Faculty Bibliography

Alzheimer's disease (AD) is the leading cause of dementia and the most common form of amyloidosis in humans. Extensive extracellular deposition of amyloid-beta (Abeta), a 40-42 amino acid degradation product of APP, is considered a hallmark feature of AD. Our attention is focused on the highly heterogeneous biochemical nature of the brain Abeta species, delving beyond Abeta40 and Abeta42, likely reflecting a complex balance between amyloidogenic and clearance pathways. We have fractionated water-soluble, detergent-soluble and formic acid soluble Abeta species from brains of transgenic mouse models of amyloid depostion and AD cases. Subsequently, we applied a combination of biochemical techniques including immunoprecipitation followed by identification of Abeta species with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Our biochemical data on the Abeta species present in sporadic AD cases and in transgenic mouse models highlight the presence of similar N-and C-terminally truncated fragments-likely reflecting the ability of multiple proteases to degrade Abeta in situ-and several post-translational modifications with still unclear roles in the amyloidogenesis mechanism. Notably, not all the brain Abeta peptides have identical solubility properties; whereas many of them are highly soluble in water-based physiologic solutions others require mild detergents or strong acids for extraction, suggesting their differential involvement in catabolic and fibrillogenic processes

Aggregation of beta-amyloid (Abeta) is implicated in the pathology of Alzheimer's disease. Development of a robust strategy to detect Abeta oligomeric intermediates, which have been identified as significant toxic agents, would be highly beneficial in the screening of drug candidates as well as enhancing our understanding of Abeta oligomerization. Rapid, specific and quantitative detection, currently unavailable, would be highly preferred for accurate and reliable probing of transient Abeta oligomers. Here, we report the development of a novel peptide probe, PG46, based on the nature of Abeta self-assembly and the conformation-sensitive fluorescence of the biarsenical dye, FlAsH. PG46 was found to bind to Abeta oligomers and displayed an increase in FlAsH fluorescence upon binding. No such event was observed when PG46 was co-incubated with Abeta low-molecular-weight species or Abeta fibrils. Abeta oligomer detection was fast, and occurred within one hour without any additional sample incubation or preparation. We anticipate that the development of a strategy for detection of amyloid oligomers described in this study will be directly relevant to a host of other amyloidogenic proteins

Patients carrying mutations within the amyloid-beta (Abeta) sequence develop severe early-onset cerebral amyloid angiopathy with some of the related variants manifesting primarily with hemorrhagic phenotypes. Matrix metalloproteases (MMPs) are typically associated with blood brain barrier disruption and hemorrhagic transformations after ischemic stroke. However, their contribution to cerebral amyloid angiopathy-related hemorrhage remains unclear. Human brain endothelial cells challenged with Abeta synthetic homologues containing mutations known to be associated in vivo with hemorrhagic manifestations (AbetaE22Q and AbetaL34V) showed enhanced production and activation of MMP-2, evaluated via Multiplex MMP antibody arrays, gel zymography, and Western blot, which in turn proteolytically cleaved in situ the Abeta peptides. Immunoprecipitation followed by mass spectrometry analysis highlighted the generation of specific C-terminal proteolytic fragments, in particular the accumulation of Abeta-(1-16), a result validated in vitro with recombinant MMP-2 and quantitatively evaluated using deuterium-labeled internal standards. Silencing MMP-2 gene expression resulted in reduced Abeta degradation and enhanced apoptosis. Secretion and activation of MMP-2 as well as susceptibility of the Abeta peptides to MMP-2 degradation were dependent on the peptide conformation, with fibrillar elements of AbetaE22Q exhibiting negligible effects. Our results indicate that MMP-2 release and activation differentially degrades Abeta species, delaying their toxicity for endothelial cells. However, taking into consideration MMP ability to degrade basement membrane components, these protective effects might also undesirably compromise blood brain barrier integrity and precipitate a hemorrhagic phenotype

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