Faculty Bibliography

Abeta1-42 vaccination trials were recently terminated because of cerebral inflammation, which may be due to Abeta toxicity and/or autoimmunity. Abeta forms inflammatory/toxic fibrils, may seed fibril formation and crosses the blood brain barrier (BBB) in experimental animals. Because of attenuated immune response, the elderly may not clear injected Abeta1-42, which may then initiate and/or enhance amyloid angiopathy and plaque formation. Therefore, it is safer to use immunogenic Abeta derivatives, which are less likely to be toxic. Unlike Abeta1-42, K6Abeta1-30 is non-fibrillogenic and non-toxic in human cell culture but diminishes amyloid burden to a similar extent as reported for Abeta1-42. Additionally, ramified IL-1beta positive microglia, associated with the plaques, are absent in the immunized mice indicating reduced inflammation in these animals. We are currently comparing the therapeutic potential of these two compounds in alum adjuvants, which are approved for human use. Our behavioral findings in a year old Tg2576 mice show no difference between these groups and controls in various sensorimotor tasks, linear maze and water maze. However, in the radial arm maze, vaccinated Tg mice and their non-Tg littermates performed equally well and had fewer errors than Tg controls (p=0.008). These groups are being evaluated at a higher amyloid burden and subsequently their brain pathology will be assessed. Overall, the use of nontoxic Abeta derivatives and/or Abeta clearing compounds with very limited access into the CNS, such as IgM, may prove to have reduced side effects compared to Abeta and/or IgG-based immunization

The outbreak of new variant Creutzfeldt-Jakob disease has raised the specter of a potentially large population being at risk to develop this prionosis. None of the prionoses currently have an effective treatment. Recently, vaccination has shown therapeutic potential in mouse models of another neurodegenerative condition, namely Alzheimers disease. Here we report that immunization with recombinant mouse prion protein delays the onset of prion disease in mice (Am. J. Pathol., in press). Vaccination was performed both prior to and after peripheral exposure to the mouse-adapted scrapie strain 139A. A delay in disease onset was seen in both groups, but was more prolonged in animals immunized prior to exposure (p = 0.040-0.002). The increase in the incubation period closely correlated with the anti-prion antibody titer (p = 0.017-0.0001). Histological and Western blot evaluations of the brains of the treated-and control groups did not reveal any apparent differences in the degree of spongiform change or levels of scrapie prion. This was expected because the mice were killed when they scored positive for three consecutive weeks for behavioral signs of prion infection. Overall, the vaccination-mediated delay in prion disease onset is highly reproducible, correlates well with antibody titer and indicates that a similar approach may work in humans or other mammalian species at risk for prion disease

The prion diseases are a rapidly fatal group of neurodegenerative disorders, which currently have no effective therapy. Recently we have shown that active immunization with recombinant PrP protein increases the incubation period in mice exposed peripherally to the 139A strain of scrapie agent (Am.J.Pathol., in press). The antibody titers correlated with the increased incubation. We have extended these observations by using 6 different monoclonal anti-mouse PrP antibodies for passive immunization, with epitopes that span the murine PrP protein. Intraperitoneal antibody injections were performed weekly, starting immediately after and 1 month following peripheral exposure to scrapie strain 139A at two different dilutions. We found a statistically significant prolongation of the incubation period from scrapie exposure to the onset of clinical symptoms. These initial findings suggest that passive immunization can be used to prolong the incubation period among individuals with a known exposure to the prion agent

Transgenic mice with brain amyloid-beta (Abeta) plaques immunized with aggregated Abeta1-42 have reduced cerebral amyloid burden. However, the use of Abeta1-42 in humans may not be appropriate because it crosses the blood brain barrier, forms toxic fibrils, and can seed fibril formation. We report that immunization in transgenic APP mice (Tg2576) for 7 months with a soluble nonamyloidogenic, nontoxic Abeta homologous peptide reduced cortical and hippocampal brain amyloid burden by 89% (P = 0.0002) and 81% (P = 0.0001), respectively. Concurrently, brain levels of soluble Abeta1-42 were reduced by 57% (P = 0.0019). Ramified microglia expressing interleukin-1beta associated with the Abeta plaques were absent in the immunized mice indicating reduced inflammation in these animals. These promising findings suggest that immunization with nonamyloidogenic Abeta derivatives represents a potentially safer therapeutic approach to reduce amyloid burden in Alzheimer's disease, instead of using toxic Abeta fibrils

Variant human cystatin C (L68Q) is an amyloidogenic protein. It deposits in the cerebral vasculature of Icelandic patients with cerebral amyloid angiopathy, leading to stroke. Wild-type and variant cystatin C are cysteine proteinase inhibitors which form concentration dependent inactive dimers; however, variant cystatin C dimerizes at lower concentrations and has an increased susceptibility to a serine protease. We studied the effect of the L68Q amino acid substitution on cystatin C properties, utilizing full length cystatin C purified in mild conditions from media of cells stably transfected with either the wild-type or variant cystatin C genes. The variant cystatin C forms fibrils in vitro detectable by electron microscopy in conditions in which the wild-type protein forms amorphous aggregates. We also show by circular dichroism, steady-state fluorescence and Fourier-transformed infrared spectroscopy that the amino acid substitution modifies cystatin C structure by destabilizing alpha-helical structures and exposing the tryptophan residue to a more polar environment, yielding a more unfolded molecule. These spectral changes demonstrate that variant cystatin C has a three-dimensional structure different from that of the wild-type protein. The structural differences between variant and wild-type cystatin C account for the susceptibility of the variant protein to unfolding, proteolysis and fibrillogenesis

An important event in the pathogenesis of Alzheimer's disease (AD) is the deposition of the amyloid beta (Abeta)1-40 and 1-42 peptides in a fibrillar form, with Abeta42 typically having a greater propensity to undergo this conformational change. A major risk factor for late-onset AD is the inheritance of the apolipoprotein E (apoE) 4 allele [3,14,31]. We previously proposed that apoE may function as a 'pathological chaperone' in the pathogenesis of AD (i.e. modulate the structure of Abeta, promoting or stabilizing a beta-sheet conformation), prior to the discovery of this linkage [7,40,41,42]. Data from apoE knockout / AbetaPP^(V717F) mice, has shown that the presence of apoE is necessary for cerebral amyloid formation [1,2], consistent with our hypothesis. However, in betaPP^(V717F) mice expressing human apoE3 or E4 early Abeta deposition at 9 months is suppressed, but by 15 months both human apoE expressing mice had significant fibrillar Abeta deposits with the apoE4 expressing mice having a 10 fold greater amyloid burden [8,9]. This and other data has suggested that apoE, in addition to having a facilitating role in fibril formation, may also influence clearance of Abeta peptides. In order to address if apoE affects the clearance of Abeta peptides across the blood-brain barrier (BBB) and whether there are differences in the clearance of Abeta40 versus Abeta42, we performed stereotactic, intra-ventricular micro-injections of Abeta40, Abeta42 or control peptides in wild-type, apoE knock-out (KO) or human apoE3 or apoE4 expressing transgenic mice. We found that consistent with other studies [5], Abeta40 is rapidly cleared from the brain across the BBB; however, Abeta42 is cleared much less effectively. This clearance of exogenous Abeta peptides across the BBB does not appear to be affected by apoE expression. This data suggests that Abeta42 production may favor amyloid deposition due to a reduced clearance across the BBB, compared to Abeta40. In addition, our experiments support a role of apoE as a pathological chaperone, and do not suggest an isotype specific role of apoE in exogenous Abeta peptide clearance from the CSF across the BBB

Transgenic mice with brain amyloid-beta (Abeta) plaques immunized with aggregated Abeta1-42 have reduced cerebral amyloid burden. However, the use of Abeta1-42 in humans may not be appropriate because it crosses the blood brain barrier, forms toxic fibrils, and it can seed fibril formation. We report that immunization in 11-12 months old Tg2576 APP mice for 7 months, with K6Abeta1-30, a highly soluble, non-amyloidogenic and non-toxic Abeta homologous peptide, reduced cortical and hippocampal brain amyloid burden by 89% (p=0.0002) and 81% (p=0.0001), respectively. Concurrently, brain levels of soluble Abeta1-42 were reduced by 57% (p=0.0019). Ramified microglia expressing interleukin-1beta associated with the Abeta plaques were absent in the immunized mice, indicating reduced inflammation in these animals. We are currently performing a long-term study on the histological, biochemical and behavioral effects of K6Abeta1-30 vaccination, where the mice received their first immunization at 2-4 months of age. Our preliminary results are that mice immunized with K6Abeta1-30 or Abeta1-42 in aluminum adjuvants have comparable titers although the former is much more soluble. Overall, our present findings suggest that immunization with soluble Abeta derivatives represents a potentially safer therapeutic approach to reduce amyloid burden in Alzheimer's disease, instead of using toxic Abeta aggregates

Neurodegenerative conditions are increasing in prevalence as the average human life expectancy rises. Alzheimer's disease (AD) is the fourth commonest cause of death in the United States; the recent outbreak of new variant Creutzfeldt-Jakob disease (nvCJD) has raised the specter of a large population being at risk to develop this prionosis. The pathogenesis of many neurodegenerative diseases is now recognized to be associated with abnormalities of protein conformation. A common theme in these disorders is the conversion of a soluble normal precursor protein into an insoluble, aggregated, ?-sheet rich form that is toxic. In AD, a critical event is the conversion of the normal, soluble A? (sA?) peptide into fibrillar A?, within neuritic plaques and congophilic angiopathy (1). Similarly, in the prionoses, the central event is the conversion of the normal prion protein, PrPC, to PrPSc (2). An increased ?-sheet content characterizes both A? and PrPSc.