A bidirectional switch in the Shank3 phosphorylation state biases synapses toward up- or downscaling.

Homeostatic synaptic plasticity requires widespread remodeling of synaptic signaling and scaffolding networks, but the role of post-translational modifications in this process has not been systematically studied. Using deep-scale quantitative analysis of the phosphoproteome in mouse neocortical neurons, we found widespread and temporally complex changes during synaptic scaling up and down. We observed 424 bidirectionally modulated phosphosites that were strongly enriched for synapse-associated proteins, including S1539 in the autism spectrum disorder-associated synaptic scaffold protein Shank3. Using a parallel proteomic analysis performed on Shank3 isolated from rat neocortical neurons by immunoaffinity, we identified two sites that were persistently hypophosphorylated during scaling up and transiently hyperphosphorylated during scaling down: one (rat S1615) that corresponded to S1539 in mouse, and a second highly conserved site, rat S1586. The phosphorylation status of these sites modified the synaptic localization of Shank3 during scaling protocols, and dephosphorylation of these sites via PP2A activity was essential for the maintenance of synaptic scaling up. Finally, phosphomimetic mutations at these sites prevented scaling up but not down, while phosphodeficient mutations prevented scaling down but not up. These mutations did not impact baseline synaptic strength, indicating that they gate, rather than drive, the induction of synaptic scaling. Thus, an activity-dependent switch between hypo- and hyperphosphorylation at S1586 and S1615 of Shank3 enables scaling up or down, respectively. Collectively, our data show that activity-dependent phosphoproteome dynamics are important for the functional reconfiguration of synaptic scaffolds and can bias synapses toward upward or downward homeostatic plasticity.

Autism-Associated Shank3 Is Essential for Homeostatic Compensation in Rodent V1.

Mutations in Shank3 are strongly associated with autism spectrum disorders and neural circuit changes in several brain areas, but the cellular mechanisms that underlie these defects are not understood. Homeostatic forms of plasticity allow central circuits to maintain stable function during experience-dependent development, leading us to ask whether loss of Shank3 might impair homeostatic plasticity and circuit-level compensation to perturbations. We found that Shank3 loss in vitro abolished synaptic scaling and intrinsic homeostatic plasticity, deficits that could be rescued by treatment with lithium. Further, Shank3 knockout severely compromised the in vivo ability of visual cortical circuits to recover from perturbations to sensory drive. Finally, lithium treatment ameliorated a repetitive self-grooming phenotype in Shank3 knockout mice. These findings demonstrate that Shank3 loss severely impairs the ability of central circuits to harness homeostatic mechanisms to compensate for perturbations in drive, which, in turn, may render them more vulnerable to such perturbations.

Upregulation of µ3A Drives Homeostatic Plasticity by Rerouting AMPAR into the Recycling Endosomal Pathway.

Synaptic scaling is a form of homeostatic plasticity driven by transcription-dependent changes in AMPA-type glutamate receptor (AMPAR) trafficking. To uncover the pathways involved, we performed a cell-type-specific screen for transcripts persistently altered during scaling, which identified the µ subunit (µ3A) of the adaptor protein complex AP-3A. Synaptic scaling increased µ3A (but not other AP-3 subunits) in pyramidal neurons and redistributed dendritic µ3A and AMPAR to recycling endosomes (REs). Knockdown of µ3A prevented synaptic scaling and this redistribution, while overexpression (OE) of full-length µ3A or a truncated µ3A that cannot interact with the AP-3A complex was sufficient to drive AMPAR to REs. Finally, OE of µ3A acted synergistically with GRIP1 to recruit AMPAR to the dendritic membrane. These data suggest that excess µ3A acts independently of the AP-3A complex to reroute AMPAR to RE, generating a reservoir of receptors essential for the regulated recruitment to the synaptic membrane during scaling up.

Activity-dependent synaptic GRIP1 accumulation drives synaptic scaling up in response to action potential blockade.

Synaptic scaling is a form of homeostatic plasticity that stabilizes neuronal firing in response to changes in synapse number and strength. Scaling up in response to action-potential blockade is accomplished through increased synaptic accumulation of GluA2-containing AMPA receptors (AMPAR), but the receptor trafficking steps that drive this process remain largely obscure. Here, we show that the AMPAR-binding protein glutamate receptor-interacting protein-1 (GRIP1) is essential for regulated synaptic AMPAR accumulation during scaling up. Synaptic abundance of GRIP1 was enhanced by activity deprivation, directly increasing synaptic GRIP1 abundance through overexpression increased the amplitude of AMPA miniature excitatory postsynaptic currents (mEPSCs), and shRNA-mediated GRIP1 knockdown prevented scaling up of AMPA mEPSCs. Furthermore, knockdown and replace experiments targeting either GRIP1 or GluA2 revealed that scaling up requires the interaction between GRIP1 and GluA2. Finally, GRIP1 synaptic accumulation during scaling up did not require GluA2 binding. Taken together, our data support a model in which activity-dependent trafficking of GRIP1 to synaptic sites drives the forward trafficking and enhanced synaptic accumulation of GluA2-containing AMPAR during synaptic scaling up.

How to scale down postsynaptic strength.

Synaptic scaling is a form of synaptic plasticity that contributes to the homeostatic regulation of neuronal activity both in vitro and in vivo, by bidirectionally and proportionally adjusting postsynaptic AMPA receptor (AMPAR) abundance to compensate for chronic perturbations in activity. This proportional regulation of synaptic strength allows synaptic scaling to normalize activity without disrupting the synapse-specific differences in strength thought to underlie memory storage, but how such proportional scaling of synaptic strength is accomplished at the biophysical level is unknown. Here we addressed this question in cultured rat visual cortical pyramidal neurons. We used photoactivation and fluorescence recovery after photobleaching of fluorescently tagged AMPAR to show that scaling down, but not up, decreases the steady-state accumulation of synaptic AMPAR by increasing the rate at which they unbind from and exit the postsynaptic density (Koff). This increase in Koff was not diffusion limited, was independent of AMPAR endocytosis, and was prevented by a scaffold manipulation that specifically blocks scaling down, suggesting that it is accomplished through enhanced dissociation of AMPAR from synaptic scaffold tethers. Finally, simulations show that increasing Koff decreases synaptic strength multiplicatively, by reducing the fractional occupancy of available scaffold "slots." These data demonstrate that scaling down is accomplished through a regulated increase in Koff, which in turn reduces the fractional occupancy of synaptic scaffolds to proportionally reduce synaptic strength.

Conditional knockout of tumor overexpressed gene in mouse neurons affects RNA granule assembly, granule translation, LTP and short term habituation.

In neurons, specific RNAs are assembled into granules, which are translated in dendrites, however the functional consequences of granule assembly are not known. Tumor overexpressed gene (TOG) is a granule-associated protein containing multiple binding sites for heterogeneous nuclear ribonucleoprotein (hnRNP) A2, another granule component that recognizes cis-acting sequences called hnRNP A2 response elements (A2REs) present in several granule RNAs. Translation in granules is sporadic, which is believed to reflect monosomal translation, with occasional bursts, which are believed to reflect polysomal translation. In this study, TOG expression was conditionally knocked out (TOG cKO) in mouse hippocampal neurons using cre/lox technology. In TOG cKO cultured neurons granule assembly and bursty translation of activity-regulated cytoskeletal associated (ARC) mRNA, an A2RE RNA, are disrupted. In TOG cKO brain slices synaptic sensitivity and long term potentiation (LTP) are reduced. TOG cKO mice exhibit hyperactivity, perseveration and impaired short term habituation. These results suggest that in hippocampal neurons TOG is required for granule assembly, granule translation and synaptic plasticity, and affects behavior.

Polarization of actin cytoskeleton is reduced in dendritic protrusions during early spine development in hippocampal neuron.

Dendritic spines are small protrusions that receive synaptic signals in neuronal networks. The actin cytoskeleton plays a key role in regulating spine morphogenesis, as well as in the function of synapses. Here we report the first quantitative measurement of F-actin retrograde flow rate in dendritic filopodia, the precursor of dendritic spines, and in newly formed spines, using a technique based on photoactivation localization microscopy. We found a fast F-actin retrograde flow in the dendritic filopodia but not in the spine necks. The quantification of F-actin flow rates, combined with fluorescence recovery after photobleaching measurements, allowed for a full quantification of spatially resolved kinetic rates of actin turnover, which was not previously feasible. Furthermore we provide evidences that myosin II regulates the actin flow in dendritic filopodia and translocates from the base to the tip of the protrusion upon spine formation. Rac1 inhibition led to mislocalization of myosin II, as well as to disruption of the F-actin flow. These results provide advances in the quantitative understanding of F-actin remodeling during spine formation.

Single-molecule imaging of translational output from individual RNA granules in neurons.

Dendritic RNAs are localized and translated in RNA granules. Here we use single-molecule imaging to count the number of RNA molecules in each granule and to record translation output from each granule using Venus fluorescent protein as a reporter. For RNAs encoding activity-regulated cytoskeletal-associated protein (ARC) or fragile X mental retardation protein (FMRP), translation events are spatially clustered near individual granules, and translational output from individual granules is either sporadic or bursty. The probability of bursty translation is greater for Venus-FMRP RNA than for Venus-ARC RNA and is increased in Fmr1-knockout neurons compared to wild-type neurons. Dihydroxyphenylglycine (DHPG) increases the rate of sporadic translation and decreases bursty translation for Venus-FMRP and Venus-ARC RNAs. Single-molecule imaging of translation in individual granules provides new insight into molecular, spatial, and temporal regulation of translation in granules.

Investigating sub-spine actin dynamics in rat hippocampal neurons with super-resolution optical imaging.

Morphological changes in dendritic spines represent an important mechanism for synaptic plasticity which is postulated to underlie the vital cognitive phenomena of learning and memory. These morphological changes are driven by the dynamic actin cytoskeleton that is present in dendritic spines. The study of actin dynamics in these spines traditionally has been hindered by the small size of the spine. In this study, we utilize a photo-activation localization microscopy (PALM)-based single-molecule tracking technique to analyze F-actin movements with approximately 30-nm resolution in cultured hippocampal neurons. We were able to observe the kinematic (physical motion of actin filaments, i.e., retrograde flow) and kinetic (F-actin turn-over) dynamics of F-actin at the single-filament level in dendritic spines. We found that F-actin in dendritic spines exhibits highly heterogeneous kinematic dynamics at the individual filament level, with simultaneous actin flows in both retrograde and anterograde directions. At the ensemble level, movements of filaments integrate into a net retrograde flow of approximately 138 nm/min. These results suggest a weakly polarized F-actin network that consists of mostly short filaments in dendritic spines.

Multiplexed RNA trafficking in oligodendrocytes and neurons.

In oligodendrocytes and neurons genetic information is transmitted from the nucleus to dendrites in the form of RNA granules. Here we describe how transport of multiple different RNA molecules in individual granules is analogous to the process of multiplexing in telecommunications. In both cases multiple messages are combined into a composite signal for transmission on a single carrier. Multiplexing provides a mechanism to coordinate local expression of ensembles of genes in myelin in oligodendrocytes and at synapses in neurons.

Multiplexed dendritic targeting of alpha calcium calmodulin-dependent protein kinase II, neurogranin, and activity-regulated cytoskeleton-associated protein RNAs by the A2 pathway.

In neurons, many different RNAs are targeted to dendrites where local expression of the encoded proteins mediates synaptic plasticity during learning and memory. It is not known whether each RNA follows a separate trafficking pathway or whether multiple RNAs are targeted to dendrites by the same pathway. Here, we show that RNAs encoding alpha calcium calmodulin-dependent protein kinase II, neurogranin, and activity-regulated cytoskeleton-associated protein are coassembled into the same RNA granules and targeted to dendrites by the same cis/trans-determinants (heterogeneous nuclear ribonucleoprotein [hnRNP] A2 response element and hnRNP A2) that mediate dendritic targeting of myelin basic protein RNA by the A2 pathway in oligodendrocytes. Multiplexed dendritic targeting of different RNAs by the same pathway represents a new organizing principle for coordinating gene expression at the synapse.