Faculty Bibliography

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.

The Note reports on experimental studies of ripple born fast electrons within the TORE-SUPRA facility, which were performed by means of a modified measuring head equipped with diamond detectors designed especially for recording the electron-induced Cherenkov radiation. There are presented signals produced by fast electrons in the TORE-SUPRA machine, which were recorded during two experimental campaigns performed in 2010. Shapes of these electron-induced signals are considerably different from those observed during the first measurements carried out by the prototype Cherenkov probe in 2008. An explanation of the observed differences is given.

Transgenic (Tg) mice overexpressing human amyloid precursor protein (APP) mutants reproduce features of early Alzheimer's disease (AD) including memory deficit, presence of beta-amyloid (Abeta) oligomers, and age-associated formation of amyloid deposits. In this study we used hippocampal microdialysis to characterize the signaling of N-methyl-d-aspartic acid receptors (NMDA-Rs) in awake and behaving AD Tg mice. The NMDA-R signaling is central to hippocampal synaptic plasticity underlying memory formation and several lines of evidence implicate the role of Abeta oligomers in effecting NMDA-R dysfunction. CA1 NMDA-Rs were stimulated by NMDA infused through reverse microdialysis while changes in the cyclic guanosine monophosphate (cGMP) concentration in the brain interstitial fluid (ISF) were used to determine NMDA-Rs responsiveness. While 4 months old wild type C57BL/6 mice mounted robust cGMP response to the NMDA challenge, the same stimulus failed to significantly change the cGMP level in 4 and 15months old APP(SW) and 4 months old APP(SW)/PS1(L166P) Tg mice, which were all on C57BL/6 background. Lack of response to NMDA in AD Tg mice occurred in the absence of changes in expression levels of several synaptic proteins including synaptophysin, NR1 NMDA-R subunit and postsynaptic density protein 95, which indicates lack of profound synaptic degeneration. Abeta oligomers were detected in all three AD Tg mice groups and their concentration in the hippocampus ranged from 40.5+/-3.6ng/g in 4 months old APP(SW) mice to 60.8+/-15.9ng/g in 4 months old APP(SW)/PS1(L166P) mice. Four months old APP(SW) mice had no Abeta amyloid plaques, while the other two AD Tg mice groups showed evidence of incipient Abeta amyloid plaque formation. Our studies describes a novel approach useful to study the function of NMDA-Rs in awake and behaving AD Tg mice and demonstrate impairment of NMDA-R response in the presence of endogenously formed Abeta oligomers but predating onset of Abeta amyloidosis.

A diagnostic technique based on the Cherenkov effect is proposed for detection and characterization of fast (super-thermal and runaway) electrons in fusion devices. The detectors of Cherenkov radiation have been specially designed for measurements in the ISTTOK tokamak. Properties of several materials have been studied to determine the most appropriate one to be used as a radiator of Cherenkov emission in the detector. This technique has enabled the detection of energetic electrons (70 keV and higher) and the determination of their spatial and temporal variations in the ISTTOK discharges. Measurement of hard x-ray emission has also been carried out in experiments for validation of the measuring capabilities of the Cherenkov-type detector and a high correlation was found between the data of both diagnostics. A reasonable agreement was found between experimental data and the results of numerical modeling of the runaway electron generation in ISTTOK.

Background: In AD, synapse loss more closely correlates with cognitive impairment than plaques and tangles. We and others have extensively described similar synapse-specific vulnerability in the ME7-model of prion disease. Recent evidence suggests that astrocytes closely associated with neuronal processes, synapses and blood vessels, can impact brain energetics cellular homeostasis and antioxidant levels. In prion ME7-animals astrocytes produce several complement components and ApoJ, a cholesterol carrier in cells that along with genes for PICALM and Complement receptor 1 were recently identified as having links to AD. Interestingly, they also produce other astrocyte-associated proteins, such as peroxiredoxin-6 (Prdx6) an antioxidant protein that has been demonstrated to be neuroprotective. However, it is still uncertain precisely how much astrocytes contribute to amelioration or exacerbation pathology in degenerative diseases. In this study, we set out to directly test the protective impact of astrocyte specific antioxidant protein Prdx6 on synaptic degeneration by examining behavioural and biochemical correlates in a protein misfolding animal model lacking or overexpressing Prdx6. Methods: Control C57BL6, and age-matched Prdx6-KO and Prdx6-Tg animals were intraperitoneally inoculated with the same stock of ME7 (amyloidogenic) prion agent or normal brain homogenate (NBH). We then set out to closely correlate the relationship between Prdx6 genotype, progressive accumulation of abnormally misfolded prion disease and associated behavioral and synaptic abnormalities.We used standardWestern blotting and immunohistochemistry protocols to profile brains and spleen from control, NBH-, and ME7-Prdx6-KO and Prdx6-Tg animals which were sacrificed at advanced disease states. Conclusions: We show that lack of Prdx6 contributes to impairment of cognitive behavioural correlates and exacerbation of prion neuropathology. However, our observations do not preclude that Prdx6 genotype impacts earlier neuroinvasion

Background: Progressive accumulation of amyloid-s (As) in the brain is the hallmark of Alzheimer's disease (AD). Excess As assembles into toxicoligomers or fibrils, which became deposited in the form of plaques or vascular deposits. As no compelling evidences for age related increase in As production have been provided, it is likely that impairment of As clearance plays critical role in development of sporadic AD. Multiple pathways of As clearance include proteolytic, enzymatic degradation in the extracellular space, clearance of As through the blood brain barrier, or uptake and degradation by glial cells. Indeed, astrocytes take up As in a clathrin-caveolin- dynamin-independent endocytosis through endosomes with efficiency depending on As aggregation status. Astrocytesare also the main source of apolipoprotein E (apo E) in the central nervous system. Apo E in isoform specific fashion is associated with the risk for AD occurrence. In this study we characterized uptake and degradation of As by astrocytes derived from apo E targeted replacement (TR) transgenic mice expressing human apo E isoforms E2, E3, and E4, apo E-KO mice, and wild type mice. Methods: Primary cortical astrocytes were established from 1-2 day old pups. Cells were treated with 1 to 10 mM of synthetic As40 or As42 for 0 to 96hr. The effect of As on astrocyte metabolic integrity was determined using MTT assay. The uptake of As from the medium and clearance by astrocyteswere evaluated using western-blotting analyses of cell culture medium and celllysate samples respectively. Results: All tested astrocytes were capable of clearing As40 and As42 from the culture medium with stronger effect on As40. Analysis of lysates demonstrated a low accumulation of As40 inside astrocytes while significant amount of As42 was found inside all types of astrocytes, but less robust in apo E-KO. The intra-astrocytic accumulated As42 appears to be mainly oligomeric. MTT assay demonstrated that As42, but not As40, diminished metabolic activity of astrocytes. Conclusions: Astrocytes demonstrate great capacity of As uptake and degradation. The As uptake appears to be independent from of apo E isoformstatus. Excess of As42 oligomerizes and accumulates inside astrocytes resulting in their dysfunction as determined by MTT assays

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

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