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Cited by 255Multiple Sclerosis Research StudiesNeuroinflammation and Neurodegeneration MechanismsSingle-cell and spatial transcriptomics

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Cell-specific and region-specific transcriptomics in the multiple sclerosis model: Focus on astrocytes

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Itoh, Noriko, Itoh, Yuichiro, Tassoni, Alessia, Ren, Emily, Kaito, Max, Ohno, Ai, Ao, Yan, Farkhondeh, Vista, Johnsonbaugh, Hadley, Burda, Josh, Sofroniew, Michael V., Voskuhl, Rhonda R.·2018·Proceedings of the National Academy of Sciences
DOI 10.1073/pnas.1716032115PMC PMC5777065
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Abstract

Significance: Molecular mechanisms underlying distinct disabilities during neurological diseases may differ based on the neurological pathway involved. Multiple sclerosis (MS) is multifocal, characterized by distinct disabilities affecting walking, vision, cognition, and fatigue. Neuroprotective treatments tailored for each disability may be more effective than nonspecific treatments aiming to reduce a composite of disabilities in clinical trials. Here, we use the MS model to apply a cell-specific and region-specific gene expression approach to discover targets in distinct neuroanatomic regions. Altered cholesterol synthesis gene expression in astrocytes in spinal cord and optic nerve was identified as a potential target for walking and visual disabilities, respectively. This disability-specific discovery approach represents a strategy for finding neuroprotective treatments for multifocal neurodegenerative diseases.

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Figures

Fig. 1. Enrichment of astrocyte-specific mRNA using RiboTag technology. (A–D) Double immunolabeling in mGFAP-Cre:RiboTag mice showed colocalization of ribosome associated HA-Tag (red) with the astrocyte-specific marker GFAP (green), with colocalization seen in Merge (yellow). Nuclei were counterstained with DAPI (blue) (40-μm-thick spinal cord sections). B–D are a magnification of Inset area in A. (Scale bars: 100 μm.) (E) Enrichment of astrocyte-specific gene expression and deenrichment of neuronal, oligodendroglial, and microglia-specific gene expression, shown as the log fold change calculated between spinal cord astrocyte RNAs immunoprecipitated by anti-HA antibody versus spinal cord total cell RNAs (including astrocytes) immunoprecipitated with control antibody, anti-RPL22. Asterisks show the significant enrichment or deenrichment (FDR < 0.1). (F) Principal component analysis of astrocyte-enriched RNAs from four different brain regions (Cb, cerebellum; Crx, cerebral cortex; Hippo, hippocampus; and Sc, spinal cord), as well as RNAs from heterogeneous cells from spinal cord (RNA from all cell types). The samples from all cells of spinal cord were markedly separated from astrocyte-enriched spinal cord samples. Within astrocyte-enriched samples, the samples from different brain regions were also separated.
Fig. 1. Enrichment of astrocyte-specific mRNA using RiboTag technology. (A–D) Double immunolabeling in mGFAP-Cre:RiboTag mice showed colocalization of ribosome associated HA-Tag (red) with the astrocyte-specific marker GFAP (green), with colocalization seen in Merge (yellow). Nuclei were counterstained with DAPI (blue) (40-μm-thick spinal cord sections). B–D are a magnification of Inset area in A. (Scale bars: 100 μm.) (E) Enrichment of astrocyte-specific gene expression and deenrichment of neuronal, oligodendroglial, and microglia-specific gene expression, shown as the log fold change calculated between spinal cord astrocyte RNAs immunoprecipitated by anti-HA antibody versus spinal cord total cell RNAs (including astrocytes) immunoprecipitated with control antibody, anti-RPL22. Asterisks show the significant enrichment or deenrichment (FDR < 0.1). (F) Principal component analysis of astrocyte-enriched RNAs from four different brain regions (Cb, cerebellum; Crx, cerebral cortex; Hippo, hippocampus; and Sc, spinal cord), as well as RNAs from heterogeneous cells from spinal cord (RNA from all cell types). The samples from all cells of spinal cord were markedly separated from astrocyte-enriched spinal cord samples. Within astrocyte-enriched samples, the samples from different brain regions were also separated.CC BY-NC-ND · PubMed Central
Fig. 2. Expression of cholesterol synthesis pathway genes is decreased and of immune pathway genes is increased in spinal cord astrocytes during EAE. (A) Top canonical pathways are shown in spinal cord astrocytes (Sc-astr), (B) cerebellar astrocytes (Cb-astr), and (C) spinal cord all cells (Sc-all), each in descending order of significance. Astrocyte-specific RNAs differentially expressed in EAE versus normal (FDR < 0.1) showed that genes involved in several cholesterol synthesis pathways were significantly enriched in spinal cord and cerebellum (Table S1), with gene expression decreased in EAE (Tables S2 and S4). The antigen presentation pathway and IFN signaling pathway were also significantly enriched in both regions (Table S1), with gene expression increased in EAE (Table S4). Several pathways enriched in spinal cord astrocytes (Sc-astr) were not detectable in spinal cord all cells (Sc-all). n = 9 mice (5 EAE, 4 normal). Full names of pathways are listed in Table S1. (D) Heat map of direction of change of genes in pathways. A heat map of cholesterol synthesis and immune pathway genes that were significantly different between EAE and normal (NL) in astrocyte-specific RNAs (FDR < 0.1) revealed that cholesterol synthesis pathway genes were decreased in EAE (green) while immune pathway genes were increased (red). Green color represents lower expression, and red color represents higher expression. n = 9 mice (5 EAE, 4 normal). Significant differences in gene expression in spinal cord astrocytes were confirmed in another independent set of astrocyte RiboTag mice using RNA-seq analysis with n = 10 mice (5 EAE, 5 normal).
Fig. 2. Expression of cholesterol synthesis pathway genes is decreased and of immune pathway genes is increased in spinal cord astrocytes during EAE. (A) Top canonical pathways are shown in spinal cord astrocytes (Sc-astr), (B) cerebellar astrocytes (Cb-astr), and (C) spinal cord all cells (Sc-all), each in descending order of significance. Astrocyte-specific RNAs differentially expressed in EAE versus normal (FDR < 0.1) showed that genes involved in several cholesterol synthesis pathways were significantly enriched in spinal cord and cerebellum (Table S1), with gene expression decreased in EAE (Tables S2 and S4). The antigen presentation pathway and IFN signaling pathway were also significantly enriched in both regions (Table S1), with gene expression increased in EAE (Table S4). Several pathways enriched in spinal cord astrocytes (Sc-astr) were not detectable in spinal cord all cells (Sc-all). n = 9 mice (5 EAE, 4 normal). Full names of pathways are listed in Table S1. (D) Heat map of direction of change of genes in pathways. A heat map of cholesterol synthesis and immune pathway genes that were significantly different between EAE and normal (NL) in astrocyte-specific RNAs (FDR < 0.1) revealed that cholesterol synthesis pathway genes were decreased in EAE (green) while immune pathway genes were increased (red). Green color represents lower expression, and red color represents higher expression. n = 9 mice (5 EAE, 4 normal). Significant differences in gene expression in spinal cord astrocytes were confirmed in another independent set of astrocyte RiboTag mice using RNA-seq analysis with n = 10 mice (5 EAE, 5 normal).CC BY-NC-ND · PubMed Central
Fig. 3. Cholesterol synthesis gene expression in spinal cord astrocytes during EAE. (A) Quantification of cholesterol synthesis gene expression by qPCR using astrocyte-specific RNAs from yet another set of astrocyte RiboTag mice with EAE at day 45 confirmed RNA-seq findings that Hmgcs1, Fdps, and Fdft1 expression were down-regulated in astrocytes during EAE (P = 0.0034 for Hmgcs1, P = 0.014 for Fdps, P = 0.0065 for Fdft1). (B) HMGCS1, FDPS, and FDFT1 were decreased in EAE spinal cord astrocytes of WT mice at the protein level using immunohistochemistry (P = 0.0007 for HMGCS1, P = 0.029 for FDPS, P = 0.0015 for FDFT1). (C) Colocalization of HMGCS1 (red) with GFAP (green) in astrocytes (yellow) in Merge. See Fig. S2 for colocalization of FDPS and FDFT1. Data are representative of two independent experiments. Shown are 10× images of ventral spinal cord white matter, yellow solid line Insets at 40×, and white dashed line Insets at 100×. (Scale bars: 100 μm.) ***P < 0.001, **P < 0.01, *P < 0.05. Four mice were examined for each group. Error bars indicate SEM between mice.
Fig. 3. Cholesterol synthesis gene expression in spinal cord astrocytes during EAE. (A) Quantification of cholesterol synthesis gene expression by qPCR using astrocyte-specific RNAs from yet another set of astrocyte RiboTag mice with EAE at day 45 confirmed RNA-seq findings that Hmgcs1, Fdps, and Fdft1 expression were down-regulated in astrocytes during EAE (P = 0.0034 for Hmgcs1, P = 0.014 for Fdps, P = 0.0065 for Fdft1). (B) HMGCS1, FDPS, and FDFT1 were decreased in EAE spinal cord astrocytes of WT mice at the protein level using immunohistochemistry (P = 0.0007 for HMGCS1, P = 0.029 for FDPS, P = 0.0015 for FDFT1). (C) Colocalization of HMGCS1 (red) with GFAP (green) in astrocytes (yellow) in Merge. See Fig. S2 for colocalization of FDPS and FDFT1. Data are representative of two independent experiments. Shown are 10× images of ventral spinal cord white matter, yellow solid line Insets at 40×, and white dashed line Insets at 100×. (Scale bars: 100 μm.) ***P < 0.001, **P < 0.01, *P < 0.05. Four mice were examined for each group. Error bars indicate SEM between mice.CC BY-NC-ND · PubMed Central
Fig. 4. Cholesterol synthesis gene expression is decreased in optic nerve astrocytes during EAE. (A) Top canonical pathways in EAE optic nerve astrocytes are shown in descending order of significance. Astrocyte-specific RNAs differentially expressed in EAE versus normal (FDR < 0.1) showed that several cholesterol synthesis pathways were different in EAE (Table S3), with gene expression decreased in EAE (Table S4). Genes involved in the antigen presentation pathway and complement system pathway were also significantly different in EAE, but these were in the direction of an increase in EAE (Table S4). (B) A heat map of cholesterol synthesis pathway genes that were significantly different between EAE and normal (NL) in astrocyte-specific RNAs (FDR < 0.1) revealed that cholesterol synthesis pathway genes were decreased in EAE optic nerve (green). Green color represents lower expression and red color represents higher expression. n = 8 mice (4 EAE, 4 normal).
Fig. 4. Cholesterol synthesis gene expression is decreased in optic nerve astrocytes during EAE. (A) Top canonical pathways in EAE optic nerve astrocytes are shown in descending order of significance. Astrocyte-specific RNAs differentially expressed in EAE versus normal (FDR < 0.1) showed that several cholesterol synthesis pathways were different in EAE (Table S3), with gene expression decreased in EAE (Table S4). Genes involved in the antigen presentation pathway and complement system pathway were also significantly different in EAE, but these were in the direction of an increase in EAE (Table S4). (B) A heat map of cholesterol synthesis pathway genes that were significantly different between EAE and normal (NL) in astrocyte-specific RNAs (FDR < 0.1) revealed that cholesterol synthesis pathway genes were decreased in EAE optic nerve (green). Green color represents lower expression and red color represents higher expression. n = 8 mice (4 EAE, 4 normal).CC BY-NC-ND · PubMed Central
Fig. 5. Validation of the decrease in cholesterol synthesis gene expression in optic nerve astrocytes during EAE. (A) Quantification of cholesterol synthesis gene expression by qPCR using astrocyte-specific RNAs from an additional set of astrocyte RiboTag mice with EAE at day 45 confirmed RNA-seq findings that Hmgcs1 and Fdft1 expression was down-regulated in astrocytes during EAE (P = 0.034 for Hmgcs1; P = 0.0017 for Fdft1). (B and C) HMGCS1 and FDFT1 were decreased in EAE optic nerve astrocytes of WT mice at the protein level using immunohistochemistry (P = 0.0378 for HMGCS1; P = 0.0046 for FDFT1). n = 3 to 4 mice in each group. (D) Colocalization of HMGCS1 (red) with GFAP (green) in astrocytes (yellow) in Merge. Shown are 10× images of coronal optic nerve sections. See Fig. S5 for colocalization of FDFT1. Data are representative of two independent experiments. (Scale bars: 10× images, 100 μm; Insets, 50 μm.) **P < 0.01, *P < 0.05. Error bars indicate SEM between mice.
Fig. 5. Validation of the decrease in cholesterol synthesis gene expression in optic nerve astrocytes during EAE. (A) Quantification of cholesterol synthesis gene expression by qPCR using astrocyte-specific RNAs from an additional set of astrocyte RiboTag mice with EAE at day 45 confirmed RNA-seq findings that Hmgcs1 and Fdft1 expression was down-regulated in astrocytes during EAE (P = 0.034 for Hmgcs1; P = 0.0017 for Fdft1). (B and C) HMGCS1 and FDFT1 were decreased in EAE optic nerve astrocytes of WT mice at the protein level using immunohistochemistry (P = 0.0378 for HMGCS1; P = 0.0046 for FDFT1). n = 3 to 4 mice in each group. (D) Colocalization of HMGCS1 (red) with GFAP (green) in astrocytes (yellow) in Merge. Shown are 10× images of coronal optic nerve sections. See Fig. S5 for colocalization of FDFT1. Data are representative of two independent experiments. (Scale bars: 10× images, 100 μm; Insets, 50 μm.) **P < 0.01, *P < 0.05. Error bars indicate SEM between mice.CC BY-NC-ND · PubMed Central
Fig. 6. Treatment targeting cholesterol homeostasis during EAE. EAE (blue) and normal (NL, black) mice were treated with either Vehicle (Veh, solid) or CS-6253 (CS, striped). (A) Quantification of cholesterol synthesis gene expression by qPCR using astrocyte-specific RNAs from CS or vehicle-treated astrocyte RiboTag mice with EAE at day 43 showed that Hmgcs1, Fdps, and Fdft1 expression were up-regulated in astrocytes in CS-treated normal mice (P = 0.0117 for Hmgcs1, P = 0.0324 for Fdps, P = 0.0475 for Fdft1) and in CS-treated EAE mice (P = 0.0059 for Hmgcs1, P = 0.0093 for Fdps, P = 0.005 for Fdft1). n = 5 in each group. (B) HMGCS1, FDPS, and FDFT1 were also increased in CS-treated EAE spinal cord astrocytes in WT mice at the protein level using immunohistochemistry (P = 0.0008 for HMGCS1, P = 0.0002 for FDPS, P = 0.00195 for FDFT1). n = 4 in each group. (C) Colocalization of HMGCS1 (red) with GFAP (green) in astrocytes (yellow) in Merge, showing the CS-mediated increase in HMGCS expression in astrocyte cell bodies and processes. Shown are 10× images of ventral spinal cord white matter, Insets at 40×. (Scale bars: 100 μm.) (D and E) CS treatment resulted in a reduction in clinical EAE severity scores, which assess walking disability (P = 0.005 cumulative disease index after EAE day 30, P = 0.013 all days; CS group n = 6, vehicle group n = 7), and an increase in the number of seconds that mice could stay on the rotarod (P = 0.0077), respectively, each compared with vehicle treatment in EAE. For confirmation, an additional set of EAE mice underwent treatment with CS-6253 or vehicle, again demonstrating improvement in EAE scores (P < 0.0001 cumulative disease index after EAE day 30, P = 0.0006 all days; CS group n = 7, vehicle group n = 9).
Fig. 6. Treatment targeting cholesterol homeostasis during EAE. EAE (blue) and normal (NL, black) mice were treated with either Vehicle (Veh, solid) or CS-6253 (CS, striped). (A) Quantification of cholesterol synthesis gene expression by qPCR using astrocyte-specific RNAs from CS or vehicle-treated astrocyte RiboTag mice with EAE at day 43 showed that Hmgcs1, Fdps, and Fdft1 expression were up-regulated in astrocytes in CS-treated normal mice (P = 0.0117 for Hmgcs1, P = 0.0324 for Fdps, P = 0.0475 for Fdft1) and in CS-treated EAE mice (P = 0.0059 for Hmgcs1, P = 0.0093 for Fdps, P = 0.005 for Fdft1). n = 5 in each group. (B) HMGCS1, FDPS, and FDFT1 were also increased in CS-treated EAE spinal cord astrocytes in WT mice at the protein level using immunohistochemistry (P = 0.0008 for HMGCS1, P = 0.0002 for FDPS, P = 0.00195 for FDFT1). n = 4 in each group. (C) Colocalization of HMGCS1 (red) with GFAP (green) in astrocytes (yellow) in Merge, showing the CS-mediated increase in HMGCS expression in astrocyte cell bodies and processes. Shown are 10× images of ventral spinal cord white matter, Insets at 40×. (Scale bars: 100 μm.) (D and E) CS treatment resulted in a reduction in clinical EAE severity scores, which assess walking disability (P = 0.005 cumulative disease index after EAE day 30, P = 0.013 all days; CS group n = 6, vehicle group n = 7), and an increase in the number of seconds that mice could stay on the rotarod (P = 0.0077), respectively, each compared with vehicle treatment in EAE. For confirmation, an additional set of EAE mice underwent treatment with CS-6253 or vehicle, again demonstrating improvement in EAE scores (P < 0.0001 cumulative disease index after EAE day 30, P = 0.0006 all days; CS group n = 7, vehicle group n = 9).CC BY-NC-ND · PubMed Central
Fig. 7. Cholesterol synthesis gene expression is decreased in optic chiasm in MS. (A) Top canonical pathways in MS optic chiasm tissues. Pathway analysis of optic chiasm from MS and healthy control patients showed that cholesterol synthesis pathways were significantly different. n = 10 human (5 MS, 5 normal). Full names of pathways are listed in Table S3. (B) Heat map of cholesterol synthesis pathway genes that were significantly different between MS and normal (NL) RNAs (FDR < 0.1) revealed that cholesterol synthesis pathway genes were decreased in MS optic chiasm (green). Green color represents lower expression and red color represents higher expression. n = 10 human (5 MS, 5 normal).
Fig. 7. Cholesterol synthesis gene expression is decreased in optic chiasm in MS. (A) Top canonical pathways in MS optic chiasm tissues. Pathway analysis of optic chiasm from MS and healthy control patients showed that cholesterol synthesis pathways were significantly different. n = 10 human (5 MS, 5 normal). Full names of pathways are listed in Table S3. (B) Heat map of cholesterol synthesis pathway genes that were significantly different between MS and normal (NL) RNAs (FDR < 0.1) revealed that cholesterol synthesis pathway genes were decreased in MS optic chiasm (green). Green color represents lower expression and red color represents higher expression. n = 10 human (5 MS, 5 normal).CC BY-NC-ND · PubMed Central
Fig. 8. Hypothetical effect of reduced cholesterol synthesis in astrocytes during EAE. (A) Peripheral cholesterols cannot enter into the CNS due to the blood–brain barrier; thus, cholesterols in the CNS are synthesized de novo. In adults, astrocytes are the main cells producing cholesterols (25), with transport via ATP-binding cassette transporter (ABCA1) to apolipoprotein E (ApoE) to neurons to make membranes and synapses (28, 29), and to oligodendrocytes to make myelin (25, 30). (B) In EAE, there is synaptic loss, axonal damage, and demyelination (71). Here, we hypothesize that less cholesterol synthesis in astrocytes during EAE may lead to reduced cholesterol for transport to neurons and oligodendrocytes, thereby reducing reparative synaptic plasticity and remyelination.
Fig. 8. Hypothetical effect of reduced cholesterol synthesis in astrocytes during EAE. (A) Peripheral cholesterols cannot enter into the CNS due to the blood–brain barrier; thus, cholesterols in the CNS are synthesized de novo. In adults, astrocytes are the main cells producing cholesterols (25), with transport via ATP-binding cassette transporter (ABCA1) to apolipoprotein E (ApoE) to neurons to make membranes and synapses (28, 29), and to oligodendrocytes to make myelin (25, 30). (B) In EAE, there is synaptic loss, axonal damage, and demyelination (71). Here, we hypothesize that less cholesterol synthesis in astrocytes during EAE may lead to reduced cholesterol for transport to neurons and oligodendrocytes, thereby reducing reparative synaptic plasticity and remyelination.CC BY-NC-ND · PubMed Central

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Authors
Itoh, N., Itoh, Y., Tassoni, A., Ren, E., Kaito, M., Ohno, A., Ao, Y., Farkhondeh, V., Johnsonbaugh, H., Burda, J., Sofroniew, M. V., & Voskuhl, R. R.
Affiliations
  • University of California, Los Angeles (US)
  • Department of Neurobiology, University of California, Los Angeles, CA 90095
Corresponding author
Voskuhl, Rhonda R.
ORCID
Venue
Proceedings of the National Academy of Sciences
Publisher
National Academy of Sciences
Published
2017-12-26
Volume
115
Issue
2
Pages
E302-E309
ISSN
  • 0027-8424 (ISSN-L)
  • 1091-6490
DOI
10.1073/pnas.1716032115
PMC
PMC5777065
OpenAlex
W2777702274
Cited by
253
References
71
Citations by year
2026: 10 · 2025: 22 · 2024: 25 · 2023: 38 · 2022: 37 · 2021: 42 · 2020: 40 · 2019: 27 · 2018: 12
FWCI
16.96
Type
journal article
Language
en
Open access
Licence
Locations
  • Proceedings of the National Academy of Sciences (publishedVersion, cc-by-nc-nd)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by-nc-nd)
  • PubMed Central (publishedVersion, cc-by-nc-nd)
  • PubMed (publishedVersion)
  • eScholarship (California Digital Library) (submittedVersion)
  • eScholarship (California Digital Library) (submittedVersion, cc-by-nc-nd)
Funders
  • Conrad N. Hilton Foundation (20150232)
  • Office of Extramural Research, National Institutes of Health (R01NS096748)
  • California Community Foundation (BAPP-15-118094)
Topics
  • Health Sciences › Medicine › Clinical Neurology › Multiple Sclerosis Research Studies
  • Life Sciences › Neuroscience › Neurology › Neuroinflammation and Neurodegeneration Mechanisms
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Single-cell and spatial transcriptomics
Keywords
multiple sclerosis, experimental autoimmune encephalomyelitis, astrocytes, region-specific gene expression, RiboTag, cholesterol biosynthesis, neuroinflammation, spinal cord, optic nerve, optic chiasm, cerebellum, cerebral cortex, hippocampus, multifocality
MeSH terms
Animals, Astrocytes, Cholesterol, Encephalomyelitis, Autoimmune, Experimental, Gene Expression Regulation, Homeostasis, Mice, Inbred C57BL, Multiple Sclerosis, RNA, Messenger, Down-Regulation, Up-Regulation, Mice, Transcriptome

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