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

Abstract

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

Keywords: Apoptosis; Cerebral amyloidosis; Cytochrome c; Familial British dementia; Oligomeric amyloid assemblies; Oxidative stress.

Figure 1. Circular dichroism spectroscopy of ABri pE/E and Bri1-23 pE/E

Changes in secondary structure were monitored recording the CD spectra after incubation in HFIP for 3–8 days (room temperature; peptide concentrations of 1 mg/ml) (A) and incubation for up to 48 hours in 10 mM PO₄ buffer containing 150 mM NaF (37°C; peptide concentrations of 50 μM) (B). In all cases, data represent the mean of 15 scans after subtraction of background readings of the respective buffer blanks.

Figure 2. Aggregation/fibrillization of ABri pE/E and Bri1-23 pE/E

(A) Oligomerization / fibrillization of ABri pE/E and Bri 1–23 pE/E was assessed by fluorescence evaluation of thioflavin T binding to 50 μM peptide in 1X PBS over 24 hours. (B) Exposure of hydrophobic regions of the molecule was evaluated by assessing the binding of the peptides to ANS. Left panel: Fluorescence evaluation in arbitrary units (A.U); graph illustrates mean ± SEM of three independent experiments after subtraction of blank levels. Right panel: representative scan illustrating ANS binding curve for ABri pE, ABri E, and Bri 1–23 pE/E at 24 hours and highlighting the blue shift of the maximum fluorescence values for ABri pE/E homologues. (C) Blue Native-PAGE. Peptides were incubated at 20 μM in 1X HBSS at 37°C for up to 24 hours and aliquots at different time-points were separated in a 4–13% Blue Native gel followed by Western blot analysis. (D) SDS-PAGE. Peptides were incubated at 20 μM in 1X HBSS at 37°C for up to 24 hours and samples at different time-points were separated in a 5–20% Tris-tricine SDS gel followed by Western blot analysis. All blots were probed with the anti-ABri 338 antibody (1:5,000).

Figure 3. Apoptosis induction by ABri pE

SH-SY5Y cells were challenged with 50 μM ABri pE/E, Bri 1–23 pE/E, or Aβ42 for 4, 8, or 24 hours. (A) Apoptosis was evaluated by Cell Death ELISA. Results are expressed as a fold of change of nucleosome formation compared to no-peptide controls at the respective time points. (B) The release of lactate dehydrogenate (LDH) from the cells was used as a measure of necrotic cell death. Results are expressed as a fold of change of LDH release compared to no-peptide controls at the respective time points; data represent the mean ± SEM of three independent experiments. ** = p<.01, *** = p<.001.

Figure 4. Mitochondrial cytochrome c release

SH-SY5Y cells were challenged with 50 μM ABri pE/E, Bri 1–23 pE/E, or Aβ42 for 4, 8, or 16 hours. Green fluorescence indicates cyt c immunostaining; blue fluorescence represents nuclear DNA counterstained with To-Pro. Magnification = X63 in all cases.

Figure 5. Mitochondrial dysfunction induced by ABri pE

SH-SY5Y cells were challenged with ABri pE/E or Aβ42 for 16 hours followed by staining with MitoTracker Red CM-H₂XRos and cyt c immunocytochemistry. Top panel: cyt c fluorescence shown in green; Central panel: oxidized Mitotracker highlighted in red; Bottom panel: merged images. Magnification = X63 in all cases.

Figure 6. Oxidative stress in cells challenged with ABri pE

SH-SY5Y cells were treated with 50 μM ABri pE/E for 4 hours followed by staining with the CellROX Deep Red fluorogenic probe. Top panel: Hoechst nuclear stain shown in blue; Central panel: oxidized CellROX reagent highlighted in red; Bottom panel: merged images. Magnification = X20 in all cases.

Figure 7. Activation of caspase-mediated apoptotic pathways by ABri pE

(A) SH-SY5Y cells were treated with 50 μM ABri pE/E and activation of caspase-9 monitored for up to 16 hours using a luminescent assay. Results are expressed as a fold of change compared to no-peptide controls; data represent the mean ± SEM of three independent experiments. (B) SH-SY5Y cells were treated with 50 μM ABri pE or 1 μM of the apoptosis-inducer staurosporine. Apoptosis in the presence or absence of caspase-9 inhibitor Z-LEHD-FMK or the pan-caspase inhibitor Z-VAD-FMK was evaluated by Cell Death ELISA; results are expressed as fold of change compared to no peptide controls in the absence of inhibitors and represent the mean ± SEM of three independent experiments. *** = p<.001.