Oscillations in Neuronal Activity: A Neuron-Centered Spatiotemporal Model of the Unfolded Protein Response in Prion Diseases.

Many neurodegenerative diseases (NDs) are characterized by the slow spatial spread of toxic protein species in the brain. The toxic proteins can induce neuronal stress, triggering the Unfolded Protein Response (UPR), which slows or stops protein translation and can indirectly reduce the toxic load. However, the UPR may also trigger processes leading to apoptotic cell death and the UPR is implicated in the progression of several NDs. In this paper, we develop a novel mathematical model to describe the spatiotemporal dynamics of the UPR mechanism for prion diseases. Our model is centered around a single neuron, with representative proteins P (healthy) and S (toxic) interacting with heterodimer dynamics (S interacts with P to form two S's). The model takes the form of a coupled system of nonlinear reaction-diffusion equations with a delayed, nonlinear flux for P (delay from the UPR). Through the delay, we find parameter regimes that exhibit oscillations in the P- and S-protein levels. We find that oscillations are more pronounced when the S-clearance rate and S-diffusivity are small in comparison to the P-clearance rate and P-diffusivity, respectively. The oscillations become more pronounced as delays in initiating the UPR increase. We also consider quasi-realistic clinical parameters to understand how possible drug therapies can alter the course of a prion disease. We find that decreasing the production of P, decreasing the recruitment rate, increasing the diffusivity of S, increasing the UPR S-threshold, and increasing the S clearance rate appear to be the most powerful modifications to reduce the mean UPR intensity and potentially moderate the disease progression.

The Role of Glial Cells in Neurobiology and Prion Neuropathology.

Prion diseases are rare and neurodegenerative diseases that are characterized by the misfolding and infectious spread of the prion protein in the brain, causing progressive and irreversible neuronal loss and associated clinical and behavioral manifestations in humans and animals, ultimately leading to death. The brain has a complex network of neurons and glial cells whose crosstalk is critical for function and homeostasis. Although it is established that prion infection of neurons is necessary for clinical disease to occur, debate remains in the field as to the role played by glial cells, namely astrocytes and microglia, and whether these cells are beneficial to the host or further accelerate disease. Here, we review the current literature assessing the complex morphologies of astrocytes and microglia, and the crosstalk between these two cell types, in the prion-infected brain.

Molecular signatures in prion disease: altered death receptor pathways in a mouse model.

BACKGROUND: Prion diseases are transmissible and fatal neurodegenerative diseases characterized by accumulation of misfolded prion protein isoform (PrPSc), astrocytosis, microgliosis, spongiosis, and neurodegeneration. Elevated levels of cell membrane associated PrPSc protein and inflammatory cytokines hint towards the activation of death receptor (DR) pathway/s in prion diseases. Activation of DRs regulate, either cell survival or apoptosis, autophagy and necroptosis based on the adaptors they interact. Very little is known about the DR pathways activation in prion disease. DR3 and DR5 that are expressed in normal mouse brain were never studied in prion disease, so also their ligands and any DR adaptors. This research gap is notable and investigated in the present study. METHODS: C57BL/6J mice were infected with Rocky Mountain Laboratory scrapie mouse prion strain. The progression of prion disease was examined by observing morphological and behavioural abnormalities. The levels of PrP isoforms and GFAP were measured as the marker of PrPSc accumulation and astrocytosis respectively using antibody-based techniques that detect proteins on blot and brain section. The levels of DRs, their glycosylation and ectodomain shedding, and associated factors warrant their examination at protein level, hence western blot analysis was employed in this study. RESULTS: Prion-infected mice developed motor deficits and neuropathology like PrPSc accumulation and astrocytosis similar to other prion diseases. Results from this research show higher expression of all DR ligands, TNFR1, Fas and p75NTR but decreased levels DR3 and DR5. The levels of DR adaptor proteins like TRADD and TRAF2 (primarily regulate pro-survival pathways) are reduced. FADD, which primarily regulate cell death, its level remains unchanged. RIPK1, which regulate pro-survival, apoptosis and necroptosis, its expression and proteolysis (inhibits necroptosis but activates apoptosis) are increased. CONCLUSIONS: The findings from the present study provide evidence towards the involvement of DR3, DR5, DR6, TL1A, TRAIL, TRADD, TRAF2, FADD and RIPK1 for the first time in prion diseases. The knowledge obtained from this research discuss the possible impacts of these 16 differentially expressed DR factors on our understanding towards the multifaceted neuropathology of prion diseases and towards future explorations into potential targeted therapeutic interventions for prion disease specific neuropathology.

Unmet needs of biochemical biomarkers for human prion diseases.

Although the development of aggregation assays has noticeably improved the accuracy of the clinical diagnosis of prion diseases, research on biomarkers remains vital. The major challenges to overcome are non-invasive sampling and the exploration of new biomarkers that may predict the onset or reflect disease progression. This will become extremely important in the near future, when new therapeutics are clinically evaluated and eventually become available for treatment. This article aims to provide an overview of the achievements of biomarker research in human prion diseases, addresses unmet needs in the field, and points out future perspectives.

Exploring Fundamentals of Prion Biology Using Natural Yeast Prions and Mammalian PrP.

The key postulate of the prion paradigm is that some proteins can take on unconventional conformations and pass these conformations to newly synthesized protein molecules with the same primary structure [...].

Sigma Receptor Ligands Are Potent Antiprion Compounds that Act Independently of Sigma Receptor Binding.

Prion diseases are invariably fatal neurodegenerative diseases of humans and other animals for which there are no effective treatment options. Previous work from our laboratory identified phenethylpiperidines as a novel class of anti-prion compounds. While working to identify the molecular target(s) of these molecules, we unexpectedly discovered ten novel antiprion compounds based on their known ability to bind to the sigma receptors, σ1R and σ2R, which are currently being tested as therapeutic or diagnostic targets for cancer and neuropsychiatric disorders. Surprisingly, however, knockout of the respective genes encoding σ1R and σ2R (Sigmar1 and Tmem97) in prion-infected N2a cells did not alter the antiprion activity of these compounds, demonstrating that these receptors are not the direct targets responsible for the antiprion effects of their ligands. Further investigation of the most potent molecules established that they are efficacious against multiple prion strains and protect against downstream prion-mediated synaptotoxicity. While the precise details of the mechanism of action of these molecules remain to be determined, the present work forms the basis for further investigation of these compounds in preclinical studies. Given the therapeutic utility of several of the tested compounds, including rimcazole and haloperidol for neuropsychiatric conditions, (+)-pentazocine for neuropathic pain, and the ongoing clinical trials of SA 4503 and ANAVEX2-73 for ischemic stroke and Alzheimer's disease, respectively, this work has immediate implications for the treatment of human prion disease.

Improvement of anti-prion efficacy with stearoxy conjugation of hydroxypropyl methylcellulose in prion-infected mice.

Prion diseases are fatal transmissible neurodegenerative disorders. Among known anti-prions, hydroxypropyl methylcellulose compounds (HPMCs) are unique in their chemical structure and action. They have several excellent anti-prion properties but the effectiveness depends on the prion-infected mouse model. In the present study, we investigated the effects of stearoxy-modified HPMCs on prion-infected cells and mice. Stearoxy modification improved the anti-prion efficacy of HPMCs in prion-infected cells and significantly prolonged the incubation period in a lower HPMC-responding mouse model. However, stearoxy modification showed no improvement over nonmodified HPMCs in an HPMC-responding mouse model. These results offer a new line of inquiry for use with prion-infected mice that do not respond well to HPMCs.

Anti-prion drugs do not improve survival in novel knock-in models of inherited prion disease.

Prion diseases uniquely manifest in three distinct forms: inherited, sporadic, and infectious. Wild-type prions are responsible for the sporadic and infectious versions, while mutant prions cause inherited variants like fatal familial insomnia (FFI) and familial Creutzfeldt-Jakob disease (fCJD). Although some drugs can prolong prion incubation times up to four-fold in rodent models of infectious prion diseases, no effective treatments for FFI and fCJD have been found. In this study, we evaluated the efficacy of various anti-prion drugs on newly-developed knock-in mouse models for FFI and fCJD. These models express bank vole prion protein (PrP) with the pathogenic D178N and E200K mutations. We applied various drug regimens known to be highly effective against wild-type prions in vivo as well as a brain-penetrant compound that inhibits mutant PrPSc propagation in vitro. None of the regimens tested (Anle138b, IND24, Anle138b + IND24, cellulose ether, and PSCMA) significantly extended disease-free survival or prevented mutant PrPSc accumulation in either knock-in mouse model, despite their ability to induce strain adaptation of mutant prions. Our results show that anti-prion drugs originally developed to treat infectious prion diseases do not necessarily work for inherited prion diseases, and that the recombinant sPMCA is not a reliable platform for identifying compounds that target mutant prions. This work underscores the need to develop therapies and validate screening assays specifically for mutant prions, as well as anti-prion strategies that are not strain-dependent.

Prion meeting 2023: implications of a growing field.

The history of human prion diseases began with the original description, by Hans Gerhard Creutzfeldt and by Alfons Maria Jakob, of patients with a severe brain disease that included speech abnormalities, confusion, and myoclonus, in a disease that was then named Creutzfeldt Jakob disease (CJD). Later, in Papua New Guinea, a disease characterized by trembling was identified, and given the name "Kuru". Neuropathological examination of the brains from CJD and Kuru patients, and of brains of sheep with scrapie disease revealed significant similarities and suggested a possible common mode of infection that, at the time, was thought to derive from an unknown virus that caused slow infections. John Stanley Griffith hypothesized that the agent causing these diseases was "probably a protein without nucleic acid" and, in 1982, Stanley Prusiner reported the identification of a proteinaceous infectious particle (coining the term prion) that was resistant to inactivation methods that were at the time standard for nucleic acids, and identified PrP as the major protein component of the infectious agent in scrapie and in Creutzfeldt-Jakob disease, classifying this also as a prion disease. Interestingly, the prion concept had been previously expanded to yeast proteins capable of replicating their conformation, seeding their own aggregation and transmitting phenotypic information. The prion concept has been more recently expanded to refer to misfolded proteins that are capable of converting a normal form of a protein into an abnormal form. The quest to understand and treat prion diseases has united a specific research community around the topic, and regular meetings (Prion Meetings) have taken place over the years to enable discussions, train junior researchers, and inspire research in the field.

Novel method for classification of prion diseases by detecting PrPres signal patterns from formalin-fixed paraffin-embedded samples.

Prion disease is an infectious and fatal neurodegenerative disease. Western blotting (WB)-based identification of proteinase K (PK)-resistant prion protein (PrPres) is considered a definitive diagnosis of prion diseases. In this study, we aimed to detect PrPres using formalin-fixed paraffin-embedded (FFPE) specimens from cases of sporadic Creutzfeldt-Jakob disease (sCJD), Gerstmann-Sträussler-Scheinker disease (GSS), glycosylphosphatidylinositol-anchorless prion disease (GPIALP), and V180I CJD. FFPE samples were prepared after formic acid treatment to inactivate infectivity. After deparaffinization, PK digestion was performed, and the protein was extracted. In sCJD, a pronounced PrPres signal was observed, with antibodies specific for type 1 and type 2 PrPres exhibited a strong or weak signals depending on the case. Histological examination of serial sections revealed that the histological changes were compatible with the biochemical characteristics. In GSS and GPIALP, prion protein core-specific antibodies presented as PrPres bands at 8-9 kDa and smear bands, respectively. However, an antibody specific for the C-terminus presented as smears in GSS, with no PrPres detected in GPIALP. It was difficult to detect PrPres in V180I CJD. Collectively, our findings demonstrate the possibility of detecting PrPres in FFPE and classifying the prion disease types. This approach facilitates histopathological and biochemical evaluation in the same sample and is safe owing to the inactivation of infectivity. Therefore, it may be valuable for the diagnosis and research of prion diseases.

Αnti-prion effects of anthocyanins.

Prion diseases, also known as Transmissible Spongiform Encephalopathies (TSEs), are protein-based neurodegenerative disorders (NDs) affecting humans and animals. They are characterized by the conformational conversion of the normal cellular prion protein, PrPC, into the pathogenic isoform, PrPSc. Prion diseases are invariably fatal and despite ongoing research, no effective prophylactic or therapeutic avenues are currently available. Anthocyanins (ACNs) are unique flavonoid compounds and interest in their use as potential neuroprotective and/or therapeutic agents against NDs, has increased significantly in recent years. Therefore, we investigated the potential anti-oxidant and anti-prion effects of Oenin and Myrtillin, two of the most common anthocyanins, using the most accepted in the field overexpressing PrPScin vitro model and a cell free protein aggregation model. Our results, indicate both anthocyanins as strong anti-oxidant compounds, upregulating the expression of genes involved in the anti-oxidant response, and reducing the levels of Reactive Oxygen Species (ROS), produced due to pathogenic prion infection, through the activation of the Keap1-Nrf2 pathway. Importantly, they showcased remarkable anti-prion potential, as they not only caused the clearance of pathogenic PrPSc aggregates, but also completely inhibited the formation of PrPSc fibrils in the Cerebrospinal Fluid (CSF) of patients with Creutzfeldt-Jakob disease (CJD). Therefore, Oenin and Myrtillin possess pleiotropic effects, suggesting their potential use as promising preventive and/or therapeutic agents in prion diseases and possibly in the spectrum of neurodegenerative proteinopathies.

[Clinical characteristics and diagnostics of human spongiform encephalopathies: an update]. / Klinik und Diagnostik humaner spongiformer Enzephalopathien: ein Update.

Human spongiform encephalopathies are rare transmissible neurodegenerative diseases of the brain and the nervous system that are caused by misfolding of the physiological prion protein into a pathological form and its deposition in the central nervous system (CNS). Prion diseases include Creutzfeldt-Jakob disease (CJD, sporadic or familial), Gerstmann-Straussler-Scheinker syndrome (GSS) and fatal familial insomnia (FFI). Prion diseases can be differentiated into three etiological categories: spontaneous (sporadic CJD), inherited (familial CJD, FFI, and GSS) and acquired (variant CJD and iatrogenic CJD). Most cases occur sporadically. Prion diseases can lead to a variety of neurological symptoms and always have an inevitably fatal course. Cerebrospinal fluid analysis and magnetic resonance imaging (MRI) play a crucial role in the diagnostics of prion diseases and may facilitate an early and reliable clinical diagnosis. A causal treatment or specific therapeutic agents are not yet available. In general, a palliative therapeutic concept is indicated.

Foiling deadly prions.

Can the course of fatal prion diseases be changed by removing the protein before it goes bad?

Infectious Diseases of the Brain and Spine: Parasitic and Other Atypical Transmissible Diseases.

Atypical infections of the brain and spine caused by parasites occur in immunocompetent and immunosuppressed hosts, related to exposure and more prevalently in endemic regions. In the United States, the most common parasitic infections that lead to central nervous system manifestations include cysticercosis, echinococcosis, and toxoplasmosis, with toxoplasmosis being the most common opportunistic infection affecting patients with advanced HIV/AIDS. Another rare but devastating transmittable disease is prion disease, which causes rapidly progressive spongiform encephalopathies. Familiarity and understanding of various infectious agents are a crucial aspect of diagnostic neuroradiology, and recognition of unique features can aid timely diagnosis and treatment.

Targeting Neuroinflammation by Pharmacologic Downregulation of Inflammatory Pathways Is Neuroprotective in Protein Misfolding Disorders.

Neuroinflammation plays a crucial role in the development of neurodegenerative protein misfolding disorders. This category of progressive diseases includes, but is not limited to, Alzheimer's disease, Parkinson's disease, and prion diseases. Shared pathogenesis involves the accumulation of misfolded proteins, chronic neuroinflammation, and synaptic dysfunction, ultimately leading to irreversible neuronal loss, measurable cognitive deficits, and death. Presently, there are few to no effective treatments to halt the advancement of neurodegenerative diseases. We hypothesized that directly targeting neuroinflammation by downregulating the transcription factor, NF-κB, and the inflammasome protein, NLRP3, would be neuroprotective. To achieve this, we used a cocktail of RNA targeting therapeutics (SB_NI_112) shown to be brain-penetrant, nontoxic, and effective inhibitors of both NF-κB and NLRP3. We utilized a mouse-adapted prion strain as a model for neurodegenerative diseases to assess the aggregation of misfolded proteins, glial inflammation, neuronal loss, cognitive deficits, and lifespan. Prion-diseased mice were treated either intraperitoneally or intranasally with SB_NI_112. Behavioral and cognitive deficits were significantly protected by this combination of NF-κB and NLRP3 downregulators. Treatment reduced glial inflammation, protected against neuronal loss, prevented spongiotic change, rescued cognitive deficits, and significantly lengthened the lifespan of prion-diseased mice. We have identified a nontoxic, systemic pharmacologic that downregulates NF-κB and NLRP3, prevents neuronal death, and slows the progression of neurodegenerative diseases. Though mouse models do not always predict human patient success and the study was limited due to sample size and number of dosing methods utilized, these findings serve as a proof of principle for continued translation of the therapeutic SB_NI_112 for prion disease and other neurodegenerative diseases. Based on the success in a murine prion model, we will continue testing SB_NI_112 in a variety of neurodegenerative disease models, including Alzheimer's disease and Parkinson's disease.

First report of a novel 108 bp deletion and five novel SNPs in PRNP gene of stray cats and in silico analysis of their possible relation with feline spongiform encephalopathy.

Prion diseases are fatal neurodegenerative diseases affecting humans and animals. A relationship between variations in the prion gene of some species and susceptibility to prion diseases has been detected. However, variations in the prion protein of cats that have close contact with humans and their effect on prion protein are not well-known. Therefore, this study aimed to investigate the variations of prion protein-encoding gene (PRNP gene) in stray cats and to evaluate variants detected in terms of genetic factors associated with susceptibility or resistance to feline spongiform encephalopathy using bioinformatics tools. For this, cat DNA samples were amplified by a PCR targeting PRNP gene and then sequenced to reveal the variations. Finally, the effects of variants on prion protein were predicted by bioinformatics tools. According to the obtained results, a novel 108 bp deletion and nine SNPs were detected. Among SNPs, five (c314A>G, c.454T>A, c.579G>C, c.642G>C and c.672G>C) were detected for the first time in this study. Bioinformatics findings showed that c.579G>C (Q193H), c.454T>A (Y152N) and c.457G>A (E153K) variants have deleterious effects on prion protein and c.579G>C (Q193H) has high amyloid propensities. This study demonstrates prion protein variants of stray cats and their deleterious effects on prion protein for the first time.

Detection of prions in matching post-mortem skin and cerebrospinal fluid samples using second-generation real-time quaking-induced conversion assay.

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.

A Protein Misfolding Shaking Amplification-based method for the spontaneous generation of hundreds of bona fide prions.

Prion diseases are a group of rapidly progressing neurodegenerative disorders caused by the misfolding of the endogenous prion protein (PrPC) into a pathogenic form (PrPSc). This process, despite being the central event underlying these disorders, remains largely unknown at a molecular level, precluding the prediction of new potential outbreaks or interspecies transmission incidents. In this work, we present a method to generate bona fide recombinant prions de novo, allowing a comprehensive analysis of protein misfolding across a wide range of prion proteins from mammalian species. We study more than 380 different prion proteins from mammals and classify them according to their spontaneous misfolding propensity and their conformational variability. This study aims to address fundamental questions in the prion research field such as defining infectivity determinants, interspecies transmission barriers or the structural influence of specific amino acids and provide invaluable information for future diagnosis and therapy applications.