LOCAL SUPPLEMENTATION OF BRAINDERIVED NEUROTROPHIC FACTOR FOR THE TREATMENT OF NEURONAL DAMAGE.

The importance of nerve growth factors, especially brain-derived neurotrophic factor (BDNF) in the regulation of neuronal survival and plastic changes in morphology and function has been increasingly studied during the recent years. It has been proposed that the pathogenesis of some neurological diseases may be due to an alteration in neurotrophic factor and/or Trk receptor levels. The use of neurotrophic factors as therapeutic agents is a promising approach aimed at restoring and maintaining neuronal function in the central nervous system (CNS). This study is undertaken to develop a novel stem cell-based gene therapy to deliver neurotrophic factors to vulnerable regions of the CNS. Stem cell-based gene therapy is a potential delivery option by which cells are engineered to produce neurotrophic factors in vitro and then transplanted to the target area where neurotrophic factors are secreted to exert protective and/or restorative effects on the host tissue. A recently isolated mesodermal stem cell, mesoangioblast (MAB), has a high adhesin-dependent migratory capacity and may selectively cross the blood-brain barrier and home in the lesioned areas. Therefore, MABs provide an ideal cellular source for BDNF delivery. In this study, we generated a genetically modified mesoangioblast producing BDNF (MABs-BDNF). These engineered MABs maintained transgene expression and secretion of bioactive BDNF in time. We investigated the protective effects of MABs-BDNF in vitro using primary cultures and organotypic cultures of hippocampal slices. The viability of the cultured slices was assessed in several ways: fluorescein diacetate (FDA) hydrolysis assay, lactate dehydrogenase (LDH) release assay, immunohistochemistry for MAP2, immunoblot for neurofilament 68, and field potential recordings. Direct exposure of recombinant BDNF to primary cultured neurons and adult slices resulted in a concentration-dependant protective effect. The conditioned medium from MABs-BDNF highly promoted cell survival, while the conditioned medium from control cells (MABs) or an equivalent amount of rBDNF showed beneficial effects on cell survival to a lesser extent. The protective effects of MABs-BDNF were attenuated by adding either with the TrkB receptor blocker K252a or the BDNF scavenger TrkB-IgG.. This indicates that the conditioned medium from MABs-BDNF can foster the adult slice culture through secreting the engineered BDNF and unknown pro-survival factors produced intrinsically by MABs. The MABs-BDNF conditioned medium was optimal for retention of morphologic characteristics and viability in organotypic cultures from adult hippocampal slices. Moreover, MABs-BDNF were found to promote neurogenesis and glia proliferation. Treatment with the MABs-BDNF conditioned medium was found to increase the number of BrdU-labeled and BrdU/NeuN double labeled cells in the dentate gyrus of cultured slices. These in vitro findings demonstrate the beneficial effects of MABs-BDNF on neurons and provide a rationale for transplanting MABs-BDNF in the damaged brain as a therapeutic approach. Thus, we tested the transplantation of MABs-BDNF in an animal model of neuronal loss, the hippocampal sclerosis induced by status epilepticus. So far, we have not detected a deposition of MABs-BDNF in the epileptic brain after their systemic administration. Future experiments will aim at optimizing the transplanting conditions, changing the delivering routes, and assessing their therapeutic value in other neurological diseases associated with cell death. In terms of their prominent beneficial by-stander effects on neurons, MABs-BDNF hold substantial promise as therapeutic agents in the treatment of neurological diseases.