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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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PLA2 inhibitors protect neurons against αSN-induced synapse damage—(A) The amounts of activated cPLA2 in synaptosomes incubated with αSN (○) or βSN (●) as shown. Values are means ± SD from triplicate experiments performed three times, n = 9; (B) The concentrations of PGE2 produced by synaptosomes incubated with control medium (□), 500 nM αSN (■) or 500 nM βSN (striped bar). Values are means ± SD from triplicate experiments performed three times, n = 9. * = significantly higher than in control synaptosomes. The amounts of synaptophysin (C) and CSP (D) in neurons pre-treated with control medium (□), phospholipase A2 inhibitors (1 µM AACOCF3 or 1 µM MAFP) (■) or phospholipase C inhibitors (10 µM U73122 or 10 µM ethyl-18-OCH3) (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-f002: PLA2 inhibitors protect neurons against αSN-induced synapse damage—(A) The amounts of activated cPLA2 in synaptosomes incubated with αSN (○) or βSN (●) as shown. Values are means ± SD from triplicate experiments performed three times, n = 9; (B) The concentrations of PGE2 produced by synaptosomes incubated with control medium (□), 500 nM αSN (■) or 500 nM βSN (striped bar). Values are means ± SD from triplicate experiments performed three times, n = 9. * = significantly higher than in control synaptosomes. The amounts of synaptophysin (C) and CSP (D) in neurons pre-treated with control medium (□), phospholipase A2 inhibitors (1 µM AACOCF3 or 1 µM MAFP) (■) or phospholipase C inhibitors (10 µM U73122 or 10 µM ethyl-18-OCH3) (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: Prior studies showed that synapse damage induced by prion peptides or amyloid-β (Aβ), thought to be the causative agent in the pathogenesis of Alzheimer’s disease, was associated with activation of synaptic cytoplasmic phospholipase A2 (cPLA2) [16]. Here we show that αSN, but not βSN, caused a dose-dependent activation of cPLA2 in synaptosomes (Figure 2A). The activation of cPLA2 was accompanied by the release of prostaglandin (PG)E2 (Figure 2B). The synaptophysin content of neurons was not significantly affected by treatment with cPLA2 inhibitors, 5 µM arachidonyl trifluoromethyl ketone (AACOCF3) (100 units synaptophysin ± 4 compared to 101 ± 4, n = 12, p = 0.5) or 5 µM methyl arachidonyl fluorophosphonate (MAFP) (100 ± 4 vs. 102 ± 6, n = 12, p = 0.4) showing that these drugs did not stimulate synaptogenesis, nor did they damage synapses. In primary cultures pre-treatment with either 1 µM AACOCF3 or 1 µM MAFP protected neurons against the αSN-induced reductions in synaptophysin (Figure 2C) and CSP (Figure 2D). In contrast, pre-treatment with phospholipase C inhibitors (10 µM U73122 or ethyl-18-OCH3) did not affect αSN-induced reductions in synaptophysin and CSP. Collectively these results support the hypothesis that hyperactivation of cPLA2 is involved in involved in αSN-induced synapse damage.


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

Bate C, Williams A - Biomolecules (2015)

PLA2 inhibitors protect neurons against αSN-induced synapse damage—(A) The amounts of activated cPLA2 in synaptosomes incubated with αSN (○) or βSN (●) as shown. Values are means ± SD from triplicate experiments performed three times, n = 9; (B) The concentrations of PGE2 produced by synaptosomes incubated with control medium (□), 500 nM αSN (■) or 500 nM βSN (striped bar). Values are means ± SD from triplicate experiments performed three times, n = 9. * = significantly higher than in control synaptosomes. The amounts of synaptophysin (C) and CSP (D) in neurons pre-treated with control medium (□), phospholipase A2 inhibitors (1 µM AACOCF3 or 1 µM MAFP) (■) or phospholipase C inhibitors (10 µM U73122 or 10 µM ethyl-18-OCH3) (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-f002: PLA2 inhibitors protect neurons against αSN-induced synapse damage—(A) The amounts of activated cPLA2 in synaptosomes incubated with αSN (○) or βSN (●) as shown. Values are means ± SD from triplicate experiments performed three times, n = 9; (B) The concentrations of PGE2 produced by synaptosomes incubated with control medium (□), 500 nM αSN (■) or 500 nM βSN (striped bar). Values are means ± SD from triplicate experiments performed three times, n = 9. * = significantly higher than in control synaptosomes. The amounts of synaptophysin (C) and CSP (D) in neurons pre-treated with control medium (□), phospholipase A2 inhibitors (1 µM AACOCF3 or 1 µM MAFP) (■) or phospholipase C inhibitors (10 µM U73122 or 10 µM ethyl-18-OCH3) (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: Prior studies showed that synapse damage induced by prion peptides or amyloid-β (Aβ), thought to be the causative agent in the pathogenesis of Alzheimer’s disease, was associated with activation of synaptic cytoplasmic phospholipase A2 (cPLA2) [16]. Here we show that αSN, but not βSN, caused a dose-dependent activation of cPLA2 in synaptosomes (Figure 2A). The activation of cPLA2 was accompanied by the release of prostaglandin (PG)E2 (Figure 2B). The synaptophysin content of neurons was not significantly affected by treatment with cPLA2 inhibitors, 5 µM arachidonyl trifluoromethyl ketone (AACOCF3) (100 units synaptophysin ± 4 compared to 101 ± 4, n = 12, p = 0.5) or 5 µM methyl arachidonyl fluorophosphonate (MAFP) (100 ± 4 vs. 102 ± 6, n = 12, p = 0.4) showing that these drugs did not stimulate synaptogenesis, nor did they damage synapses. In primary cultures pre-treatment with either 1 µM AACOCF3 or 1 µM MAFP protected neurons against the αSN-induced reductions in synaptophysin (Figure 2C) and CSP (Figure 2D). In contrast, pre-treatment with phospholipase C inhibitors (10 µM U73122 or ethyl-18-OCH3) did not affect αSN-induced reductions in synaptophysin and CSP. Collectively these results support the hypothesis that hyperactivation of cPLA2 is involved in involved in αSN-induced synapse damage.

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