The role of ATP citrate lyase in myelin formation and maintenance

Abstract

Formation of myelin by Schwann cells is tightly coupled to peripheral nervous system development and is important for neuronal function and long-term maintenance. Perturbation of myelin causes a number of specific disorders that are among the most prevalent diseases affecting the nervous system. Schwann cells synthesize myelin lipids de novo rather than relying on uptake of circulating lipids, yet one unresolved matter is how acetyl CoA, a central metabolite in lipid formation is generated during myelin formation and maintenance. Recent studies have shown that glucose-derived acetyl CoA itself is not required for myelination. However, the importance of mitochondrially-derived acetyl CoA has never been tested for myelination in vivo. Therefore, we have developed a Schwann cell-specific knockout of the ATP citrate lyase (Acly) gene to determine the importance of mitochondrial metabolism to supply acetyl CoA in nerve development. Intriguingly, the ACLY pathway is important for myelin maintenance rather than myelin formation. In addition, ACLY is required to maintain expression of a myelin-associated gene program and to inhibit activation of the latent Schwann cell injury program.

Keywords: Schwann; acetyl CoA; lipid; lipidomic; myelin.

FIGURE 1

Lipid synthesis and acetylation reactions utilize cytosolic acetyl CoA, which is derived from mitochondria via the citrate shuttle. The citrate shuttle is required for conversion of glucose/pyruvate to acetyl CoA for fatty acid/cholesterol synthesis. In addition, metabolism of fatty acids, amino acids, and ketone bodies in mitochondria require the citrate shuttle to generate cytosolic acetyl CoA. ACLY, ATP citrate lyase. MDH, malate dehydrogenase, ME, Malic enzyme, OAA, oxaloacetate, TCA, tricarboxylic acid

FIGURE 2

Exon 9 of the Acly gene was deleted selectively in Schwann cells using Mpz‐cre, and nerve morphology was analyzed. (a and a') Paraphenylenediamine‐stained femoral motor nerve from 5‐week‐old control (a) and Acly cKO (a') mice; scale bar = 20 μm. (b) Myelin thickness displayed as g‐ratio (inner axon diameter divided by outer myelin diameter) in control and Acly cKO femoral motor nerves. (c and d) Transmission electron microscopy (TEM) images (1600×) of femoral motor nerve from 5‐week‐old control (c) and Acly cKO (d) mice; scale bar = 8 μm. Arrows and arrowheads point to amyelinated and thinly myelinated axons, respectively. (e) TEM image (3400×) of “onion bulb” morphology in Acly cKO nerve; scale bar = 2 μm. (f) TEM image (3400×) of additional examples of pathology—thinly myelinated axon (top) and amyelinated axon (bottom)—in Acly cKO nerves; scale bar = 2 μm.

FIGURE 3

(a and b) Transmission electron microscopy images (3400×) of femoral motor nerve from 2‐week‐old control (a) and Acly cKO (b) mice; scale bar = 2 μm. Arrows point to amyelinated axons. (c) Myelin thickness shown as g‐ratio in control and Acly cKO femoral motor nerves. (d) Quantitative RT‐PCR for selected genes normalized to Actb in 2‐week‐old sciatic nerve. Each dot represents one mouse. The asterisk indicates a significant difference by t‐test (p = .0003). ACLY, ATP citrate lyase.

FIGURE 4

Motor function was assessed by the inverted wire screen test in two groups: Control (n = 18, 9 males, 9 females) and Acly cKO (n = 12, 5 males, 7 females). Mice were placed on a wire screen, inverted, and their ability to hang on was timed for a maximum of 60 s over five trials. *indicates p < 4×10⁻⁴. Error bars indicate standard error of the mean.

FIGURE 5

(a). RNA‐seq analysis of Acly cKO and control sciatic nerve was performed at 5 weeks of age. Enriched categories in up‐ and downregulated genes are shown in the volcano plot. (b) Many of the reduced genes in the Acly cKO are controlled by the EGR2 transcription factor, and (c) many of the activated genes in the Acly cKO are Schwann cell injury genes regulated by the JUN transcription factor. A selection of the EGR2‐ and JUN‐regulated target genes (150 and 131, respectively) is shown.

FIGURE 6

Lipidomics was performed on control and Acly cKO sciatic nerve (n = 3/group). Summed peak intensity shows changes in lipid classes. The asterisk denotes significant changes (p < .05).

FIGURE 7

(a) Specific lipid species within each lipid class are more (orange) or less (purple) abundant in the Acly cKO (≥2.0 fold change, p < .05). (b) Volcano plot of altered lipid species in the Acly cKO. (c) The diagram shows key enzymes in the sphingolipid synthesis pathway that are reduced in the RNA‐seq data from the Acly cKO. Sphingomyelin (SM), sphingolipids (hexosylCeramides) include sphingosine and dihydrosphingosine (NS and NDS) and sulfatides (SHexCer), phosphatidylethanolamine (PE), phosphatidylcholine (PC), lysoPC (LPC).

FIGURE 8

Mass spectrometry analysis of histone modifications in Acly cKO sciatic nerve at 12 weeks, log₂ FC cKO/control, n = 3, *p = <.05 (a) The indicated changes are shown for the various combinations of histone modifications in the histone H4 4–17 peptide (b) The overall change for each specific acetylation site in histone H4 4–17 is shown.