Leukodystrophies are rare diseases caused by defects in the genes coding for lysosomal enzymes that degrade several glycosphingolipids. Gene therapy for leukodystrophies requires efficient distribution of the missing enzymes in CNS tissues to prevent demyelination and neurodegeneration. In this work, we targeted the external capsule (EC), a white matter region enriched in neuronal projections, with the aim of obtaining maximal protein distribution from a single injection site. We used bidirectional (bd) lentiviral vectors (LV) (bdLV) to ensure coordinate expression of a therapeutic gene (beta-galactocerebrosidase, GALC; arylsulfatase A, ARSA) and of a reporter gene, thus monitoring simultaneously transgene distribution and enzyme reconstitution. A single EC injection of bdLV.GALC in early symptomatic twitcher mice (a murine model of globoid cell leukodystrophy) resulted in rapid and robust expression of a functional GALC protein in the telencephalon, cerebellum, brainstem and spinal cord. This led to global rescue of enzymatic activity, significant reduction of tissue storage and decrease of activated astroglia and microglia. Widespread protein distribution and complete metabolic correction were also observed after EC injection of bdLV.ARSA in a mouse model of metachromatic leukodystrophy. Our data indicated axonal transport, distribution through cerebrospinal fluid flow and cross-correction as the mechanisms contributing to widespread bioavailability of GALC and ARSA proteins in CNS tissues. LV-mediated gene delivery of lysosomal enzymes by targeting highly interconnected CNS regions is a potentially effective strategy that, combined with a treatment able to target the PNS and peripheral organs, may provide significant therapeutic benefit to patients affected by leukodystrophies.
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
MicroRNAs (miRNAs) have rapidly emerged as biologically important mediators of posttranscriptional and epigenetic regulation in both plants and animals. miRNAs function through a variety of mechanisms including mRNA degradation and translational repression; additionally, miRNAs may guide gene expression by serving as transcription factors. miRNAs are highly expressed in human brain. Tissue and cell type-specific enrichments of certain miRNAs within the nervous system argue for a biological significance during neurodevelopmental stages. On the other hand, a large number of studies have reported links between alterations of miRNA homeostasis and pathologic conditions such as cancer, heart diseases, and neurodegeneration. Thus, profiles of distinct or aberrant miRNA signatures have most recently surged as one of the most fascinating interests in current biology. Here, the most recent insights into the involvement of miRNAs in the biology of the nervous system and the occurrence of neuropathological disorders are reviewed and discussed.
In this work we showed that genotype-related patterns of hexosaminidase activity, isoenzyme composition, gene expression and ganglioside metabolism observed during embryonic and postnatal brain development are recapitulated during the progressive stages of neural precursor cell (NPC) differentiation to mature glia and neurons in vitro. Further, by comparing NPCs and their differentiated progeny established from Tay-Sachs (TS) and Sandhoff (SD) animal models with the wild-type counterparts, we studied the events linking the accumulation of undegraded substrates to hexosaminidase activity. We showed that similarly to what observed in brain tissues in TS NPCs and progeny, the stored GM2 was partially converted by sialidase to GA2, which can be then degraded in the lysosomes to its components. The latter can be used in a salvage pathway for the formation of GM3. Interestingly, results obtained from ganglioside feeding assays and from measurement of lysosomal sialidase activity suggest that a similar pathway might work also in the SD model.
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