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

Abstract

The structurally disordered N-terminal half of the prion protein (PrP C) is constitutively released into the extracellular space by an endogenous proteolytic cleavage event. Once liberated, this N1 fragment acts neuroprotective in ischemic conditions and interferes with toxic peptides associated with neurodegenerative diseases, such as amyloid-beta (Aβ) in Alzheimer's disease. Since analog protective effects of N1 in prion diseases, such as Creutzfeldt-Jakob disease, have not been studied, and given that the protease releasing N1 has not been identified to date, we have generated and characterized transgenic mice overexpressing N1 (TgN1). Upon intracerebral inoculation of TgN1 mice with prions, no protective effects were observed at the levels of survival, clinical course, neuropathological, or molecular assessment. Likewise, primary neurons of these mice did not show protection against Aβ toxicity. Our biochemical and morphological analyses revealed that this lack of protective effects is seemingly due to an impaired ER translocation of the disordered N1 resulting in its cytosolic retention with an uncleaved signal peptide. Thus, TgN1 mice represent the first animal model to prove the inefficient ER translocation of intrinsically disordered domains (IDD). In contrast to earlier studies, our data challenge roles of cytoplasmic N1 as a cell penetrating peptide or as a potent "anti-prion" agent. Lastly, our study highlights both the importance of structured domains in the nascent chain for proteins to be translocated and aspects to be considered when devising novel N1-based therapeutic approaches against neurodegenerative diseases.

Structural details of amyloid β oligomers in complex with human prion protein as revealed by solid-state MAS NMR spectroscopy

“…This article contains supporting information ( 13 , 19 , 24 , 25 , 26 , 27 , 32 , 33 , 34 , 40 , 41 , 47 , 48 , 53 , 54 , 55 , 75 , 76 , 77 , 78 , 95 , 96 , 97 , 98 , 99 ).…”

“…S1 ), but this has also been questioned ( 28 , 29 , 30 , 31 ). It has also been described that soluble PrP ( 32 ) and its N-terminal fragment PrP(23–111) ( 33 , 34 ) have a protective role by inhibiting Aβ fibrillation and sequestration of Aβ oligo .…”

G392E neuroserpin causing the dementia FENIB is secreted from cells but is not synaptotoxic

“…These results support the notion that neuronal death observed in FENIB is rather caused by intracellular stress and toxicity pathways activated by the accumulation of neuroserpin polymers within the ER, as reported in a neuronal model in vitro where overexpression of G392E-mutant neuroserpin led to an increase in oxidative stress 18 . Previous work from our group and others has reported synaptotoxicity on cultured neurons upon treatment with Aβ or prion protein 35 , 36 , so we evaluated the effects of the treatments described above on synaptic phenotype. Despite using an approach that has proved successful for other protein aggregates, we did not observe any changes in any synaptic parameter tested.…”

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

“…Murine neuroblastoma cells (N2a; ACC148, DSMZ Germany) and the embryonic mouse hippocampal cell line mHippoE-14 (CLU198; CELLutions Biosystems Inc.) were cultured at 37°C in an atmosphere of 5% CO 2 in Dulbecco’s modified Eagle’s medium (DMEM; Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (FBS; Thermo Fisher Scientific). Generation of PrP-depleted N2a cells ( Prnp KO) was described elsewhere [135]. For overexpression of murine wild-type (PrP-WT) or 3F4-tagged PrP (PrP-3F4), these PrP knockout cells were transiently transfected with the respective constructs [29] using Lipofectamine 2000 (Thermo Fisher Scientific) following the manufacturer’s instructions.…”