scholarly journals Tubulinopathy mutations in TUBA1A that disrupt neuronal morphogenesis and migration override XMAP215/Stu2 regulation of microtubule dynamics

2021 ◽  
Author(s):  
Katelyn J. Hoff ◽  
Jayne E. Aiken ◽  
Mark A. Gutierrez ◽  
Santos J. Franco ◽  
Jeffrey K. Moore
Keyword(s):  
Molecular Mechanisms ◽  
Ectopic Expression ◽  
Intermediate Phenotype ◽  
Neuronal Cultures ◽  
Missense Mutations ◽  
Primary Neuronal Cultures ◽  
Neuronal Morphogenesis ◽  
Brain Malformations ◽  
Wide Range ◽  
And Migration

ABSTRACTHeterozygous, missense mutations in α- or β-tubulin genes are associated with a wide range of human brain malformations, known as tubulinopathies. We seek to understand whether a mutation’s impact at the molecular and cellular levels scale with the severity of brain malformation. Here we focus on two mutations at the valine 409 residue of TUBA1A, V409I and V409A, identified in patients with pachygyria or lissencephaly, respectively. We find that ectopic expression of TUBA1A-V409I/A mutants disrupt neuronal migration in mice and promote excessive neurite branching and delayed retraction in primary neuronal cultures, accompanied by increased microtubule acetylation. To determine the molecular mechanisms, we modeled the V409I/A mutants in budding yeast and found that they promote intrinsically faster microtubule polymerization rates in cells and in reconstitution experiments with purified tubulin. In addition, V409I/A mutants decrease the recruitment of XMAP215/Stu2 to plus ends and ablate tubulin binding to TOG domains. In each assay tested, the TUBA1A-V409I mutant exhibits an intermediate phenotype between wild type and the more severe TUBA1A-V409A, reflecting the severity observed in brain malformations. Together, our data support a model in which the V409I/A mutations may limit tubulin conformational states and thereby disrupt microtubule regulation during neuronal morphogenesis and migration.

scholarly journals TUBA1A mutations identified in lissencephaly patients dominantly disrupt neuronal migration and impair dynein activity

2018 ◽  
Vol 28 (8) ◽  
pp. 1227-1243 ◽  
Author(s):  
Jayne Aiken ◽  
Jeffrey K Moore ◽  
Emily A Bates
Keyword(s):  
Motor Activity ◽  
Neuronal Migration ◽  
Ectopic Expression ◽  
Missense Mutations ◽  
Microtubule Cytoskeleton ◽  
Tubulin Genes ◽  
Dynein Motor ◽  
Brain Malformations ◽  
Developing Mouse Brain ◽  
And Migration

Abstract The microtubule cytoskeleton supports diverse cellular morphogenesis and migration processes during brain development. Mutations in tubulin genes are associated with severe human brain malformations known as ‘tubulinopathies’; however, it is not understood how molecular-level changes in microtubule subunits lead to brain malformations. In this study, we demonstrate that missense mutations affecting arginine at position 402 (R402) of TUBA1A α-tubulin selectively impair dynein motor activity and severely and dominantly disrupt cortical neuronal migration. TUBA1A is the most commonly affected tubulin gene in tubulinopathy patients, and mutations altering R402 account for 30% of all reported TUBA1A mutations. We show for the first time that ectopic expression of TUBA1A-R402C and TUBA1A-R402H patient alleles is sufficient to dominantly disrupt cortical neuronal migration in the developing mouse brain, strongly supporting a causal role in the pathology of brain malformation. To isolate the precise molecular impact of R402 mutations, we generated analogous R402C and R402H mutations in budding yeast α-tubulin, which exhibit a simplified microtubule cytoskeleton. We find that R402 mutant tubulins assemble into microtubules that support normal kinesin motor activity but fail to support the activity of dynein motors. Importantly, the level of dynein impairment scales with the expression level of the mutant in the cell, suggesting a ‘poisoning’ mechanism in which R402 mutant α-tubulin acts dominantly by populating microtubules with defective binding sites for dynein. Based on our results, we propose a new model for the molecular pathology of tubulinopathies that may also extend to other tubulin-related neuropathies.

scholarly journals Pathogenic DDX3X mutations impair RNA metabolism and neurogenesis during fetal cortical development

2018 ◽  
Author(s):  
Ashley L. Lennox ◽  
Ruiji Jiang ◽  
Lindsey Suit ◽  
Brieana Fregeau ◽  
Charles J. Sheehan ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
De Novo ◽  
Cortical Development ◽  
Rna Helicase ◽  
Mouse Genetics ◽  
Missense Mutations ◽  
Neurodevelopmental Disease ◽  
Brain Malformations ◽  
And Migration ◽  
Human And Mouse

AbstractDe novo germline mutations in the RNA helicase DDX3X account for 1-3% of unexplained intellectual disability (ID) cases in females, and are associated with autism, brain malformations, and epilepsy. Yet, the developmental and molecular mechanisms by which DDX3X mutations impair brain function are unknown. Here we use human and mouse genetics, and cell biological and biochemical approaches to elucidate mechanisms by which pathogenic DDX3X variants disrupt brain development. We report the largest clinical cohort to date with DDX3X mutations (n=78), demonstrating a striking correlation between recurrent dominant missense mutations, polymicrogyria, and the most severe clinical outcomes. We show that Ddx3x controls cortical development by regulating neuronal generation and migration. Severe DDX3X missense mutations profoundly disrupt RNA helicase activity and induce ectopic RNA-protein granules and aberrant translation in neural progenitors and neurons. Together, our study demonstrates novel mechanisms underlying DDX3X syndrome, and highlights roles for RNA-protein aggregates in the pathogenesis of neurodevelopmental disease.

scholarly journals MiR-203 Targets to the 3′-UTR of SLUG to Suppress Cerebral Infarction-Induced Endothelial Cell Growth and Motility

2021 ◽  
Vol 2021 ◽  
pp. 1-7
Author(s):  
Yunsong Li ◽  
Wei Bi ◽  
Bing Han ◽  
Tao Yuan ◽  
Long Shi ◽  
...  
Keyword(s):  
Cerebral Infarction ◽  
Molecular Mechanisms ◽  
Vascular Endothelial Cells ◽  
Umbilical Vein ◽  
Critical Role ◽  
Ectopic Expression ◽  
Serum Samples ◽  
Invasion And Migration ◽  
And Migration

Cerebral infarction is one of the leading causes of death worldwide, in which angiogenesis plays a critical role. On the other hand, accumulating evidence has demonstrated that microRNAs (miRNAs) function as key modulators in the formation and progression of cerebral infarction. However, the molecular mechanisms of miRNAs underlying cerebral infarction-associated angiogenesis remain unclear. In the present study, we indicated that the expression of miR-203 was significantly downregulated in serum samples derived from patients with cerebral infarction and in mice brain samples following middle cerebral artery occlusion (MCAO) compared with healthy controls. In vitro, the expression of miR-203 was obviously downregulated in hypoxia-induced human umbilical vein vascular endothelial cells (HUVECs). Functionally, ectopic expression of miR-203 drastically suppressed HUVEC proliferation, invasion, and migration. In addition, SLUG, a zinc finger transcriptional repressor, was identified as a direct target of miR-203 and was negatively correlated with miR-203 expression in MCAO mice and in hypoxia-induced HUVECs. Furthermore, overexpression of SLUG reversed the inhibitory effect of miR-203 on proliferation, invasion, and migration abilities of HUVECs. Taken together, our research provides a novel insight of the miR-203-SLUG axis into cerebral infarction-associated endothelial behaviors and may offer a powerful therapeutic target of cerebral ischemia.

scholarly journals Phosphorylation of p66Shc and forkhead proteins mediates Aβ toxicity

2005 ◽  
Vol 169 (2) ◽  
pp. 331-339 ◽  
Author(s):  
Wanli W. Smith ◽  
Darrell D. Norton ◽  
Myriam Gorospe ◽  
Haibing Jiang ◽  
Shino Nemoto ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Redox Regulation ◽  
Critical Role ◽  
Ectopic Expression ◽  
Amyloid Β ◽  
Dominant Negative ◽  
Neuronal Cultures ◽  
Amyloid Β Peptide ◽  
Dependent Manner ◽  
Primary Neuronal Cultures

Excessive accumulation of amyloid β-peptide (Aβ) plays an early and critical role in synapse and neuronal loss in Alzheimer's Disease (AD). Increased oxidative stress is one of the mechanisms whereby Aβ induces neuronal death. Given the lessened susceptibility to oxidative stress exhibited by mice lacking p66Shc, we investigated the role of p66Shc in Aβ toxicity. Treatment of cells and primary neuronal cultures with Aβ caused apoptotic death and induced p66Shc phosphorylation at Ser36. Ectopic expression of a dominant-negative SEK1 mutant or chemical JNK inhibition reduced Aβ-induced JNK activation and p66Shc phosphorylation (Ser36), suggesting that JNK phosphorylates p66Shc. Aβ induced the phosphorylation and hence inactivation of forkhead transcription factors in a p66Shc-dependent manner. Ectopic expression of p66ShcS36A or antioxidant treatment protected cells against Aβ-induced death and reduced forkhead phosphorylation, suggesting that p66Shc phosphorylation critically influences the redox regulation of forkhead proteins and underlies Aβ toxicity. These findings underscore the potential usefulness of JNK, p66Shc, and forkhead proteins as therapeutic targets for AD.

scholarly journals Isoflurane but not Fentanyl Causes Apoptosis in Immature Primary Neuronal Cells

2017 ◽  
Vol 11 (1) ◽  
pp. 39-47
Author(s):  
Monika Berns ◽  
Anna Christine Wolter ◽  
Christoph Bührer ◽  
Stefanie Endesfelder ◽  
Thoralf Kerner
Keyword(s):  
Cell Viability ◽  
Neuronal Cells ◽  
Neuronal Cultures ◽  
Primary Neurons ◽  
Primary Neuronal Cultures ◽  
Amino Butyric Acid ◽  
Wide Range ◽  
Primary Neuronal Cells ◽  
The Impact

Background: Anaesthetics are widely used in new-borns and preterm infants, although it is known that they may adversely affect the developing brain. Objective: We assessed the impact of the volatile anaesthetic, isoflurane, and the intravenous analgesic, fentanyl, on immature and mature embryonic neuronal cells. Methods: Primary neuronal cultures from embryonic rats (E18) cultured for 5 (immature) or 15 days (mature) in vitro (DIV), respectively, were exposed to isoflurane (1.5 Vol.%) or fentanyl (0.8 - 200 ng/ml) for 24 hours. Experiments were repeated in the presence of the γ-amino butyric acid-A (GABAA) receptor antagonists, bicuculline or picrotoxin (0.1 mmol/l), or the pancaspase inhibitor zVAD-fmk (20 nmol/l). Cell viability was assessed by methyltetrazolium (MTT) metabolism or lactate dehydrogenase (LDH) release. Results: Isoflurane reduced cell viability significantly in primary neuronal cells cultured for 5 DIV (Δ MTT -28 ±13%, Δ LDH +143 ±15%). Incubation with bicuculline, picrotoxin or zVAD-fmk protected the cells mostly from isoflurane toxicity. After 15 DIV, cell viability was not reduced by isoflurane. Viability of primary neurons cultured for 5 DIV did not change with fentanyl over the wide range of concentrations tested. Conclusion: Immature primary neurons may undergo apoptosis following exposure to isoflurane but are unaffected by fentanyl. Mature primary neurons were not affected by isoflurane exposure.

Expression of the Cell Polarity Protein PTK7 in Acute Myeloid Leukaemia.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1478-1478
Author(s):  
Thomas Prebet ◽  
Anne Catherine Lhoumeau ◽  
Christine Arnoulet ◽  
Sylvie Marchetto ◽  
Christian Chabannon ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Cell Polarity ◽  
Molecular Mechanisms ◽  
Ectopic Expression ◽  
Leukemia Cell ◽  
Tyrosine Kinase Receptor ◽  
Low Grade ◽  
Potential Implication ◽  
Wide Range ◽  
Low Levels

Abstract The planar cell polarity pathway plays a major role in embryogenesis and tissue organisation. Recent genetic studies have highlighted the role of novel receptors and signaling molecules implicated in this pathway. Amongst the receptors, the pseudo tyrosine kinase receptor 7 (PTK7) is an orphean tyrosine kinase receptor with kinase-dead activity. Knock-out of PTK7 in mice strongly affects embryonic development leading to a major neural tube defect. Presence of PTK7 was previously investigated in epithelial and endothelial cells that both express the receptor. In normal donors, we found no expression of PTK7 in peripheral blood (n=5) whereas PTK7 expression was found with low levels in PBPC after G-CSF stimulation (n=3) and high levels in normal myeloid progenitors and CD34+ CD38− bone marrow cells (n=3). Overexpression of PTK7 was already described in solid tumors including breast, lung and pancreatic cancers. We decided to study the potential implication of PTK7 in haematological malignancies. We performed a wide range multicolour immunophenotyping screen on more than 240 patient samples treated at Institut Paoli-Calmettes and 10 leukemia cell lines. In hematologic malignancies, we demonstrated that PTK7 was widely expressed in AML (136 of 195 patients) and in the most immature subsets of Acute lymphoblastic (5 of 20 patients) or biphenotypic leukaemia (3 of 3 patients). We found no expression of PTK7 in chronic disorder such low grade NHL (n=7), CLL (n=6) or Chronic Myelomonocytic Leukemia (n=3). In AML, we demonstrated that PTK7 expression mostly correlates with granulocytic lineage differentiation and that it could be partially expressed in AML 4 or 5 subsets. Flow cytometry analysis confirmed the co-expression of PTK7 with granulocytic lineage markers and that PTK7 expression in myelomonocytic leukaemia was limited to the myeloid subset of blasts. The strongest immunophenotyping correlation was found with CD117/c-Kit expression (p<0.001) and PTK7 Mean Fluorescence Intensity directly correlates with c-Kit MFI (p=0.001). Interestingly, stimulation of cultured TF1 cells (that endogenously express c-Kit and PTK7) with SCF triggered an increased expression of PTK7. Correlation between PTK7 expression and biological or clinical features was also evaluated. We demonstrated that PTK7 expression clustered with some cytogenetic subsets (high levels in CBF AML (n=19) or APL (n=13), low levels in FLT3 mutated AML(n=17) or complex karyotype (n=20)). We also found that PTK7 expression was associated with a lower WBC count at diagnosis (p=0.001) and a lower frequency of extramedullary disease (p<0.001) in whole population and in both AML1–3 and AML 4–5 subgroups. We report here novel findings that potentially implicate ptk7, a PCP gene, in hematopoiesis and AML. In vitro, we showed that ectopic expression of PTK7 promotes cell migration, cell survival and resistance to anthracyclin-induced apoptosis. Ongoing works are currently investigating which molecular mechanisms regulate PTK7 functions in normal and pathological situations.

In vivo functions of small GTPases in neocortical development

2014 ◽  
Vol 395 (5) ◽  
pp. 465-476 ◽  
Author(s):  
Bhavin Shah ◽  
Andreas W. Püschel
Keyword(s):  
Neuronal Development ◽  
Small Gtpases ◽  
Neuronal Cultures ◽  
Primary Neuronal Cultures ◽  
Cellular Processes ◽  
Neuronal Progenitors ◽  
Embryonic Nervous System ◽  
And Migration ◽  
Cytoskeletal Rearrangements

Abstract The complex mammalian cortex develops from a simple neuroepithelium through the proliferation of neuronal progenitors, their asymmetric division and cell migration. Newly generated neurons transiently assume a multipolar morphology before they polarize to form a trailing axon and a leading process that is required for their radial migration. The polarization and migration events during cortical development are under the control of multiple signaling cascades that coordinate the different cellular processes involved in neuronal differentiation. GTPases perform essential functions at different stages of neuronal development as central components of these pathways. They have been widely studied using cell lines and primary neuronal cultures but their physiological function in vivo still remains to be explored in many cases. Here we review the function of GTPases that have been studied genetically by the analysis of the embryonic nervous system in knockout mice. The phenotype of these mutants has highlighted the importance of GTPases for different steps of development by orchestrating cytoskeletal rearrangements and neuronal polarization.

scholarly journals P2Y Purinergic Regulation of the Glycine Neurotransmitter Transporters

2011 ◽  
Vol 286 (12) ◽  
pp. 10712-10724 ◽  
Author(s):  
Esperanza Jiménez ◽  
Francisco Zafra ◽  
Raquel Pérez-Sen ◽  
Esmerilda G. Delicado ◽  
Maria Teresa Miras-Portugal ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Protein Kinase ◽  
Molecular Mechanisms ◽  
Synaptic Cleft ◽  
Cyclic Guanosine Monophosphate ◽  
Guanosine Monophosphate ◽  
Neuronal Cultures ◽  
Primary Neuronal Cultures ◽  
Neurotransmitter Transporters ◽  
Stimulation Of

The sodium- and chloride-coupled glycine neurotransmitter transporters (GLYTs) control the availability of glycine at glycine-mediated synapses. The mainly glial GLYT1 is the key regulator of the glycine levels in glycinergic and glutamatergic pathways, whereas the neuronal GLYT2 is involved in the recycling of synaptic glycine from the inhibitory synaptic cleft. In this study, we report that stimulation of P2Y purinergic receptors with 2-methylthioadenosine 5′-diphosphate in rat brainstem/spinal cord primary neuronal cultures and adult rat synaptosomes leads to the inhibition of GLYT2 and the stimulation of GLYT1 by a paracrine regulation. These effects are mainly mediated by the ADP-preferring subtypes P2Y1 and P2Y13 because the effects are partially reversed by the specific antagonists N6-methyl-2′-deoxyadenosine-3′,5′-bisphosphate and pyridoxal-5′-phosphate-6-azo(2-chloro-5-nitrophenyl)-2,4-disulfonate and are totally blocked by suramin. P2Y12 receptor is additionally involved in GLYT1 stimulation. Using pharmacological approaches and siRNA-mediated protein knockdown methodology, we elucidate the molecular mechanisms of GLYT regulation. Modulation takes place through a signaling cascade involving phospholipase C activation, inositol 1,4,5-trisphosphate production, intracellular Ca2+ mobilization, protein kinase C stimulation, nitric oxide formation, cyclic guanosine monophosphate production, and protein kinase G-I (PKG-I) activation. GLYT1 and GLYT2 are differentially sensitive to NO/cGMP/PKG-I both in brain-derived preparations and in heterologous systems expressing the recombinant transporters and P2Y1 receptor. Sensitivity to 2-methylthioadenosine 5′-diphosphate by GLYT1 and GLYT2 was abolished by small interfering RNA (siRNA)-mediated knockdown of nitric-oxide synthase. Our data may help define the role of GLYTs in nociception and pain sensitization.

scholarly journals Loss of Dmrt5 Affects the Formation of the Subplate and Early Corticogenesis

2019 ◽  
Vol 30 (5) ◽  
pp. 3296-3312 ◽  
Author(s):  
Leslie Ratié ◽  
Elodie Desmaris ◽  
Fernando García-Moreno ◽  
Anna Hoerder-Suabedissen ◽  
Alexandra Kelman ◽  
...  
Keyword(s):  
Cortical Neurons ◽  
Marginal Zone ◽  
Cortical Plate ◽  
Neuronal Cultures ◽  
Primary Neuronal Cultures ◽  
Time Sensitivity ◽  
Proliferation And Differentiation ◽  
Cortical Patterning ◽  
Summary Statement ◽  
And Migration

Abstract Dmrt5 (Dmrta2) and Dmrt3 are key regulators of cortical patterning and progenitor proliferation and differentiation. In this study, we show an altered apical to intermediate progenitor transition, with a delay in SP neurogenesis and premature birth of Ctip2+ cortical neurons in Dmrt5−/− mice. In addition to the cortical progenitors, DMRT5 protein appears present in postmitotic subplate (SP) and marginal zone neurons together with some migrating cortical neurons. We observed the altered split of preplate and the reduced SP and disturbed radial migration of cortical neurons into cortical plate in Dmrt5−/− brains and demonstrated an increase in the proportion of multipolar cells in primary neuronal cultures from Dmrt5−/− embryonic brains. Dmrt5 affects cortical development with specific time sensitivity that we described in two conditional mice with slightly different deletion time. We only observed a transient SP phenotype at E15.5, but not by E18.5 after early (Dmrt5lox/lox;Emx1Cre), but not late (Dmrt5lox/lox;NestinCre) deletion of Dmrt5. SP was less disturbed in Dmrt5lox/lox;Emx1Cre and Dmrt3−/− brains than in Dmrt5−/− and affects dorsomedial cortex more than lateral and caudal cortex. Our study demonstrates a novel function of Dmrt5 in the regulation of early SP formation and radial cortical neuron migration. Summary Statement Our study demonstrates a novel function of Dmrt5 in regulating marginal zone and subplate formation and migration of cortical neurons to cortical plate.

scholarly journals Multi-model functionalization of disease-associated PTEN missense mutations identifies multiple molecular mechanisms underlying protein dysfunction

2019 ◽  
Author(s):  
Kathryn L. Post ◽  
Manuel Belmadani ◽  
Payel Ganguly ◽  
Fabian Meili ◽  
Riki Dingwall ◽  
...  
Keyword(s):  
Protein Function ◽  
Molecular Mechanisms ◽  
Autism Spectrum ◽  
Model Systems ◽  
Missense Mutations ◽  
Loss Of Function ◽  
Neuronal Morphogenesis ◽  
Full Characterization ◽  
Protein Functions ◽  
Mutation Impact

ABSTRACTFunctional variomics provides the foundation for personalized medicine by linking genetic variation to disease expression, outcome and treatment, yet its utility is dependent on appropriate assays to evaluate mutation impact on protein function. To fully assess the effects of 106 missense and nonsense variants of PTEN associated with autism spectrum disorder, somatic cancer and PHTS, we take a deep phenotypic profiling approach using 18 assays in 5 model systems spanning diverse cellular environments ranging from molecular function to neuronal morphogenesis and behavior. Variants inducing instability occurred across the protein, resulting in partial to complete loss of function (LoF), which was well correlated across models. However, assays were selectively sensitive to variants located in substrate binding and catalytic domains, which exhibited complete LoF or dominant negativity independent of effects on stability. Our results indicate that full characterization of variant impact requires assays sensitive to instability and a range of protein functions.

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