The lack of neuropeptide Y-Y1 receptor signaling modulates the chemical and mechanical properties of bone matrix.

Genetic and pharmacological functional studies have provided evidence that the lack of Neuropeptide Y-Y1  receptor (Y1 R) signaling pathway induces a high bone mass phenotype in mice. However, clinical observations have shown that drug or genetic mediated improvement of bone mass might be associated to alterations to bone extracellular matrix (ECM) properties, leading to bone fragility. Hence, in this study we propose to characterize the physical, chemical and biomechanical properties of mature bone ECM of germline NPY-Y1 R knockout (Y1 R-/- ) mice, and compare to their wild-type (WT) littermates. Our results demonstrated that the high bone mass phenotype observed in Y1 R-/- mice involves alterations in Y1 R-/-  bone ECM ultrastructure, as a result of accelerated deposition of organic and mineral fractions. In addition, Y1 R-/- bone ECM displays enhanced matrix maturation characterized by greater number of mature/highly packed collagen fibers without pathological accumulation of immature/mature collagen crosslinks nor compromise of mineral crystallinity. These unique features of Y1 R-/-  bone ECM improved the biochemical properties of Y1 R-/-  bones, reflected by mechanically robust bones with diminished propensity to fracture, contributing to greater bone strength. These findings support the future usage of drugs targeting Y1 R signaling as a promising therapeutic strategy to treat bone loss-related pathologies.

The alliance between nerve fibers and stem cell populations in bone marrow: life partners in sickness and health.

The bone marrow (BM) is the central hematopoietic organ in adult mammals, with great potential to be used as a tool to improve the efficacy of the body's response to a number of malignancies and stressful conditions. The nervous system emerges as a critical regulatory player of the BM both under homeostatic and pathologic settings, with essential roles in cellular anchorage and egress, stem cell differentiation, and endothelial cell permeability. This review collects the current knowledge on the interplay between the nervous system and the BM cell populations, with a focus on how the nervous system modulates hematopoietic stem and progenitor cell, mesenchymal stromal cell, and endothelial progenitor cell activity in BM. We have also highlighted the pathologies that have been associated with disturbances in the neuronal signaling in BM and discussed if targeting the nervous system, either by modulating the activity of specific neuronal circuits or by pharmacologically leveling the activity of sympathetic and sensorial signaling-responsive cells in BM, is a promising therapeutic approach to tackling pathologies from BM origin.-Leitão, L., Alves, C. J., Sousa, D. M., Neto, E., Conceição, F., Lamghari, M. The alliance between nerve fibers and stem cell populations in bone marrow: life partners in sickness and health.

Bone marrow cell response after injury and during early stage of regeneration is independent of the tissue-of-injury in 2 injury models.

Selectively recruiting bone marrow (BM)-derived stem and progenitor cells to injury sites is a promising therapeutic approach. The coordinated action of soluble factors is thought to trigger the mobilization of stem cells from the BM and recruit them to lesions to contribute to tissue regeneration. Nevertheless, the temporal response profile of the major cellular players and soluble factors involved in priming the BM and recruiting BM-derived cells to promote regeneration is unknown. We show that injury alters the BM cellular composition, introducing population-specific fluctuations during tissue regeneration. We demonstrate that injury causes an immediate, transient response of mesenchymal stromal cells and endothelial cells followed by a nonoverlapping increase in hematopoietic stem and progenitor cells. Moreover, BM reaction is identical whether the injury is inflicted on skin and muscle or also involves a bone defect, but these 2 injury paradigms trigger distinct systemic cytokine responses. Together, our results indicate that the BM response to injury in the early stages of regeneration is independent of the tissue-of-injury based on the 2 models used, but the injured tissue dictates the systemic cytokine response.-Leitão, L., Alves, C. J., Alencastre, I. S., Sousa, D. M., Neto, E., Conceição, F., Leitão, C., Aguiar, P., Almeida-Porada, G., Lamghari, M. Bone marrow cell response after injury and during early stage of regeneration is independent of the tissue-of-injury in 2 injury models.

Compartmentalized Microfluidic Platforms: The Unrivaled Breakthrough of In Vitro Tools for Neurobiological Research.

Microfluidic technology has become a valuable tool to the scientific community, allowing researchers to study fine cellular mechanisms with higher variable control compared with conventional systems. It has evolved tremendously, and its applicability and flexibility made its usage grow exponentially and transversely to several research fields. This has been particularly noticeable in neuroscience research, where microfluidic platforms made it possible to address specific questions extending from axonal guidance, synapse formation, or axonal transport to the development of 3D models of the CNS to allow pharmacological testing and drug screening. Furthermore, the continuous upgrade of microfluidic platforms has allowed a deeper study of the communication occurring between different neuronal and glial cells or between neurons and other peripheral tissues, both in physiological and pathological conditions. Importantly, the evolution of microfluidic technology has always been accompanied by the development of new computational tools addressing data acquisition, analysis, and modeling.

The Neuroimmune Interplay in Joint Pain: The Role of Macrophages.

Chronic pain associated with joint disorders, such as rheumatoid arthritis (RA), osteoarthritis (OA) and implant aseptic loosening (AL), is a highly debilitating symptom that impacts mobility and quality of life in affected patients. The neuroimmune crosstalk has been demonstrated to play a critical role in the onset and establishment of chronic pain conditions. Immune cells release cytokines and immune mediators that can activate and sensitize nociceptors evoking pain, through interaction with receptors in the sensory nerve terminals. On the other hand, sensory and sympathetic nerve fibers release neurotransmitters that bind to their specific receptor expressed on surface of immune cells, initiating an immunomodulatory role. Macrophages have been shown to be key players in the neuroimmune crosstalk. Moreover, macrophages constitute the dominant immune cell population in RA, OA and AL. Importantly, the targeting of macrophages can result in anti-nociceptive effects in chronic pain conditions. Therefore, the aim of this review is to discuss the nature and impact of the interaction between the inflammatory response and nerve fibers in these joint disorders regarding the genesis and maintenance of pain. The role of macrophages is highlighted. The alteration in the joint innervation pattern and the inflammatory response are also described. Additionally, the immunomodulatory role of sensory and sympathetic neurotransmitters is revised.

Maternal stress and vulnerability to depression: coping and maternal care strategies and its consequences on adolescent offspring.

Depressive mothers often find mother-child interaction to be challenging. Maternal stress may further impair mother-child attachment, which may increase the risk of negative developmental consequences. We used rats with different vulnerability to depressive-like behavior (Wistar and Kyoto) to investigate the impact of stress (maternal separation-MS) on maternal behavior and adolescent offspring cognition. MS in Kyoto dams increased pup-contact, resulting in higher oxytocin levels and lower anxiety-like behavior after weaning, while worsening their adolescent offspring cognitive behavior. Whereas MS in Wistar dams elicited higher quality of pup-directed behavior, increasing brain-derived neurotrophic factor (BDNF) in the offspring, which seems to have prevented a negative impact on cognition. Hypothalamic oxytocin seems to affect the salience of the social environment cues (negatively for Kyoto) leading to different coping strategies. Our findings highlight the importance of contextual and individual factors in the understanding of the oxytocin role in modulating maternal behavior and stress regulatory processes.

Osteoclast-derived extracellular vesicles are implicated in sensory neurons sprouting through the activation of epidermal growth factor signaling.

BACKGROUND: Different pathologies, affecting the skeletal system, were reported to display altered bone and/or cartilage innervation profiles leading to the deregulation of the tissue homeostasis. The patterning of peripheral innervation is achieved through the tissue-specific expression of attractive or repulsive axonal guidance cues in specific space and time frames. During the last decade, emerging findings attributed to the extracellular vesicles (EV) trading a central role in peripheral tissue innervation. However, to date, the contribution of EV in controlling bone innervation is totally unknown. RESULTS: Here we show that sensory neurons outgrowth induced by the bone resorbing cells-osteoclasts-is promoted by osteoclast-derived EV. The EV induced axonal growth is achieved by targeting epidermal growth factor receptor (EGFR)/ErbB2 signaling/protein kinase C phosphorylation in sensory neurons. In addition, our data also indicate that osteoclasts promote sensory neurons electrophysiological activity reflecting a possible pathway in nerve sensitization in the bone microenvironment, however this effect is EV independent. CONCLUSIONS: Overall, these results identify a new mechanism of sensory bone innervation regulation and shed the light on the role of osteoclast-derived EV in shaping/guiding bone sensory innervation. These findings provide opportunities for exploitation of osteoclast-derived EV based strategies to prevent and/or mitigate pathological uncontrolled bone innervation.

Early-life stress affects drug abuse susceptibility in adolescent rat model independently of depression vulnerability.

The development of substance abuse problems occurs due to a diverse combination of risk factors. Among these risks, studies have reported depression and early-life stress as of importance. These two factors often occur simultaneously, however, there is a lack of understanding of how their combined effect may impact vulnerability to drug abuse in adolescence. The present study used rats with different vulnerability to depression (Wistar and Wistar-Kyoto) to investigate the impact of maternal separation (MS) on emotional state and drug addiction vulnerability during the adolescence period. Mothers and their litters were subjected to MS (180 min/day) from postnatal day 2 to 14. The offspring emotional state was assessed by observing their exploratory behavior. Drug abuse vulnerability was assessed through conditioning to cocaine. MS impacted the emotional state in both strains. Wistar responded with increased exploration, while Wistar-Kyoto increased anxiety-like behaviours. Despite the different coping strategies displayed by the two strains when challenged with the behavioural tests, drug conditioning was equally impacted by MS in both strains. Early-life stress appears to affect drug abuse vulnerability in adolescence independently of a depression background, suggesting emotional state as the main driving risk factor.

Osteoblasts are inherently programmed to repel sensory innervation.

Tissue innervation is a complex process controlled by the expression profile of signaling molecules secreted by tissue-resident cells that dictate the growth and guidance of axons. Sensory innervation is part of the neuronal network of the bone tissue with a defined spatiotemporal occurrence during bone development. Yet, the current understanding of the mechanisms regulating the map of sensory innervation in the bone tissue is still limited. Here, we demonstrated that differentiation of human mesenchymal stem cells to osteoblasts leads to a marked impairment of their ability to promote axonal growth, evidenced under sensory neurons and osteoblastic-lineage cells crosstalk. The mechanisms by which osteoblast lineage cells provide this nonpermissive environment for axons include paracrine-induced repulsion and loss of neurotrophic factors expression. We identified a drastic reduction of NGF and BDNF production and stimulation of Sema3A, Wnt4, and Shh expression culminating at late stage of OB differentiation. We noted a correlation between Shh expression profile, OB differentiation stages, and OB-mediated axonal repulsion. Blockade of Shh activity and signaling reversed the repulsive action of osteoblasts on sensory axons. Finally, to strengthen our model, we localized the expression of Shh by osteoblasts in bone tissue. Overall, our findings provide evidence that the signaling profile associated with osteoblast phenotype differentiating program can regulate the patterning of sensory innervation, and highlight osteoblast-derived Shh as an essential player in this cue-induced regulation.

Axonal outgrowth, neuropeptides expression and receptors tyrosine kinase phosphorylation in 3D organotypic cultures of adult dorsal root ganglia.

Limited knowledge from mechanistic studies on adult sensory neuronal activity was generated, to some extent, in recapitulated adult in vivo 3D microenvironment. To fill this gap there is a real need to better characterize the adult dorsal root ganglia (aDRG) organotypic cultures to make these in vitro systems exploitable for different approaches, ranging from basic neurobiology to regenerative therapies, to address the sensory nervous system in adult stage. We conducted a direct head-to-head comparison of aDRG and embryonic DRG (eDRG) organotypic culture focusing on axonal growth, neuropeptides expression and receptors tyrosine kinase (RTK) activation associated with neuronal survival, proliferation and differentiation. To identify alterations related to culture conditions, these parameters were also addressed in retrieved aDRG and eDRG and compared with organotypic cultures. Under similar neurotrophic stimulation, aDRG organotypic cultures displayed lower axonal outgrowth rate supported by reduced expression of growth associated protein-43 and high levels of RhoA and glycogen synthase kinase 3 beta mRNA transcripts. In addition, differential alteration in sensory neuropeptides expression, namely calcitonin gene-related peptide and substance P, was detected and was mainly pronounced at gene expression levels. Among 39 different RTK, five receptors from three RTK families were emphasized: tropomyosin receptor kinase A (TrkA), epidermal growth factor receptors (EGFR, ErbB2 and ErbB3) and platelet-derived growth factor receptor (PDGFR). Of note, except for EGFR, the phosphorylation of these receptors was dependent on DRG developmental stage and/or culture condition. In addition, EGFR and PDGFR displayed alterations in their cellular expression pattern in cultured DRG. Overall we provided valuable information particularly important when addressing in vitro the molecular mechanisms associated with development, maturation and regeneration of the sensory nervous system.

Therapeutic Drugs in Bone Loss-Associated Disorders: Clinical Outcomes and Challenges.

Current therapeutic drugs for the treatment of bone loss-associated disorders such as osteoporosis and metastatic bone disease have limited clinical outcomes, namely in terms of efficiency and sustainability. Given the ageing of population in developed countries and the cumulative costs with treatment, bone loss-associated disorders represent a major socioeconomic burden worldwide. In this review, the therapeutic agents targeting bone loss tested in clinical and pre-clinical trials are summarized, as well as the challenges encountered by clinicians and patients. In an effort to attain costeffective clinical outcomes, potential cellular and signalling targets are disclosed.

Fracture pain-Traveling unknown pathways.

An increase of fracture incidence is expected for the next decades, mostly due to the undeniable increase of osteoporotic fractures, associated with the rapid population ageing. The rise in sports-related fractures affecting the young and active population also contributes to this increased fracture incidence, and further amplifies the economical burden of fractures. Fracture often results in severe pain, which is a primary symptom to be treated, not only to guarantee individual's wellbeing, but also because an efficient management of fracture pain is mandatory to ensure proper bone healing. Here, we review the available data on bone innervation and its response to fracture, and discuss putative mechanisms of fracture pain signaling. In addition, the common therapeutic approaches to treat fracture pain are discussed. Although there is still much to learn, research in fracture pain has allowed an initial insight into the mechanisms involved. During the inflammatory response to fracture, several mediators are released and will putatively activate and sensitize primary sensory neurons, in parallel, intense nerve sprouting that occurs in the fracture callus area is also suggested to be involved in pain signaling. The establishment of hyperalgesia and allodynia after fracture indicates the development of peripheral and central sensitization, still, the underlying mechanisms are largely unknown. A major concern during the treatment of fracture pain needs to be the preservation of proper bone healing. However, the most common therapeutic agents, NSAIDS and opiates, can cause significant side effects that include fracture repair impairment. The understanding of the mechanisms of fracture pain signaling will allow the development of mechanisms-based therapies to effectively and safely manage fracture pain.

Bone Injury and Repair Trigger Central and Peripheral NPY Neuronal Pathways.

Bone repair is a specialized type of wound repair controlled by complex multi-factorial events. The nervous system is recognized as one of the key regulators of bone mass, thereby suggesting a role for neuronal pathways in bone homeostasis. However, in the context of bone injury and repair, little is known on the interplay between the nervous system and bone. Here, we addressed the neuropeptide Y (NPY) neuronal arm during the initial stages of bone repair encompassing the inflammatory response and ossification phases in femoral-defect mouse model. Spatial and temporal analysis of transcriptional and protein levels of NPY and its receptors, Y1R and Y2R, reported to be involved in bone homeostasis, was performed in bone, dorsal root ganglia (DRG) and hypothalamus after femoral injury. The results showed that NPY system activity is increased in a time- and space-dependent manner during bone repair. Y1R expression was trigged in both bone and DRG throughout the inflammatory phase, while a Y2R response was restricted to the hypothalamus and at a later stage, during the ossification step. Our results provide new insights into the involvement of NPY neuronal pathways in bone repair.

Ablation of Y1 receptor impairs osteoclast bone-resorbing activity.

Y1 receptor (Y1R)-signalling pathway plays a pivotal role in the regulation of bone metabolism. The lack of Y1R-signalling stimulates bone mass accretion that has been mainly attributed to Y1R disruption from bone-forming cells. Still, the involvement of Y1R-signalling in the control of bone-resorbing cells remained to be explored. Therefore, in this study we assessed the role of Y1R deficiency in osteoclast formation and resorption activity. Here we demonstrate that Y1R germline deletion (Y1R(-/-)) led to increased formation of highly multinucleated (n > 8) osteoclasts and enhanced surface area, possibly due to monocyte chemoattractant protein-1 (MCP-1) overexpression regulated by RANKL-signalling. Interestingly, functional studies revealed that these giant Y1R(-/-) multinucleated cells produce poorly demineralized eroded pits, which were associated to reduce expression of osteoclast matrix degradation markers, such as tartrate-resistant acid phosphatase-5b (TRAcP5b), matrix metalloproteinase-9 (MMP-9) and cathepsin-K (CTSK). Tridimensional (3D) morphologic analyses of resorption pits, using an in-house developed quantitative computational tool (BonePit), showed that Y1R(-/-) resorption pits displayed a marked reduction in surface area, volume and depth. Together, these data demonstrates that the lack of Y1Rs stimulates the formation of larger multinucleated osteoclasts in vitro with reduced bone-resorbing activity, unveiling a novel therapeutic option for osteoclastic bone diseases based on Y1R-signalling ablation.

Abnormal immunoreactivity to serotonin in cerebellar Purkinje cells after neonatal cocaine exposure.

Neonatal cocaine is known to affect the developing serotonergic system in many brain structures, including the cerebellum. Changes in the cerebellar Purkinje cells after drug exposure are well documented and result in impairment of movement and other cerebellar disorders such as ataxia. These cells have a major postnatal developmental pattern; therefore, neonatal exposure to cocaine is likely to affect them. In this work, male and female Wistar rats were injected with 15 mg of cocaine hydrochloride/kg body weight/day, subcutaneously, in two daily doses, from postnatal day 1 (PND1) to PND29. Controls were given 0.9% of saline. On PND14, PND21, and PND30, rats were transcardially perfused, and brains removed and cryoprotected. Coronal sections from the cerebellum were processed for immunocytochemistry of cells containing serotonin (5-hydroxytryptamine, or 5-HT). At the same postnatal age, rats from at least three different litters were sacrificed by decapitation, and brains were dissected for determination of 5-HT in the cerebellum by high-performance liquid chromatography with electrochemical detection. Upon the expected distribution of immunoreactivity to 5-HT, an abnormal immunoreactivity to 5-HT was observed in the Purkinje cells of six cocaine-exposed animals, but not in control animals. Also, levels of cerebellar 5-HT in cocaine-exposed rats were significantly increased on PND21. These results, together with previously reported observations of altered patterns of motor behavior, indicate that neonatal cocaine exposure affects the serotonergic cerebellar system, altering the standard development of Purkinje cells and possibly compromising the motor function.

Sensory neurons and osteoblasts: close partners in a microfluidic platform.

Innervation has proven to be critical in bone homeostasis/regeneration due to the effect of soluble factors, produced by nerve fibers, associated with changes in the activity of bone cells. Thus, in this study, we have established and characterized a coculture system comprising sensory neurons and osteoblasts to mimic the in vivo scenario where nerve fibers can be found in a bone microenvironment. Embryonic or adult primary dorsal root ganglion (DRG) and MC3T3-E1 osteoblastic cells were cocultured in compartmentalized microfluidic platforms and morphological and functional tests were performed. The time of adhesion and readout of axonal outgrowth were improved by the alignment of DRG with the axis of microgrooves, which showed to be a crucial step for the designed experiments. Cocultures of entire DRG from adult origin with osteoblasts were performed, showing extended DRG projections towards the axonal compartment, reaching osteoblastic cells. Immunocytochemistry showed that the neurites present within the osteoblastic compartment were immunoreactive to synapsin and calcitonin gene-related peptide suggesting the presence of specialized structures involved in this crosstalk. This evidence was further confirmed by electron microscopy where varicosities were detected as well as electron dense structures in neurite membranes. Aiming to mimic the properties of tissue extracellular matrices, MC3T3-E1 cells were seeded in the axonal side upon laminin, collagen or within 3D functionalized alginate matrices and axonal outgrowth was clearly observed. In order to analyze and quantify data with reproducible image analysis, a semi-automated algorithm was also developed. The collagen and laminin substrates displayed a higher amount of axons reaching the axonal side. Overall, the established method revealed to be a suitable tool to study the interaction between the peripheral nervous system and bone cells in different contexts mimicking the in vivo scenario.

Methamphetamine mimics the neurochemical profile of aging in rats and impairs recognition memory.

Brain neurochemistry and cognition performance are thought to decline with age. Accumulating data indicate that similar events occur after prolonged methamphetamine (MA) exposure. Using the rat as a model, the present study was designed to uncover common alteration patterns in brain neurochemistry and memory performance between aging and prolonged MA exposure. To this end, animals were treated with a chronic binge MA administration paradigm (20mg/kg/day from postnatal day 91 to 100). Three-age control groups received isovolumetric saline treatment and were tested at the MA age-matched period, and at 12 and 20 months. We observed that both MA and aged animals presented a long, but not short, time impairment in novelty preference and an increased anxiety-like behavior. Neurochemical analysis indicated similar MA- and age-related impairments in dopamine, serotonin and metabolites in the striatum, prefrontal cortex and hippocampus. Thus, the present data illustrate that MA may be used to mimic age-related effects on neurotransmitter systems and advocate MA treatment as a feasible animal model to study neuronal processes associated with aging.