Calcium Phosphate Transfection of Primary Hippocampal Neurons

Calcium phosphate precipitation is a convenient and economical method for transfection of cultured cells. With optimization, it is possible to use this ...

Rutgers University

Complex I impairment with rotenone produces damage though a mechanism thought to be distinct from effects on mitochondrial respiration. The outcome of chronic rotenone on energy status in relation to toxicity, however, is unknown. To examine this, mesencephalic cultures were exposed to chronic, low-dose rotenone (5-100 nM, 8 days in vitro) or acute, high-dose rotenone (500 nM, 1-24 h), and ATP/ADP levels and toxicity were measured. Chronic exposure to 5-50 nM rotenone produced selective dopamine cell loss. High-dose rotenone produced nonselective damage at all exposure times. Chronic, low-dose rotenone (37.5 nM) decreased ATP/ADP gradually over several days to 40% of controls, whereas high-dose rotenone (500 nM, 1-6 h), collapsed ATP/ADP by 1 h of exposure. The ubiquitin proteasomal pathway, an ATP-dependent pathway, is implicated in Parkinson's disease and, thus, various rotenone exposures were examined for effects on ubiquitin proteasomal function. Chronic, low-dose rotenone (25-50 nM, 8 days), but not acute, high-dose rotenone (500 nM, 1-6 h), caused accumulation of ubiquitinated proteins, E1-ubiquitin activation, and increased proteasomal activities prior to toxicity even though both exposures increased free radical production. Findings show that selective dopamine cell loss and alterations in ubiquitin proteasomal function only occur with rotenone exposures that partially maintain ATP/ADP. High concentrations of rotenone that collapse energy status kill neurons in a nonselective manner independent of the ubiquitin proteasomal pathway.

Energy status, ubiquitin proteasomal function, and oxidative stress during chronic and acute complex I inhibition with rotenone in mesencephalic cultures.

Recent studies suggest that exposure to agrochemicals may contribute to the development of idiopathic Parkinson's disease. Maneb (MB), a widely used Mn-containing ethylene-bis-dithiocarbamate (EBDC) fungicide, has been implicated in selective dopaminergic neurotoxicity. In this study, we examine the potential neurotoxicity of mancozeb (MZ), a widely used EBDC fungicide that is structurally similar to MB, but contains both Zn and Mn. Primary mesencephalic cells isolated from Sprague-Dawley embryonic day 15 rat embryos were exposed in vitro to either MZ or MB to compare their cytotoxic potential. Exposure to 10-120 microM MZ or MB for 24h resulted in a dose-dependent toxicity in both the dopamine (DA) and GABA mesencephalic populations as assessed by a functional assay for high affinity transporter activity. Consistent with this, cell viability as well as tyrosine hydroxylase-positive neurons decreased with increasing doses of MZ or MB. Toxic potencies for MZ and MB were similar and no difference in sensitivity between the DA and GABA populations was observed with the fungicides. Exposure to ethylene thiourea, the major metabolite of either MZ or MB, was not toxic, implicating the parent compound in toxicity. Both the organic and Mn metal components of the fungicides were found to contribute to toxicity. Non-toxic exposures to the fungicides decreased ATP levels in a dose-dependent manner suggesting impairment of energy metabolism. In whole mitochondrial preparations isolated from adult rat brains, MZ and MB inhibited NADH-linked state 3 respiration. Mild to moderate mitochondrial uncoupling was also observed in response to the fungicides. In conclusion, our findings indicate that acute exposure to high doses of MZ and MB produce equipotent toxic effects in both DA and GABA neurons that may be associated with perturbations in mitochondrial respiration.

Acute neurotoxic effects of mancozeb and maneb in mesencephalic neuronal cultures are associated with mitochondrial dysfunction.

Previous in vitro studies in our laboratory have shown that mancozeb (MZ) and maneb (MB), both widely used EBDC fungicides, are equipotent neurotoxicants that produce cell loss in mesencephalic dopaminergic and GABAergic cells after an acute 24h exposure. Mitochondrial uncoupling and inhibition were associated with fungicide exposure. Inhibition of mitochondrial respiration is known to increase free radical production. Here the mechanism(s) of neuronal damage associated with MZ exposure was further explored by determining the role that reactive oxygen species (ROS) played in toxicity. Damage to mesencephalic dopamine and GABA cell populations were significantly attenuated when carried out in the presence of ascorbate or SOD, indicative of a free radical-mediated contribution to toxicity. ROS generation monitored by hydrogen peroxide (H(2)O(2)) production using Amplex Red increased in a dose-dependent manner in response to MZ. Inhibition of intracellular catalase with aminotriazole had little effect on H(2)O(2) generation, whereas exogenously added catalase significantly reduced H(2)O(2) production, demonstrating a large extracellular contribution to ROS generation. Conversely, cells preloaded with the ROS indicator dye DCF showed significant MZ-induced ROS production, demonstrating an increase in intracellular ROS. Both the organic backbone of MZ as well as its associated Mn ion, but not Zn ion, were responsible and required for H(2)O(2) generation. The functionally diverse NADPH oxidase inhibitors, diphenylene iodonium chloride, apocynin, and 4-(2-aminoethyl)benzene-sulfonyl fluoride hydrochloride significantly attenuated H(2)O(2) production by MZ. In growth medium lacking cells, MZ produced little H(2)O(2), but enhanced H(2)O(2) generation when added with xanthine plus xanthine oxidase whereas, in cultured cells, allopurinol partially attenuated H(2)O(2) production by MZ. Minocycline, an inhibitor of microglial activation, modestly reduced H(2)O(2) formation in mesencephalic cells. In contrast, neuronal-enriched cultures or cultures treated with MAC-1-SAP to kill microglia, did not show an attenuation of ROS production. These findings demonstrate that Mn-containing EBDC fungicides such as MZ and MB can produce robust ROS generation that likely occurs via redox cycling with extracellular and intracellular oxidases. The findings further show that microglia may contribute to but are not required for ROS production by MZ.

Reactive oxygen species generation by the ethylene-bis-dithiocarbamate (EBDC) fungicide mancozeb and its contribution to neuronal toxicity in mesencephalic cells.

At least 2 decades have past since the demonstration of a 40-50% deficit in total glutathione (GSH) levels in the substantia nigra in patients with Parkinson's disease (PD). The similar loss of GSH in the nigra in Incidental Lewy body disease, thought to be an early form of PD, indicates that this is one of the earliest derangements to occur in the pre-symptomatic stages of PD. Oxidative damage to lipids, protein and DNA in the nigra of PD patients is consistent with the loss of the antioxidant functions contributed by GSH. Past clinical trials that have used an antioxidant approach to treatment have used antioxidants that might substitute for GSH but these have shown modest to little benefit. More recent studies of the functions served by GSH in cells include in addition to its well-known participation in H(2)O(2) and toxin removal, such roles as modulation of protein function via thiolation which may control physiological and pathophysiological pathways to include DNA synthesis and repair, protein synthesis, amino acid transport, modulation of glutamate receptors and neurohormonal signaling. These multifunctional aspects to the workings of GSH in the cell would suggest that its loss perturbs many different processes and that replenishment and maintenance of GSH per se may be the best approach for preventing progressive damage from occurring. Despite this, few studies have been directed at specifically restoring GSH, although, as discussed herein, its unsanctioned use in PD is growing in popularity. This review will focus on glutathione in PD; the various functions carried out by glutathione and possible consequences of its depletion, as well as measures to elevate GSH in the CNS and its use in humans. Consideration of how the CNS generates and handles the substrates for GSH synthesis is also addressed with the view in mind that this may provide insights into control and maintenance of intracellular glutathione.

Glutathione and Parkinson's disease: is this the elephant in the room?

A liposomal preparation of glutathione (GSH) was investigated for its ability to replenish intracellular GSH and provide neuroprotection in an in vitro model of Parkinson's disease using paraquat plus maneb (PQMB) in rat mesencephalic cultures. In mixed neuronal/glial cultures depleted of intracellular GSH, repletion to control levels occurred over 4 h with liposomal-GSH or non-liposomal-GSH however, liposomal-GSH was 100-fold more potent; EC(50s) 4.75 μM and 533 μM for liposomal and non-liposomal-GSH, respectively. Liposomal-GSH utilization was also observed in neuronal cultures, but with a higher EC(50) (76.5 μM), suggesting that glia facilitate utilization. Blocking γ-glutamylcysteine synthetase with buthionine sulfoxamine prevented replenishment with liposomal-GSH demonstrating the requirement for catabolism and resynthesis. Repletion was significantly attenuated with endosomal inhibition implicating the endosomal system in utilization. Liposomal-GSH provided dose-dependent protection against PQMB with an EC(50) similar to that found for repletion. PQMB depleted intracellular GSH by 50%. Liposomal-GSH spared endogenous GSH during PQMB exposure, but did not require GSH biosynthesis for protection. No toxicity was observed with the liposomal preparation at 200-fold the EC(50) for repletion. These findings indicate that glutathione supplied in a liposomal formulation holds promise as a potential therapeutic for neuronal maintenance.

Liposomal-glutathione provides maintenance of intracellular glutathione and neuroprotection in mesencephalic neuronal cells.

Dendritic spines are actin-rich structures, the formation and plasticity of which are regulated by the Rho GTPases in response to synaptic input. Although several guanine nucleotide exchange factors (GEFs) have been implicated in spine development and plasticity in hippocampal neurons, it is not known how many different Rho GEFs contribute to spine morphogenesis or how they coordinate the initiation, establishment, and maintenance of spines. In this study, we screened 70 rat Rho GEFs in cultured hippocampal neurons by RNA interference and identified a number of candidates that affected spine morphogenesis. Of these, Dock180, which plays a pivotal role in a variety of cellular processes including cell migration and phagocytosis, was further investigated. We show that depletion of Dock180 inhibits spine morphogenesis, whereas overexpression of Dock180 promotes spine morphogenesis. ELMO1, a protein necessary for in vivo functions of Dock180, functions in a complex with Dock180 in spine morphogenesis through activating the Rac GTPase. Moreover, RhoG, which functions upstream of the ELMO1/Dock180 complex, is also important for spine formation. Together, our findings uncover a role for the RhoG/ELMO1/Dock180 signaling module in spine morphogenesis in hippocampal neurons.

The RhoG/ELMO1/Dock180 signaling module is required for spine morphogenesis in hippocampal neurons.

The polarity protein PAR-1 plays an essential role in many cellular contexts, including embryogenesis, asymmetric cell division, directional migration, and epithelial morphogenesis. Despite its known importance in different cellular processes, the role of PAR-1 in neuronal morphogenesis is less well understood. In particular, its role in the morphogenesis of dendritic spines, which are sites of excitatory synaptic inputs, has been unclear. Here, we show that PAR-1 is required for normal spine morphogenesis in hippocampal neurons. We further show that PAR-1 functions through phosphorylating the synaptic scaffolding protein PSD-95 in this process. Phosphorylation at a conserved serine residue in the KXGS motif in PSD-95 regulates spine morphogenesis, and a phosphomimetic mutant of this site can rescue the defects of kinase-dead PAR-1. Together, our findings uncover a role of PAR-1 in spine morphogenesis in hippocampal neurons through phosphorylating PSD-95.

The polarity protein partitioning-defective 1 (PAR-1) regulates dendritic spine morphogenesis through phosphorylating postsynaptic density protein 95 (PSD-95).

Laura P. Bernard

Department of Neuroscience and Cell Biology,
Robert Wood Johnson Medical School,
Department of Neuroscience and Cell Biology, Robert Wood Johnson Medical School

Rutgers University

Calcium Phosphate Transfection of Primary Hippocampal Neurons

Laura P. Bernard

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Rutgers University

Calcium Phosphate Transfection of Primary Hippocampal Neurons

Department of Neuroscience and Cell Biology,
Robert Wood Johnson Medical School,
Department of Neuroscience and Cell Biology, Robert Wood Johnson Medical School