Real-time Quaking-induced Conversion Research Articles

Detection of Pathognomonic Biomarker PrPSc and the Contribution of Cell Free-Amplification Techniques to the Diagnosis of Prion Diseases.

Transmissible spongiform encephalopathies or prion diseases are rapidly progressive neurodegenerative diseases, the clinical manifestation of which can resemble other promptly evolving neurological maladies. Therefore, the unequivocal ante-mortem diagnosis is highly challenging and was only possible by histopathological and immunohistochemical analysis of the brain at necropsy. Although surrogate biomarkers of neurological damage have become invaluable to complement clinical data and provide more accurate diagnostics at early stages, other neurodegenerative diseases show similar alterations hindering the differential diagnosis. To solve that, the detection of the pathognomonic biomarker of disease, PrPSc, the aberrantly folded isoform of the prion protein, could be used. However, the amounts in easily accessible tissues or body fluids at pre-clinical or early clinical stages are extremely low for the standard detection methods. The solution comes from the recent development of in vitro prion propagation techniques, such as Protein Misfolding Cyclic Amplification (PMCA) and Real Time-Quaking Induced Conversion (RT-QuIC), which have been already applied to detect minute amounts of PrPSc in different matrixes and make early diagnosis of prion diseases feasible in a near future. Herein, the most relevant tissues and body fluids in which PrPSc has been detected in animals and humans are being reviewed, especially those in which cell-free prion propagation systems have been used with diagnostic purposes.

Detection of prions in skin punch biopsies of Creutzfeldt-Jakob disease patients.

Prion real‐time quaking‐induced conversion (RT‐QuIC) is an ultrasensitive assay detecting pathological aggregates of misfolded prion protein in biospecimens. We studied 71 punch biopsy skin samples of 35 patients with Creutzfeldt–Jakob disease (CJD), including five assessed in vitam. The results confirmed the high value of skin prion RT‐QuIC for CJD diagnosis (89% sensitivity and 100% specificity) and support its use in clinical practice. Preliminary data based on a limited number of cases suggest that prion‐seeding activity in the skin varies according to the prion strain, being higher in sporadic CJD subtypes linked to the V2 strain (VV2 and MV2K) than in typical CJDMM1.

Neurodegeneration-Associated Proteins in Human Olfactory Neurons Collected by Nasal Brushing.

The olfactory neuroepithelium is located in the upper vault of the nasal cavity, lying on the olfactory cleft and projecting into the dorsal portion of the superior and middle turbinates beyond the mid-portion of the nasal septum. It is composed of a variety of cell types including olfactory sensory neurons, supporting glial-like cells, microvillar cells, and basal stem cells. The cells of the neuroepithelium are often intermingled with respiratory and metaplastic epithelial cells. Olfactory neurons undergo a constant self-renewal in the timespan of 2–3 months; they are directly exposed to the external environment, and thus they are vulnerable to physical and chemical injuries. The latter might induce metabolic perturbations and ultimately be the cause of cell death. However, the lifespan of olfactory neurons is biologically programmed, and for this reason, these cells have an accelerated metabolic cycle leading to an irreversible apoptosis. These characteristics make these cells suitable for research related to nerve cell degeneration and aging. Recent studies have shown that a non-invasive and painless olfactory brushing procedure allows an efficient sampling from the olfactory neuroepithelium. This approach allows to detect the pathologic prion protein in patients with sporadic Creutzfeldt–Jakob disease, using the real-time quaking-induced conversion assay. Investigating the expression of all the proteins associated to neurodegeneration in the cells of the olfactory mucosa is a novel approach toward understanding the pathogenesis of human neurodegenerative diseases. Our aim was to investigate the expression of α-synuclein, β-amyloid, tau, and TDP-43 in the olfactory neurons of normal subjects. We showed that these proteins that are involved in neurodegenerative diseases are expressed in olfactory neurons. These findings raise the question on whether a relationship exists between the mechanisms of protein aggregation that occur in the olfactory bulb during the early stage of the neurodegenerative process and the protein misfolding occurring in the olfactory neuroepithelium.

Amplification Techniques for the Detection of Misfolded Prion Proteins in Experimental and Clinical Samples.

This article describes two methods for amplifying prions present in experimental and clinical samples: the protein misfolding cyclic amplification (PMCA) assay and the real-time quaking-induced conversion (RT-QuIC) assay. Protocols for preparation of amplification substrate and analysis of results are included in addition to those for the individual assays. For each assay, control and suspect samples are mixed with appropriate amplification substrate, which is whole brains from mice in the case of PMCA and recombinant prion protein produced in bacteria for RT-QuIC, followed by cyclic amplification over a number of cycles of sonication (PMCA) or shaking (RT-QuIC) at a consistent incubation temperature. The resultant amplification products are then assessed either by western blotting (PMCA) or based on fluorescent emissions (RT-QuIC). The equipment and expertise necessary for successfully performing either assay vary and will be important factors for individual laboratories to consider when identifying which assay is more appropriate for their experimental design. © 2020 by John Wiley & Sons, Inc. Basic Protocol 1: Prion amplification via protein misfolding cyclic amplification Support Protocol 1: Collection of whole brains from mice and preparation of normal brain homogenate Basic Protocol 2: Prion amplification via real-time quaking-induced conversion Support Protocol 2: Preparation of recombinant truncated white-tailed-deer prion protein.

BMD42-2910, a Novel Benzoxazole Derivative, Shows a Potent Anti-prion Activity and Prolongs the Mean Survival in an Animal Model of Prion Disease.

Prion diseases are a group of neurodegenerative and fatal central nervous system disorders. The pathogenic mechanism involves the conversion of cellular prion protein (PrPC) to an altered scrapie isoform (PrPSc), which accumulates in amyloid deposits in the brain. However, no therapeutic drugs have demonstrated efficacy in clinical trials. We previously reported that BMD42-29, a synthetic compound discovered in silico, is a novel anti-prion compound that inhibits the conversion of PrPC to protease K (PK)-resistant PrPSc fragments (PrPres). In the present study, 14 derivatives of BMD42-29 were obtained from BMD42-29 by modifying in the side chain by in silico feedback, with the aim to determine whether they improve anti-prion activity. These derivatives were assessed in a PrPSc-infected cell model and some derivatives were further tested using real time-quaking induced conversion (RT-QuIC). Among them, BMD42-2910 showed high anti-prion activity at low concentrations in vitro and also no toxic effects in a mouse model. Interestingly, abundant PrPres was reduced in brains of mice infected with prion strain when treated with BMD42-2910, and the mice survived longer than control mice and even that treated with BMD42-29. Finally, high binding affinity was predicted in the virtual binding sites (Asn159, Gln 160, Lys194, and Glu196) when PrPC was combined with BMD-42-2910. Our findings showed that BMD42-2910 sufficiently reduces PrPres generation in vitro and in vivo and may be a promising novel anti-prion compound.

Progression of chronic wasting disease in white-tailed deer analyzed by serial biopsy RT-QuIC and immunohistochemistry.

Chronic wasting disease (CWD) continues to spread or be recognized in the United States, Canada, and Europe. CWD is diagnosed by demonstration of the causative misfolded prion protein (PrPCWD) in either brain or lymphoid tissue using immunodetection methods, with immunohistochemistry (IHC) recognized as the gold standard. In recent years, in vitro amplification assays have been developed that can detect CWD prion seeding activity in tissues, excreta, and body fluids of affected cervids. These methods potentially offer earlier and more facile detection of CWD, both pre- and post-mortem. Here we provide a longitudinal profile of CWD infection progression, as assessed by both real-time quaking-induced conversion (RT-QuIC) and IHC on serial biopsies of mucosal lymphoid tissues of white-tailed deer orally exposed to low doses of CWD prions. We report that detection of CWD infection by RT-QuIC preceded that by IHC in both tonsil and recto-anal lymphoid tissue (RAMALT) in 14 of 19 deer (74%). Of the 322 biopsy samples collected in post-exposure longitudinal monitoring, positive RT-QuIC results were obtained for 146 samples, 91 of which (62%) were concurrently also IHC-positive. The lower frequency of IHC positivity was manifest most in the earlier post-exposure periods and in biopsies in which lymphoid follicles were not detected. For all deer in which RT-QuIC seeding activity was detected in a tonsil or RAMALT biopsy, PrPCWD was subsequently or concurrently detected by IHC. Overall, this study (a) provides a longitudinal profile of CWD infection in deer after low yet infectious oral prion exposure; (b) illustrates the value of RT-QuIC for sensitive detection of CWD; and (c) demonstrates an ultimate high degree of correlation between RT-QuIC and IHC positivity as CWD infection progresses.

High Diagnostic Accuracy of RT-QuIC Assay in a Prospective Study of Patients with Suspected sCJD.

The early and accurate in vivo diagnosis of sporadic Creutzfeldt–Jakob disease (sCJD) is essential in order to differentiate CJD from treatable rapidly progressive dementias. Diagnostic investigations supportive of clinical CJD diagnosis include magnetic resonance imaging (MRI), electroencephalogram (EEG), 14-3-3 protein detection, and/or real-time quaking-induced conversion (RT-QuIC) assay positivity in the cerebrospinal fluid (CSF) or in other tissues. The total CSF tau protein concentration has also been used in a clinical setting for improving the CJD diagnostic sensitivity and specificity. We analyzed 182 CSF samples and 42 olfactory mucosa (OM) brushings from patients suspected of having sCJD with rapidly progressive dementia (RPD), in order to determine the diagnostic accuracy of 14-3-3, the total tau protein, and the RT-QuIC assay. A probable and definite sCJD diagnosis was assessed in 102 patients. The RT-QuIC assay on the CSF samples showed a 100% specificity and a 96% sensitivity, significantly higher compared with 14-3-3 (84% sensitivity and 46% specificity) and tau (85% sensitivity and 70% specificity); however, the combination of RT-QuIC testing of the CSF and OM samples resulted in 100% sensitivity and specificity, proving a significantly higher accuracy of RT-QuIC compared with the surrogate biomarkers in the diagnostic setting of patients with RPD. Moreover, we showed that CSF blood contamination or high protein levels might interfere with RT-QuIC seeding. In conclusion, we provided further evidence that the inclusion of an RT-QuIC assay of the CSF and OM in the diagnostic criteria for sCJD has radically changed the clinical approach towards the diagnosis.

Editors' note: Pearls & Oy-sters: Challenging diagnosis of Gerstmann-Sträussler-Scheinker disease: Clinical and imaging findings.

In the article “Pearls & Oy-sters: Challenging diagnosis of Gerstmann-Sträussler-Scheinker disease: Clinical and imaging findings,” Kang et al. presented the case of a 49-year-old man with progressive ataxia, dysarthria, and cognitive impairment who was found to have a heterozygous mutation in the prion protein (PRNP) gene, suspected to be de novo, resulting in a diagnosis of Gerstmann-Sträussler-Scheinker disease (GSS). In the accompanying discussion, they also presented a review of the clinical manifestations, diagnostic test results, and presence of family history of GSS in 86 patients (including theirs). In response, Dr. Paucar notes that the polymorphic PRNP codon 129M has disease-modifying effect on GSS and that real-time quaking-induced conversion (RT-QuIC) can be helpful in patients with suspected prion disease, especially with ambiguous family history, and that a discussion of transmissibility of GSS-associated PRNP mutations would have been helpful. Responding to these comments, Park et al. report the results of PRNP codon 129M polymorphisms in their review and that they found the methionine/valine heterozygote to have slightly earlier disease onset. They note that they could not evaluate the role of RT-QuIC but report that 87.7% of the patients with the P102L mutation (found in their patient) had a positive family history, whereas 8 other single-case mutations had a negative family history. In the article “Pearls & Oy-sters: Challenging diagnosis of Gerstmann-Sträussler-Scheinker disease: Clinical and imaging findings,” Kang et al. presented the case of a 49-year-old man with progressive ataxia, dysarthria, and cognitive impairment who was found to have a heterozygous mutation in the prion protein (PRNP) gene, suspected to be de novo, resulting in a diagnosis of Gerstmann-Sträussler-Scheinker disease (GSS). In the accompanying discussion, they also presented a review of the clinical manifestations, diagnostic test results, and presence of family history of GSS in 86 patients (including theirs). In response, Dr. Paucar notes that the polymorphic PRNP codon 129M has disease-modifying effect on GSS and that real-time quaking-induced conversion (RT-QuIC) can be helpful in patients with suspected prion disease, especially with ambiguous family history, and that a discussion of transmissibility of GSS-associated PRNP mutations would have been helpful. Responding to these comments, Park et al. report the results of PRNP codon 129M polymorphisms in their review and that they found the methionine/valine heterozygote to have slightly earlier disease onset. They note that they could not evaluate the role of RT-QuIC but report that 87.7% of the patients with the P102L mutation (found in their patient) had a positive family history, whereas 8 other single-case mutations had a negative family history.

Goats naturally devoid of PrPC are resistant to scrapie

Prion diseases are progressive and fatal, neurodegenerative disorders described in humans and animals. According to the “protein-only” hypothesis, the normal host-encoded prion protein (PrPC) is converted into a pathological and infectious form (PrPSc) in these diseases. Transgenic knockout models have shown that PrPC is a prerequisite for the development of prion disease. In Norwegian dairy goats, a mutation (Ter) in the prion protein gene (PRNP) effectively blocks PrPC synthesis. We inoculated 12 goats (4 PRNP+/+, 4 PRNP+/Ter, and 4 PRNPTer/Ter) intracerebrally with goat scrapie prions. The mean incubation time until clinical signs of prion disease was 601 days post-inoculation (dpi) in PRNP+/+ goats and 773 dpi in PRNP+/Ter goats. PrPSc and vacuolation were similarly distributed in the central nervous system (CNS) of both groups and observed in all brain regions and segments of the spinal cord. Generally, accumulation of PrPSc was limited in peripheral organs, but all PRNP+/+ goats and 1 of 4 PRNP+/Ter goats were positive in head lymph nodes. The four PRNPTer/Ter goats remained healthy, without clinical signs of prion disease, and were euthanized 1260 dpi. As expected, no accumulation of PrPSc was observed in the CNS or peripheral tissues of this group, as assessed by immunohistochemistry, enzyme immunoassay, and real-time quaking-induced conversion. Our study shows for the first time that animals devoid of PrPC due to a natural mutation do not propagate prions and are resistant to scrapie. Clinical onset of disease is delayed in heterozygous goats expressing about 50% of PrPC levels.

Role of donor genotype in RT-QuIC seeding activity of chronic wasting disease prions using human and bank vole substrates.

Chronic wasting disease is a transmissible spongiform encephalopathy of cervids. This fatal neurodegenerative disease is caused by misfolding of the cellular prion protein (PrPC) to pathogenic conformers (PrPSc), and the pathogenic forms accumulate in the brain and other tissues. Real-time Quaking Induced Conversion (RT-QuIC) can be used for the detection of prions and for prion strain discrimination in a variety of biological tissues from humans and animals. In this study, we evaluated how either PrPSc from cervids of different genotypes or PrPSc from different sources of CWD influence the fibril formation of recombinant bank vole (BV) or human prion proteins using RT-QuIC. We found that reaction mixtures seeded with PrPSc from different genotypes of white-tailed deer or reindeer brains have similar conversion efficiency with both substrates. Also, we observed similar results when assays were seeded with different sources of CWD. Thus, we conclude that the genotypes of all sources of CWD used in this study do not influence the level of conversion of PrPC to PrPSc.

Involvement of N- and C-terminal region of recombinant cervid prion protein in its reactivity to CWD and atypical BSE prions in real-time quaking-induced conversion reaction in the presence of high concentrations of tissue homogenates

ABSTRACT The real-time quaking-induced conversion (RT-QuIC) reaction is a sensitive and specific method for detecting prions. However, inhibitory factors present in tissue homogenates can easily interfere with this reaction. To identify the RT-QuIC condition under which low levels of chronic wasting disease (CWD) and bovine spongiform encephalopathy (BSE) prions can be detected in the presence of high concentrations of brain tissue homogenates, reactivities of various recombinant prion proteins (rPrPs) were tested. Among the tested rPrPs, recombinant cervid PrP (rCerPrP) showed a unique reactivity: the reactivity of rCerPrP to CWD and atypical BSE prions was not highly affected by high concentrations of normal brain homogenates. The unique reactivity of rCerPrP disappeared when the N-terminal region (aa 25–93) was truncated. Replacement of aa 23–149 of mouse (Mo) PrP with the corresponding region of CerPrP partially restored the unique reactivity of rCerPrP in RT-QuIC. Replacement of the extreme C-terminal region of MoPrP aa 219–231 to the corresponding region of CerPrP partially conferred the unique reactivity of rCerPrP to rMoPrP, suggesting the involvement of both N- and C-terminal regions. Additionally, rCerN–Mo–CerCPrP, a chimeric PrP comprising CerPrP aa 25–153, MoPrP aa 150–218, and CerPrP aa 223–233, showed an additive effect of the N- and C-terminal regions. These results provide a mechanistic implication for detecting CWD and atypical BSE prions using rCerPrP and are useful for further improvements of RT-QuIC.

Cell-free amplification of prions: Where do we stand?

Neurodegenerative diseases (NDs) such as Alzheimer's disease (AD), Parkinson's disease (PD), atypical parkinsonisms, frontotemporal dementia (FTLD) and prion diseases are characterized by the accumulation of misfolded proteins in the central nervous system (CNS). Although the cause for the initiation of protein aggregation is not well understood, these aggregates are disease-specific. For instance, AD is characterized by the intraneuronal accumulation of tau and extracellular deposition of amyloid-β (Aβ), PD is marked by the intraneuronal accumulation of α-synuclein, many FTLD are associated with the accumulation of TDP-43 while prion diseases show aggregates of misfolded prion protein. Hence, misfolded proteins are considered disease-specific biomarkers and their identification and localization in the CNS, collected postmortem, is required for a definitive diagnosis. With the development of two innovative cell-free amplification techniques named Protein Misfolding Cyclic Amplification (PMCA) and Real-Time Quaking-Induced Conversion (RT-QuIC), traces of disease-specific biomarkers were found in CSF and other peripheral tissues (e.g., urine, blood, and olfactory mucosa) of patients with different NDs. These techniques exploit an important feature shared by many misfolded proteins, that is their ability to interact with their normally folded counterparts and force them to undergo similar structural rearrangements. Essentially, RT-QuIC and PMCA mimic in vitro the same pathological processes of protein misfolding which occur in vivo in a very rapid manner. For this reason, they have been employed for studying different aspects of protein misfolding but, overall, they seem to be very promising for the premortem diagnosis of NDs.

RT-QuIC detection of tauopathies using full-length tau substrates

ABSTRACT Early detection and diagnosis of neurodegenerative diseases has been hampered by the lack of sensitive testing. Real-time quaking induced conversion (RT-QuIC) has been used for the early and sensitive detection of prion-induced neurologic disease, and has more recently been adapted to detect misfolded alpha-synuclein and tau as biomarkers for neurodegenerative disease. Here we use full-length recombinant tau substrates to detect tau seeding activity in Alzheimer’s disease and other human tauopathies.

Clinical diagnosis of human prion disease.

Human prion disease may present in a non-specific way and is often diagnosed at a relatively late stage of the illness. Until recently, clinical diagnosis has been supported by tests that are mostly non-specific and, sometimes, insensitive. Recent laboratory developments have led to a variety of tests that rely on a disease-specific mechanism. One test, the CSF RT-QuIC (Real-Time Quaking-Induced Conversion) test is very sensitive and specific for sporadic CJD and is now used in routine clinical practice. Other tests, based on other tissues, including blood and urine, have been developed and potentially could improve both clinical diagnostic accuracy and lead to earlier diagnosis. While there are yet no proven treatments for prion disease, any treatment to be developed will almost certainly require earlier diagnosis if therapeutic success is to be realized.

Cross-validation of the RT-QuIC assay for the antemortem detection of chronic wasting disease in elk

ABSTRACT Chronic wasting disease is a progressively fatal, horizontally transmissible prion disease affecting several members of the cervid species. Conventional diagnosis relies on ELISA or IHC evaluation using tissues collected post-mortem; however, recent research has focused on newly developed amplification techniques using samples collected antemortem. The present study sought to cross-validate the real-time quaking-induced conversion assay (RT-QuIC) evaluation of rectal biopsies collected from an elk herd with endemic CWD, assessing both binary positive/negative test results as well as relative rates of amplification between laboratories. We found that results were correlative in both categories across all laboratories performing RT-QuIC, as well as to conventional IHC performed at a national reference laboratory. A significantly higher number of positive samples were identified using RT-QuIC, with results seemingly unhindered by low follicle counts. These findings support the continued development and implementation of amplification assays in the diagnosis of prion diseases of veterinary importance, targeting not just antemortem sampling strategies, but post-mortem testing approaches as well.

In Vitro Conversion Assays Diagnostic for Neurodegenerative Proteinopathies.

In vitro conversion assays, including real-time quaking-induced conversion (RT-QuIC) and protein misfolding cyclic amplification (PMCA) techniques, were first developed to study the conversion process of the prion protein to its misfolded, disease-associated conformation. The intrinsic property of prion proteins to propagate their misfolded structure was later exploited to detect subfemtogram quantities of the misfolded protein present in tissues and fluids from humans and animals with transmissible spongiform encephalopathies. Currently, conversion assays are used clinically as sensitive and specific diagnostic tools for antemortem diagnosis of prion disease. In vitro conversion assays are now being applied to the development of diagnostics for related neurodegenerative diseases, including detection of misfolded α-synuclein in Parkinson disease, misfolded amyloid-β in Alzheimer disease, and misfolded tau in Pick disease. Like the predicate prion protein in vitro conversion diagnostics, these assays exploit the ability of endogenously misfolded proteins to induce misfolding and aggregation of their natively folded counterpart in vitro. This property enables biomarker detection of the underlying protein pathology. Herein, we review RT-QuIC and PMCA for (a) prion-, (b) α-synuclein-, (c) amyloid-β-, and (d) tau-opathies. Although already in routine clinical use for the detection of transmissible spongiform encephalopathies, in vitro conversion assays for other neurodegenerative disorders require further development and evaluation of diagnostic performance before consideration for clinical implementation.

Metformin reduces prion infection in neuronal cells by enhancing autophagy

Prion diseases are fatal infectious neurodegenerative disorders in human and animals that are caused by misfolding of the cellular prion protein (PrPC) into the infectious isoform PrPSc. No effective treatment is available for prion diseases. Metformin is a first-line medication for treatment of type 2 diabetes which is known to activate AMPK and induce autophagy through the inhibition of mammalian target of rapamycin (mTOR1) signaling. Metformin was reported to be beneficial in various protein misfolding and neurodegenerative diseases like Alzheimer’s and Huntington’s diseases. In this study we investigated the anti-prion effect of metformin in persistently prion-infected neuronal cells. Our data showed that metformin significantly decreased the PrPSc load in the treated cells, as shown by less PK resistant PrP in Western blots and reduced prion conversion activity in Real-Time Quaking-Induced Conversion (RT-QuIC) assay in both 22L-ScN2a and RML-ScCAD5 cells. Additionally, metformin induced autophagy as shown by higher levels of LC3-II in treated cells compared with control cells. On the other hand, our mouse bioassay showed that oral metformin at a dose of 2 mg/ml in drinking water had no effect on the survival of prion-infected mice. In conclusion, our findings describe the anti-prion effect of metformin in two persistently prion-infected neuronal cell lines. This effect can be explained at least partially by the autophagy inducing activity of metformin. This study sheds light on metformin as an anti-prion candidate for the combination therapy of prion diseases.

In vitro detection of haematogenous prions in white-tailed deer orally dosed with low concentrations of chronic wasting disease.

Infectivity associated with prion disease has been demonstrated in blood throughout the course of disease, yet the ability to detect blood-borne prions by in vitro methods remains challenging. We capitalized on longitudinal pathogenesis studies of chronic wasting disease (CWD) conducted in the native host to examine haematogenous prion load by real-time quaking-induced conversion (RT-QuIC) and protein misfolding cyclic amplification. Our study demonstrated in vitro detection of amyloid seeding activity (prions) in buffy-coat cells harvested from deer orally dosed with low concentrations of CWD positive (+) brain (1 gr and 300 ng) or saliva (300 ng RT-QuIC equivalent). These findings make possible the longitudinal assessment of prion disease and deeper investigation of the role haematogenous prions play in prion pathogenesis.

Validation of Poly(Propylene Imine) Glycodendrimers Towards Their Anti-prion Conversion Efficiency.

Prion diseases, such as the sporadic Creutzfeldt-Jakob disease (sCJD), are a class of fatal neurodegenerative disorders. Currently, there is no efficient treatment or therapy available. Hence, the search for molecules that may inhibit the conversion of the cellular prion protein (PrPC) into its pathological counterpart PrPScrapie (PrPSc) is of great urgency. Here, we report the generation- and dose-dependent biological action of dense-shell poly(propylene imine) (PPI) glycodendrimers by using scrapie-infected neuroblastoma (ScN2a) cells and the real-time quaking-induced conversion assay (RT-QuIC) for validation of anti-prion efficiencies. Whereas the 2nd and 3rd generation of PPI glycodendrimers exhibited anti-prion conversion efficiency in ScN2a cells validated by RT-QuIC analysis, we observed that the 4th generation of glycodendrimers had shown no significant effect. Translational RT-QuIC studies conducted with human prions derived from sCJD patients indicated an anti-prion conversion effect (not on PrPRes degradation) of PPI glycodendrimers against human prions with the highest inhibitory activity of the 4th generation of PPI glycodendrimers towards prion aggregation compared to the 2nd and 3rd generation. In conclusion, our study highlights the potential of PPI glycodendrimers as therapeutic compounds due to their anti-conversion activity on human prions in a PrPSc strain depending manner.

Quaking-induced conversion of prion protein on a thermal mixer accelerates detection in brains infected with transmissible spongiform encephalopathy agents.

Detection of misfolded prion protein, PrPTSE, in biological samples is important to develop antemortem tests for transmissible spongiform encephalopathies (TSEs). The real-time quaking-induced conversion (RT-QuIC) assay detects PrPTSE but requires dedicated equipment and relatively long incubation times when applied to samples containing extremely low levels of PrPTSE. It was shown that a microplate shaker with heated top (Thermomixer-C) accelerated amplification of PrPTSE in brain suspensions of 263K scrapie and sporadic Creutzfeldt-Jakob disease (sCJD). We expanded the investigation to include TSE agents previously untested, including chronic wasting disease (CWD), macaque-adapted variant CJD (vCJD) and human vCJD, and we further characterized the assays conducted at 42°C and 55°C. PrPTSE from all brains containing the TSE agents were successfully amplified using a truncated hamster recombinant protein except for human vCJD which required truncated bank vole recombinant protein. We compared assays conducted at 42°C on Thermomixer-C, Thermomixer-R (without heated top) and on a fluorimeter used for RT-QuIC. QuIC on Thermomixer-R achieved in only 18 hours assay sensitivity similar to that of RT-QuIC read at 60 hours (or 48 hours with sCJD). QuIC on Thermomixer-C required 24 hours to complete and the endpoint titers of some TSEs were 10-fold lower than those obtained with RT-QuIC and Thermomixer-R. Conversely, at 55°C, the reactions with sCJD and CWD on Thermomixer-C achieved the same sensitivity as with RT-QuIC but in shorter times. Human vCJD samples tested at higher temperatures gave rise to high reactivity in wells containing normal control samples. Similarly, reactions on Thermomixer-R were unsuitable at 55°C. The main disadvantage of Thermomixers is that they cannot track formation of PrP fibrils in real time, a feature useful in some applications. The main advantages of Thermomixers are that they need shorter reaction times to detect PrPTSE, are easier to use, involve more robust equipment, and are relatively affordable. Improvements to QuIC using thermal mixers may help develop accessible antemortem TSE tests.