Randomised, double-blind, placebo-controlled, parallel-group, multicentric, phase IIA clinical trial for evaluating the safety, tolerability, and therapeutic efficacy of daily oral administration of NFX88 to treat neuropathic pain in individuals with spinal cord injury.

STUDY DESIGN: Double-blind, randomized, placebo-controlled, parallel-group multicentric phase IIA clinical trial. OBJECTIVE: To assess the safety and tolerability of oral administration of NFX-88 in subjects with chronic spinal cord injury (SCI) and explore its efficacy in pain control. SETTING: A total of 7 spinal cord injury rehabilitation units in Spain. METHODS: A total of 61 adult with traumatic complete or incomplete spinal cord injury (C4-T12 level), were randomised 1:1:1:1 to a placebo, NFX88 1.05 g, 2.1 g, 4.2 g/day for up to 12 weeks. The placebo or NFX-88 was administered as add-on therapy to pre-existing pregabalin (150-300 mg per day). Safety and tolerability were evaluated, and the Visual Analogue Scale (VAS) was the primary measure to explore the efficacy of NFX-88 in pain control. RESULTS: No severe treatment-related adverse effects were reported for any of the four study groups. 44 SCI individuals completed the study and were analysed. The data obtained from the VAS analysis and the PainDETECT Questionnaire (PD-Q) suggested that the combination of NFX88 with pregabalin is more effective than pregabalin with placebo at reducing neuropathic pain (NP) in individuals with SCI and that the dose 2.10 g/day causes the most dramatic pain relief. CONCLUSIONS: NFX88 treatment was found to be highly safe and well tolerated, with the dose of 2.10 g/day being the most effective at causing pain relief. Thus, the promising efficacy of this first-in-class lipid mediator deserves further consideration in future clinical trials.

Critical Requirement of SOS1 for Development of BCR/ABL-Driven Chronic Myelogenous Leukemia.

We showed previously that the ABL-mediated phosphorylation of SOS1 promotes RAC activation and contributes to BCR-ABL leukemogenesis, suggesting the relevant role of SOS1 in the pathogenesis of CML. To try and obtain direct experimental evidence of the specific mechanistic implication of SOS1 in CML development, here, we combined a murine model of CML driven by a p210BCR/ABL transgene with our tamoxifen-inducible SOS1/2-KO system in order to investigate the phenotypic impact of the direct genetic ablation of SOS1 or SOS2 on the pathogenesis of CML. Our observations showed that, in contrast to control animals expressing normal levels of SOS1 and SOS2 or to single SOS2-KO mice, p210BCR/ABL transgenic mice devoid of SOS1 presented significantly extended survival curves and also displayed an almost complete disappearance of the typical hematological alterations and splenomegaly constituting the hallmarks of CML. SOS1 ablation also resulted in a specific reduction in the proliferation and the total number of colony-forming units arising from the population of bone marrow stem/progenitor cells from p210BCR/ABL transgenic mice. The specific blockade of CML development caused by SOS1 ablation in p210BCR/ABL mice indicates that SOS1 is critically required for CML pathogenesis and supports the consideration of this cellular GEF as a novel, alternative bona fide therapeutic target for CML treatment in the clinic.

Critical requirement of SOS1 RAS-GEF function for mitochondrial dynamics, metabolism, and redox homeostasis.

SOS1 ablation causes specific defective phenotypes in MEFs including increased levels of intracellular ROS. We showed that the mitochondria-targeted antioxidant MitoTEMPO restores normal endogenous ROS levels, suggesting predominant involvement of mitochondria in generation of this defective SOS1-dependent phenotype. The absence of SOS1 caused specific alterations of mitochondrial shape, mass, and dynamics accompanied by higher percentage of dysfunctional mitochondria and lower rates of electron transport in comparison to WT or SOS2-KO counterparts. SOS1-deficient MEFs also exhibited specific alterations of respiratory complexes and their assembly into mitochondrial supercomplexes and consistently reduced rates of respiration, glycolysis, and ATP production, together with distinctive patterns of substrate preference for oxidative energy metabolism and dependence on glucose for survival. RASless cells showed defective respiratory/metabolic phenotypes reminiscent of those of SOS1-deficient MEFs, suggesting that the mitochondrial defects of these cells are mechanistically linked to the absence of SOS1-GEF activity on cellular RAS targets. Our observations provide a direct mechanistic link between SOS1 and control of cellular oxidative stress and suggest that SOS1-mediated RAS activation is required for correct mitochondrial dynamics and function.

Concomitant deletion of HRAS and NRAS leads to pulmonary immaturity, respiratory failure and neonatal death in mice.

We reported previously that adult (HRAS-/-; NRAS-/-) double knockout (DKO) mice showed no obvious external phenotype although lower-than-expected numbers of weaned DKO animals were consistently tallied after crossing NRAS-KO and HRAS-KO mice kept on mixed genetic backgrounds. Using mouse strains kept on pure C57Bl/6 background, here we performed an extensive analysis of the offspring from crosses between HRAS-KO and NRAS-KO mice and uncovered the occurrence of very high rates of perinatal mortality of the resulting DKO littermates due to respiratory failure during the first postnatal 24-48 h. The lungs of newborn DKO mice showed normal organ structure and branching but displayed marked defects of maturation including much-reduced alveolar space with thick separating septa and significant alterations of differentiation of alveolar (AT1, AT2 pneumocytes) and bronchiolar (ciliated, Clara cells) cell lineages. We also observed the retention of significantly increased numbers of undifferentiated progenitor precursor cells in distal lung epithelia and the presence of substantial accumulations of periodic acid-Schiff-positive (PAS+) material and ceramide in the lung airways of newborn DKO mice. Interestingly, antenatal dexamethasone treatment partially mitigated the defective lung maturation phenotypes and extended the lifespan of the DKO animals up to 6 days, but was not sufficient to abrogate lethality in these mice. RNA microarray hybridization analyses of the lungs of dexamethasone-treated and untreated mice uncovered transcriptional changes pointing to functional and metabolic alterations that may be mechanistically relevant for the defective lung phenotypes observed in DKO mice. Our data suggest that delayed alveolar differentiation, altered sphingolipid metabolism and ceramide accumulation are primary contributors to the respiratory stress and neonatal lethality shown by DKO mice and uncover specific, critical roles of HRAS and NRAS for correct lung differentiation that are essential for neonatal survival and cannot be substituted by the remaining KRAS function in this organ.

Differential Role of the RasGEFs Sos1 and Sos2 in Mouse Skin Homeostasis and Carcinogenesis.

Using Sos1 knockout (Sos1-KO), Sos2-KO, and Sos1/2 double-knockout (Sos1/2-DKO) mice, we assessed the functional role of Sos1 and Sos2 in skin homeostasis under physiological and/or pathological conditions. Sos1 depletion resulted in significant alterations of skin homeostasis, including reduced keratinocyte proliferation, altered hair follicle and blood vessel integrity in dermis, and reduced adipose tissue in hypodermis. These defects worsened significantly when both Sos1 and Sos2 were absent. Simultaneous Sos1/2 disruption led to severe impairment of the ability to repair skin wounds, as well as to almost complete ablation of the neutrophil-mediated inflammatory response in the injury site. Furthermore, Sos1 disruption delayed the onset of tumor initiation, decreased tumor growth, and prevented malignant progression of papillomas in a DMBA (7,12-dimethylbenz[α]anthracene)/TPA (12-O-tetradecanoylphorbol-13-acetate)-induced skin carcinogenesis model. Finally, Sos1 depletion in preexisting chemically induced papillomas resulted also in decreased tumor growth, probably linked to significantly reduced underlying keratinocyte proliferation. Our data unveil novel, distinctive mechanistic roles of Sos 1 and Sos2 in physiological control of skin homeostasis and wound repair, as well as in pathological development of chemically induced skin tumors. These observations underscore the essential role of Sos proteins in cellular proliferation and migration and support the consideration of these RasGEFs as potential biomarkers/therapy targets in Ras-driven epidermal tumors.

Ras-GRF2 regulates nestin-positive stem cell density and onset of differentiation during adult neurogenesis in the mouse dentate gyrus.

Various parameters of neurogenesis were analyzed in parallel in the two neurogenic areas (the Dentate Gyrus[DG] and the Subventricular Zone[SVZ]/Rostral Migratory Stream[RMS]/Main Olfactory Bulb[MOB] neurogenic system) of adult WT and KO mouse strains for the Ras-GRF1/2 genes (Ras-GRF1-KO, Ras-GRF2-KO, Ras-GRF1/2-DKO). Significantly reduced numbers of doublecortin[DCX]-positive cells were specifically observed in the DG, but not the SVZ/RMS/MOB neurogenic region, of Ras-GRF2-KO and Ras-GRF1/2-DKO mice indicating that this novel Ras-GRF2-dependent phenotype is spatially restricted to a specific neurogenic area. Consistent with a role of CREB as mediator of Ras-GRF2 function in neurogenesis, the density of p-CREB-positive cells was also specifically reduced in all neurogenic regions of Ras-GRF2-KO and DKO mice. Similar levels of early neurogenic proliferation markers (Ki67, BrdU) were observed in all different Ras-GRF genotypes analyzed but significantly elevated levels of nestin-immunolabel, particularly of undifferentiated, highly ramified, A-type nestin-positive neurons were specifically detected in the DG but not the SVZ/RMS/MOB of Ras-GRF2-KO and DKO mice. Together with assays of other neurogenic markers (GFAP, Sox2, Tuj1, NeuN), these observations suggest that the deficit of DCX/p-CREB-positive cells in the DG of Ras-GRF2-depleted mice does not involve impaired neuronal proliferation but rather delayed transition from the stem cell stage to the differentiation stages of the neurogenic process. This model is also supported by functional analyses of DG-derived neurosphere cultures and transcriptional characterization of the neurogenic areas of mice of all relevant Ras-GRF genotypes suggesting that the neurogenic role of Ras-GRF2 is exerted in a cell-autonomous manner through a specific transcriptional program.

RASGRF2 controls nuclear migration in postnatal retinal cone photoreceptors.

Detailed immunocytochemical analyses comparing wild-type (WT), GRF1-knockout (KO), GRF2-KO and GRF1/2 double-knockout (DKO) mouse retinas uncovered the specific accumulation of misplaced, 'ectopic' cone photoreceptor nuclei in the photoreceptor segment (PS) area of retinas from GRF2-KO and GRF1/2-DKO, but not of WT or GRF1-KO mice. Localization of ectopic nuclei in the PS area of GRF2-depleted retinas occurred postnatally and peaked between postnatal day (P)11 and P15. Mechanistically, the generation of this phenotype involved disruption of the outer limiting membrane and intrusion into the PS layer by cone nuclei displaying significant perinuclear accumulation of signaling molecules known to participate in nuclear migration and cytoskeletal reorganization, such as PAR3, PAR6 and activated, phosphorylated forms of PAK, MLC2 and VASP. Electroretinographic recordings showed specific impairment of cone-mediated retinal function in GRF2-KO and GRF1/2-DKO retinas compared with WT controls. These data identify defective cone nuclear migration as a novel phenotype in mouse retinas lacking GRF2 and support a crucial role of GRF2 in control of the nuclear migration processes required for proper postnatal development and function of retinal cone photoreceptors.

Transcriptional profiling reveals functional links between RasGrf1 and Pttg1 in pancreatic beta cells.

BACKGROUND: Our prior characterization of RasGrf1 deficient mice uncovered significant defects in pancreatic islet count and size as well as beta cell development and signaling function, raising question about the mechanisms linking RasGrf1 to the generation of those "pancreatic" phenotypes. RESULTS: Here, we compared the transcriptional profile of highly purified pancreatic islets from RasGrf1 KO mice to that of WT control animals using commercial oligonucleotide microarrays. RasGrf1 elimination resulted in differential gene expression of numerous components of MAPK- and Calcium-signaling pathways, suggesting a relevant contribution of this GEF to modulation of cellular signaling in the cell lineages integrating the pancreatic islets. Whereas the overall transcriptional profile of pancreatic islets was highly specific in comparison to other organs of the same KO mice, a significant specific repression of Pttg1 was a common transcriptional alteration shared with other tissues of neuroectodermal origin. This observation, together with the remarkable pancreatic phenotypic similarities between RasGrf1 KO and Pttg1 KO mice suggested the possibility of proximal functional regulatory links between RasGrf1 and Pttg1 in pancreatic cell lineages expressing these proteins.Analysis of the mPttg1 promoter region identified specific recognition sites for numerous transcription factors which were also found to be differentially expressed in RasGrf1 KO pancreatic islets and are known to be relevant for Ras-ERK signaling as well as beta cell function. Reporter luciferase assays in BT3 insulinoma cells demonstrated the ability of RasGrf1 to modulate mPttg1 promoter activity through ERK-mediated signals. Analysis of the phenotypic interplay between RasGrf1 and Pttg1 in double knockout RasGrf1/Pttg1 mice showed that combined elimination of the two loci resulted in dramatically reduced values of islet and beta cell count and glucose homeostasis function which neared those measured in single Pttg1 KO mice and were significantly lower than those observed in individual RasGrf1 KO mice. CONCLUSIONS: The specific transcriptional profile and signaling behavior of RasgGrf1 KO pancreatic islets, together with the dominance of Pttg1 over RasGrf1 with regards to the generation of these phenotypes in mouse pancreas, suggest that RasGrf1 is an important upstream component of signal transduction pathways regulating Pttg1 expression and controlling beta cell development and physiological responses.

Pax6 is essential for the maintenance and multi-lineage differentiation of neural stem cells, and for neuronal incorporation into the adult olfactory bulb.

The paired type homeobox 6 (Pax6) transcription factor (TF) regulates multiple aspects of neural stem cell (NSC) and neuron development in the embryonic central nervous system. However, less is known about the role of Pax6 in the maintenance and differentiation of adult NSCs and in adult neurogenesis. Using the +/Sey(Dey) mouse, we have analyzed how Pax6 heterozygosis influences the self-renewal and proliferation of adult olfactory bulb stem cells (aOBSCs). In addition, we assessed its influence on neural differentiation, neuronal incorporation, and cell death in the adult OB, both in vivo and in vitro. Our results indicate that the Pax6 mutation alters Nestin(+)-cell proliferation in vivo, as well as self-renewal, proliferation, and survival of aOBSCs in vitro although a subpopulation of +/Sey(Dey) progenitors is able to expand partially similar to wild-type progenitors. This mutation also impairs aOBSC differentiation into neurons and oligodendrocytes, whereas it increases cell death while preserving astrocyte survival and differentiation. Furthermore, Pax6 heterozygosis causes a reduction in the variety of neurochemical interneuron subtypes generated from aOBSCs in vitro and in the incorporation of newly generated neurons into the OB in vivo. Our findings support an important role of Pax6 in the maintenance of aOBSCs by regulating cell death, self-renewal, and cell fate, as well as in neuronal incorporation into the adult OB. They also suggest that deregulation of the cell cycle machinery and TF expression in aOBSCs which are deficient in Pax6 may be at the origin of the phenotypes observed in this adult NSC population.

Differential glial activation during the degeneration of Purkinje cells and mitral cells in the PCD mutant mice.

Purkinje Cell Degeneration (PCD) mice harbor a nna1 gene mutation which leads to an early and rapid degeneration of Purkinje cells (PC) between the third and fourth week of age. This mutation also underlies the death of mitral cells (MC) in the olfactory bulb (OB), but this process is slower and longer than in PC. No clear interpretations supporting the marked differences in these neurodegenerative processes exist. Growing evidence suggests that either beneficial or detrimental effects of gliosis in damaged regions would underlie these divergences. Here, we examined the gliosis occurring during PC and MC death in the PCD mouse. Our results demonstrated different glial reactions in both affected regions. PC disappearance stimulated a severe gliosis characterized by strong morphological changes, enhanced glial proliferation, as well as the release of pro-inflammatory mediators. By contrast, MC degeneration seems to promote a more attenuated glial response in the PCD OB compared with that of the cerebellum. Strikingly, cerebellar oligodendrocytes died by apoptosis in the PCD, whereas bulbar ones were not affected. Interestingly, the level of nna1 mRNA under normal conditions was higher in the cerebellum than in the OB, probably related to a faster neurodegeneration and stronger glial reaction in its absence. The glial responses may thus influence the neurodegenerative course in the cerebellum and OB of the mutant mouse brain, providing harmful and beneficial microenvironments, respectively.

Types of cholecystokinin-containing periglomerular cells in the mouse olfactory bulb.

The periglomerular cells (PG) of the olfactory bulb (OB) are involved in the primary processing and the refinement of sensory information from the olfactory epithelium. The neurochemical composition of these neurons has been studied in depth in many species, and over the last decades such studies have focused mainly on the rat. The increasing use of genetic models for research into olfactory function demands a profound characterization of the mouse olfactory bulb, including the chemical composition of bulbar interneurons. Regarding both their connectivity with the olfactory nerve and their neurochemical fate, recently, two different types of PG have been identified in the mouse. In the present report, we analyze both the synaptology and the chemical composition of specific PG populations in the murine olfactory bulb, in particular, those containing the neuropeptide cholecystokinin. Our results demonstrate the existence in the mouse of non-GABAergic PG and that these establish synaptic contacts with the olfactory nerve within the glomeruli. Based on previous classifications, we propose that this population would constitute a new subtype of type 1 mouse PG. In addition, we demonstrate the partial coexistence of cholecystokinin with the calcium-binding proteins neurocalcin and parvalbumin. All these findings add further data to our knowledge of the synaptology and neurochemistry of mouse PG. The differences observed from other rodents reflect the neurochemical heterogeneity of PG in the mammalian OB.

Chemical characterization of Pax6-immunoreactive periglomerular neurons in the mouse olfactory bulb.

The Pax6 transcription factor is a key element along brain development in both the visual and olfactory systems. The involvement of Pax6 in neural fate is well documented in the visual system, whereas in the olfactory system, and in particular in the olfactory bulb (OB), its expression during adulthood has only begun to be elucidated. In the OB, the modulation of primary sensory information is first performed by periglomerular cells (PG). A considerable body of information has unveiled the neurochemical heterogeneity of these neurons. Thus it is well known that Pax6 coexists with dopaminergic/GABAergic mouse PG. However, the presence of this transcription factor in other mouse PG subpopulations has not been studied. Here, we analyzed whether Pax6 is expressed in PG containing the calcium-binding proteins neurocalcin and parvalbumin, and the neuropeptide cholecystokinin. Our results show that Pax6 is not expressed by these PG subpopulations, suggesting that it is mainly restricted to GABAergic PG populations. These findings provide new data in the chemical characterization of mouse Pax6-positive PG.

Zincergic innervation from the anterior olfactory nucleus to the olfactory bulb displays plastic responses after mitral cell loss.

Zinc ions are selectively accumulated in certain neurons (zinc-enriched neurons). The mouse olfactory bulb is richly innervated by zinc-enriched terminals. Here, the plasticity of the zincergic system was studied in the olfactory bulb of the Purkinje Cell Degeneration mutant mouse, an animal with specific postnatal neurodegeneration of the main projection neurons of the olfactory bulb. The analysis focused particularly on the anterior olfactory nucleus since most centrifugal afferents coming to the olfactory bulb arise from this structure. Zinc-enriched terminals in the olfactory bulb and zinc-enriched somata in the anterior olfactory nucleus were visualized after selenite injections. Immunohistochemistry against the vesicular zinc transporter was also carried out to confirm the distribution pattern of zinc-enriched terminals in the olfactory bulb. The mutant mice showed a clear reorganization of zincergic centrifugal projections from the anterior olfactory nucleus to the olfactory bulb. First, all zincergic contralateral neurons projecting to the olfactory bulb were absent in the mutant mice. Second, a significant increase in the number of stained somata was detected in the ipsilateral anterior olfactory nucleus. Since no noticeable changes were observed in the zinc-enriched terminals in the olfactory bulb, it is conceivable that mitral cell loss could induce a reorganization of zinc-enriched projections coming from the anterior olfactory nucleus, probably directed at balancing the global zincergic centrifugal modulation. These results show that zincergic anterior olfactory nucleus cells projecting to the olfactory bulb undergo plastic changes to adapt to the loss of mitral cells in the olfactory bulb of Purkinje Cell Degeneration mutant mice.

Changes in the connections of the main olfactory bulb after mitral cell selective neurodegeneration.

The connections of the main olfactory bulb (OB) of the mouse were studied with iontophoretic injections of biotinylated dextran amine. To sort efferences from mitral cells and tufted cells, the Purkinje cell degeneration (PCD) mouse was used. This mutant animal undergoes a specific neurodegeneration of mitral cells, whereas tufted cells do not degenerate. The unilateral tracer injections used were small and confined largely to the OB of both PCD and control mice at P120. Seven days after tracer injection, the efferences from the OB and the centrifugal afferences from secondary olfactory structures to it were studied. Although there is a large overlap of their target fields, mitral cell axons innervated more caudal regions of the olfactory cortex than tufted cell axons, thus providing definitive evidence of the differential projections of olfactory output neurons. Additionally, an important increase in retrogradely-labeled neurons was detected in the ipsilateral anterior olfactory nucleus of the mutant animals. This was not observed in any other secondary olfactory structure, suggesting a strengthening of the centrifugal input to the OB from that central area after mitral cell loss. Moreover, we recorded a complete loss of bilaterality in the olfactory connections of the PCD mice due to degeneration of the anterior commissure. These results point to an important reorganization of this essential olfactory circuit between the anterior olfactory nucleus and the OB, and hint at a transsynaptic level of plasticity not considered previously in literature.

Chemical organization of the macaque monkey olfactory bulb: III. Distribution of cholinergic markers.

The distribution patterns of choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) were studied in the olfactory bulb (OB) of three species of macaque. AChE was detected by a histochemical method and ChAT immunoreactivity by immunocytochemistry. Similar results were observed in all species analyzed. With the exception of the olfactory nerve layer, all layers of the macaque monkey OB demonstrated a dense innervation of AChE- and ChAT-positive fibers. The distribution patterns of AChE- and ChAT-labeled fibers were similar for both cholinergic markers, although the number of AChE-labeled fibers was clearly higher than the number of ChAT-immunoreactive fibers. The highest density of AChE and ChAT-stained fibers was observed in the interface between the glomerular layer and the external plexiform layer and in the internal plexiform layer. Dense bundles of labeled fibers were observed in the caudal OB, coursing from the olfactory peduncle. All ChAT-immunopositive elements were identified as centrifugal fibers, derived from neurons caudal to the OB. Neither olfactory fibers nor intrinsic neurons were observed after ChAT immunocytochemistry. However, a few AChE-positive cells were observed in the glomerular layer and in both external and internal plexiform layers. These neurons were presumably identified as periglomerular cells, superficial short-axon cells, and/or external tufted cells and deep short-axon cells. Contrary to other neurotransmitters and neuroactive substances, the distribution patterns of ChAT and AChE activities in the macaque monkey OB closely resembled the patterns described in macrosmatic mammals and showed laminar differences with the distribution pattern observed in humans.