A Compact Palmitoylated Motif (pABP-18) Drives Postsynaptic Targeting Toward Cell-Type-Specific Single-Synaptosome RNA Sequencing in SHANK3 Circuits

Abstract

Mutations in the SHANK3 gene have been identified as a risk factor for autism spectrum disorder (ASD), with known effects on synaptic structure and function. Recent work has started mapping SHANK3-related changes in non-human primate models, but the cell-type-specific molecular composition of synapses–especially local mRNA populations at postsynaptic sites–remains poorly understood. A long-term goal of this project is to develop an AAV-based, single-synaptosome RNA sequencing (SSS) pipeline that can profile synaptic mRNAs from defined neuron types. As a first step toward that goal, this thesis focuses on building and validating compact synaptic-targeting tools that can later be used in AAV constructs. I designed two fluorescent reporter constructs, pABP-18 and pABP-53, by fusing TagRFP to short N-terminal motifs derived from the palmitoylation domain of the postsynaptic protein pABP-L. These constructs were cloned into a pCAGGs backbone, expressed in primary mouse neurons, and imaged together with the postsynaptic marker PSD-95. To test whether these motifs are able to drive synaptic localization on their own, I quantified PSD-95 enrichment at pABP-positive puncta compared to nearby dendritic background using Fiji. A non-targeted pCAGGs-RFP control showed no enrichment (~1.0x), while pABP-53 produced moderate enrichment (1.3-2.4x). Surprisingly, the minimal pABP-18 construct showed the strongest effect, with PSD-95 levels ~3.5-4.2x higher at pABP puncta than in surrounding dendrite. These results show that short, palmitoylated pABP fragments–especially pABP-18–are sufficient on their own to drive robust postsynaptic targeting of a fluorescent reporter. The AAV implementation, in vivo validation, and SSS pipeline remain in progress. In the next stage of the project, these validated motifs will be placed into enhancer-driven AAV constructs to test whether they can drive cell-type-specific synaptic labeling in vivo. Because AAVs have size limits, having a compact sequence such as pABP-18 that reliably targets synapses is a major advantage. The goal is to use these AAVs to label postsynaptic terminals from defined neuronal populations and eventually isolate those synapses for single-synaptosome RNA sequencing, with the long-term goal of applying this approach to SHANK3 mutant circuits.