Maria Bergamasco

Heterozygous variants in the KAT6A gene encoding the histone lysine acetyltransferase KAT6A (MOZ, MYST3) cause Arboleda-Tham syndrome, a cognitive impairment syndrome. Histone acetylation is generally associated with active gene transcription. Genetic deletion of both alleles of the Kat6a gene in mice causes developmental defects including anterior homeotic transformation, cleft palate, interrupted aortic arch and cardiac septal defects. Loss of KAT6A impairs expression of HOX, DLX and TBX genes, which are essential for body segment identity specification, palate, heart and aortic arch development. However, the effects of loss of KAT6A on chromatin modifications and gene expression in neural cells, which are relevant to normal brain development and function, is still poorly understood. In this study, we used an automated high-throughput chromatin profiling method and RNA sequencing in mouse neural system and progenitor cells to assess the effects of loss of one or two alleles of Kat6a on gene expression, histone acetylation and methylation. We also assessed occupancy by a trithorax group protein and RNA polymerase II. Our data suggests two modes of action for KAT6A: (1) acetylation of histone H3 on lysine 23 at promoters and enhancers and (2) recruitment of the trithorax group protein MLL1 (KMT2A) to promote the expression of developmental genes, including SOX and homeodomain genes. Together, these two functions appear to be required for normal gene expression in neural progenitors and essential for proliferation and neuronal differentiation.

Messenger RNA (mRNA) therapeutics have rapidly emerged as a transformative approach for treating a range of health challenges. Accelerated by the success of mRNA-lipid nanoparticle (LNP) vaccines during the COVID-19 pandemic, this platform holds promise beyond immunisation for the transient expression of therapeutic proteins in targeted tissues. Despite this promise, non-invasive delivery of mRNA to the brain, as with most therapeutics, remains a challenge due to the impermeability of the blood brain barrier. Here, we present a novel strategy to deliver neurotrophic factors to the brain via intranasal delivery of mRNA-LNP. As a proof of concept, we demonstrate that intranasal delivery of mRNA encoding the neurogenic factor BDNF (Brain Derived Neurotrophic Factor) enhances memory performance in both aged mice and a transgenic mouse model of Alzheimers disease. This approach offers a promising platform for delivering therapeutic proteins to the brain and opens new avenues for treating age-related and neurodegenerative disorders.

Closely related genes typically display common essential functions but also functional diversification, ensuring retention of both genes throughout evolution. The histone lysine acetyltransferases KAT6A (MOZ) and KAT6B (QKF/MORF), sharing identical protein domain structure, are mutually exclusive catalytic subunits of a multiprotein complex. Mutations in either KAT6A or KAT6B result in congenital intellectual disability disorders in human patients. In mice, loss of function of either gene results in distinct, severe phenotypic consequences. Here we show that, surprisingly, 4-fold overexpression of Kat6b rescues all previously described developmental defects in Kat6a mutant mice, including rescuing the absence of hematopoietic stem cells. Kat6b restores acetylation at histone H3 lysines 9 and 23 and reverses critical gene expression anomalies in Kat6a mutant mice. Our data suggest that the target gene specificity of KAT6A can be substituted by the related paralogue KAT6B, despite differences in amino acid sequence, if KAT6B is expressed at sufficiently high levels.

Börjeson-Forssman-Lehmann syndrome (BFLS) is an X-linked intellectual disability and endocrine disorder caused by pathogenic variants of plant homeodomain finger gene 6 (PHF6). An understanding of the role of PHF6 in vivo in the development of the mammalian nervous system is required to advance our knowledge of how PHF6 mutations cause BFLS. Here, we show that PHF6 protein levels are greatly reduced in cells derived from a subset of patients with BFLS. We report the phenotypic, anatomical, cellular and molecular characterization of the brain in males and females in two mouse models of BFLS, namely loss of Phf6 in the germline and nervous system-specific deletion of Phf6. We show that loss of PHF6 resulted in spontaneous seizures occurring via a neural intrinsic mechanism. Histological and morphological analysis revealed a significant enlargement of the lateral ventricles in adult Phf6-deficient mice, while other brain structures and cortical lamination were normal. Phf6 deficient neural precursor cells showed a reduced capacity for self-renewal and increased differentiation into neurons. Phf6 deficient cortical neurons commenced spontaneous neuronal activity prematurely suggesting precocious neuronal maturation. We show that loss of PHF6 in the foetal cortex and isolated cortical neurons predominantly caused upregulation of genes, including Reln, Nr4a2, Slc12a5, Phip and ZIC family transcription factor genes, involved in neural development and function, providing insight into the molecular effects of loss of PHF6 in the developing brain.

Mutations in genes encoding chromatin modifiers are enriched among mutations causing intellectual disability. The continuing development of the brain postnatally, coupled with the inherent reversibility of chromatin modifications, may afford an opportunity for therapeutic intervention following a genetic diagnosis. Development of treatments requires an understanding of protein function and models of the disease. Here, we provide a mouse model of Say-Barber-Biesecker-Young-Simpson syndrome (SBBYSS) (OMIM 603736) and demonstrate proof-of-principle efficacy of postnatal treatment. SBBYSS results from heterozygous mutations in the KAT6B ( MYST4/MORF/QFK ) gene and is characterized by intellectual disability and autism-like behaviors. Using human cells carrying SBBYSS-specific KAT6B mutations and Kat6b heterozygous mice ( Kat6b ^+/– ), we showed that KAT6B deficiency caused a reduction in histone H3 lysine 9 acetylation. Kat6b ^+/– mice displayed learning, memory, and social deficits, mirroring SBBYSS individuals. Treatment with a histone deacetylase inhibitor, valproic acid, or an acetyl donor, acetyl-carnitine (ALCAR), elevated histone acetylation levels in the human cells with SBBYSS mutations and in brain and blood cells of Kat6b ^+/– mice and partially reversed gene expression changes in Kat6b ^+/– cortical neurons. Both compounds improved sociability in Kat6b ^+/– mice, and ALCAR treatment restored learning and memory. These data suggest that a subset of SBBYSS individuals may benefit from postnatal therapeutic interventions.

ZRANB1 (human Trabid) missense mutations have been identified in children diagnosed with a range of congenital disorders including reduced brain size, but how Trabid regulates neurodevelopment is not understood. We have characterized these patient mutations in cells and mice to identify a key role for Trabid in the regulation of neurite growth. One of the patient mutations flanked the catalytic cysteine of Trabid and its deubiquitylating (DUB) activity was abrogated. The second variant retained DUB activity, but failed to bind STRIPAK, a large multiprotein assembly implicated in cytoskeleton organization and neural development. Zranb1 knock-in mice harboring either of these patient mutations exhibited reduced neuronal and glial cell densities in the brain and a motor deficit consistent with fewer dopaminergic neurons and projections. Mechanistically, both DUB-impaired and STRIPAK-binding-deficient Trabid variants impeded the trafficking of adenomatous polyposis coli (APC) to microtubule plus-ends. Consequently, the formation of neuronal growth cones and the trajectory of neurite outgrowth from mutant midbrain progenitors were severely compromised. We propose that STRIPAK recruits Trabid to deubiquitylate APC, and that in cells with mutant Trabid, APC becomes hyperubiquitylated and mislocalized causing impaired organization of the cytoskeleton that underlie the neuronal and developmental phenotypes.

Trabid/ZRANB1 missense mutations have been identified in children diagnosed with a range of congenital disorders including reduced brain size, but how Trabid regulates neurodevelopment is not understood. We have characterised these patient mutations in cells and mice to identify a key role for Trabid in the regulation of neurite growth. One of the patient mutations flanked the catalytic cysteine of Trabid and its deubiquitylating (DUB) activity was abrogated. The second variant retained DUB activity, but failed to bind STRIPAK, a large multiprotein assembly implicated in cytoskeleton organisation and neural development. Trabid/ZRANB1 knock-in mice harbouring either of these patient mutations exhibited reduced neuronal and glial cell densities in the brain and a motor deficit consistent with fewer dopaminergic neurons and projections. Mechanistically, both DUB-impaired and STRIPAK-binding-deficient Trabid variants impeded the trafficking of adenomatous polyposis coli (APC) to microtubule plus-ends. Consequently, the formation of neuronal growth cones and the trajectory of neurite outgrowth from mutant midbrain progenitors were severely compromised. We propose that STRIPAK recruits Trabid to deubiquitylate APC, and that in cells with mutant Trabid, APC becomes hyperubiquitylated and mislocalised causing impaired organisation of the cytoskeleton that underlie the neuronal and developmental phenotypes.

Trabid/ZRANB1 missense mutations have been identified in children diagnosed with a range of congenital disorders including reduced brain size, but how Trabid regulates neurodevelopment is not understood. We have characterised these patient mutations in cells and mice to identify a key role for Trabid in the regulation of neurite growth. One of the patient mutations flanked the catalytic cysteine of Trabid and its deubiquitylating (DUB) activity was abrogated. The second variant retained DUB activity, but failed to bind STRIPAK, a large multiprotein assembly implicated in cytoskeleton organisation and neural development. Trabid/ZRANB1 knock-in mice harbouring either of these patient mutations exhibited reduced neuronal and glial cell densities in the brain and a motor deficit consistent with fewer dopaminergic neurons and projections. Mechanistically, both DUB-impaired and STRIPAK-binding-deficient Trabid variants impeded the trafficking of adenomatous polyposis coli (APC) to microtubule plus-ends. Consequently, the formation of neuronal growth cones and the trajectory of neurite outgrowth from mutant midbrain progenitors were severely compromised. We propose that STRIPAK recruits Trabid to deubiquitylate APC, and that in cells with mutant Trabid, APC becomes hyperubiquitylated and mislocalised causing impaired organisation of the cytoskeleton that underlie the neuronal and developmental phenotypes.