Tg2576 Cortical Neurons That Express Human Ab Are Susceptible to Extracellular Aβ-Induced, K+ Efflux Dependent Neurodegeneration

Background One of the key pathological features of AD is the formation of insoluble amyloid plaques. The major constituent of these extracellular plaques is the beta-amyloid peptide (Aβ), although Aβ is also found to accumulate intraneuronally in AD. Due to the slowly progressive nature of the disease, it is likely that neurons are exposed to sublethal concentrations of both intracellular and extracellular Aβ for extended periods of time. Results In this study, we report that daily exposure to a sublethal concentration of Aβ1-40 (1 µM) for six days induces substantial apoptosis of cortical neurons cultured from Tg2576 mice (which express substantial but sublethal levels of intracellular Aβ). Notably, untreated Tg2576 neurons of similar age did not display any signs of apoptosis, indicating that the level of intracellular Aβ present in these neurons was not the cause of toxicity. Furthermore, wildtype neurons did not become apoptotic under the same chronic Aβ1-40 treatment. We found that this apoptosis was linked to Tg2576 neurons being unable to maintain K+ homeostasis following Aβ treatment. Furthermore, blocking K+ efflux protected Tg2576 neurons from Aβ-induced neurotoxicity. Interestingly, chronic exposure to 1 µM Aβ1-40 caused the generation of axonal swellings in Tg2576 neurons that contained dense concentrations of hyperphosphorylated tau. These were not observed in wildtype neurons under the same treatment conditions. Conclusions Our data suggest that when neurons are chronically exposed to sublethal levels of both intra- and extra-cellular Aβ, this causes a K+-dependent neurodegeneration that has pathological characteristics similar to AD.


Introduction
Alzheimer's disease (AD) is characterised by profound synaptic loss and neuronal death, and the accumulation of a number of key pathological hallmarks; senile plaques, dystrophic neurites and neurofibrillary tangles [1]. The b-amyloid peptide (Ab) is the principle component of plaques, and is thought to contribute significantly to the pathogenesis of the disease [2]. However, the precise mechanisms that underlie the role of Ab in AD are not clearly understood.
The localisation of Ab is likely to have an important role in governing its toxic actions upon neurons. In this regard, it is well known that acute extracellular administration of aggregated forms of Ab (and in particular oligomers) to cultured neurons is neurotoxic [3]. This is in accordance with the presence of amyloid plaques in AD, of which extracellular, aggregated forms of Ab are the major constituent [4]. However, a growing body of evidence suggests that intraneuronal localisation of Ab may also play a significant role in AD. For example, Ab accumulates in processes and synapses prior to, and with the onset of extracellular Ab plaque formation [5,6], and in transgenic mice that develop Ab plaques [7]. There is also some evidence that cognitive impairment in AD patients does not always correlate to the level of Ab plaque deposition [8]. Similarly Ab immunisation studies in Tg2576 [9] or PDAPP [10] transgenic mice reversed memory loss, but had no impact upon amyloid plaque levels. These studies suggest that the intraneuronal accumulation of Ab may be important in disease progression and symptom onset.
Indeed, intracellular Ab appears to increase the susceptibility of neurons to neurodegeneration. For example, Abdul et al [11] reported that cortical neurons cultured from APP/PS1 transgenic mice were more vulnerable to oxidative stress, mitochondrial dysfunction and apoptosis. Yao and colleagues [12] have demonstrated that 3xTg-AD mice exhibit increased hydrogen peroxide production and lipid peroxidation. Furthermore, hippocampal neurons cultured from these mice exhibited significantly decreased mitochondrial respiration and increased glycolosis [12]. Notably, cultured neurons transfected with constructs expressing APP that contains familial-linked AD mutations that substantially increase levels of Ab are also susceptible to apoptosis-inducing treatments [13]. Others have linked intracellular Ab directly to apoptosis, reporting that transfection of constructs expressing Ab into neuroblastoma cells resulted in activation of a P53-dependent apoptotic pathway [14]. A similar outcome has been observed when synthetic Ab peptides were microinjected into neurons, which induced cytotoxicity via a p53-Bax apoptotic pathway [15]. Notably, exogenously applied Ab is rapidly internalised by cultured neurons, where it could subsequently act in a neurotoxic manner like endogenous Ab. Uptake of Ab by neuronal cells occurs via the low-density lipoprotein receptor LRP1 [16]. Treatment of PC12 cells [17] or primary neuron cultures [18] with exogenous Ab leads to accumulation of reactive oxygen species, and a decrease in redox activity and ATP levels [17]. Treatment with alpha-tocopherol (Vitamin E) can block these Abinduced changes in neuronal cells (see reviews by [19,20]).
AD is a progressive disease, in which neurons are likely to be exposed to both intracellular and extracellular Ab at sublethal concentrations for extended periods of time. To experimentally model this situation, we have exposed cultured neurons from Tg2576 mice (which accumulate substantial amounts of intracellular Ab) to daily treatment with exogenous Ab 1-40 for 6 days. Chronic Ab 1-40 treatment induced substantial apoptosis of cortical Tg2576 neurons but not wildtype neurons, suggesting that both intra-and extra-cellular Ab are required to induce apoptosis. Apoptosis was linked to an inability of Tg2576 neurons to maintain K + homeostasis following acute treatment with extracellular Ab  . Chronic exposure to 1 mM Ab 1-40 also caused the generation of hyperphosphorylated tau-immunoreactive axonal swellings in Tg2576 but not wildtype neurons. Our data suggest that chronic exposure to both intra-and extra-cellular Ab induces neurodegenerative changes that bear similarities to some of the pathological hallmarks of AD.

Uptake of exogenous Ab by cultured transgenic Tg2576 and wildtype cortical neurons
Mouse cortical neurons (wildtype and Tg2576) were maintained in culture for seven days in vitro (DIV), at which time they were treated with 10 mM soluble monomeric Ab 1-40 . After 24 hours, neurons were fixed and Ab detected by immunostaining. In untreated Tg2576 cortical neurons, Ab was distributed within the cytoplasm and processes, but generally not in the nucleus ( Figure 1A). In untreated wildtype cortical neurons, there was no Ab detected (results not shown). In wildtype neurons treated with Ab, Ab was detected in a punctate distribution within the cytoplasm and processes ( Figure 1B). When Ab was applied to Tg2576 neurons, the distribution of Ab resembled both of these scenarios, with both punctate and non-punctate regions of Ab immunoreactivity observed within the cytoplasm and nucleus ( Figure 1C). When fluorescently tagged Ab 1-40 (10 mM) was applied to either wildtype or Tg2576 cortical neurons, we did not observe any difference in neuronal uptake or distrubtion of Ab (results not shown).
Intraneuronal Ab increases the vulnerability of cortical neurons to neurotoxicity induced by extracellular Ab 7DIV mouse cortical neurons (wildtype and Tg2576) were treated with soluble monomeric Ab 1-40 (1-10 mM) daily for a period of six days. Measurement of neuronal viability by an Alamar Blue assay revealed that while daily treatment with 1 mM Ab 1-40 was not toxic to wildtype cortical neurons at any point in the six day timecourse, this treatment resulted in a significant reduction in intracellular metabolism of Tg2576 neuronal cultures by approximately 30% after six days of continual Ab 1-40 treatment ( Figure 2A). 10 mM Ab 1-40 was mildly neurotoxic to wildtype neurons, resulting in approximately 20% cell death after seven days of treatment ( Figure 2B). However, Tg2576 neurons were far more susceptible to Ab 1-40 ( Figure 2B). The neurotoxic actions of chronic exposure to Ab 1-40 upon Tg2576 cortical neurons were confirmed in propidium iodide uptake studies ( Figure 2C), whereby only dying cells internalise and incorporate propodium iodide in the nucleus. These results suggest that the non-toxic accumulation of intraneuronal Ab in Tg2576 cortical neurons increases their vulnerability to subsequent neurotoxicity induced by chronic exposure to normally sublethal (1-10 mM) levels of extracellular Ab. Notably, in all cases when either wildtype or Tg2576 neurons were treated with vehicle alone, no change in viability was observed (results not shown).
To investigate whether chronic exposure to both intra-and extracellular Ab had initiated an apoptotic pathway of cell death, immunostaining for activated caspase-3 was performed. Almost no caspase-3 labelled cells were observed in 14 DIV Tg2576 neuronal cultures ( Figure 3A). However, when 7 DIV Tg2576 neurons were treated with 1 mM Ab 1-40 for six consecutive days, caspase-3 immunoreactivity correlated with condensed or fragmented nuclei in approximately 30% of cells ( Figure 3B), and direct counting found that the number of caspase-3 labelled neurons was equivalent to the number of neurons that incorporated propidium iodide (results not shown), indicating that the combination of both intra-and extraneuronal Ab triggered an apoptotic pathway of neuronal death.

Treatment with Ab causes K + flux-dependent neurotoxicity in Tg2576 neurons
There are a number of reports suggesting that extracellular Ab triggers changes in ionic homeostasis of neurons, and that these changes contribute directly to neurotoxicity. To investigate whether intracellular Ab alters the ability of neurons to maintain ionic homeostasis following extracellular Ab treatment, we used a novel non-invasive microelectrode ion flux (MIFE) measuring technique. Using the MIFE approach, we directly observed that Ab treatment triggered rapid efflux of K + from wildtype neurons ( Figure 4A), which returned to homeostasis within 10 minutes after Ab 1-40 treatment. However, K + flux in Tg2576 neurons treated with Ab 1-40 did not return to homeostasis ( Figure 4B). Instead, transgenic neurons exhibited a continual efflux of potassium for more than 120 minutes after Ab 1-40 treatment ( Figure 5A). Measurement of total K + flux over 25 minutes following Ab 1-40 treatment revealed that significantly more potassium was extruded from Tg2576 neurons than wildtype neurons ( Figure 4C). Interestingly, continuous treatment of wildtype neurons for three days with 1 mM Ab 1-40 did not alter their ability to maintain K + homeostasis following Ab treatment (results not shown).
Concurrently, we also measured H + flux of neurons in response to Ab 1-40 treatment. Wildtype neurons treated with Ab 1-40 demonstrated a rapid influx of H + , which stabilised within 5 minutes and remained stable for the entire recording period ( Figure 4D). Tg2576 neurons displayed a similar response to Ab 1-40 ( Figure 4E). However, from about 10 minutes post-treatment, Tg2576 neurons underwent a slow gradual influx of H + ( Figure 4E). The latter was further increased during the next 120 min posttreatment with Ab 1-40 ( Figure 5B). Measurement of total H + flux over 25 minutes after Ab treatment revealed that Ab 1-40 induced significantly greater influx of H + into Tg2576 neurons in comparison to wildtype neurons ( Figure 4F).
It is generally accepted that excessive potassium efflux is a key early step in apoptosis. To confirm that prolonged extrusion of K + is responsible for Ab-induced neurotoxicity in Tg2576 neurons, cells were pre-treated with the classic K + channel blocker 4aminopyradine (4-AP) prior to Ab treatment. This resulted in a significant reduction of Ab-induced neuronal death in Tg2576 neurons ( Table 1).

The presence of both intra-and extracellular Ab causes the formation of axonal swellings in cultured neurons
Immunolabelling of the axonal cytoskeleton (tau) revealed that there was no discernible difference in axonal morphology between wildtype ( Figure 6A) and Tg2576 cortical neurons ( Figure 6B) over 7-14 DIV. Daily treatment of 7DIV wildtype neurons with 1 mM Ab 1-40 over the six day experimental timecourse did not markedly alter axonal morphology ( Figure 6C). However, substantial changes in tau-labelling were observed in Ab 1-40 treated Tg2576 neurons. Treatment of Tg2576 neurons with 1 mM Ab 1-40 resulted in a substantial increase in intensity of tau immunostaining after 24 hours ( Figure 6D). After two daily treatments of Tg2576 neurons with 1 mM Ab 1-40 , blebbing and axonal fragmentation was apparent ( Figure 6D), which worsened after four continuous days of Ab 1-40 treatment ( Figure 6D). Furthermore, after four consecutive days of 1 mM Ab 1-40 treatment, a number of axonal swellings were observed in Tg2576 neuron cultures ( Figure 6E), but never in wildtype neurons treated with 1 mM Ab 1-40 for the same period of time (results not shown). These intra-axonal swellings were highlighted by dense accumulations of the microtubule-associated protein tau, including dense accumulations of hyperphosphorylated (AT-8 immunoreactive) tau ( Figure 6E).

Discussion
AD is a progressive neurodegenerative disease, in which neurons are likely to be exposed to sublethal concentrations of both intracellular and extracellular Ab for extended periods of time. To experimentally model this situation, Tg2576 neurons (which accumulate substantial amounts of intracellular Ab) and wildtype neurons received daily treatment with soluble, monomeric Ab for 6 days. While this chronic exposure to Ab did not kill wildtype neurons, it caused substantial apoptosis of Tg2576 neurons. Further studies revealed that Tg2576 neurons were unable to maintain K + and H + homeostasis following Ab treatment, leading to prolonged extrusion of potassium and influx of protons into Tg2576 neurons. Furthermore, chronic exposure to 1 mM Ab for six days caused the generation of hyperphosphorylated tau-immunoreactive axonal swellings in Tg2576 but not wildtype neurons. In summary, our data suggest that chronic exposure to sublethal levels of both intra-and extra-cellular Ab induces neurodegenerative changes in cultured neurons that bear similarities to pathological hallmarks observed in AD. These changes appear to be driven by an inability of Tg2576 to maintain normal K + homeostasis in response to continual exposure to extracellular Ab.
The mechanism by which Ab causes neurotoxicity or neuronal dysfunction remains to be fully resolved. Numerous studies have used cultured neurons to investigate the neurotoxic actions of Ab, and can generally be classified into two paradigms; experiments whereby Ab is applied acutely or chronically to cultured neurons (testing the effect of extracellular Ab upon neurons), and experiments using neurons cultured from transgenic AD mice which express human Ab (to test the effect of intracellular Ab upon neurons). The former experiments have been particularly informative, revealing important information regarding the concentration and biochemical form of extracellular Ab that exhibits toxicity upon cultured neurons; from such studies it is proposed that soluble oligomeric forms of Ab at .5 mM concentrations are the most toxic form of extracellular Ab to neurons [3]. In a similar manner, experiments using neurons cultured from transgenic AD mice have reported that intraneuronal Ab increases the vulnerability of neurons to stressful cellular environments such as excitotoxicity and oxidative stress [11]. In some cases, the intraneuronal expression of Ab itself can trigger apoptotis of neurons via a p53-dependent mechanism [21,14]. It is worth noting that one simplistic manner in which exogenous Ab may induce neurodegenerative changes in Tg2576 neurons is due to uptake of Ab via LRP1 [16], which increases intracellular Ab levels above a threshold level leading to decreased viability and alterations in tau distribution and phosphorylation. However, two pieces of evidence argue against this possibility. Firstly, the distribution of endogenous Ab and internalised Ab in Tg2576 is quite different, suggesting that these two pools of Ab are not able to act in the same manner. And secondly, our MIFE studies demonstrate rapid and direct changes in ionic homeostasis of Tg2576 neurons triggered by application of exogenous Ab, indicating that exogenously applied Ab is probably acting in a different manner to intracellular endogenous Ab.
While the studies discussed above have provided important information regarding the effect of extracellular and intracellular Ab upon cultured neurons, it is important to consider that AD is a progressive condition, in which neurons are likely to be continuously exposed to sublethal concentrations of both intracellular and extracellular Ab. To more accurately model this situation, Tg2576 neurons (which accumulate Ab intraneuronally) were cultured in the presence of extracellular Ab. This combination of intra-and extracellular Ab induced caspase-3 dependent apoptosis of Tg2576 but not wildtype neurons. To elucidate the mechanisms underlying this, we used a non-invasive MIFE approach to observe changes in net ion flux of K + and H + ions in response to Ab. Using this approach, we were able to continuously observe net ion flux of K + and H + for more than 2 hours. We found that Ab treatment of wildtype neurons caused an immediate efflux of K + , which gradually returned to homeostasis within 10 minutes. However, we found that Abtreated Tg2576 neurons were unable to maintain K + homeostasis, leading to prolonged leakage of K + out of neurons. K + efflux from cells is a key early initiator of apoptosis, as a low potassium intracellular microenvironment assists apoptosome formation and the activation of caspases and endonucleases. This suggests that the prolonged extrusion of K + from Ab-treated Tg2576 neurons was the cause of apoptosis in this study. This was confirmed by undertaking the same experiments in the presence of the K+ channel blocker 4-AP, which provided significant protection against Ab-induced toxicity in Tg2576 neurons.
A novel element to the MIFE approach in this application is that it allows measurement of total flux of K + into-or out-of cells, rather than the flow of K+ through particular channels/ transporters that is observed through patch-clamp recording (electrogenic transporters) and pharmacological inhibitor studies. Figure 2. Tg2576 cortical neurons are more vulnerable to soluble Ab-induced neurotoxicity. Wildtype and Tg2576 cortical neurons were treated daily with 1 mM (A) or 10 mM (B) of monomeric Ab 1-40 for 6 days, and neuronal viability (intracellular metabolism as assessed by Alamar Blue assay) assessed every 24 hours. At 1 mM concentrations, only Tg2576 neurons were vulnerable to Ab 1-40 , resulting in approximately 30% cell death after 6 days (A). 10 mM Ab 1-40 was mildly toxic to wildtype neurons over the experimental timecourse, but killed 40% of Tg2576 neurons after 6 days (B). The Alamar Blue neurotoxicity assay produced similar results to direct counting of dying cells via propidium iodide uptake following treatment with 10 mM Ab 1-40 (C). * -p,0.05, ANOVA. Error bars represent standard error values from at least three replicates per experimental condition. This graph is representative of the results observed from 4 different experiments. doi:10.1371/journal.pone.0019026.g002 Our observations are in accordance with previous studies demonstrating that the neurotoxicity elicited by soluble Ab upon neurons involves elevated K + efflux, mediated through multiple pathways including enhanced activity of voltage-gated potassium channels [22,23] and the Na + /K + ATPase [24,25]. Furthermore, increasing the extracellular K + level to prevent K + loss is also able to block Ab-induced neuronal apoptosis [26]. We now demonstrate the direct measurement of net K + flux of neurons in response to Ab, and report that simultaneous exposure to both intra-and extracellular Ab significantly impairs the ability of neurons to regulate K + homeostasis. Given that intracellular accumulation of Ab within neurons in the AD brain is only observed later in life, our data provides a potential mechanism that could explain why neurons in the AD brain become vulnerable to apoptosis later in life despite being continuously exposed to extracellular Ab for many years.
While the toxicity of Ab has been extensively studied in cultured neurons, two considerable limitations of these approaches have been that they have often involved the use of relatively immature neuronal phenotypes (cultured for between 1-4 days in vitro), and a treatment period of up to 24 hours. In this study we have cultured neurons at relatively high density for seven days in vitro prior to experimentation. Under these conditions, neuronal cultures contained dense networks of processes more representative of mature neurons. Chronic exposure of wildtype neurons to sublethal concentrations of Ab did not alter axonal morphology of cultured neurons. However, Tg2576 neurons continuously treated with Ab displayed substantial axonal pathology, including increased intensity of tau immunolabelling, axonal fragmentation and degeneration and the formation of axonal swellings that were packed with hyperphosphorylated tau. While it is well described that Ab treatment of cultured neurons can directly cause tau hyperphosphorylation within 24 hours [27], our study is the first that we are aware of that demonstrates that Ab can cause the generation of hyperphosphorylated tau-immunoreactive axonal swellings in mature neuronal cultures. Notably, these axonal swellings took four days to develop, suggesting that they represent a slowly evolving, secondary phase of Ab-induced neurodegeneration. These dystrophic axonal manifestations resemble some of the key neuritic pathologies observed in the AD brain, suggesting that the prolonged effect of intra-and extracellular Ab exposure upon neurons is a critical step in the neurodegenerative process underlying AD.
In summary, we propose that in the AD brain, intraneuronal accumulation of Ab increases the vulnerability of neurons to subsequent chronic exposure to soluble Ab. This combined exposure to intra-and extracellular Ab leads to degenerative changes in neurons (such as axonal swelling and fragmentation) and apoptosis through a K + efflux mediated mechanism.

Ethics Statement
All animal experimentation was performed under the guidelines stipulated by the University of Tasmania Animal Ethics Committee, which is in accordance with the Australian code of practice for the care and use of animals for scientific purposes.

Cortical neuron cultures from Tg2576 and wildtype mice
Hemizygous Tg2576 male mice on a hybrid B6SjL background (Taconic) were crossed with wildtype B6SjLF1 females, and cortical tissue was removed from individual embryos from the pregnant wildtype mice (the embryos are either transgenic or wildtype), and cortical neurons isolated as we have described previously [28]. Cortical neurons from individual pups were maintained in Neurobasal medium (Gibco) containing 10% fetal calf serum, at 37uC in humidified air containing 5% CO 2 . The culture medium was replaced with serum-and glutamic acid-free culture medium after 24 hours, followed by half media changes twice weekly. To test the affect of chronic Ab treatment upon cortical neurons, 1 mM or 10 mM of soluble monomeric Ab  was added exogenously to 7DIV cultured neurons daily for 6 days with assessment of cellular viability conducted each day. Lyophilised Ab 1-40 peptide was purchased from EZBiolab, and solubilised in sterile MQ water (which served as the vehicle control for all experiments). The Ab 1-40 peptide was used in this study because in our experience the Ab 1-42 peptide induces substantial acute neurotoxicity at 10 mM concentrations, making this peptide unsuitable for long-term studies. Note that each day, the entire culture medium was replaced with fresh media to which Ab 1-40 was added. All experiments were performed without knowing the genotype identity (wildtype or transgenic) of the individual neuron cultures. We generally obtained cultures from 10 embryos per pregnant animal, and they were usually split evenly between transgenic and wildtype. At the conclusion of each experiment, immunocytochemical detection of Ab (using the 6E10 antibody, see details below) was used to genotype the cultures and reveal their identity for data analysis and interpretation. Untreated neuronal cultures at 14 days in vitro were used for this purpose, and neurons could be easily distinguished as containing either high-or low-levels of Ab.

Alamar blue viability assay
Neuronal viability was measured by the degree of cellular metabolic reduction of Alamar BlueH, as we have reported  previously [29,30]. Briefly, viability was determined by determination of the fluorescence of Alamar Blue in culture wells (excitation 535 nm, emission 595 nm), and was expressed as the percentage of the signal obtained from the vehicle-treated culture. Alamar Blue was applied at a 1:10 dilution in culture media for 30 minutes, after which time it was collected and fluorescence measured on a fluorescent plate reader (Tecan Genios). In experiments involving repeated measurements of viability over a consecutive series of days, following Alamar Blue collection fresh media was applied to the neurons and the viability assay procedure repeated each day. Alamar Blue is non-toxic, and we have not observed any decline in neuronal viability when using this technique on consecutive days for up to one week (results not shown).

Non-invasive microelectrode ion flux (MIFE) measurements in cultured neurons
The theory of MIFE measurements was reviewed recently [31] and the complete experimental procedure including ion-selective microelectrode fabrication [32] and neuronal culture preparation, immobilisation and recording were performed as described previously [30,33]. Briefly, ion selective microelectrodes were silanised and filled with commercially available ionophore cocktails (Fluke catalog no. 60031 and 95297 for for K + and H + , respectively). Microelectrodes for MIFE measurements were prepared on a daily basis and calibrated before and after measurements in a range of K + and H + concentrations. Cortical neurons for the MIFE measurements were grown for six days at a density of 1610 5 cells/well on poly-L-lysine-coated coverslips and chronically treated with soluble monomeric Ab 1-40 as described above. A cover slip with neural cells was washed in and adapted to the MIFE artificial CSF (aCSF) for one hour prior to experiments. The composition of the aCSF was: 150 mM NaCl, 0.5 mM KCl, 0.5 mM CaCl 2 , 1.5 mM MgCl 2 , 1.25 mM NaH 2 PO 4 , 5 mM NaHCO 3 , 25 mM glucose, pH 7.4. Electrodes were co-focused and positioned ,5 mm above the neuronal monolayer and moved up and down by a computer-controlled stepper motor providing a travel range between 5 and 50 mm from the cell surface at a frequency of 0.1 Hz. After recording steady state flux for ,5 min, neurons were treated with Aß and data acquired at a rate of 15 samples/sec and later averaged over 8 second intervals. For all ion flux measurements, the sign convention is 'influx positive' for a cation. The data were analysed using MIFE software with ion fluxes expressed in nmol m 22 s 21 . A total flux was calculated as area between flux curve over the indicated experimental timeframe (25 min) and the starting flux value.

Statistical analysis
For each experiment unless otherwise stated, a minimum of four wells from at least three separate cultures (derived from different animals), were used for quantification. Statistical analysis was completed using SPSS 16.0 (SPSS). When data was unequally distributed, data was transformed so that the residuals were approximately normally distributed. Statistical significance was calculated using t-test when only two experimental samples were compared, and One-Way ANOVA with Tukey's Post Hoc Test when multiple samples were compared. All graphical data is presented as mean 6 SEM, significance p,0.05.