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Detection of prion seeding activity in the olfactory mucosa of patients with Fatal Familial Insomnia

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ABSTRACT

Fatal Familial Insomnia (FFI) is a genetic prion disease caused by a point mutation in the prion protein gene (PRNP) characterized by prominent thalamic atrophy, diffuse astrogliosis and moderate deposition of PrPSc in the brain. Here, for the first time, we demonstrate that the olfactory mucosa (OM) of patients with FFI contains trace amount of PrPSc detectable by PMCA and RT-QuIC. Quantitative PMCA analysis estimated a PrPSc concentration of about 1 × 10−14 g/ml. In contrast, PrPSc was not detected in OM samples from healthy controls and patients affected by other neurodegenerative disorders, including Alzheimer’s disease, Parkinson’s disease and frontotemporal dementia. These results indicate that the detection limit of these assays is in the order of a single PrPSc oligomer/molecule with a specificity of 100%.

No MeSH data available.


Serial dilutions of FFI brain homogenate (D178N/129M) were analyzed by means of PMCA and RT-QuIC for estimating their ability to detect PrPSc.(A) Ten % bank vole brain homogenates (BvBH) were spiked with serial dilution of FFI brain homogenate (from 10−5 to 10−12). The signal of PrPSc was assessed by means of Western blotting, after PK digestion, with the 6D11 antibody. Mw refers to the molecular weight. PrPC refers to normal bank vole brain homogenate not digested with PK. (B) Serial dilutions of FFI brain homogenate were analyzed by means of RT-QuIC reactions using BvPrP (90-231) as substrate. Average ThT fluorescence were plotted against time. AD brain homogenate (dilution 10−5) was used as negative control. BvPrP refers to unseeded recombinant protein.
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f1: Serial dilutions of FFI brain homogenate (D178N/129M) were analyzed by means of PMCA and RT-QuIC for estimating their ability to detect PrPSc.(A) Ten % bank vole brain homogenates (BvBH) were spiked with serial dilution of FFI brain homogenate (from 10−5 to 10−12). The signal of PrPSc was assessed by means of Western blotting, after PK digestion, with the 6D11 antibody. Mw refers to the molecular weight. PrPC refers to normal bank vole brain homogenate not digested with PK. (B) Serial dilutions of FFI brain homogenate were analyzed by means of RT-QuIC reactions using BvPrP (90-231) as substrate. Average ThT fluorescence were plotted against time. AD brain homogenate (dilution 10−5) was used as negative control. BvPrP refers to unseeded recombinant protein.

Mentions: To assess whether PrPSc associated to FFI (FFI-PrPSc) was able to amplify by means of PMCA, we have performed spiking experiment diluting FFI brain homogenate (from 10−5 to 10−12 dilution of the brain) into 10% healthy bank vole brain homogenates (BvBH) carrying methionine at position 109 of the prion protein (M109). After one round of amplification, we could efficiently detect up to a 10−9 dilution while, after two rounds, we were able to amplify all dilutions (Fig. 1A). Similarly, dilutions of FFI brain homogenate were prepared in PBS and analyzed by means of RT-QuIC. The experiments were carried out using recombinant truncated form of the bank vole PrP (BvPrP(90-231)) as substrate and were seeded in triplicate with 2 μL of each dilution (Fig. 1B). All reactions were performed at least three times by different operators. We could detect up to 10−9 dilution of PrPSc, while we did not efficiently detect any signal from higher dilutions. In particular, lower dilutions of brain homogenate induced a faster aggregation of BvPrP(90-231) (at around 8 hours), while intermediate dilutions induced aggregation after 14 hours. A sample was considered positive when the mean of the highest two fluorescence values (AU) of the replicates was higher than 10.000 AU and at least two out of three replicates crossed this value before 30 hours (see materials and methods for details). Samples that did not cross the threshold before 30 hours were considered negative. Brain homogenates of patient with AD and FTD were used as control and induced aggregation of BvPrP(90-231) after 30 hours, thus were considered negative (Fig. 1B).


Detection of prion seeding activity in the olfactory mucosa of patients with Fatal Familial Insomnia
Serial dilutions of FFI brain homogenate (D178N/129M) were analyzed by means of PMCA and RT-QuIC for estimating their ability to detect PrPSc.(A) Ten % bank vole brain homogenates (BvBH) were spiked with serial dilution of FFI brain homogenate (from 10−5 to 10−12). The signal of PrPSc was assessed by means of Western blotting, after PK digestion, with the 6D11 antibody. Mw refers to the molecular weight. PrPC refers to normal bank vole brain homogenate not digested with PK. (B) Serial dilutions of FFI brain homogenate were analyzed by means of RT-QuIC reactions using BvPrP (90-231) as substrate. Average ThT fluorescence were plotted against time. AD brain homogenate (dilution 10−5) was used as negative control. BvPrP refers to unseeded recombinant protein.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5384244&req=5

f1: Serial dilutions of FFI brain homogenate (D178N/129M) were analyzed by means of PMCA and RT-QuIC for estimating their ability to detect PrPSc.(A) Ten % bank vole brain homogenates (BvBH) were spiked with serial dilution of FFI brain homogenate (from 10−5 to 10−12). The signal of PrPSc was assessed by means of Western blotting, after PK digestion, with the 6D11 antibody. Mw refers to the molecular weight. PrPC refers to normal bank vole brain homogenate not digested with PK. (B) Serial dilutions of FFI brain homogenate were analyzed by means of RT-QuIC reactions using BvPrP (90-231) as substrate. Average ThT fluorescence were plotted against time. AD brain homogenate (dilution 10−5) was used as negative control. BvPrP refers to unseeded recombinant protein.
Mentions: To assess whether PrPSc associated to FFI (FFI-PrPSc) was able to amplify by means of PMCA, we have performed spiking experiment diluting FFI brain homogenate (from 10−5 to 10−12 dilution of the brain) into 10% healthy bank vole brain homogenates (BvBH) carrying methionine at position 109 of the prion protein (M109). After one round of amplification, we could efficiently detect up to a 10−9 dilution while, after two rounds, we were able to amplify all dilutions (Fig. 1A). Similarly, dilutions of FFI brain homogenate were prepared in PBS and analyzed by means of RT-QuIC. The experiments were carried out using recombinant truncated form of the bank vole PrP (BvPrP(90-231)) as substrate and were seeded in triplicate with 2 μL of each dilution (Fig. 1B). All reactions were performed at least three times by different operators. We could detect up to 10−9 dilution of PrPSc, while we did not efficiently detect any signal from higher dilutions. In particular, lower dilutions of brain homogenate induced a faster aggregation of BvPrP(90-231) (at around 8 hours), while intermediate dilutions induced aggregation after 14 hours. A sample was considered positive when the mean of the highest two fluorescence values (AU) of the replicates was higher than 10.000 AU and at least two out of three replicates crossed this value before 30 hours (see materials and methods for details). Samples that did not cross the threshold before 30 hours were considered negative. Brain homogenates of patient with AD and FTD were used as control and induced aggregation of BvPrP(90-231) after 30 hours, thus were considered negative (Fig. 1B).

View Article: PubMed Central - PubMed

ABSTRACT

Fatal Familial Insomnia (FFI) is a genetic prion disease caused by a point mutation in the prion protein gene (PRNP) characterized by prominent thalamic atrophy, diffuse astrogliosis and moderate deposition of PrPSc in the brain. Here, for the first time, we demonstrate that the olfactory mucosa (OM) of patients with FFI contains trace amount of PrPSc detectable by PMCA and RT-QuIC. Quantitative PMCA analysis estimated a PrPSc concentration of about 1 × 10−14 g/ml. In contrast, PrPSc was not detected in OM samples from healthy controls and patients affected by other neurodegenerative disorders, including Alzheimer’s disease, Parkinson’s disease and frontotemporal dementia. These results indicate that the detection limit of these assays is in the order of a single PrPSc oligomer/molecule with a specificity of 100%.

No MeSH data available.