MANGANESE IS TOXIC TO RAT STRIATAL NEURONS IN PRIMARY CULTURE.

Elise A. Malecki and James R. Connor, Dept. Neuroscience & Anatomy, Penn State College of Medicine, P.O. Box 850, Hershey, PA 17033. eam10@psu.edu

ABSTRACT

In humans, manganese (Mn) exposure is associated with a neurodegenerative syndrome which presents like atypical Parkinsonism. We chose to develop a primary culture system to investigate the cellular mechanisms of Mn neurotoxicity. Striatal neurons were isolated from embryonic day 18 Wistar rat fetuses. At day 10 in culture, neurons were treated with 0, 50, or 500 µM added MnCl₂. After 48 hours of exposure to added Mn, neurons died as measured by decreased MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) uptake and oxidation. Neurons exposed to 50 and 500 µM Mn had only 75.2±17.8% and 62.2±4.6% of control MTT uptake and oxidation. In contrast, up to 2 mM Mn does not lead to a decrease in MTT uptake in primary microglial cultures. We believe that this primary culture system will be valuable in studying not only the cellular mechanism of Mn neurotoxicity, but glial-neuronal interactions as well.

INTRODUCTION

Excess Mn is associated with death of neurons in the globus pallidus, leading to a Parkinsonian-like syndrome, and can occur in persons exposed to airborne particles containing Mn (miners, ferroalloy or battery manufacture workers, automotive repair workers), or patients with chronic liver disease (Devenyi et al. 1994). Mn supplementation of total parenteral nutrition (TPN) solutions has been associated with fatal Mn neurotoxicity (Fel et al. 1996). Occupational exposure to Mn has been linked epidemiologically to Parkinson’s disease (Gorell et al. 1997).

In the few neuropathological studies of Mn toxicity that have been performed, brains of affected patients show a marked loss of neurons in the striatum, and reactive gliosis. Whether the reactive gliosis is a cause or a consequence of the neuronal loss has yet to be determined.

The differential uptake of Mn and Mn-associated cellular events between cell types will give insight into the mechanism of Mn neurotoxicity. Manganese is actively accumulated by astrocytes (Aschner et al. 1992), where it is required for glutamine synthetase activity (Carl et al. 1991). If astrocytes accumulate Mn, this my be a neuroprotective effect; alternatively, selective glial Mn uptake and reactivity may play a role in the pathology of Mn toxicity. Primary rat striatal neurons have been found more susceptible to Mn toxicity than mesencephalic neurons, as measured by loss of neurotransmitter uptake (Defazio et al. 1996). However, the differential susceptibility of neuronal vs. glial cell types to Mn toxicity has not been examined.

We hypothesized that Mn would be more directly toxic to neurons than glial cells. We established primary neural and glial cell cultures from rat and measured viability following Mn exposure with the MTT uptake assay.

MATERIALS AND METHODS

Materials. Calcium- and magnesium-free Hank’s buffered salt solution (CMF-HBSS), Dulbecco’s minimal essential medium (DMEM), and cytosine arabinoside were obtained from Sigma (St. Louis, MO). Trypsin/EDTA, antibiotic/antimycotic, neurobasal medium (NB2), and B-27 supplement were obtained from Gibco BRL (Grand Island, NY). Fetal bovine serum (FBS) was purchased from Biocell (Rancho Dominguez, CA).

Animals and cell culture. Timed pregnant Wistar rats were puchased from Charles River. Animal facilities were accredited by Association for Assessment and Accreditation of Laboratory Animal Care International, and protocols were approved by an Institutional Animal Care and Use Committee.

Neuronal cultures were prepared by the method of Greene et al. (1998). Timed pregnant female rats were killed by CO2 asphyxiation at E17 (day sperm seen = E0). Uteri were dissected free and rinsed with 70% EtOH. Embryonic striata were then dissected into CMF-HBSS under sterile conditions, minced, then incubated 10 min at 37°C with 1 x trypsin/EDTA. After three washes with CMF-HBSS, the tissue was suspended in 1 mL CMF-HBSS containing 10% FBS and 40 µg/mL DNase I. The tissue was gently triturated 15 times through a glass Pasteur pipette, then 15 times through a 22 ga needle. The suspension was then layered on 5 mL FBS and centrifuged at 500 g for 10 min. The resulting pellet was suspended in 5 mL of DMEM/10% FBS containing mM glucose, 2 mM glutamine, and 1 x antibiotic/antimycotic. Cells were counted using trypan blue exclusion, then diluted to 2.46 x 105 viable cells/mL.

For neuronal cultures, 2 mL of the striatal cell suspension was plated per 12mm round coverslip (5 x 104 cells/cm2) coated with poly-L-ornithine/laminin (BD). Twenty-four hours after plating, medium was replaced with NB2 medium containing 1 x B-27 supplement, 1 x antibiotic/antimycotic, and 2 µM cytosine arabinoside (Sigma, St. Louis, MO). Neuronal cultures were fed this medium (without cytosine arabinoside) on days 5 and 10 in culture. Experiments were conducted between 11 and 14 days in culture.

Astrocyte cultures were prepared from striata of two- to four-day old pups by the method of McCarthy and deVellis (1980). Striata were trypsinized and triturated as above. Cells were suspended in DMEM/10%FBS, and seeded at 1.5 x 107 cells per 75 cm2 flask previously coated with poly-L-lysine. After five days, the flasks were shaken to dislodge first microglia then O2A cells. The remaining astrocytes were harvested from the flasks with trypsin/EDTA, then washed three times with CMF-HBSS, suspended in DMEM/10%FBS, and plated at mL/well of 6-well plates previously coated with poly-L-lysine (4 x 104 cells/cm2). Astrocyte cultures were fed every two days with DMEM/10%FBS.

Microglia dislodged from the 1 hour shaking of the mixed glial cultures were plated on poly-L-lysine-coated 96-well plates. The media used was Ham’s F-12 supplemented with 10% FBS. O2A precursor cells were plated on 24-well plates. These cells were fed with B-104 rat neuroblastoma-conditioned N1S media.

MTT assay. The uptake and conversion of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) to crystals of purple formazan is related to mitochondrial dehydrogenase activity. Two hours before the desired endpoint, MTT (5 mg/mL in media) was added at 1/10 culture volume, and cells returned to the incubator. After 2 hours, cells were washed once with CMF-HBSS then dissolved with 0.04N HCl in isopropanol. Absorbance at 570 nm was determined with a Beckman DU-60 spectrophotometer or microplate reader (Cambridge Technologies).

RESULTS AND DISCUSSION

The MTT uptake/oxidation assay is commonly used as a crude measure of cell viability, as it is directly proportional to the total activity of mitochondrial complex I activity in a culture. Mn concentrations up to 2 mM exhibited no significant effect on cell viability (as judged by MTT assay) in microglia, O2A oligodendrocyte precursor cells, or the HAPI rat microglial cell line (Figure 1). Astrocyte viability was slightly but significantly adversely affected by increasing Mn concentrations. Viability of astrocytes exposed to 100-500 µM Mn was approximately 80% of non-exposed cultures (Figure 1). Neuronal cultures, by contrast, showed only 75.2±17.8% of control viability after exposure to 50 µM Mn and 62.2±4.6% of control viability after exposure to 500 µM Mn (Figure 1).

Figure 1. Toxicity of Mn to various neural cell types in culture. Cells were cultured as described in the Methods section. Cells were exposed to varying concentrations of MnCl₂ for 24 h (microglia) or 48 h (all other cell types). Values are means ± SEM of two experiments in triplicate (astrocytes) or one experiment in triplicate or quadruplicate (all other cell types).

Research in Parkinson’s disease and Mn neurotoxicity, and indeed most neurodegenerative diseases, has suffered from lack of suitable models for human neurons. This may change with wider availability of human neural stem cells. The rat pheochromacytoma cell line PC12 has been used as a model for Parkinson’s disease and for manganese neurotoxicity because these cells produce dopamine. However, PC12 cells exposed to 200 µM Mn for 24 or 48 hours showed no decreased viability by the MTT assay (Migheli et al. 1999). Our work shows that 50 µM Mn is lethal to rat neurons in primary culture, and therefore, primary neuron culture is a more physiologically relevant model system for the study of Mn neurotoxicity than PC12 cells.

We observed that glial cell types were less affected by exposure to Mn than neurons. Our results in rat primary microglia are consistent with observations of Chang and Liu (1999), who saw no decrease in MTT uptake by the murine microglial cell line N9 at Mn concentrations up to 500 µM. We expected little effect of Mn exposure on astrocyte viability, because work by Aschner et al. (1992) showed no decrease in protein per well in primary rat astrocytes exposed to 50 µM Mn. However, we saw significant astrocyte Mn toxicity at concentrations of 100 µM and above.Even if Mn is not as directly toxic to glial cells as neurons, glial cells may still play a role in Mn neurotoxicity. Neuroprotective and neurotoxic effects of these various glial cell types in response to Mn exposure need to be elucidated.

In conclusion, the differential susceptibility of rat primary neurons vs. glial cells to Mn exposure confirms the utility of this system as a model with which to study mechanisms of Mn toxicity. We will use this system to investigate cellular responses to Mn exposure, and glial/neuronal interactions.

ACKNOWLEDGEMENT

This work was supported by NIH NS10235 (EAM).

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