Transplantation of hNT neurons into the ischemic cortex: cell survival and effect on sensorimotor behavior.

Cell transplantation offers a potential new treatment for stroke. Animal studies using models that produce ischemic damage in both the striatum and the frontal cortex have shown beneficial effects when hNT cells (postmitotic immature neurons) were transplanted into the ischemic striatum. In this study, we investigated the effect of hNT cells in a model of stroke in which the striatum remains intact and damage is restricted to the cortex. hNT cells were transplanted into the ischemic cortex 1 week after stroke induced by distal middle cerebral artery occlusion (dMCAo). The cells exhibited robust survival at 4 weeks posttransplant even at the lesion border. hNT cells did not migrate, but they did extend long neurites into the surrounding parenchyma mainly through the white matter. Neurite extension was predominantly toward the lesion in ischemic animals but was bidirectional in uninjured animals. Extension of neurites through the cortex toward the lesion was also seen when there was some surviving cortical tissue between the graft and the infarct. Prolonged deficits were obtained in four tests of sensory-motor function. hNT-transplanted animals showed a significant improvement in functional recovery on one motor test, but there was no effect on the other three tests relative to control animals. Thus, despite clear evidence of graft survival and neurite extension, the functional benefit of hNT cells after ischemia is not guaranteed. Functional benefit could depend on other variables, such as infarct location, whether the cells mature, the behavioral tests employed, rehabilitation training, or as yet unidentified factors.

GABA signalling: therapeutic targets for epilepsy, Parkinson's disease and Huntington's disease.

Temporal lobe epilepsy (TLE), Parkinson's disease (PD) and Huntington's disease (HD) are neurodegenerative disorders that involve disruptions in gamma-amino butyric acid (GABA) signalling. GABA is the major inhibitory neurotransmitter in the central nervous system (CNS). TLE seizures reflect excess excitation, which may result from local inhibitory circuit dysfunction. PD devastates the input to striatal GABAergic neurones and HD destroys striatal GABAergic neurones. Controlling GABA delivery to specific brain areas should benefit each of these diseases. The molecules responsible for GABA release and signalling are ideal targets for new therapies. In this paper, we discuss the role of GABA in the circuitry affected by each of these diseases and suggest potential sites for intervention. GABA is unique among neurotransmitters because it can be synthesised by either of two related enzymes. Intracellular GABA is found throughout the cytosol and in synaptic vesicles. GABA can be released either through exocytosis, or via the plasma membrane transporter. The synthesising enzyme probably determines the intracellular location and hence the mechanism for GABA release. Directing GABA synthesis, degradation, transport or receptors can control GABA signalling. We propose that new drugs and devices aimed at GABA synthesis, release and binding will offer novel and highly effective treatments for neurodegenerative diseases.

Transfectable and transplantable postmitotic human neurons: a potential "platform" for gene therapy of nervous system diseases.

We have characterized a human embryonal carcinoma cell line (NTera-2 or NT2 cells) that is transfectable and capable of differentiating into postmitotic neuron-like cells (NT2N cells) following treatment with retinoic acid in order to identify a human neuronal cell line that might serve as a "platform" for gene therapy of human neurological diseases. Studies of NT2N cells transplanted into the brain or spinal cord of immunecompetent and immunodeficient rodents show that NT2N cells integrate into the host central nervous system (CNS) and establish the molecular and structural polarity of authentic neurons in vivo. Further, grafted NT2N cells acquire the molecular phenotype of fully mature neurons within 6 months postimplantation and the grafts survive > 1 year in immunodeficient mice without reverting to a neoplastic state. Although grafts of the retinoic acid-naive NT2 cells can form lethal tumors in the CNS, these cells differentiate into postmitotic neuron-like cells and do not form tumors when the grafts are confined to the caudoputamen. Based on the studies reviewed here, we conclude that grafted NT2N cells could serve as a suitable platform for the delivery of exogenous proteins into the CNS for gene therapy of human nervous system diseases.

Transplanted human neurons derived from a teratocarcinoma cell line (NTera-2) mature, integrate, and survive for over 1 year in the nude mouse brain.

Retinoic acid (RA) induces a human teratocarcinoma cell line (NTera-2 or NT2) to give rise exclusively to post-mitotic neuron-like (NT2N) cells, but NT2N cells never acquire a fully mature neuronal phenotype in vitro. To determine whether NT2N cells can mature into adult neuron-like cells in vivo, purified NT2N cells were grafted into different regions of the central nervous system (CNS) of adult and neonatal athymic mice, and the grafts were examined immunohistochemically by light, confocal, and electron microscopy using antibodies to a panel of developmentally regulated neuronal polypeptides. NT2N grafts were distinguished from endogenous mouse neurons with antibodies that recognize human or murine specific epitopes in selected neuronal polypeptides. Viable NT2N cells were identified in > 89% of graft recipients (N = 90), and some grafts survived 14 months. Within 3 weeks of implantation, grafted NT2N cells re-extended their processes, and the location of the grafts (e.g., septum versus neocortex) appeared to determine the extent to which processes were elaborated. Within the early post-transplantation period, grafted NT2N cells expressed the same neuronal polypeptides as their in vitro counterparts. However, between 6 weeks and 4-6 months post-implantation, the grafted NT2N cells progressively acquired the molecular phenotype of fully mature in vivo neurons as evidenced by dramatically increased expression of the most highly phosphorylated isoforms of the heavy neurofilament subunit, and the de novo expression of adult CNS tau. Notably, the time course for the extension of processes and the expression of neuronal polypeptides by NT2N grafts was similar in neonatal and adult mice. Although grafted NT2N cells formed synapse-like structures and elaborated dendrites and axons, these axons remained unmyelinated. Finally, none of the transplanted NT2N cells reverted to a neoplastic state. These studies demonstrate that pure populations of grafted human NT2N cells acquire a fully mature neuronal phenotype in vivo, and that these cells integrate and survive for > 1 year post-implantation in the mouse CNS. These human neuron-like cells are an attractive model system for studies of neuronal development, polarity and transplantation.

Human neurons that constitutively secrete A beta do not induce Alzheimer's disease pathology following transplantation and long-term survival in the rodent brain.

Since cultured neurons secrete beta-amyloid (A beta) peptides, and A beta forms amyloid deposits in the Alzheimer's disease (AD) brain, transplanted neurons may induce the deposition of A beta in the host brain. To assess this possibility, we studied grafted human neurons (NT2N cells) and their progenitors (NT2 cells) in the rodent brain. Although NT2N cells secrete more A beta than the NT2 cells in vitro, no A beta deposits or other AD lesions were induced in the rodent brain by grafts that survived days (NT2 and NT2N cells) to 46 weeks (NT2N cells). Thus, neither the deposition of A beta nor the induction of other AD lesions are obligatory consequences of the transplantation and long-term survival of human neurons or their progenitors in the rodent brain.