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1.
ACS Synth Biol ; 7(7): 1685-1693, 2018 07 20.
Article in English | MEDLINE | ID: mdl-29975841

ABSTRACT

Nerve growth factor/tropomyosin receptor kinase A (NGF/TrkA) signaling plays a key role in neuronal development, function, survival, and growth. The pathway is implicated in neurodegenerative disorders including Alzheimer's disease, chronic pain, inflammation, and cancer. NGF binds the extracellular domain of TrkA, leading to the activation of the receptor's intracellular kinase domain. As TrkA signaling is highly dynamic, mechanistic studies would benefit from a tool with high spatial and temporal resolution. Here we present the design and evaluation of four strategies for light-inducible activation of TrkA in the absence of NGF. Our strategies involve the light-sensitive protein Arabidopsis cryptochrome 2 and its binding partner CIB1. We demonstrate successful recapitulation of native NGF/TrkA functions by optical induction of plasma membrane recruitment and homo-interaction of the intracellular domain of TrkA. This approach activates PI3K/AKT and Raf/ERK signaling pathways, promotes neurite growth in PC12 cells, and supports survival of dorsal root ganglion neurons in the absence of NGF. This ability to activate TrkA using light bestows high spatial and temporal resolution for investigating NGF/TrkA signaling.


Subject(s)
Receptor, trkA/metabolism , Animals , Cell Membrane/metabolism , Cell Survival/genetics , Cell Survival/physiology , Ganglia, Spinal/metabolism , Nerve Growth Factor/metabolism , PC12 Cells , Phosphatidylinositol 3-Kinases/genetics , Phosphatidylinositol 3-Kinases/metabolism , Phosphorylation/genetics , Phosphorylation/physiology , Rats , Receptor, trkA/genetics , Signal Transduction
3.
PLoS One ; 11(4): e0153487, 2016.
Article in English | MEDLINE | ID: mdl-27082641

ABSTRACT

Acute brain injuries such as ischemic stroke or traumatic brain injury often cause massive neural death and irreversible brain damage with grave consequences. Previous studies have established that a key participant in the events leading to neural death is the excessive production of reactive oxygen species. Protecting neuronal cells by activating their endogenous defense mechanisms is an attractive treatment strategy for acute brain injuries. In this work, we investigate how the precise timing of the Raf/ERK and the AKT pathway activation affects their protective effects against oxidative stress. For this purpose, we employed optogenetic systems that use light to precisely and reversibly activate either the Raf/ERK or the AKT pathway. We find that preconditioning activation of the Raf/ERK or the AKT pathway immediately before oxidant exposure provides significant protection to cells. Notably, a 15-minute transient activation of the Raf/ERK pathway is able to protect PC12 cells against oxidant strike that is applied 12 hours later, while the transient activation of the AKT pathway fails to protect PC12 cells in such a scenario. On the other hand, if the pathways are activated after the oxidative insult, i.e. postconditioning, the AKT pathway conveys greater protective effect than the Raf/ERK pathway. We find that postconditioning AKT activation has an optimal delay period of 2 hours. When the AKT pathway is activated 30min after the oxidative insult, it exhibits very little protective effect. Therefore, the precise timing of the pathway activation is crucial in determining its protective effect against oxidative injury. The optogenetic platform, with its precise temporal control and its ability to activate specific pathways, is ideal for the mechanistic dissection of intracellular pathways in protection against oxidative stress.


Subject(s)
Cytoprotection , Extracellular Signal-Regulated MAP Kinases/metabolism , Oxidative Stress/physiology , raf Kinases/metabolism , Animals , Cytoprotection/drug effects , Enzyme Activation/drug effects , Hydrogen Peroxide/pharmacology , MAP Kinase Signaling System/drug effects , Mice , NIH 3T3 Cells , Oxidative Stress/drug effects , PC12 Cells , Rats , Time Factors
4.
Chem Biol ; 22(5): 671-82, 2015 May 21.
Article in English | MEDLINE | ID: mdl-25963241

ABSTRACT

Intracellular transport and distribution of organelles play important roles in diverse cellular functions, including cell polarization, intracellular signaling, cell survival, and apoptosis. Here, we report an optogenetic strategy to control the transport and distribution of organelles by light. This is achieved by optically recruiting molecular motors onto organelles through the heterodimerization of Arabidopsis thaliana cryptochrome 2 (CRY2) and its interacting partner CIB1. CRY2 and CIB1 dimerize within subseconds upon exposure to blue light, which requires no exogenous ligands and low intensity of light. We demonstrate that mitochondria, peroxisomes, and lysosomes can be driven toward the cell periphery upon light-induced recruitment of kinesin, or toward the cell nucleus upon recruitment of dynein. Light-induced motor recruitment and organelle movements are repeatable, reversible, and can be achieved at subcellular regions. This light-controlled organelle redistribution provides a new strategy for studying the causal roles of organelle transport and distribution in cellular functions in living cells.


Subject(s)
Arabidopsis Proteins/metabolism , Basic Helix-Loop-Helix Transcription Factors/metabolism , Cryptochromes/metabolism , Optogenetics , Animals , Arabidopsis/metabolism , Arabidopsis Proteins/genetics , Basic Helix-Loop-Helix Transcription Factors/genetics , Biological Transport/radiation effects , COS Cells , Cell Nucleus/metabolism , Chlorocebus aethiops , Cryptochromes/genetics , Kinesins/metabolism , Kinetics , Light , Lysosomes/metabolism , Lysosomes/radiation effects , Microscopy, Fluorescence , Mitochondria/metabolism , Mitochondria/radiation effects , Protein Binding , Recombinant Fusion Proteins/biosynthesis , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics
5.
ACS Chem Neurosci ; 6(1): 130-7, 2015 Jan 21.
Article in English | MEDLINE | ID: mdl-25544156

ABSTRACT

U0126 is a potent and selective inhibitor of MEK1 and MEK2 kinases. It has been widely used as an inhibitor for the Ras/Raf/MEK/ERK signaling pathway with over 5000 references on the NCBI PubMed database. In particular, U0126 has been used in a number of studies to show that inhibition of the Raf/MEK/ERK pathway protects neuronal cells against oxidative stress. Here, we report that U0126 can function as an antioxidant that protects PC12 cells against a number of different oxidative-stress inducers. This protective effect of U0126 is independent of its function as a MEK inhibitor, as several other MEK inhibitors failed to show similar protective effects. U0126 reduces reactive oxygen species (ROS) in cells. We further demonstrate that U0126 is a direct ROS scavenger in vitro, and the oxidation products of U0126 exhibit fluorescence. Our finding that U0126 is a strong antioxidant signals caution for its future usage as a MEK inhibitor and for interpreting some previous results.


Subject(s)
Butadienes/pharmacology , Enzyme Inhibitors/pharmacology , MAP Kinase Kinase Kinases/antagonists & inhibitors , Nitriles/pharmacology , Oxidative Stress/drug effects , Animals , Cell Death/drug effects , Dose-Response Relationship, Drug , Hydrogen Peroxide/toxicity , Magnetic Resonance Imaging , PC12 Cells , Phosphorylation/drug effects , Rats , Reactive Oxygen Species/metabolism , Time Factors
6.
Cell Res ; 22(2): 387-98, 2012 Feb.
Article in English | MEDLINE | ID: mdl-21691298

ABSTRACT

Serum inducible kinase (SNK), also known as polo-like kinase 2 (PLK2), is a known regulator of mitosis, synaptogenesis and synaptic homeostasis. However, its role in early cortical development is unknown. Herein, we show that snk is expressed in the cortical plate from embryonic day 14, but not in the ventricular/subventricular zones (VZ/SVZ), and SNK protein localizes to the soma and dendrites of cultured immature cortical neurons. Loss of SNK impaired dendritic but not axonal arborization in a dose-dependent manner and overexpression had opposite effects, both in vitro and in vivo. Overexpression of SNK also caused abnormal branching of the leading process of migrating cortical neurons in electroporated cortices. The kinase activity was necessary for these effects. Extracellular signal-regulated kinase (ERK) pathway activity downstream of brain-derived neurotrophic factor (BDNF) stimulation led to increases in SNK protein expression via transcriptional regulation, and this upregulation was necessary for the growth-promoting effect of BDNF on dendritic arborization. Taken together, our results indicate that SNK is essential for dendrite morphogenesis in cortical neurons.


Subject(s)
Brain-Derived Neurotrophic Factor/metabolism , Dendrites/physiology , Protein Kinases/metabolism , Animals , Cells, Cultured , Dendrites/metabolism , Extracellular Signal-Regulated MAP Kinases/metabolism , Protein Kinases/chemistry , Protein Kinases/genetics , Protein Serine-Threonine Kinases , RNA Interference , RNA, Small Interfering , Rats , Signal Transduction , Transcription, Genetic
7.
Nat Genet ; 43(12): 1252-5, 2011 Nov 20.
Article in English | MEDLINE | ID: mdl-22101681

ABSTRACT

Paroxysmal kinesigenic dyskinesia is the most common type of paroxysmal movement disorder and is often misdiagnosed clinically as epilepsy. Using whole-exome sequencing followed by Sanger sequencing, we identified three truncating mutations within PRRT2 (NM_145239.2) in eight Han Chinese families with histories of paroxysmal kinesigenic dyskinesia: c.514_517delTCTG (p.Ser172Argfs*3) in one family, c.649dupC (p.Arg217Profs*8) in six families and c.972delA (p.Val325Serfs*12) in one family. These truncating mutations co-segregated exactly with the disease in these families and were not observed in 1,000 control subjects of matched ancestry. PRRT2 is a newly discovered gene consisting of four exons encoding the proline-rich transmembrane protein 2, which encompasses 340 amino acids and contains two predicted transmembrane domains. PRRT2 is highly expressed in the developing nervous system, and a truncating mutation alters the subcellular localization of the PRRT2 protein. The function of PRRT2 and its role in paroxysmal kinesigenic dyskinesia should be further investigated.


Subject(s)
Chorea/genetics , Exome , Frameshift Mutation , INDEL Mutation , Adolescent , Animals , Brain/metabolism , Case-Control Studies , Female , Gene Components , Gene Frequency , Genetic Association Studies , Genetic Linkage , Heredity , Humans , Male , Membrane Proteins , Mice , Mice, Inbred C57BL , Nerve Tissue Proteins , Organ Specificity , Pedigree , Protein Structure, Tertiary , Sequence Analysis, DNA , Spinal Cord/metabolism , Transcription, Genetic
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