Brain and spinal cord imaging. At initial presentation: (A) MRI cervical spine sagittal T2-weighted image with hyperintense signal involving the central gray matter, dorsal and lateral tracts of the cervical and thoracic spinal cord (yellow arrow). (B) MRI thoracic spine axial T2-weighted image with hyperintense signal involving primarily gray matter (yellow arrow). (C) MRI cervical spine axial post-gadolinium T1-weighted image with contrast enhancement of the dorsal white matter tracts (yellow arrow). Follow-up at 6 months: (D) MRI cervical spine sagittal T2-weighted image with re-demonstration of central gray matter hyperintensity of the cervical and thoracic spinal cord with associated volume loss (white arrow). (E) MRI cervical spine axial T2-weighted image with increased hyperintense signal involving primarily gray matter and volume loss within the gray matter tracts (white arrow). (F) MRI brain axial T2-weighted FLAIR image with diffuse parenchymal volume loss and nearly symmetric bilateral periventricular and deep white matter signal abnormality in the frontal, parietal, and occipital lobes (white arrows). Obtained 2 weeks before death: (G) MRI cervical spine sagittal T2-weighted image with recurrent non-enhancing hyperintense signal involving primarily the dorsal central aspect of the spinal (red arrow). (H) MRI brain axial T1-weighted imaging post-gadolinium with contrast enhancement involving the optic chiasm, prechiasmatic optic nerves, and bilateral optic tracts within the suprasellar region (red arrows). (I) MRI brain axial T2-weighted FLAIR image with progressive bilateral periventricular white matter signal abnormality with central areas of white matter necrosis (red arrows).

(A) Zebrafish Acox1 protein is highly conserved compared to human ACOX1. The degree of amino acid similarity is indicated by color, with red being identical and blue being non-conserved, as visualized using PRALINE. (B) Schematic diagram of expression constructs used in zebrafish experiments. (C) Confocal z-stack images of the expression of the construct in live zebrafish larvae; immunohistochemistry for anti-GFP, and anti-RFP in the Tg(olig2:dsRed) line. Dorsal to top, rostral to the right; age 3 dpf. Note: the black line in the middle of the figure indicates where the image was stitched together. (D) Confocal z-stack images, spinal cord, rostral to the right, dorsal to the top. Expression of N237S is indicated by tagged GFP, and mature oligodendrocytes are labeled by olig2:dsRed. (E) Quantification shows no effect on oligodendrocyte numbers.

Characterization of zebrafish mutant experiments by overexpression of human ACOX1 N237S. (A) Schematic diagram of experiments. (B) Zebrafish show abnormal motor (swimming) behavior, which is rescued by D-NAC. ***p < 0.001. (C) qRT-PCR characterization of transcripts. Expression of mutant form does not impact myelin transcripts or general inflammatory markers but does activate the ISR (atf4).

Zebrafish peroxisomal reporter construct and characterization of effects on peroxisomes. (A) Schematic of the mCherry-SKL construct and transient expression in zebrafish. Confocal z-stack, lateral view of the trunk, live 3 dpf larva, green channel, DCFH-DA (dacetyldichlorofluorescein). (B) Live confocal images and paired IMARIS (3D reconstruction) image (below), of mCherry-SKL (red) containing peroxisomes in the hair cells (blue) of larval zebrafish neuromast at 6 dpf in zebrafish overexpressing WT or N237S ACOX1. (C) Size and distance to nearest neighbors in zebrafish overexpressing WT or N237S ACOX1, showing a decreased density of peroxisomes in these cells. (D) qRT-PCR for expression of genes that affect the abundance of peroxisome: pparab, pex11a, pex11b, pex11g, and nrb1b.