Impairing committed cholesterol biosynthesis in white matter astrocytes, but not grey matter astrocytes, enhances in vitro myelination

Abstract Remyelination is a regenerative process that is essential to recover saltatory conduction and to prevent neurodegeneration upon demyelination. The formation of new myelin involves the differentiation of oligodendrocyte progenitor cells (OPCs) toward oligodendrocytes and requires high amounts of cholesterol. Astrocytes (ASTRs) modulate remyelination by supplying lipids to oligodendrocytes. Remarkably, remyelination is more efficient in grey matter (GM) than in white matter (WM), which may relate to regional differences in ASTR subtype. Here, we show that a feeding layer of gmASTRs was more supportive to in vitro myelination than a feeding layer of wmASTRs. While conditioned medium from both gmASTRs and wmASTRs accelerated gmOPC differentiation, wmOPC differentiation is enhanced by secreted factors from gmASTRs, but not wmASTRs. In vitro analyses revealed that gmASTRs secreted more cholesterol than wmASTRs. Cholesterol efflux from both ASTR types was reduced upon exposure to pro‐inflammatory cytokines, which was mediated via cholesterol transporter ABCA1, but not ABCG1, and correlated with a minor reduction of myelin membrane formation by oligodendrocytes. Surprisingly, a wmASTR knockdown of Fdft1 encoding for squalene synthase (SQS), an enzyme essential for the first committed step in cholesterol biosynthesis, enhanced in vitro myelination. Reduced secretion of interleukin‐1β likely by enhanced isoprenylation, and increased unsaturated fatty acid synthesis, both pathways upstream of SQS, likely masked the effect of reduced levels of ASTR‐derived cholesterol. Hence, our findings indicate that gmASTRs export more cholesterol and are more supportive to myelination than wmASTRs, but specific inhibition of cholesterol biosynthesis in ASTRs is beneficial for wmASTR‐mediated modulation of myelination.

being more pronounced than WM remyelination (Chang et al., 2012;Strijbis, Kooi, Valk, & van der, Geurts, 2017). Likewise, regional differences in remyelination efficiency are observed in experimental toxin-induced demyelination models, where remyelination in the cortex, a GM area, is faster and more robust than remyelination in the corpus callosum, a WM area (Bai et al., 2016). Revealing the underlying mechanism of this apparent discrepancy between GM and WM remyelination may identify novel targets for therapy that promote remyelination.
When OLGs are unable to produce cholesterol during CNS development myelin formation is reduced, while there is no difference in the ratio of cholesterol to other lipids incorporated in myelin (Saher et al., 2005). This indicates that cholesterol is rate-limiting for myelin membrane growth and an indispensable lipid component of myelin membranes. ASTRs are the main suppliers of cholesterol in the CNS (Camargo et al., 2012(Camargo et al., , 2017Dietschy & Turley, 2004;Nieweg, Schaller, & Pfrieger, 2009;Pfrieger, 2003) and likely supply cholesterol to OLGs via lipoprotein particles (Saher et al., 2005).
Although OPCs synthesize more cholesterol upon toxin-induced demyelination (Voskuhl et al., 2019), remyelination may also benefit from ASTR-derived cholesterol (Camargo et al., 2012). Indeed, both cholesterol supplementation and enhancing cholesterol efflux in toxin-induced demyelination models, accelerates OPC differentiation and remyelination in the corpus callosum, emphasizing a role of horizontal cholesterol transfer for remyelination (Berghoff et al., 2017;Cantuti-Castelvetri et al., 2018). In addition, when remyelination is impaired, such as in experimental autoimmune encephalomyelitis and in MS, genes encoding for enzymes involved in cholesterol production, including Hmcgs1, Fdps, and Fdft1, are decreased in ASTRs (Itoh et al., 2018). Here, we aimed to address whether gmASTRs and wmASTRs differ in cholesterol production and/or efflux and thereby distinctly modulate myelination. Our findings revealed that although the availability of ASTR-derived cholesterol may play a role in OPC maturation, downregulation of committed cholesterol biosynthesis in wmASTRs but not gmASTRs, unexpectedly led to increased myelination in vitro.
or Charles River (CRL:WI(WU), RRID:RGD_2312472) and kept under standardized temperature and humidity and a 12 hr light-dark cycle with water and food ad libitum in standard cages (one pregnant female rat or one female rat with pups per cage). A total of approximately 30 pregnant female rats were used. The dissected brains or spinal cords from 4 to 12 newborn rats (both genders) of one litter were pooled and considered as one independent cell culture preparation. There were no exclusion criteria predetermined, the study was exploratory and not pre-registered. Cell treatments were not performed in a blinded manner, and the number of experiments was not predetermined.
For cholesterol assays and qPCR assays, ASTRs were cultured at a density of 1.0 × 10 6 cells/well in six-well plates in ASTR medium.
For immunoblot analysis, 1.0 × 10 6 cells were plated on 10 cm dishes (Corning,430,167). The cells were either left untreated or treated with a mixture of pro-inflammatory cytokines (IFNγ,IL1β,and TNFα) for 24 hr at 37°C. For collection of ACM, cells were plated in 6-well plates at 1.0 × 10 6 cells/well in ASTR medium. ASTRs were either left untreated or treated with a mixture of pro-inflammatory cytokines IFNγ, IL1β, and TNFα, or cultured in the presence of 10 μg/ ml bodipy-cholesterol (Avanti, 8110255, Hölttä-Vuori et al., 2008).
After 24 hr, cells were washed and cultured for another 24 hr in Sato medium. ACM was collected, filtered using a 0.45 µm filter (GE Healthcare, 10462100) and stored at −20˚C.
Briefly, a methanol-chloroform-mixture (1:2; chloroform, Merck, 1.02445; methanol, Biosolve, 136806) was added and the samples were centrifuged at 820 g for 5 min. The lower phase was collected and the upper phase was further processed by adding chloroform (1:1). After centrifugation at 820 g for 5 min, the lower phase was added to previous collected lower phase and dried with a vacuum centrifuge and/or heating to 60°C. To determine cholesterol and phosphate levels, the dried lipid extracts of the cell samples were dissolved in 0.5 ml chloroform mixture (1:1), split and dried with the vacuum centrifuge.

| Phosphate determination
As an internal control, the amount of phosphate in each sample was determined serving as an indication of the lipid amount in the sample (Smith et al., 1985). To this end, a standard curve was prepared using 0-320 nmol phosphate. Then, 0.2 ml of 70% perchloric acid (Sigma, 244252) was added and the samples were heated to 180°C for 30 min. After cooling down, 2 ml of molybdate reagent (ammonium heptamolybdate tetrahydrate (Sigma, 12054-85-2), concentrated sulfuric acid (Sigma, 7664-93-9), water) and 0.25 ml of freshly made 10% ascorbic acid (Wako Pure Chemical, 323-44822) were added and the samples were heated to 95°C for ten minutes. To stop the reaction, the samples were placed in ice and absorbance was measured at 812 nm (μQuant, Bio-Tek). For intracellular cholesterol levels, the amount of cholesterol was normalized to phosphate, that is, the ratio of cholesterol to phosphate was calculated (nmol/nmol).

| Primary cell cultures
The cells were fixed with 4% paraformaldehyde (VWR, 1.04005) for 15 min at 20 ℃, and permeabilized with ice-cold methanol for 10 min. Non-specific antibody binding was blocked with 4% BSA for   (Qin et al., 2017;Stancic et al., 2012) or in MATLAB using software programmed to recognize only linear structures, thus only including myelin and axons and excluding OLG cell bodies (Qin et al., 2017). In each experiment, five images per coverslip and one to two coverslips per condition were analyzed.

| Cultured grey matter astrocytes are more supportive to myelination than cultured white matter astrocytes
To examine whether gmASTRs and wmASTRs distinctly modulate myelination efficiency, an in vitro myelinating system of embryonic spinal cord cultures that relies on an ASTR feeding layer was employed (Qin et al., 2017;Sorensen et al., 2008). To this end, ASTRs from the cerebral cortex (GM, referred to as gmASTRs) and non-cortical parts (mainly WM, referred to as wmASTRs) of postnatal day 1-3 rat forebrains were isolated and cultured for at least 21 days in a mixed glial culture. The obtained gmASTRs and wmASTRs were positive for the ASTR marker GFAP (Figure 1a). Consistent with previous observations of cultured ASTRs isolated from different brain regions at postnatal day 5 (Goursaud, Kozlova, Maloteaux, & Hermans, 2009), gmASTRs were more protoplasmic, while wmASTRs adopted a more fibrous stellate morphology ( Figure 1a). Therefore, regional differences in morphology are likely acquired even before ASTRs are mature, that is, ASTRs are considered mature at postnatal day 14-21 relates to a difference in gmASTR-and wmASTR-derived secreted F I G U R E 1 Grey matter astrocytes are more supportive for in vitro myelination than white matter astrocytes. (a) GFAP immunocytochemistry of primary neonatal grey matter (gm) and white matter (wm) astrocytes (ASTRs). (b and c) In vitro myelinating cultures that depend on a feeding layer of ASTRs are obtained from 15-day-old rat embryo spinal cord cells and stained for the myelin marker myelin basic protein (MBP, green) and the axonal marker neurofilament-H (NF, red). Representative images are shown in (b) and quantification of the percentage of myelinated axons in (c) (n = 6 independent cell culture preparations). Note that a feeding layer of gmASTRs is more supportive for in vitro myelination than a feeding layer of wmASTRs (p = .018). (d-f) Neonatal wmOPCs (d-f) and gmOPCs (e and f) were differentiated for three or six days in the presence of non-conditioned medium (NCM) or ASTR-conditioned medium (ACM) from gmASTRs or wmASTRs. MBP immunocytochemistry is performed to assess differentiation (% MBP-positive cells of DAPI-stained cells) and myelin membrane formation (% myelin membranes formed by MBP-positive cells). Representative images of MBP-positive wmOLGs (red) in the presence of NCM, wmACM or gmACM three days after initiating differentiation are shown in (d) (arrow indicates MBP-positive cells; arrowheads point to myelin membranes). Quantification of OPC differentiation in the presence of NCM, wmACM or gmACM is shown in (e) and quantification of myelin membrane formation in (f) (n = 4-6 independent cell culture preparations). Note that upon exposure to gmACM but not wmACM, wmOPC differentiation is significantly increased three days after initiating differentiation (p = .028) compared to exposure to NCM, while gmOPC differentiation is increased upon exposure to both wmACM and gmACM (wmACM p = .002, gmACM p = .037). Bars represent absolute values (c) or relative means compared to NCM (e,f), which is set to 1 in each independent experiment. Error bars represent standard error of the mean (SEM). Statistical analyses are performed using column statistics with a one-sample t-test (*p < .05) to test for differences between ACM treatments and NCM-treated control, while a paired t-test (*p < .05) is used to test for differences between effects of gmACM and wmACM (not significant). For wmOPC differentiation absolute values of NCM are 15.7 ± 3.5% after three days and 36.5 ± 9.1% after six days and for myelin membrane formation 55.5 ± 5.6% after three days and 63.5 ± 16.9% after six days. For gmOPC differentiation absolute values of NCM are 9.0 ± 2.7% after three days and 47.5 ± 6.1% after six days and for myelin membrane formation 47.8 ± 8.9% after three days and 84.2 ± 2.1% after six days. Scale bars are 25 µm (a and d) and 50 µm (b) factors, the effect of astrocyte-conditioned medium (ACM) on OPC differentiation and myelin membrane formation was examined in monocultures. As gmOPCs and wmOPCs distinctly respond to injury signals (Lentferink et al., 2018), and as both are present in our in vitro (e and f) Neonatal wmOPCs were differentiated for three days in the absence (ctrl) or presence of 10 μg/ml cholesterol (chol). Myelin basic protein (MBP) immunocytochemistry is performed to assess differentiation (e, % MBP-positive cells of DAPI-stained cells) and myelin membrane formation (f, % myelin membranes formed by MBP-positive cells; n = 3 independent cell culture preparations). Note that exposure to cholesterol enhances wmOPC differentiation (p = .008) and myelin membrane formation (p = .048). (g) wmOPCs were exposed to ACM obtained from ASTRs that were pre-incubated with 10 μg/mL bodipy-cholesterol and analyzed three days after initiating differentiation. Note that wmOLGs take up bodipy-cholesterol secreted from ASTRs. Bars represent absolute values (c and d) or relative means compared to wmASTRs (b, wm) or untreated wmOLGs (e and f, ctrl), which are set to 1 in each independent experiment. Error bars represent standard error of the mean (SEM). Statistical analyses are performed using column statistics with a one-sample t-test (*p < .05, **p < .01) to test for differences with wmASTRs (b) or ctrl wmOLGs (e and f), while a paired student t-test (**p < .01) is used to test for differences between cholesterol efflux from wmASTRs and gmASTRs. Absolute values of ctrl OLGs for OPC differentiation are 20.5 ± 4. 1% and for myelin membrane formation 46.7 ± 6.4%. Scale bar is 10 µm

| Inhibition of committed cholesterol biosynthesis in white matter astrocytes increases in vitro myelination
To examine whether the higher secretion of cholesterol by gmASTRs contribute to enhanced myelination, cholesterol was added to the in vitro myelinating cultures that depends on an ASTR feeding layer. Upon continuous cholesterol treatment, the percentage of myelinated axons did not increase on a feeding layer of wmASTRs or gmASTRs at the end point of myelination (Figure 5a and b).
Thus, the percentage of myelinated axons on wmASTRs was still reduced compared to gmASTRs (wmASTRs 3.5 ± 0.6% vs. gmASTRs  cytokines, including IL1β, is increased by inhibition of isoprenylationdependent signaling pathways (Lindholm & Nilsson, 2007;Massonnet et al., 2009). In line with these observations, pro-IL1β levels in ACM of kdSQS gmASTRs were reduced compared to pro-IL1β levels in ACM of control gmASTRs (Figure 6c,d, p = .032). Similarly, pro-IL1β levels were substantially, but not significantly, reduced in ACM of kdSQS wmASTRs (Figure 6c and d). Notably, active IL1β was not detected in the medium, probably because of its short half-life after secretion (Kudo, Mizuno, Hirai, & Shimizu, 1990). Hence, impairing committed cholesterol biosynthesis in wmASTRs enhanced in vitro myelination, likely by increased unsaturated fatty acid biosynthesis, as well as non-sterol isoprenoid biosynthesis.

| D ISCUSS I ON
It is known that remyelination is not only more efficient in GM MS lesions than in WM MS lesions (Chang et al., 2012;Strijbis et al., 2017), but also faster in GM than in WM upon toxin-induced F I G U R E 3 A mixture of pro-inflammatory cytokines reduces cholesterol efflux from grey and white matter astrocytes. Primary neonatal grey matter (gm) and white matter (wm) astrocytes (ASTRs) were either untreated (ctrl) or treated for 24 hr with TLR4 agonist LPS (200 ng/ ml), TLR3 agonist Poly(I:C) (50 μg/ml), or a mixture of pro-inflammatory cytokines consisting of IL1β (1 ng/ml), IFNγ (500 U/ml) and TNFα (10 ng/ml). (a) Cholesterol assays on gmASTRs and wmASTRs. Relative cholesterol efflux is shown in (a) and intracellular cholesterol levels, shown as intracellular cholesterol/phosphate ratios of gmASTRs and wmASTRs, in (b) (n = 4-15 independent cell culture preparations). Note that treatment of gmASTRs with LPS (p = .033) as well as treatment of both types of ASTRs with the pro-inflammatory cytokine mix (wmASTRs p = .001; gmASTRs p ≤ 0.001) results in a decrease of cholesterol efflux, while intracellular cholesterol levels are not changed. (c and d) Neonatal wmOPCs were differentiated for three days in the presence of ASTR-conditioned medium (ACM) from untreated and cytokine (mixture)-treated gmASTRs or wmASTRs. Myelin basic protein (MBP) immunocytochemistry is performed to assess differentiation (c, % MBP-positive cells of DAPI-stained cells) and myelin membrane formation (d, % myelin membranes formed by MBP-positive cells; n = 3 independent cell culture preparations). Note that myelin membrane formation upon exposure to gmACM, but not to wmACM, is substantially reduced upon cytokine treatment of gmASTRs (p = .139, not significant). (e-h) mRNA levels of Srebf2 (e), Fdft1 (f), Srebf1c (g) or Fasn (h) in gmASTRs and wmASTRs that were either untreated (ctrl) or treated with a mixture of IL1β, IFNγ and TNFα for 24 hr (qPCR analysis of n = 3-4 independent cell culture preparations). Note that exposure to the pro-inflammatory cytokine mix decreases transcripts for Srebf1c in wmASTRs and gmASTRs (wmASTRs p = .048, gmASTRs p = .008) and for Fasn in gmASTRs (p < .001). Bars represent relative means compared to exposure to control wmACM or gmACM (c and d) or untreated (ctrl) gmASTRs or wmASTRs (e-h), which are set to 1 in each independent experiment. Error bars represent standard error of the mean (SEM). Statistical analyses are performed using column statistics with a one-sample t-test (*p < .05, **p < .01, ***p < .001) to test for differences with control wmACM or gmACM (c and d) or untreated (ctrl) gmASTRs or wmASTRs (e-h). Absolute values of OPC differentiation with control wmACM are 21.8 ± 8.9% and with control gmACM 30.0 ± 8.3%. Absolute values of myelin membrane formation with control wmACM are 59.7 ± 8.3% and with control gmACM 70.5 ± 12.0% demyelination (Bai et al., 2016). ASTRs are important players in the remyelination process, and malfunction of ASTR signaling contributes to remyelination failure in WM MS lesions (Gutowski, Newcombe, & Cuzner, 1999;Harlow & Macklin, 2014;Lau et al., 2012;Lundgaard, Osório, Kress, Sanggaard, & Nedergaard, 2014;Stoffels et al., 2013).
Therefore, we hypothesized that in addition to wmASTRs being more detrimental, gmASTRs may be more supportive for remyelination by supplying more cholesterol to developing OLGs. Our findings demonstrate that cholesterol efflux from gmASTRs was higher than Cholesterol, non-sterol isoprenoids and unsaturated fatty acids have acetyl-CoA as a common precursor making their synthesis F I G U R E 4 Pro-inflammatory cytokines inhibit cholesterol efflux from astrocytes via an ABCA1-dependent pathway. Primary neonatal grey matter (gm) and white matter (wm) astrocytes (ASTRs) were either untreated (ctrl) or treated for 24 hr with a mixture of proinflammatory cytokines consisting of IL1β (1 ng/ml), IFNγ (500 U/mL) and TNFα (10 ng/ml) in the absence or presence of ABCA1 inhibitor glibenclamide (gli; 0.1 mM) or ABCG1 inhibitor thyroxine (T4; 50 µM). Inhibitors which were added to the cells one hour prior to addition of cytokines. (a,b) Cholesterol assays on gmASTRs and wmASTRs. Relative cholesterol efflux is shown in (a) and intracellular cholesterol levels, shown as intracellular cholesterol/phosphate ratios, in (b) (n = 3-4 independent cell culture preparations). Note that glibenclamide counteracts the cytokine-induced reduction in cholesterol efflux from both wmASTRs and gmASTRs. (c-e) Western blot analyses of cholesterol transporters cholesterol transporters ABCA1 and ABCG1. Actin served as a loading control. Representative blots are shown in (c) and quantification for ABCA1 in (d) and for ABCG1 in (e) (n = 5-6 independent cell culture preparations). Note that exposure to cytokines decreases ABCA1 expression (p < .001) and increases ABCG1 expression (p = .004) in gmASTRs. Bars represent absolute values (b) or relative means (c,d,e) compared to control gmASTRs or wmASTRs, which are set to 1 in each independent experiment. Error bars represent standard error of the mean (SEM). Statistical analyses are performed using column statistics with a one-sample t-test (*p < .05, **p < .01, ***p < .001) to test for differences between control gmASTRs or wmASTRs. A one-way ANOVA with a Šidák multiple comparisons post-test (#p < .05) is used to test for differences between different treatments intertwined and tightly coordinated by the SREBP family of transcription factors (Figure 2a). Our findings revealed that transcripts of Sreb1fc, which activates unsaturated fatty acid biosynthesis, were increased in kdSQS wmASTRs but not in kdSQS gmAS-TRs compared to control ASTRs. As addition of polyunsaturated fatty acids promotes OPC differentiation (van Meeteren, Baron, Beermann, Dijkstra, & van Tol, 2006), increased unsaturated fatty acid biosynthesis may contribute the observed enhanced in vitro myelination upon a feeding layer of kdSQS wmASTRs. In addition, a previous study showed that upon restricting synthesis of both cholesterol and non-sterol isoprenoids in macrophages by the HMG-CoA reductase inhibitor simvastatin, the secretion of IL1β and IL8 increases, while the secretion of TNFα decreases, through an isoprenylation-dependent mechanism (Lindholm & Nilsson, 2007). In line with these findings, upon specific blocking of cholesterol biosynthesis in ASTRs, as established here with kdSQS, more substrate likely became available for non-sterol isoprenoid synthesis, which may explain the reduced pro-IL1β secretion from kdSQS ASTRs. Although findings on IL1β affecting OPC differentiation are conflicting (Vela, Molina-Holgado, Arévalo-Martín, Almazán, & Guaza, 2002;Xie et al., 2016), IL1β inhibits wmOPC differentiation in vivo (Xie et al., 2016). This indicates that statin-mediated inhibition of remyelination in the corpus callosum (Klopfleisch et al., 2008;Miron et al., 2009)  In line with the present findings, differences between GM and WM myelination upon inhibition of lipid biosynthesis in ASTRs has been described. ASTR specific deletion of SREBP-cleavageactivating protein (SCAP) that post-transcriptionally activates SREBPs (Shimano & Sato, 2017) results in a decrease of WM volume of approximately 60%, while the reduction in GM was only 10% (Camargo et al., 2017;Lindholm & Nilsson, 2007;Massonnet et al., 2009;Xie et al., 2016). Whether this is because of the lower amount of myelin present in GM or that wmOPC differentiation, as observed in the present study, is more affected by the reduced synthesis of ASTR-derived lipids is not studied yet. Similarly, hypomyelination in WM but not in GM may partially relate to reduced biosynthesis of non-sterol isoprenoids. As a result, increased secretion of several ASTR-derived cytokines may delay and/or reduce wmOPC differentiation and myelination (Camargo et al., 2017;Lindholm & Nilsson, 2007;Massonnet et al., 2009;Xie et al., 2016). In fact, wmOPC differentiation may depend more on secreted factors from ASTRs than gmOPC differentiation, including cholesterol, and wmOPCs are more susceptible to the effect of pro-inflammatory cytokines than gmOPCs (Lentferink et al., 2018). Of relevance, upon SQS deletion in OLGs, which makes OLGs dependent on cholesterol supply by other cells, myelin appears of normal thickness in GM, whereas it is very thin in WM (Saher et al., 2005). Although it cannot be excluded that this regional difference in the involvement of cholesterol in myelin membrane growth depends on an intrinsic difference between gmOPCs and wmOPCs, it is likely that a higher cholesterol efflux from gmASTRs, as is suggested (Saher et al., 2005) (Itoh et al., 2018). Our results further revealed that exposure to a mixture of pro-inflammatory cytokines reduced mRNA levels of Srebf1c in gmASTRs and wmASTRs. In addition, the mRNA expression of Fasn was reduced in cytokine-treated gmAS-TRs, but not in cytokine-treated wmASTRs. Hence, exposure to pro-inflammatory cytokines likely interfered with both cholesterol efflux and unsaturated fatty acid synthesis. As inflammatory activity is lower in GM MS lesions than in WM MS lesions (Chang F I G U R E 5 Inhibition of committed cholesterol biosynthesis in white matter astrocytes increases in vitro myelination. (a and b) In vitro myelinating cultures that depend on a feeding layer of astrocytes (ASTRs) are obtained from 15 days old rat embryo spinal cord cells and either left untreated (ctrl) or treated with 10 μg/ml cholesterol for the duration of the experiment. Cultures were stained for the myelin marker myelin basic protein (MBP, green) and the axonal marker neurofilament-H (NF, red). Representative images are shown in (a) and quantification of the percentage of myelinated axons in (b) (n = 4 independent cell culture preparations). Note that addition of cholesterol does not affect in vitro myelination efficiency of both types of ASTRs. (a and c-e) A lentiviral shRNA knockdown (kd) of Fdft1, encoding for squalene synthase (SQS), was established in grey matter (gm)ASTRs and in white matter (wm)ASTRs (kdSQS). A shRNA scrambled (scr) construct was used as negative control. mRNA levels of Fdft1 mRNA levels in control (ctrl), scr and kdSQS gmASTRs and wmASTRs is shown in (c) (qPCR analysis, n = 6 independent cell culture preparations), cholesterol efflux in (d) and intracellular cholesterol levels, shown as intracellular cholesterol/phosphate ratios, in (e) (n = 4 independent culture preparations). Note that Fdft1 mRNA levels (wmASTRs and gmASTRs p < .001) and cholesterol efflux (wmASTRs p = .041 and gmASTRs p = .014) are decreased in kdSQS ASTRs. (a and f) In vitro myelinating cultures on a feeding layer of ctrl, scr, and kdSQS gmASTRs and wmASTRs. Representative images are shown in (a) and quantification of the percentage of myelinated axons in (f) (n = 4 independent cell culture preparations). (g) Neonatal wmOPCs were differentiated for three days in the presence of ASTR-conditioned medium (ACM) from control, scr and kdSQS gmASTRs or wmASTRs. MBP immunocytochemistry is performed to assess differentiation (% MBP-positive cells of DAPI-stained cells) and myelin membrane formation (% myelin membranes formed by MBP-positive cells) (n = 4 independent independent cell culture preparations). Bars represent relative means compared to control wmASTRs or gmASTRs (b), which are set to 1 in each independent experiment. Error bars represent standard error of the mean (SEM). Statistical analyses are performed using column statistics with a one-sample t-test (*p < .05, **p < .01, ** p < .001) to test for differences between control wmASTRs or gmASTRs. Absolute values of the percentage of myelinated axons on a feeding layer of control ASTRs are 3.2 ± 0.9% for wmASTRs and 6.7 ± 1.5% for gmASTRs. Absolute values for control ACM of wmASTRs are 20.5 ± 5.4% for OPC differentiation and 56.1 ± 7.9% for myelin membrane formation. Absolute values for control ACM of gmASTRs are 23.7 ± 2.4% and 49.9 ± 9.1%, respectively. Scale bar is 10 µm All experiments were conducted in compliance with the ARRIVE guidelines.

CO N FLI C T S O F I NTE R E S T
The authors declare that they have no conflicts of interest.

F I G U R E 6
Inhibition of committed cholesterol biosynthesis in white matter astrocytes increased Sreb1fc RNA levels and substantially decreases pro-IL1β secretion. (a-d) A lentiviral shRNA knockdown (kd) of Fdft1, encoding for squalene synthase (SQS) was established in grey matter (gm)ASTRs and in white matter (wm)ASTRs (kdSQS). A shRNA scrambled (scr) construct was used as negative control. mRNA levels of Srebf1c and Fasn on control (ctrl), scrambled (scr), and kdSQS in gmASTRs and wmASTRS are shown in (a) and (b), respectively (qPCR analysis, n = 6 independent cell culture preparations). Representative Western blots of pro-IL1β levels in 80 µl ASTR-conditioned medium (ACM) are shown in (c) and quantification in (d) (n = 4 independent cell culture preparations). Note that the kdSQS in wmASTRs increases Srebf1c mRNA levels in wmASTRs (p = .013), but not in gmASTRs, and that pro-IL1β secretion is substantially reduced in ACM of kdSQS wmASTRs (not significant) and significantly reduced in kdSQS gmASTRs (p = .032). Bars represent relative means to control gmASTRs or wmASTRs, which are set to 1 in each independent experiment. Error bars represent standard error of the mean (SEM). Statistical analyses are performed using column statistics with a one-sample t-test (*p < .05) to test for differences between untreated control gmASTRs or wmASTRs