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Akt regulates glutamate receptor trafficking and postsynaptic membrane elaboration at the Drosophila neuromuscular junction.

Lee HG, Zhao N, Campion BK, Nguyen MM, Selleck SB - Dev Neurobiol (2013)

Bottom Line: The single Drosophila Akt family member, Akt1 selectively altered the postsynaptic targeting of one glutamate receptor subunit, GluRIIA, and was required for the expansion of a specialized postsynaptic membrane compartment, the subsynaptic reticulum (SSR).Several lines of evidence indicated that Akt1 influences SSR assembly by regulation of Gtaxin, a Drosophila t-SNARE protein (Gorczyca et al., 2007) in a manner independent of the mislocalization of GluRIIA.Our findings show that Akt1 governs two critical elements of synapse development, neurotransmitter receptor localization, and postsynaptic membrane elaboration.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, Pennsylvania, 16802.

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GluRIIA localization was modified in Akt1 mutants and animals with muscle-specific inhibition of Akt1. GluRIIA localization was examined in muscles 6 and 7 using monoclonal anti-GluRIIA antibody (red). Anti-HRP antibody detected neuronal projections (green). A and B: In wild-type animals, GluRIIA was located in the postsynaptic specialization that surrounds the motoneuron boutons. C and D: Akt11/Akt104226 mutants showed reduction of GluRIIA at synaptic boutons (see arrows) and redirection to intracellular bands (faint staining in muscles 6 and 7, and more prominent in muscles 15 and 16; see arrowheads). E–L: Akt1 function was compromised by muscle-specific expression of an Akt1RNAi construct using the GAL4-UAS system. UAS-Akt1RNAi/+ animals served as controls. GAL4 transcriptional activation shows temperature dependence, permitting a graded level of Akt1 blockade from 18°C (low level of inhibition) to 30°C (high level of inhibition). E–H: In control larvae, GluRIIA immunoreactivity was concentrated in the postsynaptic region surrounding boutons at all temperatures. H: Enlarged view of white box area in (G), arrows show the motoneuron boutons surrounded by GluRIIA. I–L: In Akt1RNAi expressing larval muscle (24B-GAL4>UAS-Akt1RNAi), GluRIIA mislocalization (arrowheads) was more severe with greater inhibition of Akt1 function at increasing temperature (larvae reared at 18°C (I), 25°C (J), or 30°C (K and L)). L Enlarged view of white box area in (K), arrows show synaptic boutons lacking GluRIIA immunoreactivity; arrowheads mark ectopic GluRIIA within intracellular bands. Scale bar in (A–G) and (I–K), 50 µm, in (H) and (L), 5 µm.
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fig02: GluRIIA localization was modified in Akt1 mutants and animals with muscle-specific inhibition of Akt1. GluRIIA localization was examined in muscles 6 and 7 using monoclonal anti-GluRIIA antibody (red). Anti-HRP antibody detected neuronal projections (green). A and B: In wild-type animals, GluRIIA was located in the postsynaptic specialization that surrounds the motoneuron boutons. C and D: Akt11/Akt104226 mutants showed reduction of GluRIIA at synaptic boutons (see arrows) and redirection to intracellular bands (faint staining in muscles 6 and 7, and more prominent in muscles 15 and 16; see arrowheads). E–L: Akt1 function was compromised by muscle-specific expression of an Akt1RNAi construct using the GAL4-UAS system. UAS-Akt1RNAi/+ animals served as controls. GAL4 transcriptional activation shows temperature dependence, permitting a graded level of Akt1 blockade from 18°C (low level of inhibition) to 30°C (high level of inhibition). E–H: In control larvae, GluRIIA immunoreactivity was concentrated in the postsynaptic region surrounding boutons at all temperatures. H: Enlarged view of white box area in (G), arrows show the motoneuron boutons surrounded by GluRIIA. I–L: In Akt1RNAi expressing larval muscle (24B-GAL4>UAS-Akt1RNAi), GluRIIA mislocalization (arrowheads) was more severe with greater inhibition of Akt1 function at increasing temperature (larvae reared at 18°C (I), 25°C (J), or 30°C (K and L)). L Enlarged view of white box area in (K), arrows show synaptic boutons lacking GluRIIA immunoreactivity; arrowheads mark ectopic GluRIIA within intracellular bands. Scale bar in (A–G) and (I–K), 50 µm, in (H) and (L), 5 µm.

Mentions: We began assessing the role of Akt1 in NMJ assembly by examining the distribution and level of glutamate receptor IIA (GluRIIA), one of the neurotransmitter receptor subunits at this glutaminergic synapse. Glutamate is the major excitatory neurotransmitter at the type I bouton of the Drosophila larval NMJ (Brunner and Okane, 1997; Collins and DiAntonio, 2007). The NMJ glutamate receptor (GluR) is a heterotetramer comprised of three invariant subunits: GluRIIC, D, and E. The fourth subunit, either GluRIIA or B, determines the type and the electrophysiological properties of the receptor (DiAntonio et al., 1999; Featherstone et al., 2005; Qin et al., 2005a; DiAntonio, 2006). Subunit GluRIIA and B competitively bind to GluRIIC; hence, the preferential expression of these two subunits constitutes one element of developmental plasticity exhibited by this synapse (Marrus et al., 2004). We examined the levels and distributions of GluRIIA using a well-characterized monoclonal antibody, anti-GluRIIA (Featherstone et al., 2002; Qin et al., 2005a; Karr et al., 2009). The specificity of this antibody has been well documented by showing that immunoreactive signal is lost in GluRIIA mutant (Marrus et al., 2004). Partial loss of Akt1 function, achieved with the heteroallelic combination Akt11/Akt104226, altered GluRIIA distributions and levels, with a reduction at postsynaptic structures and the appearance of GluRIIA immunoreactivity within repeated bands throughout the muscle cells [Fig. 2 compare (A), (B) to (C), (D)]. This latter phenotype was more prominent in muscles 15 and 16 and was observed to a lesser extent in muscles 6 and 7, the postsynaptic cells typically used for electrophysiological analysis [arrowheads in Fig. 2(C,D)].


Akt regulates glutamate receptor trafficking and postsynaptic membrane elaboration at the Drosophila neuromuscular junction.

Lee HG, Zhao N, Campion BK, Nguyen MM, Selleck SB - Dev Neurobiol (2013)

GluRIIA localization was modified in Akt1 mutants and animals with muscle-specific inhibition of Akt1. GluRIIA localization was examined in muscles 6 and 7 using monoclonal anti-GluRIIA antibody (red). Anti-HRP antibody detected neuronal projections (green). A and B: In wild-type animals, GluRIIA was located in the postsynaptic specialization that surrounds the motoneuron boutons. C and D: Akt11/Akt104226 mutants showed reduction of GluRIIA at synaptic boutons (see arrows) and redirection to intracellular bands (faint staining in muscles 6 and 7, and more prominent in muscles 15 and 16; see arrowheads). E–L: Akt1 function was compromised by muscle-specific expression of an Akt1RNAi construct using the GAL4-UAS system. UAS-Akt1RNAi/+ animals served as controls. GAL4 transcriptional activation shows temperature dependence, permitting a graded level of Akt1 blockade from 18°C (low level of inhibition) to 30°C (high level of inhibition). E–H: In control larvae, GluRIIA immunoreactivity was concentrated in the postsynaptic region surrounding boutons at all temperatures. H: Enlarged view of white box area in (G), arrows show the motoneuron boutons surrounded by GluRIIA. I–L: In Akt1RNAi expressing larval muscle (24B-GAL4>UAS-Akt1RNAi), GluRIIA mislocalization (arrowheads) was more severe with greater inhibition of Akt1 function at increasing temperature (larvae reared at 18°C (I), 25°C (J), or 30°C (K and L)). L Enlarged view of white box area in (K), arrows show synaptic boutons lacking GluRIIA immunoreactivity; arrowheads mark ectopic GluRIIA within intracellular bands. Scale bar in (A–G) and (I–K), 50 µm, in (H) and (L), 5 µm.
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fig02: GluRIIA localization was modified in Akt1 mutants and animals with muscle-specific inhibition of Akt1. GluRIIA localization was examined in muscles 6 and 7 using monoclonal anti-GluRIIA antibody (red). Anti-HRP antibody detected neuronal projections (green). A and B: In wild-type animals, GluRIIA was located in the postsynaptic specialization that surrounds the motoneuron boutons. C and D: Akt11/Akt104226 mutants showed reduction of GluRIIA at synaptic boutons (see arrows) and redirection to intracellular bands (faint staining in muscles 6 and 7, and more prominent in muscles 15 and 16; see arrowheads). E–L: Akt1 function was compromised by muscle-specific expression of an Akt1RNAi construct using the GAL4-UAS system. UAS-Akt1RNAi/+ animals served as controls. GAL4 transcriptional activation shows temperature dependence, permitting a graded level of Akt1 blockade from 18°C (low level of inhibition) to 30°C (high level of inhibition). E–H: In control larvae, GluRIIA immunoreactivity was concentrated in the postsynaptic region surrounding boutons at all temperatures. H: Enlarged view of white box area in (G), arrows show the motoneuron boutons surrounded by GluRIIA. I–L: In Akt1RNAi expressing larval muscle (24B-GAL4>UAS-Akt1RNAi), GluRIIA mislocalization (arrowheads) was more severe with greater inhibition of Akt1 function at increasing temperature (larvae reared at 18°C (I), 25°C (J), or 30°C (K and L)). L Enlarged view of white box area in (K), arrows show synaptic boutons lacking GluRIIA immunoreactivity; arrowheads mark ectopic GluRIIA within intracellular bands. Scale bar in (A–G) and (I–K), 50 µm, in (H) and (L), 5 µm.
Mentions: We began assessing the role of Akt1 in NMJ assembly by examining the distribution and level of glutamate receptor IIA (GluRIIA), one of the neurotransmitter receptor subunits at this glutaminergic synapse. Glutamate is the major excitatory neurotransmitter at the type I bouton of the Drosophila larval NMJ (Brunner and Okane, 1997; Collins and DiAntonio, 2007). The NMJ glutamate receptor (GluR) is a heterotetramer comprised of three invariant subunits: GluRIIC, D, and E. The fourth subunit, either GluRIIA or B, determines the type and the electrophysiological properties of the receptor (DiAntonio et al., 1999; Featherstone et al., 2005; Qin et al., 2005a; DiAntonio, 2006). Subunit GluRIIA and B competitively bind to GluRIIC; hence, the preferential expression of these two subunits constitutes one element of developmental plasticity exhibited by this synapse (Marrus et al., 2004). We examined the levels and distributions of GluRIIA using a well-characterized monoclonal antibody, anti-GluRIIA (Featherstone et al., 2002; Qin et al., 2005a; Karr et al., 2009). The specificity of this antibody has been well documented by showing that immunoreactive signal is lost in GluRIIA mutant (Marrus et al., 2004). Partial loss of Akt1 function, achieved with the heteroallelic combination Akt11/Akt104226, altered GluRIIA distributions and levels, with a reduction at postsynaptic structures and the appearance of GluRIIA immunoreactivity within repeated bands throughout the muscle cells [Fig. 2 compare (A), (B) to (C), (D)]. This latter phenotype was more prominent in muscles 15 and 16 and was observed to a lesser extent in muscles 6 and 7, the postsynaptic cells typically used for electrophysiological analysis [arrowheads in Fig. 2(C,D)].

Bottom Line: The single Drosophila Akt family member, Akt1 selectively altered the postsynaptic targeting of one glutamate receptor subunit, GluRIIA, and was required for the expansion of a specialized postsynaptic membrane compartment, the subsynaptic reticulum (SSR).Several lines of evidence indicated that Akt1 influences SSR assembly by regulation of Gtaxin, a Drosophila t-SNARE protein (Gorczyca et al., 2007) in a manner independent of the mislocalization of GluRIIA.Our findings show that Akt1 governs two critical elements of synapse development, neurotransmitter receptor localization, and postsynaptic membrane elaboration.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, Pennsylvania, 16802.

Show MeSH
Related in: MedlinePlus