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.

Dysregulation of the orexin/hypocretin system is not limited to narcolepsy but has far-reaching implications for neurological disorders.

Neuropeptides orexin A and B (OX-A/B, also called hypocretin 1 and 2) are released selectively by a population of neurons which projects widely into the entire central nervous system but is localized in a restricted area of the tuberal region of the hypothalamus, caudal to the paraventricular nucleus. The OX system prominently targets brain structures involved in the regulation of wake-sleep state switching, and also orchestrates multiple physiological functions. The degeneration and dysregulation of the OX system promotes narcoleptic phenotypes both in humans and animals. Hence, this review begins with the already proven involvement of OX in narcolepsy, but it mainly discusses the new pre-clinical and clinical insights of the role of OX in three major neurological disorders characterized by sleep impairment which have been recently associated with OX dysfunction, such as Alzheimer's disease, stroke and Prader Willi syndrome, and have been emerged over the past 10 years to be strongly associated with the OX dysfunction and should be more considered in the future. In the light of the impairment of the OX system in these neurological disorders, it is conceivable to speculate that the integrity of the OX system is necessary for a healthy functioning body.

Physiological time structure of the tibialis anterior motor activity during sleep in mice, rats and humans.

The validation of rodent models for restless legs syndrome (Willis-Ekbom disease) and periodic limb movements during sleep requires knowledge of physiological limb motor activity during sleep in rodents. This study aimed to determine the physiological time structure of tibialis anterior activity during sleep in mice and rats, and compare it with that of healthy humans. Wild-type mice (n = 9) and rats (n = 8) were instrumented with electrodes for recording the electroencephalogram and electromyogram of neck muscles and both tibialis anterior muscles. Healthy human subjects (31 ± 1 years, n = 21) underwent overnight polysomnography. An algorithm for automatic scoring of tibialis anterior electromyogram events of mice and rats during non-rapid eye movement sleep was developed and validated. Visual scoring assisted by this algorithm had inter-rater sensitivity of 92-95% and false-positive rates of 13-19% in mice and rats. The distribution of the time intervals between consecutive tibialis anterior electromyogram events during non-rapid eye movement sleep had a single peak extending up to 10 s in mice, rats and human subjects. The tibialis anterior electromyogram events separated by intervals <10 s mainly occurred in series of two-three events, their occurrence rate in humans being lower than in mice and similar to that in rats. In conclusion, this study proposes reliable rules for scoring tibialis anterior electromyogram events during non-rapid eye movement sleep in mice and rats, demonstrating that their physiological time structure is similar to that of healthy young human subjects. These results strengthen the basis for translational rodent models of periodic limb movements during sleep and restless legs syndrome/Willis-Ekbom disease.

Polymorphism of CD1 and SH2D2A genes in inflammatory neuropathies.

In the quest for susceptibility factors of inflammatory neuropathies, many genes implicated in the pathogenesis of autoimmune diseases have been investigated with negative or conflicting results. We studied, with a gene candidate approach, the CD1 system specialized in capturing and presenting glycolipids to antigen-specific T cells, and the SH2D2A gene encoding for a T-cell-specific adapter protein implicated in control of early T-cell activation. In Guillain-Barré syndrome, an initially positive association study with polymorphism of CD1A and CD1E genes was not confirmed. In chronic inflammatory demyelinating polyneuropathy, we did not find an association with CD1 genes, but we found an association with a homozygous genotype for a low repeat number of tandem GA in the SH2D2A gene. This genotype could result in defective control and elimination of autoreactive T cells. All the studies were performed on relatively small size populations. Confirmation in larger sized studies is required both for CD1 and SH2D2A genes. Considering the relative rarity of patients with inflammatory neuropathies, this can only be accomplished by international collaboration.

Sleep disorders in Prader-Willi syndrome, evidence from animal models and humans.

Prader-Willi Syndrome (PWS) is a complex genetic disorder with multiple cognitive, behavioral and endocrine dysfunctions. Sleep alterations and sleep disorders such as Sleep-disordered breathing and Central disorders of hypersomnolence are frequently recognized (either isolated or in comorbidity). The aim of the review is to highlight the pathophysiology and the clinical features of sleep disorders in PWS, providing the basis for early diagnosis and management. We reviewed the genetic features of the syndrome and the possible relationship with sleep alterations in animal models, and we described sleep phenotypes, diagnostic tools and therapeutic approaches in humans. Moreover, we performed a meta-analysis of cerebrospinal fluid orexin levels in patients with PWS; significantly lower levels of orexin were detected in PWS with respect to control subjects (although significantly higher than the ones of narcoleptic patients). Sleep disorders in humans with PWS are multifaceted and are often the result of different mechanisms. Since hypothalamic dysfunction seems to partially influence metabolic, respiratory and sleep/wake characteristics of this syndrome, additional studies are required in this framework.

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.

Susceptibility to chronic inflammatory demyelinating polyradiculoneuropathy is associated to polymorphic GA repeat in the SH2D2A gene.

The SH2D2A gene encodes a T-cell-specific adapter protein involved in the negative control of T-cell activation. The genotype GA13-16 homozygote of the SH2D2A gene promoter has been associated with the susceptibility to develop multiple sclerosis. Chronic inflammatory demyelinating polyradiculoneuropathy (CIDP) is an immune-mediated neuropathy sharing several pathogenetic mechanisms with multiple sclerosis. We genotyped the SH2D2A promoter region in 105 controls and 48 patients with CIDP. We found a significant association between CIDP and the genotype GA13-16 homozygote (OR 3.167; p 0.013). We hypothesize that this genotype is associated with the susceptibility to develop CIDP and may be implicated in the persistence of the disease.

Rapid eye movements sleep as a predictor of functional outcome after stroke: a translational study.

Study Objectives: Sleep disturbances are common in acute stroke patients and are linked with a negative stroke outcome. However, it is also unclear which and how such changes may be related to stroke outcome. To explore this link, we performed a sleep electroencephalogram (EEG) study in animals and humans after ischemic stroke. Methods: (1) Animal study: 12 male rats were assigned to two groups: ischemia (IS) and sham surgery (Sham). In both groups, sleep architecture was investigated 24 h before surgery and for the following 3 days. (2) Human study: 153 patients with ischemic stroke participating in the SAS-CARE prospective, multicenter cohort study had a polysomnography within 9 days after stroke onset. Functional stroke outcome was assessed by the modified Rankin Scale (mRS) at hospital discharge (short-term outcome) and at a 3-month follow-up (long-term outcome). Results: (1) Animal study: rapid eye movement (REM) sleep was significantly reduced in the IS group compared to the Sham group. (2) Human study: patients with poor short-term functional outcome had a reduction of REM sleep and prolonged REM latency during the acute phase of stroke. REM latency was the only sleep EEG variable found to be significantly related to short- and long-term functional impairment in a multiple linear regression analysis. Conclusions: Acute ischemic stroke is followed by a significant reduction of REM sleep in animals and humans. In humans, this reduction was linked with a bad stroke outcome; in addition, REM latency was found to be an independent predictor of stroke evolution. Potential explanations for this role of REM sleep in stroke are discussed. Clinical Trial Registration: http://clinicaltrials.gov. Unique identifier: NCT01097967.

Polymorphisms of CD1 genes in chronic dysimmune neuropathies.

CD1 are MCH-like glycoproteins specialized in capturing and presenting glycolipid to T cells. Expression of CD1 molecules has been observed on endoneurial machrophages in patients with chronic inflammatory demyelinating polyneuropathy (CIDP) and vasculitis and polymorphisms of CID1A and CD1E genes have been associated with susceptibility to develop Guillain-Barré syndrome. In 46 patients with CIDP, in 13 patients with multifocal motor neuropathy and in 132 controls we genotyped exon 2 of CD1A and CD1E genes. We found no association between chronic dysimmune neuropathies, with or without anti-ganglioside antibodies, and polymorphisms of CD1A and CD1E genes.

Role of REM Sleep, Melanin Concentrating Hormone and Orexin/Hypocretin Systems in the Sleep Deprivation Pre-Ischemia.

STUDY OBJECTIVES: Sleep reduction after stroke is linked to poor recovery in patients. Conversely, a neuroprotective effect is observed in animals subjected to acute sleep deprivation (SD) before ischemia. This neuroprotection is associated with an increase of the sleep, melanin concentrating hormone (MCH) and orexin/hypocretin (OX) systems. This study aims to 1) assess the relationship between sleep and recovery; 2) test the association between MCH and OX systems with the pathological mechanisms of stroke. METHODS: Sprague-Dawley rats were assigned to four experimental groups: (i) SD_IS: SD performed before ischemia; (ii) IS: ischemia; (iii) SD_Sham: SD performed before sham surgery; (iv) Sham: sham surgery. EEG and EMG were recorded. The time-course of the MCH and OX gene expression was measured at 4, 12, 24 hours and 3, 4, 7 days following ischemic surgery by qRT-PCR. RESULTS: A reduction of infarct volume was observed in the SD_IS group, which correlated with an increase of REM sleep observed during the acute phase of stroke. Conversely, the IS group showed a reduction of REM sleep. Furthermore, ischemia induces an increase of MCH and OX systems during the acute phase of stroke, although, both systems were still increased for a long period of time only in the SD_IS group. CONCLUSIONS: Our data indicates that REM sleep may be involved in the neuroprotective effect of SD pre-ischemia, and that both MCH and OX systems were increased during the acute phase of stroke. Future studies should assess the role of REM sleep as a prognostic marker, and test MCH and OXA agonists as new treatment options in the acute phase of stroke.

The role of sleep in recovery following ischemic stroke: A review of human and animal data.

Despite advancements in understanding the pathophysiology of stroke and the state of the art in acute management of afflicted patients as well as in subsequent neurorehabilitation training, stroke remains the most common neurological cause of long-term disability in adulthood. To enhance stroke patients' independence and well-being it is necessary, therefore, to consider and develop new therapeutic strategies and approaches. We postulate that sleep might play a pivotal role in neurorehabilitation following stroke. Over the last two decades compelling evidence for a major function of sleep in neuroplasticity and neural network reorganization underlying learning and memory has evolved. Training and learning of new motor skills and knowledge can modulate the characteristics of subsequent sleep, which additionally can improve memory performance. While healthy sleep appears to support neuroplasticity resulting in improved learning and memory, disturbed sleep following stroke in animals and humans can impair stroke outcome. In addition, sleep disorders such as sleep disordered breathing, insomnia, and restless legs syndrome are frequent in stroke patients and associated with worse recovery outcomes. Studies investigating the evolution of post-stroke sleep changes suggest that these changes might also reflect neural network reorganization underlying functional recovery. Experimental and clinical studies provide evidence that pharmacological sleep promotion in rodents and treatment of sleep disorders in humans improves functional outcome following stroke. Taken together, there is accumulating evidence that sleep represents a "plasticity state" in the process of recovery following ischemic stroke. However, to test the key role of sleep and sleep disorders for stroke recovery and to better understand the underlying molecular mechanisms, experimental research and large-scale prospective studies in humans are necessary. The effects of hospital conditions, such as adjusting light conditions according to the patients' sleep-wake rhythms, or sleep promoting drugs and non-invasive brain stimulation to promote neuronal plasticity and recovery following stroke requires further investigation.

Identification of Sleep-Modulated Pathways Involved in Neuroprotection from Stroke.

STUDY OBJECTIVES: Sleep deprivation (SDp) performed before stroke induces an ischemic tolerance state as observed in other forms of preconditioning. As the mechanisms underlying this effect are not well understood, we used DNA oligonucleotide microarray analysis to identify the genes and the gene-pathways underlying SDp preconditioning effects. DESIGN: Gene expression was analyzed 3 days after stroke in 4 experimental groups: (i) SDp performed before focal cerebral ischemia (IS) induction; (ii) SDp performed before sham surgery; (iii) IS without SDp; and (iv) sham surgery without SDp. SDp was performed by gentle handling during the last 6 h of the light period, and ischemia was induced immediately after. SETTINGS: Basic sleep research laboratory. MEASUREMENTS AND RESULTS: Stroke induced a massive alteration in gene expression both in sleep deprived and non-sleep deprived animals. However, compared to animals that underwent ischemia alone, SDp induced a general reduction in transcriptional changes with a reduction in the upregulation of genes involved in cell cycle regulation and immune response. Moreover, an upregulation of a new neuroendocrine pathway which included melanin concentrating hormone, glycoprotein hormones-α-polypeptide and hypocretin was observed exclusively in rats sleep deprived before stroke. CONCLUSION: Our data indicate that sleep deprivation before stroke reprogrammed the signaling response to injury. The inhibition of cell cycle regulation and inflammation are neuroprotective mechanisms reported also for other forms of preconditioning treatment, whereas the implication of the neuroendocrine function is novel and has never been described before. These results therefore provide new insights into neuroprotective mechanisms involved in ischemic tolerance mechanisms.

Evidence of an association between sleep and levodopa-induced dyskinesia in an animal model of Parkinson's disease.

Levodopa-induced dyskinesia (LID) represents a major challenge for clinicians treating patients affected by Parkinson's disease (PD). Although levodopa is the most effective treatment for PD, the remodeling effects induced by disease progression and the pharmacologic treatment itself cause a narrowing of the therapeutic window because of the development of LID. Although animal models of PD provide strong evidence that striatal plasticity underlies the development of dyskinetic movements, the pathogenesis of LID is not entirely understood. In recent years, slow homeostatic adjustment of intrinsic excitability occurring during sleep has been considered fundamental for network stabilization by gradually modifying plasticity thresholds. So far, how sleep affects on LID has not been investigated. Therefore, we measured synaptic downscaling across sleep episodes in a parkinsonian animal model showing dyskinetic movements similar to LID. Our electrophysiological, molecular, and behavioral results are consistent with an impaired synaptic homeostasis during sleep in animals showing dyskinesia. Accordingly, sleep deprivation causes an anticipation and worsening of LID supporting a link between sleep and the development of LID.

Neuroprotective effect of cathodal transcranial direct current stimulation in a rat stroke model.

Experimental focal brain ischemia generates in the penumbra recurrent depolarizations which spread across the injured cortex inducing infarct growth. Transcranial direct current stimulation can induce a lasting, polarity-specific, modulation of cortical excitability. To verify whether cathodal transcranial direct current stimulation could reduce the infarct size and the number of depolarizations, focal ischemia was induced in the rat by the 3 vessels occlusion technique. In the first experiment 12 ischemic rats received cathodal stimulation (alternating 15 min on and 15 min off) starting 45 min after middle cerebral artery occlusion and lasting 4 h. In the second experiment 12 ischemic rats received cathodal transcranial direct current stimulation with the same protocol but starting soon after middle cerebral artery occlusion and lasting 6 h. In both experiments controls were 12 ischemic rats not receiving stimulation. Cathodal stimulation reduced the infarct volume in the first experiment by 20% (p=0.002) and in the second by 30% (p=0.003). The area of cerebral infarction was smaller in animals receiving cathodal stimulation in both experiments (p=0.005). Cathodal stimulation reduced the number of depolarizations (p=0.023) and infarct volume correlated with the number of depolarizations (p=0.048). Our findings indicate that cathodal transcranial direct current stimulation exert a neuroprotective effect in the acute phase of stroke possibly decreasing the number of spreading depolarizations. These findings may have translational relevance and open a new avenue in neuroprotection of stroke in humans.

Possible role for nitric oxide dysregulation in critical illness myopathy.

Muscle fiber inexcitability and myosin loss underlie weakness in critical illness myopathy (CIM). Nitric oxide (NO) takes part in the maintenance of muscle fiber resting potential and, in pathological conditions accompanied by oxidative stress, may damage proteins through peroxynitrite generation. Sepsis and other conditions associated with CIM may differentially affect expression of NO synthases (NOSs), so that both downregulation and upregulation with excessive peroxynitrite production can be hypothesized. In six patients with CIM we studied NOS1, NOS2, and NOS3 protein expression by immunohistochemistry and Western blot. To investigate peroxynitrite production, we performed immunohistochemistry for nitrotyrosine and measured nitrotyrosine levels by enzyme-linked immunosorbent assay. In three patients, sarcolemmal staining for NOS1 was selectively absent. In the others, it was absent in atrophic fibers and absent or reduced in non-atrophic fibers. Total NOS1 protein content was reduced by 41% in patients compared to controls, whereas no significant changes were found in levels and localization of NOS2, NOS3, and nitrotyrosine. Further studies are warranted to determine whether NOS1 loss plays a role in the pathophysiology of CIM, possibly reducing the release of NO at the sarcolemma and affecting muscle fiber excitability.

Familial idiopathic hyper-CK-emia: an underrecognized condition.

Persistent elevation of serum creatine kinase (CK) in individuals with normal neurological and laboratory examinations has been called idiopathic hyperCKemia (IH). IH has been reported in rare families and was recently ascribed to caveolin-3 gene mutations. We retrospectively found that IH was familial in 13 of 28 subjects in whom serum CK was measured in relatives. These 13 families had a total of 41 subjects with IH, including six over 60 years of age. In eight families there was male-to-male transmission and a higher prevalence of males with hyperCKemia. Muscle biopsy in one member of all families was normal or showed minimal, nonspecific changes. Morphometric examination disclosed different patterns of changes in fiber size and distribution. Caveolin-3 expression was normal and in five families molecular genetics excluded caveolin-3 gene mutations. Our findings suggest that IH is familial in 46% of cases. Familial IH is a benign genetically heterogeneous condition that is autosomal-dominant in at least 60% of cases, with a higher penetrance in men.

Spinal muscular atrophy genotyping by gene dosage using multiple ligation-dependent probe amplification.

Spinal muscular atrophy (SMA) is an autosomal recessive disease characterized by degeneration of the anterior horn cells of the spinal cord, causing symmetric proximal muscle weakness. SMA is classified in three clinical types, SMA I, SMA II, and SMA III, based on the severity of the symptoms and the age of onset. About 95% of SMA cases are caused by homozygous deletion of the survival motor neuron 1 (SMN1) gene (5q13), or its conversion to SMN2. The molecular diagnosis of this disease is usually carried out by a polymerase chain reaction-restriction fragment length polymorphism approach able to evidence the absence of both SMN1 copies. However, this approach is not able to identify heterozygous healthy carriers, which show a very high frequency in general population (1:50). We used the multiple ligation-dependent probe amplification (MLPA) approach for the molecular diagnosis of SMA in 19 affected patient and in 57 individuals at risk to become healthy carriers. This analysis detected the absence of the homozygous SMN1 in all the investigated cases, and allowed to discriminate between SMN1 deletion and conversion to SMN2 on the basis of the size showed by the peaks specific for the different genes mapped within the SMA critical region. Moreover, MLPA analysis evidenced a condition of the absence of the heterozygous SMN1 in 33 out of the 57 relatives of the affected patients, demonstrating the usefulness of this approach in the identification of healthy carriers. Thus, the MLPA technique represents an easy, low cost, and high throughput system in the molecular diagnosis of SMA, both in affected patients and in healthy carriers.