CD200 is up-regulated in R6/1 transgenic mouse model of Huntington's disease

In Huntington’s disease (HD), striatal medium spiny neurons (MSNs) are particularly sensitive to the presence of a CAG repeat in the huntingtin (HTT) gene. However, there are many evidences that cells from the peripheral immune system and central nervous system (CNS) immune cells, namely microglia, play an important role in the etiology and the progression of HD. However, it remains unclear whether MSNs neurodegeneration is mediated by a non-cell autonomous mechanism. The homeostasis in the healthy CNS is maintained by several mechanisms of interaction between all brain cells. Neurons can control microglia activation through several inhibitory mechanisms, such as the CD200–CD200R1 interaction. Due to the complete lack of knowledge about the CD200–CD200R1 system in HD, we determined the temporal patterns of CD200 and CD200R1 expression in the neocortex, hippocampus and striatum in the HD mouse models R6/1 and HdhQ111/7 from pre-symptomatic to manifest stages. In order to explore any alteration in the peripheral immune system, we also studied the levels of expression of CD200 and CD200R1 in whole blood. Although CD200R1 expression was not altered, we observed and increase in CD200 gene expression and protein levels in the brain parenchyma of all the regions we examined, along with HD pathogenesis in R6/1 mice. Interestingly, the expression of CD200 mRNA was also up-regulated in blood following a similar temporal pattern. These results suggest that canonical neuronal–microglial communication through CD200–CD200R1 interaction is not compromised, and CD200 up-regulation in R6/1 brain parenchyma could represent a neurotrophic signal to sustain or extend neuronal function in the latest stages of HD as pro-survival mechanism.

Introduction HD is an autosomal dominant genetic disease caused by a CAG repeat expansion over 37 repeats in the HTT gene. Expanded CAG repeats are translated into a series of glutamine residues in the N-terminal region of the huntingtin protein producing a pleiotropic cellular impairment [1].
Although not yet well understood, MSNs in the caudate-putamen nuclei are the most severely affected type of cells in HD [2], resulting in the typical motor impairment known as chorea. However, in recent years it has been demonstrated that a broad neuronal alteration occurs in HD patient's brains. Indeed, HD causes neurodegeneration at a lesser extent also in cortical and hippocampal regions [3][4][5], which triggers cognitive impairment and psychiatric symptoms that precede motor dysfunction [6]. Although HD is considered a neurodegenerative disease, all cells in the organism are carrying the mutant Htt (mHtt) protein which may, in turn, alter the physiology of these cells. Peripheral immune system dysregulation produces an increased pro-inflammatory cytokine profile in pre-manifest HD patients, monocyte hyper-responsiveness [7] and migration/recruitment deficits [8]. In addition, kynurenine pathway inhibition in blood results in microglial de-activation in a HD mouse model with a reduced synaptic loss [9]. In the post-mortem HD brain, astrocytosis and microgliosis has been observed in caudate and the internal capsule with an increase complement biosynthesis by reactive microglia [10], which has been recently described as an important mechanism for early synaptic loss in Alzheimer's disease (AD) [11]. Similarly, microglia activation in HD patient brains is detected years before HD clinical manifestation by magnetic resonance imaging (MRI), allowing to predict disease onset and correlating with disease progression [12]. We recently showed that fingolimod (FTY720), a structural analog of sphingosine that act as an immunomodulatory drug for multiple sclerosis (MS), can also reduce astroglial reactivity in R6/1 mice acting through S1P receptor [13]. Hence, the peripheral immune system and specifically primed microglia activation are likely to play a significant role in neurodegeneration during HD pathogenesis as reported elsewhere [14]. Recently, microglial altered physiology has been proposed as a key factor in the etiology of depression [15], suggesting a multicellular approach to study the biology behind depression and alternative therapeutic strategies. Noteworthy, depression is one of the most common manifestations in the early stage of HD [16]. The highest societal burden associated with HD is due to psychiatric symptoms, which prevalence is estimated between 33% and 76% during disease progression in humans [17].
In normal conditions, neurons are constantly communicating with microglia about their status in order to maintain brain homeostasis [18]. Several cell populations communicate their state constantly in order to maintain the system stable [18][19][20]. Glial cells can sense neuronal activity in a paracrine manner and through cell-to-cell contacts. Microglia are constantly scavenging the brain parenchyma [21], sensing the surrounding environment for neuronal inputs. These inputs can be classified as "On" or "Off" signals depending on the microglial response they can induce [22]. Usually, the lack of "Off" signals determines microglial activation to reestablish brain homeostasis [22], which is a highly dynamic process in the CNS.
A well-known in vivo "Off" signaling system is the one between the transmembrane glycoprotein ligand CD200 (also known as OX-2), mainly expressed by neurons and endothelial cells, and its cognate receptor CD200R1 expressed by myeloid lineage cells, mostly microglia in the brain [23]. Some studies have also reported CD200 expression by oligodendrocytes and astrocytes in MS [24][25][26]. Interestingly, microglial CD200 expression has been reported only in the hippocampus of an excitotoxic kainic mouse model [27]. CD200 and CD200R1 are highly modulated during mouse CNS development [23], with CD200 usually showing a diffuse distribution in brain parenchyma and a higher intensity in grey matter compared to white matter areas, both in mice and humans [23,25]. Human and mouse brain express two isoforms as a product of an SF2/ASF-dependent alternative splicing mechanism of the CD200 mRNA, a full-length CD200 protein (CD200full) and a truncated isoform (CD200tr). Although CD200tr can bind to CD200R1, it does not activate the downstream signaling pathway, acting as physiological antagonist of the CD200full isoform [28,29]. Moreover, the Mus musculus Cd200r1 gene is translated into one protein while the human gene encodes four protein isoforms, with two of them lacking of transmembrane and cytoplasmic domains being secreted [30]. In activated mouse microglia, the downregulation of CD200R1 gene expression is regulated by CCAAT/enhancer-binding protein β (C/EBPβ) [31], while anti-inflammatory shift of microglia through CD200-CD200R1 is triggered by the signal transducer and activator of transcription 6 (STAT6)/forkhead box p3 (Foxp3) pathway [32].
Neuronal CD200 is a potent immunosuppressive molecule, in fact its decrease or complete absence induces microglial phagocytosis and pro-inflammatory activation [33,34], which has also been observed to impair hippocampal long term potentiation (LTP) [35] and blood-brain barrier permeability [36]. From a therapeutic point of view, the experimental use of CD200R1 agonists has proven its ability to tune down microglial innate immune response and neurotoxic side effects [37,38]. CD200 is also expressed by lymphoid cells in rats [39] and humans as part of the organism immune regulation [40].
Lack of information about neuronal-microglial communication in HD, and specifically about the CD200-CD200R1 system, prompted us to investigate expression of both CD200 and CD200R1 in HD mouse models. Since ovarian hormones can influence the expression of CD200 receptor in inflammatory conditions [41], we decide to perform the first characterization of CD200 system, in an HD context, in male mice only. Here, we examine the temporal patterns of CD200 and CD200R1 expression in neocortex, hippocampus and striatum of two HD mouse models providing further insight into the function of the neuroimmune system in HD. We also assessed the expression of CD200-CD200R1 in FTY720-treated animals, as we recently reported that the chronic treatment of this immunomodulating drug attenuates astrogliosis and prevents dendritic spines loss in the hippocampus of R6/1 mice [13]. We found a broad upregulation of CD200 expression in the R6/1 brain that increased with HD pathogenesis progression. Furthermore, we provide evidence that increased levels of CD200 in peripheral blood mimic with the increased CD200 levels observed in the CNS along HD pathogenesis.

HD mouse models
Male R6/1 transgenic mice expressing the human exon 1 of the mHtt gene under the control of 1 kb of its human promoter [42] and their corresponding wild-type littermates were obtained from the Jackson Laboratory (Bar Harbor, ME, USA) and maintained in a C57BL/ 6xCBA background. Genotypes were determined by PCR. CAG-repeat length was determined as previously described [1], and our R6/1 mouse colony carried 145 CAG repeats [43]. We also used HdhQ111/7 heterozygous mutant males and wild-type HdhQ7/7 knock-in mice (C57BL/ 6 background) generated by knocking-in the full-length chimeric human mHtt exon 1:mouse Htt under the endogenous mouse Htt promoter [44]. Mice were housed together in numerical birth order in groups of mixed genotypes, and data were recorded for analysis by microchip mouse number. The animals had access to food and water ad libitum in a colony room kept at 19-22˚C and 40-60% humidity, under a 12:12 h light/dark cycle. Animals were sacrificed by cervical dislocation and whole blood was quickly recovered after decapitation. Whole blood and brain samples derived from different animals. Analysis of FTY720 effects was performed on brain samples derived from FTY720-treated animals previously described [13].

Total protein extraction and Western blot
Total protein extracts were obtained after organic separation and homogenization of samples in TRI Reagent (T3809, Sigma-Aldrich) following the manufacturer's protocol. Protein quantification was determined by the Bradford assay (Bio-Rad Laboratories).

Immunohistochemistry
Immunohistochemical analysis was performed as previously described [13]. Animals were deeply anesthetized with pentobarbital and intracardially perfused with PBS and a 4% paraformaldehyde solution in 0.1 M sodium phosphate. Brains were removed and post-fixed overnight in the same solution, washed three times with PBS, cryoprotected with 30% sucrose in PBS and frozen in dry-ice cooled methylbutane (Sigma-Aldrich). Serial coronal sections (30 μm) of the brain were obtained using a Microm cryostate and collected in PBS as free-floating sections. The tissue was first incubated with a blocking solution containing PBS, 0.3% Triton X-100, and 5% normal goat serum (Pierce Biotechnology), for 2 h at room temperature.
Brain sections were then incubated overnight with shaking at 4˚C with the following primary antibodies diluted in the blocking solution: goat polyclonal anti-CD200 (1:200; R&D Systems) and mouse monoclonal anti-NeuN (1:100; Merck). After three washes with PBS, the tissue was incubated for 1 h 30 min at room temperature with specific fluorescent secondary antibodies: Cy2 donkey anti-goat (1:500) and Cy3 donkey anti-mouse (1:500) (Jackson ImmunoResearch). No signal was detected in control sections incubated in the absence of primary antibodies. Images at 10× and 40× magnification were acquired with a Leica SP5 confocal laser scanning microscope (Leica Microsystems).

Sampling and statistics
Data were analyzed using GraphPad Prism version 6.0c for Mac, GraphPad Software, La Jolla, CA, USA, www.graphpad.com. Outliers were identified through column analysis using a GraphPad integrated package. For the sample data reported here outliers were excluded and only biological replicas were considered. In CNS, the different proteins and the different genes were analyzed in the same set of samples. Samples were tested for normality, using D'Agostino-Pearson omnibus and Shapiro-Wilk normality tests, and equality of variance. Mann-Whitney U-test was performed for comparing samples that resulted having significantly different variances when an F-test was computed. Multiple t-tests were performed to test independent observations between two biological groups. A p-value < 0.05 was considered to be statistically significant.

CD200, but not CD200R1, gene expression is induced in R6/1 hippocampus and striatum concomitantly with motor symptoms' appearance
As CD200 protects from inflammation-mediated neurodegeneration [24] and has been recently shown to promote neuronal survival [47], we examined the expression of both CD200 and its receptor CD200R1 during HD pathogenesis in the telencephalon of R6/1 mice. The CNS regions mainly affected in R6/1 mice (i.e., the neocortex, hippocampus, and striatum) were analyzed by Q-PCR at pre-manifest stages of HD (12 weeks), when the HD motor and cognitive phenotype is evident (20 weeks), and at the latest disease stages (30 weeks).
Expression of the CD200 receptor, CD200R1, appeared unaltered in any of the regions analyzed at any time points between genotypes (data not shown). Whereas total CD200 transcript levels were significantly increased in the hippocampus and striatum at all time points, but not in the neocortex (Table 1).
In MS, CD200 can be expressed by activated astrocytes in the human [25] and mouse [24] CNS. Since we have recently shown that immunomodulating drug FTY720 can attenuate astrocytic activation in R6/1 mice [13], we investigated whether FTY720 chronic treatment in R6/1 mice may restore hippocampal and striatal CD200 gene expression. Interestingly, CD200 and CD200R1 mRNA levels were unmodified by FTY720 treatment in hippocampus and striatum in R6/1 mice (data not shown).

CD200 protein levels are elevated in late symptomatic stages of telencephalic regions in R6/1 mice
As the CD200-CD200R1 glycoprotein system has functional relevance at protein level triggering intercellular communication, we used samples prepared from neocortex, hippocampus and striatum from wild-type and R6/1 mice to analyze the expression CD200 and CD200R1 proteins by Western blot.
We studied samples from mice 8, 12, 20 and 30 weeks of age, spanning from asymptomatic to latest disease stages, and we found no differences in any region or time point for CD200R1 protein levels between wild-type and R6/1 mice (data not shown), supporting the Q-PCR results.
HD hippocampal and striatal regions showed higher levels of CD200 proteins at 30 weeks only. Whereas CD200 protein was significantly upregulated in neocortical samples of R6/1 mice from 20 weeks onwards (Fig 1A and 1B).

CD200 transcript levels are increased in peripheral blood of R6/1 mice
As CD200 is also expressed in B-and T-cells [40,49], we decided to examine CD200 mRNA expression in the whole blood of R6/1 mice along HD pathogenesis. First, we compared CD200 relative mRNA levels between brain (n = 6) and whole blood (n = 7) in wild-type mice. These analyses of CD200-CD200R1 in mouse blood showed that CD200 mRNA levels in the brain are significantly higher than in blood, which was not previously reported. We observed very low CD200 gene expression in peripheral blood compared to the brain (p = 0.0022) ( Fig  4A). Conversely, CD200R1 gene levels were similar (p = 0.23) between brain and whole blood of wild-type mice (Fig 4B).
https://doi.org/10.1371/journal.pone.0224901.g001 CD200 is up-regulated in HD MS, AD, epilepsy and Lewy body-associated dementia (see Table 2 for references). A dramatic reduction in CD200 expression levels is the common observation in most of the studies ( Table 2); whereas the expression levels of CD200R1 are altered in both directions. As reported in the literature, a biological interpretation of these results suggests that neuronal CD200 downregulation is a common feature of endangered neurons, which could activate microglia to stop disease progression. This appears to hold true from viral infection models that highlight the evolutionary conserved role of CD200, where its decrease triggers the innate immune response to stop the infection. In this view, neurodegenerative diseases are depend on the same innate immune pathway, chronically activating microglia through CD200 downregulation [50]. On the other hand, CD200R1 levels reflect different states of microglial activation. Furthermore, microglia react to the decrease of neuronal CD200 in a dynamic-and diseasedependent manner, computing an output between pro-and anti-inflammatory activation [51].
Worth noting, here we described a clear increase of CD200 levels in the neocortex and striatum of R6/1 mice with no alteration of CD200R1 expression. Together, these results suggest that a non-cell autonomous mechanism in HD differs from other neurodegenerative diseases in this animal model. To the best of our knowledge, only two studies showed a CD200 increase in mouse, one in a toxoplasma-associated encephalitis model [52] and another in an excitotoxicity mouse model [27]. Interestingly, excitotoxicity has been proposed as one of the driving MSNs neurodegeneration forces in HD [53]. In fact, mHtt MSNs are vulnerable to glutamatetriggered excitotoxicity [54] and it is suggested that an altered glutamatergic transmission in the cortico-striatal synapses can be involved in HD neurodegeneration [55]. However, R6/1 mice of 18 weeks of age have been shown to be resistant to excitotoxic insults [56]. Our findings not only suggest that increased levels of CD200 in the neocortex and striatum of R6/1 mice could reinforce the hypothesis of an active excitotoxic process in the cortico-striatal pathway, but also point to a neuroprotective role of CD200 towards neuronal excitotoxicity.

CD200 is up-regulated in HD
It is worth highlighting the differences between the two HD mouse models we screened, the human mHtt exon-1 R6/1 model and the full-length knock-in HdhQ111/7 model. Although R6/1 animals have shown a broad increase of CD200, HdhQ111/7 mice have shown only a transient upregulation in the striatum. Although we cannot exclude that CD200 can be modulated at older ages in HdhQ111/7 mice, differences between these two models in terms of behavioral and neuropathological symptoms have been reported elsewhere [58,59]. Among these differences, there are no studies describing a classical pro-inflammatory innate immune activation in R6/1 mice, while innate immune activation in YAC128 and R6/2 HD transgenic mice has been described previously [7,60]. In agreement with our results from R6/1 mice, our observations suggests that CD200 upregulation in the neocortex, and later in hippocampus and striatum, could mediate the resilience of this HD mouse model to show a clear pro-inflammatory microgliosis in the diseased parenchyma [24]. In addition, CD200 upregulation in R6/ week-old wild-type mice by qRT-PCR to compare mRNA levels between brain (n = 6) and whole blood (n = 7), and represented on a scatter dot plot (± SEM). (C) CD200 gene expression was measured in control and R6/1 mouse blood at 8 (wt = 5; R6/1 = 5), 12 (wt = 6; R6/1 = 6) and 25 (wt = 12; R6/ 1 mouse blood could also explain why these HD mice do not have a peripheral immune inflammation as shown by other HD mice models [7]. This hypothesis confirms the strong immunosuppressive and immunomodulatory properties of CD200 function, abundantly reported in the literature [61,62]. Further research in HD patients' bio-specimens or human multicellular stem cell-derived in vitro models may allow us to assess the relevance of CD200 in the human pathogenesis of HD.
In conclusion, here we described for the first time the temporal expression pattern of the CD200 and CD200R1 in the CNS and blood of the R6/1 HD model. In this study, we demonstrated that there is a transcriptional upregulation of CD200 from pre-symptomatic stages and an increase of the protein levels detectable only at the symptomatic phase of HD. No Literature search was performed using Scopus and the PubTator text-mining tool by PubMed, searching for "CD200" and "Brain". Articles focused on in vitro models, alterations of the myeloid receptor CD200R1 have been detected, suggesting that microglianeuronal cross-talk is not impaired. However, the extra CD200 ligand could function as a neurotrophic signal promoting survival of HD neurons. Hence, we suggest to further investigate CD200 as a possible pro-survival mechanism in HD pathogenesis in the R6/1 mouse model.