Proteasome mutations associated with CANDLE syndrome cause altered neuronal development by dysregulating polyamine synthesis
Clayton W. Winkler, Benjamin Schwarz, Katie Williams, Sara Alehashemi, Simote T. Foliaki, Joseph Snow, Lisa Joseph, Audrey Thurm, Christopher L. Friend, Gwendolyn Cooper, Eric Bohrnsen, Farzana Bhuyan, Nathan T. Brandes, Ruin Moaddel, Manfred Boehm, Guibin Chen, Cole D. Kimzey, Bibiana Bielekova, Joanna Kocot, Peter Kosa, Cathryn L. Haigh, Raphaela Goldbach-Mansky, Karin E. Peterson
Clayton W. Winkler, Benjamin Schwarz, Katie Williams, Sara Alehashemi, Simote T. Foliaki, Joseph Snow, Lisa Joseph, Audrey Thurm, Christopher L. Friend, Gwendolyn Cooper, Eric Bohrnsen, Farzana Bhuyan, Nathan T. Brandes, Ruin Moaddel, Manfred Boehm, Guibin Chen, Cole D. Kimzey, Bibiana Bielekova, Joanna Kocot, Peter Kosa, Cathryn L. Haigh, Raphaela Goldbach-Mansky, Karin E. Peterson
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Research Article Genetics Metabolism Neuroscience

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

Abstract

Loss-of-function mutations in PSMB8/β5i and other components of the 20S proteasome result in multiorgan diseases, such as Chronic atypical neutrophilic dermatosis with lipodystrophy and elevated temperature (CANDLE) syndrome. Neurocognitive dysfunction associated with CANDLE suggests that proteasomal mutations may impact neuronal function and development early in life. We generated cerebral organoids (COs) from induced pluripotent stem cells (iPSCs) made from patients with CANDLE. The COs from CANDLE iPSCs exhibited impaired neuronal development when compared with COs from healthy control iPSCs. Impaired neuronal maturation in CANDLE COs was correlated with increased polyamines, which were also elevated in CSF from patients with CANDLE. The proteasome-regulated ornithine decarboxylase (ODC), the rate limiting enzyme in polyamine biosynthesis, was elevated in CANDLE neurons. Inhibition of ODC reversed polyamine overproduction and repaired neuronal maturation in CANDLE COs, suggesting a potential therapeutic avenue for intervention. These findings demonstrate that dysfunction of the proteasome affects neuronal development through overproduction of polyamines via dysregulation of ODC and offer insight into potential therapeutic strategies for CNS-related proteasomal dysfunction.

Authors

Clayton W. Winkler, Benjamin Schwarz, Katie Williams, Sara Alehashemi, Simote T. Foliaki, Joseph Snow, Lisa Joseph, Audrey Thurm, Christopher L. Friend, Gwendolyn Cooper, Eric Bohrnsen, Farzana Bhuyan, Nathan T. Brandes, Ruin Moaddel, Manfred Boehm, Guibin Chen, Cole D. Kimzey, Bibiana Bielekova, Joanna Kocot, Peter Kosa, Cathryn L. Haigh, Raphaela Goldbach-Mansky, Karin E. Peterson

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Figure 6

NPCs and Neurons with CANDLE mutation have altered polyamine metabolism.

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NPCs and Neurons with CANDLE mutation have altered polyamine metabolism....
(A) Comparison of CANDLE and HC NPC metabolites via t test (technical n = 6, biological: n = 2 CANDLE, 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 cutoff for an 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. (C) Comparison of CANDLE and HC Neuron metabolites via t test (technical: n = 6, biological: n = 3 CANDLE, 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. (D) Amine metabolism map of the same neuron metabolite data displayed in C with the fold change of metabolites represented as color and the significance represented as node size. (E) Ratio metric densitometry analysis of Western blot banding of ODC protein relative to GAPDH in CANDLE versus HC iPSC-derived NPCs (left graph), neurons (middle graph), and COs (right graph). Each point represents an individual lysate generated from a single well of a confluent 12 well plate of either NPCs or neurons or from a single CO. The donor source of the iPSC-derived cultures or COs are indicated in the graph. Data were compared via a 2-tail, unpaired t test. ***P < 0.001. (F) Representative Western blot expressed of PSMB8 protein by iPSC-derived NPCs (left graph), neurons (middle graph), and COs (right graph). The immature form of PSMB8 (I) is approximately 26 kD in molecular weight and the mature form is approximately 21 kD. The same blots were reprobed for GAPDH expression at approximately 39 KD (bottom blots). Lysates from iPSC derived NPCs (HC1, HC2, CAN1, and CAN3) neurons (HC1, CAN1, CAN2, and CAN3) and COs (HC1, HC2, CAN1, CAN2, and CAN3) were all examined. Banding patterns between HC and CAN were found to be similar within each sample type. Specifically, NPCs had little-to-no PSMB8 expression in either genotypic group while neurons and COs had prominent bands present in all samples. Whole blots with all samples tested are shown in the supplement.