Sub-synaptic, multiplexed analysis of proteins reveals Fragile X related protein 2 is mislocalized in Fmr1 KO synapses

Abstract

The distribution of proteins within sub-synaptic compartments is an essential aspect of their neurological function. Current methodologies, such as electron microscopy (EM) and superresolution imaging techniques, can provide the precise localization of proteins, but are often limited to a small number of one-time observations with narrow spatial and molecular coverage. The diversity of synaptic proteins and synapse types demands synapse analysis on a scale that is prohibitive with current methods. Here, we demonstrate SubSynMAP, a fast, multiplexed subsynaptic protein analysis method using wide-field data from deconvolution array tomography (ATD). SubSynMAP generates probability distributions for that reveal the functional range of proteins within the averaged synapse of a particular class. This enables the differentiation of closely juxtaposed proteins. Using this method, we analyzed 15 synaptic proteins in normal and Fragile X mental retardation syndrome (FXS) model mouse cortex, and revealed disease-specific modifications of sub-synaptic protein distributions across synapse classes and cortical layers.

Probabilistic fluorescence-based synapse detection

“…The synapse detection methodology described here is not the first to grapple with the challenges of detecting synapses in immunofluorescence images [ 6 ] [ 7 ] [ 8 ] [ 9 ] [ 10 ]. The special utility and novelty of this tool partially lies in (1) producing outputs in the form of probability maps, reflecting the limited certainty with which synapses can be detected by most experimental modalities [ 11 ], and (2) the superior utility for both interactive and algorithmic exploration which is conferred by the query-based architecture resulting from the unsupervised framework.…”

Super-resolving Microscopy in Neuroscience

“…321 Multiplexed imaging applying a new computational method called SubSynMAP (SUB-SYNaptic, multiplexed analysis of proteins), which enables evaluation of several synaptic markers in multiple synapses in large brain areas at subdiffractional resolution, was also tested in Fmr1KO mice. 436 The method is based on a combination of deconvolution array technology, electron microscopy computation synapse classification, and synapse alignment using a variety of synaptic protein localization profiles. Using large data sets, this combination allows parallel investigation and comparison of several proteins in synaptic compartments to obtain information on their probability distribution.…”

“…FMRP deletion in Fmr1KO mice, however, leads to layer-specific deletion of FXR2P whereas localization of FXR1P remains unchanged. 436 Recent studies suggest that super-resolving microscopy may also become a valuable tool in advanced and probably also routine diagnostics of disease. In a proof-of-concept study, this was demonstrated by an increased detection rate of lowdensity cell surface antigens in myeloma cells.…”

“…Interestingly, disturbing synaptic anchoring of mGluR5 and NMDA-R by in vivo knock-down of Homer using AAV vector induced shRNA resulted in improvement of NMDA-R deficits and memory dysfunction . Multiplexed imaging applying a new computational method called SubSynMAP (SUB-SYNaptic, multiplexed analysis of proteins), which enables evaluation of several synaptic markers in multiple synapses in large brain areas at subdiffractional resolution, was also tested in Fmr1KO mice . The method is based on a combination of deconvolution array technology, electron microscopy computation synapse classification, and synapse alignment using a variety of synaptic protein localization profiles.…”

Q&A: Array tomography

“…a Nanoscale localization of multiple synaptic proteins along transynaptic axis, generated from 36,977 individual mouse cortical synapses (from Fig. 4 in [ 93 ]). b Variations with depth of volume density and size of seven molecular synapse types in mouse somatosensory cortex (from Fig.…”