# Loss of USP18 in microglia induces white matter pathology

Marius Schwabenland, Omar Mossad, Adam G Peres, Franziska Kessler, Feres Jose Mocayar Maron, Laura-Adela Harsan, Thomas Bienert, Dominik von Elverfeldt, Klaus-Peter Knobeloch, Ori Staszewski, et al.

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Marius Schwabenland, Omar Mossad, Adam G Peres, Franziska Kessler, Feres Jose Mocayar Maron, et al.. Loss of USP18 in microglia induces white matter pathology. Acta Neuropathologica Communications, 2019, 7 (1), pp.106. 10.1186/s40478-019-0757-8 . hal-04883969

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# Loss of USP18 in microglia induces white matter pathology

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Marius Schwabenland¹, Omar Mossad¹,², Adam G. Peres¹, Franziska Kessler¹, Feres Jose Mocayar Maron¹, Laura-Adela Harsan³,⁴, Thomas Bienert³, Dominik von Elverfeldt³, Klaus-Peter Knobeloch¹, Ori Staszewski¹, Frank L. Heppner⁵,⁹,¹⁰, Marije E. C. Meuwissen⁶, Grazia M. S. Mancini⁶, Marco Prinz¹,⁷,⁸ and Thomas Blank¹*

**Keywords:** Microglia, Type I interferon, Usp18, White matter, Phagocytosis, Corpus callosum, Behavior, Magnet resonance spectroscopy, Microgliosis

## Main text

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 [7]. USP18 is a negative regulator of the type 1 interferon (IFN) pathway [9]. Microglia lacking *Usp18* exhibited constitutive activation of type I IFN signaling pathways resulting in markedly elevated expression of multiple interferon-stimulated genes (ISGs) [7]. 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 [14], hereditary diffuse leukoencephalopathy with spheroids (HDLS) [15] and Pseudo-TORCH syndrome (PTS), including Aicardi–Goutières syndrome [12]. 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there were no cells which had taken up myelin in young adult mice as seen by luxol fast blue–PAS (LFB–PAS) histology (unpublished data).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 [7]. 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 [7]. 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 [4]. 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

<footnote>* Correspondence: thomas.blank@uniklinik-freiburg.de
¹Institute of Neuropathology, Faculty of Medicine, University of Freiburg, Breisacher Str. 64, 79106 Freiburg, Germany
Full list of author information is available at the end of the article</footnote>
A

Corpus callosum (4 mo)

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Corpus callosum (4 mo)

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Usp18fl/fl

Cx3cr1<sup>Cre</sup>: Usp18fl/fl

B

Corpus callosum (8 mo)

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Corpus callosum (8 mo)

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Usp18fl/fl

Cx3cr1<sup>Cre</sup>: Usp18fl/fl

C

Corpus callosum (8 mo)

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Usp18fl/fl

Cx3cr1<sup>Cre</sup>: Usp18fl/fl

**Fig. 1** (See legend on next page.)
(See figure on previous page.)

**Fig. 1** Microgliosis in corpus callosum of *Cx3cr1<sup>Cre;Usp18<sup>fl/fl</sup></sup>* mice. **a, b** Histology of corpus callosum in the cerebrum of adult *Usp18<sup>fl/fl</sup>* and *Cx3cr1<sup>Cre;Usp18<sup>fl/fl</sup></sup>* mice at 4 (a) and 8 months of age (b). Primary antibodies against Iba1 and ISG15 were used. To quantify the number of Iba1⁺ or ISG15⁺ cells at least six mice per genotype and 5 sections per mouse from two independent experiments were counted. Quantification of cells is shown next to the respective histological images. Significant differences were determined by an unpaired *t*-test or Mann-Whitney *U*-test and marked with asterisks (***P < 0.001 versus control littermates). Bars represent means ± S.E.M. Scale bars = 25 µm, 50 µm, 100 µm. **c** Immunohistochemistry for phosphorylated STAT1 (pSTAT1, red), CD11b (green) and DAPI (blue) in the corpus callosum of 8-month-old *Usp18<sup>fl/fl</sup>* and *Cx3cr1<sup>Cre;Usp18<sup>fl/fl</sup></sup>* mice. Scale bar: 20 µm. Quantification of pSTAT1⁺CD11b⁺ cells is shown next to the respective histological images. Each symbol represents one mouse. Error bars represent S.E.M. Significant differences are determined by an unpaired *t*-test and marked with asterisks (***P < 0.001)

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**Fig. 2** USP18-deficient microglia reduces structural integrity in corpus callosum. **a** Immunofluorescent histochemistry for Iba1 (red), Lamp2 (green) and DAPI (blue) in the corpus callosum of 4 months and 8 months (c) old *Usp18<sup>fl/fl</sup>* and *Cx3cr1<sup>Cre;Usp18<sup>fl/fl</sup></sup>* mice. Scale bar: 20 µm. Quantification of Iba1⁺ and percentage of Iba1⁺Lamp2⁺ cells is shown next to the respective histological images (b, d). Each symbol represents an individual mouse. Error bars represent s.e.m. Significant differences are determined by an unpaired *t*-test and marked with asterisks (***P < 0.01, ****P < 0.001). **e** DTI was performed on 4 and 8 months old *Usp18<sup>fl/fl</sup>* and *Cx3cr1<sup>Cre;Usp18<sup>fl/fl</sup></sup>* mice to measure the FA of the corpus callosum. Tensor images were collectively acquired in several horizontal planes from +2.0 to -4.0 mm from the bregma, with an interplane distance of 0.5 mm (*Usp18<sup>fl/fl</sup>*; *n* = 6; *Cx3cr1<sup>Cre;Usp18<sup>fl/fl</sup></sup>*; *n* = 4). Heat maps of the FA values showing the average (of all *Usp18<sup>fl/fl</sup>* and *Cx3cr1<sup>Cre;Usp18<sup>fl/fl</sup></sup>* animals) of one plane from each group (from anterior to posterior). Warm colors indicate fiber tracts with strong diffusion coherence. For both age groups the FA values were significantly reduced in *Cx3cr1<sup>Cre;Usp18<sup>fl/fl</sup></sup>* mice in comparison to *Usp18<sup>fl/fl</sup>* mice. Approximate locations of the regions of interest (ROIs) are indicated. Data are means ± SEM. (*P* < 0.05, **P** < 0.01, ***P** < 0.001, n.s. = non-significant). Statistical significance was determined using multiple *t* tests corrected for multiple comparisons using the Holm-Sidak method with a = 0.05. **f** Histological analysis by luxol fast blue-PAS (LFB-PAS) in 8-month-old *Usp18<sup>fl/fl</sup>* mice and *Cx3cr1<sup>Cre;Usp18<sup>fl/fl</sup></sup>* littermates. Representative of *n* = 6 *Usp18<sup>fl/fl</sup>* and *n* = 7 *Cx3cr1<sup>Cre;Usp18<sup>fl/fl</sup></sup>* mice. Circles represent individual mice. Unpaired two-tailed *t*-test
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 Cx3cr1<sup>Cre</sup>:Usp18<sup>fl/fl</sup> mice [2, 17].

Deterioration of white matter tracts, affecting brain struc-
tural (SC) and functional connectivity (FC) is often paral-
leled by behavioral declines [3, 6, 8]. We therefore tested
Cx3cr1<sup>Cre</sup>:Usp18<sup>fl/fl</sup> mice and Usp18<sup>fl/fl</sup> littermate controls
in different behavioral paradigms. While mice lacking
Usp18 in microglia performed normal in the odor avoid-
ance test at 4 months of age (Fig. 3a), 8-month old
Cx3cr1<sup>Cre</sup>:Usp18<sup>fl/fl</sup> mice showed severely impaired olfac-
tion (Fig. 3d). Similarly, learning and recognition memory
was fully intact at 4 months of age (Fig. 3b) but decreased
when Cx3cr1<sup>Cre</sup>:Usp18<sup>fl/fl</sup> mice were 8-month old com-
pared to age-matched Usp18<sup>fl/fl</sup> control mice (Fig. 3e).
Rotarod performance, which measures motor coordination
and motor learning, was also significantly impaired in 8-
month old Cx3cr1<sup>Cre</sup>:Usp18<sup>fl/fl</sup> mice (Fig. 3f) with no defi-
cits in 4 months old mice (Fig. 3c). In addition to the indi-
cated mouse model we investigated brainstem tissue
samples from three PTS patients with loss-of-function re-
ccessive mutations of USP18 [12]. 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 [11].

The data presented here indicate that in myeloid-
specific *Usp18* knockout animals, microglia in the white
matter were not only activated, but also caused advan-
cing damage to this structure with subsequent behav-
ioral impairment of the animals. *USP18*-deficiency in
humans belongs to a group of genetic disorders that are
collectively termed type I interferonopathies. These dis-
orders are first characterized by the persistent up-
regulation of type I interferon signaling [16]. There have
been at least seven possible cellular mechanisms de-
scribed, which result in sustained activation of interferon
signaling [16]. 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 pa-
tients displayed not only enhanced type I IFN signaling,
but also close contact to oligodendroglia. A direct inter-
action might indicate that activated microglia, as sug-
gested by their focally elevated cell density together with
altered morphological properties inflict damage to oligo-
dendroglia. This strongly resembles the white matter
damage observed in Cx3cr1<sup>Cre</sup>:Usp18<sup>fl/fl</sup> 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

<figure><img src="image_14.png" /></figure>
<figure><img src="image_15.png" /><figcaption>**Fig. 4** Microgliosis in white matter of Pseudo-TORCH patients. **a** Histology of white matter in Pseudo-TORCH patients (n = 3) and age-matched controls (n = 3) (**b**). Primary antibodies were used against Iba1, pStat1 and Nogo-A. Quantification of cells is shown next to the respective histological images. Significant differences were determined by an unpaired t-test or Mann-Whitney U-test and marked with asterisks (***P < 0.001 versus controls). Bars represent means ± s.e.m. Scale bars = 50 µm, 100 µm</figcaption></figure>

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 [13].

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.

**Abbreviations**

DTI: Diffusion tensor imaging; IFN: Interferon; ISG: Interferon-stimulated gene; MRI: Magnetic resonance imaging; NOR: Novel Object Recognition; PTS: Pseudo-TORCH syndrome; RT: Room temperature; STAT1: Signal transducer and activator of transcription 1; STING: Stimulator of interferon genes; USP: Ubiquitin-specific protease

**Acknowledgements**

The authors are thankful to Margarethe Ditter for excellent technical assistance.

**Authors' contributions**

TB, KPK, MECM, GMSM, OS, FLH and MP were responsible for the conception and design of experiments; TBi, LAH and DvE were responsible for MRI measurements; MS, OM, FK, AP and FJMM performed experiments, analysed and interpreted the data, they drafted the paper and revised and edited the final article. All authors read and approved the final manuscript.

**Funding**

TB was supported by the DFG (BL 1153/1-2). TB and MP are supported by the DFG (SFB/TRR167 "NeuroMac").

**Availability of data and materials**

The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

**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.

**Consent for publication**

All the authors have approved publication.

**Competing interests**

The authors declare that they have no competing interests.
## Author details

¹Institute of Neuropathology, Faculty of Medicine, University of Freiburg, Breisacher Str. 64, 79106 Freiburg, Germany. ²Faculty of Biology, University of Freiburg, Freiburg, Germany. ³Department of Radiology, Medical Physics, University Medical Center Freiburg, Faculty of Medicine, University of Freiburg, Freiburg, Germany. ⁴Engineering Science, Computer Science and Imaging Laboratory (ICube), Integrative Multimodal Imaging in Healthcare, CNRS, University of Strasbourg, Strasbourg, France. ⁵Department of Neuropathology, Charité - Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Charitéplatz 1 (Virchowweg 15), 10117 Berlin, Germany. ⁶Department of Clinical Genetics, Erasmus University Medical Center, 3015, GD, Rotterdam, the Netherlands. ⁷Signalling Research Centres BIOSS and CIBSS, University of Freiburg, Freiburg, Germany. ⁸Center for NeuroModulation, Faculty of Medicine, University of Freiburg, Freiburg, Germany. ⁹Cluster of Excellence, NeuroCure, Charitéplatz 1, 10117 Berlin, Germany. ¹⁰German Center for Neurodegenerative Diseases (DZNE) Berlin, 10117 Berlin, Germany.

Received: 16 May 2019 Accepted: 20 June 2019

Published online: 04 July 2019

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