Prader Willi locus Snord116 RNA regulates hypothalamic functions: sleep and temperature

Sleep is a complex behavior and it is hierarchically regulated involving several brain regions, neurotransmitters, and genes that co-operate in building modulatory mechanisms aimed at controlling and maintaining sleep. Specifically, this thesis attempts to address/understand how genomic imprinting, which affect a subset of genes in mammals resulting in a monoallelic expression, may regulate sleep. One of the main brain regions involved in sleep regulation is the hypothalamus. Within the hypothalamic region imprinted genes are highly expressed. Interestingly, it has been described that hypothalamic insufficiency caused by lack of paternal expression of chromosome 15q11- q13, leads to Prader-Willi syndrome (PWS). Specifically, the microdeletion of the small nuclear ribonucleic acid (RNA)-116 (SNORD116) cluster within the PWS locus plays a major role in developing the main endophenotypes that characterize this syndrome (i.e. REM sleep dysfunction, hyperphagia and temperature instability). However, what could be the role of the paternally imprinted gene Snord116 in the hypothalamic function is unknown. Additionally, is still unclear the specific contribution of the Snord116 gene in developing the PWS symptoms. Since these unresolved points my research has been split into three parts: In the first part of this research, it has been shown that the paternally imprinted gene Snord116 plays a crucial role in the formation and organization of the orexin (OX) and melanin concentrating hormone (MCH) systems, the two main neuro-modulatory systems within the lateral hypothalamus (LH). Moreover, a compromised neuronal dynamic in the LH and a sleep- wake regulation of mice with paternal deletion of Snord116 (PWScrm+/p-) is observed. This abnormal neuronal dynamic is accompanied by a significant reduction in OX neurons in the LH of mutant mice. For this reason, it is proposed that the dysregulation of rapid eye movement (REM) sleep, food intake and temperature control observed in PWS mice are potentially due to this imbalance between OX- and MCH-expressing neurons in the LH as observed in mutant mice. In the second part of this research, it has been investigated the microstructural electrophysiological components of sleep, such as REM sleep features and sleep spindles during non-REM sleep. Indeed, REM sleep is thought to contribute to neuronal network formation early in brain development, while spindles are markers of thalamocortical processes. In neurodevelopmental disorders both sleep structures (REM and sleep spindles) are often compromised and this influence functional properties of cortical neurons. These results indicate 1 that REM sleep properties and its occurrence (REM sleep episodes classified as short-and long REM sleep episode) throughout the sleep-wake cycles are selectively influenced by the Snord116 gene in mice. Moreover, the specific abnormalities in sleep spindles in PWS model systems, indicate that these sleep features may be translated as potential biomarkers in human PWS sufferers. In the third part of this research, it has been proposed a new therapeutic approach for PWS patients aiming to ameliorate the sleep phenotypes that significantly compromise the quality of life of these patients. Pitolisant (a wake-promoting drug) was orally administrated in mice carrying the paternal deletion of the Snord116 gene that are affected by REM sleep alteration coupled with a reduction of the OX neurons. Overall the results of this research show that Pitolisant ameliorates the REM sleep alteration in these mice, although other studies are needed to clarify whether this drug may be easily translated/used in clinics. In conclusion, this thesis provides support for the important role of Snord116 in the regulation of REM sleep and its propensity and its regulatory mechanisms in the hypothalamus. Finally, a new pharmacological approach for PWS by using Pitolisant has been proposed to ameliorate the sleep alteration that significantly affects the PWS patients

Human-Derived Cortical Neurospheroids Coupled to Passive, High-Density and 3D MEAs:A Valid Platform for Functional Tests

: With the advent of human-induced pluripotent stem cells (hiPSCs) and differentiation protocols, methods to create in-vitro human-derived neuronal networks have been proposed. Although monolayer cultures represent a valid model, adding three-dimensionality (3D) would make them more representative of an in-vivo environment. Thus, human-derived 3D structures are becoming increasingly used for in-vitro disease modeling. Achieving control over the final cell composition and investigating the exhibited electrophysiological activity is still a challenge. Thence, methodologies to create 3D structures with controlled cellular density and composition and platforms capable of measuring and characterizing the functional aspects of these samples are needed. Here, we propose a method to rapidly generate neurospheroids of human origin with control over cell composition that can be used for functional investigations. We show a characterization of the electrophysiological activity exhibited by the neurospheroids by using micro-electrode arrays (MEAs) with different types (i.e., passive, C-MOS, and 3D) and number of electrodes. Neurospheroids grown in free culture and transferred on MEAs exhibited functional activity that can be chemically and electrically modulated. Our results indicate that this model holds great potential for an in-depth study of signal transmission to drug screening and disease modeling and offers a platform for in-vitro functional testing

NKCC1 inhibition improves sleep quality and EEG information content in aDown syndromemouse model

In several brain disorders, the hyperpolarizing/inhibitory effects of GABA signaling through Cl-permeable GABAA receptors are compromised, leading to an imbalance between neuronal excitation and inhibition. For example, the Ts65Dn mouse model of Down syndrome (DS) exhibits increased expression of the Cl importer NKCC1, leading to depolarizing gamma aminobutyric acid (GABA) signaling in the mature hippocampus and cortex. Inhibiting NKCC1 with the Food and Drug Administration (FDA)-approved diuretic bumetanide rescues inhibitory GABAergic transmission, synaptic plasticity, and cognitive functions in adult Ts65Dn mice. Given that DS individuals and Ts65Dn mice show sleep disturbances, and considering the key role of GABAergic transmission in sleep, we investigated whether NKCC1 upregulation contributes to sleep abnormalities in adult Ts65Dn mice. Chronic oral administration of bumetanide ameliorated the spectral profile of sleep, sleep architecture, and electroencephalogram (EEG) entropy/complexity, accompanied by a lower hyperactivity in trisomic mice. These results offer a potential avenue for addressing common sleep disturbances in DS.Instituto de Física La Plat

PRELIMINAR ANALYSIS OF ENGINEERED FUNCTIONALLY ACTIVE HUMAN DERIVED CORTICAL NEUROSPHEROIDS FOR DRUG SCREENING AND PRECISION MEDICINE

The continue development of differentiation protocols to generate human neural cells in vitro, allows more accurate investigations of the functional mechanisms arising in such complex networks, and generates great expectations for new treatments in neurodegenerative diseases for which effective therapies are not yet available. The use of 3D aggregates for neuropharmacological in vitro studies has shown great potentials and the advent of human patient specific in vitro models opens new avenues in the field of drug screening and precision medicine. Moreover, Neuronal Stem Cell (NSC) transplantation has the potential to revolutionize brain disease research, but still presents limitations that hamper the use in therapeutics. It has been shown how the injection of NSCs directly into the host, leads to a random integration into the tissue, while a targeted transplant is needed in the specific area affected by degeneration. An alternative approach would be to produce an already differentiated healthy 3D tissue, that shows all the functional and morphological features suitable for transplant into the degenerated area. To this end, we optimized a fast differentiation protocol to engineer excitatory cortical neurospheres with 1:1 ratio between neurons and astrocytes. We first evaluated its morphology by imaging and then we evaluated its functionality (i.e. electrophysiological activity) with glassbased 60 and CMOS-based 4096 micro-electrode arrays (MEAs). Our preliminary results show how the generated structures are viable and functionally active throughout their development. Furthermore, CMOS-MEAs revealed network properties that did not emerge from standard MEAs. Although the obtained results are preliminary, all neurospheroids adhered to substrates and developed functionally active neuritic arborizations, suggesting their efficient use for functional drugs screening applications and for future in vivo transplantation

Post-acute administration of the GABA_A α5 antagonist S44819 promotes recovery of peripheral limb fine motor skills after permanent distal middle cerebral artery occlusion in rats

AbstractBackgroundIschemic stroke induces hypoexcitability of the peri-infarct cortex by increased tonic GABA activity, which impairs stroke recovery. The GABAA α5 antagonist S44819 has recently been shown to promote post-ischemic motor-coordination recovery after transient proximal middle cerebral artery occlusion (MCAO) in mice. The effects of S44819 on post-ischemic skilled limb movement recovery have so far not yet been assessed in rats.MethodsMale Sprague-Dawley rats were subjected to permanent distal MCAO. Starting 3 days post-stroke, vehicle or S44819 (3 or 10 mg/kg) were delivered orally twice a day for 28 days. A single pellet reaching test was performed at baseline, before treatment onset and at weekly intervals thereafter. Animals were sacrificed at 45 days post-stroke (that is, at 42 days post-treatment onset) after 14 days of drug washout. Body weight was monitored, and infarct size was determined by histology.ResultsS44819, administered at 10 mg/kg but not 3 mg/kg significantly improved single pellet reaching performance over 45 days (F(2,96)=22.43, p&lt;0.001). Body weight was not altered. S44819 had no effect on infarct size.ConclusionOur data indicate that post-acute administration of S44819 at 10 mg/kg promotes skilled forelimb movements. The effect was maintained after S44819 wash out.</jats:sec

Postacute administration of the GABA_A α5 antagonist S44819 promotes recovery of peripheral limb fine motor skills after permanent distal middle cerebral artery occlusion in rats

Background: Ischemic stroke causes hypoexcitability in the peri-infarct motor neocortex that stems from increased tonic γ-amino-butyric acid (GABA) activity in neurons. This hypoexcitability, while neuroprotective in the acute phase, may impair neuroplasticity and functional recovery in the subacute phase of stroke. The purpose of this study is to investigate the effect of delayed and prolonged administration of S44819, which is a potent and competitive selective antagonist of GABAA receptors, on the skilled reaching function in a rodent model of stroke. Methods: Male Sprague–Dawley rats ( n = 15) were subjected to permanent middle cerebral artery occlusion. Starting 3 days after stroke, a vehicle or S44819 (3 or 10 mg/kg, BID) was delivered orally twice a day for 28 days. All animals were euthanized 2 weeks later after the washout period. A single pellet reaching task (SPR) was performed before (baseline value) and after the ischemic surgery at several time points (3, 10, 17, 24, 31, 38, and 45 days) to assess the motor deficit. Infarct volume and body changes were also evaluated. Results: S44819, administered at 10 but not 3 mg/kg, significantly improves SPR results over the 45 days after the ischemic surgery. No effect was observed in the infarct size and in the body weight over time between the groups investigated. Conclusion: S44819 at 10 mg/kg significantly enhances motor recovery on a skilled reaching task after sensory-motor cortex lesion. Additionally, our study, in light of the results of the RESTORE BRAIN (Randomized Efficacy and Safety Trial of Oral GABAA α5 antagonist S44819 after Recent ischemic Event) trial, may help clinicians to design clinical studies and stratify variables and patients adequately. </jats:sec

Postacute administration of the GABA α5 antagonist S44819 promotes recovery of peripheral limb fine motor skills after permanent distal middle cerebral artery occlusion in rats

Background: Ischemic stroke causes hypoexcitability in the peri-infarct motor neocortex that stems from increased tonic γ-amino-butyric acid (GABA) activity in neurons. This hypoexcitability, while neuroprotective in the acute phase, may impair neuroplasticity and functional recovery in the subacute phase of stroke. The purpose of this study is to investigate the effect of delayed and prolonged administration of S44819, which is a potent and competitive selective antagonist of GABA A receptors, on the skilled reaching function in a rodent model of stroke. Methods: Male Sprague–Dawley rats ( n = 15) were subjected to permanent middle cerebral artery occlusion. Starting 3 days after stroke, a vehicle or S44819 (3 or 10 mg/kg, BID) was delivered orally twice a day for 28 days. All animals were euthanized 2 weeks later after the washout period. A single pellet reaching task (SPR) was performed before (baseline value) and after the ischemic surgery at several time points (3, 10, 17, 24, 31, 38, and 45 days) to assess the motor deficit. Infarct volume and body changes were also evaluated. Results: S44819, administered at 10 but not 3 mg/kg, significantly improves SPR results over the 45 days after the ischemic surgery. No effect was observed in the infarct size and in the body weight over time between the groups investigated. Conclusion: S44819 at 10 mg/kg significantly enhances motor recovery on a skilled reaching task after sensory-motor cortex lesion. Additionally, our study, in light of the results of the RESTORE BRAIN (Randomized Efficacy and Safety Trial of Oral GABA A α5 antagonist S44819 after Recent ischemic Event) trial, may help clinicians to design clinical studies and stratify variables and patients adequately