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Contribution of Orb2A stability in regulated amyloid-like oligomerization of Drosophila Orb2.

White-Grindley E, Li L, Mohammad Khan R, Ren F, Saraf A, Florens L, Si K - PLoS Biol. (2014)

Bottom Line: Mutation of LimK abolishes activity-dependent Orb2 oligomerization in the adult brain.Moreover, Tob-Orb2 association is modulated by neuronal activity and Tob activity in the mushroom body is required for stable memory formation.These observations suggest that the interplay between PP2A and Tob-LimK activity may dynamically regulate Orb2 amyloid-like oligomer formation and the stabilization of memories.

View Article: PubMed Central - PubMed

Affiliation: Stowers Institute for Medical Research, Kansas City, Missouri, United States of America.

ABSTRACT
How learned experiences persist as memory for a long time is an important question. In Drosophila the persistence of memory is dependent upon amyloid-like oligomers of the Orb2 protein. However, it is not clear how the conversion of Orb2 to the amyloid-like oligomeric state is regulated. The Orb2 has two protein isoforms, and the rare Orb2A isoform is critical for oligomerization of the ubiquitous Orb2B isoform. Here, we report the discovery of a protein network comprised of protein phosphatase 2A (PP2A), Transducer of Erb-B2 (Tob), and Lim Kinase (LimK) that controls the abundance of Orb2A. PP2A maintains Orb2A in an unphosphorylated and unstable state, whereas Tob-LimK phosphorylates and stabilizes Orb2A. Mutation of LimK abolishes activity-dependent Orb2 oligomerization in the adult brain. Moreover, Tob-Orb2 association is modulated by neuronal activity and Tob activity in the mushroom body is required for stable memory formation. These observations suggest that the interplay between PP2A and Tob-LimK activity may dynamically regulate Orb2 amyloid-like oligomer formation and the stabilization of memories.

Show MeSH
Tob-Orb2 association is regulated by neuronal activity, and Tob is present in the same synaptic compartment as Orb2.(A) Neuronal stimulation increases Tob-Orb2 association. Tob was immunoprecipitated from unstimulated (control) or 10 mM tyramine-stimulated head extracts and blotted with the anti-Orb2 antibody (Orb2A,B) that detects both forms of Orb2 or an antibody that detects only Orb2B (Orb2B). The preimmune serum (pre) from the same animal serves as control for Tob antibody specificity. (B) Tob-associated oligomeric Orb2 is amyloidogenic. Tob was immunoprecipitated from either unstimulated (control) or tyramine-stimulated head extracts, treated with 10% SDS and 2 M urea and blotted with the anti-Orb2 antibody. Western analysis of lysates indicates the expression levels of Orb2, Tob, and tubulin. (C) Tyramine increases Tob-Orb2A association. (Top panel) A schematic diagram of the genomic pCasperOrb2A construct, which expresses an EGFP-tagged Orb2A. The Orb2B-specific exons are indicated in blue, Orb2A-specific exon in red, and the common region in gray. (Bottom panel) Tob was immunoprecipitated from unstimulated (−) or tyramine-stimulated (+) total brain lysates with preimmune (pre) or immune serum (Tob) and probed with anti-EGFP antibody. The position of the monomeric Orb2AEGFP fusion protein is indicated. The EGFP-antibody reacting high molecular weight proteins are most likely the oligomeric form of the Orb2AEGFP protein. The asterisk indicates low molecular weight protein that is either a degradation product of Orb2AEGFP or a cross-reactive band. In the lower panel Tob is visible only in immune serum lane, indicating specificity of the pull-down. Orb2AEGFP is not detectable by Western analysis. (D) Stimulation of mushroom body neurons enhances Tob-Orb2 association. A schematic of the stimulation protocol is shown above the gel picture. The mushroom body neurons were stimulated using the temperature-sensitive dTrpA1 channel, which depolarizes and thereby activates neurons at a temperature >25°C. More Tob-associated Orb2 oligomers were observed at 30°C in flies harboring both the Gal4 driver and TrpA1 transgene compared to flies with just the Gal4-driver or TrpA1. The c747-Gal4 and MB247-Gal4 drive expression in all neurons of the adult Drosophila mushroom body. (E) Tob is widely distributed in the adult brain. Frontal cryosections of adult fly heads were immunostained with the preimmune serum or anti-Tob (green) antibody. Tob is present in the cell body and at a low level in the synaptic neuropil region of mushroom body (Mb lobes) Kenyon cells. Nc82 (red) marks the synaptic neuropil region. Scale bar, 20 µm. Also see Figure S4.
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pbio-1001786-g003: Tob-Orb2 association is regulated by neuronal activity, and Tob is present in the same synaptic compartment as Orb2.(A) Neuronal stimulation increases Tob-Orb2 association. Tob was immunoprecipitated from unstimulated (control) or 10 mM tyramine-stimulated head extracts and blotted with the anti-Orb2 antibody (Orb2A,B) that detects both forms of Orb2 or an antibody that detects only Orb2B (Orb2B). The preimmune serum (pre) from the same animal serves as control for Tob antibody specificity. (B) Tob-associated oligomeric Orb2 is amyloidogenic. Tob was immunoprecipitated from either unstimulated (control) or tyramine-stimulated head extracts, treated with 10% SDS and 2 M urea and blotted with the anti-Orb2 antibody. Western analysis of lysates indicates the expression levels of Orb2, Tob, and tubulin. (C) Tyramine increases Tob-Orb2A association. (Top panel) A schematic diagram of the genomic pCasperOrb2A construct, which expresses an EGFP-tagged Orb2A. The Orb2B-specific exons are indicated in blue, Orb2A-specific exon in red, and the common region in gray. (Bottom panel) Tob was immunoprecipitated from unstimulated (−) or tyramine-stimulated (+) total brain lysates with preimmune (pre) or immune serum (Tob) and probed with anti-EGFP antibody. The position of the monomeric Orb2AEGFP fusion protein is indicated. The EGFP-antibody reacting high molecular weight proteins are most likely the oligomeric form of the Orb2AEGFP protein. The asterisk indicates low molecular weight protein that is either a degradation product of Orb2AEGFP or a cross-reactive band. In the lower panel Tob is visible only in immune serum lane, indicating specificity of the pull-down. Orb2AEGFP is not detectable by Western analysis. (D) Stimulation of mushroom body neurons enhances Tob-Orb2 association. A schematic of the stimulation protocol is shown above the gel picture. The mushroom body neurons were stimulated using the temperature-sensitive dTrpA1 channel, which depolarizes and thereby activates neurons at a temperature >25°C. More Tob-associated Orb2 oligomers were observed at 30°C in flies harboring both the Gal4 driver and TrpA1 transgene compared to flies with just the Gal4-driver or TrpA1. The c747-Gal4 and MB247-Gal4 drive expression in all neurons of the adult Drosophila mushroom body. (E) Tob is widely distributed in the adult brain. Frontal cryosections of adult fly heads were immunostained with the preimmune serum or anti-Tob (green) antibody. Tob is present in the cell body and at a low level in the synaptic neuropil region of mushroom body (Mb lobes) Kenyon cells. Nc82 (red) marks the synaptic neuropil region. Scale bar, 20 µm. Also see Figure S4.

Mentions: Is Tob involved in activity-dependent oligomerization of Orb2? Previously we and others have observed that a neurotransmitter such as tyramine or dopamine regulates Orb2 oligomerization [4],[5]. Therefore, we checked whether tyramine modulates Orb2-Tob interaction. To this end, we fed-starved flies 10 mM tyramine and after 4 h immunopurified the Tob-Orb2 complex from tyramine-stimulated or -unstimulated adult fly brain using a Drosophila Tob-specific antibody (Figure S4A). Tyramine stimulation increased the Tob-bound oligomeric Orb2 nearly 4-fold (fold increase in oligomers normalized to monomer ± SEM, 3.82±0.88, n = 5, t test, p<0.05) (Figure 3A), and the oligomers are resistant to boiling in the presence of 10% SDS and 2 M urea, consistent with it being amyloid-like (Figure 3B). The neurotransmitter serotonin (5-HT) had less effect on Tob-Orb2 association (Figure S4B), consistent with our earlier observation that Orb2 oligomerization is influenced by tyramine and not by 5-HT [5]. Use of Orb2B-specific antibody (Figure 3A, right panel) indicated Tob-Orb2B association is enhanced by tyramine stimulation. To determine whether Tob-Orb2A association is also modulated by neuronal activity, we used a genomic construct that encompasses only Orb2A coding region and carries EGFP at the C-terminal end (pCasperOrb2AEGFP) [5]. In Tob immunoprecipitate from tyramine-treated samples, we see EGFP reacting bands that correspond to the size of the monomeric- (∼87 KDa) and oligomeric-Orb2AEGFP (Figure 3C). Since it is difficult to determine which neuronal populations are activated by tyramine feeding, we also directly activated the mushroom body neurons (c747-Gal4, MB247-Gal4) with the temperature-sensitive dTrpA1 channel [29]. The flies were transiently exposed to 30°C (dTrpA1 active) for 25 min and then returned to 22°C (dTrpA1 inactive). Compared to flies carrying only dTrpA1 or Gal4, flies carrying both transgenes (C747Gal4::UAS-dTrpA1 or MB247Gal4:UAS-dTrpA1), there was enhanced Tob-Orb2 association (Figure 3D). Taken together these observations suggest that neuronal activity that enhances Orb2 oligomerization also enhances Tob-Orb2 association.


Contribution of Orb2A stability in regulated amyloid-like oligomerization of Drosophila Orb2.

White-Grindley E, Li L, Mohammad Khan R, Ren F, Saraf A, Florens L, Si K - PLoS Biol. (2014)

Tob-Orb2 association is regulated by neuronal activity, and Tob is present in the same synaptic compartment as Orb2.(A) Neuronal stimulation increases Tob-Orb2 association. Tob was immunoprecipitated from unstimulated (control) or 10 mM tyramine-stimulated head extracts and blotted with the anti-Orb2 antibody (Orb2A,B) that detects both forms of Orb2 or an antibody that detects only Orb2B (Orb2B). The preimmune serum (pre) from the same animal serves as control for Tob antibody specificity. (B) Tob-associated oligomeric Orb2 is amyloidogenic. Tob was immunoprecipitated from either unstimulated (control) or tyramine-stimulated head extracts, treated with 10% SDS and 2 M urea and blotted with the anti-Orb2 antibody. Western analysis of lysates indicates the expression levels of Orb2, Tob, and tubulin. (C) Tyramine increases Tob-Orb2A association. (Top panel) A schematic diagram of the genomic pCasperOrb2A construct, which expresses an EGFP-tagged Orb2A. The Orb2B-specific exons are indicated in blue, Orb2A-specific exon in red, and the common region in gray. (Bottom panel) Tob was immunoprecipitated from unstimulated (−) or tyramine-stimulated (+) total brain lysates with preimmune (pre) or immune serum (Tob) and probed with anti-EGFP antibody. The position of the monomeric Orb2AEGFP fusion protein is indicated. The EGFP-antibody reacting high molecular weight proteins are most likely the oligomeric form of the Orb2AEGFP protein. The asterisk indicates low molecular weight protein that is either a degradation product of Orb2AEGFP or a cross-reactive band. In the lower panel Tob is visible only in immune serum lane, indicating specificity of the pull-down. Orb2AEGFP is not detectable by Western analysis. (D) Stimulation of mushroom body neurons enhances Tob-Orb2 association. A schematic of the stimulation protocol is shown above the gel picture. The mushroom body neurons were stimulated using the temperature-sensitive dTrpA1 channel, which depolarizes and thereby activates neurons at a temperature >25°C. More Tob-associated Orb2 oligomers were observed at 30°C in flies harboring both the Gal4 driver and TrpA1 transgene compared to flies with just the Gal4-driver or TrpA1. The c747-Gal4 and MB247-Gal4 drive expression in all neurons of the adult Drosophila mushroom body. (E) Tob is widely distributed in the adult brain. Frontal cryosections of adult fly heads were immunostained with the preimmune serum or anti-Tob (green) antibody. Tob is present in the cell body and at a low level in the synaptic neuropil region of mushroom body (Mb lobes) Kenyon cells. Nc82 (red) marks the synaptic neuropil region. Scale bar, 20 µm. Also see Figure S4.
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Related In: Results  -  Collection

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Show All Figures
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pbio-1001786-g003: Tob-Orb2 association is regulated by neuronal activity, and Tob is present in the same synaptic compartment as Orb2.(A) Neuronal stimulation increases Tob-Orb2 association. Tob was immunoprecipitated from unstimulated (control) or 10 mM tyramine-stimulated head extracts and blotted with the anti-Orb2 antibody (Orb2A,B) that detects both forms of Orb2 or an antibody that detects only Orb2B (Orb2B). The preimmune serum (pre) from the same animal serves as control for Tob antibody specificity. (B) Tob-associated oligomeric Orb2 is amyloidogenic. Tob was immunoprecipitated from either unstimulated (control) or tyramine-stimulated head extracts, treated with 10% SDS and 2 M urea and blotted with the anti-Orb2 antibody. Western analysis of lysates indicates the expression levels of Orb2, Tob, and tubulin. (C) Tyramine increases Tob-Orb2A association. (Top panel) A schematic diagram of the genomic pCasperOrb2A construct, which expresses an EGFP-tagged Orb2A. The Orb2B-specific exons are indicated in blue, Orb2A-specific exon in red, and the common region in gray. (Bottom panel) Tob was immunoprecipitated from unstimulated (−) or tyramine-stimulated (+) total brain lysates with preimmune (pre) or immune serum (Tob) and probed with anti-EGFP antibody. The position of the monomeric Orb2AEGFP fusion protein is indicated. The EGFP-antibody reacting high molecular weight proteins are most likely the oligomeric form of the Orb2AEGFP protein. The asterisk indicates low molecular weight protein that is either a degradation product of Orb2AEGFP or a cross-reactive band. In the lower panel Tob is visible only in immune serum lane, indicating specificity of the pull-down. Orb2AEGFP is not detectable by Western analysis. (D) Stimulation of mushroom body neurons enhances Tob-Orb2 association. A schematic of the stimulation protocol is shown above the gel picture. The mushroom body neurons were stimulated using the temperature-sensitive dTrpA1 channel, which depolarizes and thereby activates neurons at a temperature >25°C. More Tob-associated Orb2 oligomers were observed at 30°C in flies harboring both the Gal4 driver and TrpA1 transgene compared to flies with just the Gal4-driver or TrpA1. The c747-Gal4 and MB247-Gal4 drive expression in all neurons of the adult Drosophila mushroom body. (E) Tob is widely distributed in the adult brain. Frontal cryosections of adult fly heads were immunostained with the preimmune serum or anti-Tob (green) antibody. Tob is present in the cell body and at a low level in the synaptic neuropil region of mushroom body (Mb lobes) Kenyon cells. Nc82 (red) marks the synaptic neuropil region. Scale bar, 20 µm. Also see Figure S4.
Mentions: Is Tob involved in activity-dependent oligomerization of Orb2? Previously we and others have observed that a neurotransmitter such as tyramine or dopamine regulates Orb2 oligomerization [4],[5]. Therefore, we checked whether tyramine modulates Orb2-Tob interaction. To this end, we fed-starved flies 10 mM tyramine and after 4 h immunopurified the Tob-Orb2 complex from tyramine-stimulated or -unstimulated adult fly brain using a Drosophila Tob-specific antibody (Figure S4A). Tyramine stimulation increased the Tob-bound oligomeric Orb2 nearly 4-fold (fold increase in oligomers normalized to monomer ± SEM, 3.82±0.88, n = 5, t test, p<0.05) (Figure 3A), and the oligomers are resistant to boiling in the presence of 10% SDS and 2 M urea, consistent with it being amyloid-like (Figure 3B). The neurotransmitter serotonin (5-HT) had less effect on Tob-Orb2 association (Figure S4B), consistent with our earlier observation that Orb2 oligomerization is influenced by tyramine and not by 5-HT [5]. Use of Orb2B-specific antibody (Figure 3A, right panel) indicated Tob-Orb2B association is enhanced by tyramine stimulation. To determine whether Tob-Orb2A association is also modulated by neuronal activity, we used a genomic construct that encompasses only Orb2A coding region and carries EGFP at the C-terminal end (pCasperOrb2AEGFP) [5]. In Tob immunoprecipitate from tyramine-treated samples, we see EGFP reacting bands that correspond to the size of the monomeric- (∼87 KDa) and oligomeric-Orb2AEGFP (Figure 3C). Since it is difficult to determine which neuronal populations are activated by tyramine feeding, we also directly activated the mushroom body neurons (c747-Gal4, MB247-Gal4) with the temperature-sensitive dTrpA1 channel [29]. The flies were transiently exposed to 30°C (dTrpA1 active) for 25 min and then returned to 22°C (dTrpA1 inactive). Compared to flies carrying only dTrpA1 or Gal4, flies carrying both transgenes (C747Gal4::UAS-dTrpA1 or MB247Gal4:UAS-dTrpA1), there was enhanced Tob-Orb2 association (Figure 3D). Taken together these observations suggest that neuronal activity that enhances Orb2 oligomerization also enhances Tob-Orb2 association.

Bottom Line: Mutation of LimK abolishes activity-dependent Orb2 oligomerization in the adult brain.Moreover, Tob-Orb2 association is modulated by neuronal activity and Tob activity in the mushroom body is required for stable memory formation.These observations suggest that the interplay between PP2A and Tob-LimK activity may dynamically regulate Orb2 amyloid-like oligomer formation and the stabilization of memories.

View Article: PubMed Central - PubMed

Affiliation: Stowers Institute for Medical Research, Kansas City, Missouri, United States of America.

ABSTRACT
How learned experiences persist as memory for a long time is an important question. In Drosophila the persistence of memory is dependent upon amyloid-like oligomers of the Orb2 protein. However, it is not clear how the conversion of Orb2 to the amyloid-like oligomeric state is regulated. The Orb2 has two protein isoforms, and the rare Orb2A isoform is critical for oligomerization of the ubiquitous Orb2B isoform. Here, we report the discovery of a protein network comprised of protein phosphatase 2A (PP2A), Transducer of Erb-B2 (Tob), and Lim Kinase (LimK) that controls the abundance of Orb2A. PP2A maintains Orb2A in an unphosphorylated and unstable state, whereas Tob-LimK phosphorylates and stabilizes Orb2A. Mutation of LimK abolishes activity-dependent Orb2 oligomerization in the adult brain. Moreover, Tob-Orb2 association is modulated by neuronal activity and Tob activity in the mushroom body is required for stable memory formation. These observations suggest that the interplay between PP2A and Tob-LimK activity may dynamically regulate Orb2 amyloid-like oligomer formation and the stabilization of memories.

Show MeSH