Globoid cell leukodystrophy is a lysosomal storage disease characterized by the loss of galactocerebrosidase. Galactocerebrosidase loss leads to the accumulation of psychosine and subsequent oligodendrocyte cell death, demyelination, macrophage recruitment, and astroglial activation and proliferation. To date, no studies have elucidated the mechanism of glial cell activation and cytokine and chemokine up-regulation and release. We explored a novel explanation for the development of the pathological changes in the early stages of globoid cell leukodystrophy associated with toll-like receptor (TLR) 2 up-regulation in the hindbrain and cerebellum as a response to dying oligodendrocytes. TLR2 up-regulation on microglia/macrophages coincided with morphological changes consistent with activation at 2 and 3 weeks of age. TLR2 up-regulation on activated microglia/macrophages resulted in astrocyte activation and marked up-regulation of cytokines/chemokines. Because oligodendrocyte cell death is an important feature of globoid cell leukodystrophy, we tested the ability of TLR2 reporter cells to respond to oligodendrocyte cell death. These reporter cells responded in vitro to medium conditioned by psychosine-treated oligodendrocytes, indicating the likelihood that oligodendrocytes release a TLR2 ligand during apoptosis. TLRs are a member of the innate immune system and initiate immune and inflammatory events; therefore, the identification of TLR2 as a potential driver in the activation of central nervous system glial activity in globoid cell leukodystrophy may provide important insight into its pathogenesis.

The twitcher mouse is an animal model of Krabbe's disease (KD), which is a neurodegenerative lysosomal storage disorder resulting from the absence of functional lysosomal enzyme galactocerebrosidase (GALC). This disease affects the central and peripheral nervous systems and in its most severe form results in death before the age of 2 in humans and approximately 30–40 days in mice. This study evaluates the effect of intracerebroventricular administration of mesenchymal stem cells derived from adipose tissue (ASCs) and bone marrow (BMSCs) on the pathology of KD. Subsequent to the intracerebroventricular injection of ASCs or BMSCs on postnatal day (PND) 3–4, body weight, lifespan, and neuromotor function were evaluated longitudinally beginning on PND15. At sacrifice, tissues were harvested for analysis of GALC activity, presence of myelin, infiltration of macrophages, microglial activation, inflammatory markers, and cellular persistence. Survival analysis curves indicate a statistically significant increase in lifespan in stem cell‐treated twitcher mice as compared with control twitcher mice. Body weight and motor function were also improved compared with controls. The stem cells may mediate some of these benefits through an anti‐inflammatory mechanism because the expression of numerous proinflammatory markers was downregulated at both transcriptional and translational levels. A marked decrease in the levels of macrophage infiltration and microglial activation was also noted. These data indicate that mesenchymal lineage stem cells are potent inhibitors of inflammation associated with KD progression and offer potential benefits as a component of a combination approach for in vivo treatment by reducing the levels of inflammation. STEM CELLS 2011;29:67–77

Metachromatic leukodystrophy (MLD) and globoid cell leukodystrophy (GLD or Krabbe disease) are severe neurodegenerative lysosomal storage diseases (LSD) caused by arylsulfatase A (ARSA) and galactosylceramidase (GALC) deficiency, respectively. Our previous studies established lentiviral gene therapy (GT) as a rapid and effective intervention to provide pervasive supply of therapeutic lysosomal enzymes in CNS tissues of MLD and GLD mice. Here, we investigated whether this strategy is similarly effective in juvenile non‐human primates (NHP). To provide proof of principle for tolerability and biological efficacy of the strategy, we established a comprehensive study in normal NHP delivering a clinically relevant lentiviral vector encoding for the human ARSA transgene. Then, we injected a lentiviral vector coding for the human GALC transgene in Krabbe‐affected rhesus macaques, evaluating for the first time the therapeutic potential of lentiviral GT in this unique LSD model. We showed favorable safety profile and consistent pattern of LV transduction and enzyme biodistribution in the two models, supporting the robustness of the proposed GT platform. We documented moderate inflammation at the injection sites, mild immune response to vector particles in few treated animals, no indication of immune response against transgenic products, and no molecular evidence of insertional genotoxicity. Efficient gene transfer in neurons, astrocytes, and oligodendrocytes close to the injection sites resulted in robust production and extensive spreading of transgenic enzymes in the whole CNS and in CSF, leading to supraphysiological ARSA activity in normal NHP and close to physiological GALC activity in the Krabbe NHP, in which biological efficacy was associated with preliminary indication of therapeutic benefit. These results support the rationale for the clinical translation of intracerebral lentiviral GT to address CNS pathology in MLD, GLD, and other neurodegenerative LSD.

Globoid cell leukodystrophy (GLD) is a common neurodegenerative lysosomal storage disorder caused by a deficiency in galactocerebrosidase (GALC), an enzyme that cleaves galactocerebroside during myelination. Bone marrow transplantation has shown promise when administered to late-onset GLD patients. However, the side effects (e.g., graft versus host disease), harsh conditioning regimens (e.g., myelosuppression), and variable therapeutic effects make this an unsuitable option for infantile GLD patients. We previously reported modest improvements in the twitcher mouse model of GLD after intracerebroventricular (ICV) injections of a low dose of multipotent stromal cells (MSCs). Goals of this study were to improve bone marrow-derived MSC (BMSC) therapy for GLD by increasing the cell dosage and comparing cell type (e.g., transduced v. native), treatment timing (e.g., single v. weekly), and administration route (e.g., ICV v. intraperitoneal). Neonatal twitcher mice received (1) 2×105 BMSCs by ICV injection, (2) 1×106 BMSCs by intraperitoneal (IP) injection, (3) weekly IP injections of 1×106 BMSCs, or (4) 1×106 lentiviral-transduced BMSCs overexpressing GALC (GALC-BMSC) by IP injection. All treated mice lived longer than untreated mice. However, the mice receiving peripheral MSC therapy had improved motor function (e.g., hind limb strength and rearing ability), twitching symptoms, and weight compared to both the untreated and ICV-treated mice. Inflammatory cell, globoid cell, and apoptotic cell levels in the sciatic nerves were significantly decreased as a result of the GALC-BMSC or weekly IP injections. The results of this study indicate a promising future for peripheral MSC therapy as a non-invasive, adjunct therapy for patients affected with GLD.

One of a family of devastating lysosomal storage disorders, Krabbe disease is characterized by demyelination, psychosine accumulation, and inflammation. Affected infants rarely survive longer than two years. Using the twitcher mouse model of the disease, this study evaluated the potential of intrastriatal injection of adipose or bone marrow-derived mesenchymal stromal cells (MSCs) as a treatment option. Neonatal pups were injected with MSCs at 3–4 days of age and subjected to a battery of behavioral tests beginning at 15 days. While MSC injection failed to increase lifespan of twitchers, improvements in rotarod performance and twitching severity were observed at 27–38 days of age using MSCs derived from bone marrow. This study tested several different tasks developed in adult mice for evaluation of disease progression in immature twitchers. Rotarod was both reliable and extremely sensitive. Automated gait analysis using the Treadscan program was also useful for early evaluation of differences prior to overt gait dysfunction. Finally, this study represents the first use of the Stone T-maze in immature mice. Validation of rotarod and automated gait analysis for detection of subtle differences in disease progression is important for early stage efforts to develop treatments for juvenile disorders.