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.enzyme replacement ͉ globoid cell leukodystrophy ͉ myelination ͉ transplantation ͉ lysosomal storage disease G loboid cell leukodystrophy (GLD, also called Krabbe's disease) (1) is a lysosomal storage disease caused by genetic deficiency of activity of a lysosomal enzyme galactocerebrosidase (GALC) that catalyzes the digestion of its major substrate galactocerebroside in myelinating oligodendrocytes (OLs) and Schwann cells (2). GLD is characterized pathologically by rapidly progressive demyelination of the CNS and peripheral nervous system (PNS) and accumulation of macrophages in the demyelinating lesions. The majority of GLD patients (infantile form) succumb to severe neurological symptoms in the first two years of life.Homozygous twitcher (GALC twi/twi ; twi) mice (3) have a genetic defect in GALC activity (4, 5), show progressive demyelination and accumulation of macrophages in the CNS and PNS, and rarely survive Ͼ45 days. These similarities to GLD have enabled the mouse to serve as a bona fide model to explore the pathophysiology and therapy of GLD (6). It has recently been shown that twi OLs can internalize exogenous GALC in vitro, resulting in their phenotypic correction (7), raising the possibility that in vivo enzyme replacement could be used to treat GLD. However, it is essential to evaluate the effect of enzyme replacement on OL function in vivo, because in vitro correction cannot unequivocally determine the myelinating capacity of the cell or the longevity. Therefore, the aim of this study was to determine whether twi OLs form and maintain normal myelin when GALC is provided exogenously in the CNS. In search of a method of enzyme replacement in vivo, we transplanted twi mutant oligodendrocyte progenitor cells (OPCs) into the brain and spinal cord of homozygous shiverer (shi) mice in which OLs do not form compact myelin sheath...

The Myelin Mutants as Models to Study Myelin Repair in the Leukodystrophies

“…The generalized absence of myelin makes the analysis of transplant-induced myelination straightforward. First there is significantly more myelin than in controls, and the myelin is MBP positive [6,54,55]. Further confirmation that this myelin is not endogenous can be seen on electron microscope (EM), in which the myelin has a major dense line and a thicker sheath than compared to the thin myelin sheaths of the host [55].…”

“…In the second study, we transplanted OPCs into the brain at P0 to P1 and into the dorsal column of the thoracolumbar spinal cord of the same shi mice In (f), GFP-positive cells are seen at high density in the white matter and also in appropriate numbers in grey matter. These cells have completely myelinated the cord (g) compared to a segment from the cervical cord of the same mouse, which is MBP negative (h) 3 weeks later [6]. At 120 days, the mice were examined and GFP-positive OPCs were found to have extensively migrated throughout the brain, associated with MBPpositive myelin patches, and across the entire spinal cord at the site of injection (Fig.…”

“…Although a number of methods have been used to label cells prior to transplantation, the 2 methods most frequently used are to transduce the cells to express a reporter gene, usually β-galactosidase (lacZ) or GFP [6,55,83]. In the case of lacZ, the spinal cord or brain at the site of transplantation is incubated overnight in X-gal solution [23].…”

“…Because of the complexity of the pathophysiology of the disease, it appears likely that a combined therapeutic approach, perhaps including exogenous cell-based remyelination will be necessary. In most lysosomal disorders, the affected cells can function when the missing enzyme is replaced, even exogenously [6,[91][92][93]. Therefore, replacing GALC in oligodendrocytes and Schwann cells, either endogenously or exogenously, is critical as a therapeutic approach in Krabbe disease.…”

“…The alternative is to supply an exogenous source of OPCs or immature OLs to initiate repair. Cell transplantation and endogenous cell recruitment may not be mutually exclusive, however, as the cells are transplanted, they may supply the missing or defective enzyme or protein to endogenous OPCs or OLs [6].…”