Novel MECP2 gene therapy is effective in a multicenter study using two mouse models of Rett syndrome and is safe in non-human primates.

The AAV9 gene therapy vector presented in this study is safe in mice and non-human primates and highly efficacious without causing overexpression toxicity, a major challenge for clinical translation of Rett syndrome gene therapy vectors to date. Our team designed a new truncated methyl-CpG-binding protein 2 (MECP2) promoter allowing widespread expression of MECP2 in mice and non-human primates after a single injection into the cerebrospinal fluid without causing overexpression symptoms up to 18 months after injection. Additionally, this new vector is highly efficacious at lower doses compared with previous constructs as demonstrated in extensive efficacy studies performed by two independent laboratories in two different Rett syndrome mouse models carrying either a knockout or one of the most frequent human mutations of Mecp2. Overall, data from this multicenter study highlight the efficacy and safety of this gene therapy construct, making it a promising candidate for first-in-human studies to treat Rett syndrome.

CBP Expression Contributes to Neuropathic Pain via CREB and MeCP2 Regulation in the Spared Nerve Injury Rat Model.

Background and Objectives: This study aimed to investigate the relationship between neuropathic pain and CREB-binding protein (CBP) and methyl-CpG-binding protein 2 (MeCP2) expression levels in a rat model with spared nerve injury (SNI). Materials and Methods: Rat (male Sprague-Dawley white rats) models with surgical SNI (n = 6) were prepared, and naive rats (n = 5) were used as controls. The expression levels of CBP and MeCP2 in the spinal cord and dorsal root ganglion (DRG) were compared through immunohistochemistry at 7 and 14 days after surgery. The relationship between neuropathic pain and CBP/MeCP2 was also analyzed through intrathecal siRNA administration. Results: SNI induced a significant increase in the number of CBPs in L4 compared with contralateral DRG as well as with naive rats. The number of MeCP2 cells in the dorsal horn on the ipsilateral side decreased significantly compared with the contralateral dorsal horn and the control group. SNI induced a significant decrease in the number of MeCP2 neurons in the L4 ipsilateral DRG compared with the contralateral DRG and naive rats. The intrathecal injection of CBP siRNA significantly inhibited mechanical allodynia induced by SNI compared with non-targeting siRNA treatment. MeCP2 siRNA injection showed no significant effect on mechanical allodynia. Conclusions: The results suggest that CBP and MeCP2 may play an important role in the generation of neuropathic pain following peripheral nerve injury.

IRAK1 Duplication in MECP2 Duplication Syndrome Does Not Increase Canonical NF-κB-Induced Inflammation.

PURPOSE: Besides their developmental and neurological phenotype, most patients with MECP2/IRAK1 duplication syndrome present with recurrent and severe infections, accompanied by strong inflammation. Respiratory infections are the most common cause of death. Standardized pneumological diagnostics, targeted anti-infectious treatment, and knowledge of the underlying pathomechanism that triggers strong inflammation are unmet clinical needs. We investigated the influence of IRAK1 overexpression on the canonical NF-κB signaling as a possible cause for excessive inflammation in these patients. METHODS: NF-κB signaling was examined by measuring the production of proinflammatory cytokines and evaluating the IRAK1 phosphorylation and degradation as well as the IκBα degradation upon stimulation with IL-1ß and TLR agonists in SV40-immortalized fibroblasts, PBMCs, and whole blood of 9 patients with MECP2/IRAK1 duplication syndrome, respectively. RESULTS: Both, MECP2/IRAK1-duplicated patients and healthy controls, showed similar production of IL-6 and IL-8 upon activation with IL-1ß and TLR2/6 agonists in immortalized fibroblasts. In PBMCs and whole blood, both patients and controls had a similar response of cytokine production after stimulation with IL-1ß and TLR4/2/6 agonists. Patients and controls had equivalent patterns of IRAK1 phosphorylation and degradation as well as IκBα degradation upon stimulation with IL-1ß. CONCLUSION: Patients with MECP2/IRAK1 duplication syndrome do not show increased canonical NF-κB signaling in immortalized fibroblasts, PBMCs, and whole blood. Therefore, we assume that these patients do not benefit from a therapeutic suppression of this pathway.

MECP2 and the biology of MECP2 duplication syndrome.

MECP2 duplication syndrome (MDS), a rare X-linked genomic disorder affecting predominantly males, is caused by duplication of the chromosomal region containing the methyl CpG binding protein-2 (MECP2) gene, which encodes methyl-CpG-binding protein 2 (MECP2), a multi-functional protein required for proper brain development and maintenance of brain function during adulthood. Disease symptoms include severe motor and cognitive impairment, delayed or absent speech development, autistic features, seizures, ataxia, recurrent respiratory infections, and shortened lifespan. The cellular and molecular mechanisms by which a relatively modest increase in MECP2 protein causes such severe disease symptoms are poorly understood and consequently there are no treatments available for this fatal disorder. This review summarizes what is known to date about the structure and zcomplex regulation of MECP2 and its many functions in the developing and adult brain. Additionally, recent experimental findings on the cellular and molecular underpinnings of MDS based on cell culture and mouse models of the disorder are reviewed. The emerging picture from these studies is that MDS is a neurodegenerative disorder in which neurons die in specific parts of the central nervous system, including the cortex, hippocampus, cerebellum, and spinal cord. Neuronal death likely results from astrocytic dysfunction, including a breakdown of glutamate homeostatic mechanisms. The role of elevations in the expression of glial acidic fibrillary protein (GFAP) in astrocytes and the microtubule-associated protein, Tau, in neurons to the pathogenesis of MDS is discussed. Lastly, potential therapeutic strategies to potentially treat MDS are discussed.

MeCP2 inhibits proliferation and migration of breast cancer via suppression of epithelial-mesenchymal transition.

Methyl-CpG-binding protein 2 (MeCP2) is an important epigenetic regulator for normal neuronal maturation and brain glial cell function. Additionally, MeCP2 is also involved in a variety of cancers, such as breast, prostate, lung, liver and colorectal. However, whether MeCP2 contributes to the progression of breast cancer remains unknown. In the present study, we investigated the role of MeCP2 in cell proliferation, migration and invasion in vitro. We found that knockdown of MeCP2 inhibited expression of epithelial-mesenchymal transition (EMT)-related markers in breast cancer cell lines. In conclusion, our study suggests that MeCP2 inhibits proliferation and invasion through suppression of the EMT pathway in breast cancer.

Methyl-CpG-binding protein 2 (MECP2) mutation type is associated with bone disease severity in Rett syndrome.

BACKGROUND: More than 95% of individuals with RTT have mutations in methyl-CpG-binding protein 2 (MECP2), whose protein product modulates gene transcription. The disorder is caused by mutations in a single gene and the disease severity in affected individuals can be quite variable. Specific MECP2 mutations may lead phenotypic variability and different degrees of disease severity. It is known that low bone mass is a frequent and early complication of subjects with Rett syndrome. As a consequence of the low bone mass Rett girls are at an increased risk of fragility fractures. This study aimed to investigate if specific MECP2 mutations may affects the degree of involvement of the bone status in Rett subjects. METHODS: In 232 women with Rett syndrome (mean age 13.8 ± 8.3 yrs) we measured bone mineral density at whole body and at femur (BMD-FN and BMD-TH) by using a DXA machine (Hologic QDR 4500). QUS parameters were assessed at phalanxes by Bone Profiler-IGEA (amplitude dependent speed of sound: AD-SoS and bone transmission time: BTT). Moreover, ambulation capacity (independent or assisted), fracture history and presence of scoliosis were assessed. We divided the subjects with the most common point mutations in two group based on genotype-phenotype severity; in particular, there has been consensus in recognising that the mutations R106T, R168X, R255X, R270X are considered more severe. RESULTS: As aspect, BMD-WB, BMD-FN and BMD-TH were lower in subjects with Rett syndrome that present the most severe mutations with respect to subjects with Rett syndrome with less severe mutations, but the difference was statistically significant only for BMD-FN and BMD-TH (p < 0.05). Also both AD-SoS and BTT values were lower in subjects that present the most severe mutations with respect to less severe mutations but the difference was not statistically significant. Moreover, subjects with Rett syndrome with more severe mutations present a higher prevalence of scoliosis (p < 0.05) and of inability to walk (p < 0.05). CONCLUSION: This study confirms that MECP2 mutation type is a strong predictor of disease severity in subjects with Rett syndrome. In particular, the subjects with more severe mutation present a greater deterioration of bone status, and a higher prevalence of scoliosis and inability to walk.

Molecular characterization of Spanish patients with MECP2 duplication syndrome.

MECP2 duplication syndrome (MDS) is an X-linked neurodevelopmental disorder characterized by a severe to profound intellectual disability, early onset hypotonia and diverse psycho-motor and behavioural features. To date, fewer than 200 cases have been published. We report the clinical and molecular characterization of a Spanish MDS cohort that included 19 boys and 2 girls. Clinical suspicions were confirmed by array comparative genomic hybridization and multiplex ligation-dependent probe amplification (MLPA). Using, a custom in-house MLPA assay, we performed a thorough study of the minimal duplicated region, from which we concluded a complete duplication of both MECP2 and IRAK1 was necessary for a correct MDS diagnosis, as patients with partial MECP2 duplications lacked some typical clinical traits present in other MDS patients. In addition, the duplication location may be related to phenotypic severity. This observation may provide a new approach for genotype-phenotype correlations, and thus more personalized genetic counselling.

Early Postnatal Treatment with Valproate Induces Gad1 Promoter Remodeling in the Brain and Reduces Apnea Episodes in Mecp2-Null Mice.

The deletion of Mecp2, the gene encoding methyl-CpG-binding protein 2, causes severe breathing defects and developmental anomalies in mammals. In Mecp2-null mice, impaired GABAergic neurotransmission is demonstrated at the early stage of life. GABAergic dysfunction in neurons in the rostral ventrolateral medulla (RVLM) is considered as a primary cause of breathing abnormality in Mecp2-null mice, but its molecular mechanism is unclear. Here, we report that mRNA expression levels of Gad1, which encodes glutamate decarboxylase 67 (GAD67), in the RVLM of Mecp2-null (Mecp2-/y, B6.129P2(C)-Mecp2tm1.1Bird/J) mice is closely related to the methylation status of its promoter, and valproate (VPA) can upregulate transcription from Gad1 through epigenetic mechanisms. The administration of VPA (300 mg/kg/day) together with L-carnitine (30 mg/kg/day) from day 8 to day 14 after birth increased Gad1 mRNA expression in the RVLM and reduced apnea counts in Mecp2-/y mice on postnatal day 15. Cytosine methylation levels in the Gad1 promoter were higher in the RVLM of Mecp2-/y mice compared to wild-type mice born to C57BL/6J females, while VPA treatment decreased the methylation levels in Mecp2-/y mice. Chromatin immunoprecipitation assay revealed that the VPA treatment reduced the binding of methyl-CpG binding domain protein 1 (MBD1) to the Gad1 promoter in Mecp2-/y mice. These results suggest that VPA improves breathing of Mecp2-/y mice by reducing the Gad1 promoter methylation, which potentially leads to the enhancement of GABAergic neurotransmission in the RVLM.

Role of astrocytic MeCP2 in regulation of CNS myelination by affecting oligodendrocyte and neuronal physiology and axo-glial interactions.

Astrocytes perform several critical functions such as promoting neuronal maturation, neuronal survival, maintaining and supporting neurons and oligodendrocytes. Astrocytes participate in the formation of nodes of Ranvier. Recently, studies emphasizing on the role of astrocytes in regulating myelination by secreting pro-myelinating factors like growth factors, neurotrophins and ECM proteins, have been investigated by many researchers. Methyl-CpG-Binding Protein 2 (MeCP2), an epigenetic protein, binds to CpG islands in the genome and induces multiple gene regulatory functions by conforming changes in the chromatin structure and resulting in cell-specific gene expression. MeCP2 deficient astrocytes have been linked with abnormal neuronal function including decreased dendritic arborization and decreased dendritic outgrowth. However, role of astrocytic MeCP2 in central nervous system myelination is largely not known. The data from the current study indicate altered mRNA levels (Lif, Cntf, Pdgfa, Cxcl10) of astrocyte-secreted factors involved in myelination. Bdnf and Ngf mRNA levels were also altered in MeCP2 knockdown astrocytes. Moreover, the secreted BDNF levels were significantly altered whereas there were no significant changes in NGF secretion. We also observed that astrocytic MeCP2 affects the morphology, physiology and survival of oligodendrocytes and neurons-two of the key players in myelination. Further, we report that some of the axo-glial interaction genes, namely Caspr, Notch1, Nf155 and Nrg1 are under the regulation of astrocytic MeCP2 along with key myelin genes and proteins.

Experimental Autoimmune Encephalomyelitis (EAE)-Induced Elevated Expression of the E1 Isoform of Methyl CpG Binding Protein 2 (MeCP2E1): Implications in Multiple Sclerosis (MS)-Induced Neurological Disability and Associated Myelin Damage.

Multiple sclerosis (MS) is a chronic neurological disease characterized by the destruction of central nervous system (CNS) myelin. At present, there is no cure for MS due to the inability to repair damaged myelin. Although the neurotrophin brain derived neurotrophic factor (BDNF) has a beneficial role in myelin repair, these effects may be hampered by the over-expression of a transcriptional repressor isoform of methyl CpG binding protein 2 (MeCP2) called MeCP2E1. We hypothesize that following experimental autoimmune encephalomyelitis (EAE)-induced myelin damage, the immune system induction of the pathogenic MeCP2E1 isoform hampers the myelin repair process by repressing BDNF expression. Using an EAE model of MS, we identify the temporal gene and protein expression changes of MeCP2E1, MeCP2E2 and BDNF. The expression changes of these key biological targets were then correlated with the temporal changes in neurological disability scores (NDS) over the entire disease course. Our results indicate that MeCP2E1 mRNA levels are elevated in EAE animals relative to naïve control (NC) and active control (AC) animals during all time points of disease progression. Our results suggest that the EAE-induced elevations in MeCP2E1 expression contribute to the repressed BDNF production in the spinal cord (SC). The sub-optimal levels of BDNF result in sustained NDS and associated myelin damage throughout the entire disease course. Conversely, we observed no significant differences in the expression patterns displayed for the MeCP2E2 isoform amongst our experimental groups. However, our results demonstrate that baseline protein expression ratios between the MeCP2E1 versus MeCP2E2 isoforms in the SC are higher than those identified within the dorsal root ganglia (DRG). Thus, the DRG represents a more conducive environment than that of the SC for BDNF production and transport to the CNS to assist in myelin repair. Henceforth, the sub-optimal BDNF levels we report in the SC may arise from the elevated MeCP2E1 vs. MeCP2E2 ratio in the SC that creates a more hostile environment thereby preventing local BDNF production. At the level of transcript, we demonstrate that EAE-induces the pathological enhanced expression of MeCP2E1 that contributes to enhanced NDS during the entire disease course. Thus, the pathological induction of the MeCP2E1 isoform contributes to the disruption of the normal homeostatic signaling equilibrium network that exists between cytokines, neurotrophins and chemokines that regulate the myelin repair process by repressing BDNF. Our research suggests that the elevated ratio of MeCP2E1 relative to MeCP2E2 may be a useful diagnostic marker that clinicians can utilize to determine the degree of neurological disability with associated myelin damage. The elevated MeCP2E1 vs. MeCP2E2 ratios (E1/E2) in the SC prevent BDNF from reaching optimal levels required for myelin repair. Thus, the lower E1/E2 ratios in the DRG, allow the DRG to serve as a weak secondary compensatory mechanism for enhanced production and delivery of BDNF to the SC to try to assist in myelin repair.

RING finger protein 4 (RNF4) derepresses gene expression from DNA methylation.

RNF4 is an E3 ubiquitin ligase originally identified as a transcription co-activator. The mechanism by which RNF4 promotes transcription remains unclear. In this study, I found that RNF4 antagonizes transcriptional repression mediated by DNA methylation. RNF4 does not promote DNA demethylation, but mediates the ubiquitination of MeCP2, a methyl-CpG-binding domain (MBD) protein. Removal of MeCP2 from gene promoters activates transcription. This study thus not only uncovers how RNF4 functions as a transcription activator, but also reveals the mechanism by which MeCP2 protein stability is regulated.

Predominant cardiac sympathetic modulation during wake and sleep in patients with Rett syndrome.

BACKGROUND: Rett syndrome (RTT) is a rare neurological disorder primarily associated with mutations in the methyl-CpG-binding protein 2 (MECP2) gene. The syndrome is characterized by cognitive, social, and physical impairments, as well as sleep disorders and epilepsy. Notably, dysfunction of the autonomic nervous system is a key feature of the syndrome. Although Heart Rate Variability (HRV) has been used to investigate autonomic nervous system dysfunction in RTT during wakefulness, there is still a significant lack of information regarding the same during sleep. Therefore, our aim was to investigate cardiovascular autonomic modulation during sleep in subjects with RTT compared to an age-matched healthy control group (HC). METHOD: A complete overnight polysomnographic (PSG) recording was obtained from 11 patients with Rett syndrome (all females, 10 ± 4 years old) and 11 HC (all females, 11 ± 4 years old; p = 0.48). Electrocardiogram and breathing data were extracted from PSG and divided into wake, non-REM, and REM sleep stages. Cardiac autonomic control was assessed using symbolic non-linear heart rate variability analysis. The symbolic analysis identified three patterns: 0 V% (sympathetic), 2UV%, and 2LV% (vagal). RESULTS: The 0 V% was higher in the RTT group than in the HC group during wake, non-REM, and REM stages (p < 0.01), while the 2LV and 2UV% were lower during wake and sleep stages (p < 0.01). However, the 0 V% increased similarly from the wake to the REM stage in both RTT and HC groups. CONCLUSIONS: Therefore, the sympatho-vagal balance shifted towards sympathetic predominance and vagal withdrawal during wake and sleep in RTT, although cardiac autonomic dynamics were preserved during sleep.

Cardiac autonomic control in Rett syndrome: Insights from heart rate variability analysis.

Rett syndrome (RTT) is a rare and severe neurological disorder mainly affecting females, usually linked to methyl-CpG-binding protein 2 (MECP2) gene mutations. Manifestations of RTT typically include loss of purposeful hand skills, gait and motor abnormalities, loss of spoken language, stereotypic hand movements, epilepsy, and autonomic dysfunction. Patients with RTT have a higher incidence of sudden death than the general population. Literature data indicate an uncoupling between measures of breathing and heart rate control that could offer insight into the mechanisms that lead to greater vulnerability to sudden death. Understanding the neural mechanisms of autonomic dysfunction and its correlation with sudden death is essential for patient care. Experimental evidence for increased sympathetic or reduced vagal modulation to the heart has spurred efforts to develop quantitative markers of cardiac autonomic profile. Heart rate variability (HRV) has emerged as a valuable non-invasive test to estimate the modulation of sympathetic and parasympathetic branches of the autonomic nervous system (ANS) to the heart. This review aims to provide an overview of the current knowledge on autonomic dysfunction and, in particular, to assess whether HRV parameters can help unravel patterns of cardiac autonomic dysregulation in patients with RTT. Literature data show reduced global HRV (total spectral power and R-R mean) and a shifted sympatho-vagal balance toward sympathetic predominance and vagal withdrawal in patients with RTT compared to controls. In addition, correlations between HRV and genotype and phenotype features or neurochemical changes were investigated. The data reported in this review suggest an important impairment in sympatho-vagal balance, supporting possible future research scenarios, targeting ANS.

The Efficacy of a Human-Ready miniMECP2 Gene Therapy in a Pre-Clinical Model of Rett Syndrome.

Inactivating mutations and the duplication of methyl-CpG binding protein 2 (MeCP2), respectively, mediate Rett syndrome (RTT) and MECP2 duplication syndrome. These disorders underscore the conceptual dose-dependent risk posed by MECP2 gene therapy for mosaic RTT patients. Recently, a miRNA-Responsive Autoregulatory Element (miRARE) mitigated the dose-dependent toxicity posed by self-complementary adeno-associated viral vector serotype 9 (AAV9) miniMECP2 gene therapy (scAAV9/miniMECP2-myc) in mice. Here, we report an efficacy assessment for the human-ready version of this regulated gene therapy (TSHA-102) in male Mecp2-/y knockout (KO) mice after intracerebroventricular (ICV) administration at postnatal day 2 (P2) and after intrathecal (IT) administration at P7, P14 (±immunosuppression), and P28 (±immunosuppression). We also report qPCR studies on KO mice treated at P7-P35; protein analyses in KO mice treated at P38; and a survival safety study in female adult Mecp2-/+ mice. In KO mice, TSHA-102 improved respiration, weight, and survival across multiple doses and treatment ages. TSHA-102 significantly improved the front average stance and swing times relative to the front average stride time after P14 administration of the highest dose for that treatment age. Viral genomic DNA and miniMECP2 mRNA were present in the CNS. MiniMeCP2 protein expression was higher in the KO spinal cord compared to the brain. In female mice, TSHA-102 permitted survivals that were similar to those of vehicle-treated controls. In all, these pivotal data helped to support the regulatory approval to initiate a clinical trial for TSHA-102 in RTT patients (clinical trial identifier number NCT05606614).

Unexpected thermodynamic signature for the interaction of hydroxymethylated DNA with MeCP2.

Hydroxymethylated cytosine (5hmC) is a stable DNA epigenetic mark recognized by methyl-CpG binding protein 2 (MeCP2), which acts as a transcriptional regulator and a global chromatin-remodeling element. Because 5hmC triggers a gene regulation response markedly different from that produced by methylated cytosine (5mC), both modifications must affect DNA structure and/or DNA interaction with MeCP2 differently. MeCP2 is a six-domain intrinsically disordered protein (IDP) with two domains responsible for dsDNA binding: methyl-CpG binding domain (MBD) and intervening domain (ID). Here we report the detailed thermodynamic characterization of the interaction of hmCpG-DNA with MeCP2. We find that hmCpG-DNA interacts with MeCP2 in a distinctly different mode with a particular thermodynamic signature, compared to methylated or unmethylated DNA. In addition, we find evidence for Rett syndrome-associated mutations altering the interaction of MeCP2 with dsDNA in a cytosine modification-specific manner which may correlate with disease onset time and clinical severity score.

Caloric Restriction Can Ameliorate Postoperative Cognitive Dysfunction by Upregulating the Expression of Sirt1, MeCP2 and BDNF in the Hippocampal CA1 Region of Aged C57BL/6 Mice.

This study aimed to investigate the impact of caloric restriction (CR) on cognitive function in aged C57BL/6 mice after surgery, as well as the underlying mechanisms. Forty 14-month-old male C57BL/6 mice were randomly assigned to the ad libitum (AL, n = 20) group and the CR (n = 20) group. After feeding for 12 weeks, they were subdivided into four groups: AL control (ALC, n = 10), AL with surgery (ALS, n = 10), CR control (CRC, n = 10), and CR with surgery (CRS, n = 10). The Morris Water Maze (MWM) test was used to assess learning and memory capacity. By using western blot and immunofluorescence, the expression of Sirt1, MeCP2, and BDNF in the hippocampus and hippocampal CA1 region was quantified. According to the behavioral test, the CRC and CRS groups had significantly better learning and memory abilities than the ALC and ALS groups, respectively. Sirt1, MeCP2, and BDNF expression in the hippocampus and CA1 region in the hippocampus of the ALC and CRC groups of mice were correlated with cognitive improvement. In conclusion, CR could enhance the postoperative cognitive function in aged mice, most likely by increasing the expression of Sirt1, MeCP2, and BDNF in the CA1 region of the hippocampus.

The Role of MeCP2 in Regulating Synaptic Plasticity in the Context of Stress and Depression.

Methyl-CpG-binding protein 2 (MeCP2) is a transcriptional regulator that is highly abundant in the brain. It binds to methylated genomic DNA to regulate a range of physiological functions implicated in neuronal development and adult synaptic plasticity. MeCP2 has mainly been studied for its role in neurodevelopmental disorders, but alterations in MeCP2 are also present in stress-related disorders such as major depression. Impairments in both stress regulation and synaptic plasticity are associated with depression, but the specific mechanisms underlying these changes have not been identified. Here, we review the interplay between stress, synaptic plasticity, and MeCP2. We focus our attention on the transcriptional regulation of important neuronal plasticity genes such as BDNF and reelin (RELN). Moreover, we provide evidence from recent studies showing a link between chronic stress-induced depressive symptoms and dysregulation of MeCP2 expression, underscoring the role of this protein in stress-related pathology. We conclude that MeCP2 is a promising target for the development of novel, more efficacious therapeutics for the treatment of stress-related disorders such as depression.

Molecular Signatures of Response to Mecasermin in Children With Rett Syndrome.

Rett syndrome (RTT) is a devastating neurodevelopmental disorder without effective treatments. Attempts at developing targetted therapies have been relatively unsuccessful, at least in part, because the genotypical and phenotypical variability of the disorder. Therefore, identification of biomarkers of response and patients' stratification are high priorities. Administration of Insulin-like Growth Factor 1 (IGF-1) and related compounds leads to significant reversal of RTT-like symptoms in preclinical mouse models. However, improvements in corresponding clinical trials have not been consistent. A 20-weeks phase I open label trial of mecasermin (recombinant human IGF-1) in children with RTT demonstrated significant improvements in breathing phenotypes. However, a subsequent randomised controlled phase II trial did not show significant improvements in primary outcomes although two secondary clinical endpoints showed positive changes. To identify molecular biomarkers of response and surrogate endpoints, we used RNA sequencing to measure differential gene expression in whole blood samples of participants in the abovementioned phase I mecasermin trial. When all participants (n = 9) were analysed, gene expression was unchanged during the study (baseline vs. end of treatment, T0-T3). However, when participants were subclassified in terms of breathing phenotype improvement, specifically by their plethysmography-based apnoea index, individuals with moderate-severe apnoea and breathing improvement (Responder group) displayed significantly different transcript profiles compared to the other participants in the study (Mecasermin Study Reference group, MSR). Many of the differentially expressed genes are involved in the regulation of cell cycle processes and immune responses, as well as in IGF-1 signalling and breathing regulation. While the Responder group showed limited gene expression changes in response to mecasermin, the MSR group displayed marked differences in the expression of genes associated with inflammatory processes (e.g., neutrophil activation, complement activation) throughout the trial. Our analyses revealed gene expression profiles associated with severe breathing phenotype and its improvement after mecasermin administration in RTT, and suggest that inflammatory/immune pathways and IGF-1 signalling contribute to treatment response. Overall, these data support the notion that transcript profiles have potential as biomarkers of response to IGF-1 and related compounds.

Influence of the disordered domain structure of MeCP2 on its structural stability and dsDNA interaction.

Methyl-CpG binding protein 2 (MeCP2) is a transcriptional regulator and a chromatin-associated structural protein. MeCP2 deregulation results in two neurodevelopmental disorders: MeCP2 dysfunction is associated with Rett syndrome, while excess of activity is associated with MeCP2 duplication syndrome. MeCP2 is an intrinsically disordered protein (IDP) constituted by six structural domains with variable, small percentage of well-defined secondary structure. Two domains, methyl-CpG binding domain (MBD) and transcription repressor domain (TRD), are the elements responsible for dsDNA binding ability and recruitment of the gene transcription/silencing machinery, respectively. Previously we studied the influence of the completely disordered, MBD-flanking domains (N-terminal domain, NTD, and intervening domain, ID) on the structural and functional features of the MBD (Claveria-Gimeno, R. et al. Sci Rep. 2017, 7, 41,635). Here we report the biophysical study of the influence of the remaining domains (transcriptional repressor domain, TRD, and C-terminal domains, CTDα and CTDß) on the structural stability of MBD and the dsDNA binding capabilities of MBD and ID. The influence of distant disordered domains on MBD properties makes it necessary to consider the NTD-MBD-ID variant as the minimal protein construct for studying dsDNA/chromatin binding properties, while the full-length protein should be considered for transcriptional regulation studies.

Association of CaMK2A and MeCP2 signaling pathways with cognitive ability in adolescents.

The glutamatergic signaling pathway is involved in molecular learning and human cognitive ability. Specific single variants (SNVs, formerly single-nucleotide polymorphisms) in the genes encoding N-methyl-D-aspartate receptor subunits have been associated with neuropsychiatric disorders by altering glutamate transmission. However, these variants associated with cognition and mental activity have rarely been explored in healthy adolescents. In this study, we screened for SNVs in the glutamatergic signaling pathway to identify genetic variants associated with cognitive ability. We found that SNVs in the subunits of ionotropic glutamate receptors, including GRIA1, GRIN1, GRIN2B, GRIN2C, GRIN3A, GRIN3B, and calcium/calmodulin-dependent protein kinase IIα (CaMK2A) are associated with cognitive function. Plasma CaMK2A level was correlated positively with the cognitive ability of Taiwanese senior high school students. We demonstrated that elevating CaMK2A increased its autophosphorylation at T286 and increased the expression of its downstream targets, including GluA1 and phosphor- GluA1 in vivo. Additionally, methyl-CpG binding protein 2 (MeCP2), a downstream target of CaMK2A, was found to activate the expression of CaMK2A, suggesting that MeCP2 and CaMK2A can form a positive feedback loop. In summary, two members of the glutamatergic signaling pathway, CaMK2A and MeCP2, are implicated in the cognitive ability of adolescents; thus, altering the expression of CaMK2A may affect cognitive ability in youth.