Highly infectious prions are not directly neurotoxic

Significance Prions are infectious agents composed of polymers of misfolded prion protein which cause fatal brain diseases such as Creutzfeldt–Jakob disease. These diseases involve progressive loss of neuronal cells, and it has been long assumed that prions are directly toxic to cells as they propagate. However, recent studies have suggested that prion infectivity and neurotoxicity can be uncoupled and involve distinct mechanisms. Using highly purified infectious prions we demonstrate that prions are not directly neurotoxic and that toxicity present in infected brain tissue can be distinguished from infectious prions. This has fundamental implications for understanding of prion diseases and how to treat them and may have wide relevance in other neurodegenerative diseases which involve propagation and spread of proteopathic seeds.

. Primary neuronal cultures do not propagate RML prions after a short treatment with RML prion-infected brain homogenate (RML BH). Three independent cortical cultures from Prnp +/+ and Prnp 0/0 mice were treated with RML BH (brain concentration 10 -4 ) for 1 h, 72 h or 1 week. Infectivity titer was determined by Automatic Scrapie Cell Assay in PK1/2 cells after the 5 th (P5) and 6 th (P6) split, mean ± S.D. Infectivity in Prnp 0/0 cells represents residual infectivity of the inoculum. P values from 2-way ANOVA with Sidak's correction for multiple comparisons reveal no significant difference between genotypes upon any duration of treatment. .
Movie S1 (separate file) Infectious RML brain homogenate (BH) has a pronounced toxic effect on neuronal network. RML BH containing 10 4.2 infectious units (IU)/ml was added to 10 days old cortico-hippocampal mouse neurons after 20 hours of baseline recording. Note acute neurite retraction and loss of neuronal cell bodies.
Movie S2 (separate file) 10 days old neuronal culture treated with a noninfectious Prnp +/+ brain diluted to a concentration of 10 -4 (vehicle control for Movie S1). This brain homogenate was added to primary cells after 20 hours of baseline recording.
Movie S3 (separate file) High titre purified RML prions (infectivity 10 6.7 IU/ml) diluted in tissue culture medium are not toxic to primary neurons. Prions were added to 10 days old cortico-hippocampal mouse culture after 20 hours of baseline recording.
Movie S4 (separate file) Non-infectious Prnp 0/0 brain diluted to a concentration of 10 -4 reconstituted with high titre purified RML prions (10 6.7 IU/ml) is not toxic to primary neurons. Prions in brain homogenate were added to 10 days old corticohippocampal mouse neurons after 20 hours of baseline recording.
Movie S5 (separate file) 10 days old neuronal culture treated with a noninfectious Prnp 0/0 brain at a concentration of 10 -4 (vehicle control for Movie S4). This brain homogenate was added to primary cells after 20 hours of baseline recording.
Movie S6 (separate file) Non-infectious Prnp +/+ brain diluted to a concentration of 10 -4 reconstituted with high titre purified RML prions (10 6.7 IU/ml) is not toxic to primary neurons. Prions were added to 10 days old cortico-hippocampal mouse neurons after 20 hours of baseline recording.