Faculty Bibliography

Vascular deposition of Abeta in sporadic and familial Alzheimer's disease, through poorly understood molecular mechanisms, leads to alterations in cerebral blood flow, focal ischemia, and cerebral micro-/macro-hemorrhages, significantly contributing to cognitive impairment. We aimed to determine the molecular mechanisms triggering apoptosis of vessel wall cells in presence of Abeta40 or its vasculotropic variants E22Q or L34V. Challenging human brain microvascular endothelial cells with both variants and wild-type Abeta40, we showed that TRAIL death receptors DR4 and DR5 specifically mediate oligomeric Abeta induction of extrinsic apoptotic pathways. Caspase-8 activation preceded activation of caspase-9. The caspase-8 inhibitor cFLIP was downregulated, and mitochondrial paths were engaged through BID cleavage. Up-regulation of DR4 and DR5 and co-localization with Abeta at the cell membrane suggested their involvement as initiators of the apoptotic machinery. Direct binding assays using receptor chimeras confirmed the specific interaction of oligomeric Abeta with DR4 and DR5 whereas apoptosis protection achieved through RNA silencing of both receptors highlighted their active role in downstream apoptotic pathways unveiling new targets for therapeutic intervention

The vascular deposition of amyloid, known as cerebral amyloid angiopathy (CAA) is an age-associated condition and a common finding in Alzheimer's disease, in which Abeta vascular deposits are featured in[80% of the cases. Familial Abeta variants with substitutions at positions 21-23 are primarily associated with CAA, and they manifest clinically with striking phenotypes of cerebral hemorrhage or dementia. The more recently reported Piedmont AbL34V mutant, located outside the hot-spot 21-23, shows a similar hemorrhagic phenotype, albeit less aggressive than the widely studied Dutch AbetaE22Q variant. In spite of the clinical impact of CAA, very little is known about the mechanisms causing degeneration in vessel wall cells. Our studies monitored the apoptotic events triggered by Abeta in human cerebral microvascular cells. Structural analyses of the different Abeta homologues showed that apoptosis preceded fibril formation in all cases, correlating with the presence of oligomers and/ or protofibrils. Induction of analogous caspase-mediated mitochondrial pathways was elicited by all peptides, although within different time-frames and intensity. Vessel-wall cell death was prevented through pharmacological inhibition of mitochondrial cytochrome c release or by the action of pan- and pathway-specific caspase inhibitors, giving clear indication of the independent or synergistic engagement of both extrinsic and intrinsic apoptotic mechanisms. Activation of caspase 8, usually triggered by death receptors, preceded that of caspase 9 while pathway specific PCR arrays demonstrated the involvement of TNF-receptor family members. Overall, our data suggests a primary involvement of death receptors in Abeta-induced vascular cell apoptosis supporting the notion that rare genetic mutations constitute unique paradigms to understand the molecular pathogenesis of CAA

Background: The vascular deposition of amyloid known as cerebral amyloid angiopathy (CAA) - an age-associated condition and a common finding in Alzheimer's disease - compromises cerebral blood flow, causing macro/microhemorrhages and/or cognitive impairment. Very little is known about the mechanisms causing CAA-related degeneration of cerebral vascular cells. The Dutch E22Q familial amyloid-beta (Abeta) variant is primarily associated with CAA, and manifests clinically with severe cerebral hemorrhages. Objective: We aimed to determine the molecular mechanisms causing apoptosis of cerebral endothelial cells in the presence of wild-type Abeta40 or its vasculotropic E22Q variant. Methods: We challenged human brain microvascular endothelial cells with both Abeta variants, and studied the apoptotic pathways triggered by these peptides. Results: Caspase-mediated apoptotic pathways were elicited by both peptides within time frames correlating with their aggregation properties and formation of oligomeric/protofibrillar assemblies. Our data revealed a primary activation of caspase-8 (typically triggered by death receptors) with secondary engagement of caspase-9, with cytochrome C and apoptosis-inducing factor release from the mitochondria, suggesting the independent or synergistic engagement of extrinsic and intrinsic apoptotic mechanisms. Conclusion: Our data demonstrate the induction of caspase-8- and caspase-9-dependent mitochondrial-mediated apoptotic pathways by Abeta oligomers/protofibrils in vascular cells, likely implicating a primary activation of death receptors.

'Amyloid binging proteins' is a generic term used to designate proteins that interact with different forms of amyloidogenic peptides or proteins and that, as a result, may modulate their physiological and pathological functions by altering solubility, transport, clearance, degradation, and fibril formation. We describe a simple affinity chromatography protocol to isolate and characterize amyloid-binding proteins based on the use of sequential elution steps that may provide further information on the type of binding interaction. As an example, we depict the application of this protocol to the study of Alzheimer's amyloid beta (Abeta) peptide-binding proteins derived from human plasma. Biochemical analysis of the proteins eluted under different conditions identified serum amyloid P component (SAP) and apolipoprotein J (clusterin) as the main plasma Abeta-binding proteins while various apolipoproteins (apoA-IV, apoE, and apoA-I), as well as albumin (HSA) and fibulin were identified as minor contributors.

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