Prion proteins have been the cause of a few incurable neurodegenerative diseases in humans and animals, like the Creutzfeldt-Jakob Disease (CJD), Fatal Familial Insomnia, Kuru (the Laughing Death), etc. Caused when normal PrPC protein, made in the human body by the PRNP gene, changes in conformation when the infectious PrPScform binds and changes it from an α-helix prevalent to a β-sheet prevalent form, they spread through consumption of infected meat, especially affecting cattle through fomites. Prions show resistance to nearly all forms of sterilization, be it drugs, organic compounds like alcohols and chloroform, γ-radiation, X-rays, autoclaving, etc. (only except concentrated NaOH), and the diagnosis of prion disease has been traditionally difficult – it is these aspects which will be covered in this article.
Accurate Diagnosis of Prion Diseases:
For accurate detection of prions in living patients, a new method called RT-QuIC or Real-Time Quaking Induced Conversion has been developed which directly uses prion seeds or infection-causing PrPSc particles to convert normal PrPC into amyloid fibers. The extraction of seeds is done from patients infected with CJD by using a sterile nasal brush, that, when introduced into the nasal cavity along with a sheathed rhinoscope and nasal tampon after vasoconstrictor (1% epinephrine) application, gives the required prion seeds from the olfactory mucosal epithelium. Spiking experiments can be done and endogenous seeding activity is checked for the olfactory mucosa (OM) and brain homogenate (BH) samples, the kind used for the experiment. Fluorescence was the way by which prion quantification was done, and it was found that OM samples had much higher prion “seeds” than CSF (Cerebrospinal Fluid) samples. The sensitivity was much higher for OM RT-QuIC reactions (100%) than for CSF RT-QuIC reactions (77%). Also, the reactions with OM took around 55 hours to finish, against the 90 hours needed for reactions with CSF to complete. Thus the relatively shorter reaction time and high sensitivity of RT-QuIC analysis make it a novel approach to diagnosing prion diseases. The method has also proven to be more accurate than studying CSF, Tau protein, 14-3-3 protein, or similar proteins in assays for prion detection.
Fluorescence readings showing the presence of prion particles in infected Cerebrospinal Fluid & Olfactory Membrane compared to those from control samples (source: [1])
Using Lichen Extracts for Prion treatment:
In an experiment performed where aqueous & alcoholic extracts from 3 lichens (Parmelia sulcata, Cladonia rangiferina & Lobaria pulmonaria)were separately added to infected BH enriched with Phosphotungstic Acid (PTA), there was a significant reduction (almost 2 logs) of prion concentration as seen by immunoblotting. The lichens used were also tested for if their photobiont (algal) or mycobiont (fungal) parts could individually degrade prions in vitro. For this experiment, the photobiont extract from each lichen was separately mixed with misfolded or normally folded prion protein infected BH sample and subjected to immunoblotting, only to reveal that none of them showed the degrading activity seen only with the whole lichen, indicating the mycobionts’ large role. It has been observed that the presence of these lichens in their natural environment has also led to a decreased incidence of prion disease among animals, like deer, inhabiting the areas. Also, these lichen extracts are effective on both animal and fungal prions, indicating a wide range of activity, which probably results from infectious prions being essentially amyloid fibrils, no matter the Kingdom. Only when a protease inhibitor cocktail was used, the degradation activity was drastically reduced, and among them, only a serine protease could diminish the prion degrading activity displayed by the lichen extracts. Proteinase K, which has vast substrate range, failed to degrade infectious prion in vitro. Secondary metabolites of the lichens were also proven to be of no use in the degrading activity. L. pulmonaria extract displayed good activity from pH 4 to 11, whereas P. sulcata extract only did upto about pH 8, therefore indicating a possible pH dependence for this activity.
The presence of lower intensity bands on the 2nd to 4th lanes of both panels compared to the 1st (control) lane prove prion degradation by extracts of the mentioned lichens (source: [3])
Treating Prions with Camelid Antibodies:
A method of using Anti-PrP antibodies (Abs), PrioV3, has been developed that leads to the abrogation of prion replication in prion-permissive neuroblastoma cells. These were shown to easily cross the Blood-Brain-Barrier (BBB), which does not normally allow everything to pass. These Abs were raised in camels against prion-coated Dynabeads, that allowed the animal’s B-cells to develop monoclonal against it, thereafter these cells being grown in vitro, its RNA isolated and the DNA for the antibodies cloned, leading to mass production of prion-specific antibody. ICSM35 & CD71, two other Abs that can also permeate through the BBB, were also tested against PrioV3. Its relatively small size of 14 kDa, better solubility in the cell cytosol & more stability compared to conventional Anti-PrP Abs prove PrioV3 as more efficient. PrioV3 showed a significant reduction in prion replication with respect to ICSM35, CD71, Normal Camel Serum (NCS), and BRIC-126 (a CD47 antibody) treated/untreated cells.PrioV3 shows efficient binding to PrPC & PrPSc proteins in the cytosol of neurons, a character not shown by other conventional Anti-PrP antibodies, which only decorate the cell surface by attaching to the extracellular PrPC. Other antibodies like ICSM35 having binding sites on residues 91-110 of PrP are able to elicit neurotoxic responses on cross-linking with PrPC. PrioV3 having its binding sites between residues 171-190of PrPSc fails to elicit neurotoxicity, thereby proving better.
Comparison of prion replication abrogation activities by PrioV3, BRIC-126, ICSM35 & NCS, in presence and absence of Proteinase K to a control (no antibody) (source: [2])
Astemizole treatment and Autophagy:
Tacrolimus is already used as an immunosuppressant in organ transplants, and to ameliorate myasthenia gravis, an autoimmune disorder. Astemizole is used as a drug for chronic seasonal allergic rhinitis in humans. So, both drugs already have a degree of usage in the human population, meaning ill effects could be tolerable when using an appropriate dose for prion disease. Tacrolimus treated cells showed both cell surface and intracellular reduction of PrP by greater than 70%, which indicated translation inhibition of PrP. Astemizole treated cells showed less than 20% reduction of cell surface PrP levels, indicating a method other than preventing PrP translation to be its method of action. It was found that on tacrolimus treatment there was negligible induction of autophagy, the induced death of the infected cells by autophagosome-lysosome fusion, and destruction of all cell components. Astemizole has shown to also have increased the survival time of mice infected with prions treated with the drug, with the effect taking place about 20-50 days after infection. It was also noted that astemizole could inhibit prions at 2 µM dose only as compared to the 20 µM dose needed for tacrolimus to do the same at its screening dose. The small size of both molecules also makes them able to cross the BBB with relative ease. The mechanism of action of Astemizole was found to be lysosomotropic, i.e., binding to lysosomes & preventing autophagosome-lysosome fusion. Infectious prion being enzyme-resistant would simply escape cells to spread further by co-opting the autophagy pathway. Therefore Astemizole prevents this by potentially keeping autophagosome-bound infectious prion within the cell itself. Reduction of cell surface PrP leads to a reduction of Alzheimer’s symptoms as well, since Amyloid β peptide oligomers in Alzheimer’s disease bind to cell-surface PrP receptors for expression of symptoms like synaptotoxic effects, neuronal death & memory impairment. So alleviation of such symptoms can occur if treatment with astemizole and tacrolimus is done.
Survival time of mice prolonged by astemizole treatment (source: [4])
References:
Christina D. Orrú , MatildeBongianni, Giovanni Tonoli, Sergio Ferrari, Andrew G. Hughson, Bradley R. Groveman, Michele Fiorini, Maurizio Pocchiari, Salvatore Monaco, Byron Caughey, and GianluigiZanusso (2014) “A Test for CJD using Nasal Brushings” doi:10.1056/NEJMoa1315200.
Jones DR, Taylor WA, Bate C, David M, Tayebi M (2010) “A Camelid Anti-PrP Antibody Abrogates PrPSc Replication in Prion-Permissive Neuroblastoma Cell Lines.” PLoS ONE 5(3): e9804. doi:10.1371/journal.pone.0009804.
Johnson CJ, Bennett JP, Biro SM, Duque-Velasquez JC, Rodriguez CM, et al. (2011) “Degradation of the Disease-Associated Prion Protein by a Serine Protease from Lichens.” PLoS ONE 6(5): e19836. doi:10.1371/journal.pone.0019836.
Karapetyan YE, Sferraza GF, Zhou M, Ottenberg G, Spicer T, Chase P, Falahi M, Hodder P, Weismann C, Lasmezas CI (2013) “Unique drug screening approach for prion diseases identifies tacrolimus and astemizole as antiprion agents”.
Nelson, M. P., &Shacka, J. J. (2013). Autophagy Modulation in Disease Therapy: Where Do We Stand? Current Pathobiology Reports, 1(4), 239–245. http://doi.org/10.1007/s40139-013-0032-9.
Collier, L., Kellam, P. & Oxford, J. (2011). Human Virology. (4th edition).
Author: Abhranil Gangopadhayya
Hi, my name is Abhranil Gangopadhayya! I'm a 1st year MSc Virology student at the National Institute of Virology and I have an undying passion for the enigmatic realm of viruses. I'm basically into all the organisms or beings at the fringes of understanding, be it viruses, viroids, prions, or virophages, so I'd like to explore more and show the world more about these wonderful spectacles of nature. Hope you like this article, Thanks for reading :)
Comments