Faculty Bibliography

Background: Amyloid plaques are a key pathological hallmark of Alzheimer's disease (AD). Their visualization is important for the diagnosis of AD, monitoring disease progression and evaluation of the efficacy of therapeutic interventions. We were the first group to use ultrasmall superparamagnetic iron oxide (USPIO) nanoparticles coupled to an amyloid targeting peptide to visualize amyloid plaques. USPIO's have been widely used in animal and human imaging, and have been found to be very safe. However, our approach, so far, has required intra-carotid or intra-femoral injection, with mannitol to break the blood brain barrier (BBB). In the current study we sought to develop and test non-toxic, bifunctional USPIO particles which could be introduced via the femoral vein, without mannitol. Methods: 13 to 18 month old APP/PS1 transgenic mice and age-matched wild-type (C57Bl/6J) control mice were used. The USPIO nanoparticles were linked to Ab1-42 (targeting to amyloid plaques) and polyethyleneglycol (PEG, to increase BBB permeability), using EDC/NHS coupling methods. The potential neurotoxicity of USPIO-PEG-Ab42 was assessed in N2a cells using a MTS Assay. MRI scans were performed on a 7T micro- MRI system consisting of a 7-Telsa 200-mm horizontal bore magnet. All mice were scanned 6-hrs after iv injection (0.2 mmol Fe/kg body weight) of the USPIO-PEG-Ab42. Ex vivo imaging of mouse brains was also performed. Serial coronal sections were subject to anti-Ab immunohistochemistry and Perl staining to identify the USPIO. Results: The USPIO particles were non-toxic. Figure 1A showsmMRI and matching histology in an APP/ PS1 Tg mouse and wild type mouse injected with USPIO-PEG-Ab. Numerous dark spots are evident in 1A. To the right of 1A is a higher magnification of an area of the MRI which is matched to double Perl and Ab stain in figure 1B. A coronal section matching to 1A is seen in 1C. In 1D the blue areas point to Perl stain positive dots corresponding to USPIO particles while the red arrow points to the amyloid plaque. 1E shows a wild-type mouse injected with USPIO-PEG-Ab. 1F documents the lack of plaques in the WT mouse. Conclusions: Our non-toxic, non-invasive m MRI method has great potential for the longitudinal assessment of amyloid burden

HIV-ITP patients have a unique Ab against platelet GPIIIa49-66 which induces oxidative platelet fragmentation in the absence of complement (Cell 106: 551, 2001; JCI 113: 973, 2004). Using a phage display single-chain antibody (scFv) library, we developed a novel human monoclonal scFv Ab against GPIIIa49-66 (named A11), which act similarly to the parental Ab (JBC 283: 3224, 2008). We then produced a bifunctional GPIIIa49-66 agent (named SLK), that targets newly deposited fibrin strands within and surrounding the platelet thrombus and has reduced effects on non-activated circulating platelets (Blood 116: 2336, 2010). In this study, we produced another bifunctional GPIIIa49-66 agent (named APAC), which homes to activated platelets. Like SLK, APAC destroys platelet aggregates ex vivo in an identical fashion with ~85% destruction of platelet aggregates at 2 hrs. Platelet aggregate dissolution with a combination of SLK and APAC was ~2 fold greater than either agent alone at 0.025 muM. Platelet-rich clot lysis experiments demonstrated the time required for 50% platelet-rich fibrin clot lysis (T_50%) by APAC (95+/-6.1 min) was significantly longer than that by APAC+SLK (65+/-7.6 min) at a final concentration of 0.025 muM (APAC+SLK vs APAC, p<0.01). In comparison with APAC alone, the T_50% of APAC+SLK was shortened by 1.56, 1.67 and 2.1 fold at the concentrations of 0.025, 0.5 and 0.1muM, respectively. Thus these low concentrations of a combination of both agents are likely to be more effective and less toxic when used therapeutically in vivo

Amyloid plaques are one of the pathological hallmarks of Alzheimer's disease (AD). The visualization of amyloid plaques in the brain is important to monitor AD progression and to evaluate the efficacy of therapeutic interventions. Our group has developed several contrast agents to detect amyloid plaques in vivo using magnetic resonance microimaging (muMRI) in AD transgenic mice, where we used intra-carotid mannitol to enhance blood-brain barrier (BBB) permeability. In the present study, we used ultrasmall superparamagnetic iron oxide (USPIO) nanoparticles, chemically coupled with Abeta1-42 peptide to detect amyloid deposition along with mannitol for in vivo muMRI by femoral intravenous injection. A 3D gradient multi-echo sequence was used for imaging with a 100mum isotropic resolution. The amyloid plaques detected by T2*-weighted muMRI were confirmed with matched histological sections. Furthermore, two different quantitative analyses were used. The region of interest-based quantitative measurement of T2* values showed contrast-injected APP/PS1 mice had significantly reduced T2* values compared to wild-type mice. In addition, the scans were examined with voxel-based morphometry (VBM) using statistical parametric mapping (SPM) for comparison of contrast-injected AD transgenic and wild-type mice. The regional differences seen in VBM comparing USPIO-Abeta1-42 injected APP/PS1 and wild-type mice correlated with the amyloid plaque distribution histologically, contrasting with no differences between the two groups of mice without contrast agent injection in regions of the brain with amyloid deposition. Our results demonstrated that both approaches were able to identify the differences between AD transgenic mice and wild-type mice, after injected with USPIO-Abeta1-42. The feasibility of using less invasive intravenous femoral injections for amyloid plaque detection in AD transgenic mice facilitates using this method for longitudinal studies in the pathogenesis of AD

Triplication of chromosome 21 in Down syndrome (DS) results in overexpression of the minibrain kinase/dual-specificity tyrosine phosphorylated and regulated kinase 1A gene (DYRK1A). DYRK1A phosphorylates cytoplasmic tau protein and appears in intraneuronal neurofibrillary tangles (NFTs). We have previously shown significantly more DYRK1A-positive NFTs in DS brains than in sporadic Alzheimer disease (AD) brains. This study demonstrates a gene dosage-proportional increase in the level of DYRK1A in DS in the cytoplasm and the cell nucleus, and enhanced cytoplasmic and nuclear immunoreactivity of DYRK1A in DS. The results suggest that overexpressed DYRK1A may alter both phosphorylation of tau and alternative splicing factor (ASF). Two-dimensional electrophoresis revealed modification of ASF phosphorylation in DS/AD and AD in comparison to controls. Altered phosphorylation of ASF by overexpressed nuclear DYRK1A may contribute to the alternative splicing of the tau gene and an increase by 2.68 x of the 3R/4R ratio in DS/AD, and a several-fold increase in the number of 3R tau-positive NFTs in DS/AD subjects compared with that in sporadic AD subjects. These data support the hypothesis that phosphorylation of ASF by overexpressed DYRK1A may contribute to alternative splicing of exon 10, increased expression of 3R tau, and early onset of neurofibrillary degeneration in DS

Background: ChronicWasting Disease (CWD) is epidemic in deer and elk populations in parts of North American and poses a significant threat to humans. Although there has been no human transmission documented so far, CWD has been shown by two groups to be transmissible to squirrel monkeys. The CWD agent has been found in multiple biological fluids and tissues (including blood, muscle and feces) in both symptomatic and asymptomatic animals. It is highly infectious in the wild among deer and elk populations, with the likely major route of infection being oral. Previously, we were the first to show that scrapie prion infection via an oral route can be prevented in model wild-type CD-1 mice by vaccination using attenuated Salmonella expressing copies of murine PrP. Methods: We have performed oral inoculations in white tail deer, using attenuated salmonella expressing deer PrP. The animals were inoculated orally three times, and three additional boosts were performed which included tonsil and rectal inoculations with the inoculum supplemented with polymerized recombinant deer PrP. Control animals were inoculated with an attenuated salmonella not carrying any foreign protein. Results: Both groups of animals produced IgA anti-salmonella in plasma, saliva and feces. However, the vaccinated group had a low titer of anti-PrP IgG and IgA in plasma, as well as anti-PrP IgA in the saliva. Deer immunoglobulins were precipitated from plasma, saliva and feces; semi-purified and the heavy and light chains recognized in blots. The purified IgG and IgA from the plasma of a vaccinated animal reacted in Western blots strongly against polymerized PrP and salmonella antigens and to a lesser extent to monomers and dimers of mouse, sheep and deer recombinant PrP. Control deer showed a reaction only against the salmonella antigens in similar blots. Both groups were challenged with homogeneized CWD infected brains included in food bait. Conclusions: Our results show for the first time that a specific antibody response against the self-antigen PrP can be elicited in the biological fluids of large bovide mammals. We are expecting quantitative results of infection progression or protection, before the end of the year

Background: Manipulation of the immune system is becoming increasingly important for the potential treatment of Alzheimer's disease (AD). However, problems in the initial human clinical trials have included toxicity from excessive cell mediated immunity, hemorrhages and limited cognitive benefits. Our research group postulated stimulation of innate immunity, via the Toll-like receptors (TLRs), as an alternative approach to overcome these side effects. Our prior work in prion disease, suggested TLR9 to be an attractive candidate for AD prevention and therapy. We sought to determine the effectiveness of TLR9 stimulation on both amyloid and tau related pathology. Various CpG DNA drugs that are TLR9 agonist are safe in humans and rodents. Methods: We utilized type B CpG oligodeoxynucleotides (ODNs) to stimulate innate immunity in 3xTg-AD mice, which develop both plaque and tangle pathology. The mice were divided into 2 study groups treated from 11 to 17 months and from 7 to 20 months of age. After the treatment, the mice were subjected to behavioral testing. In addition, we tested the effect of the TLR9 agonist CpG ODN on microglial As42 uptake and degradation in culture. Results: The transgenic mice were less active than their mu/d-littermates, but no significant differences between Tg groups were found in any of the locomotor parameters. CpG ODN treatment led to working memory improvement in both study groups of 3xTg- AD mice as indicated by radial arm maze testing. As immunostaining showed a 58% reduction in hippocampal amyloid burden (p=0.009) in the group treated from 11 to 17 months and 48% reduction (p=0.003) in the group treated from 7 to 20 months compared to vehicle-treated Tg animals. Biochemical analyses and evaluation of the impact of our approach on tau related pathology are ongoing. In vitro results indicate that TLR9 activation enhances microglial uptake and degradation of aggregated As42. Whether TLR9 agonist can boost peripheral macrophages to clear As is being further explored in our studies using RAW 264.7 and differentiated human THP-1 macrophages cells. Conclusions: Immunomodulation targeting TLR9 signaling represents a novel immunotherapeutic approach for AD. The current results provide essential information prior to any clinical use of CpG ODN

The accumulation of amyloid-beta (Abeta) peptides as toxic oligomers, amyloid plaques, and cerebral amyloid angiopathy (CAA) is critical in the pathogenesis of Alzheimer's disease (AD). The binding of Abeta peptides to apolipoprotein E (ApoE) plays an important role in modulation of amyloid deposition and clearance. We have shown that blocking the Abeta/ApoE interaction with Abeta12-28P, a nontoxic blood-brain-barrier permeable and non-fibrillogenic synthetic peptide, constitutes a novel therapeutic approach for AD by reducing Abeta parenchymal deposition. In the present study, we investigate this therapeutic effect on CAA in the transgenic (Tg) AD mice model (TgSwDI), which expresses Swedish (K670N/M671L), Dutch (E693Q)/Iowa (D694N) AbetaPP mutations. These mice develop abundant CAA beginning at the age of 6 months. Behavioral results show that A12-28P treated TgSwDI AD mice performed the same as wild-type mice, whereas vehicle treated TgSwDI were impaired in spatial memory. Furthermore, this treatment resulted in a significant reduction of total amyloid burden, especially the fibrillar vascular amyloid burden, which importantly was accompanied by a reduction in microhemorrhages and neuroinflammation. Measurement of Abeta levels in the brain homogenate revealed a significant decrease in both the total amount of Abeta and Abeta oligomer levels in A12-28P treated TgSwDI mice. These findings suggest that blocking the Abeta/ApoE interaction is a highly effective therapeutic approach for vascular amyloid deposition, in contrast to some other therapeutic approaches

Most antithrombotic approaches target prevention rather than the more clinically relevant issue of resolution of an existing thrombus. In this study, we describe a novel and effective therapeutic strategy for ex vivo clearance of pre-existing platelet thrombus by the combination of two bifunctional platelet GPIIIa49-66 ligands that target different parts of the arterial thrombus. We produced an additional GPIIIa49-66 agent (named APAC), which homes to activated platelets. Like our previously described SLK (which targets newly deposited fibrin strands surrounding the platelet thrombus), APAC destroys platelet aggregates ex vivo in an identical fashion with 85% destruction of platelet aggregates at 2 hours. The combined application of APAC and SLK demonstrated a approximately 2 fold greater platelet thrombus dissolution than either agent alone at a low concentration (0.025 microM). Platelet-rich clot lysis experiments demonstrated the time required for 50% platelet-rich fibrin clot lysis (T(50%)) by APAC (95+/-6.1 min) or SLK (145+/-7.1 min) was much longer than that by combined APAC+SLK (65+/-7.6 min) at the final concentration of 0.025 microM (APAC+SLK vs APAC, p<0.05; APAC+SLK vs SLK, p<0.01). Thus these low concentrations of a combination of both agents are likely to be more effective and less toxic when used therapeutically in vivo

Single-stranded oligoribonucleotides (ORNs) stimulate innate immune responses through TLR7 and TLR8. Specific linkages and chemical modifications incorporated into synthetic ORN can greatly enhance nuclease stability, selectivity, and potency. In the present study, we have synthesized 15 ORN containing different sequence compositions and chemical modifications and studied their TLR7- and TLR8-mediated immune response profiles in HEK293 cells expressing human TLR7 or TLR8, human PBMCs, mDCs and pDCs, non-human primate (NHP) PBMCs, and in vivo in mice and NHPs. Based on the results obtained, eight of the ORNs containing specific chemical modifications induced immune responses through both TLR7 and TLR8, including activation of NF-kappaB in TLR7- and TLR8-transfected cell lines; induction of IFN-alpha, IL-6, TNF-alpha, IL-12, and IP-10 in human PBMCs; IFN-alpha induction in human pDCs; CD80 upregulation in human pDCs and mDCs; IL-12 induction following acute administration in mice; IFN-alpha, IP-10, IL-6, and IL-12 induction in NHP PBMCs; and IFN-alpha, IP-10, and IL-6 induction following acute administration in NHPs. Seven of the ORNs show selectivity for TLR8-induced responses; they specifically activate only TLR8-transfected cell lines, induce cytokines other than IFN-alpha in human and NHP PBMCs, activate mDCs more than pDCs, and do not induce IL-12 acutely in mice, consistent with the lack of functional TLR8 in mice. The novel TLR8-selective ORNs also induce cytokines other than IFN-alpha acutely in NHPs. In conclusion, we have designed and synthesized novel ORNs with varying sequence compositions and chemical modifications, which selectively act as agonists of TLR8 or dual agonists of TLR7 and TLR8.

Prion diseases currently have no effective therapy. These illnesses affect both animal and human populations, and are characterized by the conformational change of a normal self protein PrP(C) (C for cellular) to a pathological and infectious conformer, PrP(Sc) (Sc for scrapie). We used a well characterized tissue culture model of prion infection, where mouse neuroblastoma cells (N2a) were infected with 22L PrP(Sc), to screen compounds for anti-prion activity. In a prior study we designed a library of styryl based, potential imaging compounds which were selected for high affinity binding to Alzheimer's disease beta-amyloid plaques and good blood-brain barrier permeability. In the current study we screened this library for activity in the N2a/22L tissue culture system. We also tested the anti-prion activity of two clinically used drugs, trimipramine and fluphenazine, in the N2a/22L system. These were selected based on their structural similarity to quinacrine, which was previously reported to have anti-prion activity. All the compounds were also screened for toxicity in tissue culture and their ability to disaggregate amyloid fibrils composed of PrP and beta-amyloid synthetic peptides in vitro. Two of the imaging agents, 23I and 59, were found to be both effective at inhibiting prion infection in N2a/22L tissue culture and to be non-toxic. These two compounds, as well as trimipramine and fluphenazine were evaluated in vivo using wild-type CD-1 mice infected peripherally with 139A PrP(Sc). All four agents significantly prolonged the asymptomatic incubation period of prion infection (p<0.0001 log-rank test), as well as significantly reducing the degree of spongiform change, astrocytosis and PrP(Sc) levels in the brains of treated mice. These four compounds can be considered, with further development, as candidates for prion therapy