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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.

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Effects of cytosolic lipid depletion on α-syn binding. (A) Using chloroform extraction to fractionate cytosol into three fractions: the top fraction contains the gangliosides or small organic polar molecules, the interface layer contains the proteins and the bottom phase contains lipids solubilised in chloroform. We incubated the synaptic membrane with the two lipid free-fractions, top and interphase (protein) layers, in presence of recombinant α-syn. The lipid-free fractions did not show any significant effects on the Wt and A53T α-syn binding compared to the control condition (α-syn incubated with synaptic membranes in absence of cytosol; Student T-test, p > 0.05) while the A30P α-syn binding was increased (compared to control condition, Student T-test, p < 0.01). (B) Recombinant α-syn (Wt, A30P and A53T) were incubated with synaptosomal membranes in the presence of 1.5 mg/ml cytosol from either KO mice (KO) or from non-transgenic mice (nonTg) for 10 min at 37°C. Binding of normal and mutant human α-syn, measured by the human α-syn specific monoclonal antibody 211, is normalized to that of Wt α-syn in the presence of KO cytosol. (C) Recombinant Wt α-syn was incubated with synaptosomal membranes and C16:0 PAF (0, 10, 100 nM) in the absence (open bars) or presence of delipidated cytosol (closed bars). Inclusion of 100 nM C16:0 PAF significantly increased α-syn binding only in the presence of the delipidated cytosol (compared to corresponding condition without C16:0 PAF, Two-Way ANOVA, p < 0.01, Bonferroni's multiple comparison test p < 0.01, n = 3).
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Figure 3: Effects of cytosolic lipid depletion on α-syn binding. (A) Using chloroform extraction to fractionate cytosol into three fractions: the top fraction contains the gangliosides or small organic polar molecules, the interface layer contains the proteins and the bottom phase contains lipids solubilised in chloroform. We incubated the synaptic membrane with the two lipid free-fractions, top and interphase (protein) layers, in presence of recombinant α-syn. The lipid-free fractions did not show any significant effects on the Wt and A53T α-syn binding compared to the control condition (α-syn incubated with synaptic membranes in absence of cytosol; Student T-test, p > 0.05) while the A30P α-syn binding was increased (compared to control condition, Student T-test, p < 0.01). (B) Recombinant α-syn (Wt, A30P and A53T) were incubated with synaptosomal membranes in the presence of 1.5 mg/ml cytosol from either KO mice (KO) or from non-transgenic mice (nonTg) for 10 min at 37°C. Binding of normal and mutant human α-syn, measured by the human α-syn specific monoclonal antibody 211, is normalized to that of Wt α-syn in the presence of KO cytosol. (C) Recombinant Wt α-syn was incubated with synaptosomal membranes and C16:0 PAF (0, 10, 100 nM) in the absence (open bars) or presence of delipidated cytosol (closed bars). Inclusion of 100 nM C16:0 PAF significantly increased α-syn binding only in the presence of the delipidated cytosol (compared to corresponding condition without C16:0 PAF, Two-Way ANOVA, p < 0.01, Bonferroni's multiple comparison test p < 0.01, n = 3).

Mentions: Because Wt and A53T α-syn appear to require protease-insensitive cofactors for membrane binding, and α-syn conformation is known to be affected by lipids (Jo et al. 2002), we examined whether removal of cytosolic lipids by chloroform extraction can alter the proportion of α-syn able to bind synaptic membranes (Figure 3A). We observed that the binding of Wt α-syn and PD-linked mutants were decreased in the presence lipid-deficient cytosol, suggesting a role for cytosolic lipids in the binding of α-syn to synaptic membranes. These results are also consistent with our observation that heat-denatured cytosol retains its activity to potentiate Wt and A53T α-syn binding (data not shown). Moreover, consistent with the results in Fig 1B showing that A53T α-syn membrane binding is less dependent on cytosol, it was also the least affected by lipid extraction. It is also important to note that the chloroform extraction did not non-specifically denature cytosolic proteins because the protein-containing fraction partially rescued A30P α-syn binding, in accord with its dependence on a protease-sensitive cytosolic component.


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)

Effects of cytosolic lipid depletion on α-syn binding. (A) Using chloroform extraction to fractionate cytosol into three fractions: the top fraction contains the gangliosides or small organic polar molecules, the interface layer contains the proteins and the bottom phase contains lipids solubilised in chloroform. We incubated the synaptic membrane with the two lipid free-fractions, top and interphase (protein) layers, in presence of recombinant α-syn. The lipid-free fractions did not show any significant effects on the Wt and A53T α-syn binding compared to the control condition (α-syn incubated with synaptic membranes in absence of cytosol; Student T-test, p > 0.05) while the A30P α-syn binding was increased (compared to control condition, Student T-test, p < 0.01). (B) Recombinant α-syn (Wt, A30P and A53T) were incubated with synaptosomal membranes in the presence of 1.5 mg/ml cytosol from either KO mice (KO) or from non-transgenic mice (nonTg) for 10 min at 37°C. Binding of normal and mutant human α-syn, measured by the human α-syn specific monoclonal antibody 211, is normalized to that of Wt α-syn in the presence of KO cytosol. (C) Recombinant Wt α-syn was incubated with synaptosomal membranes and C16:0 PAF (0, 10, 100 nM) in the absence (open bars) or presence of delipidated cytosol (closed bars). Inclusion of 100 nM C16:0 PAF significantly increased α-syn binding only in the presence of the delipidated cytosol (compared to corresponding condition without C16:0 PAF, Two-Way ANOVA, p < 0.01, Bonferroni's multiple comparison test p < 0.01, n = 3).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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Figure 3: Effects of cytosolic lipid depletion on α-syn binding. (A) Using chloroform extraction to fractionate cytosol into three fractions: the top fraction contains the gangliosides or small organic polar molecules, the interface layer contains the proteins and the bottom phase contains lipids solubilised in chloroform. We incubated the synaptic membrane with the two lipid free-fractions, top and interphase (protein) layers, in presence of recombinant α-syn. The lipid-free fractions did not show any significant effects on the Wt and A53T α-syn binding compared to the control condition (α-syn incubated with synaptic membranes in absence of cytosol; Student T-test, p > 0.05) while the A30P α-syn binding was increased (compared to control condition, Student T-test, p < 0.01). (B) Recombinant α-syn (Wt, A30P and A53T) were incubated with synaptosomal membranes in the presence of 1.5 mg/ml cytosol from either KO mice (KO) or from non-transgenic mice (nonTg) for 10 min at 37°C. Binding of normal and mutant human α-syn, measured by the human α-syn specific monoclonal antibody 211, is normalized to that of Wt α-syn in the presence of KO cytosol. (C) Recombinant Wt α-syn was incubated with synaptosomal membranes and C16:0 PAF (0, 10, 100 nM) in the absence (open bars) or presence of delipidated cytosol (closed bars). Inclusion of 100 nM C16:0 PAF significantly increased α-syn binding only in the presence of the delipidated cytosol (compared to corresponding condition without C16:0 PAF, Two-Way ANOVA, p < 0.01, Bonferroni's multiple comparison test p < 0.01, n = 3).
Mentions: Because Wt and A53T α-syn appear to require protease-insensitive cofactors for membrane binding, and α-syn conformation is known to be affected by lipids (Jo et al. 2002), we examined whether removal of cytosolic lipids by chloroform extraction can alter the proportion of α-syn able to bind synaptic membranes (Figure 3A). We observed that the binding of Wt α-syn and PD-linked mutants were decreased in the presence lipid-deficient cytosol, suggesting a role for cytosolic lipids in the binding of α-syn to synaptic membranes. These results are also consistent with our observation that heat-denatured cytosol retains its activity to potentiate Wt and A53T α-syn binding (data not shown). Moreover, consistent with the results in Fig 1B showing that A53T α-syn membrane binding is less dependent on cytosol, it was also the least affected by lipid extraction. It is also important to note that the chloroform extraction did not non-specifically denature cytosolic proteins because the protein-containing fraction partially rescued A30P α-syn binding, in accord with its dependence on a protease-sensitive cytosolic component.

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