Splice-dependent trans-synaptic PTPδ-IL1RAPL1 interaction regulates synapse formation and non-REM sleep (2020)
Figure 1

Figure 1. PTPδ-tdTomato protein mainly localizes to presynaptic sites at excitatory, but not inhibitory, synapses in the mouse hippocampus.

(A) Transgenic strategy for generating PTPδ-tdTomato reporter mice used to visualize the distribution patterns of endogenous PTPδ protein. CRISPR/Cas9 was used to fuse tdTomato in frame to the C-terminus of the PTPδ protein, encoded by the last coding exon (exon 21) of the Ptprd gene. HA, homology arm; CDS, coding domain sequence; UTR, untranslated region; Ig, Immunoglobulin domain; FnIII, Fibronectin 3-like domain; D, Phosphatase domain; td, tdTomato.

(B) Verification of PTPδ-tdTomato reporter mice by Western blot analysis of whole-brain lysates from heterozygote (HT) and homozygous (Ho) mice (P21) and PTPδ using mCherry antibodies. Note that levels of endogenous PTPδ protein (~85 kDa) are decreased, and that PTPδ-tdTomato protein (~140 kDa) is detectable only in reporter mice (HT and Ho). Note also that the 85-kDa band represents the transmembrane domain + cytoplasmic fragment of the full-length PTPδ protein that undergoes proteolytic cleavage at extracellular membrane-proximal sites, which can only be recognized by the PTPδ C-terminal antibody that targets the cytoplasmic region. The PTPδ-tdTomato protein (~140 kDa) contains the membrane domain + cytoplasmic fragment of PTPδ (~85 kDa) fused to tdTomato (55 kDa). The N-terminal antibody recognizes equal amounts of the C-terminal cleavage product derived from PTPδ and PTPδ-tdTomato proteins.

(C) Representative coronal, horizontal, and sagittal sections from PTPδ-tdTomato mice. Enlarged windows are demarcated by black dotted lines (Fig EV1A-C). Red tdTomato images were converted to grayscale images for clarity. ACA, anterior cingulate area; ACB, nucleus accumbens; BLA, basolateral amygdala; BMA, basomedial amygdala; CA1, cornu ammonis area 1; CA2, cornu ammonis area 2; CA3, cornu ammonis area 3; CB, cerebellum; CC, corpus callosum; CEA, central amygdala; CP, caudate putamen; CST, corticospinal tract; DG, dentate gyrus; EP, endopiriform nucleus, HY, hypothalamus; L1, cortical layer 1; L2/3, cortical layer 2/3; LA, lateral amygdala; lENT, lateral entorhinal area; LH, lateral habenula; MB, midbrain; MEA, medial amygdala; mENT, medial entorhinal area; MH, medial habenula; PIR1, piriform molecular layer; PIR2, piriform pyramidal layer; PIR3, piriform polymorph layer; PL, prelimbic; PONS, pons; PRE, presubiculum; RSP, retrosplenial area; SS, somatosensory area; SUB, subiculum; TEa, temporal association area; TH, thalamus; VIS, visual area; Scale bar, 500 μm.

(D) PTPδ-tdTomato protein is detected in tau-positive axonal compartments, but not in MAP2-positive dendritic compartments in cultured neurons (entorhinal cortex + hippocampus; 1:2 mixture; days in vitro or DIV 24) derived from Ptprd-/- mouse embryos. Note that the tdTomato signals envelop, but not overlap with, MAP2-delineated dendrites, although this does not necessarily suggest that tdTomato signals not in dendrites. Scale bars, 10 μm in main images and 2 μm in enlarged images.

(E) Presynaptic PTPδ-tdTomato clusters colocalize more strongly with PSD-95 clusters (excitatory postsynaptic marker) relative to gephyrin (inhibitory postsynaptic marker) clusters in cultured neurons (entorhinal cortex + hippocampus; DIV24) derived from Ptprd-/- mouse embryos. See Fig EV1F for the quantification of the results. Scale bars, 10 μm in main images and 2 μm in enlarged images.

(F-I) Ultrastructural localization of PTPδ-tdTomato protein (red arrows; DAB staining) at axon terminals juxtaposed to electron-dense postsynaptic densities (purple arrows; PSDs) and also at vGlut2-positive excitatory synaptic axon terminals (green arrows; immunogold staining) but not at GAD67/65-positive inhibitory synaptic axon terminals (blue arrows; immunogold staining) in the stratum lacunosum-moleculare (SLM) region of the CA1 hippocampal region (P21). Note that ~85% of PTPδ-tdTomato signals are present in excitatory presynaptic terminals apposed to PSDs, ~10% at presynaptic terminals not apposed to PSDs, and ~5% in neuropils other than presynaptic terminals. Axon terminals are indicated by pink shades. Scale bar, 500 nm. (n = 4 areas from 2 mice [WT and KO]).