PK-resistant PrP in the index case, spontaneously sick and inoculated Tg mice.

<p>A) Presence of proteinase K (PK)-resistant PrP in different brain regions of the index case. Cb, cerebellum; FC, frontal cortex; PC, parietal cortex; WM, white matter. Black arrow indicates the position of the 8 kDa PrP fragment. 250 μg of protein was digested with 10 μg/ml of PK in a volume of 250 μl. B) Age-dependent accumulation of the 8 kDa fragment in Tg.HRdup-26 mice but not in Tg.M128V-39 control animals. 250 μg of protein was digested with 10 μg/ml of PK in a volume of 250 μl. C) Immunoreactivity of the 8 kDa fragment versus four α-PrP antibodies. PrP248 recognizes the octarepeat region (residues 55–96) of PrP and fails to detect the 8 kDa fragment. 12B2 (residues 88–92) is reactive with the 8 kDa fragment. Sha31 (residues 145–152) recognizes the 8 kDa fragment. VRQ61 recognizes the β2-α2 loop (residues 165–175) of PrP but is not immunoreactive with the 8 kDa fragment. Using these epitopes as minimal termini, the fragment maps to residues 88–155 using the numbering scheme of WT mouse PrP, which corresponds in turn to a molecular weight of ~7.2 kDa. D) Presence of protease-resistant PrP in Tg.HRdup-10 mice but not in aged Tg.M128V-39 mice. "H", healthy"; "S", sick. At the terminal stage of disease, HRdup prions are resistant to digestion by 10 μg/ml PK. E) Presence of the 8 kDa fragment in low-expresser Tg.HRdup-10 mice is hastened by inoculum with brain homogenate from a Tg.HRdup-26 mouse. Low expressing Tg.HRdup-10 animals and <i>tga20</i> (overexpressing WT PrP ~6x) animals received an intracranial inoculation with brain homogenate from a sick Tg.HRdup-26 animal. Tg.HRdup-10 animals inoculated with healthy brain homogenate were euthanized after 400 days. Tg.HRdup-10 animals inoculated with brain homogenate from a sick Tg.HRdup-26 animal succumbed to disease at 276±38 days. Brain homogenates from inoculated animals either undigested or exposed to PK is presented. Note that only Tg.HRdup-10 animals inoculated with Tg.HRdup-26 brain homogenate display the 8 kDa PK resistant fragment (black arrow) versus genotype-matched controls or animals treated with control inoculum (right four lanes). Analyses in A, B, D and E were performed with Sha31 antibody.