Autism-linked UBE3A gain-of-function mutation causes interneuron and behavioral phenotypes when inherited maternally or paternally in mice.

The E3 ubiquitin ligase Ube3a is biallelically expressed in neural progenitors and glial cells, suggesting that UBE3A gain-of-function mutations might cause neurodevelopmental disorders irrespective of parent of origin. Here, we engineered a mouse line that harbors an autism-linked UBE3AT485A (T503A in mouse) gain-of-function mutation and evaluated phenotypes in animals that inherited the mutant allele paternally, maternally, or from both parents. We find that paternally and maternally expressed UBE3AT503A results in elevated UBE3A activity in neural progenitors and glial cells. Expression of UBE3AT503A from the maternal allele, but not the paternal one, leads to a persistent elevation of UBE3A activity in neurons. Mutant mice display behavioral phenotypes that differ by parent of origin. Expression of UBE3AT503A, irrespective of its parent of origin, promotes transient embryonic expansion of Zcchc12 lineage interneurons. Phenotypes of Ube3aT503A mice are distinct from Angelman syndrome model mice. Our study has clinical implications for a growing number of disease-linked UBE3A gain-of-function mutations.

Cas9 gene therapy for Angelman syndrome traps Ube3a-ATS long non-coding RNA.

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.

Deletion of Topoisomerase 1 in excitatory neurons causes genomic instability and early onset neurodegeneration.

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.

Copine A Interacts with Actin Filaments and Plays a Role in Chemotaxis and Adhesion.

Copines make up a family of calcium-dependent, phospholipid-binding proteins found in numerous eukaryotic organisms. Copine proteins consist of two C2 domains at the N-terminus followed by an A domain similar to the von Willebrand A domain found in integrins. We are studying copine protein function in the model organism, Dictyostelium discoideum, which has six copine genes, cpnA-cpnF. Previous research showed that cells lacking the cpnA gene exhibited a cytokinesis defect, a contractile vacuole defect, and developmental defects. To provide insight into the role of CpnA in these cellular processes, we used column chromatography and immunoprecipitation to isolate proteins that bind to CpnA. These proteins were identified by mass spectrometry. One of the proteins identified was actin. Purified CpnA was shown to bind to actin filaments in a calcium-dependent manner in vitro. cpnA- cells exhibited defects in three actin-based processes: chemotaxis, cell polarity, and adhesion. These results suggest that CpnA plays a role in chemotaxis and adhesion and may do so by interacting with actin filaments.

RNA on the brain: emerging layers of post-transcriptional regulation in cerebral cortex development.

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.

Mouse models of Casc3 reveal developmental functions distinct from other components of the exon junction complex.

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.

Haploinsufficiency for Core Exon Junction Complex Components Disrupts Embryonic Neurogenesis and Causes p53-Mediated Microcephaly.

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.

Rbm8a haploinsufficiency disrupts embryonic cortical development resulting in microcephaly.

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 for Rbm8a, an exon junction complex (EJC) component within 1q21.1, causes severe microcephaly and defective neurogenesis in the mouse. At the onset of neurogenesis, Rbm8a regulates radial glia proliferation and prevents premature neuronal differentiation. Reduced Rbm8a levels 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 of Rbm8a alters embryonic neurogenesis, we overexpressed human RBM8A in two animal models. Using in utero electroporation of mouse neocortices as well as zebrafish models, we find RBM8A overexpression does not significantly perturb progenitor number or head size. Our findings demonstrate that Rbm8a is 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 of RBM8A may contribute to neurodevelopmental phenotypes associated with proximal 1q21.1 microdeletions.

Deficiency of asparagine synthetase causes congenital microcephaly and a progressive form of encephalopathy.

We analyzed four families that presented with a similar condition characterized by congenital microcephaly, 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/glutamate 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.

[Variations within OLF1/EBF-associated zinc finger protein gene confer susceptibility to lupus nephritis in Chinese population].

OBJECTIVE: To observe whether single nucleotide polymorphisms (SNPs) within the OLF1/EBF-associated zinc finger protein (OAZ) gene are associated with lupus nephritis (LN) susceptibility in Chinese population. METHODS: Eight SNPs located around the positive microsatellite marker D16S517 within OAZ gene with relatively high heterozygosity were chosen for genotyping on 184 systemic lupus erythromatosus (SLE) patients, including 101 non-LN patients and 83 LN patients, and 286 normal controls using TaqMan MGB allelic discrimination method. Data were collected by SDS 2.0 software. Haplotypes and their frequencies were constructed and estimated, and linkage disequilibrium analysis between pairs of SNPs was evaluated by calculating the D Prime using Helixtree program. Case-control study was performed between the SLE, LN, and non-LN groups and normal control group. RESULTS: (1) The frequency of SNP rs1344531 T allele was 47.0% in the SLE patients, significantly higher than that in the controls [38.1%,; chi(2) = 7.300, P = 0.008, OR (95% CI) = 1.441 (1.105 - 1.878)], which showed that the frequency of SNP rs1344531 T allele is associated with SLE susceptibility. The genotypic distribution of SNP rs1344531(CC/CT/TT) differed significantly between the SLE patients (25.5%/54.9%/19.6%) and normal controls (38.1%/47.6%/14.3%) (chi(2) = 8.394, P = 0.015). The CC genotype frequency of the SLE patients was 25.5%, significantly lower than that of the normal controls [38.1%; chi(2) = 7.976, P = 0.005, OR (95% CI) = 0.557 (0.370 - 0.838)] (2) The SNP rs1344531 T allele frequency of the SLE patients was 53.0%, significantly higher than that of the normal controls [38.1%; chi(2) = 11.769, P = 0.001, OR (95% CI) = 1.832 (1.293 - 2.596)], which showed an associated between SNP rs1344531 T allele frequency and LN susceptibility. The genotypic distribution of SNP rs1344531 (CC/CT/TT) differed significantly between the LN patients (22.9%/48.2%/28.9%) and the normal controls (38.1%/47.6%/14.3%) (chi(2) = 12.065, P = 0.002). The CC genotype frequency of the LN patients was 22.9%, significantly lower than that of the normal controls (38.1%) [chi(2) = 6.578, P = 0.013, OR (95% CI) = 0.481 (0.274 - 0.848)]. The TT genotype frequency of the LN patients was 28.9%, significantly higher than that of the normal controls (14.3%) [chi(2) = 9.423, P = 0.003, OR (95% CI) = 2.431 (1.363 - 4.334)]. No statistical significance was observed between the non-LN patients and normal controls in TT genotype frequency. (3) The frequencies of haplotypes containing rs1344531:rs1420676-rs1344531(C-T) [chi(2) = 11.731, P = 0.001, OR (95% CI) = 1.867 (1.302 - 2.676)], rs3803665-rs1420676-rs1344531(C-C-T) [chi(2) = 8.876, P = 0.004, OR (95% CI) = 1.772 (1.213 - 2.589)], and rs2292155-rs3803665-rs1420676-rs1344531(C-C-C-T) [chi(2) = 9.962, P = 0.002, OR (95% CI) = 1.915 (1.274 - 2.880)] were all significantly higher in the LN patients in comparison with the normal control group (41.0% versus 27.1%; 33.3% versus 21.8%; and 27.0% versus 16.2%); while the frequencies of other haplotypes: rs1344531-rs2080353(C-A) [chi(2) = 8.06, P = 0.005, OR (95% CI) = 0.603 (0.424 - 0.856)], rs1344531-rs2080353-rs933564 (C-A-G) [chi(2) = 7.929, P = 0.006, OR (95% CI) = 0.602 (0.422 - 0.859)] were significantly lower than those of the normal control group (39.5% versus 52.2%, and 36.6% versus 49.2%), which produced additional support for such association. CONCLUSION: SNP rs1344531 and some haplotypes containing SNP rs1344531 within OAZ are significantly associated with LN susceptibility. Genetic variants of the OAZ gene are involved in the pathogenesis of LN.