Aggregation of α-synuclein leads to the formation of oligomeric intermediates that can interact with membranes to form pores. However, it is unknown how this leads to cell toxicity in Parkinson's disease. We investigated the species-specific effects of α-synuclein on Ca2+ signalling in primary neurons and astrocytes using live neuronal imaging and electrophysiology on artificial membranes. We demonstrate that α-synuclein induces an increase in basal intracellular Ca2+ in its unfolded monomeric state as well as in its oligomeric state. Electrophysiology of artificial membranes demonstrated that α-synuclein monomers induce irregular ionic currents, whereas α-synuclein oligomers induce rare discrete channel formation events. Despite the ability of monomeric α-synuclein to affect Ca2+ signalling, it is only the oligomeric form of α-synuclein that induces cell death. Oligomer-induced cell death was abolished by the exclusion of extracellular Ca2+, which prevented the α-synuclein-induced Ca2+ dysregulation. The findings of this study confirm that α-synuclein interacts with membranes to affect Ca2+ signalling in a structure-specific manner and the oligomeric β-sheet-rich α-synuclein species ultimately leads to Ca2+ dysregulation and Ca2+-dependent cell death.
Amyloid-beta peptides (Aβ), implicated in Alzheimer’s disease (AD), interact with the cellular membrane and induce amyloid toxicity. The composition of cellular membranes changes in aging and AD. We designed multi-component lipid models to mimic healthy and diseased states of the neuronal membrane. Using atomic force microscopy (AFM), Kelvin probe force microscopy (KPFM) and black lipid membrane (BLM) techniques, we demonstrated that these model membranes differ in their nanoscale structure and physical properties, and interact differently with Aβ1–42. Based on our data, we propose a new hypothesis that changes in lipid membrane due to aging and AD may trigger amyloid toxicity through electrostatic mechanisms, similar to the accepted mechanism of antimicrobial peptide action. Understanding the role of the membrane changes as a key activating amyloid toxicity may aid in the development of a new avenue for the prevention and treatment of AD.
Mitochondrial permeability transition pore (mPTP) opening allows free movement of ions and small molecules leading to mitochondrial membrane depolarization and ATP depletion that triggers cell death. A multi-protein complex of the mitochondrial ATP synthase has an essential role in mPTP. However, the molecular identity of the central 'pore' part of mPTP complex is not known. A highly purified fraction of mammalian mitochondria containing C-subunit of ATPase (C-subunit), calcium, inorganic polyphosphate (polyP) and polyhydroxybutyrate (PHB) forms ion channels with properties that resemble the native mPTP. We demonstrate here that amount of this channel-forming complex dramatically increases in intact mitochondria during mPTP activation. This increase is inhibited by both Cyclosporine A, an inhibitor of mPTP and Ruthenium Red, an inhibitor of the Mitochondrial Calcium Uniporter. Similar increases in the amount of complex formation occurs in areas of mouse brain damaged by ischemia-reperfusion injury. These findings suggest that calcium-induced mPTP is associated with de novo assembly of a channel comprising C-subunit, polyP and PHB.
Aggregation of α -synuclein leads to the formation of oligomeric intermediates that caninteract with membranes to form pores. However it is unknown how this leads to cell toxicity in Parkinson's disease. We investigated the species-specific effects of αsynuclein on calcium signalling in primary neurons and astrocytes using live neuronal imaging and electrophysiology on artificial membranes. We demonstrate that αsynuclein induces an increase in basal intracellular calcium in its unfolded monomeric state as well as in its oligomeric state. Electrophysiology of artificial membranes demonstrated that α-synuclein monomers induce irregular ionic currents, while αsynuclein oligomers induce rare discrete channel formation. Despite the ability for monomeric α-synuclein to affect calcium signalling, it is only the oligomeric form of αsynuclein that induces cell death. Oligomer-induced cell death was abolished by the exclusion of extracellular calcium, which prevented the α-synuclein induced calcium dysregulation. The findings of this study confirm that α-synuclein interacts with membranes to affect calcium signalling in a structure-specific manner and the oligomeric beta sheet rich α-synuclein species ultimately leads to calcium dysregulation and calcium dependent cell death.
The native conformation of the 325‐residue outer membrane protein A (OmpA) of Escherichia coli has been a matter of contention. A narrow‐pore, two‐domain structure has vied with a large‐pore, single‐domain structure. Our recent studies show that Ser163 and Ser167 of the N‐terminal domain (1–170) are modified in the cytoplasm by covalent attachment of oligo‐(R)‐3‐hydroxybutyrates (cOHBs), and further show that these modifications are essential for the N‐terminal domain to be incorporated into planar lipid bilayers as narrow pores (∼ 80 pS, 1 m KCl, 22 °C). Here, we examined the potential effect(s) of periplasmic modifications on pore structure by comparing OmpA isolated from outer membranes (M‐OmpA) with OmpA isolated from cytoplasmic inclusion bodies (I‐OmpA). Chemical and western blot analysis and 1H‐NMR showed that segment 264–325 in M‐OmpA, but not in I‐OmpA, is modified by cOHBs. Moreover, a disulfide bond is formed between Cys290 and Cys302 by the periplasmic enzyme DsbA. Planar lipid bilayer studies indicated that narrow pores formed by M‐OmpA undergo a temperature‐induced transition into stable large pores (∼ 450 pS, 1 m KCl, 22 °C) [energy of activation (Ea) = 33.2 kcal·mol−1], but this transition does not occur with I‐OmpA or with M‐OmpA that has been exposed to disulfide bond‐reducing agents. The results suggest that the narrow pore is a folding intermediate, and demonstrate the decisive roles of cOHB‐modification, disulfide bond formation and temperature in folding OmpA into its native large‐pore configuration.
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