Journal: bioRxiv

Article Title: Proteasome mutations associated with CANDLE syndrome cause altered neuronal development by dysregulating polyamine synthesis

doi: 10.1101/2025.08.14.670165

Figure Lengend Snippet: (A-F) Immunohistochemical analysis of neural progenitor (Sox2) and mature neuron (Tuj1) markers in (A, B) HC1 and (C, D) CAN1 cerebral organoids. Blue signal is cell nuclei. Quantification of immunohistochemical labeling of (E) Sox2 and (F) Tuj1 analyzed on sections from n=56 CANDLE and n=31 healthy control organoids. Data are plotted as percent of each signal within each tissue section using symbols described in Table S1 to indicate COs from generated from different iPSCs. An unpaired t test between each group was performed. **** indicates p<0.0001. (G) Flow cytometry was performed on n=6 organoids generated from HC1 and CAN1 with labeling for the same Sox2 and Tuj1 markers. Data is shown as the percentage of positive live cells from whole dissociated organoid. A 2way ANOVA with multiple comparisons was performed. *** indicates p<0.001. Other p values are indicated with specific values.

Article Snippet: The HC1 iPSC line was obtained commercially from ATCC ( https://www.atcc.org/products/acs-1023 ), while the generation of HC2 iPSCs has been previously described as being generated from a skin punch biopsy and reprogrammed using ReproRNATM-OKSGM ( Foliaki et al ., 2020 ).

Techniques: Immunohistochemical staining, Labeling, Control, Generated, Flow Cytometry

Journal: bioRxiv

Article Title: Proteasome mutations associated with CANDLE syndrome cause altered neuronal development by dysregulating polyamine synthesis

doi: 10.1101/2025.08.14.670165

Figure Lengend Snippet: (A) cDNA reverse transcribed from mRNA isolated from COs from HC1, HC2, CAN1, CAN2 and CAN3 was analyzed by real-time PCR for the indicated genes. Data as analyzed as the ratio of GAPDH mRNA expression relative to the gene of interest for each organoid. A 2way ANOVA with a Sidak’s multiple comparisons test was used to compare these group. No statistical difference was observed. (B) Supernatants from HC1 and CAN1 COs at three weeks of age were measured for the concentration of cytokines related to the IFN response using a human inflammation 37 bio- plex multiplex immunoassay. No significant difference was observed in cytokine expression between groups. An unpaired t test between HC and CAN was used to compare groups. No statistical difference was observed. (C-D) HC1 and CAN1 COs were infected with 10 3 PFU of La Crosse virus (LACV) or Inkoo virus (INKV). At the indicated time points, COs were collected and mRNA extracted and reverse transcribed. Individual COs were analyzed for (C) IFNB1 and (D) IFIT1 mRNA expression. All graphs, each symbol represents data from individual organoids or supernatants with two-to-eight samples per group per time point. An unpaired t test was used to compare HC and CAN within the mock, LACV and INKV groups. No significance was observed.

Article Snippet: The HC1 iPSC line was obtained commercially from ATCC ( https://www.atcc.org/products/acs-1023 ), while the generation of HC2 iPSCs has been previously described as being generated from a skin punch biopsy and reprogrammed using ReproRNATM-OKSGM ( Foliaki et al ., 2020 ).

Techniques: Reverse Transcription, Isolation, Real-time Polymerase Chain Reaction, Expressing, Concentration Assay, Multiplex Assay, Infection, Virus

Journal: bioRxiv

Article Title: Proteasome mutations associated with CANDLE syndrome cause altered neuronal development by dysregulating polyamine synthesis

doi: 10.1101/2025.08.14.670165

Figure Lengend Snippet: (A) Comparison of CANDLE and HC NPC metabolites via t-test (technical n=6, biological n=2 CAN n=3 HC). Metabolites annotated as being associated with amine metabolism (purple dots), Acetyl-CoA metabolism (light blue dots), and nucleobase metabolism (magenta dots) are indicated. A line representing the raw p-value cut-off for a FDR or 10 % calculated via a Benjamini Hochberg correction is indicated. (B) Amine metabolism map of the same NPC data displayed in (A) with the fold change of metabolites represented as color and the significance represented as node size as indicated in the associated legend. Hatch marked nodes indicate metabolites below the limit of detection. The yellow diamond indicates Acetyl-CoA usage by SSAT for the inactivation of polyamines via acetylation. (C) Transcriptional expression of SSAT in NPCs derived from HC1 and CAN3 (n=3 replicate wells for each). Data are plotted as SSAT expression as a percent of GAPDH housekeeping gene. Data were compared by a two-tailed unpaired t-test. P value is indicated. (D) Comparison of CANDLE and HC Neuron metabolites via t-test (technical n=6, biological n=3 CAN n=3 HC). Metabolites annotated as being associated with amine metabolism (purple dots), Acetyl-CoA metabolism (light blue dots), and nucleobase metabolism (magenta dots) are indicated. (E) Amine metabolism map of the same neuron metabolite data displayed in (D ) with the fold change of metabolites represented as color and the significance represented as node size. The yellow diamond indicates Acetyl-CoA usage by SSAT for the inactivation of polyamines via acetylation. (F) Burst rate defined as the number of bursts (at least three spikes detected within 100ms) over a 2min period in cultured neurons from n=2 HC and n=2 CAN (15-37 technical replicate wells recorded for each patient). The average readout of active channels per well represented a replicate. Data were compared between cell lines by a two-tail unpaired t-test. **** indicates p<0.0001. (G) Western Blot analysis for OCD1 expression by neurons generated from HC1 and CAN2. n=5 wells from a 12 well plate with 5x10^5 cells. The monomeric ODC is ∼50kD in molecular weight in the top blot. The same blot was reprobed for GAPDH expression at ∼39KD (bottom blot) (H) Ratiometric densitometry analysis of ODC expression relative to GAPDH control.

Article Snippet: The HC1 iPSC line was obtained commercially from ATCC ( https://www.atcc.org/products/acs-1023 ), while the generation of HC2 iPSCs has been previously described as being generated from a skin punch biopsy and reprogrammed using ReproRNATM-OKSGM ( Foliaki et al ., 2020 ).

Techniques: Comparison, Expressing, Derivative Assay, Two Tailed Test, Cell Culture, Western Blot, Generated, Molecular Weight, Control