Faculty Bibliography

The vascular deposition of amyloid, known as Cerebral Amyloid Angiopathy (CAA) is an age-associated condition featured in about 90% of Alzheimer's disease cases and in the aging brain. Amyloid beta (Abeta) deposition in CAA compromises cerebral blood flow and can cause cerebral hemorrhage and cognitive impairment, by mechanisms that are still poorly understood. Our goal was to identify the molecular events underlying the apoptotic cascade generated by Abeta in cerebrovascular cells and to pinpoint new targets for drug discovery. Human brain microvascular endothelial cells were challenged with vasculotropic Abeta variants associated with cerebrovascular deposition and hemorrhagic outcome, and the resulting signaling pathways were analysed. The in vitro findings were validated in vivo in mice subjected to intrahippocampal Abeta injections and confirmed in human CAA cases. Our findings highlighted an activation of caspase-8 and -9, together with mitochondrial dysfunction and release of cytochrome C, suggesting death receptor mediated apoptosis, which was confirmed by an overexpression of the TRAIL (TNF-related apoptosis inducing ligand) death receptors DR4 and DR5. Signaling cascades typical of TRAIL death receptor-mediated pathways were activated. The same receptors colocalized with Abeta on the cell membrane after amyloid challenge, and immunoprecipitated in vitro with Abeta oligomers. SiRNAs against DR4 and DR5 and the prevention of mitochondrial dysfunction through Carbonic Anhydrase Inhibitors, significantly diminished Abeta mediated apoptosis in endothelial cells. In vivo experiments in mice injected with vasculotropic Abeta peptide and in human CAA cases confirmed the upregulation of the receptors, their colocalization with Abeta on the cerebral vasculature, and the activation of caspases, which could be prevented by carbonic anhydrase inhibitors. Our data strongly suggests that TRAIL death-receptors and mitochondrial functioning are key cellular targets for therapeutic intervention against Abeta-induced vascular cell death in CAA

Familial British dementia (FBD) is an early-onset non-amyloid-beta (Abeta) cerebral amyloidosis that presents with severe cognitive decline and strikingly similar neuropathological features to those present in Alzheimer's disease (AD). FBD is associated with a T to A single nucleotide transition in the stop codon of a gene encoding BRI2, leading to the production of an elongated precursor protein. Furin-like proteolytic processing at its C-terminus releases a longer-than-normal 34 amino acid peptide, ABri, exhibiting amyloidogenic properties not seen in its 23 amino acid physiologic counterpart Bri1-23. Deposited ABri exhibits abundant post-translational pyroglutamate (pE) formation at the N-terminus, a feature seen in truncated forms of Abeta found in AD deposits, and co-exists with neurofibrillary tangles almost identical to those found in AD. We tested the impact of the FBD mutation alone and in conjunction with the pE post-translational modification on the structural properties and associated neurotoxicity of the ABri peptide. The presence of pE conferred to the ABri molecule enhanced hydrophobicity and accelerated aggregation/fibrillization properties. ABri pE was capable of triggering oxidative stress, loss of mitochondrial membrane potential and activation of caspase-mediated apoptotic mechanisms in neuronal cells, whereas homologous peptides lacking the elongated C-terminus and/or the N-terminal pE were unable to induce similar detrimental cellular pathways. The data indicate that the presence of N-terminal pE is not in itself sufficient to induce pathogenic changes in the physiologic Bri1-23 peptide but that its combination with the ABri mutation is critical for the molecular pathogenesis of FBD.

Purpose: To better understand the biological significance of clusterin co-localization with the exfoliation deposits and provide insight into a pathogenic mechanism involving activation of the complement system and its pro-inflammatory consequences in patients with exfoliation glaucoma. Methods: Exfoliation lens deposits were analyzed by high resolution atomic force microscopy imaging and confocal immunofluorescence. Levels of clusterin and vitronectin as well as of the complement activation products C3a and soluble C5b-9 were assessed via ELISA. Results: Atomic-force microscopy examination of lenses with exfoliation syndrome revealed a dense fibrillar network on the anterior, aqueous-bathed surface of the lens while the epithelial side displayed no discernible structural features at the same resolution. Clusterin co-localized with exfoliation deposits, demonstrating integral association with the fibrils. Levels of activation-derived complement components C3a and soluble C5b-9 as well as the complement inhibitors clusterin and vitronectin were found significantly elevated (1.7-fold, p<0.05; 4.1-fold, p<0.05; 1.8-fold, p<0.01; and 3.0-fold, p<0.01, respectively) in aqueous humor from glaucoma patients with XFS compared with non-XFS glaucoma controls. Conclusions: The data provide compelling evidence for the activation of the complement system in XFS, highlighting the generation of sub-products with potent pro-inflammatory activity which are capable of triggering and chronically maintaining levels of subclinical inflammation, suggesting novel targets for therapeutic intervention. The co-localization of clusterin in XF fibrils suggests a failed attempt to prevent tissue accumulation of protein aggregates - as seen in other protein folding disorders - likely due to the abnormal high levels of misfolded proteins overwhelming its chaperone capacity.

Substantial genetic, biochemical, and in vivo data indicate that progressive accumulation of amyloid-beta (Abeta) plays a central role in the pathogenesis of Alzheimer's disease (AD). Historically centered in the importance of parenchymal plaques, the role of cerebral amyloid angiopathy (CAA)-a frequently neglected amyloid deposit present in >80% of AD cases-for the mechanism of disease pathogenesis is now starting to emerge. CAA consistently associates with microvascular modifications, ischemic lesions, micro- and macro-hemorrhages, and dementia, progressively affecting cerebral blood flow, altering blood-brain barrier permeability, interfering with brain clearance mechanisms and triggering a cascade of deleterious pro-inflammatory and metabolic events that compromise the integrity of the neurovascular unit. New evidence highlights the contribution of pre-fibrillar Abeta in the induction of cerebral endothelial cell dysfunction. The recently discovered interaction of oligomeric Abeta species with TRAIL DR4 and DR5 cell surface death receptors mediates the engagement of mitochondrial pathways and sequential activation of multiple caspases, eliciting a cascade of cell death mechanisms while unveiling an opportunity for exploring mechanistic-based therapeutic interventions to preserve the integrity of the neurovascular unit.

Mutations within the Abeta (amyloid beta) peptide, especially those clustered at residues 21-23, are linked to early-onset AD (Alzheimer's disease) and primarily associated with cerebral amyloid angiopathy. The Iowa variant, a substitution of an aspartic acid residue for asparagine at position 23 (D23N), associates with widespread vascular amyloid and abundant diffuse pre-amyloid lesions significantly exceeding the incidence of mature plaques. Brain Iowa deposits consist primarily of a mixture of mutated and non-mutated Abeta species exhibiting partial aspartate isomerization at positions 1, 7 and 23. The present study analysed the contribution of the post-translational modification and the D23N mutation to the aggregation/fibrillization and cell toxicity properties of Abeta providing insight into the elicited cell death mechanisms. The induction of apoptosis by the different Abeta species correlated with their oligomerization/fibrillization propensity and beta-sheet content. Although cell toxicity was primarily driven by the D23N mutation, all Abeta isoforms tested were capable, albeit at different time frames, of eliciting comparable apoptotic pathways with mitochondrial engagement and cytochrome c release to the cytoplasm in both neuronal and microvascular endothelial cells. Methazolamide, a cytochrome c release inhibitor, exerted a protective effect in both cell types, suggesting that pharmacological targeting of mitochondria may constitute a viable therapeutic avenue.

Alzheimer's disease (AD) is the most common form of dementia. Intraneuronal neurofibrillary tangles, extracellular Abeta amyloid deposits in the form of amyloid plaques and cerebral amyloid angiopathy, and synaptic and neuronal loss co-exist in the brain parenchyma, with the limbic areas being the most severely affected. The classic clinical findings are personality changes, progressive cognitive dysfunction, and loss of ability to perform activities of daily living. Visual impairment is common and appears related to disease severity, suggesting that visual testing may provide a method of screening and tracking AD changes. Although still not fully understood, research and clinical findings point to a possible common causal relationship between AD and glaucoma. These two chronic neurodegenerative disorders share biological and mechanistic features, among them (1) a strong age-related incidence, (2) retinal ganglion cell degeneration, and (3) extracellular fibrillar deposits in exfoliation syndrome, the most common recognizable cause of glaucoma, suggesting that both diseases may originate from similar misfolding mechanisms. A presentation of common pathogenetic pathways associated with these disorders, including cell death mechanisms, reactive oxygen species (ROS) production, mitochondrial dysfunction and vascular abnormalities, will serve as an initiation point for further exploration.

Vascular deposition of amyloid-beta (Abeta) in sporadic and familial Alzheimer's disease, through poorly understood molecular mechanisms, leads to focal ischemia, alterations in cerebral blood flow, and cerebral micro-/macro-hemorrhages, significantly contributing to cognitive impairment. Here, we show that tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) death receptors DR4 and DR5 specifically mediate oligomeric Abeta induction of extrinsic apoptotic pathways in human microvascular cerebral endothelial cells with activation of both caspase-8 and caspase-9. The caspase-8 inhibitor cellular FLICE-like inhibitory protein (cFLIP) is downregulated, and mitochondrial paths are engaged through BH3-interacting domain death agonist (Bid) cleavage. Upregulation of DR4 and DR5 and colocalization with Abeta at the cell membrane suggests their involvement as initiators of the apoptotic machinery. Direct binding assays using receptor chimeras confirm the specific interaction of oligomeric Abeta with DR4 and DR5 whereas apoptosis protection achieved through RNA silencing of both receptors highlights their active role in downstream apoptotic pathways unveiling new targets for therapeutic intervention.