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Loss of USP18 in microglia induces white matter pathology
Acta Neuropathologica Communications volume 7, Article number: 106 (2019)
Ubiquitin specific protease 18 (USP18) is a major negative regulator of the type 1 interferon (IFN) pathway. In a recent publication we showed that USP18 is a key molecule imposing microglial quiescence specifically in the white matter . USP18 is a negative regulator of the type 1 interferon (IFN) pathway . Microglia lacking Usp18 exhibited constitutive activation of type I IFN signaling pathways resulting in markedly elevated expression of multiple interferon-stimulated genes (ISGs) . Additionally, Usp18-deficient brains exhibited clusters of microglia in the white matter that strongly resembled the neuropathological state in several human microgliopathies. Human diseases in which microgliopathies play a primary role comprise Nasu-Hakola disease , hereditary diffuse leukoencephalopathy with spheroids (HDLS)  and Pseudo-TORCH syndrome (PTS), including Aicardi–Goutières syndrome . One might speculate that activated microglia in the white matter induce white matter abnormalities with functional consequences. However, there were no cells which had taken up myelin in young adult mice as seen by luxol fast blue–PAS (LFB–PAS) histology (unpublished data). Myelin uptake by other cells, like macrophages, would have been indicative of myelin damage. That is why we now characterized conditional myeloid-specific Usp18 deficient mice in more detail.
We know that Usp18 transcripts are highly expressed in unstimulated white matter microglia with only negligible expression levels in other CNS cells . In a previous study, we have confirmed by PCR analysis that Cx3cr1Cre:Usp18fl/fl mice have an Usp18 deletion in microglia but not in neuroectodermal cells of the CNS. These mice displayed a significant increase of Iba1+ microglia cell numbers in several white matter regions including the corpus callosum as young adult mice . This microgliosis persisted with increasing age and was detectable even in 4- and 8-month old mice (Fig. 1a, b). Usp18-deficient microglia exhibit constitutive expression of IFN target genes and fail to downregulate IFN-induced genes because the termination of type I IFN signaling is severely impaired. This became evident by the increase in ISG15 positive cells in the corpus callosum (Fig. 1a, b) and the elevated phosphorylation of STAT1 in Usp18-deficient microglia when compared to Usp18fl/fl mice (Fig. 1c). We next investigated animals at later ages than before by immunostainings against lysosome-associated membrane protein-2 (LAMP2) as a marker of phagocytosis . We found increased LAMP2 positive signals in microglia, which were localized in the corpus callosum of Cx3cr1Cre:Usp18fl/fl mice at an age of 4 months (Fig. 2a, b) and 8 months (Fig. 2c, d). To analyze white matter integrity, we performed high-resolution (11.7 T) diffusion tensor imaging (DTI). We calculated the fractional anisotropy (FA) values, permitting an exploration of the orientation coherence of axons in this fiber bundle. We found that the FA values were reduced in the corpus callosum, the internal and external capsule of Cx3cr1Cre:Usp18fl/fl mice (cf. Usp18fl/fl controls), suggesting diminished structural integrity of the white matter in 4- and 8-month old animals (Fig. 2e). Additionally, we found increased numbers of cells that had incorporated myelin and thereby indicate damage to the myelin sheaths (Fig. 2f). Together, these findings point to a reduction in myelination or even to a loss of fibers in Cx3cr1Cre:Usp18fl/fl mice [2, 17].
Deterioration of white matter tracts, affecting brain structural (SC) and functional connectivity (FC) is often paralleled by behavioral declines [3, 6, 8]. We therefore tested Cx3cr1Cre:Usp18fl/fl mice and Usp18fl/fl littermate controls in different behavioral paradigms. While mice lacking Usp18 in microglia performed normal in the odor avoidance test at 4 months of age (Fig. 3a), 8-month old Cx3cr1Cre:Usp18fl/fl mice showed severely impaired olfaction (Fig. 3d). Similarly, learning and recognition memory was fully intact at 4 months of age (Fig. 3b) but decreased when Cx3cr1Cre:Usp18fl/fl mice were 8-month old compared to age-matched Usp18fl/fl control mice (Fig. 3e). Rotarod performance, which measures motor coordination and motor learning, was also significantly impaired in 8-month old Cx3cr1Cre:Usp18fl/fl mice (Fig. 3f) with no deficits in 4 months old mice (Fig. 3c). In addition to the indicated mouse model we investigated brainstem tissue samples from three PTS patients with loss-of-function recessive mutations of USP18 . Immunohistochemistry showed increased STAT1 phosphorylation in microglia of PTS patients when compared to age-matched control tissue (Fig. 4a). In patients’ material there were also more microglial cells, which engulfed cells positive for Nogo-A (Fig. 4b), which represents an oligodendroglial marker .
The data presented here indicate that in myeloid-specific Usp18 knockout animals, microglia in the white matter were not only activated, but also caused advancing damage to this structure with subsequent behavioral impairment of the animals. USP18-deficiency in humans belongs to a group of genetic disorders that are collectively termed type I interferonopathies. These disorders are first characterized by the persistent up-regulation of type I interferon signaling . There have been at least seven possible cellular mechanisms described, which result in sustained activation of interferon signaling . One of them, PTS, is a group of not so well-defined genetic diseases, which can originate from USP18 deficiency. We found that microglia in PTS patients displayed not only enhanced type I IFN signaling, but also close contact to oligodendroglia. A direct interaction might indicate that activated microglia, as suggested by their focally elevated cell density together with altered morphological properties inflict damage to oligodendroglia. This strongly resembles the white matter damage observed in Cx3cr1Cre:Usp18fl/fl mice. Type I interferon can be regarded as a neurotoxin if its levels are not tightly controlled. Accordingly, experiments undertaken in mice demonstrate that overexpression of interferon in the CNS results in neuropathology reminiscent of that seen in certain type I interferonopathies [1, 10]. In the case of PTS, but also in the case of type I IFN overexpression, damage to the white matter seems to be prevalent [5, 12]. It is still unclear what the type I IFN source is in the context of interferonopathies. Likewise it is enigmatic which signals are responsible for microglia activation in the white matter. The escalating spiral of white matter damage might be initiated by type I IFN that is induced in microglia via stimulator of interferon genes (STING), and this IFN likely influences the microglial phenotype in an autocrine and paracrine fashion .
The white matter specificity of the USP18 effect on microglia is of particular interest and further developments in this area may have implications for an entire range of neurological disorders in which there is a preponderance of white matter pathology.
Availability of data and materials
The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.
Diffusion tensor imaging
Magnetic resonance imaging
Novel Object Recognition
Signal transducer and activator of transcription 1
Stimulator of interferon genes
Akwa Y, Hassett DE, Eloranta ML, Sandberg K, Masliah E, Powell H, Whitton JL, Bloom FE, Campbell IL (1998) Transgenic expression of IFN-alpha in the central nervous system of mice protects against lethal neurotropic viral infection but induces inflammation and neurodegeneration. J Immunol 161:5016–5026
Basser PJ (1995) Inferring microstructural features and the physiological state of tissues from diffusion-weighted images. NMR Biomed 8:333–344
Bells S, Lefebvre J, Prescott SA, Dockstader C, Bouffet E, Skocic J, Laughlin S, Mabbott DJ (2017) Changes in white matter microstructure impact cognition by disrupting the ability of neural assemblies to synchronize. J Neurosci 37:8227–8238. https://doi.org/10.1523/JNEUROSCI.0560-17.2017
Blank T, Goldmann T, Koch M, Amann L, Schon C, Bonin M, Pang S, Prinz M, Burnet M, Wagner JE et al (2017) Early microglia activation precedes photoreceptor degeneration in a mouse model of CNGB1-linked retinitis Pigmentosa. Front Immunol 8:1930. https://doi.org/10.3389/fimmu.2017.01930
Cuadrado E, Jansen MH, Anink J, De Filippis L, Vescovi AL, Watts C, Aronica E, Hol EM, Kuijpers TW (2013) Chronic exposure of astrocytes to interferon-alpha reveals molecular changes related to Aicardi-Goutieres syndrome. Brain 136:245–258. https://doi.org/10.1093/brain/aws321
Filley CM, Fields RD (2016) White matter and cognition: making the connection. J Neurophysiol 116:2093–2104. https://doi.org/10.1152/jn.00221.2016
Goldmann T, Zeller N, Raasch J, Kierdorf K, Frenzel K, Ketscher L, Basters A, Staszewski O, Brendecke SM, Spiess A et al (2015) USP18 lack in microglia causes destructive interferonopathy of the mouse brain. EMBO J 34:1612–1629. https://doi.org/10.15252/embj.201490791
Gullmar D, Seeliger T, Gudziol H, Teichgraber UKM, Reichenbach JR, Guntinas-Lichius O, Bitter T (2017) Improvement of olfactory function after sinus surgery correlates with white matter properties measured by diffusion tensor imaging. Neuroscience 360:190–196. https://doi.org/10.1016/j.neuroscience.2017.07.070
Honke N, Shaabani N, Zhang DE, Hardt C, Lang KS (2016) Multiple functions of USP18. Cell Death Dis 7:e2444. https://doi.org/10.1038/cddis.2016.326
Kavanagh D, McGlasson S, Jury A, Williams J, Scolding N, Bellamy C, Gunther C, Ritchie D, Gale DP, Kanwar YS et al (2016) Type I interferon causes thrombotic microangiopathy by a dose-dependent toxic effect on the microvasculature. Blood 128:2824–2833. https://doi.org/10.1182/blood-2016-05-715987
Kuhlmann T, Remington L, Maruschak B, Owens T, Bruck W (2007) Nogo-a is a reliable oligodendroglial marker in adult human and mouse CNS and in demyelinated lesions. J Neuropathol Exp Neurol 66:238–246. https://doi.org/10.1097/01.jnen.0000248559.83573.71
Meuwissen ME, Schot R, Buta S, Oudesluijs G, Tinschert S, Speer SD, Li Z, van Unen L, Heijsman D, Goldmann T et al (2016) Human USP18 deficiency underlies type 1 interferonopathy leading to severe pseudo-TORCH syndrome. J Exp Med 213:1163–1174. https://doi.org/10.1084/jem.20151529
Nazmi A, Field RH, Griffin EW, Haugh O, Hennessy E, Cox D, Reis R, Tortorelli L, Murray CL, Lopez-Rodriguez AB et al (2019) Chronic neurodegeneration induces type I interferon synthesis via STING, shaping microglial phenotype and accelerating disease progression. Glia. https://doi.org/10.1002/glia.23592
Paloneva J, Manninen T, Christman G, Hovanes K, Mandelin J, Adolfsson R, Bianchin M, Bird T, Miranda R, Salmaggi A et al (2002) Mutations in two genes encoding different subunits of a receptor signaling complex result in an identical disease phenotype. Am J Hum Genet 71:656–662. https://doi.org/10.1086/342259
Rademakers R, Baker M, Nicholson AM, Rutherford NJ, Finch N, Soto-Ortolaza A, Lash J, Wider C, Wojtas A, DeJesus-Hernandez M et al (2011) Mutations in the colony stimulating factor 1 receptor (CSF1R) gene cause hereditary diffuse leukoencephalopathy with spheroids. Nat Genet 44:200–205. https://doi.org/10.1038/ng.1027
Rodero MP, Crow YJ (2016) Type I interferon-mediated monogenic autoinflammation: the type I interferonopathies, a conceptual overview. J Exp Med 213:2527–2538. https://doi.org/10.1084/jem.20161596
Song SK, Sun SW, Ramsbottom MJ, Chang C, Russell J, Cross AH (2002) Dysmyelination revealed through MRI as increased radial (but unchanged axial) diffusion of water. Neuroimage 17:1429–1436
The authors are thankful to Margarethe Ditter for excellent technical assistance.
TB was supported by the DFG (BL 1153/1–2). TB and MP are supported by the DFG (SFB/TRR167 “NeuroMac”).
Ethics approval and consent to participate
All animal experiments were approved by the Federal Ministry for Nature, Environment and Consumers’ Protection of the state of Baden-Württemberg (G12/71; G16/107) and were performed in accordance with the respective national, federal and institutional regulations. For patients’ samples written parental consent was obtained. Genetic tests were performed according to The Erasmus University Medical Center’s local ethics board approved protocol MEC-2012387.
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The authors declare that they have no competing interests.
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Schwabenland, M., Mossad, O., Peres, A.G. et al. Loss of USP18 in microglia induces white matter pathology. acta neuropathol commun 7, 106 (2019) doi:10.1186/s40478-019-0757-8
- Type I interferon
- White matter
- Corpus callosum
- Magnet resonance spectroscopy