Sulfatide deficiency-induced astrogliosis and myelin lipid dyshomeostasis are independent of TREM2-mediated microglial activation

Domain: biology
Published: 2026-01-09
Editorial outcome: major_revision

Abstract
Disrupted lipid homeostasis and neuroinflammation often co-exist in neurodegenerative disorders, including Alzheimer’s disease (AD). However, the intrinsic connection and causal relationship between these deficits remain elusive. Our previous studies show that the loss of sulfatide (ST), a class of myelin-enriched lipids, causes AD-like neuroinflammatory responses, cognitive impairment, bladder enlargement, and lipid dyshomeostasis. To better understand the relationship between neuroinflammation and lipid disruption induced by ST deficiency, we established a ST-deficient mouse model with a constitutive Trem2 knockout. Our study demonstrates that TREM2 regulates ST deficiency-induced neuroinflammation and astrocyte activation at the transcriptomic level but does not affect stage 1 disease-associated microglia or astrogliosis at the protein level. Additionally, ST loss-induced lipidome disruption, free water retention, and cognitive impairment persist in the absence of TREM2. Further, these phenotypes are more severe in females compared to males. Collectively, these results emphasize the essential role of TREM2 in mediating lipid loss-associated microglia-mediated neuroinflammation, but not astrogliosis or myelin lipid disruption. Moreover, we demonstrated that attenuating TREM2-mediated neuroinflammation has a limited impact on brain ST loss-induced lipidome alteration or AD-like central and peripheral disorders. Our findings suggest that preserving the lipidome and astrocyte balance may be crucial in decelerating the progression of AD.

Introduction
Alzheimer’s disease (AD) is the main cause of dementia in the elderly population and is characterized by impairment of cognition and loss of memory1,2. The disease is affected by genetic, environmental, and aging factors3,4 such as age-related myelin disruption5–7 and loss of myelin lipids8–10. Among these factors, the myelin sheath is the foremost lesion site for the loss of various lipids in AD-like mouse models and patients5,9–12. Our previous studies report that sulfatide (ST) levels were markedly lost in both white matter and grey matter at the earliest stages of clinical AD13. Sulfatides, also known as sulfated galactocerebrosides, are glycosphingolipids highly enriched in the brain and constitute about 10% of myelin lipids10,13–15. ST regulates neuroinflammation by acting as an endogenous stimulator of the brain’s resident immune cells9,10,13,15–17. ST participates in various biological processes such as protein trafficking, cell growth, axon-myelin interactions, neuronal plasticity and cell adhesion17. In addition, ST is an important regulator of brain lipid homeostasis17. Studies have reported that apolipoprotein E (APOE) functions as a modulator for ST transport18, while the absence of APOE leads to ST accumulation in mouse brain10,18,19. Meanwhile, deletion of ST in mice results in major changes in brain lipid homeostasis, including severe disruption of phospholipids and sphingolipids9,10,13,15.

In addition to disrupted lipid homeostasis, enhanced neuroinflammation is another feature of AD11,20. To understand the underlying molecular mechanism of neuroinflammation and lipid dyshomeostasis that manifests in AD, our previous studies investigated the impact of ST loss using both conditional and constitutive CST (Cerebroside Sulfotransferase, the enzyme that catalyzes the last step of ST synthesis, encoded by Gal3st1) knockout mouse models. Our studies showed diverse myelin lipid disruption and neuroinflammation after ST depletion9,10,13,15,16,21. For example, ST deficiency induced an increase in a variety of microgliosis and astrogliosis markers10. Microglia, the resident macrophages in the brain and spinal cord, play a key role in AD pathogenesis by regulating neuroinflammation22,23. The neuroinflammatory response is one of the major defense mechanisms that promotes tissue repair by removing cellular debris and neurotoxins from the brain22–24. These processes involve the regulation of neuronal activity which impacts disease pathogenesis25,26. Consequently, prolonged neuroinflammation results in neuronal cell impairment that leads to synaptic dysfunction and memory deficits in the AD brain27.

TREM2 (triggering receptor expressed on myeloid cells 2) is one of the major receptors expressed by microglia in the brain and activates various microglial pathway genes28. It is also highly expressed in the spinal cord29. Evidence indicates that TREM2 plays a key role in regulating microglial responses in AD brains10,30. Various experimental and clinical studies have reported the activation of microglia during AD pathogenesis in aged mice as well as elderly humans31. During AD progression, TREM2 is highly upregulated which subsequently induces the expression of various microglial genes10,32,33. A disease-associated microglial (DAM) phenotype has been described for the microglial status in various neurodegenerative disease conditions34. The transition of microglia from homeostasis to DAM is a two-stage process, where the transition from DAM stage 1 to stage 2 is TREM2 dependent. In addition to neuroinflammation, TREM2 also mediates the induction of phagocytic pathways and regulates lipid metabolism10,30,33,35.

Activation of astrocytes in neurodegeneration is often seen concomitantly with the activation of microglia36,37. However, the relationship between astrogliosis and microglial activation, and the role of astrogliosis in disease progression are not very well described. Previous human and mouse transcriptomics studies showed that TREM2 regulates microglia- and astrocyte-mediated neuroinflammation at the mRNA level in AD37. Another study reported that activated microglia can induce the activation of astrocytes at both the transcriptomic and protein levels38. In the ST-deficient mouse model, we observed an upregulated neuroinflammatory response characterized by a strong progressive activation of microglial (Trem2) and astrocytic (Serpina3N) pathway genes accompanied by lipidome disruption in the central nervous system (CNS)10.

In this work, to delineate the relationship between microglial activation and astrocytic activation, as well as lipidome disruption, we crossed the constitutive Trem2 knockout (KO) mice with conditional ST-deficient mice along with their respective controls. Our study reveals that the presence of TREM2 is necessary to regulate the ST deficiency-induced microglial, but not astrocytic activation. Furthermore, TREM2 does not mediate ST deficiency-induced myelin lipid imbalance or AD-like behavioral deficits (e.g., cognitive impairment and peripheral free water retention).

Results
TREM2-dependent and -independent CNS transcriptomic modulation by ST loss
Upon confirming the effective disruption of CST and Trem2 expression in our mouse models using qPCR (Supplementary Fig. 1a–d), we sought to understand the TREM2 dependent and independent transcriptomic alterations in the CNS due to ST loss. NanoString gene expression analysis was used to assess spinal cord (SC) and cerebrum (CRM) mRNA levels in the following four groups of female and male mice (17–18 month): Trem2WT/CSTCre- (Cre-); Trem2WT/CSTCre+ (Cre+); Trem2 KO/CSTCre- (KO/Cre-) and Trem2 KO/CSTCre+ (KO/Cre+) mice.

Analysis of the ST deficiency-associated Differentially Expressed Genes (DEGs) (using the following thresholds: P ≤ 0.05 and Log2 (fold change) ≥ 1.2) identified genes involved in pathways that regulate microglia, astrocytes, and immune function as shown in the volcano plots of SC from female mice (Fig. 1a, b). DEGs such as Serpina3N, Mmp12, Trem2, Cd68, Cd84, Mpeg1, Fcrls, C1qc, and ApoE were upregulated in the Cre+ group compared to Cre- group, while the Gal3st1, Tmem144, Slc2a5, and P2ry12 were downregulated (Fig. 1a). This indicates that ST deficiency induces microglial, astrocytic, and immune pathways at the transcriptomic level. In the Trem2 KO background, a similar group of DEGs (Mmp12, Clec7a, Fcrls, Serpina3N, and Ccl3) were upregulated in Cre+ compared to Cre- (Fig. 1b). In the female CRM, we detected the elevation of Serpina3N, Lilrb4a, Lcn2, Vimentin (Vim), C3, Ly9, Spp1, Ccl3 in Cre+ compared to Cre- under the Trem2 wild type background (Supplementary Fig. 1e), while fewer genes were altered in KO/Cre+ mice compared to KO/Cre- (Supplementary Fig. 1f). Overall, the changes were more robust in the SC region compared to CRM. We performed similar analysis in the male CRM, from which we detected similar groups of DEGs comparing to that of females, but with fold changes of less magnitude (Supplementary Fig. 1g, h), suggesting ST loss leads to a more extensive transcriptomic impact in females than in males. Additionally, principal component analysis (PCA) of NanoString data from female tissues revealed a marked separation of the Cre+ group compared to the other three groups in both SC (Fig. 1c) and CRM tissues (Supplementary Fig. 1i). PCA analysis in male CRM tissue did not show much separation as compared to females (Supplementary Fig. 1j), suggesting that females are more prone to changes induced by ST loss. This is consistent with the known differences between male and female on AD susceptibility39. Overall, these results indicate that TREM2 is necessary for the induction of neuroinflammation and immune responses triggered by ST deficiency. Moreover, the effect of ST loss is more obvious in the SC compared to CRM, as well as females compared to males.

To identify the Trem2 independent/dependent transcriptomic modulations in the CNS, a Venn diagram was generated using the upregulated DEGs from Fig. 1a, b (Fig. 1d). Twenty-two shared genes were found to be elevated by ST loss regardless of the presence of Trem2, suggesting the induction of these genes is Trem2 independent (Fig. 1d). Meanwhile, the induction of 127 genes was abolished in the absence of Trem2; these were designated as Trem2 dependent DEGs (Fig. 1d). Using similar criteria, we identified 13 Trem2-independent and 55 Trem2-dependent DEGs from female CRM (Supplementary Fig. 1k). By further referencing the gene lists between SC and CRM tissues, we found that a combined 30 DEGs across tissues were upregulated by ST loss, independent of Trem2, among which 5 DEGs were shared between female SC and CRM (Fig. 1e). We found a combined 150 DEGs from both SC and CRM that were Trem2 dependent, among which 32 DEGs were shared by both tissues (Fig. 1f). Detailed lists of the 5 and 32 genes are included in the Supplementary Figs. (Supplementary Fig. 1l, m).

To understand the regulation and function of these genes, we analyzed the 5 shared Trem2-independent genes using the Enrichr database40–42 and identified class II transactivator (CIITA) and interferon regulatory factor 8 (IRF8) as the most possible up-stream transcription factors (TFs) (Fig. 1g). Previous studies have reported that both CIITA and IRF8 are involved in immune responses43,44. GO analysis suggested that these genes may be involved in the cell clearance process (Fig. 1h), a function important for the apoptotic process45. Similarly, we also analyzed the 32 Trem2-dependent DEGs using Enrichr. We identified nuclear receptor corepressor (NCoR) as a major TF regulating these genes (Fig. 1i), and GO analysis suggested these genes are mainly involved in phosphotyrosine residue and amino acid binding (Fig. 1j). NCoR is a major TF and critical regulator of macrophage inflammation and proliferation46. TREM2 drives the microglial response through the SYK dependent pathway, a protein tyrosine kinase which is in support of the role of TREM2 in mediating protein kinase dependent microglia-mediated neuroinflammation47. To confirm these findings, we investigated further details of the DEGs that were dependent/independent of Trem2 in our datasets. Fc receptor-like molecule (Fcrls), a Trem2-independent gene, was upregulated by ST loss in both wild type and Trem2 KO backgrounds in the SC and CRM of females (Fig. 1k and Supplementary Fig. 1n). On the contrary, the Fc fragment of IgG receptor IIb (Fcgr2b) and phospholipase C gamma 2 (Plcg2) were only upregulated in the Cre+, but not in the KO/Cre+ compared to their respective controls in the SC (Fig. 1l, m) and CRM (Supplementary Fig. 1o, p) from females. As Trem2 is a microglia-specific gene, we evaluated the impact of ST loss and Trem2 deletion on microglia in the CNS. The microglial cell score from females was elevated in the Cre+ compared to the Cre-, while deletion of Trem2 abolished this induction in both SC (Fig. 1n) and CRM (Supplementary Fig. 1q), suggesting deletion of Trem2, indeed, has greatly impaired microglial responses in ST loss.

TREM2 regulates ST deficiency-induced microglia-mediated neuroinflammation pathways in the CNS
Based on the above observed involvement of Trem2 in mediating ST deficiency-induced gene transcripts, we further analyzed the impact of Trem2 deletion in the CNS based on the mRNA levels obtained from the NanoString data. In female SC, microglia-enriched genes were markedly upregulated in the Cre+ groups compared to those of Cre-, while no induction was detected in KO/Cre+ vs KO/Cre- (Fig. 2a). This indicates that the Trem2 is necessary for the activation of microglia in ST-deficient animals. To validate this finding and provide further insight on the activation status, we evaluated protein levels of microglial markers in female SC tissue by Western blot (Fig. 2b). Despite the overall loss of induction on microglial gene mRNAs in the KO/Cre+ vs KO/Cre-, we found that the absence of TREM2 ameliorated ST deficiency-induced stage 2 DAM markers, AXL and LILRB4A (Fig. 2c, d), while it had no impact on the induction of stage 1 DAM markers, CD68 or IBA-1 (Fig. 2e, f), at the protein level. These observations were consistent with the fact that TREM2 is essential for the generation of stage 2 DAM37. To further evaluate the spatial distribution of microglial responses at the protein level, we performed immunofluorescence (IF) staining using female CRM sections and found that loss of ST stimulated the levels of microgliosis markers (IBA-1, CD68, and AXL) in multiple brain regions that are essential for memory function, including the hippocampus and the retrosplenial cortex (RSP) (Supplementary Fig. 2a–c). The most significant induction was detected in white matter-enriched regions, including the corpus callosum (CC) (Fig. 2g) and fimbria (Supplementary Fig. 2a–c). Among these microgliosis indicators, the induction of stage 2 DAM marker, AXL, by ST deficiency was abolished in the absence of TREM2 (Fig. 2g, h and Supplementary Fig. 2a). On the contrary, the induction of IBA-1 and CD68 by ST deficiency was detected both in the presence or absence of Trem2 (Fig. 2g, i, j and Supplementary Fig. 2b, c). These results indicate that TREM2 mediates ST deficiency-induced microglial activation specifically via promoting the formation of stage 2 DAM.

In line with the altered microglial genes observed in SC, the microglial pathway score from female CRM also showed an upregulation in the Cre+ group compared to the Cre- group, and no activation was observed after Trem2 deletion (Supplementary Fig. 3a). Similarly, mRNA levels of microglial genes such as Cd68 and Mmp12 were elevated in the Cre+ group, but not in the KO/Cre+ group compared to respective controls (Supplementary Fig. 3b, c). Based on our previous study10, enhanced inflammatory and immune pathways were detected when ST deficiency was present, yet it is unknown whether these effects are mediated by TREM2. By analyzing the levels of a group of inflammatory pathway genes, including Fcgr1, Ptpn6, Ifi30, Fcgr3, Lilrb4a, Gpr183, Cd72, Irf8, Mpeg1, and Trem2, we found that the induction of inflammation by ST loss was no longer observed in the Trem2 KO background (Fig. 2k). Accordingly, deletion of Trem2 abolished the upregulation of the inflammatory pathway score (Fig. 2l), as also reflected in the gene levels of Mpeg and Cd72 (Supplementary Fig. 3d–f). Similar results were found in the female CRM, where the induction of the inflammatory pathway score (Supplementary Fig. 3g) and the inflammatory genes (Mpeg, Lilrb4a) (Supplementary Fig. 3h, i) were upregulated in the Cre+ mice only in the presence of Trem2.

TREM2 regulates ST deficiency-induced adaptive and innate immune response pathways
To understand the role of TREM2 in the ST deficiency-induced adaptive and innate immune response pathways, we assessed immune-related genes in female SC and observed a clear upregulation in the Cre+ group compared to that of the Cre- group, but no induction was detected in the Trem2 KO background (Cre+ vs Cre-) (Fig. 2m). Consistently, both adaptive and innate immune response pathway scores were upregulated in Cre+, while the induction was diminished in KO/Cre+ compared to KO/Cre- (Fig. 2n, o). Similar trends of adaptive, innate immune response pathway scores (Supplementary Fig. 3j, k) were observed in the female CRM. Notably, the inflammatory, immune and microglial pathways in male CRM also showed a weakened induction from ST loss in the absence of Trem2 (Supplementary Fig. 3l).

Conclusively, TREM2 is necessary for activating the ST deficiency-induced neuroinflammation pathway and immune response at the transcriptomic level in both female SC and CRM, while male CRM showed minimal effects, suggesting the effect on female is more robust.

TREM2 regulates ST deficiency-induced astrocyte activation at the transcriptomic level but not at the protein level in the CNS
We next assessed the impact of Trem2 deletion on astrocyte activation induced by ST deficiency. A group of genes that are associated with astrocyte function was evaluated based on results from our NanoString measurements. We found that in female SC, ST deficiency induced astrocyte activation-related genes, such as Cd109, Vim, Tm4sf1, Serpina3N, C4a, Ptx3, S1pr3, Cd14, Timp1, ApoE, and Osmr, while knockout of Trem2 greatly limited these inductions (Fig. 3a). In line with this, the astrocyte pathway scores and mRNA levels of ApoE, Serpina3N, and C4a were found upregulated in Cre+ vs Cre- only in the presence of Trem2 (Fig. 3b–e). A similar trend was also observed in the female CRM, as shown in Supplementary Fig. 4a–e. In male CRM, we detected a pattern resembling that of females, but the regulation was less robust when compared to females (Supplementary Fig. 4f, g).

To further validate our findings at the transcription level, we assessed protein levels of astrocytic markers in the female SC tissues using Western blots. In contrary to mRNA levels, the protein levels of APOE, SERPINA3N, VIM, and GFAP were markedly upregulated in the Cre+ group compared to the Cre- group regardless of the presence or absence of TREM2 (Fig. 3f–j). To gain insight about the spatial distribution of astrocytic responses at the protein level, we performed IF staining using female CRM sections and detected the induction of SERPINA3N, VIM, and GFAP by ST deficiency both with and without TREM2 (Fig. 3k–p). The activation of astrogliosis was detected in brain regions including the hippocampus and cortex (Supplementary Fig. 5a, b), with more enhanced signals in the white matter-enriched area such as CC (Fig. 3k, m, o) and fimbria (Supplementary Fig. 5a, b). Collectively, these results suggest that TREM2 regulates the ST deficiency-induced astrocyte activation at the mRNA level, but not at the protein level.

Sulfatide deficiency-induced alterations in myelin lipids are independent of TREM2
Along with microglia and astrocyte activation, significant disruption of the lipidome was observed in both CRM and SC of ST-deficient mice10,16. To determine if TREM2 plays a role in ST deficiency-induced lipid disruption, we performed multi-dimensional mass spectrometry-based shotgun lipidomic analysis on both female and male CNS tissues. Significant losses of ST levels were confirmed in all regions evaluated (Fig. 4 and Supplementary Fig. 6), further confirming the effectiveness of the ST-deficient model.

In female CRM, lipidomic data showed that ST deficiency-induced losses of myelin lipids, e.g., phosphatidic acid (PA) and cerebroside (CBS), were significantly reduced in both Cre+ vs Cre- (indicated by *) and KO/Cre+ vs KO/Cre- (indicated by $) (Fig. 4a). Detailed group-wise comparisons of ST, PA, CBS, and plasmalogen decline in both Cre+ and KO/Cre+ groups compared to their respective controls were shown in Fig. 4b–e. These results indicate that ST deficiency-induced myelin lipid disruption is independent of TREM2. Meanwhile, the ST loss-driven alterations in the cardiolipin (CL) and phosphatidylinositol (PI) were present in the Cre+ vs Cre-, but not in the KO/Cre+ vs KO/Cre- (Fig. 4f, g), indicating these changes to be TREM2-dependent.

In female SC, ST deficiency induced the reduction of mitochondria-specific acylcarnitine (AC) species in both Cre+ (indicated by *) and KO/Cre+ (indicated by $) groups compared to their respective controls (Fig. 4h), resembling that of CRM. Other classes of lipids such as bis(monoacylglycero)-phosphate (BMP), cardiolipin (CL), and lysophosphatidylcholine (LPC) were elevated only in the KO/Cre+ group, indicating the above alterations were specifically responsive to ST loss when TREM2 is absent. Comparisons of ST, PA, CBS, and plasmalogen molecular species between groups are summarized in supplementary materials (Supplementary Fig. 6a–d). In male SC, ST deficiency led to a reduction of myelin lipids (e.g., PA, PS, and PE), and these alterations were independent of TREM2 (Supplementary Fig. 6e). Group-wise comparisons are shown in Supplementary Fig. 6f, g.

Overall, our lipidomic measurements suggested that ST deficiency-induced alterations in myelin lipids are mostly independent of TREM2 in both sexes.

Sulfatide deficiency-induced free water retention in females is independent of TREM2
We recently reported that prolonged ST loss leads to bladder enlargement due to elevated urine retention, reflecting impaired CNS control of peripheral organ function16. The elevated urine phenotype was measured using qMRI and displayed as free water retention. To assess if TREM2 is involved in the regulation of ST deficiency-induced free water retention, we measured the free water weight in 12- and 17-month female and male mice from all groups using quantitative MRI (qMRI). These age points were selected based on our previous observation that Cre+ mice start to show a water-retention phenotype around 17 months of age16. Significant elevation in free water retention was observed in 17 months but not at 12 months in the Cre+ and KO/Cre+ mice compared to their respective controls (Fig. 5a, b). No significant difference in free water was detected in males at 17 or 12 months (Fig. 5c, d). Note that in addition to free water, we also evaluated other parameters, including total water weight (Fig. 5e–h), lean weight (Supplementary Fig. 7a–d), fat weight (Supplementary Fig. 7e–h), and body weight (Supplementary Fig. 7i–l). No change was observed among all groups at ages 12 or 17 months. These results suggest that ST deficiency-induced urine retention is independent of TREM2.

Sulfatide deficiency induces cognitive impairment independent of TREM2
The critical clinical symptom of AD is the progressive loss of cognitive function. We have previously demonstrated that adult-onset ST-deficiency leads to impaired spatial memory function10. Here, we further evaluated if TREM2 plays any role in this process. We performed a Morris Water Maze (MWM) test, from which we observed significantly increased latency among female Cre+ mice compared to Cre- during the training phase (Fig. 6a). Meanwhile, the percent time spent in the target quadrant was greatly reduced in Cre+ vs. Cre- (Fig. 6b). These results suggest that ST loss led to impaired spatial learning function, which is consistent with our previous observation10. Notably, spatial memory function remained impaired in ST-deficient mice with Trem2 KO (Fig. 6a, b). On the probe day when the training platform was removed and the number of target crosses was recorded, ST loss significantly decreased the number of crosses in the presence of Trem2 (Fig. 6c), while no difference was detected under Trem2 KO due to large variations between groups (Fig. 6c). These findings suggest that ST deficiency induces cognitive impairment independent of TREM2.

Consistent with our previous observation, Cre+ mice exhibited a reduced swimming speed compared to Cre-, while knockout of Trem2 did not alter this behavioral phenotype (Fig. 6d), which further supports the limited involvement of TREM2 in ST deficiency-induced behavior.

To further confirm our observations, we performed the Barnes Maze test to evaluate the spatial memory formation in all genotype groups. A robust increase in latency was detected in Cre+ and KO/Cre+ compared to that of Cre- and KO/Cre-, respectively (Fig. 6e). Concordantly, ST-deficient mice spent a significantly less amount of time in the target quadrant regardless of the presence of Trem2 (Fig. 6f). For both MWM and Barnes Maze assays, we observed a similar trend but less significant behavioral phenotypes among males compared to females (Supplementary Fig. 8a–f), which is consistent with the milder transcriptomic and lipidomic changes observed in males compared to females.

Methods
Mice
Animal experiments were conducted in accordance with the ‘Guide for the Care and Use of Laboratory Animals’ (8th edition, National Research Council of the National Academies, 2011). The animal protocol was approved by the Institutional Animal Care and Use Committee of University of Texas Health San Antonio (protocol code 20180044AP and approved on 01-09-2019). The CST fl/fl mice were generated by Applied Stem Cell, Inc. using CRISPR technology to insert LoxP sequences up- and down-stream of exon 2 and 3 of the CST gene via injecting C57BL/6 embryos10,56. The Plp1-CreERT and Trem2 KO mouse lines were both purchased from JAX (stock #005975 and #027197, respectively). The CST fl/fl mice were crossed with Plp1-CreERT+ mice to generate conditional ST-deficient (Cre+) mice along with their respective (Cre-) controls10. The constitutive Trem2 KO mice were crossed with ST-deficient Cre+ mice to generate Trem2 KO ST-deficient mice (Trem2, CST fl/fl/Plp1-CreERT+) denoted as (KO/Cre+) along with the control (KO/Cre-). Mice were housed under 14-h light/10-hour dark cycles under temperature of 75°F+/- 3°F, and 35% humidity with free access to food and water. Tamoxifen (40mg/kg body weight, Sigma, Cat. #: T5648-5G) was administered at 3 months of age via intraperitoneal injections once every 24h for 4 consecutive days to male and female Trem2 WT/CST Cre- (Cre-); Trem2 WT/CST Cre+ (Cre+); Trem2 KO/CST Cre- (KO/Cre-) and Trem2 KO/CST Cre+ (KO/Cre+) mice.

Body composition
Quantitative Magnetic Resonance Imaging (qMRI) was used to assess the body composition (fat weight, lean weight, free water, and total water) of individual mouse using an EchoMRI 3-in-1 Body Composition Analyzer (Echo Medical Systems, Houston, TX, USA).

Nerve tissue collection and immunofluorescence staining
For biochemical analysis such as NanoString, Western blot and lipidomic studies, animals were anesthetized, and cardiac perfused with 1x PBS. Cerebrum (from the right hemi-brain) and spinal cord tissues were collected, and snap-frozen in liquid nitrogen. For immunofluorescence staining, left hemi-brain was fixed overnight in 4% paraformaldehyde, rinsed in 1x PBS, dehydrated sequentially in 10, 20, and 30% sucrose solutions before being embedded in optimal cutting temperature (OCT) compound. Frozen brain blocks were sectioned into 10µm thick slices and heat-induced antigen retrieval was performed in citrate buffer. Tissue sections were then blocked with 10% donkey serum in PBST (PBS containing 0.1% Triton X-100), incubated at 4°C overnight in primary antibodies (Supplementary Table 1), washed three times in PBST, incubated with secondary antibodies for 1 h at room temperature, and washed for three times in PBST before being evaluated under a fluorescence microscope. Images were collected using a BZ-800 viewer and analyzed using the BZ-800 Analyzer (KEYENCE).

Gene expression analysis
Cerebrum and spinal cord tissues were powdered in liquid nitrogen followed by RNA extraction using the Animal Tissue RNA Purification Kit (Norgen, Canada, Cat. #: 25700) according to the manufacturer’s protocol. The concentration of RNA was determined using a DS-11 Spectrophotometer (DeNovix, USA). NanoString multiplex gene expression analysis was performed using the Mouse Neuroinflammation Panel on the nCounter® Max Analysis System according to the manufacturer’s protocol (NanoString Technologies, USA). Data were analyzed using nSolver 4.0 software. Our basic analysis included the default Normalization Parameters which consisted of Positive Control Normalization Parameters and CodeSet Content. Both parameters included a geometric mean calculation that flags samples if their normalization factor is outside of the 0.3–3.0 range or 0.1–1.0 range, respectively. We selected all pairwise ratios for the Fold Change Estimation. Our advanced analysis included custom analysis with all default settings except selecting P-value adjustments (Benjamini-Hochberg) and “use all probes” under the section of differential profiling and cell type profiling.

For real time qPCR gene expression analysis, total RNA was extracted from tissue using a Direct-zol RNA Miniprep Kit (Zymo Research, USA, Cat. #: R2051). Reverse transcription and cDNA synthesis was performed using a QuantiTect Transcription Kit (Qiagen, Cat. #: 205313) according to the manufacturer’s instructions. qPCR reactions were performed using Power SYBR Green PCR Master Mix (Applied Biosystems Inc., Cat. #: 4367659) on a 7900HT Fast Real Time PCR System (Applied Biosystems Inc.) with the following primers: mTrem2_FW: 5’-CTGGAACCGTCACCATCACTC-3’; mTrem2_RV: 5’- CGAAACTCGATGACTCCTCGG -3’; mGal3st1_FW: 5’- CCTCCACTG-TATCCCAACATGG -3’; mGal3st1_RV: 5’- ATATCTCGCCGAGGTTGACACC -3’; mGapdh_FW: 5’- GGCAAATTCAACGGCACAGT -3’; mGapdh_RV: 5’- AGATGGTGATGGGCTTCCC-3’. A dissociation curve analysis was performed at the end of the qPCR cycle to check for non-specific products. Data were analyzed using the ΔΔCT method by normalizing target gene levels to endogenous housekeeping gene Gapdh levels. Data were presented as fold change relative to Cre- as the control group.

Lipid extraction and mass spectrometric analysis of lipids
Multidimensional mass spectrometry-based shotgun lipidomics was performed to identify lipid species and measure lipid content in samples of interest. Briefly, pulverized frozen CRM and SC tissues were homogenized in ice-cold phosphate-buffered saline using a Precellys® Evolution Tissue Homogenizer (Bertin, France). The protein concentration of homogenates was determined using a Pierce BCA Protein assay (Thermo Fisher, USA, Cat# 23225) according to the manufacturer’s instructions. Lipids were extracted by the modified procedure of Bligh and Dyer in the presence of internal standards that were added based on the total protein content of each sample62. Lipids were quantified by ion peak intensity comparison to the internal standard of the class of lipids as acquired by a triple-quadrupole mass spectrometer (TSQ Altis, Thermo Fisher Scientific, Waltham, MA, USA) equipped with a TriVersa NanoMate® device (Advion Interchim Scientific, USA) on an Xcalibur operating system63. Data processing including ion peak selection, baseline correction, data transfer, peak intensity comparison and quantitation was performed using a custom programmed Microsoft Excel macro10,64 before the results were normalized to the protein content.

Western blot
Frozen spinal cord tissue was powdered in liquid nitrogen, followed by homogenization in N-PER™ Neuronal Protein Extraction Reagent (Thermo Scientific, USA, Cat. #: 87792) with Halt Protease and Phosphatase Inhibitor Cocktails (Thermo Scientific, USA, Cat. #: 78430) using a Precellys® Evolution Tissue Homogenizer (Bertin, France). Homogenates were centrifuged at 12,000×g for 30min at 4°C. Supernatants were collected and their protein concentrations were determined using the Pierce TM BCA Protein Assay Kit (Thermo Scientific, USA, Cat. #: 23225) according to the manufacturer’s instructions. Forty micrograms of protein per sample were separated on NuPAGE 4–12%, Bis-Tris gels (Invitrogen, USA, Cat. #: NP0323) under reducing conditions. Separated proteins were then transferred to PVDF (Fluor) membrane (Millipore, USA, Cat. #: IPVH00010) for 1h at 100V using NuPAGE transfer buffer (Invitrogen, USA, Cat. #: NP0006). Membranes were hybridized overnight at 4°C using primary antibodies (Supplementary Table 1). All primary antibodies were followed by hybridization at room temperature for 1h in the appropriate horseradish peroxidase (HRP)-linked secondary antibodies (Supplementary Table 1). Membranes were washed 3×10min in tris buffered saline containing 0.05% Tween 20 after each antibody incubation. Pierce™ SuperSignal™ West Atto Ultimate Sensitivity ECL Substrate (Thermo Scientific, USA, Cat. #: A38556) was used to detect proteins before exposure to autoradiography film (HyBlot CL, Denville Scientific). Some membranes were re-blotted after being incubated in protein stripping buffer (Thermo Fisher Scientific, USA, Cat. #: 46430).

Morris Water Maze
Morris Water Maze was conducted in a circular, white-colored tub (120cm diameter), filled with water tinted to an opaque, milky white color using non-toxic, washable paint (Crayola). Spatial cues were positioned next to the tank above the eye level of the animals at each of the four coordinates (N, S, E, W). On each of the five training days, a platform (11cm in diameter) was placed in the northwest quadrant submerged 1cm under water. Mice were acclimated in the behavior room for 1h before testing each day. Each mouse was individually held with its nose facing the wall before carefully being placed into water at one of the four coordinates. Each animal was then given 60s to find the platform. In the event a mouse was unable to find the platform, it was guided to the platform. All mice were left on the platform for 15s before being removed, towel dried, and gently warmed in a clean cage using a warming lamp above the cage. Each mouse went through four training trials every day with each trial starting from a different coordinate. The trials were spaced at least one hour apart and the coordinate order was rotated daily. The sixth day consisted of one “probe” trial with the platform removed from the pool, and each mouse was allowed to swim for 60s before being taken out of the tank. An HD Pro Webcam C920 and the HVS Image software (version 2020) were used to track and analyze each trial. Parameters such as latency, path length, swim speed, platform crosses, and percent time spent in each quadrant were collected and further analyzed.

Barnes maze
The Barnes maze is an elevated circular platform composed of a table (91.5cm diameter) with 20 holes (5.72cm diameter) that are evenly distributed (approximately 7cm apart) along the perimeter. One of the 20 holes leads to an escape tunnel positioned underneath it. The remaining 19 holes do not have an escape chamber. Mice in this study were trained to find the escape chamber using spatial cues placed around the platform. Habituation was performed one day before the training phase, where each mouse was put in the center of the platform and allowed to explore for 180s. If a mouse located the escape chamber, a glass beaker was placed above the chamber for 30s to allow the mouse to become familiar the space. In the event a mouse was not able to locate the escape chamber, it was gently guided to the chamber before the glass beaker was placed on top. After habituation, the Barnes maze test began with a 5-day training phase with the presence of spatial cues, consisting of three trials per mouse each day spaced one hour apart. Each mouse was placed at the center of the table under an opaque plastic beaker for 10s, and then the beaker was removed to allow the mouse to explore the entire table for 180s, during which the mouse was motivated to seek the escape tunnel due to the open space atop the platform and the bright light in the room. In the event a mouse was unable to find the escape chamber, it was guided to it. Each animal was left in the escape chamber for 30s before being returned to its home cage. After the training days, a single probe trial on the sixth day was performed for 180s, with the escape tunnel removed to assess the spatial learning and memory of the mice. An HD Pro Webcam C920 and HVS Image software (version 2018) were used to track and analyze each trial. Data such as time spent in each quadrant, latency, speed, path length were collected for further analysis.

Statistics
Mice of the desired genotype were randomized for experimental group distribution. Three or more replicates per group were used for all tests, all attempts at replication were successful. No statistical method was used to predetermine sample size, the numbers of samples were decided based on our previous studies using the same mouse lines, where similar sample sizes were sufficient for detecting effects in the same tests. The investigator was blinded when performing the NanoString, lipidomic, and animal behavioral studies. Other evaluations were not blinded. No data were excluded from the analyses. Data were presented as mean ± SEM. Statistical analyses for the NanoString data were performed using NanoString nSolver software (version 4.0) according to the manufacturer’s recommendations. All other statistical analyses were performed using GraphPad Prism (version 10). Alpha Imager Software (Alpha Innotech) was used to quantify protein expression in Western blots. Heatmapper (http://www.heatmapper.ca) was used65 for creating heatmaps. Two-way ANOVA with appropriate post-hoc test (see details in each figure legend) was conducted for multiple comparisons. *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001 were used to compare the mean values across multiple groups.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "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.\nCo-Review Comments for Author:\n• What are the noteworthy results?\nIn this study, the authors investigated the causal relationship between lipid dyshomeostasis and neuroinflammation in Alzheimer’s disease, due to their previous extensive work establishing that the loss of sulfatide results in neuroinflammation, cognitive impairment, and lipid disruption that is reminiscent of Alzheimer’s disease pathology. The goal of the study was to delve into the specific neuroinflammatory mechanisms mediating lipid dyshomeostasis in Alzheimer’s disease, specifically via the examination of astrocytic and microglia-mediated pathways. The noteworthy results include the identification of Trem2 as an essential mediator of only microglia mediated neuroinflammation induced by lipid loss, and importantly that limiting of this Trem2- mediated inflammation did not have a significant impact on lipid dyshomeostasis induced by sulfatide loss. As such, the authors concluded that activated astrocytic contribution to lipid homeostasis may be a relevant avenue to slow Alzheimer’s disease progression.\n• Will the work be of significance to the field and related fields? How does it compare to the established literature? If the work is not original, please provide relevant references.\nThe Han group (authors) were the earliest to establish the negative correlation between sulfatide levels in the white/grey matter and Alzheimer’s disease. Recently, a paper was published in Cell (Zimmer, V. C., Lauer, A. A., Haupenthal, V., Stahlmann, C. P., Mett, J., Grösgen, S., ... & Grimm, M. O. W. (2024). A bidirectional link between sulfatide and Alzheimer’s disease. Cell Chemical Biology, 31(2), 265-283.) which investigated sulfatide deficiency in Alzheimer’s disease specifically through the lens of APP processing. However, overall studies investigating the contribution of sulfatide deficiency to Alzheimer’s disease are lacking. Here, the authors’ investigation of the link between sulfatide deficiency and glial response (particularly in relation to microglial Trem2, which is known to have a critical role in mediating beneficial microglial engulfment of lipid debris in AD) takes a novel approach. Additionally, the incorporation of the spinal cord in this study is highly novel, as the lipidomics of the spinal cord during AD pathology is relatively unstudied.\n• Does the work support the conclusions and claims, or is additional evidence needed?\nThe authors’ work strongly supports the conclusions made, and the molecular output and analysis is strong. Notably, the authors identified and suggested Trem2-independent microglial enriched genes that may be targeted to further parse out the mechanisms of microgliosis in Alzheimer’s disease.\n• Are there any flaws in the data analysis, interpretation and conclusions? Do these prohibit publication or require revision?\nThere are three major opportunities missed in this work.\nThe first is the lack of incorporation of AD mouse models, particularly those that have a tendency towards worsened female phenotype (as the authors mention occurs with sulfatide deficiency). The authors previously found that specific deletion of CNS sulfatide in Plp1+ myelinated tracts in the adult brain resulted in the increase in AD risk genes and showed that this induced cognitive behavioral changes. As such, the authors could have followed up on this work in this paper and investigated any AD-related upstream regulators of the Trem2 microglial-specific phenotype in sulfatide loss.\nThe second missed opportunity is the generic study of the cerebrum, without specific separation into grey and white matter regions, which can significantly alter the transcriptomic results, and the interpretation related to region-specific Alzheimer’s disease pathology.\nThe third is the lack of behavior analysis for a measure of functional output, despite the authors previously showing that the specific loss of sulfatide in myelin sheaths leads to cognitive impairment. Although the authors find that Trem2 mediated neuroinflammation is independent of alterations in myelin lipids, it is still worth investigating whether this results in cognitive impairment.\nDue to these points, the paper requires more critical validation in AD-specific contexts.\n• Is the methodology sound? Does the work meet the expected standards in your field?\nThis study incorporated limited CNS regions for analysis, which is a weakness in the methodology. The methodology would be more robust if the cerebrum was dissected into specific AD-relevant regions with sulfatide loss, such as the hippocampus, cortex, and white matter tracts. Further, there is no validation showing the efficiency of conditional Plp1-CreERT and Trem2 knockout via tamoxifen administration, which would greatly affect the results if the knockout was minimal (i.e., the degree of neuroinflammation could be less with a less efficient sulfatide knockout, and this could impact the finding that astrogliosis was not seen at the protein level). Further, mice were tamoxifen-induced for Plp1 and Trem2 knockout at three months of age but examined at 12 and 17-18 months of age, without much explanation for why these timepoints were chosen. It is well known in the literature that myelin lipids degrade naturally with age, which is a significant coinciding factor that would significantly skew the results towards greater sulfatide deficiency and neuroinflammation with the aging process.\nHowever, the gene expression analysis, lipidomics, and western blotting performed in this study, as well as the incorporation of both male and female mice and analysis of sex differences is of sound methodology. The gene expression analyses are analyzed elegantly and in detail.\nOverall, this study has a limited scope in the experiments that were performed. The study would be greatly enhanced with the incorporation of the above points, as well as 1) incorporation of AD models 2) AD-relevant regional analyses and 3) cognitive behavioral analyses for AD-relevant functional output (as mentioned above). As such, this study at present does not meet the expected standards for a Nature Communications paper.\n• Is there enough detail provided in the methods for the work to be reproduced?\nYes, there is sufficient detail provided in the methods for the work to be reproduced.\nMinor comments:\n1) The statement on lines 78-79 should have a reference.\n2) Lines 427 and 728 should be contiguous.\n3) The protein band sizes in Fig. 3F are not listed.\n4) Full western blots should be in the supplementary file.\n5) Normalized and filtered dataset of transcriptomic and lipidomics should be provided as supplementary tables.", "reviewer_2": "In the present manuscript the authors use a ST-deficient mouse model with constitutive Trem2 knockout to study the impact of Trem2 in regulating ST deficiency-induced microglia-mediated neuroinflammation, astrocyte activation and lipid dishomeostasis.\nThe authors clearly show that in Trem2 KO animals there is the prevention of ST deficiency-induced microglia-mediated neuroinflammation, but no effect on the induced astrogliosis and lipid dyshomeostasis. However, further studies are needed to conclude what authors conclude from their findings, that “preserving lipidome and astrocyte balance may be crucial in decelerating the progression of AD”, since no behavioural data from these animals is provided.\nThe present study follows previous studies from the same group where they showed that ST deficiency in the adult CND elicits an AD-like neuroinflammation and mild cognitive impairment. Here the authors by using NanoString multiplex gene expression analysis, mass spectrometric analysis of lipids, western blot data and qMRI for free water retention, described that: 1) Trem2 KO ST-deficient mice show a less neuroinflammatory gene expression, namely in females, rather than males and mostly in SC rather than CRM; 2) Trem2 is necessary for activation of microglia-related inflammatory and immune pathways in ST-deficiency animals, once again in Sc female samples; 3) Trem2 KO is not sufficient to reduce ST-deficiency associated astrocyte activation, astrocyte activation, myelin lipid alterations or female water retention. However, with the present data the authors are not able to clarify other mechanisms, rather than Trem2, that may justify the ST-deficiency associated astrocyte activation, myelin lipid alterations or female water retention.\nThe results should be further confirmed by immunohistochemical data from the different brain regions analysed (spinal cord and cerebrum), that could improve the distinct observations concerning microgliosis and astrogliosis, or even lipid homeostasis, rather than the bulk information give by the present methodology used. Also, a specific analysis of isolated cell types (i.e. microglia or astrocytes) and a scRNaseq could better identify the distinct populations of each of these DAM or DAA. Moreover, namely within the cerebrum the analysis of distinct regions mostly associated with AD, namely the hippocampus, as previously addressed in the authors previous publication (DOI: 10.1186/s13024-021-00488-7), can markedly improve the understanding of the similarities between AD-like and ST deficiency pathogenesis. Furthermore, potential changes at animal behavioural tests could corroborate whether the effect of Trem2 KO on microglia neuroinflammation could have an impact on ST-deficient mice outcomes.", "reviewer_3": "The manuscript by Mittra et al. investigated how deficiency of ST regulates in microglia-mediated neuroinflammation, astrocyte activation and lipid disruption in the presence and absence of TREM2. They provided evidence that microglial- enriched transcriptional changes caused by conditional deletion of CST in PLP1-cre model quantified by nanostring can be ameliorated by TREM2 deletion, in a female-biased manner. However, astrogliosis and myelin lipid disruption caused by ST deficiency not significantly impacted by TREM2 deletion. The study is straightforwardly designed, and the results are intriguing. The data quality is good and the conclusions are solid. The main weakness is the lack of mechanism dissection, and the use of nanostring instead of single nuclei or single cell analyses greatly limited the interpretation of the cell type-specificity. Some of the conclusions need additional evidence such as immunostaining or RNAscope.\nMajor concerns:\n1. Figure 1: Key findings need to be validated using either IHC or RNAscope, with co-immunostaining of cell type-specific markers for microglia.\n2. Figure 2: Panel A, the microglial genes are not accurate, since some of the genes are expressed on other cell types, such as Apoe. Similar to Figure 1, validation is needed for the conclusions to be made.\n3. Figure 3: Similarly, the western blot does not differentiate the cell type-specific changes, hence the conclusion that astrocytic alteration at the protein levels cannot be concluded. IHC is needed.\n4. Quantification of myelin lipids was performed in the CRM, while most of the transcriptomic studies were performed in spinal cord. It would be helpful to have the transcriptomics studies also performed in the CRM, or vice versa.\n5. The difference between male and females are intriguing. It is important to label the sex of the experiments in the panels, and state more explicitly in the abstract and conclusions. A more pointed discussion on this point would be helpful."}, "author_response": "Manuscript number: NCOMMS-24-54793-T\nTitle: Sulfatide deficiency-induced astrogliosis and myelin lipid dyshomeostasis are independent\nof Trem2-mediated microglial activation\nA Point-to-point Response to Reviewers’ comments\nDear reviewers:\nThank you for taking the time and effort to carefully examine our manuscript. We are grateful for your\nconstructive comments and suggestions, which are very helpful in improving our work. In\naccordance with the suggestions, we would like to provide additional information included in the\nupdated manuscript (indicated in golden copper color), as well as a detailed response.\nPlease find the point-to-point responses listed below to individual comment (please note that line\nand figure numbers mentioned in the responses below are in concordance with the revised version\nof manuscript):\nReviewer 1:\n1. Comments regarding noteworthy results:\nIn this study, the authors investigated the causal relationship between lipid dyshomeostasis and\nneuroinflammation in Alzheimer’s disease, due to their previous extensive work establishing that the\nloss of sulfatide results in neuroinflammation, cognitive impairment, and lipid disruption that is\nreminiscent of Alzheimer’s disease pathology. The goal of the study was to delve into the specific\nneuroinflammatory mechanisms mediating lipid dyshomeostasis in Alzheimer’s disease,\nspecifically via the examination of astrocytic and microglia-mediated pathways. The noteworthy\nresults include the identification of Trem2 as an essential mediator of only microglia mediated\nneuroinflammation induced by lipid loss, and importantly that limiting of this Trem2- mediated\ninflammation did not have a significant impact on lipid dyshomeostasis induced by sulfatide loss. As\nsuch, the authors concluded that activated astrocytic contribution to lipid homeostasis may be a\nrelevant avenue to slow Alzheimer’s disease progression.\nResponse: We appreciate the reviewer for summarizing the major findings of our study. We believe\nthese results facilitates an in-depth understanding of the intricate relationship among microglia\nactivation, astrogliosis, and lipidome dysregulation in the background of AD, which provide\nclarification of disease mechanism and point out potential therapeutic targeting strategies for future\nAD dementia prevention/treatment. Please find our responses to comments regarding specific\naspects of the study below.\n2. Comments regarding significance and originality:\nThe Han group (authors) were the earliest to establish the negative correlation between sulfatide\nlevels in the white/grey matter and Alzheimer’s disease. Recently, a paper was published in Cell\n(Zimmer, V. C., Lauer, A. A., Haupenthal, V., Stahlmann, C. P., Mett, J., Grösgen, S., ... & Grimm, M.\nO. W. (2024). A bidirectional link between sulfatide and Alzheimer’s disease. Cell Chemical Biology,\n31(2), 265-283.) which investigated sulfatide deficiency in Alzheimer’s disease specifically through\nthe lens of APP processing. However, overall studies investigating the contribution of sulfatide\ndeficiency to Alzheimer’s disease are lacking. Here, the authors’ investigation of the link between\nsulfatide deficiency and glial response (particularly in relation to microglial Trem2, which is known\nto have a critical role in mediating beneficial microglial engulfment of lipid debris in AD) takes a\nnovel approach. Additionally, the incorporation of the spinal cord in this study is highly novel, as the\nlipidomics of the spinal cord during AD pathology is relatively unstudied.\nResponse: We appreciate the reviewer’s recognition of the novelty and significance of this study.\nWhile glial activation and lipid alterations are commonly observed in AD, the precise contributions\nof each factor to disease progression remain unclear. Our group was the first to identify sulfatide loss\nas an early event in AD and to demonstrate that sulfatide deficiency is a critical trigger for\nneuroinflammation and cognitive impairment (Han X., et al., 2002, J Neurochem. doi:\n10.1046/j.1471-4159.2002.00997.x; Qiu S., et al., 2021, Mol Neurodegener. doi: 10.1186/s13024-\n021-00488-7). These findings have since been supported by other studies, as noted by the reviewer.\nIn this study, we further dissected the relationship between sulfatide deficiency, CNS lipidome\ndisruption, and glial responses. By integrating novel investigations from both cerebrum and spinal\ncord tissue, our findings reveal mechanisms of neuroinflammation and lipid dyshomeostasis that are\nrelevant across multiple CNS tissue types in AD. These insights provide a broader understanding of\nAD pathogenesis and highlight potential avenues for therapeutic intervention.\n3. Comments on if the work supports the conclusions:\nThe authors’ work strongly supports the conclusions made, and the molecular output and analysis\nis strong. Notably, the authors identified and suggested Trem2-independent microglial enriched\ngenes that may be targeted to further parse out the mechanisms of microgliosis in Alzheimer’s\ndisease.\nResponse: We appreciate the reviewer's recognition of our work's support for the conclusions. We\nalso agree that our study highlights a crucial direction for future research, emphasizing the role of\nTrem2-independent microglial actions in AD.\n4. Comments on aspects of the study that need improvement:\nThere are three major opportunities missed in this work.\n1) The first is the lack of incorporation of AD mouse models, particularly those that have a tendency\ntowards worsened female phenotype (as the authors mention occurs with sulfatide deficiency). The\nauthors previously found that specific deletion of CNS sulfatide in Plp1+ myelinated tracts in the\nadult brain resulted in the increase in AD risk genes and showed that this induced cognitive\nbehavioral changes. As such, the authors could have followed up on this work in this paper and\ninvestigated any AD-related upstream regulators of the Trem2 microglial-specific phenotype in\nsulfatide loss.\nResponse: We thank the reviewer for raising this point. We believe the incorporation of AD mouse\nmodels in our studies can be demonstrated through the following two key aspects:\na) The marked decline of sulfatide lipids has been validated in multiple AD mouse models by us\nand others previously. These include: ApoE knockout and ApoE isoform mouse models (Han\nX., et al., 2003, J Biol Chem. doi: 10.1074/jbc.M212340200); Amyloid precursor protein\ntransgenic models (Cheng H., et al., 2008, Neurobiol Aging. doi:\n10.1016/j.neurobiolaging.2008.07.020); and the APP and PSEN1 transgene model 5xFAD\n(Hong J., et al., 2016, J Lipid Res. doi: 10.1194/jlr.M057869).\nb) In addition to AD mouse models which mainly resemble familial AD cases, the mechanisms\nof the late-on set AD remain largely elusive. Notably, disruptions of lipid homeostasis and\nloss of sulfatide is the shared features between both early- and late-onset AD, highlighting\nthe importance of investigating lipid dysregulation in disease progression. In this regard, our\nrecent studies have demonstrated the critical role of sulfatide loss in driving AD-like\nphenotypes on layers of transcriptome, glial response, lipidome alterations, as well as\nbehavioral changes (Qiu S., et al., 2021, Mol Neurodegener. doi: 10.1186/s13024-\n021-00488-7). These findings have established the sulfatide-deficient mouse as a novel AD model well-\nsuited for studying disease pathogenesis.\nThe current study leverages the sulfatide-deficient AD mouse model to explore the interplay\nbetween neuroinflammation and lipid disruptions. The findings are highly relevant to AD and\ncontribute to a broader understanding of the disease’s underlying mechanisms. Thus, we\nbelieve that we have provided sufficient evidence to integrate AD models in our study.\nWe also appreciate the reviewer for the comment regarding investigating “AD-related upstream\nregulators of the Trem2 microglia-specific phenotype in sulfatide loss”. Lipids have been identified\nas Trem2 ligands, particularly, Trem2 senses and binds to a broad array of lipids which are often\nexposed during neuronal and glial cell death, this activates the Trem2 signaling and further facilitates\nmicroglial response in AD (Wang Y., et al., 2015, Cell. doi: 10.1016/j.cell.2015.01.049). We have\nevaluated the alterations of these lipids in the current study with notable changes in Trem2-\nrecognizeable species such as sulfatide and PA (revised manuscript Fig. 4a). Additionally, ApoE is\nalso widely recognized as an up-stream regulator of Trem2 (Atagi Y., et al., 2015, J Biol Chem. doi:\n10.1074/jbc.M115.679043). Based on our result, ApoE protein levels were significantly elevated\nupon the loss of sulfatide, and this induction was also apparent in the absence of Trem2 (revised\nmanuscript Figs. 3f, 3g). This suggest that the elevated ApoE could potentially act through other cell\nsurface receptors (such as low-density lipoprotein receptor (LDLR) and LDLR-related protein 1\n(LRP1)) to mediate Trem2-independent microglial response. We have included relevant discussion\nabout this point in the updated manuscript (revised manuscript lines 426~428).\n2) The second missed opportunity is the generic study of the cerebrum, without specific separation\ninto grey and white matter regions, which can significantly alter the transcriptomic results, and the\ninterpretation related to region-specific Alzheimer’s disease pathology.\nResponse: We appreciate the reviewer for this suggestion. We agree that further evidence including\nbrain region-specific characterizations would provide valuable and specific information regarding AD\nmechanism. Since we observed discrepancies between protein levels and mRNA levels of gliosis in\nthis study (Figs. 2 and 3), and considering the fact that protein levels reflect the real-time status of\nfunctional activities in the brain, we have performed additional immunofluorescence staining to\nevaluate protein levels, aiming to further demonstrate the brain regional events induced by sulfatide\ndeficiency under both Trem2 present and absent situations:\na) As shown in revised manuscript Figs. 2g-2j and Fig. S2, ST-loss in the presence of Trem2\nsignificantly elevated the microgliosis markers including Axl, Iba1, and CD68 in areas\nincluding hippocampus as well as cortex, with particular strong stimulation in white matter-\nenriched areas, such as the corpus callosum (CC) and fimbria. Consistent with bulk protein\nlevels detected by western-blots (revised manuscript Figs. 2b-2f), Trem2 KO only abolished\nthe induction of DAM 2 marker Axl, but had limited impact on the activation of Iba1 and CD68\nin all brain regions of interest (hippocampus, cortex and CC).\nb) Regarding astrogliosis, as shown in revised manuscript Figs. 3k-3n, and Fig. S5, ST loss\ninduced a wide range of astrocyte activation reflected by the elevated Gfap, Serpina3N, and\nVimentin levels in areas including hippocampus, cortex, thalamus, with particular strong\nstimulation in white matter-enriched CC area. Consistent with bulk protein levels detected\nby western-blots (revised manuscript Figs. 3f-3j), astrocytic markers remain activated\ndespite the absence of Trem2.\nWe believe these additional data further supported the conclusion that ST deficiency induces\nastrogliosis and stage 1 associated microglial activation independent of Trem2. It also emphasizes\nthe essential role of white matter-enriched area in AD development.\n3) The third is the lack of behavior analysis for a measure of functional output, despite the authors\npreviously showing that the specific loss of sulfatide in myelin sheaths leads to cognitive impairment.\nAlthough the authors find that Trem2-mediated neuroinflammation is independent of alterations in\nmyelin lipids, it is still worth investigating whether this results in cognitive impairment.\nDue to these points, the paper requires more critical validation in AD-specific contexts.\nResponse: We thank the reviewer for this comment. We agree with the reviewer that an evaluation\non cognitive behavior is essential to serve as a final readout of the mechanistic changes happed in\nthe current study. Actually, we did perform Morris Water Maze (MWM) and Barnes Maze behavioral\nevaluations during the course of this study, we had originally planned to report the behavior dataset\nseparately considering that the current manuscript heavily focuses on mechanistic interactions\nbetween glia and lipidomes. However, we also recognize that combining the behavior result in the\ncurrent manuscript will greatly strengthen the conclusions made and provide a more comprehensive\nview in terms of the contribution of Trem2-related microglial activity in AD. Thus, we have now\nincluded these behavioral data in the updated manuscript (revised manuscript Figs. 6a-6f and Fig.\nS8). In brief, we have detected significant impairment of cognitive function in Cre+ mice compared\nto Cre- regardless of the presence of Trem2, with more severe phenotype observed in females\ncompared to males. This suggest that ST deficiency induces cognitive decline via Trem2-independent\nmanner. We have included the description and interpretation in the updated manuscript (revised\nmanuscript lines 322~347).\n5. Comments on soundness of methodology:\nThis study incorporated limited CNS regions for analysis, which is a weakness in the methodology.\nThe methodology would be more robust if the cerebrum was dissected into specific AD-relevant\nregions with sulfatide loss, such as the hippocampus, cortex, and white matter tracts. Further, there\nis no validation showing the efficiency of conditional Plp1-CreERT and Trem2 knockout via tamoxifen\nadministration, which would greatly affect the results if the knockout was minimal (i.e., the degree of\nneuroinflammation could be less with a less efficient sulfatide knockout, and this could impact the\nfinding that astrogliosis was not seen at the protein level). Further, mice were tamoxifen-induced for\nPlp1 and Trem2 knockout at three months of age but examined at 12 and 17-18 months of age,\nwithout much explanation for why these timepoints were chosen. It is well known in the literature\nthat myelin lipids degrade naturally with age, which is a significant coinciding factor that would\nsignificantly skew the results towards greater sulfatide deficiency and neuroinflammation with\nthe aging process.\nHowever, the gene expression analysis, lipidomics, and western blotting performed in this study, as\nwell as the incorporation of both male and female mice and analysis of sex differences is of sound\nmethodology. The gene expression analyses are analyzed elegantly and in detail.\nResponse: We thank the reviewer for the positive comments and additional suggestions regarding\nthe methodology. In the revised version of our manuscript, we have made the following efforts to\nimprove the soundness of methodology:\na) To expand the CNS regions for analysis, we have further performed immunofluorescence\nstaining to evaluate the glial activation status of different brain regions, this allowed us to\nachieve more specific observations in hippocampus, cortex, as well as white matter tracks\nPlease refer to our response above to Reviewer #1’s comment #4-questions #2) for details.\nb) We thank the reviewer for bringing up the consideration for knockout efficiency, we strongly\nagree that this is important for interpreting the subsequent glial-related observations.\nFor validating the conditional Plp1-CreERT efficiency, we use sulfatide level as a readout, this\nis based on the fact that sulfatide is a class of myelin-specific lipids, the changes of sulfatide\nlevels directly reflects the efficiency of Gal3st1 gene depletion in oligodendrocytes mediated\nby Plp1-CreERT. Based on our lipidomic results, significant losses of sulfatide were detected\nin female cerebrum (revised Fig. 4b), female spinal cord (revised Fig. S6a), and male spinal\ncord (revised Fig. S6f), respectively. The extent of sulfatide change has reached ~50% loss in\nthe cerebrum, and 20-25% loss in the spinal cord (note that the spinal cord tissue is more\nenriched in myelin than the cerebrum, thus has a higher basal level of sulfatide, a loss of 20-\n25% is considered substantial given the absolute quantity of loss).\nFor validating the Trem2 whole-body knockout, we evaluated the mRNA levels of Trem2 from\nthe NanoString datasets and have confirmed that Trem2 expression was completely\nabolished in all groups with Trem2 -/- genotype (revised Fig. S3F for female spinal cord tissue,\nand panels below for male and female cerebrum tissues, respectively).\nWe have added description regarding the knockout efficiency in the updated manuscript\n(revised manuscript lines 229~230, and 281~282).\nc) We thank the reviewer’s comment regarding the choice of age point. We’ve chosen to induce\nsulfatide deficiency at age of 3 months based on the consideration that myelin development\nis mostly complete at this age, this ensures that the loss of sulfatide happens in adult stage,\nmimicking the sulfatide changes in human. The effectiveness of tamoxifen induction has\nbeen demonstrated in our previous study using the same mouse model (Qiu S., et al., 2021,\nMol Neurodegener. doi: 10.1186/s13024-021-00488-7). In the current study, we performed\ncognitive behavioral evaluations at age of 15 months, and evaluated water retention at age of\n12 and 17 months. These time points were determined based on our previous study using the\nsame mouse model, which showed that sulfatide-deficient mouse displays impaired spatial\nmemory function at age of 13-14 months (Qiu S., et al., 2021, Mol Neurodegener. doi:\n10.1186/s13024-021-00488-7), and exhibits bladder water retention at age of 17 months (He\nS., et al., 2023, Clin Transl Med. doi: 10.1002/ctm2.1332). We have now added description\nregarding the choice of age points in the revised paper (revised manuscript lines 312~314).\n6. Comments on details provided for reproductivity:\nYes, there is sufficient detail provided in the methods for the work to be reproduced.\nResponse: We appreciate the reviewer's recognition on our clarity of details provided for\nreproductivity.\n7. Other minor comments:\n1) The statement on lines 78-79 should have a reference.\nResponse: We thank the reviewer for this suggestion and have added relevant references to support\nthis statement (revised manuscript line 75).\n2) Lines 427 and 728 should be contiguous.\nResponse: We thank the reviewer for pointing this out and have made adjustment accordingly\n(revised manuscript line 481).\n3) The protein band sizes in Fig. 3F are not listed.\nResponse: We appreciate the reviewer for this point, we have now organized all raw data of western-\nblot panels (including Fig. 2b and 3f) which include markers to indicate each band size into a\nsupplementary file. We have submitted this file named “Source data” along with the current revision.\n4) Full western blots should be in the supplementary file.\nResponse: We thank the reviewer for this comment, please see our answer to the last question.\n5) Normalized and filtered dataset of transcriptomic and lipidomics should be provided as\nsupplementary tables.\nResponse: We thank the reviewer for this comment. As suggested, we have now organized the\nNormalized and filtered dataset for both transcriptomic and lipidomics as a supplementary file and\nhave submitted this file named “Supplementary Data 1” along with the current revision.\nReviewer 2:\n1. General comments:\nIn the present manuscript the authors use a ST-deficient mouse model with constitutive Trem2\nknockout to study the impact of Trem2 in regulating ST deficiency-induced microglia-mediated\nneuroinflammation, astrocyte activation and lipid dishomeostasis.\nResponse: We thank the reviewer for this summary. Our responses to specific comments are below.\n2. Major comments:\n1. The authors clearly show that in Trem2 KO animals there is the prevention of ST deficiency-induced\nmicroglia-mediated neuroinflammation, but no effect on the induced astrogliosis and lipid\ndyshomeostasis. However, further studies are needed to conclude what authors conclude from their\nfindings, that “preserving lipidome and astrocyte balance may be crucial in decelerating the\nprogression of AD”, since no behavioral data from these animals is provided.\nResponse: We appreciate the reviewer for this comment. As suggested, we have now included\nbehavioral data which further support the important role of lipidome and astrocyte homeostasis in\ndecelerating AD development. Please refer to our response to Reviewer #1’s comment #4-questions\n#3) for further details.\n2. The present study follows previous studies from the same group where they showed that ST\ndeficiency in the adult CND elicits an AD-like neuroinflammation and mild cognitive impairment.\nHere the authors by using NanoString multiplex gene expression analysis, mass spectrometric\nanalysis of lipids, western blot data and qMRI for free water retention, described that: 1) Trem2 KO\nST-deficient mice show a less neuroinflammatory gene expression, namely in females, rather than\nmales and mostly in SC rather than CRM; 2) Trem2 is necessary for activation of microglia-related\ninflammatory and immune pathways in ST-deficiency animals, once again in Sc female samples; 3)\nTrem2 KO is not sufficient to reduce ST-deficiency associated astrocyte activation, myelin lipid\nalterations or female water retention. However, with the present data the authors are not able to\nclarify other mechanisms, rather than Trem2, that may justify the ST-deficiency associated\nastrocyte activation, myelin lipid alterations or female water retention.\nResponse: We thank the reviewer for summarizing our findings and the comment regarding the\nmechanism. A major biological question we try to address in the current study is that, among the\nmultiple cellular/pathological features that are commonly detected in AD (microgliosis, astrogliosis,\nlipidome disruption), what are the most essential aspects that contribute to the disease, and\nwhether Trem2, a critical regulator of microglial response, is essential during this process. Our\nfindings demonstrated that ST deficiency-associated astrocyte activation, myelin lipid alterations,\nwater retention, and cognitive impairment are independent of Trem2. This also suggests that\nmechanisms other than Trem2, such as astrogliosis, lipid alterations, or Trem2-independent\nmicroglial responses may be important contributors for AD-like phenotypes induced by ST deficiency.\nNotably, a recent study from Dr. David Holtzman’s group, which demonstrated that tau-mediated\nneurodegeneration in an ApoE4 background is mediated through a Trem2-independent mechanism\n(Gratuze M., et al., 2023, Neuron. doi: 10.1016/j.neuron.2022.10.022), has reached a consistent\nconclusion with our findings in the current study. Indeed, we have reached out to Dr. Holtzman and\nhad a thorough discussion regarding what could be the mechanism responsible for disease\nprogression rather than Trem2. A hint provided by Dr. Holtzman’s study is the persistent up-regulation\nof microglial lysosomal markers despite the absence of Trem2. Interestingly, we have also observed\npersistent induction of lysosomal marker CD68 under ST-deficiency in the absence of Trem2 (revised\nmanuscript Figs. 2b, 2e), suggesting that Trem2-independent microglial response involving\nlysosomal activation could be one of the mechanisms leading to ST loss-related AD deficits.\nAlthough further studies are needed to fully elucidate the extent of involvement of each factor, we\nbelieve the current study provides a strong foundation for advancing our understanding of Trem2-\ndependent and independent microgliosis, its crosstalk with other disease features, as well as the\npotential roles of astrogliosis and lipidome disruption in AD progression. We have included relevant\ndiscussion regarding this point in the revised manuscript (revised manuscript lines 391~395).\n3. The results should be further confirmed by immunohistochemical data from the different brain\nregions analysed (spinal cord and cerebrum), that could improve the distinct observations\nconcerning microgliosis and astrogliosis, or even lipid homeostasis, rather than the bulk information\ngive by the present methodology used. Also, a specific analysis of isolated cell types (i.e. microglia\nor astrocytes) and a scRNaseq could better identify the distinct populations of each of these DAM or\nDAA. Moreover, namely within the cerebrum the analysis of distinct regions mostly associated with\nAD, namely the hippocampus, as previously addressed in the authors previous publication (DOI:\n10.1186/s13024-021-00488-7), can markedly improve the understanding of the similarities between\nAD-like and ST deficiency pathogenesis. Furthermore, potential changes at animal behavioral tests\ncould corroborate whether the effect of Trem2 KO on microglia neuroinflammation could have an\nimpact on ST-deficient mice outcomes.\nResponse: We thank the reviewer for the suggestions. We fully agree with these considerations and\nhave made the following efforts to address these aspects:\na) To gain further insights on the glial activities in different brain regions, we have performed\nimmunofluorescence staining of both microgliosis (Axl, Iba1, Cd68) and astrogliosis (Gfap,\nSerpina3n, Vimentin) markers. ST-deficiency has induced all makers in multiple brain regions\nincluding hippocampus, cortex, with particularly strong effects on white matter-enriched\nregion such as corpus callosum (revised manuscript Fig. 2g and Fig. S2). Except for Axl, all of\nthese elevations remained when Trem2 was knocked out (revised manuscript Figs. 2g-2j, 3k-\n3n, Fig. S2 and Fig. S5). This suggests that ST loss-induced astrogliosis and DAM1 microglial\nresponse are independent of Trem2. For further details, please refer to our response above\nto Reviewer #1’s comment #4-questions #2).\nb) For cell type-specific characterization, we initially considered scRNAseq, however, during\nthe course of this study, we noticed that the actual protein levels of DAM and DAA could not\nbe accurately reflected by transcriptomic measurements such as scRNAseq. For instance,\nalthough NanoString has detected no induction of astrogliosis markers (Vim, Gfap, Serpina3n)\nin Trem2 KO/Cre+ vs. Trem2/Cre- on mRNA level (revised manuscript Fig. 3a), the protein\nlevels of these markers were actually elevated (revised manuscript Fig. 3f-3j). Thus, we\nbelieve that immunofluorescence staining is more suitable for evaluating DAA and DAM in\nthis case, as it provides protein level information, cell type identification, as well as spatial\nlocalization. The relevant results have been discussed above in our point a).\nc) We thank the reviewer for the comment regarding behavioral study, we have now included\nbehavioral data of Morris Water Maze test and Barnes Maze test (revised manuscript Fig. 6\nand Fig. S8). Briefly, we have detected significant impairment of cognitive function in Cre+\nmice compared to Cre- regardless of the presence of Trem2, with more severe phenotype\nobserved in females compared to males. For further details, please refer to our response\nabove to Reviewer #1’s comment #4-questions #3). We have also included relevant\ndescription in revised manuscript lines 322~347.\nReviewer 3:\n1. General comments:\nThe manuscript by Mittra et al. investigated how deficiency of ST regulates in microglia-mediated\nneuroinflammation, astrocyte activation and lipid disruption in the presence and absence of TREM2.\nThey provided evidence that microglial-enriched transcriptional changes caused by conditional\ndeletion of CST in PLP1-cre model quantified by nanostring can be ameliorated by TREM2 deletion,\nin a female-biased manner. However, astrogliosis and myelin lipid disruption caused by ST deficiency\nnot significantly impacted by TREM2 deletion. The study is straightforwardly designed, and the\nresults are intriguing. The data quality is good and the conclusions are solid. The main weakness is\nthe lack of mechanism dissection, and the use of nanostring instead of single nuclei or single cell\nanalyses greatly limited the interpretation of the cell type-specificity. Some of the conclusions need\nadditional evidence such as immunostaining or RNAscope.\nResponse: We appreciate the reviewer for the positive comments regarding our study design and\ndata quality, we also thank the reviewer for providing comments about aspects to be improved. In\nresponse, we have performed further analysis and experiments to address these weaknesses:\na) In our effort to dissect the mechanism, we conducted a comparative evaluation and\ndiscussed our findings with Dr. David Holzman, whose group recently performed a Trem2-\nrelated study in the background of taupathy (Gratuze M., et al., 2023, Neuron. doi:\n10.1016/j.neuron.2022.10.022). Interestingly, both of our studies have suggested that AD\nprogresses independent of Trem2. One of the common observations we noticed was the\nactivation of lysosome marker, this suggests that Trem2-independent microgliosis involving\nlysosomal response could be one of the mechanisms involved in ST loss-induced disease\nphenotype. Additionally, as both astrogliosis and lipidome disruption were largely\nindependent of Trem2, we believe these factors may also have significant impacts on\npromoting AD. For more details, please refer to our response in Reviewer #2’s major\ncomment #2.\nb) In addition to Nanostring, we have previously considered using RNA scope or snRNAseq to\nevaluate transcriptional changes. However, our findings indicated that mRNA levels did not\nfully recapitulate the final protein levels (revised manuscript Fig. 2a-2f, and 3a-3j). As\nproteins represent the actual functional molecules in the samples, thus we have used\nimmunofluorescence staining instead of snRNAseq, given that it provides protein level\ninformation, cell type identification, as well as spatial localization. Please refer to our\nresponse in Reviewer #2’s major comment #3 for more information.\n2. Major comments:\n1. Figure 1: Key findings need to be validated using either IHC or RNAscope, with co-immunostaining\nof cell type-specific markers for microglia.\nResponse: We appreciate the reviewer for this suggestion. We have performed immunofluorescence\nstaining to address this concern. Our findings showed that ST deficiency induces microgliosis and\nastrogliosis in multiple brain regions including hippocampus, cortex, with particularly strong effects\non white matter-enriched regions such as corpus callosum. Further, ST loss-induced astrogliosis and\nDAM1 microglial response are independent of Trem2. Please refer to Reviewer #1’s comment #4-\nquestions #2) for further details.\n2. Figure 2: Panel A, the microglial genes are not accurate, since some of the genes are expressed on\nother cell types, such as Apoe. Similar to Figure 1, validation is needed for the conclusions to be\nmade.\nResponse: We thank the reviewer for pointing this out. We agree that some genes in the panel are\nnot exclusively expressed in microglia. To be more precise, we have modified “microglial genes” into\n“microglia-enriched genes”, see revised manuscript line 196. And have excluded Apoe gene from this\nlist.\n3. Figure 3: Similarly, the western blot does not differentiate the cell type-specific changes, hence\nthe conclusion that astrocytic alteration at the protein levels cannot be concluded. IHC is needed.\nResponse: We thank the reviewer for this comment. We have now performed co-staining of these\nmarkers with astrocytic marker GFAP to confirm the specific activation of Vimentin and Serpina3n in\nastrocytes (revised manuscript Fig. 3k-3n, and Fig. S5). Please refer to Reviewer #1’s comment #4-\nquestions #2) for further details.\n4. Quantification of myelin lipids was performed in the CRM, while most of the transcriptomic studies\nwere performed in spinal cord. It would be helpful to have the transcriptomics studies also\nperformed in the CRM, or vice versa.\nResponse: We thank the reviewer for this comment, we actually have performed transcriptomics\nstudies in CRM of both sexes (revised manuscript Figs. S1, S3, and S4), and female SC (revised\nmanuscript Fig. 1). For lipidomics study, we have provided data in SC of both sexes (revised\nmanuscript Figs. 4a and S6e), and female CRM (revised manuscript Fig. 4h). For the purpose of\nclearer presentation of these data, we have now labeled on top of each figure panel regarding the\nregion and sex of evaluation (see all revised figure panel titles).\n5. The difference between male and females are intriguing. It is important to label the sex of the\nexperiments in the panels, and state more explicitly in the abstract and conclusions. A more\npointed discussion on this point would be helpful.\nResponse: We appreciate the reviewer for this suggestion. We have now included labels of sex\ninformation in each experimental panel (see all revised figure panel titles). We also have included a\nsummary and more in-depth discussion regarding the difference between sexes in both abstract\n(revised manuscript lines 45~46) and discussion section (revised manuscript lines 437~447).\nWe hope that we have sufficiently addressed the comments raised by the editor and reviewers in\nthe revision. Hope the revised manuscript can be acceptable for publication in Nature\nCommunications. Thank you again for your time and effort in evaluating our manuscript."}, {"round": 2, "reviewer_comments": {"reviewer_3": "The authors have performed additional experiments and analyses, and significantly improved the manuscript. The revised manuscript adequately addressed this reviewer's concerns.", "reviewer_4": "Review on manuscript after first round of corrections:\nMost of the comments have been addressed satisfactorily, but the IHC images are not good quality (overexposed, cannot tell individual cell morphologies) and there are no low-magnification images to prove which region is which, and exactly where in that region the cells were observed. For example, where in the corpus callosum were the images taken? Was there a difference between the rostral and caudal brain? Same with the cortex- where were the ROI's, and why were those regions chosen? Which layer did you look at? The myelin density and thereby sulfatide content is very different in different layers and regions of the cortex, so this would be important to look at.\nNext, why was Trem2 knockdown in Plp+ cells not used as a readout of Cre knockdown efficiency? The argument that sulfatide levels can be used as the primary readout is weak, as this is an indirect mechanism that is too far removed from the Plp1-CreERT promoter to serve as sufficient proof of knockdown.\nOther than these two comments which should be addressed, the authors did good work in modifying the manuscript in a short amount of time."}, "author_response": "Manuscript number: NCOMMS-24-54793A\nTitle: Sulfatide deficiency-induced astrogliosis and myelin lipid dyshomeostasis are independent of Trem2-\nmediated microglial activation\nA Point-to-point Response to Reviewers’ comments\nDear reviewers:\nThank you for your time and effort to carefully examine our manuscript. We are grateful for your\nconstructive comments and suggestions. Accordingly, we would like to provide additional information in\nthe updated manuscript (indicated in orange color), as well as a detailed response.\nPlease find the point-to-point responses listed below to individual comment (please note that line and\nfigure numbers mentioned in the responses below are in concordance with the revised version of\nmanuscript):\nReviewer 3:\n1. The authors have performed additional experiments and analyses and significantly improved the\nmanuscript. The revised manuscript adequately addressed this reviewer's concerns.\nResponse: We appreciate the reviewer for reviewing the revised manuscript and your comments. We are\nglad to hear that the additional experiments and analyses have addressed your concerns. Thank you for\nyour constructive feedback, which helped us improve the quality and clarity of our work.\nReviewer 4:\n1. Most of the comments have been addressed satisfactorily, but the IHC images are not good quality\n(overexposed, cannot tell individual cell morphologies) and there are no low-magnification images to prove\nwhich region is which, and exactly where in that region the cells were observed. For example, where in the\ncorpus callosum were the images taken? Was there a difference between the rostral and caudal brain? Same with the cortex- where were the ROI's, and why were those regions chosen? Which layer did you look\nat? The myelin density and there by sulfatide content is very different in different layers and regions of the\ncortex, so this would be important to look.\nResponse: We appreciate the reviewer for raising this point, and we agree that further details regarding the\nlocation of the IHC images will help with data interpretation and strengthen our conclusion. To address\nthese questions, we have made the following efforts:\n1. We have now updated the IHC images with appropriate exposure as well as magnification to more\nclearly show cell morphologies (revised manuscript Figs. 2g, 3k, 3m, 3o). We have also included\nlow-magnification images with labeled brain regions to indicate the locations where the regions of\ninterest (ROIs) were captured (revised manuscript Figs. 3k, 3m, 3o, and supplementary Fig. 2a-2c).\n2. We’ve included descriptions regarding the rationale for our choices of ROIs (revised manuscript\nlines 208~219). Briefly, we evaluated the microgliosis and astrogliosis of the following areas:\na. Corpus callosum (CC). This area was chosen based on its role in memory processing by\nintegrating information between the left and right hemispheres, and its high myelin content\n(which is particularly relevant because sulfatide is concentrated in the myelin sheaths,\ntherefore changes in CC are most likely to reflect the impact of sulfatide loss on gliosis).\nBy evaluating multiple sequential coronal sections, we detected consistent elevation of gliosis\nmarkers (e.g., Iba1, Gfap, Serpina3n, Vimentin) induced by sulfatide loss throughout the caudal\nand rostral of CC, on a given coronal section, we also observed consistent gliosis pattern\nthroughout the lateral to medial of CC (revised manuscript Figs. 3k, 3m, 3o, and supplementary\nFigs. 2 and 5). For representation purpose, we showed images captured close to medial-CC\nabove the hippocampus area (revised manuscript Figs. 2g, 3k, 3m, 3o).\nb. Layer V~VI of the retrosplenial cortex (RSP) area. The RSP area was chosen based on its crucial\nroles in various cognitive functions, including memory, spatial navigation, and emotion (Li Y., et\nal., 2025, Commun Biol., doi: 10.1038/s42003-025-07463-8). We selected ROIs from layer V~VI\nbecause it is more myelin-enriched compared to other cortex layers, as shown in panel (A)\nbelow (Tomassy GS., et al., 2014, Science, doi: 10.1126/science.1249766).\nTo validate the myelin distribution of the brain in our sample, we performed a staining of myelin\nmarker MBP (Myelin basic protein) and referenced it to the brain regions denoted by “Allen Brain\nAtlas” (in which grey areas indicate white matter enriched brain regions). As shown in panel (B)\nbelow, we detected strong MBP signal in myelin-enriched regions such as corpus callosum\n(CC) and fimbria (Fi.), while relatively lower signal was detected in the hippocampus area. The\ncortex region showed a layer-dependent distribution of MBP, with layer I being the lowest, layer\nII~IV intermediate, and layer V~VI the highest.\nIn the layer V~VI of the RSP region, we also detected enhanced microgliosis in association with\nsulfatide deficiency including the elevated Iba1, CD68 and Axl staining (revised manuscript\nsupplementary Fig. 2a-2c), while the absence of Trem2 has abolished the activation of stage II\nmicroglial marker Axl (revised manuscript supplementary Fig. 2a).\nc. Fimbria (Fi.) area. This myelin-enriched area is connected to hippocampus and is closely linked\nto memory function in multiple aspects (e.g., spatial, sequential, episodic), as reported by\nprevious studies (Dahmani L., et al., 2020, Front Syst Neurosci., doi: 10.3389/fnsys.2019.00087;\nSutherland RJ., et al., 1989, Behav Brain Res., doi: 10.1016/S0166-4328(89)80059-2; Read ML.,\net al., 2023, Eur J Neurosci., doi: 10.1111/ejn.15940; Hoffman LJ., et al., 2022 Cereb Cortex.,\ndoi: 10.1093/cercor/bhac022). We observed gliosis patterns in fimbria similar to that of CC\n(revised manuscript supplementary Fig. 2a-2c).\nd. The hippocampal CA1 (Cornu Ammonis 1) region. The hippocampal formation is vital for\nlearning, memory, and spatial navigation (Anand KS., et al., 2012, Ann Indian Acad Neurol., doi:\n10.4103/0972-2327.104323). Within hippocampus, CA1 neurons are critically involved the\nformation, consolidation, and retrieval of hippocampal-dependent memories (Bartsch T., et al.,\n2011, Proc Natl Acad Sci USA., doi: 10.1073/pnas.1110266108). Despite the myelin content in\nthis region is moderate compared to other ROIs (such as CC and fimbria), we detected clear\nmicrogliosis in the CA1 area after sulfatide loss (revised manuscript supplementary Fig. 2a-2c),\nsuggesting glia within this region remain highly sensitive to local myelin disturbance and may\nalso potentially receive inflammatory signals from other brain regions.\nIn summary, we evaluated multiple brain regions (CC, Fimbria, RSP, and CA1) that are highly relevant to\nmemory function with varied myelin abundance, enhanced gliosis was consistently induced by sulfatide\nloss in these brain regions. The absence of Trem2 diminishes stage II activation of microglia but had limited\nimpact on astrogliosis. We have included relevant description in the discussion section (revised\nmanuscript lines 381~384)\n2. Next, why was Trem2 knockdown in Plp+ cells not used as a readout of Cre knockdown efficiency?\nThe argument that sulfatide levels can be used as the primary readout is weak, as this is an indirect mechanism\nthat is too far removed from the Plp1-CreERT promoter to serve as sufficient proof of knockdown.\nResponse: We thank the reviewer for this comment, please see our reasons below:\n1. We would like to clarify that the Trem2 depletion mouse used in this study is a constitutive whole-\nbody knockout model, thus the knockout was not under the control of Plp-cre. This design is\ndetermined based on the fact that Trem2 is generally specifically expressed in microglia, other than\noligodendrocytes (Plp+ cells). Thus, Trem2 levels was not used to indicate Plp-cre efficiency.\nTo validate the efficiency of Trem2 knockout, we performed qPCR analysis of all mice used in this\nstudy and have confirmed that Trem2 expression was completely abolished in all Trem2 KO mice\n(regardless of Plp-cre- or Plp-cre+ status), as shown below (significance levels of Trem2 KO are\nindicated by *).\nNote that within the Trem2 WT groups, Plp-cre+ significantly induced Trem2 expression (indicated\nby #), which is expected due to the enhanced microgliosis. This also indirectly reflects the\neffectiveness of Plp-cre+ in depleting CST (Gal3st1) gene expression.\n2. To directly address the efficiency of Plp1-CreERT. We used qPCR to quantitatively measure the\nmRNA levels of CST (Gal3st1) gene. Spinal cord tissues were used for this evaluation based on its\nhigh oligodendrocyte content (which is the major cell type for Plp1 expression and should directly\nreflect the Plp1-Cre efficacy). As shown below, mice with Plp1-Cre+ genotype showed significant\nreduction of Gal3st1 gene expression regardless of the presence or absence of Trem2. This\ndemonstrates that the tamoxifen induction of Plp1-CreERT is effective.\nWe have included these qPCR evaluation results (revised manuscript supplementary Figs. 1a-1d) and\nrelevant description (revised manuscript lines 121~122) in the updated manuscript to demonstrate the\nknockout efficiency.\n3. Other than these two comments which should be addressed, the authors did good work in modifying the\nmanuscript in a short amount of time.\nResponse: We sincerely thank the reviewer for the encouraging feedback and appreciation of our efforts in\nrevising the manuscript. We hope the updated version meets the reviewer’s expectations.\nIn summary, we hope that we have sufficiently addressed the comments raised by the reviewers in this\nrevision. We hope the revised manuscript can be acceptable for publication in Nature Communications.\nThank you again for your time and effort in evaluating our manuscript."}, {"round": 3, "reviewer_comments": {"reviewer_4": "In the second revision, the authors addressed all comments satisfactorily. No further revisions are requested."}, "author_response": "Manuscript number: NCOMMS-24-54793B\nTitle: Sulfatide deficiency-induced astrogliosis and myelin lipid dyshomeostasis are independent of\nTREM2-mediated microglial activation\nA Point-to-point Response to Reviewer’s comments\nReviewer 4:\n1. In the second revision, the authors addressed all comments satisfactorily. No further revisions are\nrequested.\nResponse: We appreciate the reviewer for reviewing the revised manuscript and providing this comment.\nWe are glad to hear that we have addressed all comments satisfactorily. Thank you again for your time and\neffort in evaluating our manuscript."}]

Decision letter