Epilepsy-related CDKL5 deficiency slows synaptic vesicle endocytosis in central nerve terminals

Cyclin-dependent kinase-like 5 (CDKL5) deficiency disorder (CDD) is a severe early-onset epileptic encephalopathy resulting mainly from de novo mutations in the X-linked CDKL5 gene. To determine whether loss of presynaptic CDKL5 function contributes to CDD, we examined synaptic vesicle (SV) recycling in primary hippocampal neurons generated from Cdkl5 knockout rat males. Using a genetically encoded reporter, we revealed that CDKL5 is selectively required for efficient SV endocytosis. We showed that CDKL5 kinase activity is both necessary and sufficient for optimal SV endocytosis, since kinase-inactive mutations failed to correct endocytosis in Cdkl5 knockout neurons, whereas the isolated CDKL5 kinase domain fully restored SV endocytosis kinetics. Finally, we demonstrated that CDKL5-mediated phosphorylation of amphiphysin 1, a putative presynaptic target, is not required for CDKL5-dependent control of SV endocytosis. Overall, our findings reveal a key presynaptic role for CDKL5 kinase activity and enhance our insight into how its dysfunction may culminate in CDD. SIGNIFICANCE STATEMENT Loss of cyclin-dependent kinase like 5 (CDKL5) function is a leading cause of monogenic childhood epileptic encephalopathy. However, information regarding its biological role is scarce. In this study, we reveal a selective presynaptic role for CDKL5 in synaptic vesicle endocytosis and that its protein kinase activity is both necessary and sufficient for this role. The isolated protein kinase domain is sufficient to correct this loss of function, which may facilitate future gene therapy strategies if presynaptic dysfunction is proven to be central to the disorder. It also reveals that a CDKL5-specific substrate is located at the presynapse, the phosphorylation of which is required for optimal SV endocytosis. </p

High glucose exposure promotes proliferation and in vivo network formation of adipose-tissue-derived microvascular fragments

High glucose concentrations have been shown to activate endothelial cells and promote angiogenesis. In the present study, it was investigated whether high glucose concentrations could improve the vascularisation capacity of adipose-tissue-derived microvascular fragments (ad-MVF). Ad-MVF were isolated from the epididymal fat pads of donor mice and cultivated for 24 h in University of Wisconsin (UW) solution supplemented with vehicle or 30 mM glucose. Protein expression, morphology, viability and proliferation of the cultivated ad-MVF were analysed by means of proteome profiler mouse angiogenesis array, scanning electron microscopy and immunohistochemistry. Additional cultivated ad-MVF were seeded on to collagen-glycosaminoglycan scaffolds to study their in vivo vascularisation capacity in the dorsal skinfold chamber model by intravital fluorescence microscopy, histology and immunohistochemistry. In vitro, high glucose exposure changed the protein expression pattern of ad-MVF with endoglin, interleukin (IL)-1β and monocyte chemoattractant protein (MCP)-1 as the most up-regulated pro-angiogenic factors. Moreover, high glucose exposure induced the formation of nanopores in the ad-MVF wall. In addition, ad-MVF contained significantly larger numbers of proliferating endothelial and perivascular cells while exhibiting a comparable number of apoptotic cells when compared to vehicle-treated controls. In vivo, scaffolds seeded with high-glucose-exposed ad-MVF exhibited an improved vascularisation and tissue incorporation. These findings demonstrated that the exposure of cultivated ad-MVF to high glucose concentrations is a promising approach to improve their in vivo performance as vascularisation units for tissue engineering and regenerative medicine

The presence of ochratoxin A in cord serum and in human milk and its correspondence with maternal dietary habits

Ochratoxin A (OTA) is a mycotoxin present in food that can be found in human blood and milk. The link between the nutritional habits of pregnant women both of Italian and foreign nationality resident in Italy and the presence of ochratoxin A in cord blood and in maternal milk was investigated. The study involved 130 pregnant women. Food consumption during pregnancy was evaluated by means of the EPIC questionnaire; OTA content was determined in cord serum and maternal milk by HPLC. The mean daily dietary intake of OTA was 1.02 +/- A 1.20 and 0.87 +/- A 0.78 ng/kg of bodyweight for Italian and non-Italian women, respectively, but this difference was not statistically significant. The incidence of positive milk samples was 73.0 and 85.0% among the Italian and non-Italian mothers, respectively. Pork meat, soft drinks, sweets and red wine showed a significant relationship with OTA level in serum. As far as milk is concerned, a positive relationship resulted for pork meat, sweets, soft drinks and seed oils. A positive relationship between serum OTA level and the ratio serum/milk OTA was found. The intake of OTA had no effect on the cord blood creatinine level. This study confirms that OTA is widely present in human milk and therefore could pose a risk for the newborn