Lisa Won

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).

Enhanced striatal cholinergic neuronal activity mediates l-DOPA–induced dyskinesia in parkinsonian mice

Treatment of Parkinson disease (PD) with l-3,4-dihydroxyphenylalanine (l-DOPA) dramatically relieves associated motor deficits, but l-DOPA–induced dyskinesias (LID) limit the therapeutic benefit over time. Previous investigations have noted changes in striatal medium spiny neurons, including abnormal activation of extracellular signal-regulated kinase1/2 (ERK). Using two PD models, the traditional 6-hydroxydopamine toxic lesion and a genetic model with nigrostriatal dopaminergic deficits, we found that acute dopamine challenge induces ERK activation in medium spiny neurons in denervated striatum. After repeated l-DOPA treatment, however, ERK activation diminishes in medium spiny neurons and increases in striatal cholinergic interneurons. ERK activation leads to enhanced basal firing rate and stronger excitatory responses to dopamine in striatal cholinergic neurons. Pharmacological blockers of ERK activation inhibit l-DOPA–induced changes in ERK phosphorylation, neuronal excitability, and the behavioral manifestation of LID. In addition, a muscarinic receptor antagonist reduces LID. These data indicate that increased dopamine sensitivity of striatal cholinergic neurons contributes to the expression of LID, which suggests novel therapeutic targets for LID.

Analysis of huntingtin-associated protein 1 in mouse brain and immortalized striatal neurons

Huntingtin, the protein product of the Huntingtons disease (HD) gene, is expressed with an expanded polyglutamine domain in the brain and in nonneuronal tissues in patients with HD. Huntingtin‐associated protein 1 (HAP‐1), a brain‐enriched protein, interacts preferentially with mutant huntingtin and thus may be important in HD pathogenesis. The function of HAP‐1 is unknown, but recent evidence supports a role in microtubule‐dependent organelle transport. We examined the subcellular localization of HAP‐1 with an antibody made against the NH2‐terminus of the protein. In immunoblot assays of mouse brain and immortalized striatal neurons, HAP‐1 subtypes A and B migrated together at about 68 kD and separately at 95 kD and 110 kD, respectively. In dividing clonal striatal cells, HAP‐1 localized to the mitotic spindle apparatus, especially at spindle poles and on vesicles and microtubules of the spindle body. Postmitotic striatal neurons had punctate HAP‐1 labeling throughout the cytoplasm. Western blot analysis of protein extracts obtained after subcellular fractionation and differential centrifugation of the clonal striatal cells showed that HAP‐1B was preferentially enriched in membrane fractions. Electron microscopic study of adult mouse basal forebrain and striatum showed HAP‐1 localized to membrane‐bound organelles including large endosomes, tubulovesicular structures, and budding vesicles in neurons. HAP‐1 was also strongly associated with an unusual large “dense” organelle. Microtubules were labeled in dendrites and axonal fibers. Results support a role for HAP‐1 in vesicle trafficking and organelle movement in mitotic cells and differentiated neurons and implicate HAP‐1B as the predominant molecular subtype associated with vesicle membranes in striatal neurons. J. Comp. Neurol. 403:421–430, 1999.

Methamphetamine concentrations in fetal and maternal brain following prenatal exposure

Levels of methamphetamine in maternal striatum and whole fetal mouse brain were assessed at 0.5, 1, 2, and 4 h postinjection on gestational day 14 (GD14) following a single, subcutaneous injection of 40 mg/kg (+)-methamphetamine hydrochloride to pregnant mice. In the dams, striatal concentrations of methamphetamine peaked at 1 h postinjection, reaching levels of approximately 510 ng/mg protein. Amphetamine, the primary metabolite of methamphetamine, increased to 77 ng/mg protein at 2 h and remained elevated by 4 h postinjection. In the fetal brain, peak methamphetamine concentrations of approximately 122 ng/mg protein were attained at 1 h. Amphetamine was only detectable in fetal brain at 2 and 4 h postinjection. Regional analysis of methamphetamine levels in fetal striatum, cortex, and brainstem revealed that the drug was not uniformly distributed. Maternal administration of methamphetamine results in fetal brain drug concentrations, which approximate those reported in human infants whose mother abused methamphetamine. This dosage regimen, therefore, serves as an appropriate animal model for assessing the potential risks to human offspring exposed to methamphetamine in utero.

Forskolin and dopamine D1 receptor activation increase Huntingtin's association with endosomes in immortalized neuronal cells of striatal origin

Huntingtin is a cytoplasmic protein of unknown function that associates with vesicle membranes and microtubules. Its protein interactions suggest that huntingtin has a role in endocytosis and organelle transport. In this study we sought to identify factors that regulate the transport of huntingtin in striatal neurons, which are the cells most affected in Huntingtons disease. In clonal striatal cells derived from fusions of neuroblastoma and embryonic striatal neurons, huntingtin localization is diffuse and slightly punctate in the cytoplasm. When these neurons were differentiated by treatment with forskolin, huntingtin redistributed to perinuclear regions, discrete puncta along plasma membranes, and branch points and terminal growth cones in neurites. Huntingtin staining overlapped with clathrin, a coat protein involved in endocytosis. Immunoblot analysis of subcellular membrane fractions separated by differential centrifugation confirmed that huntingtin immunoreactivity in differentiated neurons markedly increased in membrane fractions enriched with clathrin and with huntingtin-interacting protein 1. Dopamine treatment altered the subcellular localization of huntingtin and increased its expression in clathrin-enriched membrane fractions. The dopamine-induced changes were blocked by the D1 antagonist SCH 23390 and were absent in a clonal cell line lacking D1 receptors. Results suggest that the transport of huntingtin and its co-expression in clathrin and huntingtin-interacting protein 1-enriched membranes is influenced by activation of adenylyl cyclase and stimulation of dopamine D1 receptors.

Striatal Cholinergic Cell Ablation Attenuates l-DOPA Induced Dyskinesia in Parkinsonian Mice

3,4-Dihydroxyphenyl-l-alanine (l-DOPA)-induced dyskinesia (LID) is a debilitating side effect of long-term dopamine replacement therapy in Parkinsons Disease. At present, there are few therapeutic options for treatment of LID and mechanisms contributing to the development and maintenance of these drug-induced motor complications are not well understood. We have previously shown that pharmacological reduction of cholinergic tone attenuates the expression of LID in parkinsonian mice with established dyskinesia after chronic l-DOPA treatment. The present study was undertaken to provide anatomically specific evidence for the role of striatal cholinergic interneurons by ablating them before initiation of l-DOPA treatment and determining whether it decreases LID. We used a novel approach to ablate striatal cholinergic interneurons (ChIs) via Cre-dependent viral expression of the diphtheria toxin A subunit (DT-A) in hemiparkinsonian transgenic mice expressing Cre recombinase under control of the choline acetyltransferase promoter. We show that Cre recombinase-mediated DT-A ablation selectively eliminated ChIs when injected into striatum. The depletion of ChIs markedly attenuated LID without compromising the therapeutic efficacy of l-DOPA. These results provide evidence that ChIs play a key and selective role in LID and that strategies to reduce striatal cholinergic tone may represent a promising approach to decreasing l-DOPA-induced motor complications in Parkinsons disease.

Glial-derived neurotrophic factor (GDNF) induced morphological differentiation of an immortalized monoclonal hybrid dopaminergic cell line of mesencephalic neuronal origin.

Monoclonal hybrid cell lines derived from either mesencephalic or striatal neuronal parents were screened for the effects of glial-derived neurotrophic factor (GDNF) on morphological differentiation. The mesencephalic dopaminergic MN9D cell line responded to GDNF with a 3- to 4-fold increase in the number of differentiated solitary cells and provides an unlimited source of monoclonal cells for studies on the mechanism of action of GDNF and the nature of its receptor.

Dopaminergic neurons grown in three-dimensional reaggregate culture for periods of up to one year

The use of three-dimensional (3-D) reaggregate cultures of mesencephalic fetal dopaminergic neurons in co-culture with their target cells of the corpus striatum (CS) has permitted examination of the development and survival of such neurons for periods of up to 1 year. Dopaminergic neurons grown in dialyzed serum in reaggregate cultures show an increase in neurotransmitter level over the first 2 months in culture, and these levels of dopamine are maintained or increased over the next 4-12 months. While there is an apparent decrease in the numbers of dopaminergic neurons by 1 year in culture, many dopaminergic neurons survive and can be visualized at the light and electron microscopic level by tyrosine hydroxylase immunocytochemistry and their processes assessed by histofluorescent techniques. The survival of dopaminergic neurons in an organized culture system in which they demonstrate a normal developmental pattern and establish synaptic contact with appropriate target cells provides an approach to the experimental examination of a substantial portion of the life history of these neurons.

Enhanced histamine H2 excitation of striatal cholinergic interneurons in l-DOPA-induced dyskinesia

Levodopa is the most effective therapy for the motor deficits of Parkinsons disease (PD), but long term treatment leads to the development of L-DOPA-induced dyskinesia (LID). Our previous studies indicate enhanced excitability of striatal cholinergic interneurons (ChIs) in mice expressing LID and reduction of LID when ChIs are selectively ablated. Recent gene expression analysis indicates that stimulatory H2 histamine receptors are preferentially expressed on ChIs at high levels in the striatum, and we tested whether a change in H2 receptor function might contribute to the elevated excitability in LID. Using two different mouse models of PD (6-hydroxydopamine lesion and Pitx3(ak/ak) mutation), we chronically treated the animals with either vehicle or l-DOPA to induce dyskinesia. Electrophysiological recordings indicate that histamine H2 receptor-mediated excitation of striatal ChIs is enhanced in mice expressing LID. Additionally, H2 receptor blockade by systemic administration of famotidine decreases behavioral LID expression in dyskinetic animals. These findings suggest that ChIs undergo a pathological change in LID with respect to histaminergic neurotransmission. The hypercholinergic striatum associated with LID may be dampened by inhibition of H2 histaminergic neurotransmission. This study also provides a proof of principle of utilizing selective gene expression data for cell-type-specific modulation of neuronal activity.

Fetal exposure to (+/-)-methylenedioxymethamphetamine in utero enhances the development and metabolism of serotonergic neurons in three-dimensional reaggregate tissue culture.

Methylenedioxymethamphetamine (MDMA, Ecstasy) is a potent psychomotor stimulant with neurotoxic potential which is widely abused by females of childbearing age raising serious public health concerns in terms of exposure of the fetus to the drug. The current study was conducted using the three-dimensional reaggregate tissue culture system as an approach to the assessment of risk to fetal brain cells following exposure to MDMA during early to mid-gestation. In this culture system, the serotonergic and dopaminergic mesencephalic-striatal projections are reconstructed and develop with a time course similar to that observed in vivo. Pregnant C57Bl/6J mice were injected twice daily with 40 mg/kg MDMA or saline from gestational day 6 to 13. On gestational day 14, mesencephalic and striatal cells from MDMA- and saline-exposed embryos were used to prepare reaggregate cultures. Levels of neurotransmitters and their metabolites in the reaggregates and culture medium were assessed at 22 and 36 days of culture. There was a long-term enhancement of serotonergic development and metabolism by fetal exposure to MDMA as evidenced by increased reaggregate serotonin levels as well as the elevated production and release of 5-hydroxyindoleacetic acid in cultures prepared from MDMA-exposed embryos which persisted for up to 36 days of culture. Dopaminergic neurons in such cultures also exhibited increased metabolism as indicated by elevated levels of dihydroxyphenylacetic acid in reaggregate tissue and culture medium. The data obtained suggest that exposure to MDMA in utero during early to mid-gestation may result in more active serotonergic and dopaminergic neurons.