Memantine Enhanced Survival of DA Neurons in Rat Primary Midbrain Cultures
To assess the viability of DA neurons in rat primary midbrain neuron-glia cultures, [3H] DA uptake assay was used as a functional index, and immuno-cytochemical staining for TH-positive (a marker for DA neurons) neurons was used for both morphometric analysis and cell count. Various concentrations of memantine (0.1–30 µM) or vehicle were added to cultures seven days after seeding. One week later, [3H] DA uptake assay was performed. As shown in , memantine in the range of 3–10 µM enhanced the capacity of DA uptake in a dose-dependent manner. A higher concentration of memantine (30 µM) showed neurotoxicity (data not shown). Thus, a range of memantine concentration (0.1–10 µM) was used for the rest of the study. The increased DA uptake after memantine treatment was confirmed by the results from ICC studies (). Cell count analysis revealed that memantine increased the number of TH-positive neurons (DA neurons) in a dose-related manner. Furthermore, morphometric analysis indicated that memantine treatment promoted arborization of TH neurons, in both length and numbers of neurites per neuron, compared with vehicle controls.
Memantine enhanced the survival of DA neurons and its functional DA uptake capacity
Neurotrophic Effects of Memantine Were Astroglia-Dependent
In an effort to understand the cellular mechanism underlying the enhanced number and activity of DA neurons after memantine treatment, a series of experiments using different types of cell cultures were conducted. First, neuron-enriched cultures (>98% purity) were used to investigate whether memantine has a direct effect on DA neurons. As shown in , while memantine (10 µM) enhanced [3H] DA uptake capacity in neuron-glia cultures, it failed to increase DA uptake capacity in neuron-enriched cultures, indicating that the observed neurotrophic effect of memantine was not due to a direct effect on DA neurons.
The neurotrophic effect of memanatine was astroglia-dependent
To determine the possibility that glial cells (microglia or astroglia) mediated the memantineinduced neurotrophic effect, we used neuron-microglia co-cultures and microglia-depleted cultures (>95% purity). Neuron-enriched cultures supplemented with 7.5 × 104/well of microglia (neuron-microglia co-cultures) failed to increase DA uptake after memantine treatment (). On the other hand, microglia-depleted cultures that contain only neurons and astroglia, exhibited a [3H] DA uptake capacity with mematine that was comparable to that of neuron-glia cultures (). These three experiments suggest that astroglia, but not microglia, are potential targets for memantine-induced neurotrophic effects on DA neurons.
Conditioned Media from Memantine-Treated Astroglia Promoted Survival of DA Neurons in Neuron-Enriched Cultures
To confirm the role of astroglia in the memantine-induced survival-promoting effect and further investigate the underlying mechanism, conditioned media from astroglia-enriched cultures in the absence (ACM) or presence of 10 µM memantine (ACM-MMT) were prepared. Astroglia were incubated with or without memantine for 48 h, and conditioned media was collected and dialyzed to remove memantine. This conditioned media was then added to neuron-enriched cultures and incubated for 7 days before assays. Astroglia conditioned media significantly increased [3H] DA uptake compared with both non-conditioned media and memantine-treated cultures (). Menantine-treated astroglia conditioned media displayed a significant increase in DA uptake capacity, compared with astroglia conditioned media. Immunostaining analysis with anti-TH antibody showed higher levels of TH-positive neurons with more neurite configurations in enriched neuron cultures treated with menantine-treated astroglia conditioned media than those in control cultures ().
Memantine-Increased the Release of GDNF from Astroglia
GDNF, and other growth factors have been shown to promote DA neurons survival, induce neurite outgrowth and sprouting, upregulate TH expression, and enhance synaptic efficacy (Baquet et al, 2005
; Murer et al, 2001
). GDNF is one of major neurotrophic factors in astroglia (Lin et al, 1993
). Here, experiments were designed to test the possible involvement of GDNF in the neurotrophic effect of memantine. First, quantitative RT-PCR analysis showed that memantine treatment caused a time-dependent increase in GDNF mRNA levels in astroglial cultures. This increase reached two fold at 0.5 h, three fold at 1 h and returned to the control value at 24 h posttreatment (). Next, Western blot analysis revealed that memantine treatment increased the expression GDNF at 6 h and 12 h after treatment compared with 0 min (). A second experiment was conducted in order to provide evidence indicating GDNF was associated with the trophic effect of memantine. When the goat anti-GDNF antibody was mixed with memantine in neuron-glia cultures, the GDNF-neutralizing antibody significantly reduced memantine-enhanced DA uptake capacity; whereas treatment with goat isotype IgG control antibody has no effect (). Taken together, these two experiments strongly indicated a critical role of GDNF in mediating the neurotrophic effect of memantine.
GDNF mediated memantine-induced neurotrophic effects
Memantine-Induced Suppression of Astroglia Cellular HDAC Activity Was Associated with Chromatin Remodeling at GDNF Promoter Region
We had previously reported that VPA increased the expression of neurotrophic factors through the inhibition of HDAC activity which was associated with the increase of histone hyperacetylation in astroglia (Wu et al, 2008
). Thus, changes in histone acetylation were examined in order to further understand the molecular mechanism underlying memantine-elicited increase in the expression of GDNF. We first compared the mechanism by which these two compounds inhibit HDAC activity. HeLa nuclear extract was used as a source of HDAC enzymes for the inhibition of HDAC activity assay. As shown in , memantine failed to show any effect in inhibiting the HDAC activity. In contrast, VPA at clinically relevant concentrations, greatly inhibited the HDAC activity in a dose-dependent manner (). These findings indicate, despite their similar effects in the expression of GDNF, these two compounds are different in their effect on HDAC enzymes. Thus, we determined whether the HDAC activity is affected in the cellular levels of astroglia treated with memantine. The cellular HDAC activity in memantine-treated astroglia was significantly suppressed in a time-dependent manner and maximally at 4 hr, compared with the controls (). We also compared the effects of VAP (1 mM), a widely used HDAC inhibitor, and memantine (10 µM) on the HDAC activity of astrocglia at 4 hr after administration. The activity was almost equi-potently inhibited about 30% by either memantine or VPA. These results indicate that memantine and VPA inhibited HDAC activity in different mechanisms. Therefore, we examined if changes of histone acetylation levels occur at the GDNF promoter region by ChIP study. The result showed that markedly enhanced association of acetylated H4 for one (Pc) out of these three primer set regions was observed in the memantine-treated astroglia, indicating that memantine triggered recruitment of acetylated histone, specifically to the proximally close to the initiator site of GDNF gene ().
Inhibition of Cellular HDAC activity was associated with histone modification of GDNF promoter in memantine-treated astrglia
Neuroprotective Effects of Memantine against LPS-Induced Neurotoxicity Were Mediated through Microglia
Neuroprotective effects of memantine on DA neurons in midbrain neuron-glia cultures were determined by using LPS, an endotoxin from bacteria, as pro-inflammatory stimulus to induce neurotoxicity. Neuron-glia cultures were treated with various concentrations of memantine for 30 min prior to LPS (5 ng/ml). After 7-day incubation, LPS decreased the [3H] DA uptake capacity by 60%, whereas memantine reduced LPS-induced loss of DA uptake in a dose-dependent fashion (). Morphological observation also revealed that DA neurons exhibited less damage in memantine-treated LPS cultures when compared with cells treated with LPS alone (; upper panel). Specifically, DA neurons treated with LPS in the presence of memantine displayed much longer and more elaborate TH-positive neurites compared with those from cultures treated with LPS alone (; upper panel).
Memantine was neuroprotective against LPS-induced neurotoxcity
Memantine alone produced astroglia-dependent neurotrophic effect in neuron-glia cultures (). Therefore, astroglia will contribute to the neuroprotective effect of memantine against LPS-induced neurotoxicity. Since LPS is a potent inducer of microglial activation, the involvement of microglia would also be expected. Thus, neuron-microglia reconstituted cultures were used to determine whether microglia play a role in memantine-elicited neuroprotection. In the absence of astroglia in this culture, the role of microglia in memantine-elicited neuroprotection can be precisely evaluated. Cultures were pre-treated with various concentrations of memantine (1–10 µM) or vehicle for 30 min prior to LPS treatment (2.5 ng/ml). Lower concentration of LPS was used in neuron-microglia co-cultures because this type of culture is more sensitive to LPS treatment without the presumed protection of astroglia. Seven days later, the neurotoxic effects of LPS on DA neurons were assessed by [3H] DA uptake. In the absence of astroglia, memantine alone (10 µM) failed to increase the capacity of DA uptake in neuron-microglia co-cultures, indicating no trophic effect was observed under this condition (). However, memantine showed a pronounced neuroprotective effect on DA neurons against the LPS-induced neurotoxicity, strongly suggesting that microglia play a role in mediating the neuroprotective effect of memantine. The morphological changes elicited in TH-positive neurons in neuron-microglia co-cultures after LPS and/or memantine treatment were shown in (lower panel).
The morphology of microglia in the primary midbrain neuron-glia cultures and in neuronmicroglia co-cultures was assessed by ICC staining with an OX42 antibody against the CR3 receptor, a marker for microglia. After LPS treatment, in both types of cultures, microglia dramatically changed the morphology from resting round and small shape to rod-and/or amoeboid shape with a significant enlargement of cell size (). Pre-treatment with memantine attenuated the LPS-induced change in morphology seen in microglial cells, indicating that memantine inhibits the activation of microglia.
Memantine Reduced LPS-Induced Production of Reactive Oxygen Species and Pro- Inflammatory Factors from Microglia
Activated microglia produces an array of pro-inflammatory factors that are key mediators underling LPS-induced DA neurotoxicity (Block et al, 2004
). To provide more evidence of the anti-inflammatory effect of memantine, we determined several major inflammation-related factors released from microglia after LPS treatment. Because each factor released from microglia is different in terms of time and quantities, specific cultures and time points were tailored for each pro-inflammatory factor. Enriched microglia cultures were used for the determination of LPS-induced reactive oxygen species generation including extracellular superoxide and iROS. Memantine attenuated production of superoxide anion () and iROS () in a dose-dependent fashion compared with vehicle controls. In addition, in neuron-glia cultures, LPS-induced increase in TNF-α (4 h after LPS treatment) was significantly reduced by memantine (). The increased release of nitric oxide (NO, measured as nitrite) and PGE2
24 h after LPS stimulation was also significantly reduced in memantine-treated samples ().
Memantine inhibited LPS-induced production of reactive oxygen species (ROS), and proinflammatory factors
Supernatant Levels of Glutamate and Aspartate Remained Unchanged after LPS Treatment in Primary Midbrain Neuron-Glia Cultures
Since memantine is a NMDA receptor blocker, most of the reports attributed neuroprotective effects or clinical therapeutic benefits of this compound to its inhibitory effects on NMDA-receptor mediated excitotoxicity (Lipton, 2006
). To determine whether NMDA receptors play a role in the neuroprotective effect of memantine in LPS-induced DA neurotoxicity, we measured the concentrations of two excitatory amino acids, glutamate and aspartate, in the supernatants of primary neuron-glia cultures treated with LPS and/or memantine (). Basal levels of these two excitatory amino acids were low, around 12 µM for glutamate and 10 µM for aspartate. The levels remained unchanged for the entire time course (0.5 to 48 h) determined after LPS treatment. Supernatant concentrations of both glutamate and aspartate in cultures treated with memantine (10 µM) alone or added together with LPS did not differ from the levels of vehicle-or LPS-treated groups. These results suggested an NMDA receptor independent mechanism for glia-mediated neurotrophic and anti-inflammatory effects of memantine.
Concentrations of glutamate and asperate in supernatants of primary midbrain cultures