Cell-Autonomous Processes That Impair Xenograft Survival into the Cerebellum.

In immunocompetent animals, numerous factors including the immune system of the host regulate the survival of neuro-glial precursors transplanted into the cerebellum. We transplanted human neuro-glial precursors derived in vitro from partial differentiation of IPS cells into the developing cerebellum of mice and rats before maturation of the host immune system. These approaches should facilitate the development of immune-tolerance for the transplanted cells. However, we found that human cells survived the engraftment and integrated into the host cerebellum and brain stem up to about 1 month postnatally when they were rejected in both species. On the contrary, when we transplanted the same cells in NOD-SCID mice, they survived indefinitely. Our findings are consistent with the hypothesis that the slower pace of differentiation of human neural precursors compared to that of rodents restricts the induction of immune-tolerance to human antigens expressed before completion of the maturation of the immune system. As predicted by our hypothesis, when we engrafted the human neuro-glial precursor cells either in a more mature state or mixed with extracts from adult cerebellum, we prolonged the survival of the graft.

Blood Vessels: The Pathway Used by Schwann Cells to Colonize Nerve Conduits.

The repair of severe nerve injuries requires an autograft or conduit to bridge the gap and avoid axon dispersion. Several conduits are used routinely, but their effectiveness is comparable to that of an autograft only for short gaps. Understanding nerve regeneration within short conduits could help improve their efficacy for longer gaps. Since Schwann cells are known to migrate on endothelial cells to colonize the "nerve bridge", the new tissue spontaneously forming to connect the injured nerve stumps, here we aimed to investigate whether this migratory mechanism drives Schwann cells to also proceed within the nerve conduits used to repair large nerve gaps. Injured median nerves of adult female rats were repaired with 10 mm chitosan conduits and the regenerated nerves within conduits were analyzed at different time points using confocal imaging of sequential thick sections. Our data showed that the endothelial cells formed a dense capillary network used by Schwann cells to migrate from the two nerve stumps into the conduit. We concluded that angiogenesis played a key role in the nerve conduits, not only by supporting cell survival but also by providing a pathway for the migration of newly formed Schwann cells.

Neurogranin Regulates Adult-Born Olfactory Granule Cell Spine Density and Odor-Reward Associative Memory in Mice.

Neurogranin (Ng) is a brain-specific postsynaptic protein, whose role in modulating Ca2+/calmodulin signaling in glutamatergic neurons has been linked to enhancement in synaptic plasticity and cognitive functions. Accordingly, Ng knock-out (Ng-ko) mice display hippocampal-dependent learning and memory impairments associated with a deficit in long-term potentiation induction. In the adult olfactory bulb (OB), Ng is expressed by a large population of GABAergic granule cells (GCs) that are continuously generated during adult life, undergo high synaptic remodeling in response to the sensory context, and play a key role in odor processing. However, the possible implication of Ng in OB plasticity and function is yet to be investigated. Here, we show that Ng expression in the OB is associated with the mature state of adult-born GCs, where its active-phosphorylated form is concentrated at post-synaptic sites. Constitutive loss of Ng in Ng-ko mice resulted in defective spine density in adult-born GCs, while their survival remained unaltered. Moreover, Ng-ko mice show an impaired odor-reward associative memory coupled with reduced expression of the activity-dependent transcription factor Zif268 in olfactory GCs. Overall, our data support a role for Ng in the molecular mechanisms underlying GC plasticity and the formation of olfactory associative memory.

Striatal astrocytes produce neuroblasts in an excitotoxic model of Huntington's disease.

In the adult brain, subsets of astrocytic cells residing in well-defined neurogenic niches constitutively generate neurons throughout life. Brain lesions can stimulate neurogenesis in otherwise non-neurogenic regions, but whether local astrocytic cells generate neurons in these conditions is unresolved. Here, through genetic and viral lineage tracing in mice, we demonstrate that striatal astrocytes become neurogenic following an acute excitotoxic lesion. Similar to astrocytes of adult germinal niches, these activated parenchymal progenitors express nestin and generate neurons through the formation of transit amplifying progenitors. These results shed new light on the neurogenic potential of the adult brain parenchyma.

Quiescent neuronal progenitors are activated in the juvenile guinea pig lateral striatum and give rise to transient neurons.

In the adult brain, active stem cells are a subset of astrocytes residing in the subventricular zone (SVZ) and the dentate gyrus (DG) of the hippocampus. Whether quiescent neuronal progenitors occur in other brain regions is unclear. Here, we describe a novel neurogenic system in the external capsule and lateral striatum (EC-LS) of the juvenile guinea pig that is quiescent at birth but becomes active around weaning. Activation of neurogenesis in this region was accompanied by the emergence of a neurogenic-like niche in the ventral EC characterized by chains of neuroblasts, intermediate-like progenitors and glial cells expressing markers of immature astrocytes. Like neurogenic astrocytes of the SVZ and DG, these latter cells showed a slow rate of proliferation and retained BrdU labeling for up to 65 days, suggesting that they are the primary progenitors of the EC-LS neurogenic system. Injections of GFP-tagged lentiviral vectors into the SVZ and the EC-LS of newborn animals confirmed that new LS neuroblasts originate from the activation of local progenitors and further supported their astroglial nature. Newborn EC-LS neurons existed transiently and did not contribute to neuronal addition or replacement. Nevertheless, they expressed Sp8 and showed strong tropism for white matter tracts, wherein they acquired complex morphologies. For these reasons, we propose that EC-LS neuroblasts represent a novel striatal cell type, possibly related to those populations of transient interneurons that regulate the development of fiber tracts during embryonic life.

A GnRH neuronal population in the olfactory bulb translates socially relevant odors into reproductive behavior in male mice.

Hypothalamic gonadotropin-releasing hormone (GnRH) neurons regulate fertility and integrate hormonal status with environmental cues to ensure reproductive success. Here we show that GnRH neurons in the olfactory bulb (GnRHOB) of adult mice can mediate social recognition. Specifically, we show that GnRHOB neurons extend neurites into the vomeronasal organ and olfactory epithelium and project to the median eminence. GnRHOB neurons in males express vomeronasal and olfactory receptors, are activated by female odors and mediate gonadotropin release in response to female urine. Male preference for female odors required the presence and activation of GnRHOB neurons, was impaired after genetic inhibition or ablation of these cells and relied on GnRH signaling in the posterodorsal medial amygdala. GnRH receptor expression in amygdala kisspeptin neurons appear to be required for GnRHOB neurons' actions on male mounting behavior. Taken together, these results establish GnRHOB neurons as regulating fertility, sex recognition and mating in male mice.

Neurodynamic Treatment Promotes Mechanical Pain Modulation in Sensory Neurons and Nerve Regeneration in Rats.

BACKGROUND: Somatic nerve injuries are a rising problem leading to disability associated with neuropathic pain commonly reported as mechanical allodynia (MA) and hyperalgesia. These symptoms are strongly dependent on specific processes in the dorsal root ganglia (DRG). Neurodynamic treatment (NDT), consisting of selective uniaxial nerve repeated tension protocols, effectively reduces pain and disability in neuropathic pain patients even though the biological mechanisms remain poorly characterized. We aimed to define, both in vivo and ex vivo, how NDT could promote nerve regeneration and modulate some processes in the DRG linked to MA and hyperalgesia. METHODS: We examined in Wistar rats, after unilateral median and ulnar nerve crush, the therapeutic effects of NDT and the possible protective effects of NDT administered for 10 days before the injury. We adopted an ex vivo model of DRG organotypic explant subjected to NDT to explore the selective effects on DRG cells. RESULTS: Behavioural tests, morphological and morphometrical analyses, and gene and protein expression analyses were performed, and these tests revealed that NDT promotes nerve regeneration processes, speeds up sensory motor recovery, and modulates mechanical pain by affecting, in the DRG, the expression of TACAN, a mechanosensitive receptor shared between humans and rats responsible for MA and hyperalgesia. The ex vivo experiments have shown that NDT increases neurite regrowth and confirmed the modulation of TACAN. CONCLUSIONS: The results obtained in this study on the biological and molecular mechanisms induced by NDT will allow the exploration, in future clinical trials, of its efficacy in different conditions of neuropathic pain.

Curcumin-based-fluorescent probes targeting ALDH1A3 as a promising tool for glioblastoma precision surgery and early diagnosis.

Glioblastoma (GBM) is the most aggressive primary brain tumour for which both effective treatments and efficient tools for an early-stage diagnosis are lacking. Herein, we present curcumin-based fluorescent probes that are able to bind to aldehyde dehydrogenase 1A3 (ALDH1A3), an enzyme overexpressed in glioma stem cells (GSCs) and associated with stemness and invasiveness of GBM. Two compounds are selective versus ALDH1A3, without showing any appreciable interaction with other ALDH1A isoenzymes. Indeed, their fluorescent signal is detectable only in our positive controls in vitro and absent in cells that lack ALDH1A3. Remarkably, in vivo, our Probe selectively accumulate in glioblastoma cells, allowing the identification of the growing tumour mass. The significant specificity of our compounds is the necessary premise for their further development into glioblastoma cells detecting probes to be possibly used during neurosurgical operations.

Immune-tolerance to human iPS-derived neural progenitors xenografted into the immature cerebellum is overridden by species-specific differences in differentiation timing.

We xeno-transplanted human neural precursor cells derived from induced pluripotent stem cells into the cerebellum and brainstem of mice and rats during prenatal development or the first postnatal week. The transplants survived and started to differentiate up to 1 month after birth when they were rejected by both species. Extended survival and differentiation of the same cells were obtained only when they were transplanted in NOD-SCID mice. Transplants of human neural precursor cells mixed with the same cells after partial in vitro differentiation or with a cellular extract obtained from adult rat cerebellum increased survival of the xeno-graft beyond one month. These findings are consistent with the hypothesis that the slower pace of differentiation of human neural precursors compared to that of rodents restricts induction of immune-tolerance to human antigens expressed before completion of maturation of the immune system. With further maturation the transplanted neural precursors expressed more mature antigens before the graft were rejected. Supplementation of the immature cells suspensions with more mature antigens may help to induce immune-tolerance for those antigens expressed only later by the engrafted cells.

Fibroblasts Colonizing Nerve Conduits Express High Levels of Soluble Neuregulin1, a Factor Promoting Schwann Cell Dedifferentiation.

Conduits for the repair of peripheral nerve gaps are a good alternative to autografts as they provide a protected environment and a physical guide for axonal re-growth. Conduits require colonization by cells involved in nerve regeneration (Schwann cells, fibroblasts, endothelial cells, macrophages) while in the autograft many cells are resident and just need to be activated. Since it is known that soluble Neuregulin1 (sNRG1) is released after injury and plays an important role activating Schwann cell dedifferentiation, its expression level was investigated in early regeneration steps (7, 14, 28 days) inside a 10 mm chitosan conduit used to repair median nerve gaps in Wistar rats. In vivo data show that sNRG1, mainly the isoform α, is highly expressed in the conduit, together with a fibroblast marker, while Schwann cell markers, including NRG1 receptors, were not. Primary culture analysis shows that nerve fibroblasts, unlike Schwann cells, express high NRG1α levels, while both express NRG1ß. These data suggest that sNRG1 might be mainly expressed by fibroblasts colonizing nerve conduit before Schwann cells. Immunohistochemistry analysis confirmed NRG1 and fibroblast marker co-localization. These results suggest that fibroblasts, releasing sNRG1, might promote Schwann cell dedifferentiation to a "repair" phenotype, contributing to peripheral nerve regeneration.

Emerging pharmacological approaches to promote neurogenesis from endogenous glial cells.

Neurodegenerative disorders are emerging as leading contributors to the global disease burden. While some drug-based approaches have been designed to limit or prevent neuronal loss following acute damage or chronic neurodegeneration, regeneration of functional neurons in the adult Central Nervous System (CNS) still remains an unmet need. In this context, the exploitation of endogenous cell sources has recently gained an unprecedented attention, thanks to the demonstration that, in some CNS regions or under specific circumstances, glial cells can activate spontaneous neurogenesis or can be instructed to produce neurons in the adult mammalian CNS parenchyma. This field of research has greatly advanced in the last years and identified interesting molecular and cellular mechanisms guiding the neurogenic activation/conversion of glia. In this review, we summarize the evolution of the research devoted to understand how resident glia can be directed to produce neurons. We paid particular attention to pharmacologically-relevant approaches exploiting the modulation of niche-associated factors and the application of selected small molecules.

Inhibitors of GLUT/SLC2A Enhance the Action of BCNU and Temozolomide against High-Grade Gliomas.

Glucose transport across glioblastoma membranes plays a crucial role in maintaining the enhanced glycolysis typical of high-grade gliomas and glioblastoma. We tested the ability of two inhibitors of the glucose transporters GLUT/SLC2A superfamily, indinavir (IDV) and ritonavir (RTV), and of one inhibitor of the Na/glucose antiporter type 2 (SGLT2/SLC5A2) superfamily, phlorizin (PHZ), in decreasing glucose consumption and cell proliferation of human and murine glioblastoma cells. We found in vitro that RTV, active on at least three different GLUT/SLC2A transporters, was more effective than IDV, a specific inhibitor of GLUT4/SLC2A4, both in decreasing glucose consumption and lactate production and in inhibiting growth of U87MG and Hu197 human glioblastoma cell lines and primary cultures of human glioblastoma. PHZ was inactive on the same cells. Similar results were obtained when cells were grown in adherence or as 3D multicellular tumor spheroids. RTV treatment but not IDV treatment induced AMP-activated protein kinase (AMPKα) phosphorylation that paralleled the decrease in glycolytic activity and cell growth. IDV, but not RTV, induced an increase in GLUT1/SLC2A1 whose activity could compensate for the inhibition of GLUT4/SLC2A4 by IDV. RTV and IDV pass poorly the blood brain barrier and are unlikely to reach sufficient liquoral concentrations in vivo to inhibit glioblastoma growth as single agents. Isobologram analysis of the association of RTV or IDV and 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) or 4-methyl-5-oxo-2,3,4,6,8-pentazabicyclo[4.3.0]nona-2,7,9-triene-9-carboxamide (TMZ) indicated synergy only with RTV on inhibition of glioblastoma cells. Finally, we tested in vivo the combination of RTV and BCNU on established GL261 tumors. This drug combination increased the overall survival and allowed a five-fold reduction in the dose of BCNU.