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. 2020 Jun;57(6):2812-2829.
doi: 10.1007/s12035-020-01917-2. Epub 2020 May 4.

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

Behnam Mohammadi  1 , Luise Linsenmeier  1 , Mohsin Shafiq  1 , Berta Puig  2 , Giovanna Galliciotti  1 , Camilla Giudici  3 , Michael Willem  3 , Thomas Eden  4 , Friedrich Koch-Nolte  4 , Yu-Hsuan Lin  5 , Jörg Tatzelt  5 , Markus Glatzel  1 , Hermann C Altmeppen  6
Affiliations

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

Behnam Mohammadi et al. Mol Neurobiol. 2020 Jun.

Abstract

The structurally disordered N-terminal half of the prion protein (PrPC) 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.

Keywords: Cell-penetrating peptide; Cytosolic prions; Intrinsically disordered domains; Prion disease; Proteasome; Translocation.

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Figures

Fig. 1
Fig. 1
Upper panel: schematic representation of the prion protein. The cellular prion protein (PrPC) is composed of two structurally different parts. The rather globular shaped C-terminal half contains three α-helices, a short β-sheet, a disulfide bridge (not shown), up to two N-glycans and a GPI-anchor for attachment to the outer leaflet of the plasma membrane. The N-terminal half is largely unstructured and qualifies as an intrinsically disordered domain (IDD). However, this part contains important binding sites for interaction with various molecules, including toxic protein oligomers and proteopathic seeds (red) found in neurodegenerative diseases. Binding of the latter to membrane-bound PrPC induces neurotoxic signaling events and may lead to pore formation via insertion of the N-terminus into the membrane. A conserved proteolytic cleavage event termed α-cleavage (blue scissors) at H110 separates the two dissimilar halves of PrPC and releases the disordered N1 fragment into the extracellular space, where it exerts neuroprotective effects. Note that the N-terminal ER targeting signal peptide (SP; dotted gray stretch) is not part of mature PrPC under physiological conditions. Lower panel: linear representation of the PrPC sequence accentuating (i) known (proteolytic) cleavage events, (ii) resulting N- and C-terminal fragments (respective expected molecular weights), and (iii) (approximated) position of epitopes described for PrPC-directed antibodies (black) used in this study. Note that antibody 6D11 can be instrumental to differentiate between N-terminal fragments resulting from the α- (detection of N1) and β-cleavage (no detection of N2). GPI = position of GPI-anchor signal sequence
Fig. 2
Fig. 2
Characterization of TgN1 mice. Body size (a) and body weight measurements (b; n = 3) reveal no significant differences between TgN1 and the age-matched WT littermates. c, d RT-qPCR analysis in mouse brain homogenates showing higher mRNA expression levels in TgN1 compared to WT controls in cerebellum (c; n = 3; p = 0.031) and forebrain (d; n = 3; p = 0.028). (e, f) Western blot analyses of forebrain samples of e 8-week-old (n = 4; p = 0.00001) and f 43-week-old mice (n = 4; p = 0.0047) (measured as ratio of N1 fragment to corresponding fl-PrP; both detected by POM2 antibody) reveal a double band (arrowheads; upper band indicated by a “?”) and a strong increase in the levels of N1 fragments in TgN1 mice (note that in the case of TgN1 the double band was measured as “N1”). Taken together, these data demonstrate successful generation of transgenic mice overexpressing N1. In addition, a fragment of ~ 20 kDa, possibly representing the endogenous N3 fragment resulting from the γ-cleavage of PrPC, is readily detected in brain homogenates
Fig. 3
Fig. 3
Lack of overt morphological alterations in young TgN1 mice. a, b Neither H&E staining nor immunohistochemical detection of microglia (Iba1) and neurons (NeuN) revealed any alterations between eight-week-old WT and TgN1 mice in cortical (Cx; a) or cerebellar brain regions (Cb; b). c Likewise, neuronal density (assessed by neuronal marker NeuN) and amounts of proliferating cells (assessed by the marker Mib/Ki67) were similar between both genotypes in the hippocampus (Hc). Scale bars represent 100 μm
Fig. 4
Fig. 4
Intracerebral inoculation of TgN1 and control mice with prions. a Kaplan-Meier survival curve of mice upon intracerebral inoculation with mouse-adapted RML prions showing similar incubation times to terminal prion disease for TgN1 (n = 10) and WT littermates (n = 9), whereas the mock-inoculated control groups of each genotype (inoculated with CD1 brain homogenate) did not show any clinical signs until sacrification at 240 days post-inoculation (n = 4). b Western blot analysis of infected and non-infected mouse brain homogenates showing an altered glycopattern and presence of oligomeric PrPSc forms in prion-infected samples and increased total PrP levels (i.e., PrPC and PrPSc) in terminally diseased TgN1 mice (quantification in c; p = 0.0167; n = 3). d Western blot of PK-digested brain samples of terminally diseased mice of both genotypes (quantification was done by normalizing the PrPSc signals (POM1 antibody) against actin on the parallel blot with non-digested samples) (n = 4; p = 0.0167). Controls (on the left) include a non-digested RML-infected brain homogenate and a CD1-inoculated PK-digested brain sample. The shift in molecular weight and disappearance of the actin signal confirm successful enzymatic digestion. e, f Western blot analyses of candidate signaling pathways associated with prion disease showing e no differences in the phosphorylation state of Erk1/2, slightly elevated P-Akt and significantly increased activation of the P-Src in prion-infected mice (WT + CD1 vs. WT + RML: p = 0.027; WT + CD1 vs. TgN1 + RML: p = 0.003; n = 3) and f a significant increase in the activating phosphorylation of p38 in infected TgN1 samples compared to infected controls (p = 0.0032; n = 4)
Fig. 5
Fig. 5
Morphological assessment and Aβ42 treatment of primary neurons derived from TgN1 and control mice. a Representative images from primary neurons grown in a co-culture system conditioned with an astrocyte feeder layer for 2 weeks, indicating no difference in overall morphology and dendritic spines density (green dots) between TgN1 and WT neurons (quantification in c). SYP = synaptophysin (green); MAP2 = microtubule-associated protein 2 (red). b, c Treatment of neurons with synthetic Aβ42 resulted in a decrease in dendritic spine density compared to mock-treated controls (a, c); yet, no differences were found between treated neurons of both genotypes (b, c). Scale bar is 25 μm
Fig. 6
Fig. 6
Transgenically expressed N1 is not or only poorly secreted and rather retained with uncleaved SP in the cytosol. a Lysates and conditioned media of primary neurons (in mono-culture) from WT and TgN1 mice were biochemically analyzed for N1 and PrPC. While overexpression of N1 is confirmed in lysates of TgN1 neurons (note the double band), no increase (instead rather a tendency towards decrease (b)) in N1 is found in the respective media supernatants. Note that a band lower than N1 is detected in media which may represent “trimmed” N1 (see Suppl. Fig. 4a) or endogenous N2 resulting from the β-cleavage of PrPC. b Densitometric quantification of a; n = 3; levels of N1 signals were referred to levels of shed PrP found in media (a band possibly corresponding to N3 was detected in lysates but not in supernatants). c Primary neurons treated with an inhibitor of the proteasome (+MG132) or diluent only (+DMSO; as control) followed by Western blot analysis of lysates for levels of N1 fragments, PrPC, β-catenin, and β-actin (note that the low biostability of N1 may lead to fast degradation explaining the occasional need for longer exposure. As mentioned above, a weak band running lower than 10 kDa might reflect N2 or “trimmed” N1). Block of the proteasome results in a strong increase of an N1 fragment—most likely—N1-SP (here appearing as just one strong band) in the cytosol (as indicated in the scheme on the right)
Fig. 7
Fig. 7
Cytosolic retention of ectopically expressed N1. PrP-KO N2a cells were transfected with plasmids coding for either WT-PrP or N1 alone. Non-transfected cells served as negative controls. Immunfluorescent stainings on fixed and permeabilized cells revealed a predominant membrane-staining for WT-PrP with both PrP-directed antibodies (POM2 and POM1, red), whereas cells transfected with N1 showed a strong cytosolic staining pattern (note that POM1 does not detect N1; specificity control). GM130 (green) = Golgi marker. Scale bars represent 20 μm (upper panel) or 10 μm (lower two pictures)
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