Hyperactivity of Rac1-GTPase pathway impairs neuritogenesis of cortical neurons by altering actin dynamics.

The small-GTPase Rac1 is a key molecular regulator linking extracellular signals to actin cytoskeleton dynamics. Loss-of-function mutations in RAC1 and other genes of the Rac signaling pathway have been implicated in the pathogenesis of Intellectual Disability (ID). The Rac1 activity is negatively controlled by GAP proteins, however the effect of Rac1 hyperactivity on neuronal networking in vivo has been poorly studied. ArhGAP15 is a Rac-specific negative regulator, expressed in the main subtypes of pyramidal cortical neurons. In the absence of ArhGAP15, cortical pyramidal neurons show defective neuritogenesis, delayed axonal elongation, reduced dendritic branching, both in vitro and in vivo. These phenotypes are associated with altered actin dynamics at the growth cone due to increased activity of the PAK-LIMK pathway and hyperphosphorylation of ADF/cofilin. These results can be explained by shootin1 hypo-phosphorylation and uncoupling with the adhesion system. Functionally, ArhGAP15-/- mice exhibit decreased synaptic density, altered electroencephalographic rhythms and cognitive deficits. These data suggest that both hypo- and hyperactivation of the Rac pathway due to mutations in Rac1 regulators can result in conditions of ID, and that a tight regulation of Rac1 activity is required to attain the full complexity of the cortical networks.

Citron Kinase Deficiency Leads to Chromosomal Instability and TP53-Sensitive Microcephaly.

Mutations in citron (CIT), leading to loss or inactivation of the citron kinase protein (CITK), cause primary microcephaly in humans and rodents, associated with cytokinesis failure and apoptosis in neural progenitors. We show that CITK loss induces DNA damage accumulation and chromosomal instability in both mammals and Drosophila. CITK-deficient cells display "spontaneous" DNA damage, increased sensitivity to ionizing radiation, and defective recovery from radiation-induced DNA lesions. In CITK-deficient cells, DNA double-strand breaks increase independently of cytokinesis failure. Recruitment of RAD51 to DNA damage foci is compromised by CITK loss, and CITK physically interacts with RAD51, suggesting an involvement of CITK in homologous recombination. Consistent with this scenario, in doubly CitK and Trp53 mutant mice, neural progenitor cell death is dramatically reduced; moreover, clinical and neuroanatomical phenotypes are remarkably improved. Our results underscore a crucial role of CIT in the maintenance of genomic integrity during brain development.

Deletion of the Snord116/SNORD116 Alters Sleep in Mice and Patients with Prader-Willi Syndrome.

STUDY OBJECTIVES: Sleep-wake disturbances are often reported in Prader-Willi syndrome (PWS), a rare neurodevelopmental syndrome that is associated with paternally-expressed genomic imprinting defects within the human chromosome region 15q11-13. One of the candidate genes, prevalently expressed in the brain, is the small nucleolar ribonucleic acid-116 (SNORD116). Here we conducted a translational study into the sleep abnormalities of PWS, testing the hypothesis that SNORD116 is responsible for sleep defects that characterize the syndrome. METHODS: We studied sleep in mutant mice that carry a deletion of Snord116 at the orthologous locus (mouse chromosome 7) of the human PWS critical region (PWScr). In particular, we assessed EEG and temperature profiles, across 24-h, in PWScr (m+/p-) heterozygous mutants compared to wild-type littermates. High-resolution magnetic resonance imaging (MRI) was performed to explore morphoanatomical differences according to the genotype. Moreover, we complemented the mouse work by presenting two patients with a diagnosis of PWS and characterized by atypical small deletions of SNORD116. We compared the individual EEG parameters of patients with healthy subjects and with a cohort of obese subjects. RESULTS: By studying the mouse mutant line PWScr(m+/p-), we observed specific rapid eye movement (REM) sleep alterations including abnormal electroencephalograph (EEG) theta waves. Remarkably, we observed identical sleep/EEG defects in the two PWS cases. We report brain morphological abnormalities that are associated with the EEG alterations. In particular, mouse mutants have a bilateral reduction of the gray matter volume in the ventral hippocampus and in the septum areas, which are pivotal structures for maintaining theta rhythms throughout the brain. In PWScr(m+/p-) mice we also observed increased body temperature that is coherent with REM sleep alterations in mice and human patients. CONCLUSIONS: Our study indicates that paternally expressed Snord116 is involved in the 24-h regulation of sleep physiological measures, suggesting that it is a candidate gene for the sleep disturbances that most individuals with PWS experience.

Vascular and parenchymal lesions along with enhanced neurogenesis characterize the brain of asymptomatic stroke-prone spontaneous hypertensive rats.

BACKGROUND AND OBJECTIVES: Spontaneously hypertensive stroke-prone rats (SHRSPs) develop hypertension, cerebrovascular abnormalities and a stroke phenotype in association with higher levels of proteinuria. Here, we focus on cerebral abnormalities preceding lesions detectable by MRI. METHODS: Longitudinal assessment of brain histology was performed in salt-loaded male SHRSPs (n = 26) and Wistar-Kyoto (WKY) normotensive control animals (n = 27). Groups of rats were sacrificed at different time points: Time 0, before the salt diet administration; Time 1, when proteinuria achieved 40 mg/day; Time 2, when proteinuria exceeded 100 mg/day. RESULTS: At Time 0, no brain lesions were observed. At Time 1, changes of the cortical penetrating arteries, vasogenic oedema, lacunae and focal cell loss appeared in SHRSPs and worsened at Time 2, although no lesions were yet detected by MRI. Staining for proliferation markers revealed a significant boost of cellular mitosis in the subventricular zone (SVZ) of SHRSPs. Moreover, we observed higher immunopositivity for nestin, glial fibrillary acidic protein and doublecortin (markers for neural stem cells, astrocytes and immature neurons, respectively). At Time 2, apoptotic caspase-3 as well as 4-hydroxynonenal-positive neurons were associated to decreased nestin and doublecortin staining. High expression levels of glial fibrillary acidic protein were maintained in the SVZ. No comparative alterations and SVZ activation were recorded in WKYs. CONCLUSION: Appearance of vascular changes in SHRSPs, before any MRI-detectable brain lesion, is coupled to active neural proliferation in the SVZ. With disease progression, only newborn astrocytes can survive, likely because of the neurotoxicity triggered by brain oedema and oxidative stress.

Electrogastrographyc activity in Parkinson's disease patients with and without motor fluctuations.

BACKGROUND: Gastroenteric dysfunctions are very common in Parkinson's disease, but their relationship with dopaminergic response and motor fluctuations is still unclear. Electrogastrography is a noninvasive method for measuring gastric myoelectrical activity. METHODS: We evaluated the effects of levodopa intake on the motility of empty stomachs in Parkinson's disease patients with and without motor fluctuations. RESULTS: The electrogastrography findings showed a normal pattern not influenced by levodopa intake, unrelated to plasmatic levodopa concentrations and to clinical parameters. CONCLUSIONS: Our results suggest that at rest gastric activity of Parkinson's disease patients is normal and plasmatic levodopa variability is not influenced by gastric motility.

Baclofen-loaded solid lipid nanoparticles: preparation, electrophysiological assessment of efficacy, pharmacokinetic and tissue distribution in rats after intraperitoneal administration.

Intrathecal baclofen administration is the reference treatment for spasticity of spinal or cerebral origin, but the risk of infection or catheter dysfunctions are important limits. To explore the possibility of alternative administration routes, we studied a new preparation comprising solid lipid nanoparticles (SLN) incorporating baclofen (baclofen-SLN). We used SLN because they are able to give a sustained release and to target the CNS. Wistar rats were injected intraperitoneally with baclofen-SLN or baclofen solution (baclofen-sol group) at increasing dosages. At different times up to 4 h, efficacy was tested by the H-reflex and two scales evaluating sedation and motor symptoms due to spinal lesions. Rats were killed and baclofen concentration determined in blood and tissues. Physiological solution or unloaded SLN was used as controls. After baclofen-SLN injection, the effect, consisting in a greater and earlier reduction of the H/M ratio than baclofen-sol group and controls, was statistically significant from a dose of 5 mg/kg and was inversely correlated with dose. Clinical effect of baclofen-SLN on both the behavioral scales was greater than that of baclofen-sol and lasted until 4th hour. Compared with baclofen-sol, baclofen-SLN produced significantly higher drug concentrations in plasma from 2nd hour until 4th hour with a linear decrement and in the brain at all times. In conclusion, our study demonstrated the efficacy of a novel formulation of baclofen, which exploits the advantages of SLN preparations. However, for clinical purposes, high baclofen concentrations in brain tissue and sedation may be unwanted effects, requiring further studies and optimization of dosages.