β-Amyloid Carrying the Dutch Mutation Has Diverse Effects on Calpain-Mediated Toxicity in Hippocampal Neurons

Neurobiology of Disease

Ghiso

Rostagno

2016

Familial Danish Dementia (FDD), an early-onset non-amyloid-β (Aβ) 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 β-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.

Section: Resultsmentioning

confidence: 99%

Oxidative stress and mitochondria-mediated cell death mechanisms triggered by the familial Danish dementia ADan amyloid

Neurobiology of Disease

Ghiso

Rostagno

2016

“…The ability of the different A β variants studied in the present paper to induce DNA fragmentation, an event indicative of apoptosis, in SH-SY5Y and cerebral microvascular ECs was assessed at peptide concentrations typically used in in vitro assays measuring induction of cell death mechanisms by A β [24–27], with some studies testing concentrations even higher [9,28]. The actual levels of A β present in vivo in parenchymal and CAA lesions is very difficult to assess, but is by far much higher than the physiological concentration of soluble A β in biological fluids.…”

Section: Resultsmentioning

confidence: 99%

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

Fossati

Sotolongo

et al. 2013

Biochemical Journal

Mutations within the Aβ (amyloid β) 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 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.

“…DNA fragmentation, an event indicative of apoptosis, was evaluated by Cell Death ELISA in SH-SY5Y neuronal cells following incubation with the peptide homologues at 50 μM concentration, a dose in the lower range of those typically employed (25–300 μM) for the assessment of amyloid toxicity [38, 39, 53–57]. Although 20 μM was the lowest concentration of ABri pE that exhibited a measurable – albeit modest– effect in Cell Death ELISA in our dose-response experimental paradigm (not shown), a 50 μM concentration was selected for all the studies described below based on the well-defined 2.5 fold-change observed over untreated controls.…”

Section: Resultsmentioning

confidence: 99%

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

Biochimica et Biophysica Acta (BBA) - Molecular Basis of Diseas

Fossati

Ghiso

et al. 2014

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 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 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 peptide but that its combination with the ABri mutation is critical for the molecular pathogenesis of FBD.