Developmentally regulated mannose 6-phosphate receptor-mediated transport of a lysosomal enzyme across the blood-brain barrier

Abstract

Mucopolysaccharidosis type VII is a lysosomal storage disorder resulting from inherited deficiency of beta-glucuronidase (GUS). Mucopolysaccharidosis type VII is characterized by glycosaminoglycan storage in most tissues, including brain. In these disorders, enzyme delivery across the blood-brain barrier (BBB) is the main obstacle to correction of lysosomal storage in the CNS. Prior studies suggested mouse brain is accessible to GUS in the first 2 weeks of life but not later. To explore a possible role for the mannose 6-phosphate/insulin-like growth factor II receptor in GUS transport across the BBB in neonatal mice, we compared brain uptake of phosphorylated GUS (P-GUS) and nonphosphorylated GUS (NP-GUS) in newborn and adult mice. (131)I-P-GUS was transported across the BBB after i.v. injection in 2-day-old mice. The brain influx rate (K(in)) of (131)I-P-GUS in 2-day-old mice was 0.21 microl/g.min and decreased with age. By 7 weeks of age, transport of (131)I-P-GUS was not significant. Capillary depletion revealed that 62% of the (131)I-P-GUS in brain was in brain parenchyma in 2-day-old mice. In addition, uptake of (131)I-P-GUS into brain was significantly reduced by coinjection of unlabeled P-GUS or M6P in a dose-dependent manner. In contrast, the K(in) of (131)I-NP-GUS (0.04 microl/g.min) was significantly lower than (131)I-P-GUS in 2-day-old mice. Transcardiac brain perfusion confirmed that neither (131)I-P-GUS nor (131)I-NP-GUS crossed the BBB in adult mice. These results indicate that (131)I-P-GUS transport into brain parenchyma in early postnatal life is mediated by the mannose 6-phosphate/insulin-like growth factor II receptor. This receptor-mediated transport is not observed in adult mice.

Fig. 1.

Time courses of radioactivity in serum after i.v. coinjection of ¹³¹I-P-GUS (A) and ¹²⁵I-albumin (B) (550,000 cpm of each) in 2-day-old (•), 5-day-old (▴), 1-week-old (▪), 2-week-old (▾), and 7-week-old (♦) mice. n = 3-8 mice per point.

Fig. 2.

The percent (A) of ¹³¹I-P-GUS taken up by the brain and multiple-time regression analyses (B) of ¹³¹I-P-GUS and ¹²⁵I-albumin after i.v. injection in 2-day-old and 7-week-old mice. ¹³¹I-P-GUS and ¹²⁵I-albumin (550,000 cpm of each) were coinjected. n = 5-7 mice per point.

Fig. 3.

K_in (A) and V_i (B) values of ¹³¹I-P-GUS and ¹²⁵I-albumin for brain after i.v. coinjection at various ages. n = 3-8 mice per bar. Asterisks indicate a significant difference in comparison with 2-day-old mice: ^*, P < 0.05; ^**, P < 0.01; ^***, P < 0.001. ND, not detected (no blood-to-brain entry).

Fig. 4.

Distribution volume of ¹³¹I-P-GUS in brain parenchyma and capillary fraction 10 min after i.v. injection in 2-day-old and 7-week-old mice. n = 4-8 mice per mean fraction. Asterisk indicates a significant difference (P < 0.05) between the parenchyma and capillary fraction. ND, not detected.

Fig. 5.

Effects of unlabeled P-GUS (A), M6P, and IGF-II (B) on brain/serum ratios of ¹³¹I-P-GUS 10 min after i.v. injection in 2-day-old mice. Mice received ¹³¹I-P-GUS (550,000 cpm) i.v. with unlabeled P-GUS (1, 10, and 30 μg), M6P (0.02, 0.2, and 2 μmol), or IGF-II (1.35 nmol) and were killed 10 min later. The dashed line indicates the vascular space as measured with ¹²⁵I-albumin in the 2-day-old mice shown in Fig. 3. n = 3-12 mice per mean. Asterisks indicate a significant difference from the control value: ^*, P < 0.05; ^***, P < 0.001.

Fig. 6.

Time courses of radioactivity in serum (A) and multiple-time regression analyses (B) of ¹³¹I-P-GUS and ¹³¹I-NP-GUS 1-10 min after i.v. injection in 2-day-old mice. ¹³¹I-P-GUS (•) or ¹³¹I-NP-GUS (○) (550,000 cpm of each) was injected. n = 4 mice per point.

Fig. 7.

Time courses of the brain/perfusate ratios of ¹³¹I-P-GUS (•) and ¹³¹I-NP-GUS (○) corrected for ¹²⁵I-albumin with transcardiac brain perfusion in 7-week-old mice. Neither ¹³¹I-P-GUS nor ¹³¹I-NP-GUS had a measurable uptake by brain. n = 3-4 mice per point.

Fig. 8.

Comparison of the distributions of ¹³¹I-P-GUS and ¹³¹I-NP-GUS in various tissues 10 min after i.v. injection in 2-day-old mice. ¹³¹I-P-GUS or ¹³¹I-NP-GUS (550,000 cpm of each) was injected. The distribution volumes of the brain (A), heart (B), lung (C), liver (D), spleen (E), and kidney (F) were normalized by the percent of ¹³¹I-P-GUS in each tissue. n = 4-5 mice per mean. Asterisks indicate a significant difference from the control value: ^*, P < 0.05; ^**, P < 0.01; ^***, P < 0.001.