Altered cellular localisation and expression, together with unconventional protein trafficking, of prion protein, PrPC, in type 1 diabetes

Abstract

Aims/hypothesis: Normal cellular prion protein (PrP^C) is a conserved mammalian glycoprotein found on the outer plasma membrane leaflet through a glycophosphatidylinositol anchor. Although PrP^C is expressed by a wide range of tissues throughout the body, the complete repertoire of its functions has not been fully determined. The misfolded pathogenic isoform PrP^Sc (the scrapie form of PrP) is a causative agent of neurodegenerative prion diseases. The aim of this study is to evaluate PrP^C localisation, expression and trafficking in pancreases from organ donors with and without type 1 diabetes and to infer PrP^C function through studies on interacting protein partners.

Methods: In order to evaluate localisation and trafficking of PrP^C in the human pancreas, 12 non-diabetic, 12 type 1 diabetic and 12 autoantibody-positive organ donor tissue samples were analysed using immunofluorescence analysis. Furthermore, total RNA was isolated from 29 non-diabetic, 29 type 1 diabetic and 24 autoantibody-positive donors to estimate PrP^C expression in the human pancreas. Additionally, we performed PrP^C-specific immunoblot analysis on total pancreatic protein from non-diabetic and type 1 diabetic organ donors to test whether changes in PrP^C mRNA levels leads to a concomitant increase in PrP^C protein levels in human pancreases.

Results: In non-diabetic and type 1 diabetic pancreases (the latter displaying both insulin-positive [INS(+)] and -negative [INS(-)] islets), we found PrP^C in islets co-registering with beta cells in all INS(+) islets and, strikingly, unexpected activation of PrP^C in alpha cells within diabetic INS(-) islets. We found PrP^C localised to the plasma membrane and endoplasmic reticulum (ER) but not the Golgi, defining two cellular pools and an unconventional protein trafficking mechanism bypassing the Golgi. We demonstrate PrP^C co-registration with established protein partners, neural cell adhesion molecule 1 (NCAM1) and stress-inducible phosphoprotein 1 (STI1; encoded by STIP1) on the plasma membrane and ER, respectively, linking PrP^C function with cyto-protection, signalling, differentiation and morphogenesis. We demonstrate that both PRNP (encoding PrP^C) and STIP1 gene expression are dramatically altered in type 1 diabetic and autoantibody-positive pancreases.

Conclusions/interpretation: As the first study to address PrP^C expression in non-diabetic and type 1 diabetic human pancreas, we provide new insights for PrP^C in the pathogenesis of type 1 diabetes. We evaluated the cell-type specific expression of PrP^C in the human pancreas and discovered possible connections with potential interacting proteins that we speculate might address mechanisms relevant to the role of PrP^C in the human pancreas.

Keywords: Cellular prion protein; Protein trafficking; Stress-induced phosphoprotein 1; Type 1 diabetes; Type 1 diabetes-dependent endocrine cell expression.

Fig. 1

Cellular localisation of PrP^C in beta cells of human pancreases based on immunofluorescence analysis, (a, e, i) PrP^C was observed to be highly expressed in pancreatic islets of all three islet types [non-diabetic; type 1 diabetic INS(+); type 1 diabetic INS(−)] with low level expression in the exocrine pancreas, (a–d) PrP^C expression in non-diabetic donors localises to pancreatic islets and co-localises with beta cells (c). Co-registration of PrP^C/INS cells was confirmed using z-stack 3D image reconstruction analysis (d). (e, i) In our analysis of PrP^C in type 1 diabetic pancreas, we separated images from donors that contain both INS(+) (e) and INS(−) islets (i). (e)–h) As with non-diabetic donors, type 1 diabetic INS(+) islets show co-registration between PrP^C and INS cells (g, h). (i–l) In the absence of insulin [INS(−)], PrP^C continues to be expressed in a large number of cells (i, k). Scale bars, 20 αm. INS, insulin; ND, non-diabetic, T1D, type 1 diabetic

Fig. 2

Cellular localisation of PrP^C in alpha cells of human pancreases based on immunofluorescence analysis. (a)–j) We found no co-registration of PrP^C with glucagon in either non-diabetic or type 1 diabetic INS(+) islets (k)–o) but did find co-localisation of PrP^C with insulin (white arrowheads e and j). PrP^C was found to be co-localised with alpha cells in type 1 diabetic INS(−) islets, suggesting that PrP^C expression is activated in alpha cells in the absence of beta cells in these islets. Co-registration was confirmed using z-stack 3D image reconstruction analysis shown in (n). Scale bars, 20 μm. GCG, glucagon; INS, insulin; ND, non-diabetic, T1D, type 1 diabetic

Fig. 3

Intracellular localisation of PrP^C in the human pancreas. Specific co-staining of PrP^C and cellular markers for ER (WFS1), Golgi (GM130) and cell membrane (CD99) staining was implemented to assess PrP^C intracellular localisation within endocrine cells of the human pancreas. PrP^C was found to co-register with WFS1 (a, d, g) and CD99 (c, f, i) but not GM130 (b, e, h; ESM Fig. 2) based on 3D image reconstruction analysis (insets), indicating that PrP^C is mainly associated with the ER and plasma membrane of pancreatic endocrine cells. Scale bars, 20 μm. ND, non-diabetic, T1D, type 1 diabetic

Fig. 4

PRNP gene and PrP^C protein expression in non-diabetic, type 1 diabetic and AAb+ organ donor human pancreases. PRNP mRNA expression levels were measured by qRT-PCR from isolated total pancreatic RNA from 29 non-diabetic, 29 type 1 diabetic and 23 AAb+ donors. (a) PRNP gene expression in type 1 diabetes revealed a highly significant increase of 10.85-fold (p ≤ 0.0001) compared with non-diabetic donors. (b) Representative PrP^C-specific immunoblot analysis on total pancreatic protein isolated from non-diabetic and type 1 diabetic organ donors was performed to test changes in PrP^C mRNA levels. Right panel: the migration pattern of PrP^C in human brain extracts; left panel: immunoblot analysis of purified human pancreas extracts of four representative non-diabetic and five representative type 1 diabetic donors. Full immunoblot for PrP^C and total protein can be found in ESM Fig. 3. (c) Densitometric analysis data from immunoblot shown in (b) combined with additional data (6 more non-diabetic; 5 more type 1 diabetic donors) normalised to total protein, illustrating a concomitant increase in PrP^C levels of 2.16-fold (p = 0.0433). (d) In contrast, AAb+ donors exhibited significantly lower PRNP mRNA levels when compared with non-diabetic donors (0.579-fold) (p = 0.012; white diamond, single AAb+; black diamond, multiple AAb+). *p < 0.05. ND, non-diabetic; T1D, type 1 diabetic

Fig. 5

Assessment of possible interactions between PrP^C and protein aggregates in type 1 and type 2 diabetic donors. Among our 12 type 1 diabetic organ donors, three cases displayed amyloid-like plaques. In these three cases, we evaluated whether PrP^C co-localised with these islet-associated protein aggregates. (a)–d) The pathology stain, Congo Red, was used to identify amyloid/protein aggregates in the three type 1 diabetic donors as well as one type 2 diabetic donor (positive control). Black arrows show positivity for amyloid-like plaques (pink colour) in pancreatic islets. (e)–h) To visualise amyloid-like plaques and PrP^C expression together, consecutive slides were stained with thioflavin T (green) and PrP^C (red). We found no colocalisation of PrP^C-positive islet cells with the thioflavin T regions in either the type 1 or the type 2 diabetic pancreas samples. Scale bars, 20 μm. T1D, type 1 diabetic; T2D, type 2 diabetic

Fig. 6

STI1 co-registers with PrP^C in islets. (a, e, i) PrP^C is expressed in non-diabetic, type 1 diabetic INS(+) and type 1 diabetic INS(−) islets. (b, f, j) STI1 is highly expressed in the pancreatic islets of non-diabetic, type 1 diabetic INS(+) and type 1 diabetic INS(−) islets. (d, h, l) We found that STI1 highly co-registers with PrP^C in all three groups, as shown in z-stack 3D reconstruction images. (j, k, l) Even with no insulin expression in INS(−) islets, STI1 expression is still evident, with co-registration with glucagon-positive cells (ESM Fig. 6). Scale bars, 20 μm. (m) STIP1 total mRNA levels in type 1 diabetic donors is significantly increased compared with non-diabetic donors 13.42-fold (p ≤ 0.0001). (n) We also compared mRNA levels in AAb+ donors to non-diabetic donors and observed increased STIP1 expression of 2.09-fold (p = 0.0064) compared with non-diabetic donors (white diamond, single AAb+; black diamond, multiple AAb+). *p < 0.05. ND, non-diabetic; T1D, type 1 diabetic

Fig. 7

NCAM1 co-registers with PrP^C primarily in the cell membrane. (a, e, i) PrP^C is expressed in non-diabetic, type 1 diabetic INS(+) and type 1 diabetic INS(−) islets. (b, f, j) NCAM1 is highly expressed in the pancreatic islets of non-diabetic, type 1 diabetic INS(+) and type 1 diabetic INS(−) islets. (c, g, k) We found that NCAM1 highly co-registers with PrP^C in all three groups as shown in z-stack 3D reconstruction images (d, h, l). Scale bars, 20 μm. ND, non-diabetic; T1D, type 1 diabetic

Fig. 8

Schematic summary of human PrP^C cellular localisation, trafficking and interacting partners in beta and alpha cells in non-diabetic, type 1 diabetic and AAb+ human pancreas. (a) A summary of all the results in non-diabetic islets, including: PrP^C co-registration with beta cells; PrP^C existence in two cellular pools with interaction of PrP^C with NCAM1 on the plasma membrane and STI1 in the ER, along with the proposal that PrP^C must traffic from the ER to the plasma membrane bypassing transit through the Golgi. For illustration, the unconventional protein trafficking mechanism is depicted using vesicle-mediated transit from the ER to the plasma membrane. (a, c, g) PrP^C co-registers with beta cells in non-diabetic, type 1 diabetic INS(+) islets and AAb+ islets, with no co-registration with alpha cells (b, d, h). (e, f) PrP^C co-registers with alpha cells in type 1 diabetic INS(−) islets. (g) PrP^C is expressed at significantly lower levels in AAb+ beta cells compared with non-diabetic and INS(+) islets (a, c). Note: STI1 and NCAM1 were not measured in AAb+ (g, h). Graphic design created in biorender.com (https://biorender.com)