Prion disease is an infectious and fatal neurodegenerative disease. Human prion disease autopsy studies have revealed abnormal prion protein (PrPSc) deposits in the central nervous system and systemic organs. In deer, chronic wasting disease has also become a global problem, with PrPSc in saliva and feces. Therefore, understanding normal cellular prion proteins (PrPc) characteristics in human systemic organs is important since they could be a PrPSc source. This study used western blotting and immunohistochemistry to investigate endocrine and exocrine tissues, such as the human pituitary, adrenal, submandibular glands and the pancreas. All tissues had 30–40 kDa PrP signals, which is a slightly higher molecular weight than normal brain tissue. We also identified a 16–20 kDa PrP C-terminal fragment in the adrenal gland, pancreas, and submandibular glands. Most cytoplasmic PrP-positive adenohypophyseal cells were immunopositive for nuclear pituitary-specific positive transcription factor 1. The adrenal medulla and islet cells of the pancreas were PrP-positive and colocalized with chromogranin A. The duct epithelium in the submandibular gland and pancreas were immunopositive for PrP. This study reports the characteristic molecular properties and detailed tissue localization of PrPc in endocrine and exocrine tissues, which is important for infection control and diagnosis.
Article
Characteristic distribution and molecular properties of normal cellular prion protein in human endocrine and exocrine tissues
https://doi.org/10.21203/rs.3.rs-1598905/v1
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Human prion diseases, also known as transmissible spongiform encephalopathies, are lethal neurodegenerative disorders, including sporadic Creutzfeldt-Jakob disease (sCJD), inherited prion disease, and acquired human prion disease. Acquired human prion diseases include iatrogenic CJD (iCJD), kuru, and variant CJD. Notably, among the iCJD cases, many received human growth hormone (GH) derived from cadaveric pituitaries1.
In some prion diseases, abnormal PrP deposits have been identified in systemic organs other than the central nervous system2–4. Prion disease is characterized by the conversion of a normal cellular prion protein isoform (PrPc) into an abnormal pathogenic PrP (scrapie prion protein: PrPSc). PrPSc has high β-sheet content and is resistant to proteinase K treatment (PrPres). Therefore, it is important to clarify the histological localization of PrPc in systemic organs, which can be a source of PrPSc. Previously, we demonstrated that cytoplasm from anterior pituitary gland adenohypophyseal cells is rich in PrPc and that the pituitary gland of patients with prion diseases contains PrPres5.
There are already many reports on PrPc localized in systemic organs in animals. In particular, PrP is related to endocrine tissues (e.g., the pituitary and adrenal glands and the pancreas) and endocrine pathway crinophagy6–10. In addition, chronic wasting disease (CWD), a prion disease of deer and elk, has become an issue in the United States, Canada, and Europe11, 12. Transmission routes are thought to be prion-contaminated environments derived from saliva13, 14, urine15, and feces16.
In humans, information about the relationship between prion proteins and endocrine and exocrine organs remains limited, even regarding their histological localization. Therefore, this study clarified the histological localization and biochemical properties of prion proteins in endocrine and exocrine organs in autopsy patients with non-prion diseases.
Subjects
We obtained pancreatic and pituitary, adrenal, and submandibular gland tissues from six autopsy cases of non-prion diseases (one amyotrophic lateral sclerosis case, one spinocerebellar ataxia type 6 case, two multiple sclerosis cases, one myotonic dystrophy case, one Becker muscular dystrophy case) at Kyushu University Hospital, Fukuoka, Japan (Table 1). We obtained informed written consent for autopsies from all patients or their next of kin. All analyses were performed following the Declaration of Helsinki. The Ethics Committee of the Faculty of Medicine of Kyushu University (#2019 − 179) approved this study.
| Case No. | Diagnosis | Age at Death (y) | Sex | Brain Weight (g) | PMI (hour) |
|---|---|---|---|---|---|
| 1 | ALS | 79 | F | 1300 | 26 |
| 2 | SCA6 | 93 | M | 1161 | 24 |
| 3 | MS | 73 | M | 1192 | 21 |
| 4 | MS | 43 | F | 980 | 13 |
| 5 | MD | 63 | F | 1038 | 4 |
| 6 | BMD | 46 | M | 1386 | 2 |
| Abbreviations: ALS, amyotrophic lateral sclerosis; BMD, Becker muscular dystrophy; F, female; M, male; MD, myotonic dystrophy; MS, multiple sclerosis; PMI, postmortem interval; SCA6, spinocerebellar ataxia 6. | |||||
Molecular Characterization of PrP
The frontal cortex, pancreatic, and pituitary, adrenal and submandibular gland samples were obtained from formalin-fixed, paraffin-embedded (FFPE) specimens. The detailed protein extraction methods have been previously described17, 18. Briefly, each FFPE specimen was sectioned at 10 µm to produce 10 serial sections. Next, 0.5 mL of mineral oil (Sigma-Aldrich, USA) was added to the sectioned FFPE samples and then incubated at 95°C for 2 min to dissolve the wax. Finally, the supernatants were removed by centrifugation at 11 000 × g for 3 min at room temperature. The incubation and centrifugation steps were repeated by adding 0.1 mL of mineral oil. Then, the pellets were resuspended in 80 µL of citrate-sodium dodecyl sulfate (SDS) buffer (200 mM Tris-HCl (pH 7.5), 200 mM NaCl, 5% SDS, and 100 mM sodium citrate), and the supernatant was removed after centrifugation at 11 000 × g for 3 min at room temperature. Next, proteins were extracted from the pellet by incubation at 100°C for 20 min and then at 80°C for 2 h in citrate-SDS buffer. Finally, the supernatant was recovered after centrifugation at 11 000 × g for 15 min at room temperature. To concentrate the protein, acetone was added to the extracted protein and incubated overnight at − 20°C. Then, the supernatant was discarded after centrifugation at 10 000 × g for 15 min at room temperature. Finally, the pellets were left at room temperature for 30 min to volatilize acetone, then re-dissolved in citrate-SDS buffer to prepare the samples for western blotting. Prior to western blotting, the protein concentration of the sample was adjusted.
PrPc was detected by SDS polyacrylamide gel electrophoresis in pre-made gels (12% gel; Bio-Rad, Hercules, CA) and then transferred onto polyvinylidene difluoride membranes. PrPc was detected using two anti-PrP antibodies (mouse monoclonal 3F4 [specific for PrP at 109–112], 1:2000; BioLegend, San Diego, CA and rabbit monoclonal EP1802Y [specific for PrP at 212–230], 1:2000; Abcam, Cambridge, UK). Peroxidase-conjugated anti-mouse immunoglobulin (Ig) G (PI-2000, 1:20,000, VECTOR, Burlingame, CA, USA) and anti-rabbit IgG (PI-1000, 1:20,000, VECTOR) were used as secondary antibodies. Immunoreactivity was visualized using Immobilon Western Chemilum HRP substrate (Millipore, Billerica, MA, USA). After confirming the immunoreactivity with the 3F4 antibody, the primary antibody was removed using stripping buffer (62.5 mM Tris-HCl, pH 6.8, 2% SDS, 100 mM β-mercaptoethanol). Then, PrPc was identified again using another PrP antibody (EP1802Y).
Immunohistochemistry
Hematoxylin and eosin (HE) staining was performed for histological examination. Immunohistochemistry was performed using primary antibodies specific to anti-prion proteins: (mouse monoclonal 3F4 [1:400], mouse monoclonal 8G8 [specific for PrP at 95–110, 1:400]; Cayman, Ann Arbor, MI, USA, and rabbit monoclonal EP1802Y [1:1000]), mouse monoclonal anti-pituitary-specific positive transcription factor 1 (Pit-1; 1:100, Santa Cruz Biotechnology), rabbit polyclonal anti-T-Box Transcription Factor 19 (TBX19; 1:200, Atlas Antibodies, Bromma, Sweden), mouse monoclonal anti-steroidogenic factor 1 (SF-1; 1:100, Perseus Proteomics, Tokyo, Japan), and mouse monoclonal anti-chromogranin A (CgA; 1:100, Millipore). Double immunofluorescence staining was performed using a combination of mouse monoclonal Pit-1 and rabbit monoclonal EP1802Y antibodies or rabbit polyclonal TBX19 and mouse monoclonal 3F4 antibodies. Alexa 488-labeled anti-mouse IgG (Invitrogen, Carlsbad, CA, USA), Alexa 546-labeled anti-mouse IgG (Invitrogen), Alexa 488-labeled anti-rabbit IgG (Invitrogen), and Alexa 546-labeled anti-rabbit IgG (Invitrogen) were used as secondary antibodies. The specimens were visualized using a Nikon A1R-A1 confocal microscope (Nikon, Tokyo, Japan).
Molecular Characterization of PrP
The frontal cortex samples had unglycosylated, monoglycosylated, and diglycosylated PrP signals, ranging from 25 to 38 kDa using the 3F4 antibody (Fig. 1A). The PrP signal intensity varied per tissue depending on the PrP antibody and the case (Table 2). The pituitary samples had a PrP smear band around 30–40 kDa (Fig. 1A), and the adrenal gland samples had PrP smear bands around 16–20 kDa (Fig. 1B, arrowhead) in addition to those around 30–40 kDa. The pancreatic PrP signal pattern was nearly identical to the adrenal gland pattern, with ~ 16–20 kDa (Fig. 1C, arrowhead) and ~ 30–40 kDa smear bands (Fig. 1C). However, the signal intensity differed between cases, and case 4 was challenging to detect (Fig. 1C, asterisk). The submandibular gland samples had PrP signal patterns similar to those of the adrenal gland and pancreas with PrP signals of ~ 16–20 kDa (Fig. 1D, arrowhead). However, it was difficult to detect the PrP signal in case 1 (Fig. 1D, asterisk).
Table 2
The molecular characteristics of prion proteins by western blot with 3F4 and EP1802Y antibodies
Scale: –, negative; ±, weakly positive; +, positive; n. a., not available.
The membranes were then incubated with stripping buffer to remove the primary and secondary antibodies and chromogenic substrate. The images confirmed that almost all PrP signals were diminished (Suppl. Figure 1A-D). Next, another PrP antibody (EP1802Y) was used to re-examine the PrP signal patterns in these membranes, and the ~ 16–20 kDa and ~ 30–40 kDa smear bands were observed with the 3F4 antibody were also present with the EP1802Y antibody (Suppl. Figure 2A-D).
Histopathological Findings
The adenohypophyseal cells had a normal appearance in all cases (HE stain; Fig. 2A). However, the immunostaining intensity per tissue depended on the PrP antibody and case (Table 3). Immunohistochemistry for PrP using 3F4 (Fig. 2B) and other PrP antibodies (Fig. 2C: 8G8, 2D: EP1802Y) revealed cytoplasmic staining in several adenohypophyseal cells. Also, immunohistochemistry for Pit-1 revealed nuclear staining in many adenohypophyseal cells (Fig. 2E). Furthermore, double immunofluorescence of PrP (EP1802Y) and Pit-1 showed that cytoplasmic PrP-positive adenohypophyseal cells were immunopositive for nuclear Pit-1 (Fig. 2F–H). In addition, most cytoplasmic PrP-positive adenohypophyseal cells were immunopositive for nuclear Pit-1 (74.3%, 55/74).
Table 3
Immunopositivity after prion protein immunostaining
Scale: 0, negative; 1, mild; 2, moderate; 3, strong; n.a., not available. 3F4, 8G8, and EP1802Y: prion protein antibodies.
Immunohistochemistry for TBX19 revealed nuclear immunopositivity in some adenohypophyseal cells (Fig. 2I). Double immunofluorescence of PrP (3F4) and TBX19 (Fig. 2J-L) showed only a small number of cytoplasmic PrP-positive adenohypophyseal cells with nuclear TBX19 positivity (9.6%, 5/52). Immunohistochemistry for SF-1 revealed nuclear staining in the adenohypophyseal cells (Fig. 2M, arrowheads). However, double immunofluorescence of SF-1 and EP1802Y was difficult because of the different antigen activation methods. Therefore, we used a mirror image in the adjacent section. In the mirror image for SF-1 and PrP (3F4), most nuclear SF-1 positive adenohypophyseal cells were negative for cytoplasmic PrP (Fig. 2N, arrowheads). CgA immunopositivity was observed in most adenohypophyseal cells and was strongly expressed in some (Fig. 2O, arrowhead). Furthermore, double immunofluorescence of CgA and PrP (EP1802Y) revealed colocalization in most adenohypophyseal cells. However, highly CgA-expressing cells showed no cytoplasmic PrP (Fig. 2P, arrowhead).
HE staining indicated a normal appearance in the adrenal gland (Fig. 3A), and immunohistochemistry for PrP (3F4) revealed marked PrP immunopositivity in the adrenal medulla (Fig. 3B, 3C). In contrast, PrP immunoreactivity was faint in the zona reticularis of the adrenal gland (Fig. 3D). Immunohistochemistry for CgA revealed marked immunopositivity in the adrenal medulla (Fig. 3E), and double immunofluorescence of PrP (EP1802Y) and CgA showed substantial colocalization of PrP and CgA (Fig. 3F-H). In the pancreas, islet cells had weak cytoplasmic PrP immunostaining (Fig. 3I, J, asterisks). However, the ductal epithelia were also positive for PrP (Fig. 3I: 8G8, arrowheads; Fig. 3J: EP1802Y, arrowheads), and some ductal epithelia showed marked PrP staining (Fig. 3J, inset). Immunohistochemistry for CgA revealed marked immunopositivity in the pancreatic islet cells (Fig. 3K). In the submandibular gland, most ductal epithelia were PrP-positive (Fig. 3L: 3F4, 3M: 8G8, 3N: EP1802Y). The ductal epithelia showed various immunopositivity intensities depending on the antibody: 8G8 and EP1802Y were strong, but 3F4 was mild. In addition, some ductal epithelia showed marked PrP staining (Fig. 3N, inset).
This study demonstrated the characteristic molecular properties of PrP in several human endocrine and exocrine tissues, such as the pituitary, adrenal, and submandibular glands and the pancreas, which have different characteristics than the brain. Additionally, we detail the histological localization of PrP in these tissues.
Western blotting showed PrP smear bands ranging from 30 to 40 kDa in the pituitary gland, similar to previous reports5. These molecular weights are higher than those for the brain, and our previous study showed that this was due to excessive glycosylation. PrP signals were observed in the adrenal, pancreas, and submandibular glands at ~ 30–40 kDa and ~ 16–20 kDa. The ~ 30–40 kDa PrP smear signal was similar to that of the pituitary gland. However, a ~ 16–20 kDa PrP smear signal was not observed in the pituitary gland or the brain. The ~ 16–20 kDa PrP smear signal was recognized by the 3F4 antibody and the C-terminal antibody (EP1802Y), suggesting that it contains a C-terminal from the PrP core structure. Hachiya et al. showed that PrPc C-terminal fragments of approximately 20 kDa are present mainly on the cell surface19. However, the role of these PrPc C-terminal fragments in endocrine and exocrine tissues remains unclear.
Pit-1 activates transcription of the GH, prolactin, and beta-thyroid stimulating hormone genes20, and we found that most cytoplasmic PrPc positive adenohypophyseal cells were immunopositive for nuclear Pit-1. Conversely, cytoplasmic PrPc positive adenohypophyseal cells were essentially negative for SF-1 and TBX19. These findings suggest that the expression of PrPc in adenohypophyseal cells tents to occur in differentiated cells downstream from somatotroph stem cells rather than corticotroph cells and gonadotroph cells. A previous report showed that cytoplasmic PrPc positive adenohypophyseal cells are frequently positive for GH or prolactin5, and our results agree. However, there are no reports that suggest a direct association between PrP and Pit-1, and it is unclear when PrPc is highly expressed during cell differentiation by Pit-1.
In the adrenal gland, PrPc immunoreactivity was observed in the adrenal medulla. The adrenal medulla is derived from the neural crest, and PrPc is as abundant as in the central nervous system. For example, PrPSc was detected in the adrenal medulla in cattle affected with bovine spongiform encephalopathy and scrapie-infected sheep21, 22. In humans, we previously reported PrPSc aggregation in the adrenal medulla of sCJD patients and enhanced seeding activity on real-time quaking-induced conversion23. Our results suggested that PrPc in the adrenal medulla may be the source of PrPSc.
In the pancreas, islet cells have cytoplasmic PrP. In rats, PrPc is abundant in islet cells and is involved in regulating blood glucose homeostasis24. In addition, PrPc primarily expresses in pancreatic β cells in humans and may contribute to insulin resistance25, 26. CgA is a neuroendocrine protein localized to nerve cells and secretory vesicles of endocrine cells27. Reports have shown that PrPc and CgA colocalized in bovine islet cells.28 In our study, CgA and PrPc colocalization was noted in adenohypophyseal cells, pancreatic islet cells, and the adrenal medulla, suggesting that PrPc-expression in neuroendocrine cells could be expressed throughout the body of human. However, very high levels of CgA-expressing adenohypophyseal cells did not show cytoplasmic PrP. Therefore, even if the genes for CgA and PrPc are adjacent to each other, they do not necessarily colocalize in all tissues.
PrP immunopositivity was detected in the ductal epithelium of the submandibular gland and the pancreas. There have been no reports of PrP expression in the ductal epithelium of animals or humans. CWD is a fatal neurodegenerative prion disease that affects species of the Cervidae family and is now a widespread global problem in the United States, Canada, and Europe12, 29, 30. Saliva and feces containing PrPSc are likely sources of infection29, 30. Our results suggest that in CWD animals, PrPSc may be secreted from the ductal epithelium in saliva and from the pancreas through the gastrointestinal tract to feces.
This study detected PrPc in human endocrine and exocrine tissues using western blotting and clarified the histological details. PrPc is an element of the crinophagy mechanism (an autophagy mechanism that processes excess hormones and neurotransmitters) in secretory cells. Furthermore, PrPc is involved in the secretory pathway of secretory granules.8 The immunohistochemistry and western blotting detection sensitivities for PrPc varied between cases. This may be due to different endocrine and exocrine function states per case during the period before death. The immunohistochemistry detection sensitivities also differed depending on the PrP antibody, which may be related to antigen exposure owing to the three-dimensional structure of PrPc. This study includes several limitations. Frozen samples of endocrine and exocrine tissue could not be collected adequately at autopsy, therefore, analysis of western blots was performed from FFPE samples. It is undeniable that modification of the sample during the FFPE manufacturing process can affect the results of western blot. However, western blot results from FFPE of the brain and pituitary gland were similar to those of frozen samples. In addition, since it is a biochemical result of the tissue adjacent to the specimen used for histological observation, we think that it is also a very suitable method for examining the tissue localization of PrPc. Therefore, we believe that the characteristic signals found in western blots of the adrenal glands, pancreas and submandibular glands are reliable.
PrPSc deposition in prion diseases occurs in the central nervous system and other systemic organs, potentially indicating the source of PrPSc. Furthermore, CWD has become a global problem, and saliva and feces are sources of infection. PrPc in the submandibular gland might be one such source. Since prion diseases were eradicated from pituitary GH preparations, no human endocrine or exocrine-related infectious prion diseases have been documented, but careful monitoring for these fatal diseases should continue. Understanding the function of PrPc in endocrine and exocrine tissues in detail is important for infection control and prompt diagnoses.
Acknowledgments
This work was funded by a Grant of The Clinical Research Promotion Foundation (2021). The authors greatly appreciate the patients and their families for their collaboration with this study. We would like to thank Dr. Toru Iwaki (Department of Neuropathology, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan) and Takaaki Kanemaru (Department of Morphology Core Unit, Kyushu University Hospital, Fukuoka, Japan) for technical and conceptual assistance. We also wish to thank Editage (www. editage. com) for the English language editing.
Author contributions
S.K. and H.H. conceptualised the study, conducted histopathological examinations, performed western blotting, analysed the data, and wrote the manuscript. H.N. and K.Y. prepared the specimens for this study and conducted histopathological examinations, performed western blotting. H.H. and M.S. contributed to the data analysis and performing western blotting. M.Y., K.I. and N.S. provided the autopsy samples and clinical data. All authors have reviewed the manuscript.
Competing interest statement
The authors declare no competing interests.
Data availability statement
All data supporting the findings of this study are available within the article and its supplementary files.
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No competing interests reported.
- s1.png
Supplementary Figure 1. The primary and secondary antibodies and chromogenic substrate were removed by incubating with stripping buffer. (A, B) Almost all 3F4 signals were unidentifiable. However, a very weak prion protein signal remained in the (C) case 2 pancreas sample and (D) case 2 and 3 submandibular gland samples.
- s2.png
Supplementary Figure 2. Western blot analyses with the EP1802Y anti-prion protein (PrP) antibody. (A) The frontal cortex (FC) samples show ~25–38 kDa PrP signals, and the pituitary gland samples show weak PrP signals of ~30–40 kDa (cases 1, 2, 4, and 5). (B) The adrenal gland samples (cases 1–5) show ~30–40 kDa and ~16–20 kDa PrP signals. (C) The pancreas samples (cases 1–5) show ~30–40 kDa and ~16–20 kDa PrP signals (cases 1–3). The PrP signal was difficult to identify in cases 4 and 5. (D) The submandibular gland samples (cases 1–5) show ~30–40 kDa and ~16–20 kDa PrP signals (cases 2–5). The PrP signal was difficult to identify in case 1.
