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

Journal: Theranostics

Article Title: Microvascular endothelial metabolic dysfunction drives cerebral edema through bioenergetic failure after ischemia-reperfusion

doi: 10.7150/thno.127083

Figure Lengend Snippet: ATF4 promotes DDIT4 transcription leads to EC necroptosis and BBB injury in tMCAO mice. A, Western blot analysis of ATF4 protein levels in brain microvascular tissues isolated from 8-week-old and 20-month-old tMCAO mice, with quantification of protein levels (right panel, n = 6 per group). B, Real-time quantitative PCR analysis of ATF4 mRNA levels in brain microvascular tissues isolated from 8-week-old and 20-month-old tMCAO mice (n = 4 per group). C, Heat map illustrates the mRNA expression levels of key target genes associated with ATF4 transcriptional activation in the microvascular tissues of aged mice. Values were determined by qRT-PCR and plotted as fold change relative to control. D-E, qChIP analysis of the DDIT4 promoters was performed using antibodies against ATF4 in microvascular tissues from aged mice after ischemic-reperfusion (n = 3 per group). F, Changes in the mRNA levels of the downstream target gene DDIT4 were observed following ATF4 overexpression (n = 5 per group). G, Changes in the mRNA levels of the downstream target gene DDIT4 were observed following simultaneous ATF4 knockdown during in vitro oxygen-glucose deprivation and reperfusion (n = 5 per group). H. Schematic diagram of the AAV used for ATF4 knockdown in vivo . I, Experimental design and timeline of ischemic stroke, ATF4 knockdown, and analysis in aged mice. J, Representative images results of immunofluorescence staining for TUNEL (green, indicating apoptosis) and PI (red, indicating necrosis) in ECs within the ischemia-reperfusion cortical region following ATF4 knockdown. K, Representative images illustrating the co-staining of p-MLKL with BMECs (CD31) of the ischemia-reperfusion in cortical region following ATF4 knockdown. L, Illustration of TEER permeability assay and FITC-dextran permeability assay. M-N, TEER and FITC permeability of vehicle-treated (AdV-shNT) versus AdV-shDDIT4 treated ECs under ATF4 overexpression. (n = 5 independent experiments). Data are mean ± SEM.

Article Snippet: To specific knockdown of DDIT4 in BMECs in vivo , an adenovirus containing double-floxed Cre-inducible mir155-shRNA targeted against DDIT4 was constructed (Genechem, Shanghai, China).

Techniques: Western Blot, Isolation, Real-time Polymerase Chain Reaction, Expressing, Activation Assay, Quantitative RT-PCR, Control, Over Expression, Knockdown, In Vitro, In Vivo, Immunofluorescence, Staining, TUNEL Assay, Permeability, FITC-Dextran Permeability Assay

Journal: Theranostics

Article Title: Microvascular endothelial metabolic dysfunction drives cerebral edema through bioenergetic failure after ischemia-reperfusion

doi: 10.7150/thno.127083

Figure Lengend Snippet: Genetic knockdown of DDIT4 ameliorates EC necroptosis and BBB injury in tMCAO mice. A, Representative images of reconstructed Z-stack showing colocalization of DDIT4 and endothelial cell marker CD31 in the cortex of tMCAO mice. B, Representative images co-stained with fibrin, DDIT4 and cortical microvascular ECs (CD31) in the ischemia-reperfusion area. C, Quantitative analysis of the co-localization of DDIT4 and ECs (n = 8 per group). D, Analysis of correlation between co-localization ratio of DDIT4 and CD31 (represented by DDIT4/CD31) and Fibrinogen area. E, Schematic diagram of the AAV used for DDIT4 knockdown in vivo . F, Experimental design and timeline of ischemic stroke, DDIT4 knockdown, and analysis in aged mice. G, Representative images co-stained with fibrin and cortical microvascular ECs (CD31) in the ischemia-reperfusion area from DDIT4 conditional knockdown mice. H, Representative images results of immunofluorescence staining for TUNEL (green, indicating apoptosis) and PI (red, indicating necrosis) in ECs within the ischemia-reperfusion cortical region following DDIT4 knockdown. I, Representative images illustrating the co-staining of p-MLKL with BMECs (CD31) of the ischemia-reperfusion in cortical region following DDIT4 knockdown. J-K, Statistical analysis results of immunofluorescence staining for TUNEL (green, indicating apoptosis) and PI (red, indicating necrosis) in ECs within the ischemia-reperfusion cortical region following DDIT4 knockdown (n = 6 per group). L, Quantitative analysis of the co-localization of p-MLKL and ECs (n = 6 per group). M, CCK-8 results of cell viability of primary microvascular ECs (n = 4 independent experiments). N-O, Representative flow cytometry images and statistical analysis results of ECs stained with PI/annexin V. The necroptosis activation was represented by ratio of PI + /annexin V + (n = 5 per group). Data are mean ± SEM.

Article Snippet: To specific knockdown of DDIT4 in BMECs in vivo , an adenovirus containing double-floxed Cre-inducible mir155-shRNA targeted against DDIT4 was constructed (Genechem, Shanghai, China).

Techniques: Knockdown, Marker, Staining, In Vivo, Immunofluorescence, TUNEL Assay, CCK-8 Assay, Flow Cytometry, Activation Assay

Journal: Theranostics

Article Title: Microvascular endothelial metabolic dysfunction drives cerebral edema through bioenergetic failure after ischemia-reperfusion

doi: 10.7150/thno.127083

Figure Lengend Snippet: Knockdown of DDIT4 in BMECs ameliorates mitochondrial dysfunction and reduces mtROS release. A, Mitochondrial morphology in primary ECs with overexpression of DDIT4. B, Mitochondrial morphology in OGD/R-treated primary ECs after DDIT4 shRNA. C-D, Mean branch length (µm), mean perimeter (µm) and mean form factor (au) of mitochondria (n = 5 independent experiments). E, Representative images of LC3 co-stained with Tomm20 in primary BMECs after OGDR. F-G, The number of LC3 punch and co-location of LC3 and TOMM20 were presented (right). H-I, Mitochondrial membrane potential measured by flow cytometry of JC-1. JC-1 aggregation = normal mitochondrial membrane potential; JC-1 monomers = low membrane potential x-axis. J, Histograms display y-axis. K, Quantification of JC-1 flow cytometry (n = 5 per group). L, Representative images of staining with mitoSOX (red) and live cell nuclear stain hoechst33342 (blue). M, IntDen/cell measured using ImageJ (n = 5 per group). Data are mean ± SEM.

Article Snippet: To specific knockdown of DDIT4 in BMECs in vivo , an adenovirus containing double-floxed Cre-inducible mir155-shRNA targeted against DDIT4 was constructed (Genechem, Shanghai, China).

Techniques: Knockdown, Over Expression, shRNA, Staining, Membrane, Flow Cytometry

Journal: Theranostics

Article Title: Microvascular endothelial metabolic dysfunction drives cerebral edema through bioenergetic failure after ischemia-reperfusion

doi: 10.7150/thno.127083

Figure Lengend Snippet: DDIT4 facilitates the ubiquitination of NDUFS3 resulted in mitochondrial dysfunction. A, Mass spectrometry analysis of proteins pulled down by anti-DDIT4 antibodies identified potential DDIT4-interacting proteins. B, Volcano plot of differentially expressed proteins between mitochondria isolated from cerebral microvascular tissues before and after ischemia-reperfusion. C, Overlapping analysis of differential proteins in mitochondrial proteomic and interacting proteins of DDIT4. D, Heat map showed the expression of 11 overlapping proteins in mitochondrial proteomic. E, Co-immunoprecipitation of NDUFS3 (left) or DDIT4 (right) in ECs (IgG as a control antibody). F, Western blotting of NDUFS3 in indicated cells. Endothelial cell was treated with MG132 6h for harvest. And the quantification of protein was shown in below (n = 6 per group). G, Western blotting of NDUFS3 in cerebral microvascular tissues from conditional DDIT4 knockdown mice. The quantification of protein was shown in below (n = 6 per group). H, Co-IP of NDUFS3 and then western blotted with anti-ubiquitin in DDIT4 overexpression ECs. I, Representative images of staining with mitoSOX (red) and live cell nuclear stain hoechst33342 (blue). J, Phospho-MLKL aggregation in indicated cells. K, IntDen/cell measured using ImageJ (n = 6 per group). L, p-MLKL aggregates/nucleus quantified using Fiji (n = 6 per group). M, The OCR was measured in indicated cells. Data are mean ± SEM.

Article Snippet: To specific knockdown of DDIT4 in BMECs in vivo , an adenovirus containing double-floxed Cre-inducible mir155-shRNA targeted against DDIT4 was constructed (Genechem, Shanghai, China).

Techniques: Ubiquitin Proteomics, Mass Spectrometry, Isolation, Expressing, Immunoprecipitation, Control, Western Blot, Knockdown, Co-Immunoprecipitation Assay, Over Expression, Staining

Journal: Theranostics

Article Title: Microvascular endothelial metabolic dysfunction drives cerebral edema through bioenergetic failure after ischemia-reperfusion

doi: 10.7150/thno.127083

Figure Lengend Snippet: Interruption of glycolysis/H3K18la/ATF4-DDIT4 feedback loop of BMECs alleviate edema and brain injury in tMCAO mice. A, Western blotting of DDIT4 in primary BMECs treated with varying concentrations of lactate. The quantification of protein was shown in the right panel (n = 5 per group). B, Western blotting of DDIT4 in primary BMECs treated with lactate for different durations at the same concentration. The quantification of protein was shown in the right panel (n = 5 per group). C, Western blotting of DDIT4 in primary BMECs with or without LDHA overexpression. The quantification of protein was shown in the right panel (n = 6 per group). D, Western blotting of DDIT4 in OGDR treated primary BMECs with or without LDHA knockdown. The quantification of protein was shown in the right panel (n = 3 per group). E, Colorimetric assay for lactate levels in primary BMECs with or without DDIT4 overexpression (n = 5 per group). F, Colorimetric assay for lactate levels in OGDR treated primary BMECs with or without DDIT4 konckdown (n = 5 per group). G, Western blotting of Pan- and site-specific histone lactylation in primary BMECs with or without DDIT4 overexpression. The quantification of protein was shown in the right panel (n = 3-4 per group). H, Experimental design and timeline of ischemic stroke, DDIT4 knockdown, and analysis in tMCAO mice. I-J, Evans blue leakage of mice brains in coronal sections and extravasation (fold change relative to sham) from Sham, AAV-shControl Tie2-cre tMCAO, and AAV-shDDIT4 Tie2-cre tMCAO groups (n = 6 per group). K-L, Representative images and statistical results of TTC (n = 6 per group). M, Experimental design and timeline of ischemic stroke and analysis in LDHA flox/flox mice and LDHA flox/flox ; CDH5-CreERT mice. N-O, Representative images and statistical results of TTC (n = 5 per group). P, Statistical results of brain water content (n = 10 per group). Q, Modified neurological severity score at 3 days after tMCAO in different groups (n = 6 per group). Data are mean ± SEM.

Article Snippet: To specific knockdown of DDIT4 in BMECs in vivo , an adenovirus containing double-floxed Cre-inducible mir155-shRNA targeted against DDIT4 was constructed (Genechem, Shanghai, China).

Techniques: Western Blot, Concentration Assay, Over Expression, Knockdown, Colorimetric Assay, Modification