Abstract:
Osteocytes embedded in mineralized bone have their network disrupted when a bone injury occurs. However, their role in regeneration is still unclear. Bone substitutes, including bioceramics and bone-derived extracts, are commonly implanted in critical-sized defects due to their bioactive properties that promote healing, yet few studies have examined their effects on osteocytes in vivo. We studied early repair phases of a critical defect model in adult male mice calvaria, with or without biomaterial implantation (beta-TCP, bovine bone). Using microCT we determined that, after 14 days, bone formation had mainly occurred along the surface of existing bone, increasing its thickness by a factor of 1.6, independently of the biomaterial presence. Using HE staining and fluorescence imaging, we described the newly formed bone and showed the presence of recently embedded osteocytes. We specifically collected osteocytes close, and distant from the defect, using laser-assisted microdissection and analyzed their gene expression. We show that IL6 was mainly dependent on the delay after surgery whereas Dmp1 was spatially regulated. Thus, even with limited bone formation in the defect, bone apposition occurs on the inner and outer periosteal surfaces of the calvaria, a phenomenon that may have been overlooked in the development of bone repair strategies.

Abstract:
While blood vessels and osteocytes have been studied independently, their simultaneous changes with age remain undescribed. Our objective was to investigate the age-related evolution of both osteocyte and blood vessel networks in mouse cortical bone, and to assess the associated effects on osteocyte markers and oxygen intracellular levels. We analyzed femurs of male Flk1-GFP mice from growing, mature, middle-aged, and aged groups with techniques such as laser microdissection followed by RT-qPCR, tissue clearing and 3D fluorescence imaging. In the mature animals - when the cortical bone was thicker than in the growing animals - the osteocyte density, the number of dendrites per osteocyte and the blood vessel density were lower. This was associated with a reduced expression of Pdpn and with a smaller fraction of osteocytes exhibiting low intracellular oxygen. In aged animals - when cortical bone was thinner than in mature animals - the number of dendrites per osteocyte and the blood vessel density were lower. This was associated with a reduced Gja1 (Cx43) expression. Our results suggest that changes in the osteocyte network during maturation and aging are led by distinct mechanisms, and that the cortical bone blood vessels are not the main source of oxygen for osteocytes.

Abstract:
The activation of classical NMDA receptors (NMDARs) requires the binding of a co-agonist in addition to glutamate. Whereas astrocytic-derived d-serine was shown to play such a role at CA3-CA1 hippocampal synapses, the exact mechanism by which neurons interact with neighboring astrocytes to regulate synaptic d-serine availability remains to be fully elucidated. Considering the close anatomical apposition of astrocytic and neuronal elements at synapses, the aforementioned process is likely to involve cells adhesion molecules. One very likely candidate could be the astrocytic EphB3 receptor and its neuronal partner, ephrinB3. Here, we first showed in acute hippocampal slices from adult mice that stimulation of EphB3 receptors with exogenous ephrinB3 increased d-serine availability at CA3-CA1 synapses, resulting in an increased NMDAR activity. Conversely, inhibiting endogenous EphB3 receptors caused an impairment of both synaptic NMDAR activity and NMDAR-dependent long-term synaptic potentiation (LTP), effects that could be rescued by exogenous d-serine. Most interestingly, knocking down EphB3 receptor specifically in astrocytes yielded a similar impairment in hippocampal plasticity and, most importantly, caused a deficit in novel object recognition memory. Altogether, our data thus indicate that EphB3 receptors in hippocampal astrocytes play a key role in regulating synaptic NMDAR function, activity-dependent plasticity and memory.

Abstract:
OBJECTIVE: Steroidogenic factor-1 (SF1) neurons of the ventromedial hypothalamus play key roles in the regulation of food intake, body weight and glucose metabolism. The bile acid receptor Takeda G protein-coupled receptor 5 (TGR5) is expressed in the hypothalamus, where it determines some of the actions of bile acids on food intake and body weight through still poorly defined neuronal mechanisms. Here, we examined the role of TGR5 in SF1 neurons in the regulation of energy balance and glucose metabolism. METHODS: We used a genetic approach combined with metabolic phenotyping and molecular analyses to establish the effect of TGR5 deletion in SF1 neurons on meal pattern, body weight, body composition, energy expenditure and use of energy substrates as well as on possible changes in glucose handling and insulin sensitivity. RESULTS: Our findings reveal that TGR5 in SF1 neurons does not play a major role in the regulation of food intake or body weight under standard chow, but it is involved in the adaptive feeding response to the acute exposure to cold or to a hypercaloric, high-fat diet, without changes in energy expenditure. Notably, TGR5 in SF1 neurons hinder glucose metabolism, since deletion of the receptor improves whole-body glucose uptake through heightened insulin signaling in the hypothalamus and in the brown adipose tissue. CONCLUSIONS: TGR5 in SF1 neurons favours satiety by differently modifying the meal pattern in response to specific metabolic cues. These studies also reveal a novel key function for TGR5 in SF1 neurons in the regulation of whole-body insulin sensitivity, providing new insight into the role played by neuronal TGR5 in the regulation of metabolism.

Abstract:
Under neuroinflammatory conditions, astrocytes acquire a reactive phenotype that drives acute inflammatory injury as well as chronic neurodegeneration. We hypothesized that astrocytic Delta-like 4 (DLL4) may interact with its receptor NOTCH1 on neighboring astrocytes to regulate astrocyte reactivity via downstream juxtacrine signaling pathways. Here we investigated the role of astrocytic DLL4 on neurovascular unit homeostasis under neuroinflammatory conditions. We probed for downstream effectors of the DLL4-NOTCH1 axis and targeted these for therapy in two models of CNS inflammatory disease. We first demonstrated that astrocytic DLL4 is upregulated during neuroinflammation, both in mice and humans, driving astrocyte reactivity and subsequent blood-brain barrier permeability and inflammatory infiltration. We then showed that the DLL4-mediated NOTCH1 signaling in astrocytes directly drives IL-6 levels, induces STAT3 phosphorylation promoting upregulation of astrocyte reactivity markers, pro-permeability factor secretion and consequent blood-brain barrier destabilization. Finally we revealed that blocking DLL4 with antibodies improves experimental autoimmune encephalomyelitis symptoms in mice, identifying a potential novel therapeutic strategy for CNS autoimmune demyelinating disease. As a general conclusion, this study demonstrates that DLL4-NOTCH1 signaling is not only a key pathway in vascular development and angiogenesis, but also in the control of astrocyte reactivity during neuroinflammation.

Abstract:
Amyloid-peptide (Abeta) monomeric forms (ABM) occurring in presymptomatic Alzheimer's disease (AD) brain are thought to be devoid of neurotoxicity while the transition/aggregation of ABM into oligomers is determinant for Abeta-induced toxicity since Abeta is predominantly monomeric up to 3 microM and aggregates over this concentration. However, recent imaging and/or histopathological investigations revealed alterations of myelin in prodromal AD brain in absence of aggregated Abeta oligomers, suggesting that ABM may induce toxicity in myelin-producing cells in early AD-stages. To check this hypothesis, here we studied ABM effects on the viability of the Human oligodendrocyte cell line (HOG), a reliable oligodendrocyte model producing myelin proteins. Furthermore, to mimic closely interactions between oligodendrocytes and other glial cells regulating myelination, we investigated also ABM effects on mouse brain primary mixed-glial cell cultures. Various methods were combined to show that ABM concentrations (600 nM-1 microM), extremely lower than 3 microM, significantly decreased HOG cell and mouse brain primary mixed-glial cell survival. Interestingly, flow-cytometry studies using specific cell-type markers demonstrated that oligodendrocytes represent the most vulnerable glial cell population affected by ABM toxicity. Our work also shows that the neurosteroid 3alpha-O-allyl-allopregnanolone BR351 (250 and 500 nM) efficiently prevented ABM-induced HOG and brain primary glial cell toxicity. Bicuculline (50-100 nM), the GABA-A-receptor antagonist, was unable to block/reduce BR351 effect against ABM-induced HOG and primary glial cell toxicity, suggesting that BR351-evoked neuroprotection of these cells may not depend on GABA-A-receptor allosterically modulated by neurosteroids. Altogether, our results suggest that further exploration of BR351 therapeutic potential may offer interesting perspectives to develop effective neuroprotective strategies.

Abstract:
Among bone cells, osteocytes are the most abundant, but also the most challenging to study because they are located inside a dense mineralized matrix. Due to their involvement in bone homeostasis, diverse tools are needed to understand their roles in bone physiology and pathology. This work was aimed at establishing a laser-assisted microdissection protocol to isolate osteocytes and analyze their gene expressions. The goal was to overcome the limitations of the technique currently most used: RNA extraction from the whole bone. To perform laser microdissection and subsequent gene expression analysis, the five main steps of the protocol have been adapted for the bone tissue. After testing many parameters, we found that the best options were (1) take unfixed snap-frozen tissue, (2) cryosection with a supported tape system to improve the tissue morphology if necessary, (3) microdissect regions of interest, and (4) recover the bone pieces by catapulting, if feasible, or by gravity. Finally, RNA extraction (5) was the most efficient with a precipitation method and allowed quantifying the expression of well described osteocyte genes (Gja1/Cx43, Phex, Pdpn, Dmp1, Sost). This work describes two protocols optimized for femur and calvaria and gives an overview of the many optimization options that one could try when facing difficulties with laser microdissection.

Abstract:
Spatial omics have been evolving rapidly in recent years, and due to the richness of the data they provide, they appear to offer opportunities for understanding biological phenomena and improving healthcare. Spatial omics enable the analysis of genomic, transcriptional, proteomic, and metabolic content at very high resolution. Beyond identifying molecular cell subpopulations, these approaches also provide information about the spatial location of the identified subpopulations within tissue, their proximity to each other, and their relationship with the extracellular matrix, blood vessels, and other tissue components. Spatial transcriptomics (ST) is an emerging research field that combines high-throughput genetic analysis with the spatial localisation of cells within tissues. Unlike traditional sequencing methods, ST offers a more comprehensive understanding of the molecular and cellular organisation of tissues. Our review explores various ST profiling strategies, including laser microdissection, which isolates specific cells from tissue for in-depth omics analyses. We then present various fluorescence in situ hybridization (FISH) techniques used in ST. Some state-of-the-art techniques allow simultaneous visualisation of numerous targeted transcripts and proteins with 3D subcellular resolution. ST techniques based on higher-resolution sequencing are also described in the final section. Future challenges for ST include improving the spatial resolution of NGS-based methods and gaining an in-depth understanding of cellular networks and tissue interactions. The rapid evolution of these technologies offers exciting opportunities to better understand complex biological mechanisms in the spatial context of tissues.

Abstract:
This study investigated in male mice how age modulates the effects of acute 17beta-estradiol (E2) on dorsal CA1 (dCA1)-dependent retention of temporal associations, which are critical for declarative memory. E2 was systemically injected to young (3-4 months old) and aged (22-24 months old) adult mice either (i) 1 h before the acquisition of an auditory trace fear conditioning (TFC) procedure allowing the assessment of temporal memory retention 24 h later or (ii) during in vivo electrophysiological recordings of CA3 to dCA1 synaptic efficacy under anesthesia. In young mice, E2 induced parallel dose-dependent reductions in memory and synaptic efficacy, i.e. an impairment in TFC retention and a long-term (NMDA receptor-dependent) depression of dCA1 synaptic efficacy as assessed by field excitatory postsynaptic potentials. In contrast, E2 tended to improved TFC retention whilst failing to change synaptic efficacy in aged mice. Age-dependent effects of E2 treatment were confirmed by immunohistochemical analyses of TFC acquisition-elicited dCA1 Fos activation. Thus, such an activation was respectively reduced and enhanced in young and aged E2-treated mice, compared to vehicle treatments. Hippocampal mRNA expression of estrogen receptors by RT-PCR analyses revealed an age-related increase in each receptor mRNA expression. In keeping with the key role of the endocannabinoid system in memory processes and CA3 to dCA1 synaptic plasticity, we next examined the role of cannabinoid type 1 receptors (CB(1)-R) in the aforementioned age-dependent effects of E2. Having confirmed that mRNA expression of CB(1)-R diminishes with age, we then observed that the deleterious effects of E2 on both memory and synaptic efficacy were both prevented by the CB(1)-R antagonist Rimonabant whilst being absent in CB(1)-R knock out mice. This study (i) reveals age-dependent effects of acute E2 on temporal memory and CA3 to dCA1 synaptic efficacy and (ii) suggests a key role of CB(1)-R in mediating E2 deleterious effects in young adulthood. Aging-related reductions in CB(1)-R might thus underlie E2 paradoxical effects across age.

Abstract:
Social behavior is a basic domain affected by several neurodevelopmental disorders, including ASD and a heterogeneous set of neuropsychiatric disorders. The SCRIB gene that codes for the polarity protein SCRIBBLE has been identified as a risk gene for spina bifida, the most common type of neural tube defect, found at high frequencies in autistic patients, as well as other congenital anomalies. The deletions and mutations of the 8q24.3 region encompassing SCRIB are also associated with multisyndromic and rare disorders. Nonetheless, the potential link between SCRIB and relevant social phenotypes has not been fully investigated. Hence, we show that Scrib(crc/+) mice, carrying a mutated version of Scrib, displayed reduced social motivation behavior and social habituation, while other behavioral domains were unaltered. Social deficits were associated with the upregulation of ERK phosphorylation, together with increased c-Fos activity. Importantly, the social alterations were rescued by both direct and indirect pERK inhibition. These results support a link between polarity genes, social behaviors and hippocampal functionality and suggest a role for SCRIB in the etiopathology of neurodevelopmental disorders. Furthermore, our data demonstrate the crucial role of the MAPK/ERK signaling pathway in underlying social motivation behavior, thus supporting its relevance as a therapeutic target.