LRP1 Regulates Peroxisome Biogenesis and Cholesterol Homeostasis in Oligodendrocytes and is Required in CNS Myelin Development and Repair

The low-density lipoprotein related-receptor-1 (LRP1) is a large endocytic and signaling receptor. We show that Lrp1 is required for proper CNS myelinogensis in vivo. Either global inducible or oligodendrocyte (OL)-lineage specific ablation of Lrp1 impairs myelin development and adult white matter repair. In primary oligodendrocyte progenitor cells (OPCs), Lrp1 deficiency reduces cholesterol levels and attenuates differentiation into mature OLs. Despite a strong increase in the sterol-regulatory element-binding protein-2, Lrp1-/- OPCs are not able to maintain normal cholesterol levels, suggesting more global metabolic deficits. Mechanistic studies identified a decrease in peroxisomal biogenesis factor-2 and a reduction in peroxisomes localized to OL processes. Treatment of Lrp1-/- OPCs with cholesterol or pharmacological activation of peroxisome proliferator-activated receptor-γ with pioglitazone is not sufficient to promote differentiation; however when combined, cholesterol and pioglitazone treatment enhance OL production. Collectively, our studies identify a novel link between LRP1, peroxisomes, and OPC differentiation during white matter development and repair.

. To examine whether Lrp1 participates in the 192 remyelination process following LPC--induced axon demyelination, adult mice were subjected 193 to unilateral and focal injection of LPC into the corpus callosum. The contralateral side was 194 injected with saline (PBS) and served as an internal control (Figure 1h). At 10 and 21 days 195 post injection (DPI), mice were killed, brains extracted, and serially sectioned through the 196 lesion area. Sections were stained with FM--G, anti--GFAP, and the nuclear dye Hoechst 33342 197 (Figure 1--figure supplement 3c and 3d). Intracranial injection of PBS led to a transient 198 increase in GFAP, but not a reduction in FM--G staining (Figure 1--figure supplement 3c). 199 Independent of Lrp1 genotype, at 10 days following LPC injection, similar--sized white matter Because Mbp mRNA is strongly upregulated in myelin producing OLs and transported into 209 internodes (Ainger et al., 1993), we used Mpb in situ hybridization on serial sections to find the 210 center of the white matter lesion. The center was defined as the section with the largest 211 circumference of the intensely labeled Mbp + area (Figure 1h). The extent of initial white 212 matter lesion, the outer rim of elevated Mbp labeling (white dotted line), was comparable 213 between Lrp1 control and iKO mice (Figure 1l). However, the area that failed to undergo 214 repair, the inner rim of elevated Mbp labeling (yellow solid line), was larger in Lrp1 iKO mice 215 (Figure 1i). Quantification of lesion repair revealed a significant decrease in Lrp1 iKO mice 216 compared to controls ( Figure  1k). As an independent assessment, serial sections through the 217 lesion were stained for Pdgfrα, Plp1, and Mag transcripts and revealed fewer labeled cells 218 within the lesion (Figure 1--figure supplement 3g). Together these findings indicate that in 219 adult mice, Lrp1 is required for the timely repair of a chemically induced white matter lesion. 220 221     1b). The partial loss of LRP1β in brain lysates of OL--lineage specific Lrp1 cKO OL mice is due to 304 Lrp1 expression in several other neural cell types .

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To examine whether Lrp1 cKO OL mice exhibit defects in myelination, optic nerves were 307 isolated at P10, the onset of myelination; at P21, near completion of myelination; and at P56, 308 when myelination is thought to be largely completed. Ultrastructural analysis at P10 revealed 309 no significant difference in myelinated axons between Lrp1 control (17± 6%) and cKO OL (7± 310 2%) optic nerves (Figure  2a  and  2b). At P21 and P56, the percentile of myelinated axons in 311 the optic nerve of cKO OL mice (49± 4% and 66± 5%, respectively) is significantly reduced 312 compared to controls (70± 2% and 88± 1%, respectively) (Figure 2a and 2b). Similarly to 313 Lrp1 iKO mice (Figure 1--figure supplement 2a), in Lrp1 cKO OL mice intermediate to small 314 sized axons, 0.3--0.9µm in caliber, are more vulnerable to hypomyelination (Figure 2-- figure  315 supplement 1c, 1f, and 1i). As an independent assessment of fiber structure, the g--ratio was 316 determined. At P10, P21, and P56 the average g--ratio of Lrp1 cKO OL optic fibers is significantly 317 larger than in age--matched Lrp1 control mice (Figure 2c and    conduction. To examine whether Lrp1 in OLs is required for nodal or paranodal organization, 427 optic nerve sections of P21 Lrp1 control and cKO OL mice were immunostained for sodium 428 channels (PanNaCh) and the paranodal axonal protein (Caspr). Nodal density, the number of 429 PanNaCh + clusters in longitudinal optic nerve sections is significantly reduced in Lrp1 cKO OL 430 mice (Figure 2d and 2g). In addition, an increase in nodal structural defects, including 431 elongated nodes, heminodes, and nodes in which sodium channel staining is missing, was 432 observed in mutant nerves (Figure 2e). Quantification of nodal structural defects revealed an 433 increase from 13.7± 1.3% in Lrp1 control mice to 33.4± 2.9% in cKO OL optic nerves ( Figure  434 2h). Of the total number of PanNaCh + nodes quantified (including elongated nodes and 435 heminodes), a greater fraction is associated with large (>1 µm) and intermediate (0   OL--lineage specific ablation of Lrp1 impairs timely repair of damaged white matter. To 482 determine the cell autonomy of Lrp1 in adult white matter repair, we generated mice that 483 allow inducible Lrp1 ablation selectively in OPCs in adult mice. Inducible gene ablation was 484 necessary to rule out potential confounding effects on repair, originating from developmental 485 white matter defects observed in adult Lrp1 cKO OL mice (Figure 2a). For repair studies, 486 Lrp1 flox/flox ;PDGFRα--creER TM (Lrp1 iKO OL ) mice were generated and gene ablation was induced 487 by TM administration at 8 weeks of age (Figure 2j). One month later, mice were subjected to 488 stereotaxic injection of LPC into the corpus callosum. The contralateral side was injected with 489 PBS and served as a negative control. Twenty--one days post injection (21 DPI) brains were 490 collected and analyzed. Detection of the initial white matter lesion and quantification of the 491 extent of axon re--myelination was carried out as shown above (Figure 1h and 1i). The initial 492 size of the LPC inflicted white matter lesion is comparable between Lrp1 control and iKO OL 493 mice (Figure 2k and 2l). The extent of lesion repair was significantly decreased in Lrp1 iKO OL 494 mice (Figure 2m), demonstrating a cellautonomous role for Lrp1 in the OL--lineage for the 495 timely repair of a white matter lesion. 496

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Conditional ablation of Lrp1 in the OL--lineage attenuates OPC differentiation. CNS 518 hypomyelination in Lrp1 cKO OL mice may be the result of reduced OPC production or impaired 519 OPC differentiation into myelin producing OLs. To distinguish between these two possibilities, 520 optic nerve cross--sections were stained with anti--PDGFRα, a marker for OPCs, anti--Olig2 to 521 account for all OL lineage cells in the culture, and anti--CC1, a marker for mature OLs. No 522 change in OPC density was observed, but the number of mature OLs was significantly reduced 523 in Lrp1 cKO OL mice (Figure  3a  and  3c). Optic nerve ISH for Pdgfrα + revealed no reduction in 524 labeled cells in Lrp1 cKO OL mice, a finding consistent with anti--PDGFRα immunostaining. The 525 density of Plp and Mag expressing cells, however, is significantly reduced in the optic nerve 526 cross--sections and longitudinal--sections of Lrp1 cKO OL mice (Figure 3b and 3d). These 527 studies reveal that OPCs are present at normal density and tissue distribution in Lrp1 cKO OL 528 mice, but apparently fail to generate sufficient numbers of mature, myelin--producing OLs. 529 530 Lrp1 in OL differentiation, we isolated OPCs from brains of Lrp1 control and cKO OL pups 557 (Figure  3e). OPCs were kept in PDGF--AA containing growth medium (GM), allowing them to 558 proliferate or switched to differentiation medium (DM) containing triiodothyronine (T3). 559 Staining of cells for the proliferation marker Ki67 did not reveal any change in OPC 560 proliferation in Lrp1 cKO OL cultures after 1 or 2 days in GM (Figure 3--figure supplement 1a--561  1c). After 3 days in DM, the number of NG2 + and CNP + OLs was comparable between Lrp1 562 control and cKO OL cultures (Figure 3f and 3h). An abundant signal for LRP1β was detected in 563 Lrp1 control lysate, but LRP1 was not detectable in Lrp1 cKO OL cell lysate, demonstrating 564 efficient gene deletion in the OL linage (Figure 3g). Moreover, a significant reduction in 565 CNP, MAG, and PLP was detected in Lrp1 cKO OL cell lysates (Figure  3--figure  supplement  1d  566 and 1e). As LRP1 signaling is known to regulate Erk1/2 and AKT activity (Yoon  et  al.,  2013), 567 immunoblots were probed for pAKT (S473) and pErk1/2. When normalized to total AKT, 568 levels of pAKT are reduced in Lrp1 cKO OL lysate, while pErk1/2 levels are comparable 569 between Lrp1 control and cKO OL lysates (Figure  3--figure  supplement  1f  and  1g). Extended 570 culture of Lrp1 deficient OLs in DM for 5 days is not sufficient to restore myelin protein levels. 571 Compared to Lrp1 control cultures, mutants show significantly fewer MAG + , PLP + , and MBP + 572 cells (Figure 3i and 3k) and immunoblotting of cell lysates revealed a reduction in total CNP, 573 MAG, PLP and MBP (Figure 3j and 3l). Collectively, our studies demonstrate a cell--574 autonomous function for Lrp1 in the OL lineage, important for cell differentiation into myelin 575 sheet producing OLs. 576 577 We find that OPCs deficient for Lrp1 (Lrp1 --/--) have reduced levels of free cholesterol 626 compared to Lrp1 control OPCs (Figure  4a,b). Levels of cholesteryl--ester are very low in the 627 CNS(Bjorkhem and Meaney, 2004) and near the detection limit in the Lrp1 control and Lrp1 --/--628 OPCs (Figure 4c). Morphological studies with MBP + OLs revealed a significant reduction in 629 myelin--like membrane sheet expansion in Lrp1 --/--OLs (Figure 4d and 4e) particularly strong near the cell soma. In Lrp1 --/--OLs, filipin and MBP staining was significantly 635 reduced ( Figure  4f). Reduced filipin staining is not simply a reflection of smaller cell size, as 636 staining intensity was decreased when normalized to myelin sheet surface area (Figure  4g). 637 Thus, independent measurements revealed a dysregulation of cholesterol homeostasis in 638 Lrp1 --/--OPCs/OLs. 639 640 cholesterol directly added to the culture medium (Figure 4i and 4j). This shows the existence 677 of LRP1 independent cholesterol uptake mechanisms in Lrp1 --/--OLs and a normal 678 physiological response to elevated levels of cellular cholesterol. In Lrp1 control cultures, bath 679 application of cholesterol leads to a small, yet significant decrease in SREBP2 (Figure 4j). 680 Given the importance of cholesterol in OL maturation (Saher et  control OLs (Figure  4k--n). Cholesterol treated Lrp1 --/--OLs showed a modest increase in PLP 685 but levels remained below Lrp1 control OLs. To ask whether prolonged treatment with 686 exogenous cholesterol promotes OL differentiation in Lrp1 --/-cultures, cells were kept for 5 687 days in DM, either with or without cholesterol. Similar to the 3 day treatment, the 5 day 688 treatment failed to increase PLP levels (Figure 4o and 4p) or the number of MBP + OLs 689 (Figure  4q  and  4r). While differentiation of Lrp1 --/--OLs cannot be "rescued" by bath applied 690 cholesterol, cell are highly sensitive to a further reduction in cholesterol, as blocking of 691 cholesterol synthesis with simvastatin leads to a further reduction in MBP + OLs (Figure 4--692  figure supplement 1c and 1d). As cholesterol is only one of many lipid derivatives produced 693 by the cholesterol biosynthetic pathway (Figure 4--figure supplement 1a), we tested biological processes might be dysregulated by Lrp1 deficiency, we performed transcriptomic 736 analyses of OPCs acutely isolated from Lrp1 control and cKO OL pups. Gene ontology (GO) 737 analysis identified differences in "peroxisome organization" and "peroxisome proliferation--738 associated receptor (PPAR) signaling pathway" (Figure 5--figure supplement 1a). Six gene 739 products regulated by Lrp1 belong to peroxisome and PPAR GO terms, including Pex2, Pex5l, 740 Hrasls, Ptgis, Mavs, and Stard10 (Figure 5--figure supplement 1b). Western blot analysis of 741 Lrp1 --/--OLs further revealed a significant reduction in PEX2 after 5 days in DM (Figure 5--742  figure  supplement  1c and 1d). Because PEX2 has been implicated in peroxisome biogenesis 743 (Gootjes et al., 2004), and peroxisome biogenesis disorders (PBDs) are typically associated 744 with impaired lipid metabolism and CNS myelin defects (Krause et al., 2006), this prompted us 745 to further explore a potential link between LRP1 and peroxisomes. To assess whether the 746 observed reduction in PEX2 impacts peroxisome density in primary OLs after 5 days in DM, 747 MBP + OLs were stained with anti--PMP70, an ATP--binding cassette transporter enriched in 748 peroxisomes (Figure 5a). In Lrp1 --/--OLs, we observed reduced PMP70 staining (Figure 5b) 749 and a decrease in the total number of peroxisomes (Figure 5c). Normalization of peroxisome 750 counts to cell size revealed that the reduction in Lrp1 --/--OLs is not simply a reflection of 751 smaller cells (Figure 5d). 752 753

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The peroxisome is a highly dynamic 776 organelle, comprised of over 50 enzymes, many of 777 which participate in lipid metabolism, including 778 the pre--squalene sequence of the cholesterol 779 biosynthetic pathway (Faust and Kovacs, 2014). 780 The subcellular localization of peroxisomes is 781 thought to be important for ensuring a timely 782 response to metabolic demands (Berger et al., 783 2016). This prompted us to analyze the subcellular 784 distribution of peroxisomes in primary OLs.

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Interestingly, while the number of PMP70 positive 786 puncta near the cell soma is comparable between 787 Lrp1 control and Lrp1 --/--OLs, we observed a 788 significant drop in peroxisomes along radial processes of MBP + OLs (Figure 5e--5h). 789 790 PPARγ, a key regulator of lipid and glucose metabolism (Mao et al., 2017). Activated PPARγ 851 moves into the nucleus to control gene expression by binding to PPAR--responsive elements 852 (PPREs) on numerous target genes, including Lrp1 (Gauthier et al., 2003). In addition, PPREs 853 are found in genes important for lipid and glucose metabolism, and peroxisome biogenesis 854 (Fang et al., 2016, Hofer et al., 2017. In vitro, a 5 day treatment of Lrp1 control OPCs with 855 pioglitazone, an agonist of PPARγ, results in elevated LRP1 (Figure 6a and 6b) and 856 accelerated differentiation into MBP + OLs (Figure 6c and 6d) (Bernardo et al., 2009). This 857 stands in marked contrast to Lrp1 --/-cultures, where pioglitazone treatment fails to accelerate 858 OPC differentiation (Figure 6c and 6d). Moreover, pioglitazone does not regulate PMP70 859 staining intensity in Lrp1 control or Lrp1 --/--OLs, nor does it have any effect on total 860 peroxisome counts per cell (Figure 6e--6i). However, pioglitazone leads to a modest but 861 significant increase in the number of peroxisomes located in cellular processes of Lrp1 --/--OLs 862 (Figure 6j and 6k). Treatment of Lrp1 control OPCs with the PPARγ antagonist GW9662 863 blocks differentiation into MBP + OLs (Roth et al., 2003), but does not lead to a further 864 reduction in MBP + cells in Lrp1 --/--OL cultures (Figure 6m). This suggests that in Lrp1 --/--OLs 865 PPARγ is not active. Given LRP1's multifunctional receptor role, we asked whether 866 simultaneous treatment with pioglitazone and exogenous cholesterol is sufficient to rescue 867 the differentiation block of Lrp1 --/--OPCs. This is indeed the case, as the number of MBP + cells 868 in Lrp1 --/-cultures is significantly increased by the combination treatment, suggesting an 869 additive effect toward OPC differentiation (Figure 6n and 6o) OLs is significantly increased. Taken together, our studies identify a novel link between LRP1 886 and peroxisomes and suggest that broad metabolic dysregulation in Lrp1 --/--OPCs attenuates 887 differentiation into mature OLs (Figure 7). 888 In the embryonic neocortex, LRP1 is strongly expressed in the ventricular zone and partially 896 overlaps with nestin + cells, suggesting expression in undifferentiated neural stem and 897 precursor cells (NSPCs) (Hennen et al., 2013). In Lrp1 flox/flox neurospheres, conditional 898 ablation of Lrp1 reduces cell proliferation and survival, and also negatively impacts 899 differentiation into neurons and O4 + OLs, while astrocyte production is significantly increased 900 (Safina et al., 2016). In line with these observations, ablation of Lrp1 with the Olig2--cre driver 901 attenuates OPC differentiation, but neither neurogenesis nor astrocyte production are 902 significantly altered in these mice. A likely explanation for these discrepancies is the OL 903 lineage restricted gene ablation in Lrp1 cKO OL mice.  Figure2--source data 1: raw data and detail statistical analysis report 1564 Figure3--source data 1: raw data and detail statistical analysis report 1565 Figure4--source data 1: raw data and detail statistical analysis report 1566 Figure5--source data 1: raw data and detail statistical analysis report 1567 Figure6--source data 1: raw data and detail statistical analysis report 1568