Detection of ENFs in <i>α</i>-GalA KO males frontal paws.

<p>The DAPI immunostaining of 50 µm floating sagittal mice frontal paw section with marked region of interest (A). The immunohistochemistry of <i>α</i>-GalA KO males (n = 3) revealed the scattered expression of PGP9.5 (red) - specific marker of neuronal terminations in the epidermis of frontal paw skin in comparison to their WT controls (n = 3). The dermis and epidermis border was distinguished by staining for Collagen IV (green) and visually determined by dotted lines. Paw epidermal PGP9.5 positive fibers showed morphological abnormality such as fragmentation in <i>α</i>-GalA KO males, whereas the epidermal fibers showed a more regular morphology in WT males (white arrows) (B). Scale bar represents 100 µm. Numerical analysis of neuronal fibers terminations showed significant decrease (about 50%; <i>p = 0.0161</i>) in <i>α</i>-GalA KO males in comparison to WT (C).</p

The genotypic, anatomical and immunohistochemistry characterization of <i>α</i>-GalA KO mice.

<p>The specific primers amplified bands of 295 bp for <i>α</i>-GalA(+/+) WT and 202 bp for <i>α</i>-GalA(−/−) and <i>α</i>-GalA(−/0) KO. In case of founders females the heterozygosity was confirmed by PCR amplification of both WT and KO bands (A). The body weight of Fabry males was significantly increased just after 8 weeks (n = 15 for WT, n = 20 for KO; <i>p = 0.0111</i>). This trend was maintained and even increased after 12 week of age (n = 10 for WT, n = 5 for KO; <i>p = 0.0023</i>). Data are presented as fold of body weight increase in KO males related to the mean of WT males (B). Hematoxylin-eosin staining of 12 µm frozen frontal paws sections (C). Immunohistochemistry of frontal paw sections clearly shows the accumulation of globotriaosylceramide (Gb3; red) in the epidermis and stratum corneum of KO in comparison to WT mice (D). Scale bar represents 100 µm. Graphical data are expressed as mean±SEM.</p

The expression of Nav1.8 in epidermal neuronal fibers of <i>α</i>-GalA KO males frontal paws.

<p>The evaluation of co-localization of Nav1.8 and PGP 9.5 expression in 50 µm floating sagittal sections of WT (A) and <i>α</i>-GalA KO males (B) (n = 3) revealed similar values of Pearson's coefficient in both cases (<i>P_WT = 0.16</i>, <i>P_KO = 0.12</i>). The neuropathic pain receptor Nav1.8 is expressed in neuronal fibers of WT (C left panel) males marked by specific antibody PGP 9.5 with the same intensity as it is expressed in <i>α</i>-GalA KO (D left panel, <i>p = 0.1911</i>). Panels C, D were enhanced with 1.125 zoom respectively to square area in figures A, B. Scale bars represent 100 µm.</p

The expression of TRPV1 in epidermal neuronal fibers of <i>α</i>-GalA KO males frontal paws.

<p>The evaluation of co-localization of TRPV1 and PGP 9.5 expression in 50 µm floating sagittal sections of WT (A) and <i>α</i>-GalA KO (B) males (n = 4) revealed similar values of Pearson's coefficient in both <i>α</i>-GalA WT males (<i>P_WT = 0.35</i>) and KO (<i>P_KO = 0.29</i>), (<i>p = 0.1132</i>). The TRPV1 receptor is expressed in neuronal fibers of WT (C left panel) males marked by specific antibody PGP 9.5 with the high intensity as it is expressed in <i>α</i>-GalA KO (D left panel). The co-localization of both markers (C, D right panels) revealed similar results. Panels C, D were enhanced with 1.125 zoom respectively to square area in panels A, B. Scale bars represent 100 µm.</p

Mechanical and thermal sensitivity of <i>α</i>-GalA KO males.

<p>Basal sensitivity towards mechanical stimulation (latency time (A); applied force (B)). Comparison of the basal sensitivity of male <i>α</i>-GalA KO (n = 16) and relative WT (n = 28) in response to mechanical stimulation, <i>p<0.0001</i>. Basal sensitivity to hot and cold temperature stimuli in male <i>α</i>-GalA KO (n = 34) and relative WT (n = 32), <i>p<0.0001</i> as measured using the hot plate (C) and the acetone test (D) revealed the significant insensitivity of <i>α</i>-GalA KO (n = 19) males in comparison to their relative WT (n = 13), <i>p = 0.0008</i>. Basal insensitivity to noxious temperature of cold stimulus in male <i>α</i>-GalA KO (n = 7) and relative WT (n = 8), <i>p = 0.0466</i> as measured via cold plate assay (E). The data from plantar cold sensitivity assay confirmed the observed insensitivity of KO males (n = 10) to cold stimuli when compared to WT males (n = 10), <i>p = 0.0028</i> (F). Data are expressed as mean±SEM.</p

Pain Related Channels Are Differentially Expressed in Neuronal and Non-Neuronal Cells of Glabrous Skin of Fabry Knockout Male Mice

<div><p>Fabry disease (FD) is one of the X-linked lysosomal storage disorders caused by deficient functioning of the alpha-galactosidase A (<i>α</i>-GalA) enzyme. The <i>α</i>-GalA deficiency leads to multi-systemic clinical manifestations caused by the preferential accumulation of globotriaosylceramide in the endothelium and vascular smooth muscles. A hallmark symptom of FD patients is peripheral pain that appears in the early stage of the disease. Pain in FD patients is a peripheral small-fiber idiopathic neuropathy, with intra-epidermal fiber density and integrity being used for diagnosing FD in humans. However, the molecular correlates underlying pain sensation in FD remain elusive. Here, we have employed the <i>α</i>-GalA gene KO mouse as a model of FD in rodents to investigate molecular changes in their peripheral nervous system that may account for their algesic symptoms. The <i>α</i>-GalA null mice display neuropathic pain as evidenced by thermal hyperalgesia and mechanical allodynia, with histological analyses showing alterations in cutaneous innervation. Additionally, KO mice showed a decreased and scattered pattern of neuronal terminations consistent with the reduction in neuronal terminations in skin biopsies of patients with small fiber neuropathies. At the molecular level KO animals showed an increase in the expression of TRPV1 and Nav1.8, and a decrease in the expression of TRPM8. Notably, these alterations are observed in young animals. Taken together, our findings imply that the <i>α</i>-GalA KO mouse is a good model in which to study the peripheral small fiber neuropathy exhibited by FD patients, and provides molecular evidence for a hyperexcitability of small nociceptors in FD.</p></div

Different Ig-like domains can support L1CAM physical interaction with erbB receptors.

<p><b>a</b>) Truncated proteins ΔIg1-3L1CAM and ΔIg4-6L1CAM are normally expressed and distributed when transfected into COS-7 cells. High magnification confocal images of cells transiently transfected with the indicated constructs are shown. L1CAM was detected with an anti-L1CAM monoclonal antibody (green) and erbB3 with a polyclonal antibody (red). Nuclei were counterstained with the Hoechst stain (blue). As is shown both deletion mutants of L1CAM co-localize with erbB3 (white). <b>b</b>) Ablation of Ig-like domains 1 to 3 or 4 to 6 does not abrogate L1CAM interaction with erbB3: HEK293 cells were transfected with pcDNA3-erbB3 and the different truncated forms of L1CAM. Cell extracts were immunoprecipitated with anti-L1CAM antibody and blotted against erbB3. As shown, erbB3 was pulled down when co-expressed with ΔIg1-3L1CAM, ΔIg4-6L1CAM and full-length constructs but not when co-expressed with the ΔIg-L1CAM construct. As expected, anti-L1CAM antibody does not immunoprecipitate erbB3 in cells transfected with pcDNA3-erbB3 exclusively. Input lanes demonstrate the expression of erbB3 in the extracts. An aliquot of the immunoprecipitated was probed with anti-L1CAM to verify the adequate expression and immunoprecipitation of the truncated proteins. IgG bands show that a similar amount of immunoprecipitated was loaded. This experiment was repeated twice. A representative experiment is shown.</p

L1CAM-erbB interaction enhances neuregulin induced phosphorylation of erbB3. a

<p>) Upper panel: MCF-7 cells were transiently transfected with pcDNA3-L1CAM or pcDNA3 empty vector. 24 h later, cells were serum starved and stimulated with recombinant NRG1 (50 nM) for 15 min. Then, cells were harvested and lysed. Extracts were submitted to SDS-PAGE and blotted with anti-p-Tyr monoclonal antibody or anti-erbB3 polyclonal antibody. This experiment was repeated three times. A representative experiment is shown. Lower panel: the same approach was used in cells transfected with the ΔIg-L1CAM truncated construct. This experiment was repeated twice. One of them is shown. <b>b</b>) Quantification of western blots by densitometry. The normalized amount of phosphorylated 180 kDa band is increased in cells that express the full length but not the truncated ΔIg-L1CAM protein, suggesting that the physical interaction of L1CAM and erbB3 is needed for the enhancing effect on neuregulin receptor activation. Bars represent standard errors <b>c</b>) Proposed model: the interaction with L1CAM sensitizes erbB receptors to the activation by neuregulins. Removing the Ig-like rich region of L1CAM prevents the interaction and avoids receptor sensitization. For simplicity, only <i>cis</i>-interactions are depicted in the model.</p

The Ig-like domains but not the fibronectin repeats of L1CAM mediate the physical interaction with erbB receptors.

<p>a) Ablation of Ig-like domains abrogates L1CAM interaction with erbB3: HEK293 cells were transfected with pcDNA3-erbB3 and the different truncated forms of L1CAM. Cell extracts were immunoprecipitated with anti-L1CAM antibody and blotted against erbB3. As is shown, erbB3 was pulled down when co-expressed with the full length and ΔFn-L1CAM constructs, but not when was co-expressed with the ΔIg-L1CAM construct. As expected, anti-L1CAM antibody does not immunoprecipate cells transfected with pcDNA3-erbB3 exclusively. Input lanes demonstrate the expression of erbB3 in the extracts. An aliquot of the immunoprecipitate was probed with anti-L1CAM to verify the adequate immunoprecipitation of the truncated proteins. IgG bands show that a similar amount of immunoprecipitate was loaded. This experiment was repeated three times. A representative experiment is shown. b) To rule out sorting problems that could explain the absence of co-IP, L1CAM and deleted constructs were co-transfected with erbB3 in COS-7 cells. As is shown, the distribution of L1CAM, ΔIg-L1CAM and ΔFn-L1CAM is similar when transfected into HEK293 cells, being detectable in the plasma membrane. c) The co-localization of the deleted constructs and full length L1CAM with erbB3 was nearly complete, ruling out sorting defects for the mutant proteins. L1CAM was detected with the anti-L1CAM monoclonal antibody (green) and erbB3 with a polyclonal antibody (red). Nuclei were counterstained with the Hoechst stain (blue). Co-localization (white) was revealed with the ImageJ software and the Co-localization Finder plugin.</p

Physical interaction of L1CAM with erbB receptors. a

<p>) L1CAM co-immunoprecipitates with erbB1 (EGFR): a pcDNA3 plasmid containing the cDNA encoding for human L1CAM and the pcDNA6A-EGFR construct were transiently co-transfected into the HEK293 cells. 48 h later cells were homogenized and L1CAM immunoprecipitated (IP). Immunoprecipitates were resolved by SDS-PAGE and blotted with anti-myc antibody to detect EGFR. As is shown, EGFR was pulled down only in L1CAM expressing cells. EGFR expression was similar in both extracts (input). Immunoblot with anti-L1CAM shows that this protein was correctly immunoprecipitated. <b>b</b>) Reverse co-immunoprecipitation. IP with anti-myc antibody pulls down L1CAM only in EGFR transfected cells. L1CAM expression was similar in both extracts (input). Immunoblot with anti-myc shows that the EGFR was correctly immunoprecipitated. <b>c</b>) L1CAM co-immunoprecipitates with erbB2: pcDNA3-L1CAM and the pcDNA3-erbB2 were transiently co-transfected into the HEK293 cells. Extracts were immunoprecipitated with the anti-L1CAM antibody. erbB2 was pulled down only in L1CAM expressing cells. erbB2 expression was similar in both extracts (input). Anti-L1CAM immunoblot shows that this protein was correctly immunoprecipitated. <b>d</b>) Reverse co-immunoprecipitation. IP with anti-erbB2 antibody pulls down L1CAM only in erbB2 transfected cells. L1CAM expression was similar in both extracts (input). Anti-erbB2 WB shows that erbB2 was correctly immunoprecipitated. <b>e</b>) erbB3 co-immunoprecipitates with L1CAM: pcDNA3-L1CAM and the pcDNA3-erbB3 were transiently co-transfected. Extracts were immunoprecipitated with the anti-L1CAM antibody. erbB3 was pulled down only in L1CAM expressing cells. erbB3 expression was similar in both extracts (input). Anti-L1CAM immunoblot shows that this protein was correctly immunoprecipitated. <b>f</b>) Reverse co-immunoprecipitation. IP with anti-erbB3 antibody pulls down L1CAM only in erbB3 transfected cells. L1CAM expression was similar in both extracts (input). Anti-myc WB shows that erbB3 was correctly immunoprecipitated. <b>g</b>) Proximity ligation assay showing L1CAM-erbB3 <i>in vivo</i> interaction. HEK293 cells were enforced to express L1CAM and erbB3. To identify the transfected cells, a plasmid encoding GFP was included. As is shown, only the transfected cells (green) were positive for the PLA signal (red). Note that the interaction signal can be detected in cells that are not in contact with other transfected cells, showing that the interaction between L1CAM and erbB3 is produced in <i>cis</i>. Scale bars represent 20 µm. <b>h</b>) L1CAM (red) co-localizes with EGFR (erbB1), erbB2 and erbB3 (green) in growing axons during brain development (at E14). Images at the right correspond to the co-localization channel (white). Co-localization is evident in cortical projections. Poor co-localization of L1-CAM was detected with Notch 2, used as a control for specificity. Co-localization was revealed with the ImageJ software and the Co-localization Finder plugin (for co-localization at P3 stage see the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040674#pone.0040674.s001" target="_blank">Figure S1</a>). Images show coronal sections of E14 mouse brain incubated with the indicated antibodies and acquired at low magnification wide-field fluorescence (at left) or higher magnification under the confocal microscope. Scale bars correspond to 100 µm. <b>i</b>) L1CAM physically interacts with erbB3 <i>in vivo</i>. Whole brains of two days old rats were homogenized in RIPA buffer clarified by centrifugation and cross-linked with DTBP. Supernatants were immunoprecipitated with the anti-erbB3 antibody and blotted with anti-L1CAM. As a control of specificity an aliquot of the extract was immunoprecipitated with a non-specific anti-IgG. As shown, L1CAM was pulled down when immunoprecipitation was performed with the anti-erbB3 but not with the anti-IgG. Input shows that L1CAM is abundantly expressed in the P2 rat brains. IgG bands demonstrate a similar loading of immunoprecipitated proteins. This experiment was repeated 5 times. A representative experiment is shown. <b>j</b>) A similar result was obtained with the receptor erbB2.</p