Faculty Bibliography

Mutations within the amyloid-beta (Abeta) sequence, especially those clustered at residues 21-23, which are linked to early onset familial Alzheimer's disease (AD), are primarily associated with cerebral amyloid angiopathy (CAA). The basis for this predominant vascular amyloid burden and the differential clinical phenotypes of cerebral hemorrhage/stroke in some patients and dementia in others remain unknown. The AbetaD23N Iowa mutation is associated with progressive AD-like dementia, often without clinically manifested intracerebral hemorrhage. Neuropathologically, the disease is characterized by predominant preamyloid deposits, severe CAA, and abundant neurofibrillary tangles in the presence of remarkably few mature plaques. Biochemical analyses using a combination of immunoprecipitation, mass spectrometry, amino acid sequence, and Western blot analysis performed after sequential tissue extractions to separately isolate soluble components, preamyloid, and fibrillar amyloid species indicated that the Iowa deposits are complex mixtures of mutated and nonmutated Abeta molecules. These molecules exhibited various degrees of solubility, were highly heterogeneous at both the N- and C-termini, and showed partial aspartate isomerization at positions 1, 7, and 23. This collection of Abeta species-the Iowa brain Abeta peptidome-contained clear imprints of amyloid clearance mechanisms yet highlighted the unique neuropathological features shared by a non-Abeta cerebral amyloidosis, familial Danish dementia, in which neurofibrillary tangles coexist with extensive pre-amyloid deposition in the virtual absence of fibrillar lesions. These data therefore challenge the importance of neuritic plaques as the sole contributors for the development of dementia

Cerebral amyloid diseases are part of a complex group of chronic and progressive entities bracketed together under the common denomination of protein folding disorders and characterized by the intra- and extracellular accumulation of fibrillar aggregates. Of the more than 25 unrelated proteins known to produce amyloidosis in humans only about a third of them are associated with cerebral deposits translating in cognitive deficits, dementia, stroke, cerebellar and extrapyramidal signs, or a combination thereof. The familial forms reviewed herein, although infrequent, provide unique paradigms to examine the role of amyloid in the mechanism of disease pathogenesis and to dissect the link between vascular and parenchymal amyloid deposition and their differential contribution to neurodegeneration

Familial British and Familial Danish Dementia (FBD and FDD) are two dominantly inherited neurodegenerative diseases that present striking similarities with Alzheimer's disease. The genetic defects underlying those dementias are mutations in the gene that encodes for BRI2 protein. Cleavage of mutated BRI2 by furin releases the peptides ABri or ADan, which accumulate in the brains of patients. BRI2 normal function is yet unknown. To unwind aspects of its cellular role, we investigated the possibility that BRI2 forms dimers, based on structural elements of the protein, the GXXXG motif within its transmembrane domain and the odd number of cysteine residues. We found that BRI2 dimerizes in cells and that dimers are held via non-covalent interactions and via disulfide bridges between the cysteines at position 89. Additionally, we showed that BRI2 dimers are formed in the ER and appear at the cell surface. Finally, BRI2 dimers were found to exist in mouse brain. Revealing the physiological properties of BRI2 is critical in the elucidation of the deviations that lead to neurodegeneration

Cerebral amyloid angiopathy (CAA) is an age-associated condition and a common finding in Alzheimer's disease in which amyloid-beta (Abeta) vascular deposits are featured in >80% of the cases. Familial Abeta variants bearing substitutions at positions 21-23 are primarily associated with CAA, although they manifest with strikingly different clinical phenotypes: cerebral hemorrhage or dementia. The recently reported Piedmont L34V Abeta mutant, located outside the hot spot 21-23, shows a similar hemorrhagic phenotype, albeit less aggressive than the widely studied Dutch E22Q variant. We monitored the apoptotic events occurring after stimulation of human brain microvascular endothelial and smooth muscle cells with nonfibrillar structures of both variants and wild-type Abeta40. Induction of analogous caspase-mediated mitochondrial pathways was elicited by all peptides, although within different time frames and intensity. Activated pathways were susceptible to pharmacological modulation either through direct inhibition of mitochondrial cytochrome c release or by the action of pan- and pathway-specific caspase inhibitors, giving a clear indication of the independent or synergistic engagement of both extrinsic and intrinsic mechanisms. Structural analyses of the Abeta peptides showed that apoptosis preceded fibril formation, correlating with the presence of oligomers and/or protofibrils. The data support the notion that rare genetic mutations constitute unique paradigms to understand the molecular pathogenesis of CAA

Extracellular deposits of amyloid fibrils in the form of parenchymal plaques and cerebrovascular lesions, as well as intracellular accumulation of paired-helical filaments in the form of neurofibrillary tangles (NFT) in selected neuronal populations are the main neuropathologic hallmarks of Alzheimer's disease. Amyloid fibrils composed of polymeric structures of the amyloid-beta (Abeta) concentrate at the center of senile plaques and accumulate in the walls of cerebral blood vessels, exhibiting extensive Congo red/thioflavin S staining. Intraneuronal NFT are composed of building blocks of aberrantly hyperphosphorylated species of the microtubule-associated protein tau, which accumulate in the perinuclear cytoplasm of vulnerable neurons in the form of paired helical filaments (PHF). This unit presents a variety of protocols for the isolation, biochemical analysis, and characterization of amyloid fibrils and neurofibrillary tangles

In cerebral amyloid angiopathy (CAA), amyloid fibrils deposit in walls of arteries, arterioles and less frequently in veins and capillaries of the central nervous system, often resulting in secondary degenerative vascular changes. Although the amyloid-beta peptide is by far the commonest amyloid subunit implicated in sporadic and rarely in hereditary forms of CAA, a number of other proteins may also be involved in rare familial diseases in which CAA is also a characteristic morphological feature. These latter proteins include the ABri and ADan subunits in familial British dementia and familial Danish dementia, respectively, which are also known under the umbrella term BRI2 gene-related dementias, variant cystatin C in hereditary cerebral haemorrhage with amyloidosis of Icelandic-type, variant transthyretins in meningo-vascular amyloidosis, disease-associated prion protein (PrP(Sc)) in hereditary prion disease with premature stop codon mutations and mutated gelsolin (AGel) in familial amyloidosis of Finnish type. In this review, the characteristic morphological features of the different CAAs is described and the implication of the biochemical, genetic and transgenic animal data for the pathogenesis of CAA is discussed

The vasculotropic E22Q mutant of the amyloid-beta (Abeta) peptide is associated with hereditary cerebral hemorrhage with amyloidosis Dutch type. The cellular mechanism(s) of toxicity and nature of the AbetaE22Q toxic assemblies are not completely understood. Comparative assessment of structural parameters and cell death mechanisms elicited in primary human cerebral endothelial cells by AbetaE22Q and wild-type Abeta revealed that only AbetaE22Q triggered the Bax mitochondrial pathway of apoptosis. AbetaE22Q neither matched the fast oligomerization kinetics of Abeta42 nor reached its predominant beta-sheet structure, achieving a modest degree of oligomerization with a secondary structure that remained a mixture of beta and random conformations. The endogenous molecule tauroursodeoxycholic acid (TUDCA) was a strong modulator of AbetaE22Q-triggered apoptosis but did not significantly change the secondary structures and fibrillogenic propensities of Abeta peptides. These data dissociate the pro-apoptotic properties of Abeta peptides from their distinct mechanisms of aggregation/fibrillization in vitro, providing new perspectives for modulation of amyloid toxicity

Single point mutations of the amyloid precursor protein generate Abeta variants bearing amino acid substitutions at positions 21-23. These mutants are associated with distinct hereditary phenotypes of cerebral amyloid angiopathy, manifesting varying degrees of tropism for brain vessels, and impaired microvessel remodeling and angiogenesis. We examined the differential effects of E22Q (Dutch), and E22G (Arctic) variants in comparison to WT Abeta on brain endothelial cell proliferation, angiogenic phenotype expression triggered by fibroblast growth factor (FGF-2), pseudo-capillary sprouting, and induction of apoptosis. E22Q exhibited a potent anti-angiogenic profile in contrast to E22G, which had a much weaker effect. Investigations on the FGF-2 signaling pathway revealed the greatest differences among the peptides: E22Q and WT peptides suppressed FGF-2 expression while E22G had barely any effect. Phosphorylation of the FGF-2 receptor, FGFR-1, and the survival signal Akt were abolished by E22Q and WT peptides, but not by E22G. The biological dissimilar effect of the mutant and WT peptides on cerebral EC cannot be assigned to a particular Abeta structure, suggesting that the toxic effect of the Abeta assemblies goes beyond mere multimerization