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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: We show that co-incubation with brain cytosol significantly increases the membrane binding of normal and PD-linked mutant alpha-syn.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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(A-C) Recombinant α-syn (Wt, A30P and A53T) were incubated with ATP (1 mM), ATPγS (3 mM), Ca2+ (1 mM), ATP/Ca2+ or ATPγS/Ca2+ in absence or presence of 1.5 mg/ml of KO cytosol, for 10 min at 37°C. Incubation with ATP (Student's T-test, p < 0.001), but not ATPγS or Ca++ (Student's T-test, p > 0.05), induced a significant increase in the binding of Wt and mutant forms of α-syn (compared to control condition without added cofactors).
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Figure 6: (A-C) Recombinant α-syn (Wt, A30P and A53T) were incubated with ATP (1 mM), ATPγS (3 mM), Ca2+ (1 mM), ATP/Ca2+ or ATPγS/Ca2+ in absence or presence of 1.5 mg/ml of KO cytosol, for 10 min at 37°C. Incubation with ATP (Student's T-test, p < 0.001), but not ATPγS or Ca++ (Student's T-test, p > 0.05), induced a significant increase in the binding of Wt and mutant forms of α-syn (compared to control condition without added cofactors).

Mentions: α-Syn membrane attachment may be regulated by nerve terminal activity initiated by membrane depolarization [27], a process which results in Ca2+ influx, and elevated metabolic energy consumption. Therefore, we tested whether the addition of Ca2+ and ATP influenced α-syn binding. Our results show that ATP, but not ATPγS, significantly increased the level of membrane bound Wt α-syn and PD-linked mutants in the absence or presence of KO cytosol, whereas Ca2+ had no affect the α-syn binding (Figure 6A–C). The effect of ATP was additive to cytosol action suggesting that they act independently, and this was supported by the fact that ATPγS did not reduce the cytosol-dependent binding.


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)

(A-C) Recombinant α-syn (Wt, A30P and A53T) were incubated with ATP (1 mM), ATPγS (3 mM), Ca2+ (1 mM), ATP/Ca2+ or ATPγS/Ca2+ in absence or presence of 1.5 mg/ml of KO cytosol, for 10 min at 37°C. Incubation with ATP (Student's T-test, p < 0.001), but not ATPγS or Ca++ (Student's T-test, p > 0.05), induced a significant increase in the binding of Wt and mutant forms of α-syn (compared to control condition without added cofactors).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 6: (A-C) Recombinant α-syn (Wt, A30P and A53T) were incubated with ATP (1 mM), ATPγS (3 mM), Ca2+ (1 mM), ATP/Ca2+ or ATPγS/Ca2+ in absence or presence of 1.5 mg/ml of KO cytosol, for 10 min at 37°C. Incubation with ATP (Student's T-test, p < 0.001), but not ATPγS or Ca++ (Student's T-test, p > 0.05), induced a significant increase in the binding of Wt and mutant forms of α-syn (compared to control condition without added cofactors).
Mentions: α-Syn membrane attachment may be regulated by nerve terminal activity initiated by membrane depolarization [27], a process which results in Ca2+ influx, and elevated metabolic energy consumption. Therefore, we tested whether the addition of Ca2+ and ATP influenced α-syn binding. Our results show that ATP, but not ATPγS, significantly increased the level of membrane bound Wt α-syn and PD-linked mutants in the absence or presence of KO cytosol, whereas Ca2+ had no affect the α-syn binding (Figure 6A–C). The effect of ATP was additive to cytosol action suggesting that they act independently, and this was supported by the fact that ATPγS did not reduce the cytosol-dependent binding.

Bottom Line: We show that co-incubation with brain cytosol significantly increases the membrane binding of normal and PD-linked mutant alpha-syn.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