David N. Hammond

Immortalization of embryonic mesencephalic dopaminergic neurons by somatic cell fusion

To facilitate the study of trophic interactions between mesencephalic dopaminergic neurons and their target cells, clonal hybrid cell lines have been developed from rostral mesencephalic tegmentum (RMT) of the 14-day-old embryonic mouse employing somatic cell fusion techniques. Among the hybrid cell lines obtained, one contains a high level of dopamine (DA), another predominantly 3,4-dihydroxyphenylalanine (DOPA), and a third no detectable catecholamines. The hybrid nature of the cell lines is supported by karyotype analysis and by the expression of adhesion molecules as assessed by aggregation in rotation-mediated cell culture. The DA cell line shows neuronal properties including catecholamine-specific histofluorescence, neurite formation with immunoreactivity to neurofilament proteins, and large voltage-sensitive sodium currents with the generation of action potentials. In contrast to the pheochromocytoma cell line (PC12), the dopamine content of the DA hybrid cell line is depleted by low concentrations of N-methyl-4-phenylpyridinium ion (MPP+), the active metabolite of the neurotoxin N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP).

Development and characterization of clonal cell lines derived from septal cholinergic neurons

Studies employing primary cells to determine the molecular basis of neuronal development and selective synaptogenesis in the central nervous system are limited by cellular heterogeneity. Clonal hybrid cell lines derived from a particular region of brain, which express differentiated characteristics typical of the cells of origin, offer a potentially powerful alternative approach. We previously demonstrated the feasibility of deriving such cell lines from septal cholinergic cells. We now delineate the methods employed, and describe the development of additional cholinergic cell lines expressing neuronal and cholinergic features from later developmental stages. One cell line has been studied in detail and found to form neurites, express choline acetyltransferase (ChAT) and neurofilament protein (NFP), and display typical neuronal ultrastructural characteristics, including puncta adherens, neuritic varicosities, vesicles, and growth cones.

Immortalized young adult neurons from the septal region: generation and characterization

Studies of the development of the central nervous system would be greatly facilitated by the ability to immortalize neuronal tissue from a broad range of ages. We have previously used somatic cell fusion techniques to generate neuronal cell lines from embryonic mice. To immortalize older neuronal cells, a cell isolation technique was developed to obtain viable septal cells from postnatal day 21 mice. The septal cells were fused to N18TG2 neuroblastoma cells and then cultured in selective medium to isolate septum x neuroblastoma cell lines. The hybrid nature of the lines was verified by chromosome analysis and electrophoretic analysis of glucosephosphate isomerase isozymes. The lines express phenotypes typical of differentiated septal neurons. Many lines morphologically resemble neurons and express the high molecular weight neurofilament protein. Several lines express high levels of choline acetyltransferase activity; others synthesize nerve growth factor. These results demonstrate that young adult neuronal tissue can be immortalized and that hybrid cells express properties of the neuronal parent.

Septal cell lines derived from the trisomy 16 mouse : generation, characterization, and response to NGF

The trisomy 16 mouse is a genetic model of Down syndrome. Clonal cell lines were developed from trisomic as well as euploid embryonic mouse septal cells by introduction of thermolabile large T antigen mutant of SV 40. The cell lines underwent morphological differentiation at the non-permissive temperature and in response to a differentiating agent. Immunocytochemical staining indicated that cells of neuronal lineage were immortalized. The addition of beta-nerve growth factor (100 ng/ml) increased the survival rate of a trisomy cell line in differentiated state, as measured by Trypan blue exclusion. These cell lines may prove useful in studies of neuronal abnormalities in this mouse model of Down syndrome.

Constitutive expression of the mature array of neurofilament proteins by a CNS neuronal cell line

Neurofilament protein expression was examined immunochemically in a neuronal cell line derived from postnatal day 21 septal tissue. The SN48.1p cell line was found to constitutively synthesize an array of neurofilament proteins typical of a mature neuron. All three neurofilament subunits (NF-L, NF-M, and NF-H) as well as differentially phosphorylated isoforms (P-, P+, P++, and P ) of NF-M and NF-H were identified by immunoblot analysis. Immunofluorescence studies revealed that the neurofilament proteins were components of discrete, filamentous structures. Abnormal intracellular aggregations of neurofilament proteins were never observed. Some SN48.1p cells apportioned specific isoforms into selected intracellular regions based on the molecular weight and phosphorylation level of the protein. NF-L was preferentially localized to perikarya and proximal neurites; NF-M[P++] and NF-H[P ] were distributed to distal aspects of neurites. The expression of these differentiated features of neurofilament proteins and, presumably, the synthesis of the kinases and phosphatases required for normal neurofilament metabolism occurred in the absence of growth factors, differentiating agents, and specialized culture substrates. In addition, the non-neuronal intermediate filaments glial fibrillary acidic protein and epithelial cytokeratin proteins were absent. These data demonstrate that SN48.1p cells exhibit a neurofilament phenotype characteristic of mature neurons and provide a unique model to examine the expression and function of neurofilaments in differentiated neuronal cells.

In vitro cell cultures as a model of the basal forebrain.

The basal forebrain has attracted considerable attention because of its putative role in complex functions such as learning, memory and behavioral state control as well as its vulnerability in neurological disorders such as Alzheimers Disease (AD). The finding that nerve growth factor provides trophic support for the cholinergic basal forebrain neurons has stimulated further interest in understanding trophic interactions of basal forebrain neurons as well as in possible trophic factor therapeutic strategies for disease states. Our laboratory has utilized primary cell cultures and developed immortalized central nervous system cell lines to study the trophic interactions that establish and maintain the septohippocampal pathway, a basal forebrain component which plays an essential role in cognitive function and is prominently affected in AD. The results of our primary cell culture studies have demonstrated the importance of trophic signals elaborated by the hippocampus in mediating the development of septal cholinergic neurons. Nerve growth factor plays an important role in this process, but it cannot account for all of the trophic signals elaborated by authentic hippocampal target cells. The development by this laboratory of clonal cell lines of septal and hippocampal lineage offers the prospect of investigating both the response to and elaboration of neural trophic signals at a more precise level of resolution than can be achieved with primary cultures. The technology and information that is generated from the engineering of such cell lines will also serve as a strategy to study trophic interactions in other brain circuits in future years, and to investigate possible changes or dysfunctions that occur neurological diseases.

Neurite outgrowth and the amyloid protein precursor.

Neurite outgrowth in Alzheimers disease may well be a multifactorial phenomenon. Recent evidence suggests possible roles for the protease inhibitor domain of the 751 amino acid amyloid precursor protein (APP), as well as for the 695 amino acid APP.

The Trophic Effect of the Sympathetic Nervous System on Cells of the Septal Region of the Basal Forebrain

The sympathetic nervous system exerts a trophic-mitogenic effect on C-1300 mouse neuroblastoma. We now report that the trophic factor present in freshly excised sympathetic ganglia from newborn rats enhances survival and process formation of the cells of the septal region of the rat basal forebrain.

A Hippocampal Cell Line Expresses a Membrane-Associated Cholinergic Trophic Activity Not Attributable to Nerve Growth Factor

Basal forebrain cholinergic cells are prominently affected in Alzheimer’s disease (AD) (Hefti and Weiner, 1986). Choline acetyltransferase (ChAT) is decreased in cholinergic target areas such as the hippocampus and neocortex, and there is a loss of ChAT-positive cells in the basal forebrain, including the septal region. One of the postulated pathogenetic alterations in AD is a deficiency in the normal trophic interactions through which target neocortical and hippocampal cells promote the survival and function of projecting cholinergic neurons (Appel, 1981). Accordingly, trophic factor therapy has been proposed as a potential future approach to the management of AD. Even if abnormalities in trophic interactions do not play a specific pathogenetic role in AD, trophic agents might be expected to help maintain cholinergic innervation of cortex and thus possibly influence the clinical course of AD.

The Use of Reaggregating Cell Cultures And Immortalized Central Nervous System Cells to Study Cholinergic Trophic Mechanisms

A salient feature of Alzheimer’s and other neurodegenerative diseases is the selective vulnerability of particular neural pathways. Since the development and maintenance of neural connections is supported by neural trophic factors, trophic dysfunction represents one possible pathogenetic mechanism for such neurological and age-associated diseases. This laboratory has utilized primary reaggregating cell cultures and developed immortalized central nervous system cell lines to study the trophic interactions that establish and maintain the septohippocampal pathway, which plays an essential role in cognitive function and is prominently affected in Alzheimer’s Disease. The results of the primary cell culture studies have demonstrated the importance of trophic signals elaborated by the hippocampus in mediating the development of septal cholinergic neurons. Nerve growth factor plays an important trophic role in this pathway, but it cannot account for all of the effects of authentic hippocampal target cells. The development of clonal cell lines of septal and hippocampal lineage offers the prospect of investigating both the response to and elaboration of neural trophic signals at a more precise level of resolution than can be achieved with primary cultures. In addition, one of the hippocampal-derived cell lines, HN10, expresses what appears to be a novel cholinergic trophic activity. These cell lines represent a potential source for isolation of such factors, and also a potential “delivery system” via neural grafting techniques. The technology and information that are generated from these investigations will serve as a strategy to study trophic interactions in other brain circuits in future years, and to investigate possible changes or dysfunctions that occur both in the aging brain and in age-associated brain diseases.