Faculty Bibliography

The prion protein (PrP) binds copper and under some conditions copper can facilitate its folding into a more protease resistant form. Hence, copper levels may influence the infectivity of the scrapie form of prion protein (PrPSc). To determine the feasibility of copper-targeted therapy for prion disease, we treated mice with a copper chelator, D-(-)-penicillamine (D-PEN), starting immediately following intraperitoneal scrapie inoculation. D-PEN delayed the onset of prion disease in the mice by about 11 days (p = 0.002), and reduced copper levels in brain by 29% (p < 0.01) and in blood by 22% (p = 0.03) compared with control animals. Levels of other metals were not significantly altered in the blood or brain. Modest correlation was observed between incubation period and levels of copper in brain (p = 0.08) or blood (p = 0.04), indicating that copper levels are only one of many factors that influence the rate of progression of prion disease. In vitro, copper dose-dependently enhanced the proteinase K resistance of the prion protein, and this effect was counteracted in a dose-dependent manner by co-incubation with D-PEN. Overall, these findings indicate that copper levels can influence the conformational state of PrP, thereby enhancing its infectivity, and this effect can be attenuated by chelator-based therapy

The presence of amyloid-beta (Abeta) plaques in the brain is a hallmark pathological feature of Alzheimer's disease (AD). Transgenic mice overexpressing mutant amyloid precursor protein (APP), or both mutant APP and presenilin-1 (APP/PS1), develop Abeta plaques similar to those in AD patients, and have been proposed as animal models in which to test experimental therapeutic approaches for the clearance of Abeta. However, at present there is no in vivo whole-brain imaging method to detect Abeta plaques in mice or men. A novel method is presented to detect Abeta plaques in the brains of transgenic mice by magnetic resonance microimaging (muMRI). This method uses Abeta1-40 peptide, known for its high binding affinity to Abeta, magnetically labeled with either gadolinium (Gd) or monocrystalline iron oxide nanoparticles (MION). Intraarterial injection of magnetically labeled Abeta1-40, with mannitol to transiently open the blood-brain barrier (BBB), enabled the detection of many Abeta plaques. Furthermore, the numerical density of Abeta plaques detected by muMRI and by immunohistochemistry showed excellent correlation. This approach provides an in vivo method to detect Abeta in AD transgenic mice, and suggests that diagnostic MRI methods to detect Abeta in AD patients may ultimately be feasible

Prion disease is characterized by a conformational change of the normal form of the prion protein (PrP(C)) to the scrapie-associated form (PrP(Sc)). Since the emergence of new variant Creutzfeldt-Jakob disease a potentially large human population is at risk for developing prion disease. Currently, no effective treatment or form of post-exposure prophylaxis is available for prion disease. We recently showed that active immunization with recombinant PrP prolongs the incubation period of scrapie. Here we show that anti-PrP antibodies following prion exposure are effective at increasing the incubation period of the infection. Stimulation of the immune system is an important therapeutic target for the prion diseases, as well as for other neurodegenerative illnesses characterized by abnormal protein conformation

The prion protein (PrP) is a copper binding protein; however, the role of copper in prion infection is unclear. Under some conditions copper facilitates refolding of denatured PrPSc into a protease resistant and infectious form. Hence copper may enhance the infectivity of the prion protein. To determine the feasibility of copper targeted therapy for prion disease, we treated mice (n=10 per group) with d-penicillamine (d-PEN; 100 mg/kg, i.p.), immediately following scrapie inoculation (139A strain, i.p.). Subsequent drug injections were daily, five days per week. d-PEN delayed the onset of prion disease in the mice (p=0.002). The effect was more pronounced at the 1000-fold dilution of agent (d-PEN=179 +- 3 days, VEH=165 +- 4, p=0.006), but a trend for a delay was observed at the 10-fold dilution (d-PEN=153 +- 2, VEH=146 +- 3, p=0.1). As expected, d-PEN reduced brain copper levels (p<0.01) by 26% (10-fold dil.; p=0.04) and 32% (1000-fold dil.; p=0.02), compared to control animals. Brain levels of iron and zinc were not reduced. To further support the notion that the therapeutic effect of d-PEN was mediated through its copper chelating properties, brain homogenates from terminally ill 139A infected mice were incubated with copper and d-PEN. Following a 72 h incubation, copper sulfate increased aggregation of the prion protein in a dose dependent manner, resulting in an enhanced resistance to proteinase K. This effect was counteracted by co-incubation with d-PEN. These findings support the proposed in vivo effect of d-PEN in delaying the onset of prion disease in these mice. Copper chelator-based therapy may benefit those incubating prion disease but this approach may be more effective at higher doses and/or in a multi-targeted combinational therapy

We have reported that an amyloid-beta derivative, K6Abeta1-30-NH2 reduces amyloid burden in mice to a similar extent as previously shown for Abeta1-42 (Am J Pathol 159:439-47,2001). This derivative may be a safer alternative to Alzheimer's vaccination with Abeta1-42 because it has a low beta-sheet content while maintaining the main antigenic sites of Abeta. To determine the in vivo effect of other derivatives with similar in vitro properties, we immunized Tg2576 mice with Abeta1-30-NH2, in which amino acids 18 and 19 were substituted with glutamate (Abeta1-30E18E19). In a parallel study, mice were immunized with K6Abeta1-30E18E19. Freund's adjuvant was used to allow a comparison with our findings with K6Abeta1-30-NH2. Antibody titers were detectable, but much lower than we had observed for K6Abeta1-30-NH2 or Abeta1-42, indicating that the central hydrophobic region of Abeta may have an epitope important for modulating humoral response. Cognitive performance was assessed in a radial arm maze before sacrifice at 19-21 months. Control Tg mice had more errors than their wild-type littermates (p<0.01), and the Abeta1-30E18E19-treated mice (p<0.05). Mice receiving K6Abeta1-30E18E19 also performed better than their Tg controls (p<0.05). Histologically, no difference was observed in brain amyloid plaque burden in 6E10 stained brain sections from the Abeta1-30E18E19-vaccinated mice, compared to vehicle treated mice. Furthermore, amyloid burden did not correlate with cognitive performance. Analysis of plaque burden in the K6Abeta1-30E18E19-immunized mice is underway, as well as measurements of brain levels of Abeta to determine if these values will provide a better correlation with cognitive performance. A robust antibody response and a diminished plaque burden may not be necessary for a therapeutic effect of Abeta derived vaccines

Amyloid deposition in Alzheimer's disease (AD) occurs many years before cognitive impairment. Brain imaging techniques targeting plaques will have an important diagnostic value and may help in identifying individuals in preclinical stages of AD. Magnetic resonance imaging (MRI) has a much higher resolution than positron enhanced tomography (PET) imaging and, therefore, is a more sensitive method to detect amyloid plaques. In our initial proof-of-concept studies (Magnetic Resonance in Medicine, in press), we utilized Abeta1-40 peptide, labeled with gadolinium or monocrystalline iron oxide nanoparticles (MION). When either of these ligands is injected in vivo systemically with mannitol to transiently open the blood-brain-barrier, we are able to image ex vivo the majority of Abeta plaques in Tg mice. Using Gd labeled Abeta1-40 and in vivo muMRI, we can also detect a substantial percentage of amyloid lesions. There is a high correlation between the numerical density of Abeta plaques detected by muMRI and by immunohistochemistry. Clinical use of Abeta1-40 is not feasible because it may add to the plaque burden. As a safer approach, we are using gadolinium labeled K6Abeta1-30, a non-toxic Abeta derivative with low propensity to form beta-sheet, while maintaining high affinity for Abeta. Our initial findings indicate that this compound has a similar effect as gadolinium labeled Abeta1-40 in allowing in vivo detection of amyloid plaques in Tg mice. We are currently exploring various ways to enhance the uptake of this compound into the brain. This approach may lead to a diagnostic MRI method to detect Abetaplaques in AD patients

Presenilin 1 (PS1) mutations have been identified in many pedigrees with early-onset familial Alzheimer's disease (FAD). PS1 mutants are known to influence gamma-secretase action and increase amyloid-beta (Abeta) 1-42 production, but there is also evidence suggesting direct involvement of PS1 in the neuronal pathology of AD. Transgenic (Tg) mice expressing the M146L PS1 mutation, associated with FAD symptom onset in the forties, demonstrate no difference in the total number of neurons (fractionator method) in the CA1 sector of the cornu Ammonis comparing with wild type (wt) animals at two months of age. At the age of 9 months and 22 months PS1 M146L Tg mice demonstrated 20% and 29% neuronal dropout comparing to age-matched controls, respectively (p<0.05). Between 2 months and 22 months old wt animals did not show any significant neuronal loss; however, 22 month old M146L PS1 mice showed a 41% neuronal decline compared to 2 month old controls. PS1 M146L Tg animals also exhibited impaired performance of both learning and retention on the Morris water maze test (p<0.05), but not on locomotor testing comparing to wt mice. We have also analyzed another line of Tg mice expressing a P117L PS1 mutation associated with an onset of disease as early as 23 years. These mice at the age of 6 months demonstrate a 17.9% reduction in the total number of CA1 neurons comparing to wt mice and a 26.5% reduction comparing to mice expressing the wt form of human PS1 (p<0.05). Overall, this data suggest that PS1 mutations are directly involved in neuronal pathology which is age-dependant. This process is unrelated to Abeta deposition since PS1 Tg mice do not develop amyloid plaques

Deposition of amyloid-beta (Abeta) in form of the senile plaques and in vessel walls is a hallmark of Alzheimer's disease (AD). Apolipoprotein E (apoE) is known to act as a pathological chaperone by increasing the beta-sheet content of Abeta, promoting its fibrillization, toxicity, and deposition in the brain. ApoE binds to residues 12-28 of Abeta. We report in vitro and in vivo data on the blocking of the apoE/Abeta interaction by a synthetic peptide homologues to residues 12-28 of Abeta. To eliminate any residual toxicity and fibrillogenic potential the peptide sequence was altered by replacing a valine in position 18 by a proline (Abeta12-28P). On ELISA Abeta12-28P demonstrates high affinity binding to apoE and in competitive binding experiments inhibits the binding of apoE to Abeta42. Abeta12-28P also reduces the toxicity of Abeta in cell culture, as well as blocking the enhanced fibril formation of Abeta in the presence of apoE4, measured by the Thioflavin-T assay. The in vivo effect of Abeta12-28P was assessed in double transgenic (Tg) APP/PS1 AD mice which received 1mg of Abeta12-28P or placebo three times a week for four weeks. There was an approximately five fold reduction of the total and fibrillar Abeta in treated mice comparing to control (p<0.05). Also, Abeta40 and Abeta42 levels in the brain demonstrated a 40-60% reduction of both species in the total Abeta fraction and in the soluble Abeta fraction in treated mice comparing to controls. No significant titer of anti-Abeta antibodies in treated animals was detected, indicating that the effect of Abeta12-28P on Abeta deposition observed in vivo is not immune mediated. Overall, compounds blocking the interaction between Abeta and its pathological chaperones such as apoE (or alpha1anti-chymotrypsin, perlecan etc.) can be considered as an alternative approach for the treatment of beta-amyloidosis in AD