High Content Analysis of Hippocampal Neuron-Astrocyte Co-cultures Shows a Positive Effect of Fortasyn Connect on Neuronal Survival and Postsynaptic Maturation

Neuronal and synaptic membranes are composed of a phospholipid bilayer. Supplementation with dietary precursors for phospholipid synthesis -docosahexaenoic acid (DHA), uridine and choline- has been shown to increase neurite outgrowth and synaptogenesis bothin vivoandin vitro. A role for multi-nutrient intervention with specific precursors and cofactors has recently emerged in early Alzheimer's disease, which is characterized by decreased synapse numbers in the hippocampus. Moreover, the medical food Souvenaid, containing the specific nutrient combination Fortasyn Connect (FC), improves memory performance in early Alzheimer's disease patients, possibly via maintaining brain connectivity. This suggests an effect of FC on synapses, but the underlying cellular mechanism is not fully understood. Therefore, we investigated the effect of FC (consisting of DHA, eicosapentaenoic acid (EPA), uridine, choline, phospholipids, folic acid, vitamins B12, B6, C and E, and selenium), on synaptogenesis by supplementing it to primary neuron-astrocyte co-cultures, a cellular model that mimics metabolic dependencies in the brain. We measured neuronal developmental processes using high content screening in an automated manner, including neuronal survival, neurite morphology, as well as the formation and maturation of synapses. Here, we show that FC supplementation resulted in increased numbers of neurons without affecting astrocyte number. Furthermore, FC increased postsynaptic PSD95 levels in both immature and mature synapses. These findings suggest that supplementation with FC to neuron-astrocyte co-cultures increased both neuronal survival and the maturation of postsynaptic terminals, which might aid the functional interpretation of FC-based intervention strategies in neurological diseases characterized by neuronal loss and impaired synaptic functioning

Astrocyte lipid metabolism is critical for synapse development and function in vivo

The brain is considered to be autonomous in lipid synthesis with astrocytes producing lipids far more efficiently than neurons. Accordingly, it is generally assumed that astrocyte-derived lipids are taken up by neurons to support synapse formation and function. Initial confirmation of this assumption has been obtained in cell cultures, but whether astrocyte-derived lipids support synapses in vivo is not known. Here, we address this issue and determined the role of astrocyte lipid metabolism in hippocampal synapse formation and function in vivo. Hippocampal protein expression for the sterol regulatory element-binding protein (SREBP) and its target gene fatty acid synthase (Fasn) was found in astrocytes but not in neurons. Diminishing SREBP activity in astrocytes using mice in which the SREBP cleavage-activating protein (SCAP) was deleted from GFAP-expressing cells resulted in decreased cholesterol and phospholipid secretion by astrocytes. Interestingly, SCAP mutant mice showed more immature synapses, lower presynaptic protein SNAP-25 levels as well as reduced numbers of synaptic vesicles, indicating impaired development of the presynaptic terminal. Accordingly, hippocampal short-term and long-term synaptic plasticity were defective in mutant mice. These findings establish a critical role for astrocyte lipid metabolism in presynaptic terminal development and function in vivo. GLIA 2017;65:670-682

Oligodendroglial myelination requires astrocyte-derived lipids.

In the vertebrate nervous system, myelination of axons for rapid impulse propagation requires the synthesis of large amounts of lipids and proteins by oligodendrocytes and Schwann cells. Myelin membranes are thought to be cell-autonomously assembled by these axon-associated glial cells. Here, we report the surprising finding that in normal brain development, a substantial fraction of the lipids incorporated into central nervous system (CNS) myelin are contributed by astrocytes. The oligodendrocyte-specific inactivation of sterol regulatory element-binding protein (SREBP) cleavage-activating protein (SCAP), an essential coactivator of the transcription factor SREBP and thus of lipid biosynthesis, resulted in significantly retarded CNS myelination; however, myelin appeared normal at 3 months of age. Importantly, embryonic deletion of the same gene in astrocytes, or in astrocytes and oligodendrocytes, caused a persistent hypomyelination, as did deletion from astrocytes during postnatal development. Moreover, when astroglial lipid synthesis was inhibited, oligodendrocytes began incorporating circulating lipids into myelin membranes. Indeed, a lipid-enriched diet was sufficient to rescue hypomyelination in these conditional mouse mutants. We conclude that lipid synthesis by oligodendrocytes is heavily supplemented by astrocytes in vivo and that horizontal lipid flux is a major feature of normal brain development and myelination

mTORC1 Controls PNS Myelination along the mTORC1-RXRγ-SREBP-Lipid Biosynthesis Axis in Schwann Cells

Myelin formation during peripheral nervous system (PNS) development, and reformation after injury and in disease, requires multiple intrinsic and extrinsic signals. Akt/mTOR signaling has emerged as a major player involved, but the molecular mechanisms and downstream effectors are virtually unknown. Here, we have used Schwann-cell-specific conditional gene ablation of raptor and rictor, which encode essential components of the mTOR complexes 1 (mTORC1) and 2 (mTORC2), respectively, to demonstrate that mTORC1 controls PNS myelination during development. In this process, mTORC1 regulates lipid biosynthesis via sterol regulatory element-binding proteins (SREBPs). This course of action is mediated by the nuclear receptor RXRγ, which transcriptionally regulates SREBP1c downstream of mTORC1. Absence of mTORC1 causes delayed myelination initiation as well as hypomyelination, together with abnormal lipid composition and decreased nerve conduction velocity. Thus, we have identified the mTORC1-RXRγ-SREBP axis controlling lipid biosynthesis as a major contributor to proper peripheral nerve function.ISSN:2666-3864ISSN:2211-124

Persistent hypomyelination in glial fibrillary acidic protein (GFAP)- SREBP cleavage-activating protein (SCAP) mutant brains.

<p><b>A)</b> Electron microscopy (EM) analysis of corpus callosum myelination in cross-sections of either wild-type (WT) or GFAP-SCAP mice at P120. Bar graph shows the percentage of axons that is myelinated. <b>B)</b> Morphometric analysis of axons on corpus callosum of WT and GFAP-SCAP mice, showing g-ratio (myelinated axons) and axonal size distribution (both myelinated and non-myelinated axons) at P120. The relation between axon diameter (x) and g-ratio (y) was y = 9E − 05x + 0.7287 for WT and y = 7E − 05x + 0.8008 for GFAP-SCAP, with coefficients of determination R² = 0.25384 (WT) and 0.11285 (GFAP-SCAP). <b>C)</b> EM analysis of optic nerve myelination in cross-sections of either WT or GFAP-SCAP mice at depicted time points. Bar graph shows the percentage of axons that is myelinated. <b>D)</b> Morphometric analysis of axons on optic nerves of WT and GFAP-SCAP mice, showing g-ratio (myelinated axons), axonal size distribution (both myelinated and nonmyelinated axons), and myelin membrane thickness at P20 and P120. At P20, the relation between axon diameter (x) and g-ratio (y) was y = 8E − 05x + 0.7262 for WT and y = 5E − 05x + 0.8003 for GFAP-SCAP, with coefficients of determination R² = 0.31108 (WT) and 0.11441 (GFAP-SCAP). At P120: y = 9E − 05x + 0.7079 (WT); y = 3E − 05x + 0.825 (GFAP-SCAP), R² = 0.3288 (WT) and R² = 0.06062 (GFAP-SCAP). Scale bars, 2 μm. <i>t</i> test # <i>p</i> = 0.079, * <i>p</i> < 0.05, ** <i>p</i> < 0.01, <i>n</i> = 3. The numeric data can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002605#pbio.1002605.s006" target="_blank">S1 Data</a>.</p

Myelin from glial fibrillary acidic protein (GFAP)-SREBP cleavage-activating protein (SCAP) brains shows increased accumulation of dietary lipids.

<p>Lipid extracts of purified myelin of GFAP-SCAP brains (P42) were analyzed using liquid chromatography and mass spectrometry. <b>A)</b> Polar membrane lipid concentration and <b>(B)</b> sterol concentration per protein amount in GFAP-SCAP compared to wild-type (WT) myelin. GSL, glycosphingolipid; PI, phosphatidyl inositol; PE, phosphatidyl ethanolamine; PS, phosphatidyl serine; PC, phosphatidyl choline; SM, sphingomyelin. <b>C)</b> Fatty acid profile of phospholipids from purified myelin with the amount of different fatty acids species as the percentage of the total amount. Fatty acid species are depicted as “y:z,” with “y” representing the length of the fatty acid molecules and “z” representing the number of double bonds. Insert: depicted are proportions of saturated fatty acids (SFA), monounsaturated fatty acids (MUFA), polyunsaturated fatty acids (PUFA), and the ratio of 18:1/18:2. Data are presented as the mean percentage of WT ± SD. <i>t</i> tests: * = <i>p</i> < 0.05; ** = <i>p</i> < 0.01; *** = <i>p</i> < 0.001, <i>n</i> = 4. The numeric data can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002605#pbio.1002605.s006" target="_blank">S1 Data</a>.</p

Conditional inactivation of SREBP cleavage-activating protein (SCAP) in oligodendrocytes reduces lipogenic gene expression and causes motor control defects and reduced survival.

<p><b>A)</b> Protein levels of precursor sterol regulatory element-binding protein 2 (SREBP2) and mature (processed) SREBP2 were determined by immunoblotting of total extracts of the spinal cord of wild-type (WT) and CNP-SCAP animals at P20 (<i>n</i> = 3). Detection of mature and precursor SREBP2 was performed using different exposure times, and representative pictures are shown. Detection of beta-actin and on-blot protein stain was used to control for equal loading. <b>B)</b> Histogram shows quantification of precursor and mature SREBP2 protein levels after correction for equal loading (using on-blot stain) and subsequent normalization to WT levels in which the WT levels were set to 1, and the ratio between mature/precursor SREBP in which the ratio for WT was set to 1. All data are presented as mean levels ± SEM (<i>t</i> test: *<i>p</i> < 0.05, #<i>p</i> = 0.059). <b>C)</b> Expression of fatty acid synthase (FASN, green) in oligodendrocytes (Olig2, blue) and astrocytes (glial fibrillary acidic protein [GFAP], red) of WT or CNP-SCAP mutant mice at P20. Asterisks denote oligodendrocytes with FASN expression; arrowheads denote astrocytes with FASN expression (scale bar, 25 μm). <b>D)</b> Number of FASN-positive oligodendrocytes (Olig2⁺FASN⁺) and FASN-positive astrocytes (GFAP⁺FASN⁺) in WT and CNP-SCAP mice (<i>n</i> = 3). The values are provided as the percentage of the total number of oligodendrocytes (Olig2+ cells) or astrocytes (GFAP⁺ cells). Data are presented as mean ± SEM. **<i>p</i> < 0.01 using <i>t</i> test. <b>E)</b> Kaplan-Meier survival plot showing a strongly reduced life span of CNP-SCAP mice compared to age-matched WT mice. <b>F)</b> Body weight development of WT and CNP-SCAP mice over a 3-month period. Shown are the mean and SEM. <b>G)</b> At a grid test, CNP-SCAP mice showed limb ataxia, causing frequent slips of the hind limbs (red arrow) or front limbs. Mutant mice showed an abnormal reaction when tail lifted; they attempted to clasp their hind limbs and clench the toes of their rear feet. <b>H)</b> CNP-SCAP brains compared to control littermates at P28. The numeric data underlying Fig 1B, D, E and F can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002605#pbio.1002605.s006" target="_blank">S1 Data</a>.</p

Effects of a high-fat diet (HFD) on myelination, myelin protein levels, and white matter conduction velocity.

<p><b>A)</b> Electron microscopy (EM) analysis of optic nerve (ON) and corpus callosum (CC) myelination in cross-sections of glial fibrillary acidic protein (GFAP)-SREBP cleavage-activating protein (SCAP) mice (P120) on either a standard diet (SD) or HFD. See <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002605#pbio.1002605.g005" target="_blank">Fig 5A and 5C</a> for representative EM images of wild-type (WT) animals (P120) on SD or HFD. Scale bar, 2 μm. Bar graphs, percentage of axons that are myelinated (left). Morphometric analysis of myelinated axons showing g-ratio (middle and right). For ON of GFAP-SCAP mice, the relation between axon diameter (x) and g-ratio (y) was y = 3E − 05x + 0.825 for SD and y = 6E − 05x + 0.7687 for HFD, with coefficients of determination R² = 0.06062 (SD) and R² = 0.23735 (HFD). For CC of GFAP-SCAP mice, the relation between axon diameter (x) and g-ratio (y) was y = 7E − 05x + 0.8008 for SD and y = 0.0001x + 0.701 for HFD, with coefficients of determination R² = 0.11285 (SD) and R² = 0.26559 (HFD). <i>t</i> test, ** <i>p</i> < 0.01, * <i>p</i> < 0.05, # <i>p</i> = 0.07, <i>n</i> = 3–4. <b>B)</b> Immunoblot against depicted myelin proteins and coomassie staining of protein levels of total brain extracts of GFAP-SCAP mutant and WT mice (P120) fed with SD or HFD. Right panel: quantification of immunoblot for depicted myelin proteins for GFAP-SCAP and WT mice fed with SD or HFD (<i>n</i> = 3). Coomassie staining was used for normalization. Data are presented as mean ± SEM, in which WT-SD levels were set to 100%. <i>t</i> test * <i>p</i> < 0.05; ** <i>p</i> < 0.01). <b>C)</b> Example of compound action potential waveforms in the CC for a WT mouse on a standard diet (WT-SD), and a GFAP-SCAP mutant mouse on a standard diet (GFAP-SCAP-SD) or high fat diet (GFAP-SCAP-HFD). Right panel: individual plots of conduction velocity measurements in the CC of GFAP-SCAP mutant and WT fed with SD or HFD. Chi-square test, <i>n</i> = 12–17, * <i>p</i> < 0.05, *** < 0.001. The numeric data can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002605#pbio.1002605.s006" target="_blank">S1 Data</a>.</p

Virtually no myelin membrane synthesis in CNP-SREBP cleavage-activating protein (SCAP)/glial fibrillary acidic protein (GFAP)-SCAP brains.

<p><b>A)</b> Electron microscopy (EM) analysis of the corpus callosum (CC) and optic nerve (ON) in P20-old wild-type (WT) mice or mice carrying a deletion in both astrocytes and oligodendrocytes (CNP-SCAP/GFAP-SCAP). Bar graphs show the percentage of axons that are myelinated. <b>B)</b> Enlarged view on part of the electron micrograph of CNP-SCAP/GFAP-SCAP ON in A. <b>C)</b> morphometric analysis of myelinated axons in the ON of WT, CNP-SCAP, GFAP-SCAP, and CNP-SCAP/GFAP-SCAP mice at p20, showing myelin membrane thickness. <i>n</i> = at least 3 animals. Membrane thickness for each CNP-SCAP/GFAP-SCAP animal was determined for at least 22 axons that were wrapped by oligodendrocyte membrane, as depicted in 10B and D. <b>D)</b> Electron microscopic analysis of myelin membranes in the ON of WT, CNP-SCAP, GFAP-SCAP, and CNP-SCAP/GFAP-SCAP mice. Data are presented as mean ± SEM. <i>t</i> test * <i>p</i> < 0.05, *** <i>p</i> < 0.001, <i>n</i> ≥ 3. Scale bar, (A) 2 μm, (B) 0.75 μm, (C) 0.1 μm. The numeric data underlying Fig 10A and C can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002605#pbio.1002605.s006" target="_blank">S1 Data</a>.</p

Myelin from CNP-SREBP cleavage-activating protein (SCAP) brains shows changes in fatty acid composition.

<p>Lipid extracts of purified myelin of wild-type (WT) and CNP-SCAP brains at P56 were analyzed using liquid chromatography and mass spectrometry. <b>A)</b> Polar membrane lipid concentration and <b>B)</b> sterol concentration per protein amount in CNP-SCAP compared to WT myelin. GSL, glycosphingolipid; PI, phosphatidyl inositol; PE, phosphatidyl ethanolamine; PS, phosphatidyl serine; PC, phosphatidyl choline; SM, sphingomyelin. <b>C)</b> Fatty acid profile of phospholipids from purified myelin with the amount of different fatty acids species as percentage of the total amount. Fatty acid species are depicted as “y:z,” with “y” giving the length of the fatty acid molecules and “z” the number of double bonds. Insert: depicted are proportions of saturated fatty acids (SFA), monounsaturated fatty acids (MUFA), polyunsaturated fatty acids (PUFA), and the ratio of 18:1/18:2. Data are presented as mean percentage of WT ± SD. <i>t</i> tests: * = <i>p</i> < 0.05; ** = <i>p</i> < 0.01; *** = <i>p</i> < 0.001, <i>n</i> = 5. The numeric data can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002605#pbio.1002605.s006" target="_blank">S1 Data</a>.</p