Journal: bioRxiv

Article Title: Aberrant FICD-mediated AMPylation drives α-Synuclein pathology and overall protein dyshomeostasis in dopaminergic neurons in Parkinson’s disease

doi: 10.64898/2026.03.30.715195

Figure Lengend Snippet: a Immunofluorescence staining of tyrosine hydroxylase (TH, red) and FICD (white), as well as DAPI (blue) in rat substantia nigra. High-magnification example images display TH+FICD-, TH-FICD+, and TH+FICD+ (arrowheads) cells. Percentages of TH+FICD-, TH-FICD+, and TH+FICD+ subpopulations are presented, the unshown TH-FICD- cells comprise the remainder of the population. FICD is preferentially expressed in TH+ dopaminergic neurons (n = 4 animals / group). Scale bar: 5 µm or 20 µm (high-magnification). Statistical analysis: one-way ANOVA with Tukeýs multiple comparisons test and two-tailed Mann-Whitney-U-test. b DAB-Ni immunostaining of human substantia nigra from control individuals (Table S1), showing FICD expression (blue) and neuromelanin (NM) pigment (brown), a prototypical feature of dopaminergic neurons. FICD is strongly expressed in NM+ dopaminergic neurons. High-magnification example images display NM+FICD-, NM-FICD+, and NM+FICD+ (arrowhead) cells (n = 5 individuals / group). Scale bar: 50 µm or 20 µm (high-magnification). c DAB-Ni immunostaining of FICD in the human post-mortem substantia nigra of controls and PD patients. Quantification of NM+ and FICD+ cells demonstrates a significant reduction of NM+ neurons (neurons/mm 2 , right top) and their proportional reduction within FICD+ cells (right bottom) in PD (n = 5 individuals / group). Scale bar: 50 µm. d Western blot analysis of human post-mortem putamen from controls and PD patients (Table S2) shows no significant difference in FICD levels when normalized to GAPDH (bottom left). However, normalization to TH (bottom right) demonstrates significantly increased FICD levels in PD patients (n = 5 individuals / group). e-f Western blot analysis of midbrain tissue from Thy1-aSyn and non-transgenic WT mice. FICD levels were significantly elevated in Thy1-aSyn mice compared with WT controls when normalized to TH (n = 6 animals / group for quantification). d Western blot analysis of striatal tissue from MBP-aSyn and non-transgenic WT mice. FICD expression was significantly increased in MBP-aSyn mice compared with WT controls (n= 5-7 animals / group for quantification). Statistical analysis from b - f: two-tailed Mann-Whitney-U-test. p* < 0.05, p** < 0.01, p*** < 0.001. Bar graphs: mean ± SD. LC: loading control using revert 520 total protein stain; SN: substantia nigra .

Article Snippet: For each experiment, the signal intensity of proteins of interest was normalized to the corresponding signal of the designated loading control, such as immunointensity of glyceraldehyde-3-phosphate-dehydrogenase (GAPDH), total protein stain using Revert 520 Total Protein Stain (Li-Cor), or Coomassie Brilliant Blue G 250 (Bio-Rad) on the SDS-PAGE gel.

Techniques: Immunofluorescence, Staining, Two Tailed Test, MANN-WHITNEY, Immunostaining, Control, Expressing, Western Blot, Transgenic Assay

Journal: bioRxiv

Article Title: Aberrant FICD-mediated AMPylation drives α-Synuclein pathology and overall protein dyshomeostasis in dopaminergic neurons in Parkinson’s disease

doi: 10.64898/2026.03.30.715195

Figure Lengend Snippet: a Western blot analysis of AMPylated and total BiP in SNCA Ctrl and SNCA Dupl midbrain neurons treated with closantel or DMSO as control. Quantification of the AMPylated-to-total BiP ratio revealed a significant reduction upon closantel treatment in SNCA Dupl midbrain neurons, confirming effective AMPylation inhibition (n = 3). Statistical analysis: one-way ANOVA with Tukeýs multiple comparisons test. b Solubility assay of aSyn and phosphorylated aSyn in SNCA Dupl neurons treated with closantel or DMSO. Following closantel treatment, levels of insoluble total aSyn were reduced (p = 0.056). Importantly, insoluble phosphorylated aSyn was significantly and more robust reduced (n = 3). Statistical analysis: two-tailed unpaired t test. c Immunocytochemical analysis of aSyn using antibodies 15G7 (white) and LB509 (green, higher affinity to aggregated aSyn) in SNCA Ctrl and SNCA Dupl midbrain neurons expressing TUBB3 (red). Nuclei were stained via DAPI (blue). LB509-immunopositive aSyn (highlighted by arrows) accumulated predominantly in neurites of SNCA Dupl neurons, whereas closantel treatment significantly reduced aSyn accumulation in neurites. No effect was observed in SNCA Ctrl neurons. Quantifications represent the ratio of neuritic to somatic fluorescence intensity (n=3; n = 16, 17, 14, 17 neurons for Ctrl, Ctrl-Clo, Dupl and Dupl-Clo, respectively). Statistical analysis: one-way ANOVA with Tukeýs multiple comparisons test. Bar graphs for a-c: mean ± SD. d Analysis of neuritic morphology in SNCA Ctrl and SNCA Dupl neurons treated with closantel. Left: representative immunostaining and maximum intensity projection images of neurons labelled by TUBB3 immunostaining (black); Right: quantification shows an increased neurite diameter in both genotypes following treatment, while neurite volume remained unchanged (n = 3; for diameter: n = 210, 174, 236, 161 neurites for Ctrl, Ctrl-Clo, Dupl and Dupl-Clo conditions, respectively; for volume: n = 20 neurites / differentiation). Scale bars: 50 µm. Statistical analysis: one-way ANOVA with Tukeýs multiple comparisons test. Floating bars: min to max, line at media. p* < 0.05, p** < 0.01, p*** < 0.001. AMP-BiP: AMPylated BiP; Ctrl: SNCA Ctrl ; Dupl: SNCA Dupl ; Clo: closantel; IS: insoluble fraction; LC: loading control using revert 520 total protein stain; p-aSyn: phosphorylated aSyn; S: soluble fraction; TUBB3: βIII-tubulin .

Article Snippet: For each experiment, the signal intensity of proteins of interest was normalized to the corresponding signal of the designated loading control, such as immunointensity of glyceraldehyde-3-phosphate-dehydrogenase (GAPDH), total protein stain using Revert 520 Total Protein Stain (Li-Cor), or Coomassie Brilliant Blue G 250 (Bio-Rad) on the SDS-PAGE gel.

Techniques: Western Blot, Control, Inhibition, Solubility, Two Tailed Test, Expressing, Staining, Fluorescence, Immunostaining