Nuclear stabilization of p53 requires a functional nucleolar surveillance pathway.

The nucleolar surveillance pathway monitors nucleolar integrity and responds to nucleolar stress by mediating binding of ribosomal proteins to MDM2, resulting in p53 accumulation. Inappropriate pathway activation is implicated in the pathogenesis of ribosomopathies, while drugs selectively activating the pathway are in trials for cancer. Despite this, the molecular mechanism(s) regulating this process are poorly understood. Using genome-wide loss-of-function screens, we demonstrate the ribosome biogenesis axis as the most potent class of genes whose disruption stabilizes p53. Mechanistically, we identify genes critical for regulation of this pathway, including HEATR3. By selectively disabling the nucleolar surveillance pathway, we demonstrate that it is essential for the ability of all nuclear-acting stresses, including DNA damage, to induce p53 accumulation. Our data support a paradigm whereby the nucleolar surveillance pathway is the central integrator of stresses that regulate nuclear p53 abundance, ensuring that ribosome biogenesis is hardwired to cellular proliferative capacity.

Early 17ß-estradiol treatment reduces seizures but not abnormal behaviour in mice with expanded polyalanine tracts in the Aristaless related homeobox gene (ARX).

Children with severe intellectual disability have an increased prevalence of refractory seizures. Steroid treatment may improve seizure outcomes, but the mechanism remains unknown. Here we demonstrate that short term, daily delivery of an exogenous steroid 17ß-estradiol (40 ng/g) in early postnatal life significantly reduced the number and severity of seizures, but did not improve behavioural deficits, in mice modelling mutations in the Aristaless-related homeobox gene (ARX), expanding the first (PA1) or second (PA2) polyalanine tract. Frequency of observed seizures on handling (n = 14/treatment/genotype) were significantly reduced in PA1 (32% reduction) and more modestly reduced in PA2 mice (14% reduction) with steroid treatment compared to vehicle. Spontaneous seizures were assessed (n = 7/treatment/genotype) at 7 weeks of age coinciding with a peak of seizure activity in untreated mice. PA1 mice treated with steroids no longer present with the most severe category of prolonged myoclonic seizures. Treated PA2 mice had an earlier onset of seizures coupled with a subsequent reduction in seizures later in postnatal life, with a complete absence of any seizures during the analysis at 7 weeks of age. Despite the reduction in seizures, 17ß-estradiol treated mice showed no improvement in behavioural or cognitive outcomes in adulthood. For the first time we show that these deficits due to mutations in Arx are already present before seizure onset and do not worsen with seizures. ARX is a transcription factor and Arx PA mutant mice have deregulated transcriptome profiles in the developing embryonic brain. At postnatal day 10, treatment completion, RNAseq identified 129 genes significantly deregulated (Log2FC > ± 0.5, P-value<0.05) in the frontal cortex of mutant compared to wild-type mice. This list reflects genes deregulated in disease and was particularly enriched for known genes in neurodevelopmental disorders and those involved in signalling and developmental pathways. 17ß-estradiol treatment of mutant mice significantly deregulated 295 genes, with only 23 deregulated genes overlapping between vehicle and steroid treated mutant mice. We conclude that 17ß-estradiol treatment recruits processes and pathways to reduce the frequency and severity of seizures in the Arx PA mutant mice but does not precisely correct the deregulated transcriptome nor improve mortality or behavioural and cognitive deficits.

Heterozygous loss of function of IQSEC2/Iqsec2 leads to increased activated Arf6 and severe neurocognitive seizure phenotype in females.

Clinical presentations of mutations in the IQSEC2 gene on the X-chromosome initially implicated to cause non-syndromic intellectual disability (ID) in males have expanded to include early onset seizures in males as well as in females. The molecular pathogenesis is not well understood, nor the mechanisms driving disease expression in heterozygous females. Using a CRISPR/Cas9-edited Iqsec2 KO mouse model, we confirm the loss of Iqsec2 mRNA expression and lack of Iqsec2 protein within the brain of both founder and progeny mice. Both male (52%) and female (46%) Iqsec2 KO mice present with frequent and recurrent seizures. Focusing on Iqsec2 KO heterozygous female mice, we demonstrate increased hyperactivity, altered anxiety and fear responses, decreased social interactions, delayed learning capacity and decreased memory retention/novel recognition, recapitulating psychiatric issues, autistic-like features, and cognitive deficits present in female patients with loss-of-function IQSEC2 variants. Despite Iqsec2 normally acting to activate Arf6 substrate, we demonstrate that mice modelling the loss of Iqsec2 function present with increased levels of activated Arf6. We contend that loss of Iqsec2 function leads to altered regulation of activated Arf6-mediated responses to synaptic signalling and immature synaptic networks. We highlight the importance of IQSEC2 function for females by reporting a novel nonsense variant c.566C > A, p.(S189*) in an elderly female patient with profound intellectual disability, generalised seizures, and behavioural disturbances. Our human and mouse data reaffirm IQSEC2 as another disease gene with an unexpected X-chromosome heterozygous female phenotype. Our Iqsec2 mouse model recapitulates the phenotypes observed in human patients despite the differences in the IQSEC2/Iqsec2 gene X-chromosome inactivation between the species.