Silvia Fossati

Differential activation of mitochondrial apoptotic pathways by vasculotropic amyloid-β variants in cells composing the cerebral vessel walls

Cerebral amyloid angiopathy (CAA) is an age‐associated condition and a common finding in Alzheimers disease in which amyloid‐β (Aβ) vascular deposits are featured in >80% of the cases. Familial Aβ 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 Aβ 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 endo‐thelial and smooth muscle cells with nonfibrillar structures of both variants and wild‐type Aβ40. 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 Aβ 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.—Fossati, S., Cam, J., Meyerson, J., Mezhericher, E., Romero, I. A., Couraud, P. O., Weksler, B. B., Ghiso, J., Rostagno, A. Differential activation of mitochondrial apoptotic pathways by vasculotropic amyloid‐β variants in cells composing the cerebral vessel walls. FASEB J. 24, 229–241 (2010). www.fasebj.org

Dutch and arctic mutant peptides of β amyloid1–40 differentially affect the FGF-2 pathway in brain endothelium

Single point mutations of the amyloid precursor protein generate Abeta variants bearing amino acid substitutions at positions 21-23. These mutants are associated with distinct hereditary phenotypes of cerebral amyloid angiopathy, manifesting varying degrees of tropism for brain vessels, and impaired microvessel remodeling and angiogenesis. We examined the differential effects of E22Q (Dutch), and E22G (Arctic) variants in comparison to WT Abeta on brain endothelial cell proliferation, angiogenic phenotype expression triggered by fibroblast growth factor (FGF-2), pseudo-capillary sprouting, and induction of apoptosis. E22Q exhibited a potent anti-angiogenic profile in contrast to E22G, which had a much weaker effect. Investigations on the FGF-2 signaling pathway revealed the greatest differences among the peptides: E22Q and WT peptides suppressed FGF-2 expression while E22G had barely any effect. Phosphorylation of the FGF-2 receptor, FGFR-1, and the survival signal Akt were abolished by E22Q and WT peptides, but not by E22G. The biological dissimilar effect of the mutant and WT peptides on cerebral EC cannot be assigned to a particular Abeta structure, suggesting that the toxic effect of the Abeta assemblies goes beyond mere multimerization.

Matrix Metalloproteinase 2 (MMP-2) Degrades Soluble Vasculotropic Amyloid-β E22Q and L34V Mutants, Delaying Their Toxicity for Human Brain Microvascular Endothelial Cells

Patients carrying mutations within the amyloid-β (Aβ) 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 Aβ synthetic homologues containing mutations known to be associated in vivo with hemorrhagic manifestations (AβE22Q and AβL34V) 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 Aβ peptides. Immunoprecipitation followed by mass spectrometry analysis highlighted the generation of specific C-terminal proteolytic fragments, in particular the accumulation of Aβ-(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 Aβ degradation and enhanced apoptosis. Secretion and activation of MMP-2 as well as susceptibility of the Aβ peptides to MMP-2 degradation were dependent on the peptide conformation, with fibrillar elements of AβE22Q exhibiting negligible effects. Our results indicate that MMP-2 release and activation differentially degrades Aβ 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.

Insights into Caspase-Mediated Apoptotic Pathways Induced by Amyloid-β in Cerebral Microvascular Endothelial Cells

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-β (Aβ) 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 Aβ40 or its vasculotropic E22Q variant. Methods: We challenged human brain microvascular endothelial cells with both Aβ 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 Aβ oligomers/protofibrils in vascular cells, likely implicating a primary activation of death receptors.

Differential contribution of isoaspartate post-translational modifications to the fibrillization and toxic properties of amyloid β and the Asn23 Iowa mutation

Mutations within the Aβ (amyloid β) peptide, especially those clustered at residues 21-23, are linked to early-onset AD (Alzheimers 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 Aβ 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 Aβ providing insight into the elicited cell death mechanisms. The induction of apoptosis by the different Aβ species correlated with their oligomerization/fibrillization propensity and β-sheet content. Although cell toxicity was primarily driven by the D23N mutation, all Aβ 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.

Amyloidosis associated with cerebral amyloid angiopathy: cell signaling pathways elicited in cerebral endothelial cells.

Substantial genetic, biochemical, and in vivo data indicate that progressive accumulation of amyloid-β (Aβ) plays a central role in the pathogenesis of Alzheimers 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 Aβ in the induction of cerebral endothelial cell dysfunction. The recently discovered interaction of oligomeric Aβ 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.

The carbonic anhydrase inhibitor methazolamide prevents amyloid beta-induced mitochondrial dysfunction and caspase activation protecting neuronal and glial cells in vitro and in the mouse brain.

Mitochondrial dysfunction has been recognized as an early event in Alzheimers 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 Huntingtons disease and stroke models. Here, using neuronal and glial cell cultures, in addition to an acute model of amyloid beta (Aβ) toxicity, which replicates by intra-hippocampal injection the consequences of interstitial and cellular accumulation of Aβ, we analyzed the effects of MTZ on neuronal and glial degeneration induced by the Alzheimers amyloid. MTZ prevented DNA fragmentation, CytC release and activation of caspase 9 and caspase 3 induced by Aβ 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 Aβ 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 Aβ-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 Clearance in Alzheimer Disease Measured with Dynamic PET.

Evidence supporting the hypothesis that reduced cerebrospinal fluid (CSF) clearance is involved in the pathophysiology of Alzheimer disease (AD) comes primarily from rodent models. However, unlike rodents, in which predominant extracranial CSF egress is via olfactory nerves traversing the cribriform plate, human CSF clearance pathways are not well characterized. Dynamic PET with 18F‐THK5117, a tracer for tau pathology, was used to estimate the ventricular CSF time–activity as a biomarker for CSF clearance. We tested 3 hypotheses: extracranial CSF is detected at the superior turbinates; CSF clearance is reduced in AD; and CSF clearance is inversely associated with amyloid deposition. Methods: Fifteen subjects, 8 with AD and 7 normal control volunteers, were examined with 18F‐THK5117. Ten subjects additionally underwent 11C-Pittsburgh compound B (11C-PiB) PET scanning, and 8 were 11C-PiB–positive. Ventricular time–activity curves of 18F‐THK5117 were used to identify highly correlated time–activity curves from extracranial voxels. Results: For all subjects, the greatest density of CSF-positive extracranial voxels was in the nasal turbinates. Tracer concentration analyses validated the superior nasal turbinate CSF signal intensity. AD patients showed ventricular tracer clearance reduced by 23% and 66% fewer superior turbinate CSF egress sites. Ventricular CSF clearance was inversely associated with amyloid deposition. Conclusion: The human nasal turbinate is part of the CSF clearance system. Lateral ventricle and superior nasal turbinate CSF clearance abnormalities are found in AD. Ventricular CSF clearance reductions are associated with increased brain amyloid depositions. These data suggest that PET-measured CSF clearance is a biomarker of potential interest in AD and other neurodegenerative diseases.

Greater Specificity for Cerebrospinal Fluid P-tau231 over P-tau181 in the Differentiation of Healthy Controls from Alzheimer’s Disease

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 Alzheimers 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.

Mitochondrial dysfunction induced by a post-translationally modified amyloid linked to a familial mutation in an alternative model of neurodegeneration.

Familial British dementia (FBD) is an early-onset non-amyloid-β (Aβ) cerebral amyloidosis that presents with severe cognitive decline and strikingly similar neuropathological features to those present in Alzheimers 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 Aβ 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 peptides but that its combination with the ABri mutation is critical for the molecular pathogenesis of FBD.