in-vivo-postnatal-electroporation-time-lapse-imaging-neuroblast

<em>In vivo</em> Postnatal Electroporation and Time-lapse Imaging of Neuroblast Migration in Mouse Acute Brain Slices

The subventricular zone (SVZ) is one of the main neurogenic niches in the postnatal brain. Here, neural progenitors proliferate and give rise to ...

nucleofection-rodent-neuroblasts-to-study-neuroblast-migration

Nucleofection of Rodent Neuroblasts to Study Neuroblast Migration <em>In vitro</em>

The subventricular zone (SVZ) located in the lateral wall of the lateral ventricles plays a fundamental role in adult neurogenesis. In this restricted ...

King's College London

After birth, stem cells in the subventricular zone (SVZ) generate neuroblasts that migrate along the rostral migratory stream (RMS) to become interneurons in the olfactory bulb (OB). This migration is a fundamental event controlling the proper integration of new neurons in a pre-existing synaptic network. Many regulators of neuroblast migration have been identified; however, still very little is known about the intracellular molecular mechanisms controlling this process. Here, we show that the actin-bundling protein fascin is highly upregulated in mouse SVZ-derived migratory neuroblasts. Fascin-1ko mice display an abnormal RMS and a smaller OB. Bromodeoxyuridine labeling experiments show that lack of fascin significantly impairs neuroblast migration, but does not appear to affect cell proliferation. Moreover, fascin depletion substantially alters the polarized morphology of rat neuroblasts. Protein kinase C (PKC)-dependent phosphorylation of fascin on Ser39 regulates its actin-bundling activity. In vivo postnatal electroporation of phosphomimetic (S39D) or nonphosphorylatable (S39A) fascin variants followed by time-lapse imaging of brain slices demonstrates that the phospho-dependent modulation of fascin activity ensures efficient neuroblast migration. Finally, fluorescence lifetime imaging microscopy studies in rat neuroblasts reveal that the interaction between fascin and PKC can be modulated by cannabinoid signaling, which controls neuroblast migration in vivo. We conclude that fascin, whose upregulation appears to mark the transition to the migratory neuroblast stage, is a crucial regulator of neuroblast motility. We propose that a tightly regulated phospho/dephospho-fascin cycle modulated by extracellular signals is required for the polarized morphology and migration in neuroblasts, thus contributing to efficient neurogenesis.

Fascin regulates the migration of subventricular zone-derived neuroblasts in the postnatal brain.

Signalling through EGF, FGF and endocannabinoid (eCB) receptors promotes adult neurogenesis, and this can be modelled in culture using the Cor-1 neural stem cell line. In the present study we show that Cor-1 cells express a TGFβ receptor complex composed of the ActRIIB/ALK5 subunits and that a natural ligand for this receptor complex, GDF11, activates the canonical Smad2/3 signalling cascade and significantly alters the expression of ∼4700 gene transcripts within a few hours of treatment. Many of the transcripts regulated by GDF11 are also regulated by the EGF, FGF and eCB receptors and by the MAPK pathway - however, in general in the opposite direction. This can be explained to some extent by the observation that GDF11 inhibits expression of, and signalling through, the EGF receptor. GDF11 regulates expression of numerous cell-cycle genes and suppresses Cor-1 cell proliferation; interestingly we found down-regulation of Cyclin D2 rather than p27kip1 to be a good molecular correlate of this. GDF11 also inhibited the expression of numerous genes linked to cytoskeletal regulation including Fascin and LIM and SH3 domain protein 1 (LASP1) and this was associated with an inhibition of Cor-1 cell migration in a scratch wound assay. These data demonstrate GDF11 to be a master regulator of neural stem cell transcription that can suppress cell proliferation and migration by regulating the expression of numerous genes involved in both these processes, and by suppressing transcriptional responses to factors that normally promote proliferation and/or migration.

Martina Sonego

Wolfson Centre for Age-Related Diseases

King's College London

In vivo Postnatal Electroporation and Time-lapse Imaging of Neuroblast Migration in Mouse Acute Brain Slices

Nucleofection of Rodent Neuroblasts to Study Neuroblast Migration In vitro

Martina Sonego

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King's College London

In vivo Postnatal Electroporation and Time-lapse Imaging of Neuroblast Migration in Mouse Acute Brain Slices

Nucleofection of Rodent Neuroblasts to Study Neuroblast Migration In vitro

Wolfson Centre for Age-Related Diseases