Rett syndrome (RTT) is a severe X-linked neurodevelopmental disorder that is primarily caused by mutations in the methyl CpG binding protein 2 gene (MECP2). RTT is the second most prevalent genetic ...cause of intellectual disability in girls, and there is currently no cure for the disease. We have previously shown that gene therapy using a self-complementary AAV9 viral vector expressing a codon-optimized Mecp2 version (AAV9-MCO) significantly improved symptoms and increased survival in male Mecp2-deficient mice. Here, we pursued our studies and investigated the safety and efficacy of long-term gene therapy in the genetically relevant RTT mouse model: the heterozygous (HET) Mecp2 deficient female mouse. These mice were injected with the AAV9-MCO vector through the tail vein and an array of behavioral tests was performed. At 16- and 30-weeks post-injection, this treatment was able to rescue apneas and improved the spontaneous locomotor deficits and circadian locomotor activity in Mecp2 HET mice treated with AAV9-MCO at a dose of 5 × 1011 vg/mouse.
To examine whether a higher dose of vector could result in increased improvements, we injected Mecp2 HET mice with a higher MCO vector dose (1012 vg/mouse), which resulted in some severe, sometimes lethal, side effects. In order to confirm these effects, a new cohort of Mecp2 HET mice were administered increasing doses of MCO vector (1011, 5 × 1011 and 1012 vg/mouse). Again, two weeks after vector administration, some Mecp2 HET mice were found dead while others displayed severe side effects and had to be euthanized. These deleterious effects were not observed in Mecp2 HET mice injected with a high dose of AAV9-GFP and were directly proportionate to vector dosage (0, 23 or 54% mortality at an AAV9-MCO dose of 1011, 5 × 1011, 1012 vg/mouse, respectively), and no such lethality was observed in wild-type (WT) mice.
In the Mecp2 HET mice treated with the high and medium AAV9-MCO doses, blood chemistry analysis and post-mortem histology showed liver damage with drastically elevated levels of liver transaminases and disorganized liver architecture. Apoptosis was confirmed by the presence of TUNEL- and cleaved-caspase 3-positive cells in the Mecp2 HET mice treated with the higher doses of AAV9-MCO. We then studied the involvement of the unfolded protein response (UPR) in triggering apoptosis since it can be activated by AAV vectors. Increased expression of the C/EBP homologous protein (CHOP), one of UPR downstream effectors, was confirmed in Mecp2 HET mice after vector administration.
The toxic reaction seen in some treated mice indicates that, although gene therapy for RTT improved breathing deficits observed in Mecp2 HET mice, further studies are needed to better understand the underlying mechanisms and caution must be exercised before similar attempts are undertaken in female Rett patients.
•Treatment of female RTT mice with AAV9-MCO only rescued apneas occurrence.•Using a higher vector dose caused severe off target effects specific to RTT mice.•Post-mortem analysis revealed damaged liver with high CHOP levels and TUNEL expression.•Off target Mecp2 expression may have led to UPR activation and subsequent apoptosis.•These contrasting results underscore the need for additional work to limit side effects.
The central pattern generator (CPG) for locomotion is a set of pacemaker neurons endowed with inherent bursting driven by the persistent sodium current (INaP). How they proceed to regulate the ...locomotor rhythm remained unknown. Here, in neonatal rodents, we identified a persistent potassium current critical in regulating pacemakers and locomotion speed. This current recapitulates features of the M-current (IM): a subthreshold noninactivating outward current blocked by 10,10-bis(4-pyridinylmethyl)-9(10H)-anthracenone dihydrochloride (XE991) and enhanced by N-(2-chloro-5-pyrimidinyl)-3,4-difluorobenzamide (ICA73). Immunostaining and mutant mice highlight an important role of Kv7.2-containing channels in mediating IM. Pharmacological modulation of IM regulates the emergence and the frequency regime of both pacemaker and CPG activities and controls the speed of locomotion. Computational models captured these results and showed how an interplay between IM and INaP endows the locomotor CPG with rhythmogenic properties. Overall, this study provides fundamental insights into how IM and INaP work in tandem to set the speed of locomotion.
N-methyl-D-aspartate (NMDA) receptors mediate excitatory neurotransmission in the mammalian brain. Two glycine-binding NR1 subunits and two glutamate-binding NR2 subunits each form highly ...Ca2+-permeable cation channels which are blocked by extracellular Mg2+ in a voltage-dependent manner. Either GRIN2B or GRIN2A, encoding the NMDA receptor subunits NR2B and NR2A, was found to be disrupted by chromosome translocation breakpoints in individuals with mental retardation and/or epilepsy. Sequencing of GRIN2B in 468 individuals with mental retardation revealed four de novo mutations: a frameshift, a missense and two splice-site mutations. In another cohort of 127 individuals with idiopathic epilepsy and/or mental retardation, we discovered a GRIN2A nonsense mutation in a three-generation family. In a girl with early-onset epileptic encephalopathy, we identified the de novo GRIN2A mutation c.1845C>A predicting the amino acid substitution p.N615K. Analysis of NR1-NR2AN615K (NR2A subunit with the p.N615K alteration) receptor currents revealed a loss of the Mg2+ block and a decrease in Ca2+ permeability. Our findings suggest that disturbances in the neuronal electrophysiological balance during development result in variable neurological phenotypes depending on which NR2 subunit of NMDA receptors is affected.
Abstract Rett syndrome (RTT) is a severe X-linked neurodevelopmental disorder that is primarily caused by mutations in the methyl CpG binding protein 2 gene ( MECP2 ). RTT is the second most ...prevalent cause of intellectual disability in girls and there is currently no cure for the disease. The finding that the deficits caused by the loss of Mecp2 are reversible in the mouse has bolstered interest in gene therapy as a cure for RTT. In order to assess the feasibility of gene therapy in a RTT mouse model, and in keeping with translational goals, we investigated the efficacy of a self-complementary AAV9 vector expressing a codon-optimized version of Mecp2 (AAV9-MCO) delivered via a systemic approach in early symptomatic Mecp2 -deficient (KO) mice. Our results show that AAV9-MCO administered at a dose of 2 × 10 11 viral genome (vg)/mouse was able to significantly increase survival and weight gain, and delay the occurrence of behavioral deficits. Apneas, which are one of the core RTT breathing deficits, were significantly decreased to WT levels in Mecp2 KO mice after AAV9-MCO administration. Semi-quantitative analysis showed that AAV9-MCO administration in Mecp2 KO mice resulted in 10 to 20% Mecp2 immunopositive cells compared to WT animals, with the highest Mecp2 expression found in midbrain regions known to regulate cardio-respiratory functions. In addition, we also found a cell autonomous increase in tyrosine hydroxylase levels in the A1C1 and A2C2 catecholaminergic Mecp2 + neurons in treated Mecp2 KO mice, which may partly explain the beneficial effect of AAV9-MCO administration on apneas occurrence.
Proper brain functioning requires a fine-tuning between excitatory and inhibitory neurotransmission, a balance maintained through the regulation and release of glutamate and GABA. Rett syndrome (RTT) ...is a rare genetic disorder caused by mutations in the methyl-CpG binding protein 2 (MECP2) gene affecting the postnatal brain development. Dysfunctions in the GABAergic and glutamatergic systems have been implicated in the neuropathology of RTT and a disruption of the balance between excitation and inhibition, together with a perturbation of the electrophysiological properties of GABA and glutamate neurons, were reported in the brain of the Mecp2-deficient mouse. However, to date, the extent and the nature of the GABA/glutamate deficit affecting the Mecp2-deficient mouse brain are unclear. In order to better characterize these deficits, we simultaneously analyzed the GABA and glutamate levels in Mecp2-deficient mice at 2 different ages (P35 and P55) and in several brain areas. We used a multilevel approach including the quantification of GABA and glutamate levels, as well as the quantification of the mRNA and protein expression levels of key genes involved in the GABAergic and glutamatergic pathways. Our results show that Mecp2-deficient mice displayed regional- and age-dependent variations in the GABA pathway and, to a lesser extent, in the glutamate pathway. The implication of the GABA pathway in the RTT neuropathology was further confirmed using an in vivo treatment with a GABA reuptake inhibitor that significantly improved the lifespan of Mecp2-deficient mice. Our results confirm that RTT mouse present a deficit in the GABAergic pathway and suggest that GABAergic modulators could be interesting therapeutic agents for this severe neurological disorder.
Variants in the WD repeat 45 (WDR45) gene in human Xp11.23 have recently been identified in patients suffering from neurodegeneration with brain iron accumulation, a genetically and phenotypically ...heterogeneous condition. WDR45 variants cause a childhood-onset encephalopathy accompanied by neurodegeneration in adulthood and iron accumulation in the basal ganglia. They have been almost exclusively found in females, and male lethality was suggested. Here we describe a male patient suffering from a severe and early neurological phenotype, initially presenting early-onset epileptic spasms in clusters associated with an abnormal interictal electroencephalography showing slow background activity, large amplitude asynchronous spikes and abnormal neurological development. This patient is a carrier of a 19.9-kb microdeletion in Xp11.23 containing three genes, including WDR45. These findings reveal that males with WDR45 deletions are viable, and can present with early-onset epileptic encephalopathy without brain iron accumulation.
Abstract Mutations in the KCNQ2 gene encoding the voltage-dependent potassium M channel Kv7.2 subunit cause either benign epilepsy or early onset epileptic encephalopathy (EOEE). It has been proposed ...that the disease severity rests on the inhibitory impact of mutations on M current density. Here, we have analyzed the phenotype of 7 patients carrying the p.A294V mutation located on the S6 segment of the Kv7.2 pore domain (Kv7.2A294V ). We investigated the functional and subcellular consequences of this mutation and compared it to another mutation (Kv7.2A294G ) associated with a benign epilepsy and affecting the same residue. We report that all the patients carrying the p.A294V mutation presented the clinical and EEG characteristics of EOEE. In CHO cells, the total expression of Kv7.2A294V alone, assessed by western blotting, was only 20% compared to wild-type. No measurable current was recorded in CHO cells expressing Kv7.2A294V channel alone. Although the total Kv7.2A294V expression was rescued to wild-type levels in cells co-expressing the Kv7.3 subunit, the global current density was still reduced by 83% compared to wild-type heteromeric channel. In a configuration mimicking the patients' heterozygous genotype i.e., Kv7.2A294V /Kv7.2/Kv7.3, the global current density was reduced by 30%. In contrast to Kv7.2A294V , the current density of homomeric Kv7.2A294G was not significantly changed compared to wild-type Kv7.2. However, the current density of Kv7.2A294G /Kv7.2/Kv7.3 and Kv7.2A294G /Kv7.3 channels were reduced by 30% and 50% respectively, compared to wild-type Kv7.2/Kv7.3. In neurons, the p.A294V mutation induced a mislocalization of heteromeric mutant channels to the somato-dendritic compartment, while the p.A294G mutation did not affect the localization of the heteromeric channels to the axon initial segment. We conclude that this position is a hotspot of mutation that can give rise to a severe or a benign epilepsy. The p.A294V mutation does not exert a dominant-negative effect on wild-type subunits but alters the preferential axonal targeting of heteromeric Kv7 channels. Our data suggest that the disease severity is not necessarily a consequence of a strong inhibition of M current and that additional mechanisms such as abnormal subcellular distribution of Kv7 channels could be determinant.
Developmental and epileptic encephalopathies (DEE) are chronic neurological conditions where epileptic activity contributes to the progressive disruption of brain function, frequently leading to ...impaired motor, cognitive and sensory development.
The present study reports a clinical investigation and a molecular analysis by Next Generation Sequencing (NGS) of a large consanguineous family comprising several cases of developmental and epileptic encephalopathy. Bioinformatic prediction and molecular docking analysis were also carried out.
The majority of patients in our studied family had severe developmental impairments, early-onset seizures, brain malformations such as cortical atrophy and microcephaly, developmental delays and intellectual disabilities. The molecular investigations revealed a novel homozygous variant c.1411G>A (p.Gly471Arg) in the GRM7 gene which was segregating with the disease in the family. Bioinformatic tools predicted its pathogenicity and docking analysis revealed its potential effects on mGlu7 protein binding to its ligand.
Our results contribute to a better understanding of the impact of GRM7 variants for the newly described associated phenotype.
Mutations in the X-linked
gene are responsible for Rett syndrome (RTT), a severe neurological disorder. MECP2 is a transcriptional modulator that finely regulates the expression of many genes, ...specifically in the central nervous system. Several studies have functionally linked the loss of MECP2 in astrocytes to the appearance and progression of the RTT phenotype in a non-cell autonomous manner and mechanisms are still unknown. Here, we used primary astroglial cells from
-deficient (KO) pups to identify deregulated secreted proteins. Using a differential quantitative proteomic analysis, twenty-nine proteins have been identified and four were confirmed by Western blotting with new samples as significantly deregulated. To further verify the functional relevance of these proteins in RTT, we tested their effects on the dendritic morphology of primary cortical neurons from
KO mice that are known to display shorter dendritic processes. Using Sholl analysis, we found that incubation with Lcn2 or Lgals3 for 48 h was able to significantly increase the dendritic arborization of
KO neurons. To our knowledge, this study, through secretomic analysis, is the first to identify astroglial secreted proteins involved in the neuronal RTT phenotype in vitro, which could open new therapeutic avenues for the treatment of Rett syndrome.
Mutations in the X‐linked MECP2 gene are responsible for Rett syndrome (RTT), a severe neurological disorder for which there is no treatment. Several studies have linked the loss of MeCP2 function to ...alterations of brain‐derived neurotrophic factor (BDNF) levels, but non‐specific overexpression of BDNF only partially improves the phenotype of Mecp2‐deficient mice. We and others have previously shown that huntingtin (HTT) scaffolds molecular motor complexes, transports BDNF‐containing vesicles, and is under‐expressed in Mecp2 knockout brains. Here, we demonstrate that promoting HTT phosphorylation at Ser421, either by a phospho‐mimetic mutation or inhibition of the phosphatase calcineurin, restores endogenous BDNF axonal transport in vitro in the corticostriatal pathway, increases striatal BDNF availability and synaptic connectivity in vivo, and improves the phenotype and the survival of Mecp2 knockout mice—even though treatments were initiated only after the mice had already developed symptoms. Stimulation of endogenous cellular pathways may thus be a promising approach for the treatment of RTT patients.
Synopsis
BDNF is one of the most prominent deregulated factors in Rett syndrome, a severe neurodevelopmental disorder. This study reveals that huntingtin phosphorylation stimulates BDNF axonal transport and improves many features in a Rett mouse model, suggesting a possible therapeutic approach.
Inducing HTT phosphorylation stimulates axonal transport of BDNF vesicles in Mecp2‐deficient neurons.
HTT phosphorylation in vivo stimulates BDNF release in the striatum, improving corticostriatal synapse homeostasis in Mecp2‐deficient mice.
Phospho‐mimetic mutation of HTT or treatment by the calcineurin inhibitor FK506 increases BDNF transport in mice.
FK506 improves the phenotype and survival of Mecp2‐deficient mice through huntingtin phosphorylation.
BDNF is one of the most prominent deregulated factors in Rett syndrome, a severe neurodevelopmental disorder. This study reveals that huntingtin phosphorylation stimulates BDNF axonal transport and improves many features in a Rett mouse model, suggesting a possible therapeutic approach.
PDF
