Biosynthesis of Astrocytic Trehalose Regulates Neuronal Arborization

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Biosynthesis of astrocytic trehalose regulates neuronal arborisation in hippocampal neurons Giuseppe Martano, Laura Gerosa, Ilaria Prada, Giulia Garrone, Vittorio Krogh, Claudia Verderio, and Maria Passafaro ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.7b00177 • Publication Date (Web): 10 Jul 2017 Downloaded from http://pubs.acs.org on July 11, 2017

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ACS Chemical Neuroscience

Biosynthesis of astrocytic trehalose regulates neuronal arborisation in hippocampal neurons

Giuseppe Martano†*, Laura Gerosa†, Ilaria Prada†,‡, Giulia Garrone§, Vittorio Krogh§, Claudia Verderio†,∥, Maria Passafaro†*

Affiliations † Institute of Neuroscience, CNR, Via Luigi Vanvitelli, 32, I-20129, Milan, Italy ‡ Department of Medical Biotechnology and Translational Medicine, Università degli Studi di Milano, Via Luigi Vanvitelli, 32, I-20159, Milan, Italy § Fondazione IRCCS Istituto Nazionale dei Tumori, Via Giacomo Venezian, 1, I-20133, Milan, Italy ∥ IRCCS Humanitas, Via Manzoni 56, I-20089, Rozzano, Italy.

ACS Paragon Plus Environment


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Abstract Trehalose is a non-reducing disaccharide that has recently attracted much attention because of its ability to inhibit protein aggregation, induce autophagy, and protect against dissections and strokes. In vertebrates, the biosynthesis of trehalose was long considered absent due to the lack of annotated genes involved in this process. In contrast, trehalase (TreH), which is an enzyme required for the cleavage of trehalose, is known to be conserved and expressed. Here, we show that trehalose is present as an endogenous metabolite in the rodent hippocampus. We found that primary astrocytes were able to synthesize trehalose and release it into the extracellular space. Notably, the TreH enzyme was observed only in the soma of neurons, which are the exclusive users of this substrate. A statistical analysis of the metabolome during different stages of maturation indicated that this metabolite is implicated in neuronal maturation. A morphological analysis of primary neurons confirmed that trehalose is required for neuronal arborisation.

Keywords hippocampus, metabolism, neurodevelopment, neuroglia, trehalase, trehalose


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Introduction Trehalose

(InChIKey:

HDTRYLNUVZCQOY-LIZSDCNHSA-N)

is

a

non-reducing

disaccharide consisting of two glucose molecules that are linked by glycosidic linkage. Trehalose naturally occurs in several species in all biological kingdoms.1 In animals, the biosynthesis of trehalose was first reported in insects2 but not in vertebrates,3 even though trehalase (TreH, EC 3.2.1.28), which is an enzyme required for the cleavage of trehalose, was observed in different organs from various species. In humans, significant amounts of TreH were detected in the gastrointestinal tract,4 and its expression in other organs, such as the kidney5 and cerebral cortex,6 was lower. Trehalose is commonly consumed in the diet due to its natural presence in food (e.g., algae and mushrooms), and it is added to manufactured food products, medications and cosmetics. The role of gastrointestinal TreH is to break down and absorb trehalose.7 Mutations in the gene encoding TreH are reported to cause dysfunctions that are characterized by abdominal discomfort, bloating, diarrhoea, vomiting, and gas. This condition is commonly known as TreH deficiency.8 In contrast, there is no biological explanation for the role of TreH in other organs, and the potential function of TreH is currently unknown. However, trehalose has been widely used in investigations of neurological disorders during the previous decade mainly due to its ability to activate mechanistic Target Of Rapamycin (mTor)-independent autophagy9 and diminish protein aggregation.10 Its applications appear to be beneficial for the mitigation of several mechanisms that are deregulated in neurodegenerative diseases, e.g., by correcting maternal diabetes-induced neural tube defects,11 prolonging the survival of moto-neurons in amyotrophic lateral sclerosis models12 and accelerating the clearance of mutant huntingtin.13 However, the physiological role of this molecule has never been explored. In this study, we used multiple cellular and molecular biology techniques combined with a Liquid Chromatography Mass Spectrometry (LC-MS)-based metabolomics analysis to investigate the potential physiological role of trehalose in rodent brains. Here, we report that the enzyme TreH was present in different brain regions, and its highest expression was observed in the



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hippocampus and cortex. Furthermore, we inspected the metabolome of the hippocampus and found that trehalose was present as an endogenous metabolite that was used by neurons during neuronal maturation. Results and discussion Identification of trehalose in the hippocampal metabolome To investigate the hypothesis that trehalose can be biosynthesized in the mammalian brain, we analysed the metabolome of mouse hippocampi using a previously developed method based on highresolution MS.14 Metabolites were extracted from hippocampus and analysed using liquid chromatography coupled with electrospray ionization high-resolution MS (LC-ESI-HRMS). The acquisition was performed in the negative mode, enabling the detection of negatively charged molecules. The disaccharide was observed in all extracted samples from six different mice hippocampi (Figure 1A), and the natural isotopic distribution (Figure 1B) was consistent with the predicted isotopic distribution of C12H22O11. The samples were then spiked with

13

C uniformly for the labelling and re-

measuring of trehalose. The relative extracted ion chromatograms (EICs) of the disaccharide in the sample ([M-H]- = 341.1089) and the EICs of the spiked standard ([M-H]- = 353.1492) had the same retention time and peak geometry. Since the known trehalose biosynthetic pathways are associated with glycogen or intermediates involved in the glycogen pathway,15 such as uridine diphosphate (UDP)glucose,16 and glycogen storages are present in astrocytes but not in neurons,17 we investigated whether astrocytes were capable of releasing the disaccharide. The astrocyte culture medium was replaced on day in vitro (DIV) 14 with a new low serum medium, and the culture was incubated for 24 h at 37 °C. The conditioned supernatants were collected and directly injected into the LC-MS for analysis. We used (i) a medium incubated without the astrocytes (ii) and a medium supplemented with 50 µM propranolol, which is a non-selective beta blocker18 that also blocks the glycogen pathway,19 as controls. We found that trehalose was released by the astrocytes, although the quantified concentrations


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were highly variable (Figure 1C). Consistent with the hypothesis that this metabolite was linked to the glycogen pathway, we observed that its release was inhibited by propranolol (p = 0.025). A viability assay using calcein and propidium iodide was performed to ensure that the selected propranolol concentrations did not alter the number of metabolically active astrocytes (Figure S1A-S1C). Indeed, no significant differences were observed between the two conditions in term of viability (p = 0.9414). Then, we examined the intracellular levels of trehalose in hippocampal neuronal cultures, in which glial cells account for approximately 5-10% of the whole in vitro population. We found that the intracellular level of trehalose was significantly higher (p < 0.001) on DIV 7 than that at any other tested time point (Figure 1D). [Figure 1 about here] TreH expression and localization To determine whether trehalose could be used as a trophic substrate and whether it was a waste product of the glycogen pathway, we assessed the presence of TreH in different brain regions in the adult rat brain. The TreH quantification using a Western blot analysis revealed that this enzyme was predominantly present in the hippocampus and cortex and was less expressed in other regions, i.e., the cerebellum and olfactory bulb (Figure 2A and 2B). We repeated the experiment using astrocyte culture lysates and cortex homogenate as a control. Surprisingly, TreH was not detected in the lysate (Figure 2C). In astrocytes, the ability to release trehalose along with the absence of TreH suggest that this metabolite may be preferentially used by other cell types in the tissues, i.e., neurons. To explore this hypothesis, we immunostained hippocampal neuronal cultures for TreH, Microtubule-associated protein 2 (Map2), which is a neuronal marker, and Glial Fibrillary Acidic Protein (GFAP), which is a glial marker. We found that the enzyme was observed in neurons (Map2-positive cells), and it was expressed in both adult and developing neurons (Figure 2D). In neuronal cells, the enzyme localized in the cellular soma but was absent from dendrites and axons. The identification of TreH in neurons but



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not in astrocytes suggests that this pathway may be classified as a novel neuro-glia metabolic coupling pathway. A recent investigation discovered that trehalose can enter mammalian cells through solute carrier family 2 member 8 (Glut8) transporter.20 Glut8 is localized in the soma of neurons but is not expressed in glial cells,21 which resembled the observations of TreH. To test the ability of neurons to uptake trehalose, we supplemented primary hippocampal neuronal cultures on DIV 18 with a fresh medium with uniformly labelled

13C

trehalose (Figure 2E). The intracellular metabolome was then

extracted from the in vitro samples at different time points, i.e., 1, 5, 10 and 30 min. The analysis of the metabolome confirmed that the labelled trehalose entered the cells (Figure 2F-G). Notably, a small portion of unlabelled trehalose, which is biosynthesized in situ, was present in the samples and moderately increased over time (p(1vs10) = 0.006; p(1vs30) = 0.019). [Figure 2 about here] Metabolic variations during neurogenesis in hippocampal primary cultures Due to the different concentration levels and the presence of the enzyme during the early stages of development, we investigated the importance of this metabolite during neuronal maturation. We used a mass spectrometry-based untargeted approach to the identify common features in the samples between DIV 7 and DIV 14. Hierarchical clustering was performed using the following criteria: i. the exact mass was matched with the unique chemical formulas of the endogenous metabolites from the Human Metabolome DataBase (HMDB),22 ii. a mass tolerance of 3 parts per million was used, and iii. a minimum signal intensity of 104 (arbitrary instrumental units) was required. The number of processed features (i.e., identified chemical formulas) per sample is reported in Table S1. Integrated areas of each metabolite were calculated. The standardization of the dataset was performed by mean-subtraction and division using the standard deviation of each variable (dataset_stnd.txt in SI). The standardized dataset was used to build the statistical model. The samples were classified using a principal component analysis (PCA) (Figure S2A) and a Partial-Least Squared Discriminant Analysis (PLS-DA) (Figure 3A,


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Figure S2B). As expected, the different DIVs clustered in different areas. The major metabolites in the first component were identified using a Variable Importance in Projection (VIP) score higher than 2.0 (Figure 3B). Notably, the disaccharide appears to be a relevant component in this model. To assess the False Discovery Rate (FDR), a Significance Analysis of Microarray (SAM) was computed. The results reported in Figure 3C returned a total of 8 significant metabolites, including found the disaccharide (red dot) with a delta value of 3.47 (p < 0.001; q = 0.002). Finally, we constructed a heat map of the top 8 features with the lowest p-values calculated using an ANalysis Of VAriance (ANOVA) (Figure 3D, Figure S3, Table S2). Remarkably, the disaccharide exhibited a trend that was opposite to that shown by the other metabolites as follows: its intracellular concentration decreased during neuronal maturation, although it was present in adult neurons. To further confirm the validity of the statistical analysis, we matched the chemical formulas in the dataset with the HMDB. Multiple matches were resolved based on one or more of the following restriction criteria: endogenous, present in brain tissues, and reported as detected and quantified. The matched dataset was used for a pathway enrichment analysis of pathways with the number of detected metabolites > 3, FDR < 0.05 and p < 0.01 (Table S1). The results identified two metabolic pathways that can be associated with neuronal maturation, i.e., arginine and proline metabolism and alanine, aspartate and glutamate metabolism. These two pathways are known to be involved in neuronal development23,24 due to the synthesis of key metabolites that regulate synaptic transmission and cellular growth, such as N-acetyl-L-aspartate, L-glutamate, guanidinoacetate, creatine and phosphocreatine (Figure S3B-S3F). [Figure 3 about here] Trehalose enhances arborisation in neurons To determine whether trehalose is necessary for the correct development of neuronal cells and to confirm the results of the statistical analysis, we compared the morphological differences in primary neurons cultured under different conditions. Neurons were seeded on DIV 0 in four different minimal



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media supplemented with sucrose or trehalose and a minimal amount of glucose (0.1 mM) or a high glucose concentration (25.0 mM). On DIV 4, the neurons were transfected with a pUltra Hot plasmid expressing mCherry, and half of the media was replaced with Neurobasal.25 Regarding the choice of the second medium, the addition of glucose and pyruvate after DIV 4 was necessary to avoid transfection-induced cell death in the culture with 0.1 mM glucose. The cultures were fixed on DIV 7 with paraformaldehyde and mounted for the confocal microscopy analysis (Figure 4A). A Sholl analysis26 was performed, and the intersections between the Sholl radi were quantified in each condition. We evaluated the arborisations in each condition, i.e., the ability of neurons to form new dendrites and branches and their elongation. Regarding the neurons grown with the minimal glucose content (Figure 4B), the cultures with trehalose displayed a larger number of arborisations than those grown in sucrose. Notably, the presence of trehalose increased the number of dendrites but had a limited influence of the dendrite elongation. In fact, a larger number of intersections was observed near the soma between 20 and 40 units of distance. Multiple t-tests were performed to analyse the Sholl radius and demonstrated a p < 0.01 for all data points between 15 and 90. A 2-way repeated-measures (RM) ANOVA showed that the distance from the soma strongly influences the variation in arborisation (F(18,828) = 23.66 and p < 0.0001). Similar results were observed under less stringent conditions in which glucose was present at high concentration levels with sucrose or trehalose. Additionally, in this case, the presence of trehalose increases arborisation in neurons as shown in Figure 4C. The RM ANOVA of the function of distance confirmed the previous findings (F(18,828) = 4.073 and p < 0.0001). [Figure 4 about here] Conclusion In this study, we are the first to report that trehalose is endogenously biosynthesised in vertebrates and, more specifically, in rodent hippocampi using in vitro and ex vivo models. We


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identified that neurons are the main users of this metabolite, which underlines a divergence from insects, such as drosophila, in which trehalose is a key substrate for glial metabolism.27 This difference may stem from the fact that in vertebrates, glial cells are vascularized and can directly access other substrates, such as glucose and pyruvate, more efficiently from circulatory systems than from haemolymph systems. Notably, the role of this pathway appears to absolve a very specific task, and it cannot replace glucose as a carbon substrate. In fact, neurons were unable to grow properly when cultured under hypoglycaemic conditions, indicating that this disaccharide cannot fulfil the catabolic and anabolic needs of the cells, although the enzyme that cleaves trehalose is expressed and released glucose into the intracellular space. The presence of the TreH enzyme in neurons but not in glial cells also provides an explanation for recent findings indicating that exogenous trehalose can increase the in vitro cell viability of neurons but not of astrocytes.28 Furthermore, we show that trehalose influences the morphology of neurons in vitro by increasing arborisation during neuronal maturation. The localization of this enzyme in the soma of neurons but not in the protrusions supports the observation that this metabolite and the related pathway play a role in the formation of novel dendrites or their arborisation, but it is incapable of elongating the newly formed dendrites in the absence of glucose. Further studies investigating astrocyte metabolism are required to identify the enzymes that are responsible for the production of this disaccharide and the mechanism of actions in neurons responsible for the increased arborisation. Methods Animals All experimental procedures were performed in accordance with the European Communities Council Directive (86/809/EEC) on the care and use of animals, the Italian Legislation (L.D. no 26/2014), and the ARRIVE guidelines.29 All efforts were made to minimize the number of animals used and their suffering.



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Primary hippocampal neuronal cultures Primary hippocampal neuron cultures were prepared from Wistar rat embryos (Charles River, Calco, Italy) euthanized on E18. The preparation and dissociations were performed as described elsewhere.25 Immunohistochemistry for the detection of TreH, Map2 and GFAP The neurons were plated (75,000 cells/well) on poly-L-lysine coated 16 mm (ID) round glass coverslips in 12-well plates dipped in a medium solution composed of 1 ml Neurobasal supplemented with 0.2% B27, 10 mM glutamine, and 0.25 mM glutamate and incubated at 37 °C with 5.0% CO2. On DIV 4, 0.5 ml of the medium were replaced with a freshly prepared glutamate-free medium solution. For the immunohistochemistry staining, the coverslips were collected on different DIVs (DIVs 4-18), fixed with 4% paraformaldehyde and 4% sucrose and stained with anti-TreH, anti-Map2 and antiGFAP antibodies. The antibodies were diluted to 1:100, 1:300 and 1:300 in GDB solution (and incubated for 2 h at RT). The cells were then washed and incubated with the Alexa 488 (1:400), Alexa 555 (1:400) and DyLight650 (1:300) secondary antibodies, which were diluted in the GDB solution for 1 h at room temperature. The coverslips were then washed and mounted for analysis. GDB: 1% gelatine, 5% TritonX100, 0.1 M Na2HPO4, pH 7.4, and 2.0 M NaCl Morphological analysis in different media The neurons were plated (75,000 cells/well) on poly-L-lysine-coated 16-mm (ID) round glass coverslips in 12-well plates dipped in four different media compositions (A-D); 1 ml of each was supplemented with 0.2% B27, 10 mM L-glutamine, and 0.25 mM glutamate and incubated at 37 °C with 5.0% CO2. The HBSS final volume was adjusted by adding the required amount of medium to reach 18 µl in 1 ml of medium. On DIV 4, each coverslip was transferred to a new well containing 800 µl of fresh Neurobasal and 200 µl of the previous medium and maintained in an incubator for 15 min. Then, 200 µl of the transfection solution (Ts) were added to each well, and the cultures were incubated for 45 min at 37 °C. Finally, the medium was replaced with a new medium composed of Neurobasal


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and the relative conditioned medium of each condition (1:1, v:v) and incubated. On DIV 7, the coverslips were fixed with 4% paraformaldehyde/4% sucrose and mounted for analysis. The acquired images were analysed using the Sholl analysis module in Fiji software,30 and the statistical evaluation, i.e., multiple t-tests and 2-way RM ANOVAs were performed using the Prism 7 software (GraphPad Software, Inc., La Jolla, CA, USA). A:

8.3 g/L DMEM, 3.7 g/L NaHCO3, and 3.4 g/L sucrose

B:

8.3 g/L DMEM, 3.7 g/L NaHCO3, and 3.4 g/L trehalose

C:

8.3 g/L DMEM, 3.7 g/L NaHCO3, 3.4 g/L sucrose, and 4.5 g/L glucose

D:

8.3 g/L DMEM, 3.7 g/L NaHCO3, 3.4 g/L trehalose, and 4.5 g/L glucose

Ts:

1.5 µg DNA of pUltra Hot Plasmid expressing mCherry were added to 100 µl Neurobasal. The volume was adjusted to 197 µl and spiked with 3 µl of lipofectamine.

Dynamic labelling of trehalose Primary neurons on DIV 18 were incubated in Neurobasal supplemented with 13Ctrehalose. The samples were washed with freshly prepared Neurobasal, maintained at 37 °C and placed in a new well containing Neurobasal supplemented with 12.5 mM 13CTrehalose. The detailed protocol of the medium switch is described elsewhere.31 Metabolome extraction in vitro The neurons were plated (75,000 cells/well) on poly-L-lysine-coated 16-mm (ID) round glass coverslips in 12-well plates dipped in Neurobasal supplemented with 0.2% B27, 10 mM L-glutamine, and 0.25 mM glutamate and incubated at 37 °C with 5.0% CO2. On the DIV of interest, the cultures were washed with MilliQ water, maintained at 37 °C, transferred to 2 ml/well quenching solution composed of acetonitrile, methanol, MilliQ water (2:2:1) and 0.1% formic acid and maintained at -20 °C. The detailed protocol is described elsewhere.31 The cells were detached using a cell scraper, and the



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solution was then transferred to a 50-mL falcon tube and freeze-dry lyophilized. The dry extract was reconstituted in 200 µl MilliQ water and stored at -20 °C prior to the analysis. Metabolome extractions from brain slices Two-month-old male C57BL6 mice (bred in-house) were anaesthetized with chloroform and euthanized. 60 days old male mice were housed in polycarbonate cages with food and water ad libitum. Cob-bedding was changed weekly, and the animal house was 21°C with a 12 h light cycle (lights on at 08:00). Brain slices were prepared in oxygenated artificial cerebrospinal fluid (aCSF) and maintained at 4 °C. The slices were directly transferred to a well plate with 2 ml of quenching solutions (Qs) and maintained at – 20 °C. The hippocampus was then dissected, collected in a potter-elvehjem homogenizer, and maintained kept on ice. In total, 1 ml Qs and 0.1% formic acid were added, and the tissue was homogenized, transferred to a 50-ml falcon tube, frozen with liquid nitrogen and freeze-dry lyophilized. The dry residue was reconstituted in water, sonicated, centrifuged (10,000 × g, 3 min at 4 °C), and injected for the LC-MS analysis. aCSF: 125.00 mM NaCl, 2.5 mM KCl, 1.25 mM NaH2PO4, 26 mM NaHCO3, and 25 mM glucose Qs:

acetonitrile, methanol and MilliQ water prepared at the ratio of 2:2:1 (v:v:v).

Primary glial culture and treatments Mixed glial cell cultures, containing both astrocytes and microglial cells, were established from rat Sprague–Dawley embryos (E21) (Charles River, Calco, Italy). Briefly, after dissection, the hippocampi and cortices were dissociated using a treatment with 0.25% trypsin and DNase-I for 15 min at 37 °C, followed by fragmentation using a fire-polished Pasteur pipette. The dissociated cells were plated on poly-L-lysine-coated T75 flasks in minimal essential medium supplemented with 20% foetal bovine serum (FBS) and glucose (5.5 g/L) and maintained at 37 °C in 5% CO2. Microglial cells were harvested from 14-day-old cultures by orbital shaking for 30 min at 1300 rpm. The astrocytes were trypsinized and re-plated onto poly-L-lysine-coated 12-well plates (150,000 cells/well). The cells were


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starved overnight with a 1% FBS containing medium and then treated with 50 µM propranolol, which were previously diluted with sterile water, for 24 h. The conditioned cell supernatants were collected and cleared from cells and debris by centrifugation at 800 × g for 10 min. The cleared supernatants were harvested and stored at -20 °C prior to the LC-MS analysis. The cell cultures were maintained in a low serum (1% FBS) medium overnight, treated with 50 µM propranolol for 24 h and stained with calcein-AM, propidium iodide and Hoechst diluted to 1:100, 1:750 and 1:2000, respectively. The image acquisition was performed as described elsewhere.32 Sample preparation The extracted metabolomes from the brain slices were diluted to 1:5 with Mobile phase A and 10% methanol. For the identification, the samples were then spiked with

U13C

trehalose and injected for

the analysis. The extracted metabolomes from the primary hippocampal neurons were diluted to 1:1 with Mobile phase A and 10% methanol. The astrocyte supernatant was diluted to 1:10 with Mobile phase A and 10% methanol and injected. Western blotting The brain areas dissected from the adult rats were homogenized in RIPA buffer using a potterelvehjem tissue grinder. The primary astrocytes were scraped and lysed in RIPA buffer on DIV 15. Both the tissue homogenates and culture lysates were than centrifuged at 4 °C for 30 min at 9200 g, and the pellets were discarded. The proteins were separated on 10% SDS-PAGE and transferred onto nitrocellulose membranes in a buffer containing 0.025 M Tris-HCl, 0.192 M glycine, and 20% methanol, pH 8.3, at 80 V for 120 min. The immunoblotting reactions were performed by incubating with the following primary antibodies (4 °C, overnight, in 5% milk): rabbit anti-GAPDH (1:2000) and anti-TreH (1:400). Horseradish peroxidase-conjugated anti-rabbit (1:2000) was used as the secondary antibody (RT, 1 h in 5% milk). The immunoreactive bands were visualized by enhanced chemiluminescence. The quantification analysis was performed using ImageJ software. The intensity of



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TreH was normalized to that of GAPDH. The samples were standardized using the total signal intensity to minimize animal-to-animal variations. LC-MS method The chromatographic separation was performed using an Ultimate 3000 nanoLC system coupled with an Orbitrap Elite Hybrid Mass Spectrometer (Thermo Scientific, USA) and capillary column (IF100-100H035, NewObjective, Woburn, MA, USA). A binary gradient (Mobile phase A and B) was used starting with 10% B to 90% in 15 min and kept isocratic for 10 prior to the reequilibrations. The flow rate was 0.5 µl/min. A 1 µl injection loop was fully filled with the samples prior to the injections. The injections were performed in the low dispersion mode using 25% of the loop (250 nl). In the extracted metabolomes from the primary cultures, this volume corresponded to the nominal concentration of 94 cells per injection. The MS acquisition was performed in the negative mode, and the instrumental resolution was 60,000 in the full scan using a mass window between 100 and 1100. The nanoESI voltage was 1.9 mEV, the auxiliary and sheet gas were set to 0 and the endplate temperature was set to 200 °C. Mobile phase A: 450 ml MilliQ water, 50 µl acetic acid, and 180 µl tributylamine titrated to pH 9.2 with ammonium hydroxide. Mobile phase B: methanol MS data mining and statistics The peak piking, hierarchical clustering and peak integrations were performed using the eMZed2 software33 version 2.28.6.0. The HMDB (accession day 10/01/2017) was used as a reference database. The statistical analysis, including PCA, PLS-DA, SAM score, VIP score, ANOVA, and pathway enrichment analysis, were performed using the MetaboAnalysis 3.0 software34; t-test and multiple ttests were performed using the GraphPad Prism 7 software.


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Supporting information i. Viability assay of primary astrocytes treated with propranolol; ii. PCA and PLD-DA, iii. ANOVA analysis, iv. Metabolome and selected metabolite variations on different DIV, v. data processing results, vi. significant features, vii. pathway analysis, and vii. reagents and chemicals Abbreviations aCSF: artificial cerebrospinal fluid ANOVA: analysis of variance DIV: day in vitro EIC: extracted ion chromatograms ESI: electrospray ionization FBS: foetal bovine serum FDR: false discovery rate Glut8: solute carrier family 2 member 8 GFAP: glial fibrillary acidic protein HMDB: human metabolome database HRMS: high-resolution mass spectrometry LC: liquid chromatography Map2: microtubule-associated protein 2 MS: mass spectrometry mTor: mechanistic target of Rapamycin PCA: principal component analysis PLS-DA: partial-least squared discriminant analysis RM: repeated-measures SAM: significance analysis of microarray



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TreH: trehalose UDP: uridine diphosphate VIP: variable importance in projection Author information GM:

E-mail [email protected] Institute of Neuroscience, CNR, Via Luigi Vanvitelli, 32, I-20129, Milan, Italy

LG:


IP:

E-mail [email protected] Department of Medical Biotechnology and Translational Medicine, Università degli Studi di Milano, Via Luigi Vanvitelli, 32, I-20159, Milan, Italy

GG:

E-mail [email protected] Fondazione IRCCS Istituto Nazionale dei Tumori, Via Giacomo Venezian, 1, I-20133, Milan, Italy

VK:

E-mail [email protected] Fondazione IRCCS Istituto Nazionale dei Tumori, Via Giacomo Venezian, 1, I-20133, Milan, Italy

CV:


MP:


Corresponding authors * E-mail: [email protected] * E-mail: [email protected]


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Author contributions Conceptualization G.M.; Methodology G.M., L.G.; Formal Analysis G.M. and L.G.; Investigation G.M., L.G., I.P, and G.G.; Resources V.K., C.V., M.P.; Writing Original Draft G.M.; Supervision M.P.; Project Administration G.M.; Funding Acquisition M.P. Funding sources The financial support from Fondazione Telethon - Italy (Grant No. GGP12097) is gratefully acknowledged. Conflict of interest The authors declare no conflicts of interest. Acknowledgment The authors thank Adalberto Cavalleri from Fondazione IRCCS Istituto dei Tumori di Milano for technical support.



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Figure 1. Mass chromatograms of trehalose in the metabolome of mouse hippocampi. (A) Overlay of 3 different selected EICs. Each colour represents a different sample. (B) Recorded mass, isotopic distribution (upper part) and predicted isotopic distribution (lower part) of C12H22O11 minus a proton. (C) Average peak area ± SEM of trehalose in astrocyte supernatants (n = 4). (D) Average peak area ± SEM of trehalose in primary hippocampal neuronal cultures (n = 4).


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Figure 2. TreH expression levels in the rodent brain and trehalose uptake. (A) Representative Western blot of TreH and GAPDH from different rat brain region homogenates. (B) Relative intensity ± SEM of TreH in different brain regions (i.e., cortex, hippocampus, cerebellum and olfactory bulb) from adult rats (n = 3) and relative p-values (t-test) of the significant differences. (C) Representative Western blot of TreH and GAPDH from primary astrocyte lysates and cortex homogenates. (D) Immunostaining of TreH, Map2 and GFAP in hippocampal neuronal cultures on DIV 4, DIV 7 and DIV 18. (E) Schematic model of the uptake. (F) Selected EICs with

13C

trehalose in brown and

intracellular metabolome 30 min after the supplementation with and endogenous

12

13C

12C

trehalose in black from the

trehalose. (G)

13

C loading (brown)

C loading (black) of the intracellular trehalose that was extracted from primary

hippocampal neurons after the 13Ctrehalose supplementation.



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Figure 3. Statistical analysis based on the untargeted metabolome profile of hippocampal neuronal cultures on DIV 7, DIV 11 and DIV 14. (A) A 3D representation of the metabolome of each sample using PLS-DA. (B) Graphical representation of the VIP, which includes the disaccharide trehalose highlighted in red. (C) SAM showing the metabolites with a false discovery rate lower than 0.05 in green, and trehalose is highlighted in red. (D) Heat map showing the intensity of the top eight metabolites.


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Figure 4. Analysis of the influence of trehalose on the morphology of neurons. (A) Representative images acquired at a 40-fold magnification of neurons on DIV 7 grown in 4 different media with trehalose or sucrose as a control and glucose at 0.001 or 25 mM concentrations. (B) Sholl analysis evaluating the average number of intersections ± SEM evaluated as a function of distance from the cellular soma in neurons grown with trehalose or sucrose as a control with 0.1 mM glucose. (C) Sholl analysis evaluating the average number of intersections ± SEM evaluated as a function of distance from the cellular soma in neurons grown with trehalose or sucrose as a control with 25.0 mM glucose.



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Graphical Table of Contents


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Biosynthesis of Astrocytic Trehalose Regulates Neuronal Arborization

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