Biology subjects

Cottrell, J. R.

Publications and source records attributed to Cottrell, J. R..

4 recordsLinked to original sources

bioRxiv2022

Perturbations of a causal synaptic molecular network in autism and schizophrenia revealed with multiplexed imaging

The complex functions of neuronal synapses in the central nervous system depend on their tightly interacting, compartmentalized molecular network of hundreds of proteins spanning the pre- and post-synaptic sites. This biochemical system is implicated in the pathogenesis of autism spectrum disorders and schizophrenia, with identified common synaptopathologies and numerous risk genes associated with synaptic function. However, it remains unclear how the synaptic molecular network is altered in these disorders, and whether effects are common to distinct genetic perturbations. Here, we applied PRISM, a quantitative single-synapse multiplexed imaging technique, to systematically probe the effects of RNAi knockdown of 16 autism- and schizophrenia-associated genes on the simultaneous distribution of 10 synaptic proteins. This enabled the identification of novel phenotypes in synapse compositions and distributions. We applied Bayesian network inference to construct and validate a predictive model of causal hierarchical dependencies among eight proteins of the excitatory synapse. The resulting conditional dependence relationships could only be accessed via measurement which is both single-synapse and multiprotein, unique to PRISM. Finally, we show that central features of the network are similarly affected across distinct gene knockdowns. These results offer insight into the convergent molecular etiology of these debilitating, hereditary and highly polygenic disorders, as well as offering a novel, general framework for probing subcellular molecular networks.

neuroscience↗

bioRxiv2021

A Bidirectional Switch in the Shank3 Phosphorylation State Biases Synapses toward Up or Down Scaling

Homeostatic synaptic plasticity requires widespread remodeling of synaptic signaling and scaffolding networks, but the role of posttranslational modifications in this process has not been systematically studied. Using deepscale, quantitative analysis of the phosphoproteome in mouse neocortical neurons, we found wide-spread 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 ASD-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 hypo-phosphorylated during scaling up and hyper-phosphorylated 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. Thus, an activity-dependent switch between hypo- and hyperphosphorylation at S1586/ 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.

neuroscience↗

bioRxiv2020

Molecular diversity of glutamatergic and GABAergic synapses from multiplexed fluorescence imaging

Neuronal synapses contain hundreds of different protein species important for regulating signal transmission. Characterizing differential expression profiles of proteins within synapses in distinct regions of the brain has revealed a high degree of synaptic diversity defined by unique molecular organization. Multiplexed imaging of in vitro neuronal culture models at single synapse resolution offers new opportunities for exploring synaptic reorganization in response to chemical and genetic perturbations. Here, we combine 12-color multiplexed fluorescence imaging with quantitative image analysis and machine learning to identify novel synaptic subtypes within excitatory and inhibitory synapses based on the expression profiles of major synaptic components. We characterize differences in the correlated expression of proteins within these subtypes and we examine how the distribution of these synapses is modified following induction of synaptic plasticity. Under chronic suppression of neuronal activity, phenotypic characterization revealed coordinated increases in both excitatory and inhibitory protein levels without changes in the distribution of synaptic subtypes, suggesting concerted events targeting glutamatergic and GABAergic synapses. Our results offer molecular insight into the mechanisms of synaptic plasticity.

neuroscience↗

bioRxiv2017

Multiplexed confocal and super-resolution fluorescence imaging of cytoskeletal and neuronal synapse proteins

Neuronal synapses contain dozens of protein species whose expression levels and localizations are key determinants of synaptic transmission and plasticity. The spectral properties of fluorophores used in conventional microscopy limit the number of measured proteins to four species within a given sample. The ability to perform high-throughput confocal or super-resolution imaging of many proteins simultaneously without limitation in target number imposed by this spectral limit would enable large-scale characterization of synaptic protein networks in situ. Here, we introduce PRISM: Probe-based Imaging for Sequential Multiplexing, a method that sequentially utilizes either high affinity Locked Nucleic Acid (LNA) or low affinity DNA probes to enable diffraction-limited confocal and PAINT-based super-resolution imaging. High-affinity LNA probes offer high-throughput, confocal-based imaging compared with PAINT, which uses low affinity probes to realize localization-based super-resolution imaging. Simultaneous immunostaining of all targets is performed prior to imaging, followed by sequential LNA/DNA probe exchange that requires only minutes under mild wash conditions. We apply PRISM to quantify the co-expression levels and nanometer-scale organization of one dozen cytoskeletal and synaptic proteins within individual neuronal synapses. Our approach is scalable to dozens of target proteins and is compatible with high-content screening platforms commonly used to interrogate phenotypic changes associated with genetic and drug perturbations in a variety of cell types.

bioengineering↗