Journal: Redox Biology

Article Title: Upregulated GBP2 exacerbates Parkinson's disease pathogenesis by impairing NIX-dependent mitophagy

doi: 10.1016/j.redox.2026.104029

Figure Lengend Snippet: GBP2 expression is significantly upregulated in PD models. (A) Bioinformatic analysis of transcriptomic data from the GEO database ( GSE106608 ) showing mRNA expression levels of GBP family members in the substantia nigra of PD patients and healthy controls. (B) RT-qPCR analysis of GBP mRNA levels in the substantia nigra of MPTP-induced PD mice and control mice (n = 3 mice per group). (C) Representative western blots and quantification of TH and GBP2 protein levels in the substantia nigra of the indicated groups (n = 4 mice per group). (D) Representative immunofluorescence images showing GBP2 expression in the substantia nigra (n = 4 mice per group). Scale bar, 20 μm. (E–G) SH-SY5Y cells were treated with or without 1.5 mM MPP + for 24 h (n = 3 independent biological replicates). (E) RT-qPCR, (F) Western blot, and (G) immunofluorescence staining showing the upregulation of GBP2 in MPP + treated SH-SY5Y cells. Scale bar, 50 μm. Data are presented as mean ± SEM. (H) Representative Western blot showing GBP2 protein levels in SH-SY5Y cells after lentivirus-mediated A53T α-synuclein-overexpressing (n = 3 independent biological replicates). (I) Representative Western blot showing GBP2 protein levels in the substantia nigra of 13-month-old A53T transgenic mice and their wild-type littermates (n = 4 mice per group). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 for comparisons between the indicated groups.

Article Snippet: For GBP2 knockdown, an adeno-associated virus serotype 9 carrying GBP2 shRNA (AAV9-GBP2-shRNA; 1.48 × 10 13 vg/mL) was purchased from Sangon Biotech.

Techniques: Expressing, Quantitative RT-PCR, Control, Western Blot, Immunofluorescence, Staining, Transgenic Assay

Journal: Redox Biology

Article Title: Upregulated GBP2 exacerbates Parkinson's disease pathogenesis by impairing NIX-dependent mitophagy

doi: 10.1016/j.redox.2026.104029

Figure Lengend Snippet: Genetic knockdown of GBP2 attenuates apoptosis and ameliorates motor deficits in PD models. (A, B) SH-SY5Y cells were transfected with negative control siRNA (si-NC) or GBP2-specific siRNA (si-GBP2) and then treated with or without MPP + (n = 3 independent biological replicates). (A) RT-qPCR and (B) Western blot analysis validating the efficiency of GBP2 knockdown. (C) CCK-8 assay showing that GBP2 knockdown significantly attenuated MPP + -induced loss of cell viability (n = 3 independent biological replicates). (D) Flow cytometric analysis of apoptosis by Annexin V/PI staining, demonstrating that GBP2 knockdown reduced MPP + -induced apoptosis (n = 3 independent biological replicates). (E) Western blot analysis of GBP2 and TH protein levels in the substantia nigra of mice injected with AAV9-control (sh-NC) or AAV9-shGBP2 (sh-GBP2) and treated with MPTP or saline. GBP2 knockdown mitigated the MPTP-induced loss of TH protein (n = 6 mice per group). (F) Motor behavioral tests (open field, pole, and rotarod) showing that GBP2 knockdown ameliorated MPTP-induced motor deficits (n = 8 mice per group). (G) Representative IHC staining of the substantia nigra and striatum, showing ameliorated neuronal loss and pathological morphology in sh-GBP2 mice after MPTP treatment (n = 4 mice per group). Scale bar, 50 μm. (H) Representative TUNEL staining (red) in the substantia nigra, with DAPI (blue) counterstain for nuclei. GBP2 knockdown reduced MPTP-induced apoptosis (n = 4 mice per group). Scale bar, 20 μm. Data are presented as mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 for comparisons between the indicated groups.

Article Snippet: For GBP2 knockdown, an adeno-associated virus serotype 9 carrying GBP2 shRNA (AAV9-GBP2-shRNA; 1.48 × 10 13 vg/mL) was purchased from Sangon Biotech.

Techniques: Knockdown, Transfection, Negative Control, Quantitative RT-PCR, Western Blot, CCK-8 Assay, Staining, Injection, Control, Saline, Immunohistochemistry, TUNEL Assay

Journal: Redox Biology

Article Title: Upregulated GBP2 exacerbates Parkinson's disease pathogenesis by impairing NIX-dependent mitophagy

doi: 10.1016/j.redox.2026.104029

Figure Lengend Snippet: GBP2 accumulates in mitochondria and its knockdown ameliorates mitochondrial dysfunction and structural damage (A) Nucleocytoplasmic fractionation of SH-SY5Y cells treated with or without MPP + . β-actin and Histone H3 serve as cytoplasmic and nuclear markers, respectively (n = 3 independent biological replicates). (B) Mitochondrial and cytosolic fractions from cells treated with or without MPP + were analyzed by western blotting for GBP2. TOMM20 and β-actin serve as mitochondrial and cytosolic markers, respectively (n = 3 independent biological replicates). (C) Immunofluorescence confocal microscopy showing colocalization of GBP2 (green) with a Mitotracker (red) in SH-SY5Y cells treated with or without MPP + (n = 3 independent biological replicates). Scale bar, 10 μm. (D–F) SH-SY5Y cells transfected with si-NC or si-GBP2 were treated with MPP + (n = 3 independent biological replicates). (D) Cellular ATP levels, (E) quantification of mitochondrial respiration capacity, and (F) mitochondrial ROS levels (MitoSOX Red staining) were measured. GBP2 knockdown significantly ameliorated MPP + -induced mitochondrial dysfunction. (G) Representative DHE staining (red) for superoxide in the substantia nigra of mice from the indicated groups (n = 4 mice per group). Scale bar, 20 μm. (H) Representative transmission electron microscopy (TEM) images of mitochondria in the substantia nigra. Yellow arrows indicate damaged cristae; white arrows indicate swollen mitochondria. Scale bar, 500 nm. Data are presented as mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 for comparisons between the indicated groups.

Article Snippet: For GBP2 knockdown, an adeno-associated virus serotype 9 carrying GBP2 shRNA (AAV9-GBP2-shRNA; 1.48 × 10 13 vg/mL) was purchased from Sangon Biotech.

Techniques: Knockdown, Fractionation, Western Blot, Immunofluorescence, Confocal Microscopy, Transfection, Staining, Transmission Assay, Electron Microscopy

Journal: Redox Biology

Article Title: Upregulated GBP2 exacerbates Parkinson's disease pathogenesis by impairing NIX-dependent mitophagy

doi: 10.1016/j.redox.2026.104029

Figure Lengend Snippet: GBP2 knockdown enhances mitophagy. (A) Western blot analysis of key mitophagy markers in SH-SY5Y cells transfected with si-NC or si-GBP2 and treated with or without MPP + (n = 3 independent biological replicates). GBP2 knockdown specifically upregulated NIX and the LC3-II/LC3-I ratio, while downregulating p62 and TOMM20. (B) Western blot analysis of the indicated proteins in the substantia nigra of mice from the indicated groups. GBP2 knockdown enhanced NIX expression and the LC3-II/LC3-I ratio, while reducing p62 and TOMM20 levels under MPTP treatment (n = 6 mice per group). (C) Representative immunofluorescence images showing colocalization of LC3 (red) with the mitochondrial marker COX-IV (green) in (Left) SH-SY5Y cells (n = 3 independent biological replicates) and (Right) the substantia nigra (n = 4 mice per group) of mice under the indicated conditions. Scale bars, 10 μm (cells); 20 μm (tissue). (D, E) SH-SY5Y cells transfected with si-NC or si-GBP2 were pretreated with the mitophagy inhibitor Mdivi-1 (10 μM) or DMSO for 6 h prior to MPP + exposure (n = 3 independent biological replicates). (D) CCK-8 assay for cell viability and (E) flow cytometry for apoptosis showed that Mdivi-1 treatment partially reversed the protective effects of GBP2 knockdown (n = 3 independent biological replicates). Data are presented as mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 for comparisons between the indicated groups.

Article Snippet: For GBP2 knockdown, an adeno-associated virus serotype 9 carrying GBP2 shRNA (AAV9-GBP2-shRNA; 1.48 × 10 13 vg/mL) was purchased from Sangon Biotech.

Techniques: Knockdown, Western Blot, Transfection, Expressing, Immunofluorescence, Marker, CCK-8 Assay, Flow Cytometry

Journal: Redox Biology

Article Title: Upregulated GBP2 exacerbates Parkinson's disease pathogenesis by impairing NIX-dependent mitophagy

doi: 10.1016/j.redox.2026.104029

Figure Lengend Snippet: GBP2 directly interacts with NIX via its GTPase domain and facilitates its degradation. (A) Prediction of GBP2-interacting proteins from the BioGRID database, identifying NIX as a candidate. (B) ZDOCK prediction of the three-dimensional structural interaction between GBP2 and NIX, revealing a high-confidence binding interface. (C) Immunofluorescence confocal microscopy showing colocalization of endogenous GBP2 (green) and NIX (red) in SH-SY5Y cells. Scale bar, 10 μm. (D) Co-immunoprecipitation (Co-IP) assays in HEK293T cells overexpressing Flag-GBP2 and Myc-NIX, confirming their interaction. (E) Endogenous Co-IP validating the interaction between GBP2 and NIX in SH-SY5Y cells. (F) Co-IP assays in HEK293T cells expressing Myc-NIX and full-length Flag-GBP2, GTPase domain-deleted GBP2 (ΔLG), or C-terminal helical domain-deleted GBP2 (ΔCTHD). The interaction is dependent on the LG domain. (G–J) GBP2 knockdown rescues NIX protein expression. (G) MPP + treatment reduces NIX protein levels in SH-SY5Y cells, as shown by immunofluorescence (n = 3 independent biological replicates). Scale bar, 50 μm. (H, I) Immunofluorescence analysis of NIX in (H) MPP + -treated SH-SY5Y cells (n = 3 independent biological replicates) and (I) the substantia nigra of MPTP-treated mice (n = 4 mice per group) demonstrates that GBP2 knockdown prevents NIX downregulation. Scale bars, 50 μm (cell) and 20 μm (tissue) (J) Western blot of NIX in mitochondrial fractions from SH-SY5Y cells confirms the increase in NIX upon GBP2 knockdown (n = 3 independent biological replicates). Data are presented as mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 for comparisons between the indicated groups.

Article Snippet: For GBP2 knockdown, an adeno-associated virus serotype 9 carrying GBP2 shRNA (AAV9-GBP2-shRNA; 1.48 × 10 13 vg/mL) was purchased from Sangon Biotech.

Techniques: Binding Assay, Immunofluorescence, Confocal Microscopy, Immunoprecipitation, Co-Immunoprecipitation Assay, Expressing, Knockdown, Western Blot

Journal: Redox Biology

Article Title: Upregulated GBP2 exacerbates Parkinson's disease pathogenesis by impairing NIX-dependent mitophagy

doi: 10.1016/j.redox.2026.104029

Figure Lengend Snippet: GBP2 promotes NIX ubiquitination and proteasomal degradation, and its protective effects on mitophagy and PD are NIX-dependent. (A) NIX mRNA levels in SH-SY5Y cells transfected with si-NC or si-GBP2 were measured by RT-qPCR. GBP2 knockdown did not alter NIX mRNA expression (n = 3 independent biological replicates). (B, C) SH-SY5Y cells transfected with si-NC or si-GBP2 were treated with MPP + . (B) Cells were treated with cycloheximide (CHX, 100 μg/ml) for the indicated times (n = 3 independent biological replicates). Western blot quantification shows that GBP2 knockdown prolonged the half-life of NIX protein. (C) Cells were treated with the proteasome inhibitor MG132 (10 μM) or the lysosome inhibitor chloroquine (CQ, 10 μM) for 6 h (n = 3 independent biological replicates). MG132, but not CQ, blocked the GBP2 knockdown-induced increase in NIX protein levels. (D) Ubiquitination assays in HEK293T cells co-expressing HA-Ub, with Flag-GBP2 or pcDNA3.1, showing that GBP2 overexpression enhances NIX ubiquitination. (E–I) SH-SY5Y cells were co-transfected with si-GBP2 and si-NIX (or respective controls) and treated with MPP + (n = 3 independent biological replicates). NIX knockdown reversed the protective effects of GBP2 knockdown on: (E) mitophagy markers (LC3-II/I and p62) in mitochondrial fractions, (F) mitochondrial membrane potential, (G) mitochondrial ROS levels, (H) apoptosis, and (I) cell viability. Data are presented as mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 for comparisons between the indicated groups.

Article Snippet: For GBP2 knockdown, an adeno-associated virus serotype 9 carrying GBP2 shRNA (AAV9-GBP2-shRNA; 1.48 × 10 13 vg/mL) was purchased from Sangon Biotech.

Techniques: Ubiquitin Proteomics, Transfection, Quantitative RT-PCR, Knockdown, Expressing, Western Blot, Over Expression, Membrane

Journal: Redox Biology

Article Title: Upregulated GBP2 exacerbates Parkinson's disease pathogenesis by impairing NIX-dependent mitophagy

doi: 10.1016/j.redox.2026.104029

Figure Lengend Snippet: Geranylgeranylation is essential for GBP2 mitochondrial localization and function, and its inhibition protects against neurotoxicity. (A) Triton X-114 phase partitioning assay in SH-SY5Y cells treated with or without MPP + . MPP + treatment shifted GBP2 toward the detergent phase, indicating increased hydrophobicity (n = 3 independent biological replicates). (B, C) SH-SY5Y cells were transfected with Flag-GBP2, Flag-GBP2-ΔCAAX, or treated with the geranylgeranylation inhibitor GGTI298 (10 μM) for 48 h (n = 3 independent biological replicates). (B) Immunofluorescence of GBP2 (green) and a Mitotracker (red) shows that disrupting geranylgeranylation reduces mitochondrial localization of GBP2. Scale bar, 10 μm. (C) Western blot of GBP2 in mitochondrial and cytosolic fractions confirms the reduction in mitochondrial targeting in Flag-GBP2-ΔCAAX group or GGTI298 group. (D) Co-IP assays in HEK293T cells show that GGTI298 treatment or expression of the GBP2-ΔCAAX mutant impairs the interaction between GBP2 and NIX. (E–G) MPTP-induced PD model mice were treated with GGTI298 or saline. (E) Behavioral tests (open field, pole, rotarod) demonstrate that GGTI298 treatment ameliorates MPTP-induced motor deficits (n = 8 mice per group). (F) Western blot and (G) representative IHC for TH in the substantia nigra and striatum show that GGTI298 treatment preserves dopaminergic neurons (n = 4 mice per group). Scale bar, 50 μm. Data are presented as mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 for comparisons between the indicated groups.

Article Snippet: For GBP2 knockdown, an adeno-associated virus serotype 9 carrying GBP2 shRNA (AAV9-GBP2-shRNA; 1.48 × 10 13 vg/mL) was purchased from Sangon Biotech.

Techniques: Inhibition, Transfection, Immunofluorescence, Western Blot, Co-Immunoprecipitation Assay, Expressing, Mutagenesis, Saline