Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 11 de 11
Filter
Add more filters










Publication year range
1.
Life Sci Alliance ; 3(7)2020 07.
Article in English | MEDLINE | ID: mdl-32414840

ABSTRACT

During development, neurons adjust their energy balance to meet the high demands of robust axonal growth and branching. The mechanisms that regulate this tuning are largely unknown. Here, we show that sensory neurons lacking liver kinase B1 (Lkb1), a master regulator of energy homeostasis, exhibit impaired axonal growth and branching. Biochemical analysis of these neurons revealed reduction in axonal ATP levels, whereas transcriptome analysis uncovered down-regulation of Efhd1 (EF-hand domain family member D1), a mitochondrial Ca2+-binding protein. Genetic ablation of Efhd1 in mice resulted in reduced axonal morphogenesis as well as enhanced neuronal death. Strikingly, this ablation causes mitochondrial dysfunction and a decrease in axonal ATP levels. Moreover, Efhd1 KO sensory neurons display shortened mitochondria at the axonal growth cones, activation of the AMP-activated protein kinase (AMPK)-Ulk (Unc-51-like autophagy-activating kinase 1) pathway and an increase in autophagic flux. Overall, this work uncovers a new mitochondrial regulator that is required for axonal morphogenesis.


Subject(s)
Axons/metabolism , Calcium-Binding Proteins/genetics , Gene Expression Regulation, Developmental , Mitochondrial Proteins/genetics , Neurogenesis/genetics , Neurons/cytology , Neurons/metabolism , Adenosine Triphosphate , Animals , Base Sequence , Biomarkers , Calcium-Binding Proteins/metabolism , Cell Polarity/genetics , Cells, Cultured , Fluorescent Antibody Technique , Mice , Mice, Knockout , Mice, Transgenic , Mitochondria/genetics , Mitochondria/metabolism , Mitochondrial Proteins/metabolism , Morphogenesis/genetics , Sensory Receptor Cells/cytology , Sensory Receptor Cells/metabolism
2.
Proc Natl Acad Sci U S A ; 116(49): 24639-24650, 2019 12 03.
Article in English | MEDLINE | ID: mdl-31754024

ABSTRACT

Proteasome-mediated degradation of intracellular proteins is essential for cell function and survival. The proteasome-binding protein PI31 (Proteasomal Inhibitor of 31kD) promotes 26S assembly and functions as an adapter for proteasome transport in axons. As localized protein synthesis and degradation is especially critical in neurons, we generated a conditional loss of PI31 in spinal motor neurons (MNs) and cerebellar Purkinje cells (PCs). A cKO of PI31 in these neurons caused axon degeneration, neuronal loss, and progressive spinal and cerebellar neurological dysfunction. For both MNs and PCs, markers of proteotoxic stress preceded axonal degeneration and motor dysfunction, indicating a critical role for PI31 in neuronal homeostasis. The time course of the loss of MN and PC function in developing mouse central nervous system suggests a key role for PI31 in human neurodegenerative diseases.


Subject(s)
Carrier Proteins/metabolism , Motor Neurons/physiology , Neurodegenerative Diseases/genetics , Proteostasis/physiology , Purkinje Cells/physiology , Synapses/physiology , Animals , Axons/pathology , Axons/physiology , Behavior Observation Techniques , Carrier Proteins/genetics , Cell Survival/physiology , Disease Models, Animal , Female , Humans , Mice , Mice, Knockout , Motor Neurons/pathology , Mutation , Neurodegenerative Diseases/diagnosis , Neurodegenerative Diseases/pathology , Proteasome Endopeptidase Complex/metabolism , Proteolysis , Purkinje Cells/pathology , Synapses/pathology
3.
Dev Cell ; 50(4): 509-524.e10, 2019 08 19.
Article in English | MEDLINE | ID: mdl-31327739

ABSTRACT

Protein degradation by the ubiquitin-proteasome system is critical for neuronal function. Neurons utilize microtubule-dependent molecular motors to allocate proteasomes to synapses, but how proteasomes are coupled to motors and how this is regulated to meet changing demand for protein breakdown remain largely unknown. We show that the conserved proteasome-binding protein PI31 serves as an adaptor to couple proteasomes with dynein light chain proteins (DYNLL1/2). The inactivation of PI31 inhibited proteasome motility in axons and disrupted synaptic proteostasis, structure, and function. Moreover, phosphorylation of PI31 by p38 MAPK enhanced binding to DYNLL1/2 and promoted the directional movement of proteasomes in axons, suggesting a mechanism to regulate loading of proteasomes onto motors. Inactivation of PI31 in mouse neurons attenuated proteasome movement in axons, indicating this process is conserved. Because mutations affecting PI31 activity are associated with human neurodegenerative diseases, impairment of PI31-mediated axonal transport of proteasomes may contribute to these disorders.


Subject(s)
Neurons/metabolism , Proteins/genetics , Proteolysis , Synapses/genetics , Adaptor Proteins, Signal Transducing/genetics , Animals , Axonal Transport/genetics , Axons/metabolism , Cytoplasm/genetics , Cytoplasm/metabolism , Cytoplasmic Dyneins/genetics , Humans , Mice , Microtubules/genetics , Proteasome Endopeptidase Complex/genetics , Proteostasis/genetics , Synapses/metabolism , p38 Mitogen-Activated Protein Kinases/genetics
4.
Cancer Res ; 78(13): 3458-3468, 2018 07 01.
Article in English | MEDLINE | ID: mdl-29716915

ABSTRACT

Protein degradation by the ubiquitin-proteasome system (UPS) is central to protein homeostasis and cell survival. The active 26S proteasome is a large protease complex consisting of a catalytic 20S subunit and 19S regulatory particles. Cancer cells are exposed to considerable protein overload due to high metabolic rates, reprogrammed energy metabolism, and aneuploidy. Here we report a mechanism that facilitates the assembly of active 26S proteasomes in malignant cells. Upon tumorigenic transformation of the gut epithelium, 26S proteasome assembly was significantly enhanced, but levels of individual subunits were not changed. This enhanced assembly of 26S proteasomes increased further with tumor progression and was observed specifically in transformed cells, but not in other rapidly dividing cells. Moreover, expression of PSMD5, an inhibitor of proteasome assembly, was reduced in intestinal tumors and silenced with tumor progression. Reexpression of PSMD5 in tumor cells caused decreased 26S assembly and accumulation of polyubiquitinated proteins. These results suggest that inhibition of cancer-associated proteasome assembly may provide a novel therapeutic strategy to selectively kill cancer cells.Significance: Enhanced cancer-associated proteasome assembly is a major stress response that allows tumors to adapt to and to withstand protein overload.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/78/13/3458/F1.large.jpg Cancer Res; 78(13); 3458-68. ©2018 AACR.


Subject(s)
Cell Transformation, Neoplastic/pathology , Colorectal Neoplasms/pathology , Proteasome Endopeptidase Complex/metabolism , Adenomatous Polyposis Coli Protein/genetics , Animals , Azoxymethane/toxicity , Colorectal Neoplasms/chemically induced , Colorectal Neoplasms/genetics , Dextran Sulfate/toxicity , Disease Progression , Female , Gene Expression Regulation, Neoplastic , HEK293 Cells , HT29 Cells , Humans , Male , Mice, Transgenic , Neoplasms, Experimental/chemically induced , Neoplasms, Experimental/genetics , Neoplasms, Experimental/pathology , Proteasome Endopeptidase Complex/genetics
5.
Dev Cell ; 37(1): 1-2, 2016 Apr 04.
Article in English | MEDLINE | ID: mdl-27046823

ABSTRACT

In this issue of Developmental Cell, Aram et al. (2016) identify a mechanism that uses a Krebs cycle protein to control local activation of a ubiquitin ligase complex at the mitochondrial outer membrane for temporally and spatially restricted caspase activation during Drosophila sperm differentiation.


Subject(s)
Caspases/metabolism , Citric Acid Cycle/physiology , Cullin Proteins/metabolism , Drosophila Proteins/metabolism , Drosophila/metabolism , Mitochondria/metabolism , Spermatids/metabolism , Animals , Male
7.
Sci Signal ; 7(316): ra24, 2014 Mar 11.
Article in English | MEDLINE | ID: mdl-24619647

ABSTRACT

Guidance receptor signaling is crucial for neural circuit formation and elicits diverse cellular events in specific neurons. We found that signaling from the guidance cue semaphorin 3A diverged through distinct cytoplasmic domains in its receptor Plexin-A4 to promote disparate cellular behavior in different neuronal cell types. Plexin-A4 has three main cytoplasmic domains--C1, Hinge/RBD, and C2--and interacts with family members of the Rho guanine nucleotide exchange factor FARP proteins. We show that growth cone collapse occurred in Plexin-A4-deficient dorsal root ganglion sensory neurons reconstituted with Plexin-A4 containing either the Hinge/RBD or C2 domain, whereas both of the Hinge/RBD and C1 domains were required for dendritic arborization in cortical neurons. Although knockdown studies indicated that both the collapse and arborization responses involved FARP2, mutations in the cytoplasmic region of Plexin-A4 that reduced its interaction with FARP2 strongly inhibited semaphorin 3A-induced dendritic branching but not growth cone collapse, suggesting that different degrees of interaction are required for the two responses or that developing axons have an indirect path to FARP2 activation. Thus, our study provided insights into the multifunctionality of guidance receptors, in particular showing that the semaphorin 3A signal diverges through specific functions of the modular domains of Plexin-A4.


Subject(s)
Growth Cones/physiology , Nerve Tissue Proteins/metabolism , Nervous System/embryology , Receptors, Cell Surface/metabolism , Semaphorin-3A/metabolism , Sensory Receptor Cells/metabolism , Signal Transduction/physiology , Adaptor Proteins, Signal Transducing/metabolism , Animals , Blotting, Western , COS Cells , Chlorocebus aethiops , Cytoplasm/metabolism , Ganglia, Spinal/cytology , Guanine Nucleotide Exchange Factors/metabolism , HEK293 Cells , Humans , Immunohistochemistry , Immunoprecipitation , Mice , Protein Structure, Tertiary , RNA, Small Interfering/genetics , Sensory Receptor Cells/cytology
8.
Dev Neurobiol ; 74(3): 365-81, 2014 Mar.
Article in English | MEDLINE | ID: mdl-24127433

ABSTRACT

RNA localization is a regulatory mechanism that is conserved from bacteria to mammals. Yet, little is known about the mechanism and the logic that govern the distribution of RNA transcripts within the cell. Here, we present a novel organ culture system, which enables the isolation of RNA specifically from NGF dependent re-growing peripheral axons of mouse embryo, sensory neurons. In combination with massive parallel sequencing technology, we determine the subcellular localization of most transcripts in the transcriptome. We found that the axon is enriched in mRNAs that encode secreted proteins, transcription factors, and the translation machinery. In contrast, the axon was largely depleted from mRNAs encoding transmembrane proteins, a particularly interesting finding, since many of these gene products are specifically expressed in the tip of the axon at the protein level. Comparison of the mitochondrial mRNAs encoded in the nucleus with those encoded in the mitochondria, uncovered completely different localization pattern, with the latter much enriched in the axon fraction. This discovery is intriguing since the protein products encoded by the nuclear and mitochondrial genome form large co-complexes. Finally, focusing on alternative splice variants that are specific to axonal fractions, we find short sequence motifs that are enriched in the axonal transcriptome. Together our findings shed light on the extensive role of RNA localization and its characteristics.


Subject(s)
Axons/metabolism , Ganglia, Spinal/metabolism , RNA, Messenger/metabolism , Sensory Receptor Cells/metabolism , Transcriptome , Alternative Splicing , Animals , Cell Nucleus/metabolism , High-Throughput Nucleotide Sequencing , Mice , Mice, Inbred ICR , Mitochondria/metabolism , Presynaptic Terminals/metabolism , RNA, Mitochondrial , Tissue Culture Techniques
9.
Nature ; 495(7442): 474-80, 2013 Mar 28.
Article in English | MEDLINE | ID: mdl-23474986

ABSTRACT

CLP1 was the first mammalian RNA kinase to be identified. However, determining its in vivo function has been elusive. Here we generated kinase-dead Clp1 (Clp1(K/K)) mice that show a progressive loss of spinal motor neurons associated with axonal degeneration in the peripheral nerves and denervation of neuromuscular junctions, resulting in impaired motor function, muscle weakness, paralysis and fatal respiratory failure. Transgenic rescue experiments show that CLP1 functions in motor neurons. Mechanistically, loss of CLP1 activity results in accumulation of a novel set of small RNA fragments, derived from aberrant processing of tyrosine pre-transfer RNA. These tRNA fragments sensitize cells to oxidative-stress-induced p53 (also known as TRP53) activation and p53-dependent cell death. Genetic inactivation of p53 rescues Clp1(K/K) mice from the motor neuron loss, muscle denervation and respiratory failure. Our experiments uncover a mechanistic link between tRNA processing, formation of a new RNA species and progressive loss of lower motor neurons regulated by p53.


Subject(s)
Motor Neurons/metabolism , Motor Neurons/pathology , RNA, Transfer, Tyr/metabolism , Transcription Factors/metabolism , Amyotrophic Lateral Sclerosis , Animals , Animals, Newborn , Axons/metabolism , Axons/pathology , Cell Death , Diaphragm/innervation , Embryo Loss , Embryo, Mammalian/metabolism , Embryo, Mammalian/pathology , Exons/genetics , Female , Fibroblasts , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Mice, Transgenic , Muscular Atrophy, Spinal , Neuromuscular Diseases/metabolism , Neuromuscular Diseases/pathology , Oxidative Stress , RNA Processing, Post-Transcriptional , RNA, Transfer, Tyr/genetics , RNA-Binding Proteins , Respiration , Spinal Nerves/cytology , Transcription Factors/deficiency , Tumor Suppressor Protein p53/metabolism , Tyrosine/genetics , Tyrosine/metabolism
10.
PLoS Biol ; 9(9): e1001149, 2011 Sep.
Article in English | MEDLINE | ID: mdl-21931534

ABSTRACT

Bacterial superantigens, a diverse family of toxins, induce an inflammatory cytokine storm that can lead to lethal shock. CD28 is a homodimer expressed on T cells that functions as the principal costimulatory ligand in the immune response through an interaction with its B7 coligands, yet we show here that to elicit inflammatory cytokine gene expression and toxicity, superantigens must bind directly into the dimer interface of CD28. Preventing access of the superantigen to CD28 suffices to block its lethality. Mice were protected from lethal superantigen challenge by short peptide mimetics of the CD28 dimer interface and by peptides selected to compete with the superantigen for its binding site in CD28. Superantigens use a conserved ß-strand/hinge/α-helix domain of hitherto unknown function to engage CD28. Mutation of this superantigen domain abolished inflammatory cytokine gene induction and lethality. Structural analysis showed that when a superantigen binds to the T cell receptor on the T cell and major histocompatibility class II molecule on the antigen-presenting cell, CD28 can be accommodated readily as third superantigen receptor in the quaternary complex, with the CD28 dimer interface oriented towards the ß-strand/hinge/α-helix domain in the superantigen. Our findings identify the CD28 homodimer interface as a critical receptor target for superantigens. The novel role of CD28 as receptor for a class of microbial pathogens, the superantigen toxins, broadens the scope of pathogen recognition mechanisms.


Subject(s)
CD28 Antigens/immunology , Cytokines/genetics , Shock, Septic/immunology , Superantigens/immunology , Amino Acid Sequence , Animals , Bacterial Toxins/immunology , CD28 Antigens/genetics , Cell Line, Tumor , Cytokines/immunology , Enterotoxins/immunology , Epitope Mapping , Escherichia coli/genetics , Escherichia coli/metabolism , Female , Gene Expression Regulation , Genetic Vectors , Humans , Immunity, Cellular , Leukocytes, Mononuclear/cytology , Leukocytes, Mononuclear/immunology , Mice , Mice, Inbred BALB C , Molecular Sequence Data , Protein Binding , Receptors, Antigen, T-Cell/immunology , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/immunology , Shock, Septic/genetics , Staphylococcus aureus/genetics , Staphylococcus aureus/immunology , Superantigens/administration & dosage , Surface Plasmon Resonance
11.
J Neurosci ; 30(18): 6375-86, 2010 May 05.
Article in English | MEDLINE | ID: mdl-20445064

ABSTRACT

Selective degeneration of neuronal projections and neurite pruning are critical for establishment and maintenance of functional neural circuits in both insects and mammals. However, the molecular mechanisms that govern developmental neurite pruning versus injury-induced neurite degeneration are still mostly unclear. Here, we show that the effector caspases 6 and 3 are both expressed within axons and that, on trophic deprivation, they exhibit distinct modes of activation. Surprisingly, inhibition of caspases is not sufficient for axonal protection and a parallel modulation of a NAD(+)-sensitive pathway is required. The proapoptotic protein BAX is a key element in both pathways as its genetic ablation protected sensory axons against developmental degeneration both in vitro and in vivo. Last, we demonstrate that both pathways are also involved in developmental dendritic pruning in Drosophila. More specifically, the mouse Wld(S) (Wallerian degeneration slow) protein, which is mainly composed of the full-length sequence of the NAD(+) biosynthetic Nmnat1 enzyme, can suppress dendritic pruning in C4da (class IV dendritic arborization) sensory neurons in parallel to the fly effector caspases. These findings indicate that two distinct autodestruction pathways act separately or in concert to regulate developmental neurite pruning.


Subject(s)
Caspases/genetics , Drosophila Proteins/genetics , Drosophila , NAD/pharmacology , Nerve Degeneration/prevention & control , Nerve Tissue Proteins/genetics , Signal Transduction/genetics , bcl-2-Associated X Protein/genetics , Animals , Apoptosis Regulatory Proteins/antagonists & inhibitors , Apoptosis Regulatory Proteins/genetics , Apoptosis Regulatory Proteins/metabolism , Axons/metabolism , Caspase 3/metabolism , Caspase 6/metabolism , Caspase Inhibitors , Cells, Cultured , Dendrites/metabolism , Drosophila/drug effects , Drosophila/embryology , Drosophila/genetics , Drosophila/metabolism , Enzyme Inhibitors/pharmacology , Ganglia, Spinal/anatomy & histology , Ganglia, Spinal/embryology , Ganglia, Spinal/metabolism , Mice , Mice, Knockout , Mice, Transgenic , Nerve Degeneration/metabolism , Nerve Tissue Proteins/metabolism , Sensory Receptor Cells/metabolism , Signal Transduction/drug effects , bcl-2-Associated X Protein/metabolism
SELECTION OF CITATIONS
SEARCH DETAIL
...