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Sphingosine 1-phosphate lyase ablation disrupts presynaptic architecture and function via an ubiquitin- proteasome mediated mechanism

View Article: PubMed Central - PubMed

ABSTRACT

The bioactive lipid sphingosine 1-phosphate (S1P) is a degradation product of sphingolipids that are particularly abundant in neurons. We have shown previously that neuronal S1P accumulation is toxic leading to ER-stress and an increase in intracellular calcium. To clarify the neuronal function of S1P, we generated brain-specific knockout mouse models in which S1P-lyase (SPL), the enzyme responsible for irreversible S1P cleavage was inactivated. Constitutive ablation of SPL in the brain (SPLfl/fl/Nes) but not postnatal neuronal forebrain-restricted SPL deletion (SPLfl/fl/CaMK) caused marked accumulation of S1P. Hence, altered presynaptic architecture including a significant decrease in number and density of synaptic vesicles, decreased expression of several presynaptic proteins, and impaired synaptic short term plasticity were observed in hippocampal neurons from SPLfl/fl/Nes mice. Accordingly, these mice displayed cognitive deficits. At the molecular level, an activation of the ubiquitin-proteasome system (UPS) was detected which resulted in a decreased expression of the deubiquitinating enzyme USP14 and several presynaptic proteins. Upon inhibition of proteasomal activity, USP14 levels, expression of presynaptic proteins and synaptic function were restored. These findings identify S1P metabolism as a novel player in modulating synaptic architecture and plasticity.

No MeSH data available.


Expression of presynaptic proteins is reduced in SPLfl/fl/Nes mice.(a–f) Protein amounts were assessed by immunoblotting (unpaired t-test, PBassoon = 0.0002, PSynapsin-1 < 0.0001, Psyntaxin 1 = 0.0157, PVAMP2 = 0.0048, PSynaptophysin = 0.0099), and (g) transcript amounts were determined by qRT-PCR in hippocampi of 6-month-old mice. Immunostaining of the presynaptic marker proteins (h) Bassoon and (i) synaptophysin in cerebellar granule cells after 2 weeks in culture (unpaired t-test, PBassoon < 0.0001, PSynaptophysin = 0.0034). F-actin (red), bassoon and synaptophysin (green).
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f4: Expression of presynaptic proteins is reduced in SPLfl/fl/Nes mice.(a–f) Protein amounts were assessed by immunoblotting (unpaired t-test, PBassoon = 0.0002, PSynapsin-1 < 0.0001, Psyntaxin 1 = 0.0157, PVAMP2 = 0.0048, PSynaptophysin = 0.0099), and (g) transcript amounts were determined by qRT-PCR in hippocampi of 6-month-old mice. Immunostaining of the presynaptic marker proteins (h) Bassoon and (i) synaptophysin in cerebellar granule cells after 2 weeks in culture (unpaired t-test, PBassoon < 0.0001, PSynaptophysin = 0.0034). F-actin (red), bassoon and synaptophysin (green).

Mentions: Next, we aimed to identify the molecular mechanism underlying the observed alterations in synaptic morphology and function in SPL-deficient brains. First we assessed whether the reduced number of synaptic vesicles is also reflected in the expression of presynaptic proteins. We found that in hippocampi as well as in cultured cerebellar neurons of SPLfl/fl/Nesmice the expression of the presynaptic markers Bassoon and Synapsin-1 as well as the SNARE-proteins syntaxin and VAMP2 and the major synaptic vesicle protein synaptophysin was significantly decreased (Fig. 4a–e). However, the expression of other presynaptic proteins including synaptotagmin 1, piccolo, SNAP25, Munc18 and NCS-1 was not affected (Suppl. Fig. 4a–e). The expression of the post-synaptic marker PSD-95 (Fig. 4f) and other proteins including IDE (insulin degrading enzyme) and GAP-43 (Suppl. Fig. 4f,g) was not affected in SPLfl/fl/Nesmice. Since S1P was shown to modulate the activity of histone deacetylases20, we checked whether the decreased expression of presynaptic proteins occurred at transcriptional level. Yet, no changes in transcript amounts were found in brains of SPLfl/fl/Nesmice (Fig. 4g). In cultured cerebellar neurons of SPLfl/fl/Nesmice, reduction in presynaptic marker proteins Bassoon and synaptophysin were even more pronounced (Fig. 4h,i).


Sphingosine 1-phosphate lyase ablation disrupts presynaptic architecture and function via an ubiquitin- proteasome mediated mechanism
Expression of presynaptic proteins is reduced in SPLfl/fl/Nes mice.(a–f) Protein amounts were assessed by immunoblotting (unpaired t-test, PBassoon = 0.0002, PSynapsin-1 < 0.0001, Psyntaxin 1 = 0.0157, PVAMP2 = 0.0048, PSynaptophysin = 0.0099), and (g) transcript amounts were determined by qRT-PCR in hippocampi of 6-month-old mice. Immunostaining of the presynaptic marker proteins (h) Bassoon and (i) synaptophysin in cerebellar granule cells after 2 weeks in culture (unpaired t-test, PBassoon < 0.0001, PSynaptophysin = 0.0034). F-actin (red), bassoon and synaptophysin (green).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC5121647&req=5

f4: Expression of presynaptic proteins is reduced in SPLfl/fl/Nes mice.(a–f) Protein amounts were assessed by immunoblotting (unpaired t-test, PBassoon = 0.0002, PSynapsin-1 < 0.0001, Psyntaxin 1 = 0.0157, PVAMP2 = 0.0048, PSynaptophysin = 0.0099), and (g) transcript amounts were determined by qRT-PCR in hippocampi of 6-month-old mice. Immunostaining of the presynaptic marker proteins (h) Bassoon and (i) synaptophysin in cerebellar granule cells after 2 weeks in culture (unpaired t-test, PBassoon < 0.0001, PSynaptophysin = 0.0034). F-actin (red), bassoon and synaptophysin (green).
Mentions: Next, we aimed to identify the molecular mechanism underlying the observed alterations in synaptic morphology and function in SPL-deficient brains. First we assessed whether the reduced number of synaptic vesicles is also reflected in the expression of presynaptic proteins. We found that in hippocampi as well as in cultured cerebellar neurons of SPLfl/fl/Nesmice the expression of the presynaptic markers Bassoon and Synapsin-1 as well as the SNARE-proteins syntaxin and VAMP2 and the major synaptic vesicle protein synaptophysin was significantly decreased (Fig. 4a–e). However, the expression of other presynaptic proteins including synaptotagmin 1, piccolo, SNAP25, Munc18 and NCS-1 was not affected (Suppl. Fig. 4a–e). The expression of the post-synaptic marker PSD-95 (Fig. 4f) and other proteins including IDE (insulin degrading enzyme) and GAP-43 (Suppl. Fig. 4f,g) was not affected in SPLfl/fl/Nesmice. Since S1P was shown to modulate the activity of histone deacetylases20, we checked whether the decreased expression of presynaptic proteins occurred at transcriptional level. Yet, no changes in transcript amounts were found in brains of SPLfl/fl/Nesmice (Fig. 4g). In cultured cerebellar neurons of SPLfl/fl/Nesmice, reduction in presynaptic marker proteins Bassoon and synaptophysin were even more pronounced (Fig. 4h,i).

View Article: PubMed Central - PubMed

ABSTRACT

The bioactive lipid sphingosine 1-phosphate (S1P) is a degradation product of sphingolipids that are particularly abundant in neurons. We have shown previously that neuronal S1P accumulation is toxic leading to ER-stress and an increase in intracellular calcium. To clarify the neuronal function of S1P, we generated brain-specific knockout mouse models in which S1P-lyase (SPL), the enzyme responsible for irreversible S1P cleavage was inactivated. Constitutive ablation of SPL in the brain (SPLfl/fl/Nes) but not postnatal neuronal forebrain-restricted SPL deletion (SPLfl/fl/CaMK) caused marked accumulation of S1P. Hence, altered presynaptic architecture including a significant decrease in number and density of synaptic vesicles, decreased expression of several presynaptic proteins, and impaired synaptic short term plasticity were observed in hippocampal neurons from SPLfl/fl/Nes mice. Accordingly, these mice displayed cognitive deficits. At the molecular level, an activation of the ubiquitin-proteasome system (UPS) was detected which resulted in a decreased expression of the deubiquitinating enzyme USP14 and several presynaptic proteins. Upon inhibition of proteasomal activity, USP14 levels, expression of presynaptic proteins and synaptic function were restored. These findings identify S1P metabolism as a novel player in modulating synaptic architecture and plasticity.

No MeSH data available.