In vitro induction and in vivo expression of bcl-2 in the hNT neurons.

Bcl-2 encodes membrane-associated proteins that suppress programmed cell death in cells of various origins. Compelling evidence suggests that bcl-2 is also involved in neuronal differentiation and axonal regeneration. The human Neuro-Teratocarcinoma (hNT) neurons constitute a terminally differentiated human neuronal cell line that is derived from the Ntera-2/clone D1 (NT2) precursors upon retinoic acid (RA) treatment. After transplantation into the central nervous system (CNS), the hNT neurons survive, engraft, maintain their neuronal identity, and extend long neurite outgrowth. We were particularly interested in the intracellular determinants that confer these post-transplant characteristics to the hNT neurons. Thus, we asked whether the hNT neurons express bcl-2 after transplantation into the rat striatum and if RA induction of the neuronal lineage is mediated by bcl-2. The grafted hNT neurons were first identified using three different antibodies that recognize human-specific epitopes, anti-hMit, anti-hNuc, and NuMA. After a 1-month post-transplant survival time, NuMA immunostaining revealed that 12% of the hNT neurons survived the transplantation. These neurons extended long neuritic processes within the striatum, as demonstrated using the human-specific antibody against the midsize neurofilament subunit HO14. Importantly, we found that 85% of the implanted hNT neurons expressed bcl-2 and that the in vitro induction of the neuronal lineage from the NT2 precursors with RA resulted in an upregulation of bcl-2 expression. Together, these data suggest that the differentiation of the hNT neurons to a neuronal lineage could be mediated at least partially by bcl-2.

Apoptosis in cultured hNT neurons.

Programmed cell death (apoptosis) is an important mechanism shaping the size of different cell populations within the developing nervous system. In our study we used the NT2/D1 clone originally established from the Ntera 2 cell line to investigate the baseline levels of apoptosis in cultured postmitotic hNT (NT2-N) neurons previously treated for 3, 4 or 5 weeks with retinoic acid (RA) and compared it with apoptosis in NT2 precursors unexposed to RA. First, we examined whether different lengths of exposure to RA might affect baseline apoptotic rate in differentiating hNT neurons. Second, we investigated whether cultured hNT neurons, previously shown to possess dopaminergic characteristics, would be preferentially affected by apoptosis. Using the terminal deoxynucleotidyl transferase (tdt)-labeling technique we found that the postmitotic hNT neuronal cells exposed to RA demonstrated significantly higher numbers of apoptotic cells (12.5-15.8%) in comparison to rapidly dividing NT2 precursor cell line (3.6-4.4%) at both studied (1 and 5 days in vitro, DIV) time points. Similar apoptotic nuclear morphology, including a variable extent of nuclear fragmentation was observed in all examined hNT cultures. On the other hand, the incidence of apoptotic nuclei was rare in cultures of NT2 precursors not subjected to RA treatment. Combined immunocytochemistry for tyrosine hydroxylase (TH) and Hoechst staining revealed dopaminergic hNT neurons destined to die. Our double-labeling studies have demonstrated that only a subset of TH-positive hNT cells had condensed chromatin after 1 (approx. 15%) and 5 (approx. 20%) DIV. NT2 precursors were not TH-positive. Collectively, our results demonstrated that exposure to differentiating agent RA triggers an apoptotic commitment in a subset of postmitotic hNT neurons. These results suggest that this cell line may serve as a model of neuronal development to test various pathogenic factors implicated in the etiology of Parkinson's disease (PD), as well as to screen numerous pharmacological treatments that may slow or prevent dopaminergic deterioration.

Comparison of calcium-binding proteins expressed in cultured hNT neurons and hNT neurons transplanted into the rat striatum.

An alternative source of cells for neural transplantation and brain repair that has many characteristics of immature neurons is the hNT neuron, derived from an embryonal human teratocarcinoma (NTera2) cell line that is terminally differentiated in vitro with retinoic acid. The majority of hNT neurons are GABAergic in cell culture. We have determined the calcium-binding protein (CBP) phenotypes of hNT neurons for three CBPs, calretinin (CR), calbindin D-28K (CB), and parvalbumin (PV), in cell culture and after transplantation into the rat striatum. In cell culture, 95% of all cell profiles were human nuclear matrix antigen (NuMA) positive. PV-positive hNT neurons constituted 50% of all neuron-like profiles, with CB+ and CR+ constituting 14 and 6% of cells, respectively. In contrast, when the striatal grafts were examined after 30 days survival using confocal microscopy, only 10% of hNT neurons immunopositive for NuMA were PV+; 19% were CB+/NuMA+, approximately the same percentage as was seen in vitro, and 82% of grafted hNT neurons were CR+. These results suggest that hNT neurons can be subdivided into at least three subpopulations based on the CBP phenotype that they express and that there is a CBP phenotypic shift following transplantation. Three related hypotheses are proposed to account for this phenotypic shift of hNT neurons after transplantation: (a) selective survival of the CR+ subpopulation of hNT neurons, (b) selective transitory quiescence of the transplanted PV+ cells due to transplantation stress, or (c) dedifferentiation of the hNT neurons following transplantation, which may allow them to respond to local environmental cues during the engraftment process.

In vitro and in vivo characterization of hNT neuron neurotransmitter phenotypes.

The hNT neuron exhibits many characteristics of neuroepithelial precursor cells, making them an excellent model to study neuronal plasticity in vitro and in vivo. These cells express a number of neurotransmitters in vitro, including dopamine, gamma-aminobutyric acid and acetylcholine. However, there have been few reports of the neurotransmitters that hNT neurons express in vivo. The present study examined whether hNT neurons express the same neurotransmitters in vivo as they do in vitro. First, the expression of tyrosine hydroxylase (TH), glutamic acid decarboxylase (GAD), choline acetyltransferase (ChAT) and the human specific nuclear marker NuMA by hNT neurons was confirmed. Nineteen normal animals were then transplanted with 80,000 hNT neurons aimed at the striatum, hippocampus or cerebral cortex. Five additional animals received injections of medium. All animals received daily intraperitoneal injections of cyclosporine (10 mg/kg) and survived 30 days. Sections through the transplants were examined for NuMA-positive hNT neurons, and for the presence of the three neurotransmitter markers: TH, GAD and ChAT. The hNT neurons were found in the striatum and cortex. Of the hNT neurons found within the rat striatum, 33% were ChAT-positive. In the cortex, only 4% of the neurons expressed ChAT. No GAD-positive hNT neurons were detected at either site. No NuMA-positive neurons were found in the hippocampus. The implanted hNT neurons did not induce activation of astrocytes as determined by immunocytochemistry for glial fibrillary acidic protein (GFAP). Moreover, no hNT neuron was found to express GFAP in vivo. Together, these data suggest that the hNT neurons engraft in the new host tissue, maintain their neuronal identity and may be guided in differentiation according to local environmental cues.

Generation of tyrosine hydroxylase-producing neurons from precursors of the embryonic and adult forebrain.

We have explored the plastic ability of neuronal precursors to acquire different identities by manipulating their surrounding environment. Specifically, we sought to identify potential signals involved in the specification of forebrain dopaminergic neurons. Here we describe culture conditions under which tyrosine hydroxylase (TH) expression is induced in neuronal precursors, which were derived directly from the embryonic striatum and adult subependyma (SE) of the lateral ventricle or generated from multipotent forebrain stem cells. TH was successfully induced in all of these cell types by 24 hr exposure to basic fibroblast growth factor (FGF2) and glial cell conditioned media (CM). The greatest magnitude of the inductive action was on embryonic striatal precursors. Although FGF2 alone induced limited TH expression in striatal cells (1.1 +/- 0.2% of neurons), these actions were potentiated 17.5-fold (19.6 +/- 1.5% of neurons) when FGF2 was coadministered with B49 glial cell line CM. Of these TH-immunoreactive cells, approximately 15% incorporated bromodeoxyuridine (BrdU), indicating that they were newly generated, and 95% coexpressed the neurotransmitter GABA. To investigate whether precursors of the adult forebrain subependyma were competent to respond to the instructive actions of FGF2+CM, they were first labeled in vivo with a pulse of BrdU. Although none of the cells expressed TH in control, 0.2% of total cells showed TH immunoreactivity in FGF2+CM-treated cultures. Under these same conditions only, in vitro-generated precursors from epidermal growth factor-responsive stem cells exhibited TH expression in 10% of their total neuronal progeny. Regulation of neurotransmitter phenotype in forebrain neuronal precursors, by the synergistic action of FGF2 and glial-derived diffusible factors, may represent a first step in understanding how these cells are generated in the embryonic and adult brain and opens the prospect for their manipulation in vitro and in vivo for therapeutic use.

Activin co-operates with fibroblast growth factor 2 to regulate tyrosine hydroxylase expression in the basal forebrain ventricular zone progenitors.

Activin and its cognate receptors are expressed during embryogenesis in the rapidly dividing cells of the basal forebrain ventricular zone. This finding prompted us to study the role of activin in regulating neurotransmitter phenotype expression and other aspects of the ventricular zone-derived progenitor cell differentiation. Although virtually ineffective alone, activin co-operated with fibroblast growth factor 2 to induce a rapid tyrosine hydroxylase-immunoreactivity in cultured ventricular zone progenitors. Northern analysis indicated that the increase in tyrosine hydroxylase-immunoreactivity was associated with increased tyrosine hydroxylase gene expression. Activin and fibroblast growth factor 2 action was specific to tyrosine hydroxylase, as it did not induce the expression of choline acetyltransferase, nor enhance the expression of glutamate decarboxylase. Cultures treated with the DNA replication marker bromodeoxyuridine revealed that both proliferating ventricular zone progenitors and their post-mitotic progeny were induced to express tyrosine hydroxylase. In these cultures, activin acted to reduce fibroblast growth factor 2 stimulated mitotic activity. Furthermore, activin permitted neuronal differentiation and survival of the ventricular zone progenitors after three days in vitro. Together these data demonstrate a novel role of activin and fibroblast growth factor 2 in regulating the fate of the embryonic basal forebrain ventricular zone progenitors.

Antibodies against restricted sequences in human c-ErbA hinge domain recognize differentially natural mammalian alpha- or beta-type triiodothyronine receptors and interfere differently with hormone binding.

In our first report, rabbit antibodies directed to recombinant polypeptides of human alpha-type c-ErbA sequences recognized natural triiodothyronine (T3) receptors (TR) in adipocytes (mouse Ob 17 cell line) but not in liver (mouse, rat). Moreover, some of them, directed to the sequence 150-228, markedly interfered with hormone binding to adipocyte T3 receptors. We now raised antibodies against shorter synthetic peptides within this alpha-type 150-228 c-ErbA sequence, which encompasses part of the hinge (D) domain and N-terminus of the E domain (alpha-150-166 and alpha 172-191) and against a beta-type c-ErbA sequence (beta 204-220 aligned on alpha 150-166, and differing by eight amino acids). Our present antibodies, which bear the expected c-ErbA alpha- or beta-type specificity, immunoprecipitated the TR in nuclear extracts, with a different pattern between tissues: exclusive precipitation by anti-c-ErbA alpha antibodies in Ob 17 adipocytes; large but non-exclusive precipitation by anti-cErbA beta antibodies in rat or mouse liver, which also expresses some alpha-type TR. This pattern of discriminative immunoprecipitation, also obtained in parallel analysis using our previously described antibodies to other c-ErbA alpha or beta sequences (anti-alpha 144-162, anti-alpha 1 403-410 and anti-beta 62-82), roughly verifies results of c-erbA mRNA expression in these tissues. Slight differences appeared in the extent of alpha-type TR recognition by antibodies directed to alpha 172-191, whether TR were liganded or not to T3 before antibody addition. This evokes a different conformation of this region after hormone binding. Most interestingly, these anti-alpha 172-191 antibodies lowered the Ka for T3 and extensively dissociated the adipocyte T3-TR complexes; they interfered poorly with the binding of T3 in liver nuclear extracts. This strongly supports the concept that internal sequences in c-ErbA alpha, more precisely in a restricted C-terminal part of the D domain, are necessary for efficient T3 binding, which also need the C-terminal part of domain E.

Nuclear factors specifically favor thyroid hormone binding to c-ErbA alpha 1 protein (thyroid hormone receptor alpha) over-expressed in E. coli.

A recombinant rat thyroid hormone receptor alpha (TR alpha or c-ErbA alpha 1) was produced in E. coli as a non-mutated, nonfusioned protein and obtained as an efficient DNA and T3 binding protein that could be easily handled in a buffer-soluble state (rec-TR alpha). It was found that nuclear extracts (NE) added to rec-TR alpha markedly amplified not only DNA binding, which has been well documented, but also T3 binding (increased binding site concentration), which has not yet been reported. This T3 binding amplifying effect on rec-TR alpha occurs at low NE protein concentrations that produce no or minimal endogenous TR with respect to rec-TR, while similar concentrations of other proteins (e.g. ovalbumin or cytosol) only moderately enhanced T3 binding. The T3 binding amplifying nuclear factors, which are partly heat-labile, appeared as necessary auxiliaries in the analyses of partially purified rec-TR alpha. A protective effect of NE against a loss of affinity for T3 under the action of antibodies directed to certain sequences in the TR alpha D domain suggests that nuclear factors help rec-TR alpha to acquire and/or stabilize a conformation that allows the high affinity T3 binding. The nature of this nuclear amplifying factor is still unknown: RXR alpha which, produced in vitro, could amplify binding of the rec-TR alpha to a DNA thyroid response element, was unable to display such a rescue of high affinity binding sites.