Structural and mechanistic aspects influencing the ADAM10-mediated shedding of the prion protein

Background: Proteolytic processing of the prion protein (PrPC) by endogenous proteases generates bioactive membrane-bound and soluble fragments which may help to explain the pleiotropic roles of this protein in the nervous system and in brain diseases. Shedding of almost full-length PrPC into the extracellular space by the metalloprotease ADAM10 is of peculiar relevance since soluble PrP stimulates axonal outgrowth and is protective in neurodegenerative conditions such as Alzheimer’s and prion disease. However, molecular determinates and mechanisms regulating the shedding of PrP are entirely unknown. Methods: We produced an antibody recognizing the neo-epitope of shed PrP generated by ADAM10 in biological samples and used it to study structural and mechanistic aspects affecting the shedding. For this, we investigated genetically modified cellular and murine models by biochemical and morphological approaches. Results: We show that the novel antibody specifically detects shed PrP in cell culture supernatants and murine brain. We demonstrate that ADAM10 is the exclusive sheddase of PrPC in the nervous system and reveal that the glycosylation state and type of membrane-anchorage of PrPC severely affect its shedding. Furthermore, we provide evidence that PrP shedding can be modulated by pharmacological inhibition and stimulation and present data suggesting that shedding is a relevant part of a compensatory network ensuring PrPC homeostasis of the cell. Conclusions: With the new antibody, our study introduces a new tool to reliably investigate PrP-shedding. In addition, this study provides novel and important insight into the regulation of this cleavage event, which is likely to be relevant for diagnostic and therapeutic approaches even beyond neurodegeneration

Ligands binding to the prion protein induce its proteolytic release with therapeutic potential in neurodegenerative proteinopathies

The prion protein (PrPC) is a central player in neurodegenerative diseases, such as prion diseases or Alzheimer’s disease. In contrast to disease-promoting cell surface PrPC, extracellular fragments act neuroprotective by blocking neurotoxic disease-associated protein conformers. Fittingly, PrPC release by the metalloprotease ADAM10 represents a protective mechanism. We used biochemical, cell biological, morphological, and structural methods to investigate mechanisms stimulating this proteolytic shedding. Shed PrP negatively correlates with prion conversion and is markedly redistributed in murine brain in the presence of prion deposits or amyloid plaques, indicating a sequestrating activity. PrP-directed ligands cause structural changes in PrPC and increased shedding in cells and organotypic brain slice cultures. As an exception, some PrP-directed antibodies targeting repetitive epitopes do not cause shedding but surface clustering, endocytosis, and degradation of PrPC. Both mechanisms may contribute to beneficial actions described for PrP-directed ligands and pave the way for new therapeutic strategies against currently incurable neurodegenerative diseases

Transgenic Overexpression of the Disordered Prion Protein N1 Fragment in Mice Does Not Protect Against Neurodegenerative Diseases Due to Impaired ER Translocation

The structurally disordered N-terminal half of the prion protein (Pr

Structural and mechanistic aspects influencing the ADAM10-mediated shedding of the prion protein

Abstract Background Proteolytic processing of the prion protein (PrPC) by endogenous proteases generates bioactive membrane-bound and soluble fragments which may help to explain the pleiotropic roles of this protein in the nervous system and in brain diseases. Shedding of almost full-length PrPC into the extracellular space by the metalloprotease ADAM10 is of peculiar relevance since soluble PrP stimulates axonal outgrowth and is protective in neurodegenerative conditions such as Alzheimer’s and prion disease. However, molecular determinates and mechanisms regulating the shedding of PrP are entirely unknown. Methods We produced an antibody recognizing the neo-epitope of shed PrP generated by ADAM10 in biological samples and used it to study structural and mechanistic aspects affecting the shedding. For this, we investigated genetically modified cellular and murine models by biochemical and morphological approaches. Results We show that the novel antibody specifically detects shed PrP in cell culture supernatants and murine brain. We demonstrate that ADAM10 is the exclusive sheddase of PrPC in the nervous system and reveal that the glycosylation state and type of membrane-anchorage of PrPC severely affect its shedding. Furthermore, we provide evidence that PrP shedding can be modulated by pharmacological inhibition and stimulation and present data suggesting that shedding is a relevant part of a compensatory network ensuring PrPC homeostasis of the cell. Conclusions With the new antibody, our study introduces a new tool to reliably investigate PrP-shedding. In addition, this study provides novel and important insight into the regulation of this cleavage event, which is likely to be relevant for diagnostic and therapeutic approaches even beyond neurodegeneration

Additional file 8: of Structural and mechanistic aspects influencing the ADAM10-mediated shedding of the prion protein

(.jpg) Embryonic fibroblasts (MEF) of ADAM10 knockout mice accumulate PrPC. Representative western blot of media supernatants and lysates of wild-type (WT) and ADAM10 KO MEF. Lack of shedding and no increased compensatory release of PrP is observed in ADAM10 KO cells. ADAM10 is shown in lysates as a proof of genotypes. Increased levels of PrPC are found in lysates and quantified by referring to β-actin (n = 8; p = 0.00005). (JPEG 131 kb

Additional file 2: of Structural and mechanistic aspects influencing the ADAM10-mediated shedding of the prion protein

(.jpg) Species specificity of the sPrPG228 antibody. (A) Comparison of the C-terminal amino acid sequence of PrPC in mouse (Mus musculus), rat (Rattus norvegicus) and rabbit (Oryctolagus cuniculus) (source: www.uniprot.org ). “NH 2 -…-” indicates the N-terminal direction, “-GPI” the C-terminal GPI-anchor attachment site. Asterisks indicate position of ADAM10-mediated shedding in mice and rats with Gly228 representing the new C-terminus of shed PrP. Note the sequence difference compared with rabbit PrPC. (B) Western blot analysis of forebrain homogenates from different murine models (tga20, Prnp0/0, wild-type (C57BL/6)) as well as from rat and rabbit. As expected for its epitope, the sPrPG228 antibody detects sPrP in mouse (tga20 and wild-type) and rat, whereas the brain sample of rabbit only presents an immunoglobulin light chain (rb Ig-LC) signal at 25 kDa resulting from the anti-rabbit secondary antibody used for detection. Re-probing the blot with POM2 antibody reveals expression levels of PrPC. (JPEG 388 kb

Additional file 9: of Structural and mechanistic aspects influencing the ADAM10-mediated shedding of the prion protein

(.jpg) Quantification of fl-PrP levels and ratio of sPrP/fl-PrP in Sort1 knockout mice. These quantifications refer to main Fig. 5j. (A) Increased amounts of fl-PrP are found in brains of Sort1 KO mice (2.11 ± 0.23; p = 0.0004; n = 4) compared to controls (WT set to 1.00 ± 0.21; SD). Actin served as loading control and for reference in densitometric quantification. (B) No significant differences are detected in the ratio of sPrP to fl-PrP between Sort1 KO mouse brains (0.85 ± 0.07, p = 0.128; n = 4) and controls (WT set to 1.00 ± 0.14). (JPEG 229 kb

Additional file 3: of Structural and mechanistic aspects influencing the ADAM10-mediated shedding of the prion protein

(.jpg) Use of the sPrPG228 antibody for immunohistochemical stainings. Sagittal brain sections of a Prnp0/0, a wild-type (C57BL/6) and a tga20 mouse stained with hematoxilin/eosin (H&E), an antibody against total PrPC (SAF84), or the sPrP Ab showing the hippocampus (Hc) and parts of cortical areas (Cx) in overviews. Magnifications are shown for the corpus callosum and CA1 region (upper insets) as well as for the dentate gyros (DG) and CA3 region (lower insets) of the hippocampus. With the sPrPG228 Ab, a diffuse brownish staining of the brain parenchyma is seen for wild-type and tga20 whereas Prnp0/0 brain only shows blue counterstaining. Comparison with the SAF84 staining reveals that levels of shed PrP correlate with overall PrPC expression. Scale bars represent 100 Οm. (JPEG 1948 kb

Additional file 6: of Structural and mechanistic aspects influencing the ADAM10-mediated shedding of the prion protein

(.jpg) Side-by-side comparison of SWA and TM treatments and enzymatic deglycosylation reactions. Western blot of untreated (untr.), SWA- or TM-treated N2a cells showing lysates without (−) or with (+) enzymatic treatment for differential deglycosylation (Endo H or PNGase F). As also shown in Fig. 3a and b, TM-treatment causes a complete inhibition of PrP glycosylation, whereas SWA-treatment results in a shift in the banding pattern (compared to untreated cells) and (at least partial) Endo H sensitivity due to inhibition of complex glycosylation. Changes in the glycopattern and running behaviour support the functioning of our enzymatic deglycosylation protocols also performed for the experiments shown in Figs. 2, 3 and 4. Actin is shown as a loading control. (JPEG 221 kb