extfieldsofstudy: Biology Medicine

s2fieldsofstudy: Biology Chemistry

text:
Molecular Targets for PET Imaging of Activated Microglia: The Current Situation and Future Expectations

Microglia, as cellular mediators of neuroinflammation, are implicated in the pathogenesis of a wide range of neurodegenerative diseases. Positron emission tomography (PET) imaging of microglia has matured over the last 20 years, through the development of radiopharmaceuticals targeting several molecular biomarkers of microglial activation and, among these, mainly the translocator protein-18 kDa (TSPO). Nevertheless, current limitations of TSPO as a PET microglial biomarker exist, such as low brain density, even in a neurodegenerative setting, expression by other cells than the microglia (astrocytes, peripheral macrophages in the case of blood brain barrier breakdown), genetic polymorphism, inducing a variation for most of TSPO PET radiopharmaceuticals’ binding affinity, or similar expression in activated microglia regardless of its polarization (pro- or anti-inflammatory state), and these limitations narrow its potential interest. We overview alternative molecular targets, for which dedicated radiopharmaceuticals have been proposed, including receptors (purinergic receptors P2X7, cannabinoid receptors, α7 and α4β2 nicotinic acetylcholine receptors, adenosine 2A receptor, folate receptor β) and enzymes (cyclooxygenase, nitric oxide synthase, matrix metalloproteinase, β-glucuronidase, and enzymes of the kynurenine pathway), with a particular focus on their respective contribution for the understanding of microglial involvement in neurodegenerative diseases. We discuss opportunities for these potential molecular targets for PET imaging regarding their selectivity for microglia expression and polarization, in relation to the mechanisms by which microglia actively participate in both toxic and neuroprotective actions in brain diseases, and then take into account current clinicians’ expectations.

Microglia: From Resting to Activated Phenotypes
Microglia cells were first identified by Nissl in 1899, who already suggested that they can exert phagocytosis and migration [1]. Pio del Rio-Hortega, in 1921, defined them as cells with ramifications, able to transform after a pathologic event and to acquire ameboid morphology, as well as to migrate, proliferate, and exert phagocytosis [2]. Microglial cells are resident immune cells of the brain and the most important effector of brain innate immunity. Their capacities (motility, proliferation, phagocytosis, secretion of soluble molecules, etc.) are very close to those of macrophages, and microglial cells are often called "brain macrophages". Nevertheless, after 50 years of debate, it is now commonly recognized that microglia and macrophages have different genesis. Macrophages are produced in the bone marrow from the post-natal stage, whereas microglia are derived from yolk sac progenitors migrating in the neuroepithelium at the early stage of embryonic life [3,4]. Despite this difference, these two types of cells require the same proliferation and differentiation factors and share identical receptors (CD11b, CD14, etc.).
Microglial cells represent between 5% and 10% of adult brain cells. They are present in all brain areas, but with variable density, even in physiological conditions, up to 10-fold in the human brain [5]. Anatomically, microglia cells are more expressed in the telencephalon or diencephalon than mesencephalon [6]. Their repartition varies also between gray and white matter and according to axons' myelination, since myelinated parts of the brain have a higher density of microglial cells than non-myelinated areas in the same brain region [7]. A recent study in rhesus macaques points out that microglial cell density, especially in gray matter, is modified during the life-span and increases with aging [8]. Furthermore, as reported by Pintado and colleagues [9], in rats, following brain lipopolysaccharide (LPS) injection, differences in microglia brain distribution, density, and functionality were associated with different sites of injection. More recently, a genome-wide study on mice showed that microglia from several brain regions and at different ages presented differences of gene expression [10].
Kreutzberg [11] was the first to characterize microglia morphological changes occurring during microglia activation. In mature and healthy central nervous systems (CNS), microglia present a ramified morphology, with a small soma and thin cellular processes. This state corresponds to "quiescent" or "resting" microglia. Resting microglia express few surface markers and have been considered for years as functionally inactive. Nonetheless, in vivo study on microglia-GFP (green fluorescent protein) transgenic mice showed that these ramifications have a high motility allowing microglial cells to survey their environment in an active way [12]. Microglial cells are sensors of brain integrity, since any change of brain homeostasis will induce their activation through a modification of gene expression, morphology, and function [13,14]. Activated microglia show several morphological changes. First, the cell ramification number is increased and these ramifications are thicker, so that cells acquire an amoeboid phenotype, very close to peripheral macrophages [15].
For years, microglia activation was supposed to be a "yes or no" phenomenon, with cells only known to be resting or activated. In the last ten years, it has become clear that microglia activation processes are much more complex. As well as peripheral macrophages, microglial cell activation is a sequential process, leading to distinct phenotypes and functions of these cells depending on the stimulus leading to their activation [16,17]. The classification and characterization of the different subpopulation of microglial cells is based on these definitions for peripheral macrophages [18]. Classically or pro-inflammatory (M1) activated microglia are activated by LPS and interferon γ (IFNγ). M1 microglial cells secrete reactive oxygen species (ROS), as well as pro-inflammatory cytokines, such as interleukin (IL)-1β and tumor necrosis factor (TNF)-α. Their activation may lead to tissue inflammation [19,20]. Alternative or anti-inflammatory microglial cells (M2) are divided into three subpopulations. M2a cells are activated by IL-4 or IL-13 and produce growth factors (insulin growth factor (IGF)-1) and anti-inflammatory cytokines (IL-10). They participate in tissue reparation, and also have an important phagocytosis capacity, involving them in the cleaning of cellular debris [21,22]. M2b cells are induced by immune complexes, secrete IL-10, and regulate the immune response [23]. Finally, IL-10, or glucocorticoids, will lead to the M2c phenotype, also called "acquired deactivation", producing tumor growth factor-(TGF)β [24]. As brain immune cells, the principal function of microglia is to protect the brain against injury. They are the first actors in brain inflammation, even if other cells, mostly astrocytes, are also involved.

Involvement of Activated Microglia in Brain Disorders
Microglia activation is basically a beneficial phenomenon in response to neuron injury. Microglial cells phagocytose apoptotic neurons and secrete inflammatory factors to attract other immune cells to the site of injury. Inflammation resolution is well regulated and occurs when injury has been treated [25]. Nevertheless, it is now clearly established that neuroinflammation takes part in the process of most brain diseases, either in acute (traumatic brain injury (TBI), stroke) or in chronic, neurodegenerative diseases. Glutamate, adenosine triphosphate (ATP), chemokines, or superoxide are released by apoptotic brain cells (neurons, astrocytes) and lead to microglia activation through specific receptors.
In stroke or TBI, microglia activation appears in the hours following injury, release pro-inflammatory factors and chemoattractant molecules recruiting peripheral immune cells (macrophages and lymphocytes), and participate to neuronal death. Moreover, chronic neuronal death takes place in the weeks or months after these injuries and is strongly associated with microglia activation [26,27]. Molecules with anti-inflammatory effects have shown neuroprotection in preclinical models of stroke [28] and TBI [29]. These preclinical results lead to clinical study using anti-inflammatory molecules on stroke patients. Results of these clinical trials are variable and reviewed in Veltkamp and Gill [30].
In neurodegenerative diseases, the first involvement of neuroinflammation was described in Alzheimer's disease (AD) patients for whom activated microglial cells were detected in post-mortem nearby senile plaques [26]. Later, epidemiological studies suggest that long-term daily consumption of non-steroidal anti-inflammatory drugs (NSAIDs) reduce the risks of AD and Parkinson's (PD) diseases' development [27,[31][32][33]. Since then, neuroinflammation and microglia are a rich topic of research for neurodegenerative disease treatment and diagnosis. In these diseases, protein aggregates, such as Aβ-amyloid deposits in AD or α-synuclein in PD, will also activate microglia [34,35]. The persistence of these signals leads, in the long term, to an uncontrolled and unregulated microglia activation with high secretion of inflammatory factors which, in turn, will actively participate to neuronal death, because these cells are particularly sensitive to oxidative stress [36].
For years, targeting microglia in neurodegenerative diseases consisted in attempts to decrease cell activation and interesting results were obtained in preclinical models of AD, PD, and amyotrophic lateral sclerosis (ALS) [37][38][39][40][41] Nonetheless, targeting neuroinflammation in these diseases is even more complex and a regimen of anti-inflammatory treatments is also crucial. In fact, at the early stage of neurodegenerative diseases, microglia activation might exert a neuroprotective effect [51,52].

Imaging of Activated Microglia
Regarding this activated microglia involvement in neurodegenerative disorders, and its potential therapeutic impact [31][32][33], interest in the development of suitable imaging tools to investigate microglia as a relevant marker of neuronal damage and CNS activity [53] has risen in recent years. Thus, in vivo imaging of activated microglia can provide a non-invasive and reliable detection of early and localized neuroinflammation processes, thanks to the availability of several neuroimaging modalities. On one hand, MRI-based (magnetic resonance imaging) techniques for the detection of neuroinflammation have been developed, based on the macrophages' labelling by ultrasmall superparamagnetic iron oxide (USPIO) nanoparticles [54]. Targeting neuroinflammatory changes with USPIO has been used in the clinical setting, where no correlation was found between iron oxide-based enhancement and infarct size in human studies [55]. This approach reflects vascular integrity rather than microglial activation, and the specificity and accuracy of labelling strategies in molecular MRI remain to be validated. On the other hand, in vivo functional cerebral imaging of activated microglia has been widely explored by positron emission tomography (PET) molecular imaging, using sensitive radioactive probes targeting specific molecular mediators of the inflammation cascade (cell surface and mitochondrial receptors or transporters expressed in activated microglia). We will outline an overview of these molecular targets, focusing on their respective contribution for the understanding of microglial involvement in neurodegenerative diseases.

TSPO
The 18-kDa translocator protein (TSPO) is a hetero-oligomeric complex located on the outer mitochondrial membrane known to be involved in modulating immune response, in cholesterol transport, and in heme/steroid synthesis [56]. TSPO was first described as a peripheral benzodiazepine receptor (PBR), a secondary binding site for diazepam, but has been renamed to TSPO to reflect some of these cellular functions [57]. This protein was initially found in peripheral organs (i.e., kidneys, nasal epithelium, adrenal glands, lungs, and heart), but is also minimally expressed on microglia in healthy brains [58]. However, TSPO expression is dramatically upregulated during the microglia activation process. Its basal expression rises in several acute and degenerative disorders, including AD, PD, MS, Huntington's disease (HD) [59], and amyotrophic lateral sclerosis (ALS) [60]. As a result, TSPO has been considered a hallmark of neuroinflammation. Therefore, TSPO PET imaging has been used for both improving the knowledge regarding the role of neuroinflammation in CNS diseases and to assess the efficacy of novel anti-inflammatory therapeutic strategies. The most widely used TSPO PET radiopharmaceutical, namely 11 C-(R)-PK11195, is an isoquinoline carboxamide developed in the early 1980s [61]. However, 11 C-(R)-PK11195's clinical usefulness is narrowed by several major limitations, including the short half-life of carbon-11, a low brain bioavailability and a poor signal-to-noise ratio due to high nonspecific binding [62]. To counteract these drawbacks, there has been a great amount of effort toward the development of second-generation TSPO PET radiotracers [63][64][65], including 18 F-FEDAA1106, 11 C-PBR28, 11 C-DPA-713, and 18 F-DPA-714. TSPO PET radioligands made it possible to characterize in vivo, in numerous preclinical and clinical studies, the neuroinflammatory component of neurodegenerative disorders its spatial distribution, its intensity, and its longitudinal evolution. Thus, TSPO PET imaging is, nowadays, widely recognized as a useful biomarker of activated microglia involvement in CNS disorders that assisted in the early detection of neuroinflammation, monitor the severity and progression of the neurodegenerative diseases, and help to consider the effectiveness of emerging CNS therapies aimed at decreasing neuroinflammation.

TSPO Limitations as a Molecular Target for Activated Microglia PET Imaging
Several drawbacks limit the ability of TSPO as PET imaging molecular target. First, a genetic polymorphism in exon 4 of the TSPO gene (rs6971) has been identified, resulting in an alanine-to-threonine substitution (A147T) [66,67]. This polymorphism affects the binding affinity properties of most of PET TSPO radiopharmaceuticals for their target. Sensitivity to TSPO polymorphism is variable depending on the tracer considered , resulting in a very large heterogeneity in PET images and their associated quantitative data [66]. Three distinct binder statuses have been identified: HAB, high-(A/A;~70%), MAB, mixed-(A/T;~21%), and LAB, low-affinity binders (T/T; 9%). Then, this polymorphism, and its consequent binder status, can be identified by genetic analysis allowing stratification of subjects, and can subsequently account for binder status in the quantification of TSPO PET studies using second-generation radiotracers [68]. However, in LAB patients, TSPO PET images are of significantly lower quality, and the clinical usefulness of this approach is, therefore, limited in these patients. Even if the conclusions of clinical studies performed only on HAB or MAB patients might be extended to a whole-patient population, at least in AD [69], this polymorphism makes more difficult the design of clinical trials based on TSPO PET imaging. A third-generation TSPO radioligand, namely 11 C-ER176, sensitive to polymorphism in vivo but allowing quantification in LAB patients, has recently been proposed; nonetheless, the clinical relevance of this compound remains to be confirmed [70]. Secondly, the mathematical model usually applied to quantify a brain PET radioligand binding requires either serial blood samples or a reference region free of specific ligand binding. However, TSPO is distributed throughout the entire brain, even at very low density, and no clear reference region may exist in neurodegenerative diseases. However, the cerebellum was validated as a reference region for 11 C-PBR28-TSPO binding in AD patients [71], but regarding differences between radiotracers' pharmacokinetics and the variety of TSPO distribution depending on the considered disease, it is necessary to independently validate a reference region for each radiotracer and for each disease.
A third concern for the meaning of this PET imaging approach is the multicellular expression of TSPO in the human brain. In addition to activated microglia, astroglial expression of TSPO, and, in the case of a disrupted blood brain barrier (BBB), on infiltrating cells of mononuclear-phagocyte lineage have been reported [72]. TSPO in vivo PET imaging does not strictly reflect the activation of microglial cells, but a broader inflammatory process [73]. The PET signal can also be disrupted by TSPO peripheral vascular endothelial cells' expression, which, together with TSPO radioligands' plasma protein binding, impacts a partial volume effect, especially for cortical areas close to large blood vessels. Thus, Rizzo et al. have studied the positive impact of considering a model that includes an additional irreversible compartment from the blood to the endothelium (vascular component) on the quantification of 11 C-PBR28 data, compared to the standard two-tissue compartmental model (2TCM) [74]. Authors demonstrated that the inclusion of the vascular component in the kinetic model (2TCM-1K) provided a more precise and accurate quantification of 11 C-PBR28 brain PET data. The estimates are more than three-fold smaller, have a higher time stability and are better correlated to brain mRNA TSPO expression with 2TCM-1K model compared to 2TCM [74].
Lastly, since the characterization of the different subpopulations of microglial cells toward different phenotypes (M1, neurotoxic vs. M2, neuroprotective, see Section 1), it has been challenging to identify and differentiate these subtypes and their related pro/anti-inflammatory roles for both physiopathological understanding and new therapeutic approach designs. While the advent of TSPO PET agents has enabled the distribution of activated microglia in the brain to be imaged in vivo, TSPO ligands bind to both M1 and M2 phenotypes. TSPO PET, therefore, provides a measure of activated microglia load without information related with its specific functional role in different diseases and brain areas [75,76].

Potential Alternative Molecular Targets for Activated Microglia Imaging
Limitations of the TSPO, as described above, have led to the identification of other molecular targets to develop new tracers of activated microglia. Moreover, in order to fulfill the current clinician's expectancies regarding the microglial role in neurodegenerative diseases and associated potential novel therapeutic approaches, radiopharmaceuticals able to discriminate activated microglia according to their polarization from M1 to M2 phenotypes are, to date, highly expected. In this part, we summarize potential microglial imaging targets as follows: (1) Targets evaluated by PET in pre-clinical or clinical settings for imaging microglial cells within the CNS; (2) Targets that have been used for imaging inflammatory conditions by PET in peripheral disorders; and (3) Potential targets for which radioligands have not been tested/synthetized yet. We will also pay particular attention to the potential interest of these targets to discriminate microglia subtypes (see Table 1). However, no in vivo study using any PET radioligands allowed the study of only one subpopulation of microglia. Targeting these molecules for PET imaging of M1 or M2 microglia requires further exploration. Table 1 summarizes candidate microglial imaging targets, for which dedicated PET radiopharmaceuticals have been developed and evaluated in pre-clinical and/or clinical CNS settings. Cyclooxygenase is an enzyme producing important biological mediators, including prostaglandins, which are involved in the regulation of neuroinflammatory process in connection with neurodegenerative diseases [90][91][92].

Molecular Targets Evaluated in CNS Diseases
Among the different COX isoforms characterized, COX-1 is classically described as a constitutively expressed house-keeping enzyme, whereas COX-2 is an inflammatory inducible isoform, mainly expressed in response to neuroinflammation [93]. COX-1 immunoreactivity is enriched in the midbrain, pons, and medulla [94], whereas COX-2 immunoreactivity prevails in neurons and glial cells of the hippocampus, hypothalamus, and amygdala [95,96]. COX-2 is, therefore, considered as a key player in the pathophysiology of AD, PD [97,98], and has been identified as a molecular target of interest for pharmacological design of selective ligands for both therapy and molecular imaging. Thus, highly-selective COX-2 radioligands, such as 11 C-Celecoxib [99] or 11 C-Rofecoxib [77], have been evaluated to explore microglial activation by PET, but to date this approach remains unsuccessful, due to either non-specific bindings or low in vivo sensitivity of these radioligands [100,101]. In this context, the development of a selective COX-1 imaging probe has regained interest. The 11 C-ketoprofen methyl ester ( 11 C-KTP-Me) has been evaluated in rodent models of focal neuroinflammation (intrastriatal injection of lipopolysaccharide or quinolinic acid), and exhibited striatal accumulation corresponding to the early phase of microglia activation (from six hours and at day 1 after the lesion) [102]. In contrast, the time course of striatal accumulation of 11 C-PK11195, the gold-standard TSPO PET radioligand, started later and lasted up to 14 days afterward, corresponding to changes in activation of both microglia and astrocytes. This finding suggests the high specificity of cellular expression of COX-1 within microglia, during an acute neuroinflammatory process, and its ability to be evidenced by PET in vivo. More recently, the same group used 11 C-KTP-Me to investigate COX-1 involvement in amyloid precursor protein transgenic (APPSWE2576) mice, an animal model of AD [78]. PET images of (S)-11 C-KTP-Me specifically detected and clearly visualized the changes in COX-1 expression in activated microglia concomitantly to the formation of amyloid plaques in amyloid peptide precursor-transgenic mice (APP-Tg) mice. These preclinical data suggest that PET imaging of COX-1 with (S)-11 C-KTP-Me could be a promising approach for monitoring activated microglia in CNS diseases, including AD.

Cannabinoid Receptor
While the cannabinoid receptor type 1 (CB1R) is constitutively the most abundantly expressed G-protein-coupled receptor in the human brain [103], the inducible isoform, namely cannabinoid receptor type 2 (CB2R), is barely detectable in the healthy brain [104]. Low levels of CB2R expression have been found in microglial cells [104,105], in human fetal astrocytes [106], and in human cerebral microvascular endothelial cells [107]. Nonetheless, several studies reported an upregulation of CB2R on activated microglial cells in pathological conditions, including MS, ALS, PD, or AD [105,[108][109][110]. Moreover, selective CB2R activation results in a decrease of microglial activation in HD and ALS transgenic mouse models and appears to be effective in reducing neurodegeneration [109,110]. Neuroprotective effects of CB2 agonists are associated with suppression of microglia activation via inhibiting the release of neurotoxic factors and by decreasing neuronal cell damage in cell or tissue culture models [111]. These observations suggest that therapeutic modulation of CB2R may be a new promising treatment for neuropathogenic disorders characterized by a neuroinflammatory component. Recent findings have indicated that nicotine attenuates Aβ-induced microglial activation by shifting microglial M1 to M2 state, and cannabinoid CB2R mediates the process, thereby suggesting the CB2R involved in microglia polarization shift [112]. Several CB2R selective ligands have been developed over the past years [113] and the preliminary clinical evaluation with 11 C-NE40 showed appropriate fast brain kinetics in the healthy human brain [114]. However, 11 C-NE40 has not succeeded in highlighting microglial activation in AD subjects as compared to healthy controls. In 2015, Slavik et al. reported a novel carbon-11 radiolabeled tracer 11 C-RS-016 for CB2R imaging, which showed higher specific binding in postmortem ALS patient spinal cord tissues [79,80]. Since then, several other recently synthetized radiotracers are under preclinical investigations and seem to offer promising prospects for imaging CB2R expression [115][116][117].

Purinergic Ion Channel Receptor
Purinergic ion channel receptor (P2X) is a large family of receptors distributed in a wide variety of tissue [118]. Among them, P2X7 receptor (P2X7R) are expressed both peripherally and in the CNS, especially in microglia, astrocytes and Schwann cells [119]. P2X7R activation is associated with production of pro-inflammatory cytokines (IL-1β) and ROS by peripheral macrophages, as well as microglia or astrocytes [120,121]. Moreover, P2X7R expression is increased in microglia in animal models of neurodegenerative diseases, such as AD [122], ALS [123], or HD [124]. Recently, P2X7R has been proposed as a marker of M1 microglia. Indeed, in a mouse ALS model, P2X7R inhibition led to a diminution of microgliosis associated with a decrease of M1 (IL-1β) and an increase of M2 (IL-10) markers, leading authors to consider P2X7R as a potential maker of M1 microglia in ALS [125]. Involvement of P2X7R in microglia M1 polarization has recently been confirmed in vitro as P2X7R inhibition avoid M1 microglia polarization in ischaemic conditions [126]. Nevertheless, in vitro, P2X7R expression is also reported in M2 polarized macrophages and might so play a role in inflammation resolution [127].
The first candidate was the P2X7R antagonist A-740003, showing a high affinity and selectivity for the receptor. Preclinical in vivo study of 11 C-A-740003 showed little uptake in rat brains [81]. More recently, two other P2X7R antagonists has been radiolabeled and used in preclinical models. 11 C-JNJ-54173717 was shown to cross the BBB in rats and to have a higher binding in rat striatum injected with a viral vector expressing human P2X7R than in control rats. This compound was also used in monkeys and showed a specific binding to P2X7R, as the concomitant use of the JNJ-42253432, a P2X7R antagonist, completely block the brain fixation of the radiolabeled compound [128]. Anyway, the use of 11 C-JNJ-54173717 in a model of neuroinflammation has to be performed in order to validate its utilization as a marker of activated microglia. The other molecule recently tested is the GSK1482160, a strong P2X7R antagonist, evaluated in a phase 1 clinical study with a good BBB penetration [129]. The GSK1482160 has, thus, been labelled with carbon-11, and then evaluated in mice treated by LPS as a model of neuroinflammation, showing a significate increase of 11 C-GSK1482160 binding in treated mouse brains vs. control [82]. Therefore, 11 C-GSK1482160 appears as a promising radioligand of P2X7R, as a marker of neuroinflammation.

β-Glucuronidase
β-Glucuronidase is a lysosomal enzyme involved both in the hydrolysis of glycosaminoglycans on the cell surface and in the degradation of the extracellular matrix. Several studies have reported an increase of β-glucuronidase expression by activated microglia into the extracellular space at the site of neuroinflammation [130,131]. Elevated levels of β-glucuronidase have been reported in the temporal cortex of AD patients and in the putamen of HD patients [132] and, thus, constituting a biomarker of neuroinflammation in relation with neurodegenerative diseases. Antunes et al. [83] have designed a PET tracer for β-glucuronidase imaging, namely 18 F-FEAnGA, that, despite a moderate brain uptake, succeeded in detecting an increased release of β-glucuronidase during neuroinflammation in an encephalitis rat model.

Adenosine Receptor 2A
Adenosine receptors belong to the purinergic G-coupled family receptors and are involved in inflammatory processes. Microglia express several types of adenosine receptors (A1, A2A, A2B, A3). Among them, the 2A adenosine receptor (A2AR) seems to have an important implication in neurodegeneration [133], is overexpressed in vitro in activated microglia, and is also involved in the regulation of microglia activation [134,135]. A2AR has been considered an interesting target of activated microglia.
Several tracers of A2AR have been synthetized ( 11 C-TMSX, 11 C-preladenant, 18 F-FESCH) and clinical studies have been performed with the 11 C-TMSX [84,136,137]. 11 C-TMSX showed that A2AR is increased in MS patients, in association with neuroinflammation, with a binding correlated with the severity of symptoms and the loss of cerebral tissue [85]. However, in PD patients, the uptake of this tracer is not related to neuroinflammation, but to neuronal regulation [84]. A2AR are expressed both in neurons and astrocytes but, regarding their brain distribution, they are strongly expressed in the striatum (post-synaptic neurons) [84,138,139]. This striatal neuronal expression probably explains results obtained in PD patients [84]. However, A2AR's increase in other brain areas may be relevant to follow brain inflammation elsewhere than in the striatum and might be therefore an interesting tool in neurodegenerative diseases such as MS.

Nicotinic Acetylcholine Receptors α4β2
Nicotinic acetylcholine receptors (nAChR) are pentameric ligand gated ion channels. In the brain, several subtypes of nAChR have been identified and, among them, the heteromeric α4β2 is one of the most abundant. α4β2 receptor expression in microglia is not well characterized yet. The 2-18 F-fluoro-A85380 compound has been synthetized in order to follow α4β2's expression in neurodegenerative diseases [140]. Ex vivo studies with this tracer showed a decrease of binding in brain sections from AD patients [141], making this radiotracer a tool to follow the death of cholinergic neurons in neurodegenerative diseases. More recently, Martín et al. [86] used 2-18 F-fluoro-A85380 as a marker of neuroinflammation in a model of cerebral ischemia. Their results showed an uptake of this tracer similar to the one of PK11195 (for TSPO PET imaging), confirming the inflammatory expression of α4β2 in this model. Moreover, authors confirmed by immunohistochemistry the overexpression of α4β2 in microglia and astrocytes. Nonetheless, this overexpression of α4β2 in activated microglia is not well characterized yet and the neuronal expression of α4β2 is a limiting factor for the use of the nAChR tracer to follow microglia activation in vivo.

Matrix Metalloproteinases
Matrix metalloproteinases (MMPs) are a family of endopeptidases able to degrade the components of the extracellular matrix (collagen, gelatin, elastin) and are involved in tissue remodeling and degradation. Several subtypes of MMPs have been identified and their expression is enhanced by pro-inflammatory signals, such as cytokines (IL-1β, TNFα) or LPS. MMPs are associated in the CNS to excitotoxicity, neuronal damage, and BBB disruption, but also to the progression of neurodegenerative diseases [142,143]. Several tracers of MMPs or specific to one MMPs subtype have been synthetized in the last years.
Among them, the 18 F-BR-351, derived from a non peptidic MMP inhibitor, was used in a rat model of stroke by transient middle cerebral artery occlusion (tMCAo) [88,144]. In this study, PET imaging of MMPs and TSPO were performed with the 18 F-BR-351 and the 18 F-DPA714, respectively. Authors showed time course expression of MMPs and TSPO following tMCAo by PET imaging and confirmed the expression of MMP-9 in microglia in the infarct brain part by immunofluorescence. The same compound has very recently been used in a mice model of gliomas [145]. In parallel, other MMPs inhibitors, specific of one or several MMPs, have been labeled with 18 F or 11C and showed brain uptake in small rodent biodistribution. These tracers have not been tested in a pathological model yet [146,147].

Molecular Targets Evaluated in Other Inflammatory Diseases
Even if not tested for microglia imaging yet, the molecular targets cited here might be promising for PET microglia imaging (Table 2). For these targets, PET ligands have been developed and tested in peripheral inflammatory conditions (i.e., macrophages expression). Some of these targets could be specific to the M1 or M2 phenotypes, but this specificity has never been demonstrated by in vivo imaging. Furthermore, the capacity of their ligands to cross the BBB must be studied before any application related to microglia activation.

Inducible Nitric Oxide Synthase
Nitric oxide (NO) is a ubiquitous cellular messenger, involved in several physiological processes in peripheral systems, but also in the brain. NO production by immune cells (macrophages, microglia) participates in innate responses, and especially in cell death, through the inhibition of mitochondrial respiration [156,157]. The inducible nitric oxide synthase (iNOS) that produces NO is not expressed in the brain, or at very low concentration, in physiological conditions but, in inflammatory conditions, iNOS is overexpressed in microglia and astrocytes [158]. iNOS is also considered as specific of a M1 phenotype in both macrophages and microglia [148,159]. iNOS macrophage's expression in inflammatory condition has been assessed in vivo using the 18 F6-(2-fluoropropyl)-4-methyl-pyridin-2-amine, both in mouse and human. It was tested in vivo in a mouse model of iNOS induction by LPS injection. Results showed a significantly higher uptake of the tracer in the lung of LPS mice vs. control [160]. More recently, the same compound, renamed 18 F-NOS, was used in healthy volunteers for the first in-human evaluation. A comparison was made on subjects after endotoxin administration in the right lung, which evidenced an increase of 30% of 18 F-NOS intake in this lung vs. the left one [149].

Folate Receptor β
Folate receptor (FR) is a family of four receptor subtypes (α, β, γ or δ) that bind folic acid. FRβ expression is described in activated macrophages in the model of inflammatory diseases (rheumatoid arthritis, Crohn's disease, etc.) [161]. FRβ is not expressed in quiescent or resting microglia. As FRβ award macrophages to internalize molecules derived from folic acid, the development of tracers derived from folic acid could allowed the following of activated macrophages and microglia [162]. Moreover, in vitro studies showed that FRβ is specifically expressed by M2 polarized macrophages [163]. In an in vivo rat model of restraint stress MacDowell et al. [164] used FRβ as a specific marker of M2 microglia. Nonetheless, this seems to be the only publication describing FRβ as a specific marker of M2 microglia. On the other hand, a study on macrophages isolated from mice after bacterial infection showed a correlation between FRβ expression, ROS production, and TNFα secretion, both of which were more specific to an M1 phenotype [165]. FRβ might be involved in microglia M1/M2 polarization, but extended studies on this receptor expression in microglial cells are still required to assess its interest for PET microglia imaging.
Kularatne et al. [150] developed two conjugated molecules derived from folic acid for FRβ PET imaging: 4-18 F-fluorophenylfolate and 68 Ga-DOTA-folate. They tested these compounds to follow macrophages activation in a model of paw inflammation in the rat and showed that uptake of both radiotracers was increased in the inflamed paw. Another compound, 18 F-fluoro-PEG-folate, also gave interesting results in a rat model of rheumatoid arthritis [151]. If no clinical studies using PET tracers of FRβ have been performed yet, a single-photon emission computed tomography (SPECT) tracer, 9m Tc-EC20, was used on patients with rheumatoid arthritis [166].

Potential Targets of Microglia Activation
In this part, we regroup targets expressed in activated microglia but for which no radiotracer has been synthetized or tested yet ( Table 2). More investigations are needed to assess in vivo expression of these targets in activated microglia. The kynurenine pathway (KP) mediates the tryptophan catalyzation in both periphery and CNS. KP is stimulated by inflammatory molecules, such as IFN-γ, and, in the brain, products of KP have been identified to be neuroprotective (picolinic acid, kynurenic acid) or neurotoxic (quinolinic acid, 3-hydroxykinurenine) [167]. Indoleamine 2,3-dioxygenase-1 (IDO-1) is one of the limiting enzymes of KP, is expressed in immune peripheral cells and, in the brain, in microglia cells, astrocytes, and neurons. IDO-1 is strongly induced in primary microglial cells by LPS and IFN-γ [168] and is overexpressed during neurodegenerative disease processes, such as for MS [169], AD [170], and PD [171]. Furthermore, in AD patient brains, IDO-1 immunoreactivity is increased in microglia and astrocytes [172], making IDO-1 a serious candidate to follow microglia activation in vivo.
Several analogues of tryptophan have been synthetized, labelled, and used in vitro or in animal models with tumor grafts [173]. These molecules are promising to follow tryptophan metabolism but are not specific of IDO expression. Huang et al. [152] used another approach to develop a tracer of IDO-1 by using IDOL5, a strong antagonist of IDO-1. They successfully labeled IDOL5 with 18 F but this potential tracer has not been evaluated neither in in vitro nor in vivo tests so far.
More recently, the kynurenine-3-monooxygenase (KMO), another enzyme involved in KP, has been described as regulated by proinflammatory cytokine signals. In vivo, in the brain, KMO is predominantly expressed in microglia [153] and is involved in the regulation of quinolinic acid production. KMO is overexpressed, in vitro, in macrophages and microglia activated by IFN-γ. KMO is also increased by LPS treatment in rat brains. KMO could be an interesting target to follow microglia activation, but its characterization requires more investigation.

P2Y12 Receptor
The P2Y12 receptor (P2Y12R), a purinergic G-protein-coupled receptor, is exclusively expressed in microglia in the CNS. This receptor is not found in peripheral macrophages and is, thus, a good marker to distinguish resident microglia from infiltrated macrophages [174]. Expression of P2Y12R in activated microglia is still hard to qualify but showed that in vitro M2-polarized microglia overexpressed P2Y12R [155]. On the other hand, pathological conditions, such as AD, are associated with a decrease of P2Y12R expression in microglia, notably near the plaques or lesion sites, compared to microglial cells in other brain areas [174,175]. This decrease of P2Y12R expression in such inflammatory conditions (i.e., the presence of proteins able to activate the toll like receptor 4 (TLR4) pathway) may be associated with an increase of M1, and a diminution of M2, microglial cells in these areas. This hypothesis is supported by the P2Y12R expression in microglia of brains with parasite infections, a condition known to privilege M2 polarization of immune cells [155]. P2Y12R may, thus, be a promising target of M2 microglia but further studies are needed to confirm its in vivo expression in this microglia subpopulation.

Conclusions
It is, to date, broadly recognized that neuroinflammation, and in particular microglia activation, plays a crucial role in various brain disorders, from acute (stroke, TBI) to chronic (neurodegenerative disorders) ones. Initially, the large body of post mortem evidence of activated microglia in various CNS conditions led most of the authors to consider the microglial cells' shift from sensing activity to a reactive state as a deleterious process. This microglial activation has been first assessed in vivo thanks to PET radioligands targeting TSPO. Polymorphism and multicellular expression of TSPO, as well as the lack of a specific brain region of negative control, led to identifying other molecular biomarkers of activated microglia that would likely be complementary to TSPO PET imaging. Among the targets of interest summarized in the present review, P2X7 receptor appears to be the most promising one regarding: (1) its implication in neurodegenerative diseases' pathophysiology, highlighted by numerous current clinical trials aiming at evaluating P2X7 antagonists in CNS indications [176]; (2) the recent development of efficient dedicated radiopharmaceuticals that are currently coming available for clinical trials; (3) its implication on M1 microglial polarization. Thus, a multi-targets approach for PET imaging of microglia activation, combining TSPO radiopharmaceuticals with new probes specific of P2X7 expression may help to characterize the involvement of neuroinflammation over the CNS disorder's progression, as well as to follow the effects of clinical treatments on microglia polarization. Author Contributions: Claire Tronel wrote the manuscript, prepared the tables and had a major role in the selection and summary of published literature to which reference is made in this review. Bérenger Largeau wrote the manuscript and participated in the selection and summary of published literature to which reference is made in this review. Anne-Claire Dupont contributed in improving the article, notably according to the reviewers' comments and participated in the selection and summary of published literature to which reference is made in this review. Maria-Joao Santiago Ribeiro and Denis Guilloteau revised the paper. Nicolas Arlicot conceived the idea and designed the paper.


s2fieldsofstudy: Biology Medicine

text:
Myocardial Expression of Macrophage Migration Inhibitory Factor in Patients with Heart Failure

Macrophage migration inhibitory factor (MIF) is a pleiotropic inflammatory protein and contributes to several different inflammatory and ischemic/hypoxic diseases. MIF was shown to be cardioprotective in experimental myocardial ischemia/reperfusion injury and its expression is regulated by the transcription factor hypoxia-inducible factor (HIF)-1α. We here report on MIF expression in the failing human heart and assess myocardial MIF in different types of cardiomyopathy. Myocardial tissue samples from n = 30 patients were analyzed by quantitative Real-Time PCR. MIF and HIF-1α mRNA expression was analyzed in myocardial samples from patients with ischemic (ICM) and non-ischemic cardiomyopathy (NICM) and from patients after heart transplantation (HTX). MIF expression was elevated in myocardial samples from patients with ICM compared to NICM. Transplanted hearts showed lower MIF levels compared to hearts from patients with ICM. Expression of HIF-1α was analyzed and was shown to be significantly increased in ICM patients compared to patients with NICM. MIF and HIF-1α mRNA is expressed in the human heart. MIF and HIF-1α expression depends on the underlying type of cardiomyopathy. Patients with ICM show increased myocardial MIF and HIF-1α expression.

Introduction
Heart failure remains one of the most prevalent and challenging medical conditions with high morbidity and mortality despite advances in treatment [1]. Heart failure develops when a cardiac injury or insult impairs the ability of the heart to pump blood and maintain tissue perfusion [2,3]. A possible way to classify heart failure with reduced ejection fraction is to distinguish between ischemic (ICM) and non-ischemic cardiomyopathy (NICM) [1]. The most obvious difference between ICM and NICM is the existence of atherosclerotic lesions of the coronary arteries in patients with ICM and the absence of such lesions in patients with NICM. This leads to major differences in the initiation and the development of the cardiomyopathy and its progression to heart failure: most patients with ICM suffered at least once from myocardial infarction and they were expected to suffer from chronic ischemia due to repeated injuries occurring over time [4]. Such mechanisms do not exist in NICM. NICM consists of a heterogeneous group of etiologies including inflammatory, toxic, metabolic, genetic, valvular, hypertensive, and pericardial reasons [1]. Despite the underlying etiology, initial and ongoing myocardial injury as well as the initiation of compensatory mechanisms result in cellular damage and associated activation of an inflammatory response [5][6][7].
Macrophage migration inhibitory factor (MIF) is known as a pleiotropic inflammatory cytokine and has been recognized as a mediator of a number of inflammatory diseases including sepsis and atherosclerosis [8][9][10][11][12][13]. It has recently been described that MIF plays a role in NICM, since expression of MIF in myocardial biopsy samples predicted all-cause mortality in NICM patients and was described as a novel additional tool to predict adverse outcome in patients with NICM [14]. Importantly, MIF is not only a mediator of inflammatory processes, but also a key player in myocardial ischemia and reperfusion injury and has recently been identified as a potent cardioprotective factor [15][16][17][18][19]. Cardioprotection by MIF is a multifactorial phenomenon and is mediated by AMP-activated protein kinase (AMPK) signaling, inhibition of pro-apoptotic cascades, and attenuation of oxidative stress in the post-ischemic heart [15]. In patients with acute myocardial infarction, circulating MIF was rapidly released and MIF protein levels were found elevated [20,21].
Secretion of MIF was shown to be mediated by different stimuli like oxidative stress, ischemia, reperfusion, and hypoxia [17,19,20]. Hypoxia was not only shown to stimulate MIF protein secretion, but to influence MIF expression. MIF gene expression is subjected to induction by its transcription factor hypoxia-inducible factor (HIF)-1α [22]. HIF-1α is an oxygen-sensitive transcription factor that enables organisms to adapt to hypoxia by transcriptional activation of up to 200 genes and is considered to be the master switch of hypoxic and ischemic signaling [23]. To date, the HIF-1α/MIF axis has been investigated in multiple experimental models including cardiomyocyte and smooth muscle cell cultures after induction of hypoxia, in mouse models of myocardial ischemia/reperfusion injury, and in cancer [20,[24][25][26]. All studies shared the fact that hypoxia induced MIF gene and protein expression and in some of these studies, this was due to HIF-1α-mediated signaling. Importantly, the HIF-1α/MIF axis was not the only way to stimulate hypoxia-mediated MIF signaling, since other mediators like the transcription factors NF-κB and C/EBP were discussed [26].
To date, no studies exist that report on the HIF-1α/MIF axis in heart failure. We therefore investigated MIF and HIF-1α expression in myocardial samples from patients with heart failure and show different expression levels depending on the underlying type of cardiomyopathy.

Study Setting and Population
Thirty patients were included in this study. Of those, 10 patients presented with end-stage ICM, 10 patients presented with end-stage NICM, and 10 patients one year after heart transplantation (HTX) due to end-stage ICM served as controls. All samples were taken from retained samples that were no longer necessary for diagnosis or clinical examinations.
Samples of n = 10 patients with end-stage ICM were taken directly before left ventricular assist device (LVAD) implantation. ICM patients suffered from end-stage heart failure with severe coronary artery disease (CAD) without any possibility for revascularization. Left ventricular ejection fraction (LV-EF) among this cohort was 22 ± 3% (Table 1). ICM patients presented with symptomatic heart failure (New York Heart Association (NYHA) functional class ≥II). Myocardial biopsy samples from n = 10 patients with end-stage NICM (50% of NICM patients showed positive virus serology ( Table 2)) were compared to those from ICM patients. CAD was excluded by coronary angiography in all of them. LV-EF was 28 ± 4% (Table 1). NICM patients presented with symptomatic heart failure (NYHA functional class ≥ II). Myocardial samples from n = 10 patients after HTX served as controls. All patients underwent HTX due to end-stage ICM and myocardial biopsy was performed routinely one year after HTX. None of the HTX patients showed signs of heart failure (NYHA functional class ≤ I) and LV-EF was 56 ± 6%. None of the patients showed signs of organ rejection classified after International Society for Heart and Lung Transplantation (ISHLT) consensus report (Table 2) [27]. All patients were on a standard immunosuppressive regimen consisting of tacrolimus, MMF, and cortisone.
The study conformed to the principles outlined in the Declaration of Helsinki and was approved by local ethics committee of the University Duisburg-Essen, Germany. Key inclusion criteria were age ≥18 years. Key exclusion criteria was ongoing infectious disease with increased inflammatory parameters and/or clinical signs for infection.

Sample Preparation
Myocardial biopsy samples were taken from the septum of the right ventricle or from the excluded left ventricular apex directly before LVAD implantation patients.

RNA Isolation and Quantitative Real-Time PCR
Total RNA was extracted using RNeasy Fibrous Tissue Kit (Qiagen, Ratingen, Germany) and gene expression of MIF (primer Hs00236988_g1) and HIF-1α (primer Hs00153153_m1) was assessed by quantitative real-time PCR (qRT-PCR) using Applied Biosystems 7500 fast real-time PCR system (Applied Biosystems, Foster City, CA, USA) and normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as housekeeping gene (primer Hs03929097_g1).

Statistics
Gene expression was determined by a relative quantification method. The expression of the selected genes of interest (MIF and HIF-1α) was normalized to that of GAPDH as a housekeeping gene. The statistical significance of the differences in target mRNA expression level was analyzed with the Relative Expression Software Tool (REST ® ) as described previously [28]. REST ® calculates the relative expression ratios on the basis of group means for the target genes versus the reference genes and tests the group ratio results for significance. In the results section, the factor of up-or downregulation of MIF and HIF-1α gene expression between the designated groups is shown.
Data are expressed as mean ± standard deviation. For clinical characteristic, the Kolmogorov-Smirnov test was applied to check for normality distribution. We used Student's t-test for continuous variables. Pearson's correlation coefficient was calculated to analyze the association between MIF and HIF-1α mRNA levels after normalization to GAPDH as housekeeping gene. Statistical analysis was performed using Prism 6.0 software (GraphPad, La Jolla, CA, USA). A p-value < 0.05 was considered statistically significant.

MIF and HIF-1α Expression is Increased in Ischemic Cardiomyopathy
MIF mRNA expression in myocardial samples from patients with ICM was doubled compared to samples from patients with NICM (1.944 ± 0.63, p < 0.001, n = 10, Figure 1). Since HIF-1α is the key player in acute and chronic hypoxic signaling pathways and is one of the transcription factors to regulate MIF expression [24,29], we next analyzed HIF-1α expression. In myocardial samples from ICM patients, HIF-1α expression was increased compared to myocardial samples from NICM patients (2.056 ± 0.79, p < 0.001, n = 10, Figure 1). Data are expressed as mean ± standard deviation. For clinical characteristic, the Kolmogorov-Smirnov test was applied to check for normality distribution. We used Student's t-test for continuous variables. Pearson's correlation coefficient was calculated to analyze the association between MIF and HIF-1α mRNA levels after normalization to GAPDH as housekeeping gene. Statistical analysis was performed using Prism 6.0 software (GraphPad, La Jolla, CA, USA). A p-value < 0.05 was considered statistically significant.

MIF and HIF-1α Expression is Increased in Ischemic Cardiomyopathy
MIF mRNA expression in myocardial samples from patients with ICM was doubled compared to samples from patients with NICM (1.944 ± 0.63, p < 0.001, n = 10, Figure 1). Since HIF-1α is the key player in acute and chronic hypoxic signaling pathways and is one of the transcription factors to regulate MIF expression [24,29], we next analyzed HIF-1α expression. In myocardial samples from ICM patients, HIF-1α expression was increased compared to myocardial samples from NICM patients (2.056 ± 0.79, p < 0.001, n = 10, Figure 1).

Figure 1.
MIF and HIF-1α mRNA expression is increased in ischemic cardiomyopathy compared to non-ischemic cardiomyopathy. Myocardial MIF and HIF-1α mRNA expression were significantly increased in samples from patients with ICM compared to samples from NICM patients. Data were normalized to GAPDH and represented as relative expression ratios between samples from ICM patients and NICM patients as controls. *** p < 0.001 obtained with REST ® randomization test for MIF or HIF-1α mRNA expression in ICM samples versus samples from NICM patients.
Comparing MIF expression levels in myocardial samples from patients with end-stage ICM with samples from transplanted hearts with normal LV-EF, MIF mRNA expression was downregulated (0.552 ± 0.36, p < 0.001, n = 10, Figure 2). Similar results were found when comparing HIF-1α mRNA expression in samples from ICM patients with those after HTX (0.685 ± 0.25, p < 0.001, n = 10, Figure 2). There were no differences in MIF and HIF-1α expression levels when comparing samples from patients with NICM with those after HTX (data not shown).

Figure 1.
MIF and HIF-1α mRNA expression is increased in ischemic cardiomyopathy compared to non-ischemic cardiomyopathy. Myocardial MIF and HIF-1α mRNA expression were significantly increased in samples from patients with ICM compared to samples from NICM patients. Data were normalized to GAPDH and represented as relative expression ratios between samples from ICM patients and NICM patients as controls. *** p < 0.001 obtained with REST ® randomization test for MIF or HIF-1α mRNA expression in ICM samples versus samples from NICM patients.
Comparing MIF expression levels in myocardial samples from patients with end-stage ICM with samples from transplanted hearts with normal LV-EF, MIF mRNA expression was downregulated (0.552 ± 0.36, p < 0.001, n = 10, Figure 2). Similar results were found when comparing HIF-1α mRNA expression in samples from ICM patients with those after HTX (0.685 ± 0.25, p < 0.001, n = 10, Figure 2). There were no differences in MIF and HIF-1α expression levels when comparing samples from patients with NICM with those after HTX (data not shown). MIF and HIF-1α mRNA expression is decreased in patients after HTX compared to ischemic cardiomyopathy. Myocardial MIF and HIF-1α mRNA expression were significantly decreased in samples from patients after HTX compared to samples from ICM patients. Data were normalized to GAPDH and represented as relative expression ratios between samples from HTX patients and ICM patients as controls. *** p < 0.001 obtained with REST ® randomization test for MIF or HIF-1α mRNA expression in HTX samples versus samples from ICM patients.

MIF and HIF-1α Expression Show a Close Correlation in Myocardial Tissue Samples
MIF and HIF-1α expression levels in myocardial samples showed a close correlation (R 2 = 0.6971, p < 0.0001, n = 30, Figure 3) hinting at an association with hypoxia-induced HIF-1α expression with consecutive MIF gene regulation in cardiomyopathy.  MIF and HIF-1α mRNA expression is decreased in patients after HTX compared to ischemic cardiomyopathy. Myocardial MIF and HIF-1α mRNA expression were significantly decreased in samples from patients after HTX compared to samples from ICM patients. Data were normalized to GAPDH and represented as relative expression ratios between samples from HTX patients and ICM patients as controls. *** p < 0.001 obtained with REST ® randomization test for MIF or HIF-1α mRNA expression in HTX samples versus samples from ICM patients.

MIF and HIF-1α Expression Show a Close Correlation in Myocardial Tissue Samples
MIF and HIF-1α expression levels in myocardial samples showed a close correlation (R 2 = 0.6971, p < 0.0001, n = 30, Figure 3) hinting at an association with hypoxia-induced HIF-1α expression with consecutive MIF gene regulation in cardiomyopathy.

Figure 2.
MIF and HIF-1α mRNA expression is decreased in patients after HTX compared to ischemic cardiomyopathy. Myocardial MIF and HIF-1α mRNA expression were significantly decreased in samples from patients after HTX compared to samples from ICM patients. Data were normalized to GAPDH and represented as relative expression ratios between samples from HTX patients and ICM patients as controls. *** p < 0.001 obtained with REST ® randomization test for MIF or HIF-1α mRNA expression in HTX samples versus samples from ICM patients.

MIF and HIF-1α Expression Show a Close Correlation in Myocardial Tissue Samples
MIF and HIF-1α expression levels in myocardial samples showed a close correlation (R 2 = 0.6971, p < 0.0001, n = 30, Figure 3) hinting at an association with hypoxia-induced HIF-1α expression with consecutive MIF gene regulation in cardiomyopathy.

Discussion
The present study is the first to prove MIF mRNA expression in the failing human heart and the first to describe variable expression patterns depending on the etiology of cardiomyopathy. Myocardial MIF mRNA expression is increased in end-stage ICM compared to end-stage NICM and compared to transplanted hearts with normal LV-EF pointing at an ischemia/hypoxia-mediated regulation of MIF expression in failing human hearts.
The reason why MIF is upregulated in ICM may relate to three different properties of MIF: First, MIF is a redox-regulating myocardial enzyme [18,19] and its increased expression could be interpreted as a reaction to increased oxidative stress due to chronic ischemia and reperfusion. Second, MIF is a regulator of apoptotic processes via the inhibition of the stress kinase c-Jun N-terminal kinase (JNK) which is activated by myocardial ischemia and reperfusion [30]. Third, MIF was described as an angiogenic factor in experimental models of myocardial ischemia and reperfusion and was attributed to mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase (PI3K) signaling [31,32]. The 'angiogenesis theory' is emphasized by the parallel increase of HIF-1α expression. HIF-1α is one if the key players in hypoxic and ischemic signaling and has been shown to regulate the expression of cardiac proteins like brain and atrial natriuretic peptides [33]. Furthermore, HIF-1α acts as a transcription factor for MIF and the activation of the HIF-1α/MIF axis leads to enhanced angiogenesis [22,34]. Of course, these theories remain speculative, since our data are just delineative and the study was not designed to prove such a mechanism.
The major limitation of this trial is the lack of an appropriate internal control group, e.g., samples from patients without any signs of cardiomyopathy. Undoubtedly, direct measurement of protein expression levels in samples from healthy human hearts would be ideal, but the sampling of healthy tissue raises major ethical concerns and therefore is not feasible. In this trial, samples from transplanted hearts served as "control". They were characterized by a normal LV-EF and histological analysis showed no signs of acute rejection. To date, there is no data on the influence of pharmaceutical immunosuppression on mRNA expression of MIF, but one study showed lowered urinary MIF protein secretion after immunosuppression in patients with glomerulonephritis [35]. Nonetheless, our main conclusion-ischemia-associated increase of myocardial MIF and HIF-1α expression-is not affected by the comparison with samples from transplanted hearts.
The second study limitation concerns our focus on gene expression. It has to be clear that this only depicts one detail in the complex biochemistry of MIF and HIF-1α signaling and the limitation to gene expression data carries the risk of missing other factors involved in the regulation of important downstream pathways. To complete the examination of the role of MIF in heart failure, it would be necessary to investigate MIF protein expression by Western blot and its localization by immunohistochemistry in myocardial samples and to amend and correlate levels of circulating MIF in plasma. Third, our data are just descriptive and do neither give a mechanistical insight into the role of the HIF-1α/MIF axis in heart failure nor provide data on outcome and prognosis. Therefore, in addition to our "proof-of-concept" data, a prospective study should be designed to investigate the aforementioned parameters and to link them to prognosis and outcome of the included patients. Future studies could then form the basis for therapeutic approaches.
Despite these study limitations, our results provide important new aspects on MIF in heart failure. Besides increased MIF expression in chronic NICM [14], our data demonstrate increased MIF expression in chronic ICM, which seems to be induced by the master switch of hypoxia HIF-1α.
We can assume that MIF expression is not only relevant in the human heart suffering from NICM, but also in hearts from patients with ICM. Our study gives new insight into the role of MIF in heart failure and thereby complements prior studies to elucidate the "MIF story" in heart failure.


extfieldsofstudy: Medicine Chemistry

text:
Store-Operated Calcium Entry Is Required for mGluR-Dependent Long Term Depression in Cortical Neurons

Store-operated calcium entry (SOCE) is a Calcium (Ca2+) influx pathway activated by depletion of intracellular stores that occurs in eukaryotic cells. In neurons, the presence and functions of SOCE are still in question. Here, we show evidences for the existence of SOCE in primary mouse cortical neurons. Endoplasmic reticulum (ER)-Ca2+ depletion using thapsigargin (Tg) triggered a maintained cytosolic Ca2+ increase, which rapidly returned to basal level in the presence of the SOCE blockers 2-Aminoethoxydiphenyl borate (2-APB) and YM-58483. Neural SOCE is also engaged by activation of metabotropic glutamate receptors (mGluRs) with (S)-3,5-dihydroxyphenylglycine (DHPG) (agonist of group I mGluRs), being an essential mechanism to maintain the mGluR-driven Ca2+ signal. Activation of group I of mGluRs triggers long-term depression (LTD) in many brain regions, but the underlying mechanism and, specifically, the necessity of Ca2+ increase in the postsynaptic neuron is controversial. In primary cortical neurons, we now show that the inhibition of Ca2+ influx through SOCE impaired DHPG-LTD, pointing out a key function of calcium and SOCE in synaptic plasticity.

INTRODUCTION
Calcium (Ca 2+ ) is a universal second messenger that regulates numerous cellular processes of all eukaryotic cells, including metabolism, muscle contraction, exocytosis, transcription of numerous genes or programmed cell death (Brini et al., 2013). In neurons, it also plays a critical role in the transmission of synaptic information by controlling neurotransmitter release (Südhof, 2012). In addition, calcium is the intracellular messenger for many forms of activity-dependent synaptic plasticity acting both in the presynaptic and/or postsynaptic neuron (Zucker, 1999;Cavazzini et al., 2005). These roles are performed thanks to abundant and specific ligand-and voltage-gated Ca 2+ channels which exert a tight control on Ca 2+ dynamics (Grienberger and Konnerth, 2012;Brini et al., 2014). Ca 2+ stores also have a relevant role in the regulation of the intracellular Ca 2+ concentration (Verkhratsky, 2005).
Recently, several studies have suggested a relevant role for ER-Ca 2+ stores and SOCE in synaptic plasticity (Bardo et al., 2006;Majewski and Kuznicki, 2015;Moccia et al., 2015;Segal and Korkotian, 2016). SOCE has been shown to control neuronal Ca 2+ dynamics during synaptic activity in different neurons (Moccia et al., 2015). A direct link between SOCE and AMPA receptor-dependent Ca 2+ signal has been proposed (Gruszczynska-Biegala et al., 2016). A mechanism that links activation of postsynaptic NMDA receptors and L-type Ca 2+ channels has been linked to signaling by the ER-Ca 2+ sensor STIM1 (Dittmer et al., 2017). STIM1 has also been proposed to be a key regulator of Ca 2+ signaling downstream metabotropic glutamate receptor (mGluR) stimulation (Hartmann et al., 2014;Hou et al., 2015).
Activation of group I of mGluRs (mGluRs I) using electrical or pharmacological stimulation ((S)-3,5-dihydroxyphenylglycine (DHPG)) leads to long-term depression (LTD) in many brain regions (Lüscher and Huber, 2010). mGluRs I are canonically coupled to Gα q/11 and the activation of phospholipase Cβ (PLC), inositol triphosphate (IP 3 ) generation, release of Ca 2+ from intracellular stores, and protein kinase C (PKC) activation (Abe et al., 1992;Aramori and Nakanishi, 1992). However, depending on the induction protocol and the brain region, there are differences in the involvement of the PLC pathway and postsynaptic Ca 2+ rise in mGluR-LTD (Gladding et al., 2009;Lüscher and Huber, 2010).
Although LTD takes place in many brain regions, most studies have focused on hippocampal neurons. The aim of the present study was to investigate the role of SOCE in cortical neuron LTD, specifically upon mGluR stimulation.

Animals
Wild-type mice with a mixed C57BL6/Sv129 genetic background were used. Mice were housed in a humidity-and temperaturecontrolled room on a 12 h light/dark cycle, receiving water and food ad libitum. All the experimental protocols performed in this study were performed in accordance with procedures approved in the Directive 86/609/EEC of the European Union and with approval of the Institutional Ethical Committee of the Center of Molecular Biology Severo Ochoa and Universidad Autonoma de Madrid (CEEA-CBMSO-23/159) and Area de Protection Animal Comunidad de Madrid (PROEX 352/15). All efforts were made to minimize animal suffering.

Primary Neuronal Culture
Cortical neuronal cultures were prepared from E15 to E16 mouse embryos as described earlier (Ramos et al., 2003;Pardo et al., 2006). Neurons were plated at a density of 5 × 10 5 cells/cm 2 on poly-L-lysine and laminin-coated pretreated glass coverslips in Neurobasal medium supplemented with 2% B27, 1% glutamax (all from Gibco Invitrogen, Carlsbad, CA, USA) and 100 mg/ml penicillin-streptomycin. On the fifth day in vitro (DIV) half of the plating medium was removed from each well and replenished with BrainPhys medium (Bardy et al., 2015; Stem cell Technologies, Seattle, WA, USA) supplemented with 2% B27 and 100 mg/ml penicillin-streptomycin. Neurons represented >80% of the total cell population (Ramos et al., 2003;Pardo et al., 2006). The cultures were maintained at 37 • C in a humidified atmosphere of 5% CO 2 . The culture medium was partially replaced every other day. Cultures were used for experimentation between 9 and 11 DIV. BrainPhys was chosen as culture medium for these studies because it has an inorganic salt concentration, glucose level and osmolarity similar to those reported for the brain, and neurons grown in this culture medium show high viability and maintain synaptic network activity during long time in culture (Bardy et al., 2015), i.e., conditions adequate for the study of possible roles of SOCE in synaptic activity. However, we have verified that SOCE activity in cortical neurons was independent of the culture medium, and we have observed a similar and consistent Ca 2+ entry after ER-Ca 2+ discharge using thapsigargin (Tg) in cortical neurons cultured in both BrainPhys and Neurobasal medium.
For qPCR, 2.5 ng of the cDNA synthesized was amplified using the ABI Prism 7900HT real-time PCR System (Applied Biosystems, Foster City, CA, USA). The amplification protocol was: hot start (10 min 95 • C) and 40 amplification cycles (15 s 95 • C, 1 min 60 • C). All reactions were carried out in triplicate. Primer sequences for cDNA analysis by qRT-PCR were as follows (5 - Trpc7 forward aacgatgaagtcaatgaaggtg; Trpc7 reverse ccagctctcctgtagcctga. We performed an absolute quantification using linearized plasmids containing the cDNA fragments and the standard curve method. For quantification of the PCR products, we used the fluorescent dye SYBR-Green (SYBR Green, Biorad, Hercules, CA, USA). The parameter analyzed was the quantification cycle (Cq). A standard curve of each amplicon (from 10 to 10 8 copies) was used and the number of copies of SOCE genes was obtained by extrapolation. The PCR efficiency was calculated for each pair of specific primers and was applied where appropriate.

Electrophysiological Recordings and mEPSC Analysis
Voltage-clamp whole-cell recordings were performed in DIV 10-11 cortical neurons. Neurons at 37 • C were continuously perfused with a recording medium containing (in mM) 120 NaCl, 0.8 MgCl 2 , 5.4 KCl, 25 HEPES, 2.5 D-glucose, 2 CaCl 2 and supplemented with 1 µM TTX to prevent action potentialevoked responses; pH was adjusted to 7.4. Patch electrodes with resistances between 4-6 MΩ were filled with an internal solution containing (in mM) 115 K gluconate, 20 KCl, 10 HEPES, 2 MgCl 2 , 4 Na 2 -ATP, 0.3 Na 3 -GTP, pH adjusted to 7.3 and osmolarity ∼290 mOsm. Neurons were voltage-clamped at −60 mV. Electrophysiological recordings and data acquisition were carried out with a Multiclamp 700B amplifier and Clampfit 10.7 software (Molecular Devices, Sunnyvale, CA, USA). Miniature excitatory postsynaptic currents (mEPSC) were detected and analyzed using the Clampfit EventDetection module. Only events with an amplitude >8 pA, an exponential decay and a monotonic rising phase, which could be clearly discriminated from the background noise, were considered as mEPSCs. mEPSC amplitude and frequency were compared during a 2-min baseline period and in 2-min windows 15 min after 200 µM DHPG application (5 min). Decay time constants (τ) were calculated from single exponential fits of averaged mEPSC traces.

Statistical Analysis
Statistical analyses were performed using STATISTICA software, version 7 (StatSoft). Shapiro-Wilk test was applied to determine the distribution of the data. In normal distributions, one-way or two-way ANOVA test, following of a post hoc Bonferroni test were applied. In non-normal distributions (mEPSC parameters) Wilcoxon matched paired test was applied. Significance was * p < 0.05, * * p < 0.01, * * * p < 0.001. All data are expressed as the mean ± SEM.

Store-Operated Ca 2+ Entry Is Induced in Cortical Neurons after Store Depletion
To assess the presence of SOCE pathway in primary cortical neurons, we depleted ER stores using 1 µM Tg, the sarco/endoplasmatic reticulum Ca 2+ ATPase (SERCA) inhibitor, in a medium with 1 µM TTX, in order to prevent neuronal activity. Tg application resulted in a sustained increase in cytosolic calcium ( Figure 1A, black circles) that lasted for several minutes. To determine whether Ca 2+ entering the cell through SOC channels was contributing to this cytosolic Ca 2+ rise, we applied different widely used SOCE blockers along with Tg: 2-APB and YM-58483 (also called BTP2; Xia et al., 2014;Prakriya and Lewis, 2015). YM-58483 is highly specific and has been shown not to interfere with voltage-gated calcium channels (VGCC; Xia et al., 2014), and 2-APB is a commonly used bimodal SOCE modulator which decreases SOCE activity at high concentrations and also inhibits IP 3 signaling (Prakriya and Lewis, 2015). In neurons, it is known that ER Ca 2+ is continuously being released and replenished and consequently SOCE is active even in resting conditions (Samtleben et al., 2015). To avoid a drop in ER calcium caused by SOCE inhibition, the SOCE blockers were not preincubated but applied together with Tg. In the presence of 10 µM YM-58483 or 50 µM 2-APB, the Tg-induced cytosolic Ca 2+ signal was strongly attenuated, with a rapid decrease 1-2 min after onset (Figures 1A-C). This result is consistent with a Ca 2+ entry through SOC channel upon depletion of Ca 2+ ER stores with Tg.
In addition, to test the possibility that neuronal activity contributed to this calcium signal, cytosolic Ca 2+ levels were recorded in the presence of an inhibitor cocktail containing 10 µM CNQX (an AMPA/Kainate receptor inhibitor), 10 µM MK-801 (NMDA receptor inhibitor) and 5 µM MPEP (a mGluR inhibitor) to block excitatory action of ionotropic and metabotropic glutamate receptors, 10 µM TTX to block voltagegated sodium channels, and 50 µM NiCl 2 to block voltageactivated T-type calcium channels (the inhibition cocktail was present during the whole experiment). Figure 1D shows that the cytosolic Ca 2+ increase triggered by emptying ER-Ca 2+ was similar both in the absence and the presence of the inhibitory cocktail. This result contrasts with those recently reported in rat cortical neurons using a different SOCE induction protocol, in which AMPA receptors appeared to contribute to SOC mediated Ca 2+ entry (Gruszczynska-Biegala et al., 2016). This difference Data were obtained from three independent experiments (n = 3), and in each one a total of 25-30 cells were analyzed. (C) Representative color-coded Fura-2 [Ca 2+ ] i ratio images from basal situation (1), immediately after Tg ± SOCE blockers addition (2), and the end of the measurement. Pseudo-color ratio images were obtained using Aquacosmos 2.5 software (Hamamatsu). (D) Fura-2 [Ca 2+ ] i signals in cortical neurons in HCSS medium containing 2 mM CaCl 2 and 1 µM TTX ± inhibitory cocktail (10 µM CNQX (6-cyano-7-nitroquinoxaline-2,3-dione), 10 µM MK-801, 5 µM MPEP, 50 µM NiCl 2 ), upon addition of 1 µM Tg. Data were obtained from three independent experiments (n = 3), and in each one a total of 25-30 cells were analyzed. All data are normalized to the initial values and are expressed as mean ± SEM. Means were compared using one-way ANOVA, * p < 0.05, post hoc Bonferroni test.
Frontiers in Cellular Neuroscience | www.frontiersin.org could arise from the protocols used to measure SOCE activity in neurons. In this study, ER-Ca 2+ depletion was triggered in a medium containing physiological calcium concentration, while Gruszczynska-Biegala et al. (2016) employed the Ca 2+ addback protocol, in which addition of Ca 2+ to the external medium may activate neuronal Ca 2+ channels by changing the membrane excitability as described elsewhere (Lu and Fivaz, 2016).

SOC Channel Proteins Are Expressed in Mouse Cortical Neurons
We have studied the different candidate proteins forming SOC channels in other cell types, Orai and Trpc families as calcium channels located in PM and Stim family as calcium sensor in ER-membrane. We first investigated the mRNA levels of the different isoforms of the SOCE components, Orai, Stim and Trpc families, by RT-qPCR in 8-9 DIV cultured cortical neurons from four independent embryos. The profiles obtained for each embryo were quite similar to one another and we found that the isoforms forming the pore channel with highest mRNA levels were Orai1, Orai2, Trpc1 and Trpc4, while Stim1 and Stim2 mRNA levels were similar (Figure 2A). Then, protein levels of the SOCE components with highest mRNA levels were investigated by Western Blot. As positive controls, we used the human neuroblastoma SH-SY5Y cell line, which expresses these proteins (Olianas et al., 2014). We observed a band at 50 kDa with the ORAI1 antibody, a single band at 110 kDa with the TRPC4 antibody and a band at 90 kDa with the STIM2 antibody, but not bands corresponding to ORAI2 and TRPC1 proteins ( Figure 2B). These results show that in cultured cortical neurons, ORAI1, STIM2 and TRPC4 were the predominant members of their respective families, in agreement with previous reports in cortical and other neurons of the central nervous system (CNS; Berna-Erro et al., 2009;Steinbeck et al., 2011;. STIM1 proteins are also present in mouse cortical neurons (Hou et al., 2015;Guner et al., 2017), but STIM2 has been described as predominant isoform in cortex and hippocampus (Berna-Erro et al., 2009;Steinbeck et al., 2011), while STIM1 protein expression is higher in cerebellum (Lein et al., 2007;Hartmann et al., 2014;Kraft, 2015). Therefore, ORAI1, TRPC4 and STIM1 and 2, are probably responsible for SOC entry in mouse cortical neurons.

SOCE Is Involved in mGluR-Driven Ca 2+ Signals
After determining that ER-Ca 2+ depletion in neurons triggers Ca 2+ influx into the cytosol by SOCE, we sought to study the activation of this pathway by physiological agonists, particularly glutamate. Activation of group I mGluRs (mGluRs I), formed by mGluR1 and 5, is coupled to a variety of signaling pathways. The Gα q /Gα 11 protein/PLC/inositol-3,4,5-triphosphate (IP 3 ) signal cascades have been considered as the canonical pathway, which leads to Ca 2+ release from ER and activation of PKC. The decrease of ER-Ca 2+ levels could activate SOCE in the PM and play a role in mGluR-driven Ca 2+ signaling.
The experiments were performed at 10-12 DIV in a medium with 1 µM TTX in order to eliminate spontaneous activity. Application of 200 µM DHPG, a selective agonist of group I mGluRs, evoked a rapid increase in cytosolic Ca 2+ followed by a more moderate but sustained Ca 2+ signal that did not reach baseline during the recording period ( Figure 3A, black circles).
To determine the contribution of SOCE to the Ca 2+ response to DHPG, we applied the SOCE blockers together with the agonist of mGluR I, as in previous experiments with Tg (Figure 1). Interestingly, we found that the cytosolic Ca 2+ transient rapidly returned to baseline when 10 µM YM-58483 or 50 µM 2-APB were added together with DHPG (Figures 3A,B). This result indicates that SOCE is responsible for the sustained increase in Ca 2+ levels above baseline in response to DHPG. On the other hand, the presence of YM-58483 did not affect the initial Ca 2+ peak evoked by mGluRs, suggesting that SOC channels open secondarily to mGluRs activation and that the first peak involves the ER-Ca 2+ mobilization by PLC pathway. In contrast, the addition of 2-APB decreased the initial Ca 2+ peak, consistent with the effect of this compound on IP 3 signaling (Prakriya and Lewis, 2015).

SOCE Inhibition Impairs DHPG-LTD
Finally, we wished to determine whether SOCE-dependent calcium signal in response to mGluR activation had physiological consequences for neuronal function. Stimulation of group I mGluRs can lead to a decrease in synaptic strength at many synapses, referred to as long-term depression (mGluR-LTD; Lüscher and Huber, 2010). This form of LTD can be induced by the application of the group I selective mGluR agonist DHPG (so called DHPG-LTD; Xiao et al., 2001;Jo et al., 2008), but the underlying molecular mechanism is still poorly understood and appears to depend on the specific neuronal type. Having shown that SOCE was activated after stimulation of group I mGluRs and was involved in the maintenance of cytosolic Ca 2+ signal, we investigated whether the activation of SOCE may be involved in DHPG-LTD in cortical neurons. To this end, we first examined the effect of DHPG on synaptic transmission in primary cortical neurons. We found that bath application of 200 µM DHPG (5 min) caused a long lasting reduction of the mEPSC amplitude at 15 min after mGluR stimulation (Figures 4A,B,I). In contrast, we did not find any significant effect on the frequency of mEPSCs (Figures 4A,C,J), suggesting that this type of LTD is predominantly expressed via a postsynaptic mechanism. This interpretation is also consistent with an increase in the decay time constant of mEPSCs after DHPG application (Figures 4D,K). To study the contribution of SOCE to this form of synaptic plasticity, we evaluated the effect of the SOCE blocker YM-58483 (10 µM) Data are expressed as mean ± SEM and were obtained from 14 independent experiments for results of DHPG addition, 13 for those of DHPG addition along with YM-58483 and six for those of YM-58483 application alone. Due to non-normal distribution of mEPSCs parameters, statistics were performed using the Wilcoxon matched pairs test, * p < 0.05, * * * p < 0.001.
applied together with DHPG. Interestingly, the long lasting decrease of mEPSCs amplitude was significantly attenuated (7.7 ± 2.9%; Figures 4E,F,I), as well as the effect on mEPSC kinetics (Figures 4H,K). The frequency of mEPSCs was not affected by DHPG with or without YM-58483 (Figures 4G,J). Importantly, bath application of 10 µM YM-58483 did not have any effect on its own on the amplitude, frequency or kinetics of mEPSCs (Figures 4I-K). All these results strongly suggest that the calcium influx via SOCE after group I mGluRs stimulation is a necessary step for the development of DHPG-LTD in cortical neurons.

DISCUSSION
SOCE is the main mechanism to replenish intracellular calcium stores in non-excitable cells but its existence in neurons is under debate (Putney, 2003;Lu and Fivaz, 2016), as neurons exhibit other major Ca 2+ influx pathways through VGCCs and ionotropic glutamate receptors (Grienberger and Konnerth, 2012). Whereas Ca 2+ entry through SOC channels has been reported in different neuronal populations (Emptage et al., 2001;Berna-Erro et al., 2009;Gemes et al., 2011;Alkhani et al., 2014;Sun et al., 2014;Xia et al., 2014), other groups did not find a clear SOCE response in neurons (Park et al., 2010;Garcia-Alvarez et al., 2015a). One of the possible reasons for this discrepancy could stem from the commonly used Ca 2+ addback protocol to study SOCE, in which neuronal stores are emptied in a Ca 2+free medium, and then Ca 2+ is added to the external medium. Neurons express multiple Ca 2+ channels that can be activated in response to changes in extracellular calcium. In the present study, we investigated the existence of SOCE in cortical neurons in relatively mature state, in a medium containing physiological calcium concentrations. Our results clearly showed a small but maintained increase in cytosolic Ca 2+ after Tg application, which rapidly returned to basal levels in the presence of SOCE blockers, and was unaltered after a cocktail of channel/receptor inhibitors, i.e., all conditions consistent with a bona fide Tg-induced SOCE. The analysis of the molecular players of SOCE in mouse cortical neurons, showed that STIM2 and probably STIM1 are highly expressed at the mRNA and protein levels, in agreement with previous findings (Berna-Erro et al., 2009;Gruszczynska-Biegala et al., 2011;Guner et al., 2017). In the PM, ORAI1 seems to form the store-operated pore, and to a lesser extent, TRPC4, as reported for cortical and hippocampal neurons (Klejman et al., 2009;. A second finding of this study was that a physiological stimulus, group I mGluR activation, induces SOCE in mouse cortical neurons. Group I metabotropic receptors are linked to activation of PLC, IP 3 generation and release of Ca 2+ from intracellular stores (Niswender and Conn, 2010). Here, we show that store depletion after mGluR I stimulation using the specific agonist DHPG activates SOCE in primary cortical neurons, and SOCE was necessary to maintain the mGluR-driven cytosolic Ca 2+ signal. This result is in agreement with a previous report in which stimulation of mGluR I triggered STIM1 oligomerization and migration to PM (Ng et al., 2011), the first step needed in SOCE induction (Lewis, 2007). In fact, more recent findings point to a central role of STIM proteins as one of the intracellular links between mGluR I and its downstream effectors (Hartmann et al., 2014;Hou et al., 2015).
mGluRs are widely distributed throughout the CNS and play relevant roles for synaptic transmission, activity-dependent synaptic plasticity and higher cognitive functions (Niswender and Conn, 2010). Particularly, group I mGluRs induce a specific form of synaptic plasticity leading to long-term depression (LTD) of excitatory synaptic strength (mGluR LTD). In this work, we have observed that SOCE inhibition caused a strong impairment of DHPG-LTD in primary cortical neurons. In relation with SOCE components, STIM1 has been found to control mGluR1dependent slow excitatory potentials in Purkinje neurons through its action in maintaining ER-Ca 2+ levels (Hartmann et al., 2014), and both STIM2 knockout (Yap et al., 2017) and STIM1/STIM2 double knockout mice (Garcia-Alvarez et al., 2015b) have impaired LTP and LTD.
mGluR-LTD has been broadly studied in hippocampus and cerebellum (Jörntell and Hansel, 2006;Gladding et al., 2009;Lüscher and Huber, 2010), but less is known about other brain regions. It is still debated whether the expression mechanism of DHPG-LTD relies on presynaptic (neurotransmitter release) or postsynaptic (AMPA receptor internalization) changes (Anwyl, 2006). Moreover, in hippocampal neurons, the expression mechanism of DHPG-LTD depends on the developmental state of the synapse, being presynaptic in immature neurons but postsynaptic after neuronal maturation (Nosyreva and Huber, 2005). Our results show a clear reduction in the amplitude, but not frequency, of mEPSCs 15 min after DHPG application, which suggests a postsynaptic mechanism of expression. This agrees with the high maturation state of our neuronal cultures which has been previously documented when the BrainPhys Neuronal Medium was described (Bardy et al., 2015).
In cortical neurons, the strong impairment of DHPG-LTD and the failure to maintain DHPG-driven cytosolic Ca 2+ signals in the presence of the SOCE blocker YM-58483 suggest that SOCE-mediated Ca 2+ signal is essential to activate signaling pathways downstream mGluRs stimulation. It is particularly interesting that SOCE was required specifically for the maintenance of the calcium signal after mGluR activation, suggesting that this sustained signal is responsible for the long-term synaptic effects. SOCE may be acting directly by extracellular calcium influx through SOCs channels or by SOCE's known function in maintaining the ER-Ca 2+ levels (Samtleben et al., 2015), which are reduced by mGluRs stimulation. According to this, it has been demonstrated that ER-Ca 2+ release from internal stores via IP 3 generation after mGluR stimulation is required to induced LTD in cerebellum (Miyata et al., 2000;Kano et al., 2008). In the perirhinal cortex the induction of DHPG-LTD depends on interactions between the neuronal Ca 2+ sensor protein (NCS-1) and protein interacting with C kinase (PICK1) in a Ca 2+ -dependent manner (Jo et al., 2008). However, it is relevant to note that the molecular mechanisms seem to differ between brain regions. Indeed, in hippocampus, DHPG-LTD is considered to be independent of PLC/IP 3 pathway and postsynaptic Ca 2+ (Schnabel et al., 1999;Fitzjohn et al., 2001;Mockett et al., 2011;Kim et al., 2015), but there is also evidence of IP 3 mediated ER-Ca 2+ release from dendritic spines and calmodulin activation for group I mGluR LTD (Holbro et al., 2009;Sethna et al., 2016).
Although Ca 2+ influx through SOCs channels has been implicated in several functions in dendritic spines (Brini et al., 2014;Sun et al., 2014;, SOCE proteins may also play roles outside the canonical SOCE pathway. A SOCE-independent activation of STIM proteins was first described in non-excitable cells (Lefkimmiatis et al., 2009;Tian et al., 2012;Shinde et al., 2013), and recently, this behavior has been also found in neurons. It has been shown that, in hippocampal neurons, STIM2 mediated PKA-dependent phosphorylation and trafficking of AMPA receptors is associated with an increase in cAMP levels (Garcia-Alvarez et al., 2015a). A recent study found that STIM1 overexpression in hippocampus impairs both synaptically and chemically induced mGluR-LTD, without affecting SOCE, among other calcium parameters (Majewski et al., 2017). As to the SOCE blocker used in this study to perform the experiments of DHPG-LTD, YM-58483, it seems to exert its action on the channel itself, without interfering with STIM proteins (He et al., 2005). Therefore, our results suggest a relevant role of Ca 2+ entry through SOC channels themselves in DHPG-LTD.
In conclusion, our findings reveal a close relationship between mGluRs and SOCE in cortical neurons, and show that SOCE activation is a necessary step in the development of DHPG-driven cytosolic Ca 2+ signals. Blocking SOCE activation upon mGluR stimulation prevented DHPG-LTD. Altogether the results suggest that, contrary to what has been observed in hippocampus, in cortical neurons SOCE-mediated Ca 2+ signal could be relevant in generating downstream effects to evoke mGluR-LTD.

AUTHOR CONTRIBUTIONS
PG-S: designed and performed the experiments, analyzed the data and wrote the manuscript. AdA: designed and performed the experiments. JAE and JS: designed the study and wrote the manuscript. All authors reviewed the manuscript.

ACKNOWLEDGMENTS
This work has been funded by grants from the Spanish Ministry of Science and Innovation (Ministerio de Ciencia e Innovación) and BFU2011-30456-C02-01/BMC and SAF2014-56929 (to JS) and SAF2014-57233-R and PCIN-2016-095 (to JAE), the Consejería de Educación, Juventud y Deporte, Comunidad de Madrid S2010/BMD-2402 MITOLAB-CM (to JS), by an institutional grant from the Fundación Ramón Areces to the Centro de Biología Molecular Severo Ochoa. This work has also been funded by the CIBERER, an initiative from the Instituto de Salud Carlos III. PG-S is a recipient of a Formacion Personal Investigador-fellowship from Universidad Autónoma de Madrid (FPI-UAM). We thank Isabel Manso for technical support and Marta Navarrete and Jonathan Draffin for their help with data analysis.