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  • Macrophages Expressing GALC Improve Peripheral Krabbe Disease by a Mechanism Independent of Cross-Correction.

Macrophages Expressing GALC Improve Peripheral Krabbe Disease by a Mechanism Independent of Cross-Correction.

Neuron (2020-05-07)
Nadav I Weinstock, Daesung Shin, Narayan Dhimal, Xinying Hong, Eric E Irons, Nicholas J Silvestri, Chelsey B Reed, Duc Nguyen, Oliver Sampson, Yung-Chih Cheng, Joseph T Y Lau, Ernesto R Bongarzone, Julia Kofler, Maria L Escolar, Michael H Gelb, Lawrence Wrabetz, M Laura Feltri
ABSTRACT

Many therapies for lysosomal storage disorders rely on cross-correction of lysosomal enzymes. In globoid cell leukodystrophy (GLD), mutations in GALC cause psychosine accumulation, inducing demyelination, a neuroinflammatory "globoid" reaction and neurodegeneration. The efficiency of GALC cross-correction in vivo, the role of the GALC substrate galactosylceramide, and the origin of psychosine are poorly understood. Using a novel GLD model, we show that cross-correction does not occur efficiently in vivo and that Galc-deficient Schwann cells autonomously produce psychosine. Furthermore, macrophages require GALC to degrade myelin, as Galc-deficient macrophages are transformed into globoid cells by exposure to galactosylceramide and produce a more severe GLD phenotype. Finally, hematopoietic stem cell transplantation in patients reduces globoid cells in nerves, suggesting that the phagocytic response of healthy macrophages, rather than cross-correction, contributes to the therapeutic effect. Thus, GLD may be caused by at least two mechanisms: psychosine-induced demyelination and secondary neuroinflammation from galactosylceramide storage in macrophages.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Phosphatase Inhibitor Cocktail 3, DMSO solution
Sigma-Aldrich
Anti-GAPDH antibody produced in rabbit, ~1 mg/mL, affinity isolated antibody, buffered aqueous solution
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2,2,2-Tribromoethanol, 97%
Sigma-Aldrich
Saponin, for molecular biology, used as non-ionic surfactant
Sigma-Aldrich
Phosphatase Inhibitor Cocktail 2, aqueous solution (dark coloration may develop upon storage, which does not affect the activity)
Sigma-Aldrich
2-Mercaptoethanol, ≥99.0%
Sigma-Aldrich
D-Mannose 6-phosphate disodium salt hydrate, ≥97.0% dry basis (enzymatic)
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THI
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Psychosine from bovine brain, lyophilized powder, ≥98% (TLC)
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4-Methylumbelliferone sodium salt, ≥98% (HPLC), crystalline
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Goat Anti-Mouse IgM Antibody, µ chain, FITC conjugate, 2 mg/mL, Chemicon®
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Anti-Pan-Neurofascin Antibody, clone L11A/41, clone L11A/41, from mouse
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Anti-Choline Acetyltransferase Antibody, Chemicon®, from goat
Avanti
C8 Galactosyl(β) Ceramide (d18:1/8:0), Avanti Research - A Croda Brand 860538P, powder
Roche
Anti-HA-Peroxidase, High Affinity, from rat IgG1