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Stem-Cell-Derived Astrocytes Divulge Secrets of Mutant GFAP
Cell Stem Cell
Previews Stem-Cell-Derived Astrocytes Divulge Secrets of Mutant GFAP Michael V. Sofroniew1,* 1Department of Neurobiology, University of California Los Angeles, Los Angeles, CA, USA *Correspondence: [email protected] https://doi.org/10.1016/j.stem.2018.10.020
Gain-of-function mutations in the canonical astrocyte protein, GFAP, cause a fatal neurodevelopmental disorder with myelination abnormalities, seizures, and psychomotor disturbances. Recently, Li et al. (2018) (in Cell Stem Cell) and Jones et al. (2018) (in Cell Reports) have shown that patient iPSC-derived astrocytes with mutant GFAP have disrupted astrocyte functions, revealing disease mechanisms and potential roles of GFAP. The ability to generate tissue-specific cell types from induced pluripotent stem cells (iPSCs) derived from patients with monogenetic disorders has provided powerful new tools with which to unravel disease mechanisms and identify protein functions. This approach is particularly useful in the central nervous system (CNS), where interactions among different complex cell types, neurons, astrocytes, oligodendrocytes, and microglia may contribute to disease mechanisms. Using this approach, two recent studies by Li et al. (2018) in Cell Stem Cell and by Jones et al. (2018) in Cell Reports generated CNS astrocytes from patients with Alexander Disease (AxD) and oligodendrocyte progenitor cells (OPCs) from human controls to probe how glial fibrillary acidic protein (GFAP) with AxD mutations disrupts astrocyte functions and causes the disease. Astrocytes tile the entire CNS and play essential roles in normal CNS function and responses to injury and disease (Sofroniew and Vinters, 2010). They clear and recycle transmitters, maintain extracellular ion and water homeostasis, and are critical for normal neural circuit function. Astrocyte-secreted molecules critically regulate synapse development and contribute to adult synaptic plasticity. In addition, astrocytes respond to all forms of CNS injury or disease with diverse reactive changes that can in different contexts mediate either pro- or anti-inflammatory signaling (Sofroniew, 2015), and they can either exert critical neuroprotective functions or contribute to and exacerbate various CNS pathologies (Sofroniew and Vinters, 2010). Given the wide range of critical astrocyte
activities, the potential contributions of gain or loss of astrocyte functions are under increasing investigation in CNS afflictions ranging from autoimmune inflammatory disorders to neurodegenerative conditions and neuropsychiatric disorders. GFAP was first isolated almost 50 years ago from multiple sclerosis plaques and was found to be associated with reactive astrocytes (Eng et al., 2000). Since then, it has become a canonical astrocyte marker. In healthy CNS, GFAP is expressed at detectable levels by many, but not all, astrocytes, and it is upregulated in a graded fashion in response to all CNS insults, where it can serve as a marker for severity of tissue damage (Sofroniew and Vinters, 2010). GFAP is an intermediate filament with cellular structural roles (Hol and Pekny, 2015), but relatively little is known about other potential functions. Intermediate filaments appear to exhibit considerable functional redundancy, and as a result, GFAP / mice exhibit few detectable abnormalities except a mild effect on long-term maintenance of myelin and a somewhat increased susceptibility to autoimmune inflammatory disease (Liedtke et al., 1998). AxD is rare developmental disorder characterized by megalencephaly, hydrocephalus, psychomotor delays, and white matter inflammation and degeneration. It is caused by autosomal dominant mutations in GFAP (Brenner et al., 2001). Identification of AxD mutations led to the ability make transgenic mice expressing mutant human GFAP. Although these transgenic mice exhibited the AxD hallmarks of GFAP aggregation and forma-
630 Cell Stem Cell 23, November 1, 2018 ª 2018 Elsevier Inc.
tion of Rosenthal fibers, the mice failed to yield the gross neurological abnormalities or white matter pathology seen in human patients (Hagemann et al., 2006). Thus, mouse and other transgenic organism models have not been effective in identifying mechanisms by which mutant GFAP causes the disease, setting the stage for studies using astrocytes derived from AxD-derived iPSCs. The studies by Li et al. and Jones et al. both used astrocytes derived from AxD-iPSCs from multiple patients and compared these with astrocytes from AxD-iPSCs in which the mutant GFAP had been corrected to the wild-type (WT) genotype through CRISPR/Cas9-based gene editing, and in the case of Li et al., the AxD-iPSCs from multiple patients were also compared with astrocytes derived from human control iPSCs. In both studies, AxD-astrocytes exhibited GFAP aggregation and Rosenthal fibers, regarded as AxD hallmarks. Li et al. examined astrocyte-oligodendrocyte interactions to look for clues regarding the white matter pathology (leukodystrophy) characteristic of AxD. They found that AxD astrocytes, but not control or corrected astrocytes, reduced the proliferation of co-cultured OPCs derived from control iPSCs and caused a myelination defect in a 3D nanofiber culture system. Transcriptome analysis indicated that AxD astrocytes exhibited highly upregulated genes involved in immune cell activation, pro-inflammatory cytokine signaling, cell proliferation, and cell adhesion. AxD astrocytes exhibited markedly downregulated genes involved in synapse regulation and ion transport. Li et al. next focused on CHI3L1, a secreted protein
Cell Stem Cell
Previews expressed by astrocytes and previously linked to various neuroinflammatory conditions (Bonneh-Barkay et al., 2012). CHI3L1 was among the top differentially expressed genes by AxD astrocytes, and its high expression was confirmed in brain tissue from AxD patients. Li et al. found that a CHI3L1-neutralizing antibody blocked the inhibitory effect of AxD astrocyte culture media on OPC proliferation, and that shRNA knockdown of CHI3L1 production significantly attenuated the inhibitory effects of AxD astrocytes on OPC proliferation and myelination. Additional experiments implicated CHI3L1 binding to the OPC cell surface receptor CRTH2 in mediating these inhibitory effects. These observations provide a direct potential mechanism by which AxD-astrocytes could precipitate white matter pathology. Jones et al. examined the effects of AxD mutations on astrocyte differentiation, regulation of intracellular organelles, and calcium and ATP signaling. They also first conducted a transcriptional analysis, which in particular, broadly implicated a dysregulation of vesicle regulation and endoplasmic reticulum (ER) functions. Electron microscopic and immunofluorescent analyses confirmed these findings and further revealed that AxD-astrocytes exhibited a swollen and largely non-reticular ER, along with alterations in lysosome size and intracellular distribution. Looking for potential functional consequences of these changes, Jones et al. found that under mechanical stimulation, AxD-astrocytes exhibited an attenuated ability to propagate calcium waves, which are mediated by cell-to-cell signaling via release of ATP. Further experiments
showed that AxD-astrocytes were able to produce and respond to ATP but exhibited significantly and substantially impaired release of ATP. These findings identify critical astrocyte functions that are disrupted by AxD-GFAP mutations and point toward intracellular roles for GFAP in ER and vesicle regulation that may influence astrocyte secretion of ATP and potentially that of other molecules. Overall, the findings by Li et al. and Jones et al. have several implications. First, they identify mechanisms whereby AxD mutations in GFAP can lead to specific astrocyte dysfunctions that could be causally related to AxD neurological symptoms and tissue pathology, which can now be studied further. Second, the findings point toward potential functions of GFAP in regulating ER and vesicles in ways that could influence ATP release and astrocyte-to-astrocyte calcium signaling, which may provide hints regarding roles of the GFAP upregulation that occurs ubiquitously in reactive astrocytes and warrants further exploration. Third, the findings provide direct evidence for how specific astrocyte dysfunctions can be the primary cause of a disorder with psychomotor symptomatology, strengthening the rationale to look for astrocyte contributions to neurological diseases. Lastly, these studies further reinforce the power of tissue-specific cells derived from patient iPSC to provide novel and fundamental mechanistic information.
macrophage regulation of YKL-40 expression and cellular response in neuroinflammation. Brain Pathol. 22, 530–546. Brenner, M., Johnson, A.B., Boespflug-Tanguy, O., Rodriguez, D., Goldman, J.E., and Messing, A. (2001). Mutations in GFAP, encoding glial fibrillary acidic protein, are associated with Alexander disease. Nat. Genet. 27, 117–120. Eng, L.F., Ghirnikar, R.S., and Lee, Y.L. (2000). Glial fibrillary acidic protein: GFAP-thirty-one years (1969-2000). Neurochem. Res. 25, 1439–1451. Hagemann, T.L., Connor, J.X., and Messing, A. (2006). Alexander disease-associated glial fibrillary acidic protein mutations in mice induce Rosenthal fiber formation and a white matter stress response. J. Neurosci. 26, 11162–11173. Hol, E.M., and Pekny, M. (2015). Glial fibrillary acidic protein (GFAP) and the astrocyte intermediate filament system in diseases of the central nervous system. Curr. Opin. Cell Biol. 32, 121–130. Jones, J.R., Kong, L., Hanna, M.G., IV, Hoffman, B., Krencik, B., Bradley, R., Hagemann, T., Choi, J., Doers, M., Dubovis, M., et al. (2018). Mutations in GFAP Disrupt the Distribution and Function of Organelles in Human Astrocytes. Cell Rep. 25, 947–958. Li, L., Tian, E., Chen, X., Chao, J., Klein, J., Qu, Q., Sun, G., Sun, G., Huang, Y., Warden, C.D., et al. (2018). GFAP Mutations in Astrocytes Impair Oligodendrocyte Progenitor Proliferation and Myelination in an hiPSC Model of Alexander Disease. Cell Stem Cell 23, 239–251.e6. Liedtke, W., Edelmann, W., Chiu, F.C., Kucherlapati, R., and Raine, C.S. (1998). Experimental autoimmune encephalomyelitis in mice lacking glial fibrillary acidic protein is characterized by a more severe clinical course and an infiltrative central nervous system lesion. Am. J. Pathol. 152, 251–259. Sofroniew, M.V. (2015). Astrocyte barriers to neurotoxic inflammation. Nat. Rev. Neurosci. 16, 249–263.
REFERENCES Bonneh-Barkay, D., Bissel, S.J., Kofler, J., Starkey, A., Wang, G., and Wiley, C.A. (2012). Astrocyte and
Sofroniew, M.V., and Vinters, H.V. (2010). Astrocytes: biology and pathology. Acta Neuropathol. 119, 7–35.
Cell Stem Cell 23, November 1, 2018 631
Previews Stem-Cell-Derived Astrocytes Divulge Secrets of Mutant GFAP Michael V. Sofroniew1,* 1Department of Neurobiology, University of California Los Angeles, Los Angeles, CA, USA *Correspondence: [email protected] https://doi.org/10.1016/j.stem.2018.10.020
Gain-of-function mutations in the canonical astrocyte protein, GFAP, cause a fatal neurodevelopmental disorder with myelination abnormalities, seizures, and psychomotor disturbances. Recently, Li et al. (2018) (in Cell Stem Cell) and Jones et al. (2018) (in Cell Reports) have shown that patient iPSC-derived astrocytes with mutant GFAP have disrupted astrocyte functions, revealing disease mechanisms and potential roles of GFAP. The ability to generate tissue-specific cell types from induced pluripotent stem cells (iPSCs) derived from patients with monogenetic disorders has provided powerful new tools with which to unravel disease mechanisms and identify protein functions. This approach is particularly useful in the central nervous system (CNS), where interactions among different complex cell types, neurons, astrocytes, oligodendrocytes, and microglia may contribute to disease mechanisms. Using this approach, two recent studies by Li et al. (2018) in Cell Stem Cell and by Jones et al. (2018) in Cell Reports generated CNS astrocytes from patients with Alexander Disease (AxD) and oligodendrocyte progenitor cells (OPCs) from human controls to probe how glial fibrillary acidic protein (GFAP) with AxD mutations disrupts astrocyte functions and causes the disease. Astrocytes tile the entire CNS and play essential roles in normal CNS function and responses to injury and disease (Sofroniew and Vinters, 2010). They clear and recycle transmitters, maintain extracellular ion and water homeostasis, and are critical for normal neural circuit function. Astrocyte-secreted molecules critically regulate synapse development and contribute to adult synaptic plasticity. In addition, astrocytes respond to all forms of CNS injury or disease with diverse reactive changes that can in different contexts mediate either pro- or anti-inflammatory signaling (Sofroniew, 2015), and they can either exert critical neuroprotective functions or contribute to and exacerbate various CNS pathologies (Sofroniew and Vinters, 2010). Given the wide range of critical astrocyte
activities, the potential contributions of gain or loss of astrocyte functions are under increasing investigation in CNS afflictions ranging from autoimmune inflammatory disorders to neurodegenerative conditions and neuropsychiatric disorders. GFAP was first isolated almost 50 years ago from multiple sclerosis plaques and was found to be associated with reactive astrocytes (Eng et al., 2000). Since then, it has become a canonical astrocyte marker. In healthy CNS, GFAP is expressed at detectable levels by many, but not all, astrocytes, and it is upregulated in a graded fashion in response to all CNS insults, where it can serve as a marker for severity of tissue damage (Sofroniew and Vinters, 2010). GFAP is an intermediate filament with cellular structural roles (Hol and Pekny, 2015), but relatively little is known about other potential functions. Intermediate filaments appear to exhibit considerable functional redundancy, and as a result, GFAP / mice exhibit few detectable abnormalities except a mild effect on long-term maintenance of myelin and a somewhat increased susceptibility to autoimmune inflammatory disease (Liedtke et al., 1998). AxD is rare developmental disorder characterized by megalencephaly, hydrocephalus, psychomotor delays, and white matter inflammation and degeneration. It is caused by autosomal dominant mutations in GFAP (Brenner et al., 2001). Identification of AxD mutations led to the ability make transgenic mice expressing mutant human GFAP. Although these transgenic mice exhibited the AxD hallmarks of GFAP aggregation and forma-
630 Cell Stem Cell 23, November 1, 2018 ª 2018 Elsevier Inc.
tion of Rosenthal fibers, the mice failed to yield the gross neurological abnormalities or white matter pathology seen in human patients (Hagemann et al., 2006). Thus, mouse and other transgenic organism models have not been effective in identifying mechanisms by which mutant GFAP causes the disease, setting the stage for studies using astrocytes derived from AxD-derived iPSCs. The studies by Li et al. and Jones et al. both used astrocytes derived from AxD-iPSCs from multiple patients and compared these with astrocytes from AxD-iPSCs in which the mutant GFAP had been corrected to the wild-type (WT) genotype through CRISPR/Cas9-based gene editing, and in the case of Li et al., the AxD-iPSCs from multiple patients were also compared with astrocytes derived from human control iPSCs. In both studies, AxD-astrocytes exhibited GFAP aggregation and Rosenthal fibers, regarded as AxD hallmarks. Li et al. examined astrocyte-oligodendrocyte interactions to look for clues regarding the white matter pathology (leukodystrophy) characteristic of AxD. They found that AxD astrocytes, but not control or corrected astrocytes, reduced the proliferation of co-cultured OPCs derived from control iPSCs and caused a myelination defect in a 3D nanofiber culture system. Transcriptome analysis indicated that AxD astrocytes exhibited highly upregulated genes involved in immune cell activation, pro-inflammatory cytokine signaling, cell proliferation, and cell adhesion. AxD astrocytes exhibited markedly downregulated genes involved in synapse regulation and ion transport. Li et al. next focused on CHI3L1, a secreted protein
Cell Stem Cell
Previews expressed by astrocytes and previously linked to various neuroinflammatory conditions (Bonneh-Barkay et al., 2012). CHI3L1 was among the top differentially expressed genes by AxD astrocytes, and its high expression was confirmed in brain tissue from AxD patients. Li et al. found that a CHI3L1-neutralizing antibody blocked the inhibitory effect of AxD astrocyte culture media on OPC proliferation, and that shRNA knockdown of CHI3L1 production significantly attenuated the inhibitory effects of AxD astrocytes on OPC proliferation and myelination. Additional experiments implicated CHI3L1 binding to the OPC cell surface receptor CRTH2 in mediating these inhibitory effects. These observations provide a direct potential mechanism by which AxD-astrocytes could precipitate white matter pathology. Jones et al. examined the effects of AxD mutations on astrocyte differentiation, regulation of intracellular organelles, and calcium and ATP signaling. They also first conducted a transcriptional analysis, which in particular, broadly implicated a dysregulation of vesicle regulation and endoplasmic reticulum (ER) functions. Electron microscopic and immunofluorescent analyses confirmed these findings and further revealed that AxD-astrocytes exhibited a swollen and largely non-reticular ER, along with alterations in lysosome size and intracellular distribution. Looking for potential functional consequences of these changes, Jones et al. found that under mechanical stimulation, AxD-astrocytes exhibited an attenuated ability to propagate calcium waves, which are mediated by cell-to-cell signaling via release of ATP. Further experiments
showed that AxD-astrocytes were able to produce and respond to ATP but exhibited significantly and substantially impaired release of ATP. These findings identify critical astrocyte functions that are disrupted by AxD-GFAP mutations and point toward intracellular roles for GFAP in ER and vesicle regulation that may influence astrocyte secretion of ATP and potentially that of other molecules. Overall, the findings by Li et al. and Jones et al. have several implications. First, they identify mechanisms whereby AxD mutations in GFAP can lead to specific astrocyte dysfunctions that could be causally related to AxD neurological symptoms and tissue pathology, which can now be studied further. Second, the findings point toward potential functions of GFAP in regulating ER and vesicles in ways that could influence ATP release and astrocyte-to-astrocyte calcium signaling, which may provide hints regarding roles of the GFAP upregulation that occurs ubiquitously in reactive astrocytes and warrants further exploration. Third, the findings provide direct evidence for how specific astrocyte dysfunctions can be the primary cause of a disorder with psychomotor symptomatology, strengthening the rationale to look for astrocyte contributions to neurological diseases. Lastly, these studies further reinforce the power of tissue-specific cells derived from patient iPSC to provide novel and fundamental mechanistic information.
macrophage regulation of YKL-40 expression and cellular response in neuroinflammation. Brain Pathol. 22, 530–546. Brenner, M., Johnson, A.B., Boespflug-Tanguy, O., Rodriguez, D., Goldman, J.E., and Messing, A. (2001). Mutations in GFAP, encoding glial fibrillary acidic protein, are associated with Alexander disease. Nat. Genet. 27, 117–120. Eng, L.F., Ghirnikar, R.S., and Lee, Y.L. (2000). Glial fibrillary acidic protein: GFAP-thirty-one years (1969-2000). Neurochem. Res. 25, 1439–1451. Hagemann, T.L., Connor, J.X., and Messing, A. (2006). Alexander disease-associated glial fibrillary acidic protein mutations in mice induce Rosenthal fiber formation and a white matter stress response. J. Neurosci. 26, 11162–11173. Hol, E.M., and Pekny, M. (2015). Glial fibrillary acidic protein (GFAP) and the astrocyte intermediate filament system in diseases of the central nervous system. Curr. Opin. Cell Biol. 32, 121–130. Jones, J.R., Kong, L., Hanna, M.G., IV, Hoffman, B., Krencik, B., Bradley, R., Hagemann, T., Choi, J., Doers, M., Dubovis, M., et al. (2018). Mutations in GFAP Disrupt the Distribution and Function of Organelles in Human Astrocytes. Cell Rep. 25, 947–958. Li, L., Tian, E., Chen, X., Chao, J., Klein, J., Qu, Q., Sun, G., Sun, G., Huang, Y., Warden, C.D., et al. (2018). GFAP Mutations in Astrocytes Impair Oligodendrocyte Progenitor Proliferation and Myelination in an hiPSC Model of Alexander Disease. Cell Stem Cell 23, 239–251.e6. Liedtke, W., Edelmann, W., Chiu, F.C., Kucherlapati, R., and Raine, C.S. (1998). Experimental autoimmune encephalomyelitis in mice lacking glial fibrillary acidic protein is characterized by a more severe clinical course and an infiltrative central nervous system lesion. Am. J. Pathol. 152, 251–259. Sofroniew, M.V. (2015). Astrocyte barriers to neurotoxic inflammation. Nat. Rev. Neurosci. 16, 249–263.
REFERENCES Bonneh-Barkay, D., Bissel, S.J., Kofler, J., Starkey, A., Wang, G., and Wiley, C.A. (2012). Astrocyte and
Sofroniew, M.V., and Vinters, H.V. (2010). Astrocytes: biology and pathology. Acta Neuropathol. 119, 7–35.
Cell Stem Cell 23, November 1, 2018 631