Image_1_Cadherins Interact With Synaptic Organizers to Promote Synaptic Differentiation.PDF

<p>Classical cadherins, a set of ~20 related recognition and signaling molecules, have been implicated in many aspects of neural development, including the formation and remodeling of synapses. Mechanisms underlying some of these steps have been studied by expressing N-cadherin (cdh2), a Type 1 cadherin, in heterologous cells, but analysis is complicated because widely used lines express cdh2 endogenously. We used CRISPR-mediated gene editing to generate a Human embryonic kidney (HEK)293 variant lacking Cdh2, then compared the behavior of rodent cortical and hippocampal neurons co-cultured with parental, cdh2 mutant and cdh2-rescued 293 lines. The comparison demonstrated that Cdh2 promotes neurite branching and that it is required for three synaptic organizers, neurologin1 (NLGL1), leucine-rich repeat transmembrane protein 2 (LRRtm2), and Cell Adhesion Molecule 1 (Cadm1/SynCAM) to stimulate presynaptic differentiation, assayed by clustering of synaptic vesicles at sites of neurite-293 cell contact. Similarly, Cdh2 is required for a presynaptic organizing molecule, Neurexin1β, to promote postsynaptic differentiation in dendrites. We also show that another Type I cadherin, Cdh4, and a Type II cadherin, Cdh6, can substitute for Cdh2 in these assays. Finally, we provide evidence that the effects of cadherins require homophilic interactions between neurites and the heterologous cells. Together, these results indicate that classical cadherins act together with synaptic organizers to promote synaptic differentiation, perhaps in part by strengthening the intracellular adhesion required for the organizers to act efficiently. We propose that cadherins promote high affinity contacts between appropriate partners, which then enable synaptic differentiation.</p

MiR-133b does not regulate muscle reinnervation or ALS disease progression.

<p>(<b>A</b>) Semi-quantitative RT-PCR of cDNA from control or denervated hindlimb muscle 2 and 4 days after unilateral sciatic nerve cut. Levels of pre-miR-133b and AChRγ increase dramatically in denervated muscle, while levels of pre-miR-133a-1, pre-miR-133a-2 and GAPDH are unchanged, suggesting differential regulation of miR-133a and miR-133b. (<b>B–E</b>) Analysis of muscle reinnervation in tibialis anterior muscle from control (B) and miR-133b null mice (C) 3 weeks following nerve cut. (<b>D</b>) Percentage of tibialis anterior NMJs that were reinnervated. (<b>E</b>) Percentage of NMJs that were denerverated, partially reinnervated, or fully reinnervated. (<b>F,G</b>) Analysis of sternomastoid muscle reinnervation 9 days following accessory nerve crush. At least 6 mice were analyzed and 200 NMJs were examined per animal. Error bars indicate SEM. Scale bar  = 20 μm. (<b>H–J</b>) In the SOD1-G93A mouse model for ALS, loss of miR-133b does not exacerbate symptoms; disease onset (H), survival rate (I), and disease progression (J) are unchanged in the absence of miR-133b. Data were obtained from: 8 female, 8 male SOD1G93A; 10 female, 8 male miR-133b+/−;SOD1G93A; and 6 female, 9 male R-133b−/−;SOD1G93A mice. Error bars indicate SEM.</p

Generation and analysis of miR-206 and miR-133b double knockout mice.

<p>(<b>A</b>) <i>Trans</i>-allelic targeted meiotic recombination was used to generate mice lacking both miR-206 and miR-133b. MiR-206 and miR-133b heterozygous mice each containing a loxP site in place of the miRNA stemloop were bred together and with mice expressing Cre recombinase in the germline. Zygotes produced from sperm that underwent <i>trans</i>-allelic recombination contained one chromosome lacking miR-206 and miR-133b and one chromosome with a miR-206 and miR-133b duplication. These animals were then bred to obtain miR-206 and miR-133b double knockout mice, i.e. 7H4 knockout mice. P1, forward primer upstream of miR-206. P2, reverse primer downstream of miR-133b. (<b>B</b>) PCR using P1 and P2 primers (in A) gives a detectable product (550 bp) only in 7H4 heterozygous and knockout mice, demonstrating that the 7H4 genomic region containing the miR-206 and miR-133b stem loops is completely missing from the 7H4 null allele. (<b>C</b>) PCR using primers specific for the miR-133b allele yields a 600 bp band only when the WT allele is present, but no band for the 7H4 null allele. (<b>D</b>) Quantitative RT-PCR for the stemloop regions of miR-206 and miR-133b. As expected, miR-206 and miR-133b are absent in 7H4 knockout mice.</p

Development of NMJs in 7H4 mice.

<p>(<b>A–D</b>) Both miR-206 and miR-133b are dispensable for development of the NMJ. There is no obvious difference in the transformation of the postsynapse (stained using f-BTX, red) from a small plaque into a large pretzel between 7H4 knockout (B and D) and control mice of the same age (A and C). The formation of the presynaptic apparatus is also indistinguishable between 7H4 knockout mice and control mice of the same age, visualized using antibodies against synaptotagmin-2, green, and neurofilament, blue, in young animals (A and B) and YFP expressed in motor axons (C and D). Scale bar  = 10 μm for P9 and 20 μm for adult NMJs.</p

Lack of both miR-206 and miR-133b delays NMJ regeneration.

<p>(<b>A–D</b>) To determine whether both miRNAs, miR-206 and miR-133b (7H4), act in concert to affect muscle reinnervation, the peroneal nerve was crushed in control (A) and 7H4 knockout mice (B) and reinnervation of the extensor digitorium longus was examined 9 days post injury. In 7H4 muscles, the incidence of partially and completely denervated NMJs is higher than that in muscles from control animals (C, D). At least 6 mice were examined per genotype and 50 NMJs per mouse visualized. FI, fully innervated; PI, partially innervated; FD, fully denervated NMJs. Error bar  =  SEM. P-value (*) <0.02. Scale bar  = 50 μm. (<b>E, F</b>) Quantitative mRNA expression of pre-miR-1-1, pre-miR-1-2, pre-miR-133a-1, and pre-miR-133a-2 in EDL (E) and soleus (F) muscle of adult WT (black circles represent individual values and black line the mean) and 7H4 knockout (red circles represent individual values and red line the mean) mice. Gene expression is normalized to Gapdh and results are scaled to the average value of the WT samples.</p

Normal NMJ development in miR-133b knockout mice.

<p>(<b>A</b>) Immunofluorescence staining of axonal neurofilaments and vesicular synaptophysin (green) and BTX staining of postsynaptic nAChRs (red) to visualize axons innervating synaptic sites. Filled white arrowheads, NMJs with multiple axon innervation; empty arrowheads, retraction bulbs. Scale bar  = 20 μm. (<b>B</b>) The proportion of sternomastoid NMJs with multiple innervation decreases at a similar rate in control and knockout mice. (<b>C</b>) Proportion of developing sternomastoid NMJs with single, double or triple innervation is similar in control and knockout mice.</p

Morphological, physiological and molecular properties distinguish four αRGC types.

<p>A summary of morphological, physiological and molecular features for the four types of αRGCs, as reported in Figs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0180091#pone.0180091.g002" target="_blank">2</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0180091#pone.0180091.g004" target="_blank">4</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0180091#pone.0180091.g008" target="_blank">8</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0180091#pone.0180091.g010" target="_blank">10</a>. Dotted box indicates dim labeling. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0180091#sec014" target="_blank">Discussion</a>.</p

Molecular distinctions among αRGCs.

<p><b>A, B</b>: Retinal whole mounts were stained with antibodies to Opn (green), plus antibodies to one of 3 POU-domain transcription factors (Brn3a, Brn3b or Brn3c; red in <b>A</b>) or one of 3 calcium binding proteins (parvalbumin [PV], calbindin or calretinin; red in <b>B</b>). Brn3b and PV mark most αRGCs whereas Brn3a, Brn3c and calbindin mark subsets; most αRGCs are calretinin-negative. <b>C:</b> Whole mounts of YFP-H retina were quadruply stained for YFP, Opn, vAChT and the indicated marker. Cells that were Opn, YFP, and marker triple-positive were identified (green, cyan and red, respectively in top panels) and imaged (YFP only, middle panels). Stratification of YFP-positive dendrites was then determined with reference to that of starburst amacrines (vAChT-positive, red in bottom panels). Arrows point to the same cell displayed in each panel. Results are representative of 7–10 cells per type from 5 mice. Scale bar = 50 μm.</p

Morphology and light responses of mouse alpha retinal ganglion cells (αRGCs).

<p>A-D, Sample neurons with whole-mount views (outer images) and responses to a flashing spot (inner plots) for the Off-sustained (A), Off-transient (B), On-sustained (C), and On-transient (D) types. Raster graph illustrates action potentials on repeated trials of a spot flashing on (white background) and off (gray) every 2 s. Continuous curve is average firing rate over 10 or more trials.</p

Confirmation that On-transient KCNG4-cre neurons are a separate αRGC type.

<p>A-D: On-t cells express heavy neurofilament. A loose patch recording of a fluorescent neuron (A) revealed a transient response of the firing rate to light steps (B). After fixation and antibody staining one can identify the same cell based on YFP label (C) and confirm that it is strongly labeled with the neurofilament antibody SMI-32 (D). E-F: Two fluorescent neurons in close proximity (black and green arrowheads in <b>E</b>) showed sustained (black) and transient (green) response of the firing rate to a light step (<b>F</b>).</p