Epileptiform activity and cognitive deficits in SNAP-25(+/-) mice are normalized by antiepileptic drugs.

Synaptosomal-associated protein of 25 kDa (SNAP-25) is a protein that participates in the regulation of synaptic vesicle exocytosis through the formation of the soluble NSF attachment protein receptor complex and modulates voltage-gated calcium channels activity. The Snap25 gene has been associated with schizophrenia, attention deficit hyperactivity disorder, and bipolar disorder, and lower levels of SNAP-25 have been described in patients with schizophrenia. We used SNAP-25 heterozygous (SNAP-25(+/-)) mice to investigate at which extent the reduction of the protein levels affects neuronal network function and mouse behavior. As interactions of genotype with the specific laboratory conditions may impact behavioral results, the study was performed through a multilaboratory study in which behavioral tests were replicated in at least 2 of 3 distinct European laboratories. Reductions of SNAP-25 levels were associated with a moderate hyperactivity, which disappeared in the adult animals, and with impaired associative learning and memory. Electroencephalographic recordings revealed the occurrence of frequent spikes, suggesting a diffuse network hyperexcitability. Consistently, SNAP-25(+/-) mice displayed higher susceptibility to kainate-induced seizures, paralleled by degeneration of hilar neurons. Notably, both EEG profile and cognitive defects were improved by antiepileptic drugs. These results indicate that reduction of SNAP-25 expression is associated to generation of epileptiform discharges and cognitive dysfunctions, which can be effectively treated by antiepileptic drugs.

Nitric oxide synthase mediates PC12 differentiation induced by the surface topography of nanostructured TiO2.

BACKGROUND: Substrate nanoscale topography influences cell proliferation and differentiation through mechanisms that are at present poorly understood. In particular the molecular mechanism through which cells 'sense' and adapt to the substrate and activate specific intracellular signals, influencing cells survival and behavior, remains to be clarified. RESULTS: To characterize these processes at the molecular level we studied the differentiation of PC12 cells on nanostructured TiO2 films obtained by supersonic cluster beam deposition.Our findings indicate that, in PC12 cells grown without Nerve Growth Factor (NGF), the roughness of nanostructured TiO2 triggers neuritogenesis by activating the expression of nitric oxide synthase (NOS) and the phospho-extracellular signal-regulated kinase 1/2 (pERK1/2) signaling. Differentiation is associated with an increase in protein nitration as observed in PC12 cells grown on flat surfaces in the presence of NGF. We demonstrate that cell differentiation and protein nitration induced by topography are not specific for PC12 cells but can be regarded as generalized effects produced by the substrate on different neuronal-like cell types, as shown by growing the human neuroblastoma SH-SY5Y cell line on nanostructured TiO2. CONCLUSION: Our data provide the evidence that the nitric oxide (NO) signal cascade is involved in the differentiation process induced by nanotopography, adding new information on the mechanism and proteins involved in the neuritogenesis triggered by the surface properties.

LRRK2 controls synaptic vesicle storage and mobilization within the recycling pool.

Mutations in leucine-rich repeat kinase 2 (LRRK2) are the single most common cause of inherited Parkinson's disease. Little is known about its involvement in the pathogenesis of Parkinson's disease mainly because of the lack of knowledge about the physiological role of LRRK2. To determine the function of LRRK2, we studied the impact of short hairpin RNA-mediated silencing of LRRK2 expression in cortical neurons. Paired recording indicated that LRRK2 silencing affects evoked postsynaptic currents. Furthermore, LRRK2 silencing induces at the presynaptic site a redistribution of vesicles within the bouton, altered recycling dynamics, and increased vesicle kinetics. Accordingly, LRRK2 protein is present in the synaptosomal compartment of cortical neurons in which it interacts with several proteins involved in vesicular recycling. Our results suggest that LRRK2 modulates synaptic vesicle trafficking and distribution in neurons and in consequence participates in regulating the dynamics between vesicle pools inside the presynaptic bouton.

Nanostructured TiO2 surfaces promote polarized activation of microglia, but not astrocytes, toward a proinflammatory profile.

Activation of glial cells, including astrocytes and microglia, has been implicated in the inflammatory responses underlying brain injury and neurodegenerative diseases including Alzheimer's and Parkinson's diseases. The classic activation state (M1) is characterized by high capacity to present antigens, high production of nitric oxide (NO) and reactive oxygen species (ROS) and proinflammatory cytokines. Classically activated cells act as potent effectors that drive the inflammatory response and may mediate detrimental effects on neural cells. The second phenotype (M2) is an alternative, apparently beneficial, activation state, more related to a fine tuning of inflammation, scavenging of debris, promotion of angiogenesis, tissue remodeling and repair. Specific environmental chemical signals are able to induce these different polarization states. We provide here evidence that nanostructured substrates are able, exclusively in virtue of their physical properties, to push microglia toward the proinflammatory activation phenotype, with an efficacy which reflects the graded nanoscale rugosity. The acquisition of a proinflammatory phenotype appears specific for microglia and not astrocytes, indicating that these two cell types, although sharing common innate immune responses, respond differently to external physical stimuli.

Target sequencing, cell experiments, and a population study establish endothelial nitric oxide synthase (eNOS) gene as hypertension susceptibility gene.

A case-control study revealed association between hypertension and rs3918226 in the endothelial nitric oxide synthase (eNOS) gene promoter (minor/major allele, T/C allele). We aimed at substantiating these preliminary findings by target sequencing, cell experiments, and a population study. We sequenced the 140-kb genomic area encompassing the eNOS gene. In HeLa and HEK293T cells transfected with the eNOS promoter carrying either the T or the C allele, we quantified transcription by luciferase assay. In 2722 randomly recruited Europeans (53.0% women; mean age 40.1 years), we studied blood pressure change and incidence of hypertension in relation to rs3918226, using multivariable-adjusted models. Sequencing confirmed rs3918226, a binding site of E-twenty six transcription factors, as the single nucleotide polymorphism most closely associated with hypertension. In T compared with C transfected cells, eNOS promoter activity was from 20% to 40% (P<0.01) lower. In the population, systolic/diastolic blood pressure increased over 7.6 years (median) by 9.7/6.8 mm Hg in 28 TT homozygotes and by 3.8/1.9 mm Hg in 2694 C allele carriers (P≤0.0004). The blood pressure rise was 5.9 mm Hg systolic (confidence interval [CI], 0.6-11.1; P=0.028) and 4.8 mm Hg diastolic (CI, 1.5-8.2; P=0.0046) greater in TT homozygotes, with no differences between the CT and CC genotypes (P≥0.90). Among 2013 participants normotensive at baseline, 692 (34.4%) developed hypertension. The hazard ratio and attributable risk associated with TT homozygosity were 2.04 (CI, 1.24-3.37; P=0.0054) and 51.0%, respectively. In conclusion, rs3918226 in the eNOS promoter tags a hypertension susceptibility locus, TT homozygosity being associated with lesser transcription and higher risk of hypertension.