Fig 2: Paranodal axoglial junctions are disrupted in the TDP-43-cKO mice.(A) Immunostaining of P28 sciatic nerves for contactin-associated protein (Caspr) (green, paranode) and Nav channels (panNav, magenta, node). (B–D) Immunostaining of P28 sciatic nerves for (B) neurofascin (NFasc) (magenta, node and paranode) and Caspr (green), (C) contactin (Cntn, magenta, paranode) and NFasc (green), and (D) ankyrinB (AnkB, green, paranode) and ankyrinG (AnkG, magenta, node). Nodes are indicated by open arrowheads in B. Paranodes are indicated by solid arrowheads in B, D. (E) Immunostaining of P3 sciatic nerves for Caspr (green) and gliomedin (Gldn, magenta, node). (F) Immunostaining of P28 sciatic nerves for Kv1.1 channels (green, juxtaparanode), Caspr (red), and NFasc (blue). (G) Electron micrographs of P21 sciatic nerve longitudinal sections. (H) The time periods for which the mice remained on the rotarod at 24 or 32 rpm. Minima, first quartiles, medians, third quartiles, and maxima are plotted as box-and-whisker plots. n = 10 mice per genotype. *p=0.0115 for 24 rpm; **p=0.0036 for 32 rpm; Mann–Whitney tests. Scale bars, 5 µm (A–F) and 200 nm (G). Conditional heterozygote (cHet) and conditional knockout (cKO) by Dhh-Cre in (A–C) and (E–G), and by Mpz-Cre in (D, H).Figure 2—source data 1.Statistical summary for Figure 2H.

Fig 3: Schwann cell neurofascin (NFasc) expression is abolished in the TDP-43 conditional knockout (cKO).(A) The schema of mouse Nfasc locus showing the region from exon 13 to exon 33. The coding strand of Nfasc is located on the reverse strand of chromosome 1, as defined in the GRCm38/mm10 mouse genome assembly, and flipped here for reader convenience. Alternative exons specific to NF155 (exons 23 and 24) or NF186 (exons 28–30) are shown in blue while other alternative exons (exons 18 and 27) are shown in gray. NF140 contains neither exons 23 and 24 nor exons 28–30. The open rectangle denotes the untranslated region, and the solid ones the open reading frame. (B) Western blotting of P28 sciatic nerve homogenates probed for NFasc, contactin-associated protein (Caspr), and actin. (C) Immunostaining of P3 sciatic nerves for NFasc (green) and ankyrinG (AnkG) (magenta) showing pairs of approaching heminodes (nodes flanked by myelin sheaths only on one side). The paranodes are indicated by arrowheads. Scale bar, 5 µm. (D) RT-qPCR analysis of P28–P29 sciatic nerves shows the transcript levels relative to those of the wild-type (WT) after normalized to the internal control Polr2a. Exon 3 of Tardbp is the floxed exon, and the primer pair for Tardbp ex3 detects TDP-43 mRNA transcribed from the unrecombined allele. The primer pair for Tardbp exons 4–5 detects the nonfloxed region to confirm mRNA degradation after Cre recombination. Pan-Nfasc is detected by primers spanning exons 12–13, NF155 by primers spanning exons 22–23, NF140+ex27 by primers annealing to the junctions of exons 22/25 and exons 27/31, and NF140-ex27 by primers annealing to the junctions of exons 22/25 and exons 26/31. Bars represent mean ± SEM. n = 3 mice per genotype. *p<0.05; **p<0.01; ***p<0.001; ns, p=0.05; one-sample unpaired two-tailed t-tests (WT vs. conditional heterozygote [cHet] and WT vs. cKO). cHet and cKO by Dhh-Cre (B–D).Figure 5—source data 1.Statistical summary for Figure 5D.

Fig 4: Transmission Electron Microscopic (TEM) analysis reveals synaptic defects in Nfasc CKO (A–D). Representative electron micrographs of rod terminals of controls and Nfasc CKO mice at P30 (A). In controls, rod terminals (yellow) contain large, well-defined ribbon synapses (arrows) with invaginations from horizontal cells (blue) and ON bipolar cells (red). In contrast, Nfasc CKO display short and poorly defined ribbon synapses (arrows). Quantification of the length of ribbon synapses in controls (77 rod terminals; n = 4 mice) and Nfasc CKO (49 rod terminals; n=4 mice). Data represented as mean ± SEM. Statistical significance determined by an unpaired t-test. *p < 0.0001 (B). Rod terminals were designated as unclassified, empty, monad, dyad, or triad (C). A total of 275 rod terminals were analyzed for controls (n = 4 mice) and 301 rod terminals for Nfasc CKO (n = 4 mice). Frequency (%) shown for controls and Nfasc CKO in (D). Exact values are listed in the table. Scale bar = 0.5 µm.

Fig 5: NF155-dependent paranodal junctions can cluster Na+ channels in the PNS.(a) Immunostaining of P12 dorsal roots, ventral roots, and sciatic nerve from Nfascfl/fl and Avil-Cre;Nfascfl/fl mice using a pan Nfasc (Pan-NF, red) and Caspr (green) antibodies. Note the yellow overlap of the two labels at the paranodes in both genotypes. Scale bar, 5 µm. (b) Immunostaining of P12 dorsal and ventral roots from Nfascfl/fl and Avil-Cre;Nfascfl/fl mice using pan Na+ channel (NaCh, red) and Caspr (green) antibodies. Scale bar, 5 µm. (c) Immunostaining of P12 dorsal roots from Nfascfl/fl and Avil-Cre;Nfascfl/fl mice using panNF (red) to label nodes and paranodes, and antibodies against Nav1.6, AnkG, ßIV spectrin, or Gldn to label nodes (green). Note that the nodal panNF staining observed in the Nfascfl/fl mice is much stronger than the paranodal immunoreactivity. Thus, the paranodal panNF staining is more pronounced in the Avil-Cre;Nfascfl/fl mice. Scale bar, 5 µm. (d) Quantification of the percentage of dorsal root nodes of Ranvier with nodal Na+ channels at P9 and P12. N = 3 mice at each time point for each genotype. (e) Immunostaining of dorsal root ganglion/Schwann cell mixed cultures from wild-type and Avil-Cre;Nfascfl/fl mice using antibodies against NaCh (red) and myelin basic protein (MBP, blue). Scale bar = 10 µm. (f) Quantification of the relative Na+ channel fluorescence intensity at P9 and P12. N = 3 mice at each time point for each genotype. *p=0.016 at P9; p=0.026 at P12. The number of nodes measured at P9 were 263 and 250, at P12 were 337 and 269 in the Nfascfl/fl and Avil-Cre;Nfascfl/fl dorsal roots, respectively.DOI: http://dx.doi.org/10.7554/eLife.21392.002