ENGLISH SUMMARY thesis Anne-Lieke van Deijk

The central nervous system is considered to be autonomous in lipid synthesis. Lipids are building blocks of neuronal membranes and play key roles in brain functioning. The brain contains various different cell types, including neurons and glial cells. Astrocytes, a type of glial cell, contact neuronal synapses and actively contribute to the regulation of neuronal functioning by secreting factors that modulate the properties of synapses. The finding that astrocytes are active elements of neuronal synapses led to the concept of the tripartite synapse: a pre- and postsynaptic terminal ensheathed by an astrocyte.
Astrocytes are considered to be the main providers of lipids in the brain. It is thought that lipids secreted by astrocytes are provided to neurons, which have low capacity to synthesize lipids themselves, and are therefore important for the formation and function of synapses. Compromised lipid metabolism has been associated with several neurological disorders characterized by impaired synaptic function, such as Niemann-Pick disease type C, Alzheimer’s disease and Huntington’s disease. The finding that dietary lipid treatment successfully improved synaptic dysfunction in several of these neurological diseases, suggests that lipids play key roles in synaptic functioning.

The work described in this thesis aims to gain insight into the role of lipids derived from astrocytes and diet in hippocampal synapse formation and function. To study the effect of astrocyte-secreted lipids, we inactivated SCAP-SREBP-mediated lipid biogenesis in astrocytes, a prominent pathway of astrocyte lipid synthesis.

In Chapter 1, I review the current literature on astrocytes and lipids in the central nervous system. Astrocytes are the most abundant cell type in the mammalian CNS and serve a broad range of biological functions, such as maintaining the blood-brain barrier, supplying energy substrates to neurons, extracellular ion homeostasis, neurogenesis and synaptogenesis. Astrocytes play key roles in synaptic functioning by secreting many different factors. In the CNS, astrocytes are also important for de novo lipid synthesis. Although it is known that lipids are highly abundant in neuronal synapses and that lipids are important for pre- and postsynaptic function, the role of astrocyte lipid metabolism in synapse function remains largely unknown. Several studies show that compromised astrocyte lipid metabolism is associated with several neurological disorders characterized by impaired synaptic function. Chapter 1 gives, therefore, an overview of studies describing the effect of (astrocyte) lipid metabolism on synapse formation and function under healthy and diseased conditions and the effect of dietary lipid intervention.

In Chapter 2, we developed a new method to analyze neuronal developmental processes, such as neuronal survival, neurite outgrowth and synapse formation and maturation, in 146 English summary primary hippocampal neuron-astrocyte co-cultures in an automated manner. We used automated confocal microscopy in the Opera High Content Screening system and Columbus data analysis to differentiate between immature synapses (a single pre- or postsynaptic terminal) or mature synapses (opposing pre- and postsynaptic terminals that form a functional synapse). Next, we investigated the effect of the specific nutrient combination Fortasyn Connect (FC), which improves memory performance in early Alzheimer’s disease patients, on neuronal survival and synaptogenesis. Different concentrations of FC were supplemented to primary hippocampal neurons co-cultured with primary astrocytes before the developmental peak of synaptogenesis in vitro. In this culture system, FC increased neuronal survival and the maturation of postsynaptic terminals. These findings might contribute to the functional interpretation of FC-based intervention strategies in neurological diseases characterized by neuronal loss and impaired synapse function.

In Chapter 3, we investigated the effect of astrocyte lipid metabolism on the formation and function of hippocampal neurons in vivo. We demonstrated that astrocytes are indeed more efficient in lipid synthesis compared to neurons. Inactivation of the SCAP-SREBP pathway in astrocytes during embryonic development, by using the GFAP-SCAP mouse model, resulted in reduced synthesis and secretion of cholesterol and phospholipids, two lipids that are the main lipid components carried by lipoproteins and probably important for lipid transport from astrocytes to neurons. Moreover, we found that impaired astrocyte lipid metabolism leads to impaired synapse development and reduced number of presynaptic vesicles, both the total number of vesicles as the number of synaptic vesicles that are ready for release (docked vesicles). In accordance, mice with impaired astrocyte lipid metabolism had reduced presynaptic function and impaired long-term plasticity, possibly because the lower number of synaptic vesicles led to decreased neurotransmitter release. These findings show that astrocyte lipid metabolism is essential for proper formation and function of hippocampal synapses in vivo.

In Chapter 4, we investigated the mechanisms that underlie the regulation of synapse formation and function by astrocyte lipids. Analysis of hippocampal synapses in mice with impaired astrocyte lipid metabolism (GFAP-SCAP mice) using several –omics techniques, like lipidomics and proteomics, revealed an aberrant synaptic lipid composition and a downregulation of proteins essential for synaptic vesicle cycle. We treated GFAPSCAP mice with an experimental diet providing cholesterol and nutrients required for phospholipid synthesis (FC) to compensate for the reduced secretion of cholesterol and phosphatidylcholine by GFAP-SCAP astrocytes. This dietary lipid intervention caused integration of dietary lipids in the synaptic membrane and rescued the impaired regulation of a subset of endocytosis-mediating proteins, which was accompanied by normalization 147 English summary S of the number of docked vesicles as well as of synaptic long-term potentiation. These data suggest that astrocyte lipid metabolism has a critical role in the hippocampal synaptic vesicle cycle and demonstrate that defects in presynaptic maturation caused by impaired astrocyte lipid metabolism can be rescued by dietary supply of a specific combination of lipid-related nutrients.

In Chapter 5, we generated and analysed an inducible mouse model, the Glast-CreERT2- SCAP model, to investigate the effect of postnatal inactivation of astrocyte SCAP-SREBPmediated lipid biogenesis on myelination and synapse ultrastructure in adults. In this mouse model, tamoxifen injections lead to inactivated SCAP-SREBP-mediated lipid synthesis in cells expressing the astrocyte-specific Glast promotor. To our surprise, when mice were injected with tamoxifen during early postnatal development, not only Glast+ cells in the central nervous system were targeted, but also cells in peripheral organs. However, postnatal targeting after the peak of synaptogenesis and at the peak of myelination, resulted in specific targeting of astrocytes. Accordingly, postnatal inactivation of astrocyte SCAP-SREBP-mediated lipid biogenesis, reduced lipid biogenesis specifically in astrocytes. Immunohistochemical and electron microscopy analysis revealed that this resulted in hypomyelination and small changes in synapse ultrastructure. These findings suggest that Glast-CreERT2-SCAP mouse is a powerful tool to dissect the role of astrocyte lipid metabolism during specific postnatal developmental time points.

In Chapter 6, I discuss more in depth the role of astrocyte lipids in the formation and function of neuronal synapses by inactivating SCAP-SREBP-mediated lipid biogenesis in astrocytes during embryonic and postnatal development. Synaptic defects observed in our GFAP-SCAP mouse model are comparable with several neurological disorders associated with impaired lipid metabolism and synaptic deficits, such as Niemann-Pick disease type C, Huntington’s disease, Alzheimer’s disease and Parkinson’s disease. I also touch upon the effects of dietary lipid intervention under healthy conditions and when (astrocyte) lipid metabolism is affected. The specific-lipid enriched diet that is used in this thesis is compared with other diets described in literature and with a previously used high-fat diet. I postulate that lipid content and FA composition of supplemented diets may be used to selectively improve several processes within the diseased brain, such as myelination and synapse function. Finally, implications and future perspectives are considered.