Lethal recessive myelin toxicity of prion protein lacking its central domain

Abstract

PrP(C)-deficient mice expressing prion protein variants with large amino-proximal deletions (termed PrP(DeltaF)) suffer from neurodegeneration, which is rescued by full-length PrP(C). We now report that expression of PrP(DeltaCD), a PrP variant lacking 40 central residues (94-134), induces a rapidly progressive, lethal phenotype with extensive central and peripheral myelin degeneration. This phenotype was rescued dose-dependently by coexpression of full-length PrP(C) or PrP(C) lacking all octarepeats. Expression of a PrP(C) variant lacking eight residues (114-121) was innocuous in the presence or absence of full-length PrP(C), yet enhanced the toxicity of PrP(DeltaCD) and diminished that of PrP(DeltaF). Therefore, deletion of the entire central domain generates a strong recessive-negative mutant of PrP(C), whereas removal of residues 114-121 creates a partial agonist with context-dependent action. These findings suggest that myelin integrity is maintained by a constitutively active neurotrophic protein complex involving PrP(C), whose effector domain encompasses residues 94-134.

Figure 1

(A) Hydrophobicity plot of the full-length murine prion protein. SP: secretory signal peptide, cleaved after sorting of the precursor to endoplasmic reticulum; repeats: five repeats of eight amino acids; CC: charge cluster; HC: hydrophobic core; CD: central domain; H1, H2, H3: α-helices 1, 2, 3 of the globular carboxy-proximal domain; MA: membrane anchor of precursor protein, replaced during maturation with glycosyl phosphatidyl inositol anchor. (B) Scheme of wild-type prion protein (WT PrP) and deletion mutants utilized for generation of transgenic mice. Antibody POM1 recognizes a carboxy-proximal epitope, whereas POM11 binds to octarepeats, and POM3 binds to the charge cluster. (C) Southern blot analysis of PrP_ΔCD (lines Tg1046, Tg1047, and Tg1050) and PrP_ΔpHC transgenic mice (line Tg902). Genomic DNA was digested with EcoRI before loading. PrP_ΔCD transgenic band: 2916 bp; PrP_pHC: 3015 bp; Prnp^o: 2747 bp; Prnp⁺: 2148 bp. The lanes denoted phgΔCD and MoPrP-ΔH1 contain the plasmid constructs used for pronuclear injection. Numbers below the respective lanes indicate the numbers of transgenic copies per haploid genome, as determined by quantitation of ³²P Southern blotting signals against the respective Prnp⁺ genomic band. Line Tg1050 contained the largest number of transgenic copies. (D) Transgenic PrP transcription. RNA was isolated from PrP^wt, PrPΔ_CD^+/o (Tg1047), and PrP_ΔpHC^o/o (Tg902) brains, reverse-transcribed, and PrP-containing cDNA was amplified with specific primers. Wild-type brain and original plasmid served as molecular weight standards. DNase-treated, non-retrotranscribed samples were loaded for control.

Figure 2

Expression of transgenic proteins. (A) Similar glycosylation patterns of full-length PrP, PrP_ΔCD, and PrP_ΔpHC. Brain homogenates were subjected to PNGase F treatment as indicated and analyzed by Western blotting using anti-PrP antibody POM1 (Polymenidou et al, 2005). Arrowhead: C1 fragment. (B) Density gradient analysis of detergent-resistant membrane (DRM) preparations from the brains of wild-type, PrP_ΔCD, and PrP_ΔpHC transgenic mice. Fractions 4–16 of the step gradient were analyzed by Western blotting. Full-length PrP, PrP_ΔCD, and PrP_ΔpHC were detectable to a similar extent in the upper fractions, indicating that they all localize to DRMs similar to flotillin (48 kDa). Non-DRM contaminations were controlled for by probing blots with antibodies to GAPDH (lowest panel, 35 kDa). Non-buoyant PrP may indicate raft disruption or may represent immature protein fractions. (C, D) Expression levels of PrP_ΔCD and PrP_ΔpHC. Total brain homogenates were digested with PNGase F treatment and analyzed using the monoclonal antibody POM11 (^*PrP^wt band. ^**PrP_ΔCD band). (D) Densitometric quantification of protein expression after Western blotting. Each column represents the average of at least three individual mice from each transgenic strain coexpressing wild-type PrP from one haploid genomic allele. (E–S) POM1-immunostained brain sections. (E–I) Overview; (J–N) higher magnification of hippocampus and corpus callosum; (O–S), cerebellum. Similar spatial expression patterns for PrP^C and its variants in wild-type, PrP_ΔCD , PrP_ΔpHC, and PrP_ΔF mice. No immunoreactivity was visible in Prnp^o/o mice. Scale bar: (E–I) 10 mm; (J–N) 100 μm; (O–S) 10 μm.

Figure 3

(A–D) Survival of PrP_ΔCD mice. Longevity was dependent on their Prnp status, with Prnp^+/+ transgenic mice surviving longer than Prnp^+/o mice and Prnp^o/o mice showing the shortest survival times. Each line represents ⩾5 individuals. (E) Typical clinical phenotypes at terminal stage: kyphosis (left), ataxia (middle), and clasping when held head-down at the tail (right). See also Supplement movie online. (F) Several litters from a Tg1047 breeding pair were monitored for their weight. Transgenic mice (PrP_ΔCD^+/o) were constantly underweight as compared to their non-transgenic littermates (PrP^+/o). (G) The ratio between the expression of PrP_ΔCD and full-length PrP^C determines the life expectancy of mice. The black bars depict the ratios of steady-state level PrP_ΔCD/PrP^wt, as determined by quantitative Western blot analysis of brain homogenates of at least four transgene-positive individuals of the indicated line. The gray bars represent the mean life expectancy of ⩾4 transgenic individuals of each line.

Figure 4

White matter pathology and peripheral neuropathy in terminally sick PrP_ΔCD mice. Age-matched non-transgenic Prnp^+/+ littermates served as controls for all histological investigations. GFAP-stained pyramidal tract showed vacuolar degeneration and associated astrogliosis in a sick PrP_ΔCD mouse (B) but not in a control mouse (A). GFAP-stained cerebellum in PrP_ΔCD mice revealed moderate astrogliosis but preserved cerebellar granule cell layer (D). The same region in an age-matched control littermate (C). Luxol-Nissl-stained cross-section through the mid-thoracic spinal cord showed coarse vacuolar degeneration in descending and ascending fiber tracts (E, G). No such changes were observed in control mice (F). Paraphenylene diamine-stained semithin section of a descending myelinated fiber tract in a PrP_ΔCD mouse revealed axonal loss together with some enlarged ‘vacuolated' myelinated fibers with thinning of their myelin sheaths (asterisk) (I). Age-matched control littermate (H). Luxol-Nissl-stained longitudinal section through the sciatic nerve of a control (J) and a PrP_ΔCD mouse (K). Axonal breakdown and segmentation of myelin into digestion chambers (arrows). Axonal loss, hypomyelinated fibers, and degeneration of myelinated fibers with collapsed myelin sheaths (arrow) were observed in paraphenylene diamine-stained semithin sections of the sciatic nerve in PrP_ΔCD mice (M) but not in control mice (L). Electron microscopic analysis of the sciatic nerve showed demyelinated fibers (N) associated with macrophages filled with myelin debris (O). Some fibers showed collapsed axons surrounded by abnormal condensed multilamellar myelin sheaths (P). Normal myelinated fibers in a control mouse (Q). Scale bar: (A–E) 100 μm; (F, G, J, K) 20 μm; (H, I, L, M) 10 μm; (N–Q) 2 μm.

Figure 5

Survival of compound transgenic mice. (A–D) Survival curves of mice expressing PrP_ΔCD (Tg1046), PrP_ΔF (TgF35), or both, in the absence or presence of full-length PrP^C. Each line comprises ⩾5 individuals. (B) Mice expressing PrP_ΔCD (Tg1047) or PrP_ΔC (TgC4), or both, in the presence of one wild-type Prnp allele. Each line comprises ⩾7 individuals. (C) Mice expressing PrP_ΔpHC (Tg902), PrP_ΔF (TgF35), or both, in the absence or presence of PrP^C. Each line represents ⩾3 individual mice. (D) Mice expressing PrP_ΔCD (Tg1047) or PrP_ΔpHC (Tg902), or both, in the presence of one wild-type Prnp allele. Each line represents ⩾7 individuals. (E–G) Brain expression of PrP^C and transgenic PrP. (E) PrP_ΔF has a higher steady-state level than PrP^wt and PrP_ΔCD. Brain homogenates were optionally treated with PNGase F and Western blots were decorated with the antibody POM1. The arrows refer to the molecular weight of each protein after deglycosylation. (F) PrP_ΔC expression is stronger than that of PrP^wt and PrP_ΔCD. Brain homogenates were treated with PNGase F and Western blots were decorated with antibodies POM1, POM3, and POM11 recognizing different domains of PrP. (G) PrP_ΔpHC expression is lower than that of PrP^C and PrP_ΔF, but similar to that of some affected PrP_ΔCD lines. All homogenates were treated with PNGase F.

Figure 6

A model for the positive and negative effects of PrP^C and its variants. (A) PrP (black) consists of a globular C-terminal domain (hexagon) and an N-terminal flexible tail (arch) encompassing the ORs (circle). The model rests on the following assumptions: (1) PrP^C activates a hitherto unidentified receptor (PrP_R), which transmits myelin maintenance signals (flashes); (2) in the absence of PrP^C, PrP_R exerts some residual activity, either constitutively or by recruiting a surrogate ligand; (3) the activity of PrP^C and its mutants requires homo- or heterodimerization, and induces dimerization of PrP_R; and (4) PrP dimers containing PrP_ΔCD or PrP_ΔCD trap PrP_R in an inactive dominant-negative state. Finally, (5) the OR region stabilizes the interaction between PrP and PrP_R, but does not contribute directly to signaling. (B) By increasing complex affinity to PrP_R through its ORs, PrP_ΔpHC may displace PrP_ΔF homodimers, thereby ameliorating the phenotype of compound PrP_ΔFΔpHC transgenic mice. However, as PrP_ΔCD also contains ORs, it may more efficiently compete with PrP_ΔpHC for PrP_R and thus prevents any phenotypic improvement.