Deletion of macrophage-inflammatory protein 1 alpha retards neurodegeneration in Sandhoff disease mice

Abstract

Sandhoff disease is a prototypical lysosomal storage disorder in which a heritable deficiency of a lysosomal enzyme, beta-hexosaminidase, results in the storage of the enzyme's substrates in lysosomes. As with many of the other lysosomal storage diseases, neurodegeneration is a prominent feature. Although the cellular and molecular pathways that underlie the neurodegenerative process are not yet fully understood, macrophage/microglial-mediated inflammation has been suggested as one possible mechanism. We now show that the expanded macrophage/microglial population in the CNS of Sandhoff disease mice is compounded by the infiltration of cells from the periphery. Coincident with the cellular infiltration was an increased expression of macrophage-inflammatory protein 1alpha (MIP-1alpha), a leukocyte chemokine, in astrocytes. Deletion of MIP-1alpha expression resulted in a substantial decrease in infiltration and macrophage/microglial-associated pathology together with neuronal apoptosis in Sandhoff disease mice. These mice without MIP-1alpha showed improved neurologic status and a longer lifespan. The results indicate that the pathogenesis of Sandhoff disease involves an increase in MIP-1alpha that induces monocytes to infiltrate the CNS, expand the activated macrophage/microglial population, and trigger apoptosis of neurons, resulting in a rapid neurodegenerative course.

Fig. 1.

Localization and identification of HRP-labeled cells in Hexb^-/- mice. In 3-month-old Hexb^-/- mice, HRP-labeled cells were detected attached to the vascular wall in vessels in the spinal cord (a) and perivascular regions in the spinal cord (b). (c) In a 4-month-old Hexb^-/- mouse, HRP-labeled cells were localized in the parenchyma of the thalamic nucleus. (d) An HRP-labeled cell in the thalamic nucleus of a 4-month-old Hexb^-/- mouse was identified as Mac-1-positive (arrow). Arrowheads show Mac-1-positive cells. (Inset) A nickel-enhanced HRP-labeled cell. (Bars: 100 μmin a and b, 220 μmin c, and 60 μm in d.)

Fig. 2.

Expression of MIP-1α in Hexb^+/+ and Hexb^-/- mice. (a) The time course of relative MIP-1α mRNA expression levels in the cerebral cortex and spinal cord of Hexb^-/- mice compared with age-matched Hexb^+/+ mice. (b–g) Immunofluorescent staining of the spinal cord with MIP-1α and GFAP antibodies. (b–d) Control (Hexb^+/+) sections. (e–g) Hexb^-/- sections. b and e show MIP-1α immunostaining. c and f show GFAP immunostaining. d and g show double immunostaining for MIP-1α and GFAP. Note that the MIP-1α and GFAP double-positive astrocytes (arrows in e–g) are closely associated with vessels (*). (Bar: 60 μm.)

Fig. 3.

Preserved clinical condition of Sandhoff disease mice deficient for MIP-1α was clearly noted at 4 months of age compared with littermates. Hexb^-/- mice with different MIP-1α genotypes are indicated.

Fig. 4.

Body weight, lifespan, and behavioral testing. (a) The body weight in Hexb^-/-MIP-1α^-/- and Hexb^-/-MIP-1α^+/- mice was higher at and after 15 weeks of age compared with Hexb^-/-MIP-1α^+/+ mice. The data are means from three to five mice for each genotype and data point. (b) Survival of the Hexb^-/- mice with different MIP-1α backgrounds. Each group contained 10–15 mice. (c and d) Mean (±SEM) time to fall off the rotorod. Both Hexb^-/-MIP-1α^+/- and Hexb^-/-MIP-1α^-/- mice had significantly better (*) performances at all time points tested. The comparison was made only among the littermates. At all time points in c, n = 4 Hexb^-/-MIP-1α^+/+ mice and 4 Hexb^-/-MIP-1α^+/- mice. At all time points in d, n = 3 Hexb^-/-MIP-1α^+/+ mice and 3 Hexb^-/-MIP-1α^-/- mice. The righting reflex was tested at 17 (e) and 19 (f) weeks of age; n = 4 in each group and time point. Hexb^-/-MIP-1α^-/- mice had a significantly improved (*) righting reflex compared with other groups. All Hexb^-/-MIP-1α^+/+ mice were dead by 19 weeks old.

Fig. 5.

Lessened macrophage/microglia-associated pathology in Hexb^-/-MIP-1α^-/- mice compared with Hexb^-/-MIP-1α^+/+ mice. Hematoxylin/eosin and Mac-1 immunostaining show numerous foamy cells (a) and ameboid brain macrophages (c) in the thalamic nuclei of 4-month-old Hexb^-/-MIP-1α^+/+ mice. In the thalamic nuclei of 4-month-old Hexb^-/-MIP-1α^-/- mice foamy cells were rare (b) and ramified microglia were detected (d) instead of amoeboid brain macrophages. The arrows indicate foamy cells in the Hexb^-/-MIP-1α^+/+ mice, and the arrowheads indicate neurons with storage in both genotypes. (e and f) Reduction of HRP-labeled cells (arrow) in 4-month-old Hexb^-/-MIP-1α^-/- mice (f) compared with age-matched Hexb^-/-MIP-1α^+/+ mice (e). (g) Down-regulation of Mac-1α-subunit mRNA expression in the spinal cord of Hexb^-/-MIP-1α^-/- mice at 4 months old compared with age-matched Hexb^-/-MIP-1α^+/+ mice. (Bars: 20 μmin a–d and 120 μmin e and f.)

Fig. 6.

Reduction of TUNEL-positive cells (arrows) in Hexb^-/-MIP-1α^-/- mice at 4 months old (b) compared with age-matched Hexb^-/-MIP-1α^+/+ mice (a). (d) TUNEL-positive cells were counted (*, P < 0.05). Data are means ± SEM (n = 3–5). (c) The TNF-α mRNA expression level was reduced in the spinal cord of Hexb^-/-MIP-1α^-/- mice compared with Hexb^-/-MIP-1α^+/+ mice at 4 months old. (Bar: 40 μm.)