Prion and anti-codon usage: Does infectious PrP alter tRNA abundance to induce misfolding of PrP?

The "protein-only" hypothesis of prion diseases views the infectious agent as devoid of nucleic acids and consisting of misfolded prion proteins (PrP(Sc)) which, upon infiltration into host cells, act as a template that induces transformation of wild-type protein (PrP(C)) to the pathological form by unknown mechanisms. The two isoforms are identical in amino-acid composition. By analogy to reported "silent" mutations in which utilization of relatively rare tRNAs alter protein folding pattern, we postulate that misfolded PrP(Sc) alters tRNAs abundance in prion-infected cells and results in different rates of co-translational folding of PrP, leading to the formation of additional misfolded PrP(Sc). We analyze experiments that might link tRNAs to prions. This concept of "PrP-seed and tRNA-soil" envisages a vicious cycle in which PrP(Sc) levels govern specific tRNA usage, whose alteration subsequently transforms resident PrP(C) to PrP(Sc), causing the cycle to repeat itself ad infinitum.     

Transmissible encephalopathies: speculations and realities

Virtually all transmissible encephalopathies (TSEs), such as scrapie, CJD, and BSE, are caused by a type of infectious particle that remains enigmatic. The language of prion theory supersedes the reality of what is, and what is not known. This review questions the predictive value, consistency and accuracy of this now dominant assumption. Many people believe the normal cellular prion protein (PrP) self-converts into an infectious amyloid protein or prion. Although the amyloidogenic capacity of proteins is well established, the concept of an infectious protein without nucleic acid was "revolutionary." Diverse experiments have repeatedly shown, however, that this protein alone, in any form, is incapable of reproducing transmissible infection. In contrast, the infectious agent copurifies with many other molecules, including nucleic acids, while it separates from the majority of PrP. The infectious particle has a homogeneous viral size of ~25 nm, and infectivity is markedly reduced by conditions that disrupt viral core components but do not disrupt multimers of PrP amyloid. Additionally, the infectious agent replicates to high levels before any PrP abnormalities can be detected. Hence, we initially proposed that PrP changes are part of the host's pathologic response to high levels of infectious agent, but not the agent itself. Newer data clarifying a role for myeloid cells in the spread of infection, the unique character of two different agent strains propagated in a single animal, and the demonstration of long nucleic acids in a variety of simplified high titer preparations continue to raise serious questions for the prion hypothesis. Moreover, the epidemic spread of TSEs, and the activation of host innate immune mechanisms by infection, further indicate these agents are recognizably foreign, and probably viral.     

Generation of antibodies against bovine recombinant prion protein in various strains of mice

Transmissible spongiform encephalopathies (TSEs), also known as prion diseases, belong to a group of neurodegenerative disorders affecting humans and animals. To date, definite diagnosis of prion disease can only be made by analysis of tissue samples for the presence of protease-resistant misfolded prion protein (PrP(Sc)). Monoclonal antibodies (MAbs) to the prion protein provide valuable tools for TSE diagnosis, as well as for basic research on these diseases. In this communication, the development of antibodies against recombinant bovine prion protein (brecPrP) in four strains of mice (BALB/c, ND4, SJL, and NZB/NZW F(1)) is described. Immunization of autoimmunity-prone NZB/NZW F(1) and SJL mice with brecPrP was applied to overcome self-tolerance against the prion protein. ND4 and SJL mice did not develop an immune response to brecPrP. BALB/c mice produced antibody titers of 1:1,000 to 1:1,500 in an enzyme-linked immunosorbent assay (ELISA), while NZB/NZW F(1) mice responded with titers of 1:7,000 to 1:11,000. A panel of 71 anti-brecPrP MAbs recognizing continuous and discontinuous epitopes was established from BALB/c and NZB/NZW F(1) mice. Seven anti-brecPrP MAbs reacted with both the cellular form of PrP and protease K-resistant PrP(Sc) from sheep brain in Western blot assays. The epitope specificity of these MAbs was determined, and applicability to immunohistochemical detection of prions was studied. The MAbs generated will be useful tools in the development of TSE immunochemical diagnosis and for research. This is the first report of the development of anti-PrP MAbs by use of autoimmune NZB/NZW F(1) mice as an alternative approach for the generation of PrP-specific MAbs.     

朊蛋白相关蛋白Shadoo与朊病毒病

朊病毒病,即传染性海绵状脑病(transmissible spongiform encephalopathies,TSEs),是一类传染性、致死性神经退行性疾病.在朊病毒病的病理过程中,细胞正常朊蛋白PrPC转化为异常构象的PrPSc是至关重要的,但是PrPC的正常生理功能仍不清楚.国外学者利用比较基因组学发现了一个新的朊蛋白相关蛋白-Shadoo(Sho).Sho与PrPC在氨基酸序列和细胞定位的相似性及主要在脑组织表达,使它成为一个非常值得研究的PrP相关蛋白.对Sho可能存在的与PrPC重叠的功能甚至直接相互作用的研究工作,将对今后揭示PrPC正常生理功能以及揭示Prion病发病机制具有重要现实意义.     

Transmissible spongiform encephalopathies and the 'rogue share-holder protein' hypothesis

Transmissible spongiform encephalopathies(TSEs) or prion diseases are a closely related group of diseases, whose exact etiology is unknown, but is generally accepted to be related to protease-resistant prion protein PrP. PrPc is normally present in cells and its disease counterpart PrPsc is postulated to occur due to a rare stochastic change. The selfish gene hypothesis is a generally well accepted concept in evolutionary biology. Genes can be likened to the board of a company and proteins can be likened to share-holders. Here it is being hypothesized that a rogue share-holder protein's 'selfish' replicatory tendency might be the explanation for TSEs. The present hypothesis predicts existence of other examples of rogue share-holder protein and also predicts that examples would be found in lower life-forms as well.     

Newly discovered forms of prion diseases in ruminants

Transmissible spongiform encephalopathies (TSEs), are fatal neurodegenerative diseases caused by unconventional agents, the prions. They are characterised by the accumulation in infected tissues of an abnormally folded form of the host-encoded prion protein (PrP). This pathological form is partially resistant to protease digestion, leading to the production of so-called PrP(res) fragments. Different isolates from the same host species may show different eletrophoretic profiles, reflecting the existence of different prion strains. The active surveillance of ruminant TSEs implemented in European countries, based on a large-scale biochemical testing of brain tissue samples from carcasses, has revealed PrP(res) profiles unnoticed so far. Experimental transmission of these atypical cases to various transgenic mouse lines has led to the recognition of a novel scrapie strain in sheep and goats, called Nor98, and of two variant strains of spongiform encephalopathy in cattle. This review is aimed at summarising the current knowledge on these newly recognised forms of ruminants TSEs, and at discussing their possible origin and potential implications in terms of animal and human health.     

Clusterin expression in follicular dendritic cells associated with prion protein accumulation

Peripheral accumulation of abnormal prion protein (PrP) in variant Creutzfeldt-Jakob disease and some animal models of transmissible spongiform encephalopathies (TSEs) may occur in the lymphoreticular system. Within the lymphoid tissues, abnormal PrP accumulation occurs on follicular dendritic cells (FDCs). Clusterin (apolipoprotein J) has been recognized as one of the molecules associated with PrP in TSEs, and clusterin expression is increased in the central nervous system where abnormal PrP deposition has occurred. We therefore examined peripheral clusterin expression in the context of PrP accumulation on FDCs in a range of human and experimental TSEs. PrP was detected immunohistochemically on tissue sections using a novel highly sensitive method involving detergent autoclaving pretreatment. A dendritic network pattern of clusterin immunoreactivity in lymphoid follicles was observed in association with the abnormal PrP on FDCs. The increased clusterin immunoreactivity appeared to correlate with the extent of PrP deposition, irrespective of the pathogen strains, host mouse strains or various immune modifications. The observed co-localization and correlative expression of these proteins suggested that clusterin might be directly associated with abnormal PrP. Indeed, clusterin immunoreactivity in association with PrP was retained after FDC depletion. Together these data suggest that clusterin may act as a chaperone-like molecule for PrP and play an important role in TSE pathogenesis.     

Physiology of the prion protein

Prion diseases are transmissible spongiform encephalopathies (TSEs), attributed to conformational conversion of the cellular prion protein (PrP(C)) into an abnormal conformer that accumulates in the brain. Understanding the pathogenesis of TSEs requires the identification of functional properties of PrP(C). Here we examine the physiological functions of PrP(C) at the systemic, cellular, and molecular level. Current data show that both the expression and the engagement of PrP(C) with a variety of ligands modulate the following: 1) functions of the nervous and immune systems, including memory and inflammatory reactions; 2) cell proliferation, differentiation, and sensitivity to programmed cell death both in the nervous and immune systems, as well as in various cell lines; 3) the activity of numerous signal transduction pathways, including cAMP/protein kinase A, mitogen-activated protein kinase, phosphatidylinositol 3-kinase/Akt pathways, as well as soluble non-receptor tyrosine kinases; and 4) trafficking of PrP(C) both laterally among distinct plasma membrane domains, and along endocytic pathways, on top of continuous, rapid recycling. A unified view of these functional properties indicates that the prion protein is a dynamic cell surface platform for the assembly of signaling modules, based on which selective interactions with many ligands and transmembrane signaling pathways translate into wide-range consequences upon both physiology and behavior.     

Squirrel monkeys (Saimiri sciureus) infected with the agent of bovine spongiform encephalopathy develop tau pathology

Squirrel monkeys (Saimiri sciureus) were infected experimentally with the agent of classical bovine spongiform encephalopathy (BSE). Two to four years later, six of the monkeys developed alterations in interactive behaviour and cognition and other neurological signs typical of transmissible spongiform encephalopathy (TSE). At necropsy examination, the brains from all of the monkeys showed pathological changes similar to those described in variant Creutzfeldt-Jakob disease (vCJD) of man, except that the squirrel monkey brains contained no PrP-amyloid plaques typical of that disease. Constant neuropathological features included spongiform degeneration, gliosis, deposition of abnormal prion protein (PrP(TSE)) and many deposits of abnormally phosphorylated tau protein (p-Tau) in several areas of the cerebrum and cerebellum. Western blots showed large amounts of proteinase K-resistant prion protein in the central nervous system. The striking absence of PrP plaques (prominent in brains of cynomolgus macaques [Macaca fascicularis] with experimentally-induced BSE and vCJD and in human patients with vCJD) reinforces the conclusion that the host plays a major role in determining the neuropathology of TSEs. Results of this study suggest that p-Tau, found in the brains of all BSE-infected monkeys, might play a role in the pathogenesis of TSEs. Whether p-Tau contributes to development of disease or appears as a secondary change late in the course of illness remains to be determined.     

Validation of a luminescence immunoassay for the detection of PrP(Sc) in brain homogenate

A luminescence immunoassay (LIA) was developed for the diagnosis of bovine spongiform encephalopathy (BSE) in brain tissue using two different monoclonal antibodies for capture and detection of the protease-resistant fragment of the pathological prion protein (PrP27-30). PrP27-30 currently represents the most reliable marker for the infectious particle (denominated prion) causing transmissible spongiform encephalopathies (TSEs). Internal and official validation studies of this assay are described using brain homogenates from ascertained BSE positive and negative cows. Using more than 300 positive and 1400 negative bovine or ovine samples, an excellent sensitivity and specificity of 100% were demonstrated. More than 1000-fold dilutions of a BSE positive homogenate still resulted in a clear positive signal. In combination with a simple homogenisation procedure for the preparation of the samples, this assay lends itself for large scale screening of cattle and sheep for TSEs using complete automation of the process.     

Knockdown of prion protein (PrP) by RNA interference weakens the protective activity of wild-type PrP against copper ion and antagonizes the cytotoxicity of fCJD-associated PrP mutants in cultured cells

Development of the pathogenesis of transmissible spongiform encephalopathies (TSEs) requires the presence of both the normal host prion protein (PrPC) and the abnormal pathological proteinase-K resistant isoform (PrPSc). Reduction of PrPC levels has been shown to extend survival time after prion infection. In this report, based on analysis of the known sequences of human PrP, we constructed two small interfering RNA (siRNA) duplexes targeting the segments of amino acids (aa) 108-114 (Ri2) and aa 171-177 (Ri3). Western blot analysis results revealed that these PrP-specific siRNAs could effectively knock down the levels of either endogenous PrP in human neuroblastoma SHSY-5Y cells or recombinant PrP transfected with the plasmid expressing the full-length human PrP in human embryonic kidney (HEK) 293T cells. Meanwhile, the two siRNAs also showed a significant effect on the reduction of the expression of the PrP-PG9 and PrP-PG12 familial Creutzfeldt-Jakob disease (CJD)-associated PrP mutants with four and seven extra octarepeats, in the cells transfected with the respective expression plasmids. MTT tests identified that knockdown of wild-type PrP by Ri2 and Ri3 did not change the cell growth capacities, but significantly decreased the cell resistances against the challenge of Cu2+. Co-expression of Ri2 and Ri3 partially antagonized the cytotoxicity caused by expressing PrP-PG9 and PrP-PG12 in the two cell lines. Moreover, the rescuing effectiveness of PrP siRNAs was time-related, with the more efficient antagonism of the cytotoxicity of fCJD-associated PrP mutants occurring at the early stages after transfection. The data shown here provide useful clues for seeking potential therapeutic tools for prion diseases.     

Molecular interaction between prion protein and GFAP both in native and recombinant forms in vitro

Gliosis of glial fibrillary acidic protein (GFAP) associated astrocytes is considered to be one of the hallmarks of transmissible spongiform encephalopathies (TSEs). In the present study, remarkable GFAP-PrP(Sc) or GFAP-PrP(C) complexes were separately detected in the brain homogenates of 263 K (Scrapie)-infected or normal hamsters by co-immunoprecipitation assay. To get more exact molecular evidences for interaction between prion protein (PrP) and GFAP, various recombinant PrP or GFAP proteins were expressed using prokaryotic-expressing and in vitro translation system. Using pull down and co-immunoprecipitation assays, reliable molecular interaction between PrP and GFAP was observed, and proteinase K (PK)-digested PrP(Sc) molecules were confirmed to be able to bind the recombinant GFAP specifically as well. The region within PrP that was responsible for interaction with GFAP was narrowed to PK-resistant core of PrP (i.e. aa 91-230). The study of the association of PrP with GFAP supplies the molecular evidence for the observation of co-localization of PrP(Sc) and GFAP in the brains of TSEs and may further provide insight into a potential role of GFAP in the biological function of PrP and the pathogenesis of prion diseases.     

The complement system in prion diseases

The complement system is an essential component of the innate immune system that aids the recognition and destruction of pathogens. Despite the potent cytolytic activity of this system, some pathogens have turned interactions with complement to their advantage. Transmissible spongiform encephalopathies (TSEs), or prion diseases, also seem to use the complement system to facilitate target cell infection in lymphoid tissues. TSEs are associated with the accumulation of disease-specific prion protein in the brain, which is accompanied by progressive neurodegeneration. Unregulated activation of complement can cause host tissue damage, as observed in some neurodegenerative disorders, and active complement components have been detected in the brains of hosts with TSEs. Whether complement inhibitors will be useful to treat TSEs remains to be determined.     

Hypothesis of interference to superinfection between bovine spastic paresis and bovine spongiform encephalopathy; suggestions for experimentation, theoretical and practical interest

Sub-acute transmissible spongiform encephalopathies (TSEs) or prion diseases are diseases of little known etiology. The origin of these diseases would appear to be an abnormal protease-resistant prion protein (PrP(res)) which would be infectious by directly inducing its defective conformation to the normal native protein (PrP(C)). This hypothesis does not account for certain aspects of TSEs, such as interference to superinfection: in laboratory animals, inoculation by means of an attenuated strain with a long incubation period protects against later infection by a very virulent strain with a short incubation period. The hypothesis is put forward that there exists a possibility of interference to superinfection between neurodegenerative diseases of unknown origin, thought to be similar to TSEs, and a later infection by a TSE. The study of this interference between bovine spastic paresis (BSP) and bovine spongiform encephalopathy (BSE) could be used as a model for this hypothesis. BSP is a very rare disease among cattle, of unknown etiology; it is curable, in the very early stages, by using tryptophan and especially lithium, potentiated by copper and manganese. An etiology close to that of TSEs has been suggested on several occasions. If interference could be demonstrated between BSP and BSE, interesting data would be provided concerning the etiology, the pathogenesis and possibly the treatment and prevention of these diseases. Notably, such data could lead to the development of a treatment and a prevention with lithium and amino acids precursors of neuromediators (tryptophan, tyrosine, glutamic acid, etc.), as well as the developing of a vaccine to combat TSEs, especially BSE and scrapie.     

Fast, reversible interaction of prion protein with RNA aptamers containing specific sequence patterns

Summary. One of the unsolved problems in prion diseases relates to the physiological function of cellular prion protein (PrP), of which a misfolded isoform is the major component of the transmissible spongiform encephalopathies agent. Knowledge of the PrP-binding molecules may help in elucidating its role and understanding the pathological events underlying prion diseases. Because nucleic acids are known to bind PrP, we attempted to identify the preferred RNA sequences that bind to the ovine recombinant PrP. An in vitro selection approach (SELEX) was applied to a pool of 80-nucleotide(nt)-long RNAs containing a randomised 40-nt central region. The most frequently isolated aptamer, RM312, was also the best ligand (20 nM K_D value), according to both surface plasmon resonance and filter binding assays. The fast rates of association and dissociation of RM312 with immobilized PrP, which are reminiscent of biologically relevant interactions, could point to a physiological function of PrP towards cellular nucleic acids. The minimal sequence that we found necessary for binding of RM312 to PrP presents a striking similarity with one previously described PrP aptamer of comparable affinity. In addition, we here identify the two lysine clusters contained in the N-terminal part of PrP as its main nucleic-acid binding sites.     

Atypical Prion Protein Conformation in Familial Prion Disease with PRNP P105T Mutation

Protease‐resistant prion protein (PrP^Sc) is diagnostic of prion disease, yet its detection is frequently difficult. Here, we describe a patient with a PRNP P105T mutation and typical familial prion disease. Brain PrP^Sc was undetectable by conventional Western blotting and barely detectable after phosphotungstate precipitation, where it displayed an atypical pattern suggestive of noncanonical conformation. Therefore, we used a novel misfolded protein assay (MPA) that detects PrP aggregates independently of their protease resistance. The MPA revealed the presence of aggregated PrP in similar amounts as in typical sporadic Creutzfeldt‐Jakob disease. These findings suggest that measurements of PrP aggregation with the MPA may be potentially more sensitive than protease‐based methodologies.     

Autophagy induced by resveratrol prevents human prion protein-mediated neurotoxicity

Our previous study revealed that resveratrol blocks prion protein peptide PrP(106-126)-induced neurotoxicity. However, the mechanism of resveratrol-mediated neuroprotection in prion diseases is not clear. Resverstrol initiates neuroprotective effects via the activation of autophagy, which protects organelles, cells, and organisms against misfolded protein-disorders, including Alzheimer's disease and Parkinson's disease via regulation of mitochondrial homeostasis. Thus, we focused on elucidating the mechanisms responsible for resveratrol-mediated neuroprotection related to mitochondrial homeostasis as a result of autophagy activation. Resveratrol prevented PrP(106-126)-induced neuronal cell death by activating autophagy. Moreover, resveratrol-induced autophagy prevented the PrP(106-126)-induced reduction in mitochondrial potential and translocation of Bax to the mitochondria and cytochrome c release. Our results indicate that treatment with resveratrol appears to protect against neurotoxicity caused by prion protein peptides and the neuroprotection is induced by resveratrol-mediated autophagy signals.     

The pathogenic mechanisms of prion diseases

Transmissible spongiform encephalopathies (TSEs) or prion diseases are a group of fatal neurodegenerative diseases of humans and animals, including bovine spongiform encephalopathy (BSE) of cattle, scrapie of sheep, and Creutzfeldt-Jakob disease (CJD) of humans. Prion diseases have become an important issue in public health and in the scientific world not only due to the possible relationship between BSE and new variant CJD (nvCJD) but also due to the unique biological features of the infectious agent. Although the nature of the infectious agent and the pathogenic mechanisms of prion diseases are not fully understood, considerable evidence suggests that an abnormal form (PrP(Sc)) of a host prion protein (PrP(C)) may compose substantial parts of the infectious agent and that various factors such as oxidative stress and calcium cytotoxicity are associated with the pathogenesis of prion diseases. Here, we briefly review and discuss the pathogenic mechanisms of prion diseases. These advances in understandings of fundamental biology of prion diseases may open the possibilities for the prevention and treatment of these unusual diseases and also suggest applications in more common neurodegenerative disorders such as Alzheimer's disease (AD) and Parkinson's disease (PD).