Effects of Topical Application of CHF6467, a Mutated Form of Human Nerve Growth Factor, on Skin Wound Healing in Diabetic Mice.

Nerve growth factor (NGF) is the protein responsible for the development and maintenance of sensory skin innervation. Given the role of appropriate innervation in skin healing, NGF has been indicated as a possible prohealing treatment in pathologic conditions characterized by nerve-ending loss, such as chronic ulcers in diabetes; however, its use as a therapeutic agent is limited by its hyperalgesic effect. We tested the effect of topical application of the nonalgogenic NGF derivative hNGFP61S/R100E in two models of skin ulcer induced in dbdb diabetic mice, investigating healing time, skin histology, reinnervation, and angiogenesis using morphologic and molecular approaches. We showed that the topical administration of CHF6467, a recombinant human NGF in which an amino acid substitution (R100E) abolished the hyperalgesic effect usually associated with NGF, accelerated skin repair in experimental wounds (full-excision and pressure-ulcer) induced in diabetic mice (dbdb). CHF6467-induced acceleration of wound healing was accompanied by increased re-epithelization, reinnervation, and revascularization as assessed by histology, immunohistochemistry, and image analysis. Bioinformatic analysis of differentially expressed genes and signaling pathways in the wound tissues showed that protein kinase B-mammalian target of rapamycin was the most regulated pathway. In spite of the transdermal absorption leading to measurable, dose-dependent increases in CHF6467 plasma levels, no systemic thermal or local mechanical hyperalgesia was observed in treated mice. When tested in vitro in human cell lines, CHF6467 stimulated keratinocyte and fibroblast proliferation and tube formation by endothelial cells. Collectively, these results support a possible use of CHF6467 as a prohealing agent in skin lesions in diabetes. SIGNIFICANCE STATEMENT: Topical application of CHF6467 accelerates reinnervation, neoangiogenesis, and wound healing in diabetic mice in both full-thickness skin-excision and pressure-ulcer models through the protein kinase B/mammalian target of rapamycin pathway and does not induce hyperalgesia.

CDKL5 knockout leads to altered inhibitory transmission in the cerebellum of adult mice.

Mutations in the X-linked cyclin-dependent kinase-like 5 gene (CDKL5) are associated to severe neurodevelopmental alterations including motor symptoms. In order to elucidate the neurobiological substrate of motor symptoms in CDKL5 syndrome, we investigated the motor function, GABA and glutamate pathways in the cerebellum of CDKL5 knockout female mice. Behavioural data indicate that CDKL5-KO mice displayed impaired motor coordination on the Rotarod test, and altered steps, as measured by the gait analysis using the CatWalk test. A higher reduction in spontaneous GABA efflux, than that in glutamate, was observed in CDKL5-KO mouse cerebellar synaptosomes, leading to a significant increase of spontaneous glutamate/GABA efflux ratio in these animals. On the contrary, there were no differences between groups in K(+) -evoked GABA and glutamate efflux. The anatomical analysis of cerebellar excitatory and inhibitory pathways showed a selective defect of the GABA-related marker GAD67 in the molecular layer in CDKL5-KO mice, while the glutamatergic marker VGLUT1 was unchanged in the same area. Fine cerebellar structural abnormalities such as a reduction of the inhibitory basket 'net' estimated volume and an increase of the pinceau estimated volume were also observed in CDKL5-KO mice. Finally, the BDNF mRNA expression level in the cerebellum, but not in the hippocampus, was reduced compared with WT animals. These data suggest that CDKL5 deletion during development more markedly impairs the establishment of a correct GABAergic cerebellar network than that of glutamatergic one, leading to the behavioural symptoms associated with CDKL5 mutation.