Bio 599- Neural Migration II & III
Bio 599- Neural Migration II & III Bio 599
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This 9 page Class Notes was uploaded by Molly Burdsall on Thursday February 18, 2016. The Class Notes belongs to Bio 599 at University of South Carolina - Columbia taught by Dr. Fabienne Poulain in Winter 2016. Since its upload, it has received 6 views. For similar materials see Cellular and Molecular Biology in Biology at University of South Carolina - Columbia.
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Date Created: 02/18/16
16 February Review dynamic microtubules work with actin bundles several actin proteins regulate polarization and depolarization Family of Rho GPA regulate the cdc42 Cdk5- very important inguding Rhoa and Rac1 and Dynein very important in mechanism of centrosome Part II: Neuronal migration in the cerebral cortex Reminder: excitatory gltamertgic neurons (oriinate from progenitors in ventricular zone) and inhibitory neurons that populate the cortex from the outside and migrate tangentially to populate the cortex 6 different layers to cortex: progenitors I the ventricular and subventricule zone and produce progenitors and neurons which migrate o progenitor split into neurons and do radial migration Somal translocation as the first mode of radial migration at early stages o Video displays that the cell body translocates and brings cell body up into upper layer Glial-guided locomotion as a later mode of neuronal migration o Neurons that are formed use radial glaila process to migrate o Researchers: inject modified virus into the cortex and cut the brain into slices--- videomicroscopy of slices—neurons migrate along radial glial process o CONTACT BETWEEN NEURONS AND RADIAL GLAIAL FOR MIGRATION IS VERY IMPORTANT Glial-guided locomotion requires tight contacts between enruons and radial glia o Lead process of neuron-migrating neurons- o Molecules?: Integrins Integrins mediate adhesion between neurons and radial glia o Experiment: reproduce mode of transportation in neuron with micro glial and neuron (control) Addition of a3 integrin ibhibitor- Inhibitor placed after time one and completely blocks the migration of the neuron o Experiment: addition of a5 integrin ibhibitor- neurons do not even come intact with radial glial Integrins are transmembrane heterodimeric receptors linked to actin o Arp2/3 complex: proteins that make protein network o Integrins connected to cytoskeleton and produces traction Integrins mediate adhesion and migration o Proteins (with integrin) connected to actin cytoskeleton o Force of myosin II forces movement o PROVIDE THE TRACTION FOR MOVEMENT Gap junctions and radial migration o Connexins are required for radial migration Experiment: modified DNA injection at E16 Control: migration of neurons at E21 Cx 26 mutation (neurons don’t go as far- less migrate) Cx43 mutation (???) o Connexins form GAP junctions between cells This sytem mediates two things 1. Adhesion between two cells (binding stimulates adhesion) 2. Channel to transports molecules from one cell to another connexon is formed by 6 connexsins o connexins are at contact points between neurons and glia o The adhesion property of connexins is important for neuronal migration C-terminus: binds to other proteins and mediates transmission pathways (specific interaction) Adhesion: Channel: Experiment: Connexin-43 mutant mouse 1. Inject DNA injection at E16: GFP + connexin-43 domains In theory you should restore migration in mutatnt mouse Control: neurons migrate from ventrizclar zone to CP Mutant mouse: migration does not happen- neurons in VZ With connexin-43 domain: neurons do as they should-migrate to CP Connexin-43 domains to only channel and c-terminus but NO adhesion domain: neurons do not migrate Connext-43 domain to adhesion, c-terminus NO channel: neurons migrate as normal Connexin-43 domain to adhesion NO c-term or channel: restores migration-normal migration WHAT CAN WE CONCLUDE: ADHESION IS NEEDED FOR NORMAL MIGRATION Cues regulating radial migration o Where to stop? o The discovery of the Reeler mouse- ataxia, tremor, reeling gait The 6 layers are not distinguished in Reeler mouse— division of cortical layers is completely disrupted. Abnormal layers in the Reeler mouse Neurons are disorganized and almost reverse o Neurons are produced but migration is completely disrupted—cannot form the proper upper layers because it is disrupted o Reeling is a large secreted glycoprotein Reelin is detected in the marginal zone containing Cajal-Retzuis cells Red (reelin) is in marginal zone of cortex Special population of cells in upper region of cortex: cajal-retzius cells at marginal zone of cortex Reelin binds to VLDLR and ApoER2 receptors VLDLR: Very low density lipoprotein receptor ApoER2: apolipoprotein E Receptor 2 Effect of VLDLR and ApoER2 mutations Early-born neurons o Wild-type o Reeler mutatnt o VLDLR+ ApoER2 mutatnt: same effect as Reeler mutant Late neurons o Wild-type o Reeler mutatnt o V & A mutatnt: same effect as Reeler mutant Studying V and A independently o VLDLR mutatnt: neurons migrate TOO far in early and late migration No “stop” signal o ApoER2 mutant: early (migrate too far up) late (don’t migrate at all) No “go” signal Dual function of Reelin: migration and arrest o Reelin signaling to migration neurons The Reelin/Dab-1 transduction pathway Neurons that express VLDLR and ApoER2 Reelin arrives and binds to two receptors Leads to the activation of SFKs kineins Regulate (3) Dab-1 proteins (main regulator of Reelin) Dab-1 activates the chineses P13K P13K activates Rac 1 & Cdc42—inhibits GSK3B o Rac 1 & Cdc42- regulate actin o Rac 1 & Cdc42 modlules the polarization of actin polymerization and turnover o Inhibited GSK3B allows microtubule stabilization through APC One Dab-1 modulates Lis1 which allowd dynein and APC *** Lis1 can interacte with VLDLR but not ApoER2?? Other guidance cues involved o Semaphoring-3A guides radial migration Attracts or repels cells since it is a guidance cue Migration impaired if sema-3A is blocked Defect in migration if neuropilin-1 is blocked Review Later stages the neurons travel up radial glial Section of Reelin (cajal-Rezius cells) o Binds to VLDLR and ApoEr2 Tells Neuron migration (promotes and where to stop) and arrest Secretion of Sema-3A o Binds to Nrp1 and Plexin Promotes neuron migration and direction Contact of neurons and radial glaial is very important o 2 proteins that mediate the contact 1. Integrins 2. Connexins adhesion between neurons and radial glia, migration Case Study: Lissencephaly Lissencephaly: smooth brain, absence of folds and grooves Psychomotor retardation, seizures and epilepsy, failure to thrive, muscles spasticity, hypotonia, shorter life expectancy o Estimate 1/100,000 newborns Lissencephaly is caused by defects in neuronal migration Mutations found in these patients: o Depending on mutation there is a different class that differentiates slightly Mutation in Reelin-- Mutation in Lis 1 (named after the disease)-- TubA1a-- Dcx (double cortin)—mictrobules stabilization Feb. 18 Review Connexins make connexons which allow gap junction Tangential migration of interneurons Lateral geniculate emulance Medial geniculate emulance—neurnos mainly come from this site o Migrate transjunctinoally o Dynamic neurons- switch direction o Not guided by glial process- find their way in the environment of the cortex Steps of Neuron migration o 1. Excit from the proliferative zone in the mediagl ganglionic eminence (and POa), initiation of migration o 2. Selection of migratory route towards the cortex (pass LGE to find entry of the cortex) o 3. Choice of migratory stream within the cortex o 4. Local orientation of migration within the cortical wall o 5. Identification of final laminar location o 6. Termination of migration at the appropriate layer How do they exit the MGE? (step 1) o Ephrin-A5 is a chemotaxis cues localized in the MGE Ephrin-A5 repels interneurons Experiment: o Dissociation of neurons Culture: stripe assay (dark lines are control; light lines are ephrine-A5) Neurons tend to avoid ephrin- A%-Fc stripes and go towards control stripes o The family of Ephrins and their Eph receptors Two categories 1. Ephrin-A (encode at cell phase- no intracellular domain) 2. Ephrin-B (intracellular domain! have two domains??) o Both bind to Eph receptor (Eph-B and Eph-A receptor) Forward signaling: from ephrin to Eph Reverse signaling: from Eph to ephrin o Ephrin-A5 repels interneurons expressing EphA4 Ephrine-A5 produced in MGE which repels all of the neurons How do the migrate along the LGE and Str (Step 2) o The role of neurotropic and growth factors Family made up of four members 1. NGF 2. NT4, BDNF 3. NT3 4. NGF, BDNF, NT3, NT4 GDNF (very important) Not part of the same family since it doesn’t have the same receptor (not same category of molecule) but still a growth factor that has similar functions to the family Neurotropic factors are motogenic factors (doesn’t give direction- just tells cells to move) Experiement: o Culture of explants (addition of factors) Control: All neurons leaving MGE Add BDNG: Add NT4: Experiment: o Addition of beads coated with factors implanted near LGF Control: BSA-coasted bead: neurons stay away from bead GDNF-coasted beads: neurons surround bead In RET mutant: neurons around bead In GRFa1 mutant: not as many neurons around the bead Conclusion about experiment: o 1. GDNF contributes to interneurons migration o 2. GFRa1 allows neurons to respond to GDNF Migration of interneurons to the cortex slide- REVIEW OF WHAT WE LEARNED—Ephrin-A5--- Neurotrophic factors: GDNF, BDNF, NT-4 Review of Striatum Role of Striatum in the interneuron migration o Reward, planning of movement Neurons avoid the Striatum (step 3) o Experiment: Transplant ectopic piece of striatum o Semaphorin-3A and -3f are present in the striatum o Experiment: *interneurons shown in red-Dil Implant cells (not even brain cells-just cell) at the entry of the cortex (on each entry-2 sides) Control: produce Gfp Engineering the cell to produce Sema3A, Sema 3F, Gfp o Interneurons avoid Sema3A & Sema3F- repels interneurons o Semaphorins are big families 2 receptors: Neuropilins & Plexins o Experiment: Control: avoid striatum Neuropilin-2 mutant: cover the whole area Neuropilin (receptor of Sema-3A) is absolutely required in order to have neurons repel against the region o Semaphorins are responsible for sorting MGE interneurons to their final destination Interneurons to the cortex: Express NRp-1 and Nrp-2 Repelled by striatal Sema-3s Interneurons to the striatum: Do NOT have nrp-1 and Nrp-2 Do NOT have… Neuregulin-1 (Nrg1): one gene, different isoforms o Type 1 (Ig) o Type 2 (Ig) o Type 3 (CRD) Nrg1-Ig is detected near the cortex (step 4??) o Receptor: ErbB4- present at the face of interneurons Hypothesis: Nrg1-Ig is a magnet for neurons- attracts migrating MGE interneurons o Experiment: NrG1-Ig + Gfp into COS cells Neurons are drawn to this area o Experiment: Interneurons lacking ErbB4 lose directionality Control: neurons move towards cortex ErbB4 mutatnt: neurons still move but loose their direction Most interneurons migrate through the MZ and SVZ (few migrate to upper part of SP) o The chemokine Cxcl12 (Sdf-1) is produced by the meninges and the intermediate progenitors o Cxcl12 attracts interneurons migrating in the cortex Cxcr4 and Cxcr7 receptors Experiment: Control: neurons go to appropriate places and migrate Lack Cxcr4 and/or cxcr7: populate the cortex but do not find their way along the strings- pattern of migration is impaired Colonization of the cortical plate o Interneurons colonize the cortex in an inside-out manner Experiment: Early brdu injection: populate lower layer of the cortex Late BrdU injection: populate upper layer of the cortex o Interneurons lose their responsiveness to Cxcl12 over time (desensitization) *hang out here for awhile and then become desensitized so they stop migrating tangential and start migrating radially— me typing Interneurons switch from tangential to radial migration and migrate along radial glia (to populate different layers of the cortex) o Pyramidal neurons determine interneuron final destination Control: Migration of pyramidal neurons ibhibited By affecting pyramidal neurons you also effect the signal sent by pyramidal neurons which affects the migration of interneurons Neurons stay in I, IZ, SVZ layers because they responde to Clcx12 o Loose response to Clcx12 and switch to radial migration instead of tangital migration Populate different layers of the cortex (pyramidal cells give a signal to neurons of where to stop)
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