Cofactors facilitate bona fide prion misfolding in vitro but are not necessary for the infectivity of recombinant murine prions

Abstract

Prion diseases, particularly sporadic cases, pose a challenge due to their complex nature and heterogeneity. The underlying mechanism of the spontaneous conversion from PrPC to PrPSc, the hallmark of prion diseases, remains elusive. To shed light on this process and the involvement of cofactors, we have developed an in vitro system that faithfully mimics spontaneous prion misfolding using minimal components. By employing this PMSA methodology and introducing an isoleucine residue at position 108 in mouse PrP, we successfully generated recombinant murine prion strains with distinct biochemical and biological properties. Our study aimed to explore the influence of a polyanionic cofactor in modulating strain selection and infectivity in de novo-generated synthetic prions. These results not only validate PMSA as a robust method for generating diverse bona fide recombinant prions but also emphasize the significance of cofactors in shaping specific prion conformers capable of crossing species barriers. Interestingly, once these conformers are established, our findings suggest that cofactors are not necessary for their infectivity. This research provides valuable insights into the propagation and maintenance of the pathobiological features of cross-species transmissible recombinant murine prion and highlights the intricate interplay between cofactors and prion strain characteristics.

Fig 1. Evaluation of spontaneous misfolding capacity of 20 mouse rec-PrP variants with naturally occurring amino acid substitutions at position 108.

A) PMSA experiments were conducted on 20 substrates, each containing a distinct rec-PrP variant. After a 24-hour PMSA round (four replicates per substrate), rec-PrP^res formation was assessed via PK digestion, electrophoresis, and total protein staining. Results, displayed in grayscale, show four variants capable of spontaneous misfolding: L108I and L108H (100% positive replicates), L108K (75%), and L108M (50%). Variants without misfolding propensity showed no detectable rec-PrP^res (white). B) An electrophoresis gel illustrates rec-Pr^Pres detection for selected variants (L108 wt, L108I, L108F, L108Q, L108T, and L108V). Only L108I formed rec-PrP^res with the characteristic 16 kDa PK-resistant band expected of recombinant prions. C) Further PMSA experiments with the four misfolding-prone variants (L108I, L108H, L108K, and L108M) were conducted with shorter reaction times (3, 4, 6, and 16 hours). Results indicate that only L108I misfolded under these restrictive conditions, confirming it as the most prone to spontaneous misfolding among all variants. MW: Molecular weight marker.

Fig 2. Generation and stable propagation of four potentially distinct conformers of spontaneously misfolded mouse L108I rec-PrP^res.

Four spontaneously generated PMSA products (stMI-01, stMI-03, btMI-05, btMI-09) were selected as potentially distinct conformers. These were produced after a single 24-hour PMSA round in the presence of 0.5% dextran sulfate and assessed by PK digestion, electrophoresis, and total protein staining. All four conformers could be stably propagated through serial PMSA rounds (1:10 seed:substrate dilutions), maintaining their characteristic electrophoretic profiles over six rounds. The undigested substrate (rec-MoL108I) is included for size comparison. Despite similar fragmentation patterns, further analyses reveal distinct properties among the conformers. Conform: Conformer; MW: Molecular weight marker.

Fig 3. A) Biochemical and survival analysis of TgMoL108I mice inoculated with spontaneously misfolded PMSA products.

Proteinase K digestion, electrophoresis, and Western blot (Sha31, 1:4,000) of brain homogenates from TgMoL108I mice inoculated with the four PMSA products (stMI-01, stMI-03, btMI-05, btMI-09, using brain-derived RML and 22L prions as controls) revealed the classical three-banded PrP^Sc pattern, confirming their infectious nature as bona fide recombinant prions. Representative samples from each group and controls (brain-derived RML and 22L prions) were mostly indistinguishable, though slight electrophoretic differences were observed in stMI-03 (sample 01, higher bands) and btMI-09 (sample 02, lower bands), suggesting potential conformer mixtures. B) Kaplan-Meier survival curves for animals inoculated with PMSA products and RML and 22L strains demonstrated variability in incubation periods. stMI-01 and stMI-03 showed greater dispersion, while btMI-05 and btMI-09 exhibited more uniform profiles similar to RML/22L but with slightly longer incubation periods. These longer periods may reflect the need for adaptation or a transmission barrier between recombinant prions and brain-derived PrP^C, while higher dispersion could indicate strain-specific barriers, conformer mixtures undergoing selection, or immature prion conformers. Notably, the longest incubation for stMI-01 (~340 dpi) coincided with spontaneous atypical prion disease in the TgMoL108I model, distinct from induced TSE due to the absence of classical PrP^Sc patterns. PK: Proteinase K; NBH: Normal brain homogenate; MW: Molecular weight marker.

Fig 4. Brain lesion and PrP^res deposit distribution of the TgMoL108I-passaged recombinant PMSA products after secondary transmission in wild-type mice.

Histopathological assessment of spongiform lesions and PrP^res deposits shows mild to moderate spongiform changes upon hematoxylin and eosin staining (H&E), as seen in the thalamic region of the brains of representative animals from each group. PrP^res deposits were labeled with 6C2 mAb (1:1,000) for stMI-01 and btMI-05, and 2G11 mAb (1:100) for stMI-03 and btMI-09. Deposits were detectable only for stMI-03, btMI-05, and some of the btMI-09-inoculated animals, all showing synaptic fine punctate deposits (see digitally enlarged images on the left). Spongiform lesion profiles and PrP^res deposition profiles, shown on the right, represent the mean semi-quantitative scoring (0–4, vertical axis, ± standard error of the mean -error bars-) of the spongiform lesions (continuous line, black) and the immunohistochemical labeling of PrP^res deposits (dashed line, black) across 14 brain regions. Although there are some differences, mostly in terms of spongiform lesion intensity, stMI-03, btMI-05, and btMI-09-inoculated animals show highly coincident lesions and PrP^res staining. Animals inoculated with stMI-01 are the only ones with a clearly distinguishable spongiform lesion profile and without detectable PrP^res deposits in all areas analyzed. H&E: Hematoxylin and eosin staining; IHC: Immunohistochemistry.

Fig 5. Biochemical analysis and survival curves of wild-type mice inoculated with spontaneously misfolded PMSA products.

A) Biochemical analysis of wild-type brains inoculated with four selected PMSA products. Brain homogenates from all inoculated animals showing clinical signs of transmissible spongiform encephalopathy were analyzed by proteinase K (PK) digestion, electrophoresis, and Western blot (Sha31, 1:4,000). Results revealed the presence of classical three-banded pattern PrP^Sc, demonstrating the infectious capacity and bona fide nature of the recombinant prions generated spontaneously by PMSA in wild-type animals. The gel shows two representative samples from each group inoculated with the distinct recombinant products (except stMI-01, where all animals remained healthy after 600 dpi and were PrP^Sc negative) and the brain-derived RML and 22L prions. All the samples are indistinguishable from each other but easily differentiated from the controls. B) Kaplan-Meier survival curves of wild-type inoculated with PMSA products and classical murine prion strains. Kaplan-Meier survival curves illustrate the incubation periods following intracerebral inoculations with the distinct PMSA products, RML, and 22L. stMI-03 shows great dispersion in incubation periods, while btMI-05 Dx and btMI-09 Dx exhibit profiles more similar to brain-derived strains RML and 22L, with lower dispersion but longer incubation periods. The extended incubation periods could suggest a need for adaptation or the existence of a transmission barrier between recombinant prions and brain-derived PrP^C. Groups showing greater dispersion might indicate stronger barriers due to strain characteristics, mixtures of conformers undergoing slightly different selection process in each animal, or the formation of unstable or somewhat immature conformers. PK: Proteinase K; NBH: Undigested normal brain homogenate; MW: Molecular weight marker.

Fig 6. Adaptation of dextran sulfate-generated recombinant prions to a cofactor-free environment.

A) Electrophoretic patterns of recombinant prions propagated without cofactor. Four recombinant prions underwent four serial PMSA rounds in a substrate containing mouse L108I rec-PrP without dextran sulfate (referred to as CB). The first round used prion-coated zirconia-silicate beads, while subsequent rounds used 1:100 dilutions of previous products. Results were visualized by PK digestion, electrophoresis, and total protein staining. Unseeded control tubes were included to monitor cross-contamination or spontaneous misfolding. The adapted prions show distinct electrophoretic mobility patterns compared to their original dextran sulfate-complemented counterparts. stMI-01 CB, btMI-05 CB, and btMI-09 CB (derived from stMI-01, btMI-05, and btMI-09 generated in presence of dextran sulfate, respectively) converged to a similar profile characterized by the loss of the ~16 kDa fragment present in the originals. In contrast, stMI-03 CB (derived from stMI-03) exhibited a unique profile, distinct from both its original preparation and other adapted products, showing an intense 16 kDa band and a ladder of smaller proteolytic fragments. B) Electrophoretic pattern conservation during additional PMSA rounds. Prior to intracerebral inoculations in TgMoL108I, C57BL/6 wild-type mice, and TgVole (1x) models, the adapted products underwent further PMSA propagation to ensure stable biochemical features and eliminate any remaining original seeds. Replicate 01 of stMI-03 CB and btMI-09 CB were used as seeds for four additional 24-h serial PMSA rounds (R1 to R4), demonstrating maintenance of their distinct electrophoretic signatures. The product of the fourth serial round was used for intracerebral inoculations, as illustrated. rec-PrP: Control showing the undigested PMSA substrate containing mouse L108I rec-PrP. MW: Molecular weight marker. The drawing of the mouse shown in the figure was generated using Copilot Designer generative AI tool (Microsoft365).

Fig 7. Propagation of btMI-09 CB in dextran sulfate-complemented environment and back in a cofactor-devoid substrate and biochemical analysis of TgMoL108I brains inoculated with all the resulting inocula.

A) Electrophoretic patterns of recombinant prions propagated without cofactor (btMI-09 CB), then with dextran sulfate as cofactor (btMI-09 dex²) and back without cofactor (btMI-09 CB²). btMI-09 dex recombinant prions underwent four serial PMSA rounds (R1-R4) in a substrate containing mouse L108I rec-PrP without dextran sulfate (referred to as CB). Results were visualized by PK digestion, electrophoresis, and total protein staining. After stabilization of btMI-09 CB, this procedure was repeated: first using a dextran sulfate-complemented substrates (only 3 PMSA rounds were performed prior to coating of zirconia-silicate beads), resulting in btMI-09 dex² PMSA product and then again in a cofactor-devoid environment, giving rise to btMI-09 CB². The dextran sulfate-complemented prions, the original btMI-09 dex and btMI-09 dex², show almost indistinguishable electrophoretic mobility patterns, with subtle differences in low molecular weight fragment intensity. Analogously btMI-09 CB and btMI-09 CB² share a similar profile characterized by the loss of the ~16 kDa fragment present in the dextran sulfate-propagated products, also presenting subtle differences in the relative intensity of the low molecular weight fragments. B) Biochemical analysis of TgMoL108I mice brains inoculated with the four btMI-09 preparations adapted to dextran sulfate-complemented and cofactor-devoid substrates. Brain homogenates from all inoculated animals showing clinical signs of transmissible spongiform encephalopathy were analyzed by proteinase K (PK) digestion, electrophoresis, and Western blot (Sha31, 1:4,000). Results revealed the presence of classical three-banded pattern PrP^Sc, demonstrating the infectious capacity and bona fide nature of the four products btMI-09 dex, CB, dex² and CB², after sequential adaptation to the two distinct propagation environments. The gel shows a representative example from each group inoculated with the distinct recombinant products and one of the brain-derived RML as control of a classical prion strain. PrP^Sc from all animals inoculated with the different versions of btMI-09 are indistinguishable from each other. C) Digestion of preparations adapted to cofactor-free environment in the presence and absence of dextran sulfate during digestion. To evaluate the potential interfering effects of sulfated dextran on proteinase K digestion patterns, btMI-09 CB and btMI-09 CB² were incubated during 1 h in the absence (w/o) or presence (w) of dextran sulfate, showing that the detection of the 16 kDa proteolytic fragment depends on the presence of dextran in the reaction, likely due to its strong association to fibers. PK: Proteinase K; NBH: Undigested normal brain homogenate; MW: Molecular weight marker. The drawing of the mouse shown in the figure was generated using Copilot Designer generative AI tool (Microsoft365).

Fig 8. Characterization of spontaneously misfolded recombinant mouse L108I PrP in PMSA using cofactor -devoid substrate.

A) Electrophoretic mobility profile and stable propagation in vitro of PMSA products MoL108I-CB-01 and MoL108I-CB-02. Two PMSA products, MoL108I-CB-01 and MoL108I-CB-02, were generated via spontaneous misfolding of mouse L108I rec-PrP in a dextran sulfate-free substrate (CB). PK-resistant rec-PrP with the expected ~16 kDa fragment characteristic of recombinant prions was detected after the second 24-hour PMSA round and stably propagated for three additional rounds (1:10 seed dilution). PK digestion, electrophoresis, and total protein staining revealed indistinguishable electrophoretic profiles between the two products, consistent across rounds. An undigested CB substrate was included for size comparison. B) Biochemical analysis of TgMoL108I mice brains inoculated with MoL108I-CB-01 and MoL108I-CB-02 preparations and wild-type mice brains inoculated with MoL108I-CB-01. Brain homogenates from TgMoL108I and wild-type mice inoculated with MoL108I-CB-01 and MoL108I-CB-02 were analyzed via PK digestion, electrophoresis, and Western blot (Sha31, 1:4,000). All inoculated animals showing transmissible spongiform encephalopathy (TSE) symptoms exhibited the classical three-banded PrP^Sc pattern, confirming the infectious capacity and bona fide prion nature of the PMSA products. PrP^Sc profiles were indistinguishable across groups and comparable to brain-derived murine prion strains RML and 22L. PK: Proteinase K; NBH: Undigested normal brain homogenate; MW: Molecular weight marker. C) Brain lesion and PrP^res deposit distribution of the TgMoL108I inoculated MoL108I-CB-01 and MoL108I-CB-02 and wild-type mice inoculated with MoL108I-CB-01. Histopathological analysis revealed distinct lesion and PrP^res deposition profiles. TgMoL108I mice inoculated with MoL108I-CB-01 and MoL108I-CB-02 exhibited mild spongiform changes and PrP^res deposits primarily in the brainstem (thalamus), hippocampus, and cerebellar cortex (indicated by black arrowheads). PrP^res-positive plaques in the cerebellar cortex white matter resembled those observed with dextran sulfate-supplemented PMSA products (S11 Fig), suggesting similar strain features. In contrast, wild-type mice inoculated with MoL108I-CB-01 displayed a unique profile, similar to TgMoL108I-passaged stMI-03, btMI-05, and btMI-09 inocula (Fig 4). Spongiform lesions were prominent in the thalamus and cerebellar cortex, with intraneuronal and punctate neuropil PrPres deposits labeled with 6C2 (1:1,000) in the same regions. H&E: Hematoxylin and eosin staining; IHC: Immunohistochemistry.