Faculty Bibliography

PrP(Sc) is believed to serve as a template for the conversion of PrP(C) to the abnormal isoform. This process requires contact between the two proteins and implies that there may be critical contact sites that are important for conversion. We hypothesized that antibodies binding to either PrP(c)or PrP(Sc) would hinder or prevent the formation of the PrP(C)-PrP(Sc) complex and thus slow down or prevent the conversion process. Two systems were used to analyze the effect of different antibodies on PrP(Sc) formation: (i) neuroblastoma cells persistently infected with the 22L mouse-adapted scrapie stain, and (ii) protein misfolding cyclic amplification (PMCA), which uses PrP(Sc) as a template or seed, and a series of incubations and sonications, to convert PrP(C) to PrP(Sc). The two systems yielded similar results, in most cases, and demonstrate that PrP-specific monoclonal antibodies (Mabs) vary in their ability to inhibit the PrP(C)-PrP(Sc) conversion process. Based on the numerous and varied Mabs analyzed, the inhibitory effect does not appear to be epitope specific, related to PrP(C) conformation, or to cell membrane localization, but is influenced by the targeted PrP region (amino vs carboxy).

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

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: 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 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: 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

The amyloid cascade hypothesis remains a robust model of AD neurodegeneration. However, amyloid deposits contain proteins besides Abeta, such as apolipoprotein E (apoE). Inheritance of the apoE4 allele is the strongest genetic risk factor for late-onset AD. However, there is no consensus on how different apoE isotypes contribute to AD pathogenesis. It has been hypothesized that apoE and apoE4 in particular is an amyloid catalyst or "pathological chaperone". Alternatively it has been posited that apoE regulates Abeta clearance, with apoE4 been worse at this function compared to apoE3. These views seem fundamentally opposed. The former would indicate that removing apoE will reduce AD pathology, while the latter suggests increasing brain ApoE levels may be beneficial. Here we consider the scientific basis of these different models of apoE function and suggest that these seemingly opposing views can be reconciled. The optimal therapeutic target may be to inhibit the interaction of apoE with Abeta rather than altering apoE levels. Such an approach will not have detrimental effects on the many beneficial roles apoE plays in neurobiology. Furthermore, other Abeta binding proteins, including ACT and apo J can inhibit or promote Abeta oligomerization/polymerization depending on conditions and might be manipulated to effect AD treatment.

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