Waning efficacy in a long-term AAV-mediated gene therapy study in the murine model of Krabbe disease.

Neonatal AAV9-gene therapy of the lysosomal enzyme galactosylceramidase (GALC) significantly ameliorates central and peripheral neuropathology, prolongs survival, and largely normalizes motor deficits in Twitcher mice. Despite these therapeutic milestones, new observations identified the presence of multiple small focal demyelinating areas in the brain after 6-8 months. These lesions are in stark contrast to the diffuse, global demyelination that affects the brain of naive Twitcher mice. Late-onset lesions exhibited lysosomal alterations with reduced expression of GALC and increased psychosine levels. Furthermore, we found that lesions were closely associated with the extravasation of plasma fibrinogen and activation of the fibrinogen-BMP-SMAD-GFAP gliotic response. Extravasation of fibrinogen correlated with tight junction disruptions of the vasculature within the lesioned areas. The lesions were surrounded by normal appearing white matter. Our study shows that the dysregulation of therapeutic GALC was likely driven by the exhaustion of therapeutic AAV episomal DNA within the lesions, paralleling the presence of proliferating oligodendrocyte progenitors and glia. We believe that this is the first demonstration of diminishing expression in vivo from an AAV gene therapy vector with detrimental effects in the brain of a lysosomal storage disease animal model. The development of this phenotype linking localized loss of GALC activity with relapsing neuropathology in the adult brain of neonatally AAV-gene therapy-treated Twitcher mice identifies and alerts to possible late-onset reductions of AAV efficacy, with implications to other genetic leukodystrophies.

Inhibition of the IGF-1-PI3K-Akt-mTORC2 pathway in lipid rafts increases neuronal vulnerability in a genetic lysosomal glycosphingolipidosis.

Glycosphingolipid (GSL) accumulation is implicated in the neuropathology of several lysosomal conditions, such as Krabbe disease, and may also contribute to neuronal and glial dysfunction in adult-onset conditions such as Parkinson's disease, Alzheimer's disease and multiple sclerosis. GSLs accumulate in cellular membranes and disrupt their structure; however, how membrane disruption leads to cellular dysfunction remains unknown. Using authentic cellular and animal models for Krabbe disease, we provide a mechanism explaining the inactivation of lipid raft (LR)-associated IGF-1-PI3K-Akt-mTORC2, a pathway of crucial importance for neuronal function and survival. We show that psychosine, the GSL that accumulates in Krabbe disease, leads to a dose-dependent LR-mediated inhibition of this pathway by uncoupling IGF-1 receptor phosphorylation from downstream Akt activation. This occurs by interfering with the recruitment of PI3K and mTORC2 to LRs. Akt inhibition can be reversed by sustained IGF-1 stimulation, but only during a time window before psychosine accumulation reaches a threshold level. Our study shows a previously unknown connection between LR-dependent regulation of mTORC2 activity at the cell surface and a genetic neurodegenerative disease. Our results show that LR disruption by psychosine desensitizes cells to extracellular growth factors by inhibiting signal transmission from the plasma membrane to intracellular compartments. This mechanism serves also as a mechanistic model to understand how alterations of the membrane architecture by the progressive accumulation of lipids undermines cell function, with potential implications in other genetic sphingolipidoses and adult neurodegenerative conditions. This article has an associated First Person interview with the first author of the paper.

Long-Term Improvement of Neurological Signs and Metabolic Dysfunction in a Mouse Model of Krabbe's Disease after Global Gene Therapy.

We report a global adeno-associated virus (AAV)9-based gene therapy protocol to deliver therapeutic galactosylceramidase (GALC), a lysosomal enzyme that is deficient in Krabbe's disease. When globally administered via intrathecal, intracranial, and intravenous injections to newborn mice affected with GALC deficiency (twitcher mice), this approach largely surpassed prior published benchmarks of survival and metabolic correction, showing long-term protection of demyelination, neuroinflammation, and motor function. Bone marrow transplantation, performed in this protocol without immunosuppressive preconditioning, added minimal benefits to the AAV9 gene therapy. Contrasting with other proposed pre-clinical therapies, these results demonstrate that achieving nearly complete correction of GALC's metabolic deficiencies across the entire nervous system via gene therapy can have a significant improvement to behavioral deficits, pathophysiological changes, and survival. These results are an important consideration for determining the safest and most effective manner for adapting gene therapy to treat this leukodystrophy in the clinic.

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.

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.

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.

Notch1 and Numb genes are inversely expressed as oligodendrocytes differentiate.

The Notch1 pathway plays a fundamental role during the establishment of cell fates in the central nervous system (CNS) by regulating neural cell differentiation. In oligodendrocytes (OLs), Notch1 activity prevents these cells from becoming terminally mature, thereby influencing CNS myelination. Little is known of how OLs regulate the expression of this receptor at the gene level or if OLs have mechanisms to control the level of intracellular activity of the Notch1 pathway. In this study, we have found that Notch1 gene expression was higher in proliferative OL progenitor cells (OPCs) and was reduced when cells were forced to withdraw from the cell cycle and became mature, indicating that Notch1 gene expression is developmentally regulated in OLs. We observed that the blockade of terminal differentiation of OPCs by incubation with Delta1, an activator of Notch1, was a dominant process and OL-differentiating signals such as thyroid hormone could not overcome this inhibition in culture. This suggests that a downregulation of the Notch1 pathway might be required to allow OPCs to enter terminal differentiation. We also provide evidence that OPCs and OLs express the Numb gene, a known negative regulator of Notch1 activity. In vivo, Numb was found in postnatal OLs from cerebellar and cerebral white matter. In vitro, Numb expression showed to be inversely correlated to that of Notch1, with higher levels of Numb proteins in mature OLs, in association with myelin-like membranes.