Faculty Bibliography

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

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

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

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 are a unique category of illness, affecting both animals and humans, where the underlying pathogenesis is related to a conformational change of a normal self protein called cellular prion protein to a pathological and infectious conformer known as scrapie prion protein (PrP(Sc)). Currently, all prion diseases lack effective treatment and are universally fatal. Past experiences with bovine spongiform encephalopathy and variant Creutzfeldt-Jakob disease mainly in Europe, as well as the current epidemic of chronic wasting disease in North America, have highlighted the need to develop prophylactic and/or therapeutic approaches. In Alzheimer's disease that, like prion disease, is a conformational neurodegenerative disorder, both passive and active immunization has been shown to be highly effective in model animals at preventing disease and cognitive deficits, with emerging data from human trials suggesting that this approach is able to reduce amyloid-related pathology. However, any immunomodulatory approach aimed at a self-antigen has to finely balance an effective humoral immune response with potential autoimmune toxicity. The prion diseases most commonly acquired by infection typically have the alimentary tract as a portal of infectious agent entry. This makes mucosal immunization a potentially attractive method to produce a local immune response that partially or completely prevents prion entry across the gut barrier, while at the same time producing modulated systemic immunity that is unlikely to be associated with toxicity. Our results using an attenuated Salmonella vaccine strain expressing the prion protein showed that mucosal vaccination can protect against prion infection from a peripheral source, suggesting the feasibility of this approach. It is also possible to develop active and/or passive immunomodulatory approaches that more specifically target PrP(Sc) or target the shared pathological conformer found in numerous conformational disorders. Such approaches could have a significant impact on many of the common age-associated dementias

ABSTRACT: BACKGROUND: Alzheimer's Disease (AD) is the most common of the conformational neurodegenerative disorders characterized by the conversion of a normal biological protein into a beta-sheet-rich pathological isoform. In AD the normal soluble Abeta (sAbeta) forms oligomers and fibrils which assemble into neuritic plaques. The most toxic form of Abeta is thought to be oligomeric. A recent study reveals the cellular prion protein, PrPC, to be a receptor for Abeta oligomers. Abeta oligomers suppress LTP signal in murine hippocampal slices but activity remains when pretreated with the PrP monoclonal anti-PrP antibody, 6D11. We hypothesized that targeting of PrPC to prevent Abeta oligomer-related cognitive deficits is a potentially novel therapeutic approach. APP/PS1 transgenic mice aged 8 months were intraperitoneally (i.p.) injected with 1 mg 6D11 for 5 days/week for 2 weeks. Two wild-type control groups were given either the same 6D11 injections or vehicle solution. Additional groups of APP/PS1 transgenic mice were given either i.p. injections of vehicle solution or the same dose of mouse IgG over the same period. The mice were then subjected to cognitive behavioral testing using a radial arm maze, over a period of 10 days. At the conclusion of behavioral testing, animals were sacrificed and brain tissue was analyzed biochemically or immunohistochemically for the levels of amyloid plaques, PrPC, synaptophysin, Abeta40/42 and Abeta oligomers. RESULTS: Behavioral testing showed a marked decrease in errors in 6D11 treated APP/PS1 Tg mice compared with the non-6D11 treated Tg groups (p < 0.0001). 6D11 treated APP/PS1 Tg mice behaved the same as wild-type controls indicating a recovery in cognitive learning, even after this short term 6D11 treatment. Brain tissue analysis from both treated and vehicle treated APP/PS1 groups indicate no significant differences in amyloid plaque burden, Abeta40/42, PrPC or Abeta oligomer levels. 6D11 treated APP/PS1 Tg mice had significantly greater synaptophysin immunoreactivity in the dentate gyrus molecular layer of the hippocampus compared to vehicle treated APP/PS1 Tg mice (p < 0.05). CONCLUSIONS: Even short term treatment with monoclonal antibodies such as 6D11 or other compounds which block the binding of Abeta oligomers to PrPC can be used to treat cognitive deficits in aged AD transgenic mice

Many neurodegenerative diseases are characterized by the conformational change of normal self-proteins into amyloidogenic, pathological conformers, which share structural properties such as high beta-sheet content and resistance to degradation. The most common is Alzheimer's disease (AD) where the normal soluble amyloid beta (sAbeta) peptide is converted into highly toxic oligomeric Abeta and fibrillar Abeta that deposits as neuritic plaques and congophilic angiopathy. Currently, there is no highly effective treatment for AD, but immunotherapy is emerging as a potential disease modifying intervention. A major problem with most active and passive immunization approaches for AD is that both the normal sAbeta and pathogenic forms are equally targeted with the potential of autoimmune inflammation. In order to avoid this pitfall, we have developed a novel immunomodulatory method that specifically targets the pathological conformations, by immunizing with polymerized British amyloidosis (pABri) related peptide which has no sequence homology to Abeta or other human proteins. We show that the pABri peptide through conformational mimicry induces a humoral immune response not only to the toxic Abeta in APP/PS1 AD transgenic mice but also to paired helical filaments as shown on AD human tissue samples. Treated APP/PS1 mice had a cognitive benefit compared to controls (p<0.0001), associated with a reduction in the amyloid burden (p = 0.0001) and Abeta40/42 levels, as well as reduced Abeta oligomer levels. This type of immunomodulation has the potential to be a universal beta-sheet disrupter, which could be useful for the prevention or treatment of a wide range of neurodegenerative diseases

Alzheimer's disease (AD) is one of the categories of neurodegenerative diseases characterized by a conformational change of a normal protein into a pathological conformer with a high beta-sheet content that renders it resistant to degradation and neurotoxic. In AD, the normal soluble amyloid beta (sAbeta) peptide is converted into oligomeric/fibrillar Abeta. The oligomeric forms of Abeta are thought to be the most toxic, while fibrillar Abeta becomes deposited as amyloid plaques and congophilic angiopathy, which both serve as neuropathological markers of the disease. An additional important feature of AD is the accumulation of abnormally phosphorylated tau as soluble toxic oligomers and as neurofibrillary tangles. Many therapeutic interventions are under investigation to prevent and treat AD. The testing of these diverse approaches to ameliorate AD pathology has been made possible by the existence of numerous transgenic mouse models which each mirror specific aspects of AD pathology. None of the current murine models is a perfect match of the human disease. Perhaps the most exciting of the therapeutic approaches being developed is immunomodulation targeting the aggregating proteins, Abeta and tau. This type of AD therapy is currently being assessed in many transgenic mouse models, and promising findings have led to clinical trials. However, there is a discrepancy between results in murine models and ongoing clinical trials, which highlight the limitations of these models and also of our understanding of the underlying etiology and pathogenesis of AD. Because of these uncertainties, Tg models for AD are continuously being refined with the aim to better understand the disease and to enhance the predictive validity of potential treatments such as immunotherapies

Patients with HIV-1 immune-related thrombocytopenia have a unique antibody (Ab) against integrin GPIIIa49-66 capable of inducing oxidative platelet fragmentation via Ab activation of platelet nicotinamide adenine dinucleotide phosphate oxidase and 12-lipoxygenase releasing reactive oxygen species. Using a phage display single-chain antibody (scFv) library, we developed a novel human monoclonal scFv Ab against GPIIIa49-66 (named A11) capable of inducing fragmentation of activated platelets. In this study, we investigated the in vivo use of A11. We show that A11 does not induce significant thrombocytopenia or inhibit platelet function. A11 can prevent the cessation of carotid artery flow produced by induced artery injury and dissolve the induced thrombus 2 hours after cessation of blood flow. In addition, A11 can prevent, as well as ameliorate, murine middle cerebral artery stroke, without thrombocytopenia or brain hemorrhage. To further optimize the antithrombotic activity of A11, we produced a bifunctional A11-plasminogen first kringle agent (SLK), which homes to newly deposited fibrin strands within and surrounding the platelet thrombus, reducing effects on nonactivated circulating platelets. Indeed, SLK is able to completely reopen occluded carotid vessels 4 hours after cessation of blood flow, whereas A11 had no effect at 4 hours. Thus, a new antithrombotic agent was developed for platelet thrombus clearance