Faculty Bibliography

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

The BRI3 gene is a member of the BRI gene family, made up of at least three different genes (BRI1-3). Previous studies established the cDNA sequence and structure of the human and mouse BRI1 and BRI2 genes and we recently reported that mutations in the BRI2 isoform, located on chromosome 13, are associated with dementia in humans. In the present work, we determine the complete cDNA sequence and genomic organization of the human BRI3 gene. BRI3 codes for a polypeptide of 267 amino acids, with a Mr of 30 KDa and a pI of 8.47. The amino acid sequence is 43.7% identical to the sequence of the human BRI2, and 38.3% identical to that of human BRI1, with the highest percentage of amino acid identity being concentrated on the C-terminal half of the molecules. In Northern blots, BRI3 cDNA hybridizes only one message of approximately 2.1 kilobases, which is predominantly present in the human brain. The BRI3 gene is localized on chromosome 2 and consists of six exons spanning more than 20 kb. Homology search of EST data banks retrieved a Caenorhabditis briggsae homolog of BRI, indicating that the BRI gene belongs to a strongly conserved gene family. These studies, aimed at characterizing the members of the BRI gene family, may provide valuable clues to the understanding of their normal function and how mutations in BRI2 can cause neurodegeneration and dementia similar to Alzheimer's disease

Gerstmann-Straussler-Scheinker disease (GSS) is a cerebral amyloidosis associated with mutations in the prion protein (PrP) gene (PRNP). The aim of this study was to characterize amyloid peptides purified from brain tissue of a patient with the A117V mutation who was Met/Val heterozygous at codon 129, Val(129) being in coupling phase with mutant Val117. The major peptide extracted from amyloid fibrils was a approximately 7-kDa PrP fragment. Sequence analysis and mass spectrometry showed that this fragment had ragged N and C termini, starting mainly at Gly88 and Gly90 and ending with Arg148, Glu152, or Asn153. Only Val was present at positions 117 and 129, indicating that the amyloid protein originated from mutant PrP molecules. In addition to the approximately 7-kDa peptides, the amyloid fraction contained N- and C-terminal PrP fragments corresponding to residues 23-41, 191-205, and 217-228. Fibrillogenesis in vitro with synthetic peptides corresponding to PrP fragments extracted from brain tissue showed that peptide PrP-(85-148) readily assembled into amyloid fibrils. Peptide PrP-(191-205) also formed fibrillary structures although with different morphology, whereas peptides PrP-(23-41) and PrP-(217-228) did not. These findings suggest that the processing of mutant PrP isoforms associated with Gerstmann-Straussler-Scheinker disease may occur extracellularly. It is conceivable that full-length PrP and/or large PrP peptides are deposited in the extracellular compartment, partially degraded by proteases and further digested by tissue endopeptidases, originating a approximately 7-kDa protease-resistant core that is similar in patients with different mutations. Furthermore, the present data suggest that C-terminal fragments of PrP may participate in amyloid formation

Familial British dementia (FBD), pathologically characterized by cerebral amyloid angiopathy (CAA), amyloid plaques, and neurofibrillary degeneration, is associated with a stop codon mutation in the BRI gene resulting in the production of an amyloidogenic fragment, amyloid-Bri (ABri). The aim of this study was to assess the distribution of ABri fibrillar and nonfibrillar lesions and their relationship to neurofibrillary pathology, astroglial and microglial response using immunohistochemistry, confocal microscopy, and immunoelectron microscopy in five cases of FBD. Abnormal tau was studied with immunoblotting. We present evidence that ABri is deposited throughout the central nervous system in blood vessels and parenchyma where both amyloid (fibrillar) and pre-amyloid (nonfibrillar) lesions are formed. Ultrastructurally amyloid lesions appear as bundles of fibrils recognized by an antibody raised against ABri, whereas Thioflavin S-negative diffuse deposits consist of amorphous electron-dense material with sparse, dispersed fibrils. In contrast to nonfibrillar lesions, fibrillar ABri is associated with a marked astrocytic and microglial response. Neurofibrillary tangles and neuropil threads occurring mainly in limbic structures, are found in areas affected by all types of ABri lesions whereas abnormal neurites are present around amyloid lesions. Immunoblotting for tau revealed a triplet electrophoretic migration pattern. Our observations confirm a close link between ABri deposition and neurodegeneration in FBD

Cerebral amyloid angiopathy (CAA) is the common term used to define the deposition of amyloid in the walls of medium- and small-size leptomeningeal and cortical arteries, arterioles and, less frequently, capillaries and veins. CAA is an important cause of cerebral hemorrhages although it may also lead to ischemic infarction and dementia. It is a feature commonly associated with normal aging, Alzheimer disease (AD), Down syndrome (DS), and Sporadic Cerebral Amyloid Angiopathy. Familial conditions in which amyloid is chiefly deposited as CAA include hereditary cerebral hemorrhage with amyloidosis of Icelandic type (HCHWA-I), familial CAA related to Abeta variants, including hereditary cerebral hemorrhage with amyloidosis of Dutch origin (HCHWA-D), the transthyretin-related meningocerebrovascular amyloidosis of Hungarian and Ohio kindreds, the gelsolin-related spinal and cerebral amyloid angiopathy, familial PrP-CAA, and the recently described chromosome 13 familial dementia in British and Danish kindreds. This review focuses on the various molecules and genetic variants that target the cerebral vessel walls producing clinical features related to stroke and/or dementia, and discusses the potential role of amyloid in the mechanism of neurodegeneration

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

Familial British dementia (FBD) and familial Danish dementia (FDD), are associated with amyloid deposition in the CNS and neurodegeneration. Amyloids ABri and ADan are C-terminal degradation products of the same precursor BriPP codified by the chromosome 13 BRI2 gene bearing different genetic defects, namely a Stop-to-Arg mutation in FBD and a ten-nucleotide insertion before the stop codon in FDD. Both de novo created amyloid peptides are 34 amino acids long, share 100% identity of the first 22 residues and pyroglutamate at their N-terminus. Neuritic components and NFTs containing PHF co-localize with the amyloid deposits in both disorders and the pattern of hyperphosphorylated tau immunoreactivity is almost indistinguishable from that seen in AD. To explore the role of inflammatory factors in these familial disorders, complement activation was assessed via immunohistochemistry, hemolytic assays and ELISA. Components and activation products (i.e. C1q, iC3b, C3d, C4d, C5b-9) were found to co-localize with plaques and vascular deposits in both diseases, suggesting in situ activation. ABri and ADan synthetic peptides activated the classical pathway in vitro and resulted in the formation of the activation products iC3b, C4d and SC5b-9 at levels comparable to those generated by Abeta42. The ability of ABri and ADan to trigger the complement cascade in vitro together with the presence of complement proteins and activation products as integral components of parenchymal and vascular amyloid deposits suggest that, as indicated in AD, the complement system may contribute to the mechanism of neurodegeneration leading to dementia