Synapse microarray identification of small molecules that enhance synaptogenesis

Abstract

Synaptic function is affected in many brain diseases and disorders. Technologies for large-scale synapse assays can facilitate identification of drug leads. Here we report a “synapse microarray” technology that enables ultra-sensitive, high-throughput, and quantitative screening of synaptogenesis. Our platform enables the induction of synaptic structures in regular arrays by precise positioning of non-neuronal cells expressing synaptic proteins, while allowing neurites to grow freely around these cells. The technology increases by tenfold the sensitivity of the traditional assays, and simultaneously decreases the time required to capture synaptogenic events by an order of magnitude. It is readily incorporated into multiwell formats compatible with industrial high-throughput screening platforms. Using this technology, we screened a chemical library and identified novel histone deacetylase inhibitors that improve neuroligin-1 induced synaptogenesis via modulating class-I histone deacetylases. We also found a structure-activity relationship for designing novel potent histone deacetylase inhibitors, which can be applied towards development of new therapeutics.

Micropatterning neuronal networks

“…[9][10][11][12] Ex vivo neuronal circuits can be constructed by restricting neurons within microchannel architectures [13][14][15] or, more commonly, by micropatterning an adhesive environment against a so-called cytophobic background that resists cell adhesion. The latter approach does not physically restrict neuron development, but instead provides spatially-dened biochemical guidance cues for the directed organisation of the neuronal circuit.…”

Microfluidic construction of minimalistic neuronal co-cultures

“…A trap pitch of 13 mm was used to accommodate the 12.0-mm-diameter SH-SY5Y cells and the slightly smaller mouse cortical neurons and LUHMES cells. HEK293 cells are appropriate for synaptogenesis studies, 20 but have a significantly larger suspension diameter of 16.2 mm (SD ¡ 2.1). The arraying quality with HEK293 cells was markedly reduced, with only 38.8% of cells occupying single traps.…”

Transcellular Neuroligin-2 Interactions Enhance Insulin Secretion and Are Integral to Pancreatic β Cell Function

“…Analysis of syntaxin clustering was by analysis of the punctateness of syntaxin immunofluorescence (i.e. the syntaxin protein content at sites of clustering, as reflected by the brightness of individual puncta of syntaxin staining, evaluated by measuring the signal intensity of syntaxin-positive pixels) as has been described previously for synapsin and other secretory proteins (27)(28)(29). An illustration of the overall approach used is provided in the supplemental material (supplemental Fig.…”

“…Secretory microdomain assembly includes submembrane localization and clustering of t-SNAREs, such as syntaxin-1A, and other exocytic proteins (49,54,55). Neuroligin-induced assembly of the active zones of exocytosis in neuronal processes can be quantitatively assessed by analysis of presynaptic secretory protein clustering (27)(28)(29). As has been described, secretory protein clustering is reflected by the punctateness (intensity distribution) of secretory protein immunofluorescent staining; increased protein clustering increases the intensity of the immunofluorescent signal at the punctate sites of staining representing clustered proteins (27)(28)(29).…”

“…7B). As in prior studies of neuroligin function assessing presynaptic synapsin clustering (27,29), the immunofluorescent signal intensity of syntaxin-1A-positive sites was measured (Fig. 7C).…”