Manipulating Neuronal Circuits with Endogenous and Recombinant Cell-Surface Tethered Modulators

Neuronal circuits depend on the precise regulation of cell-surface receptors and ion channels. An ongoing challenge in neuroscience research is deciphering the functional contribution of specific receptors and ion channels using engineered modulators. A novel strategy, termed “tethered toxins”, was recently developed to characterize neuronal circuits using the evolutionary derived selectivity of venom peptide toxins and endogenous peptide ligands, such as lynx1 prototoxins. Herein, the discovery and engineering of cell-surface tethered peptides is reviewed, with particular attention given to their cell-autonomy, modular composition, and genetic targeting in different model organisms. The relative ease with which tethered peptides can be engineered, coupled with the increasing number of neuroactive venom toxins and ligand peptides being discovered, imply a multitude of potentially innovative applications for manipulating neuronal circuits and tissue-specific cell networks, including treatment of disorders caused by malfunction of receptors and ion channels

The Biochemical Anatomy of Cortical Inhibitory Synapses

Classical electron microscopic studies of the mammalian brain revealed two major classes of synapses, distinguished by the presence of a large postsynaptic density (PSD) exclusively at type 1, excitatory synapses. Biochemical studies of the PSD have established the paradigm of the synapse as a complex signal-processing machine that controls synaptic plasticity. We report here the results of a proteomic analysis of type 2, inhibitory synaptic complexes isolated by affinity purification from the cerebral cortex. We show that these synaptic complexes contain a variety of neurotransmitter receptors, neural cell-scaffolding and adhesion molecules, but that they are entirely lacking in cell signaling proteins. This fundamental distinction between the functions of type 1 and type 2 synapses in the nervous system has far reaching implications for models of synaptic plasticity, rapid adaptations in neural circuits, and homeostatic mechanisms controlling the balance of excitation and inhibition in the mature brain

Mass spectrometry identifies proteins present at tagged inhibitory synapses.

<p>(A) All peptides were evaluated individually, for their presence or absence in the sample isolated via VGABA_ARα1 or eGFP, using information from peptide fragmentation spectrum (MS/MS), peptide mass spectrum (MS), and peptide retention time in extracted ion chromatogram. An example is shown for peptide, GDDNAVTGTK, from GABA_ARβ2. V: Venus. G_AR: GABA_A receptor. (B) Schematic representation of the cortical inhibitory synaptic protein complex. These synapses contain a multitude of inhibitory receptors, as well as cell signaling and adhesion proteins, but are entirely lacking in cell signaling molecules. The localization of LHFPL4 and Neurobeachin is hypothetical. Complete information on each peptide is in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039572#pone-0039572-t001" target="_blank">Table 1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039572#pone.0039572.s002" target="_blank">Figure S2</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039572#pone.0039572.s003" target="_blank">Table S1</a>.</p

Venus-GABA_ARα1 localizes specifically to inhibitory synapses.

<p>(A) Light microscopy of fixed saggital sections from wild type and Otx1-VGABA_ARα1 transgenic mice treated with anti-GFP antibody and revealed with the DAB procedure. Transgenic, but not wild type mice express the fusion protein in layer 5/6 cortical pyramidal neurons. The fusion protein localizes to cell bodies (arrows) and processes (arrowheads) in cortex. Scale bars: 200 µm. (B) Immuno-electron microscopy shows VGABA_ARα1 expression (arrows) exclusively at inhibitory synapses by silver-intensified immunogold labeling (SIG). Inhibitory terminals immunoreactive for GAD65/67 are revealed with the DAB procedure (white asterisks). Asymmetric synapses (black asterisks) are immunonegative for both GAD and VGABA_ARα1. Scale: 500 nm. Cy: cytoplasm. Nu: nucleus. De: dendrite. V: Venus. G_AR: GABA_A receptor. (C) Within a total cortical area of 614.6 square microns 67 of the 134 inhibitory (symmetric) synapses were labeled by VGABA_ARα1, whereas none of the 200 excitatory (asymmetric) synapses were immunopositive for the fusion protein. (D) An average of 54% of inhibitory synapses were immunopositive for VGABA_ARα1, compared to 0% of the excitatory synapses. The data are presented as average ± SEM (t test).</p

Transgenic expression of Venus-GABA_ARα1.

<p>(A) Strategy for Otx1 BAC modification with VGABA_ARα1. The red line shows the Southern blot probe used in (B). Scale: 2 kb. (B) Correct incorporation of <i>Venus-GABRA1</i> cDNA into the Otx1 BAC is shown by southern blotting. The modified BAC (middle lane) contains an additional EcoR1 site. The right lane shows correct incorporation of the modified BAC into the mouse genome. The transgenic mouse genome contains a wild-type copy of the Otx1 regulatory region as well as the modified Otx1-<i>Venus-GABRA1</i> BAC. (C) Cortical protein extract from wild type and Otx1-VGABA_ARα1 mice immunoblotted with anti-GABA_ARα1 antibody. Only the transgenic mouse expresses the fusion version of the GABA_ARα1 subunit (top band). (D) VGABA_ARα1 expression in cortical layers 5 and 6 pyramidal neurons of Otx1-VGABA_ARα1 mice is shown by GFP immunoreactivity. The fusion protein is localized to pyramidal cell soma in layers 5/6 and processes in layers 2/3. Scale: 500 µm. (E) Immunofluorescence shows the colocalization of VGABA_ARα1 (green) and NeuN (red), a neuronal marker, in layers 5 and 6 pyramidal neurons of Otx1- VGABA_ARα1 transgenic mice (left). VGABA_ARα1 is mainly localized to the perikarya of the cell soma as well as dendrites. A control Otx1 BAC transgenic mouse expresses soluble eGFP (right), which fills the cell soma. Scale: 100 µm. V: Venus. G_AR: GABA_A receptor.</p

Biochemical purification of a tagged inhibitory synaptic protein complex.

<p>(A) Immunoblotting of various proteins shows that detergent solubilized protein extract S3 was enriched in both inhibitory (VGABA_ARα1, GABA_ARα1, GABA_ARβ2/3, GABA_ARγ2) and excitatory (GluR2, PSD95) synaptic proteins, as well as mitochondria (COx). Gel filtration of fraction S3 enabled enrichment of synaptic protein complexes relative to intracellular proteins, as shown by the specific exclusion of the endoplasmic reticulum marker BIP, from the high molecular weight fractions (6–10). Protein concentration of each fraction was measured (top), and the void volume determined by the elution of Blue Dextran (2000 kDa). Identical results were obtained for endogenous proteins in fractions prepared from wildtype or Otx1-eGFP cortices (not shown). (B) Fractions 6–10 (red box in A) from Otx1-VGABA_ARα1 or Otx1-eGFP control were pooled and subject to co-immunopurification using an anti-eGFP antibody. Immunoblotting confirmed the specific presence of inhibitory synaptic proteins (VGABA_ARα1, GABA_ARα1, GABA_ARβ2/3, GABA_ARγ2) and the absence of excitatory synaptic (GluR2, PSD95) and mitochondrial (COx) proteins in the material immunopurified via VGABA_ARα1. Only soluble eGFP was detected in the control sample. IN: Input. FT: Flow-through. IP: Immunoprecipitate. V: Venus. G_AR: GABA_A receptor. Further biochemical experimental results are presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039572#pone.0039572.s001" target="_blank">Figure S1</a>.</p

Inhibitory synaptic proteins identified by mass spectrometry.

<p>Proteins were purified via VGABA_ARα1 or eGFP and identified by LC-MS/MS. Two replicate datasets were analyzed using the GPM Database and those proteins identified in the eGFP controls were excluded from the list. Proteins present in the dataset were confirmed to be present in the replicate by either MS or MS/MS, as indicated. GPM E value is the probability that an assignment occurs by chance. Total peptide number is the total number of peptides that match a given protein. When more than one homologue is reported, unique peptide no. corresponds to the number of peptide matches that are unique to the homologue.</p

The inhibitory synapse affinity tag, Venus-GABA_ARα1, is functional <i>in vitro</i>.

<p>(A) Schematic of VGABA_ARα1, showing the N-terminal fusion of an affinity tag, Venus. (B) Representative GABA-evoked currents (left) and current amplitude quantification (right) in voltage clamped <i>Xenopus</i> oocytes after coinjection of the indicated <i>GABR</i> cRNA subunits. Values are expressed as mean ± SEM; n  = 5 oocytes per group (**p<0.01 t-test). (C) Schematic of the patch and stimulation electrodes used for paired-pulse recordings in cultured hippocampal neurons transduced with lentivirus encoding GABA_ARα1 (Lv-G_ARα1) or Venus-GABA_ARα1 (Lv-VG_ARα1) subunits. (D) Representative traces of GABAergic transmission in paired-pulse recordings in non-infected neurons and in neurons infected with the indicated lentivirus. Control traces in the presence of bicuculine are shown below each trace. (E) Quantification of the first eIPSC amplitudes and of the paired-pulse ratios obtained in the indicated neuronal cultures. Values are expressed as mean ± SEM; n  = 7−9 recorded cells per group.</p