Fig 1.
Structure of PSAP and the IM-1 antibody.
An anti-rat PSAP-specific antibody (IM-1) was prepared against the proteolytic portion (the intermediate portion between saposins C and D) of PSAP.
Fig 2.
Validation of the antibodies to GPR37 and GPR37L1.
Immunofluorescence light micrographs of 8-week-old rat DRG serial sections (a–c, d–f) stained with pre-immune serum (PIS; a, d), anti-GPR37 IgG (b), anti-GPR37L1 IgG (e), anti-GPR37 IgG absorbed with GPR37 antigen (c), or anti-GPR37L1 IgG absorbed with GPR37L1 antigen (f). GPR37 immunoreactivity (GPR37 IR) was observed mainly in the cytoplasm of satellite cells (arrows in b) and Schwan cells (arrowheads in b). GPR37L1 immunoreactivity (GPR37L1 IR) was observed mainly in the cytoplasm of satellite cells (arrows in e) and in the granular structures of neurons and satellite cells (e). Neither GPR37 nor GPR37L1 IR was observed in neighbouring sections stained with pre-immune serum (a, d) or absorbed IgG (c, f). The bars indicate the scale in micrometres.
Fig 3.
DAB immunohistochemical analyses (a–f) and immunofluorescence (g–i) with antibodies against PSAP (IM-1) (a, b, g), GPR37 (c, d, h), and GPR37L1(e, f, i) in the DRG of rats at 8 weeks after birth.
(b, d, and f) Higher magnification images of a, c, and e, respectively. Intense granular PSAP reactions were observed in small neurons, and large dot-like structures were observed in large neurons (b). Similar reactions were shown by immunofluorescence (g). GPR37 exhibited dot-like reactions that were not as strong as those observed with PSAP. GPR37L1 reactivity was similar to that of GPR37 in neurons, and was strong in satellite cells (arrows in e, f, and i), especially those around larger neurons. The numbers on the bars indicate the scale in μm.
Fig 4.
Immunoelectron micrographs of the DRG of rats at 8 weeks after birth stained with anti-saposin D antibody, which also reacts with PSAP.
Many immunogold particles (15 nm) were observed in lysosome-like structures in larger neurons (a, b). In smaller neurons (c, d), immunogold particles were observed in many small lysosomes (white arrowheads) and the basal lamina of satellite cells (black arrowheads in d) as well as larger lysosomes (arrows). Many immunogold particles were also observed in the lysosome-like structures of satellite cells (arrows in e). The numbers on the bars indicate the scale in μm.
Fig 5.
Immunofluorescence light micrographs of the rat DRG from day 1 to 2.5 years after birth stained with anti-PSAP (IM-1, red), anti-GPR37 (blue), and anti-GPR37L1 (green).
Before week 1 (a–c), staining was weak, except for some small and damaged neurons (arrowheads in c and white arrows in b, respectively). From 2–12 weeks (d–i), some satellite cells showed strong staining of GPR37L1 (arrowheads in d–k). The area in the rectangle in Fig 5e is shown in higher magnification view (f1–f4). After 16 weeks, these cells were rare. Asterisks indicate the nuclei of satellite cells. Arrows in Fig 5k and 5l indicate lipofuscin granules with strong fluorescence. The numbers on the bars indicate the scale in μm.
Fig 6.
The numbers of DRG neurons covered with satellite cells showing strong GPR37L1 staining were counted, and the percentages for each postnatal week are shown.
The peak occurred at 8 weeks.
Fig 7.
Immunofluorescence light micrographs of the rat DRG at 8 weeks, stained with antibodies against PSAP (red), GPR37L1 (green), and GPR37 (blue) (a–d). The neighbouring section was stained with antibodies against PSAP (red), GPR37 (green), and GPR37L1 (blue) (e–h). The intensity of GPR37L1-IR in satellite cells was stronger than that of GPR37-IR (c, h). The numbers on the bars indicate the scale in μm.
Fig 8.
Higher magnification immunofluorescence light micrographs of the rat DRG at 8 weeks show the colocalization of PSAP and its receptors (GPR37, GPR37L1).
In the lysosome-like structures in the neuronal cytoplasm, PSAP colocalized with GPR37L1 (yellow arrows), or existed in isolation (red arrows). Note that the lysosome with GPR37L1 is much bigger than the lysosome without GPR37L1(a, b). In the lysosome-like structures, colocalization of PSAP and GPR-37L1 was frequently observed (b, c), while PSAP did not colocalize with GPR-37 (b, d). In satellite cells, fine granular PSAP always colocalized with both GPR-37 and GPR-37L1, and accumulated near the boundary of neurons and satellite cells (arrowheads in b–d).
Fig 9.
Immunofluorescence light micrographs stained with commercial antibodies against GPR37 (purple)/GPR37L1 (green) at 4 and 8 weeks after birth.
The results were similar to those obtained with our autonomous antibodies; fine granular PSAP in satellite cells always colocalized with both GPR-37 and GPR-37L1 in the adult DRG (e–h); in young DRG neurons, PSAP did not always colocalize with receptors (a–d). In (b), only half of the satellite cells were outlined with fine granular PSAP (white arrowheads).
Fig 10.
(a–f) Immunofluorescence light micrographs of the rat DRG from 3 days to 8 weeks after birth, stained with anti-PSAP (red), anti-sortilin (Solt, green), and DAPI. Weak sortilin-IR was first observed at postnatal day 7 in satellite cells (arrows in b). After postnatal week 2, the number of sortilin-positive cells increased, peaking at postnatal week 4 (arrows in d) and decreasing after postnatal week 8 (f). (g–o) Higher magnification immunofluorescence light micrographs of the rat DRG at 8 weeks after birth stained with antibodies against PSAP (red), sortilin (green), and DAPI (blue). (g, j) Both the outer and inner cell surface of satellite cells were clearly outlined by sortilin, while only the inner surface of satellite cells was outlined with fine granular PSAP (white arrowheads in i, l, and o), and not the outer cell surface (black arrowheads in i, l). (m) Rare case of a thin satellite cell (S) between two neurons (N), indicating clear colocalization of sortilin (n) and PSAP (o). (p–x) The relationships of PSAP and its receptors (GPR37, GPR37L1) with sortilin was studied by double immunofluorescence of the DRG at postnatal week 4. Sortilin-IR was localized in satellite cells, especially near the boundary to nerve cells (q, t, and w), while PSAP-IR (r) and GPR37-IR (u) were dispersed in the cytoplasm of satellite cells. Only GPR37L1 clearly colocalized with sortilin (w, x). The numbers on the bars indicate the scale in μm.
Fig 11.
Schematic drawing of the interactions between neurons and satellite cells in the developing DRG.
DRG neurons produced and contained PSAP in lysosomes with or without GPR37L1. At approximately 8 weeks, the satellite cells around larger neurons showed dispersed PSAP colocalized with both receptors near the neuron boundary. Sortilin, not shown in this figure, was observed at an earlier stage (2–4 weeks), and always colocalized with GPR37L1. In conclusion, DRG neurons produce PSAP, which may be delivered to satellite cells in a paracrine manner. It is also plausible that satellite cells take up PSAP from their surrounding extracellular space and transport PSAP to neurons. GPR37L1 and sortilin may transport PSAP to stimulate the proliferation or maturation of satellite cells.