The Glia-Neuron Lactate Shuttle and Elevated ROS Promote Lipid Synthesis in Neurons and Lipid Droplet Accumulation in Glia via APOE/D

Abstract

Elevated reactive oxygen species (ROS) induce the formation of lipids in neurons that are transferred to glia, where they form lipid droplets (LDs). We show that glial and neuronal monocarboxylate transporters (MCTs), fatty acid transport proteins (FATPs), and apolipoproteins are critical for glial LD formation. MCTs enable glia to secrete and neurons to absorb lactate, which is converted to pyruvate and acetyl-CoA in neurons. Lactate metabolites provide a substrate for synthesis of fatty acids, which are processed and transferred to glia by FATP and apolipoproteins. In the presence of high ROS, inhibiting lactate transfer or lowering FATP or apolipoprotein levels decreases glial LD accumulation in flies and in primary mouse glial-neuronal cultures. We show that human APOE can substitute for a fly glial apolipoprotein and that APOE4, an Alzheimer's disease susceptibility allele, is impaired in lipid transport and promotes neurodegeneration, providing insights into disease mechanisms.

Keywords: APOE2; APOE3; APOE4; ARSAL; Aats-met; Alzheimer’s disease; CMT2A; Drosophila melanogaster; Leigh syndrome; MARS2; Marf; Mitofusin; Mus musculus; NDUFAF6; astrocytes; reactive oxygen species; sicily.

Figure 1. Cell specific screen to uncover proteins involved in lipid transfer

A) Model of the mechanism of lactate and lipid transfer and accumulation. B) ND42 RNAi under the control of the Rhodopsin promoter (Rh-ND42 IR) results in glial LD accumulation at day 1. Introducing neuronal (Elav) or glial (54C) GAL4s allows for knockdown of genes of interest (GOI) in a cell specific manner. Knockdown of proteins involved in lipid transfer should lead to a decrease in glial LD accumulation. C) A subset of proteins that may be involved in lipid transfer, their human orthologs and conservation as measured by the DIOPT score (Max score: 11). Effect of neuronal or glial protein knockdown on glial LD accumulation is depicted in a relative manner; three downward arrows (↓↓↓) indicate a dramatic decrease in LD accumulation and bar (−) indicates no significant change.

Figure 2. Reducing MCTs inhibit glial LD accumulation and delays neurodegeneration

A) a, j. Nile Red stain of whole-mount retina. a and f reveal a baseline of approximately 10 LD per ommatidium in 54C-GAL4; Rh-ND42 IR and Elav-GAL4; Rh-ND42 IR retinas. b–d. Glial knockdown of Sln (MCT), out (MCT) does not reduce glial LD accumulation in the Rh-ND42 IR background and knockdown of Bsg (MCT accessory protein) leads to a decrease of glial LD accumulation. g–i Neuronal knockdown of Sln, out and Bsg in the Rh-ND42 IR background lead to a decrease of glial LD accumulation. e,j knockdown of shakB does not alter LD accumulation. B) Quantification of A. C) Quantification of glial LD accumulation in the sicily^E and Marf^B mutant clones. D) Quantification of the number of remaining rhabdomeres after aging for 5 days in sicily^E and Marf^B mutant clones. All data are represented as mean ± SEM. Student’s t-tests were used to calculate all significance, n > 10 animals each (*P<0.05, **P<0.005, ***P<0.0005).

Figure 3. Primary neuronal-glial co-culture requires lactate transport to accumulate glial LD in response to ROS

A) a–b. Vehicle treated cells (24 hrs) do not exhibit LD accumulation. c–d. 2 μM rotenone treatment for 24 hrs leads to glial LD accumulation (arrowhead). e–f. 24 hr treatment with 2 μM rotenone and 200 nM MCT inhibitor (MCTi, AR-C155858) leads LD to accumulate in a subset of neurons. g–h. Cells treated with 2 μM rotenone for 24 hrs and 200 nM MCTi after 12 hrs exhibit LD accumulation in neurons and glia. B) Quantification of LD accumulation. (Kruskal-Wallis, followed by Dunn’s test for post-hoc analysis. C) Quantification of TUNEL staining post treatment. (Kruskal-Wallis, followed by Dunn’s test for post-hoc analysis. n = 200 cells counted per treatment, 3 replicates) D) Percentage incorporation of ¹³C into palmitoleic acid in astrocytes is decreased when extracellular lactate is added (Student’s t-test. Data point represent mean +/− standard deviation. n =3 biological and 3 technical replicates). E) Rotenone treated mice (3mg/kg/day, 8 days) exhibit significant (p = 0.001) LD accumulation colocalizing with astrocyte (GFAP) and microglia (IBA1) markers compared to vehicle treated mice and accumulate LD similar to p35 Ndufs4^−/− mutant mice (Student’s t-test. n = 5 per treatment. 3 sections per slice, 20 slices per animal.) All data points represent mean +/− SEM. *P<0.05, **P<0.005, ***P<0.0005. Scale bar: 50 μm.

Figure 4. Neuronal lactate is critical for glial LD accumulation

A) Quantification of LD. Glial (54C-GAL4) or neuronal (Elav-GAL4) specific knockdown of Ldh and Pdha results in a decrease of LD accumulation in the Rh-ND42 IR background. B) Removal of a copy of Pdha^A and Kdn^A reduces glial LD accumulation in the Rh-ND42 IR, Rh-Marf IR and Rh-Aats-met IR flies. C) Flies fed with dichloroacetate (DCA) exhibit a dose dependent increase in glial LD. D) Neuronal overexpression (N-Syb-GAL4) of SREBP or JNK leads to glial LD accumulation. Knockdown of MCTs (Sln, out) and metabolic enzymes (Ldh and Pdha) in the N-Syb-GAL4 overexpression background ameliorates glial LD accumulation. All data points represent mean +/− SEM (Student’s t-test. n > 10 animals each *P<0.05, **P<0.005, ***P<0.0005).

Figure 5. Lipids are transported via Fatty acid transport proteins

A) Decreasing Fatp levels in neuron or glia in the high ROS background (Rh-ND42 IR) led to less glial LD. B) Photoreceptor degeneration in sicily^E and Marf^B mutant clones is ameliorated with whole eye (Eyeless-GAL4) knockdown of Fatp. C) Primary co-culture derived from Rosa-Cas9-eGFP were transfected at 3DIV with lentiviral packaged FATP1 and FATP4 sgRNAs and subjected to rotenone treatment at 11DIV. a–d. Vehicle treated cells do not exhibit FATP1 knockdown or LD accumulation while (e–h) 2 μM rotenone treated cells exhibit glial LD accumulation. i–l. Cells transduced with FATP1 sgRNA lead to loss of FATP1. m–p. Loss of FATP1 leads to an inability to accumulate LD when subjected to 2 μM rotenone treatment. D) Quantification of C and Figure S4F. Data are represented as mean ± SEM. (Student’s t-test. n > 10 animals each or n = 200 cells counted per treatment, 3 replicates. *P<0.05, **P<0.005, ***P<0.0005). Scale bar: 50 μm.

Figure 6. Human APOE4 cannot functionally replace Glaz in lipid transport

A) a–b. Nile Red stain reveals a baseline of elevated LD accumulation. c–d. Glial knockdown of Glaz in the Rh-ND42 IR background leads to decreased in glial LD accumulation but neuronal knockdown does not alter LD accumulation. e–f. Glial knockdown of Nlaz in the Rh-ND42 IR background does not alter LD accumulation but neuronal knockdown leads to a decrease in glial LD accumulation. B) Quantification of A. (Student’s t-tests, n > 10 animals each). C) MiMIC insertion in the first coding intron of Glaz allows for recombination mediated cassette exchange to insert a Trojan exon containing a splice acceptor (SA), linker, and T2A sequence and a GAL4 sequence followed by a poly A tail. The insertion of the Trojan exon produces flies with a truncated Glaz protein (mutant) and GAL4 which is used to express any UAS-gene. D) a–b. Rh-ND42 IR and Rh-Marf IR exhibit glial LD accumulation. c–d. Removing one copy of Glaz by introducing Glaz^T2A-GAL4 decreases glial LD accumulation in the Rh-ND42 IR and Rh-Marf IR background. e–f. Replacing the one-copy-loss of Glaz with expression of APOE2 variant restores glial LD accumulation. g–h. Substituting one-copy-loss of Glaz with expression of APOE3 variant restores glial LD accumulation. i–j. Substituting one-copy-loss of Glaz with APOE4 variant does not restore LD accumulation. E) Quantification of D. F) Flies with APOE4 expression in place of Glaz accumulate less LD compared to APOE3 expressing flies when fed rotenone (Kruskal-Wallis test followed by Dunn’s test for significance. n > 10 animals each). Data are represented as mean ± SEM. (*P<0.05, **P<0.005, ***P<0.0005.).

Figure 7. Apoe null cells and APOE4 animals cannot accumulate glial LD in response to stress

A) Glial (54C-GAL4) overexpression of UAS-mCD8:GFP (control) does not lead to LD accumulation. Glial overexpression of Glaz, APOE2 and APOE3 variants lead to more than 10 LDs per ommatidium whereas APOE4 variant overexpression leads to significantly less glial LD accumulation. (Student’s t-tests were used to calculate significance. n > 10 animals each). B) a–b, Vehicle treated control cells did not accumulate glial LD after 12 hrs treatment. c–d, cells treated for 12 hrs with 1.5 μM rotenone accumulate glial LDs. e–h. Apoe^−/− treated for 12 hours with 1.5 μM rotenone accumulate minimal glial LDs. (Kruskal-Wallis test followed by Dunn’s test for significance. n < 200 cells per treatment, 3 replicates) C) Homozygous APOE4 expressing flies have more disrupted photoreceptors compared to APOE3 expressing flies immediately post ROS exposure. D). When aged for 10 days, Flies with APOE4 expression in place of Glaz lose a comparable number of neurons as Glaz null flies and more than APOE3 flies. (Kruskal-Wallis test followed by Dunn’s test for significance. n > 10 animals each) Data are represented as mean ± SEM. *P<0.05, **P<0.005, ***P<0.0005. Scale bar: 50 μm.