ddit4  (Proteintech)

Journal: Journal of Advanced Research

Article Title: Spike RBD drives sustained Parkinson’s disease progression via microglia-neuron crosstalk-mediated RTP801 upregulation

doi: 10.1016/j.jare.2025.07.060

Figure Lengend Snippet: Sustained upregulation of RTP801 in dopaminergic neurons depends on microglial activation with RBD. (A) Colocalization of TH with RTP801 in the SNc at day 1, 3 and 7 after treated with RBD (left, Scale bar = 200 μm). Colocalization of Iba1 with RTP801 in the SNc at day 1, 3 and 7 after treated with RBD (right, Scale bar = 50 μm). (B) Schematic of the microglial depletion with PLX5622 experiment. (C) Representative images demonstrating microglial (Iba1 + cells) depletion in the SNc following PLX5622 treatment, in comparison to vehicle mice (Scale bar = 200 μm). (D-E) Behavior analysis of the rotarod test and grip strength test for each group with or without PLX5622 treatment (n = 6). (F) ThS staining in the SNc for each group (Scale bar = 5 μm). (G) Western blot analysis of RTP801 and TH for each group (n = 4). (H) Colocalization of Iba1 and TH with His-tag RBD in the SNc at day 1, 3 and 7 after treated with RBD or vehicle (Scale bar = 5 μm). (I) Western blot analysis of RTP801 of neurons treated with RBD or MCM (n = 4). (J) Western blot analysis of RTP801 of SH-SY5Y-αSyn A53T cells treated with RBD or MCM and inhibitors (n = 4). Data are expressed as means ± SEM, * P < 0.05, ** P < 0.01, *** P < 0.001, ns, not significant.

Article Snippet: The membranes were then blocked using 5 % BSA in TBST and incubated overnight at 4 °C with the following primary antibodies: TH (1:500, Santa Cruz, sc-25269), 5G4 (1:1000, Sigma-Aldrich, MABN389), αSyn (1:1000, Abcam, ab138501), cGAS (1:500, Proteintech, 26416–1-AP), STING (1:2000, Proteintech, 19851–1-AP), TBK1 (1:1000, Proteintech, 28397–1-AP;), p-TBK1 (1:1000, Cell Signaling Technology, 5483S), IRF3 (1:5000, Proteintech, 66670–1-Ig), p-IRF3 (1:1000, Cell Signaling Technology, 29047S), RTP801 (1:1000, NOVUS, NBP1-77321) and β-actin (1:10000, ABclonal, AC026), followed by secondary antibodies (1:5000, KPL, Maryland, USA) at room temperature for 2 h. Protein bands were visualized using an Image Quant LAS 4000 mini (GE, Boston, USA), and their intensities were quantified using ImageJ software (Maryland, USA).

Techniques: Activation Assay, Comparison, Staining, Western Blot

Journal: Journal of Advanced Research

Article Title: Spike RBD drives sustained Parkinson’s disease progression via microglia-neuron crosstalk-mediated RTP801 upregulation

doi: 10.1016/j.jare.2025.07.060

Figure Lengend Snippet: RBD accelerates PD progression with significantly upregulation of RTP801. (A) Volcano plot of differentially expression genes of RBD treatment compared with vehicle in CSGS 1w model mice. Significantly upregulated genes are colored in orange, significantly downregulated genes are colored in blue, and the rest genes are colored in gray. (B) Gene Ontology enrichment was based on differentially expression genes that have a p value smaller than 0.05, n = 3. (C) Heatmap of selected genes of CSGS 1w model mice treated with RBD or vehicle. (D-E) Relative mRNA levels of the type I interferon (D) and stress-responsive genes (E) in SNc (n = 4). (F) Western blot analysis of RTP801 across groups (n = 4). (G) Colocalization of RTP801 with TH in SNc, scale bar = 200 μm. (H) Pearson’s correlation analysis between RTP801 density and TH + cells number (all values were normalized relative to CSGS 1w + Vehicle group), n = 4 per group. (I) Heatmap of RTP801 and TH colocalization in SNc. Data are expressed as means ± SEM, * P < 0.05, ** P < 0.01, *** P < 0.001, ns, not significant. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: The membranes were then blocked using 5 % BSA in TBST and incubated overnight at 4 °C with the following primary antibodies: TH (1:500, Santa Cruz, sc-25269), 5G4 (1:1000, Sigma-Aldrich, MABN389), αSyn (1:1000, Abcam, ab138501), cGAS (1:500, Proteintech, 26416–1-AP), STING (1:2000, Proteintech, 19851–1-AP), TBK1 (1:1000, Proteintech, 28397–1-AP;), p-TBK1 (1:1000, Cell Signaling Technology, 5483S), IRF3 (1:5000, Proteintech, 66670–1-Ig), p-IRF3 (1:1000, Cell Signaling Technology, 29047S), RTP801 (1:1000, NOVUS, NBP1-77321) and β-actin (1:10000, ABclonal, AC026), followed by secondary antibodies (1:5000, KPL, Maryland, USA) at room temperature for 2 h. Protein bands were visualized using an Image Quant LAS 4000 mini (GE, Boston, USA), and their intensities were quantified using ImageJ software (Maryland, USA).

Techniques: Expressing, Western Blot

Journal: Journal of Advanced Research

Article Title: Spike RBD drives sustained Parkinson’s disease progression via microglia-neuron crosstalk-mediated RTP801 upregulation

doi: 10.1016/j.jare.2025.07.060

Figure Lengend Snippet: RTP801 is critical for PD deterioration induced by RBD. (A) Schematic of the αSyn A53T + ; RTP801 −/− mice experiment. (B) Assessment of olfactory dysfunction was conducted using the BFPT and the visible BFPT. (C) A modified open field test with nuts or paprika quantified olfactory acuity. (D-F) Motor coordination and balance were evaluated using the rotarod (D) , pole (E) , and beam walking (F) tests. ( G ) Forelimb grip strength was gauged through the grip strength test. ( H ) The open field test appraised anxiety-related behaviors, tracking total distance traveled, center zone activity. (I-J) Recognition memory was assessed using Y maze test and NORT. Alternation index (% of total triplet arm entries) was quantified in (I) . New object index was quantified in ( J ) (n = 6). (K) Representative images of TH immunostaining in the SNc and striatum and quantitative assessment (all values were normalized relative to αSyn A53T group), scale bar = 500 μm, n = 4. (L) Western blot analysis of TH expression in SNc and striatum and quantitative analysis (n = 4). (M) Colocalization of RTP801 with TH, scale bar = 200 μm. (N) ThS staining quantifies aggregates in the SNc, including ThS signal density, and the proportion of cells inclusive ThS + puncta (n = 4), scale bar = 5 μm. (O) Western blot detection of αSyn aggregation using the 5G4 antibody, with quantitative analysis confirming significant changes (n = 4). ( P ) Analysis of αSyn partitioning in soluble and insoluble brain fractions by western blot, with quantitative data indicating significant alterations (n = 4). Data are expressed as means ± SEM, * P < 0.05, ** P < 0.01, *** P < 0.001, ns, not significant.

Article Snippet: The membranes were then blocked using 5 % BSA in TBST and incubated overnight at 4 °C with the following primary antibodies: TH (1:500, Santa Cruz, sc-25269), 5G4 (1:1000, Sigma-Aldrich, MABN389), αSyn (1:1000, Abcam, ab138501), cGAS (1:500, Proteintech, 26416–1-AP), STING (1:2000, Proteintech, 19851–1-AP), TBK1 (1:1000, Proteintech, 28397–1-AP;), p-TBK1 (1:1000, Cell Signaling Technology, 5483S), IRF3 (1:5000, Proteintech, 66670–1-Ig), p-IRF3 (1:1000, Cell Signaling Technology, 29047S), RTP801 (1:1000, NOVUS, NBP1-77321) and β-actin (1:10000, ABclonal, AC026), followed by secondary antibodies (1:5000, KPL, Maryland, USA) at room temperature for 2 h. Protein bands were visualized using an Image Quant LAS 4000 mini (GE, Boston, USA), and their intensities were quantified using ImageJ software (Maryland, USA).

Techniques: Olfactory, Modification, Activity Assay, Immunostaining, Western Blot, Expressing, Staining

Journal: Journal of Advanced Research

Article Title: Spike RBD drives sustained Parkinson’s disease progression via microglia-neuron crosstalk-mediated RTP801 upregulation

doi: 10.1016/j.jare.2025.07.060

Figure Lengend Snippet: RTP801 and the mtDNA-cGAS-STING-IFNβ pathway drive RBD-induced neurodegeneration. (A) Heatmap of mitochondrial genes in SNc of CSGS 1w model mice treated with RBD or vehicle. (B) Relative mRNA levels of mitochondrial function genes in SNc (n = 4). (C) Ultrastructure of the mitochondria in SNc. Scale bar = 1 µm. Representative TEM photomicrographs intact, swollen, damaged and degenerated mitochondria and the proportion of mitochondria with different forms. Scale bar = 200 nm. The red arrows represent the damaged mitochondria. (D) Western blot analysis of p-DRP1 S616 or 637/DRP1, MFN1, and MFN2 for each group (n = 4). (E) Quantification of cytosolic mtDNA via qPCR (n = 4). (F) Representative immunofluorescence of mtDNA release in SNc using anti-DNA (red) and anti-TOM20 (green) antibodies, and white arrows indicate mtDNA release events (Scale bar = 5 μm, n = 40 cells from 4 mice). (G) Western blot analysis of cGAS, STING, p-TBK1/TBK1, and p-IRF3/IRF3 for each group (n = 4). (H) Relative mRNA level of IFN-β in SNc (n = 4). (I) Serum IFN-β levels were measured (n = 4). (J) Western blot analysis of proteins expression in αSyn A53T + ; RTP801 −/− mice and quantitative analysis (n = 4). Data are expressed as means ± SEM, * P < 0.05, ** P < 0.01, *** P < 0.001, ns, not significant. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: The membranes were then blocked using 5 % BSA in TBST and incubated overnight at 4 °C with the following primary antibodies: TH (1:500, Santa Cruz, sc-25269), 5G4 (1:1000, Sigma-Aldrich, MABN389), αSyn (1:1000, Abcam, ab138501), cGAS (1:500, Proteintech, 26416–1-AP), STING (1:2000, Proteintech, 19851–1-AP), TBK1 (1:1000, Proteintech, 28397–1-AP;), p-TBK1 (1:1000, Cell Signaling Technology, 5483S), IRF3 (1:5000, Proteintech, 66670–1-Ig), p-IRF3 (1:1000, Cell Signaling Technology, 29047S), RTP801 (1:1000, NOVUS, NBP1-77321) and β-actin (1:10000, ABclonal, AC026), followed by secondary antibodies (1:5000, KPL, Maryland, USA) at room temperature for 2 h. Protein bands were visualized using an Image Quant LAS 4000 mini (GE, Boston, USA), and their intensities were quantified using ImageJ software (Maryland, USA).

Techniques: Western Blot, Immunofluorescence, Expressing

Journal: Journal of Advanced Research

Article Title: Spike RBD drives sustained Parkinson’s disease progression via microglia-neuron crosstalk-mediated RTP801 upregulation

doi: 10.1016/j.jare.2025.07.060

Figure Lengend Snippet: Spike RBD accelerates and sustains PD progression through microglia-neuron crosstalk-mediated RTP801 upregulation. Spike RBD exposure in CSGS 1w mice accelerates the progression of PD, leading to the deterioration of motor and non-motor functions, weakened neuronal connectivity, and increased dyskinesia. Mechanistically, RBD initially activates microglia (days 1–3 post-injection), which then secrete cytokines IL-6 and IL-8, leading to an upregulation of neuronal RTP801 by day 7. This process, coupled with RBD-induced mitochondrial dysfunction and mtDNA release, activates the cGAS-STING pathway in both neurons and microglia. The resulting aberrant increase in RTP801 establishes a detrimental feed-forward loop that sustains neurodegeneration.

Article Snippet: The membranes were then blocked using 5 % BSA in TBST and incubated overnight at 4 °C with the following primary antibodies: TH (1:500, Santa Cruz, sc-25269), 5G4 (1:1000, Sigma-Aldrich, MABN389), αSyn (1:1000, Abcam, ab138501), cGAS (1:500, Proteintech, 26416–1-AP), STING (1:2000, Proteintech, 19851–1-AP), TBK1 (1:1000, Proteintech, 28397–1-AP;), p-TBK1 (1:1000, Cell Signaling Technology, 5483S), IRF3 (1:5000, Proteintech, 66670–1-Ig), p-IRF3 (1:1000, Cell Signaling Technology, 29047S), RTP801 (1:1000, NOVUS, NBP1-77321) and β-actin (1:10000, ABclonal, AC026), followed by secondary antibodies (1:5000, KPL, Maryland, USA) at room temperature for 2 h. Protein bands were visualized using an Image Quant LAS 4000 mini (GE, Boston, USA), and their intensities were quantified using ImageJ software (Maryland, USA).

Techniques: Injection