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

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

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

Loss of Snord116 alters cortical neuronal activity in mice: a preclinical investigation of Prader-Willi syndrome.

Prader-Willi syndrome (PWS) is a neurodevelopmental disorder that is characterized by metabolic alteration and sleep abnormalities mostly related to rapid eye movement (REM) sleep disturbances. The disease is caused by genomic imprinting defects that are inherited through the paternal line. Among the genes located in the PWS region on chromosome 15 (15q11-q13), small nucleolar RNA 116 (Snord116) has been previously associated with intrusions of REM sleep into wakefulness in humans and mice. Here, we further explore sleep regulation of PWS by reporting a study with PWScrm+/p- mouse line, which carries a paternal deletion of Snord116. We focused our study on both macrostructural electrophysiological components of sleep, distributed among REMs and nonrapid eye movements. Of note, here, we study a novel electroencephalography (EEG) graphoelements of sleep for mouse studies, the well-known spindles. EEG biomarkers are often linked to the functional properties of cortical neurons and can be instrumental in translational studies. Thus, to better understand specific properties, we isolated and characterized the intrinsic activity of cortical neurons using in vitro microelectrode array. Our results confirm that the loss of Snord116 gene in mice influences specific properties of REM sleep, such as theta rhythms and, for the first time, the organization of REM episodes throughout sleep-wake cycles. Moreover, the analysis of sleep spindles present novel specific phenotype in PWS mice, indicating that a new catalog of sleep biomarkers can be informative in preclinical studies of PWS

Loss of Snord116 impacts lateral hypothalamus, sleep, and food-related behaviors

Imprinted genes are highly expressed in the hypothalamus; however, whether specific imprinted genes affect hypothalamic neuromodulators and their functions is unknown. It has been suggested that Prader-Willi syndrome (PWS), a neurodevelopmental disorder caused by lack of paternal expression at chromosome 15q11-q13, is characterized by hypothalamic insufficiency. Here, we investigate the role of the paternally expressed Snord116 gene within the context of sleep and metabolic abnormalities of PWS, and we report a significant role of this imprinted gene in the function and organization of the 2 main neuromodulatory systems of the lateral hypothalamus (LH) - namely, the orexin (OX) and melanin concentrating hormone (MCH) - systems. We observed that the dynamics between neuronal discharge in the LH and the sleep-wake states of mice with paternal deletion of Snord116 (PWScrm+/p-) are compromised. This abnormal state-dependent neuronal activity is paralleled by a significant reduction in OX neurons in the LH of mutant mice. Therefore, we propose that an imbalance between OX- and MCH-expressing neurons in the LH of mutant mice reflects a series of deficits manifested in the PWS, such as dysregulation of rapid eye movement (REM) sleep, food intake, and temperature control

Loss of Snord116 alters cortical neuronal activity in mice: a preclinical investigation of Prader–Willi syndrome

Prader–Willi syndrome (PWS) is a neurodevelopmental disorder that is characterized by metabolic alteration and sleep abnormalities mostly related to rapid eye movement (REM) sleep disturbances. The disease is caused by genomic imprinting defects that are inherited through the paternal line. Among the genes located in the PWS region on chromosome 15 (15q11-q13), small nucleolar RNA 116 (Snord116) has been previously associated with intrusions of REM sleep into wakefulness in humans and mice. Here, we further explore sleep regulation of PWS by reporting a study with PWScrm+/p− mouse line, which carries a paternal deletion of Snord116. We focused our study on both macrostructural electrophysiological components of sleep, distributed among REMs and nonrapid eye movements. Of note, here, we study a novel electroencephalography (EEG) graphoelements of sleep for mouse studies, the well-known spindles. EEG biomarkers are often linked to the functional properties of cortical neurons and can be instrumental in translational studies. Thus, to better understand specific properties, we isolated and characterized the intrinsic activity of cortical neurons using in vitro microelectrode array. Our results confirm that the loss of Snord116 gene in mice influences specific properties of REM sleep, such as theta rhythms and, for the first time, the organization of REM episodes throughout sleep–wake cycles. Moreover, the analysis of sleep spindles present novel specific phenotype in PWS mice, indicating that a new catalog of sleep biomarkers can be informative in preclinical studies of PWS