Glycolytically impaired glial cells fuel neural metabolism via β -oxidation

is Like of glial 1 . Here, we study how glial cells maintain sufficient nutrient supply to neurons under conditions of carbohydrate restriction. We show that glycolytically impaired glia switch to fatty acid breakdown via β -oxidation and provide ketone bodies as an alternate neuronal fuel. Moreover, flies also rely on glial β -oxidation under starvation conditions with glial loss of β -oxidation increasing susceptibility to starvation. Further, we show that glial cells act as a metabolic sensor in the brain and can induce mobilization of peripheral energy stores to ensure brain metabolic homeostasis. In summary, our study gives pioneering evidence on the importance of glial β -oxidation and ketogenesis for brain function, and survival, under adverse conditions, like malnutrition. The glial capacity to utilize lipids as an energy source seems to be conserved from flies to humans.


Introduction
The nervous system consumes a disproportionally large amount of energy compared to its size 2,3 . Carbohydrates are the preferred energy source, thus, large quantities of glucose are imported from circulation 4 . Glucose is then metabolized glycolytically. The highest glycolytic activity has been associated with glial cells, which produce lactate to fuel neuronal oxidative metabolism [5][6][7][8] .
This metabolic coupling of glial cells and neurons has been termed the "Astrocyte-Neuron Lactate Shuttle" (ANLS) and has been shown to be conserved across species 1,9 . Under optimal conditions glial cells are able to conserve energy by producing glycogen 10 that can be used to fuel peaks of neuronal energy demand or bridge short periods of malnutrition 10,11 . But is glial glycogen the only alternative fuel used in periods of glucose deprivation?
It has been suggested that under conditions of nutrient restrictions the brain takes up ketone bodies from circulation to meet its energetic demand 12,13 .
Whether the brain itself uses fatty acids (FA) to gain energy has been debated [14][15][16][17] ; even though major rate-limiting enzymes of β-oxidation are expressed in the nervous system 18,19 . Our study presents pioneering evidence of the importance of glial β-oxidation and ketogenesis for neuronal function and animal survival.
We analyze the metabolic plasticity of neurons and glia in the adult brain of Drosophila melanogaster. Glycolytically impaired glial cells indeed switch to βoxidation, allowing sustained support of neuronal metabolism by supplying ketone bodies instead of glucose-derived lactate. In addition, we found that glial β-oxidation is essential for survival upon nutrient deprivation.
Furthermore, manipulations of glial glycolytic and FA metabolism are instructive for fat storage and mobilization in the fat body. Overall, we show that in dire nutritional conditions glial cells are metabolic sensors, regulating systemic lipid traffic to ensure optimal fatty acid provision. These fats in circulation provide fuel for glial β-oxidation, producing ketone bodies, to supply the nervous system with energy.

Glycolytically impaired glial cells switch to β-oxidation to support neuronal function
Previously we showed that glial glycolysis is essential for neuronal survival in Drosophila, while neuronal glycolysis is dispensable 1 . Interestingly, glial glycolytic knockdown only reduces lifespan by about half 1 . Therefore, we speculated that glia use FA as an alternative fuel to ensure brain function and prolong survival. To analyze glial or neuronal breakdown of FA via β-oxidation, we targeted the enzymes Carnitine palmitoyltransferase 2 (CPT2, CG2107) and Mitochondrial trifunctional protein α subunit (Mtpα, CG4389). CPT2 is required to import acyl-carnitines into the mitochondria, while Mtpα possesses Enoyl-CoA hydratase and a Hydroxyacyl-CoA dehydrogenase activity and catalyzes breakdown of Acyl-CoA in the mitochondria. Knockdown of βoxidation alone in neurons or glia of adult animals did not reduce lifespan (CPT2 dsRNA , Cherry dsRNA and Mtpα dsRNA , Cherry dsRNA ). We used two UAS-dsRNA-constructs in all cases for better comparability with double knockdowns (Fig. 1A, S1A). While neuronal metabolic manipulations did not have any phenotypic consequences (Fig. S1A), simultaneous reduction of β-oxidation and pyruvate kinase (Pyk, a key enzyme of glycolysis) in adult glial cells significantly reduced lifespan compared to glycolysis knockdown alone (Fig.   1A), indicating that glial β-oxidation is essential upon glycolytic impairment.

Starvation induces a metabolic switch in the glia
Under starvation conditions, carbohydrate supply in the animal is limited 20 and alternative substrates must be used for glial energy production. To examine the effect of carbohydrate restriction in animals with impaired glial β-oxidation, we starved adult flies and assessed their lifespan using a Drosophila activity monitor (DAM) setup. Animals with glycolytically impaired glia (Pyk dsRNA ,Cherry dsRNA ) succumbed to starvation at the same rate as controls, indicating that starving animals do not rely on carbohydrate metabolism (Fig.   1B). In contrast, flies with knockdown of glial β-oxidation (CPT2 dsRNA ,Cherry dsRNA or Mtpα dsRNA ,Cherry dsRNA ) were more susceptible to starvation than control animals (Fig. 1B). Moreover, loss of glial glycolysis in addition to β-oxidation did not have any added effect (Fig. 1B). Thus, the glial switch to β-oxidation occurs under physiological conditions like nutrient restriction and is then essential to protect the brain.

Loss of glial β-oxidation in addition to glycolysis accelerates neurodegeneration
Since neurodegeneration is most likely the cause of premature death when glial glycolysis is impaired 1 , we speculated that it might be accelerated upon additional loss of β-oxidation. To test this, we assessed activity of the animals, hypothesizing that neurodegeneration correlates with a loss in coordination and mobility ( Fig. 1C,D, S1B,C). CPT2 dsRNA ,Cherry dsRNA and Mtpα dsRNA ,Cherry dsRNA flies showed wild-typic activity (Fig. 1C,D). In contrast, we found that glial Pyk dsRNA reduced activity in aged animals (22 days), indicating progressive decline of neuronal activity (Fig. 1C,D). This phenotype occurs earlier upon additional knockdown of glial β-oxidation. Pyk dsRNA ,CPT2 dsRNA or Pyk dsRNA ,Mtpα dsRNA , animals exhibited decreased mobility already after one week; and died latest at the age of three weeks. In contrast, respective neuronal metabolic manipulations did not result in abnormal phenotypes (Fig. S1B,C).
To confirm that glial loss of glycolysis and β-oxidation induces neurodegeneration, we analyzed brain morphology using semi-thin head sections. Neurodegenerative samples are characterized by areas with abnormal tissue organization 1,21 . Indeed, we detected holes in the cortical regions of glial double knockdown brains, already at two weeks of age ( and Hmgcl dsRNA ,Pyk dsRNA animals, we assayed their mobility. We found that already young (1 week) animals with inhibited glial ketogenesis and glycolysis are less active, phenocopying double β-oxidation, glycolysis knockdown animals ( Fig. 2 B, 1 C). This suggests that glia produce ketone bodies to fuel neurons in the absence of glycolytic activity.

Loss of glial glycolysis induces mitochondrial functional changes
Correct mitochondrial function is essential to provide energy; not only via the TCA-cycle and electron transport chain (ETC), but also β-oxidation. Analyzing the amounts of ATP5α, the ATPase of the ETC, in the brain revealed significant increase in all glial knockdown animals ( Fig. S3 A), indicating changes in mitochondrial function. To further study functional alterations in mitochondria, we focused on mitochondrial morphology. It has been shown that the degree of fusion or fission of mitochondria reflects their metabolic state 24 . Mitochondrial β-oxidation is more efficient when the mitochondria are in a highly fused state 25 .
To assess whether impairment of glial glycolysis changes the mitochondrial shape, we analyzed the mitochondrial appearance (using the mitochondrial reporter mitoGFP, Fig. 2 C). Indeed, mitochondria in glycolytically impaired glial cells show a much higher degree of fusion than in glial cells of control animals ( Fig. 2 C,D). Such changes in mitochondrial morphology could result in differences in the abundance of lipids important for mitochondrial function, such as phosphatidylglycerol (PG) 26,27 . To measure PG levels, we analyzed the lipid profile of adult fly brains using mass spectroscopy (MS). PG yields were reduced in Pyk dsRNA , CPT2 dsRNA animals ( Fig. 3 B) and that the ratio between phosphatidylethanolamine (PE) and phosphatidylcholine (PC) was shifted (1.6; controls = 2.1). The PE/PC ratio correlates with the cellular capacity to produce energy, and its reduction along with low PG levels indicates mitochondrial dysfunction 26,27 (Fig. 3 C).Taken together, the loss of glial glycolysis and βoxidation affects glial mitochondria supporting our hypothesis that glia switch to β-oxidation when glycolysis is impaired.

Impairment of glial metabolism induces mobilization of peripheral energy stores
β-oxidation is the breakdown of fatty acids, which are mostly stored as triacylglycerides (TAGs) in lipid droplets. Hence, higher β-oxidation rates should reduce cellular TAG stores. To test whether glial metabolic impairment changes TAG yields in brains, we measured TAG levels in lipid extracts from adult brains by MS (Fig 4 A,B). As expected, inhibited glial β-oxidation resulted in increased TAG levels in the CNS (Fig. 4 A,B). This confirms earlier work suggesting that glial loss of CPT2 induces TAG accumulation in the CNS 15 .
Interestingly, glial knockdown of glycolysis produced similar results indicating either increased fatty acid absorption from circulation or sugar to fat conversion in the glia 28,29 .

Interorgan communication between glia and fat body
Reduced glial metabolite turnover could initiate a feedback to the periphery 30 .
To test whether loss of glial glycolysis changes lipid production and/or mobilization in fat body cells (a Drosophila tissue analogous to mammalian liver and white adipose tissue 31 ), we analyzed complete head samples for their TAG content. Thin layer chromatography experiments using CNS and head samples showed that the TAG content of brains is negligible compared to the TAG content of head samples (Fig S3 A). Indeed, we found that DAG/PL ratios are very different between all genotypes tested ( Fig. S3 C,D). The fat body represents the main lipoprotein particle source and therefore, our data suggests a multi-dimensional regulation of this cell type by glia. Furthermore, our MS measurements confirmed high payload yields of lipoproteins in glycolysis, β-oxidation double knockdown animals ( Fig.   S3 C,E). However, the low levels of PLs point to low numbers of these lipid carriers (Fig S3 D,E). Taken together, our correlative results from both independent experimental approaches show that metabolic insufficiency of glia results in the mobilization of storage lipids. In addition, our data suggest an interorgan communication between glia and the periphery, responsible for controlling peripheral lipid storage.

Discussion
Neurons need a large amount of energy to function. This energy is provided with the help of glial cells that are glycolytically active, providing neurons with metabolites such as lactate 1,7,35 . Here we show that glial metabolism is not limited to glycolysis but is flexible and can switch to the use of FA to fuel neuronal metabolism when carbohydrate metabolism is not sufficient.
It has long been thought that the brain is mostly restricted to carbohydrates as an energy source 19 . However, Drosophila glial cells can also metabolize lipids via β-oxidation to produce ketone bodies to fuel neuronal metabolism ( Fig.   1,2,3). When glycolytically impaired glial cells additionally lose the capacity to perform β-oxidation, or produce ketone bodies, neurodegeneration occurs at an accelerated rate and the activity and lifespan of the animal are greatly reduced compared to loss of glycolysis alone (Fig. 1, 2). In addition, animals with a glial loss of β-oxidation are more susceptible to starvation. Thus, glial βoxidation is essential to maintain brain energy homeostasis when carbohydrate metabolism is restricted genetically or due to hypoglycemia.
In the insect CNS, lipid droplets are found in glial cells 15 . These lipid droplets are most likely initially used to fuel β-oxidation, since loss of β-oxidation leads to a glial accumulation of lipid droplets 15 . In mammals no lipid droplets are found in glial cells, but oligodendrocytes produce large amounts of lipid-rich myelin. Thus, the metabolic flexibility of glial cells to switch to β-oxidation is essential for maintaining energy homeostasis under restrictive conditions from Drosophila to humans.
In addition to using glia-intrinsic lipid stores, Drosophila glial cells can signal to the periphery to mobilize organismal energy stores to maintain adequate energy supply to neurons (Fig. 4). This indicates a new route of interorgan communication, used to maintain energy homeostasis in the brain. Also this interorgan communication is likely to be conserved, since loss of cerebral βoxidation in mammals induces changes in peripheral metabolism 17

Fly stocks
Flies were maintained at room temperature on standard food. Fly crosses were kept at 18°C for 4 weeks on standard food. After hatching, mated female progeny of the correct genotype were transferred to 29 °C and flipped onto new food every second day. Flies were counted as dead once movement ceased.

Drosophila activity monitoring (DAM)
Mated females aged for either one or three weeks at 29 °C on standard food were loaded into individual capillaries containing 5 % sucrose in agar.
Capillaries were placed in a Drosophila activity monitor with a 12-hour light dark cycle at 29 °C. The activity of the flies over a 24-hour period was recorded and difference in activity was determined using Mann-Whitney rank sum test.

Semi-thin epon sections
Heads of adult flies, aged for 17 or 38 days at 29 °C, were embedded in Epon as described previously 44 . 1 µm semi-thin sections were cut using a EM UC7 microtome (Leica), stained with toluidine blue and imaged using a Zeiss Axiophot.

Analysis of mitochondrial shape
Brains of 17 day old animals raised at 29 °C were dissected and fixed in 4% paraformaldehyde in PBS for 1.5h at RT. Images were acquired using a Leica

SDS-PAGE and Western blotting analysis:
SDS Page and western blotting was performed following published protocols: Analyzing mitochondria 46 using α-ATP5a (Abcam) and HRP (Thermo Fisher Scientific). Brain lysates were generated from 16 days old adult brains.

Protein estimation
Total protein content was examined following the manufacturer's manual (BCA Kit, Pierce).

Thin-layer chromatography
Samples were prepared as in 51,52 and lipids extracted by the BUME method 53 .

Statistics
Statistical analysis was performed using Sigma Plot. If data was normally distributed t-test was performed. For not normally distributed data rank sum test was used. ns=not significant, *p≤ 0.05, **p≤ 0.01, ***p≤ 0.001.   However, upon additional loss of glycolysis phospholipid levels are strongly reduced. N=1-3 E) DAG to PL ratios in the hemolymph.