Bone marrow transplantation increases efficacy of central nervous system-directed enzyme replacement therapy in the murine model of globoid cell leukodystrophy

Abstract

Globoid cell leukodystrophy (GLD, Krabbe disease), is an autosomal recessive, neurodegenerative disease caused by the deficiency of the lysosomal enzyme galactocerebrosidase (GALC). In the absence of GALC, the toxic metabolite psychosine accumulates in the brain and causes the death of the myelin-producing cells, oligodendrocytes. Currently, the only therapy for GLD is hematopoietic stem cell transplantation using bone marrow (BMT) or umbilical cord blood. However, this is only partially effective. Previous studies have shown that enzyme replacement therapy (ERT) provides some therapeutic benefit in the murine model of GLD, the Twitcher mouse. Experiments have also shown that two disparate therapies can produce synergistic effects when combined. The current study tests the hypothesis that BMT will increase the therapeutic effects of ERT when these two treatments are combined. Twitcher mice were treated with either ERT alone or both ERT and BMT during the first 2-4 days of life. Recombinant enzyme was delivered by intracerebroventricular (ICV) and intrathecal (IT) injections. Twitcher mice receiving ERT had supraphysiological levels of GALC activity in the brain 24h after injection. At 36 days of age, ERT-treated Twitcher mice had reduced psychosine levels, reduced neuroinflammation, improved motor function, and increased lifespan. Twitcher mice receiving both ERT and BMT had significantly increased lifespan, improved motor function, reduced psychosine levels, and reduced neuroinflammation in certain areas of the brain compared to untreated or ERT-treated Twitcher mice. Together, these results indicate that BMT enhances the efficacy of ERT in GLD.

Figure 1. GALC activity and psychosine levels

(a) GALC levels in the brains of the ERT-treated mice at 24 hours post-injection were significantly greater than in age-matched WT mice. (b) GALC levels in the spinal cords of the ERT-treated mice at 24 hours post-injection were significantly greater than in untreated Twitcher mice. (c) At 36 days of age, GALC levels in the brains of all Twitcher groups were significantly less than WT. (d) At 36 days of age, psychosine levels were significantly greater in untreated Twitcher mice than in untreated WT mice. Treated Twitcher mice had significantly reduced psychosine levels compared to untreated Twitcher mice, and the ERT+BMT group had a further significant reduction in psychosine compared to the ERT-only group. The horizontal bars represent the means, and the error bars represent one SEM. (*=p<0.05, **=p<0.01, ***=p<0.001)

Figure 2. GALC distribution

Twitcher mice treated with ERT had intense GALC staining (blue) in the ependymal lining and choroid plexus at 24 hours post-injection. In addition, there was intense staining in the periventricular region, the hippocampus, corpus callosum, and the meninges surrounding the cerebellum and olfactory bulb. There was an apparent decreasing gradient of GALC staining from the periventricular region to the more distal regions of the brain. WT mice had diffuse, less intense staining throughout the brain, and untreated Twitcher mice had little to no staining. All images are shown at 4x, except for the brainstem, which is at 20x.

Figure 3. Myelin basic protein (MBP) immunohistochemistry

(a) Representative images of MBP staining of the brain at 36 days of age. Twitcher mice had an apparent decrease in myelin staining, especially in the corpus callosum and brainstem, compared to WT controls. Twitcher mice also had regions in the cerebellum devoid of staining (arrows). Treated Twitcher mice appeared to have increased myelin staining compared to untreated Twitcher controls, as well as fewer cerebellar areas devoid of staining (arrows). The ERT+BMT group appeared to have more intense staining in the corpus callosum and more normal organization in the striatum compared to the ERT group. (b) The corpus callosum in untreated Twitcher mice was significantly thinner than in WT controls. The ERT and ERT+BMT groups had a slightly, though not significantly, thicker corpus callosum compared to untreated Twitcher mice. (c) In the striatum, untreated Twitcher mice had significantly thinner major axonal bundles than WT controls and the ERT-BMT group. Although there was an apparent increase in the major bundle thickness of Twitcher mice treated with ERT alone, it was not statistically significant. All images are shown at 20x. (*=p<0.05, **=p<0.01)

Figure 4. GFAP immunohistochemistry

(a) Representative images of GFAP staining of the brain at 36 days of age. In the cerebellum (b), hippocampus/ corpus callosum (c), and brainstem (d), Twitcher mice had significantly increased GFAP immunoreactivity (activated astrocytes) compared to WT controls and treated Twitcher mice. In the striatum (e), Twitcher mice had significantly increased GFAP compared to the WT and ERT+BMT groups, and the ERT+BMT group had a significant decrease from the ERT group. In the brainstem (d), the ERT+BMT group showed a significant decrease compared to the ERT group, and the ERT group had a significant increase from WT. All images are shown at 20x, except for hippocampus/ corpus callosum, which are at 10x. (*=p<0.05, **=p<0.01, ***=p<0.001)

Figure 5. CD68 brain immunohistochemistry

(a) Representative images of CD68 staining of the brain at 36 days of age. In the cerebellum (b), hippocampus/ corpus callosum (c), brainstem (d), and striatum (e), Twitcher mice had significantly increased CD68 immunoreactivity (activated microglia) compared to WT controls and treated Twitcher mice. The ERT+BMT group showed significantly less intense CD68 staining than the ERT group in the cerebellum (b). All images are shown at 20x, except for hippocampus/ corpus callosum, which are at 10x. (**=p<0.01, ***=p<0.001)

Figure 6. CD68 spinal cord immunohistochemistry

(a) Representative images of CD68 staining of the spinal cord at 36 days of age. In the thoracic (b) and lumbar (c) regions, Twitcher mice have significantly increased CD68 immunoreactivity (activated microglia) than WT controls and treated Twitcher mice. All images are shown at 4x. (***=p<0.001)

Figure 7. Lifespan and behavior

(a) The median lifespans for control, ERT-only, and ERT+BMT treated Twitcher mice were 40, 51, and 59 days, respectively. All WT mice were still alive at 100 days of age. All four of these groups were significantly different from each other. (b) Twitcher mice began declining in rotarod performance starting from 35–45 days of age. Both treated Twitcher groups showed a significant (p<0.001) increase in latency compared to untreated and vehicle Twitcher controls starting at 40 days. The ERT+BMT group showed a significant (p<0.05) increase in latency compared to the ERT-only group at 55 days. (c) All Twitcher groups showed a gradual decline in wire hang performance starting at 25 days, while WT mice improved. Both treated Twitcher groups showed a significant (p<0.01) increase in latency compared to the Twitcher controls. The ERT-only group showed a significant (p<0.05) increase in latency compared to the combination-treated (ERT+BMT) group at 25 and 30 days. (d) Untreated and vehicle-treated Twitcher controls stopped gaining weight at 30 days, and treated Twitcher mice stopped gaining weight at 35 days. The two treated Twitcher groups were significantly (p<0.005) different from the untreated and vehicle-treated Twitcher controls starting at 35 days of age.