Dataset Information

Splicing Mutations Impairing CDKL5 Expression and Activity Can be Efficiently Rescued by U1snRNA-Based Therapy.

ABSTRACT: Mutations in the CDKL5 gene lead to an incurable rare neurological condition characterized by the onset of seizures in the first weeks of life and severe intellectual disability. Replacement gene or protein therapies could represent intriguing options, however, their application may be inhibited by the recent demonstration that CDKL5 is dosage sensitive. Conversely, correction approaches acting on pre-mRNA splicing would preserve CDKL5 physiological regulation. Since ~15% of CDKL5 pathogenic mutations are candidates to affect splicing, we evaluated the capability of variants of the spliceosomal U1 small nuclear RNA (U1snRNA) to correct mutations affecting +1 and +5 nucleotides at the 5' donor splice site and predicted to cause exon skipping. Our results show that CDKL5 minigene variants expressed in mammalian cells are a valid approach to assess CDKL5 splicing pattern. The expression of engineered U1snRNA effectively rescued mutations at +5 but not at the +1 nucleotides. Importantly, we proved that U1snRNA-mediated splicing correction fully restores CDKL5 protein synthesis, subcellular distribution and kinase activity. Eventually, by correcting aberrant splicing of an exogenously expressed splicing-competent CDKL5 transgene, we provided insights on the morphological rescue of CDKL5 null neurons, reporting the first proof-of-concept of the therapeutic value of U1snRNA-mediated CDKL5 splicing correction.

SUBMITTER: Balestra D

PROVIDER: S-EPMC6747535 | biostudies-literature | 2019 Aug

REPOSITORIES: biostudies-literature

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Splicing Mutations Impairing CDKL5 Expression and Activity Can be Efficiently Rescued by U1snRNA-Based Therapy.

Balestra Dario D Giorgio Domenico D Bizzotto Matteo M Fazzari Maria M Ben Zeev Bruria B Pinotti Mirko M Landsberger Nicoletta N Frasca Angelisa A

International journal of molecular sciences 20190824 17

Mutations in the CDKL5 gene lead to an incurable rare neurological condition characterized by the onset of seizures in the first weeks of life and severe intellectual disability. Replacement gene or protein therapies could represent intriguing options, however, their application may be inhibited by the recent demonstration that CDKL5 is dosage sensitive. Conversely, correction approaches acting on pre-mRNA splicing would preserve CDKL5 physiological regulation. Since ~15% of ...[more]

PMID: 31450582

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Project description:Cyclin-dependent kinase-like 5 (CDKL5) deficiency disorder (CDD) is a severe X-linked neurodevelopmental encephalopathy caused by mutations in the CDKL5 gene and characterized by early-onset epilepsy and intellectual and motor impairments. No cure is currently available for CDD patients, as limited knowledge of the pathology has hindered the development of therapeutics. Cdkl5 knockout (KO) mouse models, recently created to investigate the role of CDKL5 in the etiology of CDD, recapitulate various features of the disorder. Previous studies have shown alterations in synaptic plasticity and dendritic pattern in the cerebral cortex and in the hippocampus, but the knowledge of the molecular substrates underlying these alterations is still limited. Here, we have examined for the first time synaptic function and plasticity, dendritic morphology, and signal transduction pathways in the perirhinal cortex (PRC) of this mouse model. Being interconnected with a wide range of cortical and subcortical structures and involved in various cognitive processes, PRC provides a very interesting framework for examining how CDKL5 mutation leads to deficits at the synapse, circuit, and behavioral level. We found that long-term potentiation (LTP) was impaired, and that the TrkB/PLCγ1 pathway could be mechanistically involved in this alteration. PRC neurons in mutant mice showed a reduction in dendritic length, dendritic branches, PSD-95-positive puncta, GluA2-AMPA receptor levels, and spine density and maturation. These functional and structural deficits were associated with impairment in visual recognition memory. Interestingly, an in vivo treatment with a TrkB agonist (the 7,8-DHF prodrug R13) to trigger the TrkB/PLCγ1 pathway rescued defective LTP, dendritic pattern, PSD-95 and GluA2-AMPA receptor levels, and restored visual recognition memory in Cdkl5 KO mice. Present findings demonstrate a critical role of TrkB signaling in the synaptic development alterations due to CDKL5 mutation, and suggest the possibility of TrkB-targeted pharmacological interventions.

Dataset Information

Splicing Mutations Impairing CDKL5 Expression and Activity Can be Efficiently Rescued by U1snRNA-Based Therapy.

Publications

Splicing Mutations Impairing CDKL5 Expression and Activity Can be Efficiently Rescued by U1snRNA-Based Therapy.

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