Long-term muscle-specific overexpression of DOK7 in mice using AAV9-tMCK-DOK7.

Neuromuscular junction (NMJ) dysfunction underlies several diseases, including congenital myasthenic syndromes (CMSs) and motor neuron disease (MND). Molecular pathways governing NMJ stability are therefore of interest from both biological and therapeutic perspectives. Muscle-specific kinase (MuSK) is necessary for the formation and maintenance of post-synaptic elements of the NMJ, and downstream of tyrosine kinases 7 (DOK7) is crucial for activation of the MuSK pathway. Overexpression of DOK7 using AAV9 has been shown to ameliorate neuromuscular pathology in pre-clinical disease models of CMS and MND. However, long-term consequences of DOK7 expression have been sparsely investigated and targeted overexpression of DOK7 in skeletal muscle yet to be established. Here, we developed and characterized a novel AAV9-DOK7 facilitating forced expression of DOK7 under a skeletal muscle-specific promoter. AAV9-tMCK-DOK7 was systemically delivered to newborn mice that were monitored over 6 months. DOK7 overexpression was restricted to skeletal muscles. Body weight, blood biochemistry, and histopathological assessments were unaffected by AAV9-tMCK-DOK7 treatment. In contrast, forced expression of DOK7 resulted in enlargement of both the pre- and post-synaptic components of the NMJ, without causing denervation. We conclude that muscle-specific DOK7 overexpression can be achieved in a safe manner, with the capacity to target NMJs in vivo.

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.

Convergent cerebrospinal fluid proteomes and metabolic ontologies in humans and animal models of Rett syndrome.

MECP2 loss-of-function mutations cause Rett syndrome, a neurodevelopmental disorder resulting from a disrupted brain transcriptome. How these transcriptional defects are decoded into a disease proteome remains unknown. We studied the proteome of Rett cerebrospinal fluid (CSF) to identify consensus Rett proteome and ontologies shared across three species. Rett CSF proteomes enriched proteins annotated to HDL lipoproteins, complement, mitochondria, citrate/pyruvate metabolism, synapse compartments, and the neurosecretory protein VGF. We used shared Rett ontologies to select analytes for orthogonal quantification and functional validation. VGF and ontologically selected CSF proteins had genotypic discriminatory capacity as determined by receiver operating characteristic analysis in Mecp2 -/y and Mecp2 -/+ . Differentially expressed CSF proteins distinguished Rett from a related neurodevelopmental disorder, CDKL5 deficiency disorder. We propose that Mecp2 mutant CSF proteomes and ontologies inform putative mechanisms and biomarkers of disease. We suggest that Rett syndrome results from synapse and metabolism dysfunction.

A kinome-wide screen identifies a CDKL5-SOX9 regulatory axis in epithelial cell death and kidney injury.

Renal tubular epithelial cells (RTECs) perform the essential function of maintaining the constancy of body fluid composition and volume. Toxic, inflammatory, or hypoxic-insults to RTECs can cause systemic fluid imbalance, electrolyte abnormalities and metabolic waste accumulation- manifesting as acute kidney injury (AKI), a common disorder associated with adverse long-term sequelae and high mortality. Here we report the results of a kinome-wide RNAi screen for cellular pathways involved in AKI-associated RTEC-dysfunction and cell death. Our screen and validation studies reveal an essential role of Cdkl5-kinase in RTEC cell death. In mouse models, genetic or pharmacological Cdkl5 inhibition mitigates nephrotoxic and ischemia-associated AKI. We propose that Cdkl5 is a stress-responsive kinase that promotes renal injury in part through phosphorylation-dependent suppression of pro-survival transcription regulator Sox9. These findings reveal a surprising non-neuronal function of Cdkl5, identify a pathogenic Cdkl5-Sox9 axis in epithelial cell-death, and support CDKL5 antagonism as a therapeutic approach for AKI.

Neurophysiological Signatures of Motor Impairment in Patients with Rett Syndrome.

OBJECTIVE: Rett syndrome (RTT) is an X-linked dominant neurodevelopmental disorder due to pathogenic mutations in the MECP2 gene. Motor impairment constitutes the core diagnostic feature of RTT. Preclinical studies have consistently demonstrated alteration of excitation/inhibition (E/I) balance and aberrant synaptic plasticity at the cortical level. We aimed to understand neurobiological mechanisms underlying motor deficit by assessing in vivo synaptic plasticity and E/I balance in the primary motor cortex (M1). METHODS: In 14 patients with typical RTT, 9 epilepsy control patients, and 11 healthy controls, we applied paired-pulse transcranial magnetic stimulation (TMS) protocols to evaluate the excitation index, a biomarker reflecting the contribution of inhibitory and facilitatory circuits in M1. Intermittent TMS-theta burst stimulation was used to probe long-term potentiation (LTP)-like plasticity in M1. Motor impairment, assessed by ad hoc clinical scales, was correlated with neurophysiological metrics. RESULTS: RTT patients displayed a significant increase of the excitation index (p = 0.003), as demonstrated by the reduction of short-interval intracortical inhibition and increase of intracortical facilitation, suggesting a shift toward cortical excitation likely due to GABAergic dysfunction. Impairment of inhibitory circuits was also confirmed by the reduction of long-interval intracortical inhibition (p = 0.002). LTP-like plasticity in M1 was abolished (p = 0.008) and scaled with motor disability (all p = 0.003). INTERPRETATION: TMS is a method that can be used to assess cortical motor function in RTT patients. Our findings support the introduction of TMS measures in clinical and research settings to monitor the progression of motor deficit and response to treatment. ANN NEUROL 2020;87:763-773.

Inhibition of Piezo1 attenuates demyelination in the central nervous system.

Piezo1 is a mechanosensitive ion channel that facilitates the translation of extracellular mechanical cues to intracellular molecular signaling cascades through a process termed, mechanotransduction. In the central nervous system (CNS), mechanically gated ion channels are important regulators of neurodevelopmental processes such as axon guidance, neural stem cell differentiation, and myelination of axons by oligodendrocytes. Here, we present evidence that pharmacologically mediated overactivation of Piezo1 channels negatively regulates CNS myelination. Moreover, we found that the peptide GsMTx4, an antagonist of mechanosensitive cation channels such as Piezo1, is neuroprotective and prevents chemically induced demyelination. In contrast, the positive modulator of Piezo1 channel opening, Yoda-1, induces demyelination and neuronal damage. Using an ex vivo murine-derived organotypic cerebellar slice culture model, we demonstrate that GsMTx4 attenuates demyelination induced by the cytotoxic lipid, psychosine. Importantly, we confirmed the potential therapeutic effects of GsMTx4 peptide in vivo by co-administering it with lysophosphatidylcholine (LPC), via stereotactic injection, into the cerebral cortex of adult mice. GsMTx4 prevented both demyelination and neuronal damage usually caused by the intracortical injection of LPC in vivo; a well-characterized model of focal demyelination. GsMTx4 also attenuated both LPC-induced astrocyte toxicity and microglial reactivity within the lesion core. Overall, our data suggest that pharmacological activation of Piezo1 channels induces demyelination and that inhibition of mechanosensitive channels, using GsMTx4, may alleviate the secondary progressive neurodegeneration often present in the latter stages of demyelinating diseases.

Differential Dependence of GABAergic and Glutamatergic Neurons on Glia for the Establishment of Synaptic Transmission.

In the mammalian cortex, GABAergic and glutamatergic neurons represent 2 major neuronal classes, which establish inhibitory and excitatory synapses, respectively. Despite differences in their anatomy, physiology and developmental origin, both cell types require support from glial cells, particularly astrocytes, for their growth and survival. Recent experiments indicate that glutamatergic neurons also depend on astrocytes for synapse formation. However, it is not clear if the same holds true for GABAergic neurons. By studying highly pure GABAergic cell cultures, established through fluorescent activated cell sorting, we find that purified GABAergic neurons are smaller and have reduced survival, nevertheless they establish robust synaptic transmission in the absence of glia. Support from glial cells reverses morphological and survival deficits, but does little to alter synaptic transmission. In contrast, in cultures of purified glutamatergic neurons, morphological development, survival and synaptic transmission are collectively dependent on glial support. Thus, our results demonstrate a fundamental difference in the way GABAergic and glutamatergic neurons depend on glia for the establishment of synaptic transmission, a finding that has important implications for our understanding of how neuronal networks develop.

CDKL5 variants: Improving our understanding of a rare neurologic disorder.

OBJECTIVE: To provide new insights into the interpretation of genetic variants in a rare neurologic disorder, CDKL5 deficiency, in the contexts of population sequencing data and an updated characterization of the CDKL5 gene. METHODS: We analyzed all known potentially pathogenic CDKL5 variants by combining data from large-scale population sequencing studies with CDKL5 variants from new and all available clinical cohorts and combined this with computational methods to predict pathogenicity. RESULTS: The study has identified several variants that can be reclassified as benign or likely benign. With the addition of novel CDKL5 variants, we confirm that pathogenic missense variants cluster in the catalytic domain of CDKL5 and reclassify a purported missense variant as having a splicing consequence. We provide further evidence that missense variants in the final 3 exons are likely to be benign and not important to disease pathology. We also describe benign splicing and nonsense variants within these exons, suggesting that isoform hCDKL5_5 is likely to have little or no neurologic significance. We also use the available data to make a preliminary estimate of minimum incidence of CDKL5 deficiency. CONCLUSIONS: These findings have implications for genetic diagnosis, providing evidence for the reclassification of specific variants previously thought to result in CDKL5 deficiency. Together, these analyses support the view that the predominant brain isoform in humans (hCDKL5_1) is crucial for normal neurodevelopment and that the catalytic domain is the primary functional domain.

Radically truncated MeCP2 rescues Rett syndrome-like neurological defects.

Heterozygous mutations in the X-linked MECP2 gene cause the neurological disorder Rett syndrome. The methyl-CpG-binding protein 2 (MeCP2) protein is an epigenetic reader whose binding to chromatin primarily depends on 5-methylcytosine. Functionally, MeCP2 has been implicated in several cellular processes on the basis of its reported interaction with more than 40 binding partners, including transcriptional co-repressors (for example, the NCoR/SMRT complex), transcriptional activators, RNA, chromatin remodellers, microRNA-processing proteins and splicing factors. Accordingly, MeCP2 has been cast as a multi-functional hub that integrates diverse processes that are essential in mature neurons. At odds with the concept of broad functionality, missense mutations that cause Rett syndrome are concentrated in two discrete clusters coinciding with interaction sites for partner macromolecules: the methyl-CpG binding domain and the NCoR/SMRT interaction domain. Here we test the hypothesis that the single dominant function of MeCP2 is to physically connect DNA with the NCoR/SMRT complex, by removing almost all amino-acid sequences except the methyl-CpG binding and NCoR/SMRT interaction domains. We find that mice expressing truncated MeCP2 lacking both the N- and C-terminal regions (approximately half of the native protein) are phenotypically near-normal; and those expressing a minimal MeCP2 additionally lacking a central domain survive for over one year with only mild symptoms. This minimal protein is able to prevent or reverse neurological symptoms when introduced into MeCP2-deficient mice by genetic activation or virus-mediated delivery to the brain. Thus, despite evolutionary conservation of the entire MeCP2 protein sequence, the DNA and co-repressor binding domains alone are sufficient to avoid Rett syndrome-like defects and may therefore have therapeutic utility.

Reduced axonal diameter of peripheral nerve fibers in a mouse model of Rett syndrome.

Rett syndrome (RTT) is a neurological disorder characterized by motor and cognitive impairment, autonomic dysfunction and a loss of purposeful hand skills. In the majority of cases, typical RTT is caused by de novo mutations in the X-linked gene, MECP2. Alterations in the structure and function of neurons within the central nervous system of RTT patients and Mecp2-null mouse models are well established. In contrast, few studies have investigated the effects of MeCP2-deficiency on peripheral nerves. In this study, we conducted detailed morphometric as well as functional analysis of the sciatic nerves of symptomatic adult female Mecp2+/- mice. We observed a significant reduction in the mean diameter of myelinated nerve fibers in Mecp2+/- mice. In myelinated fibers, mitochondrial densities per unit area of axoplasm were significantly altered in Mecp2+/- mice. However, conduction properties of the sciatic nerve of Mecp2 knockout mice were not different from control. These subtle changes in myelinated peripheral nerve fibers in heterozygous Mecp2 knockout mice could potentially explain some RTT phenotypes.

Improved MECP2 Gene Therapy Extends the Survival of MeCP2-Null Mice without Apparent Toxicity after Intracisternal Delivery.

Intravenous administration of adeno-associated virus serotype 9 (AAV9)/hMECP2 has been shown to extend the lifespan of Mecp2-/y mice, but this delivery route induces liver toxicity in wild-type (WT) mice. To reduce peripheral transgene expression, we explored the safety and efficacy of AAV9/hMECP2 injected into the cisterna magna (ICM). AAV9/hMECP2 (1 × 1012 viral genomes [vg]; ICM) extended Mecp2-/y survival but aggravated hindlimb clasping and abnormal gait phenotypes. In WT mice, 1 × 1012 vg of AAV9/hMECP2 induced clasping and abnormal gait. A lower dose mitigated these adverse phenotypes but failed to extend survival of Mecp2-/y mice. Thus, ICM delivery of this vector is impractical as a treatment for Rett syndrome (RTT). To improve the safety of MeCP2 gene therapy, the gene expression cassette was modified to include more endogenous regulatory elements believed to modulate MeCP2 expression in vivo. In Mecp2-/y mice, ICM injection of the modified vector extended lifespan and was well tolerated by the liver but did not rescue RTT behavioral phenotypes. In WT mice, these same doses of the modified vector had no adverse effects on survival or neurological phenotypes. In summary, we identified limitations of the original vector and demonstrated that an improved vector design extends Mecp2-/y survival, without apparent toxicity.

Development of a Novel AAV Gene Therapy Cassette with Improved Safety Features and Efficacy in a Mouse Model of Rett Syndrome.

Rett syndrome (RTT), caused by loss-of-function mutations in the MECP2 gene, is a neurological disorder characterized by severe impairment of motor and cognitive functions. The aim of this study was to investigate the impact of vector design, dosage, and delivery route on the efficacy and safety of gene augmentation therapy in mouse models of RTT. Our results show that AAV-mediated delivery of MECP2 to Mecp2 null mice by systemic administration, and utilizing a minimal endogenous promoter, was associated with a narrow therapeutic window and resulted in liver toxicity at higher doses. Lower doses of this vector significantly extended the survival of mice lacking MeCP2 or expressing a mutant T158M allele but had no impact on RTT-like neurological phenotypes. Modifying vector design by incorporating an extended Mecp2 promoter and additional regulatory 3' UTR elements significantly reduced hepatic toxicity after systemic administration. Moreover, direct cerebroventricular injection of this vector into neonatal Mecp2-null mice resulted in high brain transduction efficiency, increased survival and body weight, and an amelioration of RTT-like phenotypes. Our results show that controlling levels of MeCP2 expression in the liver is achievable through modification of the expression cassette. However, it also highlights the importance of achieving high brain transduction to impact the RTT-like phenotypes.

Clinical and biological progress over 50 years in Rett syndrome.

In the 50 years since Andreas Rett first described the syndrome that came to bear his name, and is now known to be caused by a mutation in the methyl-CpG-binding protein 2 (MECP2) gene, a compelling blend of astute clinical observations and clinical and laboratory research has substantially enhanced our understanding of this rare disorder. Here, we document the contributions of the early pioneers in Rett syndrome (RTT) research, and describe the evolution of knowledge in terms of diagnostic criteria, clinical variation, and the interplay with other Rett-related disorders. We provide a synthesis of what is known about the neurobiology of MeCP2, considering the lessons learned from both cell and animal models, and how they might inform future clinical trials. With a focus on the core criteria, we examine the relationships between genotype and clinical severity. We review current knowledge about the many comorbidities that occur in RTT, and how genotype may modify their presentation. We also acknowledge the important drivers that are accelerating this research programme, including the roles of research infrastructure, international collaboration and advocacy groups. Finally, we highlight the major milestones since 1966, and what they mean for the day-to-day lives of individuals with RTT and their families.

Characterisation of Cdkl5 transcript isoforms in rat.

CDKL5 deficiency is a severe neurological disorder caused by mutations in the X-linked Cyclin-Dependent Kinase-Like 5 gene (CDKL5). The predominant human CDKL5 brain isoform is a 9.7kb transcript comprised of 18 exons with a large 6.6kb 3'-untranslated region (UTR). Mammalian models of CDKL5 disorder are currently limited to mouse, and little is known about Cdkl5 in other organisms used to model neurodevelopmental disorders, such as rat. In this study we characterise, both bioinformatically and experimentally, the rat Cdkl5 gene structure and its associated transcript isoforms. New exonic regions, splice sites and UTRs are described, confirming the presence of four distinct transcript isoforms. The predominant isoform in the brain, which we name rCdkl5_1, is orthologous to the human hCDKL5_1 and mouse mCdkl5_1 isoforms and is the most highly expressed isoform across all brain regions tested. This updated gene model of Cdkl5 in rat provides a framework for studies into its protein products and provides a reference for the development of molecular therapies for testing in rat models of CDKL5 disorder.

Kynurenine pathway metabolism following prenatal KMO inhibition and in Mecp2+/- mice, using liquid chromatography-tandem mass spectrometry.

To quantify the full range of tryptophan metabolites along the kynurenine pathway, a liquid chromatography - tandem mass spectrometry method was developed and used to analyse brain extracts of rodents treated with the kynurenine-3-mono-oxygenase (KMO) inhibitor Ro61-8048 during pregnancy. There were significant increases in the levels of kynurenine, kynurenic acid, anthranilic acid and 3-hydroxy-kynurenine (3-HK) in the maternal brain after 5 h but not 24 h, while the embryos exhibited high levels of kynurenine, kynurenic acid and anthranilic acid after 5 h which were maintained at 24 h post-treatment. At 24 h there was also a strong trend to an increase in quinolinic acid levels (P = 0.055). No significant changes were observed in any of the other kynurenine metabolites. The results confirm the marked increase in the accumulation of some neuroactive kynurenines when KMO is inhibited, and re-emphasise the potential importance of changes in anthranilic acid. The prolonged duration of metabolite accumulation in the embryo brains indicates a trapping of compounds within the embryonic CNS independently of maternal levels. When brains were examined from young mice heterozygous for the meCP2 gene - a potential model for Rett syndrome - no differences were noted from control mice, suggesting that the proposed roles for kynurenines in autism spectrum disorder are not relevant to Rett syndrome, supporting its recognition as a distinct, independent, condition.

Characterisation of CDKL5 Transcript Isoforms in Human and Mouse.

Mutations in the X-linked Cyclin-Dependent Kinase-Like 5 gene (CDKL5) cause early onset infantile spasms and subsequent severe developmental delay in affected children. Deleterious mutations have been reported to occur throughout the CDKL5 coding region. Several studies point to a complex CDKL5 gene structure in terms of exon usage and transcript expression. Improvements in molecular diagnosis and more extensive research into the neurobiology of CDKL5 and pathophysiology of CDKL5 disorders necessitate an updated analysis of the gene. In this study, we have analysed human and mouse CDKL5 transcript patterns both bioinformatically and experimentally. We have characterised the predominant brain isoform of CDKL5, a 9.7 kb transcript comprised of 18 exons with a large 6.6 kb 3'-untranslated region (UTR), which we name hCDKL5_1. In addition we describe new exonic regions and a range of novel splice and UTR isoforms. This has enabled the description of an updated gene model in both species and a standardised nomenclature system for CDKL5 transcripts. Profiling revealed tissue- and brain development stage-specific differences in expression between transcript isoforms. These findings provide an essential backdrop for the diagnosis of CDKL5-related disorders, for investigations into the basic biology of this gene and its protein products, and for the rational design of gene-based and molecular therapies for these disorders.

Exclusive expression of MeCP2 in the nervous system distinguishes between brain and peripheral Rett syndrome-like phenotypes.

Rett syndrome (RTT) is a severe genetic disorder resulting from mutations in the X-linked MECP2 gene. MeCP2 protein is highly expressed in the nervous system and deficiency in the mouse central nervous system alone recapitulates many features of the disorder. This suggests that RTT is primarily a neurological disorder, although the protein is reportedly widely expressed throughout the body. To determine whether aspects of the RTT phenotype that originate in non-neuronal tissues might have been overlooked, we generated mice in which Mecp2 remains at near normal levels in the nervous system, but is severely depleted elsewhere. Comparison of these mice with wild type and globally MeCP2-deficient mice showed that the majority of RTT-associated behavioural, sensorimotor, gait and autonomic (respiratory and cardiac) phenotypes are absent. Specific peripheral phenotypes were observed, however, most notably hypo-activity, exercise fatigue and bone abnormalities. Our results confirm that the brain should be the primary target for potential RTT therapies, but also strongly suggest that some less extreme but clinically significant aspects of the disorder arise independently of defects in the nervous system.

Label-free imaging of thick tissue at 1550 nm using a femtosecond optical parametric generator.

We have developed a simple wavelength-tunable optical parametric generator (OPG), emitting broadband ultrashort pulses with peak wavelengths at 1530-1790 nm, for nonlinear label-free microscopy. The OPG consists of a periodically poled lithium niobate crystal, pumped at 1064 nm by a ultrafast Yb:fiber laser with high pulse energy. We demonstrate that this OPG can be used for label-free imaging, by third-harmonic generation, of nuclei of brain cells and blood vessels in a >150 µm thick brain tissue section, with very little decay of intensity with imaging depth and no visible damage to the tissue at an incident average power of 15 mW.