Journal: Nature Communications

Article Title: TNF alpha unmasks enteric malate aspartate shuttle dysfunction bridging Parkinson disease and intestinal inflammation

doi: 10.1038/s41467-026-71317-y

Figure Lengend Snippet: a Paradigm illustrating the differentiation of ENLs from iPSCs. BMP4, Recombinant human bone morphogenetic protein-4; CHIR, CHIR 99021; RA, Retinoic Acid; SB, SB431542; FGF2, Recombinant Human FGF Basic; AA, ascorbic acid; GDNF, Recombinant Human Glial Derived Neurotrophic Factor; NC, neural crest; ENC, enteric neural crest; ENL, enteric neural lineages. Created in BioRender. Winner, B. (2026) https://BioRender.com/652io1g ( b ) Heatmap showing gene expression of neuronal marker TUBB3 , glial marker GFAP and enteric neuronal markers PHOX2B , ELAVL4 and HOXB3 in iPSC-derived ENLs at days 6, 40 and 70 of differentiation using RT-qPCR. Log 2 fold change was calculated in relation to the Iso group at day 6 of differentiation and averaged for n = 3 independent SNCA 3x and 3 isogenic lines per group, from two independent differentiations. See also Fig. . Source data are provided as a Source Data file. c Immunocytochemistry characterization of iPSC-derived ENLs at day 70 after start of differentiation. Panels show the glial marker GFAP (red), the enteric neuronal marker HuC/D (green), α-syn (green, 2A7 antibody, Novus, Cat#NBP1-05194) and DAPI (blue) for cell nuclei staining for both Iso (left) and SNCA 3x (right). Scale bar = 50 μm. Figure is representative of two independent experiments. d UMAP plots obtained from scRNAseq analysis of Iso and SNCA 3x ENLs at day 70 after start of differentiation, separated by the clusters identified after annotation. e Compositional analysis plot showing the absence of significantly changed clusters between Iso and SNCA 3x. Data are presented as observed log 2 fold change (center points) +/− 95% confidence intervals (error bars). Confidence intervals were calculated using bootstrap resampling. P -values were determined via permutation testing and adjusted for multiple comparisons using the Benjamini-Hochberg method (adj. p -value). FDR=False Discovery Rate; log2FD=log 2 Fold Difference. n = 3 biological replicates per group. f Dotplot showing the average and percentage of expression of canonical marker genes for each cluster identified in (D). g Ridgeplot comparing the expression of SNCA between each cluster identified in (D). n = 3 biological replicates per group, p-values calculated by unpaired two-tailed Student’s t test.

Article Snippet: Panels show the glial marker GFAP (red), the enteric neuronal marker HuC/D (green), α-syn (green, 2A7 antibody, Novus, Cat#NBP1-05194) and DAPI (blue) for cell nuclei staining for both Iso (left) and SNCA 3x (right).

Techniques: Recombinant, Derivative Assay, Gene Expression, Marker, Quantitative RT-PCR, Immunocytochemistry, Staining, Expressing, Two Tailed Test

Journal: Nature Communications

Article Title: TNF alpha unmasks enteric malate aspartate shuttle dysfunction bridging Parkinson disease and intestinal inflammation

doi: 10.1038/s41467-026-71317-y

Figure Lengend Snippet: a Ligand-Receptor pair analysis of scRNAseq data prior to subclustering. Pairs with large mean change and high variance are purple. Pairs failing variance ( ≥ 0.3) or mean ( ≥ 0.3) cut-offs are orange and gray, respectively. b Experimental paradigm depicting iPSC-ENL stimulation with cytokines. Created in BioRender. Winner, B. (2026) https://BioRender.com/a2k344q . c ELISA of total α-syn, normalized by total protein. n = 3 independent SNCA 3x and 3 isogenic lines per group, from two independent differentiations, mean ± SEM, * p = 0.0433 by two-way ANOVA with Sidak post-hoc. d Flow cytometry analysis using Live/Dead staining. n = 3 independent SNCA 3x and 3 isogenic lines per group, from two independent differentiations, mean ± SEM. e ELISA of aggregated α-syn, normalized by total protein. n = 3 independent SNCA 3x and 3 isogenic lines per group, from two independent differentiations, mean ± SEM, * p = 0.0303 by two-way ANOVA with Sidak post-hoc. f Human cytokine array quantification of the supernatants of iPSC-ENLs previously stimulated for 24 h with 100 ng/ml TNF. Data represent the mean of n = 3 independent SNCA 3x and 3 isogenic lines per group, from one differentiation. g Experimental paradigm for the MEA experiments. Created in BioRender. Winner, B. (2026) https://BioRender.com/vwugkr4 . Quantification of the number of spikes ( h ), and active electrodes ( i ), n =wells of a CytoView MEA 48-well plate, representative of n = 3 independent SNCA 3x and 3 isogenic lines per group, from two independent differentiations, mean ± SEM, * p = 0.046, ** p = 0.0031 by two-way ANOVA with Sidak post-hoc. j Flow cytometry analysis of the percentage and total of α-syn + (2A7 antibody) enteric neurons (CD56 + CD24 high ) and enteric glia (CD56 + CD24 low ). n = 3 independent SNCA 3x and 3 isogenic lines per group, from two independent differentiations, mean ± SEM, p = 0.0419 (% α-syn + neurons); p = 0.0294 (# α-syn + neurons); p = 0.0478 (% α-syn + glia); p = 0.0123 (# α-syn + glia) by two-way ANOVA with Sidak post-hoc. k Flow cytometry analysis of the percentage and total enteric neurons (CD56 + CD24 high ) and enteric glia (CD56 + CD24 low ). n = 3 independent SNCA 3x and 3 isogenic lines per group, from two independent differentiations, mean ± SEM. Source data are provided as a Source Data file in c – f , h , I , j , k . Basal: DPBS + 0.1% BSA.

Article Snippet: Panels show the glial marker GFAP (red), the enteric neuronal marker HuC/D (green), α-syn (green, 2A7 antibody, Novus, Cat#NBP1-05194) and DAPI (blue) for cell nuclei staining for both Iso (left) and SNCA 3x (right).

Techniques: Enzyme-linked Immunosorbent Assay, Flow Cytometry, Staining

Journal: Nature Communications

Article Title: TNF alpha unmasks enteric malate aspartate shuttle dysfunction bridging Parkinson disease and intestinal inflammation

doi: 10.1038/s41467-026-71317-y

Figure Lengend Snippet: a Paradigm describing the proximity ligation assay (PLA) and measurement of mitochondrial ROS experiments. Created in BioRender. Winner, B. (2026) https://BioRender.com/vb5oi60 . b Assessment of PLA results using immunocytochemistry. Panels are separated in basal and TNF stimulated for both Iso and SNCA 3x groups. Panels show the nuclear marker DAPI (blue), the mitochondrial marker TOM20 (gray), α-syn (green) and PLA dots for TOM20-α-syn (red). Scale bar = 50 μm. Basal refers to cells treated with vehicle used to dilute the TNF (DPBS + 0.1% BSA). c Quantification of the PLA dots in the cytoplasm. n=number of total individual cells analyzed per condition indicated in the figure, from 3 independent SNCA 3x and 3 isogenic lines per group, from one differentiation, with mean ± SEM, **** p < 0.0001, by one-way ANOVA with Tukey’s post-hoc. Basal refers to cells treated with vehicle used to dilute the TNF (DPBS + 0.1% BSA). Data were normalized to the number of nuclei per image. Source data are provided as a Source Data file. Concatenated t-SNE plots from flow cytometry data of all lines highlighting Mitosox high populations across genotypes and conditions and quantification of the percentage and total mitosox high cells in enteric neurons (CD56 + CD24 high ) ( d ) and in enteric glia (CD56 + CD24 low ) ( e ). n = 3 independent SNCA 3x and 3 isogenic lines per group, from two independent differentiations, mean ± SEM. Source data are provided as a Source Data file.

Article Snippet: Panels show the glial marker GFAP (red), the enteric neuronal marker HuC/D (green), α-syn (green, 2A7 antibody, Novus, Cat#NBP1-05194) and DAPI (blue) for cell nuclei staining for both Iso (left) and SNCA 3x (right).

Techniques: Proximity Ligation Assay, Immunocytochemistry, Marker, Flow Cytometry

Journal: Nature Communications

Article Title: TNF alpha unmasks enteric malate aspartate shuttle dysfunction bridging Parkinson disease and intestinal inflammation

doi: 10.1038/s41467-026-71317-y

Figure Lengend Snippet: a Paradigm of meta-analysis of independent UC patient cohorts, responders or non-responders to infliximab therapy and controls ( GSE16879 , GSE12251 , GSE73661 ). Created in BioRender. Winner, B. (2026) https://BioRender.com/jkisazh . b GOT1 expression (log2 normalized) across datasets. Analyzed by Student’s t -test or one-way ANOVA (Tukey’s post-hoc). GSE73661 : Control vs. UC NR before, p = 0.0002; Control vs. UC R before, p = 0.035; UC R before vs. UC R after, p = 0.0421. GSE16879 : Control vs. UC NR before, p = 0.0007. GSE12251 : p = 0.0158. c SNCA expression (log2 normalized) across datasets. Analyzed by t-test or ANOVA. GSE73661 : Control vs. UC NR before, p = 0.0237; UC NR after vs. UC R after, p = 0.0112. d Bioinformatic analysis paradigm of UC cohorts (inflamed/non-inflamed) using bulk RNA-seq (IBDome, https://ibdome.org/ ) and scRNAseq (scIBD, http://scibd.cn/ ). Created in BioRender. Winner, B. (2026) https://BioRender.com/h6tkzor . e Expression of MAS genes ( GOT1 , GOT2 , MDH1 , MDH2 ) in inflamed vs. non-inflamed UC tissue (IBDome, n = 66 inflamed, n = 28 non-inflamed). TPM normalized, log10 scale. Two-sided Wilcoxon-Mann-Whitney test. Error bars: 95% CI. Exact p-values are indicated in the figure. f SNCA expression in inflamed vs. non-inflamed UC tissue (IBDome, n = 66 inflamed, n = 28 non-inflamed). TPM normalized, log10 scale. Two-sided Wilcoxon–Mann–Whitney test. Error bars: 95% CI. g Correlation analysis between GOT1 and SNCA in inflamed and non-inflamed tissue (IBDome, n = 66 inflamed, n = 28 non-inflamed). Pearson test. The shaded area denotes the 95% confidence interval of the slope of the linear model. h UMAP plot generated by integrating the 12 scRNAseq studies from the scIBD platform, highlighting the neural cluster in red. i Mean expression value of the MAS enzymes GOT1 , GOT2 , MDH1 and MDH2 in healthy, UC non-inflamed and UC inflamed tissue. Data came from the scIBD platform. j Immunocytochemistry of healthy controls and UC patient gut tissue: β-tubulin III (orange), total α-syn (red), AATC (green), DAPI (blue). Scale bar: 50 μm. k Quantification of the immunocytochemistry shown is (J) for n = 3 controls and n = 3 UC patients, mean ± SEM. Integrated density was calculated using FIJI and normalized to the total cell count per image. Data analyzed by unpaired two-tailed t -tests. AATC, * p = 0.04; α-syn, * p = 0.01. Source data are provided as a Source Data file in b , c , e , f , g , k .

Article Snippet: Panels show the glial marker GFAP (red), the enteric neuronal marker HuC/D (green), α-syn (green, 2A7 antibody, Novus, Cat#NBP1-05194) and DAPI (blue) for cell nuclei staining for both Iso (left) and SNCA 3x (right).

Techniques: Expressing, Control, RNA Sequencing, MANN-WHITNEY, Generated, Immunocytochemistry, Cell Characterization, Two Tailed Test

Journal: Nature Communications

Article Title: TNF alpha unmasks enteric malate aspartate shuttle dysfunction bridging Parkinson disease and intestinal inflammation

doi: 10.1038/s41467-026-71317-y

Figure Lengend Snippet: Graphical summary of findings. SNCA 3x drives inflammatory-metabolic vulnerability in the ENS. Our multi-omics and functional approach using iPSC-derived ENLs revealed that α-syn accumulation induces basal mitochondrial and synaptic dysfunction, altering cell composition and transcriptional profiles in both enteric neurons and glia. Challenging these cells with the proinflammatory cytokine TNF acts as a second-hit, unmasking selective vulnerability in SNCA 3x ENLs, marked by enhanced α-syn-mitochondria interactions and a corresponding surge in oxidative stress-associated populations. This functional collapse is mechanistically driven by a disruption of the malate-aspartate shuttle and the TCA cycle, forcing cells to become critically reliant on glutamine oxidation for energy. We demonstrate that the glutamate metabolism modulator Chicago Sky Blue 6B effectively restores mitochondrial function, reverses the TNF-driven glutamine dependency, and enhances metabolic flexibility, suggesting a targeted therapeutic strategy for α-syn-driven enteric pathology. Finally, we validate this core mechanism by showing that MAS suppression, linked to α-syn accumulation, is a conserved signature in inflamed human gut tissue. Created in BioRender. Winner, B. (2026) https://BioRender.com/d91886y .

Article Snippet: Panels show the glial marker GFAP (red), the enteric neuronal marker HuC/D (green), α-syn (green, 2A7 antibody, Novus, Cat#NBP1-05194) and DAPI (blue) for cell nuclei staining for both Iso (left) and SNCA 3x (right).

Techniques: Biomarker Discovery, Functional Assay, Derivative Assay, Disruption