Immortalized human dorsal root ganglion cells differentiate into neurons with nociceptive properties.

A renewable source of human sensory neurons would greatly facilitate basic research and drug development. We had established previously conditionally immortalized human CNS cell lines that can differentiate into functional neurons (). We report here the development of an immortalized human dorsal root ganglion (DRG) clonal cell line, HD10.6, with a tetracycline-regulatable v-myc oncogene. In the proliferative condition, HD10.6 cells have a doubling time of 1.2 d and exhibit a neuronal precursor morphology. After differentiation of clone HD10.6 for 7 d in the presence of tetracycline, v-myc expression was suppressed, and >50% of the cells exhibited typical neuronal morphology, stained positively for neuronal cytoskeletal markers, and fired action potentials in response to current injection. Furthermore, this cell line was fate-restricted to a neuronal phenotype; even in culture conditions that promote Schwann cell or smooth muscle differentiation of neural crest stem cells, HD10.6 differentiated exclusively into neurons. Moreover, differentiated HD10.6 cells expressed sensory neuron-associated transcription factors and exhibited capsaicin sensitivity. Taken together, these data indicate that we have established an immortalized human DRG cell line that can differentiate into sensory neurons with nociceptive properties. The cell line HD10.6 represents the first example of a human sensory neuronal line and will be valuable for basic research, as well as for the discovery of novel drug targets and clinical candidates.

Application of ex vivo gene therapy in the treatment of Parkinson's disease.

Ex vivo gene therapy approaches hold great promise for the treatment of neurodegenerative diseases where there is currently no cure or adequate treatment for affected individuals. In this review we have focused on the use of ex vivo gene transfer techniques in Parkinson's disease models; however, the issues and approaches outlined are applicable to other neurodegenerative disorders. In utilizing the ex vivo strategy two considerations are critical for delivery of therapeutic levels of transgene product to the target: (i) the vector system and (ii) the cell type for grafting. We describe herein different vector systems that are currently available and briefly review the various cell types that have been transduced and grafted into the striatum of animals with experimental Parkinson's disease. The strategies for application of gene therapy techniques to a treatment for Parkinson's disease have expanded beyond the classical dopamine replacement toward the use of neurotrophic factors in enhancing cell function or preventing cell death. In addition, we explore the utility of CNS-derived neural progenitors as alternative cell types for ex vivo gene therapy in an animal model of Parkinson's disease.

Expression of alpha 2 adrenoceptors during rat brain development--I. Alpha 2A messenger RNA expression.

The distribution of alpha 2A adrenoceptor messenger RNA expression in developing rat brain was characterized using in situ hybridization with 35S-labeled riboprobes. Intense hybridization signal was detected as early as embryonic day 14 in several areas adjacent to the forebrain and hindbrain germinal zones and in central noradrenergic neurons. A marked increase in messenger RNA expression was observed throughout the brain during late prenatal development, consistent with the migration and maturation of neurons in developing brain structures. In embryonic brain, there was a temporal and spatial correspondence in the appearance of alpha 2A messenger RNA expression and binding sites labeled with [3H]idazoxan or p-[125I]iodoclonidine, indicating translation into receptor protein at an early stage of development. Whereas the presynaptic expression remained constant throughout development, there was an early postnatal decline of alpha 2A receptor expression in many brain regions, including the olfactory bulb, cortex, caudate-putamen, hippocampus, thalamus, hypothalamus and medulla. Thereafter, messenger RNA expression increased, establishing an adult-like pattern during the second postnatal week, but remained low in areas such as the caudate-putamen, thalamus and hippocampus, which do not exhibit extensive expression in the adult. The transient perinatal expression of this alpha 2 adrenoceptor type, which coincides with a period of hyperreactivity to sensory stimuli in the locus coeruleus, may indicate a specific functional role for the alpha 2A receptor in the developing rat brain. The early and intense expression in olfactory structures suggests an involvement in early olfactory learning. The pattern of widespread, transient expression of alpha 2A receptors in the fetal brain is in marked contrast to the postnatal development of the alpha 2C receptor type.

Expression of alpha 2 adrenoceptors during rat brain development--II. Alpha 2C messenger RNA expression and [3H]rauwolscine binding.

The distributions of alpha 2C adrenoceptor messenger RNA and high-affinity [3H]rauwolscine binding sites were characterized in developing rat brain. Using in situ hybridization with 35S-labeled riboprobes directed against the third intracellular loop, alpha 2C messenger RNA expression appeared in an adult-like pattern during the first and second postnatal weeks, in the anterior olfactory nucleus, caudate-putamen, olfactory tubercles, islands of Calleja and hippocampus, following the time-course of maturation of these structures. Only in the cerebellum was alpha 2C messenger RNA transiently expressed during the critical period of granule cell development. High-affinity [3H]rauwolscine binding sites were detected using receptor autoradiography and revealed a similar spatial and temporal time-course of appearance during rat brain development. The highest numbers of binding sites were detected in the olfactory tubercles and islands of Calleja, and moderate numbers in the anterior olfactory nucleus, caudate-putamen and hippocampus. Like alpha 2C messenger RNA expression, high-affinity [3H]rauwolscine binding sites were transiently expressed in the cerebellum. In some areas (e.g., the substantia nigra), [3H]rauwolscine binding sites were detected even though alpha C2 messenger RNA expression was absent. The strong spatial and temporal correspondence between messenger RNA expression and radioligand binding supports the conclusion that [3H]rauwolscine selectively labels alpha 2C adrenoceptors in the rat brain. The developmental pattern which was observed is in marked contrast to the early, transient expression of the alpha 2A adrenoceptor. Thus, the alpha 2A and alpha 2C receptor types may serve distinct functional roles in the developing brain.

Regional differences in responsiveness of adult CNS axons to grafts of cells expressing human neurotrophin 3.

Neurotrophin 3 (NT3) belongs to the neurotrophin family, which also includes nerve growth factor, brain-derived neurotrophic factor, and neurotrophin 4/5. NT3 mRNA is widely expressed in the rodent nervous system, but the physiological function of the native protein is still unclear. Genetically modified cell lines that produce physiological amounts of NT3 can provide a useful tool in the elucidation of the NT3 effects in the adult central nervous system (CNS). Genetically modified rat primary skin fibroblasts expressing and secreting human NT3 (hNT3) were prepared and characterized. In vitro, cell lines derived from different retroviral constructs expressed hNT3 mRNA, as determined by PCR and RNA blot analysis. Secretion of biologically active hNT3 was confirmed by specific elicitation of neurite outgrowth from cultured chick primary sympathetic and sensory neurons and from rat fetal locus coeruleus neurons in the presence of hNT3-producing cell conditioned media. In vivo, implanted fibroblasts survived well up to the maximal experimental time points of 6 weeks (brain) and 4 weeks (spinal cord) and continued to express hNT3 mRNA in vivo. As early as 2 weeks postgrafting, specific sprouting of host sensory neurites in response to hNT3-producing grafts was observed in the spinal cord. In contrast, hNT3-producing cerebral grafts did not induce a sprouting response different from that observed with control grafts. These findings establish the existence of a regionally different responsiveness of the CNS axons to local hNT3 overexpression.

Basic fibroblast growth factor increases dopaminergic graft survival and function in a rat model of Parkinson's disease.

The clinical use of fetal neural grafts as an intracerebral source of dopamine for patients with Parkinson's disease has met with limited success. Since basic fibroblast growth factor (bFGF) enhances the survival and growth of dopaminergic neurons in vitro, we explored whether cells genetically modified to produce bFGF would improve the functional efficacy of dopaminergic neurons implanted into rats with experimental Parkinson's disease. Results show that bFGF-producing cells grafted together with fetal dopamine neurons have potent growth-promoting effects on the implanted neurons in vivo. Moreover, rats implanted with such co-grafts display the most pronounced behavioural improvements post-grafting. These findings not only provide insight into the function of bFGF in situ, but also suggest an approach for enhancing the survival and function of dopamine neurons grafted into the damaged brain.

Opioid effects on [3H]norepinephrine release from dissociated embryonic locus coeruleus cell cultures.

The acute and chronic effects of opioid exposure on [3H]norepinephrine ([3H]NE) release were examined in cell cultures of embryonic rat locus coeruleus (LC). Initial morphological and biochemical characterization of the cultures indicated that the cells exhibited properties similar to those observed in situ. Specific [3H]NE uptake was saturable with a Km value of 222 +/- 52 nM. [3H]NE accumulated by LC cells was released in response to 20 mM K+ stimulation, in a calcium-dependent manner. Both components of neurotransmitter release, spontaneous and K+ evoked, were significantly inhibited by beta-endorphin, with the latter being maintained in the presence of tetrodotoxin. The pharmacology of the opioid effect was consistent with that of mu-receptor activation. The effect of chronic exposure to the mu-selective agonist fentanyl (1 microM) was examined following 4 days of drug treatment. Although there was no significant effect of fentanyl on K(+)-evoked [3H]NE release, these cells were tolerant to the acute inhibitory effect of beta-endorphin. These results indicate that this is an appropriate system for examining the effects of acute and chronic opioid treatment on noradrenergic cells in vitro. In addition, this system may be useful as a CNS model for examining mechanisms that underlie tolerance and dependence following chronic opioid exposure.

Cells engineered to produce acetylcholine: therapeutic potential for Alzheimer's disease.

Alzheimer's disease (AD) is a debilitating disorder of the central nervous system which may affect up to 50% of the population over the age of 85 years. The etiology of AD is unknown and there is currently no cure for the disease. Well-documented losses in cholinergic and other neurotransmitter systems have provided a focal point for attempting pharmacological interventions in AD to ameliorate some of the cognitive deficits that occur. However, current systemic strategies have met with limited success. An alternative strategy, that has been pursued in animal models of neurodegenerative disease, is to augment neurotransmitter function within the brain through tissue transplantation. Such implants have an advantage over conventional drug therapies in that the cells can be precisely placed within compromised areas of the brain. We have pursued a strategy of designing cells, through the use of molecular biology techniques, to produce neurotrophic factors and neurotransmitters. Recently, we developed a primary fibroblast cell line that was genetically modified to express choline acetyltransferase (ChAT). In vitro, these cells produced and released acetylcholine at levels that varied with the amount of choline in the culture media. When implanted into the hippocampus of rats, the in vivo microdialysis technique revealed that the ChAT-expressing fibroblasts continued to produce and release acetylcholine after grafting. Most importantly, the levels of acetylcholine synthesized by the cells could be regulated by the localized infusion of choline in the vicinity of the grafts. These results confirmed previous work which indicated that engineered fibroblasts provide an effective delivery vehicle of different substances to the brain. While the intracerebral implantation of genetically modified cells will not cure AD, the continuing development of this strategy may ultimately provide a powerful approach for ameliorating the devastating cognitive impairments which are a hallmark of this disease.

Further pharmacological characterization of [3H]idazoxan binding sites in rat brain: evidence for predominant labeling of alpha 2-adrenergic receptors.

In addition to binding to alpha 2-adrenergic receptors, the antagonist [3H]idazoxan has been reported to bind to non-adrenergic sites in a number of tissues and species. In the present study, the pharmacological nature of [3H]idazoxan binding sites in rat brain slices has been examined using radioligand binding and autoradiographic techniques. In Na2KHPO4 buffer, four drugs with high affinity for alpha 2-adrenergic binding sites were potent inhibitors of [3H]idazoxan binding, with the rank order of potency being RX821002 greater than phentolamine greater than yohimbine greater than (-)epinephrine. Non-linear regression analysis resolved all competition curves into two components, with the high affinity site representing the majority of total [3H]idazoxan binding. In autoradiographic studies performed in Na2KHPO4 buffer, all alpha 2-selective ligands displaced greater than or equal to 75% of total [3H]idazoxan binding to most brain regions. These findings indicate that the major component of [3H]idazoxan binding was to sites that are alpha 2-adrenergic in nature. [3H]Idazoxan binding was also examined in glycylglycine buffer. In contrast to binding in Na2KHPO4 buffer, the proportion of low affinity sites was significantly increased in glycylglycine buffer. Autoradiographic studies confirmed these findings. These pharmacological data are consistent with our previously reported conclusions that, under appropriate assay conditions, [3H]idazoxan predominantly labels alpha 2-adrenergic binding sites in rat brain. These sites are widely distributed and have pharmacological characteristics consistent with those previously reported for alpha 2A-adrenergic receptors.

Epidermal growth factor and basic fibroblast growth factor: effects on an overlapping population of neocortical neurons in vitro.

Epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) have trophic effects on rat neocortical neurons in vitro. Concentration-response studies reveal that EGF maximally stimulates neuronal survival and process outgrowth at approximately 10 ng/ml, while the maximal effect of bFGF is seen at 10-30 ng/ml. Treatment with maximal concentrations of bFGF results in cultures containing a greater number of neurons with long processes, as well as greater branching of processes, than does treatment with EGF. When EGF and bFGF are added together to cultures the effects are not additive. In addition, bFGF is capable of supporting the survival of neurons previously treated with EGF. These findings indicate that EGF and bFGF affect a largely overlapping population of neocortical neurons, but that bFGF may be a more effective trophic agent for these cells.