Dor activation in mature oligodendrocytes regulates α-ketoglutarate metabolism leading to enhanced remyelination in aged mice

ABSTRACT The decreased ability of mature oligodendrocytes to produce myelin negatively affects remyelination in demyelinating diseases and aging, but the underlying mechanisms are

incompletely understood. In the present study, we identify a mature oligodendrocyte-enriched transcriptional coregulator diabetes- and obesity-related gene (DOR)/tumor protein p53-inducible

nuclear protein 2 (TP53INP2), downregulated in demyelinated lesions of donors with multiple sclerosis and in aged oligodendrocyte-lineage cells. _Dor_ ablation in mice of both sexes results

in defective myelinogenesis and remyelination. Genomic occupancy in oligodendrocytes and transcriptome profiling of the optic nerves of wild-type and _Dor_ conditional knockout mice reveal

that DOR and SOX10 co-occupy enhancers of critical myelinogenesis-associated genes including _Prr18_, encoding an oligodendrocyte-enriched, proline-rich factor. We show that DOR targets

regulatory elements of genes responsible for α-ketoglutarate biosynthesis in mature oligodendrocytes and is essential for α-ketoglutarate production and lipid biosynthesis. Supplementation

with α-ketoglutarate restores oligodendrocyte-maturation defects in _Dor_-deficient adult mice and improves remyelination after lysolecithin-induced demyelination and cognitive function in

17-month-old wild-type mice. Our data suggest that activation of α-ketoglutarate metabolism in mature oligodendrocytes can promote myelin production during demyelination and aging. Access

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SGK1 DRIVES HIPPOCAMPAL DEMYELINATION AND DIABETES-ASSOCIATED COGNITIVE DYSFUNCTION IN MICE Article Open access 17 February 2025 MEDIATOR MED23 CONTROLS OLIGODENDROGENESIS AND MYELINATION

BY MODULATING SP1/P300-DIRECTED GENE PROGRAMS Article Open access 15 October 2024 ENDOGENOUS SOX8 IS A CRITICAL FACTOR FOR TIMELY REMYELINATION AND OLIGODENDROGLIAL CELL REPLETION IN THE

CUPRIZONE MODEL Article Open access 14 December 2023 DATA AVAILABILITY All the RNA-seq and Cut&Tag-seq data produced in the present study have been deposited in the NCBI GEO under

accession no. GSE233593. Other expression data used in the present study are publicly available under GEO accession nos. GSE38010 and GSE134765. Source data are provided with this paper.

CODE AVAILABILITY No customized code was used in the present study. Open-source algorithms were used as detailed in analysis methods including MACS2 (v.2.2.7.1)

(https://pypi.org/project/MACS2), Bowtie 2 (v.2.4.4) (https://bowtie-bio.sourceforge.net/bowtie2/manual.shtml), Sambamba (v.0.8.1) (https://lomereiter.github.io/sambamba), RSeQC (v.4.0.0)

(https://rseqc.sourceforge.net), ChIPseeker (v.1.28.3) (https://guangchuangyu.github.io/software/ChIPseeker), MAnorm (v.1.3.0) (https://anaconda.org/bioconda/manorm), clusterProfiler

(v.4.0.5) (https://bioconductor.org/packages/release/bioc/html/clusterProfiler.html), HOMER (v.4.11.1) (http://homer.ucsd.edu/homer), deepTools (v.3.5.1)

(https://github.com/deeptools/deepTools) and Mochiview (v.1.46) (https://www.johnsonlab.ucsf.edu/mochiview-downloads). CHANGE HISTORY * _ 24 SEPTEMBER 2024 A Correction to this paper has

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ACKNOWLEDGEMENTS We thank C. Liu and Y. Wang for _Cspg4_-_Cre__ERT_ mice, K. A. Nave for the _Cnp_-Cre line, R. Dutta and B. Trapp (supported by National Institutes of Health, grant no.

R35NS097303) for MS tissue samples and E. Hurlock for comments and suggestions. X.L.H. is supported by the National Key R&D Program of China (grant no. 2022YFA1105500), the National

Science Foundation of China (grant nos. 82171356 and 82203832) and the Fundamental Research Funds for the Central Universities (grant no. YJ202325), and Q.R.L. is supported in part by the

National Multiple Sclerosis Society award (grant no. RG-2110-38554). AUTHOR INFORMATION Author notes * These authors contributed equally: Guojiao Huang, Zhidan Li, Xuezhao Liu. * These

authors jointly supervised this work: Xuelian He, Q. Richard Lu. AUTHORS AND AFFILIATIONS * Center for Translational Medicine, Key Laboratory of Birth Defects and Related Disease of Women

and Children of MOE, State Key Laboratory of Biotherapy, West China Second University Hospital, Sichuan University, Chengdu, China Guojiao Huang, Zhidan Li, Menglong Guan, Tao Zheng, Dezhi

Mu, Yingkun Guo, Lin Zhang & Xuelian He * Department of Pediatrics, Division of Experimental Hematology and Cancer Biology, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH,

USA Xuezhao Liu, Xiaowen Zhong, Dazhuan Xin & Q. Richard Lu * Key Laboratory of Neuroregeneration of Jiangsu and Ministry of Education, NMPA Key Laboratory for Research and Evaluation

of Tissue Engineering Technology Products, Jiangsu Clinical Medicine Center of Tissue Engineering and Nerve Injury Repair, Co-Innovation Center of Neuroregeneration, Nantong University,

Nantong, China Songlin Zhou & Xiaosong Gu * Department of Biotherapy, State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University, Chengdu, China Liguo

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CONTRIBUTIONS X.H. and Q.R.L. conceptualized the study and designed the experiments and wrote the manuscript. G.H., Z.L., X.L., M.G., S.Z., X.Z., T.Z., D.X. and Liguo Z. carried out most of

the experiments and data processing. Z.L. performed bioinformatic analysis. X.G., D.M., Y.G. and Lin Z. provided resources and inputs and edited the manuscript. CORRESPONDING AUTHOR

Correspondence to Xuelian He. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no competing interests. PEER REVIEW PEER REVIEW INFORMATION _Nature Neuroscience_ thanks Aiman Saab

and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. ADDITIONAL INFORMATION PUBLISHER’S NOTE Springer Nature remains neutral with regard to

jurisdictional claims in published maps and institutional affiliations. EXTENDED DATA EXTENDED DATA FIG. 1 MATURE OLS WERE ABUNDANTLY DETECTED WITHIN HUMAN MS LESIONS. (A) Schematic diagram

showing an MS brain tissue. (B, C) Representative immunostaining for MBP and CC1 (arrow) in the normal-appearing white matter (NAWM, b) and MS lesion (active and chronic active lesion types,

c) from autopsies of 6 MS subjects of either sex. (D) Quantification of CC1+ cells in NAWM and MS lesions from 6 MS tissues (Supplementary Table 1). Each point represents one case. (E)

Left: representative image of CC1+OLIG2+ cells (arrow) in MS lesions. Right: quantification of CC1+/OLIG2+ cells in in NAWM and MS lesions from 6 MS tissues. Each point represents one case.

(F) Left: co-immunolabeling (arrow) of DOR with PDGFRα in the NAWM. Right: the percentages of CC1+ cells and PDGFRα+ cells among total DOR+ cells in the NAWM (n = 6 cases). Error bars

indicate mean ± SEM. unpaired two-tailed t-test. Source data EXTENDED DATA FIG. 2 DYNAMIC DOR EXPRESSION IN THE CNS AT DEVELOPMENTAL AND ADULT STAGES. (A) Left: co-localization of DOR with

OLIG2 (arrows) from P7 to P14 by immunofluorescence labeling in corpus callosum. Right: quantification of DOR+/OLIG2+ cells on the corpus callosum of wild-type mice of either sex. n = 5

mice/stage, each point represents one mouse. One-way ANOVA (F = 9.068) with Tukey’s multiple comparisons test. (B) The percentages of DOR+ cells among CC1+ cells or PDGFRα+ cells in the

corpus callosum at P14 wild-type mice (n = 3 mice). (C) DOR and CC1 immunostaining in the corpus callosum of wild-type brain (n = 5 mice) at the indicated stages. (D) Left: DOR and CC1

immunostaining at the indicated stages in the spinal cord. Right, quantification of DOR+ CC1+cells in the ventral white matter of spinal cord. n = 3 mice/stage. (E) Left: DOR with PDGFRα

immunostaining at the indicated stages in the spinal cord. Right: the percentage of PDGFRα+ cells among DOR+ cells in the ventral white matter of spinal cord. n = 3 mice/stage. (F)

Immunostaining of the corpus callosum of P14 _Dor_ cKO mice for DOR and PDGFRα (left) or CC1 (right). n = 3 mice. (G) Photographs of optic nerves of control and _Dor_ cKO mice at P10 (left)

and P60 (right). (H, I) Representative RNA in situ hybridization for _Mbp_ (h) and _Plp1_ (i) in P2 and P14 spinal cord sections from control and _Dor_ cKO mice. n = 3 mice/group. Error bars

indicate mean ± SEM. Source data EXTENDED DATA FIG. 3 _DOR_ DELETION IN THE OL LINEAGE DOES NOT AFFECT OPCS OR OTHER CELL TYPES IN THE BRAIN. (A) Left: RNA in situ hybridization for

_PDGFRa_ in control and _Dor_ cKO brains at P2 and P7. Right: quantification of _PDGFRα_+ OPCs in the cortex. (B) Left: immunostaining for PDGFRα and Ki67 in the corpus callosum from P7 Ctrl

and _Dor_ cKO mice of either sex. Right: quantification of Ki67+ cells as a percentage of PDGFRα+ OPCs. (C) Immunolabeling (left) and quantification for the percentages of Ki67+PDGFRα+

cells (middle) and PDGFRα+ cells (right) in cortex from P60 control and _Dor_ cKO mice. (D, E) Immunolabeling and quantification for GFAP (d) and IBA1(e) in control and _Dor_ cKO mice at

P14. (F) Immunolabeling (left) and quantification (right) for cleaved caspase 3 (CC3) and CC1 in the corpus callosum of control and _Dor_ cKO mice at P14. (G) Left: quantification of g

ratios of myelinated axons in the corpus callosum from control and _Dor_ cKO mice at P14. n = 300 axons from 3 mice for each group. Each point represents one axon. Right: average g-ratio

values for different fiber diameters in control and _Dor_ cKO mice at P14. (H) Quantification of the percentage of axons with different diameters in the optic nerve (left) and corpus

callosum (right). (I) Immunostaining for GST-\(\pi\) (left) and quantification of GST-\(\pi\)+ OLs (right) in control and _Dor_ cKO brains at 7 months (7 M). (J) Immunostaining for OLIG2

(left) and quantification of OLIG2+ cells (right) in control and _Dor_ cKO cortices at 7 months. (K) EM (left) and quantification of myelinated axons (right) in control and _Dor_ cKO optic

nerves at 7 months. Error bars indicate mean ± SEM. a-g(right), h-k, n = 3 animals/genotype. Each point represents one mouse. Unpaired two-tailed t-test. Source data EXTENDED DATA FIG. 4

_DOR_ DELETION IMPAIRS MYELINOGENESIS BY OLS. (A) The brains of Ctrl (_Cnp-Cre__+/−_) and _Dor-_cKO_Cnp_ (_Dor_fl/fl;_Cnp-Cre__+/−_) mcie of either sex at P11 immunolabeled for MBP and

OLIG2. n = 3 mice/genotype. (B, C) Immunolabeling for CC1 and OLIG2 (b) and quantification (c) for CC1+ cells in Ctrl and _Dor-_cKO_Cnp_ brains at P11. (D) Top, schematic of tamoxifen (TAM)

administration and harvest at P28. Bottom, immunolabeling of corpus callosum of control (Ctrl, _Plp-Cre__ERT_) and _Dor_-iKO mice for DOR and tdTomato (tdTOM). Arrows indicate the absence of

DOR in tdTOM+ cells. Right: quantification of tdTOM+ cells. (E) Representative image of MBP immunostaining of Ctrl and _Dor_-iKO cortices at P28. Arrows, superficial cortex. n = 3

mice/genotype. (F) Immunofluorescence staining (left) and quantification (right) in Ctrl and _Dor_-iKO corpus callosum at P28. (G) Representative EM of transverse optic nerve sections from

Ctrl and _Dor_ iKO mice at P28. Middle: quantification of myelinated axons. Right: average g-ratio values for different fiber diameters in control and _Dor-_iKO mice at P28. (H)

Representative EM of corpus callosum from Ctrl and _Dor_ iKO mice at P28. Middle: quantification of myelinated axons. Right: average g-ratio values for different fiber diameters in corpus

callosum from Ctrl and _Dor_-iKO mice at P28. (I) P28 Ctrl and _Dor_-iKO brains (n = 3 mice/group) immunolabeled for PDGFRα and OLIG2. (J) Quantification of OLIG2+ and PDGFRα+ cells in

corpus callosum or cortex from Ctrl and _Dor_-iKO mice at P28. (K) The percentage of CC3+ cells among total CC1+ OLs in corpus callosum of control and _Dor_-iKO at P28. Error bars indicate

mean ± SEM. c, d, f-h, j, k, n = 3 animals/genotype. Each point represents one mouse. Unpaired two-tailed t-test. Source data EXTENDED DATA FIG. 5 DELETION OF _DOR_ IMPAIRS REMYELINATION

AFTER LPC-INDUCED DEMYELINATION. (A) Schematic of tamoxifen (TAM) administration (top). Representative image of CC1 and OLIG2 in the spinal cord of Ctrl and _Dor_-iKO mice of either sex

(bottom). Quantification of CC1+ OLIG2+ cells (right). (B) Left: EM in P120 spinal cords. Right: percentage of myelinated axons. (C) Top: schematic of TAM administration and harvest

one-month after TAM administration. Bottom: immunolabeling of spinal cords of adult mice for DOR, CC1 and tdTomato (tdTOM). Arrows indicate the absence of DOR in tdTOM+ cells in _Dor_-iKO

mice. Right: quantification of CC1+ cells among tdTomato+ cells in ventral spinal white matter. (D) Left: representative images of control and _Dor_-iKO spinal cord stained for cleaved

caspase 3 (CC3). Right: quantification for CC3. (E) ASPA immunostaining in LPC lesions from control and _Dor_-iKO spinal cords at 21 dpl. n = 5 mice/group. (F) Left: quantification of ASPA+

cells among total tdTOM+ OLs in LPC lesion sites at 21 dpl. Right: quantification of tdTOM+ cells. (G) Left: PDGFRα and OLIG2 immunostaining in LPC lesions from control and _Dor_-iKO spinal

cords at 21 dpl. Right: quantification of PDGFRα+ cells among total OLIG2+ OLs in LPC lesion sites at 21 dpl. (H) EM of LPC lesions from control and _Dor_-iKO spinal cords at 21 dpl. (I)

Left: the percentage of remyelinated axons in LPC-induced lesions of control and _Dor_-iKO spinal cords at 21 dpl. Right: scatter plot of g-ratio from LPC-induced lesions at 21dpl. n > 

300 axons were counted from 5 mice/group. Each point represents one axon. Error bars indicate mean ± SEM. a-d, n = 3 mice/group, each point represents one mouse; f, g, i(left), n = 5

mice/group, each point represents one mouse; a–d, f, g, i, unpaired two-tailed t-test. Source data EXTENDED DATA FIG. 6 DOR ACTIVATES THE TRANSCRIPTIONAL PROGRAM FOR OL MATURATION. (A)

qRT-PCR analysis of WNT signaling pathway genes in O4+ OLs from P14 cortices of control and _Dor_ cKO mice of either sex. (B) qRT-PCR analysis of indicated autophagy-related genes in O4+ OLs

from P14 cortices of control and Dor cKO. (C) Left: schematic of TAM administration and OL isolation at P14. Right: heatmap representing expression of myelination–related genes in O4+ OLs

from the transcriptome of P14 cortices of control and _Dor_ iKO mice. (D) The gene ontology (GO) enrichment of the significantly downregulated genes between control and _Dor_ iKO mice.

Significance was determined by P-value derived from ClusterProfiler. (E) Binding profiles of H3K27ac around DOR peak summits in iOL and mOL. (F) Pie chart of relative percentages of

DOR-target genes in mOLs that were significantly upregulated or downregulated in _Dor_ cKO. (G, H) Genomic track visualization of DOR, H3K27ac, and SOX10-binding profiles in iOLs and mOLs on

the regulatory elements of indicated myelin-associated genes loci (g) and loci of OL-enriched genes (h). RNA abundance represented as FPKM in indicated neural cell types. n = 2 biological

replicates. Error bars indicate mean ± SEM. a, b, n = 3 independent experiments per group, each point represents one experiment, unpaired two-tailed t-test. Source data EXTENDED DATA FIG. 7

DOR-REGULATED PRR18 IS REQUIRED FOR OL MATURATION AND MYELINATION. (A) Expression profile in different human organs. (B) The cortex of control and _Prr18_ cKO mice of either sex at P60 was

immunostained with MBP and OLIG2. Right, quantification of MBP-positive myelin areas and OLIG2+ cells in the layers I–IV of the cortex. n = 3 mice/group, each point represents one mouse. (C)

Immunolabeled for PDGFRα and OLIG2 (left) and quantification of OLIG2+ or PDGFRα+ cells (right) in the corpus callosum of control and _Prr18_ cKO mice at P60. n = 3 mice/group, each point

represents one mouse. Error bars indicate mean ± SEM. Unpaired two-tailed t-test. ns, not significant (p > 0.05). Source data EXTENDED DATA FIG. 8 DOR REGULATES THE GENES CRITICAL FOR ΑKG

BIOSYNTHESIS. (A) Expression of indicated genes quantified by real-time qPCR in rat OPCs treated with non-targeted control or _Dor_-targeted siRNA for 48 h (left). αKG measured in rat OPCs

transfected with siRNAs against _Dor_ under T3-induced differentiation condition (right). (B) qRT-PCR analyses of indicated genes (left) and αKG measurement (right) in primary rat OPCs

transfected with control (pCIG) and vector-expressing _Dor_. (C) Top: qRT-PCR analyses of TCA–associated genes following treatments with scrambled control and _Dor_-targeted siRNAs. Bottom:

DOR, SOX10, and H3K27ac occupancy on loci of genes associated with TCA cycle. (D) Heatmap of the differential metabolites (P < 0.05) between P14 Ctrl and Dor-cKOCnp O4+ OLs. n = two

independent experiments. (E) Schematic of DMKG administration (left). MBP and OLIG2 immunostaining in Veh or DMKG treated mice (middle). Right, quantification of MBP+/OLIG2+ cells. n = 3

mice/group, each point represents one mouse. Error bars indicate mean ± SEM. a-c, n = 3 independent experiments per group, each point represents one experiment. a-c, e, unpaired two-tailed

t-test. Source data EXTENDED DATA FIG. 9 ΑKG SUPPLEMENTATION ENHANCES MYELIN FORMATION. (A) A schematic of DMKG treatment (left). Immunolabeling for DOR (middle) and quantification of

DOR+/DAPI+ cells (right) in the corpus callosum of mice of either sex at P12. (B) Left: a schematic of DMKG treatment. Middle: representative EM of corpus callosum sections taken at P30 from

mice treated with Veh and DMKG. Right: quantification of g ratios and myelinated axons in corpus callosum. (C) A schematic of DMKG treatment (left). Immunolabeling for GST-\(\pi\) and OLIG2

(middle) and quantification of GST\(\pi\)+ cells (right) in the corpus callosum sections at P60. (D) A schematic of TAM and DMKG treatment (left). ASPA immunostaining in the cortex of

vehicle and DMKG treated _Dor_ iKO (middle) mice. Right, quantification of mGFP+ area and ASPA+ cells in the cortical layers I-IV. (E) qRT-PCR analysis of _Prr18_ expression in Ctrl

(_Olig1-Cre_) and _Prr18_ cKO (Prr18fl/fl;_Olig1-Cre_) optic nerves at P10. n = 3 tissues per group, each point represents one experiment. (F) Heat map representing the expression of

myelination–related genes and αKG biosynthesis-associated genes from the transcriptomic profiles of P10 optic nerves of control and _Prr18_ cKO mice. n = two independent experiments. (G) GO

enrichment of the significantly downregulated genes between control and _Prr18_ cKO. Significance was determined by P-value derived from ClusterProfiler. (H) Top: a schematic of DMKG

treatment of control and _Prr18_ cKO mice. Bottom: representative EM of optic nerve sections taken at P15 from mice treated with Veh and DMKG. Right: quantification of myelinated axons in

optic nerves. Error bars indicate mean ± SEM. a-d, h, n = 3 mice/group, each point represents one mouse. a-c, h, one-way ANOVA with Tukey’s multiple comparisons test (a, F = 20.53; b,

Fg-ratio = 34.61, F%myelinated axons = 22.07; c, F = 20.56; h, F = 27.08); d, e, unpaired two-tailed t-test. Source data EXTENDED DATA FIG. 10 MEMORY IMPAIRMENT IN _DOR_ CKO MICE AND

DMKG-INDUCED REMYELINATION IN AGED MICE. (A, B) Performance of adult control and _Dor_ cKO mice of either sex. after platform removal as measured by paths traveled (a) and number of platform

crossings, and time spent in the southwest quadrant (b). (C) Left: in situ hybridization analysis of _Mbp_ and _Plp1_ in the lesions at 14 dpl in spinal cords of aged mice treated with Veh

and DMKG. Right: quantification of the numbers of _Plp1_+ cells at 14 dpl in spinal cords of aged mice treated with Veh and DMKG. (D) Left: immunolabeling of Ki67 in the lesion regions of

aged mice carrying a PDGFRα-H2B-GFP reporter and treated with Veh and DMKG at 21 dpl. Right: quantification of GFP+Ki67+ cells. Nuclei are counterstained with DAPI. Error bars indicate mean 

± SEM. b, n = 6 mice/group; c, d, n = 5 mice/group; each point represents one mouse. b, c, d, unpaired two-tailed t-test. Source data SUPPLEMENTARY INFORMATION SUPPLEMENTARY INFORMATION

Supplementary Tables 1 and 2. REPORTING SUMMARY SUPPLEMENTARY TABLE 3 Supplementary Table 3 Metabolome datasets. SOURCE DATA SOURCE DATA FIGS. 1–7 Statistical source data for Figs.1–7.

SOURCE DATA FIGS. 2, 5 AND 7 Unprocessed western blots for Figs. 2, 5 and 7. SOURCE DATA EXTENDED DATA FIGS. 1–10 Statistical source data for Extended Data Figs. 1–10. RIGHTS AND PERMISSIONS

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CITE THIS ARTICLE Huang, G., Li, Z., Liu, X. _et al._ DOR activation in mature oligodendrocytes regulates α-ketoglutarate metabolism leading to enhanced remyelination in aged mice. _Nat

Neurosci_ 27, 2073–2085 (2024). https://doi.org/10.1038/s41593-024-01754-9 Download citation * Received: 20 July 2023 * Accepted: 07 August 2024 * Published: 12 September 2024 * Issue Date:

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