Symptomatic SOD1^G93A mice, phrenic nerve.

<p>Anti-ribosomal P-protein immunofluorescence (red) is present in myelinated axons outlined by anti-myelin basic protein antibody (arrows). Bar, 5 µm.</p

Quantification of increase of polyribosomes in myelinated axons in the phrenic nerve of SOD1^G93A mutant mice.

<p>Both the percentage of myelinated axons containing ribosomes (A) as well as the absolute number of ribosomes in axons (B) has increased dramatically in both presymptomatic (12 weeks PN, n = 5, *p<0.01) as well as symptomatic (17 weeks PN n = 5, *p<0.01) compared to their littermate controls (Ntg, n = 5) and SOD1 Wt animals, (n = 5) whereas there are no statistical differences between the presymptomatic and symptomatic groups.</p

Symptomatic SOD1^G93A mice, sciatic nerve.

<p>Non-radioactive in situ hybridization for rRNA (green) yields strong signals in 2 myelinated axons (arrows), delineated by Nile red fluorescence of myelin (red). In the left axon, the small signal inside the myelin sheath (arrowhead) most likely corresponds to ribosomal RNA localized in ad-axonal Schwann cell cytoplasm or in Schwann cell cytoplasm between the myelin membranes (e.g. clefts of Schmidt-Lantermann), which has previously been suggested as a transfer route for ribosomes from Schwann cells to axons (Court et al, J Neurosci 2008, 28∶11024–11029). The signal outside the myelin sheets (arrowheads) corresponds to ribosomes in the peri-axonal Schwann cell cytoplasm. Please note that most axons do not show a hybridization signal, but show a strong Schwann cell signal (arrowheads), confirming the specificity of the ISH. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087255#pone.0087255.s002" target="_blank">Figure S2</a> for optical sections of this area. Bar, 5 µm.</p

Increased axonal ribosome numbers in human ALS.

<p>A, sural nerve biopsy of a control person having no neurological symptoms, showing a myelinated axon in which no polyribosomes can be discerned. Polyribosomes are detected in the Schwann cell (Sc), either freely in the cytoplasm, or associated with the rough endoplasmic reticulum (arrowhead). Inset shows detail of axoplasm containing many cross sectioned microtubules (Mt). B, sural nerve autopsy from a patient diagnosed with sporadic ALS. In the axoplasm, clusters of polyribosomes are presented. Left inset: arrowhead points to ribosomes in the cytoplasm of a Schwann cell, which show similar morphology as those in the axoplasm (right inset). Bar, 100 nm.</p

Increased Axonal Ribosome Numbers Is an Early Event in the Pathogenesis of Amyotrophic Lateral Sclerosis

<div><p>Myelinating glia cells support axon survival and functions through mechanisms independent of myelination, and their dysfunction leads to axonal degeneration in several diseases. In amyotrophic lateral sclerosis (ALS), spinal motor neurons undergo retrograde degeneration, and slowing of axonal transport is an early event that in ALS mutant mice occurs well before motor neuron degeneration. Interestingly, in familial forms of ALS, Schwann cells have been proposed to slow disease progression. We demonstrated previously that Schwann cells transfer polyribosomes to diseased and regenerating axons, a possible rescue mechanism for disease-induced reductions in axonal proteins. Here, we investigated whether elevated levels of axonal ribosomes are also found in ALS, by analysis of a superoxide dismutase 1 (SOD1)^G93A mouse model for human familial ALS and a patient suffering from sporadic ALS. In both cases, we found that the disorder was associated with an increase in the population of axonal ribosomes in myelinated axons. Importantly, in SOD1^G93A mice, the appearance of axonal ribosomes preceded the manifestation of behavioral symptoms, indicating that upregulation of axonal ribosomes occurs early in the pathogenesis of ALS. In line with our previous studies, electron microscopy analysis showed that Schwann cells might serve as a source of axonal ribosomes in the disease-compromised axons. The early appearance of axonal ribosomes indicates an involvement of Schwann cells early in ALS neuropathology, and may serve as an early marker for disease-affected axons, not only in ALS, but also for other central and peripheral neurodegenerative disorders.</p></div

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

Drebrin induced dendritic protrusions do not require PI3K.

<p>Rat hippocampal neurons were transfected at 7 DIV with YFP or YFP-DBN. The PI3K inhibitor wortmannin was applied at 100 nM following transfection. At 17 DIV, neurons were fixed and the length of individual dendrite protrusions was determined from the base to the tip of the spine head using Neuron-J. To avoid spine variability, we restricted the analysis to the primary and secondary dendrites 100-150 µm away from the soma. Each data point represent the relative length of at least 220 protrusions measured over 3 independent experiments <u>+</u> sem. ***p<0.001. Scale bar: 3 µm.</p

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

pS647-Drebrin is de-phosphorylated in dependence of the PTEN phosphatase activity, but independent of PI3K signalling.

<p>(A) Alignment of S647 DBN. DBN comprises of an N-terminal ADF/cofilin homology domain (ADF), a coiled-coil (C–C) domain, a proline-rich stretch (P) and an unstructured C-terminus. (B) Adult rat brain homogenate was analysed by western blot analysis and probed with anti-DBN antibody or with the affinity purified pS647-DBN antibody. (C) Brain homogenate was incubated on ice or at 37°C for 1 hour to activate endogenous phosphatases, before western blotting and analyses. (D) Cortical neurons were nucleofected with control or DBN specific shRNA. Neuronal lysate was analysed at 3 DIV. (E) Bacterially expressed and purified His-DBN was probed for anti-DBN and anti-pS647-DBN antibodies. (F) Cell lysates prepared from YFP-DBN or YFP-S647A-DBN expressing HEK293 cells were analysed by western blotting using indicated antibodies. (G) 19 DIV hippocampal neurons were stained with pan-drebin (mouse) and pS647-DBN antibodies (rabbit) in the presence of control serum or with the pS647-DBN peptide. Scale bar: 10 µm. (H) GFP-PTEN or the GFP-PTEN CS mutant was co-expressed with Flag-DBN in U87MG cells. Bar graph represents the average band density of pS647-DBN/DBN in 3 independent experiments <u>+</u> sem. *p<0.05. (I) Flag-DBN expressing U87MG cells were treated with wortmannin (wm) for 1 hour at 100 nM. (J) As in (H), but cells were co-transfected with Flag-DBN and GFP-PTEN, or with Flag-DBN and GFP-PTENΔD. Bar graph (right) represents the average band density of pS647-DBN/DBN in 3 independent experiments <u>+</u> sem. *p<0.05.</p