Globoid cell leukodystrophy (GLD) is a lysosomal storage disease caused by deficient activity of β-galactocerebrosidase (GALC). The infantile forms manifest with rapid and progressive central and peripheral demyelination, which represent a major hurdle for any treatment approach. We demonstrate here that neonatal lentiviral vector-mediated intracerebral gene therapy (IC GT) or transplantation of GALC-overexpressing neural stem cells (NSC) synergize with bone marrow transplant (BMT) providing dramatic extension of lifespan and global clinical–pathological rescue in a relevant GLD murine model. We show that timely and long-lasting delivery of functional GALC in affected tissues ensured by the exclusive complementary mode of action of the treatments underlies the outstanding benefit. In particular, the contribution of neural stem cell transplantation and IC GT during the early asymptomatic stage of the disease is instrumental to enhance long-term advantage upon BMT. We clarify the input of central nervous system, peripheral nervous system and periphery to the disease, and the relative contribution of treatments to the final therapeutic outcome, with important implications for treatment strategies to be tried in human patients. This study gives proof-of-concept of efficacy, tolerability and clinical relevance of the combined gene/cell therapies proposed here, which may constitute a feasible and effective therapeutic opportunity for children affected by GLD.

Murine neural stem cells (mNSCs), either naive or genetically modified to express supranormal levels of β-galactocerebrosidase (GALC), were transplanted into the brain of Twitcher mice, a murine model of globoid cell leukodystrophy, a severe sphingolipidosis. Cells engrafted long-term into the host cytoarchitecture, producing functional GALC. Levels of enzyme activity in brain and spinal cord tissues were enhanced when GALC-overexpressing NSC were used. Enzymatic correction correlated with reduced tissue storage, decreased activation of astroglia and microglia, delayed onset of symptoms, and longer lifespan. Mechanisms underlying the therapeutic effect of mNSC included widespread enzyme distribution, cross-correction of host cells, anti-inflammatory activity, and neuroprotection. Similar cell engraftment and metabolic correction were reproduced using human NSC. Thus, NSC gene therapy rapidly reconstitutes sustained and long-lasting enzyme activity in central nervous system tissues. Combining this approach with treatments targeting the systemic disease associated with leukodystrophies may provide significant therapeutic benefit. Stem Cells 2011;29:1559–1571

We report a novel role for the lysosomal galactosylceramidase (GALC), which is defective in globoid cell leukodystrophy (GLD), in maintaining a functional post-natal subventricular zone (SVZ) neurogenic niche. We show that proliferation/self-renewal of neural stem cells (NSCs) and survival of their neuronal and oligodendroglial progeny are impaired in GALC-deficient mice. Using drugs to modulate inflammation and gene transfer to rescue GALC expression and activity, we show that lipid accumulation resulting from GALC deficiency acts as a cell-autonomous pathogenic stimulus in enzyme-deficient NSCs and progeny before upregulation of inflammatory markers, which later sustain a non-cell-autonomous dysfunction. Importantly, we provide evidence that supply of functional GALC provided by neonatal intracerebral transplantation of NSCs ameliorates the functional impairment in endogenous SVZ cells. Insights into the mechanism/s underlying GALC-mediated regulation of early post-natal neurogenic niches improve our understanding of the multi-component pathology of GLD. The occurrence of a restricted period of SVZ neurogenesis in infancy supports the implications of our study for the development of therapeutic strategies to treat this severe pediatric neurodegenerative disorder.

Globoid cell leukodystrophy (GLD) or Krabbe disease is a lysosomal storage disorder caused by genetic defects in the expression and activity of galactosylceramidase, a key enzyme in the catabolism of myelin-enriched sphingolipids. While there are several histologic, biochemical, and functional studies on GLD, correlations between morphologic and biochemical alterations in central nervous system (CNS) tissues during disease progression are lacking. Here, we combined immunohistochemistry and metabolic analysis using (1)H and (13)C magnetic resonance (MR) spectra of spinal cord, cerebellum, and forebrain to investigate glial-neuronal metabolic interactions and dysfunction in a GLD murine model that recapitulates the human pathology. In order to assess the temporal- and region-dependent disease progression and the potential metabolic correlates, we investigated CNS tissues at mildly symptomatic and fully symptomatic stages of the disease. When compared with age-matched controls, GLD mice showed glucose hypometabolism, alterations in neurotransmitter content, N-acetylaspartate, N-acetylaspartylglutamate, and osmolytes levels. Notably, age- and region-dependent patterns of metabolic disturbances were in close agreement with the progression of astrogliosis, microglia activation, apoptosis, and neurodegeneration. We suggest that MR spectroscopy could be used in vivo to monitor disease progression, as well as ex vivo and in vivo to provide criteria for the outcome of experimental therapies.

Schwann cells (SCs) generate the myelin wrapping of peripheral nerve axons and are promising candidates for cell therapy. However, to date a renewable source of SCs is lacking. In this study, we show the conversion of skin fibroblasts into induced Schwann cells (iSCs) by driving the expression of two transcription factors, Sox10 and Egr2. iSCs resembled primary SCs in global gene expression profiling and PNS identity. In vitro, iSCs wrapped axons generating compact myelin sheaths with regular nodal structures. Conversely, iSCs from Twitcher mice showed a severe loss in their myelinogenic potential, demonstrating that iSCs can be an attractive system for in vitro modelling of PNS diseases. The same two factors were sufficient to convert human fibroblasts into iSCs as defined by distinctive molecular and functional traits. Generating iSCs through direct conversion of somatic cells offers opportunities for in vitro disease modelling and regenerative therapies.

Globoid cell leukodystrophy (GLD), or Krabbe's disease, is a lysosomal storage disorder resulting from deficiency of the lysosomal hydrolase galactosylceramidase. The infantile forms are characterized by a unique relentless and aggressive progression with a wide range of neurological symptoms and complications. Here we review and discuss the basic concepts and the novel mechanisms identified as key contributors to the peculiar GLD pathology, highlighting their therapeutic implications. Then, we evaluate evidence from extensive experimental studies on GLD animal models that have highlighted fundamental requirements to obtain substantial therapeutic benefit, including early and timely intervention, high levels of enzymatic reconstitution, and global targeting of affected tissues. Continuous efforts in understanding GLD pathophysiology, the interplay between various therapies, and the mechanisms of disease correction upon intervention may allow advancing research with innovative approaches and prioritizing treatment strategies to develop more efficacious treatments. © 2016 Wiley Periodicals, Inc.

The activities of plasma membrane associated sialidase Neu3, total β-glucosidase, CBE-sensitive β-glucosidase, non-lysosomal β-glucosyl ceramidase GBA2, β-galactosidase, β-hexosaminidase and sphingomyelinase were determined at three different stages of differentiation of murine neural stem cell cultures, corresponding to precursors, commited progenitors, and differentiated cells. Cell immunostaining for specific markers of the differentiation process, performed after 7 days in culture in presence of differentiating agents, clearly showed the presence of oligodendrocytes, astrocytes and neurons. Glial cells were the most abundant. Sialidase Neu3 after a decrease from progenitors to precursors, showed an increase parallel to the differentiation process. All the other glycosidases increased their activity along differentiation. The activity of CBE-sensitive β-glucosidase and GBA2 were very similar at the precursor stage, but CBE-sensitive β-glucosidase increased 7 times while GBA2 only two in the differentiated cells. In addition, we analysed also sphingomyelinase as enzyme specifically associated to sphingolipids. The activity of this enzyme increased from precursors to differentiated cells.

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