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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%.

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Amplified OM-PrPSc comparison with that of FFI or sCJD-type 1 and sCJD-type 2 brain homogenates.(A) Western blot of the final products of RT-QuIC reaction seeded with brain homogenates or olfactory mucosa from sCJD, iCJD, FFI, AD, PD patients and healthy controls (HC). Samples were digested with PK [50 μg/mL] and immunoblot was probed with the C-terminal antibody SAF-84. Number in the right indicates the position of Mw. (B) Biochemical profile of amplified products (both brain homogenates and olfactory mucosa) was compared to that of FFI, sCJD-type 1 and sCJD-type 2 brain homogenates assessed before and after PMCA. FFI brain homogenate was concentrated 20-fold for PrPSc detection. The signal of PrPSc was assessed by means of Western blotting, after PK digestion, with the 6D11 antibody. Numbers in the right indicate the position of Mw: 21 kDa refers to type 1 PrPSc, while 19 kDa refers to type 2 PrPSc.
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f3: Amplified OM-PrPSc comparison with that of FFI or sCJD-type 1 and sCJD-type 2 brain homogenates.(A) Western blot of the final products of RT-QuIC reaction seeded with brain homogenates or olfactory mucosa from sCJD, iCJD, FFI, AD, PD patients and healthy controls (HC). Samples were digested with PK [50 μg/mL] and immunoblot was probed with the C-terminal antibody SAF-84. Number in the right indicates the position of Mw. (B) Biochemical profile of amplified products (both brain homogenates and olfactory mucosa) was compared to that of FFI, sCJD-type 1 and sCJD-type 2 brain homogenates assessed before and after PMCA. FFI brain homogenate was concentrated 20-fold for PrPSc detection. The signal of PrPSc was assessed by means of Western blotting, after PK digestion, with the 6D11 antibody. Numbers in the right indicate the position of Mw: 21 kDa refers to type 1 PrPSc, while 19 kDa refers to type 2 PrPSc.

Mentions: OM samples (n = 28) collected from patients with FFI (n = 2), AD (n = 6), PD (n = 6), FTD (n = 4) and HC (n = 10) were blindly analyzed by means of PMCA and RT-QuIC. After three rounds of PMCA, we could detect PrPSc only in FFI-OM samples (Fig. 2A). All the other samples remained negative even after 7 rounds of amplification (Supplementary Fig. 1). Similarly, through RT-QuIC analysis we could detect prion seeding activity in both FFI-OM samples, while PrPSc was never detected in any of the OM belonging to the other patients analyzed (Fig. 2B). The final RT-QuIC reaction products were collected (at 30 hours) digested with PK and analyzed by means of Western blotting. As previously observed24, seeds collected from OM of patients with FFI, sporadic or iatrogenic forms of CJD were able to produce final RT-QuIC aggregates partially resistant to PK digestion and characterized by a molecular weight ranging from 14 to 18 kDa (Fig. 3A). On the contrary, OM samples collected from patients with PD, AD or healthy subjects did not produce any PK resistant aggregate. Brain homogenate of patients with FFI and iCJD were used as positive controls.


Detection of prion seeding activity in the olfactory mucosa of patients with Fatal Familial Insomnia
Amplified OM-PrPSc comparison with that of FFI or sCJD-type 1 and sCJD-type 2 brain homogenates.(A) Western blot of the final products of RT-QuIC reaction seeded with brain homogenates or olfactory mucosa from sCJD, iCJD, FFI, AD, PD patients and healthy controls (HC). Samples were digested with PK [50 μg/mL] and immunoblot was probed with the C-terminal antibody SAF-84. Number in the right indicates the position of Mw. (B) Biochemical profile of amplified products (both brain homogenates and olfactory mucosa) was compared to that of FFI, sCJD-type 1 and sCJD-type 2 brain homogenates assessed before and after PMCA. FFI brain homogenate was concentrated 20-fold for PrPSc detection. The signal of PrPSc was assessed by means of Western blotting, after PK digestion, with the 6D11 antibody. Numbers in the right indicate the position of Mw: 21 kDa refers to type 1 PrPSc, while 19 kDa refers to type 2 PrPSc.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC5384244&req=5

f3: Amplified OM-PrPSc comparison with that of FFI or sCJD-type 1 and sCJD-type 2 brain homogenates.(A) Western blot of the final products of RT-QuIC reaction seeded with brain homogenates or olfactory mucosa from sCJD, iCJD, FFI, AD, PD patients and healthy controls (HC). Samples were digested with PK [50 μg/mL] and immunoblot was probed with the C-terminal antibody SAF-84. Number in the right indicates the position of Mw. (B) Biochemical profile of amplified products (both brain homogenates and olfactory mucosa) was compared to that of FFI, sCJD-type 1 and sCJD-type 2 brain homogenates assessed before and after PMCA. FFI brain homogenate was concentrated 20-fold for PrPSc detection. The signal of PrPSc was assessed by means of Western blotting, after PK digestion, with the 6D11 antibody. Numbers in the right indicate the position of Mw: 21 kDa refers to type 1 PrPSc, while 19 kDa refers to type 2 PrPSc.
Mentions: OM samples (n = 28) collected from patients with FFI (n = 2), AD (n = 6), PD (n = 6), FTD (n = 4) and HC (n = 10) were blindly analyzed by means of PMCA and RT-QuIC. After three rounds of PMCA, we could detect PrPSc only in FFI-OM samples (Fig. 2A). All the other samples remained negative even after 7 rounds of amplification (Supplementary Fig. 1). Similarly, through RT-QuIC analysis we could detect prion seeding activity in both FFI-OM samples, while PrPSc was never detected in any of the OM belonging to the other patients analyzed (Fig. 2B). The final RT-QuIC reaction products were collected (at 30 hours) digested with PK and analyzed by means of Western blotting. As previously observed24, seeds collected from OM of patients with FFI, sporadic or iatrogenic forms of CJD were able to produce final RT-QuIC aggregates partially resistant to PK digestion and characterized by a molecular weight ranging from 14 to 18 kDa (Fig. 3A). On the contrary, OM samples collected from patients with PD, AD or healthy subjects did not produce any PK resistant aggregate. Brain homogenate of patients with FFI and iCJD were used as positive controls.

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.


Related in: MedlinePlus