ABSTRACT
Heterozygous deletions of 17p13.3 result in the human neuronal migration disorders isolated lissencephaly sequence (ILS) and the more severe Miller-Dieker syndrome (MDS). Mutations in PAFAH1B1 (the gene encoding LIS1) are responsible for ILS and contribute to MDS, but the genetic causes of the greater severity of MDS are unknown. Here, we show that the gene encoding 14-3-3epsilon (YWHAE), one of a family of ubiquitous phosphoserine/threonine-binding proteins, is always deleted in individuals with MDS. Mice deficient in Ywhae have defects in brain development and neuronal migration, similar to defects observed in mice heterozygous with respect to Pafah1b1. Mice heterozygous with respect to both genes have more severe migration defects than single heterozygotes. 14-3-3epsilon binds to CDK5/p35-phosphorylated NUDEL and this binding maintains NUDEL phosphorylation. Similar to LIS1, deficiency of 14-3-3epsilon results in mislocalization of NUDEL and LIS1, consistent with reduction of cytoplasmic dynein function. These results establish a crucial role for 14-3-3epsilon in neuronal development by sustaining the effects of CDK5 phosphorylation and provide a molecular explanation for the differences in severity of human neuronal migration defects with 17p13.3 deletions.
Subject(s)
Abnormalities, Multiple/pathology , Brain Diseases/pathology , Brain/abnormalities , Cell Cycle Proteins/metabolism , Cell Movement , Enzyme Inhibitors/metabolism , Tyrosine 3-Monooxygenase/metabolism , 1-Alkyl-2-acetylglycerophosphocholine Esterase , 14-3-3 Proteins , Abnormalities, Multiple/genetics , Abnormalities, Multiple/metabolism , Animals , Brain Diseases/genetics , Brain Diseases/metabolism , Cells, Cultured , Coatomer Protein/metabolism , Cyclin-Dependent Kinase 5 , Cyclin-Dependent Kinases/metabolism , Dyneins/metabolism , Female , Green Fluorescent Proteins , Humans , Immunoenzyme Techniques , Luminescent Proteins/metabolism , Male , Mice , Mice, Inbred C57BL , Microtubule-Associated Proteins/metabolism , Neurons/cytology , Phosphoprotein Phosphatases/metabolism , Protein Kinase C/antagonists & inhibitors , Syndrome , Tyrosine 3-Monooxygenase/geneticsABSTRACT
Phosphorylation of neurotransmitter receptors can modify their activity and regulate neuronal excitability. Cyclin-dependent kinase 5 (cdk5) is a proline-directed serine/threonine kinase involved not only in neuronal development, but also in synaptic function and plasticity. Here we demonstrate that group I metabotropic glutamate receptors (mGluRs), which modulate post-synaptic signaling by coupling to intracellular signal transduction pathways, are phosphorylated by cdk5. In vitro kinase assays reveal that cdk5 phosphorylates mGluR5 within the domain of the receptor that interacts with the scaffolding protein homer. Using a novel phosphospecific mGluR antibody, we show that the homer-binding domain of both mGluR1 and mGluR5 are phosphorylated in vivo, and that inhibition of cdk5 with siRNA decreases the amount of phosphorylated receptor. Furthermore, kinetic binding analysis, by surface plasmon resonance, indicates that phosphorylation of mGluR5 enhances its association with homer. Homer protein complexes in the post-synaptic density, and their disruption by an activity-dependent short homer 1a isoform, have been shown to regulate the trafficking and signaling of the mGluRs and impact many neuroadaptive processes. Phosphorylation of the mGluR homer-binding domain, in contrast to homer 1a induction, provides a novel mechanism for potentially regulating a subset of homer interactions.
Subject(s)
Carrier Proteins/metabolism , Cyclin-Dependent Kinase 5/metabolism , Receptors, Metabotropic Glutamate/metabolism , Amino Acid Sequence , Animals , COS Cells , Carrier Proteins/chemistry , Cells, Cultured , Chlorocebus aethiops , Cyclin-Dependent Kinase 5/chemistry , Homer Scaffolding Proteins , Humans , Mice , Mice, Knockout , Molecular Sequence Data , Phosphorylation/physiology , Protein Binding/physiology , Rats , Rats, Sprague-Dawley , Receptors, AMPA/metabolism , Receptors, Kainic Acid/metabolism , Receptors, Metabotropic Glutamate/chemistryABSTRACT
Correct neuronal migration and positioning during cortical development are essential for proper brain function. Mutations of the LIS1 gene result in human lissencephaly (smooth brain), which features misplaced cortical neurons and disarrayed cerebral lamination. However, the mechanism by which LIS1 regulates neuronal migration remains unknown. Using RNA interference (RNAi), we found that the binding partner of LIS1, NudE-like protein (Ndel1, formerly known as NUDEL), positively regulates dynein activity by facilitating the interaction between LIS1 and dynein. Loss of function of Ndel1, LIS1, or dynein in developing neocortex impairs neuronal positioning and causes the uncoupling of the centrosome and nucleus. Overexpression of LIS1 partially rescues the positioning defect caused by Ndel1 RNAi but not dynein RNAi, whereas overexpression of Ndel1 does not rescue the phenotype induced by LIS1 RNAi. These results provide strong evidence that Ndel1 interacts with LIS1 to sustain the function of dynein, which in turn impacts microtubule organization, nuclear translocation, and neuronal positioning.
Subject(s)
Carrier Proteins/metabolism , Cell Movement/physiology , Dyneins/metabolism , Microtubule-Associated Proteins/physiology , Neocortex/embryology , Neurons/cytology , 1-Alkyl-2-acetylglycerophosphocholine Esterase , Animals , Blotting, Western , COS Cells , Chlorocebus aethiops , Cytoplasm/metabolism , Mice , Models, Neurological , Signal TransductionABSTRACT
Cyclin-dependent kinase 5 (Cdk5) is activated on binding of activator proteins p35 and p39. A N-terminally truncated p35, termed p25, is generated through cleavage by the Ca(2+)-dependent protease calpain after induction of ischemia in rat brain. p25 has been shown to accumulate in brains of patients with Alzheimer's disease and may contribute to A-beta peptide-mediated toxicity. Studies from transfected neurons as well as p35 and p25 transgenic mice have indicated that Cdk5, when activated by p25, gains some toxic function compared with p35/Cdk5. It remains unclear, however, whether p25/Cdk5 signaling additionally channels into pathways usually used by p35/Cdk5 and whether p25 is associated with a loss of p35 function. To clarify these issues, we have generated p25-transgenic mice in a p35-null background. We find that low levels of p25 during development induce a partial rescue of the p35-/- phenotype in several brain regions analyzed, including a rescue of cell positioning of a subset of neurons in the neocortex. In accordance with the partial rescue of brain anatomy, phosphorylation of the Cdk5 substrate mouse disabled 1 is partially restored during development. Besides this, p25/Cdk5 fails to phosphorylate other substrates that are normally phosphorylated by p35/Cdk5. Our results show that p25 can substitute for p35/Cdk5 under certain circumstances during development. In addition, they suggest that p25 may have lost some functions of p35.
Subject(s)
Brain/cytology , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/physiology , Phosphopyruvate Hydratase/genetics , Aging , Animals , Brain/embryology , Brain/enzymology , Brain/pathology , Cerebellum/cytology , Cerebral Cortex/cytology , Corpus Callosum/cytology , Cyclin-Dependent Kinase 5 , Cyclin-Dependent Kinases/metabolism , Gene Expression , Hippocampus/cytology , Mice , Mice, Knockout , Mice, Transgenic , Nerve Tissue Proteins/analysis , Nerve Tissue Proteins/metabolism , Neurodegenerative Diseases/pathology , Neurons/classification , Neurons/enzymology , Phenotype , Phosphorylation , Signal TransductionABSTRACT
In this study, we developed human immune system (HIS) mice that possess functional human CD4+ T cells and B cells, named HIS-CD4/B mice. HIS-CD4/B mice were generated by first introducing HLA class II genes, including DR1 and DR4, along with genes encoding various human cytokines and human B cell activation factor (BAFF) to NSG mice by adeno-associated virus serotype 9 (AAV9) vectors, followed by engrafting human hematopoietic stem cells (HSCs). HIS-CD4/B mice, in which the reconstitution of human CD4+ T and B cells resembles to that of humans, produced a significant level of human IgG against Plasmodium falciparum circumsporozoite (PfCS) protein upon immunization. CD4+ T cells in HIS-CD4/B mice, which possess central and effector memory phenotypes like those in humans, are functional, since PfCS protein-specific human CD4+ T cells secreting IFN-γ and IL-2 were detected in immunized HIS-CD4/B mice. Lastly, PfCS protein-immunized HIS-CD4/B mice were protected from in vivo challenge with transgenic P. berghei sporozoites expressing the PfCS protein. The immune sera collected from protected HIS-CD4/B mice reacted against transgenic P. berghei sporozoites expressing the PfCS protein and also inhibited the parasite invasion into hepatocytes in vitro. Taken together, these studies show that our HIS-CD4/B mice could mount protective human anti-malaria immunity, consisting of human IgG and human CD4+ T cell responses both specific for a human malaria antigen.
Subject(s)
Disease Models, Animal , Immunity, Humoral/immunology , Malaria, Falciparum/immunology , Mice, Transgenic/immunology , Animals , Enzyme-Linked Immunosorbent Assay , Heterografts , Histocompatibility Antigens Class II , Humans , Malaria Vaccines , Mice , Protozoan Proteins/immunologyABSTRACT
The circumsporozoite protein (CSP) of Plasmodium falciparum is a major surface protein, which forms a dense coat on the sporozoite's surface. Preclinical research on CSP and clinical evaluation of a CSP fragment-based RTS, S/AS01 vaccine have demonstrated a modest degree of protection against P. falciparum, mediated in part by humoral immunity and in part by cell-mediated immunity. Given the partial protective efficacy of the RTS, S/AS01 vaccine in a recent Phase 3 trial, further improvement of CSP-based vaccines is crucial. In this report, we describe the preclinical development of a full-length, recombinant CSP (rCSP)-based vaccine candidate against P. falciparum malaria suitable for current Good Manufacturing Practice (cGMP) production. Utilizing a novel high-throughput Pseudomonas fluorescens expression platform, we demonstrated greater efficacy of full-length rCSP as compared to N-terminally truncated versions, rapidly down-selected a promising lead vaccine candidate, and developed a high-yield purification process to express immunologically active, intact antigen for clinical trial material production. The rCSP, when formulated with various adjuvants, induced antigen-specific antibody responses as measured by enzyme-linked immunosorbent assay (ELISA) and immunofluorescence assay (IFA), as well as CD4+ T-cell responses as determined by ELISpot. The adjuvanted rCSP vaccine conferred protection in mice when challenged with transgenic P. berghei sporozoites containing the P. falciparum repeat region of CSP. Furthermore, heterologous prime/boost regimens with adjuvanted rCSP and an adenovirus type 35-vectored CSP (Ad35CS) showed modest improvements in eliciting CSP-specific T-cell responses and anti-malarial protection, depending on the order of vaccine delivery. Collectively, these data support the importance of further clinical development of adjuvanted rCSP, either as a stand-alone product or as one of the components in a heterologous prime/boost strategy, ultimately acting as an effective vaccine candidate for the mitigation of P. falciparum-induced malaria.
Subject(s)
Malaria Vaccines/administration & dosage , Malaria, Falciparum/prevention & control , Plasmodium falciparum/immunology , Protozoan Proteins/metabolism , Pseudomonas fluorescens/genetics , Recombinant Proteins/metabolism , Adjuvants, Immunologic/pharmacology , Animals , Disease Models, Animal , Female , Hep G2 Cells , Humans , Malaria Vaccines/chemistry , Malaria, Falciparum/immunology , Mice , Mice, Inbred C57BL , Organisms, Genetically Modified , Protozoan Proteins/genetics , Pseudomonas fluorescens/metabolism , Recombinant Proteins/genetics , Recombinant Proteins/immunology , Vaccination/methodsABSTRACT
The availability of high quality, well-characterized antibodies for molecular and cellular neuroscience studies is important. However, not all available antibodies are rigorously evaluated, nor are limitations of particular antibodies often reported. We have examined a panel of currently available mGluR1 antibodies and have identified which ones are selective for use by western blots and immunocytochemistry. We have also specifically determined whether the antibodies cross-react to recognize mGluR5, by examining (1) tissue from both mGluR1 and mGluR5 knock-out mice and (2) primary cortical cultures, in which mGluR5 is widely expressed but mGluR1 is not. Together, these data provide a baseline characterization of antibodies that can and cannot be reliably used in these types of studies, and will hopefully facilitate and positively impact the research efforts of others studying mGluR1.
Subject(s)
Antibodies/metabolism , Antibody Specificity , Neurons/metabolism , Receptors, Metabotropic Glutamate/immunology , Animals , Animals, Newborn , Cells, Cultured , Cerebral Cortex/cytology , Gene Expression/genetics , Immunohistochemistry , Indoles , Mice , Mice, Knockout , Receptor, Metabotropic Glutamate 5 , Receptors, Metabotropic Glutamate/deficiencyABSTRACT
The effective utilization of stem cells in regenerative medicine critically relies upon our understanding of the intricate interactions between cells and their extracellular environment. While bulk mechanical and chemical properties of the matrix have been shown to influence various cellular functions, the role of matrix interfacial properties on stem cell behavior is unclear. Here, we report the striking effect of matrix interfacial hydrophobicity on stem cell adhesion, motility, cytoskeletal organization, and differentiation. This is achieved through the development of tunable, synthetic matrices with control over their hydrophobicity without altering the chemical and mechanical properties of the matrix. The observed cellular responses are explained in terms of hydrophobicity-driven conformational changes of the pendant side chains at the interface leading to differential binding of proteins. These results demonstrate that the hydrophobicity of the extracellular matrix could play a considerably larger role in dictating cellular behaviors than previously anticipated. Additionally, these tunable matrices, which introduce a new control feature for regulating various cellular functions offer a platform for studying proliferation and differentiation of stem cells in a controlled manner and would have applications in regenerative medicine.
Subject(s)
Hydrogels/chemistry , Mesenchymal Stem Cells/cytology , Tissue Scaffolds/chemistry , Biocompatible Materials/chemistry , Cell Adhesion , Cell Differentiation , Cell Movement , Humans , Hydrophobic and Hydrophilic InteractionsABSTRACT
Lis1 and Ndel1 are essential for animal development. They interact directly with one another and with cytoplasmic dynein. The developing brain is especially sensitive to reduced Lis1 or Ndel1 levels, as both proteins influence spindle orientation, neural cell fate decisions, and neuronal migration. We report here that Lis1 and Ndel1 reduction in a mitotic cell line impairs prophase nuclear envelope (NE) invagination (PNEI). This dynein-dependent process facilitates NE breakdown (NEBD) and occurs before the establishment of the bipolar spindle. Ndel1 phosphorylation is important for this function, regulating binding to both Lis1 and dynein. Prophase cells in the ventricular zone (VZ) of embryonic day 13.5 Lis1(+/-) mouse brains show reduced PNEI, and the ratio of prophase to prometaphase cells is increased, suggesting an NEBD delay. Moreover, prophase cells in the VZ contain elevated levels of Ndel1 phosphorylated at a key cdk5 site. Our data suggest that a delay in NEBD in the VZ could contribute to developmental defects associated with Lis1-Ndel1 disruption.
Subject(s)
1-Alkyl-2-acetylglycerophosphocholine Esterase/metabolism , Carrier Proteins/metabolism , Microtubule-Associated Proteins/metabolism , Neurons , Nuclear Envelope/metabolism , Stem Cells , 1-Alkyl-2-acetylglycerophosphocholine Esterase/genetics , Animals , COS Cells , Carrier Proteins/genetics , Cell Cycle/physiology , Cell Line , Chlorocebus aethiops , Dynactin Complex , Dyneins/metabolism , Female , Humans , Male , Mice , Microtubule-Associated Proteins/genetics , Microtubules/metabolism , Neurons/cytology , Neurons/physiology , Nocodazole/metabolism , Phosphorylation , Protein Binding , Rats , Stem Cells/cytology , Stem Cells/metabolism , Tubulin Modulators/metabolismABSTRACT
Ndel1, the mammalian homologue of the Aspergillus nidulans NudE, is emergently viewed as an integrator of the cytoskeleton. By regulating the dynamics of microtubules and assembly of neuronal intermediate filaments (IFs), Ndel1 promotes neurite outgrowth, neuronal migration, and cell integrity (1-6). To further understand the roles of Ndel1 in cytoskeletal dynamics, we performed a tandem affinity purification of Ndel1-interacting proteins. We isolated a novel Ndel1 molecular complex composed of the IF vimentin, the molecular motor dynein, the lissencephaly protein Lis1, and the cis-Golgi-associated protein alphaCOP. Ndel1 promotes the interaction between Lis1, alphaCOP, and the vimentin-dynein complex. The functional result of this complex is activation of dynein-mediated transport of vimentin. A loss of Ndel1 functions by RNA interference fails to incorporate Lis1/alphaCOP in the complex, reduces the transport of vimentin, and culminates in IF accumulations and altered neuritogenesis. Our findings reveal a novel regulatory mechanism of vimentin transport during neurite extension that may have implications in diseases featuring transport/trafficking defects and impaired regeneration.
Subject(s)
Dyneins/metabolism , Microtubule-Associated Proteins/metabolism , Neurites/metabolism , Vimentin/metabolism , 1-Alkyl-2-acetylglycerophosphocholine Esterase/metabolism , Animals , Chromatography, Affinity , HeLa Cells , Humans , Mice , Microtubule-Associated Proteins/isolation & purification , Microtubules/metabolism , Models, Biological , Nerve Tissue Proteins/metabolism , Protein Binding , Protein Transport , RNA Interference , Two-Hybrid System TechniquesABSTRACT
The correct positioning of neurons during development--achieved through directed migration--is the basis for proper brain function. Several decades of research have yielded a comprehensive map illustrating the temporal and spatial events underlying neurogenesis and neuronal migration during development. The discovery of distinct migration modes and pathways has been accompanied by the identification of a large interwoven molecular network that transmits extracellular signals into the cell. Moreover, recent work has shed new light on how the cytoskeleton is regulated and coordinated at the molecular and cellular level to execute neuronal migration.