In Vitro Effects of Chronic Spirolide Treatment on Human Neuronal

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IN VITRO EFFECTS OF CHRONIC SPIROLIDE TREATMENT ON HUMAN NEURONAL STEM CELL DIFFERENTIATIATION AND CHOLINERGIC SYSTEM DEVELOPMENT Andrea Boente Juncal, Aida G Mendez, Carmen Vale, Mercedes R. Vieytes, and Luis M. Botana ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.8b00036 • Publication Date (Web): 08 Mar 2018 Downloaded from http://pubs.acs.org on March 9, 2018

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ACS Chemical Neuroscience

IN VITRO EFFECTS OF CHRONIC SPIROLIDE TREATMENT ON HUMAN NEURONAL STEM CELL DIFFERENTIATIATION AND CHOLINERGIC SYSTEM DEVELOPMENT Andrea Boente-Juncal†, Aida G. Méndez†, Carmen Vale†*, Mercedes R. Vieytes‡ and Luis M. Botana†* †

Departamento de Farmacología, Farmacia y Tecnología Farmacéutica,

Facultad de Veterinaria, Universidad de Santiago de Compostela, Lugo, Spain ‡

Departamento de Fisiología, Facultad de Veterinaria, Universidad de Santiago de Compostela, Lugo, Spain * Email: [email protected]; [email protected]

Key words: spirolides, CTX0E16 cell line, acetylcholine, neuronal stem cell; nicotinic receptors, neuroprotection.

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Graphical Table of Contents


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Abstract Spirolides are marine toxins, produced by dinoflagellates that act as potent antagonists of nicotinic acetylcholine receptors. These compounds are not toxic for humans and since there are no reports of human intoxications caused by this group of toxins they are not yet currently regulated in Europe. Currently 13desmethyl spirolide C, 13,19-didesmethyl spirolide C and 20-methyl spirolide G are commercially available as reference materials. Previous work in our laboratory has demonstrated that after 4 days of treatment of primary mice cortical neurons with 13-desmethyl spirolide C, the compound ameliorated the glutamate induced toxicity and increased acetylcholine levels and the expression of the acetylcholine synthesizing enzyme being useful both in vitro and in vivo to decrease the brain pathology associated with Alzheimer´s disease. In this work we aimed to extend the study of the neuronal effects of spirolides in human neuronal cells. To this end, human neuronal progenitor cells CTX0E16 were employed to evaluate the in vitro effect of spirolides on neuronal development. The results presented here indicate that long term exposure (30 days) of human neuronal stem cells to SPX compounds, at concentrations up to 50 nM, ameliorated the MPP+-induced neurotoxicity, increased the expression of neuritic and dendritic markers, the levels of the choline acetyltransferase enzyme and the protein levels of the α7 subunit of nicotinic acetylcholine receptors. These effects are presumably due to the previously described interaction of these compounds with nicotinic receptors containing both α7 and α4 subunits. All together these data emphasize the idea that SPX could be attractive lead molecules against neurodegenerative disorders.



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Introduction Spirolides (SPX) are secondary metabolites produced by the dinoflagellates

Alexandrium Ostenfeldii and Alexandrium Peruvianum (1-3). The first knowledge about the presence of SPX in shellfish occurred during a routine monitoring for biotoxins in bivalve molluscs in Nova Scotia, Canada, in 1991(2). Currently, these toxins have a worldwide distribution and spirolides are also found on European coasts and have been identified in a number of European countries bordering the Mediterranean Sea, Atlantic coast and the North Sea (4). These compounds are not toxic for humans and since there are no reports of human intoxications caused by SPX, this group of toxins is not yet currently regulated in Europe (5). SPX share a common chemical structure characterized by a macrocycle ring of 14-27 carbon atoms, a seven-membered cyclic imine group, which constitutes a key pharmacophore element, and a spiroketal cyclic, which can be 6,5,5-spiroketal (spirolides A–F), 6,5-spiroketal (spirolides H and I), or 6,6,5-spiroketal in the case of spirolide G. It is widely accepted that the cyclic imine moiety is responsible for the toxicity of SPX, since spirolides E and F, which have an acyclic aminoketone, exhibit no toxicity (6, 7). SPX are lipophilic compounds belonging to the group of cyclic imines such as pinnatoxins,

pterotoxins,

prorocentrolides,

spiro-propocentrimine

and

gymnodimine (2). Since their discovery, 16 analogues of SPX have been reported (6). The SPX group of toxins is divided into 3 subgroups (8). The first one includes spirolides from A to D, the second SPX E to F, and the third subgroup spirolides G (1). Among the SPX group of toxins, 20-methyl spirolideG (20-MeSPXG) was the main spirolide toxin identified both in shellfish and plankton samples from Norway (9). Besides its chemical diversity, nowadays, only 3 compounds from the SPX group are available as reference materials: 13-desmethyl spirolide C (13-desMeC), 13,19-didesmethyl spirolide C (13,19-didesMeC) and 20-MeSPXG. All the SPX toxins act on the cholinergic system and previous work has demonstrated that SPX compounds such as 13-desMeC and 13,19-didesMeC are potent antagonists of nicotinic acetylcholine receptors (nAChRs) acting both on muscular and neuronal receptors (6, 10, 11). Due to its effect on nAChR, we


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have previously demonstrated that 13-desMeC exhibited beneficial in vitro and

in vivo effects against neurodegenerative diseases decreasing the amyloid beta load and the tau hyperphosphorylation in primary cortical neurons and in the brain of triple transgenic mice for Alzheimer´s disease (12, 13). Moreover, SPXs are rapidly absorbed from the intestinal tract appearing in blood at 15 min and in urine after 1 h of oral administration (14). In addition, we have also demonstrated that 13-desMeC crossed the blood brain barrier and reached the brain only 2 minutes after intraperitoneal injection, being detectable even 24 hours post administration (12). Our previous work in primary mouse cortical neurons has demonstrated no toxicity of 13-desMeC after short exposures of 4 days to the compound. Additionally, at 50 nM, 13-desMeC ameliorated the glutamate induced toxicity, increased acetylcholine levels in primary cortical neurons from the murine Alzheimer´s disease model and also increased the expression of the acetylcholine synthesizing enzyme (ChAT) in these cultures (13). Besides, although so far no other reports on the neuroprotective effects of SPX on neurons have been released, the beneficial effect of this compound has also been demonstrated in vivo in a murine Alzheimer´s disease model (12). Recently, emerging lines of evidence have suggested that nAChR are expressed on stem cells, where they likely mediate crucial effects of cholinergic signaling on stem cell survival/apoptosis, proliferation, differentiation and maturation (15). Conditionally immortalized human neural progenitor cells (hNPCs) constitute a robust source of native neural cells to investigate physiological mechanisms and toxicity both in health and disease (16, 17). Previous work has fully characterized the conditionally immortalized, corticallyderived hNPC line CTX0E16, showing that this cell line provides a robust source of cortical neurons with functional properties and a glutamatergic phenotype

and

afford

and

ideal

model

system

to

investigate

neurodevelopmental mechanisms in native human cells (16). This previous work has also demonstrated that CTX0E16 NPCs express a select number of neurotransmitter-targeted G-protein-coupled receptors and ionotropic receptor



subunits,

including

those

belonging

to

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glutamatergic,

dopaminergic,

serotonergic and cholinergic receptor families. During the last years several reports have demonstrated that nAChR are implicated in neuronal development. Thus, in rodent neuronal progenitor cells, cultured for 12 days in the presence of cholinergic modulators, the nAChRs agonist nicotine decreased neurosphere area in the neocortex and this decrease was reverted by the nAChRs antagonist mecamylamine (18). Moreover, nAChRs mediate cholinergic modulations in stem cell apoptosis, a type of programmed cell death (15, 19, 20). However, although a variety of nAChR subtypes are expressed on stem cells, it has been suggested that the

α7 nAChR should be relevant for neuronal differentiation since this subtype showed high Ca2+ permeability and the calcium increase after α7 nAChR activation affected stem cell function and pathology (15). Recent findings have contributed to reinforce the idea that nAChRs play important roles in the mediation

of

cholinergic

modulations

of

stem

cell

survival/apoptosis,

proliferation and differentiation. In this sense, it has been recently demonstrated that human neurons differentiated from hippocampal neural stem/progenitor cells express functional α7 and α4β2 nAChRs, as well as M1 and M3 muscarinic acetylcholine receptors (mAChRs) (21). Likewise, it was also evidenced that α7 nAChRs are involved in the neurogenesis of cholinergic neurons in the mouse subventricular zone (22). In view of the potent effect of SPX as nAChRs antagonists in several systems and the importance of these receptors in human neuronal stem cell differentiation, in this work, we aimed to investigate the chronic effect of SPX on human neuronal differentiation. For this, the influence of the three commercially available SPX compounds over the expression of several neural fate markers and their potential impact on cholinergic system development has been evaluated.

Results and Discussion We have previously demonstrated the ability of 13-desMeC to prevent the neuronal degeneration induced by glutamate in primary cultures of cortical


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neurons obtained from a mouse model of Alzheimer´s disease (12, 13), reverting the decrease in acetylcholine levels in Alzheimer´s disease as well as beta-amyloid accumulation and tau hyperphosphorylation. Therefore, in this study we aimed to evaluate the effect of these compounds on human neuronal differentiation using a human neuronal stem cell line.

Conditionally immortalized, cortically-derived, human NPC line, CTX0E16 differentiates to form a mixed neuronal and glial population First we analyzed the neuronal differentiation of the CTX0E16 cells, to confirm the previous data regarding the neuronal phenotype of this cells line (16). In order to do this, cells cultured in glass coverslips were fixed and stained with neuronal and glial markers. Figure 1 shows, in red, β3-tubulin (early neuronal marker) and glial fibrillar acidic protein (GFAP, glial marker) at different stages of differentiation of the cell line. As shown in Figures 1A and 1B, at 3 days of differentiation (dd) cells showed low levels of β3-tubulin and GFAP staining while at 15 dd most of the cells stained positively for β3-tubulin and also GFAP staining was detected (Figures 1C and D). A similar situation was observed after 30 days of differentiation (Figures 1E and F). In all the microphotographs cell nuclei were stained with DAPI (4',6-diamidino-2-phenylindole) in blue.

Long term exposure of CTX0E16 to SPX compounds did not alter either cell viability or morphology In order to have functional receptors expressed (16), all the experiments evaluating the chronic effect of SPX in human neuronal stem cells were performed in cells differentiated for at least 15 to 21 days. After this time, cells were exposed to 13-desMeC, 13,19-didesMeC or 20-MeSPXG. The chemical structures of these compounds are shown in Figure 2. With the aim to evaluate the effect of SPX on human neuronal cells, differentiated cells were exposed to SPX concentrations ranging from 1 to 100 nM for 5 days in culture and the cell viability was assessed by the MTT test. As shown in Figure 3 none of the compounds affected cell viability after 5 days of exposure to the compounds, a fact that is in agreement with the effect observed



previously in mice cortical neurons where no toxicity of 13-desMeC was detected after exposure of primary cortical neurons to the toxin from 3 to 7 days in culture (13). Since, the purpose of the present work was to investigate the chronic effect of SPX on human neuronal differentiation, next, CTX0E16 cells were exposed for 30 days in vitro to 13,19-didesMeC at 25 nM, replacing half of the medium with toxin every 2 or 3 days. As shown in Figure 4, immunocytochemical staining with the neuronal nuclear protein (NeuN, in green), a protein whose expression is exclusively associated with the nervous tissue (23), and the cytoskeletal neuronal marker (β3-tubulin, red) showed no morphological differences amongst control neurons (Figure 4A) and neurons treated with 25 nM 13,19-didesMeC (Figure 4B). As expected, NeuN immunoreactivity was mainly associated with cell nuclei in both control and treated cells (24) while β3-tubulin stained neuronal processes (25).

SPX treatment partially prevented the changes in neuronal morphology elicited by the neurotoxin MPP+ Inasmuch as we have previously demonstrated a neuroprotective effect of 50 nM 13-desMeC in murine primary cortical neurons (13), first we tried to determine the neuroprotective effect of SPXs in human neuronal stem cells. In order to evaluate the potential cytoprotective effect of spirolides in human neuronal stem cells, different neurotoxic approaches were assessed. As shown in supplementary Figure 1, we found that the viability of the CTX0E16 cell line was not affected by treatment for 7 days with L-glutamate (Supplementary Figure 1A) and was only slightly decreased by treatment with the glutamate agonist N-methyl-D-aspartate (NMDA) as depicted in Supplementary Figure 1B. In view of the low toxicity of glutamate and NMDA in this cell line, the toxicity of the kainate receptor agonist domoic acid was also evaluated. As displayed in Supplementary Figure 1C, after 7 days of treatment domoic acid, at 100 µM, decreased cell viability by about 40%. However, the domoic acid toxicity was much lower in the human CTX0E16 cell line than that previously described in murine neuronal cultures (26). Thus, we hypothesized that ionotropic glutamate receptors could not be expressed in this cell line. However, western blot analysis showed expression for the AMPAR subunits GluR2,3,4 and for the


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NMDAR subunit NMDA2A/B after 30 days of differentiation (Supplementary Figure 1D) accordingly with the earlier demonstration of the presence of RNA for ionotropic receptors in differentiated cells (16). Therefore, the low toxicity of glutamate receptor agonists in differentiated CTX0E16 neurons was not related with the lack of expression of glutamate receptors. But rather it could be in agreement with the lower toxicity of several neurotoxins on immortalized cmycERTAM human neuronal stem cells versus rat neuronal stem cells or primary neuronal cultures(22). Moreover, glutamate has been previously reported to increase proliferation and neurogenesis in human neuronal progenitor cells derived from fetal cortex (27). However, the low toxicity of ionotropic glutamate receptor agonists on this cell line could also be related with the immaturity of the cells since in some human stem cells glutamate toxicity was only observed after 6 weeks in culture but not before (28). Differentiated CTX0E16 cells have previously been shown to respond to different neurotransmitters, including dopamine (16). In dopaminergic neurons neurotoxins

such

as

6-hydroxydopamine

or

1-methyl-4-phenyl-1,2,3,6-

tetrahydropyridine (MPTP) and its active derivative 1-methyl-4-phenylpyridinium ion (MPP+) induce degeneration in human neuronal stem cells (29).Hence, the toxicity of MPP+ was evaluated in CTX0E16 cells. As shown in supplementary Figure 2, 24 h treatment of CTX0E16 cells with MPP+ induced a decrease in cell viability that was concentration dependent with and IC50 of 2.6 mM (95 % confidence intervals: 1.9 to 3.5 mM). Consequently, in order to assess the neuroprotective effect of SPXs compounds, human neuronal stem cells were incubated with MPP+ for either 8 or 24 hours in culture, alone or in the simultaneous presence of SPXs, and the cellular morphology was observed with double staining for the cytoskeletal marker β tubulin (red) and the nuclear neuronal marker NeuN (green). Figure 5 shows that the typical staining of nuclei and neuronal processes by NeuN and β-tubulin, respectively, was drastically altered by treatment with 2 mM MPP+ for 8h. Figure 5A displays the typical pyramidal cell morphology of control CTX0E16 neurons, with processes stained in red with β-tubulin and the nuclei stained in green with NeuN. The neuronal morphology of differentiated CTX0E16 neurons was drastically altered after exposure of the cells to the neurotoxin MPP+ at 2 mM during 8 hours, which



lead to nuclei condensation and loss of cellular processes (Figure 5B). The effect of MPP+ in neuronal morphology was similar to that previously described in dopaminergic neurons and human neuroblastoma cells (28, 30). However, incubation of the cells with 50 nM 13-desMeC (Figure 5C) or 50 nM 20MeSPXG (Figure 5D) simultaneously with MPP+ somehow prevented the change in cell morphology elicited by the MPP+ treatment causing a partial reduction in the loss of neuronal processes elicited by MPP+ alone. A similar neuroprotective effect of SPX compounds against the MPP+-induced toxicity was observed when the cells were treated with MPP+ for 24 hours (Figure 6). In this case, the loss in neuronal processes elicited by MPP+ was more pronounced after exposure of the cells to the neurotoxin at 2 mM for 24 hours in culture. Figure 6 shows that the neuronal morphology of control neurons (Figure 6A) was completely altered by the MPP+ treatment (Figure 6B). Under this condition MPP+ exposure led to a complete loss of neuronal processes stained with β-tubulin (red) and nuclear condensation of β-tubulin as indicated by the overlapping fluorescence of the cytoskeletal marker β-tubulin and the nuclear neuronal marker NeuN in the nuclei of MPP+-treated cells. Also, in this case, cotreatment of the cells with either 50 nM 13-desMeC (Figure 6C) or 50 nM 13,19-didesMeC (Figure 6D) partially reverted the neurotoxicity of MPP+. In the simultaneous presence of each SPX and MPP+ neuronal processes were still observed, however the compounds were not able to fully prevent the alterations in neuronal morphology caused by MPP+ alone.

Chronic SPX treatment did not affect the expression of the neuronal differentiation and neuronal recruitment markers nestin and doublecortin In order to evaluate the effect of long term SPX treatment on human neuronal stem cells differentiation, CTX0E16 cells were exposed to the different SPX compounds for 30 days in culture. The neuronal differentiation markers evaluated were nestin and doublecortin (DCX). Nestin, the neuroepithelial stem cell protein, is a class IV and VI intermediate filament protein expressed in undifferentiated central nervous system (CNS) cells during development. Nestin is required for self-renewal of stem cells (31). Besides been frequently used as a marker of neuronal stem cells, both in embryonic and adult brain, nestin is


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expressed transiently in adult neural stem cells and immature neural progenitor cells and vanishes upon cell differentiation (32). As shown in Figure 7A, nestinband intensity was not affected after 30 days of exposure of cortical neurons to each SPX at 50 nM. Representative western blot bands showing the protein level for nestin in control cells of 45 days in vitro and in cells treated with SPX for 30 days in culture are shown in Figure 7A (left panel). The corresponding quantifications of band intensities are shown on the right. Nestin band intensity was 100 ± 0.4 (n = 7) in control cells, 100 ± 13 (n = 7; t = 0.03; df = 12) in cells treated with 20-MeSPXG, 110 ± 18 (n = 7; t = 0.56; df = 12) after treatment with 13,19-didesMeC and 102 ± 10 (n = 7; t = 0.23; df = 12) after exposure of the cells to 13-desMeC. Similarly, the neuronal marker DCX was not modified after long term exposure of the cells to SPX, as depicted in Figure 7B. DCX is a microtubule associated protein utilized as a marker for detecting the processes of neural recruitment (30). DCX facilitates microtubule polymerization and is expressed in migrating neuroblasts and immature neurons (32). Representative western blot bands for DCX immunoreactivity in control and treated cells are shown on the left panel of Figure 7B and the corresponding quantifications of the DCX band immunoreactivity are shown on the right panel. As indicated in this figure, DCX band intensity did not differ between control cells and cells treated for 30 days with each of the SPX at 50 nM. In control cells DCX band intensity was 100 ± 5, while in cells treated with 20-MeSPXG the corresponding DCX band intensity was 120 ± 16 (n = 3; t = 1.26; df = 4). Similarly, for cells treated with 13,19-didesMeC, DCX band intensity was 122 ± 13 (n = 3; t = 1.69; df = 4) and 93 ± 20 in cells treated with 13-desMeC (n = 3; t = 0.35; df = 4). Thus, the data presented here indicates that none of the SPX toxins, at 50 nM, affected the neuronal recruitment of human neuronal stem cells.

Chronic SPX treatment increased the expression of TUC4 and MAP2 In the next set of experiments, the effects of SPX on markers related to axonal and

dendritic

differentiation

were

also

evaluated.

The TUC

(TOAD-

64/Ulip/CRMP) family of proteins has been implicated in axon guidance and outgrowth. TUC4 (Ulip-1) was identified as a protein that is regulated during neuronal differentiation in the cerebral cortex and in neuronal cell lines in



response to nerve growth factor (NGF) or retinoic acid (33, 34). Furthermore, TUC4 has been initially suggested to participate in the regulation of neurite extension and branching through a mechanism that could involve membrane transport in the growth cone (34). Later on, it was demonstrated that TUC4 was sufficient and necessary for dendrite outgrowth in hippocampal neurons through the interaction with the actin cytoskeleton (35). Recently, the protein has been associated also with autism related disorders (36). Since our initial immunocytochemistry studies with tubulin suggested an increase in neuronal branching, the effect of SPX treatment on TUC4 protein expression was analyzed (Figure 8A). The left panel of Figure 8A shows representative western blot bands for TUC4 immunoreactivity in control and treated cells and the corresponding band quantifications on the right. In control cells TUC4 band intensity was 100 ± 1 (n = 6) and increased by about 20 % after treatment of the cells with 50 nM 20-MeSPXG yielding a band intensity of 119 ± 16 (n = 6) although this increase did not reach statistical significance (t = 1.18; df = 10; p = 0.26). However, treatment with 50 nM 13,19-didesMeC increased band intensity to 178 ± 26 (n = 6; t = 3; df = 10; p < 0.05). Similarly, although in lower proportion, treatment of the cells with 50 nM 13-desMeC increased TUC4 band intensity to 121 ± 6 (n = 4; t = 3.97; df = 8; p < 0.005). Since TUC4 protein expression is sufficient and necessary for dendritic growth and maturation in cultured hippocampal neurons and its overexpression increases dendritic tips, total dendritic length, spine density, and the frequency but not amplitude of miniature excitatory synaptic currents (35), this finding indicates that 13,19didesMeC and 13-desMeC probably increased dendritic arborization in neurons. In order to reinforce this observation, the effects of SPX on neuronal microtubule associated protein MAP2 levels were also evaluated. MAP2 is known to interact with both microtubules and F-actin and its role seems to be critical for neuromorphogenic processes including neurite initiation due to its ability to coordinate the reorganization of microtubules and F-actin (37). Again, CTX0E16 cells were treated with each of the three SPX toxins at 50 nM during 30 days in culture. Figure 8B shows, in the left, representative western blot bands for MAP2 immunoreactivity in control and treated cells and their corresponding band intensities on the right. In control cells band intensity was 100 ± 8 (n = 4), and it was increased to 137 ± 22 (n = 3) by treatment with 20-


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MeSPXG, although this increase did not reach statistical significance (t = 1.79; df = 5). However, the increase was 95 ± 14 % (n = 3; t = 6.35; df = 5; p < 0.005) in the presence of 13,19-didesMeC and 210 ± 82 % (n = 4; t = 2.54; df = 6; p < 0.05) in cells cultured in the presence of 13-desMeC. As indicated above, MAP2 is highly expressed in neurons where it interacts with microtubules playing a role in the initiation of neurite growth and its expression is restricted to neuronal somata and dendrites in adult neurons (37). Moreover, MAP2 expression is normally associated with neuronal differentiation (38). Therefore, our results indicate that both 13,19-didesMeC and 13-desMeC, but in less proportion the 20-MeSPXG

analogue

promoted

dendritic

branching

and

neuronal

differentiation of human neuronal stem cells. The effects of SPX on TUC4 and MAP2 are in agreement with the in vivo potency of the three compounds since earlier data has demonstrated higher toxicity for 13-desMeC than for 13,19didesmethylC and absence of toxicity for 20-MeSPXG administrated by the intraperitoneal route (14).

SPX increased cholinergic transmission in human neuronal stem cells Several reports have demonstrated that nicotinic acetylcholine receptors are the main target of SPX toxins (6, 10, 39). Recently, it has been reported that both 13,19-didesMeC and 13-desMeC blocked the acetylcholine induced currents through human α7-nicotinic receptors with IC50 (95% confidence intervals) of 0.25 (0.239–0.272) and 0.18 (0.16–0.21) nM while their IC50 for human α4β2 acetylcholine receptors were 6.26 (4.7–8.3) and 3.9 (2.9–5.1) nM respectively (6). Moreover, we have previously found that 13-desMeC abolished the decrease in α7 nicotinic receptors in primary neurons from a mouse model of Alzheimer´s

disease

without

affecting

the

expression

of

the choline

acetyltransferase enzyme (ChAT), an enzyme implicated in the final production of ACh (13). Since nAChRs are involved in stem cell differentiation and survival (21, 22), we evaluated the effect of long-term treatment of human neuronal stem cells with SPX on the cholinergic system. In order to do this, cells were exposed to 50 nM 20-MeSPXG, 50 nM 13-desMeC or 25 nM 13,19-didesMeC during 30 days in culture and the protein expression for ChAT and α7 nAChRs were evaluated. Figure 9A shows representative western blot bands for ChAT



on the left panel and the corresponding band intensity quantification on the right. As shown in Figure 9A, both 20-MeSPXG and 13-desMeC significantly increased ChAT band intensity from 100 ± 2 in control conditions to 191 ± 37 % (t = 2.45; df = 8; p < 0.05) and 150 ± 17 (t = 2.99; df = 8; p < 0.05;) while in the case of 13,19-didesMeC the ChAT band intensity was similar to control conditions yielding a value of 100 ± 13 % (t = 0.019; df = 7). With an identical treatment the effects of SPX on α7 protein expression were also evaluated. Figure 9B shows, in the left panel, representative α7 immunoreactive bands in control and treated neurons. The quantification of the α7 band intensity (right panel) yielded a value of 100 ± 3 % (n = 9) in control neurons, 127 ± 11 % (n=5; t = 2.923; df = 12; p < 0.05) in cells exposed to 50 nM 20-MeSPXG, 140 ± 14 (n = 9; t = 2.688 df = 16; p < 0.05) in neurons treated with 25 nM 13,19-didesMeC and 174 ± 19 (n=8; t = 4.11; df = 15; p < 0.001) in cells treated with 50 nM 13desMeC. In summary, the data presented here indicate that chronic SPX treatment affects human neuronal stem cell differentiation. Cyclic imine toxins are among the few organic compounds produced by dinoﬂagellates known to interact with the major neuronal nAChRs, as assessed by functional and ligand-binding assays (40). Furthermore, 13-desMeC is known to inhibit nicotine-mediated dopamine release from rat striatal synaptosomes (41) with high potency (IC50 = 0.2 nM). Our results indicate that the interaction of spirolides with multiple neuronal Ach receptors may be responsible for their effects on human neuronal cell differentiation. Thus, the expression of ChAT during brain development has been extensively used as an indicator for the development of the cholinergic system (42). Furthermore, the increase in ChAT expression elicited by SPX could be related with the effects of SPX on neurite and dendritic markers such as TUC4 and MAP2, since the increase in acetylcholine induces dendrite outgrowth in primary neuronal cultures (43). Moreover, in order to understand the effects of SPXs on neuronal differentiation, it seems necessary to point out that variations in the expression of particular nAChR subtypes profoundly influences neuronal development (42). For instance, cell survival is modulated by nAChRs, and both neurotoxic and neuroprotective effects of nAChRs


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activation, that also are influenced by the developmental state, have been described, with α4β2 nAChRs promoting neuronal survival and α7 nAChRs mediating the neurotoxic effects of the agonist nicotine (42, 44). All together, the results presented here constitute the first finding indicating that prolonged nicotinic receptor blockade elicited by SPX influences human neuronal development increasing the number of α7 nAChRs, and the expression of acethylcholine synthesizing enzyme ChAT, a fact that can account for the increase in the expression of neuritic and dendritic markers. Overall, the data presented here constitute the first report on the long term effects of nAChRs antagonists on human neuronal stem cells. The main effects of this group of toxins were the decrease in the neuronal damage induced by the neurotoxin MPP+, the increase in neuritic and dendritic neuronal markers and in the expression of α7 nAChRs and presumably in the acetylcholine levels due to the increase in the expression of the acetylcholine synthesizing enzyme ChAT. Therefore, these findings reinforce the idea that SPX are attractive lead molecules for the development of therapies against neurodegenerative disorders.

Methods Cortical human neural stem cell line The human neuronal stem cells line, CTX0E16, was obtained from the cerebral cortex of a fetus of 12 weeks of gestation and immortalized by the ectopic expression of the c-mycERTAM transgene and kindly provided by a material transfer agreement with ReNeuron Limited (Guildford, Surrey GU2 7AF, U.K). Human neural progenitor cells CTX0E16 hNPCs were cultured following the provider instructions as previously reported (16). Briefly, proliferating cells were maintained in reduced modified medium (RMM) containing DMEM:F12 with 15 mM HEPES and sodium bicarbonate (Sigma) supplemented with 0.03% human serum albumin (Sigma), 100 µg/ml apotransferrin (Scipac Ltd , Kent, UK), 16.2 µg/ml putrescine (Sigma), 5 µg/ml human insulin (Sigma), 60 ng/ml progesterone (Sigma), 2 mM L-glutamine (Sigma) and 40 ng/ml of sodium selenite (Sigma). Under proliferative conditions cells were cultured in the



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presence of 10 ng/ml of human fibroblast growth factor (FGF2), 20 ng/ml of human epidermal growth factor (EGF), both from PeproTech, Rocky Hill, NJ and 100 nM hydroxy tamoxifen (4-OHT, Sigma). CTX0E16 hNPCs were seeded onto Poly-D-lysine (PDL, 5 µg/cm2, Sigma,) and laminin-coated (1 µg/cm2; Sigma) tissue culture flasks, with full media changes occurring every 2–3 days. Cells were passaged once they reached 70–80 % confluence using accutase (Sigma) and maintained between 25 and 30 passages; all experiments were carried out using cells from passages 12 to 30. Neural progenitor cell differentiation For

differentiation

CTX0E16

cultures

were

washed

twice

with

non-

supplemented DMEM:F12 medium and passaged onto PDL and laminin-coated tissue culture plates or glass coverslips at a density of 50,000 cells per ml. Cells were

then

washed

in

warm

Dulbecco’s

phosphate-buffered

saline

(DPBS;Thermofisher) and maintained in neuronal differentiation media (NDM: Neurobasal Medium (Thermofisher) supplemented with human serum albumin, apotransferrin, putrescine, human insulin, progesterone, L-glutamine, and sodium selenite at the concentrations used for proliferation and 1 × B27 serumfree supplement (Thermofisher). Half medium changes were performed every 2–3 days and cultures were differentiated for up to 60 days. Chemicals and solution Plastic tissue-culture dishes were purchased from Thermofisher (Madrid, Spain). The marine phycotoxins SPX, available as quality controlled standards (13-desMeC, 13,19-didesMeC and 20-MeSPXG) were obtained from CIFGA laboratory (Lugo, Spain). All toxins had purity higher than 99%. The final concentration of compound solvent (methanol with trifluoroacetic acid 0.05% v/v) was less than 0.5%. All other chemicals were of reagent grade and purchased from Sigma. Chronic SPX treatment Cell cultures differentiated during 15-21 days were treated with the SPX toxins at the concentrations indicated in the text and figure legends with half medium ACS Paragon Plus Environment

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change every two or three days and full medium change every 7 days. In all the treatments the solvent concentration was less than 0.5% and added to the control wells. Toxin treatment was maintained during 5 days for viability experiments and during 30 days for the rest of the experiments. Fresh toxins were added in each medium change. Determination of cellular viability Cell viability was assessed by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide) test as previously described (45). This test evaluates the ability of mitochondrial dehydrogenases to reduce the MTT dye and there is a linear correlation between mitochondrial function and the cell ability to reduce the MTT (46). For viability assays cells differentiated during 30 days were cultured in 48 well plates, and treated with 13-desMeC, 13,19didesMeC or 20-MeSPXG at concentrations ranging of 1 nM to 100 nM for 5 days. After treatment, cells were incubated with a solution of 500 µg/ml of MTT dissolved in Locke’s buffer containing (in mM): 154 NaCl, 5.6 KCl, 1.3 CaCl2, 1 MgCl2, 10 HEPES, and 5.6 glucose, (pH 7.4), for 1 hour at 37 ºC. After washing off excess MTT, cells were disaggregated with 5% sodium dodecyl sulfate overnight, and the absorbance of the colored formazan salt was measured at 595 nm in a spectrophotometer plate reader. DMSO at 10% was used as death control and its absorbance was subtracted from the other data. Immunocytochemistry For immunocytochemistry, control and SPX treated cells were fixed in 4% formaldehyde and subsequently labeled with primary antibodies for β3 tubulin (MAB1637: 1:500), NeuN (ABN78, 1:1000) or glial fibrilar acid protein (GFAP: AB5804, 1:1000) all from Millipore and fluorescent secondary antibodies. Primary antibodies were added in Phosphate Buffered Saline (PBS) containing 2.5 % BSA overnight at 4 °C, followed by 3 washes in PBS. Afterwards, cells were incubated with fluorescent secondary antibody (Thermofisher) diluted in PBS with 2.5% BSA for one hour at room temperature. After washing, the coverslips were then mounted in 50% glycerol dissolved in phosphate buffered saline and sealed with nail polish. Cells were analyzed using a confocal laser



Page 18 of 38

scanning microscope (Nikon, Mellville, NY) with a Hamamatsu ORCA-ER camera (Hamamatsu Photonics KK, Japan). Immunocytochemistry controls for the specificity of the detection methods were carried out by omitting the incubation step with the primary antibodies in one of each four consecutive coverslips. None of the coverslips run without primary antibody showed specific labeling comparable to that obtained with the primary antibodies. For confocal analysis, all laser parameters were first adjusted in control neurons and left unchanged for the analysis of the corresponding SPX treated neurons. Western blotting Cell cultures were maintained for 30 days in the presence of toxins, Afterwards, the cells were washed three times with cold PBS and cell lysates were prepared in RIPA lysis buffer (Thermofisher), containing 25 mM Tris-HCl (pH 7.6), 150 mM

NaCl,

1%

NP

(nonyl

phenoxypoliethoxylethanol)-40,

1%

sodium

deoxycholate and 0.1% SDS supplemented with commercial phosphatase and protease inhibitors (Halt Phosphatase Inhibitor Cocktail and Halt Protease Inhibitor Cocktail Kit, respectively, Thermofisher), and stored at -20ºC when needed. Total protein concentration of each lysate was determined in triplicate by the Bradford assay, using BSA as standard. Samples of cell lysates containing 25 µg of total protein were denaturalized in commercial 4x Laemmli sample buffer (BioRad, containing 277.8 mM Tris-HCl, pH 6.8, 4.4 % SDS, 44.4% glycerol, and 0.02 % bromophenol blue, supplemented with 2.5% 2mercaptoethanol), resolved by SDS-PAGE electrophoresis in 4-20% or 10% polyacycrilamide gels at a constant voltage of 200 V for 45 minutes and transferred from the gel to nitrocellulose or PVDF membranes at a voltage of 10 V for 30 min using a semi-dry transfer cell (Biorad). The membranes were blocked for 1 hour with 5% non fat milk or 5 % BSA, following the antibody manufactures recommendations, and incubated overnight at 4 ºC with the primary antibodies listed in Table 1. The immunoreactive bands were detected using the supersignal west pico or fento chemiluminescent substrate (Pierce) and the Diversity 4 gel documentation and analysis system (Syngene, Cambridge, UK). Chemiluminescence was measured with the Diversity GeneSnap software (Syngene). β-actin was used as control for lane loading


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and to normalize chemiluminescence values. Each condition was analyzed in duplicate per each experiment, and at least four experiments were performed per antibody. Statistical Analysis Student’s t-test was used to assess significant differences between control and SPX treated neurons. All values are expressed as mean ±SEM of at least 3 independent experiments. P values

In Vitro Effects of Chronic Spirolide Treatment on Human Neuronal

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