Aurka-Bhlhe41 axis prevents premature aging-like microglial dysfunction and promotes remyelination

Domain: biology
Published: 2026-03-27
Editorial outcome: major_revision

Abstract
Aging accelerates central nervous system remyelination failure and neurodegeneration. Microglia promote remyelination by phagocytosing myelin debris, but this function is impaired by aging-related CD22 upregulation. However, the molecular mechanisms counteracting premature aging-related microglial dysfunction and remyelination impairment remain unclear. Here, we report that Aurka-Bhlhe41 axis prevents premature aging-like microglial dysfunction and promotes remyelination by restraining progressive CD22 upregulation. We identified that microglia-enriched Bhlhe41 was negatively autoregulated and inhibited by Aurka loss. Bhlhe41- or Aurka-deficient young mice exhibited aging-like microglial morphology, phagocytic deficits, progressive CD22 upregulation, and remyelination impairment in cuprizone-induced demyelination model. Conversely, ectopic Bhlhe41 expression induced hypertrophic microglia, and counteracted phagocytic deficits and CD22 upregulation in Aurka-deficient microglia. CD22 blockade restored phagocytic function and remyelination in Bhlhe41-deficient mice. Notably, a conserved pattern of CD22 upregulation was observed in human PCDH9high microglia subsets with BHLHE41 downregulation. These findings offer insights into potential therapeutic strategies to combat aging-related neurodegeneration and central nervous system functional decline.

Introduction
Aging is a primary risk factor for central nervous system (CNS) degeneration and neurodegenerative diseases, including Alzheimer’s disease (AD), Parkinson’s disease (PD), and multiple sclerosis (MS)1. A key pathological feature of these age-related CNS disorders is the accumulation of neurotoxic material, which contributes to neuronal damage and CNS dysfunction1, 2. In the context of normal aging and MS, inhibitory myelin debris accumulated from demyelination inhibits the maturation of myelinating oligodendrocytes, leading to remyelination failure and neurodegeneration2. Microglia, the CNS resident macrophages, play a crucial role in promoting CNS remyelination by phagocytic clearance of myelin debris, thereby restoring neuronal health2, 3. However, this function deteriorates with aging, which is increasingly recognized as essential to age-related CNS remyelination failure and neurodegeneration2.

Microglia, which originate from yolk sac myeloid progenitors before embryonic day 8 (E8) and reside in the brain between E9.5 and E14.5, are self-maintained in adulthood under physiological conditions4-6. With aging, microglia exhibit progressive dystrophy along with functional decline7. It is well documented that microglial phagocytic function is crucial for maintaining CNS homeostasis and is positively regulated by multiple phagocytic receptors, including triggering receptor expressed on myeloid cells 2 (TREM2), MER proto-oncogene, tyrosine kinase (MERTK) and C-X3-C motif chemokine receptor 1 (CX3CR1), along with Toll-like receptors (TLRs) and scavenger receptors8-13. Remarkably, mutations in TREM2 have been associated with multiple neurodegenerative diseases14, 15. In contrast, CD22, which is barely detected in young microglia, is upregulated during aging and impairs microglial phagocytic clearance of myelin debris16. Targeting CD22 with neutralizing antibodies restores aging microglial function and promotes CNS homeostasis16. Nevertheless, the intrinsic molecular mechanisms that counteract premature aging-related microglial dysfunction and CNS remyelination impairment remain poorly understood.

Transcription repressor basic helix-loop-helix family member e41(Bhlhe41), also known as Dec2, has been reported to regulate molecular clock and sleep length in mammals17, 18. In the immune system, Bhlhe41 is highly expressed in B-1a cells, alveolar macrophages (AM) and microglia19-21. Bhlhe41 regulates the self-renewal of B-1a cells and AM19, 20, but its function in microglia remain unknown.

In this work, we dissect the intrinsic role of Bhlhe41 in microglial biology by integrating genetic mouse models and a cuprizone (CPZ)-induced demyelination model. We show that loss of Bhlhe41 leads to premature aging-like microglial dysfunction, including dystrophic-like morphology, phagocytic deficits, progressive CD22 upregulation and remyelination impairment. Unexpectedly, we identify the mitotic kinase aurora kinase A (Aurka) as an upstream regulator of Bhlhe41, establishing an Aurka–Bhlhe41 axis that counteracts premature aging-like microglial dysfunction and promotes remyelination by restraining progressive CD22 upregulation.

Results
Bhlhe41 is microglia-enriched and negatively autoregulated

We and others have reported that Bhlhe41 is preferentially expressed in mouse microglia, alveolar macrophages (AM) and B-1 lymphocytes in the immune system19-21. To further elucidate Bhlhe41 expression in microglia during development, we first analyzed public RNA sequencing data from yolk sac myeloid progenitors and microglia across embryonic, postnatal, and adult stages22. Bhlhe41 is barely detected in yolk sac myeloid progenitors, but notably, is observed in microglia from embryonic days 11.5 (E11.5) and increases progressively during development (Supplementary Fig. 1a). To validate this finding, we used Bhlhe41 reporter mice, Bhlhe41dTomato-Cre, in which a dTomato-2A-Cre-polyA cassette was inserted into the upstream region of the start codon of Bhlhe41 locus. This resulted in Bhlhe41 promoter-driven co-expression of dTomato and Cre with Bhlhe41 translation terminated. In agreement with the RNA sequencing data, we failed to detect dTomato expression in yolk sac myeloid progenitors during E10.5 and E14.5 (Supplementary Fig. 1b). However, dTomato in microglia (Supplementary Fig. 1c) from Bhlhe41dTomato-Cre/+ mice (hereafter referred to as B41HET mice) increased in an age-dependent manner (Fig. 1a). Of note, Bhlhe41 deficiency in Bhlhe41dTomato-Cre/dTomato-Cre mice (hereafter referred to as B41KO mice) increased dTomato levels by 5-10 times in microglia, compared to a twofold increase in AM and peritoneal B-1 cells (Fig. 1a, Supplementary Fig. 1d-e), indicating potential microglia-specific self-repression of Bhlhe41 promoter activity. Further Western blot (WB) analysis was consistent with potential microglial self-repression of Bhlhe41, with B41HET microglia showing only a slight reduction rather than the expected 50%, and complete loss in B41KO microglia (Fig. 1b).

To assess whether Bhlhe41 was specific to microglia in the mouse CNS, we first performed immunofluorescence (IF) analysis of Bhlhe41+ cells (dTomato+) in the CNS of B41KO mice as dTomato levels in B41HET microglia were too low to be detected by IF. Notably, Bhlhe41 expression was enriched in microglia (Iba-1+) rather than astrocytes (Gfap+) or neurons (NeuN+), and was observed to a much lesser extent in pan-oligodendrocyte lineage cells (Olig2+) (Fig. 1c). Bhlhe41 deficiency may affect the analysis of its expression profile in the CNS. To further corroborate this finding, we analyzed B41HETRosa26EYFP/+ mice (P7 and P30), in which Bhlhe41-expressing cells were irreversibly and persistently labeled as EYFP+ cells. More than 99% of microglia, but only a few other CNS-resident cells and approximately 10% of CNS-associated macrophages (CAM) including perivascular and leptomeningeal macrophages (CD206+) were EYFP+ (Fig. 1d-e, Supplementary Fig. 2a). Further analysis revealed more than 80% of EYFP+ fetal microglia in B41HETRosa26EYFP/+ mice at E15.5 (Fig. 1f, Supplementary Fig. 2b), corroborating Bhlhe41 expression early in fetal microglia. Altogether, these data demonstrate that Bhlhe41 is microglia-enriched and negatively autoregulated in the mouse CNS, and B41HET mice may be a useful genetic tool for targeting and tracing microglia in vivo.

Bhlhe41-deficient young microglia exhibit aging-like deficits

Bhlhe41 regulates the self-renewal of B-1a cells and AM19, 20. To examine whether Bhlhe41 was required in maintaining microglia, we assessed microglial populations in young and middle-aged B41KO mice. Bhlhe41 deficiency did not affect microglia numbers at either age (Fig. 2a, Supplementary Fig. 3a). However, while wildtype (WT) and B41HET microglia displayed a typical ramified morphology, young Bhlhe41-deficient microglia exhibited aging-like morphology characterized by shorter processes and fewer branch intersections, which became more evident in middle-aged microglia (Fig. 2b, Supplementary Fig. 3b) 7. To confirm whether this abnormality was due to intrinsic Bhlhe41 deficiency in microglia, we generated a Bhlhe41 conditional knockout (KO) mouse, Bhlhe41fl/fl, in which a 2.4 kb genomic region containing the entire Bhlhe41 coding sequence was loxP flanked. Using microglia-specific Cre mice Cx3cr1Cre, we obtained microglia-specific Bhlhe41 knockout mice, Cx3cr1Cre/+Bhlhe41fl/fl (hereafter referred to as B41cKO), as well as heterozygous controls Cx3cr1Cre/+Bhlhe41fl/+ (hereafter referred to as B41cHET), and Cre controls, Cx3cr1Cre/+ (hereafter referred to as WT). WB analysis showed efficient Bhlhe41 ablation in B41cKO microglia, while B41cHET microglia exhibited only a slight, less-than-expected 50% reduction, consistent with potential microglial self-repression of Bhlhe41 (Fig. 2c). Microglia numbers were comparable across middle-aged WT, B41cHET and B41cKO mice (Supplementary Fig. 3c). While WT and B41cHET microglia retained a ramified morphology, middle-aged B41cKO microglia exhibited a dystrophic-like features, including spheroidal swelling, de-ramified and fragmented processes23 (Supplementary Fig. 3d).

Microglial dystrophy suggests potential functional impairment24. We therefore assessed whether Bhlhe41 regulates microglial phagocytic function using pHrodo Green–labeled E. coli and Alexa fluor 488 (AF488)-labeled Zymosan A (S. cerevisiae) as sensitive, generalizable screening targets. pHrodo Green fluoresces only in acidic compartments such as phagosomes, enabling assessment of particle internalization, whereas AF488 is constitutively fluorescent and was used to evaluate overall particle clearance in vivo.

In vitro phagocytosis assay revealed that B41cKO microglia exhibit impaired uptake of pHrodo Green–labeled E. coli, as evidenced by both a reduced fraction of pHrodo Green+ microglia and lower geometric mean fluorescence intensity (gMFI) per cell compared to WT microglia (Fig. 2d). To confirm this finding in vivo, we performed stereotactic injection of AF488-Zymosan into the cerebral cortex of B41KO and B41cKO mice (Fig. 2e). Unlike the pHrodo-based assay, AF488 is not restricted to acidic compartments and therefore reflects overall particle clearance rather than specific internalization. The injected Zymosan recruit and activate microglia, which phagocytose the Zymosan bioparticles25. However, five hours after injection, ablation of Bhlhe41 in both B41KO and B41cKO mice resulted in fewer microglia phagocytosing Zymosan (Fig. 2f-g). Consistently, accumulated Zymosan was detected at the injection site of both Bhlhe41-deficient models (Fig. 2h-i), indicating impaired Zymosan clearance due to loss of Bhlhe41.

Taken together, these results demonstrate that Bhlhe41 is essential for maintaining microglial morphology and phagocytic function.

Bhlhe41 loss disrupts microglial responses to remyelination

During aging and MS, microglia promote CNS remyelination by facilitating the phagocytic clearance of myelin debris12. To examine whether Bhlhe41 deficiency affects CNS myelination, we utilized the CPZ-induced demyelination model, a toxin-induced, non-immune-mediated system in which demyelination occurs without peripheral immune cell infiltration26. Five weeks of CPZ diet exposure induces the loss of myelinating oligodendrocytes, microglial activation and demyelination in the corpus callosum (CC). Subsequent exposure to a CPZ-free diet for two weeks leads to remyelination (Fig. 3a). We analyzed myelination in the CC of WT and B41cKO mice using Black Gold II (BGII) staining to assess intact myelin, IF analysis of major basic protein (Mbp) to detect total myelin, and transmission electron microscopy (TEM) analysis to examine unmyelinated and myelinated axons. Despite dystrophic-like morphology and phagocytic deficits in B41cKO microglia, myelination in the CC of WT and B41cKO mice was comparable under steady-state conditions (Fig. 3b-d). After 5 weeks of CPZ treatment, both WT and B41cKO mice exhibited significant but similar demyelination, evidenced by reduced BGII staining and Mbp levels, and increased unmyelinated axons (Fig. 3b-d). However, while WT mice displayed remyelination in the CC after being fed a CPZ-free diet for an additional two weeks, B41cKO mice exhibited delayed remyelination in the CC, characterized by a higher proportion of unmyelinated axons (Fig. 3d).

Microglial clearance of myelin debris is critical for remyelination by promoting the maturation of myelinating oligodendrocytes11. We therefore analyzed pan-oligodendrocyte lineage cells (Olig2+) and mature oligodendrocytes (CC-1+) in the CC of WT and B41cKO mice in the CPZ-induced demyelination model. While loss of oligodendrocytes was observed in both WT and B41cKO mice during the demyelination phase, mature oligodendrocytes in the CC of B41KO mice failed to repopulate during the recovery phase (Fig. 3e-f). We next co-stained degraded Mbp (dMbp), a marker of myelin debris, with Iba-1 to assess microglial phagocytosis. Although overall demyelination was comparable between WT and B41cKO mice (Fig. 3b-d), B41cKO mice accumulated more myelin debris (dMbp+) during the demyelination phase, accompanied by markedly reduced colocalization with microglia (Iba-1+), indicating impaired debris uptake (Fig. 3g). During recovery, WT mice exhibited low levels of residual myelin debris, whereas B41cKO mice retained elevated debris (Fig. 3g). Notably, increased colocalization of Iba-1 with dMbp in B41cKO mice indicates delayed microglial clearance and impaired remyelination (Fig. 3g).

Altogether, these data demonstrate that Bhlhe41 promotes remyelination by facilitating phagocytic clearance of myelin debris.

Aurka loss impairs Bhlhe41, microglial function and remyelination

In a B cell study, we unexpectedly identified that Bhlhe41 expression in B-1 cells is inhibited by loss of Aurka (Supplementary Figure 4a), a mitotic kinase that regulates mitosis and multiple other cellular events in mitotic kinase-dependent and independent manners27, 28. To examine whether Aurka regulated Bhlhe41 expression in microglia, we analyzed the Bhlhe41 expression (dTomato+) in B41HETAurkafl/fl microglia, in which loxP flanked exon 3 of Aurka was deleted by Bhlhe41-promoter driven Cre recombinase29. Compared to B41HETAurkafl/+ microglia, the gMFI of dTomato was markedly reduced in Aurka-deficient microglia, reaching the background level observed in WT microglia (Supplementary Fig. 4b). Consistently, WB analysis confirmed that Aurka ablation inhibited Bhlhe41 expression in microglia (Supplementary Fig. 4c). To further validate this finding, we generated independent microglia-specific Aurka knockout strain, Cx3cr1Cre/+Aurkafl/fl, in which Bhlhe41 expression was similarly diminished (Fig. 4a).

To explore whether inhibited Bhlhe41 expression in Aurka-deficient microglia was associated with disrupted mitosis, we analyzed Bhlhe41 expression in microglia deficient in Aurkb, another key mitotic enzyme that regulates chromosome segregation and cytokinesis30. Loss of Aurkb had no effect on Bhlhe41 expression in microglia, suggesting that Bhlhe41 expression is independent of mitosis (Supplementary Fig. 4d). These data demonstrate that Aurka controls Bhlhe41 expression in microglia.

This finding led us to examine the role of Aurka in regulating microglia number, morphology and phagocytic function. We first investigated microglia numbers and morphology in both Cx3cr1Cre/+Aurkafl/fl and B41HETAurkafl/fl mice. Compared to WT mice, both young and middle-aged Aurka-deficient mice exhibited decreased microglia numbers and a rod-like microglial morphology with diminished processes and branch intersections (Fig. 4b-c, Supplementary Fig. 5). Aurka deficiency leads to stalled mitosis with a consequence of arrested proliferation and increased apoptosis31. Therefore, we sought to examine if decreased microglia by Aurka deficiency was due to impaired mitosis and cell apoptosis. Considering that microglia proliferate explosively during the first two weeks32, we performed IF analysis of microglia in B41HETAurkafl/fl mice at P13. Aurka deficiency at P13 had no effect on microglia numbers yet (Supplementary Fig. 6a). However, this resulted in disturbed microglial proliferation in the cortex and increased apoptotic microglia in the cortex, subventricular zone (SVZ) and CC (Supplementary Fig. 6a-b). Loss of Aurka impaired microglial mitosis, identified by increased microglia positive for phospho-Histone H3, a marker for mitosis (Supplementary Fig. 6c). Despite unchanged microglia population, B41HETAurkafl/fl microglia at P13 displayed a rod-like morphology with reduced processes and branch intersections (Supplementary Fig. 6d). Notably, compared to infant B41HETAurkafl/fl mice and adult Cx3cr1Cre/+Aurkafl/fl mice, less rod-like microglia were observed in young and middle-aged B41HETAurkafl/fl mice (Fig. 4b, Supplementary Fig. 5-6). Altogether, Aurka regulates microglia development in a mitotic kinase-dependent manner.

We next examined whether Aurka regulated microglial phagocytic function and responses to remyelination. To test this, we performed in vivo phagocytosis analysis by stereotactic injection of Zymosan-AF488 into the cortex of WT and Cx3cr1Cre/+Aurkafl/fl mice, followed by IF analysis 48 hours post injection. Aurka deficiency resulted in increased extracellular accumulation of Zymosan (AF488+), suggesting that Aurka promotes microglial phagocytosis (Fig. 4d).

Using Cx3cr1Cre/+Aurkafl/fl mice, we further examined whether Aurka deficiency impaired remyelination in CPZ-indued demyelination model, following the experimental design in Fig. 3a. At steady-state condition, the myelination was unaffected by Aurka deficiency (Fig. 4e-f). After CPZ treatment, both WT and Aurka-deficient mice exhibited significant but comparable demyelination in the CC (Fig. 4e-f). During recovery, Aurka-deficient mice remained exacerbated demyelination (Fig. 4e-f). Consistently, mature oligodendrocytes (CC-1+) failed to repopulate in the CC of Aurka-deficient mice during recovery (Fig. 4g). Further co-staining of dMbp with Iba-1 showed that, similar to B41cKO mice, Aurka-deficient mice accumulated myelin debris (dMbp+) during both demyelination and recovery (Fig. 4h). Colocalization of dMbp with Iba-1 was initially reduced during demyelination but increased during recovery (Fig. 4h), indicating delayed microglial clearance of myelin debris and impaired remyelination. These data collectively suggest that Aurka is required for remyelination by sustaining microglial clearance of myelin debris.

Premature CD22 upregulation in Bhlhe41- or Aurka-KO microglia

To understand the molecular mechanisms by which Bhlhe41 maintained microglial phagocytosis, we performed gene expression microarray (GEM) analysis of young B41HET and B41KO microglia. Next, we identified reliable differentially expressed (DE) genes by comparing in-house GEM data with public bulk RNA sequencing data from Bhlhe40/Bhlhe41 double knockout microglia33. This led to the identification of 122 shared DE genes (Supplementary Data 1, Fig. 5a). Cacng1, which regulates calcium ion transmembrane transport, and Upk1b, which encodes a microglia-specific membrane receptor34, 35, ranked as the top upregulated and downregulated genes, respectively. Several DE genes including Plaur, Ptprg, Ccr5, Fcgr2b and Ciita have been associated with microglial biology (Supplementary Data 1, Fig. 5a)36-40.

Notably, Cd22, an aging-related phagocytic repressor16, was upregulated in young B41KO microglia (Supplementary Data 1, Fig. 5a). In line with prior reports, we confirmed that CD22 expression in the mouse CNS was microglia-specific, being barely detectable in young microglia but prominent in aged populations (Supplementary Fig. 7a). This observation prompted us to hypothesize that Bhlhe41 represses CD22 expression in young microglia. Flow cytometric analysis validated this hypothesis, showing that CD22 was barely detected on both young and middle-aged (10 – 14 months) WT and B41HET microglia (Fig. 5b). In contrast, CD22 was prematurely upregulated on young B41KO microglia, and this upregulation was accelerated on middle-aged B41KO microglia (Fig. 5b, Supplementary Fig. 7b). IF analysis confirmed that B41KO microglia with dystrophic-like morphology exhibited CD22 upregulation (Fig. 5c). Further studies using B41cKO mice corroborated an intrinsic effect of Bhlhe41 on CD22 suppression in microglia (Fig. 5d-e, Supplementary Fig. 7c). Nevertheless, CD22 expression was unaffected on Bhlhe41-deficient AM and B-1 cells (Supplementary Fig. 8), indicating microglia-specific CD22 suppression by Bhlhe41.

Considering inhibited Bhlhe41 expression in Aurka-deficient microglia, we further analyzed CD22 expression on B41HETAurkafl/fl microglia. Indeed, Aurka deficiency induced premature and progressive CD22 upregulation on young and middle-aged microglia (Fig. 5f). Given that CD22 upregulation on B41HETAurkafl/fl microglia might be affected by heterozygous deletion of Bhlhe41, we analyzed CD22 expression on Cx3cr1Cre/+Aurkafl/fl microglia. Consistently, CD22 upregulation was observed on rod-like Cx3cr1Cre/+Aurkafl/fl microglia (Fig. 5g). To further examine whether CD22 was upregulated on Aurka-deficient microglia that bypassed neonatal developmental deficits, we generated tamoxifen-inducible microglia-specific Aurka knockout model, Cx3cr1CreER/+Aurkafl/fl (Fig. 5h). CD22 was upregulated upon tamoxifen-induced ablation of Aurka in adult microglia, despite a modest reduction in microglial numbers (Fig. 5i-j, Supplementary Fig. 9).

Bhlhe41 represses transcription through binding to the E-box motif CACGTG19, 20. Notably, the promoters of human and murine CD22 and BHLHE41 contain the CACGTG motif (Fig. 5k). Therefore, we performed chromatin immunoprecipitation (ChIP)-qPCR analysis to determine whether Bhlhe41 is bound to the three sites, P1 in the Cd22 promoter, and P2 and P3 in the Bhlhe41 promoter (Fig. 5l). While a negative control DNA fragment located in intron 11 of Cd22 was not enriched, ChIP-qPCR analysis revealed significant enrichment of P1, P2, and P3 in fragmented chromatin from purified microglia immunoprecipitated with anti-Bhlhe41 polyclonal antibody (Fig. 5m), indicating that Bhlhe41 directly binds to its own promoter and the Cd22 promoter.

Taken together, Aurka and Bhlhe41 prevents premature aging-related CD22 upregulation in microglia.

Bhlhe41 reverses Aurka-loss-induce CD22 and microglial deficits

To address whether Aurka-deficient microglial deficits was dependent on Bhlhe41, we generated Bhlhe41 transgenic mice, Rosa26Bhlhe41-EYFP (hereafter referred to as R26B41-EYFP). In R26B41-EYFP mice, a CAG promoter-loxP-PGK-Neo-6xSV40 pA-loxP-Kozak-Bhlhe41 CDS-P2A-EYFP-WPRE-BGH pA cassette was integrated into the Rosa26 locus (Supplementary Fig. 10a). Upon Cre recombinase-mediated removal of the loxP-flanked DNA cassette, which prevents the expression of the downstream bicistronic sequences, Bhlhe41 and EYFP were independently co-expressed. WB analysis confirmed ectopic Bhlhe41 expression in microglia from microglia-specific Bhlhe41 transgenic mice, B41HETR26B41-EYFP/+ (Supplementary Fig. 10b).

Ectopic Bhlhe41 expression mildly increased microglia numbers in the CA1 and CC of B41HETR26B41-EYFP/+ mice (Supplementary Fig. 11a). Of note, B41HETR26B41-EYFP/+ microglia displayed enlarged somas with thicker, shorter and more ramified processes, a typical hypertrophic morphology24, 41, 42 (Fig. 6a). Additionally, CD68, an activation marker of microglia, was induced in Bhlhe41 transgenic microglia (Supplementary Fig. 11b). Using R26B41-EYFP mice, we next rescued Bhlhe41 expression in Aurka-deficient microglia by generating B41HETAurkafl/flR26B41-EYFP/+ mice. In Aurka-deficient microglia, ectopic Bhlhe41 expression resulted in a morphology change from rod-like microglia to ramified microglia with long and branched processes (Fig. 6b). Collectively, these data reinforce the role of Bhlhe41 in maintaining microglia morphology and homeostasis and demonstrate that Bhlhe41 is sufficient to restore morphology deficits in Aurka-deficient microglia.

Bhlhe41 deficiency enhanced Bhlhe41 promoter activity and CD22 expression (Fig. 1a, Fig. 5b-e). Hence, we investigated whether ectopic Bhlhe41 expression inhibited Bhlhe41 promoter activity and CD22 expression. The gMFI of dTomato in B41HETR26B41-EYFP/+ microglia was decreased to background levels observed in WT microglia, supporting potential Bhlhe41 self-repression in microglia (Fig. 6c). In contrast to CD22 upregulation on B41KO microglia, ectopic Bhlhe41 expression suppressed CD22 expression (Fig. 6c-d), corroborating that Bhlhe41 represses its own expression as well as CD22 expression in microglia.

To assess whether Aurka-mediated CD22 suppression was dependent on Bhlhe41, we analyzed B41HETAurkafl/flR26B41-EYFP/+ microglia. More than 97% of EYFP+ microglia from B41HETAurkafl/flR26B41-EYFP/+ mice corroborated the fact that B41HETAurkafl/fl microglia have undergone efficient DNA recombination (Fig. 6e). As expected, ectopic Bhlhe41 expression reversed CD22 upregulation on Aurka-deficient microglia (Fig. 6e-f), suggesting that Aurka suppresses CD22 expression on microglia through Bhlhe41.

To further investigate whether ectopic Bhlhe41 expression restored microglial phagocytic function in Aurka-deficient mice, we performed in vivo phagocytosis analysis by stereotactic injection of purified myelin conjugated with Dil (Myelin-Dil) into the cortex of B41HETAurkafl/fl and B41HETAurkafl/flR26B41-EYFP/+ mice. After 48 hours post injection, the injection site of B41HETAurkafl/flR26B41-EYFP/+ mice displayed reduced accumulation of injected myelin (Dil+), suggesting that Aurka-mediated microglial phagocytosis is dependent on Bhlhe41 (Fig. 6g).

Altogether, these data demonstrate that Aurka maintains microglial morphology, phagocytic function and CD22 suppression through Bhlhe41.

CD22 blockade restores CNS repair in Bhlhe41-deficient mice

To investigate whether phagocytic deficits in Bhlhe41- or Aurka- deficient microglia were ascribed to CD22 upregulation, we conducted in vivo phagocytosis by stereotaxic co-injection of myelin-Dil with either an IgG isotype control or anti-CD22 blocking antibody into opposite brain hemispheres of the same B41cKO or B41HETAurkafl/fl mice, respectively (Fig. 7a). After 48 hours post injection, CD22 blockade robustly reduced the accumulation of injected myelin (Dil+) in B41cKO or B41HETAurkafl/fl mice (Fig. 7b-c) , suggesting that phagocytic deficits in Bhlhe41- or Aurka-deficient microglia are dependent on CD22 upregulation.

To further assess whether long-term CD22 inhibition restored remyelination in Bhlhe41-deficient mice, we stereotaxically infused either an IgG isotype control or anti-CD22 blocking antibody every three days into the brain of B41cKO mice, which were meanwhile fed CPZ-enriched diet for 5 weeks, or followed by CPZ-free normal diet for an additional 2 weeks (Fig. 7d). During demyelination, BGII staining showed comparable overall demyelination between IgG- and anti-CD22-treated mice, although Mbp signal was slightly reduced, indicating that anti-CD22 did not significantly affect myelin loss (Fig. 7e–f). In contrast, during the recovery phase, anti-CD22 treatment markedly enhanced remyelination, as evidenced by increased BGII and Mbp staining and elevated numbers of mature oligodendrocytes (CC-1+), compared to IgG-treated controls (Fig. 7e–h). Colocalization analyses further revealed that anti-CD22 treatment enhanced microglia-mediated clearance of myelin debris (Fig.7i). Collectively, these results indicate that long-term CD22 blockade facilitates microglia-mediated clearance of myelin debris and promotes remyelination in Bhlhe41-deficient mice.

Taken together, CD22 blockade restores phagocytic function in Bhlhe41- and Aurka-deficient microglia and remyelination in Bhlhe41-deficient mice.

Elevated CD22 in human PCDH9high microglia with low BHLHE41

To extend our studies from mouse to human microglia, we analyzed whether BHLHE41 is specific to microglia in human CNS using public single-cell RNA sequencing (scRNA-seq) data from healthy white matter43. BHLHE41 is highly expressed in human microglia, with lower expression levels observed in certain oligodendrocytes, neurons, and astrocytes (Fig. 8a-b). Additionally, co-expression of BHLHE41 with microglia-specific genes (P2RY12 and TMEM119) was observed in a small fraction of CAM (Fig. 8a). Furthermore, analysis of two independent scRNA-seq datasets from human fetal brains revealed high BHLHE41 expression in fetal microglia (Supplementary Fig. 12)44, 45. These findings suggest that BHLHE41 is highly expressed in human CNS microglia starting early in development.

Nevertheless, analysis of public scRNA-seq data from either healthy white matter43 or brains of patients with Alzheimer’s disease (AD)46, Parkinson’s disease (PD) and Lewy body dementia (LBD)47 showed that CD22 is barely expressed in human microglia (Fig. 7b-c). Intriguingly, oligodendrocytes from both cognitively healthy subjects and patients with neurodegenerative diseases, which weakly express BHLHE41, exhibit high CD22 expression (Fig. 7b-c). These data suggest an inverse expression pattern of CD22 and BHLHE41 in human microglia and oligodendrocytes.

To further refine the analysis of BHLHE41 and CD22 expression in microglia, we performed sub-cluster analysis of 3690 microglia from healthy white matter43. A PCDH9high microglia sub-population, which express multiple microglia marker genes APBB1IP48, TBXAS149, SLCO2B150, DOCK851, CSF1R and P2RY1252, exhibits decreased BHLHE41 expression but upregulated CD22 expression (Fig. 8d-e, Supplementary Fig. 13a). Given that oligodendrocytes also highly express PCDH9 and CD22 (Supplementary Fig. 13a), we further validated this finding using independent scRNA-seq data from the dorsolateral prefrontal cortex of patients with AD and cognitively healthy subjects, which covers 40754 microglia (Supplementary Fig. 13b-c). Consistently, the sub-cluster analysis identified PCDH9high microglia sub-population, in which BHLHE41 is downregulated while CD22 is upregulated, independent of health condition (Fig. 8f-g, Supplementary Fig. 13d).

Taken together, BHLHE41 is downregulated in human PCDH9high microglia subsets with CD22 upregulation.

Methods
Mice
Cx3cr1Cre mice (Stock ID: 025524, JAX) and Cx3cr1CreER mice (Stock ID: 021160, JAX) were obtained from the Jackson Laboratory. Bhlhe41dTomato-Cre/+ (referred to as B41HET) and Aurkafl/fl mice were generated as previously reported21, 29. Bhlhe41fl/fl, Aurkbfl/fl, and Rosa26Bhlhe41-EYFP/+ (referred to as R26B41-EYFP/+) mice were generated by Cyagen Biosciences Inc. using CRISPR-Cas9 technology. To generate Bhlhe41fl/+ mice, a targeting vector with homologous arms and a 2.4 kb genomic region containing the entire Bhlhe41 coding sequence flanked by loxP sites was constructed by PCR using BAC clone RP23-133A9 as the template. To generate Aurkbfl/+mice, a targeting vector with homologous arms and exons 2-6 flanked by loxP sites was constructed by PCR using BAC clone RP24-281K2 as the template. To generate Bhlhe41 transgenic mice, R26B41-EYFP/+, a targeting vector with homologous arms and a CAG promoter-loxP-PGK-Neo-6xSV40 pA-loxP-Kozak-Bhlhe41 CDS-P2A-EYFP-WPRE-BGH pA cassette was integrated into the intron 1 of Rosa26 locus. Mice production was performed by co-injection of ribonucleoprotein complexes and the targeting vector into fertilized eggs. Primers for in-house generated mouse genotyping were designed in-house, synthesized by Sangon Biotech and listed in Supplementary Table 1. In-house generated mice in this study are available upon request.

Bhlhe41fl/fl mice were crossed with Cx3cr1Cre mice for generating microglia-specific Bhlhe41 knockout mice Cx3cr1Cre/+Bhlhe41fl/fl mice (referred to as B41cKO). Aurkafl/fl mice were crossed with B41HET, Cx3cr1Cre, or Cx3cr1CreER to generate microglia-specific Aurka knockout mice, B41HETAurkafl/fl, Cx3cr1Cre/+Aurkafl/fl and Cx3cr1CreER/+Aurkafl/fl. R26B41-EYFP/+ mice were crossed with B41HET, or B41HETAurkafl/+ mice to generate B41HETR26B41-EYFP/+ and B41HETAurkafl/flR26B41-EYFP/+mice. Aurkbfl/fl mice were crossed with B41HET mice to generate microglia-specific Aurkb knockout mice B41HETAurkbfl/fl. To avoid germline recombination, only female Bhlhe41dTomato-Cre mice or female offspring carrying the Cre allele were used for breeding with floxed mice. Wildtype (WT), Cx3cr1Cre/+, B41HET, Cx3cr1Cre/+Bhlhe41fl/+ (referred to as B41cHET), B41HETAurkafl/+, Cx3cr1Cre/+Aurkafl/+, Cx3cr1CreER/+Aurkafl/+, or B41HETAurkbfl/+ littermates were used as control mice. Cx3cr1CreER/+Aurkafl/fl mice were injected with tamoxifen (TAM, 75 mg/kg) for five consecutive days at 3 months and 6 months of age, respectively.

All mice were maintained on the C57BL/6 genetic background and maintained in a specific pathogen-free (SPF) conditions in plastic cages (22 ± 2 °C; 50 ± 10% humidity; 12-h light/dark cycle) with ad libitum access to food and water. Sex-matched mice of different genotypes were used in all experiments, except for cuprizone-induced demyelination and in vivo phagocytosis assays, which were performed exclusively in male mice to minimize biological variability. Age of mice used in each experiment is specified in figure legends. All mouse experiments were performed in accordance with the guidelines and regulations of Xuzhou Medical University and were approved by the Animal Experimental Ethics Committee of Xuzhou Medical University (Approval no: 202211S011).

Cuprizone-induced demyelination model
For the cuprizone (CPZ)-induced demyelination model, male WT and B41cKO littermates, or Cx3cr1Cre/+Aurka+/+ and Cx3cr1Cre/+Aurkafl/fl littermates, were fed regular chow mixed with 0.2% CPZ (Sigma, Cat: 14690) for 5 weeks to induce demyelination (denoted as the CPZ group), or followed by a CPZ-free normal diet for an additional 2 weeks to induce remyelination (denoted as the Recovery group). Mice fed a CPZ-free normal diet were sacrificed as the control group and sacrificed at the end of the experiment. The corpus callosum (CC), identified as the white matter tract between the lateral ventricles in coronal brain sections, was analyzed for myelination by immunofluorescence analysis of intact (Mbp+) and degraded (dMbp+) myelin, Black Gold II staining of intact myelin, and electron microscopy assessment of myelinated and unmyelinated axons. For Mbp and BGII staining, myelin density was quantified as the total staining intensity within the CC. Myelin debris was defined as the dMbp+ immunostained area above background threshold and expressed as a percentage of the total CC area. Microglial engulfment of myelin debris was assessed by quantifying the area of dMbp+ signal colocalized within Iba-1+ microglia. Three sections containing the CC per mouse were analyzed. For immunofluorescence quantifications, two non-overlapping fields of view (FOV) were acquired per section at identical settings. Values from multiple FOV were averaged to generate a single value per mouse, which was used for statistical analysis. All image acquisition and quantifications were performed in a blinded manner.

In vitro phagocytosis assay
For in vitro phagocytosis assays, microglia were enriched from the brain of WT and B41cKO mice by 40% Percoll. Enriched microglia were seeded in duplicated 96-well plates (1×105 cells per well) and in vitro cultured in RPMI 1640 complete medium containing 10% FBS and 200 μg/mL E.coli Bioparticles conjugated with pHrodo Green (ThermoFisher Scientific, Cat: P35366). One plate was cultured at 37 ℃ and the other plate at 4°C as a negative control. After one-hour, enriched microglia were washed with pre-cold phosphate buffer solution (PBS) and stained with antibodies for microglial surface markers. Microglia phagocytosis was assessed by flow cytometric analysis of pHrodo Green+ microglia by pre-gating live microglia (CD45loCD11b+Cx3cr1+).

In vivo phagocytosis assays
In vivo phagocytosis assays were performed by stereotaxic injection of phagocytic target material, Zymosan A conjugated with Alexa fluor 488 (Zymosan-AF488, ThermoFisher Scientific, Cat: Z23373) or myelin conjugated with Dil (Myelin-Dil) into indicated mice using a stereotaxic apparatus (RWD Life Science, Cat: 68507).

For the preparation of Myelin-Dil, CNS myelin was isolated as previously described68, 69. Briefly, crude brain homogenates from WT mice underwent sucrose density gradient ultracentrifugation and hypoosmotic shocks in hypotonic buffers and low-speed centrifugation to remove myelin sheaths from axons, followed by repeated sucrose density gradient ultracentrifugation. Myelin was quantified by BCA Protein Assay Kit (Beyotime Biotechnology, Cat: P0012). Next, purified myelin pellets were conjugated with Dil by incubation of 10 mg of myelin with 5 µg/mL Dil (Yuanye, Cat: S25170) in PBS for 10 min at room temperature, followed by washing with PBS three times and centrifugation to remove free dye.

For the in vivo analysis of microglial phagocytosis in Bhlhe41- or Aurka-deficient mice, 2 µL of Zymosan-AF488 (2 mg/mL) was stereotactically injected into male WT and B41cKO littermates, or male Cx3cr1Cre/+Aurka+/+ and Cx3cr1Cre/+Aurkafl/fl littermates. For the in vivo phagocytosis analysis of Aurka-deficient microglia with Bhlhe41 overexpression, 1 µL of Myelin-Dil was stereotactically injected into male B41HETAurkafl/fl and B41HETAurkafl/flR26B41-EYFP/+mice. For in vivo phagocytosis assays by CD22 blockade, 1 µL of Myelin-Dil (20 mg/mL) pre-mixed with 1 µL either an IgG isotype control (1 mg/mL) or anti-CD22 antibody (1 mg/mL) were injected into opposite brain hemispheres of male B41cKO or B41HETAurkafl/fl mice, respectively.

Briefly, mice pre-anesthetized with pentobarbital sodium (90 mg/kg body weight) were positioned on a stereotactic apparatus. Subsequently, the skulls were exposed with a hole drilled at the injection site using aseptic technique. The phagocytic target solution, or the antibody–phagocytic target solution was injected into the cerebral cortex of the mice through a micro-syringe pump (Quintessential Injector, Stoelting, Cat: 53311) at ±2 mm lateral, 0 mm anterior–posterior, and -1.5 mm deep relative to the bregma at a rate of 200 nL/min. The needle was slowly withdrawn after remaining in place for an additional 10 min to allow for diffusion. For flow cytometric analysis of phagocytic microglia, mice were sacrificed and transcardially perfused with PBS after 5 hours, followed by flow cytometric analysis of 40% Percoll-enriched microglia from the entire brain. For IF analysis of the clearance of phagocytic target material, mice were euthanized after 48 hours and were transcardially perfused with 4% paraformaldehyde (PFA). The entire injection site was sectioned at 40 μm in thickness using vibrating microtome (Leica, Cat: VT100S). The entire injection site was sectioned using vibrating microtome for IF analysis. Six to eight consecutive sections, separated by 40 µm, were then quantified to determine the overall target clearance over the injection site. All quantitative analyses were performed in a double-blind analysis, during which sections without significant Iba-1+ microgliosis at the injection site margin were excluded. Each dot represents an average fluorescence intensity of at least six consecutive slides from one mouse.

In vivo BrdU assay Analysis
Infant WT, B41HETAurkafl/+ and B41HETAurkafl/fl mice (P10) were intraperitoneally injected with 100 µL of BrdU solution (10 mg/mL, Bestbio, Cat: BB-4261) once every 12 h for 3 consecutive days. Eight hours post last injection, mice were sacrificed for the analysis of microglial proliferation.

Immunofluorescence analysis
Mice pre-anesthetized with pentobarbital sodium were perfused with 4% PFA. Brains were dissected out and post-fixed in 4% PFA overnight, followed by treatment with 30% sucrose for 48 hours. Next, brains were sectioned at 20 μm in thickness using freezing microtome (Leica, Cat: CM1950). Sections were blocked with 10% goat serum, 1% Bovine Serum Albumin (BSA), 0.3% Triton-X in PBS for one hour at room temperature before applying the primary antibody mix diluted in blocking buffer overnight at 4°C. To assay Bhlhe41 expression in CNS, sections were labeled with mouse Anti-NeuN (1:100, Millipore, Cat: MAB377), mouse anti-Gfap (1:200, Biolegend, Cat: 644702) and rabbit anti-Olig2 (1:500, Proteintech, Cat: 13999-1-AP). To examine microglia morphology and numbers, sections were stained with rabbit anti-Iba-1 (1:500, Wako, Cat: 019-19741). To further characterize microglia, sections were labeled with goat anti-dTomato (1:100, Biorbyt, Cat: orb182397) or rabbit anti-RFP (1:200, Rockland, Cat: 600-401-379S), rat anti-CD206 (1:200, Bio-Rad, Cat: MCA2235), rat anti-BrdU (1:400, Abcam, Cat: ab6326), mouse anti-phospho-Histone H3(Ser10) (1:200, Cell Signaling Technology, Cat: 9706S), rat anti-CD68 (1:500, Abcam, Cat: ab53444,) and mouse anti-CD22 (1:500, Abcam, Cat: ab181771). For the analysis of brains from mice following cuprizone-induced demyelination, sections were stained with rat anti-Mbp (1:1000, Abcam, Cat: ab7349), mouse anti-APC (1:100, CC-1, Merck, Cat: OP80), rabbit anti-degraded myelin basic protein antibody (1:2000, Merck, Cat: AB5864) and rabbit anti-Olig2 (1:500, Proteintech, Cat: 13999-1-AP). Sections were stained overnight and then washed five times at room temperature. Next, sections were stained with matched AF488 (1:500, ThermoFisher Scientific, Cat: A-21206/A-11006), AF555 (1:500, ThermoFisher Scientific, Cat: A-31572/A-21422), AF647 (1:500, ThermoFisher Scientific, Cat: A-32733)-conjugated secondary antibody for 2 hours at room temperature. TUNEL staining was performed according to manufacturer's instructions (Roche, Cat: 12156792910) after incubation with secondary antibodies. Following washing five times, sections were mounted with DAPI mounting medium (Abcam, Cat: ab104139) and fluorescent images were acquired using a confocal laser-scanning microscope (Zeiss LSM710 and Leica STELLARIS 5) and processed with ZEISS ZEN (v3.9) and Leica Application Suite X (v3.9), respectively.

Long-term CD22 blockade assay
Mice pre-anesthetized with pentobarbital sodium were positioned in a stereotaxic device. To continuously inject antibodies into the lateral ventricle, a guide cannula (RWD Life Science, Cat: 62003) was implanted at +1 mm lateral, −0.3 mm anterior–posterior, and −3 mm deep relative to bregma. The guide cannula and anchoring screws were fixed with dental cement and capped by a threaded plastic cap (RWD Life Science, Cat: 62102) to prevent clogging. After surgery, mice received antibiotic treatment (Penicillin V potassium, 200 U/g, Solarbio, Cat: p7480) and were allocated a recovery period of one week. Then the mice were given 2 µL of anti-CD22 antibody (100 µg/mL, BioXCell, Cat: BE0011) or IgG isotype control antibody (100 µg/mL, BioXCell, Cat: BE0083) every 3 days for 7 weeks, using an injector cannula (RWD Life Science, Cat: 62203) connected to the guide cannula, while being fed a 0.2% CPZ-enriched diet for 5 weeks followed by a CPZ-free normal diet for an additional 2 weeks.

Transmission electron microscopy
Mice from CPZ-induced demyelination model were deeply anesthetized using sodium pentobarbital and subsequently transcardially perfused with PBS to remove blood, followed by perfusion with EM fixative (Cat: G1102-100 mL, Servicebio). The central region of the CC was dissected (1 mm in thickness), promptly fixed in EM fixative and washed with PBS. Post-fixation was conducted using 1% osmium tetroxide for two hours. The tissue was subsequently rinsed with double-distilled water and dehydrated through a graded series of ethanol and acetone. The samples were then infiltrated with varying concentrations of epoxy resin and polymerized sequentially at 37℃ for 24 hours, 45℃ for 24 hours, and finally at 60℃ for 24 hours. Ultrathin sections with a thickness of 80 nm were prepared and treated with uranyl acetate and lead citrate before being examined using a transmission electron microscope (TEM, Hitachi HT7800) and processed with the Hitachi TEM system. TEM images of the CNC were used to identify myelinated axons, defined by the presence of intact multilamellar myelin sheaths, and unmyelinated axons, including axons with unstructured myelin exhibiting lamellar splitting, disintegration or vacuolar formation70. For each mouse, three non-overlapping FOV per section were analyzed at identical magnification. The numbers of myelinated and unmyelinated axons were manually counted, averaged per mouse and used to assess demyelination and remyelination.

Flow cytometric analysis
Prior to flow cytometric analysis, cell suspensions were made from yolk sac, brain, spinal cord, bronchoalveolar lavage fluid (BALF) and peritoneal cavity. Briefly, whole brains from embryos, newborn and adult mice were minced and mechanically dissociated by gentle homogenization, followed by suspension in 40% Percoll and centrifugation at 800 g for 30 minutes at room temperature. Yolk sac from embryos were minced and incubated with 0.5 mg/mL Collagenase IV (ThermoFisher Scientific, Cat: 17104019) in RPMI 1640 medium with 1% FBS for one hour. BALF were collected by inserting a canula into the trachea following opening the chest cavity. 1 mL of pre-chilled PBS with 2 mM EDTA was used to flush the lung three times. Cell pellets were collected and washed using PBS with 1% FBS and 2 mM EDTA and filtered through 70 μm mesh prior to labeling for flow cytometric analysis. Data were acquired on BD FACSAria III using BD FACS DIVA software (v9)

For the surface staining, Anti-CD16/CD32 antibody was used as Fc blocker, while Zombie Aqua™ Fixable Viability Dye (FVD, Biolegend, Cat: 423102) was used to label dead cells. The following markers were used for cell identification: yolk sac myeloid cells (FVD-CD45+CD11b+Cx3cr1+), microglia (FVD-CD45loCx3cr1+CD11b+), alveolar macrophage (FVD-SSChiCD45+CD11bloCD11c+CD24-) and peritoneal B-1 cells (FVD-CD45+CD19+B220lo). For the analysis of CD22 expression, microglia from the brains of young mice (2 – 4 months) and middle-aged mice (10 – 14 months) of different genotypes were enriched by 40% Percoll and labeled with antibodies for microglia markers and APC anti-mouse CD22 Antibody (clone OX-97, BioLegend, Cat: 126110), or matching APC Rat IgG1, κ Isotype Ctrl Antibody (BioLegend, Cat: 400411). For the validation of CD22 expression, InVivoMab anti-mouse CD22 Antibody (clone Cy34.1, BioXcell, Cat: BE0011) were in-house labeled with PacBlue succinimidyl ester (AAT, Cat: 570) according to the instructions by the manufacturers. Excess PacBlue dye was removed using Bio-Spin P-6 gel desalting column (Bio-Rad, Cat: 7326227). Flow cytometric data were analyzed using Flowjo (v10.8.1). For visualization in histogram overlays, fluorescence intensity data were normalized to the mode of each distribution to standardize peak heights, thereby enabling direct visual comparison of expression distribution shapes across samples independent of cell count.

Western blot
For the analysis of Bhlhe41 and Aurka expression, microglia were purified by flow cytometric sorting of FVD-CD45loCx3cr1+CD11b+ cells on FACSAria III (BD Biosciences). For the validation of R26B41-EYFP mice, primary microglia from adult WT, B41KO and B41HETR26B41-EYFP/+ mice were purified by flow cytometric sorting. Purified cells were lysed with RIPA lysis buffer with Protease and phosphatase inhibitor cocktail (Beyotime Biotechnology, Cat: P1045). The supernatant was collected after centrifugation at 12000 g for 5 minutes. The protein concentration of the supernatant was assayed using BCA protein assay kit (Beyotime Biotechnology, Cat: P0012S). Equal amounts of protein from each group were mixed with 5× loading buffer and heated at 95°C for 5 minutes prior to loading onto the SDS-PAGE gel. The separated proteins were then transferred to a nitrocellulose membrane, which was blocked with 5% BSA at room temperature for 2 hours and stained with rabbit anti-Bhlhe41 polyclonal Antibody (1:1000, Proteintech, Cat: 12688-1-AP), anti-β-actin (1:50000, Proteintech, Cat: 66009-1), rabbit anti-Aurka polyclonal Antibody (1:1000, Novus, Cat: NBP-1-51843) and Anti-GAPDH (1:5000, Sangon Biotech, Cat: D190090) at 4 °C for overnight. The membranes were washed three times (5 min each) by Tris-buffered saline with Tween 20 (TBST) and stained with Goat Anti-Mouse/Rabbit lgG Dylight 800 (1:10000, Abbkin, Cat: A23910/A23920) at room temperature for 2 hours. The membranes were scanned using a Western blot imaging system (Odyssey CLXL1-CDR). Image acquisition was performed using Image Studio (V5.5). Band intensities were quantified using ImageJ (v1.54).

Histological analysis
Black-Gold II myelin staining was used to assess myelination in the corpus callosum of mice subjected to the CPZ-induced demyelination model. Briefly, coronal brain sections (20 μm) were cut on a Leica CM1950 cryostat, mounted on gelatin-coated slides and oven-dried at 60 °C for 30 minutes. Sections were rehydrated in deionized water (2 minutes), stained in pre-heated Black-Gold II solution (Biosensis, Cat. BSS-TR-100-BG) at 60 °C for 12 minutes, rinsed in deionized water (3 × 5 minutes) and immersed in stop solution (2 minutes). Samples were dehydrated in graded ethanol (70%, 95%, 100%, each for 3 minutes), cleared in xylene (3 minutes) and coverslipped with neutral balsam mounting medium. Myelinated fibers were imaged by brightfield microscopy. Quantification of myelinated fibers was performed by measuring optical density in the CC using ImageJ

Gene expression microarray analysis
Microglia were purified by flow cytometric sorting of FVD-CD45loCx3cr1+CD11b+ cells from 40% percoll-enriched cells from the brains of young B41HET and B41KO mice (10 - 15 mice per sample). Total RNA was extracted using a miRneasy Mini Kit (Qiagen, Cat: 217084) and quantified with NanoDrop 2000 Spectrophotometer (ThermoFisher Scientific). The cRNA was prepared using a LowInput QuickAmp Labeling Kit (Agilent, Cat: 5190-2305). Gene expression microarray (GEM) analysis was performed using SurePrint G3 Mouse Gene Expression v2 8x60K Microarray (Agilent, Cat: G4852B). The GEM data was quantile normalized, and the differential expression analysis was performed using R package Limma (v3.54.2)71. In-house generated GEM data was compared with public bulk RNA sequencing data from Bhlhe40/Bhlhe41 double knockout microglia. Shared differentially expressed genes were defined as genes with an absolute fold-change (logFC) ≥ 0.5 and an adjusted P value ≤ 0.05.

Chromatin immunoprecipitation coupled with qPCR (ChIP-qPCR)
Putative BHLHE41-binding sites in the promoters of human and mouse BHLHE41 and CD22, which were retrieved from UCSC Genome Browser, were identified using JASPAR database. For the validation of putative Bhlhe41-binding sites, chromatin immunoprecipitation coupled with qPCR (ChIP-qPCR) analysis was performed with microglia enriched by 40% Percoll from WT mice using a ChIP Assay Kit (Beyotime, Cat: P2078). Briefly, enriched microglia were fixed with 1% PFA for 10 min before quenching with 1× glycine solution for 5 min. The nuclei were isolated using 1× SDS lysis buffer with 1× PMSF protease inhibitor and sonicated on ultrasonic homogenizer to result in chromatin fragments ranging from 200 bp to 800 bp. 1% of total chromatin fragments were preserved as Input DNA for qPCR analysis. chromatin fragments were blocked with salmon sperm DNA and immunoprecipitated using 2 μg of rabbit anti-Bhlhe41 polyclonal antibody (Proteintech, Cat: 12688-1-AP) or rabbit IgG (Sangon Biotech, Cat: D110502) overnight at 4 °C. Protein-DNA complexes bound to Protein A/G agarose were washed, and the crude DNA was used for qPCR analysis. Enrichment of the putative Bhlhe41-binding sites in the promoters of Bhlhe41 and CD22, as well as negative control regions was analyzed by qPCR analysis using Taq Pro Universal SYBR qPCR Master Mix (Vazyme, Cat: Q712) on a LightCycler 480 (Roche). The specific qPCR primers were designed in-house, synthesized by Sangon Biotech and are shown in Supplementary Table 2. All IP signals were normalized relative to the signal from the input chromatin.

scRNA-seq data analysis
For the analysis of BHLHE41 expression in microglia and oligodendrocytes from human post-mortem white matter43, the dorsolateral prefrontal cortex from patients with Alzheimer’s disease (AD)46, the substantia nigra pars compacta from patients with Parkinson’s disease (PD) and Lewy body dementia (LBD)47, and fetal cerebellum44, 45, dot plots were generated using Cellxgene platform (https://cellxgene.cziscience.com/gene-expression)72. For the analysis of BHLHE41 and marker genes of microglia and oligodendrocytes from patients with AD, UMAP plots were generated Cellxgene (https://cellxgene.cziscience.com/e/100c6145-7b0e-4ba6-81c1-ffebed0d1ac4.cxg/)46.

The scRNA-seq data from human post-mortem white matter and the dorsolateral prefrontal cortex from patients with AD were retrieved from Cellxgene 43, 46.The public scRNA-seq data were preprocessed and normalized using the ln(CPTT + 1) transformation of raw counts, where CPTT is Counts Per Ten Thousand72. Cells with more than 10% mitochondrial genes and fewer than 200 detected genes per cell were removed. Genes expressed in fewer than three cells were excluded before identifying the top 2000 variable genes in the dataset.

For the analysis of scRNA-seq data from human post-mortem white matter from healthy brains (n = 20 donors)43, two rounds of clustering were performed. In the first round, all cell types from the white matter were included to study the distribution of genes of interest across different cell types. In the second round, microglia were subclustered with a resolution of 0.05. For the analysis of scRNA-seq data from microglia from patients with AD (n = 83 donors)46, non-microglia contaminants characterized by high expression of LYVE1, MRC1, and CD163 (cluster 13), SKAP1 and ETS1 (cluster 15), and FCN1, VCAN, and FGR (cluster 16) were removed prior to subcluster analysis. Microglia subcluster analysis was performed with a resolution of 0.4. The mean expression of genes of interest was visualized using dot plots and UMAP plots. A BHLHE41-CD22 Tendency Score was calculated for each cell as the log-transformed expression of BHLHE41 minus that of CD22. Positive values indicated a bias toward BHLHE41 expression, negative values toward CD22, and values near zero reflected balanced expression. Donor-level pseudobulk scores were obtained by averaging the tendency score across all cells within each donor × cluster combination. For comparisons between two clusters, donor-level scores were tested using a two-sided Wilcoxon rank-sum test. The scRNA-seq data analysis was performed using the Scanpy v1.11.5 package in Python (v3.12)73.

Statistical analysis
Statistical analyses were performed using R (v4.2.2) with the following packages: lme4 (v1.1-33), lmerTest (v3.1-3), emmeans (v1.11.1), DHARMa (v0.4.7), MASS (v7.3-60), nlme (v3.1-162), rstatix (v0.7.2) and ARTool (v0.11.2). Data are presented as mean ± SEM unless stated otherwise. Genotype, treatment/model phase, CNS region and axon status were treated as categorical variables. Prior to analysis, data distributions were assessed using Q-Q plots and/or Shapiro-Wilk test, and homogeneity of variance was evaluated using Levene’s test. When appropriate, outcome variables were log2-transformed to meet normality assumptions.

For bounded or non-normal continuous outcomes, non-parametric methods were applied. For two-group comparisons of continuous outcomes meeting parametric assumptions, Student’s t-test or paired t-test was used when variances were equal; Welch’s t-test was applied if variances were unequal. When normality assumptions were violated or sample sizes were very small, Wilcoxon rank-sum tests were used. For comparisons involving more than two groups, one-way or two-way ANOVA was applied, including interaction terms when multiple factors were modeled. When assumptions of normality or homogeneity were violated, or for bounded continuous data, Kruskal-Wallis tests (non-factorial designs) or Aligned Rank Transform (ART) ANOVA (factorial designs) were applied.

Group differences in count outcomes were analyzed using generalized linear models (GLMs) with either a Poisson or negative binomial distribution, selected based on the presence of overdispersion. Model fit was assessed using Pearson residuals, and overdispersion was evaluated. For experiments with repeated measurements from individual animals, linear mixed-effects models (LMMs) were applied. Mouse identity was included as a random intercept to account for within-animal correlation, while genotype, treatment/model phase, axon status, and their interactions were treated as fixed effects. Model assumptions were evaluated using DHARMa residual diagnostics, including tests for goodness-of-fit (testUniformity) and dispersion (testDispersion). In the presence of heteroscedasticity, weighted mixed-effects models were fitted using the nlme package.

All post hoc pairwise comparisons were performed using estimated marginal means (EMMeans) with Bonferroni correction to adjust for multiple testing. All statistical tests were two-sided, and a P value or adjusted P value < 0.05 was considered statistically significant. Detailed statistical results, including exact P values, are provided in the figure legends and the Source Data file.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "The authors investigated the role of the transcription factor Bhlhe41 and its coregulator Aurora kinase A (Aurka) in regulating microglial morphology, phagocytosis, and both de- and remyelination. They propose that Aurka mitigates the self-repressive activity of Bhlhe41 in microglia, thereby preventing the upregulation of CD22—a phagocytic receptor and marker of aged microglia. Loss of either Bhlhe41 or Aurka results in CD22 upregulation, leading to a premature aging phenotype characterized by dystrophic microglial morphology, phagocytic impairments, and defective remyelination.\nThe authors employ multiple mouse models, including both global and conditional knockouts of Bhlhe41 and Aurka, and provide mechanistic insights supported by a rescue experiment in which ectopic Bhlhe41 expression restores the phenotype in Aurka-deficient mice.\nWe appreciate the substantial effort that went into this study, and we recognize its potential relevance to the field of microglial biology and myelin repair. However, in its current form, the manuscript contains major weaknesses in experimental design, model validation, and statistical interpretation. Below, we outline the key concerns.\n# Summary of Major Issues\n## Lack of Wild-Type Controls\nVirtually all experiments compare only B41HET and B41KO mice. Without inclusion of WT animals, the direction and magnitude of Bhlhe41’s effects cannot be established. This omission is particularly problematic given the hypothesized autoregulatory loop of Bhlhe41. WT controls are essential for proper interpretation of gene dosage effects.\n## Incomplete Knockout and Model Validation\nThe Bhlhe41^dTomato‑Cre line used for microglia-specific ablation retains substantial Bhlhe41 protein in “KO” cells (Fig. S1D), undermining the validity of the conclusions drawn from most of the experiments. The authors should instead use the floxed null allele (KO) and respective WT control mice that they generated in this study, after confirmation of microglia specificity and estimation of recombination efficiency.\n## Underuse of the Floxed Bhlhe41 Model\nThe Bhlhe41^fl/fl allele is a powerful tool that could enable definitive comparisons across WT, HET, and KO genotypes. Yet it is used only once, and without WT controls. This limits the ability to assess gene dosage effects and the plausibility of the proposed self-repression mechanism.\n## Lack of Statistical Details and Pseudoreplication\nStatistical analyses appear to treat repeated measurements as independent replicates. For example, n=16 data points in Figures 2B and S3B/D arise from only three mice. This inflates statistical significance and violates assumptions of independence. Reanalysis using the correct unit of replication (e.g., individual animals) or appropriate hierarchical models is required. Another major issue is the lack of statistical details like test statistics with degrees of freedom, confidence intervals, and multiple testing correction used.\n# Additional Comments and Questions\n## Use of B41HET Mice as Controls\n- What is the rationale for using B41HET as a default comparator in all experiments? If HET mice already exhibit haploinsufficient phenotypes, this invalidates conclusions drawn from HET vs KO comparisons.\n- Supplementary Figure 1D suggests persistent Bhlhe41 expression in KO cells. Could this reflect Cre leakiness or incomplete recombination?\n- Figure 1A suggests no increase in dTomato in B41HET relative to WT, arguing against self-repression at the heterozygous state. Is autoregulation only observed in complete KO?\n- Is B41Het an appropriate tool to study microglia in vivo, as stated in line 187, given that these cells still retain 50% Bhlhe41?\n- What does remyelination look like in WT mice? Are B41HET mice already impaired?\n## Validation of Mouse Models\n- Have the authors validated microglia-specific recombination using lineage markers (e.g., P2RY12, CX3CR1) in their conditional KO (B41cKO, and Cx3cr1-Aurka fl/fl))?\n- Has Bhlhe41 deletion been confirmed at the mRNA/protein level in sorted microglia?\n- Similar validation is needed for Aurka deletion in B41HET;Aurka^fl/fl and Cx3cr1-Aurka^fl/fl backgrounds (microglial identity, Aurka loss via flow or western blot.\n## Clarification of MBP and Myelin Analyses (Figure 3D)\n- Does “MBP reduction” refer to total intensity, area coverage, or puncta count? Consider plotting raw MBP intensities.\n- Lines 257–259 mention increased myelin debris and unstructured sheaths; however, Figure 3D does not clearly differentiate between MBP puncta (fragmented myelin) vs intact sheaths.\n- Have authors considered temporal delay in remyelination rather than failure? Are later time points (e.g., 2 weeks) available?\n## Phagocytosis Assays\n- Lines 282–283 overstate the conclusions. Phagocytosis of myelin debris has not been directly measured. E. coli and zymosan beads do not capture endogenous phagocytosis of myelin debris.\n- Co-staining for CD68 or LAMP1 with MBP could confirm in vivo internalization.\n- Morphological assessments alone are insufficient to support claims of defective clearance (Line 349). Use of CD68/LAMP1 co-staining may be a better choice.\n## Minor Issues and Clarifications\n- Supplementary Figure 1: Show gMFI and percent positivity for all populations (microglia, B-1 cells, alveolar macrophages). Quantify blots for all genotypes.\n- Figure 1A: CD206 alone is insufficient to distinguish BAM subtypes; consider Lyve1 (PVM) and MHC-II (LMM). Iba1 is not specific for microglia—prefer P2RY12, Sall1, or Tmem119.\n- Figures 2C, 2E: Include gMFI for phagocytosis markers to assess per-cell capacity.\n- Line 306: Revise “microglial identity”—the authors are measuring function and morphology.\n- Supplementary Figure Mismatch: Correct citation errors (e.g., Lines 311–321).\n- Line 311: Should refer to Supplementary Figure 6, not 5.\n- Line 317: Supplementary Figure 7A, not 6A.\n- Line 319: Supplementary Figure 7A-B, not 6A-B.\n- Line 321: Supplementary Figure 7C, not 6C.\n- Line 894: Clarify method for microglial isolation (mechanical vs enzymatic) as this can influence microglial activation. Indicate whether whole brains were used or regions excluded.\n- Supplementary Figure 12: CD68 signal is faint and hard to interpret.", "reviewer_2": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.", "reviewer_3": "Li and colleagues use several new and interesting mouse lines to investigate the transcription factor Bhlhe41. There are some novel and interesting findings in this manuscript regarding how Bhlhe41 impacts microglia morphology and phagocytosis. They find that Bhlhe41 restrains CD22, a known inhibitor of phagocytosis that increases with age. Overall, there is essential findings in this work. However, I do have some serious concerns about how conclusions are made without appropriate analyses or data to support these conclusions.\nMajor concerns\n-the claim that B41KO recovery in the EAE is not due to hyperactive T-cells is not well backed up by the data. Figure 3B measures the proportion of CD4 T-cells that express IFN-g and IL17. These measurements are too superficial for this conclusion. They should measure the proportion of leukocytes/ CD4 and CD8 T-cells as a whole. Many other immune cells could also contribute to this recovery, so the link with microglia is extremely tenuous.\n-For the cuprizone work in Figures 3 and 4, the MBP images are (for me) blurry and unclear). By convention, the dorsal side of the brain should be up, but I suspect these are flipped. It is also unclear where these images are taken in the corpus callosum. MBP will label both myelin and myelin debris, so the claims of more or less demyelination/ remyelination with MBP are problematic. These analyses should be confirmed with compact myelin stains that are more sensitive to intact myelin, such as Sudan black or black gold staining.\n-While g-ratios are a good metric for EM analyses, they do not directly inform the degree of demyelination and remyelination in this model. These EM images should be quantified and the proportion of unmyelinated and myelinated axons should be reported in the corpus callosum. The authors should also make it clear how they know they were quantifying axons in the corpus callosum\nThe methods for the cuprizone section should outline how they analyzed the various data (olig2/ MBP, etc.). For example, how many images were taken, what region was quantified, how were these images blinded, etc.\n-The authors talk about myelin debris but offer no data about how much debris is inside of microglia during demyelination and remyelination in this model. The authors need to examine debris uptake directly to conclude in line 263 “that Bhlhe41 is required to promote clearance of myelin debris”. The authors should also not use enlarged cell bodies of microglia as indications of “sustained phagocytic activity.” This should be measured more directly.\n-as there may or may not (it is unclear from the data) be differences in baseline demyelination in this model, and differences in demyelination will impact remyelination, the authors are not in a position to make any conclusion on remyelination with the data they provide. They do not differentiate between unmyelinated and myelinated axons, spared myelin and myelin debris, or remyelination and myelin debris.\n-The authors use Olig2 as a marker of oligodendrocyte. Olig2 is found in both OPCs and oligodendrocytes. They should redo these quantifications with markers of both OPCs and oligodendrocytes.\nI do not see CD22 in all the immunofluorescence images. The authors need to re-examine the CD22 representative images.\nFor the phagocytosis assays, it is important to understand how the anti-CD22 antibody impacts control animals, not just Aukura cKO and B41 KO mice.\n-given that they treat cuprizone mice with anti-CD22 throughout the demyelination phase, it is not possible to differentiate between the possibility that this works to limit demyelination or promote remyelination.\nSmaller points\n-In figure 1c, I do not limited tdTom in the pvM and mM as the images are too zoomed out and of a low quality\n-in line 185, you call Bhlhe41 microglia specific, but it is in a population of BAM and likely other myeloid cells, so this language should be tempered to something like microglia enriched\n-In Figures 2A and 2B, you refer to microglia in the Bhlhe41 deficient group as being dystrophic. These do not look dystrophic but more shrunken with shorter, fatter processes. I did not see blebbing or fragmented processes from the images, although my images were of a low quality.\n-The rationale behind doing an EAE study was problematic. Line 229:”To examine whether Bhlhe41 deficiency affects CNS myelination, we established an inflammatory demyelinating model…”. EAE is an inflammatory model, so it is unlikely to impact myelination, such as developmental myelination. Also, no myelin metric was investigated here. EAE is not a model (with these mice) to distinguish remyelination, so a new rationale should be included.\n-Line 319 may refer to Supp Fig. 7, not Supp Fig. 6."}, "author_response": "Response to Reviewers\nManuscript ID: NCOMMS-25-19678A-Z\nTitle: Aurka-Bhlhe41 axis prevents premature aging-like microglial dysfunction and\npromotes remyelination\nWe thank the reviewers for their time and insightful comments, which have\nsignificantly improved our manuscript. We have addressed all points raised in a point-\nby-point manner below. All changes in the manuscript have been tracked, and new data\nhave been incorporated into the figures. In addition, we have provided a high-resolution\nPDF version of all images as a separate file to ensure optimal clarity for visualization\nand publication.\nReviewers' comments:\nReviewer #1 (Remarks to the Author):\nThe authors investigated the role of the transcription factor Bhlhe41 and its coregulator\nAurora kinase A (Aurka) in regulating microglial morphology, phagocytosis, and both\nde- and remyelination. They propose that Aurka mitigates the self-repressive activity of\nBhlhe41 in microglia, thereby preventing the upregulation of CD22—a phagocytic\nreceptor and marker of aged microglia. Loss of either Bhlhe41 or Aurka results in CD22\nupregulation, leading to a premature aging phenotype characterized by dystrophic\nmicroglial morphology, phagocytic impairments, and defective remyelination.\nThe authors employ multiple mouse models, including both global and conditional\nknockouts of Bhlhe41 and Aurka, and provide mechanistic insights supported by a\nrescue experiment in which ectopic Bhlhe41 expression restores the phenotype in\nAurka-deficient mice.\nWe appreciate the substantial effort that went into this study, and we recognize its\npotential relevance to the field of microglial biology and myelin repair. However, in its\ncurrent form, the manuscript contains major weaknesses in experimental design, model\nvalidation, and statistical interpretation. Below, we outline the key concerns.\n# Summary of Major Issues\n## Lack of Wild-Type Controls\nVirtually all experiments compare only B41HET and B41KO mice. Without inclusion\nof WT animals, the direction and magnitude of Bhlhe41’s effects cannot be established.\nThis omission is particularly problematic given the hypothesized autoregulatory loop\nof Bhlhe41. WT controls are essential for proper interpretation of gene dosage effects.\n- Response:We thank the reviewer for this critical and insightful comment. We fully\nagree that the inclusion of WT controls is essential to establish a baseline and properly\ninterpret gene dosage effects, especially for Bhlhe41 that is self-repressed in microglia.\nIn our initial investigation, we did include WT controls for the foundational\ncharacterization of microglia number and morphology under homeostatic conditions.\nThose data confirmed that B41HET and B41cHET microglia were indistinguishable from\nWT. This is supported by comparable CD22 expression between WT and B41HET\nmicroglia (Fig 5b, Fig 6c and Fig S7). However, we agree that WT controls should be\nincluded for all key experiments as a true baseline. We apologize for this in our initial\nsubmission.\nTo address this fundamental concern, we have included the WT data from our initial\nanalysis and re-performed key experiments using WT mice as controls. This includes:\n1. Western blot (WB) analysis of Bhlhe41 expression, revealing its partial but not\nhalved reduction in both B41HET (Fig.1b) and B41cHET (Fig.2c) microglia compared\nto WT, supporting its auto-repressive mechanism.\n2. Immunofluorescence (IF) analysis of microglia number and morphology in both\nBhlhe41 global (WT, B41HET and B41KO mice, Fig.2a-b, S3a-b) and conditional\n(WT, B41cHET and B41cKO mice, Fig.S3c-d) knockout models, demonstrating a\ndystrophic-like phenotype only in KO mice, with WT and HET mice being\nindistinguishable.\n3. WB analysis of Aurka and Bhlhe41 expression in WT, B41HETAurkafl/+,\nB41HETAurkafl/fl (Fig.4b) and Cx3cr1Cre/+Aurka+/+, Cx3cr1Cre/+Aurkafl/+ and\nCx3cr1Cre/+Aurkafl/fl microglia (Fig. 4c), revealing successful knockout of Aurka in\na gene-dosage-dependent manner. While Aurka expression was halved in Aurka\ncHET microglia and ablated in Aurka cKO microglia, Bhlhe41 expression was\nsignificantly reduced only in the Aurka cKO group but slightly reduced in Aurka\ncHET microglia.\n4. IF analysis of microglia number and morphologyin microglial-specific Aurka\nknockout mice models (B41HETAurka+/+, B41HETAurkafl/+and B41HETAurkafl/fl mice,\nFig.S5a; Cx3cr1Cre/+Aurka+/+, Cx3cr1Cre/+Aurkafl/+ and Cx3cr1Cre/+Aurkafl/fl. Fig.\nS5b), demonstrating impaired microglia number and morphology in Aurka cKO\nmice, with WT and cHET mice being indistinguishable.\n5. Key functional assays, includingin vitroandin vivophagocytosis (Fig.2d-i and Fig.\n4f) and the CPZ-induced demyelination model (Fig.3 and Fig.4g-j), were re-\nperformed using B41cKO and AurkacKO mice, and Cx3cr1Cre/+mice as WT controls.\n## Incomplete Knockout and Model Validation\nThe Bhlhe41^dTomato-Cre line used for microglia-specific ablation retains substantial\nBhlhe41 protein in “KO” cells (Fig. S1D), undermining the validity of the conclusions\ndrawn from most of the experiments. The authors should instead use the floxed null\nallele (KO) and respective WT control mice that they generated in this study, after\nconfirmation of microglia specificity and estimation of recombination efficiency.\n- Response:We thank the reviewer for raising this important point regarding the\nvalidation of our genetic model. We agree that the faint band observed in the WB\n(previously Fig. S1D, nowFig.1b in the revised manuscript) warrants careful\ninterpretation.\nWe would like to clarify that our Bhlhe41dTomato-Cre knock-in allele\n(designatedB41HET in heterozygotes and B41KO in homozygotes) was designed to\ndisrupt native Bhlhe41 protein expression by replacing its initiation codon with\nadTomato-2A-Cre-polyA cassette. It is well-established that Bhlhe41 is essential for B-\n1a cell self-renewal1. In our previous publication2, we functionally validated this allele\nby demonstrating a severe impairment of B-1a cell populations inB41KOmice, with no\neffect observed in WT or B41HET mice (as shown in Fig. 1C-D of the referenced work;\nan observation supported by an impaired B-1 cell ratio in now Fig.S1d in the revised\nmanuscript). This result is consistent with the established role of Bhlhe41 and confirms\nthat our allele acts as a functional null.\nTo further address this concern directly, we performed additional WB analysis of B41KO\nand B41cKO microglia alongside WT and HET controls. Although a faint residual band\nwas observed in both knockout models, quantitative densitometric analysis (ImageJ)\nrevealed a significant reduction in Bhlhe41 protein levels in B41HO and B41cKO\nmicroglia compared to WT controls (new data presented in Fig.1b and Fig.2c).\nWe acknowledge that the faint residual signal could potentially stem from nonspecific\nbinding of the anti-Bhlhe41 polyclonal antibody used. However, the profound reduction\nin Bhlhe41 protein levels, coupled with the severe functional deficits across all our\nassays, provides compelling evidence that our model effectively ablates Bhlhe41\nfunction in microglia.\n## Underuse of the Floxed Bhlhe41 Model\nThe Bhlhe41^fl/fl allele is a powerful tool that could enable definitive comparisons\nacross WT, HET, and KO genotypes. Yet it is used only once, and without WT controls.\nThis limits the ability to assess gene dosage effects and the plausibility of the proposed\nself-repression mechanism.\n- Response:We thank the reviewer for this insightful comment, which we fully agree\nwith. We have now performed extensive new experiments using B41cKOmice\nwith WTcontrols to provide definitive comparisons.\nOur initial molecular and phenotypic analyses (in both the original submission and the\nrevised data) consistently demonstrated that microglia fromB41HETandB41cHETmice\nare functionally and morphologically indistinguishable from WT controls (Fig.1b and\nFig.2C for Bhlhe41 expression; Fig.2a-b and Fig.S3a-d for morphology; Fig. 5b and\nFig.6c for CD22 expression).\nTherefore, to most efficiently address the reviewer's concern while maximizing the\nbiological relevance of our comparisons, we focused our efforts on the most informative\ncontrast: direct comparison betweenB41cKOand WTcontrols in key functional and\ndisease models.\nSpecifically, we have now performed new experiments for:\n1. In vitro and in vivophagocytosis assays (revised Fig.2d, 2g and 2I, and Fig.4f\nfor AurkacKO) clearly demonstrate phagocytic deficits inB41cKOand AurkacKO\ngroup compared toWT controls\n2. The CPZ-induced demyelination/remyelination model (revised Fig. 3 and\nFig.4g-j for AurkacKO), confirming that remyelination impairment is\nspecifically caused byloss of Bhlhe41 or Aurka in microglia.\n3. CD22 blockade experiments, including in vivo phagocytosis assays (Fig.7b)\nand the CPZ-induced demyelination model (Fig.7d-i) using B41cKO mice.\n## Lack of Statistical Details and Pseudoreplication\nStatistical analyses appear to treat repeated measurements as independent replicates.\nFor example, n=16 data points in Figures 2B and S3B/D arise from only three mice.\nThis inflates statistical significance and violates assumptions of independence.\nReanalysis using the correct unit of replication (e.g., individual animals) or appropriate\nhierarchical models is required. Another major issue is the lack of statistical details like\ntest statistics with degrees of freedom, confidence intervals, and multiple testing\ncorrection used.\n- Response: We thank the reviewer for this important comment. We agree that treating\nindividual cells as independent replicates inflates statistical significance. We have re-\nanalyzed the Sholl data using a linear mixed-effects model, treating individual cells as\nrepeated measurements nested within each mouse. This approach properly accounts for\nintra-mouse correlation, and the results remain consistent with our original conclusions.\nWe have thoroughly revised the manuscript to include detailed descriptions of all\nstatistical tests in both the figure legends and the Methods section, ensuring full\ntransparency and reproducibility.\n# Additional Comments and Questions\n## Use of B41HET Mice as Controls\n- What is the rationale for using B41HET as a default comparator in all experiments? If\nHET mice already exhibit haploinsufficient phenotypes, this invalidates conclusions\ndrawn from HET vs KO comparisons.\n- Response:We thank the reviewer for raising this critical point regarding the choice of\ncontrols, which we acknowledge was not sufficiently justified in our original\nsubmission.\nThe decision to useB41HETmice as controls in many experiments was based on our\ninitial phenotypic characterization under homeostatic conditions. This analysis, which\nincluded WT, B41HET and B41KOmice, consistently demonstrated that microglia\nnumber and morphology in B41HETmice werephenotypically indistinguishable from\nWT controls. Furthermore, the use of littermateB41HET andB41KOmice was intended\nto minimize experimental variation in subsequent disease models.\nHowever, we fully agree with the reviewer that establishing a true WT baseline is\nessential for all key experiments. Therefore, as detailed in our response to the previous\ncomment on \"## Lack of Wild-Type Controls\", we have now systematically included\nWT control data into all critical experiments in the revised manuscript.\nThe new data now provides a clear and consistent narrative: whileB41HET microglia\nare functionally and morphologically normal,B41KOmicroglia exhibit severe deficits\nacross all assays. The inclusion of WT controls throughout the study firmly establishes\nthis baseline and confirms that the conclusions drawn from\ntheHETvs. KOcomparisons are indeed valid.\nWe sincerely thank the reviewer for prompting us to provide this comprehensive\nanalysis, which has significantly strengthened the rigor of our study.\n- Supplementary Figure 1D suggests persistent Bhlhe41 expression in KO cells. Could\nthis reflect Cre leakiness or incomplete recombination?\n- Response:We thank the reviewer for this question, which is closely related to the\nprevious point regarding model validation (##Incomplete Knockout and Model\nValidation). We believe there may be a misunderstanding regarding the fundamental\ndesign of our Bhlhe41dtomato-Cre genetic model, which is crucial for interpreting the data.\nAs detailed in our response to the previous comment (##Incomplete Knockout and\nModel Validation), this allele isnot a conditional (floxed) allele, but rather a knock-in\nnull allele.The genetic design involves replacing the endogenousBhlhe41 initiation\ncodon with adTomato-2A-Cre-polyAcassette. This manipulation abrogates the\nproduction of native Bhlhe41 protein, regardless of Cre activity. Therefore, the concepts\nof \"Cre leakiness\" or \"incomplete recombination\" do not apply to the core mechanism\nofBhlhe41gene inactivation in this model.\nHowever, the reviewer's question remains highly relevant to the interpretation of the\nWB data. Given that the allele is a definitive null, the most plausible explanations for\nthe faint residual band in theB41KOlanes (previous Fig. S1D, now Fig.1b) are non-\nspecific binding by the anti-Bhlhe41 polyclonal antibodyto other proteins of similar\nmolecular weight.\nWe have comprehensively addressed the concerns about protein expression and model\nvalidity in our previous response (##Incomplete Knockout and Model Validation),\nwhich we kindly refer the reviewer to.\n- Figure 1A suggests no increase in dTomato in B41HET relative to WT, arguing against\nself-repression at the heterozygous state. Is autoregulation only observed in complete\nKO?\n- Response:We thank the reviewer for this comment, which allows us to clarify a key\naspect of our experimental design and the interpretation of the dTomato reporter.\nThere is no dTomato signal in WT microglia, as they lack the reporter allele entirely. In\nourBhlhe41dTomato-Cre knock-in model, the dTomato reporter is expressedonly in cells\nthat have one or two copies of the knocked-in allele(B41HETorB41KO), replacing the\nnativeBhlhe41gene. Therefore, the critical comparison for assessingBhlhe41's\nautoregulation of its own promoteris not betweenWT (which lacks the dTomato\nreporter entirely) andB41HET (which possesses one copy of the reporter), but rather\nbetweenB41HET andB41KOmicroglia. Both B41HETandB41KO microglia possess the\nknocked-in dTomato reporter and therefore exhibit fluorescence driven by the Bhlhe41\npromoter. The key difference is that B41HETmicroglia can produce functional Bhlhe41\nprotein to repress this promoter, whereas B41KO cells cannot.\nAs we show in Fig.1a,deleting both alleles ofBhlhe41(B41KO) causes a dramatic ~10-\nfold increase in dTomato signal compared toB41HET microglia.This magnitude of\nchange far exceeds the expected 2-fold increase from gene dosage alone and provides\nstrong evidence for a positive feedback loop where Bhlhe41 protein represses its own\npromoter. Consistently, ectopic expression of Bhlhe41 from the Rosa26 promoter\n(B41HETR26mB41-EYFP/+) suppressed dTomato expression, further supporting the model\nof Bhlhe41-mediated self-repression (Fig. 6c).\nFurthermore, this finding of Bhlhe41 self-repression is independently corroborated by\nWB analysis. We found that Bhlhe41 protein levels in B41HET and B41cHET microglia\nare not reduced by half, but instead show only a partial reduction compared to WT\n(Fig.1b and Fig.2c). This clear deviation from the expected gene dosage effect further\nsupports the conclusion that Bhlhe41 limits its own expression.\n- Is B41Het an appropriate tool to study microglia in vivo, as stated in line 187, given\nthat these cells still retain 50% Bhlhe41?\n- Response:We thank the reviewer for this important question. B41HETmicroglia retain\none functional Bhlhe41 allele, and as we show in the revised manuscript possess\nsubstantial Bhlhe41 protein activity (Fig.1b and Fig.2c). We agree that for studying\nthecell-autonomous functionsof Bhlhe41, the homozygous knockout (B41KO) would\nbe the more appropriate model. However, our statement that \"B41HET mice may be a\nuseful tool to study microgliain vivo\" was made in a specific context: its utility as\nahighly specific genetic tool for targeting and tracing microglia.\nThe key evidence supporting this utility is:\n1. Exceptional cellular specificity: In B41HETR26EYFP mice, we observed\nirreversible EYFP labeling in >99% of microglia, with minimal labeling of other\nCNS cell types (~10% in CNS-associated macrophages) (Fig. 1e-f and\nFig.S2A).\n2. Early and Sustained Expression: This microglia-specific labeling is established\nas established\nas early as E15.5 in the fetus (Fig.1g and Fig.S2b) and persists into adulthood,\nenabling fate-mapping and longitudinal studies.\nIn summary, we propose that theB41HETmouse is not a tool for interrogating the loss\nof Bhlhe41 function, but rather ahighly specific and sensitive genetic tool for targeting,\ntracking, and manipulating microgliain vivoacross developmental and adult stages. We\nhave revised the wording in the manuscript (now Line 206-207) from \"a useful tool\" to\n\"a useful genetic tool for targeting and tracing microgliain vivo\" to eliminate any\npotential ambiguity regarding its intended application.\n- What does remyelination look like in WT mice? Are B41HET mice already impaired?\n- Response: We thank the reviewer for this critical question regarding the baseline of\nremyelination and the potential phenotype of B41HET mice.\nIn the CPZ-induced demyelination model, WT mice exhibit the expected sequence of\nevents: robust demyelination after 5 weeks of CPZ diet, followed by efficient\nspontaneous remyelination upon return to a normal diet. To assess myelination in the\ncorpus callosum, we analyzed WT and B41cKO mice using complementary approaches:\nBlack Gold II (BGII) staining to visualize intact myelin, immunofluorescence for\nmyelin basic protein (Mbp) to detect total myelin, and transmission electron\nmicroscopy (TEM) to quantify myelinated and unmyelinated axons. Our revised data\nclearly recapitulate this process in WT mice, showing substantial myelin loss during\ndemyelination and significant recovery after a 2-week recovery period (revised Fig.\n3b–d).\nRegarding the reviewer's specific question aboutB41HET mice, our initial and revised\nanalyses consistently demonstrate thatB41HETandB41cHETmicroglia are molecularly\nand phenotypically indistinguishable from WT controls. This is evidenced by: 1) only\na partial, non-50% reduction in Bhlhe41 protein levels (Fig. 1b and Fig.2c), consistent\nwith its self-repressive mechanism. 2) comparable microglial morphology (Fig. 2a-b\nand Fig.S3) and 3) no significant difference in the expression of CD22 (Fig.5b and\nFig.6c).\nTherefore, we focused our analysis on the comparison between WT andB41cKO(Fig.\n3), which revealed a profound and specific requirement for Bhlhe41 in promoting\nmicroglial clearance of myelin debris and supporting remyelination.\n## Validation of Mouse Models\n- Have the authors validated microglia-specific recombination using lineage markers\n(e.g., P2RY12, CX3CR1) in their conditional KO (B41cKO, and Cx3cr1-Aurka fl/fl))?\n- Has Bhlhe41 deletion been confirmed at the mRNA/protein level in sorted microglia?\n- Similar validation is needed for Aurka deletion in B41HET;Aurka^fl/fl and Cx3cr1-\nAurka^fl/fl backgrounds (microglial identity, Aurka loss via flow or western blot.\n- Responses: We thank the reviewer for raising this important point regarding the\nvalidation of cell type specificity in our conditional knockout models. We fully agree\nthat demonstrating microglia-specific recombination and gene deletion at the tissue\nlevel would be ideal. Unfortunately, due to the lack of reliable immunofluorescence-\ngrade antibodies for Bhlhe41 and Aurka, we were unable to directly assess protein loss\nin situ within brain sections. Regarding specificity, the Cx3cr1-Cre driver line is widely\nused and well-established for achieving efficient recombination in microglia within the\nCNS, although partial recombination in peripheral myeloid cells has been reported. Our\nphenotypic analyses were performed on homeostatic microglia and the CPZ-induced\ndemyelination model without peripheral immune cells infiltration, minimizing\nconfounding effects from peripheral subsets.\nTo validate our KO mouse models, we validated gene deletion in sorted microglia by\nWB, which confirmed efficient loss of Bhlhe41 in B41KO (Fig.1b) and B41cKO (Fig.2c)\nmice, and of Aurka in B41HETAurkafl/fl (Fig.4b), Cx3cr1Cre/+Aurkafl/fl (Fig.4c) and\nCx3cr1CreER/+Aurkafl/fl (Fig.S9a) mice, compared with WT and HET mice. These results\nverify effective deletion at the protein level in microglia. We have clarified these points\nin the revised manuscript and included our microglia validation data to strengthen\nconfidence in the knockout specificity.\n## Clarification of MBP and Myelin Analyses (Figure 3D)\n- Does “MBP reduction” refer to total intensity, area coverage, or puncta count?\n- Lines 257–259 mention increased myelin debris and unstructured sheaths; however,\nFigure 3D does not clearly differentiate between MBP puncta (fragmented myelin) vs\nintact sheaths.\n- Responses: We thank the reviewer for this important comment. In the original version\nof the manuscript, “MBP reduction” referred to the quantification of total Mbp\nfluorescence intensity, which we acknowledge does not fully distinguish between intact\nmyelin sheaths and fragmented myelin debris.\nTo address this limitation, in the revised manuscript we have repeated the analysis using\nWT and B41cKO mice. In addition, we introduced staining for degraded Mbp (dMbp) to\nspecifically identify myelin debris, thereby complementing the Mbp analysis.\nQuantification was still performed on total intensity, but the inclusion of dMbp now\nallows us to better differentiate between intact myelin and myelin debris.\nAdditionally, we performed Black-Gold II staining, which selectively labels compact\nmyelin sheaths. This provided an independent validation of myelin loss and confirmed\nthe conclusions drawn from our Mbp/dMbp analyses.\nThese new data are presented in Fig 3b-c,g and the corresponding text has been updated\naccordingly.\n- Have authors considered temporal delay in remyelination rather than failure? Are later\ntime points (e.g., 2 weeks) available?\n- Responses: We thank the reviewer for this thoughtful comment. We agree that a\ntemporal delay in remyelination is a possible explanation for the observed phenotype.\nIn the current study, our analyses were limited to 2-week recovery after cuprizone\nwithdrawal. At this stage, myelin restoration was markedly reduced compared with WT\ncontrols, but we observed ongoing myelin repair in the B41cKO and AurkacKO mice,\nevidenced by increased colocalization of dMbp with Iba-1 (Fig 3g and Fig 4j). Thus,\nthe phenotype may reflect a delayed rather than a completely failed remyelination\nprocess. We have clarified this interpretation in the revised Results sections.\n## Phagocytosis Assays\n- Lines 282–283 overstate the conclusions. Phagocytosis of myelin debris has not been\ndirectly measured. E. coli and zymosan beads do not capture endogenous phagocytosis\nof myelin debris.\n- Co-staining for CD68 or LAMP1 with MBP could confirm in vivo internalization.\n- Morphological assessments alone are insufficient to support claims of defective\nclearance (Line 349). Use of CD68/LAMP1 co-staining may be a better choice.\n- Responses: We thank the reviewer for pointing out this overstatement. We agree that\nour original assays using E. coli and zymosan particles did not directly capture\nendogenous phagocytosis of myelin debris. To address this concern, in the revised\nmanuscript we performed co-staining of Iba-1 with dMbp, which specifically labels\nmyelin debris. In WT mice subjected to cuprizone, we observed robust colocalization\nof Iba-1+ microglia with dMbp, whereas this colocalization was markedly reduced in\nB41cKO mice, indicating impaired uptake of myelin debris.\nAlthough we were unable to perform additional co-staining of CD68 with Mbp due to\nboth antibodies being rat-derived, the Iba-1/dMbp analysis nonetheless provides direct\nin vivo evidence of myelin debris internalization by microglia. These new data are\nincluded in Fig.3g, which reinforce our interpretation of defective myelin debris\nclearance in B41cKO mice.\n## Minor Issues and Clarifications\n- Supplementary Figure 1: Show gMFI and percent positivity for all populations\n(microglia, B-1 cells, alveolar macrophages). Quantify blots for all genotypes.\n- Responses:We thank the reviewer for this suggestion. Our goal in Fig.1 and Fig. S1\nwas to quantitatively assess Bhlhe41 expression levels, particularly in populations\nsuch as microglia that exhibit continuous rather than binary expression. For this\npurpose, geometric mean fluorescence intensity (gMFI) provides a more informative\nmeasure of expression intensity than percent positivity. Regarding the WB data, all\nblots included in the manuscript were quantified across the relevant genotypes, and\nthese quantifications are presented in the corresponding figures (Fig.1b, Fig.2c,\nFig.4b-c, Fig.9a and Fig.10b).\n- Figure 1A: CD206 alone is insufficient to distinguish BAM subtypes; consider Lyve1\n(PVM) and MHC-II (LMM). Iba1 is not specific for microglia—prefer P2RY12, Sall1,\nor Tmem119.\n- Responses: We thank the reviewer for this comment and agree that CD206 alone is\ninsufficient to distinguish BAM subtypes. In Fig.1d and 1f, CD206 was combined with\nanatomical localization to distinguish perivascular from leptomeningeal macrophages,\nwhich is commonly used 3. We also agree that Iba-1 is not strictly microglia-specific4.\nHowever, parenchymal Iba-1+ cells predominantly correspond to microglia.\nImportantly, our flow cytometric analysis of homeostatic microglia using\nCD45loCD11b+Cx3cr1+ gating confirmed microglial identity and allowed subsequent\nquantification analysis of Bhlhe41/Aurka expression and recombination efficiency,\nsupporting the conclusions drawn from Fig.1d and 1f.\n- Figures 2C, 2E: Include gMFI for phagocytosis markers to assess per-cell capacity.\n- Responses: We thank the reviewer for this insightful suggestion. For original Fig. 2C\n(now Fig. 2d, in vitro phagocytosis of E coli-pHrodo Green), we have now included\ngMFI analysis to assess per-cell phagocytic capacity, as the fluorescence intensity of\npHrodo Green reflects both particle uptake and phagosome acidification.\nFor original Figure 2E (now Fig. 2f-g, in vivo phagocytosis of Zymosan- AF488), the\nAF488 signal does not change with phagosome acidification and only reports particle\nuptake. Therefore, gMFI is not an accurate measure of per-cell phagocytic activity in\nthis context. Instead, we quantified the percentage of phagocytic microglia.\n- Line 306: Revise “microglial identity”—the authors are measuring function and\nmorphology.\n- Responses: We thank the reviewer for pointing this out. We agree that “microglial\nidentity” was not precise, as the experiments measured microglial number,\nmorphology and phagocytic activity rather than identity per se. Accordingly, we have\nrevised the sentence to:\n“This finding led us to examine the role of Aurka in regulating microglial number,\nmorphology and phagocytic function.”, line 358-359.\n- Supplementary Figure Mismatch: Correct citation errors (e.g., Lines 311–321).\n- Line 311: Should refer to Supplementary Figure 6, not 5.\n- Line 317: Supplementary Figure 7A, not 6A.\n- Line 319: Supplementary Figure 7A-B, not 6A-B.\n- Line 321: Supplementary Figure 7C, not 6C.\n- Responses: We thank the reviewer for pointing out the citation errors. These have been\ncorrected in the revised manuscript. We have double-checked all figure citations\nthroughout the manuscript to ensure their accuracy.\n- Line 894: Clarify method for microglial isolation (mechanical vs enzymatic) as this\ncan influence microglial activation. Indicate whether whole brains were used or\nregions excluded.\n- Responses: We used mechanical dissociation to isolate microglia from whole brains.\nWe have now clarified this in the Methods section, line 1150-1151.\n- Supplementary Figure 12: CD68 signal is faint and hard to interpret.\n- Responses: We acknowledge that the CD68 signal appeared relatively faint in the\ninitial submission. In the revised manuscript, we have adjusted brightness and contrast\nto improve visibility and provided high-resolution PDF figures.\nReviewer #2 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports.\nThis is part of the Nature Communications initiative to facilitate training in peer review\nand to provide appropriate recognition for Early Career Researchers who co-review\nmanuscripts.\nReviewer #3 (Remarks to the Author):\nLi and colleagues use several new and interesting mouse lines to investigate the\ntranscription factor Bhlhe41. There are some novel and interesting findings in this\nmanuscript regarding how Bhlhe41 impacts microglia morphology and phagocytosis.\nThey find that Bhlhe41 restrains CD22, a known inhibitor of phagocytosis that\nincreases with age. Overall, there is essential findings in this work. However, I do have\nsome serious concerns about how conclusions are made without appropriate analyses\nor data to support these conclusions.\nMajor concerns\n-the claim that B41KO recovery in the EAE is not due to hyperactive T-cells is not\nwell backed up by the data. Figure 3B measures the proportion of CD4 T-cells that\nexpress IFN-g and IL17. These measurements are too superficial for this conclusion.\nThey should measure the proportion of leukocytes/ CD4 and CD8 T-cells as a whole.\nMany other immune cells could also contribute to this recovery, so the link with\nmicroglia is extremely tenuous.\n-The rationale behind doing an EAE study was problematic. Line 229:”To examine\nwhether Bhlhe41 deficiency affects CNS myelination, we established an inflammatory\ndemyelinating model…”. EAE is an inflammatory model, so it is unlikely to\nimpact myelination, such as developmental myelination. Also, no myelin metric was\ninvestigated here. EAE is not a model (with these mice) to distinguish remyelination,\nso a new rationale should be included.\n- Response: We address the above two related comments together. We thank the\nreviewer for raising concerns regarding our EAE experiments. We agree that the\nrationale for using EAE to assess the role of Bhlhe41 in CNS myelination was\nproblematic. As the reviewer correctly pointed out, EAE is primarily an inflammatory\ndemyelination model and does not reliably reflect developmental myelination or\nremyelination. Moreover, our original data on the proportion of IFN-γ+ and IL-17+\nCD4+ T cells were insufficient to conclude that recovery in B41KO mice is independent\nof hyperactive T cells. In fact, we did examine overall leukocyte, CD4+ T, CD8+ T,\nneutrophil, macrophage and microglia proportions and numbers, and found no\nsignificant differences. We agree that EAE is not an ideal model to link microglial\nfunction with remyelination.\nTo better address the core focus of our study—microglial regulation of\nremyelination—we have removed the EAE data from the manuscript. Instead, we re-\nperformed the CPZ-induced demyelination model using WT controls and B41cKO mice.\nMoreover, we performed Black Gold II staining and co-staining of dMbp with Iba-1\nto assess myelination and microglial phagocytic clearance of myelin debris (Fig.3).\nThese new analyses provide direct and robust evidence supporting our finding that\nBhlhe41 regulates microglial responses during remyelination.\n-For the cuprizone work in Figures 3 and 4, the MBP images are (for me) blurry and\nunclear). By convention, the dorsal side of the brain should be up, but I suspect these\nare flipped. It is also unclear where these images are taken in the corpus callosum.\nMBP will label both myelin and myelin debris, so the claims of more or less\ndemyelination/ remyelination with MBP are problematic. These analyses should be\nconfirmed with compact myelin stains that are more sensitive to intact myelin, such as\nSudan black or black gold staining.\n- Response: We thank the reviewer for the constructive suggestions regarding our CPZ\nexperiments. To address these concerns and taking into account the reviewer’s\ncomment on appropriate controls, we re-performed the CPZ-induced demyelination\nstudy using WT mice as controls alongside B41cKO and AurkacKO mice. As suggested,\nwe employed Black Gold II staining to more accurately visualize compact myelin.\nIn addition, we performed co-staining of dMbp with Iba-1 to examine microglial\ninternalization with myelin debris. These new analyses provide clearer and more\nreliable evidence for the impact of Bhlhe41 and Aurka deficiency in microglia on\ndemyelination and remyelination.\n-While g-ratios are a good metric for EM analyses, they do not directly inform the\ndegree of demyelination and remyelination in this model. These EM images should be\nquantified and the proportion of unmyelinated and myelinated axons should be\nreported in the corpus callosum. The authors should also make it clear how they know\nthey were quantifying axons in the corpus callosum\n- Response: We thank the reviewer for this excellent suggestion regarding the EM\nanalysis. We agree that quantifying the proportion of myelinated axons provides a\nmore direct measure of remyelination efficiency. In the updated Fig.3d and Fig.4h,\nwe analyzed the EM images by quantifying the proportion of myelinated vs.\nunmyelinated axons in the corpus callosum. This new analysis confirms that B41cKO\nand AurkacKO mice exhibit a significantly higher percentage of unmyelinated axons\ncompared to WT controls during the recovery phase.\nFurthermore, as the reviewer rightly points out, we have now made it explicit in the\nMethods section how the corpus callosum was identified for EM sampling.\n“The corpus callosum (CC), identified as the white matter tract between the lateral\nventricles in coronal brain sections,” line 997-999.\nThe methods for the cuprizone section should outline how they analyzed the various\ndata (olig2/ MBP, etc.). For example, how many images were taken, what region was\nquantified, how were these images blinded, etc.\n- Response: We thank the reviewer for this important comment. We agree that a\ndetailed description of the quantitative image analysis is essential for rigor and\nreproducibility. We have now thoroughly revised the Methods section for the\ncuprizone model analyses. Please see line 1006-1010; line 1142-1145 in the revised\nmanuscript.\n-The authors talk about myelin debris but offer no data about how much debris is inside\nof microglia during demyelination and remyelination in this model. The authors need\nto examine debris uptake directly to conclude in line 263 “that Bhlhe41 is required to\npromote clearance of myelin debris”. The authors should also not use enlarged cell\nbodies of microglia as indications of “sustained phagocytic activity.” This should be\nmeasured more directly.\n- Response: We thank the reviewer for this insightful comment, which is in line with\na similar concern raised by Reviewer #1. We fully agree that direct evidence of myelin\ndebris uptake is required to substantiate our conclusion.\nTo address this point, we performed new experiments in the revised manuscript to\nquantify myelin debris internalization by microglia in vivo. Specifically, we conducted\nco-staining of the microglial marker Iba-1 with dMbp in brain sections from cuprizone-\ntreated mice.\nThis new analysis, now presented in Fig.3g, Fig.4j and Fig.7i, demonstrates that:\nWT Iba1+ microglia exhibit robust colocalization with dMbp+ myelin debris during the\ndemyelination phase. In contrast, this colocalization is markedly reduced in\nB41cKO/AurkacKO mice during demyelination, indicating impaired uptake of myelin\ndebris (Fig.3g, Fig.4j). In anti-CD22-treated B41cKO mice, this colocalization is\nincreased, indicating improved uptake of myelin debris (Fig.7i).\nRegarding the point on microglial morphology, we thank the reviewer for the helpful\nfeedback. We have removed the text in the revised manuscript and rely on the Iba-\n1/dMbp colocalization data as the primary evidence for the uptake of myelin debris.\n-as there may or may not (it is unclear from the data) be differences in baseline\ndemyelination in this model, and differences in demyelination will impact\nremyelination, the authors are not in a position to make any conclusion on\nremyelination with the data they provide. They do not differentiate between\nunmyelinated and myelinated axons, spared myelin and myelin debris, or\nremyelination and myelin debris.\n- Response: We thank the reviewer for this important comment. We agree that potential\ndifferences in baseline demyelination must be carefully evaluated, as they can strongly\ninfluence the interpretation of remyelination.\nTo address this, in the revised manuscript we performed new experiments using\nB41cKO, AurkacKO and WT mice subjected to the cuprizone-induced demyelination\nmodel. To examine myelinated axon, unmyelinated axon, intact myelin and myelin\ndebris, Black-Gold II staining, Mbp/dMbp/Iba-1 staining and TEM were employed.\nUnder homeostatic conditions, Black-Gold II staining, Mbp/dMbp staining and TEM\nanalyses revealed no differences in myelination between cKO and WT mice. After 5\nweeks of cuprizone treatment, Black-Gold II and TEM analyses showed comparable\nlevels of demyelination between cKO and WT groups, despite slight increased Mbp\nstaining observed. However, dMbp staining, which selectively labels myelin debris,\nrevealed increased debris accumulation in cKO mice compared to WT mice. This\nsuggests that while the extent of demyelination is similar, impaired debris clearance\nmay already be evident during the demyelination phase. We fully acknowledge that\nmicroglial regulation of remyelination is a dynamic process that begins with efficient\ndebris removal and proceeds to subsequent remyelination.\nOur revised text now carefully reflects this distinction by interpreting our findings\nprimarily in terms of defective debris clearance, while noting that this impairment is\nlikely to impact the efficiency of the remyelination phase.\n-The authors use Olig2 as a marker of oligodendrocyte. Olig2 is found in both OPCs\nand oligodendrocytes. They should redo these quantifications with markers of both\nOPCs and oligodendrocytes.\n- Response: We thank the reviewer for this helpful comment. We agree that Olig2\nalone does not distinguish between oligodendrocyte precursor cells (OPCs) and mature\noligodendrocytes. In the revised manuscript, we therefore performed additional\nstaining and quantification using CC-1 in addition to Olig2, which allows specific\nidentification of mature oligodendrocytes. These new data are now included in Fig. 3f,\n4i and 7h, which confirm our original conclusion.\nI do not see CD22 in all the immunofluorescence images. The authors need to re-\nexamine the CD22 representative images.\n- Response: We thank the reviewer for this careful observation. We apologize for the\ndifficulty in visualizing CD22 in the immunofluorescence images. CD22 was detected\nin the green channel, which may have been less apparent in the original figure.\nColocalization of CD22 (Green) and Iba-1(red) can be observed as yellow spots in the\nmerged image (Fig.5-6). To address this, we have reprocessed the images by adjusting\nthe contrast and brightness of the CD22 (green) channel, and white arrows were used\nto indicate areas of colocalization appearing as yellow. A high-resolution PDF version\nof the images has also been included to ensure that CD22 staining can be clearly\nvisualized.\nFor the phagocytosis assays, it is important to understand how the anti-CD22 antibody\nimpacts control animals, not just Aukura cKO and B41 KO mice.\n- Response:We thank the reviewer for this important comment. Indeed, it is critical to\nunderstand the effect of anti-CD22 treatment in control animals. As demonstrated in the\nNature 2019 study 5(Fig.3g of referenced paper), anti-CD22 treatment did not exert\nsignificant effects on young control mice, consistent with the very low baseline\nexpression of CD22 in young microglia. Therefore, our interpretation focuses on the\ndifferential impact observed in the B41cKO and AurkacKO models, which show\nupregulated CD22 expression on young microglia.\n-given that they treat cuprizone mice with anti-CD22 throughout the demyelination\nphase, it is not possible to differentiate between the possibility that this works to limit\ndemyelination or promote remyelination.\n- Response:We thank the reviewer for raising this important point. To address this, we\nperformed additional experiments including both the cuprizone demyelination phase\nand the recovery phase. In the cuprizone-treated groups, despite a slight increase of\nMbp staining, Black-Gold II staining revealed no overt differences between control and\nanti-CD22–treated mice, suggesting that global demyelination was comparable (Fig. e-\nf). However, staining for dMbp showed a reduction in the anti-CD22–treated group,\nindicating that CD22 blockade may already facilitate microglial clearance of myelin\ndebris during the demyelination phase.\nWe agree with the reviewer that this raises the possibility that anti-CD22 acts not only\nduring remyelination but also earlier during demyelination by enhancing debris\nremoval. Given that microglial regulation of remyelination is a dynamic process that\nbegins with efficient clearance of myelin debris, our data suggest that anti-CD22\ntreatment may contribute to both phases. We have revised the text to carefully reflect\nthis nuance without overstating the conclusions.\nSmaller points\n-In figure 1c, I do not limited tdTom in the pvM and mM as the images are too zoomed\nout and of a low quality\n- Response: We thank the reviewer for the comment and apologize for the low quality\nof the original JPG images. As the average proportion of tdTomato⁺ cells is only ~10%,\nwe have now provided enlarged views highlighting the positive cells, together with\nhigh-resolution PDF versions of the figures to allow clearer visualization.\n-in line 185, you call Bhlhe41 microglia specific, but it is in a population of BAM and\nlikely other myeloid cells, so this language should be tempered to something like\nmicroglia enriched\n- Response: We thank the reviewer for this insightful comment. We agree that\n\"microglia-specific\" was an overstatement and have revised the text throughout the\nmanuscript to describe Bhlhe41 as \"microglia-enriched\". This change ensures our\nterminology precisely aligns with the expression data.\n-In Figures 2A and 2B, you refer to microglia in the Bhlhe41 deficient group as being\ndystrophic. These do not look dystrophic but more shrunken with shorter, fatter\nprocesses. I did not see blebbing or fragmented processes from the images, although\nmy images were of a low quality.\n- Response: We thank the reviewer for this careful observation and apologize for the\nlow clarity of the original images. To better illustrate microglial morphology, we have\nnow provided single-cell 3D reconstructions of young microglia in Fig.2b and middle-\naged microglia in Fig.S3, from WT, HET, B41KO, and B41cKO mice. Dystrophic\nmicroglia, as described in the literature6, are characterized by beaded and discontinuous\nprocesses. Our analyses show that Bhlhe41-deficient microglia exhibit similar features,\nincluding shorter and discontinuous processes, and fewer branch intersections. To\nensure rigor and avoid overinterpretation, we have revised the text throughout the\nmanuscript to describe these cells as displaying dystrophic-like morphology.\n-Line 319 may refer to Supp Fig. 7, not Supp Fig. 6.\n- Response: We thank the reviewer for pointing this out. Following the removal of one\nsupplementary figure during revision, the numbering of subsequent figures has shifted.\nThe correct citation has now been updated to Supplementary Fig.6. We have double-\nchecked all figure citations throughout the manuscript to ensure their accuracy.\nReferences\n1. Kreslavsky, T., Vilagos, B., Tagoh, H., Poliakova, D.K., Schwickert, T.A.,\nWohner, M., Jaritz, M., Weiss, S., Taneja, R., Rossner, M.J., and Busslinger, M.\n(2017). Essential role for the transcription factor Bhlhe41 in regulating the\ndevelopment, self-renewal and BCR repertoire of B-1a cells. Nat. Immunol. 18,\n442-455. 10.1038/ni.3694.\n2. Li, H., Tang, Y., Ren, J., Bai, R., Hu, L., Jia, W., Cao, Y., Hong, L., Xu, M., Gao,\nS., et al. (2022). Identification of novel B-1 transitional progenitors by B-1\nlymphocyte fate-mapping transgenic mouse model Bhlhe41dTomato-Cre. Front.\nImmunol. 13. 10.3389/fimmu.2022.946202.\n3. Brioschi, S., Belk, J.A., Peng, V., Molgora, M., Rodrigues, P.F., Nguyen, K.M.,\nWang, S., Du, S., Wang, W.L., Grajales-Reyes, G.E., et al. (2023). A Cre-deleter\nspecific for embryo-derived brain macrophages reveals distinct features of\nmicroglia and border macrophages. Immunity 56, 1027-1045 e1028.\n10.1016/j.immuni.2023.01.028.\n4. Eme-Scolan, E., and Dando, S.J. (2020). Tools and Approaches for Studying\nMicroglia In vivo. Frontiers in immunology 11, 583647.\n10.3389/fimmu.2020.583647.\n5. Pluvinage, J.V., Haney, M.S., Smith, B.A.H., Sun, J., Iram, T., Bonanno, L., Li,\nL., Lee, D.P., Morgens, D.W., Yang, A.C., et al. (2019). CD22 blockade restores\nhomeostatic microglial phagocytosis in ageing brains. Nature 568, 187-192.\n10.1038/s41586-019-1088-4.\n6. Shahidehpour, R.K., Higdon, R.E., Crawford, N.G., Neltner, J.H., Ighodaro,\nE.T., Patel, E., Price, D., Nelson, P.T., and Bachstetter, A.D. (2021). Dystrophic\nmicroglia are associated with neurodegenerative disease and not healthy aging\nin the human brain. Neurobiol Aging 99, 19-27.\n10.1016/j.neurobiolaging.2020.12.003."}, {"round": 2, "reviewer_comments": {"reviewer_1": "See attached pdf document with my comments for the authors", "reviewer_2": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.", "reviewer_4": "This is a revised manuscript aimed at studying the function of Aurka/Bhlhe41 in the context of myelin repair. I want to commend the authors for their work on this revised manuscript; overall, I feel that it has been significantly strengthened. The study used an impressive combination of genetic mouse models and pharmaceutical intervention to show that Bhlhe41- or Aurka-deficient mice have microglia that exhibit aging characteristics, consisting of altered morphology, phagocytic deficits, progressive CD22 upregulation, leading to remyelination impairment in cuprizone-induced demyelination. I have a few minor suggestions for the authors.\nThe diversity of phagocytic cargo used is confusing. Why do the authors utilize E. coli and zymosan, which are not biologically relevant to the study of remyelination? A refocus on myelin debris and apoptotic cells could be more informative.\nThe authors should be careful about their conclusions regarding phagocytosis; only Phrodo-labeled molecules (Fig. 2d for example) will allow the team to discern between internalization versus binding.\nThe authors may want to include in the text that OLIG2 stains all cells of the oligodendrocyte lineage (OPCs and oligodendrocytes).\nFig 4a-b should be moved to a supplementary figure."}, "author_response": "Response to Reviewers\nManuscript ID: NCOMMS-25-19678A-Z\nTitle: Aurka-Bhlhe41 axis prevents premature aging-like microglial dysfunction and\npromotes remyelination\nWe thank the reviewers for their time and insightful comments, which have\nsignificantly improved our manuscript. We have addressed all points raised in a point-\nby-point manner below. All changes in the manuscript have been tracked, and new data\nhave been incorporated into the figures."}, {"round": 3, "reviewer_comments": {"reviewer_1": "I thank the authors for their careful and thorough responses to my previous comments. The revised manuscript has satisfactorily addressed all of my major concerns.\nI have no further substantive comments and consider the manuscript suitable for publication.\n(Remarks on code availability)\nI did not independently run the code. However, the repository appears well organized and publicly accessible, with sufficient documentation to allow inspection and reuse by the community. The availability of the code supports transparency and reproducibility.", "reviewer_2": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": "Response to referees\nREVIEWERS' COMMENTS\nReviewer #1 (Remarks to the Author):\nI thank the authors for their careful and thorough responses to my previous comments.\nThe revised manuscript has satisfactorily addressed all of my major concerns.\nI have no further substantive comments and consider the manuscript suitable for\npublication.\nReviewer #1 (Remarks on code availability):\nI did not independently run the code. However, the repository appears well organized\nand publicly accessible, with sufficient documentation to allow inspection and reuse by\nthe community. The availability of the code supports transparency and reproducibility.\nResponses: We thank the reviewer again for their constructive comments, which have\ngreatly improved the rigor of our work.\nReviewer #2 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports.\nThis is part of the Nature Communications initiative to facilitate training in peer review\nand to provide appropriate recognition for Early Career Researchers who co-review\nmanuscripts.\nResponses: We thank the reviewer again for their constructive comments, which have\ngreatly improved the rigor of our work.\nThe authors did a good job revising the manuscript and addressed most of my concerns regarding\nWT controls, statistical analysis, and demyelination assays. There are a few minor issues that\nshould be addressed prior to publication:\n1. Flow cytometry histograms: The Y-axes on the gMFI histograms are labeled\ninconsistently as “Normalized to Mode” or “Normalized to Model.” This typo should be\ncorrected, and the meaning of “Mode” or “Model” should be explained in the figure\nlegend.\n2. Figures 1a and 4a: Two-way ANOVA was used to analyze the data. Were the three\ngroups compared at each time point, or were AUC values compared between genotypes?\n3. Figure 3: Two-way ANOVA was employed, most likely to test interactions between\nphase and genotype, but the interaction results were not reported.\n4. Supplementary Figure 1c: This figure does not seem to show an age-dependent increase\nin Bhlhe41 expression as stated in line 180. Instead it shows a gating strategy to sort or\nanalyze microglia.\n5. Sholl measurements: It is appreciated that the authors used mixed models, which allow\nthem to fully leverage their dataset. However, the statistical details provided are too\nminimal. The authors report using the lme4 package, but the package version (v.X.X.X) is\nmissing. What were considered fixed and random effects? Ideally, the authors should\nshare the model formula and raw data for reproducibility.\n6. Lines 184–185: Increased TdTomato intensity in B41KO is not sufficient to claim\nself-repression of Bhlhe41. The authors should consider adding a citation that\nexperimentally validates the self-repression mechanism (e.g., using dual-luciferase assays\nor H3K27ac data). Even if such validation is performed in other immune cell types, this\nwould provide stronger support for a self-repressing mechanism in microglia. While\nself-repression likely explains the observed pattern of Bhlhe41 expression across\ngenotypes and models, it has not been directly tested in this study. This limitation should\nbe clearly stated.\n7. Figure 3b: The scale bar is missing.\n8. Figure 3d: Individual data points are obscured within bar graphs, and the data were\nanalyzed using two-way ANOVA. Linear mixed-effects models could also be applied\nhere.\n9. Line 334: Microglial morphology cannot be “impaired”, altered or similar adjectives\nwould be more appropriate.\n10. Figure 4a: Quantification shows a 50% reduction of Aurka in Cx3cr1Cre/+ Aurka fl/+\nmicroglia, but the representative Western blot image does not support this. Is Aurka also\nself-repressing?\n11. Supplementary Figure 6: WT controls are missing. Aurka fl/+ shows 50% Aurka\nexpression and reduced Bhlhe41 expression and therefore cannot serve as proper\ncontrols. WT samples should be included.\n12. Line 516: “Aurka-medicated” should be corrected to “Aurka-mediated.”\n13. Supplementary Figure 10b: Validation of ectopic Bhlhe41 expression should be\nperformed in microglial cells rather than B cells.\n14. Figure 6d: How does the negative control (secondary antibody only) look? Including this\nwould help distinguish specific signal from background.\n15. CD22 blockade: Is the restoration of microglial phagocytosis and improved recovery\nduring remyelination driven specifically by CD22 blockade in microglia? Given that\nCD22 is highly expressed in oligodendrocytes and minimally in microglia (as shown in\nthis and cited human scRNA-seq studies), a multicellular mechanism may be involved.\nThis should be discussed.\nOne major issue remains related to data sharing and reproducibility, and in particular re: the use\nand reporting of statistical results and procedures that, while greatly improved compared to the\noriginal submission, is still lacking critical details, and revealed mis-application of statistical\ntests in several of the reported analyses.\nCritical statistical details are still missing: for each contrast presented in the main text and\nfigures not only the level of significance but also the exact p-value, effect size with unit (unless\nstandardized) and 95% confidence interval, test statistic with df, etc. depending on test used (see\nfor example statistical reports generated with the report R package that follows APA standards).\nAlso, when reporting the use of a linear model it is not sufficient to just say “a linear model was\nused”. Instead, the exact model specification should be reported, e.g., using the R formula syntax\nor conventional statistical model notation (the aforementioned R package can also help with the\nreporting of linear model fits and contrasts).\nIf the authors prefer to avoid crowding figure legends with statistical details, one option is to\nhave “lean” figure legends reporting only the most important contrasts with full details, and the\nsame figure legends or a spreadsheet with all statistical details in Suppl. Information.\nPlease also note that sample size should report what “n” refers to (e.g., n = 4 mice per\ngenotype, or n = 4 fields of view per mouse, 3 mice per genotype, when repeated measures are involved)\nand that figure 1 is missing the statistical procedures used for panels c-g.\nMis-application of statistical tests: Most if not all the statistical tests used, given the small\nsample sizes, assume normally distributed, independent, unbiased, and homoskedastic residuals\nand an unbounded, continuous outcome variable. Great care needs to be taken when using these\ntest with bounded and/or discrete outcome variables, and/or the model assumptions are clearly\nviolated (e.g., when using counts and proportions as outcome variables and data points are near\nthe lower and/or upper bounds, e.g., figures 1f-g, 3g, 4b-c, or when pseudoreplicates are fed to a\ntest like the t-test, which assumes independence of the data points, e.g., figure 8e and g). Because\nof these concerns, I would like to request a statistical review of this manuscript.\nData sharing and reproducibility: All raw data, metadata, and computational notebooks/scripts\nthat were used to generate all statistical results and plots shown in the article (not just those\nrelated to transcriptomic data), should be made available as Supplementary Info. and/or shared in\npublicly accessible repositories at the time of publication. Data available upon request is no\nlonger an acceptable stance. The code that is currently available on the github repository is not\nsufficient."}]

Decision letter