Ampa Receptor Transport Along The Cytoskeleton In Dendrites And In Spines

The long-range dendritic trafficking of AMPARs depends on the microtubular cytoskeleton that runs along dendritic shafts. This can be demonstrated by monitoring AMPAR dendritic trafficking in neurons in which the tubulin cytoskeleton is destabilized pharmacologically. As shown in Figure 24.1 (Colorplate 11), incubation with a low concentration of vincristine (a microtubule destabilizer) impairs the dendritic transport of newly synthesized green fluorescence protein (GFP)-tagged AMPARs, to the point that their accumulation in distal dendrites is severely reduced. As a control, the same treatment does not affect the dendritic distribution of a co-expressed cytosolic protein, such as red fluorescence protein (RFP) (Figure 24.1; Colorplate 11).

Figure 24.1. Dendritic Trafficking of AMPA Receptors is Impaired by Destabilization of the Microtubular Cytoskeleton. (A) The GFP-tagged GluR2 subunit of the AMPA receptor and a cytosolic red fluorescence protein (RFP) were co-expressed in organotypic hippocampal slice cultures1 with ("VCST") or without ("—") 5 nM vincristine. Confocal fluorescence images were acquired 36 h after transfection. Scale bar: 20 |m. (B) Quantification of GluR2-GFP and RFP fluorescence 170-200 |m away from the cell soma. Fluorescence intensity values are normalized to those found at the cell soma. Plotted are average values and standard error of the mean from four experiments as the one shown in (A). GluR2-GFP presence in distal dendrites was drastically reduced in neurons incubated with vincristine. See Colorplate 11.

Figure 24.1. Dendritic Trafficking of AMPA Receptors is Impaired by Destabilization of the Microtubular Cytoskeleton. (A) The GFP-tagged GluR2 subunit of the AMPA receptor and a cytosolic red fluorescence protein (RFP) were co-expressed in organotypic hippocampal slice cultures1 with ("VCST") or without ("—") 5 nM vincristine. Confocal fluorescence images were acquired 36 h after transfection. Scale bar: 20 |m. (B) Quantification of GluR2-GFP and RFP fluorescence 170-200 |m away from the cell soma. Fluorescence intensity values are normalized to those found at the cell soma. Plotted are average values and standard error of the mean from four experiments as the one shown in (A). GluR2-GFP presence in distal dendrites was drastically reduced in neurons incubated with vincristine. See Colorplate 11.

The transport of membrane organelles on microtubule tracks is an active process powered mainly by motor proteins of the kinesin and dynein superfamilies22. Therefore, membrane compartments bearing AMPARs are likely to be recognized and transported by some of these motor proteins. The molecular mechanisms underlying these processes are just being uncovered (the role of motor proteins in the transport of synaptic proteins is also discussed in Chapter 13 of this book).

The PDZ domain-containing protein GRIP1/ABP interacts directly with the heavy chain of conventional kinesin23 and with the C-terminal PDZ motif of GluR2 and GluR324. Therefore, GRIP1/ABP may serve as the link between AMPARs and microtubular motor proteins. In addition, liprin-a associates with both the GluR2-GRIP1/ABP complex25 and with the kinesin family member KIF126, suggesting that the GRIP 1 -AMPAR complex can be transported along dendrites by multiple kinesin motor proteins. It seems likely that additional links between AMPARs and microtubular motor proteins will be discovered in the future, possibly mediated by adaptor scaffolding molecules.

Most excitatory synapses in the adult brain are formed on small dendritic protuberances called spines. Dendritic spines lack microtubular cytoskeleton, but they are rich in highly motile actin filaments. Therefore, at some point, AMPAR-containing organelles trafficking along microtubular tracks, must be transferred to the spine actin-based cytoskeleton for their final delivery into synapses. In fact, pharmacological depolymerization of actin filaments leads to the removal of AMPARs from dendritic spines27. So far, two molecular links between AMPARs and the actin cytoskeleton have been described: 4.1N and RIL. The different members of the protein 4.1 family are known to link the spectrin-actin cytoskeleton to different membrane-associated proteins28. The neuronal isoform 4.1N directly interacts with GluR129 and GluR430, and this interaction is important for the surface expression of these AMPAR subunits in heterologous systems. RIL is a PDZ domain-containing protein that links GluR1 with a-actinin, and it is important for the transport of AMPA receptors into dendritic spines31.

The transport of AMPARs along the spine actin cytoskeleton is likely to be bidirectional, since AMPARs are known to move in and out of synapses in a very dynamic manner. This expectation has recently been confirmed by the identification of an actin-based motor protein, myosin VI, as a mediator of the endocytic removal of AMPARs from synapses32. Interestingly, myosin VI interacts with the GluR1-binding protein SAP9733, providing a mechanistic link between AMPARs and a motor protein that drives their internalization. Undoubtedly, further studies will be required to unravel what is likely to be a network of interactions mediating the bidirectional transport of AMPARs along the actin cytoskeleton at dendritic spines.

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