Aberrant Enhanced NLRP3 Inflammasomes and Cell Pyroptosis in the Brains of Prion-Infected Rodent Models Are Largely Associated with the Proliferative Astrocytes.

Neuroinflammation is a common pathological feature in a number of neurodegenerative diseases, which is mediated primarily by the activated glial cells. Nucleotide-binding oligomerization domain-like receptor pyrin domain-containing-3 (NLRP3) inflammasome-associated neuroinflammatory response is mostly considered. To investigate the situation of the NLRP3-related inflammation in prion disease, we assessed the levels of the main components of NLRP3 inflammasome and its downstream biomarkers in the scrapie-infected rodent brain tissues. The results showed that the transcriptional and expressional levels of NLRP3, caspase-1, and apoptosis-associated speck-like protein (ASC) in the brains of scrapie-infected rodents were significantly increased at terminal stage. The increased NLPR3 overlapped morphologically well with the proliferated GFAP-positive astrocytes, but little with microglia and neurons. Using the brain samples collected at the different time-points after infection, we found the NLRP3 signals increased in a time-dependent manner, which were coincidental with the increase of GFAP. Two main downstream cytokines, IL-1ß and IL-18, were also upregulated in the brains of prion-infected mice. Moreover, the gasdermin D (GSDMD) levels, particularly the levels of GSDMD-NT, in the prion-infected brain tissues were remarkably increased, indicating activation of cell pyroptosis. The GSDMD not only co-localized well with the astrocytes but also with neurons at terminal stage, also showing a time-dependent increase after infection. Those data indicate that NLRP3 inflammasomes were remarkably activated in the infected brains, which is largely mediated by the proliferated astrocytes. Both astrocytes and neurons probably undergo a pyroptosis process, which may help the astrocytes to release inflammatory factors and contribute to neuron death during prion infection.

Emodin treatment of papillary thyroid cancer cell lines in vitro inhibits proliferation and enhances apoptosis via downregulation of NF­κB and its upstream TLR4 signaling.

Thyroid cancer is one of the most common types of endocrine malignancy. In addition to surgical treatment, it is very important to find new treatment methods. The aim of the present study was to evaluate the effect of 1,3,8-trihydroxy-6-methylanthraquinone (emodin) on cellular NF-κB components and the upstream regulatory pathway of toll-like receptor 4 (TLR4) signaling, as well as the invasion and migration of papillary thyroid carcinoma (PTC) cells. The protein expression of NF-κB components p65 and p50 and their phosphorylated (p-) forms in the sections of PTC tissues was measured by individual immunohistochemical assays. PTC cell lines TPC-1 and IHH4 were exposed to 20 and 40 µM emodin for 24 h. The levels of the NF-κB components p65, p50, c-Rel, p-p65 and p-p50, elements in TLR4 signaling, including TLR4, MYD88 innate immune signal transduction adaptor (MyD88), interferon regulatory factor 3, AKT and MEK, and proliferative and apoptotic biomarkers, including c-Myc, cyclin D1, proliferating cell nuclear antigen, Bcl-2 and Bax, were evaluated by western blotting and immunofluorescent assays. The invasion and migration of PTC cell lines exposed to emodin were tested by plate colony and wound healing assay. Compared with hyperplasia tissue, the expression levels of NF-κB components p65 and p50, and p-p65 and p-p50 in PTC tissue were significantly increased. Treatment of PTC cell lines with emodin lead to significantly reduced levels of the aforementioned NF-κB components, accompanied by markedly downregulated TLR4 signaling. MYD 88-dependent and -independent pathways, are also significantly down-regulated. Downregulation of proliferative factors and activation of apoptotic factors were observed in the cell lines following treatment with emodin. Consequently, inhibition of the invasion and migration activities were observed in the emodin-treated PTC cells. Emodin could inhibit proliferation and promote apoptosis of PTC cells, which is dependent on the downregulation of cellular NF-κB and the TLR4 signaling pathway.

Increased Gal-3 Mediates Microglia Activation and Neuroinflammation via the TREM2 Signaling Pathway in Prion Infection.

Galectin 3 (Gal-3) is one of the major elements for activating microglia and mediating neuroinflammation in some types of neurodegenerative diseases. However, its role in the pathogenesis of prion disease is seldom addressed. In this study, markedly increased brain Gal-3 was identified in three scrapie-infected rodent models at the terminal stage. The increased Gal-3 was mainly colocalized with the activated microglia. Coincidental with the increased brain Gal-3 in prion-infected animals, the expression of brain trigger receptor expressed in myeloid cell 2 (TREM2), one of the Gal-3 receptors, and some components in the downstream pathway also significantly increased, whereas Toll-like receptor 4 (TLR4), another Gal-3 receptor, and the main components in its downstream signaling were less changed. The increased Gal-3 signals were distributed at the areas with PrPSc deposit but looked not to colocalize directly with PrPSc/PrP signals. Similar changing profiles of Gal-3, the receptors TREM2 and TLR4, as well as the proteins in the downstream pathways were also observed in prion-infected cell line SMB-S15. Removal of PrPSc replication in SMB-S15 cells reversed the upregulation of cellular Gal-3, TREM2, and the relevant proteins. Moreover, we presented data for interactions of Gal-3 with TREM2 and with TLR4 morphologically and molecularly in the cultured cells. Stimulation of prion-infected cells or their normal partner cells with recombinant mouse Gal-3 in vitro induced obvious responses for activation of TREM2 signaling and TLR4 signaling. Our data here strongly indicate that prion infection or PrPSc deposit induces remarkably upregulated brain Gal-3, which is actively involved in the microglia activation and neuroinflammation mainly via TREM2 signaling.

Accumulation of Prion Triggers the Enhanced Glycolysis via Activation of AMKP Pathway in Prion-Infected Rodent and Cell Models.

Mitochondrial dysfunction is one of the hallmarks in the pathophysiology of prion disease and other neurodegenerative diseases. Various metabolic dysfunctions are identified and considered to contribute to the progression of some types of neurodegenerative diseases. In this study, we evaluated the status of glycolysis pathway in prion-infected rodent and cell models. The levels of the key enzymes, hexokinase (HK), phosphofructokinase (PFK), and pyruvate kinase (PK) were significantly increased, accompanying with markedly downregulated mitochondrial complexes. Double-stained IFAs revealed that the increased HK2 and PFK distributed widely in GFAP-, Iba1-, and NeuN-positive cells. We also identified increased levels of AMP-activated protein kinase (AMPK) and the downstream signaling. Changes of AMPK activity in prion-infected cells by the AMPK-specific inhibitor or activator induced the corresponding alterations not only in the downstream signaling, but also the expressions of three key kinases in glycolysis pathway and the mitochondrial complexes. Transient removal or complete clearance of prion propagation in the prion-infected cells partially but significantly reversed the increases of the key enzymes in glycolysis, the upregulation of AMPK signaling pathway, and the decreases of the mitochondrial complexes. Measurements of the cellular oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) showed lower OCR and higher ECAR in prion-infected cell line, which were sufficiently reversed by clearance of prion propagation. Those data indicate a metabolic reprogramming from oxidative phosphorylation to glycolysis in the brains during the progression of prion disease. Accumulation of PrPSc is critical for the switch to glycolysis, largely via activating AMPK pathway.

Spontaneous prion disease in homozygous and heterozygous transgenic mouse models of T188K genetic Creutzfeldt-Jakob disease.

Genetic Creutzfeldt-Jakob disease with T188K mutation (T188K gCJD) is the most frequent genetic prion disease in China. To explore the penetration of T188K mutation and the pathogenesis of T188K gCJD, we constructed 2 lines of transgenic mouse models: homozygous Tg188K+/+ mice containing T188K mutation in 2 alleles of human PRNP background and heterozygous Tg188K+/- mice containing 1 allele of T188K human PRNP and 1 allele of the wild-type mouse PRNP. Spontaneous neurological illnesses were identified in all Tg188K mice at their old ages (750-800 days old). About half of the Tg188K mice died prior to the final observation (930 days old). Extensive spongiosis, PrPSc deposit, and reactive gliosis of astrocytes and microglia are neuropathologically identified, showing time-dependent exacerbation. Proteinase K-resistant PrP was detected in the brain, muscle, and intestine tissues, and positive real-time quaking-induced conversion reactions were elicited by the brain and muscle tissues of Tg188K mice. Those data verify that the constructed Tg188K mice highly mimic the clinicopathology of human T188K gCJD, strongly indicating the pathogenicity of T188K mutated PrP.

Abnormal Changes of IL3/IL3R and Its Downstream Signaling Pathways in the Prion-Infected Cell Line and in the Brains of Scrapie-Infected Rodents.

Interleukin 3 (IL-3) plays an important role in hematopoiesis and immune regulation, brain IL-3/IL-3R signaling has been shown to involve in the physiological and pathological processes of a variety of neurodegenerative diseases, but its role in prion diseases is rarely described. Here, the changes of IL-3/IL-3R and its downstream signaling pathways in a scrapie-infected cell line and in the brains of several scrapie-infected rodent models were evaluated by various methods. Markedly decreased IL-3Rα were observed in the brains of scrapie-infected rodents at terminal stage and in the prion-infected cell model, which showed increased in the brain samples collected at early and middle stage of infection. The IL-3 levels were almost unchanged in the brains of scrapie-infected mice and in the prion-infected cell line. Morphological assays identified close co-localization of the increased IL-3Rα signals with NeuN- and Iba1-positive cells, whereas co-localization of IL-3 signals with NeuN- and GFAP-positive cells in the scrapie-infected brain tissues. Some downstream components of IL-3/IL-3R pathways, including JAK2-STAT5 and PI3K/AKT/mTOR pathways, were downregulated in the brains of scrapie-infected rodents at terminal stage and in the prion-infected cells. Stimulation of recombinant IL-3 on the cultured cells showed prion that the prion-infected cells displayed markedly more reluctant responses of JAK2-STAT5 and PI3K/AKT/mTOR pathways than the normal partner cells. These data suggest that although prion infection or PrPSc accumulation in brain tissues does not affect IL-3 expression, it significantly downregulates IL-3R levels, thereby inhibiting the downstream pathways of IL-3/IL-3R and blocking the neuroregulatory and neuroprotective activities of IL-3.

Aberrant SENP1-SUMO-Sirt3 Signaling Causes the Disturbances of Mitochondrial Deacetylation and Oxidative Phosphorylation in Prion-Infected Animal and Cell Models.

Post-translational modifications of proteins, such as acetylation and SUMOylation, play important roles in regulation of protein functions and pathophysiology of different diseases including neurodegenerative diseases. Our previous studies have identified aberrant acetylation profiles and reduced deacetylases Sirt3 and Sirt1 in the brains of prion-infected mouse models. In this study, we have found that the levels of acetylated forms of AceCS2 and LCAD, the key enzymes regulating lipid metabolism, CS and IHD2, the key enzymes regulating complete oxidative metabolism, GDH, the key enzyme regulating the oxidative decomposition of glutamate into the tricarboxylic acid (TCA) cycle, and NDUFA9, the essential component in the complex I of respiratory chain activity, were significantly upregulated in the prion-infected animal and cell models, along with the decrease of Sirt3 activity and mitochondrial cytochrome c oxidase activity. Meanwhile, the increases of SUMO1 modifications and SUMO1-Sirt3 and decrease of SENP1 were identified in the brains and the cultured cells with prion infections. Removal of prion propagation in the cultured cells partially, but significantly, reversed the aberrant situations. Moreover, similar abnormal phenomena were also observed in the cultured 293 T cells transiently expressing cytosolic form PrP (Cyto-PrP), including decreased SENP1, increased SUMO1, decreased Sirt3 activity, increased acetylated forms of the key enzymes, and decreased cytochrome c oxidase activity. Attenuation of the accumulation of Cyto-PrP by co-expression of the p62 protein sufficiently diminished those abnormalities. The data here strongly indicate that deposits of prions in brains or accumulations of Cyto-PrP in cells trigger dysregulation of the SENP1-SUMO1-Sirt pathway and subsequently induce aberrant mitochondrial deacetylation and the mitochondrial respiratory chain.

Global Profiles of Acetylated Proteins in Brains of Scrapie Agents 139A- and ME7-Infected Mice Collected at Mid-Early, Mid-Late, and Terminal Stages.

Objective: To describe the global profiles of acetylated proteins in the brains of scrapie agents 139A- and ME7-infected mice collected at mid-early, mid-late, and terminal stages. Methods: The acetylated proteins from the cortex regions of scrapie agent (139A- and ME7)-infected mice collected at mid-early (80 days postinfection, dpi), mid-late (120 dpi), and terminal (180 dpi) stages were extracted, and the global profiles of brain acetylated proteins were assayed with proteomic mass spectrometry. The proteins in the infected mice showing 1.5-fold higher or lower levels than that of age-matched normal controls were considered as differentially expressed acetylated peptides (DEAPs). Results: A total of 118, 42, and 51 DEAPs were found in the brains of 139A-80, 139A-120, and 139A-180 dpi mice, respectively. Meanwhile, 390, 227, and 75 DEAPs were detected in the brains of ME7-80, ME7-120, and ME7-180 dpi mice, respectively. The overwhelming majority of DEAPs in the mid-early stage were down-regulated, and more portions of DEAPs in the mid-late and late stages were up-regulated. Approximately 22.1% (328/1,485) of acetylated peptides mapped to 74 different proteins were mitochondrial associated. Kyoto Encyclopedia of Genes and Genomes pathway analysis identified 39 (80 dpi), 13 (120 dpi), and 10 (180 dpi) significantly changed pathways in 139A-infected mice. Meanwhile, 55, 25, and 18 significantly changed pathways were observed in the 80, 120, and 180 dpi samples of 139A- and ME7-infected mice ( P < 0.05), respectively. Six pathways were commonly involved in all tested samples. Moreover, many steps in the citrate cycle (tricarboxylic acid cycle) were affected, represented by down-regulated acetylation for relevant enzymes in the mid-early stage and up-regulated acetylation in the mid-late and late stages. Conclusion: Our data here illustrated the changes in the global profiles for brain acetylated proteins during prion infection, showing remarkably inhibited acetylation in the early stage and relatively enhanced acetylation in the late stage.

Enhanced M-CSF/CSF1R Signaling Closely Associates with PrPSc Accumulation in the Scrapie-Infected Cell Line and the Brains of Scrapie-Infected Experimental Rodents.

Activation and proliferation of microglia are one of the hallmarks of prion disease and is usually accompanied by increased levels of various cytokines and chemokines. Our previous study demonstrated that the level of brain macrophage colony-stimulating factor (M-CSF) was abnormally elevated during prion infection, but its association with PrPSc is not completely clear. In this study, colocalization of the increased M-CSF with accumulated PrPSc was observed by IHC with serial brain sections. Reliable molecular interaction between total PrP and M-CSF was observed in the brain of 263 K-infected hamsters and in cultured prion-infected cell line. Immunofluorescent assays showed that morphological colocalization of M-CSF with neurons and microglia, but not with astrocytes in brains of scrapie-infected animals. The transcriptional and expressing levels of CSF1R were also significantly increased in prion-infected cell line and mice, and colocalization of CSF1R with neurons and microglia was observed in the brains of prion-infected mouse models. Removal of PrPSc replication by resveratrol in SMB-S15 cells induced limited reductions of cellular levels of M-CSF and CSF1R. In addition, we found that the level of IL-34, another ligand of CSF1R, did not change significantly after prion infection, but its distribution on the cell types in the brains shifted from neurons in healthy mice to the proliferated astrocytes and microglia in scrapie-infected mice. Our data demonstrate activation of M-CSF/IL-34/CSF1R signaling in the microenvironment of prion infection, strongly indicating its vital role in the pathophysiology of prions. It provides solid scientific evidence for the therapeutic potential of inhibiting M-CSF/CSF1R signaling in prion diseases.

Different Aberrant Changes of mGluR5 and Its Downstream Signaling Pathways in the Scrapie-Infected Cell Line and the Brains of Scrapie-Infected Experimental Rodents.

Metabotropic glutamate receptor subtype 5 (mGluR5) is a G-protein-coupled receptor found widely in the central nervous system. It has been involved in the development and progression of some neurodegenerative diseases, but its role in prion diseases is rarely described. In this study, the changes of mGluR5 and its downstream signaling pathways in prion-infected cell line SMB-S15 and the brains of scrapie-infected experimental rodents were evaluated by various methodologies. We found the levels of mGluR5 were significantly increased in a prion-infected cell line SMB-S15 and the cultured cells transiently express an abnormal form PrP (Cyto-PrP). Using immunoprecipitation tests and immunofluorescent assays (IFA), molecular interaction and morphological colocalization between PrP and mGluR5 were observed in the cultured cells. We identified that the (GPCRs)-IP3-IP3R-Ca2+ pathway was activated and the levels of the downstream kinases p38, ERK, and JNK were increased in SMB-S15 cells. After treated with mGluR5 antagonist (MTEP) or the removal of prion replication by resveratrol in SMB-S15 cells, the upregulations of mGluR5 and the downstream kinases were restored in a certain degree. Moreover, increased mGluR5 contributes to the cell damage in prion-infected cells. Contrarily, the levels of mGluR5 in the brains of several scrapie-infected rodent models were decreased at terminal stage. IFA of the brain sections of scrapie-infected rodents demonstrated that the signals of mGluR5 were preferentially colocalized with the NeuN-positive cells, accompanying with severe neuron losses in Nissl staining, which might be a reason for the decrease of mGluR5. Our data indicate the different aberrant alterations of mGluR5 and the downstream signaling pathways during prion infection in vivo and in vitro.

Difference of geographic distributions of the Chinese patients with prion diseases in the permanent resident places and referring places.

Human prion diseases (PrDs) are a group of transmissible neurodegenerative diseases that can be clarified as sporadic, genetic and iatrogenic forms. In this study, we have analysed the time and geographic distributions of 2011 PrD cases diagnosed by China National Surveillance for Creutzfeldt-Jakob disease (CNS-CJD) since 2006, including 1792 sporadic CJD (sCJD) cases and 219 gPrD cases. Apparently, the cases numbers of both sCJD and gPrD increased along with the surveillance years, showing a stepping up every five years. The geographic distributions of the PrDs cases based on the permanent residences were wide, distributing in 30 out of 31 provincial-level administrative divisions in Chinese mainland. However, the case numbers in the provincial level varied largely. The provinces in the eastern part of China had much more cases than those in the western part. Normalized the case numbers with the total population each province revealed higher incidences in six provinces. Further, the resident and referring places of all PrD cases were analysed, illustrating a clear concentrating pattern of referring in the large metropolises. Five provincial-level administrative divisions reported more PrD cases from other provinces than the local ones. Particularly, BJ reported not only more than one-fourth of all PrDs cases in Chinese mainland but also 3.64-fold more PrDs cases from other provinces than its local ones. We believed that good medical resources, well-trained programmes and knowledge of PrDs in the clinicians and the CDC staffs contributed to well-referring PrD cases in those large cities.

PrPSc Inhibition and Cellular Protection of DBL on a Prion-Infected Cultured Cell via Multiple Pathways.

Prion diseases are kinds of fatal neurodegenerative diseases without effective therapeutic and prophylactic tools currently. In this study, the inhibition of PrPSc propagation and cellular protectivity of 3,4-dihydroxybenzalacetone (DBL), a small catechol-containing compound isolated and purified from the ethanol extract of Inonotus obliquus, upon a prion-infected cell line SMB-S15 were evaluated. Western blots showed that after incubation with 10 µM of DBL for 14 days, the level of PrPSc in SMB-S15 cells was significantly decreased. Meanwhile, the levels of ROS and hydrogen peroxide were decreased with a dose-dependent manner, whereas the levels of some antioxidant factors, such as HO-1, GCLC and GCLM, were significantly increased. The activities of total glutathione and SOD were up-regulated. DBL-treated SMB-S15 cells also showed the up-regulation of UPR-related proteins, including PERK, IRE1α, ATF6 and GRP78, and activation of autophagy system. Furthermore, the SIRT3 abnormalities caused by prion infection were relieved by DBL treatment. On the contrary, these comprehensive changes were not significantly noticed in the normal partner cell line SMB-PS under the same experimental condition. Those data indicate that treatment of DBL on prion-infected cells can reduce PrPSc level, activate UPR and autophagy system and meanwhile relieve intracellular oxidative stress, endoplasmic reticulum stress and mitochondrial dysfunction by raising the levels of multiple antioxidant factors. The PrPSc inhibition and protective effectiveness of DBL upon the prion-infected cells in vitro make it worthy of further study.

Characteristics of Chinese patients with genetic CJD who have E196A or E196K mutation in PRNP: comparative analysis of patients identified in the Chinese National CJD Surveillance System.

OBJECTIVE: Two different mutations at codon 196, namely E196A and E196K, have been reported to be related to genetic Creutzfeldt-Jakob disease (CJD). We aimed to comparatively analyse the features of Chinese patients with these two mutations from the CJD surveillance system in China. DESIGN AND SETTING: Comparative analysis of patients identified via the Chinese National CJD Surveillance System during the period 2006-2020. PARTICIPANTS: 16 Chinese patients with genetic CJD with E196A mutation and 5 with E196K mutation. METHODS: Neurological examination, EEG and MRI, western blot, gene sequence, and RT-QuIC. RESULTS: The age of onset of E196K genetic CJD cases (median of 61 years) was older than the E196A cases (median of 67 years). Generally, these two subtypes of genetic CJD were more like sporadic Creutzfeldt-Jakob disease (sCJD) clinically. The E196A cases showed more major symptoms, while those of E196K cases were restricted to dementia and mental problems. During progression, more sCJD-associated symptoms and signs gradually appeared, but none of the E196K cases showed cerebellum and visual disturbances. Typical periodic sharp wave complexes on MRI were recorded in 25% of E196A cases but not in E196K cases. sCJD-associated abnormalities on MRI, positive cerebrospinal fluid (CSF) 14-3-3 and increased CSF total tau were observed frequently, ranging from two out of three cases to four out of five cases, without a difference. Positive CSF RT-QuIC was detected in 37.5% (6 of 16) of E196A cases and 60% (3 of 5) of E196K cases. The duration of survival of E196K cases (median of 4.5 months) was shorter than the E196A cases (median of 6.5 months). Moreover, female cases and cases with young age of onset (<60 years) in E196A displayed longer survival time than male patients and cases with older age of onset (≥60 years). CONCLUSIONS: This is the largest comprehensive report of genetic CJD with mutations at codon 196 to date, describing the similarity and diversity in clinical and laboratory tests between patients with E196A and with E196K mutations.

Accumulation of Prion and Abnormal Prion Protein Induces Hyperphosphorylation of α-Synuclein in the Brain Tissues from Prion Diseases and in the Cultured Cells.

Prion disease (PrD) and Parkinson's disease (PD) are neurodegenerative diseases characterized by aggregation of misfolded proteins in brain tissues, including protease-resistant prion protein (PrPSc) in PrD and α-synuclein in PD. In recent years, overlap of these two proteins has attracted increased attention, and cross-seeding of prion proteins by aggregated α-synuclein has been proposed. However, the changes in α-synuclein after prion infection are still unclear. In this study, we showed that α-synuclein expression was significantly decreased in the brains of prion-infected rodent models, in the SMB-S15 cell line, which exhibits persistent prion replication, and in the brains of humans with PrDs. Meanwhile, α-synuclein phosphorylated at serine 129(p(S129)-α-synuclein) was significantly increased in the brains of scrapie-infected mice and prion-infected SMB-S15 cells. The increased p(S129)-α-synuclein colocalized with GFAP- and NeuN-positive cells in the brains of scrapie-infected mice. p(S129)-α-synuclein was also observed in the cytoplasm of SMB-S15 and HEK-293 cells transiently expressing an abnormal form of prion protein (Cyto-PrP). Molecular interactions between PrP and α-synuclein were detected in recombinant proteins, normal and prion-infected brain tissues, and cultured cells. The increased p(S129)-α-synuclein colocalized with PrP signals from prion-infected SMB-S15 and HEK-293 cells expressing Cyto-PrP. Moreover, increased morphological colocalization of p(S129)-α-synuclein with mitochondrial markers was also detected in the two cell types. Our results indicate that prion replication and accumulation in cells and brains induce hyperphosphorylation of α-synuclein, particularly at S129, which may aggravate mitochondrial damage and facilitate α-synuclein aggregation in the central nervous system tissues from PrDs.

Genetic Prion Disease: Insight from the Features and Experience of China National Surveillance for Creutzfeldt-Jakob Disease.

Human genetic prion diseases (gPrDs) are directly associated with mutations and insertions in the PRNP (Prion Protein) gene. We collected and analyzed the data of 218 Chinese gPrD patients identified between Jan 2006 and June 2020. Nineteen different subtypes were identified and gPrDs accounted for 10.9% of all diagnosed PrDs within the same period. Some subtypes of gPrDs showed a degree of geographic association. The age at onset of Chinese gPrDs peaked in the 50-59 year group. Gerstmann-Sträussler-Scheinker syndrome (GSS) and fatal familial insomnia (FFI) cases usually displayed clinical symptoms earlier than genetic Creutzfeldt-Jakob disease (gCJD) patients with point mutations. A family history was more frequently recalled in P105L GSS and D178N FFI patients than T188K and E200K patients. None of the E196A gCJD patients reported a family history. The gCJD cases with point mutations always developed clinical manifestations typical of sporadic CJD (sCJD). EEG examination was not sensitive for gPrDs. sCJD-associated abnormalities on MRI were found in high proportions of GSS and gCJD patients. CSF 14-3-3 positivity was frequently detected in gCJD patients. Increased CSF tau was found in more than half of FFI and T188K gCJD cases, and an even higher proportion of E196A and E200K gCJD patients. 63.6% of P105L GSS cases showed a positive reaction in cerebrospinal fluid RT-QuIC. GSS and FFI cases had longer durations than most subtypes of gCJD. This is one of the largest studies of gPrDs in East Asians, and the illness profile of Chinese gPrDs is clearly distinct. Extremely high proportions of T188K and E196A occur among Chinese gPrDs; these mutations are rarely reported in Caucasians and Japanese.

Analysis of the Electrophoretic Profiles of Prion Protein in Carcinous and Pericarcinous Lysates of Six Different Types of Cancers.

OBJECTIVE: To find the different electrophoretic profiles of prion protein in carcinous and individual pericarcinous tissues in lysates of gastric, colon, liver, lung, thyroid, and laryngeal cancers. METHODS: Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot were used to test the amounts and electrophoretic patterns of total PrP and the tolerance of PK (protease K) digestion among six various cancer tissue types. RESULTS: A mass of PrP signals with a large molecular weight were identified in the homogenates of peripheral tissues. The amounts and electrophoretic patterns of total PrP did not differ significantly between carcinous and pericarcinous tissues. PrPs in all types of the tested cancer samples were PK sensitive but showed diversity in the tolerance of PK digestion among various tissue types. CONCLUSIONS: The study revealed that the included electrophoretic patterns of carcinous and pericarcinous tissues were almost similar. Unlike PrP-specific immunohistochemical assay, evaluation of PrP electrophoretic patterns in the peripheral organs and tissues by Western blot does not reflect tumor malignancy.

The low levels of nerve growth factor and its upstream regulatory kinases in prion infection is reversed by resveratrol.

Resveratrol shows ability to eliminate prion replication, but the exact mechanism for prion eradication was not clear yet. Our previous studies demonstrate a downregulation of brain-derived nerve growth factor (BDNF) during prion infection, meanwhile recovery of cerebral nerve growth factor (NGF) level by resveratrol treatment has been reported in other neurodegenerative models. To obtain the possible changes of brain NGF and its upstream regulatory cascade during prion infection and after removal of prion propagation, the levels of NGF and its upstream regulatory factors in various prion-infected and prion-eradicated SMB cell lines and mice brains inoculated with various SMB cellular lysates were assessed with various methodologies. The levels of NGF were significantly decreased during prion replication, while recovered after removal of PrPSc by resveratrol in vitro. Morphological assays revealed that the NGF signals mainly colocalized within neurons, but not in the proliferative astrocytes and microglia. The upstream positive regulatory kinases, such as p-CREB, p-CaMKIV, CaMKK2 were decreased in the prion infected cells and mice brains, whereas the negative regulatory one, p-CaMKK2, was increased. The aberrant situations of those kinases in prion infected cell lines or mice brains could be also partially reversed by removal of prion agent. Moreover, we demonstrated that the signals of CaMKK2 and p-CaMKK2 were also distributed predominately in neurons in the brain tissues. The data illustrate a direct linkage of abnormally repressive NGF and its upstream regulatory kinases with prion infection. Resveratrol has not only the ability to inhibit prion replication, but also to improve the expression of NGF via CaMKK2/CaMKIV cascade, which might benefit the microenvironment in brains.

Clinical and Laboratory Features of Three Rare Chinese V210I gCJD Patients.

Genetic human prion diseases are a group of inherited encephalopathies directly associated with different mutations in PrP-encoding gene PRNP, including more than 50 different mutations worldwide. Some genotypes of mutations show ethno-correlation, and among them, genetic Creutzfeldt-Jacob disease (gCJD) with V210I mutation is frequent in European countries but rare in East Asia. Here, we comparatively analyzed the clinical and laboratory features of three Chinese patients with V210I mutant identified via the Chinese National CJD Surveillance System (CNS-CJD) in 2019. Two cases were Han Chinese and one was Hui Chinese, without blood kinship. The onset ages of three cases were 69, 64, and 59 years old, respectively. The clinical features of V210I gCJD were similar to sporadic CJD (sCJD), displaying typical clinical symptoms and signs, except that Case 3 did not show myoclonic movement. All three cases displayed sCJD-associated abnormalities on MRI and positive CSF 14-3-3, while two cases recorded typical EEG abnormalities. Only one case was positive in CSF real-time quaking-induced conversion (RT-QuIC). Appearances of mutism in three cases were relatively fast, with the intervals of 30 to 50 days after onset. Family history was not reported in all three cases. Those V210I gCJD cases are rare in China, and probably the first three in East Asia.

Preparation of PrP-specific Polyclonal Antibody via Immunization of PRNP-knockout Mice with Recombinant Human PrP Protein.

OBJECTIVE: The definite diagnosis of human and animal prion diseases depends on the examination of special pathological changes and/or detection of PrP Sc in the brain tissues of suspected cases. Thus, developing methods to obtain PrP antibody with good specificity and sensitivity is fundamental for prion identification. METHODS: We prepared a PrP-specific polyclonal antibody (pAb P54) in a PRNP-knockout mouse model via immunization with recombinant full-length human PrP protein residues 23-231. Thereafter, we verified that pAb in Western blot, immunohistochemistry (IHC), and immunofluorescent (IFA) assays. RESULTS: Western blot illustrated that the newly prepared pAb P54 could react with recombinant PrP protein, normal brain PrP C from healthy rodents and humans, and pathological PrP Sc in the brains of experimental rodents infected with scrapie and humans infected with different types of prion diseases. The electrophoretic patterns of brain PrP C and PrP Sc observed after their reaction with pAb P54 were nearly identical to those produced by commercial PrP monoclonal antibodies. Three glycosylated PrP molecules in the brain homogenates were clearly demonstrated in the reactions of these molecules with pAb P54. IHC assay revealed apparent PrP deposits in the GdnCl-treated brain slices of 139A-infected mice and 263K-infected hamsters. IFA tests with pAb P54 also showed clear green signals surrounding blue-stained cell nuclei. CONCLUSION: The newly prepared pAb P54 demonstrated reliable specificity and sensitivity and, thus, may have potential applications not only in studies of prion biology but also in the diagnosis of human and experimental rodent prion diseases.

Stilbene Compounds Inhibit the Replications of Various Strains of Prions in the Levels of Cell Culture, PMCA, and RT-QuIC Possibly via Molecular Binding.

Resveratrol shows the ability to block prion replication in a scrapie-infected cell line, SMB-S15, and remove the infectivity of the treated cell lysates in an experimental bioassay. In this study, we compared the effectiveness of three stilbene compounds, resveratrol (Res), pterostilbene (Pte), and piceatannol (Pic), on inhibiting prion propagations in the levels of cell culture, PMCA, and RT-QuIC. All three chemicals showed active suppressions on PrPSc replication in SMB-S15 cells, in which Res seemed to be the most active one, followed by Pic and Pte. Mouse PrP-based PMCA tests using the lysates of SMB-S15 cells and brain homogenates of scrapie agents S15-, 139A-, or ME7-infected mice verified that Res, Pte, and Pic inhibited the amplifications of PK-resistant signals. Res was also the most effective one. Mouse PrP-based RT-QuIC using the above seeds demonstrated that three stilbenes efficiently inhibited the fibril formation. However, Pic was the most effective one, followed by Res and Pte. Furthermore, the inhibition activities of the three stilbenes on the brain-derived prion from a 263K-infected hamster were tested with hamster PrP-based PMCA and RT-QuIC. The results indicated that Pic was the most effective one apparently, followed by Res and Pte. According to the results of Biacore, Res showed binding affinities much stronger than those of Pte, whereas both revealed markedly stronger binding affinities with mouse PrP. Our data here indicate that different stilbenes have the ability to block PrPSc replication in vitro with different prion species. The suppressive effects of stilbene compounds are likely associated with their molecular binding activities with PrPs.