Faculty Bibliography

The endocytic pathway is important in amyloid precursor protein (APP) processing and beta-amyloid formation. Our studies have shown that endocytic pathway activation is a prominent and early feature of neurons in vulnerable regions of the brain in sporadic Alzheimer's disease. We report that endocytic pathway abnormalities are present not only in neurons, but in cerebral endothelia in Alzheimer's disease caused by certain APP mutations. The presence or absence of endocytic abnormalities distinguish subtypes of familial Alzheimer's disease linked to APP mutations from presenilin mutations, supporting the notion that different cellular pathways are involved in the altered processing of APP leading to increased beta-amyloid generation in certain of these different Alzheimer's disease subtypes.

Amyloid proteins and peptides comprise a diverse group of molecules that vary both in size and amino-acid sequence, yet assemble into amyloid fibrils that have a common core structure. Kinetic studies of amyloid fibrillogenesis have revealed that certain amyloid proteins form oligomeric intermediates prior to fibril formation. We have investigated fibril formation with a peptide corresponding to residues 195-213 of the human prion protein. Through a combination of kinetic and equilibrium studies, we have found that the fibrillogenesis of this peptide proceeds as an all-or-none reaction where oligomeric intermediates are not stably populated. This variation in whether oligomeric intermediates are stably populated during fibril formation indicates that amyloid proteins assemble into a common fibrillar structure; however, they do so through different pathways

Transgenic mouse models exist for the major neurodegenerative diseases, including Alzheimer's disease, tauopathy and amyotrophic lateral sclerosis. Although many of the mice do not completely replicate the human disease they are intended to model, they have provided insight into the mechanisms that underlie disease etiology. In the case of the Alzheimer's disease and amyotrophic lateral sclerosis models, the mice have also provided a therapeutic testing ground for the testing of agents that have been shown to have considerable clinical promise

Amyloid plaques are the principal features of Alzheimers disease (AD) pathology and are considered to be a major factor in the disease process. These fibrillar deposits are composed primarily of the 40-42 residue amyloid-beta (Abeta) peptide which is a proteolytic product of a larger membrane precursor protein. Electron microscopy and X-ray diffraction have revealed that the mature amyloid fibrils are assembled as a highly beta-sheet polymer that has a well-defined protofilament quaternary structure. This organization is observed for amyloid fibrils from a wide variety of disorders and appears to represent a structural superfamily. Amyloid plaques also contain a number of other components such as proteoglycans that contain highly sulfated glycosaminoglycan (GAG) chains. These amyloid-associated elements may contribute to the aggregation and/or stabilization of Abeta as insoluble fibrils. We have recently developed an aggressive model for Abeta plaque formation in transgenic mice that exhibits an 'early-onset' phenotype. Immunocytochemistry has demonstrated that even with this rapid progression, Abeta deposits within the neuropil and cerebrovascular system all co-localize with heparan sulfate proteoglycans (HSPG). These findings indicate a number of structural features that can be targeted as potential sites for the development of amyloid inhibitors. In addition, the use of small compounds that interfere with the proteoglycan-amyloid pathway may be effective therapeutic agents that can be assessed through the use of these transgenic models

A prominent feature of neurodegenerative diseases is a loss of specific neuronal populations. The pathophysiological mechanisms responsible are, however, poorly understood. Primary cultures of rodent embryonic neurons represent a useful experimental system for investigation of molecular pathways of neurodegeneration and mechanisms of cell death. Here, we report a technique utilizing triple-label immunocytochemistry with confocal immunofluorescence detection designed to simultaneously assess multiple parameters of cell injury in individual hippocampal neurons in primary culture. This method combines detection of DNA damage (TUNEL or Klenow assay) with double-label immunocytochemistry for the activated form of caspase-3 or, alternatively, caspase-cleaved actin (fractin), and microtubule-associated protein-2 (MAP-2) or beta-tubulin. The combined evaluation of the form of nuclear damage (karyorrhexis, pyknosis), the presence or absence of activated caspase-3, and the extent of the damage to cell cytoskeleton, allows for precise assessment of the extent of injury and the mode of cell death (apoptosis, oncosis) for individual neurons

We have created early-onset transgenic (Tg) models by exploiting the synergistic effects of familial Alzheimer's disease mutations on amyloid beta-peptide (Abeta) biogenesis. TgCRND8 mice encode a double mutant form of amyloid precursor protein 695 (KM670/671NL+V717F) under the control of the PrP gene promoter. Thioflavine S-positive Abeta amyloid deposits are present at 3 months, with dense-cored plaques and neuritic pathology evident from 5 months of age. TgCRND8 mice exhibit 3,200-4,600 pmol of Abeta42 per g brain at age 6 months, with an excess of Abeta42 over Abeta40. High level production of the pathogenic Abeta42 form of Abeta peptide was associated with an early impairment in TgCRND8 mice in acquisition and learning reversal in the reference memory version of the Morris water maze, present by 3 months of age. Notably, learning impairment in young mice was offset by immunization against Abeta42 (Janus, C., Pearson, J., McLaurin, J., Mathews, P. M., Jiang, Y., Schmidt, S. D., Chishti, M. A., Horne, P., Heslin, D., French, J., Mount, H. T. J., Nixon, R. A., Mercken, M., Bergeron, C., Fraser, P. E., St. George-Hyslop, P., and Westaway, D. (2000) Nature 408, 979-982). Amyloid deposition in TgCRND8 mice was enhanced by the expression of presenilin 1 transgenes including familial Alzheimer's disease mutations; for mice also expressing a M146L+L286V presenilin 1 transgene, amyloid deposits were apparent by 1 month of age. The Tg mice described here suggest a potential to investigate aspects of Alzheimer's disease pathogenesis, prophylaxis, and therapy within short time frames

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