Role of the epigenetic factor Sirt7 in neuroinflammation and neurogenesis

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Role of the epigenetic factor Sirt7 in neuroinflammation and neurogenesis Nicolas Burg, Stefan Bittner, Erik Ellwardt ∗ Focus Program Translational Neurosciences (FTN) and Immunology (FZI), Rhine Main Neuroscience Network (rmn2), Department of Neurology, University Medical Center of the Johannes Gutenberg University Mainz, Mainz, Germany

a r t i c l e

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Article history: Received 31 May 2017 Received in revised form 7 September 2017 Accepted 14 September 2017 Available online xxx Keywords: Sirt7 EAE Epigenetic Multiple sclerosis Neuroinflammation Adult neurogenesis

a b s t r a c t Epigenetic regulators are increasingly recognized as relevant modulators in the immune and nervous system. The class of sirtuins consists of NAD+ -dependent histone deacetylases that regulate transcription. Sirtuin family member Sirt1 has already been shown to influence the disease course in an animal model of autoimmune neuroinflammation (experimental autoimmune encephalomyelitis (EAE). A role of Sirt7, a related epigenetic regulator, on immune system regulation has been proposed before, as these mice are more susceptible to develop inflammatory cardiomyopathy. Sirt7−/− animals showed no differences in clinical score compared to wild-type littermates after EAE induction with myelin oligodendrocyte glycoprotein (MOG) peptide 35-55 , although we found subtle immune alterations at different phases of EAE and decreased survival of newly generated neurons in the hippocampus. Our data indicate that Sirt7 has a slight protective impact on both the adaptive immune system and neurogenesis. However, overall this epigenetic factor is not capable of impacting the acute or chronic phase of neuroinflammation. © 2017 Elsevier Ireland Ltd and Japan Neuroscience Society. All rights reserved.

1. Introduction Multiple sclerosis (MS) is an autoimmune demyelinating disease of the central nervous system (CNS). Epigenetic regulation is known to be involved in inflammatory responses in MS pathogenesis (Kong et al., 2012; Staszewski and Prinz, 2014; van den Elsen et al., 2014). It is involved in differentiation and cytokine production following chromatin remodeling of T cells, which infiltrate the CNS in MS and in the murine model of MS, experimental autoimmune encephalomyelitis (EAE) (Agarwal and Rao, 1998). Histone deacetylation, one mechanism of epigenetic regulation, is known to promote remyelination capacities of oligodendrocytes in rodents and humans (Copray et al., 2009; Pedre et al., 2011) and represents a potential therapeutic target in MS. Sirtuins, which form part of the Sir2 (silent information regulator 2) protein family originally discovered in yeast, are NAD+ -dependent histone deacetylases that regulate transcription of genes (North and Verdin, 2004). There are 7 sirtuins described so far. Sirtuin 7 (Sirt7) itself is involved in cellular proliferation,

∗ Corresponding author at: Focus Program Translational Neurosciences (FTN), Rhine Main Neuroscience Network (rmn2 ), Department of Neurology, University Medical Center of the Johannes-Gutenberg University Langenbeckstraße 1, 55131 Mainz, Germany. E-mail address: [email protected] (E. Ellwardt).

RNA regulation, cell homeostasis and genome regulation (Kiran et al., 2015). Sirt7 expression is ubiquitous, including in the blood, heart, brain and hippocampus (Ford et al., 2006; Wang et al., 2012; Wronska et al., 2016). It might have pro-oncogenic properties as it facilitates tumor formation (Barber et al., 2012). Nevertheless, Sirt7-deficient mice show a reduced maximum lifespan and develop inflammatory hypertrophic cardiomyopathy spontaneously (Vakhrusheva et al., 2008). They have increased numbers of granulocytes and T lymphocytes in blood and show inflammatory infiltrates in the myocardium. This suggests an impact on inflammation and cell proliferation for Sirt7 with a mainly antiinflammatory effect. For the related Sirt1 opposing data exists. One group showed a protective role for Sirt1 in neuroinflammation (Nimmagadda et al., 2013). In contrast, recent data suggests that the related Sirt1 has a pro-inflammatory role, as Sirt1-deficient mice showed an ameliorated EAE disease course and increased oligodendrocyte expansion (Lim et al., 2015; Rafalski et al., 2013). Treatment with the Sirt1 activator resveratrol led to exacerbated demyelination and inflammation (Sato et al., 2013). MS patients can suffer from mild cognitive deficits already at early disease stages (Chiaravalloti and DeLuca, 2008; Lucchinetti et al., 2011). Immune cell infiltration into the CNS seems to influence the generation of new neurons in the adult dentate gyrus of the hippocampus (Huehnchen et al., 2011). Epigenetic factors modu-

http://dx.doi.org/10.1016/j.neures.2017.09.005 0168-0102/© 2017 Elsevier Ireland Ltd and Japan Neuroscience Society. All rights reserved.

Please cite this article in press as: Burg, N., et al., Role of the epigenetic factor Sirt7 in neuroinflammation and neurogenesis. Neurosci. Res. (2017), http://dx.doi.org/10.1016/j.neures.2017.09.005

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Fig. 1. Characterization of Sirt7 in acute EAE A: Disease course and prevalence after immunization with MOG35-55 showed no difference between Sirt7−/− and littermates (n = 6 animals per group). The experiment was terminated at the peak of disease (day 21) for further immune cell characterization. B: FACS analysis of cell types isolated from CNS (brains and spinal cord) and spleen at disease peak, gated on CD45+ cells. The percentages of the individual cell types did not differ between Sirt7−/− and wild-type littermates. C: Cytokine profiling of CNS immune cells showed similar distributions for both groups for CD4 and CD8 cells; cytokine production by peripheral spleen CD4 and CD8 cells was much lower than in the CNS. Moreover, IFN-␥ production was significantly decreased in Sirt7−/− spleen CD8+ cells (*p < 0.05, unpaired two-sided students t-test, n = 3-4 animals per group in B-E); also see FACS plots (D) for IFN-␥ of spleen cells gated on CD45+ and CD8+ cells. E: Percentages of FoxP3 expressing regulatory CD4+ T cells were similar for both groups.

late neurogenesis during development, as well as during adulthood within the neurogenic niches (Ma et al., 2010), in addition to the established extrinsic regulators like running and enriched environment (Kempermann et al., 1997; van Praag et al., 1999). For sirtuins, especially Sirt1, there is evidence for regulation of adult hippocampal neurogenesis and neuronal progenitor cells which was partially

shown by our group (Ma et al., 2014; Prozorovski et al., 2008; Saharan et al., 2013). We here addressed the question whether Sirt7 regulates the EAE disease course and in vitro T cell proliferation. Additionally, we assessed the impact of Sirt7 on adult hippocampal neurogenesis and show an impact on the adaptive immune system and a

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positive influence on survival of adult-born neurons in the dentate gyrus. 2. Material and methods 2.1. Mice Sirt7 knockout mice were kindly provided by the group of Thomas Braun (Max Planck Institute Bad Nauheim, Germany). Female animals were bred under germ-free conditions with access to food and water ad libitum. Animals were backcrossed to a C57Bl/6 genetic background, for five generations. For all experiments, age-matched female littermates were used as controls. All experiments were conducted in accordance with animal protection guidelines and approved by the local authorities. 2.2. EAE induction and scoring For EAE induction, approximately 10-week-old female mice were immunized with myelin oligodendrocyte glycoprotein (MOG)35-55 in complete Freund’s adjuvant (CFA; Hooke laboratories, USA). A total volume of 200 ␮l MOG35-55 /CFA was injected subcutaneously (s.c.) at two sites according to the company’s protocol. Intraperitoneal (i.p.) injection of 200 ng pertussis toxin diluted in 100 ␮l PBS was performed on the same day as immunization and again the next day. Following immunization, animals were scored daily for motor symptoms using the following scale: 0: healthy, 1: tail paralysis, 2: hind limb paresis, 3: hind limb paralysis, 4: fore limb paralysis, 5: death. 2.3. Isolation of leukocytes from CNS and spleen Isolation of leukocytes from the CNS and spleen was performed at the peak of disease and during the chronic phase. For lymphocyte isolation from the CNS, lethally anesthetized animals were transcardially perfused with cold PBS. The brain and spinal cord were isolated, cut into small pieces, and transferred into IMDM (Life Technologies) substituted with 360 U/ml collagenase, 200 U/ml DNase, and 5 ␮g/ml collagenase/dispase (all R&D). After incubation for 30 min at 37 ◦ C under continuous movement, the CNS tissue was filtered through a mesh (70 ␮m) and mononuclear cells were separated by conventional 40/70 Percoll centrifugation. For lymphocyte isolation from the spleen, tissue was filtered through a 70 ␮m mesh and lysis of erythrocytes was performed. 2.4. Cytokine staining and flow cytometry Cytokine analysis of isolated lymphocytes was preceded by plate-bound anti-CD3 and anti-CD28 (2.5 ␮g/ml and 3 ␮g/ml, respectively; both R&D) stimulation for 4 h; brefeldin A was added after 2 h. Antibodies used for FACS surface and intracellular stainings were as follows: CD45R-AF 700, CD11c-AF 647, TNF␣-AF700 (Life Technologies); CD4-Horizon, CD4-FITC, CD8-FITC, CD8-APC, CD11b-PeCy7, MHCII-PE, IFN-␥ Horizon, IL-17 PE (BD Biosciences); and CD45-eflour 605, FoxP3-PeCy7 (eBioscience). Samples were measured with a FACSCanto II (BD Bioscience) and analysis was performed with FlowJo Software.

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staining and populations with a purity of >95% were used. CD4 T cells were then incubated for 30 min at 37 ◦ C in culture medium and subsequently washed twice in pre-warmed RPMI + 1% HEPES (RPMI/H). Afterwards, cells were incubated in pre-warmed RPMI/H containing 2.5 ␮M CFSE for 10 min at 37 ◦ C in the dark. The CFSElabeled cells were washed twice with cold culture medium and cultured in 96-well plates for 3 days (200,000 cells per 200 ␮l) and activated with plate-bound anti-CD3 and anti-CD28 antibodies (2.5 ␮g/ml and 3 ␮g/ml, respectively; both R&D). Cells were harvested, washed with FACS buffer, stained with anti-CD4-Horizon fluorescent antibody (BD Biosciences) and measured on a FACSCanto II (BD Germany).

2.6. BrdU-Injection, tissue preparation and DAB staining For neurogenesis assessment, animals were injected i.p. with 50 mg/kg bodyweight 5-bromo-2-deoxyuridine (BrdU, Sigma, Hamburg, Germany) on three consecutive days (for exact experimental design please see Figs. 4 and 5) dissolved in 0.9% NaCl. For tissue preparation, mice were perfused first with PBS followed by ice cold 4% paraformaldehyde. Brains were stored in 4% paraformaldehyde overnight and the transferred to a 30% sucrose solution for 72 h. Brains were cut into 40 ␮m thick coronal sections. For determination of absolute cell numbers, every sixth section was stained with a rat anti-BrdU antibody (1:750, Harlan Laboratories, Indianapolis, Indiana, USA) and a secondary anti-rat antibody which was biotinylated (1:1000, Vector Laboratories, Burlingame, CA, USA). We then applied the ABC Vectastain kit (Vector Laboratories, Burlingame, CA, USA) to visualize labeled cells. Quantification was performed via light microscopy (Olympus, Germany, 20 x objective/NA 0.75); positive cells in the subgranular and granular zone only of the dentate gyrus were counted and then multiplied by six.

2.7. Immunofluorescent staining of hippocampal neurons For phenotypic analysis of BrdU-positive cells immunofluorescent stainings were performed with guinea pig anti-DCX (doublecortin, 1:1000, Merck Millipore, Billerica, Massachusetts, United States), rabbit anti-NeuN (neuronal nuclei, 1:1000, Merck Millipore, Billerica, Massachusetts, United States), rabbit antihistone H3 (acetyl K18, 1:500, Abcam, Germany) and rabbit anti-GFAP (glial fibrillary acidic protein, 1:1000, Merck Millipore, Billerica, Massachusetts, United States) primary antibodies. For co-labeling, at least 50 positive BrdU cells were analyzed using confocal microscopy (Leica TCS SP8, DM 6000CS) and sequential scans with a 63-fold objective (Leica, NA 1.4) and resolution of 1024 × 1024 pixels were performed. The percentage of double positive cells was multiplied by the absolute number of BrdU-positive cells to determine absolute NeuN, GFAP and DCX cell numbers.

2.5. Cell proliferation assay

2.8. Statistical analyses

To measure murine CD4 T cell proliferation, spleens and lymph nodes were extracted. Following cell collection using a cell strainer and lysis of erythrocytes, CD4 cells were isolated after labeling cells with magnetic beads and separating all non-CD4 cells from the ® probe (negative CD4 sort using MACS Microbeads from Miltenyi Biotec including CD8a-Ly2). Purity of sorting was checked by FACS

For statistical analysis, we performed unpaired two-sided students t-test or Mann-Whitney U when appropriate, or KruskalWallis analyses with Dunn’s post-hoc test depending of the experimental setup using GraphPad Prism Version 6. Statistical significance was defined as p < 0.05. All results are expressed as mean ± s.e.m (standard error of mean).

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Fig. 2. Characterization of Sirt7 in chronic EAE A: No significant differences were observed in disease course, onset, or prevalence between Sirt7 knockout animals and littermates over 7 weeks after immunization (n = 10 animals per group). The experiment was terminated at the end (day 37) for further immune cell characterization. B: FACS analysis of cell types isolated from CNS (brains and spinal cord) and spleen during the chronic phase (day 37), gated on CD45+ cells, showed no difference between Sirt7−/− animals and littermates. C: Intracellular stainings for IL-17, IFN-␥ and TNF␣ showed similar characteristics in both groups for CNS and spleen CD4+ and CD8+ cells. D: FACS plots for FoxP3 of CNS cells gated on CD45+ and CD4+ cells. E: Intracellular staining of isolated CNS cells revealed that CD4+ cells had lower co-localization with the regulatory transcription factor FoxP3 in Sirt7−/− (*p < 0.05, unpaired two-sided students t-test). (n = 10 animals in A, n = 3-4 animals per group in B–E).

3. Results 3.1. Sirt7 does not influence disease course or prevalence of MOG35-55 -induced EAE To determine the impact of the sirtuine Sirt7 on neuroinflammation, we first backcrossed Sirt7+/− animals to a C57Bl/6 background and then used animals that were at least in generation five for this

study.1 After EAE induction with MOG35-55 , Sirt7−/− animals and littermates showed no difference in disease onset, disease severity or remission score for a total of 3 independent EAE experiments (n = 610 animals per group and experiment), which were terminated at different time points. Two of these disease courses are shown in

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All results in this study are part of the thesis of N. Burg.

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Fig. 3. A: The percentage of CD4+ cells from naïve mice after sorting was >95%. B: CSFE proliferation analysis of CD4+ cells isolated from healthy non-immunized animals revealed similar proliferation patterns for both groups (n = 8 per group from 2 independent experiments).

Figs. 1A and 2A , focusing on the acute and chronic phases of the disease, respectively. The prevalence of EAE, defined as a score of 1 or higher, was similar in Sirt7−/− animals and littermates. Thus, Sirt7 knockout did not influence the prevalence or disease course of our EAE model. 3.2. Sirt7−/− mice show subtle differences in peripheral cytokine profiles without impact on CNS inflammation in the acute phase of EAE To assess whether Sirt7 influences the characteristics of infiltrating CNS cells and immune cells in the periphery in acute EAE (day 21 of EAE, terminal experiment), cells were isolated from the CNS and spleen and stained for surface and intracellular markers. In the acute phase (Fig. 1), Sirt7-deficient animals and littermates showed a similar distribution of CD4+ lymphocytes, CD8+ lymphocytes, CD11b+ myeloid cells, CD11c+ dendritic cells, and CD45R B cells when gated to CD45+ leukocytes in FACS analysis (all FACS data acquired from n = 3-4 animals per group), both in the CNS and spleen (Fig. 1B). MHC II, an activity marker on e.g. CD11b+ and CD11c+ cells did not show any differences. In the acute phase, predominantly CD4+ cells and CD11b+ myeloid cells were present in the CNS, whereas in the spleen mainly CD11b+ myeloid cells and CD45R+ B cells were detected. The cytokine profiles of CD4+ cells did not reveal differences between Sirt7−/− and littermates (Fig. 1C). Interleukin (IL)-17 is the most abundant cytokine of CNSinfiltrating CD4+ cells, whereas TNF␣ (tumor necrosis factor alpha) is predominantly produced in peripheral CD4+ cells isolated from the spleen. A large percentage of CD4+ cells express FoxP3, a transcription factor with regulatory properties (Fig. 1E). Amongst the CD8+ cells, mainly interferon gamma (IFN-␥) and TNF␣ are produced at much higher percentages in cells infiltrating the CNS than those in the periphery. However, Sirt7−/− animals showed significantly lower percentages of IFN-␥-producing CD8 cells in the spleen compared to controls (Sirt7−/− : 4.17 ± 0.78%, littermates: 7.23 ± 0.93%, *p < 0.05, unpaired two sided students t-test, Fig. 1D) suggesting a modest shift of peripheral immune cell properties in this EAE model due to Sirt7 knockout. 3.3. Sirt7−/− animals do not show an accumulation of Tregs in the CNS in the chronic phase of EAE As seen in the acute phase, the distribution of cell types (CD4+ lymphocytes, CD8+ lymphocytes, CD11b+ myeloid cells, CD11c+ dendritic cells, and CD45R B cells) in the CNS or spleen did not differ between Sirt7-deficient animals and littermates in the chronic phase (Fig. 2B, day 37 of EAE, terminal experiment). In the CNS of both groups, mainly CD4+ lymphocytes, CD11b+ myeloid cells and CD45R+ B cells were found. In the spleen, cells from the adaptive immune system (CD4+ and CD8+ cells, CD45R+ B cells) were more prevalent. Only about 15% of CD4+ cells in the CNS produce IL-17

during the chronic phase while the percentage of TNF␣ producers increased in relation to the acute phase (Fig. 2C). The cytokine profile of CNS and peripheral CD8+ cells did not reveal any significant differences. Interestingly, the percentage of regulatory CD4+ FoxP3+ cells in the CNS in Sirt7-deficient mice was significantly lower than in littermates (Fig. 2D and E; Sirt7−/− : 31.55 ± 2.16, littermates: 40.87 ± 2.39, *p < 0.05, unpaired two sided students t-test). Thus, the normal recruitment of Tregs into the CNS during EAE (Korn et al., 2007; Koutrolos et al., 2014) in Sirt7−/− was not as efficient as in our littermates control (compare Figs. 1E and 2E). 3.4. No change in proliferation capacity of CD4-sorted cells in Sirt7-deficient animals To investigate if CD4+ cells, the prominent subset in experimental neuroinflammation (Siffrin et al., 2010), show a Sirt7-dependent proliferation in vitro, we performed a CSFE proliferation assay after a CD4 sort of cells isolated from spleens and lymph nodes. The purity of CD4+ cells after sorting was nearly 100% (Fig. 3A). The proliferation pattern of CD4+ cells did not differ between Sirt7−/− mice and littermates (n = 8 animals per group). Both groups showed around 10% of cells in the 4th generation (three divisions) after three days in culture with polyclonal anti-CD3 and anti-CD28 stimulation (Fig. 3B). Therefore, in vivo and in vitro proliferation of CD4 cells does not seem to be influenced by Sirt7. 3.5. Sirt7 positively regulates survival of adult-born hippocampal neurons and down regulates H3k18ac As for Sirt1 there have been reported effects on adult neurogenesis (Ma et al., 2014; Saharan et al., 2013), we assessed the influence of Sirt7 on adult hippocampal neurogenesis. First, we looked at neural proliferation in hippocampus (experimental design depicted in Fig. 4A, n = 3–4 animals per group) both in chronic EAE animals and healthy non EAE animals and found no significant difference in absolute numbers of BrdU positive cells per hippocampus in Sirt7−/− animals and their respective littermates control (Fig. 4B, Kruskal-Wallis analyses with Dunn’s post-hoc test). However, the proliferation in EAE animals was significantly decreased compared to non-immunized animals (2672 ± 203 vs. 1652 ± 175, **p < 0.01, two way ANOVA test) which is in contrast to existing literature on the influence of EAE on adult neurogenesis in wild-type animals (Giannakopoulou et al., 2016; Huehnchen et al., 2011). The percentages of BrdU and DCX or NeuN double positive cells did not differ between all groups (Fig. 4C and D, Kruskal-Wallis analyses with Dunn’s post-hoc test) although the absolute number of DCXgenerated cells was again significantly decreased in EAE animals compared to healthy non-EAE animals (1657 ± 147 vs. 1201 ± 105, *p < 0.05, two way ANOVA test). Similarly a tendency towards less NeuN-generated cells in EAE was observed, although not significant (p = 0.07).

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Fig. 4. Sirt7 and adult hippocampal neurogenesis A: Experimental design for investigating proliferation in the hippocampus. EAE animals (Sirt7−/− and littermates) received BrdU on day 34, 35 and 36 before they were sacrificed on day 37. Non-EAE age-matched animals (Sirt7−/− and littermates) also received BrdU on three consecutive days before they were sacrificed one day later (n = 3-4 animals for each group). B: Absolute BrdU+ cell numbers per hippocampus did not show differences between Sirt7−/− and littermates in EAE or healthy animals (Kruskal-Wallis analyses with Dunn’s post-hoc test). Pooled data from EAE and non-EAE animals revealed a significant decrease in EAE animals (**p < 0.01, two way ANOVA test). Middle and right panels show representative light microscope pictures of DAB BrdU staining of the hippocampus (scale bar = 150 ␮m) in non-EAE and EAE Sirt7−/− animals. C: Relative numbers of newly born BrdU+ and doublecortin (DCX) positive cells displayed no significant differences (Kruskal-Wallis analyses with Dunn’s post-hoc test); absolute DCX numbers are again reduced in pooled EAE animals (*p < 0.05, two way ANOVA test). D: Relative and absolute numbers of newly born neuronal nuclei (NeuN) positive cells showed no difference in the percentage of BrdU+ /NeuN+ double labeled cells but a tendency towards decreased absolute NeuN+ cells in pooled EAE animals compared to non-EAE although not significant (p = 0.07, two way ANOVA test). E: Confocal images for BrdU (green), DCX (red) and NeuN (cyan) in the dentate gyrus. ML-molecular layer, GCL-granular cell layer, SGZ-subgranular zone. Scale bar = 75 ␮m. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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Fig. 5. Survival and net neurogenesis in non-EAE Sirt7−/− A: Experimental design for assessment of survival and net neurogenesis in Sirt7−/− animals. BrdU injections took place on three consecutive days and mice were sacrificed on day 8 or 23. B: left panel Significant decrease in survival of BrdU+ cells per hippocampus was found in Sirt7−/− animals (588 ± 131 vs. 941 ± 41, *p < 0.05, unpaired two sided students t-test). No difference in net neurogenesis at day 23 (n = 5 animals per group). right panel percentage of BrdU+ cells that also express H3k18ac, the main target of Sirt7 with significant reduction in littermates (0.82 ± 0.023 vs. 0.46 ± 0.077, **p < 0.01, unpaired two sided students t-test). C: Confocal images for cells (Sirt7−/− animal from survival experiment) stained with BrdU, NeuN and H3k18ac (scale bar upper panel = 75 ␮m). Arrow head points at a cell that expresses BrdU, NeuN and H3k18ac at the lower panel (scale bar = 15 ␮m). D: The relative percentages of BrdU+ cells (from survival experiment) that also expressed DCX or NeuN (left part of diagram) were similar between knock out animals and littermates whereas absolute cell numbers of newborn (BrdU+ ) DCX and NeuN cells (right part of diagram) were decreased in Sirt7−/− animals (DCX 443 ± 96 vs. 749 ± 25, *p < 0.05, NeuN 516 ± 115 vs. 895 ± 31, n = 5 per group, unpaired two sided students t-test). E: Confocal micrographs (animal from survival experiment) of BrdU (green), DCX (red) and NeuN (cyan) in the dentate gyrus (upper panel, scale bar = 75 ␮m) and zoom of one BrdU+ cells which also expresses DCX and NeuN (lower panel, scale bar = 25 ␮m). F: Confocal micrographs (animal from neurogenesis experiment) of BrdU (green), GFAP (red) and NeuN (cyan) in the dentate gyrus with one enlarged BrdU+ cell which expresses NeuN but not GFAP. Scale bar upper panel = 30 ␮m, lower panel 10 ␮m. G: Co-staining for astrocytic marker GFAP and neuronal marker NeuN showed equal relative (left part) and absolute amounts (right part of diagram) of newborn cells in the hippocampus. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

To assess the impact of sirtuine 7 on survival and net neurogenesis of adult-born neurons in the dentate gyrus we conducted two additional experiments in non EAE animals (experimental design illustrated in Fig. 5A, n = 5 animals per group). The survival and net

outcome of initially labeled cells can be determined based on different intervals between BrdU injections and perfusion/analysis of animals. Here, we found a significantly decreased survival of adultborn neurons in Sirt7-deficient animals (588 ± 131 vs. 941 ± 41,

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*p < 0.05, unpaired two sided students t-test, Fig. 5B, left panel) after 8 days. However, net neurogenesis was unaltered and showed nearly identically results for newborn cells in Sirt7−/− and Sirt7+/+ littermates after 23 days. The percentage of BrdU+ cells (survival) that were acetylated at the histone protein H3k18, one of the main targets of Sirt7 (Barber et al., 2012) was significantly increased in Sirt7−/− as expected (Fig. 5B right panel and Fig. 5C). Immunofluorescent assessment of animals that had been euthanized 8 days after first BrdU injection (survival) revealed similar co-expression with DCX and NeuN whereas absolute numbers for newborn (BrdU labeled) DCX+ (443 ± 96 vs. 749 ± 25, * p < 0.05, unpaired two sided students t-test) and NeuN+ (516 ± 115 vs. 895 ± 31, * p < 0.05, unpaired two sided students t-test) cells were significantly reduced in Sirt7 deficient animals (Fig. 5D and E). 23 days after first BrdU injection only few newborn cells expressed the astrocytic marker GFAP with no differences between Sirt7−/− and littermates (Fig. 5F and G) indicating an unaltered differentiation.

4. Discussion Histone deacetylases such as the sirtuins have a broad impact on gene regulation and many cellular processes as they act as epigenetic regulators, for instance in neurogenesis (Ma et al., 2014; Saharan et al., 2013) and autoimmune diseases like MS (Tegla et al., 2014). Their ability to regulate proliferation and cytokine expression of immune cells suggests a prominent role in inflammatory diseases such as MS or EAE (Pedre et al., 2011). The known effect of histone deacetylases on oligodendrocyte differentiation and remyelination (Copray et al., 2009; Rafalski et al., 2013) could be used to modify disease progression in demyelinating diseases. As Sirt7−/− animals show enhanced inflammation in a mouse model for cardiomyopathy (Vakhrusheva et al., 2008) we addressed the question whether Sirt7 influences the process of neuroinflammation in EAE. Ultimately, we found no significant impact on disease course and prevalence of EAE. This is in clear contrast to Sirt1, which was shown to worsen EAE symptoms and whose knockout ameliorates disease symptoms (Lim et al., 2015). In the CNS, IFN-␥ exerts both regulatory and pro-inflammatory functions (Ottum et al., 2015). In our study, Sirt7−/− animals showed similar IFN-␥ levels in CD4+ and CD8+ cells in the CNS. However, we saw a reduction of IFN-␥ producing CD8+ cells in the spleen in Sirt7−/− in the acute phase which is interesting as peripheral IFN-␥ is necessary for proper blood-brain barrier functioning (Ni et al., 2014). Moreover, IFN-␥ deficiency leads to an exacerbation of disease symptoms in EAE and negatively regulates GM-CSF producing CD4 cells, underlining its anti-inflammatory properties in EAE (Chu et al., 2000; Codarri et al., 2011). On the other hand, IFN␥ and CD8+ cells are found in humans in MS lesions emphasizing their significant pathophysiological role in MS (Friese and Fugger, 2009; Traugott and Lebon, 1988). The reduced peripheral IFN-␥ in CD8+ cells in the acute EAE phase together with the fact that we found fewer FoxP3+ expressing Tregs in Sirt7−/− animals during the chronic phase in the CNS suggests a rather protective role of the epigenetic factor Sirt7 in EAE, although this was not reflected in the clinical score (Mangalam et al., 2014). FoxP3+ Treg cells suppress CD8+ effector cells and control the motility of effector T cells in the CNS. During EAE they accumulate in the CNS and limit the ongoing inflammation. In Sirt7−/− they failed to accumulate in the chronic phase which might be due to less migration through the blood brain barrier (Göbel et al., 2012; Korn et al., 2007; Koutrolos et al., 2014). These results are partially in line with the enhanced inflammation found in Sirt7−/− animals in the murine cardiomyopathy model (Vakhrusheva et al., 2008). However, there are also peripheral induced regulatory T cells which do not express FoxP3 (Korn et al., 2007; Pennati et al., 2016; Zohar et al., 2014). Those Tr1

cells and the net amount of secreted cytokines of regulatory T cells are increasingly recognized and should be investigated in future studies regarding Sirt7. Furthermore, rather general functions of Sirt7, for instance in tumor maintenance and chromatin remodeling, have been described (Barber et al., 2012). However, we here showed that the in vitro proliferation of CD4+ cells was not influenced by Sirt7 in healthy animals. Neuroinflammation, neurodegeneration and adult neurogenesis are processes which are partly linked (Ellwardt and Zipp, 2014; Huehnchen et al., 2011). Generation of new hippocampal neurons in adults facilitates learning and memorization (Kempermann, 2008) which might be relevant in neuroinflammation as neurons can undergo cell death (Siffrin et al., 2010). We found no difference in hippocampal proliferation between Sirt7−/− animals and littermates under either healthy or EAE-diseased conditions. However, Sirt7 promoted survival of new adult-born neurons in the hippocampus in healthy animals as the number of neurons in Sirt7−/− animals was 37% lower than in littermates. This was reflected in increased newborn DCX+ and NeuN+ cells in healthy littermates. At the same time the acetylation of the histone protein H3k18 was decreased in new born hippocampal neurons in littermate mice which dispose of Sirt7. H3k18 is one main target of Sirt7 (Barber et al., 2012). The overall net neurogenesis, measured 23 days after first BrdU injection, and the differentiation into neuronal and glial lineage were not affected. Therefore, the effect of Sirt7 on neurogenesis seems to be transient. Our findings are contrary to reported findings on Sirt1, which rather negatively regulates adult neurogenesis (Ma et al., 2014; Prozorovski et al., 2008; Saharan et al., 2013). The impact of NAD+ -dependent histone deacetylases like sirtuins on neurogenesis is evident, but each sirtuin has to be investigated separately. Interestingly, the cell numbers for hippocampal proliferation in EAE were decreased 38% compared to healthy non-immunized animals. So far, there was a positive effect reported on proliferation in EAE due to enhanced gliogenesis (Huehnchen et al., 2011) in wildtype animals. Contrary, general neuroinflammation has been shown to reduce neurogenesis (Monje et al., 2003). Qualitative stainings for the neuronal progenitor marker DCX and the mature neuronal marker NeuN did not reveal any differences between healthy Sirt7−/− animals and littermates or between EAE affected Sirt7−/− animals and littermates. However, the decreased number of newly born cells in EAE was also reflected in reduced numbers of DCX+ and NeuN+ cells. Taken together, we show that the epigenetic factor Sirt7 regulates cell differentiation and cytokine production especially by reduced peripheral IFN-␥ production and failed accumulation of regulatory T cells in the CNS in the EAE in the knockout model. This effect is not strong enough to be mirrored in the clinical course of EAE. Furthermore, Sirt7 positively regulates survival of adultborn neurons but does not impact the proliferation of hippocampal neurons. Thus, it can be concluded that the epigenetic factor Sirt7 influences the immune and nervous system, but it is too weak to be of modulating relevance in the clinical score in experimental autoimmune neuroinflammation. Funding This work was supported by the German Research Foundation (DFG; CRC-TR-128 to SB). The authors declare no relevant conflicts of interest. Acknowledgements We thank Christine Oswald, Heike Ehrengard and Jerome Birkenstock for excellent technical assistance and Cheryl Ernest for proofreading and editing the manuscript.

Please cite this article in press as: Burg, N., et al., Role of the epigenetic factor Sirt7 in neuroinflammation and neurogenesis. Neurosci. Res. (2017), http://dx.doi.org/10.1016/j.neures.2017.09.005

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Please cite this article in press as: Burg, N., et al., Role of the epigenetic factor Sirt7 in neuroinflammation and neurogenesis. Neurosci. Res. (2017), http://dx.doi.org/10.1016/j.neures.2017.09.005