RESUMO
The cell orchestrates the dance of chromosome segregation with remarkable speed and fidelity. The mitotic spindle is built from scratch after interphase through microtubule (MT) nucleation, which is dependent on the γ-tubulin ring complex (γ-TuRC), the universal MT template. Although several MT nucleation pathways build the spindle framework, the question of when and how γ-TuRC is targeted to these nucleation sites in the spindle and subsequently activated remains an active area of investigation. Recent advances facilitated the discovery of new MT nucleation effectors and their mechanisms of action. In this review, we illuminate each spindle assembly pathway and subsequently consider how the pathways are merged to build a spindle.
Assuntos
Proteínas Associadas aos Microtúbulos , Tubulina (Proteína) , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Tubulina (Proteína)/genética , Tubulina (Proteína)/metabolismo , Microtúbulos/genética , Microtúbulos/metabolismo , Fuso Acromático/genética , Fuso Acromático/metabolismo , Centro Organizador dos Microtúbulos/metabolismoRESUMO
Primary microcephaly is a rare neurogenic and genetically heterogeneous disorder characterized by significant brain size reduction that results in numerous neurodevelopmental disorders (NDD) problems, including mild to severe intellectual disability (ID), global developmental delay (GDD), seizures and other congenital malformations. This disorder can arise from a mutation in genes involved in various biological pathways, including those within the brain. We characterized a recessive neurological disorder observed in nine young adults from five independent consanguineous Pakistani families. The disorder is characterized by microcephaly, ID, developmental delay (DD), early-onset epilepsy, recurrent infection, hearing loss, growth retardation, skeletal and limb defects. Through exome sequencing, we identified novel homozygous variants in five genes that were previously associated with brain diseases, namely CENPJ (NM_018451.5: c.1856A > G; p.Lys619Arg), STIL (NM_001048166.1: c.1235C > A; p.(Pro412Gln), CDK5RAP2 (NM_018249.6 c.3935 T > G; p.Leu1312Trp), RBBP8 (NM_203291.2 c.1843C > T; p.Gln615*) and CEP135 (NM_025009.5 c.1469A > G; p.Glu490Gly). These variants were validated by Sanger sequencing across all family members, and in silico structural analysis. Protein 3D homology modeling of wild-type and mutated proteins revealed substantial changes in the structure, suggesting a potential impact on function. Importantly, all identified genes play crucial roles in maintaining genomic integrity during cell division, with CENPJ, STIL, CDK5RAP2, and CEP135 being involved in centrosomal function. Collectively, our findings underscore the link between erroneous cell division, particularly centrosomal function, primary microcephaly and ID.
Assuntos
Proteínas de Ciclo Celular , Deficiência Intelectual , Microcefalia , Linhagem , Humanos , Microcefalia/genética , Deficiência Intelectual/genética , Masculino , Feminino , Proteínas de Ciclo Celular/genética , Adulto , Proteínas Cromossômicas não Histona/genética , Proteínas do Tecido Nervoso/genética , Divisão Celular/genética , Mutação , Peptídeos e Proteínas de Sinalização Intracelular/genética , Genômica , Adulto Jovem , Consanguinidade , Sequenciamento do Exoma , Homozigoto , Deficiências do Desenvolvimento/genética , Adolescente , Paquistão , Proteínas Associadas aos MicrotúbulosRESUMO
BACKGROUND/AIM: Autosomal recessive primary microcephaly (MCPH) is a rare and genetically heterogeneous group of disorders characterized by intellectual disability and microcephaly at birth, classically without further organ involvement. MCPH3 is caused by biallelic variants in the cyclin-dependent kinase 5 regulatory subunit-associated protein 2 gene CDK5RAP2. In the corresponding Cdk5rap2 mutant or Hertwig's anemia mouse model, congenital microcephaly as well as defects in the hematopoietic system, germ cells and eyes have been reported. The reduction in brain volume, particularly affecting gray matter, has been attributed mainly to disturbances in the proliferation and survival of early neuronal progenitors. In addition, defects in dendritic development and synaptogenesis exist that affect the excitation-inhibition balance. Here, we studied proteomic changes in cerebral cortices of Cdk5rap2 mutant mice. MATERIAL AND METHODS: We used large-gel two-dimensional gel (2-DE) electrophoresis to separate cortical proteins. 2-DE gels were visualized by a trained observer on a light box. Spot changes were considered with respect to presence/absence, quantitative variation and altered mobility. RESULT: We identified a reduction in more than 30 proteins that play a role in processes such as cell cytoskeleton dynamics, cell cycle progression, ciliary functions and apoptosis. These proteome changes in the MCPH3 model can be associated with various functional and morphological alterations of the developing brain. CONCLUSION: Our results shed light on potential protein candidates for the disease-associated phenotype reported in MCPH3.
Assuntos
Microcefalia , Humanos , Camundongos , Animais , Microcefalia/genética , Proteoma/genética , Proteômica , Proteínas de Ciclo Celular/genética , Mutação , Proteínas do Tecido Nervoso/genéticaRESUMO
BACKGROUND & OBJECTIVES: Primary Microcephaly (MCPH) is a rare neurogenetic disease, manifesting congenitally reduced head circumference and non-progressive intellectual disability (ID). To date, twenty-eight genes with biallelic mutations have been reported for this disorder. The study aimed for molecular genetic characterization of Pakistani families segregating MCPH. METHODS: We studied two unrelated consanguineous families (family A and B) presenting >2 patients with diagnostic symptoms of MCPH, born to asymptomatic parents. We employed whole-exome sequencing (WES) of probands to find putative causal mutations. The candidate variants were further confirmed and analyzed for co-segregation by Sanger sequencing of all available members of each family. This study was conducted at Government College University, Faisalabad, Pakistan, and Cologne Center for Genomics (CCG), University of Cologne, Germany; during 2017-2020. RESULTS: We identified a novel homozygous variant c.10097_10098delGA, p.(Gly3366Glufs*19) in exon 26 of ASPM gene in family A which presents with moderate intellectual disability, speech impairment, visual abnormalities, seizures, and ptyalism. Family B was found to segregate nonsense, homozygous variant c.448C>T p.(Arg150*) in CDK5RAP2. The patients also exhibited mild to severe seizures without ptyalism that has not been previously reported in patients with mutations in the CDK5RAP2 gene. CONCLUSION: We report a novel mutation in ASPM and ultra-rare mutation in the CDK5RAP2 gene, both causing primary microcephaly. The study expands the mutational spectrum of the ASPM gene to 212, and also adds to the clinical spectrum of CDK5RAP2 mutations. It also demonstrated the utility of WES in the investigation and genetic diagnosis of genetically heterogeneous disorders like MCPH. These findings would aid in diagnostic and preventive strategies including carrier screening, cascade testing, and genetic counselling.
RESUMO
Studies in differentiating skeletal muscle cells in vitro have revealed that the microtubule-organizing center shifts from the centrosome to the perinuclear sites. As the Golgi apparatus surrounds the nucleus in a myotube, it is unclear whether microtubules are nucleated at the nuclear envelope or at the surrounding Golgi apparatus. In this study, we investigated the positional relationship between the microtubule nucleating sites and the Golgi apparatus in C2C12 myotubes and in primary cultured mouse skeletal myotubes. We focused on gaps in the perinuclear Golgi apparatus where the nuclear envelope was not covered with the Golgi apparatus. In microtubule regrowth assay, microtubule regrowth after cold-nocodazole depolymerization of preexisting microtubules was not found at the gap of the perinuclear Golgi apparatus. Most of the microtubule regrowth was detected at the CDK5RAP2 (CDK5 regulatory subunit-associated protein 2)-rich spots on the perinuclear Golgi apparatus. Disruption of the perinuclear Golgi apparatus with brefeldin A treatment eliminated the perinuclear microtubule regrowth. The Golgi apparatus of undifferentiated myoblasts and those at the cytoplasm of myotubes were also the microtubule nucleating sites. From these observations, we concluded that most of the perinuclear microtubule nucleation occurs on the Golgi apparatus surrounding the nucleus.
Assuntos
Complexo de Golgi/metabolismo , Centro Organizador dos Microtúbulos/metabolismo , Músculo Esquelético/metabolismo , Animais , Diferenciação Celular , Linhagem Celular , Núcleo Celular/metabolismo , Camundongos , Músculo Esquelético/citologiaRESUMO
BACKGROUND: Primary hereditary microcephaly (MCPH) comprises a large group of autosomal recessive disorders mainly affecting cortical development and resulting in a congenital impairment of brain growth. Despite the identification of >25 causal genes so far, it remains a challenge to distinguish between different MCPH forms at the clinical level. METHODS: 7 patients with newly identified mutations in CDK5RAP2 (MCPH3) were investigated by performing prospective, extensive and systematic clinical, MRI, psychomotor, neurosensory and cognitive examinations under similar conditions. RESULTS: All patients displayed neurosensory defects in addition to microcephaly. Small cochlea with incomplete partition type II was found in all cases and was associated with progressive deafness in 4 of them. Furthermore, the CDK5RAP2 protein was specifically identified in the developing cochlea from human fetal tissues. Microphthalmia was also present in all patients along with retinal pigmentation changes and lipofuscin deposits. Finally, hypothalamic anomalies consisting of interhypothalamic adhesions, a congenital midline defect usually associated with holoprosencephaly, was detected in 5 cases. CONCLUSION: This is the first report indicating that CDK5RAP2 not only governs brain size but also plays a role in ocular and cochlear development and is necessary for hypothalamic nuclear separation at the midline. Our data indicate that CDK5RAP2 should be considered as a potential gene associated with deafness and forme fruste of holoprosencephaly. These children should be given neurosensory follow-up to prevent additional comorbidities and allow them reaching their full educational potential. TRIAL REGISTRATION NUMBER: NCT01565005.
Assuntos
Proteínas de Ciclo Celular/genética , Doenças Cocleares/genética , Microcefalia/genética , Proteínas do Tecido Nervoso/genética , Criança , Pré-Escolar , Cóclea/diagnóstico por imagem , Cóclea/metabolismo , Cóclea/patologia , Doenças Cocleares/diagnóstico por imagem , Doenças Cocleares/patologia , Anemia de Fanconi/genética , Anemia de Fanconi/patologia , Feminino , Humanos , Hipotálamo/diagnóstico por imagem , Hipotálamo/patologia , Lactente , Imageamento por Ressonância Magnética , Masculino , Microcefalia/diagnóstico por imagem , Microcefalia/patologia , Mutação , Neurogênese/genética , Linhagem , Retina/diagnóstico por imagem , Retina/patologiaRESUMO
Biallelic mutations in the cyclin-dependent kinase 5 regulatory subunit-associated protein 2 gene CDK5RAP2 cause autosomal recessive primary microcephaly type 3 (MCPH3). MCPH is characterized by intellectual disability and microcephaly at birth, classically without further organ involvement. Only recently, congenital cataracts were reported in four patients of one pedigree with MCPH3. Given the lack of a further pedigree with this phenotype, it remained unclear whether this was a true causal relationship. Here we support the link between CDK5RAP2 and eye development by showing that most Cdk5rap2 mutant mice (an/an) exhibit eye malformations ranging from reduced size of one or both eyes (microphthalmia) to total absence of both eyes (anophthalmia). We also detected increased apoptosis in the an/an retinal progenitor cells associated with more mitotic cells. This indicates an important role of Cdk5rap2 in physiologic eye development.
Assuntos
Anoftalmia/patologia , Proteínas de Ciclo Celular/genética , Olho/embriologia , Olho/metabolismo , Microcefalia/fisiopatologia , Microftalmia/patologia , Mutação , Animais , Anoftalmia/etiologia , Proteínas de Ciclo Celular/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Camundongos , Camundongos Endogâmicos C57BL , Microftalmia/etiologiaRESUMO
Congenital microcephaly is highly associated with intellectual disability. Features of autosomal recessive primary microcephaly subtype 3 (MCPH3) also include hyperactivity and seizures. The disease is caused by biallelic mutations in the Cyclin-dependent kinase 5 regulatory subunit-associated protein 2 gene CDK5RAP2. In the mouse, Cdk5rap2 mutations similar to the human condition result in reduced brain size and a strikingly thin neocortex already at early stages of neurogenesis that persists through adulthood. The microcephaly phenotype in MCPH arises from a neural stem cell proliferation defect. Here, we report a novel role for Cdk5rap2 in the regulation of dendritic development and synaptogenesis of neocortical layer 2/3 pyramidal neurons. Cdk5rap2-deficient murine neurons show poorly branched dendritic arbors and an increased density of immature thin spines and glutamatergic synapses in vivo. Moreover, the excitatory drive is enhanced in ex vivo brain slice preparations of Cdk5rap2 mutant mice. Concurrently, we show that pyramidal neurons receive fewer inhibitory inputs. Together, these findings point towards a shift in the excitation - inhibition balance towards excitation in Cdk5rap2 mutant mice. Thus, MCPH3 is associated not only with a neural progenitor proliferation defect but also with altered function of postmitotic neurons and hence with altered connectivity.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Microcefalia/fisiopatologia , Neocórtex/fisiopatologia , Vias Neurais/fisiopatologia , Neurogênese/fisiologia , Animais , Proteínas de Ciclo Celular/genética , Diferenciação Celular/fisiologia , Camundongos , Camundongos Mutantes , Microcefalia/genética , Microcefalia/metabolismo , Mutação , Neocórtex/metabolismo , Vias Neurais/metabolismo , Células Piramidais/metabolismo , Células Piramidais/patologia , Transmissão Sináptica/fisiologiaRESUMO
Nonsense mutations in the ASPM gene have been most frequently identified among familial microcephaly patients. Depletion of the Drosophila orthologue (asp) causes spindle pole unfocusing during mitosis in multiple cell types. However, it remains unknown whether human ASPM has a similar function. Here, by performing CRISPR-based gene knockout (KO) and RNA interference combined with auxin-inducible degron, we show that ASPM functions in spindle pole organisation during mitotic metaphase redundantly with another microcephaly protein, CDK5RAP2 (also called CEP215), in human tissue culture cells. Deletion of the ASPM gene alone did not affect spindle morphology or mitotic progression. However, when the pericentriolar material protein CDK5RAP2 was depleted in ASPM KO cells, spindle poles were unfocused during prometaphase, and anaphase onset was significantly delayed. The phenotypic analysis of CDK5RAP2-depleted cells suggested that the pole-focusing function of CDK5RAP2 is independent of its known function to localise the kinesin-14 motor HSET (also known as KIFC1) or activate the γ-tubulin complex. Finally, a hypomorphic mutation identified in ASPM microcephaly patients similarly caused spindle pole unfocusing in the absence of CDK5RAP2, suggesting a possible link between spindle pole disorganisation and microcephaly.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/genética , Cinesinas/genética , Proteínas do Tecido Nervoso/genética , Polos do Fuso/metabolismo , Anáfase , Sistemas CRISPR-Cas , Proteínas de Ciclo Celular , Edição de Genes , Regulação da Expressão Gênica , Técnicas de Inativação de Genes , Células HCT116 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Cinesinas/metabolismo , Metáfase , Microcefalia/genética , Microcefalia/metabolismo , Microcefalia/patologia , Modelos Biológicos , Proteínas do Tecido Nervoso/deficiência , Proteínas do Tecido Nervoso/metabolismo , Transdução de Sinais , Polos do Fuso/ultraestrutura , Tubulina (Proteína)/genética , Tubulina (Proteína)/metabolismoRESUMO
phr2AB is the regulatory subunit of the Dictyostelium discoideum phosphatase PP2A and is the ortholog of the human B55 regulatory subunit of PP2A. phr2AB was isolated as a binding partner of the centrosomal protein CEP161, an ortholog of mammalian CDK5RAP2. CEP161 is presumably a phosphoprotein and a component of the Hippo pathway. The interaction site was located in the N-terminal half of CEP161 which encompasses the γTURC binding domain in CEP161. This binding domain is responsible for binding of the γ-tubulin ring complex which allows microtubule nucleation at the centrosome. GFP-tagged phr2AB is diffusely distributed throughout the cell and enriched at the centrosome. Ectopic expression of phr2AB as GFP fusion protein led to multinucleation, aberrant nucleus centrosome ratios and an altered sensitivity to okadaic acid. Some of these features were also affected in cells over-expressing domains of CEP161 and in cells from patients suffering from primary microcephaly, which carried a mutated CDK5RAP2 gene.
Assuntos
Dictyostelium/genética , Dictyostelium/metabolismo , Fosfoproteínas/metabolismo , Animais , Proteínas de Ciclo Celular , Centrossomo/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Microtúbulos/metabolismo , Proteínas do Tecido Nervoso , Monoéster Fosfórico Hidrolases/metabolismo , Ligação Proteica , Tubulina (Proteína)/metabolismoRESUMO
Microtubules are polar cytoskeleton filaments that extend via growth at their plus ends. Microtubule plus-end-tracking proteins (+TIPs) accumulate at these growing plus ends to control microtubule dynamics and attachment. The +TIP end-binding protein 1 (EB1) and its homologs possess an autonomous plus-end-tracking mechanism and interact with other known +TIPs, which then recruit those +TIPs to the growing plus ends. A major +TIP class contains the SXIP (Ser-X-Ile-Pro, with X denoting any amino acid residue) motif, known to interact with EB1 and its homologs for plus-end tracking, but the role of SXIP in regulating EB1 activities is unclear. We show here that an interaction of EB1 with the SXIP-containing +TIP CDK5 regulatory subunit-associated protein 2 (CDK5RAP2) regulates several EB1 activities, including microtubule plus-end tracking, dynamics at microtubule plus ends, microtubule and α/ß-tubulin binding, and microtubule polymerization. The SXIP motif fused with a dimerization domain from CDK5RAP2 significantly enhanced EB1 plus-end-tracking and microtubule-polymerizing and bundling activities, but the SXIP motif alone failed to do so. An SXIP-binding-deficient EB1 mutant displayed significantly lower microtubule plus-end tracking than the wild-type protein in transfected cells. These results suggest that EB1 cooperates with CDK5RAP2 and perhaps other SXIP-containing +TIPs in tracking growing microtubule tips. We also generated plus-end-tracking chimeras of CDK5RAP2 and the adenomatous polyposis coli protein (APC) and found that overexpression of the dimerization domains interfered with microtubule plus-end tracking of their respective SXIP-containing chimeras. Our results suggest that disruption of SXIP dimerization enables detailed investigations of microtubule plus-end-associated functions of individual SXIP-containing +TIPs.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Multimerização Proteica/fisiologia , Proteína da Polipose Adenomatosa do Colo/genética , Proteína da Polipose Adenomatosa do Colo/metabolismo , Motivos de Aminoácidos , Proteínas de Ciclo Celular , Linhagem Celular Tumoral , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas Associadas aos Microtúbulos/genética , Microtúbulos/genética , Proteínas do Tecido Nervoso/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismoRESUMO
Microtubules in animal cells assemble (nucleate) from both the centrosome and the cis-Golgi cisternae. A-kinase anchor protein 350 kDa (AKAP350A, also called AKAP450/CG-NAP/AKAP9) is a large scaffolding protein located at both the centrosome and Golgi apparatus. Previous findings have suggested that AKAP350 is important for microtubule dynamics at both locations, but how this scaffolding protein assembles microtubule nucleation machinery is unclear. Here, we found that overexpression of the C-terminal third of AKAP350A, enhanced GFP-AKAP350A(2691-3907), induces the formation of multiple microtubule-nucleation centers (MTNCs). Nevertheless, these induced MTNCs lacked "true" centriole proteins, such as Cep135. Mapping analysis with AKAP350A truncations demonstrated that AKAP350A contains discrete regions responsible for promoting or inhibiting the formation of multiple MTNCs. Moreover, GFP-AKAP350A(2691-3907) recruited several pericentriolar proteins to MTNCs, including γ-tubulin, pericentrin, Cep68, Cep170, and Cdk5RAP2. Proteomic analysis indicated that Cdk5RAP2 and Cep170 both interact with the microtubule nucleation-promoting region of AKAP350A, whereas Cep68 interacts with the distal C-terminal AKAP350A region. Yeast two-hybrid assays established a direct interaction of Cep170 with AKAP350A. Super-resolution and deconvolution microscopy analyses were performed to define the association of AKAP350A with centrosomes, and these studies disclosed that AKAP350A spans the bridge between centrioles, co-localizing with rootletin and Cep68 in the linker region. siRNA-mediated depletion of AKAP350A caused displacement of both Cep68 and Cep170 from the centrosome. These results suggest that AKAP350A acts as a scaffold for factors involved in microtubule nucleation at the centrosome and coordinates the assembly of protein complexes associating with the intercentriolar bridge.
Assuntos
Proteínas de Ancoragem à Quinase A/metabolismo , Centrossomo/metabolismo , Proteínas do Citoesqueleto/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Centro Organizador dos Microtúbulos/metabolismo , Modelos Moleculares , Proteínas do Tecido Nervoso/metabolismo , Fosfoproteínas/metabolismo , Proteínas de Ancoragem à Quinase A/antagonistas & inibidores , Proteínas de Ancoragem à Quinase A/química , Proteínas de Ancoragem à Quinase A/genética , Biomarcadores/metabolismo , Proteínas de Ciclo Celular , Linhagem Celular , Centrossomo/ultraestrutura , Proteínas do Citoesqueleto/antagonistas & inibidores , Proteínas do Citoesqueleto/química , Proteínas do Citoesqueleto/genética , Humanos , Imageamento Tridimensional , Peptídeos e Proteínas de Sinalização Intracelular/química , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas Luminescentes/química , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Microscopia Eletrônica de Transmissão , Proteínas Associadas aos Microtúbulos/química , Proteínas Associadas aos Microtúbulos/genética , Centro Organizador dos Microtúbulos/ultraestrutura , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/genética , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Fosfoproteínas/química , Fosfoproteínas/genética , Domínios e Motivos de Interação entre Proteínas , Mapeamento de Interação de Proteínas , Multimerização Proteica , Proteômica/métodos , Interferência de RNA , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Técnicas do Sistema de Duplo-HíbridoRESUMO
INTRODUCTION: Primary microcephaly type 3 is a genetically heterogeneous condition caused by a homozygous or compound heterozygous mutation in CDK5 regulatory subunit associated protein 2 (CDK5RAP2) and characterized by reduced head circumference (<5th percentile) with additional phenotypes varying from pigmentary abnormalities to sensorineural hearing loss. Until now, congenital cataracts have not been reported in patients with primary microcephaly type 3. CLINICAL REPORT: We report multiple affected family members from a consanguineous Saudi family with microcephaly and congenital cataracts. We utilized a next-generation sequencing-based microcephaly gene panel that revealed a CDK5RAP2 variant (c.4055A>G; p.Glu1352Gly) as the most plausible candidate for the likely etiology in this family. Then we performed family segregation analysis using Sanger sequencing, autozygosity mapping, and whole exome sequencing, all of which revealed no other possible disease-causing variants. CONCLUSION: Here we report on a new clinical manifestation of CDK5RAP2 and expand the phenotype of primary microcephaly type 3.
Assuntos
Catarata/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Microcefalia/genética , Proteínas do Tecido Nervoso/genética , Adolescente , Catarata/congênito , Proteínas de Ciclo Celular , Criança , Pré-Escolar , Consanguinidade , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Linhagem , Fenótipo , Arábia Saudita , Sequenciamento do ExomaRESUMO
INTRODUCTION: Because currently known Alzheimer's disease (AD) single-nucleotide polymorphisms only account for a small fraction of the genetic variance in this disease, there is a need to identify new variants associated with AD. METHODS: Our team performed a genome-wide association study in the Quebec Founder Population isolate to identify novel protective or risk genetic factors for late-onset sporadic AD and examined the impact of these variants on gene expression and AD pathology. RESULTS: The rs10984186 variant is associated with an increased risk of developing AD and with a higher CDK5RAP2 mRNA prevalence in the hippocampus. On the other hand, the rs4837766 variant, which is among the best cis-expression quantitative trait loci in the CDK5RAP2 gene, is associated with lower mild cognitive impairment/AD risk and conversion rate. DISCUSSION: The rs10984186 risk and rs4837766 protective polymorphic variants of the CDK5RAP2 gene might act as potent genetic modifiers for AD risk and/or conversion by modulating the expression of this gene.
Assuntos
Doença de Alzheimer/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas do Tecido Nervoso/genética , Proteínas tau/metabolismo , Idoso , Doença de Alzheimer/patologia , Proteínas de Ciclo Celular , Disfunção Cognitiva/genética , Feminino , Predisposição Genética para Doença , Estudo de Associação Genômica Ampla , Hipocampo/patologia , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Masculino , Proteínas do Tecido Nervoso/metabolismo , Polimorfismo de Nucleotídeo Único , Locos de Características QuantitativasRESUMO
Autosomal recessive primary microcephaly (MCPH) is a rare and heterogeneous genetic disorder characterized by reduced head circumference, low cognitive prowess and, in general, architecturally normal brains. As many as 14 different loci have already been mapped. We recruited 35 MCPH families in Pakistan and could identify the genetic cause of the disease in 31 of them. Using homozygosity mapping complemented with whole-exome, gene panel or Sanger sequencing, we identified 12 novel mutations in 3 known MCPH-associated genes - 9 in ASPM, 2 in MCPH1 and 1 in CDK5RAP2. The 2 MCPH1 mutations were homozygous microdeletions of 164,250 and 577,594 bp, respectively, for which we were able to map the exact breakpoints. We also identified four known mutations - three in ASPM and one in WDR62. The latter was initially deemed to be a missense mutation but we demonstrate here that it affects splicing. As to ASPM, as many as 17 out of 27 MCPH5 families that we ascertained in our sample were found to carry the previously reported founder mutation p.Trp1326*. This study adds to the mutational spectra of four known MCPH-associated genes and updates our knowledge about the genetic heterogeneity of MCPH in the Pakistani population considering its ethnic diversity.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/genética , Microcefalia/genética , Proteínas do Tecido Nervoso/genética , Proteínas de Ciclo Celular , Proteínas do Citoesqueleto , Feminino , Predisposição Genética para Doença , Homozigoto , Humanos , Masculino , Microcefalia/epidemiologia , Microcefalia/fisiopatologia , Mutação , Paquistão/epidemiologia , Linhagem , Sequenciamento do ExomaRESUMO
CDK5RAP2 is a centrosomal protein that regulates the recruitment of a γ-tubulin ring complex (γ-TuRC) onto centrosomes and microtubules (MTs) dynamics as a member of MT plus-end-tracking proteins (+TIPs). In our previous report, we found mammalian Cep169 as a CDK5RAP2 binding partner, and Cep169 accumulates at the distal ends of MTs and centrosomes, and coincides with CDK5RAP2. Depletion of Cep169 induces MT depolymerization, indicating that Cep169 targets MT tips and regulates stability and dynamics of MTs. However, how Cep169 contributes to the stabilization of MT remains unclear. Here we show that Cep169 is able to stabilize MTs and induces formation of long MT bundles with intense acetylation of MTs with CDK5RAP2, when expressed at higher levels in U2OS cells. In addition, we demonstrated that Cep169 forms homodimers through its N-terminal domain and directly interacts with MTs through its C-terminal domain. Interestingly, Cep169 mutants, which lack each domains, completely abolished the activity, respectively. Therefore, Cep169 bundles MTs and induces solid structure of MTs by crosslinking each adjacent MTs as a homodimer.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Sítios de Ligação , Proteínas de Ciclo Celular/genética , Linhagem Celular , Centrossomo/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Associadas aos Microtúbulos/genética , Mutação , Proteínas do Tecido Nervoso/metabolismo , Multimerização Proteica , Estrutura Terciária de Proteína , Tubulina (Proteína)/metabolismoRESUMO
Homozygous mutations in the cyclin-dependent kinase-5 regulatory subunit-associated protein 2 gene CDK5RAP2 cause primary autosomal recessive microcephaly (MCPH). MCPH is characterized by a pronounced reduction of brain volume, particularly of the cerebral cortex, and mental retardation. Though it is a rare developmental disorder, MCPH has moved into the spotlight of neuroscience because of its proposed central role in stem-cell biology and brain development. Investigation of the neural basis of genetically defined MCPH has been limited to animal studies and neuroimaging of affected patients as no neuropathological studies have been published. In the present study, we depict the spatiotemporal expression of CDK5RAP2 in the developing brain of mouse and human. We found intriguing concordance between regions of high CDK5RAP2 expression in the mouse and sites of pathology suggested by neuroimaging studies in humans and mouse. Our findings in human tissue confirm those in mouse tissues, underlining the function of CDK5RAP2 in cell proliferation and arguing for a conserved role of this protein in the development of the mammalian cerebral cortex.
Assuntos
Encéfalo/embriologia , Encéfalo/metabolismo , Proteínas de Ciclo Celular/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismo , Animais , Feto/metabolismo , Humanos , Camundongos , Microcefalia/embriologia , Microcefalia/metabolismoRESUMO
Autosomal recessive primary microcephaly type 3 (MCPH3) caused by pathogenic variations in CDK5RAP2, is characterized by sensorineural hearing loss, abnormality of skin pigmentation, ocular defects and severe microcephaly associated with neurodevelopmental delay. In this study, we expand the phenotype of MCPH3 as we describe a 10-year-old girl with a biallelic exonic frameshift variant in CDK5RAP2 displaying previously unreported features usually associated with Meier-Gorlin and microcephalic osteodysplastic primordial dwarfism type II (MOPDII). We further describe the clinical phenotype of this form of centrosomal-based primary microcephaly and emphasize the importance of skeletal defect screening in affected individuals.
Assuntos
Nanismo , Microcefalia , Osteocondrodisplasias , Feminino , Humanos , Microcefalia/patologia , Nanismo/genética , Nanismo/diagnóstico , Osteocondrodisplasias/genética , Osteocondrodisplasias/diagnóstico , Retardo do Crescimento Fetal/genética , Retardo do Crescimento Fetal/diagnóstico , Proteínas do Tecido Nervoso , Proteínas de Ciclo Celular/genéticaRESUMO
To establish the microtubule cytoskeleton, the cell must tightly regulate when and where microtubules are nucleated. This regulation involves controlling the initial nucleation template, the γ-tubulin ring complex (γTuRC). Although γTuRC is present throughout the cytoplasm, its activity is restricted to specific sites including the centrosome and Golgi. The well-conserved γ-tubulin nucleation activator (γTuNA) domain has been reported to increase the number of microtubules (MTs) generated by γTuRCs. However, previously we and others observed that γTuNA had a minimal effect on the activity of antibody-purified Xenopus γTuRCs in vitro (Thawani et al., eLife, 2020; Liu et al., 2020). Here, we instead report, based on improved versions of γTuRC, γTuNA, and our TIRF assay, the first real-time observation that γTuNA directly increases γTuRC activity in vitro, which is thus a bona fide γTuRC activator. We further validate this effect in Xenopus egg extract. Via mutation analysis, we find that γTuNA is an obligate dimer. Moreover, efficient dimerization as well as γTuNA's L70, F75, and L77 residues are required for binding to and activation of γTuRC. Finally, we find that γTuNA's activating effect opposes inhibitory regulation by stathmin. In sum, our improved assays prove that direct γTuNA binding strongly activates γTuRCs, explaining previously observed effects of γTuNA expression in cells and illuminating how γTuRC-mediated microtubule nucleation is regulated.
Assuntos
Microtúbulos , Tubulina (Proteína) , Animais , Tubulina (Proteína)/metabolismo , Microtúbulos/metabolismo , Centro Organizador dos Microtúbulos/metabolismo , Centrossomo/metabolismo , Xenopus laevis/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismoRESUMO
Centromeres are chromosomal loci where kinetochores assemble to ensure faithful chromosome segregation during mitosis. CENP-A defines the loci by serving as an epigenetic marker that recruits other centromere components for a functional structure. However, the mechanism that controls CENP-A regulation of centromeric chromatin integrity remains to be explored. Separate studies have shown that loss of CENP-A or the Cdk5 regulatory subunit associated protein 2 (Cdk5rap2), a key player in mitotic progression, triggers the occurrence of lagging chromosomes. This prompted us to investigate a potential link between CENP-A and Cdk5rap2 in the maintenance of centromeric chromatin integrity. Here, we demonstrate that loss of Cdk5rap2 causes reduced CENP-A expression while exogenous Cdk5rap2 expression in cells depleted of endogenous Cdk5rap2 restores CENP-A expression. Indeed, we show that Cdk5rap2 is a nuclear protein that acts as a positive transcriptional regulator of CENP-A. Cdk5rap2 interacts with the CENP-A promoter and upregulates CENP-A transcription. Accordingly, loss of Cdk5rap2 causes reduced level of centromeric CENP-A. Exogenous CENP-A expression partially inhibits the occurrence of lagging chromosomes in Cdk5rap2 knockdown cells, indicating that lagging chromosomes induced by loss of Cdk5rap2 is due, in part, to loss of CENP-A. Aside from manifesting lagging chromosomes, cells depleted of Cdk5rap2, and thus CENP-A, show increased micronuclei and chromatin bridge formation. Altogether, our findings indicate that Cdk5rap2 serves to maintain centromeric chromatin integrity partly through CENP-A.