Faculty Bibliography

There is increasing recognition that numerous neurodegenerative conditions have the same underlying pathogenetic mechanism, namely a change in protein conformation, where the beta-sheet content is increased. In Alzheimer's disease, amyloid deposition in the form of neuritic plaques and congophilic angiopathy is driven by the conversion of normal soluble amyloid-beta peptide (sA beta) to A beta plaques; while in the prionoses the critical event is the conversion of normal prion protein, PrP(C), to the disease-associated form, PrP(Sc). This common theme in the pathogenesis of these disorders and the extracellular localization of the accumulating abnormal protein make them highly amenable to therapeutic approaches based on experimental manipulation of protein conformation and clearance. A number of different approaches under current development include drugs which affect the processing of the precursor proteins drugs the clearance of the amyloidogenic protein, and which inhibit or prevent the conformation change and immunological approaches. Particularly interesting are compounds termed 'beta-sheet breakers' that directly target the abnormal conformational change both for A beta- and PrP(Sc)-related deposits. In addition, immune system activation can serve as beta-sheet breakers and/or to increase the clearance of the disease-associated proteins. These conformation-based approaches appear to hold the best promise for therapies for this devastating group of disorders

There is growing realization that many neurodegenerative conditions have the same underlying pathogenetic mechanism: a change in protein conformation, where the beta-sheet content is increased. In Alzheimer's disease (AD), amyloid deposition in the form of neuritic plaques and congophilic angiopathy is driven by the conversion of normal soluble amyloid beta (sAbeta) to Abeta plaques, whereas in the prionoses the critical event is the conversion of normal prion protein, PrP(C), to PrP(Sc). This common theme in the pathogenesis of these disorders and the extracellular localization of the accumulating abnormal protein make them highly amenable to therapeutic approaches based on experimental manipulation of protein conformation and clearance. Different approaches under development include drugs that affect the processing of the precursor proteins, enhance clearance of the amyloidogenic protein, and inhibit or prevent the conformation change. Particularly interesting are recent studies of immune system activation, which appear to increase the clearance of the disease-associated protein. These immunologically based approaches are highly effective in animal models of these disorders, and in these model systems are associated with no obvious side effects. In transgenic mice with AD-related pathology, immunization has also been shown to prevent age-related cognitive impairment. However, the first clinical trial of this approach in AD patients was associated with unacceptable toxicity. These immune-based treatment approaches have great potential as rational therapies for this devastating group of disorders, but additional development is needed before they can be safely applied to humans

Smart molecular probes for both diagnostic and therapeutic purposes are expected to provide significant advances in clinical medicine and biomedical research. We describe such a probe that targets beta-amyloid plaques of Alzheimer's disease and is detectable by magnetic resonance imaging (MRI) because of contrast imparted by gadolinium labeling. Three properties essential for contrast enhancement of beta-amyloid plaques on MRI exist in this smart molecular probe, putrescine-gadolinium-amyloid-beta peptide: (1) transport across the blood-brain barrier following intravenous injection conferred by the polyamine moiety, (2) binding to plaques with molecular specificity by putrescine-amyloid-beta, and (3) magnetic resonance imaging contrast by gadolinium. MRI was performed on ex vivo tissue specimens at 7 T at a spatial resolution approximating plaque size (62.5 microm(3)), in order to prove the concept that the probe, when administered intravenously, can selectively enhance plaques. The plaque-to-background tissue contrast-to-noise ratio, which was precisely correlated with histologically stained plaques, was enhanced more than nine-fold in regions of cortex and hippocampus following intravenous administration of this probe in AD transgenic mice. Continuing engineering efforts to improve spatial resolution are underway in MRI, which may enable in vivo imaging at the resolution of individual plaques with this or similar contrast probes. This could enable early diagnosis and also provide a direct measure of the efficacy of anti-amyloid therapies currently being developed

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

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.

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