Faculty Bibliography

Background: Due to well-known markedly increased susceptibility contrast at ultra-high-field MR, this work is using 7T MR susceptibiltyweighted imaging (SWI) to better identify the histopathologic correlate of amyloid plaques containing iron and otherwise invisible subhippocampal structures of human post-mortem brain in patients with Alzheimer's disease. Methods: Post-mortem brain specimens of the frontal lobe and hippocampus were obtained from 8 patients (mean age 71.266.2 years) with clinically diagnosed AD and 6 age-matched healthy controls (72.4 66.6 years) without AD. Coronal 1w3 cm thick brain slices were preserved and fixed in 2% agar or formalin for this study. Imaging was performed on a 7.0T MR. A 24- element phased array head coil was used. High resolution 3D SWI was obtained with isotropic voxel size 150w320I=m. For imaging optimization to better visualize amyloid plaques, we varied TR, TE, bandwidth and flip angle from 30-100ms, 12-36ms, 60-140Hz/pixel and 10-40-; respectively. Results: Compared to controls, 7T SWI revealed a largely increased number of hypointense foci inADbrain samples along the cortical mantle of the frontal and entorhinal cortex due to enhanced susceptibility effects and superb signal. Figure 1 shows amyloid plaques identified with histologic slice (A) and in post-processed SWI image of the frontal cortex of an AD patient (B) as compared to a healthy control (C). The average phase value in the cortex region of AD data (2296 6 72) is significantly higher than that of control data (2032 6 64), which indicates higher iron amount in AD samples. In addition, 7T SWI also provides high resolution images for subregional hippocampal structures including CA1 CA2 CA3 subiculum, and dentate gyrus with significant atrophy of these structures in AD patients. Conclusions: Our findings suggest that SWI with optimization at ultra-high-field strength MR (i.e. 7T) has exhibited the capability to detect diminutive susceptibility contrast associated with iron deposition and otherwise invisible fine hippocampal structures with near histopathologic resolution. Therefore, SWI has great potential for direct detection and quantification of amyloid plaques in live human brain, and may become feasible in vivo in the near future

Background: Alzheimer's Disease (AD) is the most common of the conformational neurodegenerative disorders (NDD), while prion diseases are the most transmissible NDD. No highly effective treatment is currently available for any NDD. Previously, we have shown that mutated Ab peptides soluble and devoid of T-cell epitopes, produced a response which correlates with amelioration of AD pathology in animal models. Our lab has demonstrated that active immunization with antigens mimicking abnormal conformations such as polymerized ABri (pABri), could elicit antibody responses to oligomerized Ab and conformational aberrant hyperphosphorylated tau (ptau).We have now tested a combined vaccination using pBri and polymerized mutated Ab (pAb) in 3xTg mice and human PrP-Tg animals. Methods: We produced pBri and pAb1-30K18K19, which by electron microscopy formed oligomer-like structures. The pBri alone was used as an immunogen in HuPrPTg animals and in conjunction with pAb1-30KK in AD 3xTg mice using alum as an adjuvant. At the age of 15-16 months the AD mice were subjected to behavioral testing followed by histological and biochemical analysis. The HuPrPTg were assessed for the presence of antibodies against PrPRes. Results: 3xTg mice immunized animals showed no difference in locomotor activity compared to controls but a significant behavioral rescue by radial arm maze testing. The animals produced significant titers of IgM and IgG against Ab42 oligomers. Histological analysis showed a significant reduction of both amyloid and tau pathology. Biochemically, Ab42 and soluble ptau were significantly reduced. In HuPrPTg animals a systemic IgM and IgG response against PrPRes was observed, similar to the systemic response obtained in our prior successful mucosal PrP vaccinations. Conclusions: pBri conformational vaccination can be used to elicit antibodies that recognize multiple pathological proteins, including PrPRes, oligomer Ab and ptau. In 3x Tg mice with both Ab and tau pathology which were vaccinated with the combined pABri+pAb1-30KK, each of these pathologies were reduced histologically and biochemically, in association with a behavioral rescue. Such a combined approach targeting both pathological Ab and tau is more likely to be efficacious in AD patients

Background: Prionoses are transmissible, neurodegenerative disorders. Bovine spongioform encephalopathy (BSE) has crossed the species barrier to infect humans. Chronic wasting disease (CWD) infects large numbers of deer and elk, with the potential to infect humans. Currently no prionoses has an effective treatment. Previously, we have demonstrated we could prevent transmission of prions in a proportion of susceptible mice with a mucosal vaccine. Currently, we have attempted to produce an immune response in animals naturally susceptible to CWD, white tail deer, which could inhibit transmission. Methods: White tail deer were orally inoculated with attenuated Salmonella carrying deer or mouse PrP. Once established the mucosal response, the animals were boosted orally and locally in the tonsils and rectum with polymerized recombinant PrP. Blood, saliva and feces were collected regularly to assess IgA, IgM and IgG titers to PrP. The vaccinated and control animals were then challenged orally with CWD infected brains. Tonsil and rectal biopsies plus blood, saliva, feces and urine were collected every three months post-infection until the 9 month; and every 45 days thereafter. Results: Two vaccinated animals produced low antibody titers, two intermediate titers and one high titers of IgA and IgG anti-PrP. Both groups produced high titers of IgA and IgG against Salmonella. Six months post-infection 5 out of 6 controls and 3 out of 5 vaccinated showed histologically prion structures in the tonsils. Ayear post-infection one of the vaccinated animals remained prion free, with all controls being infected. The negative animal has the highest titers of IgA in saliva and IgG systemic against PrP. Immunoglobulins purified from saliva, feces and serum of this vaccinated deer reacts to PrPRes. Conclusions: Oral immunization can be used to overcome tolerance to self-PrP protein and produce a mucosal IgA and systemic IgG response to normal and conformational modified PrP in large mammals. High antibody titers might be enough to prevent transmission or to retard progression of PrP infection. This approach may lead to an effective anti-prion vaccine

Background: Alzheimer's disease (AD) is the most common cause of dementia and has a major societal and economic impact. Immunomodulation has shown great promise as an AD therapy, even though the initial clinical trial was associated with severe adverse effects in a minority of patients. Our research group postulated stimulation of the innate immune system, via the Toll-like receptor 9 (TLR9), as a possible alternative method for ameliorating AD pathology, without associated toxicity. A significant concern with immunotherapy is clearance of vascular amyloid and associated microhemorrhages. This is an important issue, since cerebral amyloid angiopathy (CAA) is a common feature in AD and cognitively normal elderly individuals. We tested the efficacy of TLR9 signaling stimulation for reducing parenchymal and vascular amyloid, as well as tau related pathology. Methods: We utilized TLR9 agonist type B CpG oligonucleotides (ODNs) to stimulate innate immunity in 3xTg-AD mice, which develop both plaque and tangle pathology, and in Tg-SwDI mice with extensive CAA. Animals were divided into 2 study groups treated prior to or after the onset of AD pathology. Results: After treatment the mice were behaviorally tested. No statistical differences were observed between the groups in any of the locomotor parameters measured. CpG ODN treatment improved working memory in 3xTg-AD mice as indicated by radial arm maze testing. TLR9 stimulation was also effective at improving short-term memory in Tg-SwDI mice as evidenced by novel-object recognition testing. In 3xTg-AD mice TLR9 signaling reduced both amyloid deposits and hyperphosphorylated tau pathology. Semiquantitative analysis of hippocampal CA1 neurons revealed reduction in AT8 and PHF1 immunoreactivity in CpG ODN-treated 3xTg-AD mice. The reduction of plaque and tangle pathology was paralleled by an overall reduction in the numbers of activated microglia. There were no group differences in the levels of CNS astrocytosis; hence there was no evidence of encephalitis in the brains of treated mice. Further histological, biochemical analyses and characterization of immune responses are ongoing. Conclusions: Overall, stimulation of the TLR9 and thus innate immunity with CpG ODN (currently used in clinical trials for a variety of other diseases) represents a novel immunotherapeutic approach for AD

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