Elimination of [PSI+] Prion and Influence on Yeast Cell Growth

Background Prions are proteinaceous infectious particles that act as pathogens and cause the development of lethal neurodegenerative diseases in humans and other animals. Yeast Saccharomyces cerevisiae is a widespread model system in which mechanisms of prion induction and elimination have been identified. New and safe substances and methods are being sought to cure cells of prion proteins. It is particularly important that by treating cells from prions and restoring them from the [PSI+] to the [psi−] form, the primary growth of the cells is restored. One of the main objectives of this study was to determine the growth dynamics of S. cerevisiae cells with different [PSI+] prion variants, cells that have lost [PSI+] prion variants, and cells that never had [PSI+] prion variants. Results In this research, we applied GuHCl and combined GuHCl and PEF treatment against [PSI+] prion. We evaluated cells culture growth dynamics – optical density and doubling time and determined that method of [PSI+] prion elimination does not affect cell doubling time. Also, we found that both elimination methods affect the optical density reached by [psi−] cells. However, the cells in which the [PSI+] prion has been eliminated by GuHCl alone are able to reach the same optical density as unaffected [psi−] cells and higher optical density than the affected [psi−] cells by GuHCl alone. Conclusions These findings indicate the potential long-term positive effect of [PSI+] prion on cell growth, which persists after [PSI+] removal.

Heterologous Overexpression of Sup35 in E. coli Leads to Both Monomer and Complex States

The heterologous overexpression states of prion proteins play a critical role in understanding the mechanisms of prion-related diseases. We report herein the identification of soluble monomer and complex states for a bakers’ yeast prion, Sup35, when expressed in E. coli. Two peaks are apparent with the elution of His-tagged Sup35 by imidazole from a Ni affinity column. Peak I contains Sup35 in both monomer and aggregated states. Sup35 aggregate is abbreviated as C-aggregate and includes a non-fibril complex comprising Sup35 aggregate-HSP90-Dna K, ATP synthase β unit (chain D), 30S ribosome subunit, and Omp F. The purified monomer and C-aggregate can remain stable for an extended period of time. Peak II contains Sup35 also in both monomer and aggregated (abbreviated as S-aggregate) states, but the aggregated states are caused by the formation of inter-Sup35 disulfide bonds. This study demonstrates that further assembly of Sup35 non-fibril C-aggregate can be interrupted by the chaperone repertoire system in E. coli.

Immunotherapeutic Approaches for the Treatment of Neurodegenerative Diseases: Challenges and Outcomes

Background: Neurodegenerative diseases, being rapidly increasing disorders and the seventh leading cause of death worldwide, have been a great challenge for researchers, affecting cognition, motor activity and other body functioning due to neurodegeneration. Several neurodegenerative diseases are caused by aggregation of proteins which induce the alteration of neuronal function leading to cell death. These proteins are amyloid-β peptide, tau, α-synuclein, and mHTT, which cause Alzheimer’s disease, Frontotemporal dementia, Corticobasal degeneration, Progressive supranuclear palsy, Parkinson’s disease, Multiple system atrophy, Dementia with Lewy-body and Huntington’s disease. Currently available treatments only reduce symptoms and increase life sustainability; however, they possess side effects and are ineffective in curing the diseases. Objectives: Literature survey of neurodegenerative diseases and immunotherapeutic approaches used to evaluate their pharmacological effects and future endeavours. Methods: A literature search was performed to find the relevant articles related to neurodegenerative diseases and immunotherapies. Clinical trials data were analysed from clinicaltrial.com. Result: According to literature study, it was found that researchers have explored the effect of active and passive vaccines generated against amyloid-β, tau, α-synuclein and mHTT. Few clinical trials have shown severe side effects and terminated, despite of that, few of them produced desirable effects for the treatment of AD and PD. Conclusion: Several immunotherapeutic trials have shown promising outcomes against amyloid-β, tau and α-synuclein. In addition, various preclinical studies against mHTT and prion proteins are under scrutinization. These clinical outcomes indicate promising role of immunotherapies against neurodegenerative diseases.

The Role of the Nasal Cavity in the Pathogenesis of Prion Diseases

Prion diseases, or transmissible spongiform encephalopathies (TSEs), are a class of fatal neurodegenerative diseases caused by the entry and spread of infectious prion proteins (PrPSc) in the central nervous system (CNS). These diseases are endemic to certain mammalian animal species that use their sense of smell for a variety of purposes and therefore expose their nasal cavity (NC) to PrPSc in the environment. Prion diseases that affect humans are either inherited due to a mutation of the gene that encodes the prion protein, acquired by exposure to contaminated tissues or medical devices, or develop without a known cause (referred to as sporadic). The purpose of this review is to identify components of the NC that are involved in prion transport and to summarize the evidence that the NC serves as a route of entry (centripetal spread) and/or a source of shedding (centrifugal spread) of PrPSc, and thus plays a role in the pathogenesis of the TSEs.

Hofmeister Effect in RT-QuIC Seeding Activity of Chronic Wasting Disease Prions

Chronic wasting disease (CWD) is a transmissible spongiform encephalopathy (TSE) that causes a fatal neurodegenerative disease in cervids. Cases of CWD are rapidly increasing in North America among wild and farmed cervid populations, and potential for zoonotic transmission is not yet determined. Therefore, in order to manage the disease, it is imperative to devise a system that can detect CWD during its early phases to prevent spread to new captive herds through introduction of CWD-affected animals into otherwise CWD-free herds. Real-time quaking–induced conversion (RT-QuIC) assays have been applied to detect the presence of disease-associated prions from various samples in both animals and humans. In this study, we have tested the use of five Hofmeister anions that range from weakly hydrating to strongly hydrating: Na3citrate, Na2SO4, NaCl, NaI, and NaClO4 in RT-QuIC reactions for CWD seeding activity using different recombinant prion proteins as substrates. This work shows how the ionic environment of the RT-QuIC reaction can enhance or diminish the seeding activity. The use of Na2SO4 or NaI as the sodium salt for RT-QuIC using bank vole recombinant prion substrate for the detection of CWD using brain samples reduces the lag time to detect with reasonable specificity. For detection of the CWD in fecal samples, only NaI showed comparable reduction in lag time relative to NaCl but required reduced temperature to alleviate spontaneous fibril formation in negative control samples. Selection of the proper ion environment and recombinant prion protein substrate will make RT-QuIC a powerful diagnostic tool for early detection of CWD prions, further supporting CWD surveillance in wild and captive cervids.

Development of α-Synuclein Real-Time Quaking-Induced Conversion as a Diagnostic Method for α-Synucleinopathies

Parkinson’s disease, dementia with Lewy bodies, and multiple system atrophy are characterized by aggregation of abnormal α-synuclein (α-syn) and collectively referred to as α-synucleinopathy. Because these diseases have different prognoses and treatments, it is desirable to diagnose them early and accurately. However, it is difficult to accurately diagnose these diseases by clinical symptoms because symptoms such as muscle rigidity, postural dysreflexia, and dementia sometimes overlap among these diseases. The process of conformational conversion and aggregation of α-syn has been thought similar to that of abnormal prion proteins that cause prion diseases. In recent years, in vitro conversion methods, such as real-time quaking-induced conversion (RT-QuIC), have been developed. This method has succeeded in amplifying and detecting trace amounts of abnormal prion proteins in tissues and central spinal fluid of patients by inducing conversion of recombinant prion proteins via shaking. Additionally, it has been used for antemortem diagnosis of prion diseases. Recently, aggregated α-syn has also been amplified and detected in patients by applying this method and many clinical studies have examined diagnosis using tissues or cerebral spinal fluid from patients. In this review, we discuss the utility and problems of α-syn RT-QuIC for antemortem diagnosis of α-synucleinopathies.

Exploring Drug and Antibody-Based Treatment Options for Creutzfeldt-Jakob Disease

Creutzfeldt-Jakob Disease (CJD) is a neurodegenerative disease characterized by mutant PrP prion proteins, which accumulates and impairs the function of wild-type PrPc proteins. The interaction of prion proteins with wild-type proteins converts the PrPc proteins to mutant PrP proteins. These mutant prion proteins lead to neural tissue degradation and other nervous system problems that can eventually lead to death. The use of antibodies to target and destroy prion proteins can be used to decrease PrP levels that can stop CJD progression. The binding affinities of different anti-PrP Fab antibodies are analyzed to determine which antibody best binds to PrP proteins and targets them for destruction. Through antibody-based targeting of prion proteins, potential treatment methods could be developed for CJD. In addition, the use of drugs, such as quinacrine and doxycycline, also show short-term effects in decreasing the progression of CJD. These drugs extend the average lifespan of tested subjects with CJD but also lead to the development of drug-resistant prion proteins that eventually cause the death of the subject affected by CJD.

MicroRNAs in Prion Diseases—From Molecular Mechanisms to Insights in Translational Medicine

MicroRNAs (miRNAs) are small non-coding RNA molecules able to post-transcriptionally regulate gene expression via base-pairing with partially complementary sequences of target transcripts. Prion diseases comprise a singular group of neurodegenerative conditions caused by endogenous, misfolded pathogenic (prion) proteins, associated with molecular aggregates. In humans, classical prion diseases include Creutzfeldt–Jakob disease, fatal familial insomnia, Gerstmann–Sträussler–Scheinker syndrome, and kuru. The aim of this review is to present the connections between miRNAs and prions, exploring how the interaction of both molecular actors may help understand the susceptibility, onset, progression, and pathological findings typical of such disorders, as well as the interface with some prion-like disorders, such as Alzheimer’s. Additionally, due to the inter-regulation of prions and miRNAs in health and disease, potential biomarkers for non-invasive miRNA-based diagnostics, as well as possible miRNA-based therapies to restore the levels of deregulated miRNAs on prion diseases, are also discussed. Since a cure or effective treatment for prion disorders still pose challenges, miRNA-based therapies emerge as an interesting alternative strategy to tackle such defying medical conditions.

Proteasome control of [URE3] prion propagation by degradation of anti-prion proteins Cur1 and Btn2 in Saccharomyces cerevisiae

[URE3] is a prion of the nitrogen catabolism controller, Ure2p, and [PSI+] is a prion of the translation termination factor Sup35p in S. cerevisiae. Btn2p cures [URE3] by sequestration of Ure2p amyloid filaments. Cur1p, paralogous to Btn2p, also cures [URE3], but by a different (unknown) mechanism. We find that an array of mutations impairing proteasome assembly or MG132 inhibition of proteasome activity result in loss of [URE3]. In proportion to their prion—curing effects, each mutation affecting proteasomes elevates the cellular concentration of the antiprion proteins Btn2 and Cur1. Of > 4600 proteins detected by SILAC, Btn2p was easily the most overexpressed in a pre9Δ (α3 core subunit) strain. Indeed, deletion of BTN2 and CUR1 prevents the prion—curing effects of proteasome impairment. Surprisingly, the 15 most unstable yeast proteins are not increased in pre9Δ cells suggesting altered proteasome specificity rather than simple inactivation. Hsp42, a chaperone that cooperates with Btn2 and Cur1 in curing [URE3], is also necessary for the curing produced by proteasome defects, although Hsp42p levels are not substantially altered by a proteasome defect. We find that pre9Δ and proteasome chaperone mutants that most efficiently lose [URE3], do not destabilize [PSI+] or alter cellular levels of Sup35p. A tof2 mutation or deletion likewise destabilizes [URE3], and elevates Btn2p, suggesting that Tof2p deficiency inactivates proteasomes. We suggest that when proteasomes are saturated with denatured/misfolded proteins, their reduced degradation of Btn2p and Cur1p automatically upregulates these aggregate-handling systems to assist in the clean-up.