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Immunomodulation targeting abnormal protein conformation reduces pathology in a mouse model of Alzheimer's disease

Goni, Fernando; Prelli, Frances; Ji, Yong; Scholtzova, Henrieta; Yang, Jing; Sun, Yanjie; Liang, Feng-Xia; Kascsak, Regina; Kascsak, Richard; Mehta, Pankaj; Wisniewski, Thomas
Many neurodegenerative diseases are characterized by the conformational change of normal self-proteins into amyloidogenic, pathological conformers, which share structural properties such as high beta-sheet content and resistance to degradation. The most common is Alzheimer's disease (AD) where the normal soluble amyloid beta (sAbeta) peptide is converted into highly toxic oligomeric Abeta and fibrillar Abeta that deposits as neuritic plaques and congophilic angiopathy. Currently, there is no highly effective treatment for AD, but immunotherapy is emerging as a potential disease modifying intervention. A major problem with most active and passive immunization approaches for AD is that both the normal sAbeta and pathogenic forms are equally targeted with the potential of autoimmune inflammation. In order to avoid this pitfall, we have developed a novel immunomodulatory method that specifically targets the pathological conformations, by immunizing with polymerized British amyloidosis (pABri) related peptide which has no sequence homology to Abeta or other human proteins. We show that the pABri peptide through conformational mimicry induces a humoral immune response not only to the toxic Abeta in APP/PS1 AD transgenic mice but also to paired helical filaments as shown on AD human tissue samples. Treated APP/PS1 mice had a cognitive benefit compared to controls (p<0.0001), associated with a reduction in the amyloid burden (p = 0.0001) and Abeta40/42 levels, as well as reduced Abeta oligomer levels. This type of immunomodulation has the potential to be a universal beta-sheet disrupter, which could be useful for the prevention or treatment of a wide range of neurodegenerative diseases
PMID: 20967130
ISSN: 1932-6203
CID: 114051

Anti-PrP Mab 6D11 suppresses PrP(Sc) replication in prion infected myeloid precursor line FDC-P1/22L and in the lymphoreticular system in vivo

Sadowski, Martin J; Pankiewicz, Joanna; Prelli, Frances; Scholtzova, Henrieta; Spinner, Daryl S; Kascsak, Regina B; Kascsak, Richard J; Wisniewski, Thomas
The pathogenesis of prion diseases is related to conformational transformation of cellular prion protein (PrP(C)) into a toxic, infectious, and self-replicating conformer termed PrP(Sc). Following extracerebral inoculation, the replication of PrP(Sc) is confined for months to years to the lymporeticular system (LRS) before the secondary CNS involvement results in occurrence of neurological symptoms. Therefore, replication of PrP(Sc), in the early stage of infection can be targeted by therapeutic approaches, which like passive immunization have limited blood-brain-barrier penetration. In this study, we show that 6D11 anti-PrP monoclonal antibody (Mab) prevents infection on a FDC-P1 myeloid precursor cell line stably infected with 22L mouse adapted scrapie strain. Passive immunization of extracerebrally infected CD-1 mice with Mab 6D11 resulted in effective suppression of PrP(Sc) replication in the LRS. Although, a rebound of PrP(Sc) presence occurred when the Mab 6D11 treatment was stopped, passively immunized mice showed a prolongation of the incubation period by 36.9% (pb0.0001) and a significant decrease in CNS pathology compared to control groups receiving vehicle or murine IgG. Our results indicate that antibody-based therapeutic strategies can be used, even on a short-term basis, to delay or prevent disease in subjects accidentally exposed to prions
PMID: 19385058
ISSN: 1095-953x
CID: 101114

Mechanistic Insights into Prion Curing by Novel Anti-Prion Compounds

Webb, Sarah; Lekishvili, Tamuna; Loeschner, Corinna; Sellarajah, Shane; Prelli, Frances; Wisniewski, Thomas; Gilbert, Ian H; Brown, David R
Prion diseases are fatal neurodegenerative disorders. Identification of possible therapeutic tools is important in the search for a potential treatment for these diseases. Congo Red is an azo dye that has been used for many years to detect abnormal prion protein in the brains of diseased patients or animals. Congo Red has little therapeutic potential for the treatment of these diseases due to toxicity and poor permeation of the blood-brain-barrier. We have prepared two congo red derivatives designing out these liabilities with potent activity in cellular models of prion disease. One of these compounds cured cells of the transmissible agent. The mechanism of action of these compounds is possibly multifactorial. The high affinity of both ineffective and effective prion-curing Congo Red derivatives for abnormally folded prion protein suggests that the amyloidophylic property of these derivatives is not as critical to the mechanism of action as other effects. Effective prion-curing Congo Red derivatives increased degradation of abnormal PrP by the proteasome. Therefore the principal mechanism of action of the congo red analogues was to prevent inhibition of proteasomal activity by PrP(Sc)
PMID: 17652397
ISSN: 0022-538x
CID: 73169

Novel prion protein conformation and glycotype in Creutzfeldt-Jakob disease [Case Report]

Zanusso, Gianluigi; Polo, Alberto; Farinazzo, Alessia; Nonno, Romolo; Cardone, Franco; Di Bari, Michele; Ferrari, Sergio; Principe, Serena; Gelati, Matteo; Fasoli, Elisa; Fiorini, Michele; Prelli, Frances; Frangione, Blas; Tridente, Giuseppe; Bentivoglio, Marina; Giorgi, Alessandra; Schinina, Maria Eugenia; Maras, Bruno; Agrimi, Umberto; Rizzuto, Nicola; Pocchiari, Maurizio; Monaco, Salvatore
OBJECTIVE: To describe a novel molecular and pathological phenotype of Creutzfeldt-Jakob disease. Patient A 69-year-old woman with behavioral and personality changes followed by rapidly evolving dementia. RESULTS: Postmortem examination of the brain showed intracellular prion protein deposition and axonal swellings filled with amyloid fibrils. Biochemical analysis of the pathological prion protein disclosed a previously unrecognized PrP(Sc) tertiary structure lacking diglycosylated species. Genetic analysis revealed a wild-type prion protein gene. The prion agent responsible for this atypical phenotype was successfully passaged to bank voles. CONCLUSION: To our knowledge, our results define a new human prion disorder characterized by intracellular accumulation of a novel type of pathological prion protein
PMID: 17420324
ISSN: 0003-9942
CID: 96089

Blocking the apolipoprotein E/amyloid-{beta} interaction as a potential therapeutic approach for Alzheimer's disease

Sadowski, Martin J; Pankiewicz, Joanna; Scholtzova, Henrieta; Mehta, Pankaj D; Prelli, Frances; Quartermain, David; Wisniewski, Thomas
The amyloid-beta (Abeta) cascade hypothesis of Alzheimer's disease (AD) maintains that accumulation of Abeta peptide constitutes a critical event in the early disease pathogenesis. The direct binding between Abeta and apolipoprotein E (apoE) is an important factor implicated in both Abeta clearance and its deposition in the brain's parenchyma and the walls of meningoencephalic vessels as cerebral amyloid angiopathy. With the aim of testing the effect of blocking the apoE/Abeta interaction in vivo as a potential novel therapeutic target for AD pharmacotherapy, we have developed Abeta12-28P, which is a blood-brain-barrier-permeable nontoxic, and nonfibrillogenic synthetic peptide homologous to the apoE binding site on the full-length Abeta. Abeta12-28P binds with high affinity to apoE, preventing its binding to Abeta, but has no direct effect on Abeta aggregation. Abeta12-28P shows a strong pharmacological effect in vivo. Its systemic administration resulted in a significant reduction of Abeta plaques and cerebral amyloid angiopathy burden and a reduction of the total brain level of Abeta in two AD transgenic mice models. The treatment did not affect the levels of soluble Abeta fraction or Abeta oligomers, indicating that inhibition of the apoE/Abeta interaction in vivo has a net effect of increasing Abeta clearance over deposition and at the same time does not create conditions favoring formation of toxic oligomers. Furthermore, behavioral studies demonstrated that treatment with Abeta12-28P prevents a memory deficit in transgenic animals. These findings provide evidence of another therapeutic approach for AD
PMID: 17116874
ISSN: 0027-8424
CID: 69282

Clearance and prevention of prion infection in cell culture by anti-PrP antibodies

Pankiewicz, Joanna; Prelli, Frances; Sy, Man-Sun; Kascsak, Richard J; Kascsak, Regina B; Spinner, Daryl S; Carp, Richard I; Meeker, Harry C; Sadowski, Marcin; Wisniewski, Thomas
Prion diseases are transmissible and invariably fatal neurodegenerative disorders associated with a conformational transformation of the cellular prion protein (PrP(C)) into a self-replicating and proteinase K (PK)-resistant conformer, scrapie PrP (PrP(Sc)). Humoral immunity may significantly prolong the incubation period and even prevent disease in murine models of prionoses. However, the mechanism(s) of action of anti-PrP monoclonal antibodies (Mabs) remain(s) obscure. The murine neuroblastoma N2a cell line, infected with the 22L mouse-adapted scrapie strain, was used to screen a large library of Mabs with similar binding affinities to PrP, to identify those antibodies which could clear established infection and/or prevent infection de novo. Three Mabs were found capable of complete and persistent clearing of already-infected N2a cells of PrP(Sc). These antibodies were 6D11 (generated to PK-resistant PrP(Sc) and detecting PrP residues 93-109), and 7H6 and 7A12, which were raised against recombinant PrP and react with neighbouring epitopes of PrP residues 130-140 and 143-155, respectively. Mabs were found to interact with PrP(Sc) formation both on the cell surface and after internalization in the cytosol. Treatment with Mabs was not associated with toxicity nor did it result in decreased expression of PrP(C). Both preincubation of N2a cells with Mabs prior to exposure to 22L inoculum and preincubation of the inoculum with Mabs prior to infecting N2a cells resulted in a significant reduction in PrP(Sc) levels. Information provided in these studies is important for the rational design of humoral immune therapy for prion infection in animals and eventually in humans
PMID: 16817866
ISSN: 0953-816X
CID: 65120

Studies on the first described Alzheimer's disease amyloid beta mutant, the Dutch variant

Levy, Efrat; Prelli, Frances; Frangione, Blas
Amyloid protein deposited in cerebral vessel walls and diffuse plaques of patients with hereditary cerebral hemorrhage with amyloidosis, Dutch type (HCHWA-D), is similar to the 40-42 residues amyloid beta (Abeta) in vessel walls and senile plaques in brains of patients with Alzheimer's disease (AD), Down's syndrome, and familial and sporadic cerebral amyloid angiopathy (CAA). In 1990 we sequenced the amyloid beta-protein precursor (AbetaPP) gene from HCHWA-D patients revealing a single mutation that results in an amino acid substitution, Abeta E22Q. Subsequent identification of additional mutations in the AbetaPP gene in familial AD (FAD) pedigrees revealed that whereas substitutions in the middle of Abeta, residues Abeta21-23, are predominantly vasculotropic, those found amino- or carboxyl-terminal to the Abeta sequence within AbetaPP enhance amyloid parenchymal plaque deposition. Studies of transfected cells showed that substitutions amino- or carboxyl-terminal to Abeta lead to either greater Abeta production or to enhanced secretion of the more hydrophobic thus more fibrillogenic Abeta1-42. Substitutions in the center of Abeta facilitate rapid aggregation and fibrillization, slower clearance across the blood-brain barrier and perivascular drainage to the systemic circulation, possibly higher resistance to proteolysis, and enhanced toxicity towards endothelial and smooth muscle cells. However, most AD patients have no genetic defects in AbetaPP, indicating that other factors may alter Abeta production, conformation, and/or clearance initiating the disease process
PMID: 16914871
ISSN: 1387-2877
CID: 68936

Purification of human wild-type or variant cystatin C from conditioned media of transfected cells

Prelli, Frances; Pawlik, Monika; Frangione, Blas; Levy, Efrat
The characterization of proteins in their native state is essential for the understanding of patho-genic isoforms. A variant of the cysteine protease inhibitor cystatin C is the major constituent of the amyloid deposited in the cerebral vasculature of patients with the Icelandic form of hereditary cerebral hemorrhage with amyloidosis (HCHWA-I). In order to study the nature of the bio-physical changes owing to the Leu68Gln substitution in cystatin C, we have developed a purification procedure of human cystatin C in its native state. The protein is isolated from media of stably transfected tissue culture cells using physiological conditions that preclude protein denaturation. The importance of mild purification conditions is underscored by the finding that denaturation of the wild-type and variant proteins facilitates a similar folding of both molecules, diminishing their differences in structure and biophysical properties. Following native purification conditions, variant cystatin C has a distinct structure compared to the wild-type protein
PMID: 15980605
ISSN: 1064-3745
CID: 56366

Identification of distinct N-terminal truncated forms of prion protein in different Creutzfeldt-Jakob disease subtypes

Zanusso, Gianluigi; Farinazzo, Alessia; Prelli, Frances; Fiorini, Michele; Gelati, Matteo; Ferrari, Sergio; Righetti, Pier Giorgio; Rizzuto, Nicolo; Frangione, Blas; Monaco, Salvatore
In prion diseases, the cellular prion protein (PrP(C)) is converted to an insoluble and protease-resistant abnormal isoform termed PrP(Sc). In different prion strains, PrP(Sc) shows distinct sites of endogenous or exogenous proteolysis generating a core fragment named PrP27-30. Sporadic Creutzfeldt-Jakob disease (sCJD), the most frequent human prion disease, clinically presents with a variety of neurological signs. As yet, the clinical variability observed in sCJD has not been fully explained by molecular studies relating two major types of PrP27-30 with unglycosylated peptides of 21 (type 1) and 19 kDa (type 2) and the amino acid methionine or valine at position 129. Recently, smaller C-terminal fragments migrating at 12 and 13 kDa have been detected in different sCJD phenotypes, but their significance remains unclear. By using two-dimensional immunoblot with anti-PrP antibodies, we identified two novel groups of protease-resistant PrP fragments in sCJD brain tissues. All sCJD cases with type 1 PrP27-30, in addition to MM subjects with type 2 PrP27-30, were characterized by the presence of unglycosylated PrP fragments of 16-17 kDa. Conversely, brain homogenates from patients VV and MV with type 2 PrP27-30 contained fully glycosylated PrP fragments, which after deglycosylation migrated at 17.5-18 kDa. Interestingly, PrP species of 17.5-18 kDa matched deglycosylated forms of the C1 PrP(C) fragment and were associated with tissue PrP deposition as plaque-like aggregates or amyloid plaques. These data show the presence of multiple PrP(Sc) conformations in sCJD and, in addition, shed new light on the correlation between sCJD phenotypes and disease-associated PrP molecules
PMID: 15247220
ISSN: 0021-9258
CID: 101671

Copper chelation delays the onset of prion disease

Sigurdsson, Einar M; Brown, David R; Alim, Muhammad A; Scholtzova, Henrieta; Carp, Richard; Meeker, Harry C; Prelli, Frances; Frangione, Blas; Wisniewski, Thomas
The prion protein (PrP) binds copper and under some conditions copper can facilitate its folding into a more protease resistant form. Hence, copper levels may influence the infectivity of the scrapie form of prion protein (PrPSc). To determine the feasibility of copper-targeted therapy for prion disease, we treated mice with a copper chelator, D-(-)-penicillamine (D-PEN), starting immediately following intraperitoneal scrapie inoculation. D-PEN delayed the onset of prion disease in the mice by about 11 days (p = 0.002), and reduced copper levels in brain by 29% (p < 0.01) and in blood by 22% (p = 0.03) compared with control animals. Levels of other metals were not significantly altered in the blood or brain. Modest correlation was observed between incubation period and levels of copper in brain (p = 0.08) or blood (p = 0.04), indicating that copper levels are only one of many factors that influence the rate of progression of prion disease. In vitro, copper dose-dependently enhanced the proteinase K resistance of the prion protein, and this effect was counteracted in a dose-dependent manner by co-incubation with D-PEN. Overall, these findings indicate that copper levels can influence the conformational state of PrP, thereby enhancing its infectivity, and this effect can be attenuated by chelator-based therapy
PMID: 14519758
ISSN: 0021-9258
CID: 48185