Y225A induces long-range conformational changes in human prion protein that are protective in Drosophila.

Prion protein (PrP) misfolding is the key trigger in the devastating prion diseases. Yet the sequence and structural determinants of PrP conformation and toxicity are not known in detail. Here, we describe the impact of replacing Y225 in human PrP with A225 from rabbit PrP, an animal highly resistant to prion diseases. We first examined human PrP-Y225A by molecular dynamics simulations. We next introduced human PrP in Drosophila and compared the toxicity of human PrP-WT and Y225A in the eye and in brain neurons. Y225A stabilizes the ß2-α2 loop into a 310-helix from six different conformations identified in WT and lowers hydrophobic exposure. Transgenic flies expressing PrP-Y225A exhibit less toxicity in the eye and in brain neurons and less accumulation of insoluble PrP. Overall, we determined that Y225A lowers toxicity in Drosophila assays by promoting a structured loop conformation that increases the stability of the globular domain. These findings are significant because they shed light on the key role of distal α-helix 3 on the dynamics of the loop and the entire globular domain.

Large-scale validation of skin prion seeding activity as a biomarker for diagnosis of prion diseases.

Definitive diagnosis of sporadic Creutzfeldt-Jakob disease (sCJD) relies on the examination of brain tissues for the pathological prion protein (PrPSc). Our previous study revealed that PrPSc-seeding activity (PrPSc-SA) is detectable in skin of sCJD patients by an ultrasensitive PrPSc seed amplification assay (PrPSc-SAA) known as real-time quaking-induced conversion (RT-QuIC). A total of 875 skin samples were collected from 2 cohorts (1 and 2) at autopsy from 2-3 body areas of 339 cases with neuropathologically confirmed prion diseases and non-sCJD controls. The skin samples were analyzed for PrPSc-SA by RT-QuIC assay. The results were compared with demographic information, clinical manifestations, cerebrospinal fluid (CSF) PrPSc-SA, other laboratory tests, subtypes of prion diseases defined by the methionine (M) or valine (V) polymorphism at residue 129 of PrP, PrPSc types (#1 or #2), and gene mutations in deceased patients. RT-QuIC assays of the cohort #1 by two independent laboratories gave 87.3% or 91.3% sensitivity and 94.7% or 100% specificity, respectively. The cohort #2 showed sensitivity of 89.4% and specificity of 95.5%. RT-QuIC of CSF available from 212 cases gave 89.7% sensitivity and 94.1% specificity. The sensitivity of skin RT-QuIC was subtype dependent, being highest in sCJDVV1-2 subtype, followed by VV2, MV1-2, MV1, MV2, MM1, MM1-2, MM2, and VV1. The skin area next to the ear gave highest sensitivity, followed by lower back and apex of the head. Although no difference in brain PrPSc-SA was detected between the cases with false negative and true positive skin RT-QuIC results, the disease duration was significantly longer with the false negatives [12.0 ± 13.3 (months, SD) vs. 6.5 ± 6.4, p < 0.001]. Our study validates skin PrPSc-SA as a biomarker for the detection of prion diseases, which is influenced by the PrPSc types, PRNP 129 polymorphisms, dermatome sampled, and disease duration.

Potential role of BAY11-7082, a NF-κB blocker inhibiting experimental autoimmune encephalomyelitis in C57BL/6J mice via declining NLRP3 inflammasomes.

Multiple sclerosis (MS) is an inflammatory autoimmune demyelinating disease of the central nervous system. NOD-like receptor pyrin domain-containing 3 (NLRP3) inflammasome is implicated in the pathogenesis of MS and its animal model, experimental autoimmune encephalomyelitis (EAE). However, the exact mechanism by which NLRP3 inflammasome is involved in the development of MS and EAE is not clear. NF-kappaB (NF-κB) is associated with the activity of NLRP3 inflammasomes, but the role of NF-κB is controversial. We sought to demonstrate that both NF-κB and NLRP3 contribute to development of MS and EAE, and NF-κB pathway is positively correlated with NLRP3 activation in EAE. The inhibitor of NF-κB and NLRP3, BAY11-7082, can prevent and treat EAE. BAY11-7082 (5 and 20 mg/kg/i.p.) was intraperitoneally administered to EAE mice at the time of second injection of pertussis toxin (BAY11-7082 prevention group) or at the onset of symptoms (BAY11-7082 treatment group). mRNA expressions of NLRP3 were determined by qPCR. Protein expressions of NLRP3, NF-κB p65, and phosphorylated p65 were determined by western blotting. Serum levels of inflammatory cytokines were measured by cytometric bead array. Mice treated with BAY11-7082 (both prevention and treatment groups) showed lower clinical scores and attenuated pathological changes. NLRP3 inflammasome and activity of NF-κB in spinal cord of EAE mice was higher than that in control group. However, the level of NLRP3 inflammasome decreased in BAY11-7082 prevention and treatment groups. BAY11-7082 is a promising therapeutic agent for MS. NLRP3 activation in EAE maybe related with NF-κB pathway.

Clinical characteristics and prognostic analysis of anti-gamma-aminobutyric acid-B (GABA-B) receptor encephalitis in Northeast China.

OBJECTIVE: To investigate the clinical characteristics and prognosis of anti-gamma-aminobutyric acid-B (GABA-B) receptor encephalitis. METHODS: This retrospective study enrolled nineteen patients with anti-GABA-B receptor encephalitis. Clinical manifestations, radiological and electroencephalogram features, treatment and outcomes were collected and analyzed. The neurological function was evaluated according to the modified Rankin Scale (mRS). RESULTS: There were eleven patients in the favorable-prognosis group (mRS ≤ 2) and eight patients in the poor-prognosis group (mRS > 2). In the favorable-prognosis group, clinical symptoms included memory deterioration (n = 10; 90.9%), epileptic seizures (n = 9; 81.8%), psychiatric disorders (n = 9; 81.8%), and conscious disturbance (n = 5; 45.5%); magnetic resonance imaging (MRI) indicated an involvement of the limbic system in three (27.3%) cases in this group. Lung cancer was detected in one patient (9.1%). After an average follow-up period of 11.7 months, four (36.4%) patients were cured, and seven (63.6%) patients showed significant improvements. In the poor-prognosis group, all patients presented with memory deterioration, epileptic seizures, psychiatric disorders, and conscious disturbance; five (62.5%) patients had convulsive status epilepticus, and five (62.5%) patients developed respiratory failure; MRI indicated an involvement of the limbic system in seven (87.5%) cases. Malignant tumors were detected in five (62.5%) patients. After an average follow-up period of 14.8 months, seven (87.5%) patients died and one (12.5%) patient remained dependent in daily life. CONCLUSIONS: The clinical manifestations of anti-GABA-B receptor encephalitis include epileptic seizures, cognitive impairment and psychiatric disorders. Patients with convulsive status epilepticus or respiratory failure have poor outcomes. In anti-GABA-B receptor encephalitis, limbic system involvement is associated with a poor prognosis in and radiological examinations can reflect disease progression. Early diagnosis and appropriate treatment should be highlighted.

Role of Inflammasomes in Neuroimmune and Neurodegenerative Diseases: A Systematic Review.

Inflammasomes are multiprotein complexes that can sense pathogen-associated molecular patterns and damage-associated molecular signals. They are involved in the initiation and development of inflammation via activation of IL-1ß and IL-18. Many recent studies suggest a strong correlation between inflammasomes and neurological diseases, such as multiple sclerosis (MS), Alzheimer's disease (AD), and Parkinson's disease (PD). Several components of inflammasomes, such as nucleotide-binding oligomerization domain- (NOD-) like receptor, absent in melanoma 2- (AIM2-) like receptors (ALRs), apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), and caspase-1, as well as the upstream factors and downstream effectors, are associated with the initiation and development of MS and its animal model, experimental autoimmune encephalomyelitis. Additionally, inflammasomes affect the efficacy of interferon-ß therapy in patients with MS. Finally, the strong association of inflammasomes with AD and PD needs to be further studied. In this review of latest literatures, we comprehensively tease out diverse roles of different kinds of inflammasomes in neuroimmune and neurodegenerative diseases, especially in the perspective of double roles involved in pathogenesis, and identify future research priorities.

Exploring the Relationship Between Sporadic Creutzfeldt-Jakob Disease and Gut Microbiota Through a Mendelian Randomization Study.

Observational studies have shown gut microbiota changes in sporadic Creutzfeldt-Jakob disease patients, but the causal relationship remains unknown. We aimed to determine any causal links between gut microbiota and this prion disease. Using Mendelian randomization analysis, we examined the causal relationship between gut microbiota composition and sporadic Creutzfeldt-Jakob disease. Data on gut microbiota (N = 18,340) and disease cases (5208) were obtained. Various analysis methods were used, including inverse variance weighted, Mendelian randomization-Egger, weighted median, simple mode, and weighted mode. In addition, MR-PRESSO was used to evaluate horizontal pleiotropy and detect outliers. Pleiotropy and heterogeneity were assessed, and reverse analysis was conducted. Negative associations were found between sporadic Creutzfeldt-Jakob disease and family Defluviitaleaceae, family Ruminococcaceae, genus Butyricicoccus, genus Desulfovibrio, and genus Eubacterium nodatum. Genus Lachnospiraceae UCG010 showed a positive correlation. Reverse analysis indicated genetic associations between the disease and decreased levels of family Peptococcaceae, genus Faecalibacterium, and genus Phascolarctobacterium, as well as increased levels of genus Butyrivibrio. No pleiotropy, heterogeneity, outliers, or weak instrument bias were observed. This study revealed bidirectional causal effects between specific gut microbiota components and sporadic Creutzfeldt-Jakob disease. Certain components demonstrated inhibitory effects on disease pathogenesis, while others were positively associated with the disease. Modulating gut microbiota may provide new insights into prion disease therapies. Further research is needed to clarify mechanisms and explore treatments for sporadic Creutzfeldt-Jakob disease.

Causal relationship between gut microbiota and myasthenia gravis: a bidirectional mendelian randomization study.

BACKGROUND: Observational studies have demonstrated an association between gut microbiota and myasthenia gravis; however, the causal relationship between the two still lacks clarity. Our goals are to ascertain the existence of a bidirectional causal relationship between gut microbiota composition and myasthenia gravis, and to investigate how gut microbiota plays a role in reducing the risk of myasthenia gravis. METHODS: We acquired gut microbiota data at the phylum, class, order, family, and genus levels from the MiBioGen consortium (N = 18,340) and myasthenia gravis data from the FinnGen Research Project (426 cases and 373,848 controls). In the two-sample Mendelian randomization analysis, we assessed the causal relationship between the gut microbiota and myasthenia gravis. We also conducted bidirectional MR analysis to determine the direction of causality. The inverse variance weighted, mendelian randomization-Egger, weighted median, simple mode, and weighted mode were used to test the causal relationship between the gut microbiota and severe myasthenia gravis. We used MR-Egger intercept and Cochran's Q test to assess for pleiotropy and heterogeneity, respectively. Furthermore, we utilized the MR-PRESSO method to evaluate horizontal pleiotropy and detect outliers. RESULTS: In the forward analysis, the inverse-variance weighted method revealed that there is a positive correlation between the genus Lachnoclostridium (OR = 2.431,95%CI 1.047-5.647, p = 0.039) and the risk of myasthenia gravis. Additionally, the family Clostridiaceae1 (OR = 0.424,95%CI 0.202-0.889, p = 0.023), family Defluviitaleaceae (OR = 0.537,95%CI 0.290-0.995, p = 0.048), family Enterobacteriaceae (OR = 0.341,95%CI 0.135-0.865, p = 0.023), and an unknown genus (OR = 0.407,95%CI 0.209-0.793, p = 0.008) all demonstrated negative correlation with the risk of developing myasthenia gravis. Futhermore, reversed Mendelian randomization analysis proved a negative correlation between the risk of myasthenia gravis and genus Barnesiella (OR = 0.945,95%CI 0.906-0.985, p = 0.008). CONCLUSION: Our research yielded evidence of a causality connection in both directions between gut microbiota and myasthenia gravis. We identified specific types of microbes associated with myasthenia gravis, which offers a fresh window into the pathogenesis of this disease and the possibility of developing treatment strategies. Nonetheless, more studies, both basic and clinical, are necessary to elucidate the precise role and therapeutic potential of the gut microbiota in the pathogenesis of myasthenia gravis.

Characteristics and Management of Autoimmune Disease-Associated Cerebral Venous Sinus Thrombosis.

Cerebral venous sinus thrombosis (CVST) is a central nervous system disease characterised by thrombosis in cerebral venous or dural sinuses. Autoimmune diseases, a series of diseases caused by immune responses to autoantigens, are important causes of CVST. The most common diseases that lead to CVST are Behçet's syndrome, systemic lupus erythematosus, antiphospholipid syndrome, and Sjögren's syndrome. Each of these diseases have different clinical and imaging manifestations and treatment for CVST varies by aetiology. This review summarises the characteristics and the current management strategies for autoimmune disease-associated CVST and emphasises controversial therapeutic strategies to provide informative reference information for diagnosis and treatment. Risk factors of autoimmune antigens should not be neglected when unconventional CVST occurs, and both drugs and interventional therapy need further standardisation and discussion with more prospective clinical studies.

Further Characterization of Glycoform-Selective Prions of Variably Protease-Sensitive Prionopathy.

Prion is an infectious protein (PrPSc) that is derived from a cellular glycoprotein (PrPC) through a conformational transition and associated with a group of prion diseases in animals and humans. Characterization of proteinase K (PK)-resistant PrPSc by western blotting has been critical to diagnosis and understanding of prion diseases including Creutzfeldt-Jakob disease (CJD) and Gerstmann-Sträussler-Scheinker (GSS) disease in humans. However, formation as well as biochemical and biological properties of the glycoform-selective PrPSc in variably protease-sensitive prionopathy (VPSPr) remain poorly understood. Here we reveal that formation of the ladder-like PrPSc in VPSPr is a PK-dependent two-step process, which is enhanced by basic pH. Two sets of PrPSc fragments can be identified with antibodies directed against an intermediate or a C-terminal domain of the protein. Moreover, antibodies directed against specific PrP glycoforms reveal faster electrophoretic migrations of PrP fragments mono-glycosylated at residue 181 and 197 in VPSPr than those in sporadic CJD (sCJD). Finally, RT-QuIC assay indicates that PrPSc-seeding activity is lower and its lag time is longer in VPSPr than in sCJD. Our results suggest that the glycoform-selective PrPSc in VPSPr is associated with altered glycosylation, resulting in different PK-truncation and aggregation seeding activity compared to PrPSc in sCJD.

Decrease in Skin Prion-Seeding Activity of Prion-Infected Mice Treated with a Compound Against Human and Animal Prions: a First Possible Biomarker for Prion Therapeutics.

Previous studies have revealed that the infectious scrapie isoform of prion protein (PrPSc) harbored in the skin tissue of patients or animals with prion diseases can be amplified and detected through the serial protein misfolding cyclic amplification (sPMCA) or real-time quaking-induced conversion (RT-QuIC) assays. These findings suggest that skin PrPSc-seeding activity may serve as a biomarker for the diagnosis of prion diseases; however, its utility as a biomarker for prion therapeutics remains largely unknown. Cellulose ethers (CEs, such as TC-5RW), widely used as food and pharmaceutical additives, have recently been shown to prolong the lifespan of prion-infected mice and hamsters. Here we report that in transgenic (Tg) mice expressing hamster cellular prion protein (PrPC) infected with the 263K prion, the prion-seeding activity becomes undetectable in the skin tissues of TC-5RW-treated Tg mice by both sPMCA and RT-QuIC assays, whereas such prion-seeding activity is readily detectable in the skin of untreated mice. Notably, TC-5RW exhibits an inhibitory effect on the in vitro amplification of PrPSc in both skin and brain tissues by sPMCA and RT-QuIC. Moreover, we reveal that TC-5RW is able to directly decrease protease-resistant PrPSc and inhibit the seeding activity of PrPSc from chronic wasting disease and various human prion diseases. Our results suggest that the level of prion-seeding activity in the skin may serve as a useful biomarker for assessing the therapeutic efficacy of compounds in a clinical trial of prion diseases and that TC-5RW may have the potential for the prevention/treatment of human prion diseases.

Characterization of Anchorless Human PrP With Q227X Stop Mutation Linked to Gerstmann-Sträussler-Scheinker Syndrome In Vivo and In Vitro.

Alteration in cellular prion protein (PrPC) localization on the cell surface through mediation of the glycosylphosphatidylinositol (GPI) anchor has been reported to dramatically affect the formation and infectivity of its pathological isoform (PrPSc). A patient with Gerstmann-Sträussler-Scheinker (GSS) syndrome was previously found to have a nonsense heterozygous PrP-Q227X mutation resulting in an anchorless PrP. However, the allelic origin of this anchorless PrPSc and cellular trafficking of PrPQ227X remain to be determined. Here, we show that PrPSc in the brain of this GSS patient is mainly composed of the mutant but not wild-type PrP (PrPWt), suggesting pathological PrPQ227X is incapable of recruiting PrPWt in vivo. This mutant anchorless protein, however, is able to recruit PrPWt from humanized transgenic mouse brain but not from autopsied human brain homogenates to produce a protease-resistant PrPSc-like form in vitro by protein misfolding cyclic amplification (PMCA). To further investigate the characteristics of this mutation, constructs expressing human PrPQ227X or PrPWt were transfected into neuroblastoma cells (M17). Fractionation of the M17 cells demonstrated that most PrPWt is recovered in the cell lysate fraction, while most of the mutant PrPQ227X is recovered in the medium fraction, consistent with the results obtained by immunofluorescence microscopy. Two-dimensional gel-electrophoresis and Western blotting showed that cellular PrPQ227X spots clustered at molecular weights of 22-25 kDa with an isoelectric point (pI) of 3.5-5.5, whereas protein spots from the medium are at 18-26 kDa with a pI of 7-10. Our findings suggest that the role of GPI anchor in prion propagation between the anchorless mutant PrP and wild-type PrP relies on the cellular distribution of the protein.

Sjögren's Syndrome with Cerebral Venous Sinus Thrombosis: A Case Report and Literature Review.

Cerebral venous sinus thrombosis (CVST) is a cerebrovascular disease that is caused by a number of factors, including hypercoagulability and vessel wall damage. Sjögren's syndrome (SS) is a chronic inflammatory autoimmune disease characterized by lymphocyte infiltration of the exocrine glands. CVST could be caused by autoimmune diseases. According to previous reports, the most frequently reported autoimmune diseases which could cause CVST are systemic lupus erythematosus and antiphospholipid syndrome. Reports of SS leading to CVST are scarce. Here, we present a case of a 51-year-old woman who was diagnosed with SS-induced CVST. We tease out knowledge about the pathogenesis, feature of clinic symptom, treatment, and prognosis of SS-associated CVST, and illustrates how a detailed patient history can contribute to an accurate diagnosis.

1H nuclear magnetic resonance-based metabolic profiling of cerebrospinal fluid to identify metabolic features and markers for tuberculosis meningitis.

BACKGROUND: Tuberculosis meningitis (TBM) is the most severe form of tuberculosis, and currently lacks efficient diagnostic approaches. Metabolomics has the potential to differentiate patients with TBM from those with other forms of meningitis and meningitis-negative individuals. However, no systemic metabolomics research has compared the cerebrospinal fluid (CSF) of these patients. METHODS: 1H nuclear magnetic resonance (NMR) was used for CSF metabolic profiling. Principal component analysis and orthogonal signal correction-partial least squares-discriminant analysis (OPLS-DA) were used to screen for important variables. The Human Metabolome Database was used to identify metabolites, and MetaboAnalyst 4.0 was used for pathway analysis and over-representation analysis. RESULTS: OPLS-DA modeling could distinguish TBM from other forms of meningitis, and several significantly changed metabolites were identified. Additionally, 23, 6, and 21 metabolites were able to differentiate TBM from viral meningitis, bacterial meningitis, and meningitis-negative groups, respectively. Pathway analysis indicated that these metabolites were mainly involved in carbohydrate and amino acid metabolism, and over-representation analysis indicated that some of these pathways were over-represented. CONCLUSIONS: The metabolites identified have the potential to serve as biomarkers for TBM diagnosis, and carbohydrate and amino acid metabolism are perturbed in the CSF of patents with TBM. Metabolomics is a valuable approach for screening TBM biomarkers. With further investigation, the metabolites identified in this study could aid in TBM diagnosis.

Mass spectrometry-based metabolomics for tuberculosis meningitis.

Tuberculosis meningitis (TBM) is a prevalent form of extra-pulmonary tuberculosis that causes substantial morbidity and mortality. Diagnosis of TBM is difficult because of the limited sensitivity of existing laboratory techniques. A metabolomics approach can be used to investigate the sets of metabolites of both bacteria and host, and has been used to clarify the mechanisms underlying disease development, and identify metabolic changes, leadings to improved methods for diagnosis, treatment, and prognostication. Mass spectrometry (MS) is a major analysis platform used in metabolomics, and MS-based metabolomics provides wide metabolite coverage, because of its high sensitivity, and is useful for the investigation of Mycobacterium tuberculosis (Mtb) and related diseases. It has been used to investigate TBM diagnosis; however, the processes involved in the MS-based metabolomics approach are complex and flexible, and often consist of several steps, and small changes in the methods used can have a huge impact on the final results. Here, the process of MS-based metabolomics is summarized and its applications in Mtb and Mtb-related diseases discussed. Moreover, the current status of TBM metabolomics is described.