Faculty Bibliography

Binding of EphB receptors to ephrinB ligands on the surface of adjacent cells initiates signaling cascades that regulate angiogenesis, axonal guidance and neuronal plasticity. These functions require processing of EphB receptors and removal of EphB-ephrinB complexes from the cell surface but the mechanisms involved are poorly understood. Here we show that the ectodomain of EphB2 receptor is released to extracellular space following cleavage after EphB2 residue 543. The remaining membrane-associated fragment is cleaved by the presenilin (PS)-dependent -secretase activity after EphB2 residue 569 releasing an intracellular peptide that contains the cytoplasmic domain of EphB2. This cleavage is inhibited by PS1 familial Alzheimer's disease mutations. Processing of EphB2 receptor depends on specific treatments: ephrinB ligand-induced processing requires endocytosis and the ectodomain cleavage is sensitive to peptide inhibitor ZVLL but insensitive to metalloproteinase inhibitor GM6001. The ligand-induced processing takes place in endosomes and involves the rapid degradation of the extracellular EphB2. EphrinB ligand stimulates ubiquitination of EphB2 receptor. Calcium influx- and NMDA-induced processing of EphB2 is inhibited by GM6001 and ADAM10 inhibitors but not by ZVLL. This processing requires no endocytosis and promotes rapid shedding of extracellular EphB2 indicating it takes place at the plasma membrane. Our data identify novel cleavages and modifications of EphB2 receptor and indicate that specific conditions determine the proteolytic systems and subcellular sites involved in the processing of this receptor

Prion diseases are characterized by conversion of the cellular prion protein (PrP) to a protease-resistant conformer, the srapie form of PrP (PrP(Sc)). Humoral immune responses to nondenatured forms of PrP(Sc) have never been fully characterized. We investigated whether production of antibodies to PrP(Sc) could occur in PrP null (Prnp(-/-)) mice and further, whether innate immune stimulation with the TLR9 agonist CpG oligodeoxynucleotide (ODN) 1826 could enhance this process. Whether such stimulation could raise anti-PrP(Sc) antibody levels in wild-type (Prnp(+/+)) mice was also investigated. Prnp(-/-) and Prnp(+/+) mice were immunized with nondenatured 139A scrapie-associated fibrils (SAF), with or without ODN 1826, and were tested for titers of PrP-specific antibodies. In Prnp(-/-) mice, inclusion of ODN 1826 in the immunization regime increased anti-PrP titers more than 13-fold after two immunizations and induced, among others, antibodies, which were only present in the immune repertoire of mice receiving ODN 1826, to an N-terminal epitope. mAb 6D11, derived from such a mouse, reacted with the N-terminal epitope QWNK in native and denatured forms of PrP(Sc) and recombinant PrP and exhibited a Kd in the 10(-11) M range. In Prnp(+/+) mice, ODN 1826 increased anti-PrP levels as much as 84% after a single immunization. Thus, ODN 1826 potentiates adaptive immune responses to PrP(Sc) in 139A SAF-immunized mice. These results represent the first characterization of humoral immune responses to nondenatured, infectious PrP(Sc) and suggest methods for optimizing the generation of mAb to PrP(Sc), many of which could be used for diagnosis and treatment of prion diseases

Amyloid beta (Abeta) immunoreactivity in neurons was examined in brains of 32 control subjects, 31 people with Down syndrome, and 36 patients with sporadic Alzheimer's disease to determine if intraneuronal Abeta immunoreactivity is an early manifestation of Alzheimer-type pathology leading to fibrillar plaque formation and/or neurofibrillary degeneration. The appearance of Abeta immunoreactivity in neurons in infants and stable neuron-type specific Abeta immunoreactivity in a majority of brain structures during late childhood, adulthood, and normal aging does not support this hypothesis. The absence or detection of only traces of reaction with antibodies against 4-13 aa and 8-17 aa of Abeta in neurons indicated that intraneuronal Abeta was mainly a product of alpha- and gamma-secretases (Abeta(17-40/42)). The presence of N-terminally truncated Abeta(17-40) and Abeta(17-42) in the control brains was confirmed by Western blotting and the identity of Abeta(17-40) was confirmed by mass spectrometry. The prevalence of products of alpha- and gamma -secretases in neurons and beta- and gamma-secretases in plaques argues against major contribution of Abeta-immunopositive material detected in neuronal soma to amyloid deposit in plaques. The strongest intraneuronal Abeta(17-42) immunoreactivity was observed in structures with low susceptibility to fibrillar Abeta deposition, neurofibrillary degeneration, and neuronal loss compared to areas more vulnerable to Alzheimer-type pathology. These observations indicate that the intraneuronal Abeta immunoreactivity detected in this study is not a predictor of brain amyloidosis or neurofibrillary degeneration. The constant level of Abeta immunoreactivity in structures free from neuronal pathology during essentially the entire life span suggests that intraneuronal amino-terminally truncated Abeta represents a product of normal neuronal metabolism

Prion diseases are a unique category of illness, affecting both animals and humans, where the underlying pathogenesis is related to a conformation change of the cellular form of a normal, self-protein called a prion protein (PrP(c) [C for cellular]) to a pathological and infectious conformation known as scrapie form (PrPsc [Sc for scrapie]). Currently, all prion diseases are without effective treatment and are universally fatal. The emergence of bovine spongiform encephalopathy and variant Creutzfeldt-Jakob disease has highlighted the need to develop possible therapies. In Alzheimer's disease (AD), which has similarities to prion diseases, both passive and active immunisation have been shown to be highly effective at preventing disease and cognitive deficits in model animals. In a human trial of active vaccination in AD, despite indications of cognitive benefits in patients with an adequate humoral response, 6% of patients developed significant complications related to excessive cell-mediated immunity. This experience highlights that immunotherapies designed to be directed against a self-antigen have to finely balance an effective humoral immune response with potential autoimmune toxicity. Many prion diseases have the gut as a portal of infectious agent entry. This makes mucosal immunisation a potentially very attractive method to partially or completely prevent prion entry across the gut barrier and to also produce a modulated immune response that is unlikely to be associated with any toxicity. The authors' recent results using an attenuated Salmonella vaccine strain expressing the prion protein show that mucosal vaccination can partially protect against prion infection from a peripheral source, suggesting the feasibility of this approach.

We have identified and synthesized a series of diaryl substituted pyrazoles as potent antagonists of the chemokine receptor subtype 2. Structure-activity relationship studies directed toward improving the potency led to the discovery of 23 (IC50 = 6 nM).

We have developed a sensitive in vitro assay for detecting disease associated prion aggregates by combining an aggregation specific enzyme-linked immunosorbent assay (AS-ELISA) with a Fluorescent Amplification Catalyzed by T7 RNA polymerase Technique (FACTT). The new assay, named AS-FACTT, is much more sensitive than AS-ELISA and could detect prion aggregates in the brain of mice as early as 7 days after an intra-peritoneal inoculation of PrP(Sc). However, AS-FACTT was still unable to detect prion aggregates in blood of infected mice. To further improve the detection limit of AS-FACTT, we added an additional prion amplification step (Am) and developed a third generation assay, termed Am-A-FACTT. Am-A-FACTT has 100% sensitivity and specificity in detecting disease-associated prion aggregates in blood of infected mice at late but still asymptomatic stages of disease. At a very early stage, Am-A-FACTT had a sensitivity of 50% and specificity of 100%. Most importantly, Am-A-FACTT also detects prion aggregates in blood of mule deer infected with a naturally occurring prion disease, chronic wasting disease. Application of this assay to cattle, sheep, and humans could safeguard food supplies and prevent human contagion

Alzheimer's disease (AD) is the most common age-associated neurodegenerative disease in the world. The major neuropathological features of AD are synaptic loss, neuronal loss, neurofibrillary tangles and the deposition of amyloid-beta (Abeta) as plaques and in cerebral blood vessels. Numerous Abeta targeting therapeutic approaches have been shown to prevent amyloid deposition and resulting in cognitive improvement in transgenic mouse models of AD. Some of these approaches are currently in early clinical trials. It remains to be seen if these approaches will be proven effective in patients. Future anti-AD therapies will likely be multi-modal and individually tailored depending on the patient's immune status, genetic background and their amyloid burden, as determined by imaging studies using Abeta specific labeling ligands. Pre-clinical data suggests that it will be much more feasible to prevent AD related pathology, then to clear existing pathology, making early diagnosis critically important.

The amyloid-beta (Abeta) cascade hypothesis of Alzheimer's disease (AD) maintains that accumulation of Abeta peptide constitutes a critical event in the early disease pathogenesis. The direct binding between Abeta and apolipoprotein E (apoE) is an important factor implicated in both Abeta clearance and its deposition in the brain's parenchyma and the walls of meningoencephalic vessels as cerebral amyloid angiopathy. With the aim of testing the effect of blocking the apoE/Abeta interaction in vivo as a potential novel therapeutic target for AD pharmacotherapy, we have developed Abeta12-28P, which is a blood-brain-barrier-permeable nontoxic, and nonfibrillogenic synthetic peptide homologous to the apoE binding site on the full-length Abeta. Abeta12-28P binds with high affinity to apoE, preventing its binding to Abeta, but has no direct effect on Abeta aggregation. Abeta12-28P shows a strong pharmacological effect in vivo. Its systemic administration resulted in a significant reduction of Abeta plaques and cerebral amyloid angiopathy burden and a reduction of the total brain level of Abeta in two AD transgenic mice models. The treatment did not affect the levels of soluble Abeta fraction or Abeta oligomers, indicating that inhibition of the apoE/Abeta interaction in vivo has a net effect of increasing Abeta clearance over deposition and at the same time does not create conditions favoring formation of toxic oligomers. Furthermore, behavioral studies demonstrated that treatment with Abeta12-28P prevents a memory deficit in transgenic animals. These findings provide evidence of another therapeutic approach for AD