CRMP5 Regulates Generation and Survival of Newborn Neurons in Olfactory and Hippocampal Neurogenic Areas of the Adult Mouse Brain

The Collapsin Response Mediator Proteins (CRMPs) are highly expressed in the developing brain, and in adult brain areas that retain neurogenesis, ie: the olfactory bulb (OB) and the dentate gyrus (DG). During brain development, CRMPs are essentially involved in signaling of axon guidance and neurite outgrowth, but their functions in the adult brain remain largely unknown. CRMP5 has been initially identified as the target of auto-antibodies involved in paraneoplasic neurological diseases and further implicated in a neurite outgrowth inhibition mediated by tubulin binding. Interestingly, CRMP5 is also highly expressed in adult brain neurogenic areas where its functions have not yet been elucidated. Here we observed in both neurogenic areas of the adult mouse brain that CRMP5 was present in proliferating and post-mitotic neuroblasts, while they migrate and differentiate into mature neurons. In CRMP5−/− mice, the lack of CRMP5 resulted in a significant increase of proliferation and neurogenesis, but also in an excess of apoptotic death of granule cells in the OB and DG. These findings provide the first evidence that CRMP5 is involved in the generation and survival of newly generated neurons in areas of the adult brain with a high level of activity-dependent neuronal plasticity

Specific alteration in the expression of glial fibrillary acidic protein, glutamate dehydrogenase, and glutamine synthetase in rats with genetic absence epilepsy

International audienceAstrocytes play a predominant role in energy metabolism and in the catabolism of gamma-aminobutyric acid (GABA) and glutamate, neurotransmitters critically involved in epileptic processes. We show specific astrocytic alterations in the genetic absence epilepsy rats from Strasbourg (GAERS). Spontaneous absence seizures appear in this strain in the cortex and thalamus after the age of 1 month. In these brain structures, we demonstrate increased GFAP expression in both adult and young GAERS, suggesting that reactive astrocytes are already present before the onset of seizures. Glutamate dehydrogenase (GDH) and glutamine synthetase (GS), which are localized mainly in astrocytes and involved in glutamate catabolism, are shown to be differentially altered. GDH expression was increased in the thalamus of both young and adult GAERS and in the cortex of young GAERS. GS expression was slightly decreased in the thalamus of young GAERS. These astrocytic modifications are not adaptive responses to seizures, as the modifications appear before the development of absence seizures. Thus, astrocytes might be involved in the neuronal processes giving rise to epileptic seizures in this strain

Immunofluorescence labeling of CRMP5 expression in neuroblasts of the mouse hippocampal neurogenic area.

<p>(<b>A,B</b>) Confocal microscopy of immuno-labelled sections showing CRMP5-positive cells in the subgranular zone (SGZ) of the dentate gyrus (DG) in the hippocampus. (<b>C–N</b>) Double immunolabeling in the DG of CRMP5 with the glial markers GFAP (<b>C–E</b>) and S100 (<b>F–H</b>) demonstrates that glial cells never expressed CRMP5 (2E and 2F, arrows). In contrast, all CRMP5-positive cells co-expressed doublecortin (DCX) a selective marker of immature neurons (or neuroblasts) (<b>I–K</b>, arrow). Finally, double immunolabeling of CRMP5 and KI67, a marker of cycling cells, shows that some CRMP5-positive cells are still mitotic (<b>L–N</b>, arrow), whereas others are no longer proliferative. Granular cell layer (GCL); Hilus (Hi). Optical thickness of the confocal planes = 1 µm. Scale bars 50 µm (A,B); 20 µm (C–E); 15 µm (F–N).</p

Immunofluorescence labeling of CRMP5 expression in neuroblasts of the mouse olfactory neurogenic area.

<p>(<b>A–C</b>) Confocal microscopy of immuno-labelled sections showing numerous CRMP5-positive cells at three levels of the olfactory neurogenic area, ie: the subependymal layer of the OB (SEL-OB), the rostral migratory stream (RMS) and the subventricular zone (SVZ) of the forebrain. (<b>D–O</b>) Characterization of CRMP5-positive cells in the SVZ. Double immunolabeling of CRMP5 and the glial markers GFAP (<b>D–F</b>) or S100 (<b>G–I</b>, arrow) revealed the lack of CRMP5 expression in glial cells. In contrast, all CRMP5-positive cells co-expressed doublecortin (DCX) a selective marker of immature neurons (or neuroblasts) (<b>J–L</b>, arrow). Finally, double immunolabeling of CRMP5 and KI67, a marker of cycling cells, shows that some CRMP5-positive cells are still mitotic (<b>M–O</b>, arrow), whereas the majority are no longer proliferative. Lateral ventricles (LV). Optical thickness of the confocal planes = 1 µm. Scale bars 50 µm (A–C); 15 µm (D–I); 10 µm (J–L); 20 µm (M–O).</p

Suppression of CRMP5 expression in CRMP5^−/− mice increases cell proliferation in the adult brain neurogenic areas.

<p>(<b>A,B</b>) Immunofluorescence detection of CRMP5 in the dentate gyrus (3A) and SVZ (3B) confirms the lack of CRMP5 expression in CRMP5^−/− mice in comparison to wild type mice (WT). (<b>C</b>) Immunocytochemical labeling of BrdU-positive cells in the SGZ of the dentate gyrus (DG) of WT and CRMP5^−/− mice, 2 hours after BrdU injections. Hilus (Hi); Granular cell layer (GCL). Scale bar 100 µm. (<b>D</b>) Increased total number of BrdU positive cells in the DG of CRMP5^−/− mice. Values are expressed as mean + SEM. Student t-test. * p<0.05; ** p<0.01. (<b>E</b>) Immunocytochemical labeling of BrdU-positive cells in the subventricular zone (SVZ) of WT and CRMP5^−/− mice, 2 hours after BrdU injections. Lateral ventricles (LV). Scale bar 100 µm. (<b>F</b>) Increased total number of BrdU positive cells in the SVZ of CRMP5^−/− mice. Values are expressed as mean + SEM. Student t-test. * p<0.05; ** p<0.01.</p

Suppression of CRMP5 expression increases neuronal cell death in the olfactory bulb and dentate gyrus of CRMP5^−/− mice.

<p>(<b>A,B</b>) Stereological measurements of the volume of granular cell layers in the OB (A) and the DG (B) of wild type (WT) and CRMP5^−/− mice. In the OB (A), the lack of CRMP5 in CRMP5^−/− mice induced a significant reduction of the granule cell layer thickness (*** p<0.005) when compared to the wild type. DAPI-stained coronal sections of the OB failed to reveal other major morphological alterations in KO mice (Scale bar 400 µm). In the DG (B), the thickness of the granule cell layer was similar in the CRMP5^−/− and wild type mice. DAPI-stained coronal sections of the dorsal hippocampus failed to reveal any major morphological alterations in CRMP5^−/− mice (Scale bar 400 µm). All values are expressed as mean + SEM. Student t-test: *** p<0.005. (<b>C,D</b>). Increased number of caspase-3 positive (apoptotic) cells in the OB (<b>C</b>) and in the DG (<b>D</b>) of CRMP5^−/− mice when compared to the wild type (WT). Values are expressed as mean + SEM. Student t-test. * p<0.05; *** p<0.005. (<b>E</b>). Confocal microscopy of a caspase-3/NeuN immunolabeled cell (apoptotic neuron) in the DG of WT mouse (arrow, step size of confocal Z planes = 1 µm). Scale bar 15 µm. (<b>F</b>). Increased total number of caspase-3/NeuN immunoreactive cells in the OB of CRMP5^−/− mice when compared to wild type (WT) mice. Values are expressed as mean + SEM. Student t-test. ** p<0.01.</p

Suppression of CRMP5 expression increases the rate of newly generated neurons in the adult olfactory bulb and dentate gyrus of CRMP5^−/− mice.

<p>(<b>A,B</b>) Immunocytochemical labeling of BrdU-positive cells in the DG (A) and in the OB (B) of WT and CRMP5^−/− mice, 21 days after BrdU injections. Hilus (Hi); Granular cell layer (GCL); Subependymal layer (SEL). Scale bar 100 µm. (<b>C,D</b>) Increased total number of BrdU positive cells in the DG (<b>C</b>) and in the OB (<b>D</b>) of CRMP5^−/− mice. Values are expressed as mean + SEM. Student t-test. * p<0.05; ** p<0.01. (<b>E</b>). Immunocytochemical labeling of BrdU-NeuN double-labeled cell in the DG of WT mouse (arrow, step size of confocal Z planes = 1 µm). Scale bar 15 µm. (<b>F</b>). Total number of BrdU-NeuN positive cells is increased in the DG of CRMP5^−/− mice. Values are expressed as mean + SEM. Student t-test. * p<0.05; ** p<0.01. (<b>G</b>). Immunocytochemical labeling of BrdU-NeuN double-labeled cell in the GCL of the OB of WT mouse (arrow, step size of confocal Z planes = 1 µm). Scale bar 15 µm. (<b>H</b>). Total number of BrdU-NeuN positive cells is increased in the OB of CRMP5^−/− mice. Values are expressed as mean + SEM. Student t-test. * p<0.05; ** p<0.01.</p

Matrix-binding vascular endothelial growth factor (VEGF) isoforms guide granule cell migration in the cerebellum via VEGF receptor Flk1.

Vascular endothelial growth factor (VEGF) regulates angiogenesis, but also has important, yet poorly characterized roles in neuronal wiring. Using several genetic and in vitro approaches, we discovered a novel role for VEGF in the control of cerebellar granule cell (GC) migration from the external granule cell layer (EGL) toward the Purkinje cell layer (PCL). GCs express the VEGF receptor Flk1, and are chemoattracted by VEGF, whose levels are higher in the PCL than EGL. Lowering VEGF levels in mice in vivo or ectopic VEGF expression in the EGL ex vivo perturbs GC migration. Using GC-specific Flk1 knock-out mice, we provide for the first time in vivo evidence for a direct chemoattractive effect of VEGF on neurons via Flk1 signaling. Finally, using knock-in mice expressing single VEGF isoforms, we show that pericellular deposition of matrix-bound VEGF isoforms around PC dendrites is necessary for proper GC migration in vivo. These findings identify a previously unknown role for VEGF in neuronal migration.Journal ArticleResearch Support, Non-U.S. Gov'tSCOPUS: ar.jinfo:eu-repo/semantics/publishe