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Severe loss of cholinergic neurons in the basal forebrain nuclei and of noradrenergic neurons in the locus coeruleus are almost invariant histopathological hallmarks of Alzheimer’s disease. However, the role of these transmitter systems in the spectrum of cognitive dysfunctions typical of the disease is still unclear, nor is it yet fully known whether do these systems interact and how. Selective ablation of either neuronal population, or both of them combined, were produced in developing animals to investigate their respective and/or concurrent contribution to spatial learning and memory, known to be severely affected in Alzheimer’s disease. Single or double lesions were created in 4–8 days old rats by bilateral intraventricular infusion of two selective immunotoxins. At about 16 weeks of age, the animals underwent behavioural tests specifically designed to evaluate reference and working memory abilities, and their brains were later processed for quantitative morphological analyses. Animals with lesion to either system alone showed no significant reference memory deficits which, by contrast, were evident in the double-lesioned subjects. These animals could not adopt an efficient search strategy on a given testing day and were unable to transfer all relevant information to the next day, suggesting deficits in acquisition, storage and/or recall. Only animals with single noradrenergic or double lesions exhibited impaired working memory. Interestingly, ablation of cholinergic afferents to the hippocampus stimulated a robust ingrowth of thick fibres from the superior cervical ganglion which, however, did not appear to have contributed to the observed cognitive performance. Ascending cholinergic and noradrenergic afferents to the hippocampus and neocortex appear to be primarily involved in the regulation of different cognitive domains, but they may functionally interact, mainly at hippocampal level, for sustaining normal learning and memory. Moreover, these transmitter systems are likely to compensate for each other, but apparently not via ingrowing sympathetic fibres.

Amyotrophic lateral sclerosis (ALS) is one of the most common motoneuronal disease, characterized by motoneuronal loss and progressive paralysis. Despite research efforts, ALS remains a fatal disease, with a survival of 2-5 years after disease onset. Numerous gene mutations have been correlated with both sporadic (sALS) and familiar forms of the disease, but the pathophysiological mechanisms of ALS onset and progression are still largely uncertain. However, a common profile is emerging in ALS pathological features, including misfolded protein accumulation and a cross-talk between neuroinflammatory and degenerative processes. In particular, astrocytes and microglial cells have been proposed as detrimental influencers of perineuronal microenvironment, and this role may be exerted via gap junctions (GJs)- and hemichannels (HCs)-mediated communications. Herein we investigated the role of the main astroglial GJs-forming connexin, Cx43, in human ALS and the effects of focal spinal cord motoneuronal depletion onto the resident glial cells and Cx43 levels. Our data support the hypothesis that motoneuronal depletion may affect glial activity, which in turn results in reactive Cx43 expression, further promoting neuronal suffering and degeneration.

Motoneuronal loss is the main feature of amyotrophic lateral sclerosis, although pathogenesis is extremely complex involving both neural and muscle cells. In order to translationally engage the sonic hedgehog pathway, which is a promising target for neural regeneration, recent studies have reported on the neuroprotective effects of clobetasol, an FDA-approved glucocorticoid, able to activate this pathway via smoothened. Herein we sought to examine functional, cellular, and metabolic effects of clobetasol in a neurotoxic mouse model of spinal motoneuronal loss. We found that clobetasol reduces muscle denervation and motor impairments in part by restoring sonic hedgehog signaling and supporting spinal plasticity. These effects were coupled with reduced pro-inflammatory microglia and reactive astrogliosis, reduced muscle atrophy, and support of mitochondrial integrity and metabolism. Our results suggest that clobetasol stimulates a series of compensatory processes and therefore represents a translational approach for intractable denervating and neurodegenerative disorders.

Multiple myeloma (MM) is a clonal B-cell malignancy characterized by an accumulation of plasma cells (PC) in the bone marrow (BM), leading to bone loss and BM failure. Osteolytic bone disease is a common manifestation observed in MM patients and represents the most severe cause of morbidity, leading to progressive skeletal damage and disabilities. Pathogenetic mechanisms of MM bone disease are closely linked to PCs and osteoclast (OCs) hyperactivity, coupled with defective osteoblasts (OBs) function that is unable to counteract bone resorption. The aim of the present study was to investigate the effects of Ixazomib, a third-generation proteasome inhibitor, on osteoclastogenesis and osteogenic differentiation. We found that Ixazomib was able to reduce differentiation of human monocytes into OCs and to inhibit the expression of OC markers when added to the OC medium. Concurrently, Ixazomib was able to stimulate osteogenic differentiation of human mesenchymal stromal cells (MSCs), increasing osteogenic markers, either alone or in combination with the osteogenic medium. Given the key role of Sonic Hedgehog (SHH) signaling in bone homeostasis, we further investigated Ixazomib-induced SHH pathway activation. This set of experiments showed that Ixazomib, but not Bortezomib, was able to bind the Smoothened (SMO) receptor leading to nuclear translocation of GLI1 in human MSCs. Moreover, we demonstrated that PCs act as GLI1 suppressors on MSCs, thus reducing the potential of MSCs to differentiate in OBs. In conclusion, our data demonstrated that Ixazomib regulates bone remodeling by decreasing osteoclastogenesis and prompting osteoblast differentiation via the canonical SHH signaling pathway activation, thus, representing a promising therapeutic option to improve the complex pathological condition of MM patients.

Neuropathic pain is caused by primary lesion or dysfunction of either peripheral or central nervous system. Due to its complex pathogenesis, often related to a number of comorbidities, such as cancer, neurodegenerative and neurovascular diseases, neuropathic pain still represents an unmet clinical need, lacking long-term effective treatment and complex case-by-case approach. We analyzed the recent literature on the role of selective voltage-sensitive sodium, calcium and potassium permeable channels and non-selective gap junctions (GJs) and hemichannels (HCs) in establishing and maintaining chronic neuropathic conditions. We finally focussed our review on the role of extracellular microenvironment modifications induced by resident glial cells and on the recent advances in cell-to-cell and cell-to-extracellular environment communication in chronic neuropathies. In this review, we provide an update on the current knowledge of neuropathy chronicization processes with a focus on both neuronal and glial ion channels, as well as on channel-mediated intercellular communication.

Simple Summary The biological pathways underlying glioblastoma malignancy and radioresistance are still unclear. In this review, we describe the role of the hypoxic microenvironment and SRC proto-oncogene non-receptor tyrosine kinase in the activation of radioresistance and invasion pathways of glioblastoma. We also highlight the hypoxia- and ionizing radiation-induced infiltration, providing updated evidences on the involvement of SRC in these processes. Optimizing radiotherapy and identifying druggable molecular players are crucial steps to improve current glioblastoma therapeutic strategies. Abstract Advances in functional imaging are supporting neurosurgery and radiotherapy for glioblastoma, which still remains the most aggressive brain tumor with poor prognosis. The typical infiltration pattern of glioblastoma, which impedes a complete surgical resection, is coupled with a high rate of invasiveness and radioresistance, thus further limiting efficient therapy, leading to inevitable and fatal recurrences. Hypoxia is of crucial importance in gliomagenesis and, besides reducing radiotherapy efficacy, also induces cellular and molecular mediators that foster proliferation and invasion. In this review, we aimed at analyzing the biological mechanism of glioblastoma invasiveness and radioresistance in hypoxic niches of glioblastoma. We also discussed the link between hypoxia and radiation-induced radioresistance with activation of SRC proto-oncogene non-receptor tyrosine kinase, prospecting potential strategies to overcome the current limitation in glioblastoma treatment.

Extensive loss of noradrenaline-containing neurons and fibers is a nearly invariant feature of Alzheimer’s Disease (AD). However, the exact noradrenergic contribution to cognitive and histo- pathological changes in AD is still unclear. Here, this issue was addressed following selective lesioning and intrahippocampal implantation of embryonic noradrenergic progenitors in developing rats. Starting from about 3 months and up to 12 months post-surgery, animals underwent behav- ioral tests to evaluate sensory-motor, as well as spatial learning and memory, followed by post- mortem morphometric analyses. At 9 months, Control, Lesioned and Lesion1Transplant animals exhibited equally efficient sensory-motor and reference memory performance. Interestingly, working memory abilities were seen severely impaired in Lesion-only rats and fully recovered in Transplanted rats, and appeared partly lost again 2 months after ablation of the implanted neuroblasts. Morphological analyses confirmed the almost total lesion-induced noradrenergic neuronal and terminal fiber loss, the near-normal reinnervation of the hippocampus promoted by the transplants, and its complete removal by the second lesion. Notably, the noradrenergic-rich transplants normalized also the nuclear expression of the transactive response DNA-binding protein 43 (TDP- 43) in various hippocampal subregions, whose cytoplasmic (i.e., pathological) occurrence appeared dramatically increased as a result of the lesions. Thus, integrity of ascending noradrenergic inputs to the hippocampus may be required for the regulation of specific aspects of learning and memory and to prevent TDP-43 tissue pathology.

It is known that exists a relationship between listening to music and food intake. Hypothalamus appears to integrate the orexigenic properties of a novel peptide, ghrelin (Ghre) that induces food intake through neuropeptide Y (NPY). Ghre stimulates appetite by acting on the ventral hypothalamus, which controls food intake. Ghre is secreted from the stomach and circulates in the bloodstream under fasting conditions, sending a hunger signal from the periphery to the Central Nervous System. The aim of this study was to evaluate, in the rat, the effects of different musical frequencies (432 and 440 Hz) on the Ghre and NPY expression in the hypothalamic neurons through immunohistochemistry; in addition, we investigated on the different production of Ghre in the serum through Western blot assay (Wb), in relation to the body weight of animals. Ghre-immunopositive cells were counted, showing a significant increase in music-treated compared to the control (CTR) group. Similarly, music-treated rats showed increased levels of Ghre in the serum compared to CTR animals. Finally, the body weight of animals was affected by music. In particular, music exposure was able to stimulate the body weight increase and, interestingly, the increase was higher when animals were exposed to music at 440 Hz. Together, the results strongly support the hypothesis that different musical frequencies could affect food intake by modulating the hypothalamic Ghre expression and its release.

Despite the relevant research efforts, the causes of amyotrophic lateral sclerosis (ALS) are still unknown and no effective cure is available. Many authors suggest that ALS is a multi-system disease caused by a network failure instead of a cell-autonomous pathology restricted to motoneurons. Although motoneuronal loss is the critical hallmark of ALS given their specific vulnerability, other cell populations, including muscle and glial cells, are involved in disease onset and progression, but unraveling their specific role and crosstalk requires further investigation. In particular, little is known about the plastic changes of the degenerating motor system. These spontaneous compensatory processes are unable to halt the disease progression, but their elucidation and possible use as a therapeutic target represents an important aim of ALS research. Genetic animal models of disease represent useful tools to validate proven hypotheses or to test potential therapies, and the conception of novel hypotheses about ALS causes or the study of pathogenic mechanisms may be advantaged by the use of relatively simple in vivo models recapitulating specific aspects of the disease, thus avoiding the inclusion of too many confounding factors in an experimental setting. Here, we used a neurotoxic model of spinal motoneuron depletion induced by injection of cholera toxin-B saporin in the gastrocnemius muscle to investigate the possible occurrence of compensatory changes in both the muscle and spinal cord. The results showed that, following the lesion, the skeletal muscle became atrophic and displayed electromyographic activity similar to that observed in ALS patients. Moreover, the changes in muscle fiber morphology were different from that observed in ALS models, thus suggesting that some muscular effects of disease may be primary effects instead of being simply caused by denervation. Notably, we found plastic changes in the surviving motoneurons that can produce a functional restoration probably similar to the compensatory changes occurring in disease. These changes could be at least partially driven by glutamatergic signaling, and astrocytes contacting the surviving motoneurons may support this process.

Sonic hedgehog (Shh) signaling is a key pathway within the central nervous system (CNS), during both development and adulthood, and its activation via the 7-transmembrane protein Smoothened (Smo) may promote neuroprotection and restoration during neurodegenerative disorders. Shh signaling may also be activated by selected glucocorticoids such as clobetasol, fluocinonide and fluticasone, which therefore act as Smo agonists and hold potential utility for regenerative medicine. However, despite its potential role in neurodegenerative diseases, the impact of Smo-modulation induced by these glucocorticoids on adult neural stem cells (NSCs) and the underlying signaling mechanisms are not yet fully elucidated. The aim of the present study was to evaluate the effects of Smo agonists (i.e., purmorphamine) and antagonists (i.e., cyclopamine) as well as of glucocorticoids (i.e., clobetasol, fluocinonide and fluticasone) on NSCs in terms of proliferation and clonal expansion. Purmorphamine treatment significantly increased NSC proliferation and clonal expansion via GLI-Kruppel family member 1 (Gli1) nuclear translocation and such effects were prevented by cyclopamine co-treatment. Clobetasol treatment exhibited an equivalent pharmacological effect. Moreover, cellular thermal shift assay suggested that clobetasol induces the canonical Smo-dependent activation of Shh signaling, as confirmed by Gli1 nuclear translocation and also by cyclopamine co-treatment, which abolished these effects. Finally, fluocinonide and fluticasone as well as control glucocorticoids (i.e., prednisone, corticosterone and dexamethasone) showed no significant effects on NSCs proliferation and clonal expansion. In conclusion, our data suggest that Shh may represent a druggable target system to drive neuroprotection and promote restorative therapies.

Plastic changes have been reported in the SOD1-G93A mouse model of amyotrophic lateral sclerosis, a disorder characterized by progressive motoneuronal loss; however, whether these changes related with the onset and development of motor impairments is still unclear. Here, the functional and anatomical changes taking place in SOD1-G93A mice and their time course were investigated during ongoing motoneuronal degeneration. Starting from about 4 postnatal weeks, SOD1-G93A and wild-type (WT) mice were evaluated in the rotarod test, to be sacrificed at about 12-13 or 19 weeks of age, and their lumbar spinal cords were processed for histo- and immunohistochemistry. Compared to age-matched WT controls, 12 weeks-old SOD1-G93A mice exhibited relatively mild or no motor impairments in the rotarod test, in spite of a dramatic (≈60%, as estimated by stereology) loss of choline acetyl-transferase (ChAT)-immunoreactive motoneurons which remained virtually unchanged in SOD1-G93A mice surviving up to 19 weeks. Notably, the functional sparing in SOD1-G93A mice at 12 weeks was paralleled by a marked ≈50% increase in motoneuron volume and a near-normal density of acetylcholinesterase-positive process arborization, which was significantly increased when analyzed as ratio to the decreased number of ChAT-positive motoneurons. By contrast, at 19 weeks, when motor deficits had become dramatically evident, both measures were found reverted to about 50-60% of control values. Thus, at specific stages during the progression of the disease, robust compensatory events take place in surviving motoneurons of SOD1-G93A mice, which sustain motor performance, and whose full understanding may highlight a valuable therapeutic opportunity window.

Olfactory Ensheathing Cells (OECs), glial cells of the olfactory system, are able to secrete several neurotrophic growth factors, promoting axonal growth and supporting remyelination of damaged axons. In the olfactory system, during the development, they drive the non-myelinated axons of the Olfactory Receptor Neurons towards the Olfactory bulbs. OEC transplantation has emerged as a possible experimental therapy to induce repair of nervous system injury, even if the functional recovery is still limited. Several studies suggest that into all the olfactory system play an important role Ghrelin (Ghre), an orexigenic peptide that acts not only in the olfactory cerebral areas, but also on the most peripheral part of the olfactory system. It is a polypeptide and a gut hormone produced from the stomach, it is able to reach the Central Nervous System by regulating metabolism and by performing neuroprotection. Our previous investigations have shown that OB mitral cells express Ghre, thus being able to play an important role in regulating food behavior in response to odors. The aim of this study was to investigate on the secretion and the presence of Ghre peptide and its receptors in OECs. Primary OECs were obtained from 2-d old mice pup (P2) olfactory bulbs, the cells were grown in Dulbecco's Modified Eagle's medium supplemented with 10% Foetal Bovine Serum. Immunocytochemical and Western blot analysis were used. Our results, for the first time, showed that OECs were able to secrete the Ghre peptide and that OECs express GHS-R 1a receptors. Moreover, being known that Ghre has a neurotrophic action, both in promoting axonal growth and synaptogenesis probably through the secreting factors, it could be hypothesized that even the OECs through the Ghre, can improve the growth of neurites and promote alignment. Therefore, it can be hypothesized that they can interact with reinforcement function, in the peripheral olfactory circuit, providing a neurotrophic support to the synaptic interaction between the ORNs and the mitral cells.

Neuropathic pain is a chronic condition triggered by lesions to the somatosensory nervous system in which pain stimuli occur spontaneously or as pathologically amplified responses. In this scenario, the exchange of signaling molecules throughout cell-to-cell and cell-to-extracellular environment communications plays a key role in the transition from acute to chronic pain. As such, connexin 43 (Cx43), the core glial gap junction and hemichannel-forming protein, is considered a triggering factor for disease chronicization in the central nervous system (CNS). Drugs targeting μ opioid receptors (MOR) are currently used for moderate to severe pain conditions, but their use in chronic pain is limited by the tolerability profile. δ opioid receptors (DOR) have become attractive targets for the treatment of persistent pain and have been associated with the inhibition of pain-sustaining factors. Moreover, it has been shown that simultaneous targeting of MOR and DOR leads to an improved pharmacological fingerprint. Herein, we aimed to study the effects of the benzomorphan ligand LP2, a multitarget MOR/DOR agonist, in an experimental model of neuropathic pain induced by the unilateral sciatic nerve chronic constriction injury (CCI) on male Sprague-Dawley rats. Results showed that LP2 significantly ameliorated mechanical allodynia from the early phase of treatment up to 21 days post-ligatures. We additionally showed that LP2 prevented CCI-induced Cx43 alterations and pro-apoptotic signaling in the CNS. These findings increase the knowledge of neuropathic pain development and the role of spinal astrocytic Cx43, suggesting new approaches for the treatment of neuropathic pain.

In recent years, microRNAs (miRNAs) have received increasing attention for their important role in tumor initiation and progression. MiRNAs are a class of endogenous small non-coding RNAs that negatively regulate the expression of several oncogenes or tumor suppressor genes. MiR-19a, a component of the oncogenic miR-17-92 cluster, has been reported to be highly expressed only in anaplastic thyroid cancer, the most undifferentiated, aggressive and lethal form of thyroid neoplasia. In this work, we evaluated the putative contribution of miR-19a in de-differentiation and aggressiveness of thyroid tumors. To this aim, we induced miR-19a expression in the well-differentiated follicular thyroid cancer cell line and evaluated proliferation, apoptosis and gene expression profile of cancer cells. Our results showed that miR-19a overexpression stimulates cell proliferation and alters the expression profile of genes related to thyroid cell differentiation and aggressiveness. These findings not only suggest that miR-19a has a possible involvement in de-differentiation and malignancy, but also that it could represent an important prognostic indicator and a good therapeutic target for the most aggressive thyroid cancer.

Olfactory Ensheathing Cells (OECs) are glial cells able to secrete different neurotrophic growth factors and thus promote axonal growth, also acting as a mechanical support. In the olfactory system, during development, they drive the non-myelinated axons of the Olfactory Receptor Neurons (ORNs) towards the Olfactory Bulb (OB). Ghrelin (Ghre), a gut-brain peptide hormone, and its receptor (GHS-R 1a) are expressed in different parts of the central nervous system. In the last few years, this peptide has stimulated particular interest as results show it to be a neuroprotective factor with antioxidant, anti-inflammatory and anti-apoptotic properties. Our previous studies showed that OB mitral cells express Ghre, thus being able to play an important role in regulating food behavior in response to odors. In this study, we investigated the presence of Ghre and GHS-R 1a in primary mouse OECs. The expression of both Ghre and its receptor was assessed by an immunocytochemical technique, Western Blot and Polymerase Chain Reaction (PCR) analysis. Our results demonstrated that OECs are able to express both Ghre and GHS-R 1a and that these proteins are detectable after extensive passages in vitro; in addition, PCR analysis further confirmed these data. Therefore, we can hypothesize that Ghre and GHS-R 1a interact with a reinforcement function, in the peripheral olfactory circuit, providing a neurotrophic support to the synaptic interaction between ORNs and mitral cells.

Multiple myeloma (MM) is a clonal B-cell malignancy characterized by an accumulation of clonal plasma cells (PC) in the bone marrow (BM) leading to bone destruction and BM failure. Osteolytic bone disease is a common manifestation of MM that leads to progressive skeleton destruction and is the most severe cause of morbidity in MM patients. Pathogenetic mechanisms of MM bone destruction are closely linked to MM PC and osteoclasts (OCs) hyperactivity coupled with defective osteoblast (OB) function unable to counteract bone resorption. We recently demonstrated that the proteasome inhibitor (PI) Bortezomib, commonly used to treat MM, was capable to inhibit osteoclastic differentiation modulating chitinase family genes. In this work we investigated the effect of Ixazomib (IXA), a third generation PI, on osteoclastogenesis and osteogenic differentiation. Human monocytes were differentiated in OCs in presence of OC medium (supplemented with RANKL and M-CSF), and/or 10nM IXA. We observed that IXA was able to inhibit the expression of different OCs markers such as RANK, CTSK, TRAP, and MMP9 when added in OC medium in respect to OC medium alone (p Disclosures Conticello: Celgene: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Honoraria, Research Funding. Palumbo:Amgen: Honoraria; Celgene: Honoraria; Hospira: Honoraria; Janssen: Honoraria; Novartis: Honoraria; Teva: Honoraria. Di Raimondo:Amgen: Consultancy, Honoraria, Research Funding; Takeda: Consultancy; Celgene: Consultancy, Honoraria, Research Funding.

A better understanding of pediatric tumor biology is needed to allow the development of less toxic and more efficient therapies, as well as to provide novel reliable biomarkers for diagnosis and risk stratification. The emerging role of microRNAs in controlling key pathways implicated in tumorigenesis makes their use in diagnostics a powerful novel tool for the early detection, risk assessment and prognosis, as well as for the development of innovative anticancer therapies. This perspective would be more urgent for the clinical management of pediatric cancer. In this review, we focus on the involvement of microRNAs in the biology of the main childhood tumors, describe their clinical significance and discuss their potential use as novel therapeutic tools and targets.

Transactive response DNA-binding protein of 43 kDa (TDP-43) is a nuclear DNA/RNA-binding protein involved in gene transcription and mRNA processing. Recently, TDP-43 has been found in the cytoplasmic inclusions observed in amyotrophic lateral sclerosis. Substantial attention has been devoted to the toxic effects of the cytoplasmic TDP-43 aggregates, whereas the functional role of this protein remains poorly investigated. Interestingly, TDP-43 could be localized in the synapse and affect synaptic plasticity and locomotion in Drosophila. Here, we would like to understand if TDP-43 could modulate spinal cord plasticity in a mouse model of neurotoxic motoneuron depletion. Therefore, the expression levels of TDP- 43 and synaptic proteins such as synapsin-I and the α-amino-3-hydroxy-5-methyl-4- isoxazolepropionate (AMPA) receptor subunits GluR1, GluR2 and GluR4 were measured by western blotting. By using multivariate regression models, protein expression levels were correlated each other as well as with the motor performance. The results suggested that motor performance could be linked to the expression of synapsin-I, and that the latter could depend on TDP-43, which in turn could interact with AMPA receptors. In conclusion, our results suggest that TDP-43 is likely involved in the modulation of synaptic plasticity. Given the increasing interest in mouse models of TDP-43 gain or loss of function in neurodegenerative diseases, the elucidation of the role of TDP-43 in the spinal cord is mandatory. More generally, given the recently increased knowledge about spinal cord plasticity, we postulate that the stimulation of the intrinsic plastic potential of spinal cord would be a successful repairing strategy.

Traumatic injury or surgical excision of diseased bone tissue usually require the reconstruction of large bone defects unable to heal spontaneously, especially in older individuals. This is a big challenge requiring the development of biomaterials mimicking the bone structure and capable of inducing the right commitment of cells seeded within the scaffold. In particular, given their properties and large availability, the human adipose-derived stem cells are considered as the better candidate for autologous cell transplantation. In order to evaluate the regenerative potential of these cells along with an osteoinductive biomaterial, we have used collagen/hydroxyapatite scaffolds to test ectopic bone formation after subcutaneous implantation in mice. The process was analysed both in vivo, by Fluorescent Molecular Tomography (FMT), and ex vivo, to evaluate the formation of bone and vascular structures. The results have shown that the biomaterial could itself be able of promoting differentiation of host cells and bone formation, probably by means of its intrinsic chemical and structural properties, namely the microenvironment. However, when charged with human mesenchymal stem cells, the ectopic bone formation within the scaffold was increased. We believe that these results represent an important advancement in the field of bone physiology, as well as in regenerative medicine.

Degeneration of noradrenergic neurons in the locus coeruleus and loss of fiber terminals in the neocortical and hippocampal target regions represent prominent and early features of Alzheimer’s disease, however, whether these events are causally linked to cognitive decline in Alzheimer’s disease is still unclear. In the present study, the noradrenergic contribution to the regulation of hippocampus-dependent spatial learning and memory was investigated. Postnatal day 4 rats underwent selective immunolesioning of hippocampal noradrenergic afferents and, 4 days later, the bilateral intrahippocampal implantation of locus coeruleus noradrenergic neuroblasts. Starting from 4 weeks and up to about 9 months postsurgery, sensory-motor and spatial navigation abilities were evaluated, followed by postmortem tissue analyses. All animals in the Control, Lesion, and Lesion+Transplant groups exhibited normal sensory-motor function and were equally efficient in the reference memory version of the water maze task, whereas working memory abilities were seen consistently impaired in the Lesion-only rats. Notably, the noradrenergic reinnervation promoted by the grafted progenitors reinstated a fairly normal working memory performance, suggesting a primary role for coeruleo-hippocampal noradrenergic inputs in the maintenance of specific aspects of cognition.

Recently, multipotent mesenchymal stem cells (MSCs) have attracted much attention in the field of regenerative medicine due to their ability to give rise to different cell types, including chondrocytes. Damaged articular cartilage repair is one of the most challenging issues for regenerative medicine, due to the intrinsic limited capability of cartilage to heal because of its avascular nature. While surgical approaches like chondral autografts and allografts provide symptoms and function improvement only for a short period, MSC based stimulation therapies, like microfracture surgery or autologous matrix-induced chondrogenesis demonstrate to be more effective. The use of adult chondrocytes, which are the main cellular constituent of cartilage, in medical practice, is indeed limited due to their instability in monolayer culture and difficulty to collect donor tissue (articular and nasal cartilage). The most recent cartilage engineering approaches combine cells, biomaterial scaffold and bioactive factors to promote functional tissue replacements. Many recent evidences demonstrate that scaffolds providing specific microenvironmental conditions can promote MSCs differentiation toward a functional phenotype. In the present work, the chondrogenic potential of a new Collagen I based 3D scaffold has been assessed in vitro, in combination with human adipose-derived MSCs which possess a higher chondrogenic potential compared to MSCs isolated from other tissues. Our data indicate that the scaffold was able to promote the early stages of chondrogenic commitment and that supplementation of specific soluble factors was able to induce the complete differentiation of MSCs in chondrocytes as demonstrated by the appearance of cartilage distinctive markers (Sox 9, Aggrecan, Matrilin-1, and Collagen II), as well as by the cartilage-specific Alcian Blue staining and by the acquisition of typical cellular morphology. Such evidences suggest that the investigated scaffold formulation could be suitable for the production of medical devices that can be beneficial in the field of articular cartilage engineering, thus improving the efficacy and durability of the current therapeutic options.

The bone grafting is the classical way to treat large bone defects. Among the available techniques, autologous bone grafting is still the most used but, however, it can cause complications such as infection and donor site morbidity. Alternative and innovative methods rely on the development of biomaterials mimicking the structure and properties of natural bone. In this study, we characterized a cell-free scaffold, which was subcutaneously implanted in mice and then analyzed both in vivo and ex vivo after 1, 2, 4, 8 and 16 weeks, respectively. Two types of biomaterials, made of either collagen alone or collagen plus magnesium-enriched hydroxyapatite have been used. The results indicate that bone augmentation and angiogenesis could spontaneously occur into the biomaterial, probably by the recruitment of host cells, and that the composition of the scaffolds is crucial. In particular, the biomaterial more closely mimicking the native bone drives the process of bone augmentation more efficiently. Gene expression analysis and immunohistochemistry demonstrate the expression of typical markers of osteogenesis by the host cells populating the scaffold. Our data suggest that this biomaterial could represent a promising tool for the reconstruction of large bone defects, without using exogenous living cells or growth factors.

Sonic hedgehog and Noggin are morphogenetic factors involved in neural induction and ventralization of the neural tube, but recent findings suggest that they could participate in regeneration and functional recovery after injury. Here, in order to verify if these mechanisms could occur in the spinal cord and involve synaptic plasticity, we measured the expression levels of Sonic hedgehog, Noggin, Choline Acetyltransferase, Synapsin-I and Glutamate receptor subunits (GluR1, GluR2, GluR4), in a motoneuron-depleted mouse spinal cord lesion model obtained by intramuscular injection of Cholera toxin-B saporin. The lesion caused differential expression changes of the analyzed proteins. Moreover, motor performance was found correlated with Sonic hedgehog and Noggin expression in lesioned animals. The results also suggest that Sonic hedgehog could collaborate in modulating synaptic plasticity. Together, these findings confirm that the injured mammalian spinal cord has intrinsic potential for repair and that some proteins classically involved in development, such as Sonic hedgehog and Noggin could have important roles in regeneration and functional restoration, by mechanisms including synaptic plasticity.

Neural stem cells (NSCs) have become promising tools for basic research and regenerative medicine. Intracerebral transplantation studies have suggested that these cells may be able to adopt neuronal phenotypes typical of their engraftment site and to establish appropriate connections in the recipient circuitries. Here, we examined the in vivo neurogenic competence of well-characterized NSC lines subjected to in vitro priming and subsequent implantation into the adult intact mouse brain. Upon implantation into the hippocampus and, less frequently, in the striatum and in the cerebral cortex, numerous green fluorescent protein (GFP)-tagged cells acquired differentiated features indistinguishable from resident neurons. Upon closer examination, however, we found that this outcome resulted from fusion of donor cells with local neuronal elements generating long-term persistent GFP+ neuronal hybrids. This fusogenic behavior of NSCs was unexpected and also observed in coculture with E18 hippocampal immature neural cells, but not with microglia or astrocytes. Similar findings were consistently obtained with different NSC lines, mouse recipients, and donor cell-labeling methods. The frequent and cell type-specific fusion of donor NSCs with host neurons highlights a previously underestimated biological property of the nervous tissue that might prove profitable for basic and therapeutically oriented studies. © Copyright 2013, Mary Ann Liebert, Inc. 2013.

Noradrenergic neurons in the locus coeruleus play a role in learning and memory, and their loss is an early event in Alzheimer’s disease pathogenesis. Moreover, noradrenaline may sustain hippocampal neurogenesis; however, whether are these events related is still unknown. Four to five weeks following the selective immunotoxic ablation of locus coeruleus neurons, young adult rats underwent reference and working memory tests, followed by postmortem quantitative morphological analyses to assess the extent of the lesion, as well as the effects on proliferation and/or survival of neural progenitors in the hippocampus. When tested in the Water Maze task, lesioned animals exhibited no reference memory deficit, whereas working memory abilities were seen significantly impaired, as compared with intact or sham-lesioned controls. Stereological analyses confirmed a dramatic noradrenergic neuron loss associ- ated to reduced proliferation, but not survival or differentiation, of 5-bromo-20deoxyuridineepositive progenitors in the dentate gyrus. Thus, ascending noradrenergic afferents may be involved in more complex aspects of cognitive performance (i.e., working memory) possibly via newly generated pro- genitors in the hippocampus.

Mesenchymal stem cells (MSCs) play a crucial role in regulating normal skeletal homeostasis and, in case of injury, in bone healing and reestablishment of skeletal integrity. Recent scientific literature is focused on the development of bone regeneration models where MSCs are combined with biomimetic three-dimensional scaffolds able to direct MSC osteogenesis. In this work the osteogenic potential of human MSCs isolated from adipose tissue (hADSCs) has been evaluated in vitro in combination with collagen/Mg doped hydroxyapatite scaffolds. Results demonstrate the high osteogenic potential of hADSCs when cultured in specific differentiation induction medium, as revealed by the Alizarin Red S staining and gene expression profile analysis. In combination with collagen/hydroxyapatite scaffold, hADSCs differentiate into mature osteoblasts even in the absence of specific inducing factors; nevertheless, the supplement of the factors markedly accelerates the osteogenic process, as confirmed by the expression of specific markers of pre-osteoblast and mature osteoblast stages, such as osterix, osteopontin (also known as bone sialoprotein I), osteocalcin and specific markers of extracellular matrix maturation and mineralization stages, such as ALPL and osteonectin. Hence, the present work demonstrates that the scaffold per se is able to induce hADSCs differentiation, while the addition of osteo-inductive factors produces a significant acceleration of the osteogenic process. This observation makes the use of our model potentially interesting in the field of regenerative medicine for the treatment of bone defects.

The possibility to selectively remove noradrenergic neurons in the locus coeruleus/subcoeruleus (LC/SubC) complex by the immunotoxin anti-dopamine-beta-hydroxylase (DBH)-saporin has offered a powerful tool to study the functional role of this projection system. In the present study, the anatomical consequences of selective lesions of the LC/SubC on descending noradrenergic projections during early postnatal development have been investigated following bilateral intraventricular injections of anti-DBH-saporin or 6-hydroxydopamine to immature (4 day old) rats. Administration of increasing doses (0.25-1.0 microg) of the immunotoxin produced, about 5 weeks later, a dose-dependent loss of DBH-immunoreactive neurons in the LC/SubC complex (approximately 45-90%) paralleled by a similar reduction of noradrenergic innervation in the terminal territories in the lumbar spinal cord. Even at the highest dose used (1.0 microg) the immunotoxin did not produce any detectable effects on dopaminergic, adrenergic, serotonergic or cholinergic neuronal populations, which, by contrast, were markedly reduced after administration of 6-hydroxydopamine. The approximately 90% noradrenergic depletion induced by 0.5 and 1.0 microg of anti-DBH-saporin remained virtually unchanged at 40 weeks post-lesion. Conversely, the approximately 45% reduction of spinal innervation density estimated at 5 weeks in animals injected with the lowest dose (0.25 microg) of the immunotoxin was seen recovered up to near-normal levels at 40 weeks, possibly as a result of the intrinsic plasticity of the developing noradrenergic system. A similar reinnervation in the lumbar spinal cord was also seen promoted by grafts of fetal LC tissue implanted at the postnatal day 8 (i.e. 4 days after the lesion with 0.5 microg of anti-DBH-saporin). In these animals, the number of surviving neurons in the grafts and the magnitude of the reinnervation, with fibers extending in both the grey and white matter for considerable distances, were seen higher than those reported in previous studies using adult recipients. This would suggest that the functional interactions between the grafted tissue and the host may recapitulate the events normally occurring during the ontogenesis of the coeruleo-spinal projection system, and can therefore be developmentally regulated. Thus, the neonatal anti-DBH-saporin lesion model, with the possibility to produce graded noradrenergic depletions, holds promises as a most valuable tool to address issues of compensatory reinnervation and functional recovery in the severed CNS as well as to elucidate the mechanisms governing long-distance axon growth from transplanted neural precursors.

Fetal spinal cord (SC) tissue grafts can survive and develop into the lesioned SC, but no conclusive data are available concerning the long-term fate of transplanted material and the relation between the graft fate and the donor embryo age. Here, pre-labelled suspensions of ventral SC from E12 or E17 rat fetuses were grafted to the lumbar SC of adult rats with motoneuron depletion induced by perinatal injection of volkensin. E12 and E17 are presumably the stages when motoneuron development starts and terminates, respectively. Four or 10 months post-grafting, SCs were analyzed to check the graft survival rate and to follow the differentiation and spatial distributions of grafted cells. Neurotoxic lesion produced a 61% motoneuronal loss in the lumbar SC. In transplanted animals, all E12 fetal grafts survived until the observed time-points and developed various mature cell phenotypes. Many motoneuron-like labelled cells were found within the graft area or adjacent to it. Conversely, none of the E17 fetal grafts survived, since no graft-derived elements with neuronal morphology were found either in the site of graft placement or adjacent to it. The present findings indicate that spinal neuroblasts can survive for a long time and develop within the motoneuron-depleted SC, and that the donor embryo age is crucial for successful engraftment.