Promoting sleep health during pregnancy for enhancing women's health: a longitudinal randomized controlled trial combining biological, physiological and psychological measures, Maternal Outcome after THERapy for Sleep (MOTHERS).

BACKGROUND: Sleep is vital for maintaining individuals' physical and mental health and is particularly challenged during pregnancy. More than 70% of women during the gestational period report insomnia symptoms. Sleep dysfunction in the peripartum increases the risk for a cascade of negative health outcomes during late pregnancy, birth, and postpartum. While psychological interventions are considered the first line treatment for sleep difficulties, they are still scarcely offered during pregnancy and there is a lack of longitudinal research combining psychological and physiological indices. METHODS: The present protocol outlines a randomized controlled trial aimed at testing the long-term effectiveness of an automatized digitalized psychoeducational intervention for insomnia for expectant mothers complaining insomnia symptoms without comorbidity. Outcomes include physiological, hormonal, and subjective indices of maternal psychopathology, stress, and emotional processes, and sleep and wellbeing of the family system. The trial is part of a longitudinal study evaluating expectant mothers from early pregnancy (within the 15th gestational week) to 6-months postpartum through 6 observational phases: baseline (BSL), 6- and 12-weeks from BSL (FU1-FU2), 2-to-4 weeks after delivery (FU3), and 3- and 6-months after delivery (FU4-5). We plan to recruit 38 women without sleep difficulties (Group A) and 76 women with sleep difficulties (Group B). Group B will be randomly assigned to digital psychological control intervention (B1) or experimental psychoeducational intervention targeting insomnia (B2). At 3 time points, an ecological-momentary-assessment (EMA) design will be used to collect data on sleep and emotions (diaries), sleep-wake parameters (actigraphy) and stress reactivity (salivary cortisol). We will also test the DNA methylation of genes involved in the stress response as biomarkers of prenatal poor sleep. Information on partner's insomnia symptoms and new-borns' sleep will be collected at each stage. DISCUSSION: The proposed protocol aims at testing an easily accessible evidence-based psychoeducational intervention for expectant mothers to help them improving sleep, health, and wellbeing in the peripartum. The results could improve the understanding and management of sleep difficulties and peripartum depression. TRIAL REGISTRATION: The study protocol has been registered on 22 April 2024 with ClinicalTrials.gov Protocol Registration and Results System (PRS), ID: NCT06379074. PROTOCOL VERSION: April 23, 2024.

How to study sleep apneas in mouse models of human pathology.

Sleep apnea, the most widespread sleep-related breathing disorder (SBD), consists of recurrent episodes of breathing cessation during sleep. This condition can be classified as either central (CSA) or obstructive (OSA) sleep apnea, with the latest being the most common and toxic. Due to the complexity of living organisms, animal models and, particularly, mice still represent an essential tool for the study of SBD. In the present review we first discuss the methodological pros and cons in the use of whole-body plethysmography to coupling respiratory and sleep measurements and to characterize CSA and OSA in mice; then, we draw an updated and objective picture of the methods used so far in the study of sleep apnea in mice. Most of the studies present in the literature used intermittent hypoxia to mimic OSA in mice and to investigate consequent pathological correlates. On the contrary, few studies using genetic manipulation or high-fat diets investigated the pathogenesis or potential treatments of sleep apnea. To date, mice lacking orexins, hemeoxygenase-2, monoamine oxidase A, Phox2b or Cdkl5 can be considered validated mouse models of sleep apnea. Moreover, genetically- or diet-induced obese mice, and mice recapitulating Down syndrome were proposed as OSA models. In conclusion, our review shows that despite the growing interest in the field and the need of new therapeutical approaches, technical complexity and inter-study variability strongly limit the availability of validated mouse of sleep apnea, which are essential in biomedical research.

Cardiac Functional and Structural Abnormalities in a Mouse Model of CDKL5 Deficiency Disorder.

CDKL5 (cyclin-dependent kinase-like 5) deficiency disorder (CDD) is a severe neurodevelopmental disease that mostly affects girls, who are heterozygous for mutations in the X-linked CDKL5 gene. Mutations in the CDKL5 gene lead to a lack of CDKL5 protein expression or function and cause numerous clinical features, including early-onset seizures, marked hypotonia, autistic features, gastrointestinal problems, and severe neurodevelopmental impairment. Mouse models of CDD recapitulate several aspects of CDD symptomology, including cognitive impairments, motor deficits, and autistic-like features, and have been useful to dissect the role of CDKL5 in brain development and function. However, our current knowledge of the function of CDKL5 in other organs/tissues besides the brain is still quite limited, reducing the possibility of broad-spectrum interventions. Here, for the first time, we report the presence of cardiac function/structure alterations in heterozygous Cdkl5 +/- female mice. We found a prolonged QT interval (corrected for the heart rate, QTc) and increased heart rate in Cdkl5 +/- mice. These changes correlate with a marked decrease in parasympathetic activity to the heart and in the expression of the Scn5a and Hcn4 voltage-gated channels. Interestingly, Cdkl5 +/- hearts showed increased fibrosis, altered gap junction organization and connexin-43 expression, mitochondrial dysfunction, and increased ROS production. Together, these findings not only contribute to our understanding of the role of CDKL5 in heart structure/function but also document a novel preclinical phenotype for future therapeutic investigation.

Expression of a Secretable, Cell-Penetrating CDKL5 Protein Enhances the Efficacy of Gene Therapy for CDKL5 Deficiency Disorder.

Although delivery of a wild-type copy of the mutated gene to cells represents the most effective approach for a monogenic disease, proof-of-concept studies highlight significant efficacy caveats for treatment of brain disorders. Herein, we develop a cross-correction-based strategy to enhance the efficiency of a gene therapy for CDKL5 deficiency disorder, a severe neurodevelopmental disorder caused by CDKL5 gene mutations. We created a gene therapy vector that produces an Igk-TATk-CDKL5 fusion protein that can be secreted via constitutive secretory pathways and, due to the cell-penetration property of the TATk peptide, internalized by cells. We found that, although AAVPHP.B_Igk-TATk-CDKL5 and AAVPHP.B_CDKL5 vectors had similar brain infection efficiency, the AAVPHP.B_Igk-TATk-CDKL5 vector led to higher CDKL5 protein replacement due to secretion and penetration of the TATk-CDKL5 protein into the neighboring cells. Importantly, Cdkl5 KO mice treated with the AAVPHP.B_Igk-TATk-CDKL5 vector showed a behavioral and neuroanatomical improvement in comparison with vehicle or AAVPHP.B_CDKL5 vector-treated Cdkl5 KO mice. In conclusion, we provide the first evidence that a gene therapy based on a cross-correction approach is more effective at compensating Cdkl5-null brain defects than gene therapy based on the expression of the native CDKL5, opening avenues for the development of this innovative approach for other monogenic diseases.

Pilot Study of the Effects of Chronic Intracerebroventricular Infusion of Human Anti-IgLON5 Disease Antibodies in Mice.

BACKGROUND: Anti-IgLON5 disease is a rare late-onset neurological disease associated with autoantibodies against IgLON5, neuronal accumulation of phosphorylated Tau protein (p-Tau), and sleep, respiratory, and motor alterations. PURPOSE: We performed a pilot study of whether the neuropathological and clinical features of anti-IgLON5 disease may be recapitulated in mice with chronic intracerebroventricular infusion of human anti-IgLON5 disease IgG (Pt-IgG). METHODS: Humanized transgenic hTau mice expressing human Tau protein and wild-type (WT) control mice were infused intracerebroventricularly with Pt-IgG or with antibodies from a control subject for 14 days. The sleep, respiratory, and motor phenotype was evaluated at the end of the antibody infusion and at least 30 days thereafter, followed by immunohistochemical assessment of p-Tau deposition. RESULTS: In female hTau and WT mice infused with Pt-IgG, we found reproducible trends of diffuse neuronal cytoplasmic p-Tau deposits in the brainstem and hippocampus, increased ventilatory period during sleep, and decreased inter-lick interval during wakefulness. These findings were not replicated on male hTau mice. CONCLUSION: The results of our pilot study suggest, but do not prove, that chronic ICV infusion of mice with Pt-IgG may elicit neuropathological, respiratory, and motor alterations. These results should be considered as preliminary until replicated in larger studies taking account of potential sex differences in mice.

Early-life nicotine or cotinine exposure produces long-lasting sleep alterations and downregulation of hippocampal corticosteroid receptors in adult mice.

Early-life exposure to environmental toxins like tobacco can permanently re-program body structure and function. Here, we investigated the long-term effects on mouse adult sleep phenotype exerted by early-life exposure to nicotine or to its principal metabolite, cotinine. Moreover, we investigated whether these effects occurred together with a reprogramming of the activity of the hippocampus, a key structure to coordinate the hormonal stress response. Adult male mice born from dams subjected to nicotine (NIC), cotinine (COT) or vehicle (CTRL) treatment in drinking water were implanted with electrodes for sleep recordings. NIC and COT mice spent significantly more time awake than CTRL mice at the transition between the rest (light) and the activity (dark) period. NIC and COT mice showed hippocampal glucocorticoid receptor (GR) downregulation compared to CTRL mice, and NIC mice also showed hippocampal mineralocorticoid receptor downregulation. Hippocampal GR expression significantly and inversely correlated with the amount of wakefulness at the light-to-dark transition, while no changes in DNA methylation were found. We demonstrated that early-life exposure to nicotine (and cotinine) concomitantly entails long-lasting reprogramming of hippocampal activity and sleep phenotype suggesting that the adult sleep phenotype may be modulated by events that occurred during that critical period of life.

Obstructive sleep apneas naturally occur in mice during REM sleep and are highly prevalent in a mouse model of Down syndrome.

STUDY OBJECTIVES: The use of mouse models in sleep apnea study is limited by the belief that central (CSA) but not obstructive sleep apneas (OSA) occur in rodents. We aimed to develop a protocol to investigate the presence of OSAs in wild-type mice and, then, to apply it to a validated model of Down syndrome (Ts65Dn), a human pathology characterized by a high incidence of OSAs. METHODS: In a pilot study, nine C57BL/6J wild-type mice were implanted with electrodes for electroencephalography (EEG), neck electromyography (nEMG), and diaphragmatic activity (DIA), and then placed in a whole-body-plethysmographic (WBP) chamber for 8 h during the rest (light) phase to simultaneously record sleep and breathing activity. CSA and OSA were discriminated on the basis of WBP and DIA signals recorded simultaneously. The same protocol was then applied to 12 Ts65Dn mice and 14 euploid controls. RESULTS: OSAs represented about half of the apneic events recorded during rapid-eye-movement-sleep (REMS) in each experimental group, while the majority of CSAs were found during non-rapid eye movement sleep. Compared with euploid controls, Ts65Dn mice had a similar total occurrence rate of apneic events during sleep, but a significantly higher occurrence rate of OSAs during REMS, and a significantly lower occurrence rate of CSAs during NREMS. CONCLUSIONS: Mice physiologically exhibit both CSAs and OSAs. The latter appear almost exclusively during REMS, and are highly prevalent in Ts65Dn. Mice may, thus, represent a useful model to accelerate the understanding of the pathophysiology and genetics of sleep-disordered breathing and to help the development of new therapies.

Orexin/Hypocretin and Histamine Cross-Talk on Hypothalamic Neuron Counts in Mice.

The loss of hypothalamic neurons that produce wake-promoting orexin (hypocretin) neuropeptides is responsible for narcolepsy type 1 (NT1). While the number of histamine neurons is increased in patients with NT1, results on orexin-deficient mouse models of NT1 are inconsistent. On the other hand, the effect of histamine deficiency on orexin neuron number has never been tested on mammals, even though histamine has been reported to be essential for the development of a functional orexin system in zebrafish. The aim of this study was to test whether histamine neurons are increased in number in orexin-deficient mice and whether orexin neurons are decreased in number in histamine-deficient mice. The hypothalamic neurons expressing L-histidine decarboxylase (HDC), the histamine synthesis enzyme, and those expressing orexin A were counted in four orexin knock-out mice, four histamine-deficient HDC knock-out mice, and four wild-type C57BL/6J mice. The number of HDC-positive neurons was significantly higher in orexin knock-out than in wild-type mice (2,502 ± 77 vs. 1,800 ± 213, respectively, one-tailed t-test, P = 0.011). Conversely, the number of orexin neurons was not significantly lower in HDC knock-out than in wild-type mice (2,306 ± 56 vs. 2,320 ± 120, respectively, one-tailed t-test, P = 0.459). These data support the view that orexin peptide deficiency is sufficient to increase histamine neuron number, supporting the involvement of the histamine waking system in the pathophysiology of NT1. Conversely, these data do not support a significant role of histamine in orexin neuron development in mammals.

Autonomic mechanisms of blood pressure alterations during sleep in orexin/hypocretin-deficient narcoleptic mice.

STUDY OBJECTIVES: Increase in arterial pressure (AP) during sleep and smaller differences in AP between sleep and wakefulness have been reported in orexin (hypocretin)-deficient mouse models of narcolepsy type 1 (NT1) and confirmed in NT1 patients. We tested whether these alterations are mediated by parasympathetic or sympathetic control of the heart and/or resistance vessels in an orexin-deficient mouse model of NT1. METHODS: Thirteen orexin knock-out (ORX-KO) mice were compared with 12 congenic wild-type (WT) mice. The electroencephalogram, electromyogram, and AP of the mice were recorded in the light (rest) period during intraperitoneal infusion of atropine methyl nitrate, atenolol, or prazosin to block muscarinic cholinergic, ß 1-adrenergic, or α 1-adrenergic receptors, respectively, while saline was infused as control. RESULTS: AP significantly depended on a three-way interaction among the mouse group (ORX-KO vs WT), the wake-sleep state, and the drug or vehicle infused. During the control vehicle infusion, ORX-KO had significantly higher AP values during REM sleep, smaller decreases in AP from wakefulness to either non-rapid-eye-movement (non-REM) sleep or REM sleep, and greater increases in AP from non-REM sleep to REM sleep compared to WT. These differences remained significant with atropine methyl nitrate, whereas they were abolished by prazosin and, except for the smaller AP decrease from wakefulness to REM sleep in ORX-KO, also by atenolol. CONCLUSIONS: Sleep-related alterations of AP due to orexin deficiency significantly depend on alterations in cardiovascular sympathetic control in a mouse model of NT1.

Tibialis anterior electromyographic bursts during sleep in histamine-deficient mice.

Antihistamine medications have been suggested to elicit clinical features of restless legs syndrome. The available data are limited, particularly concerning periodic leg movements during sleep, which are common in restless legs syndrome and involve bursts of tibialis anterior electromyogram. Here, we tested whether the occurrence of tibialis anterior electromyogram bursts during non-rapid eye movement sleep is altered in histidine decarboxylase knockout mice with congenital histamine deficiency compared with that in wild-type control mice. We implanted six histidine decarboxylase knockout and nine wild-type mice to record neck muscle electromyogram, bilateral tibialis anterior electromyogram, and electroencephalogram during the rest (light) period. The histidine decarboxylase knockout and wild-type mice did not differ significantly in terms of sleep architecture. In both histidine decarboxylase knockout and wild-type mice, the distribution of intervals between tibialis anterior electromyogram bursts had a single peak for intervals < 10 s. The total occurrence rate of tibialis anterior electromyogram bursts during non-rapid eye movement sleep and the occurrence rate of the tibialis anterior electromyogram bursts separated by intervals < 10 s were significantly lower in histidine decarboxylase knockout than in wild-type mice. These data do not support the hypothesis that preventing brain histamine signalling may promote restless legs syndrome. Rather, the data suggest that limb movements during sleep, including those separated by short intervals, are a manifestation of subcortical arousal requiring the integrity of brain histamine signalling.

The physiological signature of daily torpor is not orexin dependent.

Under conditions of scarce food availability and cool ambient temperature, the mouse (Mus Musculus) enters into torpor, a state of transient metabolic suppression mediated in part by the autonomic nervous system. Hypothalamic orexins are involved in the coordination of behaviors and autonomic function. We tested whether orexins are necessary for the coordinated changes in physiological variables, which underlie torpor and represent its physiological signature. We performed simultaneous measurements of brain temperature, electroencephalographic, and electromyographic activity allowing objective assessment of wake-sleep behavior, and cardiovascular, respiratory, and metabolic variables in orexin knockout mice (ORX-KO) and wild-type mice (WT) during torpor bouts elicited by caloric restriction and mild cold stress. We found that torpor bouts in WT are characterized by an exquisitely coordinated physiological signature. The characteristics of torpor bouts in terms of duration and rate of change of brain temperature and electromyographic activity at torpor entrance and exit did not differ significantly between ORX-KO and WT, and neither did the cardiovascular, respiratory, and metabolic characteristics of torpor. ORX-KO and WT also had similar wake-sleep state changes associated with torpor bouts, with the exception of a significantly higher rapid-eye movement sleep time in ORX-KO at torpor entrance. Our results demonstrate that orexins are not necessary either for the normal physiological adaptations occurring during torpor in mice or for their coordination, suggesting that mechanisms different from orexin peptide signaling may be involved in the regulation and the coordination of these physiological responses.

Effect of ambient temperature on sleep breathing phenotype in mice: the role of orexins.

The loss of orexinergic neurons, which release orexins, results in narcolepsy. Orexins participate in the regulation of many physiological functions, and their role as wake-promoting molecules has been widely described. Less is known about the involvement of orexins in body temperature and respiratory regulation. The aim of this study was to investigate if orexin peptides modulate respiratory regulation as a function of ambient temperature (Ta) during different sleep stages. Respiratory phenotype of male orexin knockout (KO-ORX, N=9) and wild-type (WT, N=8) mice was studied at thermoneutrality (Ta=30°C) or during mild cold exposure (Ta=20°C) inside a whole-body plethysmography chamber. The states of wakefulness (W), non-rapid eye movement sleep (NREMS) and rapid eye movement sleep (REMS) were scored non-invasively, using a previously validated technique. In both WT and KO-ORX mice, Ta strongly and significantly affected ventilatory period and minute ventilation values during NREMS and REMS; moreover, the occurrence rate of sleep apneas in NREMS was significantly reduced at Ta=20°C compared with Ta=30°C. Overall, there were no differences in respiratory regulation during sleep between WT and KO-ORX mice, except for sigh occurrence rate, which was significantly increased at Ta=20°C compared with Ta=30°C in WT mice, but not in KO-ORX mice. These results do not support a main role for orexin peptides in the temperature-dependent modulation of respiratory regulation during sleep. However, we showed that the occurrence rate of sleep apneas critically depends on Ta, without any significant effect of orexin peptides.

Author Correction: Neural control of fasting-induced torpor in mice.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Stress & sleep: A relationship lasting a lifetime.

Stress is an adaptative response aimed at restoring body homeostasis. The classical neuroendocrine stress response involving the activation of the hypothalamic-pituitary-adrenal (HPA) axis modulates many physiological aspects, such as the wake-sleep cycle. In the present review, we will first report a series of human and rodent studies showing that each actor of the HPA axis has the potential to interfere with sleep homeostasis and, then, we will highlight how acute or chronic stress differently modulates the wake-sleep cycle. Moreover, we will present new and interesting studies dealing with the relationship between sleep and stress on a different (longer) time scale. Particularly, we will discuss how the exposure to perinatal stress, probably through epigenetic modulations, is sufficient to cause persistent sleep derangements during adult life. In light of this evidence, the main message of the present review is that the complex relationship between sleep and stress changes dramatically on the basis of the time scale considered and, consequently, "time" should be considered as a critical factor when facing this topic.

Autonomic effects induced by pharmacological activation and inhibition of Raphe Pallidus neurons in anaesthetized adult pigs.

The Raphe Pallidus (RPa) is a region of the brainstem that was shown to modulate the sympathetic outflow to many tissues and organs involved in thermoregulation and energy expenditure. In rodents, the pharmacological activation of RPa neurons was shown to increase the activity of the brown adipose tissue, heart rate, and expired CO2 , whereas their inhibition was shown to induce cutaneous vasodilation and a state of hypothermia that, when prolonged, leads to a state resembling torpor referred to as synthetic torpor. If translatable to humans, this synthetic torpor-inducing procedure would be advantageous in many clinical settings. A first step to explore such translatability, has been to verify whether the neurons within the RPa play the same role described for rodents in a larger mammal such as the pig. In the present study, we show that the physiological responses inducible by the pharmacological stimulation of RPa neurons are very similar to those observed in rodents. Injection of the GABAA agonist GABAzine in the RPa induced an increase in heart rate (from 99 to 174 bpm), systolic (from 87 to 170 mm Hg) and diastolic (from 51 to 98 mm Hg) arterial pressure, and end-tidal CO2 (from 49 to 62 mm Hg). All these changes were reversed by the injection in the same area of the GABAA agonist muscimol. These results support the possibility for RPa neurons to be a key target in the research for a safe and effective procedure for the induction of synthetic torpor in humans.

Neural control of fasting-induced torpor in mice.

Torpor is a peculiar mammalian behaviour, characterized by the active reduction of metabolic rate, followed by a drop in body temperature. To enter torpor, the activation of all thermogenic organs that could potentially defend body temperature must be prevented. Most of these organs, such as the brown adipose tissue, are controlled by the key thermoregulatory region of the Raphe Pallidus (RPa). Currently, it is not known which brain areas mediate the entrance into torpor. To identify these areas, the expression of the early gene c-Fos at torpor onset was assessed in different brain regions in mice injected with a retrograde tracer (Cholera Toxin subunit b, CTb) into the RPa region. The results show a network of hypothalamic neurons that are specifically activated at torpor onset and a direct torpor-specific projection from the Dorsomedial Hypothalamus to the RPa that could putatively mediate the suppression of thermogenesis during torpor.

Validation of 'Somnivore', a Machine Learning Algorithm for Automated Scoring and Analysis of Polysomnography Data.

Manual scoring of polysomnography data is labor-intensive and time-consuming, and most existing software does not account for subjective differences and user variability. Therefore, we evaluated a supervised machine learning algorithm, SomnivoreTM, for automated wake-sleep stage classification. We designed an algorithm that extracts features from various input channels, following a brief session of manual scoring, and provides automated wake-sleep stage classification for each recording. For algorithm validation, polysomnography data was obtained from independent laboratories, and include normal, cognitively-impaired, and alcohol-treated human subjects (total n = 52), narcoleptic mice and drug-treated rats (total n = 56), and pigeons (n = 5). Training and testing sets for validation were previously scored manually by 1-2 trained sleep technologists from each laboratory. F-measure was used to assess precision and sensitivity for statistical analysis of classifier output and human scorer agreement. The algorithm gave high concordance with manual visual scoring across all human data (wake 0.91 ± 0.01; N1 0.57 ± 0.01; N2 0.81 ± 0.01; N3 0.86 ± 0.01; REM 0.87 ± 0.01), which was comparable to manual inter-scorer agreement on all stages. Similarly, high concordance was observed across all rodent (wake 0.95 ± 0.01; NREM 0.94 ± 0.01; REM 0.91 ± 0.01) and pigeon (wake 0.96 ± 0.006; NREM 0.97 ± 0.01; REM 0.86 ± 0.02) data. Effects of classifier learning from single signal inputs, simple stage reclassification, automated removal of transition epochs, and training set size were also examined. In summary, we have developed a polysomnography analysis program for automated sleep-stage classification of data from diverse species. Somnivore enables flexible, accurate, and high-throughput analysis of experimental and clinical sleep studies.

Post-sigh sleep apneas in mice: Systematic review and data-driven definition.

Sleep apneas can be categorized as post-sigh (prevailing in non-rapid eye movement sleep) or spontaneous (prevailing in rapid eye movement sleep) according to whether or not they are preceded by an augmented breath (sigh). Notably, the occurrence of these apnea subtypes changes differently in hypoxic/hypercapnic environments and in some genetic diseases, highlighting the importance of an objective discrimination. We aim to: (a) systematically review the literature comparing the criteria used in categorizing mouse sleep apneas; and (b) provide data-driven criteria for this categorization, with the final goal of reducing experimental variability in future studies. Twenty-two wild-type mice, instrumented with electroencephalographic/electromyographic electrodes, were placed inside a whole-body plethysmographic chamber to quantify sleep apneas and sighs. Wake-sleep states were scored on 4-s epochs based on electroencephalographic/electromyographic signals. Literature revision showed that highly different criteria were used for post-sigh apnea definition, the intervals for apnea occurrence after sigh ranging from 1 breath up to 20 s. In our data, the apnea occurrence rate during non-rapid eye movement sleep was significantly higher than that calculated before the sigh only in the 1st and 2nd 4-s epochs following a sigh. These data suggest that, in mice, apneas should be categorized as post-sigh only if they start within 8 s from a sigh; the choice of shorter or longer time windows might underestimate or slightly overestimate their occurrence rate, respectively.

Heterozygous CDKL5 Knockout Female Mice Are a Valuable Animal Model for CDKL5 Disorder.

CDKL5 disorder is a severe neurodevelopmental disorder caused by mutations in the X-linked CDKL5 (cyclin-dependent kinase-like five) gene. CDKL5 disorder primarily affects girls and is characterized by early-onset epileptic seizures, gross motor impairment, intellectual disability, and autistic features. Although all CDKL5 female patients are heterozygous, the most valid disease-related model, the heterozygous female Cdkl5 knockout (Cdkl5 +/-) mouse, has been little characterized. The lack of detailed behavioral profiling of this model remains a crucial gap that must be addressed in order to advance preclinical studies. Here, we provide a behavioral and molecular characterization of heterozygous Cdkl5 +/- mice. We found that Cdkl5 +/- mice reliably recapitulate several aspects of CDKL5 disorder, including autistic-like behaviors, defects in motor coordination and memory performance, and breathing abnormalities. These defects are associated with neuroanatomical alterations, such as reduced dendritic arborization and spine density of hippocampal neurons. Interestingly, Cdkl5 +/- mice show age-related alterations in protein kinase B (AKT) and extracellular signal-regulated kinase (ERK) signaling, two crucial signaling pathways involved in many neurodevelopmental processes. In conclusion, our study provides a comprehensive overview of neurobehavioral phenotypes of heterozygous female Cdkl5 +/- mice and demonstrates that the heterozygous female might be a valuable animal model in preclinical studies on CDKL5 disorder.

CDKL5 protein substitution therapy rescues neurological phenotypes of a mouse model of CDKL5 disorder.

Cyclin-dependent kinase like-5 (CDKL5) disorder is a rare neurodevelopmental disease caused by mutations in the CDKL5 gene. The consequent misexpression of the CDKL5 protein in the nervous system leads to a severe phenotype characterized by intellectual disability, motor impairment, visual deficits and early-onset epilepsy. No therapy is available for CDKL5 disorder. It has been reported that a protein transduction domain (TAT) is able to deliver macromolecules into cells and even into the brain when fused to a given protein. We demonstrate that TAT-CDKL5 fusion protein is efficiently internalized by target cells and retains CDKL5 activity. Intracerebroventricular infusion of TAT-CDKL5 restored hippocampal development, hippocampus-dependent memory and breathing pattern in Cdkl5-null mice. Notably, systemically administered TAT-CDKL5 protein passed the blood-brain-barrier, reached the CNS, and rescued various neuroanatomical and behavioral defects, including breathing pattern and visual responses. Our results suggest that CDKL5 protein therapy may be an effective clinical tool for the treatment of CDKL5 disorder.