be used to clarify the different stages of BSE in cattle.

Actions of the Japanese Pancreas and Islet Transplantation Association Regarding Transplanted Human Islets Isolated Using Liberase HI

Purpose The potential for introducing transmissible spongiform encephalopathy (TSE) into islet cells was indicated by recognizing that Liberase HI is isolated from Clostridium histolyticum grown in media containing brain–heart infusion broth. A national team within the Japanese Pancreas and Islet Transplantation Association implemented an islet transplantation program in Japan using Liberase HI. The program comprised 65 islet isolations from non–heart-beating donors and 34 transplants into 18 patients. Herein, we have summarized how the Association followed these recipients over the long term. Procedures We established an ad hoc committee to follow recipients transplanted with islets isolated using Liberase HI after becoming informed of the associated dangers of using this enzyme. We also stopped islet transplantations using Liberase. The committee addressed the major concerns of the risk of the collagenase being contaminated with TSE and of the recipient follow-up. All recipients were examined by diffusion MRI and EEG and then scheduled for evaluation and follow-up by specialists in Creutzfeldt-Jakob disease (CJD). Bioassays of bovine spongiform encephalopathy prions in the enzyme proceeded using knock-in mice expressing bovine prion protein. These assays could detect contaminating prions at a dilution of 1 × 10 4. After inactivating its collagenase activity, Liberase HI was injected into the abdominal cavities of knock-in mice. Four months later, prion infectivity in Liberase HI was evaluated by immunohistochemical staining and Western blotting of spleen homogenates using anti-prion protein antibodies. Main findings Western blotting and immunohistochemical staining did not detect prions in Liberase HI. Diffusion MRI and EEG evaluations performed by CJD specialists confirmed that none of the transplanted recipients had CJD. Conclusions Three years of follow-up revealed that none of the Japanese recipients of islet transplants developed CJD. Prion bioassays showed that the Liberase HI used to isolate islets for transplantation was free of infectious TSE prions.

Conditional Expression of Full-Length Humanized Anti-Prion Protein Antibodies in Chinese Hamster Ovary Cells

Because of their high antigen specificity and metabolic stability, genetically engineered human monoclonal antibodies are on the way to becoming one of the most promising medical diagnostics and therapeutics. In order to establish an in vitro system capable of producing such biosimilar antibodies, we used human constant chain sequences to design the novel human antibody expressing vector cassette pMAB-ABX. A bidirectional tetracycline (tet)-controllable promotor was used for harmonized expression of immunoglobulin type G (IgG) heavy and light chains. As an example we used anti-prion protein (anti-PrP) IgGs. Therefore, the variable heavy (V(H)) and light chain (V(L)) sequences of anti-PrP antibodies, previously generated in our laboratory by DNA immunization of prion protein knock-out mice, were isolated from murine hybridoma cell lines and inserted into pMAB-ABX vector. After transfection of Chinese hamster ovary (CHO) cells, a number of stable antibody producing cell clones were selected. One cell line (pMAB-ABX-13F10/3B5) stably expressing the recombinant humanized antibody (rechuAb) 13F10/3B5 was selected for detailed characterization by Western blot, immunofluorescence, and flow cytometric analyses. The full-length recombinant humanized IgG antibody showed a high level of expression in the cytoplasm. In conclusion, the new cell system described here is a suitable tool to produce functional intact full-length humanized IgG antibodies.

Large‐scale immunohistochemical examination for lymphoreticular prion protein in tonsil specimens collected in Britain

There have been 173 cases of variant Creutzfeldt-Jakob disease (vCJD) in the UK, as of 5 July 2010, as a result of the bovine spongiform encephalopathy epidemic. The number of individuals subclinically infected with vCJD, and thus the eventual number of cases, remains, however, uncertain. In an attempt to address this problem, 63,007 tonsil tissue specimens were previously tested by enzyme immunoassay (EIA) for the presence of disease-related prion protein (PrP(res)) and found to be negative. To confirm the reliability of this result, all those in the birth cohort most at risk (1961-1985) and a few others, including controls, have now been tested by immunohistochemistry (IHC). Histological slides were prepared from 10,075 anonymized formalin-fixed, paraffin-embedded tissues and examined for PrP(res) with two anti-prion protein antibodies, ICMS35 and KG9. One specimen showed a single strongly positive follicle with both antibodies, on two slides from adjacent sections. As this specimen was negative when it was further investigated by EIA, IHC, and immunoblotting, it is unclear whether the patient from whom the tonsil came will go on to develop vCJD. If, however, this is the case, then a finding of 1 out of 9160 gives a prevalence of disease-related prion protein in the British population of 109 per million, with a 95% confidence interval (CI) of 3-608 per million, which is not statistically different (exact p = 0.63) from population prevalence estimates based on finding three positives out of 10 278 in a previous IHC study of appendix tissue. If this is not the case, a finding of 0 out of 9160 gives a prevalence of 0-403 per million (95% CI) for the 1961-1985 cohort, which is also not different (exact p = 0.25) from previous population prevalence estimates. Therefore, the results of this work could be summarized as finding, by IHC, no or one vCJD-positive individual.

Anti-PrP antibodies detected at terminal stage of prion-affected mouse

The causative agent of prion diseases is the pathological isoform (PrP Sc) of the host-encoded cellular prion protein (PrP C). PrP Sc has an identical amino acid sequence to PrP C; thus, it has been assumed that an immune response against PrP Sc could not be found in prion-affected animals. In this study, we found the anti-prion protein (PrP) antibody at the terminal stage of mouse scrapie. Several sera from mice in the terminal stage of scrapie reacted to the recombinant mouse PrP (rMPrP) molecules and brain homogenates of mouse prion diseases. These results indicate that mouse could recognize PrP C or PrP Sc as antigens by the host immune system. Furthermore, immunization with rMPrP generates high titers of anti-PrP antibodies in wild-type mice. Some anti-PrP antibodies immunized with rMPrP prevent PrP Sc replication in vitro. The mouse sera from terminal prion disease have several wide epitopes, although mouse sera immunized with rMPrP possess narrow epitopes.

Human anti-prion protein antibodies block A117V PrP peptide fibril formation and prevent A117V PrP peptide-induced neurotoxicity

Human anti-prion protein antibodies block A117V PrP peptide fibril formation and prevent A117V PrP peptide-induced neurotoxicity

Antibodies to a Nonconjugated Prion Protein Peptide 95-123 Interfere with PrP Sc Propagation in Prion-Infected Cells

1. Vaccination-induced anti-prion protein antibodies are presently regarded as a promising approach toward treatment of prion diseases. Here, we investigated the ability of five peptides corresponding to three different regions of the bovine prion protein (PrP) to elicit antibodies interfering with PrP(Sc) propagation in prion-infected cells.2. Rabbits were immunized with free nonconjugated peptides. Obtained immune sera were tested in enzyme-linked immunosorbent assay (ELISA) and immunoblot for their binding to recombinant PrP and cell-derived pathogenic isoform (PrP(Sc)) and normal prion protein (PrP(c)), respectively. Sera positive in all tests were chosen for PrP(Sc) inhibition studies in cell culture.3. All peptides induced anti-peptide antibodies, most of them reacting with recombinant PrP. Moreover, addition of the serum specific to peptide 95-123 led to a transient reduction of PrP(Sc) levels in persistently prion-infected cells.4. Thus, anti-PrP antibodies interfering with PrP(Sc) propagation were induced with a prion protein peptide nonconjugated to a protein carrier. These results point to the potential application of the nonconjugated peptide 95-123 for the treatment of prion diseases.

A solid-phase immunoassay of protease-resistant prion protein with filtration blotting involving sodium dodecyl sulfate

The precise diagnosis for bovine spongiform encephalopathy (BSE) is crucial for preventing new transmission to humans. Several testing procedures are reported for determining protease-resistant prion protein in various tissues as a major hallmark of prion diseases such as BSE, scrapie, and Creutzfeldt–Jakob disease. However, contamination of materials from tissues or degradation of the specimens sometimes disturbs the accuracy of the assay. Here, we have developed a novel method for solid-phase immunoassay of the disease-specific conformational isoform, PrP Sc, using filtration blotting of protein in the presence of sodium dodecyl sulfate (SDS) followed by a filtration-based immunoassay with a single anti-prion protein antibody, together with the improved fractionation procedure involving high concentrations of surfactant/detergent. The SDS/heat treatment renders unfolded PrP Sc quantitative retention on a polyvinylidene difluoride filter and allows enhancement of the analyte signal with immunodetection; thus, all of the tested specimens are determined with 100% accuracy. In addition, the immunoassay is completed in approximately 1 h, indicating its usefulness not only for the screening of BSE specimens but probably also for the postmortem BSE diagnosis of fallen stock as the antibody recognizes the core part of PrP Sc. The solid-phase immunoassay method, including the filtration blotting with SDS, would be applicable to determining even more sensitively proteins other than PrP Sc, especially those having rigid conformations.

Update on Creutzfeldt-Jakob disease

Prion diseases are transmissible fatal neurodegenerative disorders in which infectivity is associated with the accumulation of PrP(Sc), a disease-related isoform of normal cellular prion protein. The recent emergence of variant Creutzfeldt-Jakob disease has led to major public health concerns, and the need for the development of effective treatments. As PrP(Sc) is associated both with pathology and infectivity, therapeutic approaches to date have largely aimed at preventing its accumulation, but this strategy has produced only modest results in animal models. The link between PrP(Sc) and neurotoxicity is unclear, and alternative pathological processes need to be considered. Here we focus on the latest progress in therapeutic strategies and potential mechanisms of prion neurotoxicity. Passive immunisation with anti-prion protein antibodies prevents peripheral prion replication and blocks progression to clinical disease in peripherally infected mice. A new approach, in which neuronal cellular prion protein is depleted in mice with established neuroinvasive prion infection, prevents the onset of clinical disease, blocks neuronal cell loss and reverses early spongiform pathology. This dramatic protective effect occurs despite the continued build-up of extraneuronal PrP(Sc) and continued replication of prion infectivity, effectively producing a sub-clinical state. New insights into the mechanisms of neurotoxicity in prion diseases support the concept that PrP(Sc) itself is not directly neurotoxic. They suggest that neuronal prion propagation results in the production of a toxic intermediate or depletion of a key constituent. Prevention of the formation of such a species rather than PrP(Sc) accumulation itself is a clear target for prion therapeutics.

Labeling of the scrapie-associated prion protein in vitro and in vivo

Prion diseases are a group of infectious neurodegenerative diseases that affect both animals and humans. A characteristic of prion diseases is the aggregation and accumulation of a disease-associated isoform of the prion protein in the brains of infected individuals. The amyloid imaging probe ( trans, trans)-1-bromo-2,5-bis-(3-hydroxycarbonyl-4-hydroxy)styrylbenzene (BSB) has shown potential in the diagnosis of other amyloid disorders and we hypothesized that this compound would be effective in labeling prion protein plaques in vitro and in vivo. To test this, we compared BSB fluorescence to prion protein immunostaining on infected and uninfected brain tissue sections from scrapie-infected hamsters. We found that both methods labeled the same plaques in infected tissues while not substantially staining uninfected tissues. To test the potential of BSB as an in vivo label for prion aggregates, we perfused scrapie-infected animals with BSB and observed BSB labeled plaques co-stained with an anti-prion protein antibody. These results suggest that BSB may have use as a diagnostic tool for prion diseases. We were unable to detect BSB staining in preclinical scrapie-infected hamsters suggesting that the diagnostic potential of BSB could be limited in cases of prion disease that do not have plaques either due to a preclinical lack of pathology or disease agents like sporadic Creutzfeldt–Jakob disease (CJD), which generally lack prion plaques. However, BSB may be a useful for prion diseases where plaques are present, such as clinical variant CJD.

Immunoseparation of Prion protein-enriched domains from other detergent-resistant membrane fractions, isolated from neuronal cells

The possibility of coexistence of different subtypes of membrane lipid rafts has been investigated in cerebellar granule cells, by submitting detergent-resistant membrane fractions to immunoprecipitation. Among the proteins and lipids present in detergent-resistant fractions, almost all Prion protein, GAP43 and PKC were present in the immunoprecipitate obtained with anti-GAP43 or anti-Prion protein antibody at 4°C, together with a small fraction of cholesterol and sphingolipids, suggesting that they belong to a distinct subset of membranes. On the contrary, all Fyn and almost all MARCKS remained in the supernatant. Fluorescence microscopy experiments showed that Fyn and Prion protein were mostly not colocalized within a single neuron. Our results suggest that granule cells membranes contains different subtypes of detergent-resistant fractions, possibly deriving from different lipid rafts.