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Topoisomerase 1 (TOP1) relieves torsional stress in DNA during transcription and facilitates the expression of long (>100 kb) genes, many of which are important for neuronal functions. To evaluate how loss of Top1 affected neurons in vivo, we conditionally deleted (cKO) Top1 in postmitotic excitatory neurons in the mouse cerebral cortex and hippocampus. Top1 cKO neurons develop properly, but then show biased transcriptional downregulation of long genes, signs of DNA damage, neuroinflammation, increased poly(ADP-ribose) polymerase-1 (PARP1) activity, single-cell somatic mutations, and ultimately degeneration. Supplementation of nicotinamide adenine dinucleotide (NAD+) with nicotinamide riboside partially blocked neurodegeneration, and increased the lifespan of Top1 cKO mice by 30%. A reduction of p53 also partially rescued cortical neuron loss. While neurodegeneration was partially rescued, behavioral decline was not prevented. These data indicate that reducing neuronal loss is not sufficient to limit behavioral decline when TOP1 function is disrupted., Topoisomerase 1 (TOP1) relieves DNA torsional stress during transcription and facilitates the expression of long neuronal genes. Here we show that deletion of Top1 in excitatory neurons leads to early onset neurodegeneration that is partially dependent on p53/PARP1 activation and NAD+ depletion.

Angelman syndrome (AS) is a severe neurodevelopmental disorder caused by a mutation or deletion of the maternally inherited UBE3A allele. In neurons, the paternally inherited UBE3A allele is silenced in cis by a long non-coding RNA called UBE3A-ATS. Here, as part of a systematic screen, we found that Cas9 can be used to activate ('unsilence') paternal Ube3a in cultured mouse and human neurons when targeted to Snord115 genes, which are small nucleolar RNAs that are clustered in the 3′ region of Ube3a-ATS. A short Cas9 variant and guide RNA that target about 75 Snord115 genes were packaged into an adeno-associated virus and administered to a mouse model of AS during the embryonic and early postnatal stages, when the therapeutic benefit of restoring Ube3a is predicted to be greatest1,2. This early treatment unsilenced paternal Ube3a throughout the brain for at least 17 months and rescued anatomical and behavioural phenotypes in AS mice. Genomic integration of the adeno-associated virus vector into Cas9 target sites caused premature termination of Ube3a-ATS at the vector-derived polyA cassette, or when integrated in the reverse orientation, by transcriptional collision with the vector-derived Cas9 transcript. Our study shows that targeted genomic integration of a gene therapy vector can restore the function of paternally inherited UBE3A throughout life, providing a path towards a disease-modifying treatment for a syndromic neurodevelopmental disorder. Genomic integration of an adeno-associated virus vector in a mouse model of Angelman syndrome unsilences paternal Ube3a and rescues anatomical and behavioural phenotypes, suggesting a pathway towards the treatment of this neurodevelopmental disorder.

The cerebral cortex is built during embryonic neurogenesis, a period when excitatory neurons are generated from progenitors. Defects in neurogenesis can cause acute neurodevelopmental disorders, such as microcephaly (reduced brain size). Altered dosage of the 1q21.1 locus has been implicated in the etiology of neurodevelopmental phenotypes; however, the role of 1q21.1 genes in neurogenesis has remained elusive. Here, we show that haploinsufficiency forRbm8a, an exon junction complex (EJC) component within 1q21.1, causes severe microcephaly and defective neurogenesis in the mouse. At the onset of neurogenesis,Rbm8aregulates radial glia proliferation and prevents premature neuronal differentiation. ReducedRbm8alevels result in subsequent apoptosis of neurons, and to a lesser extent, radial glia. Hence, compared to control,Rbm8a-haploinsufficient brains have fewer progenitors and neurons, resulting in defective cortical lamination. To determine whether reciprocal dosage change ofRbm8aalters embryonic neurogenesis, we overexpressed humanRBM8Ain two animal models. Usingin uteroelectroporation of mouse neocortices as well as zebrafish models, we findRBM8Aoverexpression does not significantly perturb progenitor number or head size. Our findings demonstrate thatRbm8ais an essential neurogenesis regulator, and add to a growing literature highlighting roles for EJC components in cortical development and neurodevelopmental pathology. Our results indicate that disruption ofRBM8Amay contribute to neurodevelopmental phenotypes associated with proximal 1q21.1 microdeletions.

Embryonic development is a critical period during which neurons of the brain are generated and organized. In the developing cerebral cortex, this requires complex processes of neural progenitor proliferation, neuronal differentiation, and migration. Each step relies upon highly regulated control of gene expression. In particular, RNA splicing, stability, localization, and translation have emerged as key post-transcriptional regulatory nodes of mouse corticogenesis. Trans-regulators of RNA metabolism, including microRNAs (miRs) and RNA-binding proteins (RBPs), orchestrate diverse steps of cortical development. These trans-factors function either individually or cooperatively to influence RNAs, often of similar classes, termed RNA regulons. New technological advances raise the potential for an increasingly sophisticated understanding of post-transcriptional control in the developing neocortex. Many RNA-binding factors are also implicated in neurodevelopmental diseases of the cortex. Therefore, elucidating how RBPs and miRs converge to influence mRNA expression in progenitors and neurons will give valuable insights into mechanisms of cortical development and disease. WIREs Dev Biol 2018, 7:e290. doi: 10.1002/wdev.290 This article is categorized under: Gene Expression and Transcriptional Hierarchies > Regulatory RNA Nervous System Development > Vertebrates: Regional Development Adult Stem Cells, Tissue Renewal, and Regeneration > Stem Cells and Disease.

The exon junction complex (EJC) is a multiprotein complex integral to mRNA metabolism. Biochemistry and genetic studies have concluded that the EJC is composed of four core proteins, MAGOH, EIF4A3, RBM8A, and CASC3. Yet recent studies in Drosophila indicate divergent physiological functions for Barentsz, the mammalian Casc3 ortholog, raising the question as to whether CASC3 is a constitutive component of the EJC. This issue remains poorly understood, particularly in an in vivo mammalian context. We previously found that haploinsufficiency for Magoh, Eif4a3, or Rbm8a disrupts neuronal viability and neural progenitor proliferation, resulting in severe microcephaly. Here, we use two new Casc3 mouse alleles to demonstrate developmental phenotypes that sharply contrast those of other core EJC components. Homozygosity for either null or hypomorphic Casc3 alleles led to embryonic and perinatal lethality, respectively. Compound embryos lacking Casc3 expression were smaller with proportionately reduced brain size. Mutant brains contained fewer neurons and progenitors, but no apoptosis, all phenotypes explained by developmental delay. This finding, which contrasts with severe neural phenotypes evident in other EJC mutants, indicates Casc3 is largely dispensable for brain development. In the developing brain, CASC3 protein expression is substoichiometric relative to MAGOH, EIF4A3, and RBM8A. Taken together, this argues that CASC3 is not an essential EJC component in brain development and suggests it could function in a tissue-specific manner.

The exon junction complex (EJC) is an RNA binding complex comprised of the core components Magoh, Rbm8a, and Eif4a3. Human mutations in EJC components cause neurodevelopmental pathologies. Further, mice heterozygous for either Magoh or Rbm8a exhibit aberrant neurogenesis and microcephaly. Yet despite the requirement of these genes for neurodevelopment, the pathogenic mechanisms linking EJC dysfunction to microcephaly remain poorly understood. Here we employ mouse genetics, transcriptomic and proteomic analyses to demonstrate that haploinsufficiency for each of the 3 core EJC components causes microcephaly via converging regulation of p53 signaling. Using a new conditional allele, we first show that Eif4a3 haploinsufficiency phenocopies aberrant neurogenesis and microcephaly of Magoh and Rbm8a mutant mice. Transcriptomic and proteomic analyses of embryonic brains at the onset of neurogenesis identifies common pathways altered in each of the 3 EJC mutants, including ribosome, proteasome, and p53 signaling components. We further demonstrate all 3 mutants exhibit defective splicing of RNA regulatory proteins, implying an EJC dependent RNA regulatory network that fine-tunes gene expression. Finally, we show that genetic ablation of one downstream pathway, p53, significantly rescues microcephaly of all 3 EJC mutants. This implicates p53 activation as a major node of neurodevelopmental pathogenesis following EJC impairment. Altogether our study reveals new mechanisms to help explain how EJC mutations influence neurogenesis and underlie neurodevelopmental disease., Author Summary The mammalian neocortex is the brain structure responsible for higher cognition, abstract thought, and language. One process critical for brain development is neurogenesis, in which neural stem cell populations generate neurons. Alterations in neurogenesis can lead to neurodevelopmental disorders affecting brain size and function, such as microcephaly, in which the brain is significantly smaller than normal. Therefore, understanding the genes and processes controlling normal brain development is of strong clinical relevance. Here we studied proteins of the RNA binding exon junction complex, which are strongly implicated in several neurodevelopmental pathologies, but whose functions in brain development remain largely unknown. Using mouse models, we find that reduced levels of any of three essential proteins of this complex results in altered embryonic neurogenesis and microcephaly. We demonstrate that mutant mice show common alterations in p53 activation, expression of ribosomal components and splice variants for RNA processing factors. Interestingly we find that genetic suppression of p53 significantly rescues microcephaly in mutant mice. Given that patients harboring mutations in exon junction complex components present with neurodevelopmental deficits, our findings highlight molecular pathways which could underlie disease pathogenesis.

International audience; We analyzed four families that presented with a similar condition characterized by congenital micro-cephaly, intellectual disability, progressive cerebral atrophy, and intractable seizures. We show that recessive mutations in the ASNS gene are responsible for this syndrome. Two of the identified missense mutations dramatically reduce ASNS protein abundance, suggesting that the mutations cause loss of function. Hypomorphic Asns mutant mice have structural brain abnormalities, including enlarged ventricles and reduced cortical thickness, and show deficits in learning and memory mimicking aspects of the patient phenotype. ASNS encodes asparagine synthetase, which catalyzes the synthesis of asparagine from glutamine and aspartate. The neurological impairment resulting from ASNS deficiency may be explained by asparagine depletion in the brain or by accumulation of aspartate/gluta-mate leading to enhanced excitability and neuronal damage. Our study thus indicates that asparagine synthesis is essential for the development and function of the brain but not for that of other organs.