Protein aggregation causes α-synuclein to switch from its physiological role to a pathological toxic gain of function. Under physiological conditions, monomeric α-synuclein improves ATP synthase efficiency. Here, we report that aggregation of monomers generates beta sheet-rich oligomers that localise to the mitochondria in close proximity to several mitochondrial proteins including ATP synthase. Oligomeric α-synuclein impairs complex I-dependent respiration. Oligomers induce selective oxidation of the ATP synthase beta subunit and mitochondrial lipid peroxidation. These oxidation events increase the probability of permeability transition pore (PTP) opening, triggering mitochondrial swelling, and ultimately cell death. Notably, inhibition of oligomer-induced oxidation prevents the pathological induction of PTP. Inducible pluripotent stem cells (iPSC)-derived neurons bearing SNCA triplication, generate α-synuclein aggregates that interact with the ATP synthase and induce PTP opening, leading to neuronal death. This study shows how the transition of α-synuclein from its monomeric to oligomeric structure alters its functional consequences in Parkinson’s disease.

During apoptosis, proapoptotic factors are released from mitochondria by as yet undefined mechanisms. Patch-clamping of mitochondria and proteoliposomes formed from mitochondrial outer membranes of mammalian (FL5.12) cells has uncovered a novel ion channel whose activity correlates with onset of apoptosis. The pore diameter inferred from the largest conductance state of this channel is ∼4 nm, sufficient to allow diffusion of cytochrome c and even larger proteins. The activity of the channel is affected by Bcl-2 family proteins in a manner consistent with their pro- or antiapoptotic properties. Thus, the channel activity correlates with presence of proapoptotic Bax in the mitochondrial outer membrane and is absent in mitochondria from cells overexpressing antiapoptotic Bcl-2. Also, a similar channel activity is found in mitochondrial outer membranes of yeast expressing human Bax. These findings implicate this channel, named mitochondrial apoptosis–induced channel, as a candidate for the outer-membrane pore through which cytochrome c and possibly other factors exit mitochondria during apoptosis.

Polyphosphate (poly-P) is an important metabolite and signaling molecule in prokaryotes and eukaryotes. DAPI (4',6-diamidino-2-phenylindole), a widely used fluorescent label for DNA, also interacts with polyphosphate. Binding of poly-P to DAPI, shifts its peak emission wavelength from 475 to 525 nm (excitation at 360 nm), allowing use of DAPI for detection of poly-P in vitro, and in live poly-P accumulating organisms. This approach, which relies on detection of a shift in fluorescence emission, allows use of DAPI only for qualitative detection of relatively high concentrations of poly-P, in the microg/ml range. Here, we report that long-wavelength excitation (> or = 400 nm) of the DAPI-poly-P complex provides a dramatic increase in the sensitivity of poly-P detection. Using excitation at 415 nm, fluorescence of the DAPI-poly-P complex can be detected at a higher wavelength (550 nm) for as little as 25 ng/ml of poly-P. Fluorescence emission from free DAPI and DAPI-DNA are minimal at this wavelength, making the DAPI-poly-P signal highly specific and essentially independent of the presence of DNA. In addition, we demonstrate the use of this protocol to measure the activity of poly-P hydrolyzing enzyme, polyphosphatase and demonstrate a similar signal from the mitochondrial region of cultured neurons.

Inorganic polyphosphate (poly P) is a polymer made from as few as 10 to several hundred phosphate molecules linked by phosphoanhydride bonds similar to ATP. Poly P is ubiquitous in all mammalian organisms, where it plays multiple physiological roles. The metabolism of poly P in mammalian organisms is not well understood. We have examined the mechanism of poly P production and the role of this polymer in cell energy metabolism. Poly P levels in mitochondria and intact cells were estimated using a fluorescent molecular probe, 4,6-diamidino-2-phenylindole. Poly P levels were dependent on the metabolic state of the mitochondria. Poly P levels were increased by substrates of respiration and in turn reduced by mitochondrial inhibitor (rotenone) or an uncoupler (carbonyl cyanide p-trifluoromethoxyphenylhydrazone). Oligomycin, an inhibitor of mitochondrial ATP-synthase, blocked the production of poly P. Enzymatic depletion of poly P from cells significantly altered the rate of ATP metabolism. We propose the existence of a feedback mechanism where poly P production and cell energy metabolism regulate each other. Inorganic polyphosphate (poly P)2 is found in all living organisms ranging from bacteria to mammals (1). Poly P performs multiple physiological functions, which are distinct and dependent on the type of organism and the subcellular localization of the polymer. In microorganisms, poly P primarily plays a role in transcription. Additionally, poly P serves as an energy store (2) and as a reserve pool of inorganic phosphates (3). However, in mammalian organisms, poly P plays predominantly a regulatory role (4) and has been implicated in the regulation of enzyme activity in cancer cells (5), stimulation of blood coagulation (6), regulation of mitochondrial ion transport (7), and regulation of respiratory chain activity (8).Although a specific enzyme(s) responsible for poly P production in mammals is currently not known (1), poly P synthesis has been detected in intact mammalian cells. Lysis of mammalian cells leads to loss of poly P synthesis activity, suggesting that poly P synthesis in mammalian cells is likely an energy-dependent process linked to membrane transport and integrity (1, 9). Taking into account that the membrane potential generated at the mitochondrial inner membrane is a major energy source for cellular metabolism, we hypothesized that mitochondria may be the likely source of poly P production in mammalian cells.Poly P is found in mammalian cells at significantly lower levels when compared with microorganisms (9); therefore, it is very difficult to adapt poly P measurement methods developed for bacterial studies for the study of mammalian cells. Recently we developed a protocol, which we optimized for suitability for measuring low amounts of poly P using the fluorescent probe 4Ј,6-diamidino-2-phenylindole (DAPI) (10). In our previous study we used this method to confirm poly P hydrolyzing activity of yeast polyphosphatase expressed in mitochondria of mammalian cultured cells (8). Here we take advantag...

Polyphosphate (polyP) consists of tens to hundreds of phosphates, linked by ATP-like high-energy bonds. Although polyP is present in mammalian mitochondria, its physiological roles there are obscure. Here, we examine the involvement of polyP in mitochondrial energy metabolism and ion transport. We constructed a vector to express a mitochondrially targeted polyphosphatase, along with a GFP fluorescent tag. Specific reduction of mitochondrial polyP, by polyphosphatase expression, significantly modulates mitochondrial bioenergetics, as indicated by the reduction of inner membrane potential and increased NADH levels. Furthermore, reduction of polyP levels increases mitochondrial capacity to accumulate calcium and reduces the likelihood of the calcium-induced mitochondrial permeability transition, a central event in many types of necrotic cell death. This confers protection against cell death, including that induced by ␤-amyloid peptide, a pathogenic agent in Alzheimer's disease. These results demonstrate a crucial role played by polyP in mitochondrial function of mammalian cells. mitochondria ͉ permeability transition ͉ polyphosphate ͉ ␤-amyloid peptide ͉ necrosis T he chemical and physical properties of polyphosphate (polyP), including its high negative charge and its ability to form complexes with Ca 2ϩ and to form high energy bonds, underlie its potential to play an important role in cell metabolism. Significant amounts of polyP have been found in bacteria and in lower eukaryotes. In those organisms, it provides energy storage and a reserve pool of inorganic phosphate, participates in regulation of gene expression, protects cells from the toxicity of heavy metals by forming complexes with them, and participates in channel formation through assembly into complexes with Ca 2ϩ and polyhydroxybutyrate (PHB) (polyP/Ca 2ϩ /PHB complex) (1, 2) and possibly through interaction with channelforming proteins (3).PolyP has also been found in all higher eukaryotic organisms tested, where it is localized in various subcellular compartments, including mitochondria (4). Furthermore, mitochondrial polyP can form polyP/Ca 2ϩ /PHB complexes (5) with ion-conducting properties similar to those of native mitochondrial permeability transition pore (mPTP) (6). mPTP opening or formation in the mitochondrial inner membrane is believed to underlie the Ca 2ϩ -induced permeability transition (PT), a phenomenon that causes inner membrane depolarization and disruption of ATP synthesis and plays a central role during various types of necrotic and apoptotic cell death (7). The molecular composition of the conducting pathway of mPTP is currently not well defined.Recently, we have raised the possibility that, in vivo, the polyP/ Ca 2ϩ /PHB complex might comprise the ion-conducting part of the mPTP complex (6). If so, mitochondrial polyP should be essential for mPTP opening/formation. Here, we examine the involvement of polyP in normal mitochondrial function and in PT development during stress. To this end, we specifically reduced levels of mitocho...

Protein aggregation and abnormal lipid homeostasis are both implicated in neurodegeneration through unknown mechanisms. Here we demonstrate that aggregate-membrane interaction is critical to induce a form of cell death called ferroptosis. Importantly, the aggregate-membrane interaction that drives ferroptosis depends both on the conformational structure of the aggregate, as well as the oxidation state of the lipid membrane. We generated human stem cell-derived models of synucleinopathy, characterized by the intracellular formation of α-synuclein aggregates that bind to membranes. In human iPSC-derived neurons with SNCA triplication, physiological concentrations of glutamate and dopamine induce abnormal calcium signaling owing to the incorporation of excess α-synuclein oligomers into membranes, leading to altered membrane conductance and abnormal calcium influx. α-synuclein oligomers further induce lipid peroxidation. Targeted inhibition of lipid peroxidation prevents the aggregate-membrane interaction, abolishes aberrant calcium fluxes, and restores physiological calcium signaling. Inhibition of lipid peroxidation, and reduction of iron-dependent accumulation of free radicals, further prevents oligomer-induced toxicity in human neurons. In summary, we report that peroxidation of polyunsaturated fatty acids underlies the incorporation of β-sheet-rich aggregates into the membranes, and that additionally induces neuronal death. This suggests a role for ferroptosis in Parkinson’s disease, and highlights a new mechanism by which lipid peroxidation causes cell death.

Highlights d Deletion of the c-subunit leads to loss of the mPTP channel d C-subunit KO mitochondria contain a CSA-sensitive channel d The c-subunit KO channel has lower conductance compared to mPTP d The c-subunit KO channel is sensitive to ANT inhibitors

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.