Adaptation of the protein misfolding cyclic amplification (PMCA) technique for the screening of anti-prion compounds.

Prion diseases result from the misfolding of the physiological prion protein (PrPC) to a pathogenic conformation (PrPSc). Compelling evidence indicates that prevention and/or reduction of PrPSc replication are promising therapeutic strategies against prion diseases. However, the existence of different PrPSc conformations (or strains) associated with disease represents a major problem when identifying anti-prion compounds. Efforts to identify strain-specific anti-prion molecules are limited by the lack of biologically relevant high-throughput screening platforms to interrogate compound libraries. Here, we describe adaptations to the protein misfolding cyclic amplification (PMCA) technology (able to faithfully replicate PrPSc strains) that increase its throughput to facilitate the screening of anti-prion molecules. The optimized PMCA platform includes a reduction in sample and reagents, as well as incubation/sonication cycles required to efficiently replicate and detect rodent-adapted and cervid PrPSc strains. The visualization of PMCA products was performed via dot blots, a method that contributed to reduced processing times. These technical changes allowed us to evaluate small molecules with previously reported anti-prion activity. This proof-of-principle screening was evaluated for six rodent-adapted prion strains. Our data show that these compounds targeted either none, all or some PrPSc strains at variable concentrations, demonstrating that this PMCA system is suitable to test compound libraries for putative anti-prion molecules targeting specific PrPSc strains. Further analyses of a small compound library against deer prions demonstrate the potential of this new PMCA format to identify strain-specific anti-prion molecules. The data presented here demonstrate the use of the PMCA technique in the selection of prion strain-specific anti-prion compounds.

A versatile tissue-rolling technique for spatial-omics analyses of the entire murine gastrointestinal tract.

Tissues are dynamic and complex biological systems composed of specialized cell types that interact with each other for proper biological function. To comprehensively characterize and understand the cell circuitry underlying biological processes within tissues, it is crucial to preserve their spatial information. Here we report a simple mounting technique to maximize the area of the tissue to be analyzed, encompassing the whole length of the murine gastrointestinal (GI) tract, from mouth to rectum. Using this method, analysis of the whole murine GI tract can be performed in a single slide not only by means of histological staining, immunohistochemistry and in situ hybridization but also by multiplexed antibody staining and spatial transcriptomic approaches. We demonstrate the utility of our method in generating a comprehensive gene and protein expression profile of the whole GI tract by combining the versatile tissue-rolling technique with a cutting-edge transcriptomics method (Visium) and two cutting-edge proteomics methods (ChipCytometry and CODEX-PhenoCycler) in a systematic and easy-to-follow step-by-step procedure. The entire process, including tissue rolling, processing and sectioning, can be achieved within 2-3 d for all three methods. For Visium spatial transcriptomics, an additional 2 d are needed, whereas for spatial proteomics assays (ChipCytometry and CODEX-PhenoCycler), another 3-4 d might be considered. The whole process can be accomplished by researchers with skills in performing murine surgery, and standard histological and molecular biology methods.

Elderly mice with history of acetaminophen intoxication display worsened cognitive impairment and persistent elevation of astrocyte and microglia burden.

Acetaminophen (APAP) is a leading cause of acute liver failure. The effect of APAP metabolite's effects in the periphery are well characterized; however, associated consequences in the brain remain poorly understood. Animal studies on this subject are few and reveal that frequent APAP intake can trigger cerebral abnormalities that vary depending on the subject's age. Alarmingly, experimental efforts have yet to examine associated consequences in elderly hosts, who correspond to the highest risk of medication overload, impaired drug clearance, and cognitive deficits. Here, we interrogated the cerebral and peripheral pathology of elderly mice submitted to monthly episodes of APAP intoxication since a young adult age. We found that weeks after the final episode of recurrent APAP exposure, mice exhibited worsened non-spatial memory deficit whereas spatial memory performance was unaltered. Interestingly, one month after the period of APAP intoxication, these mice showed increased glial burden without associated drivers, namely, blood-brain barrier disruption, cholesterol accumulation, and elevation of inflammatory molecules in the brain and/or periphery. Our experimental study reveals how recurrent APAP exposure affects the cognitive performance and cellular events in elderly brains. These data suggest that APAP-containing pharmacological interventions may foreshadow the elevated risk of neuropsychiatric disorders that afflict elderly populations.

Subcellular functions of tau mediates repair response and synaptic homeostasis in injury.

Injury responses in terminally differentiated cells such as neurons is tightly regulated by pathways aiding homeostatic maintenance. Cancer patients subjected to neuronal injury in brain radiation experience cognitive declines similar to those seen in primary neurodegenerative diseases. Numerous studies have investigated the effect of radiation in proliferating cells of the brain, yet the impact in differentiated, post-mitotic neurons, especially the structural and functional alterations remain largely elusive. We identified that microtubule-associated tau is a critical player in neuronal injury response via compartmentalized functions in both repair-centric and synaptic regulatory pathways. Ionizing radiation-induced injury acutely induces increase in phosphorylated tau in the nucleus and directly interacts with histone 2AX (H2AX), a DNA damage repair (DDR) marker. Loss of tau significantly reduced H2AX after irradiation, indicating that tau may play an important role in neuronal DDR response. We also observed that loss of tau increases eukaryotic elongation factor levels after irradiation, the latter being a positive regulator of protein translation. This cascades into a significant increase in synaptic proteins, resulting in disrupted homeostasis. Consequently, novel object recognition test showed decrease in learning and memory in tau-knockout mice after irradiation, and electroencephalographic activity showed increase in delta and theta band oscillations, often seen in dementia patients. Our findings demonstrate tau's previously undefined, multifunctional role in acute responses to injury, ranging from DDR response in the nucleus to synaptic function within a neuron. Such knowledge is vital to develop therapeutic strategies targeting neuronal injury in cognitive decline for at risk and vulnerable populations.

Chronic Wasting Disease: State of the Science.

Chronic wasting disease (CWD) is a prion disease affecting cervid species, both free-ranging and captive populations. As the geographic range continues to expand and disease prevalence continues to increase, CWD will have an impact on cervid populations, local economies, and ecosystem health. Mitigation of this "wicked" disease will require input from many different stakeholders including hunters, landowners, research biologists, wildlife managers, and others, working together. The NC1209 (North American interdisciplinary chronic wasting disease research consortium) is composed of scientists from different disciplines involved with investigating and managing CWD. Leveraging this broad breadth of expertise, the Consortium has created a state-of-the-science review of five key aspects of CWD, including current diagnostic capabilities for detecting prions, requirements for validating these diagnostics, the role of environmental transmission in CWD dynamics, and potential zoonotic risks associated with CWD. The goal of this review is to increase stakeholders', managers', and decision-makers' understanding of this disease informed by current scientific knowledge.

Nasal bots carry relevant titers of CWD prions in naturally infected white-tailed deer.

Chronic wasting disease (CWD) is a prion disease affecting farmed and free-ranging cervids. CWD is rapidly expanding across North America and its mechanisms of transmission are not completely understood. Considering that cervids are commonly afflicted by nasal bot flies, we tested the potential of these parasites to transmit CWD. Parasites collected from naturally infected white-tailed deer were evaluated for their prion content using the protein misfolding cyclic amplification (PMCA) technology and bioassays. Here, we describe PMCA seeding activity in nasal bot larvae collected from naturally infected, nonclinical deer. These parasites efficiently infect CWD-susceptible mice in ways suggestive of high infectivity titers. To further mimic environmental transmission, bot larvae homogenates were mixed with soils, and plants were grown on them. We show that both soils and plants exposed to CWD-infected bot homogenates displayed seeding activity by PMCA. This is the first report describing prion infectivity in a naturally occurring deer parasite. Our data also demonstrate that CWD prions contained in nasal bots interact with environmental components and may be relevant for disease transmission.

Plants as vectors for environmental prion transmission.

Prions cause fatal neurodegenerative diseases and exhibit remarkable durability, which engenders a wide array of potential exposure scenarios. In chronic wasting disease of deer, elk, moose, and reindeer and in scrapie of sheep and goats, prions are transmitted via environmental routes and the ability of plants to accumulate and subsequently transmit prions has been hypothesized, but not previously demonstrated. Here, we establish the ability of several crop and other plant species to take up prions via their roots and translocate them to above-ground tissues from various growth media including soils. We demonstrate that plants can accumulate prions in above-ground tissues to levels sufficient to transmit disease after oral ingestion by mice. Our results suggest plants may serve as vectors for prion transmission in the environment-a finding with implications for wildlife conservation, agriculture, and public health.

Blue-shift photoconversion of near-infrared fluorescent proteins for labeling and tracking in living cells and organisms.

Photolabeling of intracellular molecules is an invaluable approach to studying various dynamic processes in living cells with high spatiotemporal precision. Among fluorescent proteins, photoconvertible mechanisms and their products are in the visible spectrum (400-650 nm), limiting their in vivo and multiplexed applications. Here we report the phenomenon of near-infrared to far-red photoconversion in the miRFP family of near infrared fluorescent proteins engineered from bacterial phytochromes. This photoconversion is induced by near-infrared light through a non-linear process, further allowing optical sectioning. Photoconverted miRFP species emit fluorescence at 650 nm enabling photolabeling entirely performed in the near-infrared range. We use miRFPs as photoconvertible fluorescent probes to track organelles in live cells and in vivo, both with conventional and super-resolution microscopy. The spectral properties of miRFPs complement those of GFP-like photoconvertible proteins, allowing strategies for photoconversion and spectral multiplexed applications.

Dynamics of CWD prion detection in feces and blood from naturally infected white-tailed deer.

Chronic wasting disease (CWD) is a prion disease affecting cervids. Confirmatory testing of CWD is currently performed postmortem in obex and lymphoid tissues. Extensive evidence demonstrates the presence of infectious prions in feces of CWD-infected deer using in vitro prion-amplification techniques and bioassays. In experimental conditions, this has been achieved as soon as 6-month post-inoculation, suggesting this sample type is a candidate for antemortem diagnosis. In the present study, we optimized the detection of CWD-prions in fecal samples from naturally infected, pre-clinical white-tailed deer by comparing protocols aiming to concentrate CWD-prions with direct spiking of the sample into the PMCA reactions. Results of this screening were compared with similar analyses made in blood. Our data shows that CWD-prion detection in feces using PMCA is best in the absence of sample pre-treatments. We performed a screening of 169 fecal samples, detecting CWD-prions with diagnostic sensitivity and specificity of 54.81% and 98.46%, respectively. In addition, the PMCA seeding activity of 76 fecal samples was compared with that on blood of matched deer. Our findings, demonstrate that CWD-prions in feces and blood are increased at late pre-clinical stages, exhibiting similar detection in both sample types (> 90% sensitivity) when PrP96GG animals are tested. Our findings contribute to understand prion distribution across different biological samples and polymorphic variants in white-tailed deer. This information is also relevant for the current efforts to identify platforms to diagnose CWD.

PMCA screening of retropharyngeal lymph nodes in white-tailed deer and comparisons with ELISA and IHC.

Chronic wasting disease (CWD) is a prion disease affecting cervids. CWD diagnosis is conducted through enzyme-linked immunosorbent assay (ELISA) and immunohistochemistry (IHC) in retropharyngeal lymph nodes. Unfortunately, these techniques have limited sensitivity against the biomarker (CWD-prions). Two in vitro prion amplification techniques, real-time quaking-induced conversion (RT-QuIC) and protein misfolding cyclic amplification (PMCA), have shown promise in detecting CWD-prions in tissues and bodily fluids. Recent studies have demonstrated that RT-QuIC yields similar results compared to ELISA and IHC. Here, we analyzed 1003 retropharyngeal lymph nodes (RPLNs) from Texas white-tailed deer. PMCA detected CWD at a higher rate compared to ELISA/IHC, identified different prion strains, and revealed the presence of CWD-prions in places with no previous history. These findings suggest that PMCA exhibits greater sensitivity than current standard techniques and could be valuable for rapid and strain-specific CWD detection.

Identification of chronic wasting disease prions in decaying tongue tissues from exhumed white-tailed deer.

Chronic wasting disease (CWD) prions cause fatal neuropathies in farmed and free-ranging cervids. The deposition of prions in natural and humanmade environmental components has been implicated as a major mechanism mediating CWD spread in wild and captive populations. Prions can be deposited in the environment through excreta, tissues, and carcasses from pre-clinical and clinical animals. Furthermore, burial of CWD-positive animals may reduce but not completely mitigate prion spread from carcasses into the surrounding environment. Here, we analyzed exhumed, decaying deer carcasses for the presence of CWD prions. By analyzing tongue tissues through the protein misfolding cyclic amplification (PMCA) technique, we were able to identify seven out of 95 exhumed white-tailed deer carcasses as CWD prions carriers. Confirmatory analyses were performed using the real-time quaking-induced conversion (RT-QuIC) technique. In addition, we evaluated the potential contamination of the pens that housed these animals by swabbing feeders and waterers. PMCA analyses of swabs confirmed CWD contamination on farming equipment. This work demonstrates the usefulness of PMCA to detect CWD prions in a variety of contexts, including exhumed/decaying tissues. In addition, this is the first report demonstrating swabbing coupled with PMCA as a method for the detection of prion seeding activity on naturally exposed surfaces. Considering that this study was focused on a single site, further studies should confirm whether prion amplification assays are useful to identify CWD prions not only in animals but also in the environment that contains them. IMPORTANCE Environmental contamination is thought to be a major player in the spread of chronic wasting disease (CWD), a fatal prion disease affecting a wide variety of cervid species. At present, there are no officially approved methods allowing for the detection of prion infectivity in environmental components. Importantly, animal as well as anthropogenic activities are thought to contribute to prion environmental contamination. Here, we detected CWD prions in exhumed white-tailed deer carcasses by using the protein misfolding cyclic amplification (PMCA) assay. In addition, we identified CWD prions in feeders used within the infected facility. These results highlight the potential role of PMCA in identifying prion infectivity in a variety of scenarios, ranging from decaying tissues to farming equipment.

Two structurally defined Aß polymorphs promote different pathological changes in susceptible mice.

Misfolded Aß is involved in the progression of Alzheimer's disease (AD). However, the role of its polymorphic variants or conformational strains in AD pathogenesis is not fully understood. Here, we study the seeding properties of two structurally defined synthetic misfolded Aß strains (termed 2F and 3F) using in vitro and in vivo assays. We show that 2F and 3F strains differ in their biochemical properties, including resistance to proteolysis, binding to strain-specific dyes, and in vitro seeding. Injection of these strains into a transgenic mouse model produces different pathological features, namely different rates of aggregation, formation of different plaque types, tropism to specific brain regions, differential recruitment of Aß40 /Aß42 peptides, and induction of microglial and astroglial responses. Importantly, the aggregates induced by 2F and 3F are structurally different as determined by ssNMR. Our study analyzes the biological properties of purified Aß polymorphs that have been characterized at the atomic resolution level and provides relevant information on the pathological significance of misfolded Aß strains.

Low-grade intestinal inflammation two decades after pelvic radiotherapy.

BACKGROUND: Radiotherapy is effective in the treatment of cancer but also causes damage to non-cancerous tissue. Pelvic radiotherapy may produce chronic and debilitating bowel symptoms, yet the underlying pathophysiology is still undefined. Most notably, although pelvic radiotherapy causes an acute intestinal inflammation there is no consensus on whether the late-phase pathophysiology contains an inflammatory component or not. To address this knowledge gap, we examined the potential presence of a chronic inflammation in mucosal biopsies from irradiated pelvic cancer survivors. METHODS: We biopsied 24 cancer survivors two to 20 years after pelvic radiotherapy, and four non-irradiated controls. Using tandem mass tag (TMT) mass spectrometry and mRNA sequencing (mRNA-seq), we charted proteomic and transcriptomic profiles of the mucosal tissue previously exposed to a high or a low/no dose of radiation. Changes in the immune cell populations were determined with flow cytometry. The integrity of the protective mucus layers were determined by permeability analysis and 16S rRNA bacterial detection. FINDINGS: 942 proteins were differentially expressed in mucosa previously exposed to a high radiation dose compared to a low radiation dose. The data suggested a chronic low-grade inflammation with neutrophil activity, which was confirmed by mRNA-seq and flow cytometry and further supported by findings of a weakened mucus barrier with bacterial infiltration. INTERPRETATION: Our results challenge the idea that pelvic radiotherapy causes an acute intestinal inflammation that either heals or turns fibrotic without progression to chronic inflammation. This provides a rationale for exploring novel strategies to mitigate chronic bowel symptoms in pelvic cancer survivors. FUNDING: This study was supported by the King Gustav V Jubilee Clinic Cancer Foundation (CB), The Adlerbertska Research Foundation (CB), The Swedish Cancer Society (GS), The Swedish State under the ALF agreement (GS and CB), Mary von Sydow's foundation (MA and VP).

Sepsis exacerbates Alzheimer's disease pathophysiology, modulates the gut microbiome, increases neuroinflammation and amyloid burden.

While our understanding of the molecular biology of Alzheimer's disease (AD) has grown, the etiology of the disease, especially the involvement of peripheral infection, remains a challenge. In this study, we hypothesize that peripheral infection represents a risk factor for AD pathology. To test our hypothesis, APP/PS1 mice underwent cecal ligation and puncture (CLP) surgery to develop a polymicrobial infection or non-CLP surgery. Mice were euthanized at 3, 30, and 120 days after surgery to evaluate the inflammatory mediators, glial cell markers, amyloid burden, gut microbiome, gut morphology, and short-chain fatty acids (SCFAs) levels. The novel object recognition (NOR) task was performed 30 and 120 days after the surgery, and sepsis accelerated the cognitive decline in APP/PS1 mice at both time points. At 120 days, the insoluble Aß increased in the sepsis group, and sepsis modulated the cytokines/chemokines, decreasing the cytokines associated with brain homeostasis IL-10 and IL-13 and increasing the eotaxin known to influence cognitive function. At 120 days, we found an increased density of IBA-1-positive microglia in the vicinity of Aß dense-core plaques, compared with the control group confirming the predictable clustering of reactive glia around dense-core plaques within 15 µm near Aß deposits in the brain. In the gut, sepsis negatively modulated the α- and ß-diversity indices evaluated by 16S rRNA sequencing, decreased the levels of SCFAs, and significantly affected ileum and colon morphology in CLP mice. Our data suggest that sepsis-induced peripheral infection accelerates cognitive decline and AD pathology in the AD mouse model.

Clinical evidence of human pathogens implicated in Alzheimer's disease pathology and the therapeutic efficacy of antimicrobials: an overview.

A wealth of pre-clinical reports and data derived from human subjects and brain autopsies suggest that microbial infections are relevant to Alzheimer's disease (AD). This has inspired the hypothesis that microbial infections increase the risk or even trigger the onset of AD. Multiple models have been developed to explain the increase in pathogenic microbes in AD patients. Although this hypothesis is well accepted in the field, it is not yet clear whether microbial neuroinvasion is a cause of AD or a consequence of the pathological changes experienced by the demented brain. Along the same line, the gut microbiome has also been proposed as a modulator of AD. In this review, we focus on human-based evidence demonstrating the elevated abundance of microbes and microbe-derived molecules in AD hosts as well as their interactions with AD hallmarks. Further, the direct-purpose and potential off-target effects underpinning the efficacy of anti-microbial treatments in AD are also addressed.

G-quadruplexes and associated proteins in aging and Alzheimer's disease.

Aging is a prominent risk factor for many neurodegenerative disorders, such as Alzheimer's disease (AD). Alzheimer's disease is characterized by progressive cognitive decline, memory loss, and neuropsychiatric and behavioral symptoms, accounting for most of the reported dementia cases. This disease is now becoming a major challenge and burden on modern society, especially with the aging population. Over the last few decades, a significant understanding of the pathophysiology of AD has been gained by studying amyloid deposition, hyperphosphorylated tau, synaptic dysfunction, oxidative stress, calcium dysregulation, and neuroinflammation. This review focuses on the role of non-canonical secondary structures of DNA/RNA G-quadruplexes (G4s, G4-DNA, and G4-RNA), G4-binding proteins (G4BPs), and helicases, and their roles in aging and AD. Being critically important for cellular function, G4s are involved in the regulation of DNA and RNA processes, such as replication, transcription, translation, RNA localization, and degradation. Recent studies have also highlighted G4-DNA's roles in inducing DNA double-strand breaks that cause genomic instability and G4-RNA's participation in regulating stress granule formation. This review emphasizes the significance of G4s in aging processes and how their homeostatic imbalance may contribute to the pathophysiology of AD.

Stress levels, psychological symptoms, and C-reactive protein levels in COVID-19: A cross-sectional study.

BACKGROUND: Although many studies have pointed out a possible relationship between COVID-19 and the presence of psychiatric disorders, the majority of the studies have significant limitations. This study investigates the influence of COVID-19 infection on mental health. METHODS: This cross-sectional study included an age- and sex-matched sample of adult individuals positive (cases) or negative (controls) for COVID-19. We evaluated the presence of psychiatric conditions and C-reactive protein (CRP). RESULTS: Findings showed greater severity of depressive symptoms, higher levels of stress, and greater CRP in cases. The severity of depressive and insomnia symptoms, as well as the CRP were more remarkable in individuals with moderate/severe COVID-19. We found a positive correlation between stress and severity of anxiety, depression, and insomnia in individuals with or without COVID-19. There was a positive correlation between CRP levels and severity of depressive symptoms in cases and controls, and a positive correlation between CRP levels and the severity of anxiety symptoms and stress levels only in individuals with COVID-19. Individuals with COVID-19 and depression had greater CRP than those with COVID-19 without current major depressive disorder. LIMITATIONS: We cannot infer causality because this is a cross-sectional study, and the majority of COVID-19 sample was asymptomatic or had mild symptoms, which may limit the generalizability of our findings for moderate/severe cases. CONCLUSIONS: Individuals with COVID-19 showed greater severity of psychological symptoms, which may impact on the development of psychiatric disorders in the future. CPR seem to be a promising biomarker for earlier detection of post-COVID depression.