Effect of Inammatory Stress on Neural Stem Cells Behaviour: A Rescue Effect of Phosphatidylcholine by Modulating Neuronal Plasticity.

The balances between NSCs growth and differentiation, and between glial and neuronal differentiation play a key role for brain regeneration after any pathological conditions. It is well known that the nervous tissue shows a poor recovery after injury due to the factors present in the wounded microenvironment, particularly inammatory factors, that prevent neuronal differentiation. Thus, it is essential to generate a favourable condition for NSCs and conduct them to differentiate towards functional neurons. Here, we show that neuroinammation has no effect on NSCs proliferation but induces an aberrant neuronal differentiation that gives rise to dystrophic, non-functional neurons. This is perhaps the initial step of brain failure associate to many neurological disorders. Interestingly, we demonstrate that phosphatidylcholine (PtdCho)-enriched media enhances neuronal differentiation even under inammatory stress by modifying the commitment of post-mitotic cells. The pro-neurogenic effect of PtdCho increases the population of healthy normal neurons. In addition, we provide evidences that this phospholipid ameliorates the damage of neurons and, in consequence, modulates neuronal plasticity. These results contribute to our understanding of NSCs behaviour under inammatory conditions, opening up new venues to improve neurogenic capacity in the brain.


Introduction
Despite its diverse presentation, in ammation is a common feature across several neuropathological processes and has been implicated as a critical mechanism responsible for the progression of neurodegenerative disorders including Parkinson's disease, Alzheimer's disease multiple sclerosis 1, 2, 3 as well as traumatic brain injury 4, 5 and stroke 2, 6, 7 . Neuroin ammation is considered a double-edged sword, with protective as well as detrimental effects on the nervous system, especially during repair and recovery. In response to different types of injuries that cause neurons and oligodendrocytes death, activated astrocytes and the resident immune-like glial cells, the microglia, proliferate and generate proin ammatory cytokines (such as IL-1, IL-6, IFN-γ and TNF-α), chemokines, prostaglandins, and free oxygen radicals, often leading to the development of cerebral damage, and promoting macrophages in ltration 8 . Both kinds of cells act as a host defence mechanism eliminating cellular debris and releasing in ammatory factors. These factors nally activate astrocytes, which proliferate and form the glia scar to de ne a dense limiting border between the healthy and damaged tissue. Two major niches of neural stem cells (NSCs) that support neurogenesis are in the subventricular zone and in the dentate gyrus of the hippocampus of the adult mammalian brain 9,10 . NSCs are multipotent self-renewing cells that have a regenerative potential because they can proliferate, migrate and differentiate into neurons, astrocytes or oligodendrocytes and thus, promote functional and structural repair of the injured tissue.
Several studies have evidenced a cross-talk between immune modulators and NSCs fate 11,12,13 . In response to in ammatory reactions, it was shown that the glia scar could prevent tissue regeneration by NSCs 8 , and that LPS-induced neuroin ammation caused synapse loss by a mechanism dependent of microglia activation and IL-1β secretion 14 . In this scenario, understanding the NSCs response to these conditions and mechanisms involved in the integration into the injured brain will be critical for the development of effective therapeutic strategies using stem cells.
We have previously demonstrated that phospholipids affect the fate of post-mitotic neural precursors; speci cally, phosphatidylcholine (PtdCho) promotes neuronal differentiation at expenses of astroglial and unspeci ed precursors 15 . As the loss of neurons is the detrimental outcome of brain injuries and neurodegenerative diseases, we asked whether PtdCho could still favour neuronal differentiation under in ammatory conditions, and thus prevent or restore tissue damage in this context. By different approaches we have demonstrated that under pro-in ammatory culturing conditions there is an increase in neuronal differentiation that could support a renewal mechanism needed for tissue reparation.
Strikingly, under the same conditions, neurons also display an aberrant morphology that could re ect the deleterious effect of neuroin ammation. Interestingly, addition of liposome of egg-source PtdCho further induces neuronal differentiation and also rescues the morphological and functional de cit by modulating neuronal plasticity.

Effect of in ammatory stress on NSCs proliferation
The balance between NSCs proliferation and differentiation is essential for tissue repair 16 , and up to know is not clear how it is affected by in ammation. To that end, we incubated NSCs under normal proliferative condition (in the presence of EGF and FGF, neurosphere culture) supplemented with different concentrations (% V/V) of macrophages-activated media (AM) or with media without activation as a control (UM). We con rmed by RT-PCR that macrophages activated with LPS express and, as a consequence, secrete IL-1β, IL-6 and TNF-α to the media as previously demonstrated 17,18 (Supplementary gure 1A). Treated cells were incubated for 96 hours, and after this time, cell viability was analysed by MTT assay; UM plus LPS was also included to evaluate LPS toxicity as control (Supplementary gure 1B). To induce a moderate stress, cells were treated with AM (20% V/V) and, after 96 hours, the neurosphere's diameter was measured as a growth parameter ( Figure 1B). As Figures 1A, B and D show, there is no signi cant change in the proliferation rate in the presence of different concentrations (V/V) of activated media respect to media without activation and control. To evaluate the role of each IL individually, similar analyses were performed in UM supplemented with IL-1β and/or IL-6. Effect of in ammatory stress on neuronal differentiation of NSCs Neuronal differentiation is key in neural tissue regeneration after injuries 19 . To investigate this process under pro-in ammatory conditions, we analysed neuronal differentiation by immunocytochemistry using βIII-tubulin as neuronal lineage marker. Cells were incubated for 72 hours in media supplemented with macrophages-activated media (AM-20% V/V), media without LPS activation (UM-20% V/V) or control media. The quanti cation analysis demonstrated that in the presence of AM there is a signi cant increase in the percentage of neurons in comparison with the UM or the control (Figure 2A). Similar results were observed at shorter time points (Supplementary gure 2). However, a morphological observation revealed that neurons incubated with AM display a different shape than neurons at the control or neurons incubated with UM, with apparent tubulin disorganization, dystrophy with increased soma size ( Figure 4) and presence of vacuoles 20,21 . We theref ore hypothesized that neuronal differentiation could be aberrant under pro-in ammatory stress, leading to cell dystrophy. To determine whether the observed effect is a direct action of LPS or the in ammatory components (IL-1β and IL-6), we evaluated neuronal differentiation in the presence or absence of each molecule individually. As Figure 3 shows, treatment with UM supplemented with the indicated concentration of LPS, IL-1β and/or IL-6 did not affect the rate of neuronal differentiation nor the morphology of the neurons.

Phosphatidylcholine enhances neuronal differentiation and ameliorates neuronal alterations caused by in ammatory conditions
We have previously demonstrated that PtdCho, as liposomes supplemented in the media, regulates the fate of post-mitotic precursor cells, inducing neurogenesis 15 . To test the effect of this molecule under proin ammatory stress conditions, we incubated NSCs under each condition in the presence of egg-source PtdCho (50 μM) and counted the resulting βIII-tubulin expressing cells. As shown in Figure 2A, the proneurogenic effect of PtdCho shown in normal and UM control conditions, is also observed under in ammation, reaching the highest levels of neuronal differentiation. Interestingly, the aberrant phenotype observed in AM was ameliorated when cells were incubated in the presence of PtdCho ( Figure 2B, lower panel). A detailed morphometric analysis demonstrated that the soma size was restored in the presence of PtdCho ( Figure 4A and B). More interestingly, the percentage of dystrophic neurons, decreases with PtdCho treatment, resulting in a signi cant increase in the phenotypically normal neurons ( Figure 4B) and suggesting that under in ammatory conditions, PtdCho not only regulates the fate of post-mitotic cells increasing neuronal differentiation 15 , but also rescues the dystrophic neurons, turning its morphology back to the normal.

PtdCho restores synaptic defect caused by in ammation
We next examined whether the observed defect in neuronal morphology is associated with alteration in neuronal functionality. The amount and localization of synaptophysin protein plays a critical role in synapse formation 22,23 , exo-endocytosis of synaptic vesicles 24 , neural plasticity 25,26 , memory 27 , motor development, behavioural features and cognitive impairments 28 . Neurons were immunostained for the presynaptic protein synaptophysin, and the relative number of signal puncta on neurites was counted and analysed by western blot. As Figure 5 shows, incubation with AM clearly decreases the number of synaptophysin-containing vesicles and its levels of expression relative to the control. Interestingly, incubation with PtdCho restores the level of expression and location of synaptophysin under in ammatory conditions. This result clearly suggests that PtdCho has two effects: induces neurogenesis and improves function of sick/damaged neurons.
We have previously demonstrated that lipid treatment 24 hours after plating the cells did not affect neuronal differentiation, indicating a narrow time-window of response in post-mitotic cells 15 . To con rm the effect of PtdCho independent of the promotion of neurogenesis, we quanti ed the percentage of neurons and the morphology adding PtdCho 24 hours post in ammatory condition. As expected, PtdCho does not increase the percentage of neurons ( Figure 6A) 15 , but clearly altered the balance between healthy/normal neurons and dystrophic, pushing it to the normal population ( Figure 6B).

Discussion
NSCs have a fundamental role after nervous tissue damage as they have the potential for regeneration owing to their capacity of self-renewal and differentiation into neurons 8, 29 . However, this extraordinary capacity is limited under pathological conditions due to the factors present in the wounded microenvironment that can affect NSCs survival, proliferation and differentiation 30,31,32,33 .
In this report, we provide details of the NSCs behaviour under in ammatory condition, a common scenario of many acute and chronic brain diseases. Furthermore, we provide evidence that PtdCho treatment could target NSCs conducting them towards functional neurons and also restoring the morphological de cit caused by in ammation.
Neuroin ammation can either affect the niche or the NSCs directly, with the end result of altered NSCs proliferation and/or differentiation 1,34 . We demonstrated that after incubation of NSCs with AM or with individual cytokines, cell viability ( Supplementary Fig. 1) and the rate of NSCs proliferation are not affected (Fig. 1). These results differ from previous demonstrating that proin ammatory cytokines reduce the number of new born neurons in the dentate gyrus in adult mice due to the restrain of the cell cycle 35 .
As NSCs proliferation depends on the cell progression, we assumed that under our study condition cell cycle progression is not affected. In addition, the observed discrepancy could base on the different origin of the NSCs utilized.
The in deep study of the cellular mechanism leading to neuronal dysfunction under in ammatory condition is essential for the development of novel therapies. These experiments demonstrated that incubation of NSCs with 20% V/V of AM, but not with ILs individually (Fig. 3), induces aberrant neuronal differentiation, that give rise to dystrophic neurons (Fig. 2). The relatively constant number of neurons in AM-treated cultures during different periods of times (day 1 to day 3) (Supplementary Fig. 2) suggests that AM does not affect speci c step of neuronal differentiation process. Rather, it seems to be a very dynamic sequence of morphological changes with a constant progression to dystrophic morphology ( Fig. 2 and Supplementary Fig. 2). It is well known that LPS activates microglia, and the consequent ILs secretion affects neuronal differentiation 36, 37, 38 , we discard this effect as ILs and LPS alone did not affect neuronal differentiation of NSCs, nor the morphology of the neurons (Fig. 3).
Currently, lipids are taking a leading role in the nervous system. They have been shown to intervene in cellular functions such as proliferation, differentiation, cell cycle and act as pro-resolution lipid mediators in in ammatory events 15,39,40,41,42 . As this maladaptive neuronal plasticity that takes place under in ammation could be the reason of many brain failures, we evaluated the effect of PtdCho on NSCs differentiation. We demonstrated that PtdCho induces neuronal differentiation under in ammatory condition increasing the percentage of healthy non-dystrophic neurons (Fig. 4). Therefore, PtdCho changes the fate of post-mitotic cells increasing neurogenesis by turning on the PKA/CREB signalling pathway even under in ammatory conditions (AM). In fact, the percentage of βIII-tubulin positive cells decrease in the presence of PKA inhibitor (KT5720) (Supplementary Fig. 3). This speci c effect of PtdCho could favour and increase the replacement of damaged neurons favouring NSCs-dependant neurogenesis. More interestingly, this phospholipid ameliorates the damage of neurons and, in consequence, modulates neuronal plasticity: in fact, treatment with PtdCho even 24 hours post in ammatory condition, restores the soma diameter and increases synaptophysin expression and distribution in neurons (Figs. 4 and 5). Hence, it decreases the amount of dystrophic neurons by a mechanism independent of PKA activity and NSCs differentiation ( Fig. 6 and Supplementary Fig. 3). This capacity to modulate neuronal plasticity was also described for choline in the treatment of Rett  Fig. 3); however, PtdCho activates neurogenesis even under inhibition of these enzymes. This results clearly demonstrate that choline need to be converted in PtdCho to affect the fate of NSCs.
In conclusion, more research need to be done to understand the molecular mechanism of PtdCho as modulator of neuronal plasticity, but, considering that loss and damage of neurons are the major consequence of acute and chronic neuroin ammation, these results might open a door to develop new therapeutic approaches. Louis, MO, USA). As speci ed in product information, they have a purity over 99% and a fatty acid content of approximately 33% palmitic, 13% stearic, 31% oleic, and 15% linoleic. In addition the detailed fatty acid composition of the mixture of egg yolk phosphatidylcholine and phosphatidylethanolamine has been recently described 50, 51 . Animal studies and fetal neural stem cells culture.
All animal experiments and related experimental protocols were approved by the Bioethics Commission for the Management and Use of Laboratory Animals of National University of Rosario, Argentina (N 6060/89). All procedures were carried out in accordance with the approved guidelines (Guide for the care and use of Laboratory Animals-8° edition-e National Academies Press-Washington DC 2011 and in compliance with the ARRIVE guidelines). Time pregnant female C57/BL6 mice (gestation day 13) were sacri ced by cervical dislocation under supervision of the Animal Care and Use Committee. Neurospheres were obtained from E13 cortical cells as previously described 52 . Brie y, the lateral portion of the dorsal telencephalon (cortex) of embryonic day 13 mouse C57/BL6 was isolated. The cortices were chemically disrupted adding trypsin (0.05% w/v) for 5 minutes and then mechanically disrupted into single cells by repeated pipetting in medium DMEM/F12 (1:1) containing 10% FBS, penicillin G (100 units/ml) and streptomycin (100 μg/ml). Cells were centrifugated at 1000 rpm for 5 min and the pellet resuspended in serum-free medium DMEM/F12 (1:1). Dissociated cells were cultured at a density of 5 × 10 4 cells/ml in medium DMEM/F12 (1:1) supplemented with B27, 10 ng/ml bFGF and 10 ng/ml EGF, at 37 °C in a humidi ed 5% CO 2 incubator. Within 7 days, cells grew as free coating neurospheres that were then collected by centrifugation, and chemically and mechanically dissociated to obtain a new passage. For cell differentiation, neurospheres were chemically and mechanically dissociated. After counting, 2.5 10 5 cells were plated on poly-D-lysine (PDL) (10 μg/ml)-coated 24 well plates, or 5 × 10 4 cells were plated on PDL (10 μg/ ml)-coated 96 well plates in medium DMEM/F12 (1:1) supplemented with B27.
Macrophages culture and LPS-induced stimulation.
The mouse cell line Raw 264.7 (ATCC® TIB-71™) was cultured in DMEM 10% FBS supplemented with penicillin G (100 units/ml), streptomycin (100 μg/ml) (proliferation conditions) and maintained in a 5% CO 2 humidi ed incubator at 37 °C. For activation, cells were grown to 80% con uence in petri dishes with DMEM medium supplemented with 10% FBS. At this time, cells were centrifuged at 1500 rpm for 10 minutes. The cell pellet was resuspended in 1 ml of DMEM/F12 medium and cells were transferred to a new plate containing DMEM/F12 stem cell medium (in the absence of FBS, B-27 and growth factors). For stimulation, pure LPS was added in a nal concentration of 1 µg/ml. After 18 hours of incubation, cells were centrifugated at 1000 rpm for 5 minutes and the culture medium was ltered through 0.22 μm lters (Sartorius) and stored immediately at -80 °C.
Total RNA isolation, DNase treatment and retrotranscription reaction Murine macrophage RAW 264.7 total RNA was extracted in Quick-Zol (Kalium) following the supplier's speci cations. Brie y, cells were resuspended in 1 ml Quick-Zol and incubated for 5 minutes at room temperature. Then, 0.2 ml of chloroform was added and they were centrifuged at 12,000 rpm for 10 minutes at 4 °C. Then, the aqueous phase was transferred to a new tube, 0.5 ml of isopropanol was added and the samples were incubated for 24 hours at -20 °C. The following day, they were centrifuged at 12,000 rpm for 10 minutes at 4 °C, the pellet was washed with 75% ethanol and centrifuged again at 12,000 rpm for 5 minutes at 4 °C. To evaluate the quality and quantity of the RNA obtained, absorbance measurements were made at 230, 260 and 280 nm (NanoVue Plus, General Electrics). Next, 1 µg of RNA was seeded on a 1.8% agarose gel to evaluate its integrity. To remove DNA from the samples, 10 μg of the RNA / DNA mixture was treated for 2 hours with the RNase-free RQ1 DNase enzyme (Promega) at 37°C . The reaction was then stopped by incubating the sample at 65 °C for 10 minutes. Once the treatment was completed, the RNA concentration was determined spectrophotometrically (NanoVue Plus, General Electrics). Cell viability and proliferation assays Cell viability was assessed by MTT-reduction assay. After cell treatment, MTT (5 mg/ml) was added to the cell culture medium at a nal concentration of 0.5 mg/ml and incubated for 4 hours at 37°C, 5% CO 2 .
The assay was stopped by replacing the MTT-containing medium with DMSO. The extent of MTT reduction was measured spectrophotometrically at 570 nm 53 . Results are expressed as a percentage of the control.
Proliferation of NSCs was assayed by measuring neurosphere's diameter 54 . Brie y, 5000 living cells were seeded per well in 24-well plates and cultured for up to 96 hours to evaluate the expansion rates. Size of 100 neurospheres (expressed as neurospheres diameters) was measured in three independent experiments. Images were taken with a microscope Olympus BH-2 and analysed using the freeware image J (National Institutes of Health, freeware).

Liposome preparation
Concentrated lipid stocks were prepared as previously described 55 . Brie y, pure lipids were diluted in chloroform and dried in acid-washed glass centrifuge tubes under a stream of nitrogen. Phospholipid samples were suspended at 2-6 mM in phosphate-buffered saline at pH 7.2 and sonicated twice for 5 min at power setting 0.2-0.5% amplitude. All samples were sterilized with 0.22 μm-pore lters (Sartorius).
The recovery of phospholipids after ltration was typically 90% or more.
After different time of incubation, cells were xed in 4% (w/v) paraformaldehyde-sucrose for 30 min at room temperature, permeabilized with 0.2% Triton X100 and blocked for 1 hour in 5% BSA. Cells were incubated with the primary antibody overnight at 4 °C followed by incubation with the uorescently labelled secondary antibody for 1 hour at room temperature. Primary and secondary antibodies were diluted as follows: rabbit anti-βIII-tubulin (1:500) mouse anti-synaptophysin (1:300), anti-rabbit Alexa Fluor ® 488-labeled (1:500) and anti-mouse Cy3-labeled (1:300). To visualize nuclei, cells were counterstained and mounted with ProLong Gold antifade reagent containing DAPI (Molecular probes, Life technologies).

Microscopy and Image Analysis
Micrographs were acquired using a confocal microscope (Zeiss LSM 880) or the Nikon Model Eclipse 800 microscope and quantitative analyses were performed with Image J" (NIH). Cells were counted from twenty randomly selected elds per well for each individual experiment. At least three independent experiments were performed. The percentage of neuronal cell population was calculated against the DAPI-positive total cell number which includes undifferentiated stem cells and differentiated neurons. Cells bearing at least one neurite equal or longer than the soma diameter were considered to be differentiated. Soma size and number of synaptophysin containing vesicles were measured and counted manually. To differentiate dystrophic and normal neuronal populations, 100 neurons from each experiment were manually selected and analysed.

Western blot analysis
Western blot experiment was developed following protocols previously described 15,42 . Neurospherederived cells were plated at a density of 1.5 × 10 5 and cultured on PDL-coated 35 mm culture dishes under differentiation conditions. Three days later, cells were collected, resuspended in lysis buffer (   NSCs differentiation is affected by in ammation and restored by PtdCho. a) Percentage of β-III tubulin positive cells analysed by immunocytochemistry coupled to uorescence microscopy of NSCs exposed to 20% V/V of AM and UM in the presence or in the absence of PtdCho (50 μM) during 72 hours. Graph represents the percentage of neuronal differentiation measured in ve independent experiments. Data were presented as mean ± SEM. ***p < 0.001; **p < 0.01; *p < 0.05. b) Representative images (40X) of the immuno uorescence assays with the neuronal marker (β-III Tubulin, green) nuclei (DAPI, blue). Yellow arrow indicates normal neuron and red arrow indicates dystrophic neuron. Scale bars: 20 μm.

Figure 3
NSCs differentiation is not affected by ILs. a) Percentage of β-III tubulin positive cells analysed by immunocytochemistry coupled to uorescence microscopy of NSCs exposed 72 hours to 20% V/V of UM supplemented with the indicated ILs (50 ng/ml) or LPS (1 µg/ml). Graph represents the percentage of neuronal differentiation measured in three independent experiments. b) Representative images (40X) of the immuno uorescence assays with the neuronal marker (β-III tubulin, green) and nuclei (DAPI, blue).
Scale bars: 1 μm. c) Representative image of Western Blot showing synaptophysin level in NSCs exposed to 20% V/V of AM in the presence or in the absence of PtdCho (50 μM) during 72 hours. The gels/blots displayed here are cropped, and without high-contrast (overexposure). The full length gels and blots are included in a Supplementary Information le. Densitometric analysis, *p < 0.05 (Student's T-test).

Figure 6
PtdCho restores morphology of dystrophic neurons. a) Percentage of β-III Tubulin positive cells analysed by immunocytochemistry coupled to uorescence microscopy of NSCs exposed to 20% V/V of AM when PtdCho was added later on, after 24 h of culture and incubated for 72 hours. Graph represents the percentage of neuronal differentiation measured in three independent experiments. Data were presented