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. 2015 Oct;78(4):540-53.
doi: 10.1002/ana.24463. Epub 2015 Aug 25.

Modulation of Creutzfeldt-Jakob disease prion propagation by the A224V mutation

Joel C Watts  1   2 , Kurt Giles  1   2 , Ana Serban  1 , Smita Patel  1 , Abby Oehler  3 , Sumita Bhardwaj  1 , Shenheng Guan  1   4 , Michael D Greicius  5 , Bruce L Miller  6   2 , Stephen J DeArmond  1   3 , Michael D Geschwind  6   2 , Stanley B Prusiner  1   2   7
Affiliations

Modulation of Creutzfeldt-Jakob disease prion propagation by the A224V mutation

Joel C Watts et al. Ann Neurol. 2015 Oct.

Abstract

Objective: Mutations in the gene encoding the prion protein (PrP) are responsible for approximately 10 to 15% of cases of prion disease in humans, including Creutzfeldt-Jakob disease (CJD). Here, we report on the discovery of a previously unreported C-terminal PrP mutation (A224V) in a CJD patient exhibiting a disease similar to the rare VV1 subtype of sporadic (s) CJD and investigate the role of this mutation in prion replication and transmission.

Methods: We generated transgenic (Tg) mice expressing human PrP with the V129 polymorphism and A224V mutation, denoted Tg(HuPrP,V129,A224V) mice, and inoculated them with different subtypes of sCJD prions.

Results: Transmission of sCJD VV2 or MV2 prions was accelerated in Tg(HuPrP,V129,A224V) mice, compared to Tg(HuPrP,V129) mice, with incubation periods of ∼110 and ∼210 days, respectively. In contrast, sCJD MM1 prions resulted in longer incubation periods in Tg(HuPrP,V129,A224V) mice, compared to Tg(HuPrP,V129) mice (∼320 vs. ∼210 days). Prion strain fidelity was maintained in Tg(HuPrP,V129,A224V) mice inoculated with sCJD VV2 or MM1 prions, despite the altered replication kinetics.

Interpretation: Our results suggest that A224V is a risk factor for prion disease and modulates the transmission behavior of CJD prions in a strain-specific manner, arguing that residues near the C-terminus of PrP are important for controlling the kinetics of prion replication.

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Conflict of interest statement

Potential conflicts of interest

The Institute for Neurodegenerative Diseases has a research collaboration with Daiichi Sankyo (Tokyo, Japan).

Figures

Figure 1
Figure 1. Characterization of the CJD(A224V) patient’s brain
(A) 1.5T brain MRI with DWI sequences at 10.5 months following disease onset showing hyperintensity with reduced diffusion involving the cortex of the middle and superior frontal gyri, bilateral insula, bilateral hippocampi (solid arrows), bilateral cingulate gyri (black arrowheads), bilateral caudate and anterior putamen (dashed arrows), bilateral occipital (white arrowheads) and temporal cortex (dotted arrows). (B–I) Neuropathological analysis of the CJD(A224V) patient. Sections of the prefrontal cortex (B–E) and the cerebellar cortex (F–I) show features of severe CJD. (B, F) H&E stain reveals status spongiosis in cortical layers 1–3 and marked nerve cell loss in layers 2 and 3 (B) and spongiform degeneration of the molecular layer, Purkinje nerve cell loss, and at least 50% granule cell neuron loss (F). Arrow indicates a torpedo along the proximal portion of a Purkinje cell axon. (C, G) Immunostaining with 3F4 reveals dense finely granular staining for PrPSc with less intense staining in layer 1 (C) and abundant PrPSc deposition in the molecular layer and granule cell layer (G). (D, H) GFAP immunostaining shows severe reactive astrocytic gliosis in all layers (D), and reactive Bergman radial glia in the molecular layer and reactive astrocytes in the granule cell layer and underlying white matter (H). Arrow indicates a torpedo. (E) The Nissl stain verifies marked nerve cell loss in layers 2 and 3. (I) The Bielschowsky silver stain strongly stains a Purkinje cell torpedo (arrow). Bar in B represents 100 μm and applies to all photomicrographs. In B, numbers denote cerebral cortical layers. In F, regions of the cerebellar cortex are indicated: m, molecular layer; p, Purkinje cell layer; g, granule cell layer; w, white matter. (J) Immunoblot of brain homogenates from a control (non-neurodegenerative disease) patient and the CJD(A224V) patient with (+) or without (−) digestion with PK. (K) Immunoblot of PK-resistant PrPSc in the brain of the CJD(A224V) patient reveals a Type-1 signature (migration to ~21 kDa) similar to that observed in an sCJD VV1 patient. In J and K, PrP was detected using the antibody HuM-P. (L) Quantification of the relative amounts of wt (A224) and mutant (V224) PK-resistant PrPSc in the brain of the CJD(A224V) patient. Approximately equal proportions of wt and mutant PrPSc were found (no significant difference: P = 0.08 by the t-test). Data (mean ± SEM) from 4 technical replicates are shown.
Figure 2
Figure 2. Transmission of CJD(A224V) to transgenic mice
(A) Kaplan-Meier survival curves for Tg(HuPrP,V129)152 mice inoculated with brain homogenate from either the CJD(A224V) patient (solid black line, n = 7) or an sCJD VV1 patient (dashed black line, n = 7). The curves were not significantly different by the Log-rank test (P = 0.56). (B) Immunoblot of PK-resistant PrPSc in the brains of clinically ill Tg152 mice at the indicated days postinoculation (dpi) with either CJD(A224V) or sCJD VV1 brain homogenate. PrPSc profiles in the human brain homogenates used for inoculation (“Inoc.”) are shown for comparison. PK-resistant PrP was detected using the antibody HuM-P. (C–J) Neuropathological characterization of CJD-inoculated Tg152 mice. Brain sections from clinically ill mice at 313–337 dpi with either CJD(A224V) (C–F) or sCJD VV1 (G–J) brain homogenate were analyzed by H&E staining (C, D, G, and H) or by immunostaining for PrP (E, F, I, and J). Prominent spongiform degeneration was observed in the parietal cortex (C, G) and striatum (D, H), and abundant granular PrPSc deposition was observed in the granule cell layer of the cerebellum (E, I) as well as the thalamus (F, J). PrPSc deposits were visualized using the antibody 3F4. Scale bar in C represents 100 μm and also applies to panels D, G, and H; scale bar in E represents 50 μm and also applies to panels F, I, and J.
Figure 3
Figure 3. Tg(HuPrP,V129,A224V) mice do not develop spontaneous disease
(A) Immunoblot of PrP in undigested brain homogenates from wt FVB mice and four distinct lines of Tg(HuPrP,V129,A224V) mice. (B) Immunoblot of total PrP in undigested (−) brain homogenates or insoluble PrP following PK digestion (+) of brain homogenates from aged Tg(HuPrP,V129)152 and Tg(HuPrP,V129,A224V)8422 mice. Note that 100 times more material was loaded for the PK-digested samples. (C) Immunoblot of PK-resistant PrPSc in the brains of clinically ill Tg8422 mice at the indicated dpi with sCJD VV1 prions (left) or asymptomatic Tg18807 mice at the indicated days postinoculation (dpi) with CJD(A224V) prions (right). The PrPSc profiles in the human brain homogenates used for inoculation (“Inoc.”) are shown for comparison. In all panels, PrP was detected using the antibody HuM-P.
Figure 4
Figure 4. Accelerated transmission of sCJD VV2 and MV2 prions to Tg(HuPrP,V129,A224)8422 mice
(A) Kaplan-Meier survival curves for Tg152 mice (black lines) and Tg8422 mice (red lines) inoculated with sCJD VV2 case i (solid lines, n = 8 and 15, respectively), sCJD VV2 case ii (dashed lines, n = 8 each), or sCJD MV2 (dotted lines, n = 8 each) prions. The presence of the A224V mutation significantly accelerated the onset of neurologic illness in VV2- and MV2-inoculated mice (P < 0.001 by the Log-rank test). (B) Immunoblot of PK-resistant PrPSc in the brains of clinically ill Tg152 or Tg8422 mice injected with sCJD MM1 or sCJD VV2 (case i) prions (1st or 2nd passage) at the indicated days postinoculation (dpi). (C) Kaplan-Meier survival curves for Tg152 mice (black line, n = 7) and Tg8422 mice (red line; n = 7) inoculated with brain homogenate from an sCJD MM1 patient. The presence of the A224V mutation significantly delayed the onset of neurologic illness in MM1-inoculated mice (P < 0.001 by the Log-rank test). (D) Correlation of PrP expression levels and incubation periods following inoculation of Tg(HuPrP,V129,A224V) mice with sCJD MM1 prions (dotted line) or VV2 case i prions (solid line). As determined by linear regression, there was a direct relationship (positive slope) between PrP expression level and incubation period in MM1-inoculated mice and an inverse relationship (negative slope) in VV2-inoculated mice. (E) Immunoblot of PK-resistant PrPSc in the brains of clinically ill Tg152 or Tg8422 mice injected with sCJD VV2 (case ii) or sCJD MV2 prions at the indicated dpi. PrPSc profiles in the human brain homogenates used for inoculation (“Inoc.”) are shown for comparison. In panels B and E, PK-resistant PrP was detected using the antibody HuMP.
Figure 5
Figure 5. Passage of sCJD VV2 case i prions in Tg(HuPrP,V129,A224V) mice did not alter the prion strain properties
(A–C) Immunohistochemical detection of PrPSc deposition in the brains of sCJD VV2-inoculated Tg mice at the indicated days postinoculation (dpi). Plaque-like PrPSc deposits in the corpus callosum were observed in Tg152 mice (A) and Tg8422 mice (B, C) following one (A, B) or two (C) passages of sCJD VV2 prions. PrPSc deposits were detected using the antibody 3F4. Scale bar in A represents 50 μm and also applies to panels B and C. (D) Representative immunoblots and (E) denaturation curves (n = 3 brains per group) of residual PK-resistant PrPSc levels after conformational stability assays in brain homogenates from clinically ill Tg152 and Tg8422 mice inoculated with sCJD VV2 prions. No significant difference in the [GdnHCl]1/2 values was observed for sCJD VV2 prions upon propagation in Tg152 or Tg8422 mice (P = 0.57 by the Extra sum-of-squares F-test). (F) Kaplan-Meier survival curves for sCJD VV2-inoculated Tg152 (solid black line; n = 8) or Tg18807 (red line; n = 30) mice as well as Tg152 mice inoculated with sCJD VV2 prions that had been passaged once in Tg18807 mice (dashed black line; n = 8). There was no significant difference in the survival curves for the two groups of inoculated Tg152 mice (P = 0.15 by the Log-rank test). (G) Immunoblots of PK-resistant PrPSc in the brains of clinically ill Tg152 or Tg18807 mice infected with sCJD VV2 prions as well as Tg152 mice inoculated with sCJD VV2 prions passaged once in Tg18807 mice. The incubation period for each mouse is indicated as days postinoculation (dpi). The PrPSc profile in the sCJD VV2 human brain homogenate used for the original inoculation (“Inoc.”) is shown for comparison. In panels D and G, PrP was detected using the antibody HuM-P.
Figure 6
Figure 6. Replication of sCJD VV2 prions in bigenic mice coexpressing wt and mutant A224V HuPrP alleles
(A) Immunoblot of total PrP in undigested (−) brain homogenates or insoluble PrP following PK digestion (+) of brain homogenates from aged Tg8422/152 mice coexpressing mutant HuPrP(V129,A224V) and wt HuPrP(V129) and Tg8422/2669 mice coexpressing mutant HuPrP(V129,A224V) and wt HuPrP(M129). Note that 100 times more material was loaded for the PK-digested samples. (B) Kaplan-Meier survival curves for Tg8422/152 mice (red line; n = 7) and Tg8422/2669 mice (black line; n = 8) following inoculation with sCJD VV2 case i prions. The presence of wt HuPrP(M129) in trans significantly delayed the onset of neurologic illness in VV2-inoculated mice (P < 0.001 by the Log-rank test). (C) Immunoblot of PK-resistant PrPSc in the brains of clinically ill, sCJD VV2-inoculated Tg8422/152 and Tg8422/2669 mice at the indicated days postinoculation (dpi). PK-resistant PrPSc in the sCJD VV2 inoculum (“Inoc.”) is shown for comparison. In panels A and C, PrP was detected using the antibody HuM-P.
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