Faculty Bibliography

Alzheimer's disease (AD) is associated with accumulation of beta-amyloid (Abeta). A major genetic risk factor for sporadic AD is inheritance of the apolipoprotein (apo) E4 allele. ApoE can act as a pathological chaperone of Abeta, promoting its conformational transformation from soluble Abeta into toxic aggregates. We determined if blocking the apoE/Abeta interaction reduces Abeta load in transgenic (Tg) AD mice. The binding site of apoE on Abeta corresponds to residues 12 to 28. To block binding, we synthesized a peptide containing these residues, but substituted valine at position 18 to proline (Abeta12-28P). This changed the peptide's properties, making it non-fibrillogenic and non-toxic. Abeta12-28P competitively blocks binding of full-length Abeta to apoE (IC(50) = 36.7 nmol). Furthermore, Abeta12-28P reduces Abeta fibrillogenesis in the presence of apoE, and Abeta/apoE toxicity in cell culture. Abeta12-28P is blood-brain barrier-permeable and in AD Tg mice inhibits Abeta deposition. Tg mice treated with Abeta12-28P for 1 month had a 63.3% reduction in Abeta load in the cortex (P = 0.0043) and a 59.5% (P = 0.0087) reduction in the hippocampus comparing to age-matched control Tg mice. Antibodies against Abeta were not detected in sera of treated mice; therefore the observed therapeutic effect of Abeta12-28P cannot be attributed to an antibody clearance response. Our experiments demonstrate that compounds blocking the interaction between Abeta and its pathological chaperones may be beneficial for treatment of beta-amyloid deposition in AD

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

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

A significant percentage of Alzheimer's disease (AD) patients exhibit concomitant vascular pathology. Epidemiological evidence suggest that vascular disease may not only add to global cognitive impairment but also exacerbate the course of AD pathology. The goal of this study was to analyze the impact of global ischemia on the cellular and amyloid-beta pathology in AD murine transgenic (Tg) models. Seven month old double Tg mice, expressing Swedish amyloid precursor protein (APP) and M146L presenilin 1 (PS1) mutations and single Tg mice (PS1 mutation alone) were subjected to 45 minutes bilateral common carotid artery occlusion or sham surgery. Behavioral testing performed two weeks after the surgery showed impaired learning and memory retention on Morris water maze and Hebb-Williams tests in both single PS1 and double PS1/APP Tg mice which underwent ischemia comparing to sham operated animals (p<0.05). Double Tg mice scored worse than single Tg mice. Animals were sacrificed two months after ischemia. The total brain volume was decreased by 6.5% and 5% and the ventricular volume was increased by 33.7% and 46.4% in single and double operated Tg mice, respectively comparing to sham animals. Unbiased stereological analysis demonstrated a 23% neuronal dropout in the CA1 sector of the cornu Ammonis after ischemia. Increased Abetaburden and plaque density was also observed in APP/PS1 animals which underwent ischemia comparing to sham operated ones. Overall, this data indicate that global ischemia exacerbate both neuronal and Abetarelated pathology in AD Tg animal models

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