Search

During development of the vertebrate CNS, the basic helix-loop-helix (bHLH) transcription factor Olig2 sustains replication competence of progenitor cells that give rise to neurons and oligodendrocytes. A pathological counterpart of this developmental function is seen in human glioma, wherein Olig2 is required for maintenance of stem-like cells that drive tumor growth. The mitogenic/gliomagenic functions of Olig2 are regulated by phosphorylation of a triple serine motif (S10, S13, and S14) in the amino terminus. Here, we identify a set of three serine/threonine protein kinases (glycogen synthase kinase 3α/β [GSK3α/β], casein kinase 2 [CK2], and cyclin-dependent kinases 1/2 [CDK1/2]) that are, collectively, both necessary and sufficient to phosphorylate the triple serine motif. We show that phosphorylation of the motif itself serves as a template to prime phosphorylation of additional serines and creates a highly charged "acid blob" in the amino terminus of Olig2. Finally, we show that small molecule inhibitors of this forward-feeding phosphorylation cascade have potential as glioma therapeutics.

RASopathies are a family of related syndromes caused by mutations in regulators of the RAS/Extracellular Regulated Kinase 1/2 (ERK1/2) signaling cascade that often result in neurological deficits. RASopathy mutations in upstream regulatory components, such as NF1, PTPN11/SHP2, and RAS have been well-characterized, but mutation-specific differences in the pathogenesis of nervous system abnormalities remain poorly understood, especially those involving mutations downstream of RAS. Here, we assessed cellular and behavioral phenotypes in mice expressing a Raf1L613V gain-of-function mutation associated with the RASopathy, Noonan Syndrome. We report that Raf1L613V/wt mutants do not exhibit a significantly altered number of excitatory or inhibitory neurons in the cortex. However, we observed a significant increase in the number of specific glial subtypes in the forebrain. The density of GFAP+ astrocytes was significantly increased in the adult Raf1L613V/wt cortex and hippocampus relative to controls. OLIG2+ oligodendrocyte progenitor cells were also increased in number in mutant cortices, but we detected no significant change in myelination. Behavioral analyses revealed no significant changes in voluntary locomotor activity, anxiety-like behavior, or sociability. Surprisingly, Raf1L613V/wt mice performed better than controls in select aspects of the water radial-arm maze, Morris water maze, and cued fear conditioning tasks. Overall, these data show that increased astrocyte and oligodendrocyte progenitor cell (OPC) density in the cortex coincides with enhanced cognition in Raf1L613V/wt mutants and further highlight the distinct effects of RASopathy mutations on nervous system development and function., Author summary The RASopathies are a large and complex family of syndromes caused by mutations in the RAS/MAPK signaling cascade with no known cure. Individuals with these syndromes often present with heart defects, craniofacial differences, and neurological abnormalities, such as developmental delay, cognitive changes, epilepsy, and an increased risk of autism. However, there is wide variation in the extent of intellectual ability between individuals. It is currently unclear how different RASopathy mutations affect brain development. Here, we describe the cellular and behavioral consequences of a mutation in a gene called Raf1 that is associated with a common RASopathy, Noonan Syndrome. We find that mice harboring a mutation in Raf1 show moderate increases in the number of two subsets of glial cells, which is also observed in a number of other RASopathy brain samples. Surprisingly, we found that Raf1 mutant mice show improved performance in several learning and memory tasks. Our work highlights potential mutation-specific changes in RASopathy brain function and helps set the framework for future personalized therapeutic approaches.

GSK-3 is an essential mediator of several signaling pathways that regulate cortical development. We therefore created conditional mouse mutants lacking both GSK-3α and GSK-3β in newly born cortical excitatory neurons. Gsk3-deleted neurons expressing upper layer markers exhibited striking migration failure in all areas of the cortex. Radial migration in hippocampus was similarly affected. In contrast, tangential migration was not grossly impaired after Gsk3 deletion in interneuron precursors. Gsk3-deleted neurons extended axons and developed dendritic arbors. However, the apical dendrite was frequently branched while basal dendrites exhibited abnormal orientation. GSK-3 regulation of migration in neurons was independent of Wnt/β-catenin signaling. Importantly, phosphorylation of the migration mediator, DCX, at ser327, and phosphorylation of the semaphorin signaling mediator, CRMP-2, at Thr514 were markedly decreased. Our data demonstrate that GSK-3 signaling is essential for radial migration and dendritic orientation and suggest that GSK-3 mediates these effects by phosphorylating key microtubule regulatory proteins. DOI: http://dx.doi.org/10.7554/eLife.02663.001, eLife digest In the brain, one of the most striking features of the cerebral cortex is that its neurons are organized into different layers that are specifically connected to one another and to other regions of the brain. How newly generated neurons find their appropriate layer during the development of the brain is an important question; and, in humans, when this process goes awry, it can often result in seizures and mental retardation. An enzyme called GSK-3 regulates several major signaling pathways important to brain development. The GSK-3 enzyme switches other proteins on or off by adding phosphate groups to them. Morgan-Smith et al. set out to better understand the role of GSK-3 in brain development by deleting the genes for this enzyme specifically in the cerebral cortex of mice. Mice have two genes that encode slightly different forms of the GSK-3 enzyme. Deleting both of these in different groups of neurons during brain development revealed that a major group of neurons need GSK-3 in order to migrate to the correct layer. Specifically, the movement of neurons from where they arise in the central region of the brain to the outermost layer (a process called radial migration) was disrupted when the GSK-3 genes were deleted. Morgan-Smith et al. further found that cortical neurons without GSK-3 were unable to develop the shape needed to undertake radial migration because they failed to switch from having many branches to having just two main branches. Additional experiments revealed that these abnormalities did not depend on certain signaling pathways, such as the Wnt-signaling pathway or the PI3K signaling pathway that can control GSK-3 activity. Instead, Morgan-Smith et al. found that two proteins that are normally targeted by the GSK-3 enzyme have fewer phosphate groups than normal in the cortical neurons that did not contain the enzyme: both of these proteins regulate the shape of neurons by interacting with the molecular ‘scaffolding’ within the cell. The GSK-3 enzyme was already known to modify the activities of many other proteins that affect the migration of cells. Thus, the findings of Morgan-Smith et al. suggest that this enzyme may coordinate many of the mechanisms thought to underlie this process during brain development. DOI: http://dx.doi.org/10.7554/eLife.02663.002

Laminin trimers composed of alpha, beta, and gamma chains are major components of basal laminae (BLs) throughout the body. To date, three alpha chains (alpha1-3) have been shown to assemble into at least seven heterotrimers (called laminins 1-7). Genes encoding two additional alpha chains (alpha4 and alpha5) have been cloned, but little is known about their expression, and their protein products have not been identified. Here we generated antisera to recombinant alpha4 and alpha5 and used them to identify authentic proteins in tissue extracts. Immunoprecipitation and immunoblotting showed that alpha4 and alpha5 assemble into four novel laminin heterotrimers (laminins 8-11: alpha4beta1gamma1, alpha4beta2gamma1, alpha5beta1gamma1, and alpha5beta2gamma1, respectively). Using a panel of nucleotide and antibody probes, we surveyed the expression of alpha1-5 in murine tissues. All five chains were expressed in both embryos and adults, but each was distributed in a distinct pattern at both RNA and protein levels. Overall, alpha4 and alpha5 exhibited the broadest patterns of expression, while expression of alpha1 was the most restricted. Immunohistochemical analysis of kidney, lung, and heart showed that the alpha chains were confined to extracellular matrix and, with few exceptions, to BLs. All developing and adult BLs examined contained at least one alpha chain, all alpha chains were present in multiple BLs, and some BLs contained two or three alpha chains. Detailed analysis of developing kidney revealed that some individual BLs, including those of the tubule and glomerulus, changed in laminin chain composition as they matured, expressing up to three different alpha chains and two different beta chains in an elaborate and dynamic progression. Interspecific backcross mapping of the five alpha chain genes revealed that they are distributed on four mouse chromosomes. Finally, we identified a novel full-length alpha3 isoform encoded by the Lama3 gene, which was previously believed to encode only truncated chains. Together, these results reveal remarkable diversity in BL composition and complexity in BL development.

SummaryWe have defined functions of MEK in regulating gliogenesis in developing cerebral cortex using loss- and gain-of-function mouse genetics. Radial progenitors deficient in both Mek1 and Mek2 fail to transition to the gliogenic mode in late embryogenesis, and astrocyte and oligodendroglial precursors fail to appear. In exploring mechanisms, we found that the key cytokine-regulated gliogenic pathway is attenuated. Further, the Ets transcription family member Etv5/Erm is strongly regulated by MEK and Erm overexpression can rescue the gliogenic potential of Mek-deleted progenitors. Remarkably, Mek1/2-deleted mice surviving postnatally exhibit cortices almost devoid of astrocytes and oligodendroglia and exhibit neurodegeneration. Conversely, expression of constitutively active MEK1 leads to a major increase in numbers of astrocytes in the adult brain. We conclude that MEK is essential for acquisition of gliogenic competence by radial progenitors and that levels of MEK activity regulate gliogenesis in the developing cortex.

Studies on the pathogenesis of human motor neurone disease have suffered from the absence of models of motor neurone degeneration in adult animals. Normally in adult rodents, transection of motor neurone axons results in only a modest degree of neuronal death. We reasoned that axotomy-induced motor neurone death might be enhanced by modulating glutamatergic transmission. By axotomizing the facial nerve in adult rats and then administering MK-801 for the first week of a 4-week or 8-week post-lesion survival period, we induced a 67% motor neurone loss by 8 weeks as compared with a 19% loss in controls. A possible explanation for the increased motor neurone loss after MK-801 treatment is that transient blockade of NMDA receptors may upregulate synthesis of NMDA receptor components. In order to test this idea, we employed quantitativein situhybridization to determine the response of NMDAR1 mRNA to axotomy and axotomy + MK-801 treatment. Quantification of the percentage of area occupied by NMDAR1 silver grains per motor neurone somata indicated that axotomy alone did not provoke a change in NMDAR1 mRNA. However, axotomy and MK-801 combined treatment resulted in a highly significant upregulation of NMDAR1 mRNA when compared with controls or animals treated solely with axotomy. Our results suggest that motor neurone death in adult animals can be enhanced after axotomy in association with the upregulation of NMDA receptor mRNA. Thus, abnormalities in glutamate receptor signalling may lead to subacute motor neurone deathin vivo. Furthermore these results indicate that transient treatment with MK-801 is a convenient method for enhancing the degree of motor neurone death after axotomy in adult animals.

SummaryWe have established functions of the stimulus-dependent MAPKs, ERK1/2 and ERK5, in DRG, motor neuron, and Schwann cell development. Surprisingly, many aspects of early DRG and motor neuron development were found to be ERK1/2 independent, and Erk5 deletion had no obvious effect on embryonic PNS. In contrast, Erk1/2 deletion in developing neural crest resulted in peripheral nerves that were devoid of Schwann cell progenitors, and deletion of Erk1/2 in Schwann cell precursors caused disrupted differentiation and marked hypomyelination of axons. The Schwann cell phenotypes are similar to those reported in neuregulin-1 and ErbB mutant mice, and neuregulin effects could not be elicited in glial precursors lacking Erk1/2. ERK/MAPK regulation of myelination was specific to Schwann cells, as deletion in oligodendrocyte precursors did not impair myelin formation, but reduced precursor proliferation. Our data suggest a tight linkage between developmental functions of ERK/MAPK signaling and biological actions of specific RTK-activating factors.

Conditional deletion of APC leads to marked disruption of cortical development and to excessive axonal branching of cortical neurons. However, little is known about the cell biological basis of this neuronal morphological regulation. Here we show that APC deficient cortical neuronal growth cones exhibit marked disruption of both microtubule and actin cytoskeleton. Functional analysis of the different APC domains revealed that axonal branches do not result from stabilized β-catenin, and that the C-terminus of APC containing microtubule regulatory domains only partially rescues the branching phenotype. Surprisingly, the N-terminus of APC containing the oligomerization domain and the armadillo repeats completely rescues the branching and cytoskeletal abnormalities. Our data indicate that APC is required for appropriate axon morphological development and that the N-terminus of APC is important for regulation of the neuronal cytoskeleton.

Background: Regulator of G-protein signaling (RGS) proteins have been well-described as accelerators of Ga-mediated GTP hydrolysis (‘‘GTPase-accelerating proteins’’ or GAPs). However, RGS proteins with complex domain architectures are now known to regulate much more than Ga GTPase activity. RGS14 contains tandem Ras-binding domains that have been reported to bind to Rap- but not Ras GTPases in vitro, leading to the suggestion that RGS14 is a Rap-specific effector. However, more recent data from mammals and Drosophila imply that, in vivo, RGS14 may instead be an effector of Ras. Methodology/Principal Findings: Full-length and truncated forms of purified RGS14 protein were found to bind indiscriminately in vitro to both Rap- and Ras-family GTPases, consistent with prior literature reports. In stark contrast, however, we found that in a cellular context RGS14 selectively binds to activated H-Ras and not to Rap isoforms. Co- transfection / co-immunoprecipitation experiments demonstrated the ability of full-length RGS14 to assemble a multiprotein complex with components of the ERK MAPK pathway in a manner dependent on activated H-Ras. Small interfering RNA-mediated knockdown of RGS14 inhibited both nerve growth factor- and basic fibrobast growth factor- mediated neuronal differentiation of PC12 cells, a process which is known to be dependent on Ras-ERK signaling. Conclusions/Significance: In cells, RGS14 facilitates the formation of a selective Ras?GTP-Raf-MEK-ERK multiprotein complex to promote sustained ERK activation and regulate H-Ras-dependent neuritogenesis. This cellular function for RGS14 is similar but distinct from that recently described for its closely-related paralogue, RGS12, which shares the tandem Ras- binding domain architecture with RGS14.

SummaryRadial glia are highly polarized cells that serve as neuronal progenitors and as scaffolds for neuronal migration during construction of the cerebral cortex. How radial glial cells establish and maintain their morphological polarity is unknown. Using conditional gene targeting in mice, we demonstrate that adenomatous polyposis coli (APC) serves an essential function in the maintenance of polarized radial glial scaffold during brain development. In the absence of APC, radial glial cells lose their polarity and responsiveness to the extracellular polarity maintenance cues, such as neuregulin-1. Elimination of APC further leads to marked instability of the radial glial microtubule cytoskeleton. The resultant changes in radial glial function and loss of APC in radial glial progeny lead to defective generation and migration of cortical neurons, severely disrupted cortical layer formation, and aberrant axonal tract development. Thus, APC is an essential regulator of radial glial polarity and is critical for the construction of cerebral cortex in mammals.

The development of the brain requires the exquisite coordination of progenitor proliferation and differentiation to achieve complex circuit assembly. It has been suggested that glycogen synthase kinase 3 (GSK-3) acts as an integrating molecule for multiple proliferation and differentiation signals because of its essential role in the RTK, Wnt and Shh signaling pathways. We created conditional mutations that deleted both the alpha and beta forms of GSK-3 in mouse neural progenitors. GSK-3 deletion resulted in massive hyperproliferation of neural progenitors along the entire neuraxis. Generation of both intermediate neural progenitors and postmitotic neurons was markedly suppressed. These effects were associated with the dysregulation of beta-catenin, Sonic Hedgehog, Notch and fibroblast growth factor signaling. Our results indicate that GSK-3 signaling is an essential mediator of homeostatic controls that regulate neural progenitors during mammalian brain development.

Regulator of G-protein signaling (RGS) proteins accelerate GTP hydrolysis by heterotrimeric G-protein alpha subunits and thus inhibit signaling by many G protein-coupled receptors. Several RGS proteins have a multidomain architecture that adds further complexity to their roles in cell signaling in addition to their GTPase-accelerating activity. RGS12 contains a tandem repeat of Ras-binding domains but, to date, the role of this protein in Ras-mediated signal transduction has not been reported. Here, we show that RGS12 associates with the nerve growth factor (NGF) receptor tyrosine kinase TrkA, activated H-Ras, B-Raf, and MEK2 and facilitates their coordinated signaling to prolonged ERK activation. RGS12 is required for NGF-mediated neurite outgrowth of PC12 cells, but not outgrowth stimulated by basic fibroblast growth factor. siRNA-mediated knockdown of RGS12 expression also inhibits NGF-induced axonal growth in dissociated cultures of primary dorsal root ganglia neurons. These data suggest that RGS12 may play a critical, and receptor-selective, role in coordinating Ras-dependent signals that are required for promoting and/or maintaining neuronal differentiation.

Glycogen synthase kinase-3beta (GSK-3beta) is thought to mediate morphological responses to a variety of extracellular signals. Surprisingly, we found no gross morphological deficits in nervous system development in GSK-3beta null mice. We therefore designed an shRNA that targeted both GSK-3 isoforms. Strong knockdown of both GSK-3alpha and beta markedly reduced axon growth in dissociated cultures and slice preparations. We then assessed the role of different GSK-3 substrates in regulating axon morphology. Elimination of activity toward primed substrates only using the GSK-3 R96A mutant was associated with a defect in axon polarity (axon branching) compared to an overall reduction in axon growth induced by a kinase-dead mutant. Consistent with this finding, moderate reduction of GSK-3 activity by pharmacological inhibitors induced axon branching and was associated primarily with effects on primed substrates. Our results suggest that GSK-3 is a downstream convergent point for many axon growth regulatory pathways and that differential regulation of primed versus all GSK-3 substrates is associated with a specific morphological outcome.

The functions of neurotrophins in relation to axon growth and branching during development of the nervous system are unknown. In order to address this question, we have investigated the influences of systemically administered mouse nerve growth factor (mNGF) and human recombinant neurotrophin-3 (hrNT-3) on dorsal root axon growth in the spinal cord of embryonic rats. As anticipated, mNGF has a marked influence on growth of dorsal root axons. In mNGF-treated animals, dorsal root axons in the developing dorsal funiculi and axon collaterals in developing gray matter are substantially longer than those of age-matched controls. Furthermore, growth cones of some dorsal root axons have more than twice the surface area of controls. These effects of NGF are highly selective. Dorsal root axons that occupy a lateral position in white matter and that normally give off collaterals to superficial dorsal horn are prominently affected. Axons that run medially in dorsal columns and that give off collaterals to laminae III and IV and the ventral horn are not demonstrably influenced by treatment with exogenous mNGF. In contrast to the striking effects of mNGF on dorsal root axon growth, the influences of hrNT-3 were considerably more complex. Administration of hrNT-3 increased the mean soma area of DRG neurons, particularly those at the larger end of the size spectrum, consistent with its hypothesized role as a growth factor for proprioceptive sensory neurons. However, in striking contrast to the actions of mNGF, hrNT-3 consistently inhibited axon collateral growth in gray matter at early developmental stages. At later stages, we could not discern a clear-cut influence of hrNT-3 on dorsal root axon growth and branching. We conclude that the ability of mNGF to stimulate axon growth in both white and gray matter is consistent with the idea that mNGF regulates the developing axonal projections of DRG neurons in vivo. In contrast, systemically administered hrNT-3 inhibits the axon collateralizations of DRG neurons in gray matter at early developmental stages. We hypothesize that this inhibitory effect may be related to disruption of a chemotropic gradient of NT-3, or to the widespread expression of the NT-3 receptor trkC, on non-neuronal cells.

Extrinsic cues activate intrinsic signaling mechanisms to pattern neuronal shape and connectivity. We showed previously that three cytoplasmic Ser/Thr kinases, LKB1, SAD-A, and SAD-B, control early axon-dendrite polarization in forebrain neurons. Here, we assess their role in other neuronal types. We found that all three kinases are dispensable for axon formation outside of the cortex but that SAD kinases are required for formation of central axonal arbors by subsets of sensory neurons. The requirement for SAD kinases is most prominent in NT-3 dependent neurons. SAD kinases transduce NT-3 signals in two ways through distinct pathways. First, sustained NT-3/TrkC signaling increases SAD protein levels. Second, short-duration NT-3/TrkC signals transiently activate SADs by inducing dephosphorylation of C-terminal domains, thereby allowing activating phosphorylation of the kinase domain. We propose that SAD kinases integrate long- and short-duration signals from extrinsic cues to sculpt axon arbors within the CNS.

SummaryDeletion of UBE3A causes the neurodevelopmental disorder Angelman syndrome (AS), while duplication or triplication of UBE3A is linked to autism. These genetic findings suggest that the ubiquitin ligase activity of UBE3A must be tightly maintained to promote normal brain development. Here, we found that protein kinase A (PKA) phosphorylates UBE3A in a region outside of the catalytic domain at residue T485 and inhibits UBE3A activity toward itself and other substrates. A de novo autism-linked missense mutation disrupts this phosphorylation site, causing enhanced UBE3A activity in vitro, enhanced substrate turnover in patient-derived cells, and excessive dendritic spine development in the brain. Our study identifies PKA as an upstream regulator of UBE3A activity and shows that an autism-linked mutation disrupts this phosphorylation control. Moreover, our findings implicate excessive UBE3A activity and the resulting synaptic dysfunction to autism pathogenesis.

Despite the fact that NGF was discovered more than 40 years ago, an understanding of neurotrophin regulation of sensory functions is only now emerging. Strong evidence links NGF to regulation of pain transmission, both in mediating acute responses to inflammation and other noxious stimuli and in regulating gene expression that may lead to chronically enhanced nociceptor transmission (14xLewin, G.R and Mendell, L.M. Trends Neurosci. 1993; 16: 353–359Abstract | Full Text PDF | PubMed | Scopus (397)See all References, 16xMcMahon, S.B, Bennett, D.L.H, Priestley, J.V, and Shelton, D.L. Nature Med. 1995; 1: 774–780Crossref | PubMed | Scopus (314)See all References). From a clinical standpoint, this powerful regulation of nociception is likely to become the most important pharmacological action of this family of molecules. Studies of neurotrophin regulation of other sensory modalities are only beginning. The continued expression of NT-3 by muscle spindles in adult animals suggests the potential for regulation of proprioceptive function by NT-3. The study of Airaksinen et al. now suggests tactile sensation as an additional target for NT-3 regulation. We will look forward to future studies that will further clarify the roles of neurotrophins in sensory physiology.

Little is known about how nerve growth factor (NGF) signaling controls the regulated assembly of microtubules that underlies axon growth. Here we demonstrate that a tightly regulated and localized activation of phosphatidylinositol 3-kinase (PI3K) at the growth cone is essential for rapid axon growth induced by NGF. This spatially activated PI3K signaling is conveyed downstream through a localized inactivation of glycogen synthase kinase 3β (GSK-3β). These two spatially coupled kinases control axon growth via regulation of a microtubule plus end binding protein, adenomatous polyposis coli (APC). Our results demonstrate that NGF signals are transduced to the axon cytoskeleton via activation of a conserved cell polarity signaling pathway.

I have compared the dendritic complexity and innervation of homologous parasympathetic ganglion cells in several closely related species of mammals. In the smaller of these species (mouse, hamster, and rat), submandibular ganglion cells generally lack dendrites altogether and are innervated by a single axon. In the guinea pig, a somewhat larger species, these neurons possess rudimentary dendritic arbors and are innervated by 2 axons, on average. In the largest species investigated, the rabbit, submandibular ganglion cells have moderately complex dendritic arbors and receive innervation from several axons. These findings, together with a previous study of sympathetic ganglion cells in these same species (Purves and Lichtman, 1985a), indicate that relationships among neuronal morphology, convergent innervation, and animal size are widespread in the autonomic nervous system of mammals.

Axon regeneration failure accounts for permanent functional deficits following CNS injury in adult mammals. However, the underlying mechanisms remain elusive. In analyzing axon regeneration in different mutant mouse lines, we discovered that deletion of suppressor of cytokine signaling 3 (SOCS3), in adult retinal ganglion cells (RGCs), promotes robust regeneration of injured optic nerve axons. This regeneration-promoting effect is efficiently blocked in SOCS3-gp130 double knockout mice, suggesting that SOCS3 deletion promotes axon regeneration via a gp130-dependent pathway. Consistently, a transient up-regulation of ciliary neurotrophic factor (CNTF) was observed within the retina following optic nerve injury. Intravitreal application of CNTF further enhances axon regeneration from SOCS3-deleted RGCs. Together, our results suggest that compromised responsiveness to injury-induced growth factors in mature neurons contributes significantly to regeneration failure. Thus, developing strategies to modulate negative signaling regulators may be an efficient strategy of promoting axon regeneration after CNS injury.