Search Results (189)

Prion diseases are classically attributed to the accumulation of protease-resistant prion protein (PrP^Sc); however, recent evidence suggests that alternative misfolded prion conformers and systemic inflammatory factors may also contribute to neurodegeneration. This study investigated whether recombinant moPrP^Res, generated by incubating wild-type mouse PrP^C with bacterial lipopolysaccharide (LPS), can induce prion-like disease in FVB/N female mice, whether LPS alone causes neurodegeneration, and how LPS modulates disease progression in mice inoculated with the Rocky Mountain Laboratory (RML) strain of prions. Wild-type female FVB/N mice were randomized into six subcutaneous treatment groups: saline, LPS, moPrP^Res, moPrP^Res + LPS, RML, and RML + LPS. Animals were monitored longitudinally for survival, body weight, and clinical signs. Brain tissues were analyzed histologically and immunohistochemically for vacuolar degeneration, PrP^Sc accumulation, reactive astrogliosis, and amyloid-β plaque deposition. Recombinant moPrP^Res induced a progressive spongiform encephalopathy characterized by widespread vacuolation and astrogliosis, yet with no detectable PrP^Sc by Western blot or immunohistochemistry. LPS alone triggered a distinct neurodegenerative phenotype, including cerebellar amyloid-β plaque accumulation and terminal-stage spongiosis, with approximately 40% mortality by the end of the study. Co-administration of moPrP^Res and LPS resulted in variable regional pathology and intermediate survival (50% at 750 days post-inoculation). Interestingly, RML + LPS co-treatment led to earlier clinical onset and mortality compared to RML alone; however, vacuolation levels were not significantly elevated and, in some brain regions, were reduced. These results demonstrate that chronic endotoxemia and non-infectious misfolded PrP conformers can independently or synergistically induce key neuropathological hallmarks of prion disease, even in the absence of classical PrP^Sc. Targeting inflammatory signaling and toxic prion intermediates may offer novel therapeutic strategies for prion and prion-like disorders. Full article

Background: Inflammatory indices have been proposed as simple and routinely obtainable markers of systemic inflammation in cardiac disease. This study investigated whether the neutrophil-to-lymphocyte ratio (NLR), the monocyte-to-lymphocyte ratio (MLR), and the pan-immune inflammation value (PIV) serve as biomarkers for risk stratification and outcomes measures in patients with severe aortic stenosis (AS) following valve replacement (AVR). Methods: In this retrospective analysis (January 2017–June 2022), patients with AS underwent pre-procedural cardiovascular magnetic resonance (CMR) imaging and were assigned a treatment strategy by a multidisciplinary Heart Team: (1) transcatheter AVR, (2) surgical AVR, or (3) no valvular intervention. Kaplan–Meier estimates and regression analyses were used to demonstrate associations between the NLR, MLR, and PIV with myocardial fibrosis—assessed by late gadolinium enhancement (LGE) and extracellular volume (ECV) on CMR—and a combined endpoint of heart failure hospitalizations and all-cause mortality. Results: A total of 356 patients (median age: 80 years, 50% male) were followed for a median of 40 months, during which 162 (46%) reached the combined endpoint. Linear regression identified C-reactive protein, but not the presence of LGE or elevated ECV, as the only independent predictor of all three inflammatory indices (p for all <0.001). After multivariable adjustment for clinical (EuroSCORE II), laboratory (baseline N-terminal prohormone of brain natriuretic peptide [NT-proBNP] and C-reactive protein), and imaging parameters (AV mean pressure gradient, right ventricular ejection fraction, and ECV), the above-the-upper-quartile NLR (adjusted hazard ratio [aHR]: 1.45, 95%-confidence interval [CI]: 1.01–1.92, p = 0.042), MLR (aHR: 1.48, 95%-CI: 1.05–2.09, p = 0.025), and PIV (aHR: 1.56, 95%-CI: 1.11–2.21, p = 0.011) remained significantly associated with adverse outcomes. Following AVR, the median NLR (3.5 to 3.4) and PIV (460 to 376) showed a significant post-procedural decline compared to baseline (p ≤ 0.019 for both). Conclusions: Inflammatory indices are readily available biomarkers independently associated with adverse outcomes in severe AS following AVR. However, no significant relationship was observed between the NLR, MLR, PIV, and myocardial fibrosis on CMR. Full article

The essential SUP35 gene encodes yeast translation termination factor Sup35/eRF3. The N-terminal domain of Sup35 is also responsible for Sup35 prionization that leads to generation of the [PSI⁺] prion. Previously we isolated different types of sup35 mutations (missense and nonsense) and demonstrated that sup35 nonsense mutations (sup35-n) are incompatible with the [PSI⁺] prion, leading to lethality of sup35-n [PSI⁺] haploid cells. Here, we show that sup35 missense mutations (sup35-m) within conservative regions of the Sup35 C-domain result in lethality of [PSI⁺] cells because of weak activity of Sup35/eRF3 as a translation termination factor. Mutant Sup35 maintain their ability to be incorporated into pre-existing [PSI⁺] aggregates and to form amyloid aggregates in vitro, while sup35-m mutations do not influence the [PSI⁺] prion induction and stability. All these mutations (D363N, R372K, T378I) are located in the conservative GTPase region of Sup35, decreasing the GTPase activity of mutated proteins. We propose that such low activity of mutant Sup35 combined with aggregation of Sup35 constituting the [PSI⁺] prion is not sufficient to maintain the viability of yeast cells. Full article

Autophagy impairments have been implicated in various aging conditions. Previous studies in cervical motor neurons show an age-dependent increase in the key autophagy proteins LC3 and p62, reflecting autophagy impairment and autophagosome accumulation. Chloroquine is commonly used to inhibit autophagy by preventing autophagosome–lysosome fusion and may thus emulate the effects of aging on the neuromuscular system. Indeed, acute chloroquine administration in old mice decreases maximal transdiaphragmatic pressure generation, consistent with aging effects. We hypothesized that chloroquine alters diaphragm muscle neuromuscular junction (NMJ) morphology and increases denervation. Adult male and female C57BL/6 × 129J mice between 5 and 8 months of age were used to examine diaphragm muscle NMJ morphology and denervation following daily intraperitoneal injections of chloroquine (10 mg/kg/d) or vehicle for 7 days. The motor end-plates and pre-synaptic terminals were fluorescently labeled with α-bungarotoxin and anti-synaptophysin, respectively. Confocal microscopy was used to assess pre- and post-synaptic morphology and denervation. At diaphragm NMJs, chloroquine treatment decreased pre-synaptic volume by 12% compared to the vehicle (p < 0.05), with no change in post-synaptic volume. Chloroquine treatment increased the proportion of partially denervated NMJs by 2.7-fold compared to vehicle treatment (p < 0.05). The morphological changes observed were similar to those previously reported in the diaphragm muscles of 18-month-old mice. These findings highlight the importance of autophagy in the maintenance of the structural properties at adult NMJs in vivo. Full article

Background/Objectives: A mouse antibody directed against truncated Annexin A1 showed high tumor retention in pre-clinical cancer models and was approved by the National Cancer Institute Experimental Therapeutics (NExT) program for humanization and large batch cGMP production for toxicology and clinical trials. In this process, a contractor for Leidos accidentally produced a mutated version of humanized AnnA1 (hAnnA1-mut) with a single nucleotide deletion in the terminal Fc coding region that increased the translated size by eight amino acids with random alterations in the final twenty-four amino acids. We investigated the tissue distribution of hAnnA1-mut, hAnnA1, mAnnA1, and isotope-matched human IgG1 under various injection and conjugation conditions with C57BL/6, FVB, and BALB/c nude mice strains. Methods: Biodistribution studies were performed 24 h after injection of Tc-99m-HYNIC radiolabeled antibodies (purity > 98%). Non-reducing gel electrophoresis studies were conducted with IR680 labeled antibodies incubated with various mouse sera. Results: Our results showed that Tc-99m-HYNIC-hAnnA1 had low spleen and liver retention not statistically different from Tc-99m-HYNIC-IgG1 and Tc-99m-HYNIC-mAnnA1, with corresponding higher blood levels; however, Tc-99m-HYNIC-hAnnA1-mut had high levels in the spleen and liver with differences identified among the mouse strains, radiolabeling conditions, and injection routes. Histopathology showed no morphological change in the liver or spleen from any conditions. Gel electrophoresis showed an upward shift of hAnnA1-mut, consistent with the binding of blood serum protein. Conclusions: The changes in the Fc region of hAnnA1-mut led to higher liver and spleen uptake, suggesting the antibody’s recognition by the innate immune system (likely complement protein binding) and subsequent clearance. Future clinical translation using hAnnA1 and other antibodies needs to limit protein modifications that could drastically reduce blood clearance. Full article

Learning and memory formation rely on synaptic plasticity, the process that changes synaptic strength in response to neuronal activity. In the tripartite synapse concept, molecular signals that affect synapse strength and morphology originate not only from the pre- and post-synaptic neuronal terminals but also from astrocytic processes ensheathing many synapses. Despite significant progress made in understanding astrocytic contribution to synaptic plasticity, only a few astrocytic plasticity-related proteins have been identified so far. In this study, we present evidence indicating the role of astrocyte-secreted Lipocalin-2 (Lcn2) in neuronal plasticity. We show that Lcn2 expression is induced in hippocampal astrocytes in a kainate-evoked aberrant plasticity model. Next, we demonstrate that chemically induced long-term potentiation (cLTP) similarly increases Lcn2 expression in astrocytes of neuronal–glial co-cultures, and that glutamate causes the immediate release of Lcn2 from these cultures. Additionally, through experiments in primary astrocytic cultures, we reveal that Lcn2 release is triggered by calcium signaling, and we demonstrate that a brief treatment of neuronal–glial co-cultures with Lcn2 alters the morphology of dendritic spines. Based on these findings, we propose Lcn2 as an activity-dependent molecule released by astrocytes that influences dendritic spine morphology. Full article

Neurodegenerative diseases are characterized by progressive loss of neurons and persistent inflammation. Neurons are terminally differentiated cells, and lost neurons cannot be replaced since neurogenesis is restricted to only two neurogenic niches in the adult brain, whose neurogenic potential decreases with age. In this regard, the astrocytes reprogramming into neurons may represent a promising strategy for restoring the lost neurons and rebuilding neural circuits. To date, many anti-inflammatory agents have been shown to reduce neuroinflammation; however, their potential to restore neuronal loss was poorly investigated. This study investigates the anti-inflammatory effects of lactoferrin on DI-TNC1 astrocyte cell line and its ability to induce astrocyte reprogramming in a context of sustained inflammation. For this purpose, astrocytes were pre-treated with lactoferrin (4 μg/mL) for 24 h, then with lipopolysaccharide (LPS) (400 ng/mL), and examined 2, 9 and 16 days from treatment. The results demonstrate that lactoferrin attenuates astrocyte reactivity by reducing Toll-like receptor 4 (TLR4), Glial fibrillary acidic protein (GFAP) and IL-6 expression, as well as by upregulating Interleukin-10 (IL-10) cytokine and NRF2 expression. Moreover, lactoferrin promotes the reprogramming of reactive astrocytes into proliferative neuroblasts by inducing the overexpression of the Sex determining region Y/SRY-box 2 (SOX2) reprogramming transcription factor. Overall, this study highlights the potential effects of lactoferrin to attenuate neuroinflammation and improve neurogenesis, suggesting a future strategy for the treatment of neurodegenerative disorders. Full article

Extremely premature infants are at significant risk for developing bronchopulmonary dysplasia (BPD) and neurodevelopmental impairment (NDI). Although BPD is a predictor of poor neurodevelopmental outcomes, it is currently unknown how BPD contributes to brain injury and long-term NDI in pre-term infants. Extracellular vesicles (EVs) are small, membrane-bound structures released from cells into the surrounding environment. EVs are involved in inter-organ communication in diverse pathological processes. Inflammasomes are large, multiprotein complexes that are part of the innate immune system and are responsible for triggering inflammatory responses and cell death. Apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) is pivotal in inflammasome assembly and activating inflammatory caspase-1. Activated caspase-1 cleaves gasdermin D (GSDMD) to release a 30 kD N-terminal domain that can form membrane pores, leading to lytic cell death, also known as pyroptosis. Activated caspase-1 can also cleave pro-IL-1β and pro-IL-18 to their active forms, which can be rapidly released through the GSDMD pores to induce inflammation. Recent evidence has emerged that activation of inflammasomes is associated with neonatal lung and brain injury, and inhibition of inflammasomes reduces hyperoxia-induced neonatal lung and brain injury. Additionally, multiple studies have demonstrated that hyperoxia stimulates the release of lung-derived EVs that contain inflammasome cargos. Adoptive transfer of these EVs into the circulation of normal neonatal mice and rats induces brain inflammatory injury. This review focuses on EV–inflammasomes’ roles in mediating lung-to-brain crosstalk via EV-dependent and EV-independent mechanisms critical in BPD, brain injury, and NDI pathogenesis. EV–inflammasomes will be discussed as potential therapeutic targets for neonatal lung and brain injury. Full article

The conserved role of juvenile hormone (JH) signals in preventing larvae from precocious metamorphosis has been confirmed in insects. Crustaceans have different metamorphosis types from insects; we previously proved that methyl farnesoate (MF) can prohibit larvae metamorphosis in mud crabs, but the molecular signal of this process still needs to be elucidated. In this study, methoprene-tolerant (Met) of Scylla paramamosain was obtained and characterized, which we named Sp-Met. Sp-Met contains a 3360 bp ORF that encodes 1119 amino acids; the predicted protein sequences of Sp-Met include one bHLH, two PAS domains, one PAC domain, and several long unusual Gln repeats at the C-terminal. AlphaFold2 was used to predict the 3D structure of Sp-Met and the JH binding domain of Met. Furthermore, the binding properties between Sp-Met and MF were analyzed using CD-DOCK2, revealing a putative high affinity between the receptor and ligand. In silico site-directed mutagenesis suggested that insect Mets may have evolved to exhibit a higher affinity for both MF or JH III compared to the Mets of crustaceans. In addition, we found that the expression of Sp-Met was significantly higher in female reproductive tissues than in males but lower in most of the other examined tissues. During larval development, the expression variation in Sp-Met and Sp-Kr-h1 was consistent with the immersion effect of MF. The most interesting finding is that knockdown of Sp-Met blocked the inhibitory effect of MF on metamorphosis in the fifth zoea stage and induced pre-metamorphosis phenotypes in the fourth zoea stage. The knockdown of Sp-Met significantly reduced the expression of Sp-Kr-h1 and two ecdysone signaling genes, Sp-EcR and Sp-E93. However, only the reduction in Sp-Kr-h1 could be rescued by MF treatment. In summary, this study provides the first evidence that MF inhibits crustacean larval metamorphosis through Met and that the MF-Met→Kr-h1 signal pathway is conserved in mud crabs. Additionally, the crosstalk between MF and ecdysteroid signaling may have evolved differently in mud crabs compared to insects. Full article

Huntington’s disease (HD) is an autosomal dominant neurodegenerative disease caused by a single mutation in the huntingtin gene (HTT). Normal HTT has a CAG trinucleotide repeat at its N-terminal within the range of 36. However, once the CAG repeats exceed 37, the mutant gene (mHTT) will encode mutant HTT protein (mHTT), which results in neurodegeneration in the brain, specifically in the striatum and other brain regions. Since the mutation was discovered, there have been many research efforts to understand the mechanism and develop therapeutic strategies to treat HD. HTT is a large protein with many post-translational modification sites (PTMs) and can be modified by phosphorylation, acetylation, methylation, sumoylation, etc. Some modifications reduced mHTT toxicity both in cell and animal models of HD. We aimed to find the known kinase inhibitors that can modulate the toxicity of mHTT. We performed an in vitro kinase assay using HTT peptides, which bear different PTM sites identified by us previously. A total of 368 kinases were screened. Among those kinases, cyclin-dependent kinases (CDKs) affected the serine phosphorylation on the peptides that contain S1181 and S1201 of HTT. We explored the effect of CDK1 and CDK5 on the phosphorylation of these PTMs of HTT and found that CDK5 modified these two serine sites, while CDK5 knockdown reduced the phosphorylation of S1181 and S1201. Modifying these two serine sites altered the neuronal toxicity induced by mHTT. Roscovitine, a CDK inhibitor, reduced the p-S1181 and p-S1201 and had a protective effect against mHTT toxicity. We further investigated the feasibility of the use of roscovitine in HD mice. We confirmed that roscovitine penetrated the mouse brain by IP injection and inhibited CDK5 activity in the brains of HD mice. It is promising to move this study to in vivo for pre-clinical HD treatment. Full article

Pre-harvest sprouting (PHS) is a complex abiotic stress caused by multiple exogenous and endogenous variables that results in random but significant quality and yield loss at the terminal crop stage in more than half of the wheat-producing areas of the world. Systematic research over more than five decades suggests that addressing this challenge requires tools beyond the traditional genetic manipulation approach. Previous molecular studies indicate a possible role of epigenetics in the regulation of seed dormancy and PHS in crops, especially through RNA-directed DNA methylation (RdDM) pathways mediated by Argonaute (AGO) proteins. In this study, we explore the role of the AGO802B gene associated with PHS resistance in wheat, through the presence of a SINE retrotransposon insertion. The current study found the SINE insertion at 3′UTR of the TaAGO802B present in 73.2% of 41 cultivars analyzed and in 92.6% of the resistant cultivar subset. The average expression of TaAGO802B in cultivars with the SINE insertion was 73.3% lower than in cultivars without insertion. This study also indicated a significant positive correlation between the PHS score and methylation levels in the cultivars. The resistant cultivars with the SINE insertion recorded 54.7% lower methylation levels than susceptible cultivars. Further analysis of a DH population (Sadash × P2711) reveals that SINE insertion co-segregates with PHS resistance. This sets forth the SINE insertion in TaAGO802B as a genetic marker for screening wheat germplasm and as an efficient tool for breeding PHS-resistant wheat cultivars. Full article

Background: Approximately 10–20% of subjects vaccinated with HBsAg-based hepatitis B virus (HBV) vaccines are non-responders. BM32 is a recombinant grass pollen allergy vaccine containing the HBV-derived preS surface antigen as an immunological carrier protein. PreS includes the binding site of HBV to its receptor on hepatocytes. We investigated whether immunological non-responsiveness to HBV after repeated HBsAg-based vaccinations could be overcome by immunization with VVX001 (i.e., alum-adsorbed BM325, a component of BM32). Methods: A subject failing to develop protective HBV-specific immunity after HBsAg-based vaccination received five monthly injections of 20 µg VVX001. PreS-specific antibody responses were measured by enzyme-linked immunosorbent assay (ELISA) and micro-array technology. Serum reactivity to subviral particles of different HBV genotypes was determined by sandwich ELISA. PreS-specific T cell responses were monitored by carboxyfluorescein diacetate succinimidyl ester (CFSE) staining and subsequent flow cytometry. HBV neutralization was assessed using cultured HBV-infected HepG2 cells. Results: Vaccination with VVX001 induced a strong and sustained preS-specific antibody response composed mainly of the IgG₁ subclass. PreS-specific IgG antibodies were primarily directed to the N-terminal part of preS containing the sodium taurocholate co-transporting polypeptide (NTCP) attachment site. IgG reactivity to subviral particles as well as to the N-terminal preS-derived peptides was comparable for HBV genotypes A–H. A pronounced reactivity of CD3⁺CD4⁺ lymphocytes specific for preS after the complete injection course remaining up to one year after the last injection was found. Maximal HBV neutralization (98.4%) in vitro was achieved 1 month after the last injection, which correlated with the maximal IgG reactivity to the N-terminal part of preS. Conclusions: Our data suggest that VVX001 may be used as a preventive vaccination against HBV even in non-responders to HBsAg-based HBV vaccines. Full article

Photoreceptors in the mammalian retina convert light signals into electrical and molecular signals through phototransduction and transfer the visual inputs to second-order neurons via specialized ribbon synapses. Two kinds of photoreceptors, rods and cones, possess distinct morphology and function. Currently, we have limited knowledge about rod versus (vs.) cone synapse development and the associated genes. The transcription factor neural retina leucine zipper (NRL) determines the rod vs. cone photoreceptor cell fate and is critical for rod differentiation. Nrl knockout mice fail to form rods, generating all cone or S-cone-like (SCL) photoreceptors in the retina, whereas ectopic expression of Nrl using a cone-rod homeobox (Crx) promoter (CrxpNrl) forms all rods. Here, we examined rod and cone pre-synapse development, including axonal elongation, terminal shaping, and synaptic lamination in the outer plexiform layer (OPL) in the presence or absence of Nrl. We show that NRL loss and knockdown result in delayed OPL maturation and plasticity with aberrant dendrites of bipolar neurons. The integrated analyses of the transcriptome in developing rods and SCLs with NRL CUT&RUN and synaptic gene ontology analyses identified G protein subunit beta (Gnb) 1 and p21 (RAC1) activated kinase 5 (Pak5 or Pak7) transcripts were upregulated in developing rods and down-regulated in developing SCLs. Notably, Gnb1 and Gnb5 are rod dominant, and Gnb3 is enriched in cones. NRL binds to the genes of Gnb1, Gnb3, and Gnb5. NRL also regulates pre-synapse ribbon genes, and their expression is altered in rods and SCLs. Our study of histological and gene analyses provides new insights into the morphogenesis of photoreceptor pre-synapse development and regulation of associated genes in the developing retina. Full article

Hepatitis B virus (HBV) infection remains a major health threat with limited treatment options. One of various new antiviral strategies is based on a fusion of Staphylococcus aureus nuclease (SN) with the capsid-forming HBV core protein (HBc), termed coreSN. Through co-assembly with wild-type HBc-subunits, the fusion protein is incorporated into HBV nucleocapsids, targeting the nuclease to the encapsidated viral genome. However, coreSN expression was based on transfection of a plasmid vector. Here, we explored whether introducing protein transduction domains (PTDs) into a fluorescent coreSN model could confer cell-penetrating properties for direct protein delivery into cells. Four PTDs were inserted into two different positions of the HBc sequence, comprising the amphiphilic translocation motif (TLM) derived from the HBV surface protein PreS2 domain and three basic PTDs derived from the Tat protein of human immunodeficiency virus-1 (HIV-1), namely Tat4, NP, and NS. To directly monitor the interaction with cells, the SN in coreSN was replaced with the green fluorescent protein (GFP). The fusion proteins were expressed in E. coli, and binding to and potential uptake by human cells was examined through flow cytometry and fluorescence microscopy. The data indicate PTD-dependent interactions with the cells, with evidence of uptake in particular for the basic PTDs. Uptake was enhanced by a triplicated Simian virus 40 (SV40) large T antigen nuclear localization signal (NLS). Interestingly, the basic C terminal domain of the HBV core protein was found to function as a novel PTD. Hence, further developing cell-permeable viral capsid protein fusions appears worthwhile. Full article

Background: A drug repositioning effort supported the possible use of the anti-HIV drug etravirine as a disease-modifying drug for Friedreich ataxia (FRDA). Etravirine increases frataxin protein and corrects the biochemical defects in cells derived from FRDA patients. Because of these findings, and since etravirine displays a favorable safety profile, we conducted a pilot open-label phase 2 clinical trial assessing the safety and potential efficacy of etravirine in FRDA patients. Methods: Thirty-five patients were stratified into three severity groups and randomized to etravirine 200 mg/day or 400 mg/day. They were treated for 4 months. Safety endpoints were the number and type of adverse events and number of dropouts. Efficacy endpoints were represented by changes in peak oxygen uptake and workload as measured by incremental exercise test, SARA score, cardiac measures, measures of QoL and disability. Data were collected 4 months before the start of the treatment (T − 4), at the start (T0), at the end (T4) and 4 months after the termination of the treatment (T + 4). Results: Etravirine was reasonably tolerated, and adverse events were generally mild. Four months of etravirine treatment did not significantly increase the peak oxygen uptake but was associated with a change in the progression of the SARA score (p value < 0.001), compared to the 4 months pre- and post-treatment. It also significantly increased peak workload (p value = 0.021). No changes in the cardiac measures were observed. Health and QoL measures showed a worsening at the suspension of the drug. Conclusions: In this open trial etravirine treatment was safe, reasonably well tolerated and appreciably improved neurological function and exercise performance. Even though a placebo effect cannot be ruled out, these results suggest that etravirine may represent a potential therapeutic agent in FRDA deserving testing in a randomized placebo-controlled clinical trial. Full article