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    • br Results We set out to identify in an

    2022-11-24


    Results We set out to identify in an unbiased manner a set of candidate “scaling factors” that might participate in transcription-dependent synaptic scaling up. The ex vivo slice and profiling studies were conducted on HsCt5 mice (described later) at postnatal day (P) 14 to P15, an age at which synaptic scaling can be induced within L4 star pyramidal neurons by 2 days of optic nerve blockade using intraocular TTX (Figure 1A; Desai et al., 2002). In vitro experiments were performed on postnatal visual cortical cultures after 7–8 days in vitro, and pyramidal neurons were targeted using standard morphological features as described (Watt et al., 2000).
    Discussion To generate insight into the transcription-dependent processes that induce synaptic scaling up, we devised a cell-type-specific screen to identify a set of candidate scaling factors with altered Hydroxysafflor yellow A during scaling. This screen identified μ3A, the cargo-recognition subunit of the APC family member AP-3, as important for sorting and trafficking of membrane-bound cargo between endosomal compartments. Because of the known association of μ3A with AMPAR, we set out to determine whether μ3A has a role in the regulated changes in AMPAR trafficking that drive synaptic scaling. We found that activity blockade reroutes both μ3A and GluA2 into RE, without affecting the δ3 subunit, which is obligatory for complex formation. KD of μ3A prevented synaptic scaling and the redistribution of GluA2 into RE, while OE of either full-length μ3A or a truncated form that cannot interact with the AP-3A complex was sufficient to drive GluA2 to REs. Finally, OE of μ3A acted synergistically with GRIP1 to recruit GluA2 to the cell surface. Taken together, these data support a model in which excess μ3A acts independently of the AP-3A complex to reroute AMPAR to RE, from which they are recruited to the synapse to enhance synaptic strength during scaling up (Figure S1). We used an unbiased cell-type-specific profiling approach (Sugino et al., 2006) that allowed us to probe for persistent transcriptional changes in a population of neurons (L4 star pyramidal neurons from V1) known to undergo synaptic scaling in response to visual deprivation during early postnatal development (Desai et al., 2002). None of the small number of candidates identified (Table 1) had previously been associated with synaptic scaling. There is no overlap between this candidate set and transcripts previously found to be regulated by visual deprivation in V1 of rodents or primates (Nedivi et al., 1996; Lachance and Chaudhuri, 2004; Majdan and Shatz, 2006; Tropea et al., 2006). This is likely due to large methodological differences in approach; most importantly, these previous studies probed whole V1 extracts, while here we probe changes in a specific cell type as these neurons are undergoing synaptic scaling. Because of the complexity of neocortical circuits and cell types and the diversity of plasticity mechanisms present (Feldman, 2009; Nelson and Turrigiano, 2008), this cell-type-specific approach is likely critical for identifying candidates that are tied to particular forms of neocortical plasticity, rather than general circuit-wide responses to deprivation or other activity paradigms. Interestingly, none of the signaling and trafficking proteins that have previously been linked to synaptic scaling through a candidate approach came up in our screen (Table S1). As an example, the essential scaling factor GRIP1 is not transcriptionally regulated, consistent with the observation that although GRIP1 increases at synapses during scaling, this is not accompanied by an increase in total GRIP1 protein (Gainey et al., 2015). This underscores the point that synaptic scaling involves the activity-dependent regulation of several AMPAR trafficking steps, only some of which are regulated at the level of transcription. We chose to focus on μ3A because AP-3 plays a role in trafficking of receptors into dendrites and/or to the neuronal surface (Matsuda et al., 2008; Bendor et al., 2010) and, more recently, AP-3A has been shown to interact with AMPAR through an indirect association involving μ3A binding to the TARP stargazin (STG), an association that is important for the induction of long-term depression (LTD) (Matsuda et al., 2013). None of the tetrameric APC family member subunits were previously known to exhibit activity-dependent transcriptional regulation, whereas here we find that both μ3A and μ4 (but not any other subunits of either APC) were upregulated by day 2 of visual deprivation. AP-3A is primarily known for a role in sorting cargo from the Golgi or EE to lysosomes or lysosome-related organelles (LROs) (Dell’Angelica, 2009; Dell’Angelica et al., 1997; Peden et al., 2004), and during LTD, the association of μ3A with stargazin enhances the trafficking of AMPAR to lysosomes (Matsuda et al., 2013). Thus, it was a surprise to find that during synaptic scaling, the upregulation of μ3A leads to enhanced accumulation of μ3A and AMPAR within the RE compartment and a reduction in the association of μ3A with lysosomes. These data show that the localization of μ3A is dynamic and can be regulated by activity to redirect cargo (in particular, AMPAR) into the recycling endocytic pathway.