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α-Synuclein-induced synapse damage in cultured neurons is mediated by cholesterol-sensitive activation of cytoplasmic phospholipase A2.

Bate C, Williams A - Biomolecules (2015)

Bottom Line: The accumulation of aggregated forms of the α-synuclein (αSN) is associated with the pathogenesis of Parkinson's disease (PD) and Dementia with Lewy Bodies.The loss of synapses is an important event in the pathogenesis of these diseases.They also indicate that αSN-induced activation of cPLA2 is influenced by the cholesterol content of membranes.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology and Pathogen Biology, Royal Veterinary College, Hawkshead Lane, North Mymms, Herts AL9 7TA, UK. cbate@rvc.ac.uk.

ABSTRACT
The accumulation of aggregated forms of the α-synuclein (αSN) is associated with the pathogenesis of Parkinson's disease (PD) and Dementia with Lewy Bodies. The loss of synapses is an important event in the pathogenesis of these diseases. Here we show that aggregated recombinant human αSN, but not βSN, triggered synapse damage in cultured neurons as measured by the loss of synaptic proteins. Pre-treatment with the selective cytoplasmic phospholipase A2 (cPLA2) inhibitors AACOCF3 and MAFP protected neurons against αSN-induced synapse damage. Synapse damage was associated with the αSN-induced activation of synaptic cPLA2 and the production of prostaglandin E2. The activation of cPLA2 is the first step in the generation of platelet-activating factor (PAF) and PAF receptor antagonists (ginkgolide B or Hexa-PAF) also protect neurons against αSN-induced synapse damage. αSN-induced synapse damage was also reduced in neurons pre-treated with the cholesterol synthesis inhibitor (squalestatin). These results are consistent with the hypothesis that αSN triggered synapse damage via hyperactivation of cPLA2. They also indicate that αSN-induced activation of cPLA2 is influenced by the cholesterol content of membranes. Inhibitors of this pathway that can cross the blood brain barrier may protect against the synapse damage seen during PD.

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Platelet-activating factor (PAF) antagonists protect neurons against αSN-induced synapse damage—The amounts of synaptophysin (A) and CSP (B) in neurons pre-treated with a vehicle control (□) or PAF antagonists (1 µM H-PAF, ginkgolide B or CV6209 (■)) and incubated with αSN. *= significantly higher than in control neurons incubated with αSN. Values are means ± SD from triplicate experiments performed four times, n = 12; (C) The concentrations of PGE2 produced by synaptosomes pre-treated with incubated with a vehicle control (□) or PAF antagonists (1 µM H-PAF, ginkgolide B or CV6209 (■)) and incubated with 500 nM αSN. Values are means ± SD from triplicate experiments performed three times, n = 9. * = significantly lower than control synaptosomes incubated with 500 nM αSN. The amounts of synaptophysin (D) and CSP (E) in neurons pre-treated with a vehicle control (□), the prostanoid EP receptor antagonist AH13205 (■) or the prostanoid DP receptor antagonist BWA868C (striped bars) and incubated with 1 μM αSN. Values are means ± SD from triplicate experiments performed four times, n = 12. * = significantly higher than in control neurons incubated with αSN.
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biomolecules-05-00178-f004: Platelet-activating factor (PAF) antagonists protect neurons against αSN-induced synapse damage—The amounts of synaptophysin (A) and CSP (B) in neurons pre-treated with a vehicle control (□) or PAF antagonists (1 µM H-PAF, ginkgolide B or CV6209 (■)) and incubated with αSN. *= significantly higher than in control neurons incubated with αSN. Values are means ± SD from triplicate experiments performed four times, n = 12; (C) The concentrations of PGE2 produced by synaptosomes pre-treated with incubated with a vehicle control (□) or PAF antagonists (1 µM H-PAF, ginkgolide B or CV6209 (■)) and incubated with 500 nM αSN. Values are means ± SD from triplicate experiments performed three times, n = 9. * = significantly lower than control synaptosomes incubated with 500 nM αSN. The amounts of synaptophysin (D) and CSP (E) in neurons pre-treated with a vehicle control (□), the prostanoid EP receptor antagonist AH13205 (■) or the prostanoid DP receptor antagonist BWA868C (striped bars) and incubated with 1 μM αSN. Values are means ± SD from triplicate experiments performed four times, n = 12. * = significantly higher than in control neurons incubated with αSN.

Mentions: The activation of PLA2 is also the first step in the production of platelet-activating factor (PAF) [20] that has been shown to cause synapse degeneration in vitro [21]. The addition of PAF receptor antagonists (1-O-Hexadecyl-2-acetyl-sn-glycerol-3-phospho-(N,N,N-trimethyl)-hexanolamine (Hexa-PAF), CV6209 or ginkgolide B), in the range 0.1–10 µM, did not affect the amount of synaptophysin or CSP in cortical neurons. However, pre-treatment with 2 µM Hexa-PAF, 2 µM CV6209 or 1 µM ginkgolide B provided protection against αSN-induced loss of synaptophysin (Figure 4A) or CSP (Figure 4B). Prior studies showed that PAF facilitates the production of prostaglandins [22] suggesting that one or more of the prostaglandins produced in response to αSN are responsible for synapse degeneration. Here we show that pre-treatment of synaptosomes with PAF antagonists significantly reduced the αSN-induced production of PGE2 by synaptosomes (Figure 4C). Prostaglandin E2, acts via specific prostanoid E receptors [23] and pre-treatment with the prostanoid E receptor antagonist AH13205, but not the prostanoid D receptor antagonist BWA868C, prevented the loss of synaptophysin and CSP in cortical neurons incubated with αSN (Figure 4D,E). Collectively, these results show that the effects of αSN on synapses were ultimately mediated through prostanoid E receptors.


α-Synuclein-induced synapse damage in cultured neurons is mediated by cholesterol-sensitive activation of cytoplasmic phospholipase A2.

Bate C, Williams A - Biomolecules (2015)

Platelet-activating factor (PAF) antagonists protect neurons against αSN-induced synapse damage—The amounts of synaptophysin (A) and CSP (B) in neurons pre-treated with a vehicle control (□) or PAF antagonists (1 µM H-PAF, ginkgolide B or CV6209 (■)) and incubated with αSN. *= significantly higher than in control neurons incubated with αSN. Values are means ± SD from triplicate experiments performed four times, n = 12; (C) The concentrations of PGE2 produced by synaptosomes pre-treated with incubated with a vehicle control (□) or PAF antagonists (1 µM H-PAF, ginkgolide B or CV6209 (■)) and incubated with 500 nM αSN. Values are means ± SD from triplicate experiments performed three times, n = 9. * = significantly lower than control synaptosomes incubated with 500 nM αSN. The amounts of synaptophysin (D) and CSP (E) in neurons pre-treated with a vehicle control (□), the prostanoid EP receptor antagonist AH13205 (■) or the prostanoid DP receptor antagonist BWA868C (striped bars) and incubated with 1 μM αSN. Values are means ± SD from triplicate experiments performed four times, n = 12. * = significantly higher than in control neurons incubated with αSN.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4384118&req=5

biomolecules-05-00178-f004: Platelet-activating factor (PAF) antagonists protect neurons against αSN-induced synapse damage—The amounts of synaptophysin (A) and CSP (B) in neurons pre-treated with a vehicle control (□) or PAF antagonists (1 µM H-PAF, ginkgolide B or CV6209 (■)) and incubated with αSN. *= significantly higher than in control neurons incubated with αSN. Values are means ± SD from triplicate experiments performed four times, n = 12; (C) The concentrations of PGE2 produced by synaptosomes pre-treated with incubated with a vehicle control (□) or PAF antagonists (1 µM H-PAF, ginkgolide B or CV6209 (■)) and incubated with 500 nM αSN. Values are means ± SD from triplicate experiments performed three times, n = 9. * = significantly lower than control synaptosomes incubated with 500 nM αSN. The amounts of synaptophysin (D) and CSP (E) in neurons pre-treated with a vehicle control (□), the prostanoid EP receptor antagonist AH13205 (■) or the prostanoid DP receptor antagonist BWA868C (striped bars) and incubated with 1 μM αSN. Values are means ± SD from triplicate experiments performed four times, n = 12. * = significantly higher than in control neurons incubated with αSN.
Mentions: The activation of PLA2 is also the first step in the production of platelet-activating factor (PAF) [20] that has been shown to cause synapse degeneration in vitro [21]. The addition of PAF receptor antagonists (1-O-Hexadecyl-2-acetyl-sn-glycerol-3-phospho-(N,N,N-trimethyl)-hexanolamine (Hexa-PAF), CV6209 or ginkgolide B), in the range 0.1–10 µM, did not affect the amount of synaptophysin or CSP in cortical neurons. However, pre-treatment with 2 µM Hexa-PAF, 2 µM CV6209 or 1 µM ginkgolide B provided protection against αSN-induced loss of synaptophysin (Figure 4A) or CSP (Figure 4B). Prior studies showed that PAF facilitates the production of prostaglandins [22] suggesting that one or more of the prostaglandins produced in response to αSN are responsible for synapse degeneration. Here we show that pre-treatment of synaptosomes with PAF antagonists significantly reduced the αSN-induced production of PGE2 by synaptosomes (Figure 4C). Prostaglandin E2, acts via specific prostanoid E receptors [23] and pre-treatment with the prostanoid E receptor antagonist AH13205, but not the prostanoid D receptor antagonist BWA868C, prevented the loss of synaptophysin and CSP in cortical neurons incubated with αSN (Figure 4D,E). Collectively, these results show that the effects of αSN on synapses were ultimately mediated through prostanoid E receptors.

Bottom Line: The accumulation of aggregated forms of the α-synuclein (αSN) is associated with the pathogenesis of Parkinson's disease (PD) and Dementia with Lewy Bodies.The loss of synapses is an important event in the pathogenesis of these diseases.They also indicate that αSN-induced activation of cPLA2 is influenced by the cholesterol content of membranes.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology and Pathogen Biology, Royal Veterinary College, Hawkshead Lane, North Mymms, Herts AL9 7TA, UK. cbate@rvc.ac.uk.

ABSTRACT
The accumulation of aggregated forms of the α-synuclein (αSN) is associated with the pathogenesis of Parkinson's disease (PD) and Dementia with Lewy Bodies. The loss of synapses is an important event in the pathogenesis of these diseases. Here we show that aggregated recombinant human αSN, but not βSN, triggered synapse damage in cultured neurons as measured by the loss of synaptic proteins. Pre-treatment with the selective cytoplasmic phospholipase A2 (cPLA2) inhibitors AACOCF3 and MAFP protected neurons against αSN-induced synapse damage. Synapse damage was associated with the αSN-induced activation of synaptic cPLA2 and the production of prostaglandin E2. The activation of cPLA2 is the first step in the generation of platelet-activating factor (PAF) and PAF receptor antagonists (ginkgolide B or Hexa-PAF) also protect neurons against αSN-induced synapse damage. αSN-induced synapse damage was also reduced in neurons pre-treated with the cholesterol synthesis inhibitor (squalestatin). These results are consistent with the hypothesis that αSN triggered synapse damage via hyperactivation of cPLA2. They also indicate that αSN-induced activation of cPLA2 is influenced by the cholesterol content of membranes. Inhibitors of this pathway that can cross the blood brain barrier may protect against the synapse damage seen during PD.

Show MeSH
Related in: MedlinePlus