RESUMO
VPS13B/COH1 is the only known causative factor for Cohen syndrome, an early-onset autosomal recessive developmental disorder with intellectual inability, developmental delay, joint hypermobility, myopia, and facial dysmorphism as common features, but the molecular basis of VPS13B/COH1 in pathogenesis remains largely unclear. Here, we identify Sec23 interacting protein (Sec23IP) at the ER exit site (ERES) as a VPS13B adaptor that recruits VPS13B to ERES-Golgi interfaces. VPS13B interacts directly with Sec23IP via the VPS13 adaptor binding domain (VAB), and the interaction promotes the association between ERES and the Golgi. Disease-associated missense mutations of VPS13B-VAB impair the interaction with Sec23IP. Knockout of VPS13B or Sec23IP blocks the formation of tubular ERGIC, an unconventional cargo carrier that expedites ER-to-Golgi transport. In addition, depletion of VPS13B or Sec23IP delays ER export of procollagen, suggesting a link between procollagen secretion and joint laxity in patients with Cohen disease. Together, our study reveals a crucial role of VPS13B-Sec23IP interaction at the ERES-Golgi interface in the pathogenesis of Cohen syndrome.
Assuntos
Retículo Endoplasmático , Complexo de Golgi , Deficiência Intelectual , Microcefalia , Hipotonia Muscular , Miopia , Proteínas de Transporte Vesicular , Humanos , Complexo de Golgi/metabolismo , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/genética , Proteínas de Transporte Vesicular/metabolismo , Proteínas de Transporte Vesicular/genética , Deficiência Intelectual/genética , Deficiência Intelectual/metabolismo , Deficiência Intelectual/patologia , Hipotonia Muscular/genética , Hipotonia Muscular/metabolismo , Hipotonia Muscular/patologia , Miopia/metabolismo , Miopia/genética , Miopia/patologia , Microcefalia/genética , Microcefalia/metabolismo , Microcefalia/patologia , Dedos/anormalidades , Células HeLa , Transporte Proteico , Ligação Proteica , Células HEK293 , Mutação de Sentido Incorreto , Deficiências do Desenvolvimento , Obesidade , Degeneração RetinianaRESUMO
The WD repeat-containing protein 4 (WDR4) has repeatedly been associated with primary microcephaly, a condition of impaired brain and skull growth. Often, faulty centrosomes cause microcephaly, yet aberrant cilia may also be involved. Here, we show using a combination of approaches in human fibroblasts, zebrafish embryos and patient-derived cells that WDR4 facilitates cilium formation. Molecularly, we associated WDR4 loss-of-function with increased protein synthesis and concomitant upregulation of proteasomal activity, while ubiquitin precursor pools are reduced. Inhibition of proteasomal activity as well as supplementation with free ubiquitin restored normal ciliogenesis. Proteasome inhibition ameliorated microcephaly phenotypes. Thus, we propose that WDR4 loss-of-function impairs head growth and neurogenesis via aberrant cilia formation, initially caused by disturbed protein and ubiquitin homeostasis.
Assuntos
Cílios , Complexo de Endopeptidases do Proteassoma , Ubiquitina , Peixe-Zebra , Complexo de Endopeptidases do Proteassoma/metabolismo , Humanos , Cílios/metabolismo , Cílios/patologia , Animais , Ubiquitina/metabolismo , Microcefalia/genética , Microcefalia/metabolismo , Microcefalia/patologia , Fibroblastos/metabolismo , NeurogêneseRESUMO
Polycomb repressive complex 1 (PRC1) modifies chromatin through catalysis of histone H2A lysine 119 monoubiquitination (H2AK119ub1). RING1 and RNF2 interchangeably serve as the catalytic subunit within PRC1. Pathogenic missense variants in PRC1 core components reveal functions of these proteins that are obscured in knockout models. While Ring1a knockout models remain healthy, the microcephaly and neuropsychiatric phenotypes associated with a pathogenic RING1 missense variant implicate unappreciated functions. Using an in vitro model of neurodevelopment, we observe that RING1 contributes to the broad placement of H2AK119ub1, and that its targets overlap with those of RNF2. PRC1 complexes harboring hypomorphic RING1 bind target loci but do not catalyze H2AK119ub1, reducing H2AK119ub1 by preventing catalytically active complexes from accessing the locus. This results in delayed DNA damage repair and cell cycle progression in neural progenitor cells (NPCs). Conversely, reduced H2AK119ub1 due to hypomorphic RING1 does not generate differential expression that impacts NPC differentiation. In contrast, hypomorphic RNF2 generates a greater reduction in H2AK119ub1 that results in both delayed DNA repair and widespread transcriptional changes. These findings suggest that the DNA damage response is more sensitive to H2AK119ub1 dosage change than is regulation of gene expression.
Assuntos
Reparo do DNA , Histonas , Mutação de Sentido Incorreto , Neurogênese , Complexo Repressor Polycomb 1 , Ubiquitinação , Animais , Humanos , Camundongos , Cromatina/metabolismo , Dano ao DNA , Histonas/metabolismo , Histonas/genética , Microcefalia/genética , Microcefalia/metabolismo , Células-Tronco Neurais/metabolismo , Complexo Repressor Polycomb 1/metabolismo , Complexo Repressor Polycomb 1/genética , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina-Proteína Ligases/genéticaRESUMO
Cockayne Syndrome B (CSB) is a hereditary multiorgan syndrome which-through largely unknown mechanisms-can affect the brain where it clinically presents with microcephaly, intellectual disability and demyelination. Using human induced pluripotent stem cell (hiPSC)-derived neural 3D models generated from CSB patient-derived and isogenic control lines, we here provide explanations for these three major neuropathological phenotypes. In our models, CSB deficiency is associated with (i) impaired cellular migration due to defective autophagy as an explanation for clinical microcephaly; (ii) altered neuronal network functionality and neurotransmitter GABA levels, which is suggestive of a disturbed GABA switch that likely impairs brain circuit formation and ultimately causes intellectual disability; and (iii) impaired oligodendrocyte maturation as a possible cause of the demyelination observed in children with CSB. Of note, the impaired migration and oligodendrocyte maturation could both be partially rescued by pharmacological HDAC inhibition.
Assuntos
Síndrome de Cockayne , Células-Tronco Pluripotentes Induzidas , Oligodendroglia , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/patologia , Síndrome de Cockayne/genética , Síndrome de Cockayne/metabolismo , Síndrome de Cockayne/patologia , Oligodendroglia/metabolismo , Oligodendroglia/citologia , Movimento Celular , Enzimas Reparadoras do DNA/metabolismo , Enzimas Reparadoras do DNA/genética , Neurônios/metabolismo , Neurônios/patologia , Autofagia , Encéfalo/metabolismo , Encéfalo/patologia , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/genética , Ácido gama-Aminobutírico/metabolismo , DNA Helicases/metabolismo , DNA Helicases/genética , Microcefalia/patologia , Microcefalia/metabolismo , Microcefalia/genética , Doenças Desmielinizantes/patologia , Doenças Desmielinizantes/metabolismo , Diferenciação CelularRESUMO
Mutations in the IER3IP1 (Immediate Early Response-3 Interacting Protein 1) gene can give rise to MEDS1 (Microcephaly with Simplified Gyral Pattern, Epilepsy, and Permanent Neonatal Diabetes Syndrome-1), a severe condition leading to early childhood mortality. The small endoplasmic reticulum (ER)-membrane protein IER3IP1 plays a non-essential role in ER-Golgi transport. Here, we employed secretome and cell-surface proteomics to demonstrate that the absence of IER3IP1 results in the mistrafficking of proteins crucial for neuronal development and survival, including FGFR3, UNC5B and SEMA4D. This phenomenon correlates with the distension of ER membranes and increased lysosomal activity. Notably, the trafficking of cargo receptor ERGIC53 and KDEL-receptor 2 are compromised, with the latter leading to the anomalous secretion of ER-localized chaperones. Our investigation extended to in-utero knock-down of Ier3ip1 in mouse embryo brains, revealing a morphological phenotype in newborn neurons. In summary, our findings provide insights into how the loss or mutation of a 10 kDa small ER-membrane protein can cause a fatal syndrome.
Assuntos
Retículo Endoplasmático , Complexo de Golgi , Microcefalia , Retículo Endoplasmático/metabolismo , Animais , Microcefalia/genética , Microcefalia/metabolismo , Microcefalia/patologia , Camundongos , Complexo de Golgi/metabolismo , Humanos , Mutação , Transporte Proteico , Proteínas de Membrana/metabolismo , Proteínas de Membrana/genética , Neurônios/metabolismo , Neurônios/patologiaRESUMO
MCPH1 has been identified as the causal gene for primary microcephaly type 1, a neurodevelopmental disorder characterized by reduced brain size and delayed growth. As a multifunction protein, MCPH1 has been reported to repress the expression of TERT and interact with transcriptional regulator E2F1. However, it remains unclear whether MCPH1 regulates brain development through its transcriptional regulation function. This study showed that the knockout of Mcph1 in mice leads to delayed growth as early as the embryo stage E11.5. Transcriptome analysis (RNA-seq) revealed that the deletion of Mcph1 resulted in changes in the expression levels of a limited number of genes. Although the expression of some of E2F1 targets, such as Satb2 and Cdkn1c, was affected, the differentially expressed genes (DEGs) were not significantly enriched as E2F1 target genes. Further investigations showed that primary and immortalized Mcph1 knockout mouse embryonic fibroblasts (MEFs) exhibited cell cycle arrest and cellular senescence phenotype. Interestingly, the upregulation of p19ARF was detected in Mcph1 knockout MEFs, and silencing p19Arf restored the cell cycle and growth arrest to wild-type levels. Our findings suggested it is unlikely that MCPH1 regulates neurodevelopment through E2F1-mediated transcriptional regulation, and p19ARF-dependent cell cycle arrest and cellular senescence may contribute to the developmental abnormalities observed in primary microcephaly.
Assuntos
Pontos de Checagem do Ciclo Celular , Senescência Celular , Inibidor p16 de Quinase Dependente de Ciclina , Microcefalia , Animais , Camundongos , Pontos de Checagem do Ciclo Celular/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Senescência Celular/genética , Inibidor p16 de Quinase Dependente de Ciclina/genética , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Inibidor p16 de Quinase Dependente de Ciclina/deficiência , Fator de Transcrição E2F1/genética , Fator de Transcrição E2F1/metabolismo , Fibroblastos/metabolismo , Camundongos Knockout , Microcefalia/genética , Microcefalia/metabolismo , Microcefalia/patologiaRESUMO
Entosis, a form of cell cannibalism, is a newly discovered pathogenic mechanism leading to the development of small brains, termed microcephaly, in which P53 activation was found to play a major role. Microcephaly with entosis, found in Pals1 mutant mice, displays P53 activation that promotes entosis and apoptotic cell death. This previously unappreciated pathogenic mechanism represents a novel cellular dynamic in dividing cortical progenitors which is responsible for cell loss. To date, various recent models of microcephaly have bolstered the importance of P53 activation in cell death leading to microcephaly. P53 activation caused by mitotic delay or DNA damage manifests apoptotic cell death which can be suppressed by P53 removal in these animal models. Such genetic studies attest P53 activation as quality control meant to eliminate genomically unfit cells with minimal involvement in the actual function of microcephaly associated genes. In this review, we summarize the known role of P53 activation in a variety of microcephaly models and introduce a novel mechanism wherein entotic cell cannibalism in neural progenitors is triggered by P53 activation.
Assuntos
Apoptose , Entose , Microcefalia , Proteína Supressora de Tumor p53 , Microcefalia/genética , Microcefalia/metabolismo , Microcefalia/patologia , Proteína Supressora de Tumor p53/metabolismo , Proteína Supressora de Tumor p53/genética , Animais , Humanos , Camundongos , Modelos Animais de DoençasRESUMO
Raine syndrome (RNS) is a rare autosomal recessive osteosclerotic dysplasia. RNS is caused by loss-of-function disease-causative variants of the FAM20C gene that encodes a kinase that phosphorylates most of the secreted proteins found in the body fluids and extracellular matrix. The most common RNS clinical features are generalized osteosclerosis, facial dysmorphism, intracerebral calcifications and respiratory defects. In non-lethal RNS forms, oral traits include a well-studied hypoplastic amelogenesis imperfecta (AI) and a much less characterized gingival phenotype. We used immunomorphological, biochemical, and siRNA approaches to analyze gingival tissues and primary cultures of gingival fibroblasts of two unrelated, previously reported RNS patients. We showed that fibrosis, pathological gingival calcifications and increased expression of various profibrotic and pro-osteogenic proteins such as POSTN, SPARC and VIM were common findings. Proteomic analysis of differentially expressed proteins demonstrated that proteins involved in extracellular matrix (ECM) regulation and related to the TGFß/SMAD signaling pathway were increased. Functional analyses confirmed the upregulation of TGFß/SMAD signaling and subsequently uncovered the involvement of two closely related transcription cofactors important in fibrogenesis, Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ). Knocking down of FAM20C confirmed the TGFß-YAP/TAZ interplay indicating that a profibrotic loop enabled gingival fibrosis in RNS patients. In summary, our in vivo and in vitro data provide a detailed description of the RNS gingival phenotype. They show that gingival fibrosis and calcifications are associated with, and most likely caused by excessed ECM production and disorganization. They furthermore uncover the contribution of increased TGFß-YAP/TAZ signaling in the pathogenesis of the gingival fibrosis.
Assuntos
Anormalidades Múltiplas , Proteínas Adaptadoras de Transdução de Sinal , Fissura Palatina , Hipoplasia do Esmalte Dentário , Exoftalmia , Fibroblastos , Fibrose , Gengiva , Osteosclerose , Proteômica , Transdução de Sinais , Fatores de Transcrição , Fator de Crescimento Transformador beta , Proteínas de Sinalização YAP , Humanos , Fator de Crescimento Transformador beta/metabolismo , Gengiva/metabolismo , Gengiva/patologia , Proteômica/métodos , Fibrose/metabolismo , Proteínas de Sinalização YAP/metabolismo , Proteínas de Sinalização YAP/genética , Osteosclerose/metabolismo , Osteosclerose/genética , Osteosclerose/patologia , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Hipoplasia do Esmalte Dentário/metabolismo , Hipoplasia do Esmalte Dentário/genética , Hipoplasia do Esmalte Dentário/patologia , Fibroblastos/metabolismo , Fibroblastos/patologia , Microcefalia/metabolismo , Microcefalia/genética , Microcefalia/patologia , Feminino , Proteínas com Motivo de Ligação a PDZ com Coativador Transcricional/metabolismo , Masculino , Transativadores/metabolismo , Transativadores/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Caseína Quinase I/metabolismo , Caseína Quinase I/genética , Proteínas da Matriz Extracelular/metabolismo , Proteínas da Matriz Extracelular/genética , Amelogênese Imperfeita/metabolismo , Amelogênese Imperfeita/genética , Amelogênese Imperfeita/patologia , Células CultivadasRESUMO
Copy number variants (CNVs) represent the genetic cause of about 15-20% of neurodevelopmental disorders (NDDs). We identified a ~67 kb de novo intragenic deletion on chromosome 2q22.3 in a female individual showing a developmental encephalopathy characterised by epilepsy, severe intellectual disability, speech delay, microcephaly, and thin corpus callosum with facial dysmorphisms. The microdeletion involved exons 5-6 of GTDC1, encoding a putative glycosyltransferase, whose expression is particularly enriched in the nervous system. In a previous study, a balanced de novo translocation encompassing GTDC1 was reported in a male child with global developmental delay and delayed speech and language development. Based on these premises, we explored the transcriptomic profile of our proband to evaluate the functional consequences of the novel GTDC1 de novo intragenic deletion in relation to the observed neurodevelopmental phenotype. RNA-seq on the proband's lymphoblastoid cell line (LCL) showed expression changes of glycine/serine and cytokine/chemokine signalling pathways, which are related to neurodevelopment and epileptogenesis. Subsequent analysis by ELISA (enzyme-linked immunosorbent assay) and HPLC (high-performance liquid chromatography) revealed increased levels of glycine in the proband's LCL and serum compared to matched controls. Given that an increased level of glycine has been observed in the plasma samples of individuals with Rett syndrome, a condition sharing epilepsy, microcephaly, and intellectual disability with our proband, we proposed that the GTDC1 downregulation is implicated in neurodevelopmental impairment by altering glycine metabolism. Furthermore, our findings expanded the phenotypic spectrum of the novel GTDC1-related condition, including microcephaly and epilepsy among relevant clinical features.
Assuntos
Glicina , Transtornos do Neurodesenvolvimento , Criança , Pré-Escolar , Humanos , Deleção Cromossômica , Cromossomos Humanos Par 2/genética , Epilepsia/genética , Epilepsia/metabolismo , Epilepsia/patologia , Glicina/metabolismo , Glicina/genética , Deficiência Intelectual/genética , Deficiência Intelectual/patologia , Deficiência Intelectual/metabolismo , Microcefalia/genética , Microcefalia/patologia , Microcefalia/metabolismo , Transtornos do Neurodesenvolvimento/genética , Transtornos do Neurodesenvolvimento/metabolismo , Transtornos do Neurodesenvolvimento/patologia , FemininoRESUMO
Mutations of the human TRAFFICKING PROTEIN PARTICLE COMPLEX SUBUNIT 9 (TRAPPC9) cause a neurodevelopmental disorder characterised by microcephaly and intellectual disability. Trappc9 constitutes a subunit specific to the intracellular membrane-associated TrappII complex. The TrappII complex interacts with Rab11 and Rab18, the latter being specifically associated with lipid droplets (LDs). Here we used non-invasive imaging to characterise Trappc9 knock-out (KO) mice as a model of the human hereditary disorder. KOs developed postnatal microcephaly with many grey and white matter regions being affected. In vivo magnetic resonance imaging (MRI) identified a disproportionately stronger volume reduction in the hippocampus, which was associated with a significant loss of Sox2-positive neural stem and progenitor cells. Diffusion tensor imaging indicated a reduced organisation or integrity of white matter areas. Trappc9 KOs displayed behavioural abnormalities in several tests related to exploration, learning and memory. Trappc9-deficient primary hippocampal neurons accumulated a larger LD volume per cell following Oleic Acid stimulation, and the coating of LDs by Perilipin-2 was much reduced. Additionally, Trappc9 KOs developed obesity, which was significantly more severe in females than in males. Our findings indicate that, beyond previously reported Rab11-related vesicle transport defects, dysfunctions in LD homeostasis might contribute to the neurobiological symptoms of Trappc9 deficiency.
Assuntos
Microcefalia , Animais , Feminino , Humanos , Masculino , Camundongos , Imagem de Tensor de Difusão , Gotículas Lipídicas , Camundongos Knockout , Microcefalia/genética , Microcefalia/metabolismo , Neurônios/metabolismoRESUMO
Microcephaly is a rare neurodevelopmental disorder characterized by reduced skull circumference and brain volume that occurs sporadically in farm animals. We investigated an early-onset neurodegenerative disorder observed in seven lambs of purebred Kerry Hill sheep. Clinical signs included inability to stand or severe ataxia, convulsions, and early death. Diagnostic imaging and brain necropsy confirmed microcephaly. The pedigree of the lambs suggested monogenic autosomal recessive inheritance. We sequenced the genome of one affected lamb, and comparison with 115 control genomes revealed a single private protein-changing variant. This frameshift variant, MFSD2A: c.285dupA, p.(Asp96fs*9), represents a 1-bp duplication predicted to truncate 80% of the open reading frame. MFSD2A is a transmembrane protein that is essential for maintaining blood-brain barrier homeostasis and plays a key role in regulating brain lipogenesis. Human MFSD2A pathogenic variants are associated with a neurodevelopmental disorder with progressive microcephaly, spasticity, and brain imaging abnormalities (NEDMISBA, OMIM 616486). Here we present evidence for the occurrence of a recessively inherited form of microcephaly in sheep due to a loss-of-function variant in MFSD2A (OMIA 002371-9940). To the best of our knowledge, this is the first report of a spontaneous MFSD2A variant in domestic animals.
Assuntos
Microcefalia , Doenças dos Ovinos , Simportadores , Humanos , Ovinos/genética , Animais , Microcefalia/genética , Microcefalia/veterinária , Microcefalia/metabolismo , Encéfalo/metabolismo , Barreira Hematoencefálica/metabolismo , Barreira Hematoencefálica/patologia , Mutação da Fase de Leitura , Carneiro Doméstico/genética , Carneiro Doméstico/metabolismo , Animais Domésticos/genética , Linhagem , Simportadores/genética , Doenças dos Ovinos/genéticaRESUMO
The coordination of cellular behaviors during neurodevelopment is critical for determining the form, function, and size of the central nervous system (CNS). Mutations in the vertebrate Abnormal Spindle-Like, Microcephaly Associated (ASPM) gene and its Drosophila melanogaster ortholog abnormal spindle (asp) lead to microcephaly (MCPH), a reduction in overall brain size whose etiology remains poorly defined. Here, we provide the neurodevelopmental transcriptional landscape for a Drosophila model for autosomal recessive primary microcephaly-5 (MCPH5) and extend our findings into the functional realm to identify the key cellular mechanisms responsible for Asp-dependent brain growth and development. We identify multiple transcriptomic signatures, including new patterns of coexpressed genes in the developing CNS. Defects in optic lobe neurogenesis were detected in larval brains through downregulation of temporal transcription factors (tTFs) and Notch signaling targets, which correlated with a significant reduction in brain size and total cell numbers during the neurogenic window of development. We also found inflammation as a hallmark of asp mutant brains, detectable throughout every stage of CNS development, which also contributes to the brain size phenotype. Finally, we show that apoptosis is not a primary driver of the asp mutant brain phenotypes, further highlighting an intrinsic Asp-dependent neurogenesis promotion mechanism that is independent of cell death. Collectively, our results suggest that the etiology of the asp mutant brain phenotype is complex and that a comprehensive view of the cellular basis of the disorder requires an understanding of how multiple pathway inputs collectively determine tissue size and architecture.
Assuntos
Microcefalia , Animais , Encéfalo/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Imunidade , Microcefalia/genética , Microcefalia/metabolismo , Mutação , Proteínas do Tecido Nervoso/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
BACKGROUND: Telomeres inhibit DNA damage response at the ends of the chromosome to suppress cell cycle arrest as well as ensure genome stability. Dyskeratosis congenita (DC), a telomere-related disease, includes the classical triad involving oral leukoplakia, dysplastic nails, and lacy reticular pigment in the neck and/or upper chest. Hoyeraal-Hreidarrson syndrome (HHS), a severe manifestation of DC, frequently occurs during childhood, and patients with HHS often show short-term survival and thus do not exhibit all mucocutaneous manifestations or syndromic features. CASE: We report here a patient with HHS characterized by the proband`s clinical attributes, such as growth delay, bone marrow failure, microcephaly, defects in body development, and the absence of cerebellar hypoplasia combined with Blake`s pouch cyst. By using exome sequencing, novel compound heterozygous mutations (c.1451C > T and c.1266+3del78bp) were detected in the RTEL1 (regulator of telomere elongation helicase 1) gene. CONCLUSIONS: The DNA helicase RTEL1 plays a role in genome stability, DNA replication, telomere maintenance, and genome repair. Terminal restriction fragment length analysis revealed a significantly shorter telomere length of the proband. Our findings provided evidence that compound heterozygous RTEL1 mutations cause HHS.
Assuntos
Bolsas Cólicas , Disceratose Congênita , Deficiência Intelectual , Microcefalia , Humanos , DNA Helicases/genética , Disceratose Congênita/genética , Disceratose Congênita/metabolismo , Retardo do Crescimento Fetal , Instabilidade Genômica , Deficiência Intelectual/genética , Microcefalia/genética , Microcefalia/metabolismo , Mutação , Telômero/metabolismoRESUMO
Loss of function in transport protein particles (TRAPP) links a new set of emerging genetic disorders called "TRAPPopathies". One such disorder is NIBP syndrome, characterized by microcephaly and intellectual disability, and caused by mutations of NIBP/TRAPPC9, a crucial and unique member of TRAPPII. To investigate the neural cellular/molecular mechanisms underlying microcephaly, we developed Nibp/Trappc9-deficient animal models using different techniques, including morpholino knockdown and CRISPR/Cas mutation in zebrafish and Cre/LoxP-mediated gene targeting in mice. Nibp/Trappc9 deficiency impaired the stability of the TRAPPII complex at actin filaments and microtubules of neurites and growth cones. This deficiency also impaired elongation and branching of neuronal dendrites and axons, without significant effects on neurite initiation or neural cell number/types in embryonic and adult brains. The positive correlation of TRAPPII stability and neurite elongation/branching suggests a potential role for TRAPPII in regulating neurite morphology. These results provide novel genetic/molecular evidence to define patients with a type of non-syndromic autosomal recessive intellectual disability and highlight the importance of developing therapeutic approaches targeting the TRAPPII complex to cure TRAPPopathies.
Assuntos
Deficiência Intelectual , Microcefalia , Animais , Camundongos , Deficiência Intelectual/genética , Deficiência Intelectual/metabolismo , Microcefalia/genética , Microcefalia/metabolismo , Neuritos/fisiologia , Neurônios/metabolismo , Peixe-ZebraRESUMO
Primary microcephalies (PMs) are defects in brain growth that are detectable at or before birth and are responsible for neurodevelopmental disorders. Most are caused by biallelic or, more rarely, dominant mutations in one of the likely hundreds of genes encoding PM proteins, i.e., ubiquitous centrosome or microtubule-associated proteins required for the division of neural progenitor cells in the embryonic brain. Here, we provide an overview of the different types of PMs, i.e., isolated PMs with or without malformations of cortical development and PMs associated with short stature (microcephalic dwarfism) or sensorineural disorders. We present an overview of the genetic, developmental, neurological, and cognitive aspects characterizing the most representative PMs. The analysis of phenotypic similarities and differences among patients has led scientists to elucidate the roles of these PM proteins in humans. Phenotypic similarities indicate possible redundant functions of a few of these proteins, such as ASPM and WDR62, which play roles only in determining brain size and structure. However, the protein pericentrin (PCNT) is equally required for determining brain and body size. Other PM proteins perform both functions, albeit to different degrees. Finally, by comparing phenotypes, we considered the interrelationships among these proteins.
Assuntos
Encefalopatias , Microcefalia , Humanos , Microcefalia/genética , Microcefalia/metabolismo , Centrossomo/metabolismo , Encéfalo/metabolismo , Encefalopatias/metabolismo , Tamanho Corporal , Proteínas do Tecido Nervoso/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismoRESUMO
Microcephaly with pontine and cerebellar hypoplasia (MICPCH) syndrome is a neurodevelopmental disorder caused by the deficiency of the X-chromosomal gene CASK. However, the molecular mechanisms by which CASK deficiency causes cerebellar hypoplasia in this syndrome remain elusive. In this study, we used CASK knockout (KO) mice as models for MICPCH syndrome and investigated the effect of CASK mutants. Female CASK heterozygote KO mice replicate the progressive cerebellar hypoplasia observed in MICPCH syndrome. CASK KO cultured cerebellar granule (CG) cells show progressive cell death that can be rescued by co-infection with lentivirus expressing wild-type CASK. Rescue experiments with CASK deletion mutants identify that the CaMK, PDZ, and SH3, but not L27 and guanylate kinase domains of CASK are required for the survival of CG cells. We identify missense mutations in the CaMK domain of CASK derived from human patients that fail to rescue the cell death of cultured CASK KO CG cells. Machine learning-based structural analysis using AlphaFold 2.2 predicts that these mutations disrupt the structure of the binding interface with Liprin-α2. These results suggest that the interaction with Liprin-α2 via the CaMK domain of CASK may be involved in the pathophysiology of cerebellar hypoplasia in MICPCH syndrome.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Cerebelo , Guanilato Quinases , Proteínas de Membrana , Deficiência Intelectual Ligada ao Cromossomo X , Microcefalia , Cerebelo/metabolismo , Cerebelo/patologia , Deficiência Intelectual Ligada ao Cromossomo X/genética , Deficiência Intelectual Ligada ao Cromossomo X/metabolismo , Deficiência Intelectual Ligada ao Cromossomo X/patologia , Microcefalia/genética , Microcefalia/metabolismo , Microcefalia/patologia , Guanilato Quinases/química , Guanilato Quinases/genética , Guanilato Quinases/metabolismo , Humanos , Proteínas de Membrana/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Camundongos Knockout , Animais , Camundongos , Feminino , Células Cultivadas , Mutação , Domínios Proteicos , Aprendizado de Máquina , Software , ApoptoseRESUMO
Patients with autosomal recessive microcephaly 15 caused by deficiency in the sodium-dependent lysophosphatidylcholine (LPC) transporter major facilitator superfamily domain-containing 2a (Mfsd2a) present with both microcephaly and hypomyelination, suggesting an important role for LPC uptake by oligodendrocytes in the process of myelination. Here we demonstrate that Mfsd2a is specifically expressed in oligodendrocyte precursor cells (OPCs) and is critical for oligodendrocyte development. Single-cell sequencing of the oligodendrocyte lineage revealed that OPCs from OPC-specific Mfsd2a-KO mice (2aOKO mice) underwent precocious differentiation into immature oligodendrocytes and impaired maturation into myelinating oligodendrocytes, correlating with postnatal brain hypomyelination. 2aOKO mice did not exhibit microcephaly, a finding consistent with the notion that microcephaly is the consequence of an absence of LPC uptake at the blood-brain barrier rather than a deficiency in OPCs. Lipidomic analysis showed that OPCs and iOLs from 2aOKO mice had significantly decreased levels of phospholipids containing omega-3 fatty acids, with a corresponding increase in unsaturated fatty acids, the latter being products of de novo synthesis governed by Srebp-1. RNA-Seq indicated activation of the Srebp-1 pathway and defective expression of regulators of oligodendrocyte development. Taken together, these findings indicate that the transport of LPCs by Mfsd2a in OPCs is important for maintaining OPC state to regulate postnatal brain myelination.
Assuntos
Ácidos Graxos Ômega-3 , Microcefalia , Simportadores , Animais , Camundongos , Microcefalia/metabolismo , Proteína de Ligação a Elemento Regulador de Esterol 1/metabolismo , Linhagem da Célula , Simportadores/metabolismo , Camundongos Knockout , Proteínas de Membrana Transportadoras/metabolismo , Ácidos Graxos Ômega-3/metabolismo , Oligodendroglia/metabolismo , Diferenciação CelularRESUMO
The Drosophila abnormal spindle (asp) gene was discovered about 40 years ago and shown to be required for both mitotic and meiotic cell division. Subsequent studies showed that asp is highly conserved and that mutations in its human ortholog ASPM (Abnormal Spindle-like Microcephaly-associated; or MCPH5) are the most common cause of autosomal recessive primary microcephaly. This finding greatly stimulated research on ASPM and its fly and mouse (Aspm) orthologs. The three Asp orthologous proteins bind the microtubules (MTs) minus ends during cell division and also function in interphase nuclei. Investigations on different cell types showed that Asp/Aspm/ASPM depletion disrupts one or more of the following mitotic processes: aster formation, spindle pole focusing, centrosome-spindle coupling, spindle orientation, metaphase-to-anaphase progression, chromosome segregation, and cytokinesis. In addition, ASPM physically interacts with components of the DNA repair and replication machineries and is required for the maintenance of chromosomal DNA stability. We propose the working hypothesis that the asp/Aspm/ASPM genes play the same conserved functions in Drosophila, mouse, and human cells. Human microcephaly is a genetically heterogeneous disorder caused by mutations in 30 different genes that play a variety of functions required for cell division and chromosomal DNA integrity. Our hypothesis postulates that ASPM recapitulates the functions of most human microcephaly genes and provides a justification for why ASPM is the most frequently mutated gene in autosomal recessive primary microcephaly.
Assuntos
Microcefalia , Animais , Humanos , Camundongos , DNA , Drosophila/metabolismo , Microcefalia/genética , Microcefalia/metabolismo , Mitose , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismoRESUMO
Biallelic loss-of-function variants in SMPD4 cause a rare and severe neurodevelopmental disorder with progressive congenital microcephaly and early death. SMPD4 encodes a sphingomyelinase that hydrolyses sphingomyelin into ceramide at neutral pH and can thereby affect membrane lipid homeostasis. SMPD4 localizes to the membranes of the endoplasmic reticulum and nuclear envelope and interacts with nuclear pore complexes (NPC). We refine the clinical phenotype of loss-of-function SMPD4 variants by describing five individuals from three unrelated families with longitudinal data due to prolonged survival. All individuals surviving beyond infancy developed insulin-dependent diabetes, besides presenting with a severe neurodevelopmental disorder and microcephaly, making diabetes one of the most frequent age-dependent non-cerebral abnormalities. We studied the function of SMPD4 at the cellular and organ levels. Knock-down of SMPD4 in human neural stem cells causes reduced proliferation rates and prolonged mitosis. Moreover, SMPD4 depletion results in abnormal nuclear envelope breakdown and reassembly during mitosis and decreased post-mitotic NPC insertion. Fibroblasts from affected individuals show deficient SMPD4-specific neutral sphingomyelinase activity, without changing (sub)cellular lipidome fractions, which suggests a local function of SMPD4 on the nuclear envelope. In embryonic mouse brain, knockdown of Smpd4 impairs cortical progenitor proliferation and induces premature differentiation by altering the balance between neurogenic and proliferative progenitor cell divisions. We hypothesize that, in individuals with SMPD4-related disease, nuclear envelope bending, which is needed to insert NPCs in the nuclear envelope, is impaired in the absence of SMPD4 and interferes with cerebral corticogenesis and survival of pancreatic beta cells.
Assuntos
Diabetes Mellitus , Microcefalia , Humanos , Animais , Camundongos , Membrana Nuclear/química , Membrana Nuclear/metabolismo , Microcefalia/genética , Microcefalia/metabolismo , Esfingomielina Fosfodiesterase/análise , Esfingomielina Fosfodiesterase/genética , Esfingomielina Fosfodiesterase/metabolismo , Poro Nuclear/metabolismo , Mitose , Diabetes Mellitus/metabolismoRESUMO
Variants in the AUTS2 gene are associated with a broad spectrum of neurological conditions characterized by intellectual disability, microcephaly, and congenital brain malformations. Here, we use a human cerebral organoid model to investigate the pathophysiology of a heterozygous de novo missense AUTS2 variant identified in a patient with multiple neurological impairments including primary microcephaly and profound intellectual disability. Proband cerebral organoids exhibit reduced growth, deficits in neural progenitor cell (NPC) proliferation and disrupted NPC polarity within ventricular zone-like regions compared to control cerebral organoids. We used CRISPR-Cas9-mediated gene editing to correct this variant and demonstrate rescue of impaired organoid growth and NPC proliferative deficits. Single-cell RNA sequencing revealed a marked reduction of G1/S transition gene expression and alterations in WNT-ß-catenin signalling within proband NPCs, uncovering a novel role for AUTS2 in NPCs during human cortical development. Collectively, these results underscore the value of cerebral organoids to investigate molecular mechanisms underlying AUTS2 syndrome.