Limits...
Differential regulation of wild-type and mutant alpha-synuclein binding to synaptic membranes by cytosolic factors.

Wislet-Gendebien S, Visanji NP, Whitehead SN, Marsilio D, Hou W, Figeys D, Fraser PE, Bennett SA, Tandon A - BMC Neurosci (2008)

Bottom Line: However, the regulation of alpha-syn's cellular localization in neurons and the effects of the PD-linked mutations are poorly understood.We further show that 1-O-hexadecyl-2-acetyl-sn-glycero-3-phosphocholine (C16:0 PAF) is one of the principal lipids found in complex with cytosolic proteins and is required to enhance alpha-syn interaction with synaptic membrane.In addition, the impaired membrane binding observed for A30P alpha-syn was significantly mitigated by the presence of protease-sensitive factors in brain cytosol.

View Article: PubMed Central - HTML - PubMed

Affiliation: Centre for Research in Neurodegenerative Diseases, University of Toronto, Toronto, Ontario, M5S 3H2 Canada. s.wislet@ulg.ac.be

ABSTRACT

Background: Alpha-Synuclein (alpha-syn), a 140 amino acid protein associated with presynaptic membranes in brain, is a major constituent of Lewy bodies in Parkinson's disease (PD). Three missense mutations (A30P, A53T and E46K) in the alpha-syn gene are associated with rare autosomal dominant forms of familial PD. However, the regulation of alpha-syn's cellular localization in neurons and the effects of the PD-linked mutations are poorly understood.

Results: In the present study, we analysed the ability of cytosolic factors to regulate alpha-syn binding to synaptic membranes. We show that co-incubation with brain cytosol significantly increases the membrane binding of normal and PD-linked mutant alpha-syn. To characterize cytosolic factor(s) that modulate alpha-syn binding properties, we investigated the ability of proteins, lipids, ATP and calcium to modulate alpha-syn membrane interactions. We report that lipids and ATP are two of the principal cytosolic components that modulate Wt and A53T alpha-syn binding to the synaptic membrane. We further show that 1-O-hexadecyl-2-acetyl-sn-glycero-3-phosphocholine (C16:0 PAF) is one of the principal lipids found in complex with cytosolic proteins and is required to enhance alpha-syn interaction with synaptic membrane. In addition, the impaired membrane binding observed for A30P alpha-syn was significantly mitigated by the presence of protease-sensitive factors in brain cytosol.

Conclusion: These findings suggest that endogenous brain cytosolic factors regulate Wt and mutant alpha-syn membrane binding, and could represent potential targets to influence alpha-syn solubility in brain.

Show MeSH

Related in: MedlinePlus

Effect of cytosol on binding α-syn. (A) Recombinant α-syn (Wt, A30P and A53T) were incubated in presence of different concentrations of KO cytosol (0.5, 1.5, and 3 mg/ml), for 10 min at 37°C. Compared to the control condition (without cytosol), all cytosol concentrations had a significant effect on Wt and A30P α-syn binding, but only the highest concentration of cytosol had a significant effect on A53T α-syn binding (One way ANOVA test, p < 0.0001, Bonferroni's multiple comparison post-test). (B) KO synaptic membranes and α-syn were pre-incubated for 15 minutes at room temperature with KO cytosol. Membranes were then centrifuged at 14000 × g and washed with KOAc buffer to remove unbound factors. Binding of purified α-syn to KO membranes in the absence of cytosol (ctrl) was compared to its binding to cytosol-treated membranes without added cytosol (memb), and to cytosol-treated α-syn incubated with KO membranes (α-syn). No significant difference was observed between the two pre-incubated condition (Student T-test, p > 0.05). (C) KO cytosol was pre-incubated with trypsin or proteinase K for 15 min at 37°C. Enzymes were then respectively inactivated with trypsin inhibitor and PMSF for 10 min at room temperature. Compared to the cytosol condition (cyt) which, as a control, was incubated with the enzyme pre-inactivated by the inhibitor, only A30P α-syn binding was significantly affected by the cytosolic protein digestion (Student T-test, p < 0.0001), whereas no significant differences were observed for Wt and A53T proteins (Student T-test, p > 0.05).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2562387&req=5

Figure 2: Effect of cytosol on binding α-syn. (A) Recombinant α-syn (Wt, A30P and A53T) were incubated in presence of different concentrations of KO cytosol (0.5, 1.5, and 3 mg/ml), for 10 min at 37°C. Compared to the control condition (without cytosol), all cytosol concentrations had a significant effect on Wt and A30P α-syn binding, but only the highest concentration of cytosol had a significant effect on A53T α-syn binding (One way ANOVA test, p < 0.0001, Bonferroni's multiple comparison post-test). (B) KO synaptic membranes and α-syn were pre-incubated for 15 minutes at room temperature with KO cytosol. Membranes were then centrifuged at 14000 × g and washed with KOAc buffer to remove unbound factors. Binding of purified α-syn to KO membranes in the absence of cytosol (ctrl) was compared to its binding to cytosol-treated membranes without added cytosol (memb), and to cytosol-treated α-syn incubated with KO membranes (α-syn). No significant difference was observed between the two pre-incubated condition (Student T-test, p > 0.05). (C) KO cytosol was pre-incubated with trypsin or proteinase K for 15 min at 37°C. Enzymes were then respectively inactivated with trypsin inhibitor and PMSF for 10 min at room temperature. Compared to the cytosol condition (cyt) which, as a control, was incubated with the enzyme pre-inactivated by the inhibitor, only A30P α-syn binding was significantly affected by the cytosolic protein digestion (Student T-test, p < 0.0001), whereas no significant differences were observed for Wt and A53T proteins (Student T-test, p > 0.05).

Mentions: We recently reported that the dissociation of the α-syn from synaptic membranes requires cytosolic proteins as defined by sensitivity to proteases. To further characterize cytosol action on α-syn binding, and because the data in Fig 1C suggests that cytosol activity becomes saturated at high α-syn concentration, we analysed α-syn binding with varying cytosol concentrations over a 6-fold range that we have previously shown to be effective in mobilizing reserve neurotransmitter from permeabilized synaptosomes [19] (Figure 2A). In accord with the data in figure 1B, both Wt and A30P α-syn binding was strongly up-regulated by increasing cytosol concentration, whereas only high cytosol concentration resulted in increased A53T α-syn binding. To determine whether the cytosolic factors act on α-syn or on the acceptor synaptic membranes, we first pre-incubated α-syn or synaptic membranes separately with KO cytosol. The membranes were subsequently washed briefly to remove unbound cytosolic factors. As shown on Figure 2B, exposure of the membranes alone to cytosol was sufficient to potentiate α-syn binding, which was equivalent to α-syn binding to membranes in the presence of cytosol. These results suggest that cytosolic activity can be mediated by affecting the acceptor membrane rather than soluble α-syn.


Differential regulation of wild-type and mutant alpha-synuclein binding to synaptic membranes by cytosolic factors.

Wislet-Gendebien S, Visanji NP, Whitehead SN, Marsilio D, Hou W, Figeys D, Fraser PE, Bennett SA, Tandon A - BMC Neurosci (2008)

Effect of cytosol on binding α-syn. (A) Recombinant α-syn (Wt, A30P and A53T) were incubated in presence of different concentrations of KO cytosol (0.5, 1.5, and 3 mg/ml), for 10 min at 37°C. Compared to the control condition (without cytosol), all cytosol concentrations had a significant effect on Wt and A30P α-syn binding, but only the highest concentration of cytosol had a significant effect on A53T α-syn binding (One way ANOVA test, p < 0.0001, Bonferroni's multiple comparison post-test). (B) KO synaptic membranes and α-syn were pre-incubated for 15 minutes at room temperature with KO cytosol. Membranes were then centrifuged at 14000 × g and washed with KOAc buffer to remove unbound factors. Binding of purified α-syn to KO membranes in the absence of cytosol (ctrl) was compared to its binding to cytosol-treated membranes without added cytosol (memb), and to cytosol-treated α-syn incubated with KO membranes (α-syn). No significant difference was observed between the two pre-incubated condition (Student T-test, p > 0.05). (C) KO cytosol was pre-incubated with trypsin or proteinase K for 15 min at 37°C. Enzymes were then respectively inactivated with trypsin inhibitor and PMSF for 10 min at room temperature. Compared to the cytosol condition (cyt) which, as a control, was incubated with the enzyme pre-inactivated by the inhibitor, only A30P α-syn binding was significantly affected by the cytosolic protein digestion (Student T-test, p < 0.0001), whereas no significant differences were observed for Wt and A53T proteins (Student T-test, p > 0.05).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2562387&req=5

Figure 2: Effect of cytosol on binding α-syn. (A) Recombinant α-syn (Wt, A30P and A53T) were incubated in presence of different concentrations of KO cytosol (0.5, 1.5, and 3 mg/ml), for 10 min at 37°C. Compared to the control condition (without cytosol), all cytosol concentrations had a significant effect on Wt and A30P α-syn binding, but only the highest concentration of cytosol had a significant effect on A53T α-syn binding (One way ANOVA test, p < 0.0001, Bonferroni's multiple comparison post-test). (B) KO synaptic membranes and α-syn were pre-incubated for 15 minutes at room temperature with KO cytosol. Membranes were then centrifuged at 14000 × g and washed with KOAc buffer to remove unbound factors. Binding of purified α-syn to KO membranes in the absence of cytosol (ctrl) was compared to its binding to cytosol-treated membranes without added cytosol (memb), and to cytosol-treated α-syn incubated with KO membranes (α-syn). No significant difference was observed between the two pre-incubated condition (Student T-test, p > 0.05). (C) KO cytosol was pre-incubated with trypsin or proteinase K for 15 min at 37°C. Enzymes were then respectively inactivated with trypsin inhibitor and PMSF for 10 min at room temperature. Compared to the cytosol condition (cyt) which, as a control, was incubated with the enzyme pre-inactivated by the inhibitor, only A30P α-syn binding was significantly affected by the cytosolic protein digestion (Student T-test, p < 0.0001), whereas no significant differences were observed for Wt and A53T proteins (Student T-test, p > 0.05).
Mentions: We recently reported that the dissociation of the α-syn from synaptic membranes requires cytosolic proteins as defined by sensitivity to proteases. To further characterize cytosol action on α-syn binding, and because the data in Fig 1C suggests that cytosol activity becomes saturated at high α-syn concentration, we analysed α-syn binding with varying cytosol concentrations over a 6-fold range that we have previously shown to be effective in mobilizing reserve neurotransmitter from permeabilized synaptosomes [19] (Figure 2A). In accord with the data in figure 1B, both Wt and A30P α-syn binding was strongly up-regulated by increasing cytosol concentration, whereas only high cytosol concentration resulted in increased A53T α-syn binding. To determine whether the cytosolic factors act on α-syn or on the acceptor synaptic membranes, we first pre-incubated α-syn or synaptic membranes separately with KO cytosol. The membranes were subsequently washed briefly to remove unbound cytosolic factors. As shown on Figure 2B, exposure of the membranes alone to cytosol was sufficient to potentiate α-syn binding, which was equivalent to α-syn binding to membranes in the presence of cytosol. These results suggest that cytosolic activity can be mediated by affecting the acceptor membrane rather than soluble α-syn.

Bottom Line: However, the regulation of alpha-syn's cellular localization in neurons and the effects of the PD-linked mutations are poorly understood.We further show that 1-O-hexadecyl-2-acetyl-sn-glycero-3-phosphocholine (C16:0 PAF) is one of the principal lipids found in complex with cytosolic proteins and is required to enhance alpha-syn interaction with synaptic membrane.In addition, the impaired membrane binding observed for A30P alpha-syn was significantly mitigated by the presence of protease-sensitive factors in brain cytosol.

View Article: PubMed Central - HTML - PubMed

Affiliation: Centre for Research in Neurodegenerative Diseases, University of Toronto, Toronto, Ontario, M5S 3H2 Canada. s.wislet@ulg.ac.be

ABSTRACT

Background: Alpha-Synuclein (alpha-syn), a 140 amino acid protein associated with presynaptic membranes in brain, is a major constituent of Lewy bodies in Parkinson's disease (PD). Three missense mutations (A30P, A53T and E46K) in the alpha-syn gene are associated with rare autosomal dominant forms of familial PD. However, the regulation of alpha-syn's cellular localization in neurons and the effects of the PD-linked mutations are poorly understood.

Results: In the present study, we analysed the ability of cytosolic factors to regulate alpha-syn binding to synaptic membranes. We show that co-incubation with brain cytosol significantly increases the membrane binding of normal and PD-linked mutant alpha-syn. To characterize cytosolic factor(s) that modulate alpha-syn binding properties, we investigated the ability of proteins, lipids, ATP and calcium to modulate alpha-syn membrane interactions. We report that lipids and ATP are two of the principal cytosolic components that modulate Wt and A53T alpha-syn binding to the synaptic membrane. We further show that 1-O-hexadecyl-2-acetyl-sn-glycero-3-phosphocholine (C16:0 PAF) is one of the principal lipids found in complex with cytosolic proteins and is required to enhance alpha-syn interaction with synaptic membrane. In addition, the impaired membrane binding observed for A30P alpha-syn was significantly mitigated by the presence of protease-sensitive factors in brain cytosol.

Conclusion: These findings suggest that endogenous brain cytosolic factors regulate Wt and mutant alpha-syn membrane binding, and could represent potential targets to influence alpha-syn solubility in brain.

Show MeSH
Related in: MedlinePlus