Expression of BRI2 mRNA and protein in normal human brain and familial British dementia: its relevance to the pathogenesis of disease.

INTRODUCTION: Two different disease-specific mutations in the BRI2 gene, situated on chromosome 13, have been identified as giving rise to familial British dementia (FBD) and familial Danish dementia (FDD). Each mutation results in extension of the open reading frame generating the disease-specific precursor proteins which are cleaved by furin-like proteolysis releasing the amyloidogenic C-terminal peptides ABri and ADan in FBD and FDD, respectively. MATERIAL AND METHODS: To understand the mechanism of the formation of amyloid lesions in FBD, we studied the origin of the precursor proteins and furin in the human brain. We used control brains, cases of sporadic Alzheimer's disease (AD), variant AD with cotton wool plaques and FBD to study BRI2 mRNA expression using in situ hybridization. Furin and BRI2 protein expression was investigated using Western blotting and immunohistochemistry. RESULTS: BRI2 mRNA and BRI2 protein are widely expressed primarily by neurones and glia and are deposited in the amyloid lesions in FBD. They were, however, not expressed by cerebrovascular components. Furin expression showed a similar pattern except that it was also present in cerebrovascular smooth muscle cells. CONCLUSIONS: These findings suggest that neurones and glia and are a major source of BRI2 protein and that in FBD, the mutated precursor protein may undergo furin cleavage within neurones to produce the amyloid peptide ABri. The failure to demonstrate BRI2 in blood vessels under the conditions tested suggests that vascular amyloid peptide production does not contribute significantly to cerebral amyloid angiopathy (CAA) in FBD and FDD, lending indirect support to the drainage hypothesis of CAA.

Molecular chaperons, amyloid and preamyloid lesions in the BRI2 gene-related dementias: a morphological study.

Molecular chaperons or amyloid-associated proteins (AAPs) are deposited in vascular and parenchymal amyloid lesions in Alzheimer's disease (AD) and other amyloidoses. AAPs, such as apolipoprotein E (ApoE) or apolipoprotein J (ApoJ) have been strongly implicated in the pathogenesis of AD in vitro and in vivo. Furthermore the possession of the ApoE in4 allele is a well-studied risk factor for AD. In view of the similarities between AD and both familial British dementia (FBD) and familial Danish dementia (FDD), we investigated the presence of AAPs in these two diseases to understand better their role in the general process of amyloidogenesis. Immunohistochemistry for ApoE, ApoJ, serum amyloid P (SAP), alpha-1-antichymotrypsin, cystatin C, heparan sulphate proteoglycans, such as agrin, perlecan, syndecans, glypican-1 and for heparan sulphate glycosaminoglycan (HS GAG) side chains was carried out together with immunohistochemical preparations specific to the amyloid subunits. Significant or extensive staining for ApoE, ApoJ, agrin, glypican-1 and HS GAG side chains was found in both amyloid (fibrillar) and preamyloid (nonfibrillar) deposits in FBD and FDD. The remaining AAPs, including SAP, were predominantly found in amyloid lesions. Only very weak staining was present in a small proportion of the amyloid lesions using perlecan immunohistochemistry. These findings suggest that the deposition patterns of AAPs in FBD and FDD are mostly similar to those in AD. The presence of AAPs in the preamyloid lesions supports the notion that chaperon molecules may play a role in the early steps of fibrillogenesis.

Genetic alterations of the BRI2 gene: familial British and Danish dementias.

Classic arguments sustaining the importance of amyloid in the pathogenesis of dementia are usually centered on amyloid beta (Abeta) and its role in neuronal loss characteristic of Alzheimer disease, the most common form of human cerebral amyloidosis. Two non-Abeta cerebral amyloidoses, familial British and Danish dementias, share many aspects of Alzheimer disease, including the presence of neurofibrillary tangles, parenchymal pre-amyloid and amyloid deposits, cerebral amyloid angiopathy, and a widespread inflammatory response. Both early-onset conditions are linked to specific mutations in the BRI2 gene, causing the generation of longer-than-normal protein products and the release of 2 de novo created peptides ABri and ADan, the main components of amyloid fibrils in these inherited dementias. Although the molecular mechanisms and signal transduction pathways elicited by the amyloid deposits and their relation to cognitive impairment remain to be clarified, new evidence indicates that, independent of the differences in their primary structures, Abeta, ABri, and ADan subunits are able to form morphologically compatible ion-channel-like structures and elicit single ion-channel currents in reconstituted lipid membranes. These findings reaffirm the notion that non-Abeta amyloidosis constitute suitable alternative models to study the role of amyloid deposition in the mechanism of neuronal cell death.

Chromosome 13 dementias.

The importance of cerebral amyloid deposition in the mechanism of neurodegeneration is still debatable. Classic arguments are usually centered on amyloid beta(Abeta) and its role in the neuronal loss characteristic of Alzheimer's disease, the most common form of human cerebral amyloidosis. Two non-Abeta cerebral amyloidoses, familial British and Danish dementias (FBD and FDD), share many aspects of Alzheimer's disease, including the presence of neurofibrillary tangles, parenchymal preamyloid and amyloid deposits, cerebral amyloid angiopathy and a variety of amyloid-associated proteins and inflammatory components. Both early-onset conditions are linked to specific mutations at or near the stop codon of the chromosome 13 gene BRI2 that cause generation of longer-than-normal protein products. Furin-like processing of these longer precursors releases two de novo-created peptides, ABri and ADan, which deposit as amyloid fibrils in FBD and FDD, respectively. Due to the similar pathology generated by completely unrelated amyloid subunits, FBD and FDD, collectively referred to as chromosome 13 dementias, constitute alternative models for studying the role of amyloid deposition in the mechanism of neuronal cell death.

Substitution at codon 22 reduces clearance of Alzheimer's amyloid-beta peptide from the cerebrospinal fluid and prevents its transport from the central nervous system into blood.

A point mutation of G to C at codon 693 of the amyloid-beta (Abeta) precursor protein gene results in Glu to Gln substitution at position 22 of the Abeta (AbetaQ22) associated with hereditary cerebrovascular amyloidosis with hemorrhage Dutch type. Factors that regulate AbetaQ22 levels in the central nervous system (CNS) are largely unknown. By using ventriculo-cisternal perfusion technique in guinea pigs, we demonstrated that clearance from the cerebrospinal fluid and transport from the CNS to blood of [(125)I]-AbetaQ22 (1 nM) were reduced by 36% and 52%, respectively, in comparison to the wild type Abeta(1-40) peptide. In contrast to significant uptake and transport of Abeta(1-40) across the brain capillaries and leptomeningeal vessels, AbetaQ22 was not taken up at these CNS vascular transport sites, which was associated with its 53% greater accumulation in the brain. The CNS clearance of Abeta(1-40) was half-saturated at 23.6 nM; AbetaQ22 had about 6.8-fold less affinity for the CNS efflux transporters and its elimination relied mainly on transport across the choroid plexus. Thus, the Dutch mutation impairs elimination of Abeta from brain by reducing its rapid transport across the blood-brain barrier and the vascular drainage pathways, which in turn may result in accumulation of the peptide around the blood vessels and in brain.

Melatonin reverses the profibrillogenic activity of apolipoprotein E4 on the Alzheimer amyloid Abeta peptide.

Inheritance of apoE4 is a strong risk factor for the development of late-onset sporadic Alzheimer's disease (AD). Several lines of evidence suggest that apoE4 binds to the Alzheimer Abeta protein and, under certain experimental conditions, promotes formation of beta-sheet structures and amyloid fibrils. Deposition of amyloid fibrils is a critical step in the development of AD. We report here that addition of melatonin to Abeta in the presence of apoE resulted in a potent isoform-specific inhibition of fibril formation, the extent of which was far greater than that of the inhibition produced by melatonin alone. This effect was structure-dependent and unrelated to the antioxidant properties of melatonin, since it could be reproduced neither with the structurally related indole N-acetyl-5-hydroxytryptamine nor with the antioxidants ascorbate, alpha-tocophenol, and PBN. The enhanced inhibitory effects of melatonin and apoE were lost when bovine serum albumin was substituted for apoE. In addition, Abeta in combination with apoE was highly neurotoxic (apoE4 > apoE3) to neuronal cells in culture, and this activity was also prevented by melatonin. These findings suggest that reductions in brain melatonin, which occur during aging, may contribute to a proamyloidogenic microenvironment in the aging brain.

Familial cerebral amyloid angiopathy related to stroke and dementia.

The term cerebral amyloid angiopathy (CAA) refers to the specific deposition of amyloid fibrils in the walls of leptomeningeal and cortical arteries, arterioles and, although less frequently in capillaries and veins. It is commonly associated with Alzheimers disease, Down's syndrome and normal aging, as well as with a variety of familial conditions related to stroke and/or dementia: hereditary cerebral hemorrhage with amyloidosis of Icelandic type (HCHWA-I), various inherited disorders linked to Abeta mutants (including the Dutch variant of HCHWA), and the recently described chromosome 13 familial dementia in British and Danish kindreds. This review focuses on four different types of hereditary CAA, emphasizing the notion that CAA is not only related to stroke but also to neurodegeneration and dementia of the Alzheimer's type.

Systemic amyloid deposits in familial British dementia.

Familial British dementia (FBD) is an early onset inherited disorder that, like familial Alzheimer's disease (FAD), is characterized by progressive dementia, amyloid deposition in the brain, and neurofibrillary degeneration of limbic neurons. The primary structure of the amyloid subunit (ABri) extracted from FBD brain tissues (Vidal, R., Frangione, B., Rostagno, A., Mead, S., Revesz, T., Plant, G., and Ghiso, J. (1999) Nature 399, 776-781) is entirely different and unrelated to any previously known amyloid protein. Patients with FBD have a single nucleotide substitution at codon 267 in the BRI2 gene, resulting in an arginine replacing the stop codon and a longer open reading frame of 277 amino acids instead of 266. The ABri peptide comprises the 34 C-terminal residues of the mutated precursor ABriPP-277 and is generated via furin-like proteolytic processing. Here we report that carriers of the Stop-to-Arg mutation have a soluble form of the amyloid peptide (sABri) in the circulation with an estimated concentration in the range of 20 ng/ml, several fold higher than that of soluble Abeta. In addition, ABri species identical to those identified in the brain were also found as fibrillar components of amyloid deposits predominantly in the blood vessels of several peripheral tissues, including pancreas and myocardium. We hypothesize that the high concentration of the soluble de novo created amyloidogenic peptide and/or the insufficient tissue clearance are the main causative factors for the formation of amyloid deposits outside the brain. Thus, FBD constitutes the first documented cerebral amyloidosis associated with neurodegeneration and dementia in which the amyloid deposition is also systemic.

Immunization with a nontoxic/nonfibrillar amyloid-beta homologous peptide reduces Alzheimer's disease-associated pathology in transgenic mice.

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.

Sequence, genomic structure and tissue expression of Human BRI3, a member of the BRI gene family.

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.

Distinct properties of wild-type and the amyloidogenic human cystatin C variant of hereditary cerebral hemorrhage with amyloidosis, Icelandic type.

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.

Regional distribution of amyloid-Bri deposition and its association with neurofibrillary degeneration in familial British dementia.

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.

A 7-kDa prion protein (PrP) fragment, an integral component of the PrP region required for infectivity, is the major amyloid protein in Gerstmann-Sträussler-Scheinker disease A117V.

Gerstmann-Sträussler-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-Sträussler-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.

Chromosome 13 dementia syndromes as models of neurodegeneration.

Two hereditary conditions, familial British dementia (FBD) and familial Danish dementia (FDD), are associated with amyloid deposition in the central nervous system and neurodegeneration. The two amyloid proteins, ABri and ADan, are degradation products of the same precursor molecule BriPP bearing different genetic defects, namely a Stop-to-Arg mutation in FBD and a ten-nucleotide duplication-insertion immediately before the stop codon in FDD. Both de novo created amyloid peptides have the same length (34 amino acids) and the same post-translational modification (pyroglutamate) at their N-terminus. Neurofibrillary tangles containing the classical paired helical filaments as well as neuritic components in many instances co-localize with the amyloid deposits. In both disorders, the pattern of hyperphosphorylated tau immunoreactivity is almost indistinguishable from that seen in Alzheimer's disease. These issues argue for the primary importance of the amyloid deposits in the mechanism(s) of neuronal cell loss. We propose FBD and FDD, the chromosome 13 dementia syndromes, as models to study the molecular basis of neurofibrillary degeneration, cell death and amyloid formation in the brain.

Conformation as therapeutic target in the prionoses and other neurodegenerative conditions.

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.

Clearance of Alzheimer's amyloid-ss(1-40) peptide from brain by LDL receptor-related protein-1 at the blood-brain barrier.

Elimination of amyloid-ss peptide (Ass) from the brain is poorly understood. After intracerebral microinjections in young mice, (125)I-Ass(1-40) was rapidly removed from the brain (t(1/2)

Apolipoprotein J (clusterin) and Alzheimer's disease.

Apolipoprotein J (clusterin) is a ubiquitous multifunctional glycoprotein capable of interacting with a broad spectrum of molecules. In pathological conditions, it is an amyloid associated protein, co-localizing with fibrillar deposits in systemic and localized amyloid disorders. In Alzheimer's disease, the most frequent form of amyloidosis in humans and the major cause of dementia in the elderly, apoJ is present in amyloid plaques and cerebrovascular deposits but is rarely seen in NFT-containing neurons. ApoJ expression is up-regulated in a wide variety of insults and may represent a defense response against local damage to neurons. Four different mechanisms of action could be postulated to explain the role of apoJ as a neuroprotectant during cellular stress: (1) function as an anti-apoptotic signal, (2) protection against oxidative stress, (3) inhibition of the membrane attack complex of complement proteins locally activated as a result of inflammation, and (4) binding to hydrophobic regions of partially unfolded, stressed proteins, and therefore avoiding aggregation in a chaperone-like manner. This review focuses on the association of apoJ in biological fluids with Alzheimer's soluble Abeta. This interaction prevents Abeta aggregation and fibrillization and modulates its blood-brain barrier transport at the cerebrovascular endothelium.

Senile dementia associated with amyloid beta protein angiopathy and tau perivascular pathology but not neuritic plaques in patients homozygous for the APOE-epsilon4 allele.

Amyloid beta protein deposition in cortical and leptomeningeal vessels, causing the most common type of cerebral amyloid angiopathy, is found in sporadic and familial Alzheimer's disease (AD) and is the principal feature in the hereditary cerebral hemorrhage with amyloidosis, Dutch type. The presence of the Apolipopriotein E (APOE)-epsilon4 allele has been implicated as a risk factor for AD and the development of cerebral amyloid angiopathy in AD. We report clinical, pathological and biochemical studies on two APOE-epsilon4 homozygous subjects, who had senile dementia and whose main neuropathological feature was a severe and diffuse amyloid angiopathy associated with perivascular tau neurofibrillary pathology. Amyloid beta protein and ApoE immunoreactivity were observed in leptomeningeal vessels as well as in medium-sized and small vessels and capillaries in the parenchyma of the neocortex, hippocampus, thalamus, cerebellum, midbrain, pons, and medulla. The predominant peptide form of amyloid beta protein was that terminating at residue Val40, as determined by immunohistochemistry, amino acid sequence and mass spectrometry analysis. A crown of tau-immunopositive cell processes was consistently present around blood vessels. DNA sequence analysis of the Amyloid Precursor Protein gene and Presenilin-1 (PS-1) gene revealed no mutations. In these APOE-epsilon4 homozygous patients, the pathological process differed from that typically seen in AD in that they showed a heavy burden of perivascular tau-immunopositive cell processes associated with severe amyloid beta protein angiopathy, neurofibrillary tangles, some cortical Lewy bodies and an absence of neuritic plaques. These cases emphasize the concept that tau deposits may be pathogenetically related to amyloid beta protein deposition.