Early axonal loss accompanied by impaired endocytosis, abnormal axonal transport, and decreased microtubule stability occur in the model of Krabbe's disease.

In Krabbe's disease (KD), a leukodystrophy caused by ß-galactosylceramidase deficiency, demyelination and a myelin-independent axonopathy contributes to the severe neuropathology. Beyond axonopathy, we show that in Twitcher mice, a model of KD, a decreased number of axons both in the PNS and in the CNS, and of neurons in dorsal root ganglia (DRG), occurred before the onset of demyelination. Despite the early axonal loss, and although in vitro Twitcher neurites degenerated over time, Twitcher DRG neurons displayed an initial neurite overgrowth and, following sciatic nerve injury, Twitcher axons were regeneration-competent, at a time point where axonopathy was already ongoing. Psychosine, the toxic substrate that accumulates in KD, induced lipid raft clustering. At the mechanistic level, TrkA recruitment to lipid rafts was dysregulated in Twitcher neurons, and defective activation of the ERK1/2 and AKT pathways was identified. Besides defective recruitment of signaling molecules to lipid rafts, the early steps of endocytosis and the transport of endocytic and synaptic vesicles were impaired in Twitcher DRG neurons. Defects in axonal transport, specifically in the retrograde component, correlated with decreased levels of dynein, abnormal levels of post-translational tubulin modifications and decreased microtubule stability. The identification of the axonal defects that precede demyelination in KD, together with the finding that Twitcher axons are regeneration-competent when axonopathy is already installed, opens new windows of action to effectively correct the neuropathology that characterizes this disorder.

Primary bone marrow mesenchymal stromal cells rescue the axonal phenotype of Twitcher mice.

Krabbe's disease (KD) is a demyelinating disorder caused by the deficiency of lysosomal galactocerebrosidase (GALC), affecting both the central (CNS) and the peripheral nervous system (PNS). A current therapy, hematopoietic stem cell transplantation (HSCT), is ineffective at correcting the PNS pathology. We have previously shown that systemic delivery of immortalized bone marrow-derived murine mesenchymal stromal cells (BM-MSCs) diminishes the neuropathology of transplanted Twitcher mice, a murine model of KD. In this study, to move one step closer to clinical application, the effectiveness of a systematic delivery of primary BM-MSCs to promote recovery of the Twitcher PNS was assessed. Primary BM-MSCs grafted to the Twitcher sciatic nerve led to increased GALC activity that was not correlated to decreased psychosine (the toxic GALC substrate) accumulation. Nevertheless, BM-MSC transplantation rescued the axonal phenotype of Twitcher mice in the sciatic nerve, with an increased density of both myelinated and unmyelinated axons in transplanted animals. Whereas no increase in myelination was observed, upon transplantation an increased proliferation of Schwann cell precursors occurred. Supporting these findings, in vitro, BM-MSCs promoted neurite outgrowth of Twitcher sensory neurons and proliferation of Twitcher Schwann cells. Moreover, BM-MSCs expressed nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) and promoted increased BDNF synthesis by neighboring Schwann cells. Besides their action in neurons and glia, BM-MSCs led to macrophage activation in Twitcher sciatic nerves. In summary, primary BM-MSCs diminish the neuropathology of Twitcher sciatic nerves by coordinately affecting neurons, glia, and macrophages.

Advances and pitfalls of cell therapy in metabolic leukodystrophies.

Leukodystrophies are a group of disorders characterized by myelin dysfunction, either at the level of myelin formation or maintenance, that affect the central nervous system (CNS) and also in some cases, to a lesser extent, the peripheral nervous system (PNS). Although these genetic-based disorders are generally rare, all together they have a significant impact in the society, with an estimated overall incidence of 1 in 7,663 live births. Currently, there is no cure for leukodystrophies, and the development of effective treatments remains challenging. Not only leukodystrophies generally progress very fast, but also most are multifocal needing the simultaneous targeting at multiple sites. Moreover, as the CNS is affected, the blood-brain barrier (BBB) limits the efficacy of treatment. Recently, interest on cell therapy has increased, and the leukodystrophies for which metabolic correction is needed have become first-choice candidates for cell-based clinical trials. In this review, we present and discuss the available cell transplantation therapies in metabolic leukodystrophies including fucosidosis, X-linked adrenoleukodystrophy, metachromatic leukodystrophy, Canavan disease, and Krabbe's disease. We will discuss the latest advances of cell therapy and its pitfalls in this group of disorders, taking into account, among others, the limitations imposed by reduced cell migration in multifocal conditions, the need to achieve corrective enzyme threshold levels, and the growing awareness that not only myelin but also the associated axonopathy needs to be targeted in some leukodystrophies.

Systemic delivery of bone marrow-derived mesenchymal stromal cells diminishes neuropathology in a mouse model of Krabbe's disease.

In Krabbe's disease, a demyelinating disorder, add-on strategies targeting the peripheral nervous system (PNS) are needed, as it is not corrected by bone-marrow (BM) transplantation. To circumvent this limitation of BM transplantation, we assessed whether i.v. delivery of immortalized EGFP(+) BM-derived murine mesenchymal stromal cells (BM-MSC(TERT-EGFP) ) targets the PNS of a Krabbe's disease model, the Twitcher mouse. In vitro, BM-MSC(TERT-EGFP) retained the phenotype of primary BM-MSC and did not originate tumors upon transplantation in nude mice. In vivo, undifferentiated EGFP(+) cells grafted the Twitcher sciatic nerve where an increase in Schwann cell precursors and axonal number was detected. The same effect was observed on BM-MSC(TERT-EGFP) i.v. delivery following sciatic nerve crush, a model of axonal regeneration. Reiterating the in vivo findings, in a coculture system, BM-MSC(TERT-EGFP) induced the proliferation of Twitcher-derived Schwann cells and the neurite outgrowth of both Twitcher-derived neurons and wild-type neurons grown in the presence of psychosine, the toxic substrate that accumulates in Krabbe's disease. In vitro, this neuritogenic effect was blocked by K252a, an antagonist of Trk receptors, and by antibody blockage of brain derived neurotrophic factor, a neurotrophin secreted by BM-MSC(TERT-EGFP) and induced in neighboring Schwann cells. In vivo, BM-MSC(TERT-EGFP) surmounted the effect of K252a, indicating their ability to act through a neurotrophin-independent mechanism. In summary, i.v. delivery of BM-MSC(TERT-EGFP) exerts a multilevel effect targeting neurons and Schwann cells, coordinately diminishing neuropathology. Therefore, to specifically target the PNS, MSC should be considered an add-on option to BM transplantation in Krabbe's disease and in other disorders where peripheral axonal loss occurs.