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Commentary Free access | 10.1172/JCI134842
Department of Medical Microbiology and Immunology, School of Medicine, Creighton University, Omaha, Nebraska, USA.
Address correspondence to: Jason C. Bartz, Department of Medical Microbiology and Immunology, 2500 California Plaza, Omaha, Nebraska 68178, USA. Phone: 402.280.1811; Email: [email protected].
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Published January 27, 2020 - More info
Posttranslational modifications (PTMs) are common among proteins that aggregate in neurodegenerative disease, yet how PTMs impact the aggregate conformation and disease progression remains unclear. By engineering knockin mice expressing prion protein (PrP) lacking 2 N-linked glycans (Prnp180Q/196Q), we provide evidence that glycans reduce spongiform degeneration and hinder plaque formation in prion disease. Prnp180Q/196Q mice challenged with 2 subfibrillar, non–plaque-forming prion strains instead developed plaques highly enriched in ADAM10-cleaved PrP and heparan sulfate (HS). Intriguingly, a third strain composed of intact, glycophosphatidylinositol-anchored (GPI-anchored) PrP was relatively unchanged, forming diffuse, HS-deficient deposits in both the Prnp180Q/196Q and WT mice, underscoring the pivotal role of the GPI-anchor in driving the aggregate conformation and disease phenotype. Finally, knockin mice expressing triglycosylated PrP (Prnp187N) challenged with a plaque-forming prion strain showed a phenotype reversal, with a striking disease acceleration and switch from plaques to predominantly diffuse, subfibrillar deposits. Our findings suggest that the dominance of subfibrillar aggregates in prion disease is due to the replication of GPI-anchored prions, with fibrillar plaques forming from poorly glycosylated, GPI-anchorless prions that interact with extracellular HS. These studies provide insight into how PTMs impact PrP interactions with polyanionic cofactors, and highlight PTMs as a major force driving the prion disease phenotype.
Alejandro M. Sevillano, Patricia Aguilar-Calvo, Timothy D. Kurt, Jessica A. Lawrence, Katrin Soldau, Thu H. Nam, Taylor Schumann, Donald P. Pizzo, Sofie Nyström, Biswa Choudhury, Hermann Altmeppen, Jeffrey D. Esko, Markus Glatzel, K. Peter R. Nilsson, Christina J. Sigurdson
The prion agent is unique in biology and is comprised of prion protein scrapie (PrPSc), a self-templating conformational variant of the host encoded prion protein cellular (PrPC). The deposition patterns of PrPSc in the CNS can vary considerably from a diffuse synaptic pattern to large plaque-like aggregates. Alterations of PrPC posttranslational processing can change PrPSc deposition patterns; however, the mechanism underlying these observations is unclear. In this issue of the JCI, Sevillano and authors determined that parenchymal PrPSc plaques of the mouse brain preferentially incorporated underglycosylated PrPC that had been liberated from the cell surface by the metalloproteinase, ADAM-10, in combination with heparan sulfate. These results provide mechanistic insight into the formation of PrPSc plaques and suggest that PrP posttranslational modifications direct pathogenicity as well as the rate of disease progression.
Prions are protein-only infectious agents that subvert the normal form of the host cellular prion protein, PrPC, to the self-templating scrapie prion form, PrPSc (1, 2). Due to the unique nature of prions, the properties of both the host PrPC and agent PrPSc contribute to the pathogenesis of disease. Specifically, strains of prions result in distinct patterns of PrPSc deposition and neurodegeneration in the CNS that are encoded by strain-specific conformations of PrPSc (3, 4). Interestingly, changes in the host PrPC can result in alteration of the phenotype of disease, including neuropathology, in the absence of altering the prion strain. For example, murine PrPC is polymorphic with two alleles, and inoculation of ME7 from one PrPC genetic background to the other resulted in a change in both the incubation period of disease and the distribution of neurodegeneration (5). Importantly, back passage of ME7 to the original PrPC genetic background resulted in an incubation period and neuropathology indistinguishable from the original PrPC allele (i.e., class 1 strain) (5). This seminal work indicated that differences in the amino acid sequence of PrPC contribute to the pathogenesis of disease independently of the prion strain.
Posttranslational modifications of PrPC can alter prion pathogenesis. PrPC is tethered to the cell membrane by a glycophosphatidylinositol (GPI) anchor (6). In transgenic mice, deleting the PrPC GPI anchor results in PrPC retrafficking into the intracellular space. Subsequent prion inoculation resulted in PrPSc formation, indicating that prion conversion is independent of the GPI anchor (7). Importantly, these mice showed substantial PrPSc distribution differences in the whole body as well as differences in the CNS. In the CNS, the pattern of PrPSc deposition was characterized by dense plaque-like formations, compared with the more diffuse synaptic pattern of PrPSc deposition in nontransgenic WT animals. While prion infection of PrPC GPI anchorless mice resulted in prion formation, these animals did not develop clinical signs of prion disease. It is unclear if their healthy phenotype is due to a failure of PrPSc to gain access to the areas of the CNS responsible for the onset of clinical signs, if the PrPSc generated was less neurotoxic, or if the absence of symptoms was a property of the strain tested.
In addition to a GPI anchor, PrPC contains two N-linked glycosylation sites that have variable occupancy (8, 9). Transgenic mice in which the PrPC lacked both N-linked glycosylation sites still supported prion formation. PrPSc from these mice transmits prion disease to WT mice, indicating that glycosylation is not required for prion formation or transmission (10). In the strains tested, however, the absence of PrPC glycosylation resulted in an increase in incubation period and PrPSc amyloid plaque deposits compared with WT animals that have a full repertoire of PrPC glycosylation. Shifting of PrPSc to a more amyloid plaque structure can reduce the pathogenicity of PrPSc by extraneuronal routes of infection, consistent with the hypothesis that small, soluble PrPSc oligomers are the preferred species of PrPSc for retrograde transport in the peripheral nervous system that facilitates neuroinvasion (11, 12). Both GPI anchorless PrPC and underglycosylated PrPC share a common neuropathological feature of parenchymal PrPSc plaques. These studies indicate that alteration of PrPC posttranslational modifications can result in profound changes in the neuropathology of prion disease. The mechanism responsible for these observations is, however, unclear.
The work of Sevillano et al. in this issue of the JCI examines the mechanism of how PrPC posttranslational modifications alter neuropathology (13). Mice expressing unglycosylated PrPC were generated (Prnp180Q/196Q) and were shown to have normal PrPC expression patterns that, importantly, failed to spontaneously develop neurodegeneration. The Prnp180Q/196Q mice were challenged with four murine-adapted prion strains, three of which (RML, 22L, and ME7) are categorized as subfibrillar strains with a predominately diffuse synaptic pattern of PrPSc deposition in the CNS, and one of which (mCWD) is a fibrillar strain that is characterized by large plaques of PrPSc in the CNS. All of the strains tested resulted in the Prnp180Q/196Q animals developing prion disease. Notably, the mice inoculated with the subfibrillar strains developed plaque-like deposits. This altered neuropathology is consistent with previous studies indicating that reduced PrPC glycosylation results in an increase in PrPSc plaque formation (10). To examine the composition of these plaque-like deposits in the Prnp180Q/196Q mice, the authors used an anti-PrP antibody that specifically recognizes PrP that has been cleaved from the cell surface by the metalloproteinase ADAM-10 (14). It was determined that the PrPSc plaques were enriched for ADAM-10–cleaved PrP. Next, the authors identified that heparan sulfate (HS) colocalized to the plaques but was restricted from areas with diffuse aggregates of PrPSc. Consistently, HS specifically bound to PrP that had been cleaved by ADAM-10. Further, the HS/ADAM-10–cleaved PrP interaction was influenced by the glycosylation state of PrP where a progressive reduction in the glycosylation of PrP resulted in a corresponding increase in affinity of PrP to bind HS. These data suggest that underglycosylated PrPC that is cleaved from the cell surface in combination with HS produces large plaque-like structures.
To test the hypothesis that glycosylation influences plaque-like pathology, Sevillano and colleagues generated novel transgenic mice that expressed PrPC with three N-linked glycosylation sites (Prnp187N) (13). Inoculation of the Prnp187N mice with the plaque-forming strain mCWD resulted in a neuropathology that lacked plaques and instead was comprised of diffuse PrPSc deposits compared with WT mice that contain 2 N-linked PrPC glycosylation sites. This observation is consistent with the premise that PrP glycosylation inhibits PrPSc fibril formation by interfering with binding to HS. Overall, a model for prion formation suggests that underglycosylated PrP released from the cell surface associates with HS to form large parenchymal deposits, whereas cell-bound fully glycosylated PrPSc preferentially forms small diffuse aggregates (Figure 1 and ref. 13).
Role of posttranslational processing of PrPC in the formation of prion aggregates. Fibrillar prion strains such as mCWD preferentially incorporate underglycosylated PrPC (blue) that is released from the cell surface by ADAM-10 into plaques that form in association with HS in the parenchyma. Subfibrillar prion stains, such as RML, preferentially incorporate glycosylated PrPC into diffuse synaptic deposits of PrPSc (red) without a HS scaffold. Genetic ablation of N-linked glycosylation sites (N) can switch in nonfibrillar strain pathology from synaptic diffuse deposition to plaque-like deposition of PrPSc in the parenchyma.
The role of the prion strain in the preponderance to form plaques versus diffuse deposits of PrPSc is only partially understood. Transmission of all four prion strains to Prnp180Q/196Q mice resulted in an increase in plaque-like PrPSc deposits in the CNS. However, the degree to which plaque-like deposits increased was not uniform between the strains tested. The mechanism behind this observation remains unclear (13). It is possible that the strain-specific differences in the preferred PrPC substrate for conversion, as has been identified using in vitro studies, may play a role in this observation (15). In addition, strain-specific rates of formation and clearance may favor specific patterns of prion accumulation (16).
Strain-specific responses in the alteration of the incubation period of disease in the Prnp180Q/196Q mice were observed. Specifically, the incubation period of RML-infected WT or Prnp180Q/196Q mice was similar. This was in contrast to that of 22L-infected animals, where the incubation period of Prnp180Q/196Q-infected mice was longer compared with WT mice. Importantly, the extended incubation period of RML-infected Prnp180Q/196Q mice was preserved upon second passage. One potential explanation for this observation is that differences in the PrP amino acid sequence between the WT and Prnp180Q/196Q mice may cause a species barrier–like phenomenon that can alter strain properties. Alternatively, while evidence suggests that glycosylation is not required on the inoculum PrPSc to maintain strain properties (17), it is possible the host PrPC glycosylation directly or indirectly via interactions with cellular cofactors plays a role in maintenance of strain properties during prion conversion (18). The small differences in incubation period between WT and Prnp180Q/196Q mice infected with RML suggest that glycosylation may not substantially affect prion formation in contrast to what has been observed in vitro where glycosylation inhibits prion formation (19, 20). Several scenarios could explain this discrepancy. It is possible that the strains tested in this study respond differently to changes in glycosylation compared with strains that were used in the in vitro studies (13, 19, 20). Many factors in addition to the kinetics of PrPSc accumulation can influence the incubation period of disease. The relative rate of PrPSc formation, clearance, and transport to populations of neurons that are susceptible to PrPSc neurotoxicity are only a few of the factors that contribute to the incubation period of disease.
When Sevillano and colleagues introduced a third glycosylation site in PrPC, the incubation period shortened. Further, there was a corresponding increase in diffuse PrPSc aggregates and an absence of PrPSc plaques (13). These findings suggest that a more rapid disease course corresponds with diffuse PrPSc deposits. While more work is needed to further characterize the contributions of posttranslational modifications of PrPC and the prion strain to prion pathogenesis, the Sevillano et al. study illustrates that shifting diffuse synaptic PrPSc deposits to plaques is a viable therapeutic modality to slow the progression of prion disease.
JCB is supported by the National Institute of Allergy and Infectious Diseases and the National Institute of Neurological Disorders and Stroke of the NIH via grants P01 0011877A, R01 NS103763, and R01 NS107246, and by award CWD20-018 from the State of Michigan.
Address correspondence to: Jason C. Bartz, Department of Medical Microbiology and Immunology, 2500 California Plaza, Omaha, Nebraska 68178, USA. Phone: 402.280.1811; Email: [email protected].
Conflict of interest: The author has declared that no conflict of interest exists.
Copyright: © 2020, American Society for Clinical Investigation.
Reference information: J Clin Invest. 2020;130(3):1087–1089. https://doi.org/10.1172/JCI134842.
See the related article at Prion protein glycans reduce intracerebral fibril formation and spongiosis in prion disease.