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    • The RE compartment has been shown to be

    2024-03-27

    The RE compartment has been shown to be critical for supplying AMPAR during LTP (Hanley, 2010; Park et al., 2004), but whether they also supply AMPAR for synaptic scaling has been less clear (Gainey et al., 2015; Tan et al., 2015). Interestingly, although an increase in μ3A is necessary for scaling, and is necessary and sufficient to drive AMPAR to RE, it is not sufficient to increase mEPSC amplitude. This suggests that once AMPAR are rerouted into the recycling pathway by μ3A, they must still be recruited to the synaptic membrane in a second activity-dependent trafficking step. The GluA2-interacting protein GRIP1 plays a critical role in recruiting AMPAR from internal endosomal compartments to the synaptic membrane during synaptic scaling (Gainey et al., 2015; Tan et al., 2015). Here we show that when μ3A and GRIP1 are OE together, they can act synergistically to enhance surface AMPAR accumulation, providing direct experimental support for a two-step trafficking model in which μ3A recruits AMPAR into the recycling pathway, where they can then be recruited to and stabilized by GRIP1 at the synaptic membrane (Figure S1). Systemic loss of functional AP-3 causes endosomal trafficking defects, Hermansky-Pudlak syndrome, and neurological symptoms (Kantheti et al., 1998; Lane and Deol, 1974; Peden et al., 2002; Seong et al., 2005; Sirkis et al., 2013; Swank et al., 2000; Yang et al., 2000). These have mainly been ascribed to defects in lysosomal trafficking and/or biogenesis of LROs. Our data suggest that the μ3A subunit traffics AMPAR away from alkaline phosphatase inhibitor and into the recycling pathway by acting independently of the full AP-3 complex. Three main pieces of evidence support this idea. First, while μ3A is upregulated during synaptic scaling, the δ3 subunit is not. Because the δ3 subunit is obligatory for formation of both the AP-3A and the AP-3B complexes (Kantheti et al., 1998; Peden et al., 2002), this suggests that either μ3A is limiting for complex formation or that activity-induced μ3A is not acting as part of the complex. The latter interpretation is favored by the observation that the intensity of the μ3A signal increases even in endosomal compartments with no detectible δ3. Second, we find that OE of μ3A alone is sufficient to recruit AMPAR to RE and to enhance the ability of GRIP1 to recruit AMPAR to the dendritic membrane. Finally, OE of a truncated μ3A that cannot interact with the AP-3 complex is also able to recruit AMPAR to RE. These data show that selectively increasing μ3A is able redirect AMPAR into the recycling pathway, possibly by protecting AMPAR from association with the full AP-3 complex. Once in the recycling pathway, they can be recruited to the synapse during a subsequent trafficking step, likely involving GRIP1 (Figure S1). Taken together, our data identify μ3A as an essential transcription-dependent switch point that can redirect AMPAR to RE during synaptic scaling. These data show that both scaling up and LTP share a common reliance on AMPAR trafficking to the RE compartment (Ehlers, 2000; Park et al., 2004). Furthermore, AP-3 is important for trafficking AMPAR to lysosomes during LTD (Matsuda et al., 2013). This raises the possibility that competition between AP-3 and μ3A for the binding and sorting of AMPAR is a key mechanism underlying several distinct forms of synaptic plasticity.
    Experimental Procedures All experiments were approved by the Brandeis Animal Care and Use Committee and were in accordance with NIH guidelines. Experiments were performed on animals of both sexes. Detailed methods are provided in Supplemental Experimental Procedures. Briefly, cultures were prepared and transfected 72 hr before recording or staining as described (Pratt et al., 2003). For culture physiology, mEPSC recordings were obtained from visually identified pyramidal neurons and analyzed as described previously (Turrigiano et al., 1998; Wierenga et al., 2005). At P12 or P13, HsCt5 mice were subjected to monocular deprivation by intraocular injection of TTX (1 mM) as described (Desai et al., 2002; Maffei and Turrigiano, 2008); injections were performed twice (at P12 or P13 and then again at P13 or P14) to maintain the block for 48 hr. For slice electrophysiology, coronal brain slices from HsCt5 mice (300 μm) containing monocular primary visual cortex (V1m) were prepared from control and deprived hemispheres (P14–P15) and recordings were obtained from labeled neurons as described (Maffei et al., 2006; Loebrich et al., 2013).