Hyperactivity of Rac1-GTPase pathway impairs neuritogenesis of cortical neurons by altering actin dynamics.

The small-GTPase Rac1 is a key molecular regulator linking extracellular signals to actin cytoskeleton dynamics. Loss-of-function mutations in RAC1 and other genes of the Rac signaling pathway have been implicated in the pathogenesis of Intellectual Disability (ID). The Rac1 activity is negatively controlled by GAP proteins, however the effect of Rac1 hyperactivity on neuronal networking in vivo has been poorly studied. ArhGAP15 is a Rac-specific negative regulator, expressed in the main subtypes of pyramidal cortical neurons. In the absence of ArhGAP15, cortical pyramidal neurons show defective neuritogenesis, delayed axonal elongation, reduced dendritic branching, both in vitro and in vivo. These phenotypes are associated with altered actin dynamics at the growth cone due to increased activity of the PAK-LIMK pathway and hyperphosphorylation of ADF/cofilin. These results can be explained by shootin1 hypo-phosphorylation and uncoupling with the adhesion system. Functionally, ArhGAP15-/- mice exhibit decreased synaptic density, altered electroencephalographic rhythms and cognitive deficits. These data suggest that both hypo- and hyperactivation of the Rac pathway due to mutations in Rac1 regulators can result in conditions of ID, and that a tight regulation of Rac1 activity is required to attain the full complexity of the cortical networks.

COUP-TFI controls activity-dependent tyrosine hydroxylase expression in adult dopaminergic olfactory bulb interneurons.

COUP-TFI is an orphan nuclear receptor acting as a strong transcriptional regulator in different aspects of forebrain embryonic development. In this study, we investigated COUP-TFI expression and function in the mouse olfactory bulb (OB), a highly plastic telencephalic region in which continuous integration of newly generated inhibitory interneurons occurs throughout life. OB interneurons belong to different populations that originate from distinct progenitor lineages. Here, we show that COUP-TFI is highly expressed in tyrosine hydroxylase (TH)-positive dopaminergic interneurons in the adult OB glomerular layer (GL). We found that odour deprivation, which is known to downregulate TH expression in the OB, also downregulates COUP-TFI in dopaminergic cells, indicating a possible correlation between TH- and COUP-TFI-activity-dependent action. Moreover, we demonstrate that conditional inactivation of COUP-TFI in the EMX1 lineage results in a significant reduction of both TH and ZIF268 expression in the GL. Finally, lentiviral vector-mediated COUP-TFI deletion in adult-generated interneurons confirmed that COUP-TFI acts cell-autonomously in the control of TH and ZIF268 expression. These data indicate that COUP-TFI regulates TH expression in OB cells through an activity-dependent mechanism involving ZIF268 induction and strongly argue for a maintenance rather than establishment function of COUP-TFI in dopaminergic commitment. Our study reveals a previously unknown role for COUP-TFI in the adult brain as a key regulator in the control of sensory-dependent plasticity in olfactory dopaminergic neurons.

Eps8 involvement in neuregulin1-ErbB4 mediated migration in the neuronal progenitor cell line ST14A.

Stable expression of the tyrosine kinase receptor ErbB4 confers increased migratory behavior to the neuronal progenitor cell line ST14A, in response to neuregulin 1 (NRG1) stimulation. We used gene expression profiling analysis to identify transcriptional changes associated with higher migratory activity caused by the activation of a specific ErbB4 isoform, and found constitutive up-regulation of the epidermal growth factor receptor pathway substrate 8 (Eps8), a multimodular regulator of actin dynamics. We confirmed the increase of Eps8, both at the mRNA and at the protein level, in stable clones expressing two different ErbB4 isoforms, both characterized by high migratory activity. Using Transwell assays and experimental manipulation of Eps8 expression level, we demonstrated that Eps8 synergizes with ErbB4 to increase both basal and ligand induced cell migration, whereas siRNA mediated Eps8 silencing strongly impairs cell motility and NRG1 induced actin cytoskeleton remodeling. By transient knockdown of Eps8 through in vivo siRNA electroporation, followed by explant primary cultures, we demonstrated that Eps8 down-regulation affects migration of normal neuronal precursors. In conclusion, our data demonstrate that Eps8 is a key regulator of motility of neuronal progenitor cells expressing ErbB4, both in basal conditions and in response to external motogenic cues.

Wnt5a is a transcriptional target of Dlx homeogenes and promotes differentiation of interneuron progenitors in vitro and in vivo.

During brain development, neurogenesis, migration, and differentiation of neural progenitor cells are regulated by an interplay between intrinsic genetic programs and extrinsic cues. The Dlx homeogene transcription factors have been proposed to directly control the genesis and maturation of GABAergic interneurons of the olfactory bulb (OB), subpallium, and cortex. Here we provide evidence that Dlx genes promote differentiation of olfactory interneurons via the signaling molecule Wnt5a. Dlx2 and Dlx5 interact with homeodomain binding sequences within the Wnt5a locus and activate its transcription. Exogenously provided Wnt5a promotes GABAergic differentiation in dissociated OB neurons and in organ-type brain cultures. Finally, we show that the Dlx-mutant environment is unfavorable for GABA differentiation, in vivo and in vitro. We conclude that Dlx genes favor interneuron differentiation also in a non-cell-autonomous fashion, via expression of Wnt5a.

Hepatocyte growth factor regulates migration of olfactory interneuron precursors in the rostral migratory stream through Met-Grb2 coupling.

The olfactory bulb is one of the few structures in the mammalian forebrain in which continuous neurogenesis takes place throughout life. Neuronal precursors originate from progenitors located in the subventricular zone (SVZ) of the lateral ventricles, move tangentially in chains through the rostral migratory stream (RMS), and reach the olfactory bulb (OB), where they finally differentiate into granule and glomerular interneurons. Multiple molecular factors are involved in controlling the various steps of this neurogenic process. Here, we show that hepatocyte growth factor (HGF) and its receptor Met protein are expressed in vivo in the OB and throughout the migratory pathway, implying that HGF might mediate migratory signals in this system. By using primary in vitro cultures, we demonstrate that HGF promotes migration of RMS neuroblasts, acting both as an inducer and attractant. HGF stimulation on RMS tissue explants selectively induces MAP kinase pathway activation. Furthermore, in vitro analysis of mice with a point mutation in the Met receptor that impairs signal transduction through the Ras/MAP kinase pathway (Met(Grb2/Grb2)) shows that without Met-Grb2 binding, neuroblast migration is reduced. Overall, these findings indicate that HGF signaling via Met-Grb2 coupling influences olfactory interneuron precursor migration along the RMS.

Generation of distinct types of periglomerular olfactory bulb interneurons during development and in adult mice: implication for intrinsic properties of the subventricular zone progenitor population.

The subventricular zone (SVZ) of the lateral ventricle develops from residual progenitors of the embryonic lateral ganglionic eminence (LGE) and maintains neurogenic activity throughout life. Precursors from LGE/SVZ migrate to the olfactory bulb (OB) where they differentiate into local interneurons, principally in the granule layer and glomerular layer (GL). By in situ dye labeling, we show that neonatal and adult SVZ progenitors differentially contribute to neurochemically distinct types of periglomerular interneurons in the GL. Namely, calbindin-positive periglomerular cells are preferentially generated during early life, whereas calretinin- and tyrosine hydroxylase-expressing neurons are mainly produced at later ages. Furthermore, homochronic/heterochronic transplantation demonstrates that progenitor cells isolated from the LGE or SVZ at different stages (embryonic day 15 and postnatal days 2 and 30) engraft into the SVZ of neonatal or adult mice, migrate to the OB, and differentiate into local interneurons, including granule and periglomerular cells as well as other types of interneurons. The total number of integrated cells and the relative proportion of granule or periglomerular neurons change, according to the donor age, whereas they are weakly influenced by the recipient age. Analysis of the neurochemical phenotypes acquired by transplanted cells in the GL shows that donor cells of different ages also differentiate according to their origin, regardless of the host age. This suggests that progenitor cells at different ontogenetic stages are intrinsically directed toward specific lineages. Neurogenic processes occurring during development and in adult OB are not equivalent and produce different types of periglomerular interneurons as a consequence of intrinsic properties of the SVZ progenitors.

ErbB4 expression in neural progenitor cells (ST14A) is necessary to mediate neuregulin-1beta1-induced migration.

Activation of the receptor tyrosine kinase ErbB4 leads to various cellular responses such as proliferation, survival, differentiation, and chemotaxis. Two pairs of naturally occurring ErbB4 isoforms differing in their juxtamembrane (JMa/JMb) and C termini (cyt1/cyt2) have been described. To examine the role of ErbB4 in neuron migration, we cloned and stably transfected each of the four ErbB4 isoforms in ST14A cells (a neural progenitor cell line derived from the striatum of embryonic day 14 rats) endogenously expressing the other members of the ErbB family: ErbB1, ErbB2, and ErbB3. Using immunoprecipitation assays, we showed that the neuregulin-1beta1 (NRG1beta1) stimulus induced ErbB4 tyrosine phosphorylation and phosphatidylinositol 3-kinase (PI3K) recruitment and activation (as demonstrated by Akt phosphorylation) either directly (ErbB4 cyt1 isoform) or indirectly (ErbB4 cyt2 isoform). We examined the ability of the four ErbB4 isoforms to induce chemotaxis and cell proliferation in response to NRG1beta1 stimulation. Using migration assays, we observed that only ErbB4-expressing cells stimulated with NRG1beta1 showed a significant increase in migration, whereas the growth rate remained unchanged. Additional assays showed that inhibition of PI3K (but not of phospholipase Cgamma) dramatically reduced migratory activity. Our data show that ErbB4 signaling via PI3K activation plays a fundamental role in controlling NRG1beta1-induced migration.