Galactocerebrosidase-deficient oligodendrocytes maintain stable central myelin by exogenous replacement of the missing enzyme in mice

Abstract

Globoid cell leukodystrophy (GLD) is a lysosomal storage disease caused by genetic deficiency of galactocerebrosidase (GALC) activity. Failure in catalyzing the degradation of its major substrate, galactocerebroside, in oligodendrocytes (OLs) and Schwann cells leads to death of these myelinating cells, progressive demyelination, and early demise of GLD patients. Transplantation of bone marrow cells and umbilical cord blood have been attempted as a means of enzyme replacement and have shown limited success. It remains unknown whether or how these therapies support survival of GALC-deficient OLs and myelin maintenance. We report that, upon transplantation, GALC-deficient OLs from the twitcher mouse, a model of GLD, achieved widespread myelination in the brain and spinal cord of the myelin-deficient shiverer mouse, which was preserved for the life of the host. GALC immunohistochemistry showed direct evidence for GALC transfer from the shiverer environment to the engrafted mutant OLs in vivo. These findings suggest that the mutant OLs can internalize exogenous GALC and maintain stable myelin, demonstrating that exogenous enzyme replacement will be a key strategy in the therapy of GLD.

Fig. 1.

Widespread twi/CNP-EGFP grafts in the shi mouse brain 4 months after transplantation. Extensive distribution of twi mutant OLs/myelin in coronal (A) and sagittal (B) sections of shi brains revealed by EGFP signal (green), MBP immunolabeling (red), and their merged images. (C-E) Merged images of CNP-EGFP and MBP immunolabeling. (C) The twi/CNP-EGFP OL-derived myelination extending toward the ventral part of the external capsule at the midbrain level, showing long-distance migration of donor cells. The grafts were also found in areas distant from the injection sites, such as optic chiasm (D) and cerebellar white matter (E). C and D are coronal sections, and E is a sagittal section. [Scale bars: 500 μm (A-C) and 200 μm (D and E).]

Fig. 2.

Widespread twi/CNP-EGFP grafts in the shi spinal cord at 4 months of age. (A) Distribution of twi/CNP-EGFP OL/myelin was visible to the naked eye as white patches in the spinal cord of shi mice. Epifluorescence at 488 nm excitation (Insets) corresponds to the white patches, confirming that the myelinating cells were of donor origin. (B) In the cross section of A, the donor OLs/myelin spread over almost the whole white matter, shown by EGFP fluorescence (green), MBP immunolabeling (red), and their merged image. (C) MBP immunolabeling of the thoracolumbar spinal cord of 45-day-old moribund twi, 4-month-old wild-type, and 4-month-old untransplanted shi mice. (D) Toluidine blue myelin staining of the ventral column of thoracolumbar spinal cord. Despite severe demyelination in the 42-day-old moribund twi mouse, the transplanted twi/CNP-EGFP OLs myelinated numerous axons in the shi host. Myelin sheaths were absent in the untransplanted shi mouse. twi/CNP-EGFP OLs myelinated shi axons extensively in the dorsal (E), lateral (F), and ventral (G) columns. (H) Representative electron micrographs from the engrafted ventral column of shi spinal cord. (Inset) A higher magnification of the area indicated with *, showing well compacted myelin. [Scale bars: 1 mm (A), 200 μm (B and C), 20 μm (D), 10 μm (E-G), 5 μm (H), and 200 nm (H Inset).]

Fig. 3.

Stable integration of twi/CNP-EGFP OLs in the shi spinal cord. (A) The number of TUNEL⁺ cells was frequent in twi and rare in wild-type, shi, and the transplanted shi mice. (B-D) Data are the mean + SD number of TUNEL⁺ cells per 20-μm section. TUNEL⁺ cells (red) were found mostly in the white matter of twi mice (B) and were negligible in the shi mice with twi/CNP-EGFP graft (C) (TUNEL in red and CNP-EGFP in green) where, if found, 40% of TUNEL⁺ cells were the twi OLs (arrowhead in D), and the rest were not (arrow). [Scale bars: 200 μm (B and C) and 20 μm (D).]

Fig. 4.

Increased activity and expression of GALC in the shi spinal cord and their reduction after twi/CNP-EGFP OPC transplantation. (A) GALC activities of the excised dorsal column of the thoracolumbar spinal cord from twi, normal (MBP^shi/+), shi, shi with dead twi/CNP-EGFP OPC transplantation (sham surgery), and shi with twi/CNP-EGFP OPC transplantation. Data are mean + SD (n = 3-6 in each group). (B) GALC immunolabeling of the dorsal spinal cord of twi, wild-type, shi, and the transplanted shi mice. (C) Optical density of GALC immunoreactivity in the dorsal column was expressed as relative intensity to the optical density for twi mice. Data are the mean + SD. (n = 3 in each group). (D) Confocal images of GALC immunolabeling (red) showed the localization of GALC in a twi/CNP-EGFP OL (arrows) in the graft. Reconstructed orthogonal images are presented as viewed in the x-z (Lower) and y-z (Right) planes. [Scale bars: 100 μm (B) and 10 μm (D).]

Fig. 5.

Absence of macrophage accumulation in the twi/CNP-EGFP graft. CD45 immunolabeling (red) of the dorsal column of thoracolumbar spinal cord in 45-day-old moribund twi mice (A), 60-day-old wild-type mice (B), and 4-month-old untransplanted shi mice (C). Microglia in the engrafted dorsal column of shi mice were sporadically further activated; however, there was no macrophage accumulation (D). (Scale bar: 100 μm.)