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. 2017 Apr 21;5(1):32.
doi: 10.1186/s40478-017-0430-z.

Enhanced neuroinvasion by smaller, soluble prions

Cyrus Bett  1   2 , Jessica Lawrence  1 , Timothy D Kurt  1 , Christina Orru  3 , Patricia Aguilar-Calvo  1 , Anthony E Kincaid  4 , Witold K Surewicz  5 , Byron Caughey  3 , Chengbiao Wu  6 , Christina J Sigurdson  7   8
Affiliations

Enhanced neuroinvasion by smaller, soluble prions

Cyrus Bett et al. Acta Neuropathol Commun. .

Abstract

Infectious prion aggregates can propagate from extraneural sites into the brain with remarkable efficiency, likely transported via peripheral nerves. Yet not all prions spread into the brain, and the physical properties of a prion that is capable of transit within neurons remain unclear. We hypothesized that small, diffusible aggregates spread into the CNS via peripheral nerves. Here we used a structurally diverse panel of prion strains to analyze how the prion conformation impacts transit into the brain. Two prion strains form fibrils visible ultrastructurally in the brain in situ, whereas three strains form diffuse, subfibrillar prion deposits and no visible fibrils. The subfibrillar strains had significantly higher levels of soluble prion aggregates than the fibrillar strains. Primary neurons internalized both the subfibrillar and fibril-forming prion strains by macropinocytosis, and both strain types were transported from the axon terminal to the cell body in vitro. However in mice, only the predominantly soluble, subfibrillar prions, and not the fibrillar prions, were efficiently transported from the tongue to the brain. Sonicating a fibrillar prion strain increased the solubility and enabled prions to spread into the brain in mice, as evident by a 40% increase in the attack rate, indicating that an increase in smaller particles enhances prion neuroinvasion. Our data suggest that the small, highly soluble prion particles have a higher capacity for transport via nerves. These findings help explain how prions that predominantly assemble into subfibrillar states can more effectively traverse into and out of the CNS, and suggest that promoting fibril assembly may slow the neuron-to-neuron spread of protein aggregates.

Keywords: Amyloid; Axonal transport; Fibrils; Neurodegeneration; Prion disease; Prion strains.

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Figures

Fig. 1
Fig. 1
Fibrillar prion strains rarely neuroinvade following intra-tongue inoculation. a Schematic of time course experiment following IT exposure. T1, and T2: 0.50 and 0.75 of the expected incubation period; T3: terminal disease (1.0). b Paraffin-embedded tissue (PET) blot of brainstem (T2 timepoint) shows subfibrillar prions in the facial, reticular, and deep cerebellar nuclei (strain RML, left panel, arrows), whereas fibrillar prions were not detected at any early timepoint (strain 87V, right panel). c Survival curves of WT or d tga20 mice (overexpress mouse PrPC) inoculated IT or IC with subfibrillar (blue - 22L, RML, ME7) or fibrillar (red - 87V, mCWD) prion strains. e Brain immunolabelled for PrP shows diffuse prion aggregates or large dense plaques in mice exposed to highly or poorly neuroinvasive prions, respectively. No plaques were detected in mice inoculated with mCWD IT (red box highlights brain sections from mice inoculated with mCWD by the IC versus IT route). Percentage of IT-inoculated mice that developed terminal prion disease is noted. Brain regions shown are as follows: cerebral cortex (IC: 22L); corpus callosum (IC: mCWD); thalamus (IC: 87V, RML, ME7; IT: 22L); hypothalamus (IT: ME7); brainstem (IT: 87V, RML, mCWD). f Immunoblots of brain from T1, T2, and T3 timepoints post IT inoculation. “p” and “n” refer to prion positive and negative brain samples (controls), and PK indicates proteinase K treatment. For IC inoculated mice, n = 4 (22L), 5 (87V, ME7, mCWD), or 6 (RML). For IT inoculated mice, n = 8 (22L), 9 (87V), 6 (RML), 4 (ME7), or 5 (mCWD). Scale bar = 100 μm
Fig. 2
Fig. 2
Subfibrillar and fibrillar prion strains are internalized and transported from the axon terminal to the soma. a Representative western blot shows subfibrillar (22L, RML) or fibrillar (87V, mCWD) prion internalization over time. Mean and SE from four (22L, 87V) or three (RML, mCWD) independent experiments. b Schematic of the microfluidic chamber in which the cells bodies reside in a large chamber and axons grow through fine grooves into side chambers where prions are introduced. c RT-QuIC analysis reveals PrPSc in the soma of neurons whose axons were exposed to subfibrillar (22L) or fibrillar (87V) prions, but not uninfected WT brain (WT). Shown are the mean and SE thioflavin T fluorescence signal for the positive cell body samples collected two weeks after prion exposure (4 of 8 positive samples per strain). The 87V prions were detected by RT-QuIC at slightly earlier times, which is not indicative of differences in the prion levels. Dashed line represents the threshold for positivity (see Methods)
Fig. 3
Fig. 3
Increasing PrPSc concentration or sonicating prions increases neuroinvasion of fibrillar prion (strain 87V). a Survival curves of mice inoculated with a 10-fold higher concentration of fibrillar 87V prions (high dose: solid red line) (10% IT: n = 13; 1% IT: n = 9; 1% IC: n = 5) (1% IC and 1% IT mice are the same shown in Fig. 1c). b Representative western blot of brain shows proteinase-K (PK) resistant PrPSc in 3 of 5 mice exposed IT to high dose 87V prions. c Representative images of recombinant PrP fibril ultrastructure show that sonication results in short homogenous fibrils (mean length of unsonicated = 524 nm versus sonicated = 222 nm; P < 0.0001, Student’s t test). d Solubility assay on sonicated and unsonicated fibrils shows that sonication increases the solubility of the fibrils. e Disaggregation assay. Western blots show the increase of PK-resistant 22L and 87V prions in the supernatant following sonication of prion-infected brain homogenate. Quantification shows the results of all strains. f Survival curve of mice inoculated with sonicated, low dose (1%) fibrillar 87V prions. n = 10 (1% sonic. IT), 9 (1% IT), 4 (1% sonic. IC). The 9 mice inoculated with 1% prions IT are also shown in panel a and Fig. 1c. g Representative western blot of brain from mice exposed IT to sonicated fibrillar 87V prions shows PK-resistant PrPSc in 3 of 5 mice. h PrPSc shows large dense plaques in the brains of mice (cerebral cortex) exposed IC or IT to sonicated 87V prions. “n”: mock-inoculated brain control. Scale bars = 500 nm (c) and 100 μm (h)
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