Conceived and designed the experiments: MJZ. Performed the experiments: BTB. Analyzed the data: BTB MJZ. Wrote the paper: MJZ.
The authors have declared that no competing interests exist.
Thiamine monophosphatase (TMPase, also known as Fluoride-resistant acid phosphatase or FRAP) is a classic histochemical marker of small- to medium-diameter dorsal root ganglia (DRG) neurons and has primarily been studied in the rat. Previously, we found that TMPase was molecularly identical to Prostatic acid phosphatase (PAP) using mice. In addition, PAP was expressed in a majority of nonpeptidergic, isolectin B4-binding (IB4+) nociceptive neurons and a subset of peptidergic, calcitonin gene-related peptide-containing (CGRP+) nociceptive neurons. At the time, we were unable to determine if PAP was present in rat DRG neurons because the antibody we used did not cross-react with PAP in rat tissues. In our present study, we generated a chicken polyclonal antibody against the secretory isoform of mouse PAP. This antibody detects mouse, rat and human PAP protein on western blots. Additionally, this antibody detects PAP in mouse and rat small- to medium-diameter DRG neurons and axon terminals in lamina II of spinal cord. In the rat, 92.5% of all PAP+ cells bind the nonpeptidergic marker IB4 and 31.8% of all PAP+ cells contain the peptidergic marker CGRP. Although PAP is found in peptidergic and nonpeptidergic neurons of mice and rats, the percentage of PAP+ neurons that express these markers differs between species. Moreover, PAP+ axon terminals in the rat partially overlap with Protein kinase Cγ (PKCγ+) interneurons in dorsal spinal cord whereas PAP+ axon terminals in the mouse terminate dorsal to PKCγ+ interneurons. Collectively, our studies highlight similarities and differences in PAP localization within nociceptive neurons of mice and rats.
It has long been known that small- to medium-diameter DRG neurons contain an acid phosphatase called Thiamine monophosphatase (TMPase; also known as Fluoride-resistant acid phosphatase or FRAP)
In general, nociceptive (“pain-sensing”) neurons can be divided into peptidergic and nonpeptidergic subsets that differ molecularly, anatomically, developmentally and functionally
Recently, we found that TMPase was molecularly identical to the transmembrane isoform of Prostatic acid phosphatase (PAP; also known as ACPP). PAP was expressed in a majority of all nonpeptidergic neurons and a subset of peptidergic nociceptive neurons in the mouse
There are two isoforms of PAP, a secreted isoform and a transmembrane isoform
PAP was discovered over 70 years ago and was used as a diagnostic marker for prostate cancer in humans
% Identity | ||||
mPAP | rPAP | hPAP | ||
mPAP | ----- | 88 | 83 | |
% Similarity | rPAP | 94 | ----- | 81 |
hPAP | 91 | 89 | ----- |
Calculated using BLASTP with GenBank accession #'s NP_062781.2 (mPAP), NP_064457.1 (rPAP) and NP_001090.2 (hPAP). The less conserved N- and C-terminal regions were not included in these alignments, resulting in higher percent identity values relative to a previous study with rPAP and hPAP
To generate a polyclonal antibody that reliably detects PAP in mouse tissues, we immunized chickens with the secretory isoform of full-length recombinant mPAP protein, purified as described previously
(A) Western blot containing purified recombinant mPAP protein and pure hPAP protein probed with chicken (Ck) anti-PAP antibody. (B) Duplicate gel stained with GelCode blue to confirm that equivalent amounts of protein were loaded. (C) Western blot of cell lysates from untransfected HEK 293 cells and HEK 293 cells transfected with rTM-PAP or mTM-PAP.
We previously found that PAP was co-localized in a majority of all IB4+ nonpeptidergic neurons and a subset of all CGRP+ peptidergic neurons in the mouse using the Biømeda antibody
(A–D) Sections from mouse L4-L6 DRG and (E–L) lumbar spinal cord were stained with chicken anti-PAP antibodies (red) and with antibodies against various sensory neuron markers and spinal interneuron marker PKCγ (blue, green). (D, H, L) Merged images. All images were acquired by confocal microscopy. Scale bar in (D) 50 µm, (H) 100 µm.
We next triple-immunostained lumbar DRG and spinal cord sections from the rat to determine if our polyclonal antibody also recognized PAP in rat tissues. The chicken anti-PAP antibody labeled a subset of small- to medium-diameter neurons in rat DRG and labeled axon terminals in lamina II of the spinal cord (
(A–D) Sections from rat L4-L6 DRG and (E–L) lumbar spinal cord were stained with chicken anti-PAP antibodies (red) and with antibodies against various sensory neuron markers and spinal interneuron marker PKCγ (blue, green). (D, H, L) Merged images. All images were acquired by confocal microscopy. Scale bar in (D) 50 µm, (H) 100 µm.
In the dorsal spinal cord, there was extensive co-localization between PAP+ and IB4+ axon terminals in lamina II and partial overlap with CGRP+ axon terminals (
Taken together, our studies indicate that PAP is found in peptidergic and nonpeptidergic neurons of the rat, although the extent of co-localization (both in DRG neurons and dorsal spinal cord) differs with species. Notably, a larger percentage (31.8%) of PAP+ neurons in the rat contains the peptidergic marker CGRP when compared to mouse (14.8%). This correlates with a more extensive overlap between PAP+ and CGRP+ axon terminals in spinal cord of rat (compare
Recombinant mPAP protein containing a C-terminal thrombin-hexahistidine epitope tag was purified using the baculovirus expression system as previously described
Recombinant mPAP protein and hPAP protein (Millipore/Chemicon Cat. #AG60) were separated by SDS-PAGE (0.2–1.0 µg protein/lane). HEK 293 cells were transfected as described
All procedures involving vertebrate animals were approved by the Institutional Animal Care and Use Committee at the University of North Carolina at Chapel Hill.
Adult male mice (C57BL/6, 6–8 weeks) were sacrificed by decapitation. Adult male Sprague-Dawley rats were sacrificed by overdosing with pentobarbital. Lumbar DRG and spinal cord were removed and immersed in 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) for 4 h (mouse DRG), 8 h (mouse spinal cord) or 12 h (rat tissues). Tissues were cryoprotected in 30% sucrose in 0.1 M phosphate buffer after immersion-fixation. DRG were sectioned frozen at 20 µm, collected on Superfrost plus slides then immunostained on slides. Spinal cords were sectioned at 30 µm and processed free-floating. Sections were treated with 1% hydrogen peroxide in phosphate-buffered saline (pH 7.4) for 30 min to reduce endogenous peroxidase. A high-salt (2.7%) Tris-buffered saline containing 0.3% Triton-X (TBS/TX) was used for all subsequent steps. Sections were incubated overnight at 4°C with primary antibodies diluted in 10% normal donkey serum in TBS/TX. Primary antibodies included: chicken anti-PAP (1∶4,000), rabbit anti-CGRP (1∶750; Bachem/Peninsula, T-4032) and rabbit anti-PKCγ (1∶750; Santa Cruz, C-19, sc-211). Chicken anti-PAP staining was revealed through the use of a biotinylated secondary (Jackson ImmunoResearch; 703-065-155), ABC complex (Standard Elite, Vector Laboratories, PK-6100) and TSA-Cy3 amplification (PerkinElmer, SAT704A). CGRP and PKCγ staining were revealed through the use of Cy5-coupled secondary antibodies. Sections were treated with IB4-Alexa Fluor-488 (Invitrogen; I21412) after TSA-Cy3 amplification because IB4-staining was difficult to detect if tissues were incubated with IB4 before TSA-Cy3 amplification. Images were obtained using a Zeiss LSM 510 confocal microscope.
We thank Yvette Chuang for excellent technical assistance, Nick Moss for providing rats, Pirkko Vihko for providing a rat TM-PAP plasmid, Jost Vielmetter and Michael Anaya at the Caltech Protein Expression Center for purifying mPAP protein and Aves Labs for making our chicken anti-PAP antibody commercially available.