Faculty Bibliography

Background: Disturbance of beta-amyloid (Abeta) homeostasis and its accumulation in the brain underlie early Alzheimer's disease (AD) pathogenesis. Apolipoprotein (apo) E is a lipid carrier protein, which directly interacts with Abeta promoting its oligomerization, deposition in parenchymal plaques and in the brain vasculature, and also influences the rate of Abeta brain clearance. APOE alleles are well-recognized genetic risk factors for sporadic AD and correlate with severity of beta-amyloidosis. APOE4 increases AD risk, while APOE2 shows a relative protective effect. Here, we investigated effects of the treatment with a pharmacological inhibitor of the apoE/Abeta binding on AD pathology in APP SW/PS1 dE9/apoE2-TR (TR=targeted replacement) and APP SW/PS1 dE9/apoE4-TR mice. Methods: APP SW/ PS1 dE9/apoE-TR mice were generated by crossing APP SW/PS1 dE9 transgenic mice to human apoE2-TR and apoE4-TR mice. Abeta12-28P-a synthetic peptide antagonist of the apoE/Abeta binding, which was modified for in vivo administration, or vehicle were intraperitoneally administered to the mice between the age of six and 10 months. Before conclusion of the experiment animals' memory was tested using object recognition (OR) and radial arm maze (RAM) tests. Brain Abeta load was analyzed by biochemical and morphometric methods. Results: There was no significant behavioral deficit in vehicle treated APP SW/PS1 dE9/apoE2-TR mice as compared to age and sex matched apoE2-TR mice. Abeta12-28P treatment of APP SW/PS1 dE9/ apoE2-TR animals also had no significant effect on their OR and RAM performance. In contrast, vehicle treated APP SW/PS1 dE9/apoE4-TR mice showed impaired OR behavior and on RAM test performed significantly worse than apoE4-TR mice. Abeta12-28P treatment of APP SW/PS1 dE9/ apoE4-TR mice normalized OR behavior and improved RAM performance to the level of apoE4-TR mice. Levels of soluble and insoluble Abeta x-40 and Abeta x-42 measured by ELISA in the whole brain extract and the burden of Thioflavin-S po!

Accumulation of beta-amyloid (Abeta) in the brain is a key event in Alzheimer disease pathogenesis. Apolipoprotein (Apo) E is a lipid carrier protein secreted by astrocytes, which shows inherent affinity for Abeta and has been implicated in the receptor-mediated Abeta uptake by neurons. To characterize ApoE involvement in the intraneuronal Abeta accumulation and to investigate whether blocking the ApoE/Abeta interaction could reduce intraneuronal Abeta buildup, we used a noncontact neuronal-astrocytic co-culture system, where synthetic Abeta peptides were added into the media without or with cotreatment with Abeta12-28P, which is a nontoxic peptide antagonist of ApoE/Abeta binding. Compared with neurons cultured alone, intraneuronal Abeta content was significantly increased in neurons co-cultured with wild-type but not with ApoE knockout (KO) astrocytes. Neurons co-cultured with astrocytes also showed impaired intraneuronal degradation of Abeta, increased level of intraneuronal Abeta oligomers, and marked down-regulation of several synaptic proteins. Abeta12-28P treatment significantly reduced intraneuronal Abeta accumulation, including Abeta oligomer level, and inhibited loss of synaptic proteins. Furthermore, we showed significantly reduced intraneuronal Abeta accumulation in APPSW/PS1dE9/ApoE KO mice compared with APPSW/PS1dE9/ApoE targeted replacement mice that expressed various human ApoE isoforms. Data from our co-culture and in vivo experiments indicate an essential role of ApoE in the mechanism of intraneuronal Abeta accumulation and provide evidence that ApoE/Abeta binding antagonists can effectively prevent this process.

Prionoses are a group of neurodegenerative diseases characterized by misfolding of cellular prion protein (PrP(C)) and accumulation of its diseases specific conformer PrP(Sc) in the brain and neuropathologically, they can be associated with presence or absence of PrP amyloid deposits. Functional molecular chaperones (MCs) that constitute the unfolded protein response include heat shock proteins and glucose-regulated protein families. They protect intracellular milieu against various stress conditions including accumulation of misfolded proteins and oxidative stress, typical of neurodegenerative diseases. Little is known about the role of MCs in pathogenesis of prionoses in mammalian prion model systems. In this study we characterized MCs response pattern in mice infected with various mouse adapted scrapie strains. Rather than uniform upregulation of MCs, we encountered two distinctly different patterns of MCs response distinguishing ME7 and 87V strains from 22L and 139A strains. ME7 and 87V strains are known for the induction of amyloid deposition in infected animals, while in mice infected with 22L and 139A strains amyloid deposits are absent. MCs response pattern similar to that associated with amyloidogenic ME7 and 87V strains was also observed in APPPS1-21 Alzheimer's transgenic mice, which represent an aggressive model of cerebral amyloidosis caused by beta-amyloid deposition. Our results highlight the probability that different mechanisms of MCs regulation exist driven by amyloidogenic and non-amyloidogenic nature of prion strains.

Background: Longstanding accumulation of a toxic and prone to self-aggregation s-amyloid peptide (As) in the brain is considered a leading pathological mechanism of Alzheimer's disease (AD). Our goal is to develop a bloodbrain barrier (BBB) permeable, small molecule inhibitor of the As aggregation into toxic oligomers and fibrils, which could attenuate accumulation and toxicity of As in brains of AD patients. Methods: Several libraries of compounds encompassing multi-aryl and heteroaryl groups were developed and screened for toxicity in SK-N-SH human neuroblastoma cells and against As aggregation using assays specifically designed to promote assembly of synthetic As into oligomers or fibrils. The neuroprotective effects of the lead compound on excitatory synapses were investigated in 18 day in vitro (DIV) primary hippocampal neuronal cultures. The pharmacokinetic profile, including bioavailability and BBB penetration, were established in mice following oral and intravenous administration using a combined liquid chromatographymass spectrometry (LC-MS) approach. Results: Our screen has identified the lead compound dubbed ARN 4261, [(E)-2-(pyridin-2-ylmethyleneamino) phenol;MW=198.2Da], which is not toxic and at low micromolar concentration shows strong effects against both oligomerization and fibrillization of synthetic As peptide. Treatment of primary hippocampal neurons exposed to As with ARN4261 restored loss of synaptic proteins expression caused by As oligomers. We have subsequently modified the structure of ARN 4261 producing ARN 2966 (2-[(pyridine-2-ylmethyl)-amino]-phenol) which shows comparable anti-aggregation potency to ARN 4261, but it is more stable in acidic environment, hence it is suitable for oral administration. Pharmacokinetic experiments in mice showed that t_1/2 of ARN 2966 is 6.13hr and 64.2% of orally administered dose is absorbed from the alimentary tract and passes the portal circulation. Preliminary studies show that ARN 2966 penetrates the BBB after oral and intravenous administration. Conclusions: Pyridin-2-ylmethylamine derivatives are a class of novel promising AD therapeutics. They are non toxic, have strong anti-As aggregation and neuroprotective properties and can be easily modified chemically for enhanced oral bioavailability and BBB penetration. Experiments in AD transgenic mice characterizing their effect on AD pathology in vivo are currently ongoing

Background: Conformational transformation of a cellular prion protein (PrP^C) into the misfolded, proteinase K (PK)-resistant and self-replicating conformer PrP^Sc is considered to be the main pathological mechanism of prionoses. We and others have demonstrated that treatment with anti-PrP monoclonal antibodies (Mabs) stops replication of PrP^Sc in prion infected cell lines and significantly delays the clinical onset of disease in prion infected mice. This research was conducted to elucidate the mechanism(s) determining therapeutic efficacy of Mabs, which remains unknown. Methods: The effects of anti-PrP Mab on kinetics of PrP^Sc disappearance were investigated in N2A murine neuroblastoma line infected with 22L prion strain (N2A/22L), and in N2A/22L transfected with siRNA targeting Prnp mRNA. Trafficking of Cy3-6D11 in N2A and N2A/22L was tracked by a time-lapse fluorescent microscopy. Effects of Mabs and 3F4 on PrP solubility were studied in N2A clones over-expressing wild-type (WT) human PrP and Gerstmann-Straussler-Scheinker (GSS) mutant with either valine (V) or methionine (M) at position 129. Results: Mab treatment of N2A/ 22L resulted in total disappearance of PrP^Sc within 48hr, with PrP^Sc t_{1/ 2}=10.7hr. Mab treatment was associated with initial reduction in the level of total PrP and its unglycosylated fraction, which subsequently increased at 48hr, when PrP^Sc was no longer observed. Transfection of N2A and N2A/22L with siRNA resulted in 50% reduction of the total PrP after 17hr and 19.5hr, respectively. PrP^Sc level was diminished by 50% after 10.7hr in siRNA transfected N2A/22L, whereas Mab co-treatment shortened PrP^Sc t_1/2 to 7hr. Massive intracellular penetration of Cy3-6D11 was observed in N2A/22L but not in N2A cells, indicating that directs surface expressed PrP^Sc for intracellular degradation. Treatment of N2A clones over-expressing WT-M129 and WT-V129 human PrP and A117V/ M129, A117V/V129, and F198S/V129 GSS mutants with Mabs 3F4 and increased the detergent soluble PrP fraction from two to five folds (p< 0.05). Conclusions: Anti-PrP Mabs appear to exert their therapeutic effect by promoting degradation of PrP^Sc, but they do not affect PrP production. Anti- PrP Mabs increase the solubility of PrP aggregates, which likely exerts a complementary effect in promoting PrP^Sc degradation

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