Drebrin regulates neuroblast migration in the postnatal Mammalian brain

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 crucial for the proper integration of newborn neurons in a pre-existing synaptic network and is believed to play a key role in infant human brain development. Many regulators of neuroblast migration have been identified; however, still very little is known about the intracellular molecular mechanisms controlling this process. Here, we have investigated the function of drebrin, an actin-binding protein highly expressed in the RMS of the postnatal mammalian brain. Neuroblast migration was monitored both in culture and in brain slices obtained from electroporated mice by time-lapse spinning disk confocal microscopy. Depletion of drebrin using distinct RNAi approaches in early postnatal mice affects neuroblast morphology and impairs neuroblast migration and orientation in vitro and in vivo. Overexpression of drebrin also impairs migration along the RMS and affects the distribution of neuroblasts at their final destination, the OB. Drebrin phosphorylation on Ser142 by Cyclin-dependent kinase 5 (Cdk5) has been recently shown to regulate F-actin-microtubule coupling in neuronal growth cones. We also investigated the functional significance of this phosphorylation in RMS neuroblasts using in vivo postnatal electroporation of phosphomimetic (S142D) or non-phosphorylatable (S142A) drebrin in the SVZ of mouse pups. Preventing or mimicking phosphorylation of S142 in vivo caused similar effects on neuroblast dynamics, leading to aberrant neuroblast branching. We conclude that drebrin is necessary for efficient migration of SVZ-derived neuroblasts and propose that regulated phosphorylation of drebrin on S142 maintains leading process stability for polarized migration along the RMS, thus ensuring proper neurogenesis

Drebrin phosphorylation on S142 regulates neuroblast morphology and orientation <i>in vivo</i>.

<p>(A) Sagittal brain slices were immunostained for YFP 5 days after <i>in vivo</i> electroporation of plasmids encoding YFP-tagged wt, S142A or S142D drebrin and imaged with a confocal microscope. The empty vector encoding only YFP served as control. The majority of control cells display a single leading process oriented towards the OB (yellow asterisk). Many neuroblasts overexpressing wt drebrin display a bipolar morphology with two diametrically opposite protrusions (one towards the OB and one towards the SVZ) (wt, arrowheads and D). Overexpression of S142A or S142D drebrin significantly increases the percentage of misoriented cells (pointing towards the SVZ instead of the OB, arrowheads) compared to control or wt drebrin (C). (B) Quantitative morphological analysis also shows a decrease in leading process length for wt, S142A and S142D compared to the control (mean ± SEM; n = 8 brains for empty vector; n = 5 brains for wt, S142A, and S142D drebrin; *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001). Scale bar: 50 μm.</p

Altering levels of drebrin and pS142 phosphorylation of drebrin affects neuroblast distribution in the OB.

<p>P2 mice were electroporated in the lateral ventricle with plasmids encoding YFP or YFP-tagged wt, S142A or S142D drebrin. Sagittal brain slices were prepared 14 days later and immunostained for YFP. (A, top row) Representative confocal projections of sagittal brain slices showing overall labeling in the OB and overlaid with the mask used for analysis (area A, inner OB; area B, outer OB). High magnification pictures of the OB insets are shown in the bottom row. (B) Schematic diagrams of a typical quantification analysis using all OB sections from one brain for each condition. Cell bodies in areas A and B are represented as dots. (C) Overexpression of wt, S142A and S142D drebrin caused a significant cell accumulation in the inner OB compared to GFP (mean ± SEM; n = 3 brains for each construct; *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001). Scale bars: (A, top row), 500 μm; (A, bottom row), 100 μm.</p

Localization of pS142-drebrin.

<p>(A) Drebrin phosphorylated on S142 can be detected in RMS and OB homogenates from P7 rat pups by Western blot using an anti-pS142-drebrin antibody. Actin is shown as a loading control. (B, top) Rat RMS neuroblasts were immunostained for pS142 drebrin (green) and βIII tubulin (red). pS142 drebrin is found along the membrane of the leading process and close to the tip of the leading process. (B, bottom) Neuroblasts were immunostained for drebrin pS142 (green) and fascin (red). Fascin and drebrin show colocalisation at the basal region of filopodia. Nuclei are stained with Hoechst (blue). Scale bars: 10 μm.</p

Intracellular localisation of drebrin in migratory neuroblasts.

<p>Immunostaining of cultured rat RMS neuroblasts embedded in Matrigel. (Top row) Drebrin (red) is present along the leading process but mostly excluded from peripheral filopodia, visualized by Life-Act GFP (green, arrowhead). Nuclei are stained with Hoechst dye (blue). (Middle row) Colocalization between drebrin (red) and fascin (green), which decorates peripheral filopodia, is limited to the basal region of filopodia. (Bottom row) Drebrin (green) is found along the membrane and especially concentrated towards the tip of leading processes (arrowheads), which are immunostained for ßIII tubulin-positive microtubules (red). Nuclei are stained with Hoechst dye (blue). Scale bars: (top), 10 μm; (middle and bottom), 20 μm.</p

Drebrin is highly expressed in SVZ-derived migratory neuroblasts.

<p>(A) Immunostained sagittal brain sections from P7 (left) and P90 (right) mice show strong drebrin expression (brown) in the subventricular zone (SVZ), rostral migratory stream (RMS), and olfactory bulb (OB). (B) Confocal images from P7 mice SVZ sagittal sections showing that drebrin immunostaining overlaps with DCX+ migrating neuroblasts (top row), but is almost completely excluded from GFAP+ stem cells and astrocytes (middle row). Very little colocalization is observed with Mash-1+ transit-amplifying progenitors (bottom row). Merge panels include DAPI staining to visualize cell nuclei and higher magnification insets. Scale bars: (A), 200 μm; (B), 10 μm; insets, 5 μm.</p

Drebrin regulates RMS neuroblast migration <i>in vitro</i>.

<p>(A) Reaggregated rat neuroblasts were embedded in Matrigel 52 h after nucleofection of control or drebrin shRNA-GFP and allowed to migrate for 24 h. Representative images of fixed reaggregates immunostained for GFP. The GFP channel is shown as a grayscale image. (B) Drebrin-depleted neuroblasts (black column) show a ~20% decrease in migration distance compared to control shRNA-nucleofected cells (white column). GFP-negative, untransfected cells (hatched columns) served as an internal control (mean ± SEM; n = 3 independent experiments; 15 to 20 explants analysed per experiment; *<i>P</i><0.05). (C) Reaggregated rat neuroblasts were embedded in Matrigel 52 h after nucleofection with control shRNA-GFP and m-cherry-empty vector or drebrin shRNA-GFP and m-cherry-human drebrin (shRNA-resistant) and allowed to migrate for 24 h before immunostaining for GFP (green) and m-cherry (red). Cell nuclei were visualized by Hoechst staining (blue). (D) The impaired migration caused by drebrin knockdown was rescued by co-transfection with the shRNA-resistant human drebrin (mean ± SEM; n = 3 independent experiments; 15 to 20 explants analysed per experiment). Scale bars: 50 μm.</p

Altering levels of drebrin and pS142 phosphorylation affects neuroblast migration along the RMS.

<p>P2 mice were electroporated <i>in vivo</i> with plasmids encoding YFP, or YFP-tagged wt, S142A, or S142D drebrin. Fluorescently-labelled RMS neuroblasts were imaged 5 days later in acute brain slice cultures over a period of 3 hours. (A) Representative migration paths of neuroblasts expressing YFP empty vector, YFP-tagged wt, S142A, or S142D drebrin. Yellow asterisks mark the location of the OB. Quantitative tracking analysis reveals that neuroblasts overexpressing any one of the drebrin versions display a lower migratory distance (B), displacement (C), speed (D) and a trend towards a lower migratory index (E) compared to control cells expressing only YFP (mean ± SEM; n = 6 brains for control; n = 5 brains for wt; and n = 4 brains for S142A and n = 5 for S142D; **<i>P</i><0.01, ***<i>P</i><0.001).</p