Journal: Neural Regeneration Research

Article Title: FUBP3 mediates the amyloid-β-induced neuronal NLRP3 expression

doi: 10.4103/NRR.NRR-D-23-01799

Figure Lengend Snippet: NLRP3 is expressed in neurons and regulates tau phosphorylation. (A) Brain sections from 3-month-old wild-type mice were costained with antibodies to NLRP3 (red, Alexa Fluor 564) and the microglial marker Iba1 (green, Alexa Fluor 488). (B) Brain sections from 8-month-old APP/PS1ΔE9 AD model mice were costained with anti-NLRP3 (red, Alexa Fluor 564) and anti-Iba1 (green, Alexa Fluor 488) antibodies. Arrowheads indicate NLRP3-positive microglia, and the neuritic plaque is circled. (C) Brain sections from 8-month-old APP/PS1ΔE9 AD model mice were costained with anti-NLRP3 (red, Alexa Fluor 564) and anti-NeuN (green, Alexa Fluor 488) antibodies. The arrowhead indicates an NLRP3-positive neuron. (D) Hydrogen peroxide (H 2 O 2 ) was injected into the hippocampus of APP/PS1ΔE9 mice, and brain sections from these mice were costained with antibodies to NLRP3 and the neuronal marker NeuN. Arrowheads indicate NeuN-positive, NLRP3-negative neurons in the peri-injection area. The dashed boxes indicate the areas that are enlarged in the lower panels. (E) Western blot analysis of lysates from the microglial cell line BV2 and primary neurons from wild-type mice (PN WT ). (F) PN WT from wild-type mice and cultured primary neurons from APP Swedish mutant transgenic mice (PN APP ) expressing human APP with the Swedish mutation were costained with anti-NLRP3 (red, Alexa Fluor 564) and anti-NeuN (green, Alexa Fluor 488) antibodies. NLRP3 was not clearly detectable beyond the background staining in PN WT , whereas the NLRP3 signal in PN APP was markedly stronger than in nonneuronal cells. Asterisks indicate nonneuronal (NeuN-negative) cells. (G) Western blot for NLRP3 in Neuro2A cell lysates. (H) Total RNA was extracted from Neuro2A cells and reverse transcribed, and the indicated genes were PCR-amplified. RNA was used as negative control template. (I) Mouse brains were lysed and treated with or without λ-protein phosphatase. The treated lysates were subjected to western blot for p-tau181 and p-tau202/205 to determine the specificity of the antibodies. PN APP were treated with the NLRP3 inhibitors CY-09 and CORM3 for 4 hours. The cell lysates were subjected to western blot for p-tau181 and p-tau202-205. Data are expressed as mean ± SD ( n = 3 independent repeats). * P < 0.05, ** P < 0.01 (one-way analysis of variance with Tukey’s post hoc test). (J) Brains lysates from APP/PS1ΔE9 mice were treated with phosphatase inhibitor (P.I.) or λ-protein phosphatase (λPP), and subjected to western blot to detect the indicated proteins. AD: Alzheimer’s disease; APP: amyloid precursor protein; DAPI: 4′,6-diamidino-2-phenylindole; DMSO: dimethyl sulfoxide; DNA: nuclear stained by DAPI that labels DNA; GSDMD: gasdermin D; GSDMD-FL: full-length GSDMD without being cleaved by active caspase-1; GSDMD-N: N-terminal fragment of GSDMD due to the cleavage of GSDMD-FL by active caspase-1; Iba1: ionized calcium binding adaptor molecule 1; NLRP3: NOD-, LRR- and pyrin domain-containing protein 3; ns: not significant; PCR: polymerase chain reaction; pro-caspase-1: The holo protein of caspase-1 without being cleaved; WT: wild-type.

Article Snippet: APPswe transgenic mice (Stock No. C001076, RRID: IMSR_JAX:001076) were purchased from Cyagen Biosciences Inc. (Suzhou, Jiangsu, China).

Techniques: Marker, Injection, Western Blot, Cell Culture, Mutagenesis, Transgenic Assay, Expressing, Staining, Reverse Transcription, Amplification, Negative Control, Binding Assay, Polymerase Chain Reaction

Journal: Neural Regeneration Research

Article Title: FUBP3 mediates the amyloid-β-induced neuronal NLRP3 expression

doi: 10.4103/NRR.NRR-D-23-01799

Figure Lengend Snippet: FUBP3 is required for NLRP3 expression in neurons in the presence of Aβ. (A) Control siRNA or mouse FUBP3 -specific siRNA and pNLRP3-Del5 were transfected into Neu2A cells for the luciferase assay. The promoter activities after normalization to the pGL3-basic vector were plotted. (B) Neuro2A cells were transfected with control siRNA or FUBP3-specific siRNA, and treated with DMSO as a control or with 1 μM Aβ overnight. The NRKP3 mRNA levels in these cells were determined by quantitative PCR, and the proteins were examined by western blot. FUBP3 and NLRP3 expression levels were quantified. (C) Primary neurons from wild-type mice (PN WT ) were infected with viruses expressing control shRNA or FUBP3 -targeting shRNA under the syn1 promoter, and treated with or without Aβ. FUBP3 and NLRP3 expression levels were compared among the different treatment groups. (D) PN WT were infected with viruses expressing control shRNA or shFUBP3, and the cells were treated with cerebrospinal fluid from patients with viral encephalitis. FUBP3 and NLRP3 were detected by western blot, and their expression levels were quantified. (E) PN APP were infected with viruses expressing control shRNA or shFUBP3, and FUBP3 and NLRP3 were detected by western blot, and their expression levels were quantified. (F) PN WT and primary neurons from APP Swedish mutant transgenic mice (PN APP ) growing on coverslips were stained with antibodies to FUBP3 (green, Alexa Fluor 488) and NeuN (red, Alexa Fluor 564), a neuronal cell marker. There was no discernable difference in FUBP3 expression levels between PN WT and PN APP . Asterisks indicate contaminating cells in the neuronal culture. (G) Brains sections from young wild-type mice, APP/PS1ΔE9 AD model mice, and aged wild-type mice were stained for FUBP3 (green, Alexa Fluor 488) and NeuN (red, Alexa Fluor 564), a neuronal marker. (H) FUBP3 was also expressed in the microglial cell line BV2. All data are expressed as mean ± SEM ( n = 3 independent repeats). * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 (two-tailed Student’s t -test). Aβ: Amyloid-β; B.E.: brightness enhanced; CSF: cerebrospinal fluid; DAPI: 4′,6-diamidino-2-phenylindole; DMSO: dimethyl sulfoxide; DNA: nuclear stained by DAPI that labels DNA; FUBP3: far upstream binding protein 3; NLRP3: NOD-, LRR- and pyrin domain-containing protein 3; ns: not significant; PCR: polymerase chain reaction; shRNA: short hairpin RNA.

Article Snippet: APPswe transgenic mice (Stock No. C001076, RRID: IMSR_JAX:001076) were purchased from Cyagen Biosciences Inc. (Suzhou, Jiangsu, China).

Techniques: Expressing, Control, Transfection, Luciferase, Plasmid Preparation, Real-time Polymerase Chain Reaction, Western Blot, Infection, shRNA, Mutagenesis, Transgenic Assay, Staining, Marker, Two Tailed Test, Binding Assay, Polymerase Chain Reaction

Journal: Neural Regeneration Research

Article Title: FUBP3 mediates the amyloid-β-induced neuronal NLRP3 expression

doi: 10.4103/NRR.NRR-D-23-01799

Figure Lengend Snippet: FUBP3 is required for IL1β secretion and tau phosphorylation in PN APP . (A) PN APP were infected with AAV expressing shFUBP3 shRNA to suppress FUBP3 expression. IL1β levels in the conditioned media were determined by ELISA (PN APP from 10 different embryos). (B) PN APP were infected with AAV expressing shFUBP3 or control shRNAs. The cell lysates were subjected to western blot for indicated proteins ( n = 6). Data are expressed as mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001 (two-tailed Student’s t -test). AAV: Adeno-associated virus; CaMK2α: calmodulin-dependent kinase IIα; ELISA: enzyme-linked immunosorbent assay; FUBP3: far upstream binding protein 3; IL1β: interleukin-1β; NLRP3: NOD-, LRR- and pyrin domain-containing protein 3; PME1: phosphatase methylesterase 1; PN APP : primary neurons of APP Swedish mutant single transgenic mouse; shRNA: short hairpin RNA.

Article Snippet: APPswe transgenic mice (Stock No. C001076, RRID: IMSR_JAX:001076) were purchased from Cyagen Biosciences Inc. (Suzhou, Jiangsu, China).

Techniques: Infection, Expressing, shRNA, Enzyme-linked Immunosorbent Assay, Control, Western Blot, Two Tailed Test, Virus, Binding Assay, Mutagenesis, Transgenic Assay

Journal: Neural Regeneration Research

Article Title: FUBP3 mediates the amyloid-β-induced neuronal NLRP3 expression

doi: 10.4103/NRR.NRR-D-23-01799

Figure Lengend Snippet: FUBP3 is involved in the immune response in Neuro2A cells. RNA was extracted from three paired Neuro2A cell lines expressing control- or siFUBP3-siRNA. (A) FPKM analysis of the effects of FUBP3 inhibition on gene expression compared with control cells. (B) Venn diagram showing the number of differentially expressed genes in each group. (C) PCR validation of the RNA-seq results. The expression levels of NLRP6 , GSDMD , PPP1R1A , and IL1b were examined. (D) PPP1R1A expression in primary neurons from APP Swedish mutant transgenic mice (PN APP ) in which FUBP3 was knocked down was examined by western blot ( N = 3 independent repeats, n = 5 (PN APP from five different embryos)). Data are expressed as mean ± SEM. ** P < 0.01 (two-tailed Student’s t -test). (E) KEGG analysis of key pathways highly enriched in differentially expressed genes. The red arrows indicate that genes involved in the immune response, as well as human diseases-infectious diseases viral/bacteria pathways, were highly enriched. (F) Histogram showing the number of differentially expressed genes in each KEGG pathway classification. The red arrows indicate immune system and infectious diseases (viral), and the green arrows indicate the endocrine system. (G) Gene set enrichment analysis (GSEA) using the Reactome database showed that the ATR and HDR pathways were negatively regulated in the NAF group compared with the NAC group. Aβ: Amyloid-β; DMSO: dimethyl sulfoxide; FPKM: fragments per kilobase of transcript per million fragments mapped; FUBP3: far upstream binding protein 3; GSDMD: gasdermin D; IL1b: interleukin-1b; KEGG: Kyoto Encyclopedia of Genes and Genomes; NAC: Neuro2A expressing control siRNA treated with Aβ; NAF: Neuro2A expressing siFUBP3 siRNA treated with Aβ; NDC: Neuro2A expressing control siRNA treated with DMSO; NDF: Neuro2A expressing siFUBP3 siRNA treated with DMSO; NLRP6: NOD-, LRR- and pyrin domain-containing protein 6; ns: not significant; PCR: polymerase chain reaction; PPP1R1A: protein phosphatase 1 regulatory inhibitor subunit 1A; siRNA: small interfering RNA.

Article Snippet: APPswe transgenic mice (Stock No. C001076, RRID: IMSR_JAX:001076) were purchased from Cyagen Biosciences Inc. (Suzhou, Jiangsu, China).

Techniques: Expressing, Control, Inhibition, RNA Sequencing Assay, Mutagenesis, Transgenic Assay, Western Blot, Two Tailed Test, Bacteria, Binding Assay, Polymerase Chain Reaction, Small Interfering RNA