Pip5k1γ regulates axon formation by limiting Rap1 activity.

During their differentiation, neurons establish a highly polarized morphology by forming axons and dendrites. Cortical and hippocampal neurons initially extend several short neurites that all have the potential to become an axon. One of these neurites is then selected as the axon by a combination of positive and negative feedback signals that promote axon formation and prevent the remaining neurites from developing into axons. Here, we show that Pip5k1γ is required for the formation of a single axon as a negative feedback signal that regulates C3G and Rap1 through the generation of phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2). Impairing the function of Pip5k1γ results in a hyper-activation of the Fyn/C3G/Rap1 pathway, which induces the formation of supernumerary axons. Application of a hyper-osmotic shock to modulate membrane tension has a similar effect, increasing Rap1 activity and inducing the formation of supernumerary axons. In both cases, the induction of supernumerary axons can be reverted by expressing constitutively active Pip5k. Our results show that PI(4,5)P2-dependent membrane properties limit the activity of C3G and Rap1 to ensure the extension of a single axon.

An isoform-specific function of Cdc42 in regulating mammalian Exo70 during axon formation.

The highly conserved GTPase Cdc42 is an essential regulator of cell polarity and promotes exocytosis through the exocyst complex in budding yeast and Drosophila In mammals, this function is performed by the closely related GTPase TC10, whereas mammalian Cdc42 does not interact with the exocyst. Axon formation is facilitated by the exocyst complex that tethers vesicles before their fusion to expand the plasma membrane. This function depends on the recruitment of the Exo70 subunit to the plasma membrane. Alternative splicing generates two Cdc42 isoforms that differ in their C-terminal 10 amino acids. Our results identify an isoform-specific function of Cdc42 in neurons. We show that the brain-specific Cdc42b isoform, in contrast to the ubiquitous isoform Cdc42u, can interact with Exo70. Inactivation of Arhgef7 or Cdc42b interferes with the exocytosis of post-Golgi vesicles in the growth cone. Cdc42b regulates exocytosis and axon formation downstream of its activator Arhgef7. Thus, the function of Cdc42 in regulating exocytosis is conserved in mammals but specific to one isoform.

The balance of mitochondrial fission and fusion in cortical axons depends on the kinases SadA and SadB.

Neurons are highly polarized cells that display characteristic differences in the organization of their organelles in axons and dendrites. The kinases SadA and SadB (SadA/B) promote the formation of distinct axonal and dendritic extensions during the development of cortical and hippocampal neurons. Here, we show that SadA/B are required for the specific dynamics of axonal mitochondria. Ankyrin B (AnkB) stimulates the activity of SadA/B that function as regulators of mitochondrial dynamics through the phosphorylation of tau. Suppression of SadA/B or AnkB in cortical neurons induces the elongation of mitochondria by disrupting the balance of fission and fusion. SadA/B-deficient neurons show an accumulation of hyper-fused mitochondria and activation of the integrated stress response (ISR). The normal dynamics of axonal mitochondria could be restored by mild actin destabilization. Thus, the elongation after loss of SadA/B results from an excessive stabilization of actin filaments and reduction of Drp1 recruitment to mitochondria.

On the role of vesicle transport in neurite growth: Modeling and experiments.

The processes that determine the establishment of the complex morphology of neurons during development are still poorly understood. Here, we focus on the question how a difference in the length of neurites affects vesicle transport. We performed live imaging experiments and present a lattice-based model to gain a deeper theoretical understanding of intracellular transport in neurons. After a motivation and appropriate scaling of the model we present numerical simulations showing that initial differences in neurite length result in phenomena of biological relevance, i.e. a positive feedback that enhances transport into the longer neurite and oscillation of vesicles concentrations that can be interpreted as cycles of extension and retraction observed in experiments. Thus, our model is a first step towards a better understanding of the interplay between the transport of vesicles and the spatial organization of cells.

Igf1R/InsR function is required for axon extension and corpus callosum formation.

One of the earliest steps during the development of the nervous system is the establishment of neuronal polarity and the formation of an axon. The intrinsic mechanisms that promote axon formation have been extensively analyzed. However, much less is known about the extrinsic signals that initiate axon formation. One of the candidates for these signals is Insulin-like growth factor 1 (Igf1) that acts through the Igf1 (Igf1R) and insulin receptors (InsR). Since Igf1R and InsR may act redundantly we analyzed conditional cortex-specific knockout mice that are deficient for both Igf1r and Insr to determine if they regulate the development of the cortex and the formation of axons in vivo. Our results show that Igf1R/InsR function is required for the normal development of the embryonic hippocampus and cingulate cortex while the lateral cortex does not show apparent defects in the Igf1r;Insr knockout. In the cingulate cortex, the number of intermediate progenitors and deep layer neurons is reduced and the corpus callosum is absent at E17. However, cortical organization and axon formation are not impaired in knockout embryos. In culture, cortical and hippocampal neurons from Igf1r;Insr knockout embryos extend an axon but the length of this axon is severely reduced. Our results indicate that Igf1R/InsR function is required for brain development in a region-specific manner and promotes axon growth but is not essential for neuronal polarization and migration in the developing brain.

The Sema3A receptor Plexin-A1 suppresses supernumerary axons through Rap1 GTPases.

The highly conserved Rap1 GTPases perform essential functions during neuronal development. They are required for the polarity of neuronal progenitors and neurons as well as for neuronal migration in the embryonic brain. Neuronal polarization and axon formation depend on the precise temporal and spatial regulation of Rap1 activity by guanine nucleotide exchange factors (GEFs) and GTPases-activating proteins (GAPs). Several Rap1 GEFs have been identified that direct the formation of axons during cortical and hippocampal development in vivo and in cultured neurons. However little is known about the GAPs that limit the activity of Rap1 GTPases during neuronal development. Here we investigate the function of Sema3A and Plexin-A1 as a regulator of Rap1 GTPases during the polarization of hippocampal neurons. Sema3A was shown to suppress axon formation when neurons are cultured on a patterned substrate. Plexin-A1 functions as the signal-transducing subunit of receptors for Sema3A and displays GAP activity for Rap1 GTPases. We show that Sema3A and Plexin-A1 suppress the formation of supernumerary axons in cultured neurons, which depends on Rap1 GTPases.

The guanine nucleotide exchange factor Arhgef7/ßPix promotes axon formation upstream of TC10.

The characteristic six layers of the mammalian neocortex develop sequentially as neurons are generated by neural progenitors and subsequently migrate past older neurons to their final position in the cortical plate. One of the earliest steps of neuronal differentiation is the formation of an axon. Small GTPases play essential roles during this process by regulating cytoskeletal dynamics and intracellular trafficking. While the function of GTPases has been studied extensively in cultured neurons and in vivo much less is known about their upstream regulators. Here we show that Arhgef7 (also called ßPix or Cool1) is essential for axon formation during cortical development. The loss of Arhgef7 results in an extensive loss of axons in cultured neurons and in the developing cortex. Arhgef7 is a guanine-nucleotide exchange factor (GEF) for Cdc42, a GTPase that has a central role in directing the formation of axons during brain development. However, active Cdc42 was not able to rescue the knockdown of Arhgef7. We show that Arhgef7 interacts with the GTPase TC10 that is closely related to Cdc42. Expression of active TC10 can restore the ability to extend axons in Arhgef7-deficient neurons. Our results identify an essential role of Arhgef7 during neuronal development that promotes axon formation upstream of TC10.

The loss of the kinases SadA and SadB results in early neuronal apoptosis and a reduced number of progenitors.

The neurons that form the mammalian neocortex originate from progenitor cells in the ventricular (VZ) and subventricular zone (SVZ). Newborn neurons are multipolar but become bipolar during their migration from the germinal layers to the cortical plate (CP) by forming a leading process and an axon that extends in the intermediate zone (IZ). Once they settle in the CP, neurons assume a highly polarized morphology with a single axon and multiple dendrites. The AMPK-related kinases SadA and SadB are intrinsic factors that are essential for axon formation during neuronal development downstream of Lkb1. The knockout of both genes encoding Sad kinases (Sada and Sadb) results not only in a loss of axons but also a decrease in the size of the cortical plate. The defect in axon formation has been linked to a function of Sad kinases in the regulation of microtubule binding proteins. However, the causes for the reduced size of the cortical plate in the Sada-/-;Sadb-/- knockout remain to be analyzed in detail. Here we show that neuronal cell death is increased and the number of neural progenitors is decreased in the Sada-/-;Sadb-/- CP. The reduced number of progenitors is a non-cell autonomous defect since they do not express Sad kinases. These defects are restricted to the neocortex while the hippocampus remains unaffected.

PlexinA2 Forward Signaling through Rap1 GTPases Regulates Dentate Gyrus Development and Schizophrenia-like Behaviors.

Dentate gyrus (DG) development requires specification of granule cell (GC) progenitors in the hippocampal neuroepithelium, as well as their proliferation and migration into the primordial DG. We identify the Plexin family members Plxna2 and Plxna4 as important regulators of DG development. Distribution of immature GCs is regulated by Sema5A signaling through PlxnA2 and requires a functional PlxnA2 GTPase-activating protein (GAP) domain and Rap1 small GTPases. In adult Plxna2-/- but not Plxna2-GAP-deficient mice, the dentate GC layer is severely malformed, neurogenesis is compromised, and mossy fibers form aberrant synaptic boutons within CA3. Behavioral studies with Plxna2-/- mice revealed deficits in associative learning, sociability, and sensorimotor gating-traits commonly observed in neuropsychiatric disorder. Remarkably, while morphological defects are minimal in Plxna2-GAP-deficient brains, defects in fear memory and sensorimotor gating persist. Since allelic variants of human PLXNA2 and RAP1 associate with schizophrenia, our studies identify a biochemical pathway important for brain development and mental health.

Membrane-binding and activation of LKB1 by phosphatidic acid is essential for development and tumour suppression.

The serine/threonine kinase LKB1 regulates various cellular processes such as cell proliferation, energy homeostasis and cell polarity and is frequently downregulated in various tumours. Many downstream pathways controlled by LKB1 have been described but little is known about the upstream regulatory mechanisms. Here we show that targeting of the kinase to the membrane by a direct binding of LKB1 to phosphatidic acid is essential to fully activate its kinase activity. Consequently, LKB1 mutants that are deficient for membrane binding fail to activate the downstream target AMPK to control mTOR signalling. Furthermore, the in vivo function of LKB1 during development of Drosophila depends on its capacity to associate with membranes. Strikingly, we find LKB1 to be downregulated in malignant melanoma, which exhibit aberrant activation of Akt and overexpress phosphatidic acid generating Phospholipase D. These results provide evidence for a fundamental mechanism of LKB1 activation and its implication in vivo and during carcinogenesis.

Rap1 GTPases Are Master Regulators of Neural Cell Polarity in the Developing Neocortex.

During the development of the mammalian neocortex, the generation of neurons by neural progenitors and their migration to the final position are closely coordinated. The highly polarized radial glial cells (RGCs) serve both as progenitor cells to generate neurons and as support for the migration of these neurons. After their generation, neurons transiently assume a multipolar morphology before they polarize and begin their migration along the RGCs. Here, we show that Rap1 GTPases perform essential functions for cortical organization as master regulators of cell polarity. Conditional deletion of Rap1 GTPases leads to a complete loss of cortical lamination. In RGCs, Rap1 GTPases are required to maintain their polarized organization. In newborn neurons, the loss of Rap1 GTPases prevents the formation of axons and leading processes and thereby interferes with radial migration. Taken together, the loss of RGC and neuronal polarity results in the disruption of cortical organization.

Regulation of Rap GTPases in mammalian neurons.

Small GTPases are central regulators of many cellular processes. The highly conserved Rap GTPases perform essential functions in the mammalian nervous system during development and in mature neurons. During neocortical development, Rap1 is required to regulate cadherin- and integrin-mediated adhesion. In the adult nervous system Rap1 and Rap2 regulate the maturation and plasticity of dendritic spine and synapses. Although genetic studies have revealed important roles of Rap GTPases in neurons, their regulation by guanine nucleotide exchange factors (GEFs) that activate them and GTPase activating proteins (GAPs) that inactivate them by stimulating their intrinsic GTPase activity is just beginning to be explored in vivo. Here we review how GEFs and GAPs regulate Rap GTPases in the nervous system with a focus on their in vivo function.

C3G/Rapgef1 Is Required in Multipolar Neurons for the Transition to a Bipolar Morphology during Cortical Development.

The establishment of a polarized morphology is essential for the development and function of neurons. During the development of the mammalian neocortex, neurons arise in the ventricular zone (VZ) from radial glia cells (RGCs) and leave the VZ to generate the cortical plate (CP). During their migration, newborn neurons first assume a multipolar morphology in the subventricular zone (SVZ) and lower intermediate zone (IZ). Subsequently, they undergo a multi-to-bipolar (MTB) transition to become bipolar in the upper IZ by developing a leading process and a trailing axon. The small GTPases Rap1A and Rap1B act as master regulators of neural cell polarity in the developing mouse neocortex. They are required for maintaining the polarity of RGCs and directing the MTB transition of multipolar neurons. Here we show that the Rap1 guanine nucleotide exchange factor (GEF) C3G (encoded by the Rapgef1 gene) is a crucial regulator of the MTB transition in vivo by conditionally inactivating the Rapgef1 gene in the developing mouse cortex at different time points during neuronal development. Inactivation of C3G results in defects in neuronal migration, axon formation and cortical lamination. Live cell imaging shows that C3G is required in cortical neurons for both the specification of an axon and the initiation of radial migration by forming a leading process.

Rassf5 and Ndr kinases regulate neuronal polarity through Par3 phosphorylation in a novel pathway.

The morphology and polarized growth of cells depend on pathways that control the asymmetric distribution of regulatory factors. The evolutionarily conserved Ndr kinases play important roles in cell polarity and morphogenesis in yeast and invertebrates but it is unclear whether they perform a similar function in mammalian cells. Here, we analyze the function of mammalian Ndr1 and Ndr2 (also known as STK38 or STK38L, respectively) in the establishment of polarity in neurons. We show that they act downstream of the tumor suppressor Rassf5 and upstream of the polarity protein Par3 (also known as PARD3). Rassf5 and Ndr1 or Ndr2 are required during the polarization of hippocampal neurons to prevent the formation of supernumerary axons. Mechanistically, the Ndr kinases act by phosphorylating Par3 at Ser383 to inhibit its interaction with dynein, thereby polarizing the distribution of Par3 and reinforcing axon specification. Our results identify a novel Rassf5-Ndr-Par3 signaling cascade that regulates the transport of Par3 during the establishment of neuronal polarity. Their role in neuronal polarity suggests that Ndr kinases perform a conserved function as regulators of cell polarity.

STI1 antagonizes cytoskeleton collapse mediated by small GTPase Rnd1 and regulates neurite growth.

Rnd proteins comprise a branch of the Rho family of small GTP-binding proteins, which have been implicated in rearrangements of the actin cytoskeleton and microtubule dynamics. Particularly in the nervous system, Rnd family proteins regulate neurite formation, dendrite development and axonal branching. A secreted form of the co-chaperone Stress-Inducible Protein 1 (STI1) has been described as a prion protein partner that is involved in several processes of the nervous system, such as neurite outgrowth, neuroprotection, astrocyte development, and the self-renewal of neural progenitor cells. We show that cytoplasmic STI1 directly interacts with the GTPase Rnd1. This interaction is specific for the Rnd1 member of the Rnd family. In the COS collapse assay, overexpression of STI1 prevents Rnd1-plexin-A1-mediated cytoskeleton retraction. In PC-12 cells, overexpression of STI1 enhances neurite outgrowth in cellular processes initially established by Rnd1. Therefore, we propose that STI1 participates in Rnd1-induced signal transduction pathways that are involved in the dynamics of the actin cytoskeleton.