Modulating membrane binding of α-synuclein as a therapeutic strategy Andre´ Pinedaa and Jacqueline Burre´ a,1
α-Synuclein aggregation is a pathological hallmark of Parkinson’s disease (PD), Lewy body dementia, multiple system atrophy, and a variety of other synucleinopathies (1). The occurrence of familial PD due to α-synuclein mutations, gene duplication and triplication, as well as polymorphisms in regulatory elements of the α-synuclein gene, supports a causative role of α-synuclein in these neurodegenerative diseases (reviewed in ref. 2). In addition, a prion-like spread of α-synuclein pathology has been proposed, by propagation of neurotoxic α-synuclein aggregates from one neuron to the other (3). Attenuating or stopping aggregation of α-synuclein is thus a highly pursued strategy to combat pathology in these diseases (Fig. 1). A number of factors have been reported to influence the aggregation propensity of α-synuclein, including oxidative stress, posttranslational modifications in α-synuclein, and increased local concentration. Proposed strategies to stop or reduce α-synuclein aggregation include enhancing the levels of heat shock proteins to stabilize protein folding, using compounds with antioxidant or antiaggregant activity, promoting intracellular degradation of α-synuclein, or immunotherapies to clear α-synuclein (reviewed in ref. 4). However, none of these approaches has had success in translation to the clinic, and treatments for PD remain symptomatic, focusing on treating the movement disorder symptoms associated with loss of dopaminergic neurons in the substantia nigra pars compacta. Treatment of the nonmotor symptoms is much more challenging and less established, and no therapy is available that slows down or stops the progression of PD. In PNAS, Perni et al. (5) report an inhibitory effect of squalamine—an antimicrobial agent derived from the dogfish shark—on α-synuclein aggregation in vitro and toxicity in vivo, via displacing α-synuclein from phospholipid membranes. Physiologically, α-synuclein exists in a dynamic equilibrium between a natively unfolded cytosolic state and a multimeric membrane-bound state on synaptic vesicles (6, 7). The N-terminal sequence of α-synuclein forms an amphipathic α-helix that mediates association of α-synuclein with lipid membranes (8). This region also contains the non–amyloid-β component (NAC), an area believed
Fig. 1. Physiological and pathological conformations of α-synuclein and strategies to combat α-synuclein aggregation and toxicity. Physiologically, α-synuclein exists in an equilibrium between α-helical multimers bound to synaptic vesicles, and a natively unfolded monomeric state. Membranous α-synuclein clusters synaptic vesicles and chaperones SNARE complex assembly to maintain neurotransmitter release. Under pathological conditions, partial membrane binding of α-synuclein increases the local concentration of the aggregation-prone NAC region, allowing seeding of α-synuclein aggregation. Similarly, cytosolic monomeric α-synuclein spontaneously forms oligomers, which eventually build up as amyloid fibrils and spread to neurons and glia in a prion-like manner. Various therapeutic strategies are being pursued to prevent α-synuclein–mediated pathology, by modulating α-synuclein aggregation, transsynaptic spread, or membrane binding.
to be responsible for α-synuclein aggregation (9). The interaction between α-synuclein and synaptic membranes is believed to be a key feature for mediating its proposed cellular functions: the presynaptic localization of α-synuclein, its interaction with synaptic vesicles and synaptobrevin-2, its SNARE complex-chaperoning activity, its effects on vesicle clustering, and its changes during periods of song acquisition-related synaptic rearrangements in birds (reviewed in ref. 10) strongly suggest that α-synuclein plays a role in neurotransmitter
a Appel Institute for Alzheimer’s Disease Research, Brain and Mind Research Institute, Weill Cornell Medicine, New York, NY 10021 Author contributions: A.P. and J.B. wrote the paper. The authors declare no conflict of interest. See companion article on page E1009. 1 To whom correspondence should be addressed. Email: [email protected].
www.pnas.org/cgi/doi/10.1073/pnas.1620159114
PNAS | February 7, 2017 | vol. 114 | no. 6 | 1223–1225
COMMENTARY
COMMENTARY
release and synaptic plasticity (Fig. 1), although its precise function remains unclear. In contrast, under pathological conditions, α-synuclein adopts a β-sheet–rich amyloid conformation, which is associated with α-synuclein aggregation, fibril formation, and deposition into Lewy bodies (11). Although these β-sheet–rich oligomers are thought to lead to neurodegeneration, the exact nature of the toxic species of α-synuclein remains unknown. In addition, it remains controversial whether aggregation of α-synuclein initiates from its lipid-bound α-helical form or from its unstructured cytosolic state. Membranes have been reported to both accelerate (5, 12, 13) and inhibit α-synuclein fibril formation (14–16). Identification of the aggregation-prone pool of α-synuclein is important to mechanistically understand how aggregates form and how aggregation can be prevented. The presence of α-synuclein on membranes may drastically alter its aggregation dynamics and propensity, via increasing the local concentration of α-synuclein, and long-range intramolecular interactions between the N and C termini of α-synuclein have been shown to be crucial for the process of fibril formation (17). However, binding of the 45 C-terminal residues of α-synuclein to synaptobrevin-2 on the synaptic vesicle membrane (18) shields the C terminus and may prevent contacts between the N and C termini of α-synuclein, thereby impeding aggregation. Strikingly, all identified familial PD mutations are located within the lipid-binding domain of α-synuclein, suggesting that changes in lipid binding may be linked to α-synuclein pathology. E46K and A53T do not affect lipid binding of α-synuclein but increase its aggregation, A30P reduces lipid binding and increases aggregation, A53E and G51D reduce lipid binding and aggregation of α-synuclein, and H50Q increases aggregation of α-synuclein with unclear effects on lipid binding (reviewed in ref. 19), suggesting that lipid binding and aggregation are not tightly correlated. However, partial membrane binding of α-synuclein where the NAC domain remains unbound (20, 21) may result in local crowding of the aggregation-prone NAC domain and subsequently trigger seeding of α-synuclein aggregation (Fig. 1). Thus, abolishing these misfolded states of α-synuclein on lipid membranes, as proposed by Perni et al. (5), could reduce α-synuclein aggregation and toxicity in vivo. Applying biophysical techniques, Perni et al. (5) measured the ability of recombinant α-synuclein to associate with artificial liposomes in the presence of increasing concentrations of squalamine, and found that squalamine displaced α-synuclein from highly charged liposomes in a concentration-dependent manner. This displacement of α-synuclein from liposomes correlated with a decrease in α-synuclein aggregation. The authors mention that squalamine displayed a high degree of binding to α-synuclein fibrils, which may at least partially explain the observed inhibition of squalamine on the lipid-induced aggregation of α-synuclein. The authors then went on to test squalamine in a neuroblastoma cell line treated with recombinant α-synuclein oligomers and found that squalamine decreased mitochondrial damage and reactive oxygen species
production in these cells, accompanied by a reduction in α-synuclein oligomer binding to the outer leaflet of the plasma membrane. Finally, the authors demonstrated in the nematode Caenorhabditis elegans an improvement in motility of α-synuclein-YFP–overexpressing worms and a reduction in α-synuclein inclusion formation in aged worms upon administration of squalamine. From these data, the
In PNAS, Perni et al. report an inhibitory effect of squalamine—an antimicrobial agent derived from the dogfish shark—on α-synuclein aggregation in vitro and toxicity in vivo, via displacing α-synuclein from phospholipid membranes. authors suggest that squalamine has a specific effect against α-synuclein aggregation, and propose a competitive binding model where squalamine and toxic α-synuclein oligomers compete for binding sites at the surface of vesicles and neurons. Squalamine is an interesting compound. Originally discovered in the tissues of the dogfish shark (Squalus acanthias) as a broad-spectrum antimicrobial agent in 1993 (22), it has been reported to have systemic antiviral properties with therapeutic potential (23). Its positive net charge neutralizes the negative charge of anionic phospholipids, resulting in the displacement of proteins that are associated with the inner face of the cytoplasmic membrane through electrostatic interactions (23), thereby making the cell less capable of supporting the replication of certain viruses. Squalamine exits cells and is cleared from circulation within hours, and dosing and toxicology are established for treating pathological angiogenesis in humans (24). However, squalamine cannot enter the brain after systemic administration, has been shown to result in permeabilization of large phospholipid vesicles (25) and altered liposome fusion, diameter, and melting temperature (5), and may have nonspecific effects on other proteins that associate with membranes by electrostatic forces. Nonetheless, the study by Perni et al. (5) reveals a striking effect of squalamine on reducing α-synuclein–mediated aggregation and toxicity in vitro and in vivo. Although displacing α-synuclein from its physiologically active location on synaptic vesicle membranes could be detrimental for the long-term operation of the nervous system and may even further promote neuropathology in synucleinopathies, squalamine may be particularly useful for inhibiting the prion-like spread of α-synuclein, by acting on α-synuclein oligomers in the extracellular space and blocking their binding to neuronal membranes and thereby their uptake into neurons. Thus, modulating the membrane binding of α-synuclein is an important strategy in disease intervention, but a greater understanding is needed to inhibit the pathological activities of α-synuclein while preserving its physiological functions on the synaptic vesicle membrane.
Acknowledgments Our research is supported by the American Parkinson Disease Association (J.B.).
1 Spillantini MG, et al. (1997) Alpha-synuclein in Lewy bodies. Nature 388(6645):839–840. 2 Benskey MJ, Perez RG, Manfredsson FP (2016) The contribution of alpha synuclein to neuronal survival and function—implications for Parkinson’s disease. J Neurochem 137(3):331–359. 3 Desplats P, et al. (2009) Inclusion formation and neuronal cell death through neuron-to-neuron transmission of alpha-synuclein. Proc Natl Acad Sci USA 106(31): 13010–13015. 4 Dehay B, et al. (2015) Targeting α-synuclein for treatment of Parkinson’s disease: Mechanistic and therapeutic considerations. Lancet Neurol 14(8):855–866. 5 Perni M, et al. (2017) A natural product inhibits the initiation of α-synuclein aggregation and suppresses its toxicity. Proc Natl Acad Sci USA 114:E1009–E1017. 6 Burre´ J, Sharma M, Südhof TC (2014) α-Synuclein assembles into higher-order multimers upon membrane binding to promote SNARE complex formation. Proc Natl Acad Sci USA 111(40):E4274–E4283.
1224 | www.pnas.org/cgi/doi/10.1073/pnas.1620159114
Pineda and Burre´
7 Weinreb PH, Zhen W, Poon AW, Conway KA, Lansbury PT, Jr (1996) NACP, a protein implicated in Alzheimer’s disease and learning, is natively unfolded. Biochemistry 35(43):13709–13715. 8 Davidson WS, Jonas A, Clayton DF, George JM (1998) Stabilization of alpha-synuclein secondary structure upon binding to synthetic membranes. J Biol Chem 273(16):9443–9449. 9 Giasson BI, Murray IV, Trojanowski JQ, Lee VM (2001) A hydrophobic stretch of 12 amino acid residues in the middle of alpha-synuclein is essential for filament assembly. J Biol Chem 276(4):2380–2386. 10 Burre´ J (2015) The synaptic function of α-synuclein. J Parkinsons Dis 5(4):699–713. 11 Conway KA, Harper JD, Lansbury PT, Jr (2000) Fibrils formed in vitro from alpha-synuclein and two mutant forms linked to Parkinson’s disease are typical amyloid. Biochemistry 39(10):2552–2563. 12 Cole NB, et al. (2002) Lipid droplet binding and oligomerization properties of the Parkinson’s disease protein alpha-synuclein. J Biol Chem 277(8):6344–6352. 13 Lee HJ, Choi C, Lee SJ (2002) Membrane-bound alpha-synuclein has a high aggregation propensity and the ability to seed the aggregation of the cytosolic form. J Biol Chem 277(1):671–678. 14 Narayanan V, Scarlata S (2001) Membrane binding and self-association of alpha-synucleins. Biochemistry 40(33):9927–9934. 15 Zhu M, Fink AL (2003) Lipid binding inhibits alpha-synuclein fibril formation. J Biol Chem 278(19):16873–16877. 16 Burre´ J, Sharma M, Südhof TC (2015) Definition of a molecular pathway mediating α-synuclein neurotoxicity. J Neurosci 35(13):5221–5232. 17 Ulrih NP, Barry CH, Fink AL (2008) Impact of Tyr to Ala mutations on alpha-synuclein fibrillation and structural properties. Biochim Biophys Acta 1782(10):581–585. 18 Burre´ J, et al. (2010) Alpha-synuclein promotes SNARE-complex assembly in vivo and in vitro. Science 329(5999):1663–1667. 19 Snead D, Eliezer D (2014) Alpha-synuclein function and dysfunction on cellular membranes. Exp Neurobiol 23(4):292–313. 20 Bodner CR, Maltsev AS, Dobson CM, Bax A (2010) Differential phospholipid binding of alpha-synuclein variants implicated in Parkinson’s disease revealed by solution NMR spectroscopy. Biochemistry 49(5):862–871. 21 Drescher M, et al. (2008) Spin-label EPR on alpha-synuclein reveals differences in the membrane binding affinity of the two antiparallel helices. ChemBioChem 9(15):2411–2416. 22 Moore KS, et al. (1993) Squalamine: An aminosterol antibiotic from the shark. Proc Natl Acad Sci USA 90(4):1354–1358. 23 Zasloff M, et al. (2011) Squalamine as a broad-spectrum systemic antiviral agent with therapeutic potential. Proc Natl Acad Sci USA 108(38):15978–15983. 24 Bhargava P, et al. (2001) A phase I and pharmacokinetic study of squalamine, a novel antiangiogenic agent, in patients with advanced cancers. Clin Cancer Res 7(12):3912–3919. 25 Selinsky BS, et al. (1998) The aminosterol antibiotic squalamine permeabilizes large unilamellar phospholipid vesicles. Biochim Biophys Acta 1370(2):218–234.
Pineda and Burre´
PNAS | February 7, 2017 | vol. 114 | no. 6 | 1225
α-Synuclein aggregation is a pathological hallmark of Parkinson’s disease (PD), Lewy body dementia, multiple system atrophy, and a variety of other synucleinopathies (1). The occurrence of familial PD due to α-synuclein mutations, gene duplication and triplication, as well as polymorphisms in regulatory elements of the α-synuclein gene, supports a causative role of α-synuclein in these neurodegenerative diseases (reviewed in ref. 2). In addition, a prion-like spread of α-synuclein pathology has been proposed, by propagation of neurotoxic α-synuclein aggregates from one neuron to the other (3). Attenuating or stopping aggregation of α-synuclein is thus a highly pursued strategy to combat pathology in these diseases (Fig. 1). A number of factors have been reported to influence the aggregation propensity of α-synuclein, including oxidative stress, posttranslational modifications in α-synuclein, and increased local concentration. Proposed strategies to stop or reduce α-synuclein aggregation include enhancing the levels of heat shock proteins to stabilize protein folding, using compounds with antioxidant or antiaggregant activity, promoting intracellular degradation of α-synuclein, or immunotherapies to clear α-synuclein (reviewed in ref. 4). However, none of these approaches has had success in translation to the clinic, and treatments for PD remain symptomatic, focusing on treating the movement disorder symptoms associated with loss of dopaminergic neurons in the substantia nigra pars compacta. Treatment of the nonmotor symptoms is much more challenging and less established, and no therapy is available that slows down or stops the progression of PD. In PNAS, Perni et al. (5) report an inhibitory effect of squalamine—an antimicrobial agent derived from the dogfish shark—on α-synuclein aggregation in vitro and toxicity in vivo, via displacing α-synuclein from phospholipid membranes. Physiologically, α-synuclein exists in a dynamic equilibrium between a natively unfolded cytosolic state and a multimeric membrane-bound state on synaptic vesicles (6, 7). The N-terminal sequence of α-synuclein forms an amphipathic α-helix that mediates association of α-synuclein with lipid membranes (8). This region also contains the non–amyloid-β component (NAC), an area believed
Fig. 1. Physiological and pathological conformations of α-synuclein and strategies to combat α-synuclein aggregation and toxicity. Physiologically, α-synuclein exists in an equilibrium between α-helical multimers bound to synaptic vesicles, and a natively unfolded monomeric state. Membranous α-synuclein clusters synaptic vesicles and chaperones SNARE complex assembly to maintain neurotransmitter release. Under pathological conditions, partial membrane binding of α-synuclein increases the local concentration of the aggregation-prone NAC region, allowing seeding of α-synuclein aggregation. Similarly, cytosolic monomeric α-synuclein spontaneously forms oligomers, which eventually build up as amyloid fibrils and spread to neurons and glia in a prion-like manner. Various therapeutic strategies are being pursued to prevent α-synuclein–mediated pathology, by modulating α-synuclein aggregation, transsynaptic spread, or membrane binding.
to be responsible for α-synuclein aggregation (9). The interaction between α-synuclein and synaptic membranes is believed to be a key feature for mediating its proposed cellular functions: the presynaptic localization of α-synuclein, its interaction with synaptic vesicles and synaptobrevin-2, its SNARE complex-chaperoning activity, its effects on vesicle clustering, and its changes during periods of song acquisition-related synaptic rearrangements in birds (reviewed in ref. 10) strongly suggest that α-synuclein plays a role in neurotransmitter
a Appel Institute for Alzheimer’s Disease Research, Brain and Mind Research Institute, Weill Cornell Medicine, New York, NY 10021 Author contributions: A.P. and J.B. wrote the paper. The authors declare no conflict of interest. See companion article on page E1009. 1 To whom correspondence should be addressed. Email: [email protected].
www.pnas.org/cgi/doi/10.1073/pnas.1620159114
PNAS | February 7, 2017 | vol. 114 | no. 6 | 1223–1225
COMMENTARY
COMMENTARY
release and synaptic plasticity (Fig. 1), although its precise function remains unclear. In contrast, under pathological conditions, α-synuclein adopts a β-sheet–rich amyloid conformation, which is associated with α-synuclein aggregation, fibril formation, and deposition into Lewy bodies (11). Although these β-sheet–rich oligomers are thought to lead to neurodegeneration, the exact nature of the toxic species of α-synuclein remains unknown. In addition, it remains controversial whether aggregation of α-synuclein initiates from its lipid-bound α-helical form or from its unstructured cytosolic state. Membranes have been reported to both accelerate (5, 12, 13) and inhibit α-synuclein fibril formation (14–16). Identification of the aggregation-prone pool of α-synuclein is important to mechanistically understand how aggregates form and how aggregation can be prevented. The presence of α-synuclein on membranes may drastically alter its aggregation dynamics and propensity, via increasing the local concentration of α-synuclein, and long-range intramolecular interactions between the N and C termini of α-synuclein have been shown to be crucial for the process of fibril formation (17). However, binding of the 45 C-terminal residues of α-synuclein to synaptobrevin-2 on the synaptic vesicle membrane (18) shields the C terminus and may prevent contacts between the N and C termini of α-synuclein, thereby impeding aggregation. Strikingly, all identified familial PD mutations are located within the lipid-binding domain of α-synuclein, suggesting that changes in lipid binding may be linked to α-synuclein pathology. E46K and A53T do not affect lipid binding of α-synuclein but increase its aggregation, A30P reduces lipid binding and increases aggregation, A53E and G51D reduce lipid binding and aggregation of α-synuclein, and H50Q increases aggregation of α-synuclein with unclear effects on lipid binding (reviewed in ref. 19), suggesting that lipid binding and aggregation are not tightly correlated. However, partial membrane binding of α-synuclein where the NAC domain remains unbound (20, 21) may result in local crowding of the aggregation-prone NAC domain and subsequently trigger seeding of α-synuclein aggregation (Fig. 1). Thus, abolishing these misfolded states of α-synuclein on lipid membranes, as proposed by Perni et al. (5), could reduce α-synuclein aggregation and toxicity in vivo. Applying biophysical techniques, Perni et al. (5) measured the ability of recombinant α-synuclein to associate with artificial liposomes in the presence of increasing concentrations of squalamine, and found that squalamine displaced α-synuclein from highly charged liposomes in a concentration-dependent manner. This displacement of α-synuclein from liposomes correlated with a decrease in α-synuclein aggregation. The authors mention that squalamine displayed a high degree of binding to α-synuclein fibrils, which may at least partially explain the observed inhibition of squalamine on the lipid-induced aggregation of α-synuclein. The authors then went on to test squalamine in a neuroblastoma cell line treated with recombinant α-synuclein oligomers and found that squalamine decreased mitochondrial damage and reactive oxygen species
production in these cells, accompanied by a reduction in α-synuclein oligomer binding to the outer leaflet of the plasma membrane. Finally, the authors demonstrated in the nematode Caenorhabditis elegans an improvement in motility of α-synuclein-YFP–overexpressing worms and a reduction in α-synuclein inclusion formation in aged worms upon administration of squalamine. From these data, the
In PNAS, Perni et al. report an inhibitory effect of squalamine—an antimicrobial agent derived from the dogfish shark—on α-synuclein aggregation in vitro and toxicity in vivo, via displacing α-synuclein from phospholipid membranes. authors suggest that squalamine has a specific effect against α-synuclein aggregation, and propose a competitive binding model where squalamine and toxic α-synuclein oligomers compete for binding sites at the surface of vesicles and neurons. Squalamine is an interesting compound. Originally discovered in the tissues of the dogfish shark (Squalus acanthias) as a broad-spectrum antimicrobial agent in 1993 (22), it has been reported to have systemic antiviral properties with therapeutic potential (23). Its positive net charge neutralizes the negative charge of anionic phospholipids, resulting in the displacement of proteins that are associated with the inner face of the cytoplasmic membrane through electrostatic interactions (23), thereby making the cell less capable of supporting the replication of certain viruses. Squalamine exits cells and is cleared from circulation within hours, and dosing and toxicology are established for treating pathological angiogenesis in humans (24). However, squalamine cannot enter the brain after systemic administration, has been shown to result in permeabilization of large phospholipid vesicles (25) and altered liposome fusion, diameter, and melting temperature (5), and may have nonspecific effects on other proteins that associate with membranes by electrostatic forces. Nonetheless, the study by Perni et al. (5) reveals a striking effect of squalamine on reducing α-synuclein–mediated aggregation and toxicity in vitro and in vivo. Although displacing α-synuclein from its physiologically active location on synaptic vesicle membranes could be detrimental for the long-term operation of the nervous system and may even further promote neuropathology in synucleinopathies, squalamine may be particularly useful for inhibiting the prion-like spread of α-synuclein, by acting on α-synuclein oligomers in the extracellular space and blocking their binding to neuronal membranes and thereby their uptake into neurons. Thus, modulating the membrane binding of α-synuclein is an important strategy in disease intervention, but a greater understanding is needed to inhibit the pathological activities of α-synuclein while preserving its physiological functions on the synaptic vesicle membrane.
Acknowledgments Our research is supported by the American Parkinson Disease Association (J.B.).
1 Spillantini MG, et al. (1997) Alpha-synuclein in Lewy bodies. Nature 388(6645):839–840. 2 Benskey MJ, Perez RG, Manfredsson FP (2016) The contribution of alpha synuclein to neuronal survival and function—implications for Parkinson’s disease. J Neurochem 137(3):331–359. 3 Desplats P, et al. (2009) Inclusion formation and neuronal cell death through neuron-to-neuron transmission of alpha-synuclein. Proc Natl Acad Sci USA 106(31): 13010–13015. 4 Dehay B, et al. (2015) Targeting α-synuclein for treatment of Parkinson’s disease: Mechanistic and therapeutic considerations. Lancet Neurol 14(8):855–866. 5 Perni M, et al. (2017) A natural product inhibits the initiation of α-synuclein aggregation and suppresses its toxicity. Proc Natl Acad Sci USA 114:E1009–E1017. 6 Burre´ J, Sharma M, Südhof TC (2014) α-Synuclein assembles into higher-order multimers upon membrane binding to promote SNARE complex formation. Proc Natl Acad Sci USA 111(40):E4274–E4283.
1224 | www.pnas.org/cgi/doi/10.1073/pnas.1620159114
Pineda and Burre´
7 Weinreb PH, Zhen W, Poon AW, Conway KA, Lansbury PT, Jr (1996) NACP, a protein implicated in Alzheimer’s disease and learning, is natively unfolded. Biochemistry 35(43):13709–13715. 8 Davidson WS, Jonas A, Clayton DF, George JM (1998) Stabilization of alpha-synuclein secondary structure upon binding to synthetic membranes. J Biol Chem 273(16):9443–9449. 9 Giasson BI, Murray IV, Trojanowski JQ, Lee VM (2001) A hydrophobic stretch of 12 amino acid residues in the middle of alpha-synuclein is essential for filament assembly. J Biol Chem 276(4):2380–2386. 10 Burre´ J (2015) The synaptic function of α-synuclein. J Parkinsons Dis 5(4):699–713. 11 Conway KA, Harper JD, Lansbury PT, Jr (2000) Fibrils formed in vitro from alpha-synuclein and two mutant forms linked to Parkinson’s disease are typical amyloid. Biochemistry 39(10):2552–2563. 12 Cole NB, et al. (2002) Lipid droplet binding and oligomerization properties of the Parkinson’s disease protein alpha-synuclein. J Biol Chem 277(8):6344–6352. 13 Lee HJ, Choi C, Lee SJ (2002) Membrane-bound alpha-synuclein has a high aggregation propensity and the ability to seed the aggregation of the cytosolic form. J Biol Chem 277(1):671–678. 14 Narayanan V, Scarlata S (2001) Membrane binding and self-association of alpha-synucleins. Biochemistry 40(33):9927–9934. 15 Zhu M, Fink AL (2003) Lipid binding inhibits alpha-synuclein fibril formation. J Biol Chem 278(19):16873–16877. 16 Burre´ J, Sharma M, Südhof TC (2015) Definition of a molecular pathway mediating α-synuclein neurotoxicity. J Neurosci 35(13):5221–5232. 17 Ulrih NP, Barry CH, Fink AL (2008) Impact of Tyr to Ala mutations on alpha-synuclein fibrillation and structural properties. Biochim Biophys Acta 1782(10):581–585. 18 Burre´ J, et al. (2010) Alpha-synuclein promotes SNARE-complex assembly in vivo and in vitro. Science 329(5999):1663–1667. 19 Snead D, Eliezer D (2014) Alpha-synuclein function and dysfunction on cellular membranes. Exp Neurobiol 23(4):292–313. 20 Bodner CR, Maltsev AS, Dobson CM, Bax A (2010) Differential phospholipid binding of alpha-synuclein variants implicated in Parkinson’s disease revealed by solution NMR spectroscopy. Biochemistry 49(5):862–871. 21 Drescher M, et al. (2008) Spin-label EPR on alpha-synuclein reveals differences in the membrane binding affinity of the two antiparallel helices. ChemBioChem 9(15):2411–2416. 22 Moore KS, et al. (1993) Squalamine: An aminosterol antibiotic from the shark. Proc Natl Acad Sci USA 90(4):1354–1358. 23 Zasloff M, et al. (2011) Squalamine as a broad-spectrum systemic antiviral agent with therapeutic potential. Proc Natl Acad Sci USA 108(38):15978–15983. 24 Bhargava P, et al. (2001) A phase I and pharmacokinetic study of squalamine, a novel antiangiogenic agent, in patients with advanced cancers. Clin Cancer Res 7(12):3912–3919. 25 Selinsky BS, et al. (1998) The aminosterol antibiotic squalamine permeabilizes large unilamellar phospholipid vesicles. Biochim Biophys Acta 1370(2):218–234.
Pineda and Burre´
PNAS | February 7, 2017 | vol. 114 | no. 6 | 1225
