Loss of GluN2A‐containing NMDA receptors impairs extra‐dimensional set‐shifting

Glutamate neurotransmission via the N‐methyl‐d ‐aspartate receptor (NMDAR) is thought to mediate the synaptic plasticity underlying learning and memory formation. There is increasing evidence that deficits in NMDAR function are involved in the pathophysiology of cognitive dysfunction seen in neuropsychiatric disorders and addiction. NMDAR subunits confer different physiological properties to the receptor, interact with distinct intracellular postsynaptic scaffolding and signaling molecules, and are differentially expressed during development. Despite these known differences, the relative contribution of individual subunit composition to synaptic plasticity and learning is not fully elucidated. We have previously shown that constitutive deletion of GluN2A subunit in the mouse impairs discrimination and re‐learning phase of reversal when exemplars are complex picture stimuli, but spares acquisition and extinction of non‐discriminative visually cued instrumental response. To investigate the role of GluN2A containing NMDARs in executive control, we tested GluN2A knockout (GluN2A^KO), heterozygous (GluN2A^HET) and wild‐type (WT) littermates on an attentional set‐shifting task using species‐specific stimulus dimensions. To further explore the nature of deficits in this model, mice were tested on a visual discrimination reversal paradigm using simplified rotational stimuli. GluN2A^KO were not impaired on discrimination or reversal problems when tactile or olfactory stimuli were used, or when visual stimuli were sufficiently easy to discriminate. GluN2A^KO showed a specific and significant impairment in ventromedial prefrontal cortex‐mediated set‐shifting. Together these results support a role for GluN2A containing NMDAR in modulating executive control that can be masked by overlapping deficits in attentional processes during high task demands.     

Oxidative stress in Rett syndrome: Natural history,genotype, and variants

Abstract

Objectives

Rett syndrome (RTT) is an X-linked autism spectrum disorder caused by mutations in the MeCP2 gene in the great majority of cases. Evidence suggests a potential role of oxidative stress (OS) in its pathogenesis. Here, we investigated the potential value of OS markers (non-protein-bound iron (NPBI) and F₂-isoprostanes (F₂-IsoPs)) in explaining natural history, genotype-phenotype correlation, and clinical heterogeneity of RTT, and gauging the response to omega-3 polyunsaturated fatty acids (ω-3 PUFAs).

Methods

RTT patients (n = 113) and healthy controls were assayed for plasma NPBI and F₂-IsoPs, and intraerythrocyte NPBI. Forty-two patients with typical RTT were randomly assigned to ω-3 PUFAs supplementation for 12 months. NPBI was measured by HPLC and F₂-IsoPs using a gas chromatography/negative ion chemical ionization tandem mass spectrometry (GC/NICI-MS/MS) technique.

Results

F₂-IsoPs were significantly higher in the early stages as compared with the late natural progression of classic RTT. MeCP2 mutations related to more severe phenotypes exhibited higher OS marker levels than those of milder phenotypes. Higher OS markers were observed in typical RTT and early seizure variant as compared with the preserved speech and congenital variants. Significant reduction in OS markers levels and improvement of severity scores were observed after ω-3 PUFAs supplementation.

Discussion

OS is a key modulator of disease expression in RTT.     

Acetyl-L-Carnitine Improves Behavior and Dendritic Morphology in a Mouse Model of Rett Syndrome

Rett syndrome (RTT) is a devastating neurodevelopmental disorder affecting 1 in 10,000 girls. Approximately 90% of cases are caused by spontaneous mutations in the X-linked gene encoding methyl-CpG-binding protein 2 (MeCP2). Girls with RTT suffer from severe motor, respiratory, cognitive and social abnomalities attributed to early deficits in synaptic connectivity which manifest in the adult as a myriad of physiological and anatomical abnormalities including, but not limited to, dimished dendritic complexity. Supplementation with acetyl-L-carnitine (ALC), an acetyl group donor, ameliorates motor and cognitive deficits in other disease models through a variety of mechanisms including altering patterns of histone acetylation resulting in changes in gene expression, and stimulating biosynthetic pathways such as acetylcholine. We hypothesized ALC treatment during critical periods in cortical development would promote normal synaptic maturation, and continuing treatment would improve behavioral deficits in the Mecp2^1lox mouse model of RTT. In this study, wildtype and Mecp2^1lox mutant mice received daily injections of ALC from birth until death (postnatal day 47). General health, motor, respiratory, and cognitive functions were assessed at several time points during symptom progression. ALC improved weight gain, grip strength, activity levels, prevented metabolic abnormalities and modestly improved cognitive function in Mecp2 null mice early in the course of treatment, but did not significantly improve motor or cognitive functions assessed later in life. ALC treatment from birth was associated with an almost complete rescue of hippocampal dendritic morphology abnormalities with no discernable side effects in the mutant mice. Therefore, ALC appears to be a promising therapeutic approach to treating early RTT symptoms and may be useful in combination with other therapies.     

Anaplerotic Triheptanoin Diet Enhances Mitochondrial Substrate Use to Remodel the Metabolome and Improve Lifespan,Motor Function,and Sociability in MeCP2-Null Mice

Rett syndrome (RTT) is an autism spectrum disorder (ASD) caused by mutations in the X-linked MECP2 gene that encodes methyl-CpG binding protein 2 (MeCP2). Symptoms range in severity and include psychomotor disabilities, seizures, ataxia, and intellectual disability. Symptom onset is between 6-18 months of age, a critical period of brain development that is highly energy-dependent. Notably, patients with RTT have evidence of mitochondrial dysfunction, as well as abnormal levels of the adipokines leptin and adiponectin, suggesting overall metabolic imbalance. We hypothesized that one contributor to RTT symptoms is energy deficiency due to defective nutrient substrate utilization by the TCA cycle. This energy deficit would lead to a metabolic imbalance, but would be treatable by providing anaplerotic substrates to the TCA cycle to enhance energy production. We show that dietary therapy with triheptanoin significantly increased longevity and improved motor function and social interaction in male mice hemizygous for Mecp2 knockout. Anaplerotic therapy in Mecp2 knockout mice also improved indicators of impaired substrate utilization, decreased adiposity, increased glucose tolerance and insulin sensitivity, decreased serum leptin and insulin, and improved mitochondrial morphology in skeletal muscle. Untargeted metabolomics of liver and skeletal muscle revealed increases in levels of TCA cycle intermediates with triheptanoin diet, as well as normalizations of glucose and fatty acid biochemical pathways consistent with the improved metabolic phenotype in Mecp2 knockout mice on triheptanoin. These results suggest that an approach using dietary supplementation with anaplerotic substrate is effective in improving symptoms and metabolic health in RTT.     

A heterozygous mutation in tubulin,beta 2B ( Tubb2b ) causes cognitive deficits and hippocampal disorganization

Development of the mammalian forebrain requires a significant contribution from tubulin proteins to physically facilitate both the large number of mitoses in the neurogenic brain (in the form of mitotic spindles) as well as support cellular scaffolds to guide radial migration (radial glial neuroblasts). Recent studies have identified a number of mutations in human tubulin genes affecting the forebrain, including TUBB2B . We previously identified a mouse mutation in Tubb2b and we show here that mice heterozygous for this missense mutation in Tubb2b have significant cognitive defects in spatial learning and memory. We further showed reduced hippocampal long‐term potentiation consistent with these defects. In addition to the behavioural and physiological deficits, we show here abnormal hippocampal morphology. Taken together, these phenotypes suggest that heterozygous mutations in tubulin genes result in cognitive deficits not previously appreciated. This has implications for design and interpretation of genetic testing for humans with intellectual disability disorders.     

Effects of the Rho GTPase-activating toxin CNF1 on fibroblasts derived from Rett syndrome patients: A pilot study

The bacterial product CNF1, through its action on the Rho GTPases, is emerging as a modulator of crucial signalling pathways involved in selected neurological diseases characterized by mitochondrial dysfunctions. Mitochondrial impairment has been hypothesized to have a key role in paramount mechanisms underlying Rett syndrome (RTT), a severe neurologic rare disorder. CNF1 has been already reported to have beneficial effects in mouse models of RTT. Using human RTT fibroblasts from four patients carrying different mutations, as a reliable disease-in-a-dish model, we explored the cellular and molecular mechanisms, which can underlie the CNF1-induced amelioration of RTT deficits. We found that CNF1 treatment modulates the Rho GTPases activity of RTT fibroblasts and induces a considerable re-organization of the actin cytoskeleton, mainly in stress fibres. Mitochondria of RTT fibroblasts show a hyperfused morphology and CNF1 decreases the mitochondrial mass leaving substantially unaltered the mitochondrial dynamic. From a functional perspective, CNF1 induces mitochondrial membrane potential depolarization and activation of AKT in RTT fibroblasts. Given that mitochondrial quality control is altered in RTT, our results are suggestive of a reactivation of the damaged mitochondria removal via mitophagy restoration. These effects can be at the basis of the beneficial effects of CNF1 in RTT.     

Graded and pan-neural disease phenotypes of Rett Syndrome linked with dosage of functional MeCP2

Rett syndrome (RTT) is a progressive neurodevelop-mental disorder,mainly caused by mutations in MeCP2 and currently with no cure.We report here that neurons from R106W MeCP2 RTT human iPSCs as well as human embryonic stem cells after MeCP2 knockdown exhibit consistent and long-lasting impairment in maturation as indicated by impaired action potentials and passive membrane properties as well as reduced soma size and spine density.Moreover,RTT-inherent defects in neu-ronal maturation could be pan-neuronal and occurred in neurons with both dorsal and ventral forebrain features.Knockdown of MeCP2 led to more severe neuronal deficits as compared to RTT iPSC-derived neurons,which appeared to retain partial function.Strikingly,consistent deficits in nuclear size,dendritic complexity and circuitry-dependent spontaneous postsynaptic currents could only be observed in MeCP2 knockdown neurons but not RTT iPSC-derived neurons.Both neu-ron-intrinsic and circuitry-dependent deficits of MeCP2-deficient neurons could be fully or partially rescued by re-expression of wild type or T158M MeCP2,strengthening the dosage dependency of MeCP2 on disease phenotypes and also the partial function of the mutant.Our findings thus reveal stable neuronal matu-ration deficits and unexpectedly,graded sensitivities of neuron-inherent and neural transmission phenotypes towards the extent of MeCP2 deficiency,which is infor-mative for future therapeutic development.     

Genetic and behavioral characterization of a Kmt2d mouse mutant,a new model for Kabuki Syndrome

The recessive mutant mice bate palmas (bapa) ‐ claps in Portuguese arose from N‐ethyl‐N‐nitrosourea mutagenesis. A single nucleotide, T > C, change in exon 13, leading to a Thr₁₂₈₉Ala substitution, was identified in the lysine (K)‐specific methyltransferase 2D gene (Kmt2d) located on chromosome 15. Mutations with a loss‐of‐function in the KMT2D gene on chromosome 12 in humans are responsible for Kabuki syndrome (KS). Phenotypic characterization of the bapa mutant was performed using a behavioral test battery to evaluate the parameters related to general activity, the sensory nervous system, the psychomotor system, and the autonomous nervous system, as well as to measure motor function and spatial memory. Relative to BALB/cJ mice, the bapa mutant showed sensory and psychomotor impairments, such as hypotonia denoted by a surface righting reflex impairment and hindquarter fall, and a reduction in the auricular reflex, suggesting hearing impairment. Additionally, the enhanced general activity showed by the increased rearing and grooming frequency, distance traveled and average speed possibly presupposes the presence of hyperactivity of bapa mice compared with the control group. A slight motor coordination dysfunction was showed in bapa mice, which had a longer crossing time on the balance beam compared with BALB/cJ controls. Male bapa mice also showed spatial gait pattern changes, such as a shorter stride length and shorter step length. In conclusion, the bapa mouse may be a valuable animal model to study the mechanisms involved in psychomotor and behavior impairments, such as hypotonia, fine motor coordination and hyperactivity linked to the Kmt2d mutation.     

A BDNF loop-domain mimetic acutely reverses spontaneous apneas and respiratory abnormalities during behavioral arousal in a mouse model of Rett syndrome

Reduced levels of brain-derived neurotrophic factor (BDNF) are thought to contribute to the pathophysiology of Rett syndrome (RTT), a severe neurodevelopmental disorder caused by loss-of-function mutations in the gene encoding methyl-CpG-binding protein 2 (MeCP2). In Mecp2 mutant mice, BDNF deficits have been associated with breathing abnormalities, a core feature of RTT, as well as with synaptic hyperexcitability within the brainstem respiratory network. Application of BDNF can reverse hyperexcitability in acute brainstem slices from Mecp2-null mice, suggesting that therapies targeting BDNF or its receptor, TrkB, could be effective at acute reversal of respiratory abnormalities in RTT. Therefore, we examined the ability of LM22A-4, a small-molecule BDNF loop-domain mimetic and TrkB partial agonist, to modulate synaptic excitability within respiratory cell groups in the brainstem nucleus tractus solitarius (nTS) and to acutely reverse abnormalities in breathing at rest and during behavioral arousal in Mecp2 mutants. Patch-clamp recordings in Mecp2-null brainstem slices demonstrated that LM22A-4 decreases excitability at primary afferent synapses in the nTS by reducing the amplitude of evoked excitatory postsynaptic currents and the frequency of spontaneous and miniature excitatory postsynaptic currents. In vivo, acute treatment of Mecp2-null and -heterozygous mutants with LM22A-4 completely eliminated spontaneous apneas in resting animals, without sedation. Moreover, we demonstrate that respiratory dysregulation during behavioral arousal, a feature of human RTT, is also reversed in Mecp2 mutants by acute treatment with LM22A-4. Together, these data support the hypothesis that reduced BDNF signaling and respiratory dysfunction in RTT are linked, and establish the proof-of-concept that treatment with a small-molecule structural mimetic of a BDNF loop domain and a TrkB partial agonist can acutely reverse abnormal breathing at rest and in response to behavioral arousal in symptomatic RTT mice.KEY WORDS: Mecp2, Brain-derived neurotrophic factor (BDNF), Respiration, Brainstem, Arousal     

MeCP2 R168X male and female mutant mice exhibit Rett‐like behavioral deficits

Rett syndrome (RTT) is a regressive developmental disorder characterized by motor and breathing abnormalities, anxiety, cognitive dysfunction and seizures. Approximately 95% of RTT cases are caused by more than 200 different mutations in the X‐linked gene encoding methyl‐CpG‐binding protein 2 (MeCP2). While numerous transgenic mice have been created modeling common mutations in MeCP2, the behavioral phenotype of many of these male and, especially, female mutant mice has not been well characterized. Thorough phenotyping of additional RTT mouse models will provide valuable insight into the effects of Mecp2 mutations on behavior and aid in the selection of appropriate models, ages, sexes and outcome measures for preclinical trials. In this study, we characterize the phenotype of male and female mice containing the early truncating MeCP2 R168X nonsense point mutation, one of the most common in RTT individuals, and compare the phenotypes to Mecp2 null mutants. Mecp2^R168X mutants mirror many clinical features of RTT. Mecp2^R168X/y males exhibit impaired motor and cognitive function and reduced anxiety. The behavioral phenotype is less severe and with later onset in Mecp2^R168X/+ females. Seizures were noted in 3.7% of Mecp2^R168X mutant females. The phenotype in Mecp2^R168X/y mutant males is remarkably similar to our previous characterizations of Mecp2 null males, whereas Mecp2^R168X/+ females exhibit a number of phenotypic differences from females heterozygous for a null Mecp2 mutation. This study describes a number of highly robust behavioral paradigms that can be used in preclinical drug trials and underscores the importance of including Mecp2 mutant females in preclinical studies .     

Different effects of neuropeptide Y on proliferation of vascular smooth muscle cells via regulation of Geminin

Rett syndrome (RTT) is a neurodevelopmental disorder, mainly affecting females, which is associated to a mutation on the methyl-CpG-binding protein 2 gene. In the pathogenesis and progression of classic RTT, red blood cell (RBC) morphology has been shown to be an important biosensor for redox imbalance and chronic hypoxemia. Here we have evaluated the impact of oxidation and redox imbalance on several functional properties of RTT erythrocytes. In particular, we report for the first time a stopped-flow measurement of the kinetics of oxygen release by RBCs and the analysis of the intrinsic affinity of the hemoglobin (Hb). According to our experimental approach, RBCs from RTT patients do not show any intrinsic difference with respect to those from healthy controls neither in Hb’s oxygen-binding affinity nor in O₂ exchange processes at 37 °C. Therefore, these factors do not contribute to the observed alteration of the respiratory function in RTT patients. Moreover, the energy metabolism of RBCs, from both RTT patients and controls, was evaluated by ion-pairing HPLC method and related to the level of malondialdehyde and to the oxidative radical scavenging capacity of red cells. Results have clearly confirmed significant alterations in antioxidant defense capability, adding important informations concerning the high-energy compound levels in RBCs of RTT subjects, underlying possible correlations with inflammatory tissue alterations.     

Association by guilt: identification of DLX5 as a target for MeCP2 provides a molecular link between genomic imprinting and Rett syndrome

Rett syndrome (RTT) is an X-linked dominant neurodevelopmental disorder affecting almost exclusively girls. Although mutations in methyl-CpG-binding protein (MeCP2) are known to be associated with RTT, gene expression patterns are not significantly altered in MeCP2-deficient cells. A recent study1 identified MeCP2-mediated histone modification and formation of a higher-order chromatin loop structure specifically associated with silent chromatin at the Dlx5-Dlx6 locus in normal cells, and its absence thereof in RTT patients. This altered expression of Dlx5 through loss of silent chromatin loop formation provides a molecular mechanism underlying RTT and proposes a novel role for MeCP2 in chromatin organization and imprinting.     

Oxidative stress,serotonergic changes and decreased ultrasonic vocalizations in a mouse model of Smith–Lemli–Opitz syndrome

Smith–Lemli–Opitz syndrome is an inherited monogenic disorder in which mutations to the 7‐dehydrocholesterol (7‐DHC) reductase (Dhcr7) gene lead to deficits in cholesterol synthesis. As a result, many patients suffer from gross physiological and neurological deficits. The purpose of this study was to identify a potential abnormal behavioral phenotype in a compound mutant mouse model for Smith–Lemli–Opitz disease (Dhcr7 ^{Δ3 –5/ T93M} ) to further validate the model and to provide potential targets for future therapeutic interventions. We also sought to identify some of the underlying changes in brain function that may be responsible for behavioral differences among groups. The Dhcr7 compound mutant mice were smaller than their single mutant littermates. Both single and compound heterozygous mice made fewer ultrasonic vocalizations when separated from the dam, which may suggest a communication deficit in these animals. Striking increases of the highly oxidizable 7‐DHC were observed in the compound mutant mice. 7‐Dehydrocholesterol is the precursor to cholesterol and builds up because of decreased function of the mutated Dhcr7 enzyme. Additionally, several differences were noted in the serotonergic system including increased expression of the serotonin transporter and increased uptake of serotonin by isolated synaptosomes. We propose that changes to the oxidative environment during development can have a significant impact on the development of serotonergic function and that this contributes to behavioral differences observed in the mutant mice.     

PIRIN2 stabilizes cysteine protease XCP2 and increases susceptibility to the vascular pathogen Ralstonia solanacearum in Arabidopsis

PIRIN (PRN) is a member of the functionally diverse cupin protein superfamily. There are four members of the Arabidopsis thaliana PRN family, but the roles of these proteins are largely unknown. Here we describe a function of the Arabidopsis PIRIN2 (PRN2) that is related to susceptibility to the bacterial plant pathogen Ralstonia solanacearum. Two prn2 mutant alleles displayed decreased disease development and bacterial growth in response to R.  solanacearum infection. We elucidated the underlying molecular mechanism by analyzing PRN2 interactions with the papain‐like cysteine proteases (PLCPs) XCP2, RD21A, and RD21B, all of which bound to PRN2 in yeast two‐hybrid assays and in Arabidopsis protoplast co‐immunoprecipitation assays. We show that XCP2 is stabilized by PRN2 through inhibition of its autolysis on the basis of PLCP activity profiling assays and enzymatic assays with recombinant protein. The stabilization of XCP2 by PRN2 was also confirmed in planta. Like prn2 mutants, an xcp2 single knockout mutant and xcp2 prn2 double knockout mutant displayed decreased susceptibility to R. solanacearum, suggesting that stabilization of XCP2 by PRN2 underlies susceptibility to R. solanacearum in Arabidopsis.