Faculty Bibliography

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: 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: 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: Hyperglycemia is a metabolic abnormality defining diabetes mellitus (DM). Misfolding and accumulation of abnormally conformed amyloid-s (As) and associated neurodegenerative cascades underpins Alzheimer's disease (AD) pathogenesis. Epidemiological studies have repeatedly demonstrated increased coincidence of AD pathology in DM patients, and the clinical course of AD is often hastened in subjects with concomitant DM. Despite these associations, it remains unclear how DM mechanistically impacts AD. We have focused on defining the relationship between hyperglycemia (hGly) and the As pathology in AD transgenic (Tg) animals. Methods: Chronic hGly was induced in male Thy-1-APPKM670/671NL/PS1L166PAD Tg mice by intraperitoneal injection of streptozotocin (STZ) (200mg/kg), which is selectively toxic to insulin-producing pancreatic s-cells. STZ or vehicle (n=8) were injected at the age of two months; prior to appearance of As plaques and cerebral amyloid angiopathy (CAA) in this model. Animals were given ad libitum access to food and water. Blood glucose levels were monitored monthly. At eight months, animals were sacrificed, their brains extracted and portioned for biochemical analysis and immunohistochemistry. Statistical analysis was performed using Mann-Whitney U test. Results: Average non-fasted blood glucose in hGly-Tg mice was 398625mg/dL compared to 159612mg/dL in normoglycemic (nGly)- Tg mice. Total Formic acid extracted As40 and As42 levels were significantly elevated by 28.8% and 17.1% (p < 0.05), respectively in hGly-Tg mice compared to age/sex matched nGly-Tg mice. Aggregated As-specific ELISA revealed that hGly-Tg mice had 50.1% (p < 0.01) increases in As oligomers compared to nGly-Tg mice. Morphometric analysis revealed significantly increased CAA load in hGly-Tg mice by 61.9%(p < 0.05), associated with increased incidence and severity of perivascular haemorrhages 75.8% (p < 0.01) compared to nGly-Tg mice. There was a non-statistically significant increase in hippocampal (19.1%) and neocortical (16.9%) As plaque load in hGly-Tg mice. Conclusions: Chronic hyperglycemia is strongly associated with exacerbation of As pathology in ADTg mice. Primarily aggravatingCAA,which is allied with increased damage to blood vessel integrity. It also promotes formation of As oligomers which enhances As toxicity. Efforts to elucidate the mechanistic relationship between chronic hyperglycemia and exacerbation of As misfolding and accumulation is under way

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

A diagnostics capable of characterizing the runaway and superthermal electrons has been developing on the ISTTOK tokamak. In previous paper, a use of single-channel Cherenkov-type detector with titanium filter for runaway electron studies in ISTTOK was reported. To measure fast electron populations with different energies, a prototype of a four-channel detector with molybdenum filters was designed. Test-stand studies of filters with different thicknesses (1, 3, 7, 10, 20, 50, and 100 μm) have shown that they should allow the detection of electrons with energies higher than 69, 75, 87, 95, 120, 181, and 260 keV, respectively. First results of measurements with the four-channel detector revealed the possibility to measure reliably different fast electrons populations simultaneously.

While cerebrospinal fluid (CSF) biomarkers are of use in the prediction and diagnosis of Alzheimer's disease our understanding of the background effects of age and the ApoE genotype is limited. Seventy-eight community-based normal volunteers (mean age 60+/-10 years, range 36-86) were examined to determine the relationships between CSF measures of total tau (T-tau), hyperphosphorylated tau (P-tau 231), amyloid beta (Abeta42/Abeta40 ratio), and isoprostane (IP) with age and ApoE genotype. The results showed that age by epsilon4 genotype interactions were found for P-tau231 (beta=1.82; p<0.05) and IP (beta=1.6; p<0.05). T-tau CSF concentration increased with age. The increasing CSF concentrations of P-tau and IP in epsilon4 carriers suggest that early tauopathy and oxidative stress may be related to the increased risk for AD. The data also suggest that T-tau changes are more age dependent than Abeta changes. The evidence that P-tau231 and IP are the earliest markers for the neuronal damage related to AD awaits longitudinal study.

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