Mouse neuronal CAD 5 cell line effectively propagates various strains of prions. Previously, we have shown that it can also be differentiated into the cells morphologically resembling neurons. Here, we demonstrate that CAD 5 cells chronically infected with prions undergo differentiation under the same conditions. To make our model more realistic, we triggered the differentiation in the 3D culture created by gentle rocking of CAD 5 cell suspension. Spheroids formed within 1 week and were fully developed in less than 3 weeks of culture. The mature spheroids had a median size of ~300 μm and could be cultured for up to 12 weeks. Increased expression of differentiation markers GAP 43, tyrosine hydroxylase, β-III-tubulin and SNAP 25 supported the differentiated status of the spheroid cells. The majority of them were found in the G0/G1 phase of the cell cycle, which is typical for differentiated cells. Moreover, half of the PrPC on the cell membrane was N-terminally truncated, similarly as in differentiated CAD 5 adherent cells. Finally, we demonstrated that spheroids could be created from prion-infected CAD 5 cells. The presence of prions was verified by immunohistochemistry, western blot and seed amplification assay. We also confirmed that the spheroids can be infected with the prions de novo. Our 3D culture model of differentiated CAD 5 cells is low cost, easy to produce and cultivable for weeks. We foresee its possible use in the testing of anti-prion compounds and future studies of prion formation dynamics.
- MeSH
- buněčná diferenciace * fyziologie MeSH
- buněčné kultury metody MeSH
- buněčné linie MeSH
- buněčné sféroidy * metabolismus MeSH
- myši MeSH
- neurony metabolismus MeSH
- prionové nemoci * metabolismus patologie MeSH
- priony metabolismus MeSH
- techniky 3D buněčné kultury metody MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Real-time quaking-induced conversion assay (RT-QuIC) exploits templating activity of pathogenic prion protein for ultrasensitive detection of prions. We have utilized second generation RT-QuIC assay to analyze matching post-mortem cerebrospinal fluid and skin samples of 38 prion disease patients and of 30 deceased neurological controls. The analysis of cerebrospinal fluid samples led to 100% sensitivity and 100% specificity, but some samples had to be diluted before the analysis to alleviate the effect of present RT-QuIC inhibitors. The analysis of the corresponding skin samples provided 89.5% sensitivity and 100% specificity. The median seeding dose present in the skin was one order of magnitude higher than in the cerebrospinal fluid, despite the overall fluorescent signal of the skin samples was comparatively lower. Our data support the use of post-mortem cerebrospinal fluid for confirmation of prion disease diagnosis and encourage further studies of the potential of skin biopsy samples for intra-vitam prion diseases ́ diagnostics.
- MeSH
- biotest MeSH
- Creutzfeldtova-Jakobova nemoc * diagnóza mozkomíšní mok MeSH
- kůže metabolismus MeSH
- lidé MeSH
- prionová bílkovina MeSH
- prionové nemoci * diagnóza MeSH
- priony * metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Diagnostika prionových onemocnění je obtížná vzhledem k jejich heterogenitě a překryvu klinických příznaků s jinými neurodegenerativními chorobami. Doposud dostupné diagnostické metody neměly dostatečnou citlivost a specificitu. V roce 2018 byla do diagnostických kritérií (WHO) zařazena metoda RT‐QuIC (Real Time Quacking Induced Conformation assay), která využívá pro detekci prionů jejich schopnost agregovat nativní rekombinantní prionový protein. Agregace je sledována v reálném čase pomocí fluorescenční sondy. Metoda je extrémně citlivá a specifická. Naše výsledky potvrzují, že umožňuje detekci prionů v mozkomíšním moku pacientů za jejich života, krátce po objevení příznaků onemocnění.
Diagnostics of prion diseases is difficult due to their heterogeneity and overlap of clinical symptoms with other neurodegenerative disorders. Till now utilized diagnostics methods did not have sufficient sensitivity and specificity. In 2018 WHO diagnostic criteria were updated by the inclusion of RT-QuIC assay (Real Time Quacking Induced Conformation), which utilizes the ability of prions to aggregate native recombinant prion protein for their detection. The aggregation is monitored in real time using fluorescent probe. The assay is extremely sensitive and specific. Our results confirms that it allows detection of prions in cerebrospinal fluid of living patients shortly after the appearance of symptoms.
- MeSH
- Creutzfeldtova-Jakobova nemoc diagnóza MeSH
- diferenciální diagnóza MeSH
- klinické laboratorní techniky metody MeSH
- lidé MeSH
- prionové nemoci * diagnóza MeSH
- priony analýza mozkomíšní mok MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- přehledy MeSH
Prions are responsible for a number of lethal neurodegenerative and transmissible diseases in humans and animals. Extracellular vesicles, especially small exosomes, have been extensively studied in connection with various diseases. In contrast, larger microvesicles are often overlooked. In this work, we compared the ability of large extracellular vesicles (lEVs) and small extracellular vesicles (sEVs) to spread prions in cell culture. We utilized CAD5 cell culture model of prion infection and isolated lEVs by 20,000×g force and sEVs by 110,000×g force. The lEV fraction was enriched in β-1 integrin with a vesicle size starting at 100 nm. The fraction of sEVs was partially depleted of β-1 integrin with a mean size of 79 nm. Both fractions were enriched in prion protein, but the lEVs contained a higher prion-converting activity. In addition, lEV infection led to stronger prion signals in both cell cultures, as detected by cell and western blotting. These results were verified on N2a-PK1 cell culture. Our data suggest the importance of lEVs in the trafficking and spread of prions over extensively studied small EVs.
Prion' is a term used to describe a protein infectious particle responsible for several neurodegenerative diseases in mammals, e.g., Creutzfeldt-Jakob disease. The novelty is that it is protein based infectious agent not involving a nucleic acid genome as found in viruses and bacteria. Prion disorders exhibit, in part, incubation periods, neuronal loss, and induce abnormal folding of specific normal cellular proteins due to enhancing reactive oxygen species associated with mitochondria energy metabolism. These agents may also induce memory, personality and movement abnormalities as well as depression, confusion and disorientation. Interestingly, some of these behavioral changes also occur in COVID-19 and mechanistically include mitochondrial damage caused by SARS-CoV-2 and subsequenct production of reactive oxygen species. Taken together, we surmise, in part, long COVID may involve the induction of spontaneous prion emergence, especially in individuals susceptible to its origin may thus explain some of its manesfestions post-acute viral infection.
- MeSH
- COVID-19 * MeSH
- lidé MeSH
- postakutní syndrom COVID-19 MeSH
- priony * metabolismus MeSH
- reaktivní formy kyslíku MeSH
- SARS-CoV-2 MeSH
- savci metabolismus MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- přehledy MeSH
Emerging experimental evidence suggests tau pathology spreads between neuroanatomically connected brain regions in a prion-like manner in Alzheimer's disease (AD). Tau seeding, the ability of prion-like tau to recruit and misfold naïve tau to generate new seeds, is detected early in human AD brains before the development of major tau pathology. Many antitumour drugs have been reported to confer protection against neurodegeneration, supporting the repurposing of approved and experimental or investigational oncology drugs for AD therapy. In this study, we evaluated whether antitumour drugs that abrogate the generation of seed-competent aggregates of tau Repeat 3 (R3) domain peptides can prevent tau seeding and toxicity in Tau-RD P301S FRET Biosensor cells and Caenorhabditis elegans. We demonstrate that drugs that interact with the N-terminal VQIVYK or the C-terminal region housing the Cys322 prevent R3 dimerisation, abolishing the generation of prion-like R3 seeds. Preformed R3 seeds (fibrils) capped with, or R3 seeds formed in the presence of VQIVYK- or Cys322-targeting drugs have a reduced potency to cause aggregation of naïve tau in biosensor cells and protect worms from aggregate toxicity. These findings indicate that VQIVYK- or Cys322-targeting drugs may act as prophylactic agents against tau seeding.
Závěrečná zpráva o řešení grantu Agentury pro zdravotnický výzkum MZ ČR
nestr.
Prion diseases are fatal neurodegenerative disorders connected with accumulation of aggregated misfolded prion protein in the brain of affected individuals. The diseases are transmissible, no therapy is available and at present the diagnosis is confirmed only after neuropathological assessment post mortem. Our project is aimed on utilization of a new revolutionary method, Real-Time Quaking Induced Conversion (RT-QuIC) assay, for intravital diagnostics of prion diseases. The method already showed superior sensitivity and specificity in analysis of cerebrospinal fluid which led to its recent inclusion into internationally recognized diagnostic criteria for Creutzfeldt-Jakob disease. Our project specifically aims to assess diagnostic potential of RT-QuIC in analysis of easily accessible samples of urine and skin. The importance of early diagnosis confirmation using minimally invasive procedure lays in mitigation of the risk of prion nosocomial transmission, in immediate implementation of special patient care and in securing a room for therapeutic intervention in the future.
Prionové choroby jsou smrtelná neurodegenerativní onemocnění spojená s hromaděním agregovaného konformačně pozměněného prionového proteinu v mozku postižených jedinců. Onemocnění je přenosné, neexistuje účinná léčba a v současnosti lze jeho diagnózu potvrdit pouze neuropatologickým vyšetřením post mortem. Náš projekt je zaměřen na využití nové revoluční metody RT-QuIC (Real-Time Quaking Induced Conversion) pro potvrzení diagnózy onemocnění za života pacienta. Metoda RT-QuIC již prokázala vysokou specificitu a senzitivitu při analýze mozkomíšního moku, která tento rok vedla k jejímu zahrnutí do mezinárodních diagnostických kritérií Cretzfeldtovy-Jakobovy nemoci. Náš projekt specificky cílí na posouzení diagnostického potenciálu RT-QuIC u snadno dostupných vzorků moči a kůže. Důležitost rychlého potvrzení diagnózy pomocí minimálně invazivního postupu tkví nejen v prevenci rizika nozokomiálního přenosu prionů a včasné aplikaci specializované péče o pacienta, ale i ve vytvoření nezbytného prostoru pro vyvíjené terapeutické zásahy v budoucnosti.
- Klíčová slova
- RT-QuIC,
- MeSH
- Creutzfeldtova-Jakobova nemoc diagnóza MeSH
- klinické chemické testy metody MeSH
- kůže chemie MeSH
- moč chemie MeSH
- mozkomíšní mok chemie MeSH
- prionová bílkovina chemie MeSH
- prionové nemoci diagnóza MeSH
- Konspekt
- Patologie. Klinická medicína
- NLK Obory
- neurologie
- NLK Publikační typ
- závěrečné zprávy o řešení grantu AZV MZ ČR
Proteiny 14-3-3 se řadí mezi vysoce konzervované kyselé homologní proteiny, které se podílí na celé řadě důležitých procesů v lidském těle, např. na řízení buněčného cyklu, apoptóze, neuronálním vývoji, buněčném růstu nebo signální transdukci a fosforylaci. Skládají se ze sedmi izoforem, které se nachází ve všech eukaryotních buňkách. Nejvyšší exprese proteinů 14 3 3 je však vykazována v mozku. Z tohoto důvodu má detekce/stanovení proteinů 14-3-3 význam u pacientů s neurologickým onemocněním, převážně u rychle progredující demence s neurologickými příznaky. Nejčastěji používanou metodou je Western blot s chromogenní nebo chemiluminiscenční detekcí, možnou variantou je i ELISA stanovení jednotlivých izoforem.
14-3-3 proteins are among the highly conserved acidic homologous proteins that are involved in several important processes in the human body, such as cell cycle control, apoptosis, neuronal development, cell growth, or signal transduction and phosphorylation. They consist of seven isoforms found in all eukaryotic cells. However, the highest expression of 14-3-3 proteins is shown in the brain. For this reason, the detection/determination of 14-3-3 proteins is important in patients with neurological diseases, predominantly in rapidly progressive dementia with neurological symptoms. The most commonly used method is Western blot with chromogenic or chemiluminescent detection. A possible variant is the ELISA determination of individual isoforms.
- MeSH
- biologické markery MeSH
- Creutzfeldtova-Jakobova nemoc diagnóza MeSH
- demence diagnóza MeSH
- lidé MeSH
- prionová bílkovina analýza MeSH
- proteiny 14-3-3 * analýza MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- přehledy MeSH
Prion disorders, or transmissible spongiform encephalophaties (TSE), are fatal neurodegenerative diseases affecting mammals. Prion-infectious particles comprise of misfolded pathological prion proteins (PrPTSE). Different TSEs are associated with distinct PrPTSE folds called prion strains. The high resistance of prions to conventional sterilization increases the risk of prion transmission in medical, veterinary and food industry practices. Recently, we have demonstrated the ability of disulfonated hydroxyaluminum phthalocyanine to photodynamically inactivate mouse RML prions by generated singlet oxygen. Herein, we studied the efficiency of three phthalocyanine derivatives in photodynamic treatment of seven mouse adapted prion strains originating from sheep, human, and cow species. We report the different susceptibilities of the strains to photodynamic oxidative elimination of PrPTSE epitopes: RML, A139, Fu-1 > mBSE, mvCJD > ME7, 22L. The efficiency of the phthalocyanine derivatives in the epitope elimination also differed (AlPcOH(SO3)2 > ZnPc(SO3)1-3 > SiPc(OH)2(SO3)1-3) and was not correlated to the yields of generated singlet oxygen. Our data suggest that the structural properties of both the phthalocyanine and the PrPTSE strain may affect the effectiveness of the photodynamic prion inactivation. Our finding provides a new option for the discrimination of prion strains and highlights the necessity of utilizing range of prion strains when validating the photodynamic prion decontamination procedures.
- MeSH
- fotochemoterapie metody MeSH
- fotosenzibilizující látky farmakologie MeSH
- indoly chemie MeSH
- lidé MeSH
- mozek účinky léků metabolismus účinky záření MeSH
- myši MeSH
- organokovové sloučeniny chemie MeSH
- ovce MeSH
- oxidace-redukce MeSH
- prionová bílkovina metabolismus MeSH
- prionové nemoci farmakoterapie metabolismus patologie MeSH
- sbalování proteinů MeSH
- singletový kyslík MeSH
- skot MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- skot MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
