Mutations in several genes encoding centrosomal proteins dramatically decrease the size of the human brain. We show that Aspm (abnormal spindle-like, microcephaly-associated) and Wdr62 (WD ...repeat-containing protein 62) interact genetically to control brain size, with mice lacking Wdr62, Aspm, or both showing gene dose-related centriole duplication defects that parallel the severity of the microcephaly and increased ectopic basal progenitors, suggesting premature delamination from the ventricular zone. Wdr62 and Aspm localize to the proximal end of the mother centriole and interact physically, with Wdr62 required for Aspm localization, and both proteins, as well as microcephaly protein Cep63, required to localize CENPJ/CPAP/Sas-4, a final common target. Unexpectedly, Aspm and Wdr62 are required for normal apical complex localization and apical epithelial structure, providing a plausible unifying mechanism for the premature delamination and precocious differentiation of progenitors. Together, our results reveal links among centrioles, apical proteins, and cell fate, and illuminate how alterations in these interactions can dynamically regulate brain size.
•Wdr62 and Aspm interact genetically to control brain size in mice•Loss of Wdr62 or Aspm causes centriole duplication defects proportional to microcephaly•WDR62 and ASPM are maternal centriolar proteins that recruit CPAP to the centrosome•Loss of Wdr62 and Aspm causes gene dose-dependent disruption of the apical complex
Jayaraman et al. show that microcephaly proteins Wdr62 and Aspm localize to the maternal centriole and physically interact. Mice lacking Wdr62, Aspm, or both show gene dose-dependent defects in brain size, centriole duplication, centrosomal localization of CPAP, and the apical complex, which plays a critical role in cell fate.
Progressive microcephaly is a heterogeneous condition with causes including mutations in genes encoding regulators of neuronal survival. Here, we report the identification of mutations in QARS ...(encoding glutaminyl-tRNA synthetase QARS) as the causative variants in two unrelated families affected by progressive microcephaly, severe seizures in infancy, atrophy of the cerebral cortex and cerebellar vermis, and mild atrophy of the cerebellar hemispheres. Whole-exome sequencing of individuals from each family independently identified compound-heterozygous mutations in QARS as the only candidate causative variants. QARS was highly expressed in the developing fetal human cerebral cortex in many cell types. The four QARS mutations altered highly conserved amino acids, and the aminoacylation activity of QARS was significantly impaired in mutant cell lines. Variants p.Gly45Val and p.Tyr57His were located in the N-terminal domain required for QARS interaction with proteins in the multisynthetase complex and potentially with glutamine tRNA, and recombinant QARS proteins bearing either substitution showed an over 10-fold reduction in aminoacylation activity. Conversely, variants p.Arg403Trp and p.Arg515Trp, each occurring in a different family, were located in the catalytic core and completely disrupted QARS aminoacylation activity in vitro. Furthermore, p.Arg403Trp and p.Arg515Trp rendered QARS less soluble, and p.Arg403Trp disrupted QARS-RARS (arginyl-tRNA synthetase 1) interaction. In zebrafish, homozygous qars loss of function caused decreased brain and eye size and extensive cell death in the brain. Our results highlight the importance of QARS during brain development and that epilepsy due to impairment of QARS activity is unusually severe in comparison to other aminoacyl-tRNA synthetase disorders.
Genes disrupted in human microcephaly (meaning “small brain”) define key regulators of neural progenitor proliferation and cell-fate specification. In comparison, genes mutated in human lissencephaly ...(lissos means smooth and cephalos means brain) highlight critical regulators of neuronal migration. Here, we report two families with extreme microcephaly and grossly simplified cortical gyral structure, a condition referred to as microlissencephaly, and show that they carry homozygous frameshift mutations in NDE1, which encodes a multidomain protein that localizes to the centrosome and mitotic spindle poles. Both human mutations in NDE1 truncate the C-terminal NDE1domains, which are essential for interactions with cytoplasmic dynein and thus for regulation of cytoskeletal dynamics in mitosis and for cell-cycle-dependent phosphorylation of NDE1 by Cdk1. We show that the patient NDE1 proteins are unstable, cannot bind cytoplasmic dynein, and do not localize properly to the centrosome. Additionally, we show that CDK1 phosphorylation at T246, which is within the C-terminal region disrupted by the mutations, is required for cell-cycle progression from the G2 to the M phase. The role of NDE1 in cell-cycle progression probably contributes to the profound neuronal proliferation defects evident in Nde1-null mice and patients with NDE1 mutations, demonstrating the essential role of NDE1 in human cerebral cortical neurogenesis.
Genetic microcephaly and lissencephaly are 2 of the most common brain malformations. Each of them is a heterogeneous group of disorders caused by mutations of many different genes. They are a ...significant cause of neurological morbidity in children worldwide, responsible for many cases of mental retardation, cerebral palsy, and epilepsy. Recent advances in molecular genetics have led to the identification of several genes causing these disorders, and thus accurate molecular diagnosis and improved genetic counseling has become available for many patients and their families. More recently identified genes include STIL , causing primary autosomal recessive microcephaly (microcephaly vera), and TUBA1A , causing lissencephaly. Numerous other disease genes are likely still to be identified. Functional studies of genes that cause microcephaly and lissencephaly have provided valuable insight into the molecular mechanisms of human brain development.
Aminoacyl‐transfer RNA (tRNA) synthetases ligate amino acids to specific tRNAs and are essential for protein synthesis. Although alanyl‐tRNA synthetase (AARS) is a synthetase implicated in a wide ...range of neurological disorders from Charcot‐Marie‐Tooth disease to infantile epileptic encephalopathy, there have been limited data on their pathogenesis. Here, we report loss‐of‐function mutations in AARS in two siblings with progressive microcephaly with hypomyelination, intractable epilepsy, and spasticity. Whole‐exome sequencing identified that the affected individuals were compound heterozygous for mutations in AARS gene, c.2067dupC (p.Tyr690Leufs*3) and c.2738G>A (p.Gly913Asp). A lymphoblastoid cell line developed from one of the affected individuals showed a strong reduction in AARS abundance. The mutations decrease aminoacylation efficiency by 70%–90%. The p.Tyr690Leufs*3 mutation also abolished editing activity required for hydrolyzing misacylated tRNAs, thereby increasing errors during aminoacylation. Our study has extended potential mechanisms underlying AARS‐related disorders to include destabilization of the protein, aminoacylation dysfunction, and defective editing activity.
Mutations in alanyl‐tRNA synthetase (AARS) are implicated in neurological disorders, but there have been limited data on the pathogenesis. We identified loss‐of‐function mutations in AARS in two siblings with progressive microcephaly with hypomyelination, intractable epilepsy and spasticity. A lymphoblastoid cell line derived from an affected indivual showed a strong reduction in AARS protein abundance. Functional analysis of the mutations broadened the spectrum of potential mechanisms underlying AARS‐related disorders to include destabilization of the protein, aminoacylation dysfunction, and defective editing activity.
Despite recent advances in understanding the genetic bases of microcephaly, a large number of cases of microcephaly remain unexplained, suggesting that many microcephaly syndromes and associated ...genes are yet to be identified. Here we report mutations in PYCR2, which encodes an enzyme in the proline biosynthesis pathway, as the cause of a unique syndrome characterized by postnatal microcephaly, hypomyelination, and reduced cerebral white matter volume. Linkage mapping and whole-exome sequencing identified homozygous mutations in PYCR2 (c.355C>T p.Arg119Cys and c.751C>T p.Arg251Cys) in the affected individuals of two consanguineous families. A lymphoblastoid cell line from one affected individual showed a strong reduction in PYCR2 level. When mutant cDNAs were transfected into HEK293FT cells, the mutant protein retained normal mitochondrial localization but the level was lower than the wild-type protein, suggesting that mutant protein is less stable. A PYCR2-deficient HEK293FT cell line generated by clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 genome-editing showed that PYCR2 loss of function led to decreased mitochondrial membrane potential and increased susceptibility to apoptosis under oxidative stress. Morpholino-based knockdown of the zebrafish PYCR2 homolog, pycr1b, recapitulated the human microcephaly phenotype, which was rescued by wild-type human PYCR2 mRNA, but not by mutant mRNAs, further supporting the pathogenicity of the identified variants. Hypomyelination and the absence of lax, wrinkly skin distinguishes this condition from that caused by previously reported mutations in the gene encoding PYCR2’s isozyme, PYCR1, suggesting a unique and indispensable role for PYCR2 in the human central nervous system during development.
Katanin is a microtubule-severing complex whose catalytic activities are well characterized, but whose in vivo functions are incompletely understood. Human mutations in KATNB1, which encodes the ...noncatalytic regulatory p80 subunit of katanin, cause severe microlissencephaly. Loss of Katnb1 in mice confirms essential roles in neurogenesis and cell survival, while loss of zebrafish katnb1 reveals specific roles for katnin p80 in early and late developmental stages. Surprisingly, Katnb1 null mutant mouse embryos display hallmarks of aberrant Sonic hedgehog signaling, including holoprosencephaly. KATNB1-deficient human cells show defective proliferation and spindle structure, while Katnb1 null fibroblasts also demonstrate a remarkable excess of centrioles, with supernumerary cilia but deficient Hedgehog signaling. Our results reveal unexpected functions for KATNB1 in regulating overall centriole, mother centriole, and cilia number, and as an essential gene for normal Hedgehog signaling during neocortical development.
•Human mutations in KATNB1, encoding katanin p80, cause severe microlissencephaly•Katanin p80 is required for embryogenesis and neocortical development•Katnb1 null mice display few cortical progenitors and nearly absent neurons•Loss of katanin p80 causes excess centrioles and cilia, and disrupts Shh signaling
Hu et al. identify human mutations in KATNB1, encoding the p80 subunit of microtubule-severing complex katanin, as a cause of microlissencephaly and show that katanin p80 regulates centriole and cilia duplication, and Shh signaling during neocortical development.
The tight junction, or zonula occludens, is a specialized cell-cell junction that regulates epithelial and endothelial permeability, and it is an essential component of the blood-brain barrier in the ...cerebrovascular endothelium. In addition to functioning as a diffusion barrier, tight junctions are also involved in signal transduction. In this study, we identified a homozygous mutation in the tight-junction protein gene
JAM3 in a large consanguineous family from the United Arab Emirates. Some members of this family had a rare autosomal-recessive syndrome characterized by severe hemorrhagic destruction of the brain, subependymal calcification, and congenital cataracts. Their clinical presentation overlaps with some reported cases of pseudo-TORCH syndrome as well as with cases involving mutations in occludin, another component of the tight-junction complex. However, massive intracranial hemorrhage distinguishes these patients from others. Homozygosity mapping identified the disease locus in this family on chromosome 11q25 with a maximum multipoint LOD score of 6.15. Sequence analysis of genes in the candidate interval uncovered a mutation in the canonical splice-donor site of intron 5 of
JAM3. RT-PCR analysis of a patient lymphoblast cell line confirmed abnormal splicing, leading to a frameshift mutation with early termination. JAM3 is known to be present in vascular endothelium, although its roles in cerebral vasculature have not been implicated. Our results suggest that JAM3 is essential for maintaining the integrity of the cerebrovascular endothelium as well as for normal lens development in humans.
Characterizing perturbation of molecular pathways in congenital Zika virus (ZIKV) infection is critical for improved therapeutic approaches. Leveraging integrative systems biology, proteomics, and ...RNA-seq, we analyzed embryonic brain tissues from an immunocompetent, wild-type congenital ZIKV infection mouse model. ZIKV induced a robust immune response accompanied by the downregulation of critical neurodevelopmental gene programs. We identified a negative correlation between ZIKV polyprotein abundance and host cell cycle-inducing proteins. We further captured the downregulation of genes/proteins, many of which are known to be causative for human microcephaly, including Eomesodermin/T-box Brain Protein 2 (EOMES/TBR2) and Neuronal Differentiation 2 (NEUROD2). Disturbances of distinct molecular pathways in neural progenitors and post-mitotic neurons may contribute to complex brain phenotype of congenital ZIKV infection. Overall, this report on protein- and transcript-level dynamics enhances understanding of the ZIKV immunopathological landscape through characterization of fetal immune response in the developing brain.
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•Multi-omics analysis revealed key molecular pathways in congenital Zika syndrome•JAK/STAT activation is a potential driver of deranged brain development•Genes associated with microcephaly were downregulated in ZIKV-infected mouse brains•ZIKV load and cell cycle-inducing protein levels showed a negative correlation
Immunology; Virology; Developmental neuroscience
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