Wendy R. Galpern

Transplanted xenogeneic neural cells in neurodegenerative disease models exhibit remarkable axonal target specificity and distinct growth patterns of glial and axonal fibres

Clinical trials are under way using fetal cells to repair damaged neuronal circuitry. However, little is known about how transplanted immature neurons can grow anatomically correct connections in the adult central nervous system (CNS). We transplanted embryonic porcine neural cells in vivo into adult rat brains with neuronal and axonal loss typical of Parkinsons or Huntingtons disease. Using complementary species-specific cellular markers, we found donor axons and CD44+ astroglial fibres in host white matter tracts up to 8 mm from CNS transplant sites, although only donor axons were capable of reaching correct gray matter target regions. This work demonstrates that adult host brain can orient growth of transplanted neurons and that there are differences in transplant donor glial and axonal growth patterns in cellular repair of the mature CNS.

Embryonic stem cells differentiated in vitro as a novel source of cells for transplantation

The controlled differentiation of mouse embryonic stem (ES) cells into near homogeneous populations of both neurons and skeletal muscle cells that can survive and function in vivo after transplantation is reported. We show that treatment of pluripotent ES cells with retinoic acid (RA) and dimethylsulfoxide (DMSO) induce differentiation of these cells into highly enriched populations of gamma-aminobutyric acid (GABA) expressing neurons and skeletal myoblasts, respectively. For neuronal differentiation, RA alone is sufficient to induce ES cells to differentiate into neuronal cells that show properties of postmitotic neurons both in vitro and in vivo. In vivo function of RA-induced neuronal cells was demonstrated by transplantation into the quinolinic acid lesioned striatum of rats (a rat model for Huntingtons disease), where cells integrated and survived for up to 6 wk. The response of embryonic stem cells to DMSO to form muscle was less dramatic than that observed for RA. DMSO-induced ES cells formed mixed populations of muscle cells composed of cardiac, smooth, and skeletal muscle instead of homogeneous populations of a single muscle cell type. To determine whether the response of ES cells to DMSO induction could be further controlled, ES cells were stably transfected with a gene coding for the muscle-specific regulatory factor, MyoD. When induced with DMSO, ES cells constitutively expressing high levels of MyoD differentiated exclusively into skeletal myoblasts (no cardiac or smooth muscle cells) that fused to form myotubes capable of spontaneous contraction. Thus, the specific muscle cell type formed was controlled by the expression of MyoD. These results provided evidence that the specific cell type formed (whether it be muscle, neuronal, or other cell types) can be controlled in vitro. Further, these results demonstrated that ES cells can provide a source of multiple differentiated cell types that can be used for transplantation.

Xenotransplantation of Porcine Fetal Ventral Mesencephalon in a Rat Model of Parkinson's Disease: Functional Recovery and Graft Morphology

Neurotransplantation of human fetal dopamine (DA) neurons is currently being investigated as a therapeutic modality for Parkinsons disease (PD). However, the practical limitations of human fetal transplantation indicate a need for alternative methodologies. Using the 6-hydroxydopamine rat model of PD, we transplanted dopaminergic neurons derived from Embryonic Day 27 porcine fetuses into the denervated striatum of cyclosporine-A (CyA)-treated or non-CyA-treated rats. Functional recovery was assessed by amphetamine-induced rotation, and graft survival and morphology were analyzed using neuronal and glial immunostaining as well as in situ hybridization with a porcine repeat element DNA probe. A significant, sustained reduction in amphetamine-induced rotational asymmetry was present in the CyA-treated rats whereas the non-CyA-treated rats showed a transient behavioral recovery. The degree of rotational recovery was highly correlated to the number of surviving transplanted porcine dopaminergic neurons. TH+ neuronal survival and graft volume were significantly greater in the CyA-treated group as compared to the non-CyA group. By donor-specific neuronal and glial immunostaining as well as donor-specific DNA labeling, we demonstrate that porcine fetal neuroblasts are able to survive in the adult brain of immunosuppressed rats, mediate functional recovery, and extensively reinnervate the host striatum. These findings suggest that porcine DA neurons may be a suitable alternative to the use of human fetal tissue in neurotransplantation for PD.

Detection of dopaminergic cell loss and neural transplantation using pharmacological MRI, PET and behavioral assessment.

We demonstrate the use of magnetic resonance imaging (MRI) for detection of neurotransmitter stimulation using the dopamine transporter ligands amphetamine and CFT (2beta-carbomethoxy-3beta-(4-fluorophenyl)tropane) as pharmacological challenges. We demonstrate that the unilateral loss of a hemodynamic response to either amphetamine or CFT challenge by unilateral 6-hydroxydopamine lesioning is restored by transplantation of fetal dopamine neurons in the striatum. The time course for the hemodynamic changes parallels the time courses for dopamine release, measured by prior microdialysis studies, and also for the rotational behavior in the unilaterally lesioned animals. Transplantation of the fetal cells results in hemodynamic time courses after CFT or amphetamine challenges at the graft site that are identical to those induced both before transplantation and on the intact contralateral side. The transplantation also results in complete behavioral recovery. The spatial extent of the dopaminergic recovery in the lesioned striatum is the same when measured using either PET of tracer levels of [11C]CFT binding or MRI. These results show great promise for the application of pharmacological MRI for application to studies of dopamine cell loss and potential recovery in Parkinsons disease.

Cell-mediated delivery of brain-derived neurotrophic factor enhances dopamine levels in an MPP+ rat model of substantia nigra degeneration

Brain-derived neurotrophic factor (BDNF) promotes the survival of fetal mesencephalic dopaminergic cells and protects dopaminergic neurons against the toxicity of MPP+ in vitro. Supranigral implantation of fibroblasts genetically engineered to secrete BDNF attenuates the loss of substantia nigra pars compacta (SNc) dopaminergic neurons associated with striatal infusion of MPP+ in the adult rat. Using this MPP+ rat model of nigral degeneration, we evaluated the neurochemical effects of supranigral, cell-mediated delivery of BDNF on substantia nigra (SN) dopamine (DA) content and turnover. Genetically engineered BDNF-secreting fibroblasts (approximately 12 ng BDNF/24 h) were implanted dorsal to the SN 7 days prior to striatal MPP+ administration. The present results demonstrate that BDNF-secreting fibroblasts, as compared to control fibroblasts, enhance SN DA levels ipsilateral as well as contralateral to the graft without altering DA turnover. This augmentation of DA levels suggests that local neurotrophic factor delivery by genetically engineered cells may provide a therapeutic strategy for preventing neuronal death or enhancing neuronal function in neurodegenerative diseases characterized by dopaminergic neuronal dysfunction, such as Parkinsons disease.

NGF attenuates 3-nitrotyrosine formation in a 3-NP model of Huntington's disease

Nerve growth factor (NGF)-secreting fibroblasts are able to protect against the Huntington-like striatal neurodegeneration induced by the mitochondrial toxin 3-nitropropionic acid (3-NP). In the present study, we investigated whether the neuroprotective effects of NGF are mediated through antioxidative mechanisms. Rats were grafted in the corpus callosum with NGF[+] or NGF[-] fibroblasts 7 days before administration of 3-NP. The generation of peroxynitrite was evaluated by measuring the striatal levels of 3-nitrotyrosine. NGF significantly decreased the 3-NP induced generation of 3-nitrotyrosine, presumably by decreasing peroxynitrite formation. These findings suggest that NGF might protect against neuronal death by inhibiting the production of nitric oxide or decreasing the levels of superoxide radicals, thereby decreasing the generation of oxidative agents such as peroxynitrite.

Effects of striatal excitotoxicity on huntingtin-like immunoreactivity.

&NA; The relationship between the specific neuronal loss observed in Huntingtons disease and the mutation in the IT15 gene responsible for this disease remains obscure. Using an antipeptide antibody against amino acids 3114‐3141 of the human huntingtin protein, we demonstrate that striatal injection of quinolinic acid in mice induces increased immunoreactivity for huntingtin in some remaining neurons but not in glial cells. This increase is apparent in both neuronal cell bodies and in cell processes in the white matter six hours after excitotoxic challenge. This finding suggests that huntingtin may be involved in the response to excitotoxic stress in these neurons.

Neurotrophic Factor Protection against Excitotoxic Neuronal Death

Neurotrophic factors are polypeptides capable of promoting neuronal survival in both the developing and the adult brain. In addition to the neurotrophins, NGF, brain-derived neurotropic factor, and NT-3 to -6, other neurotrophic factors include ciliary neurotrophic factor, fibroblast growth factors, insulin-like growth factors, members of the transforming growth factor superfamily, members of the epidermal growth factor family, and other cytokines such as leukemia inhibitory factor, oncostatin M, and interleukins-6 and -11. One condition under which these factors promote survival is the challenge of neurons with analogs of excitatory amino acid transmitters. Such analogs, including quinolinic acid, kainic acid, and ibotenic acid, are frequently employed as models of neurological diseases such as Huntingtons disease, Parkinsons disease, Alzheimers disease, epilepsy, cerebellar degenerations, and amyotrophic lateral sclerosis. Excitotoxicity also plays a role in neu ronal death caused by focal ischemia, hypoglycemia, or trauma. Although much has been learned about the mechanisms of both the action of neurotrophic factors and of cell death in response to excitotoxins, the mechanism of protection by these factors remains uncertain. This review explores the biochemical and phys iological changes mediated by neurotrophic factors that may underlie their ability to protect against excito toxic cell death. Second messenger pathways used degenerately by both excitotoxins and neurotrophic factors are discussed as a potential site of interaction mediating the protective effects of neurotrophic factors. Particular attention is also paid to the importance of the route of neurotrophic factor delivery in conferring neuroprotection in particular excitotoxic models. The Neuroscientist 1:286-297, 1995

Transplanting Fetal Neural Xenogeneic Cells in Parkinson’s and Huntington’s Disease Models

Fetal allogeneic neural transplants have been repeatedly shown to cause functional recovery in animal models of neurodegenerative diseases (1–8), improve symptoms, and reduce drug-induced side effects in patients with Parkinson’s disease (9–13). It has, however, proven difficult to obtain human fetal tissue for neural transplantation, both from a practical and ethical standpoint (14–16). Thus, although a therapeutic rationale exists for using allogeneic neural transplantation in Parkinson’s (and Huntington’s) disease, the limited availability of human fetal neural tissue poses serious limitations on clinical application (17). Neural xenotransplantation, which is transplantation of fetal neuroblasts obtained from homologous neural structures of a different (mammalian) species into the adult brain, overcomes many of the limitations associated with the use of human fetuses. For clinical use, neuroblasts at precisely defined embryonic ages can be prepared in large quantities and in a sterile fashion from the fetuses of especially bred pathogen-free xenogeneic donors.