Faculty Bibliography

Mitochondrial dysfunction has been recognized as an early event in Alzheimer's disease (AD) pathology, preceding and inducing neurodegeneration and memory loss. The presence of cytochrome c (CytC) released from the mitochondria into the cytoplasm is often detected after acute or chronic neurodegenerative insults, including AD. The carbonic anhydrase inhibitor (CAI) methazolamide (MTZ) was identified among a library of drugs as an inhibitor of CytC release and proved to be neuroprotective in Huntington's disease and stroke models. Here, using neuronal and glial cell cultures, in addition to an acute model of amyloid beta (Abeta) toxicity, which replicates by intra-hippocampal injection the consequences of interstitial and cellular accumulation of Abeta, we analyzed the effects of MTZ on neuronal and glial degeneration induced by the Alzheimer's amyloid. MTZ prevented DNA fragmentation, CytC release and activation of caspase 9 and caspase 3 induced by Abeta in neuronal and glial cells in culture through the inhibition of mitochondrial hydrogen peroxide production. Moreover, intraperitoneal administration of MTZ prevented neurodegeneration induced by intra-hippocampal Abeta injection in the mouse brain and was effective at reducing caspase 3 activation in neurons and microglia in the area surrounding the injection site. Our results, delineating the molecular mechanism of action of MTZ against Abeta-mediated mitochondrial dysfunction and caspase activation, and demonstrating its efficiency in a model of acute amyloid-mediated toxicity, provide the first combined in vitro and in vivo evidence supporting the potential of a new therapy employing FDA-approved CAIs in AD.

Cerebrospinal fluid (CSF) measures of phosphorylated-tau (P-tau) 231 and P-tau181 are two biomarkers for the identification of tau pathology as related to Alzheimer's disease (AD). While both are pathologically validated, their relative diagnostic performances are not well known. This cross-sectional diagnostic study of 87 normal (NL) subjects and 28 AD subjects compared CSF P-tau231 with CSF P-tau181. Logistic regression modeling demonstrated that the P-tau231 was superior to the P-tau181 in the diagnostic classifications. At a fixed 85% sensitivity cutoff, the ROC analysis shows that P-tau231 has greater overall specificity than P-tau181. While both P-tau analytes demonstrated equivalent negative predictive accuracies, P-tau231 yielded significantly fewer false positives. Moreover, P-tau231, but not P-tau181, demonstrated sensitivity to the E4 genotype. A postmortem validation with 9 AD subjects confirmed the superiority of the CSF P-tau231 specificity. This study suggests that P-tau231 has the potential to improve the CSF tau biomarker diagnosis of AD.

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

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.

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.

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.

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

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.