Glial glucose metabolism- a global metabolic sensor governing decision-making in Drosophila melanogaster larvae

Metabolic coupling between glial cells and neurons is essential for neuronal function. It is a well-conserved and vital feature of the bilaterian nervous system as well. Under normal and adverse conditions, glial cells act as a major metabolic hub fueling neuronal oxidative metabolism by producing lactate or ketone bodies. I now ask the question of whether such metabolic coupling is only necessary for preserving brain homeostasis or if it could also have implications in decision making such as food choice behavior. Choosing an appropriate food source is key for the survival of an organism. Carbohydrates are the preferred source of energy and thus evaluation of their nutritive content is essential. Several studies have demonstrated that Drosophila melanogaster larvae and adults, as mammals, can distinguish between nutritious and non-nutritious carbohydrates independent of their taste. Two groups of neurons, Diuretic Hormone 44 (Dh44)-expressing neurons and gustatory receptor 43a (Gr43a)-expressing neurons, have been implicated in postprandial sugar sensing in adult flies. Gr43a- expressing neurons are narrowly fine-tuned for sensing fructose in both adults and larvae. Nonetheless, in the larva, central nervous system (CNS) Gr43a neurons have been shown to act as the main sugar sensor. This raises the question of how CNS fructose-sensing neurons are involved in sensing non-fructose dietary sugars. To decipher this post-ingestive mechanism, I used frustrated total internal reflection (FTIR) - based larval tracking to investigate larval food choice behavior. The results present in this thesis suggest that besides Gr43a-expressing neurons, glial cells are indispensable for sensing nutritive non-fructose dietary sugars such as glucose and sorbitol. I show that post-ingestive carbohydrate sensing involves carbohydrate conversion into fructose locally in the glial cells. Glia-derived fructose then enables Gr43a-dependent postprandial carbohydrate sensing in the CNS and drives carbohydrate preferences with the help of the downstream signalling peptide corazonin. Thus, in post-ingestive carbohydrate sensing, the glial cells act as a master metabolite sensor and provide a fructose stimulus to neurons to regulate behavior.:Table of Contents Abstract 1 Zusammenfassung 2 1. Introduction 3 1.1. External Nutrient Sensing in mammals and Drosophila melanogaster 4 1.1.1. Taste detection in mammals 4 1.1.2. Taste detection in Drosophila melanogaster 6 1.1.3 Sweet and Umami Taste in mammals 8 1.1.4. Sweet Sensing in Drosophila melanogaster 8 1.1.5. Bitter Taste in mammals 9 1.1.6. Bitter Sensing in Drosophila melanogaster 9 1.1.7. Sour, Carbonation, Fatty acid and Salty taste in mammals 9 1.1.8. Amino acid, Fatty acid and Salt Sensing in Drosophila melanogaster 10 1.2 Post-ingestive nutrient sensing in mammals and Drosophila melanogaster 11 1.2.1. Post-ingestive amino acid sensing in mammals 11 1.2.2. Post-ingestive amino acid sensing in Drosophila melanogaster 12 1.2.3. Post-ingestive lipid sensing in mammals 13 1.2.4. Post-ingestive carbohydrate sensing in mammals 13 1.2.4.1. The role of the nervous system in post-ingestive carbohydrate sensing mammals 16 1.2.5. Post-ingestive carbohydrate sensing in Drosophila melanogaster 16 1.2.5.1. The role of the nervous system in post-ingestive carbohydrate sensing in flies 18 1.2.5.2. The role of the nervous system in post-ingestive carbohydrate sensing in larvae 19 1.3 The cellular architecture of the larval nervous system 20 1.3.1 The Blood-brain barrier (BBB) and carbohydrate transport 23 1.4 Aim of study 25 2. Experimental Procedures 26 2.1 Materials 26 2.1.1 Chemicals 26 2.1.2 Media 27 2.1.3 Buffer and Solution 27 2.1.4 Antibodies 28 2.1.5 Flystocks 29 2.2 Methods 33 2.2.1 Fly genetics 33 2.2.1.1 Maintenance and crosses 33 2.2.2 Immunohistochemistry & Microscopy 33 2.2.2.1 Immunohistochemistry of larval filets 33 2.2.2.2 Confocal Microscopy 33 2.3 Experimental Design 34 2.3.1 Studying food choice in Drosophila larvae 34 2.3.1.1 Concept 34 2.3.1.2 Experimental setup 36 2.3.1.3 Analysis 36 2.3.1.4 MATLAB Script 38 3. Results 40 3.1. Frustrated total internal reflection based two-choice assay (TCA) 40 3.1.1 Investigating larval sugar preference 40 3.2 The role of diuretic hormone 44 (Dh44) neurons in post-ingestive glucose sensing in third instar larvae 47 3.2.1 Immunohistochemical analysis of Dh44 in third instar larval brains 47 3.2.2 Role of Dh44 in glucose sensing 49 3.3 The role of gustatory receptor 43a (Gr43a) neurons in post-ingestive nutritive carbohydrate sensing in third instar larvae 51 3.3.1 Immunohistochemical analysis of Gr43a in third instar larval brains 51 3.4. Investigating the role of polyol pathway in post-ingestive carbohydrate sensing 56 3.4.1 Identifying the key polyol pathway enzyme in Drosophila melanogaster 56 3.4.2 Examining RNAi mediated neuronal and glial knockdown of polyol pathway enzymes in postprandial sugar sensing 57 3.4.2.1 CG9436 57 3.4.2.2 CG6084 and CG10863 60 3.4.2.3 Sodh-2 66 3.5 Determining the glia subtypes vital for conducting polyol pathway 69 3.6 The role of the blood brain barrier (BBB) in post-ingestive carbohydrate sensing 74 4. Discussion 80 4.1. Gr43a is the only sugar sensor in Drosophila larvae 80 4.2. Polyol Pathway is crucial for glucose and sorbitol sensing 82 4.3. Glia, the master metabolic sensor 84 4 4. Transport over BBB: a prerequisite for postprandial carbohydrate sensing85 4.5. Proposed model for larval post-ingestive nutritive carbohydrate sensing... 86 5. References 89 6. Abbreviation List 100 7. Appendix 10

Glial glucose metabolism- a global metabolic sensor governing decision-making in Drosophila melanogaster larvae

Metabolic coupling between glial cells and neurons is essential for neuronal function. It is a well-conserved and vital feature of the bilaterian nervous system as well. Under normal and adverse conditions, glial cells act as a major metabolic hub fueling neuronal oxidative metabolism by producing lactate or ketone bodies. I now ask the question of whether such metabolic coupling is only necessary for preserving brain homeostasis or if it could also have implications in decision making such as food choice behavior. Choosing an appropriate food source is key for the survival of an organism. Carbohydrates are the preferred source of energy and thus evaluation of their nutritive content is essential. Several studies have demonstrated that Drosophila melanogaster larvae and adults, as mammals, can distinguish between nutritious and non-nutritious carbohydrates independent of their taste. Two groups of neurons, Diuretic Hormone 44 (Dh44)-expressing neurons and gustatory receptor 43a (Gr43a)-expressing neurons, have been implicated in postprandial sugar sensing in adult flies. Gr43a- expressing neurons are narrowly fine-tuned for sensing fructose in both adults and larvae. Nonetheless, in the larva, central nervous system (CNS) Gr43a neurons have been shown to act as the main sugar sensor. This raises the question of how CNS fructose-sensing neurons are involved in sensing non-fructose dietary sugars. To decipher this post-ingestive mechanism, I used frustrated total internal reflection (FTIR) - based larval tracking to investigate larval food choice behavior. The results present in this thesis suggest that besides Gr43a-expressing neurons, glial cells are indispensable for sensing nutritive non-fructose dietary sugars such as glucose and sorbitol. I show that post-ingestive carbohydrate sensing involves carbohydrate conversion into fructose locally in the glial cells. Glia-derived fructose then enables Gr43a-dependent postprandial carbohydrate sensing in the CNS and drives carbohydrate preferences with the help of the downstream signalling peptide corazonin. Thus, in post-ingestive carbohydrate sensing, the glial cells act as a master metabolite sensor and provide a fructose stimulus to neurons to regulate behavior.:Table of Contents Abstract 1 Zusammenfassung 2 1. Introduction 3 1.1. External Nutrient Sensing in mammals and Drosophila melanogaster 4 1.1.1. Taste detection in mammals 4 1.1.2. Taste detection in Drosophila melanogaster 6 1.1.3 Sweet and Umami Taste in mammals 8 1.1.4. Sweet Sensing in Drosophila melanogaster 8 1.1.5. Bitter Taste in mammals 9 1.1.6. Bitter Sensing in Drosophila melanogaster 9 1.1.7. Sour, Carbonation, Fatty acid and Salty taste in mammals 9 1.1.8. Amino acid, Fatty acid and Salt Sensing in Drosophila melanogaster 10 1.2 Post-ingestive nutrient sensing in mammals and Drosophila melanogaster 11 1.2.1. Post-ingestive amino acid sensing in mammals 11 1.2.2. Post-ingestive amino acid sensing in Drosophila melanogaster 12 1.2.3. Post-ingestive lipid sensing in mammals 13 1.2.4. Post-ingestive carbohydrate sensing in mammals 13 1.2.4.1. The role of the nervous system in post-ingestive carbohydrate sensing mammals 16 1.2.5. Post-ingestive carbohydrate sensing in Drosophila melanogaster 16 1.2.5.1. The role of the nervous system in post-ingestive carbohydrate sensing in flies 18 1.2.5.2. The role of the nervous system in post-ingestive carbohydrate sensing in larvae 19 1.3 The cellular architecture of the larval nervous system 20 1.3.1 The Blood-brain barrier (BBB) and carbohydrate transport 23 1.4 Aim of study 25 2. Experimental Procedures 26 2.1 Materials 26 2.1.1 Chemicals 26 2.1.2 Media 27 2.1.3 Buffer and Solution 27 2.1.4 Antibodies 28 2.1.5 Flystocks 29 2.2 Methods 33 2.2.1 Fly genetics 33 2.2.1.1 Maintenance and crosses 33 2.2.2 Immunohistochemistry & Microscopy 33 2.2.2.1 Immunohistochemistry of larval filets 33 2.2.2.2 Confocal Microscopy 33 2.3 Experimental Design 34 2.3.1 Studying food choice in Drosophila larvae 34 2.3.1.1 Concept 34 2.3.1.2 Experimental setup 36 2.3.1.3 Analysis 36 2.3.1.4 MATLAB Script 38 3. Results 40 3.1. Frustrated total internal reflection based two-choice assay (TCA) 40 3.1.1 Investigating larval sugar preference 40 3.2 The role of diuretic hormone 44 (Dh44) neurons in post-ingestive glucose sensing in third instar larvae 47 3.2.1 Immunohistochemical analysis of Dh44 in third instar larval brains 47 3.2.2 Role of Dh44 in glucose sensing 49 3.3 The role of gustatory receptor 43a (Gr43a) neurons in post-ingestive nutritive carbohydrate sensing in third instar larvae 51 3.3.1 Immunohistochemical analysis of Gr43a in third instar larval brains 51 3.4. Investigating the role of polyol pathway in post-ingestive carbohydrate sensing 56 3.4.1 Identifying the key polyol pathway enzyme in Drosophila melanogaster 56 3.4.2 Examining RNAi mediated neuronal and glial knockdown of polyol pathway enzymes in postprandial sugar sensing 57 3.4.2.1 CG9436 57 3.4.2.2 CG6084 and CG10863 60 3.4.2.3 Sodh-2 66 3.5 Determining the glia subtypes vital for conducting polyol pathway 69 3.6 The role of the blood brain barrier (BBB) in post-ingestive carbohydrate sensing 74 4. Discussion 80 4.1. Gr43a is the only sugar sensor in Drosophila larvae 80 4.2. Polyol Pathway is crucial for glucose and sorbitol sensing 82 4.3. Glia, the master metabolic sensor 84 4 4. Transport over BBB: a prerequisite for postprandial carbohydrate sensing85 4.5. Proposed model for larval post-ingestive nutritive carbohydrate sensing... 86 5. References 89 6. Abbreviation List 100 7. Appendix 10

Glial glucose metabolism- a global metabolic sensor governing decision-making in Drosophila melanogaster larvae

Metabolic coupling between glial cells and neurons is essential for neuronal function. It is a well-conserved and vital feature of the bilaterian nervous system as well. Under normal and adverse conditions, glial cells act as a major metabolic hub fueling neuronal oxidative metabolism by producing lactate or ketone bodies. I now ask the question of whether such metabolic coupling is only necessary for preserving brain homeostasis or if it could also have implications in decision making such as food choice behavior. Choosing an appropriate food source is key for the survival of an organism. Carbohydrates are the preferred source of energy and thus evaluation of their nutritive content is essential. Several studies have demonstrated that Drosophila melanogaster larvae and adults, as mammals, can distinguish between nutritious and non-nutritious carbohydrates independent of their taste. Two groups of neurons, Diuretic Hormone 44 (Dh44)-expressing neurons and gustatory receptor 43a (Gr43a)-expressing neurons, have been implicated in postprandial sugar sensing in adult flies. Gr43a- expressing neurons are narrowly fine-tuned for sensing fructose in both adults and larvae. Nonetheless, in the larva, central nervous system (CNS) Gr43a neurons have been shown to act as the main sugar sensor. This raises the question of how CNS fructose-sensing neurons are involved in sensing non-fructose dietary sugars. To decipher this post-ingestive mechanism, I used frustrated total internal reflection (FTIR) - based larval tracking to investigate larval food choice behavior. The results present in this thesis suggest that besides Gr43a-expressing neurons, glial cells are indispensable for sensing nutritive non-fructose dietary sugars such as glucose and sorbitol. I show that post-ingestive carbohydrate sensing involves carbohydrate conversion into fructose locally in the glial cells. Glia-derived fructose then enables Gr43a-dependent postprandial carbohydrate sensing in the CNS and drives carbohydrate preferences with the help of the downstream signalling peptide corazonin. Thus, in post-ingestive carbohydrate sensing, the glial cells act as a master metabolite sensor and provide a fructose stimulus to neurons to regulate behavior.:Table of Contents Abstract 1 Zusammenfassung 2 1. Introduction 3 1.1. External Nutrient Sensing in mammals and Drosophila melanogaster 4 1.1.1. Taste detection in mammals 4 1.1.2. Taste detection in Drosophila melanogaster 6 1.1.3 Sweet and Umami Taste in mammals 8 1.1.4. Sweet Sensing in Drosophila melanogaster 8 1.1.5. Bitter Taste in mammals 9 1.1.6. Bitter Sensing in Drosophila melanogaster 9 1.1.7. Sour, Carbonation, Fatty acid and Salty taste in mammals 9 1.1.8. Amino acid, Fatty acid and Salt Sensing in Drosophila melanogaster 10 1.2 Post-ingestive nutrient sensing in mammals and Drosophila melanogaster 11 1.2.1. Post-ingestive amino acid sensing in mammals 11 1.2.2. Post-ingestive amino acid sensing in Drosophila melanogaster 12 1.2.3. Post-ingestive lipid sensing in mammals 13 1.2.4. Post-ingestive carbohydrate sensing in mammals 13 1.2.4.1. The role of the nervous system in post-ingestive carbohydrate sensing mammals 16 1.2.5. Post-ingestive carbohydrate sensing in Drosophila melanogaster 16 1.2.5.1. The role of the nervous system in post-ingestive carbohydrate sensing in flies 18 1.2.5.2. The role of the nervous system in post-ingestive carbohydrate sensing in larvae 19 1.3 The cellular architecture of the larval nervous system 20 1.3.1 The Blood-brain barrier (BBB) and carbohydrate transport 23 1.4 Aim of study 25 2. Experimental Procedures 26 2.1 Materials 26 2.1.1 Chemicals 26 2.1.2 Media 27 2.1.3 Buffer and Solution 27 2.1.4 Antibodies 28 2.1.5 Flystocks 29 2.2 Methods 33 2.2.1 Fly genetics 33 2.2.1.1 Maintenance and crosses 33 2.2.2 Immunohistochemistry & Microscopy 33 2.2.2.1 Immunohistochemistry of larval filets 33 2.2.2.2 Confocal Microscopy 33 2.3 Experimental Design 34 2.3.1 Studying food choice in Drosophila larvae 34 2.3.1.1 Concept 34 2.3.1.2 Experimental setup 36 2.3.1.3 Analysis 36 2.3.1.4 MATLAB Script 38 3. Results 40 3.1. Frustrated total internal reflection based two-choice assay (TCA) 40 3.1.1 Investigating larval sugar preference 40 3.2 The role of diuretic hormone 44 (Dh44) neurons in post-ingestive glucose sensing in third instar larvae 47 3.2.1 Immunohistochemical analysis of Dh44 in third instar larval brains 47 3.2.2 Role of Dh44 in glucose sensing 49 3.3 The role of gustatory receptor 43a (Gr43a) neurons in post-ingestive nutritive carbohydrate sensing in third instar larvae 51 3.3.1 Immunohistochemical analysis of Gr43a in third instar larval brains 51 3.4. Investigating the role of polyol pathway in post-ingestive carbohydrate sensing 56 3.4.1 Identifying the key polyol pathway enzyme in Drosophila melanogaster 56 3.4.2 Examining RNAi mediated neuronal and glial knockdown of polyol pathway enzymes in postprandial sugar sensing 57 3.4.2.1 CG9436 57 3.4.2.2 CG6084 and CG10863 60 3.4.2.3 Sodh-2 66 3.5 Determining the glia subtypes vital for conducting polyol pathway 69 3.6 The role of the blood brain barrier (BBB) in post-ingestive carbohydrate sensing 74 4. Discussion 80 4.1. Gr43a is the only sugar sensor in Drosophila larvae 80 4.2. Polyol Pathway is crucial for glucose and sorbitol sensing 82 4.3. Glia, the master metabolic sensor 84 4 4. Transport over BBB: a prerequisite for postprandial carbohydrate sensing85 4.5. Proposed model for larval post-ingestive nutritive carbohydrate sensing... 86 5. References 89 6. Abbreviation List 100 7. Appendix 10

tRNA overexpression rescues peripheral neuropathy caused by mutations in tRNA synthetase

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tRNA overexpression rescues peripheral neuropathy caused by mutations in tRNA synthetase.

[Figure: see text]