Effects of irradiation on the postnatal development of the brain in a genetic mouse model of globoid cell leukodystrophy.

Irradiation is one way to condition Twitcher mice--a natural model of globoid cell leukodystrophy (GLD)--prior to receive bone marrow transplantation (BMT). BMT showed to delay but not to completely prevent GLD disease in treated mutants. The reasons why BMT is not completely preventive in Twitchers are unclear but we speculate that irradiation might contribute to worsen the neurological impairments generated by the disease by altering postnatal neurogenesis. To test this hypothesis, we examined proliferation, migration and differentiation of neural precursors in neurogenic areas of the Twitcher brain after exposure of 5 day-old mutant pups to 620 rad, a non-lethal dose that leads to 80-90% of bone-marrow engraftment in classic BMT. Twitchers showed to be sensitive to irradiation, leading to a severe retardation of body growth of irradiated mutants. Irradiated Twitchers had reduced proliferation of neural precursors and increased astrogliosis and microgliosis, with reduced numbers of migratory neuroblasts and significantly less brain myelination. These effects were accompanied by caspase-3 activation and appeared largely irreversible in the lifespan of the Twitcher. Our work confirms that exposure of the neonatal brain to irradiation conditions such as those performed prior to BMT, can lead to long-lasting alterations of postnatal neurogenesis and myelination, which might contribute to worsen the progression of disease in these myelin mutants and to reduce the success of BMT.

Notch signaling in astrocytes and neuroblasts of the adult subventricular zone in health and after cortical injury.

The postnatal subventricular zone (SVZ) is a niche for continuous neurogenesis in the adult brain and likely plays a fundamental role in self-repair responses in neurodegenerative conditions. Maintenance of the pool of neural stem cells within this area depends on cell-cell communication such as that provided by the Notch signaling pathway. Notch1 receptor mRNA has been found distributed in different areas of the postnatal brain including the SVZ. Although the identity of Notch1-expressing cells has been established in the majority of these areas, it is still unclear what cell types within the SVZ are expressing components of this pathway. Here we demonstrate that most of expression of Notch1 in the adult SVZ occurs in polysialylated neural cell adhesion molecule (PSA-NCAM)-positive neural precursors and in glial fibrillary acidic protein-positive SVZ astrocytes. Notch1 was also found in PSA-NCAM-positive neuroblasts located within the rostral migratory stream (RMS) but much less in those that have reached the olfactory bulb. We show that two of the naturally occurring Notch1 activators, Jagged1 and Delta1, are also expressed in the SVZ and within the RMS in the adult mouse brain. Finally, using a model of cortical stab wound, we show that the astrogliogenic response of the SVZ to injury is accompanied by activation of the Notch pathway.

Oligodendroglial progenitor cell therapy limits central neurological deficits in mice with metachromatic leukodystrophy.

This work describes the first successful oligodendrocyte-based cell therapy for presymptomatic arylsulfatase A (ARSA) null neonate mice, a murine model for human metachromatic leukodystrophy (MLD). We found that oligodendrocyte progenitors (OLPs) engrafted and survived into adulthood when transplanted in the neonatal MLD brain. Transplanted cells integrated nondisruptively, did not produce tumors, and survived as proteolipid protein- and MBP-positive postmitotic myelinating oligodendrocytes (OLs) intermingled with endogenous MLD OLs within the adult MLD white matter. Transplanted MLD mice had reduced sulfatide accumulation in the CNS, increased brain ARSA activity, and full prevention of the electrophysiological and motor deficits that characterize untreated MLD mice. Our results provide direct evidence that healthy OLPs can tolerate the neurotoxic accumulation of sulfatides that evolves during the postnatal development of the MLD brain and contribute to OL cell replacement to limit the accumulation of sulfatides and the evolution of CNS defects in this lysosomal storage disease mouse model.

IL-3 or IL-7 increases ex vivo gene transfer efficiency in ADA-SCID BM CD34+ cells while maintaining in vivo lymphoid potential.

To improve maintenance and gene transfer of human lymphoid progenitors for clinical use in gene therapy of adenosine deaminase (ADA)-deficient SCID we investigated several gene transfer protocols using various stem cell-enriched sources. The lymphoid differentiation potential was measured by an in vitro clonal assay for B/NK cells and in the in vivo SCID-hu mouse model. Ex vivo culture with the cytokines TPO, FLT3-ligand, and SCF (T/F/S) plus IL-3 or IL-7 substantially increased the yield of transduced bone marrow (BM) CD34(+) cells purified from ADA-SCID patients or healthy donors, compared to T/F/S alone. Moreover, the use of IL-3 or IL-7 significantly improved the maintenance of in vitro B cell progenitors from ADA-SCID BM cells and allowed the efficient transduction of B and NK cell progenitors. Under these optimized conditions transduced CD34(+) cells were efficiently engrafted into SCID-hu mice and gave rise to B and T cell progeny, demonstrating the maintenance of in vivo lymphoid reconstitution capacity. The protocol based on the T/F/S + IL-3 combination was included in a gene therapy clinical trial for ADA-SCID, resulting in long-term engraftment of stem/progenitor cells. Remarkably, gene-corrected BM CD34(+) cells obtained from one patient 4 and 11 months after gene therapy were capable of repopulating the lymphoid compartment of SCID-hu hosts.