Faculty Bibliography

Familial Danish Dementia (FDD), an early-onset non-amyloid-beta (Abeta) cerebral amyloidosis, is neuropathologically characterized by widespread cerebral amyloid angiopathy, parenchymal amyloid and preamyloid deposits, as well as neurofibrillary degeneration indistinguishable to that seen in Alzheimer's disease (AD). The main amyloid subunit composing FDD lesions, a 34-amino acid de-novo generated peptide ADan, is the direct result of a genetic defect at the 3'-end of the BRI2 gene and the physiologic action of furin-like proteolytic processing at the C-terminal region of the ADan precursor protein. We aimed to study the impact of the FDD mutation, the additional formation of the pyroglutamate (pE) posttranslational modification as well as the relevance of C-terminal truncations -all major components of the heterogeneous FDD deposits- on the structural and neurotoxic properties of the molecule. Our data indicates that whereas the mutation generated a beta-sheet-rich hydrophobic ADan subunit of high oligomerization/fibrillization propensity and the pE modification further enhanced these properties, C-terminal truncations had the opposite effect mostly abolishing these features. The potentiation of pro-amyloidogenic properties correlated with the initiation of neuronal cell death mechanisms involving oxidative stress, perturbation of mitochondrial membrane potential, release of mitochondrial cytochrome c, and downstream activation of caspase-mediated apoptotic pathways. The amyloid-induced toxicity was inhibited by targeting specific components of these detrimental cellular pathways, using reactive oxygen scavengers and monoclonal antibodies recognizing the pathological amyloid subunit. Taken together, the data indicate that the FDD mutation and the pE posttranslational modification are both primary elements driving intact ADan into an amyloidogenic/neurotoxic pathway while truncations at the C-terminus eliminate the pro-amyloidogenic characteristics of the molecule, likely reflecting effect of physiologic clearance mechanisms.

Amyloid beta (Abeta) is the major constituent of the brain deposits found in parenchymal plaques and cerebral blood vessels of patients with Alzheimer's disease (AD). Several lines of investigation support the notion that synaptic pathology, one of the strongest correlates to cognitive impairment, is related to the progressive accumulation of neurotoxic Abeta oligomers. Since the process of oligomerization/fibrillization is concentration-dependent, it is highly reliant on the homeostatic mechanisms that regulate the steady state levels of Abeta influencing the delicate balance between rate of synthesis, dynamics of aggregation, and clearance kinetics. Emerging new data suggest that reduced Abeta clearance, particularly in the aging brain, plays a critical role in the process of amyloid formation and AD pathogenesis. Using well-defined monomeric and low molecular mass oligomeric Abeta1-40 species stereotaxically injected into the brain of C57BL/6 wild-type mice in combination with biochemical and mass spectrometric analyses in CSF, our data clearly demonstrate that Abeta physiologic removal is extremely fast and involves local proteolytic degradation leading to the generation of heterogeneous C-terminally cleaved proteolytic products, while providing clear indication of the detrimental role of oligomerization for brain Abeta efflux. Immunofluorescence confocal microscopy studies provide insight into the cellular pathways involved in the brain removal and cellular uptake of Abeta. The findings indicate that clearance from brain interstitial fluid follows local and systemic paths and that in addition to the blood-brain barrier, local enzymatic degradation and the bulk flow transport through the choroid plexus into the CSF play significant roles. Our studies highlight the diverse factors influencing brain clearance and the participation of various routes of elimination opening up new research opportunities for the understanding of altered mechanisms triggering AD pathology and for the potential design of combined therapeutic strategies.

Background: Neurodegeneration and memory loss in Alzheimer's disease (AD) have been associated in many reports with mitochondrial dysfunction. Molecular pathways triggered by mitochondrial deregulation, with associated production of reactive oxygen species and release of pro-apoptotic factors, are thought to be early events in the pathogenesis of the disease. Carbonic anhydrase inhibitors such as methazolamide (MTZ) are FDA approved for glaucoma as well as other indications, and have been considered as potential therapeutic strategies in models of Huntington's disease, stroke, muscular dystrophy and diabetes. Methods: We analyzed the effects of MTZ on neuronal and glial degeneration induced by the Alzheimer's amyloid in vitro and by intra-hippocampal injection resulting in interstitial and cellular accumulation of Abeta in the mouse brain. Results:MTZ prevented CytC release and activation of caspase 9 and caspase 3 induced by Abeta in neuronal and glial cells through the inhibition of mitochondrial hydrogen peroxide production, and inhibited DNA fragmentation indicative of apoptosis. MTZ administered IP before intra-hippocampal Abeta injection was effective at reducing caspase 3 activation and neurodegeneration in the mouse brain. Conclusions: Our results demonstrate the efficacy of MTZ in in vitro and in vivo models of amyloid-mediated toxicity, delineating the molecular mechanism of action of the compound and providing the first evidence in support of the possibility of a new therapeutic approach for AD

Matrix metalloproteases MMP-2 and MMP-9 have been implicated in the physiologic catabolism of Alzheimer amyloid-beta (Abeta). Conversely, their association with vascular amyloid deposits, blood-brain barrier disruption, and hemorrhagic transformations after ischemic stroke also highlights their involvement in pathologic processes. To better understand this dichotomy, recombinant human (rh) MMP-2 and MMP-9 were incubated with Abeta40 and Abeta42 and the resulting proteolytic fragments assessed via immunoprecipitation and quantitative mass spectrometry. Both MMPs generated Abeta fragments truncated only at the C-terminus, ending at positions 34, 30 and 16. Using deuterated homologues as internal standards, we observed limited and relatively slow degradation of Abeta42 by rhMMP-2 while the enzyme cleaved >80% of Abeta40 during the first hour of incubation. rhMMP-9 was significantly less effective, particularly in degrading Abeta1-42, although the targeted peptide bonds were identical. Using Abeta1-34 and Abeta1-30, we demonstrated that these peptides are also substrates for both MMPs, cleaving Abeta1-34 to produce Abeta1-30 first and Abeta1-16 subsequently. Consistent with the kinetics observed with full-length Abeta, rhMMP-9 degraded only a minute fraction of Abeta1-34 and was even less effective in producing Abeta1-16. Further degradation of Abeta1-16 by either MMP-2 or MMP-9 was not observed even after prolonged incubation times. Notably, all MMP-generated C-terminally truncated Abeta fragments were highly soluble, did not exhibit fibrillogenic properties or induce cytotoxicity in human cerebral microvascular endothelial or neuronal cells supporting the notion that these truncated Abeta species are associated with clearance mechanisms rather than being key elements in the fibrillogenesis process.

Objective: Vascular deposition of amyloid, a universal feature in Alzheimer's disease (AD), severely compromises the integrity of the neurovascular unit, a dynamic entity encompassing functional interactions among cells of the microvasculature, neurons, and astrocytic/glial populations. The complexity of the cellular mechanisms elicited by amyloid in astrocytic/glial cells and their relationship to the induction of pro-inflammatory conditions capable of affecting microvessel function/permeability remain to be fully elucidated. In this work we aimed to provide insight into the molecular pathways affected and identify potential new targets for drug discovery. Methods: Astrocytic/glioma cells were challenged with wild-type Abeta and the E22Q vasculotropic variant associated with cerebrovascular deposition and hemorrhagic clinical phenotypes. A combination of FACs-analyzed bead arrays, ELISA, zymography, and confocal studies were employed to evaluate production of pro-inflammatory cytokines, activation of MMPs, and ROS generation whereas the vitamin-E analog Trolox was tested for prevention/amelioration of these detrimental cellular pathways. Results: Oligomeric-Abeta triggered elevated production of the pro-inflammatory mediators IL-6, IL-8, and IFN-gamma, enhanced activation of MMP-2, exacerbated ROS production, and cell death. In all cases, challenge with E22Q translated into a more pronounced response, in agreement with the high oligomerization tendency of the variant and the aggressiveness and early onset of the clinical phenotype. Trolox not only inhibited ROS production and MMP-2 activation, but also preserved cellular integrity and viability, highlighting the primary role of ROS in the initiation of amyloid-induced cell death pathways. Conclusions: Our data emphasizes the detrimental role of astrocyte/glia-initiated Abeta-mediated pro-inflammatory pathways for the integrity of the neurovascular unit

Objective: A heterogeneous group of amyloid beta (Abeta) species constitute the parenchymal and cerebrovascular amyloid deposits in Alzheimer's disease (AD). Besides the classic full-length peptides, biochemical and proteomic analysis of AD deposits revealed high degree of Abeta heterogeneity at both N- and C-terminal ends likely resulting from the local action of multiple proteolytic enzymes. Interestingly, many of these fragments are also normal components of cerebrospinal fluid, suggesting their active participation in clearance mechanisms. Increasing evidence indicates that deficient brain clearance largely contributes to Abeta accumulation; thus, we compared the biophysical properties and lesion distribution of various N- and C-terminally degraded Abeta fragments to better understand their biological importance. Methods: Synthetic homologues of in vivo identified truncated peptides were used to compare solubility properties, formation of beta-sheet-rich structures, binding to thioflavin T, self-oligomerization and formation of amyloid-like fibrils. Novel antibodies recognizing specific N- and C-terminal truncations were generated and employed to immunolabel amyloid deposits and conduct biochemical analysis in transgenic models and AD brains. Results: N- and C-terminally truncated peptides exhibited completely different biophysical properties and brain tissue distribution. C-terminally degraded Abeta fragments were extremely soluble, did not convert to beta-sheet-rich structures, failed to aggregate or form fibrils and did not co-localize with plaque deposits. Contrastingly, those degraded at the N-terminus were poorly soluble, with high tendency to aggregate and fibrillize and specifically co-localize with Congo-red-positive plaque cores. Conclusions: Degradation at the C-terminal-end of Abeta generates fragments likely associated to catabolic/clearance mechanisms while truncations at the N-terminus favor the process of amyloidogenesis

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.