PACAP decides neuronal laminar fate via PKA signaling in the developing cerebral cortex

Laminar formation in the developing cerebral cortex requires the precisely regulated generation of phenotype-specified neurons. To test the possible involvement of pituitary adenylate cyclase-activating polypeptide (PACAP) in this formation, we investigated the effects of PACAP administered into the telencephalic ventricular space of 13.5-day-old mouse embryos. PACAP partially inhibited the proliferation of cortical progenitors and altered the position and gene-expression profiles of newly generated neurons otherwise expected for layer IV to those of neurons for the deeper layers, V and VI, of the cerebral cortex. The former and latter effects were seen only when the parent progenitor cells were exposed to PACAP in the later and in earlier G1 phase, respectively; and these effects were suppressed by co-treatment with a protein kinase A (PKA) inhibitor. These observations suggest that PACAP participates in the processes forming the neuronal laminas in the developing cortex via the intracellular PKA pathway.     

JNK phosphorylates Ser332 of doublecortin and regulates its function in neurite extension and neuronal migration

Doublecortin (DCX) is expressed in young neurons and functions as a microtubule‐associated protein. DCX is essential for neuronal migration because humans with mutations in the DCX gene exhibit cortical lamination defects known as lissencephaly in males and subcortical laminar heterotopia (or double cortex syndrome) in females. Phosphorylation of DCX alters its affinity for tubulin and may modulate neurite extension and neuronal migra tion. Previous in vitro phosphorylation experiments revealed that cyclin‐dependent kinase 5 (Cdk5) phosphorylates multiple sites of DCX, including Ser332, (S332). However, phosphorylation at only Ser297 has been shown in vivo. In the present study, we examined phosphorylation of S332 of DCX in the Cdk5?/? mouse brain and results found, unexpectedly, indicate an increased DCX phosphorylation at S332. We found that JNK, not Cdk5, phosphorylates DCX at S332 in vivo. To examine the physiological significance of S332 phosphorylation of DCX in neuronal cells, we transfected cells with either GFP, GFP‐DCX‐WT, or GFP‐DCX‐S332A and analyzed neurite extension and migration. Introduction of GFP‐DCX‐WT enhanced neurite extension and migration. These effects of DCX introduction were suppressed when we used GFP‐DCX‐S332A. Treatment of neurons with JNK inhibitor increased the amount of DCX that bound to tubulin. Interestingly, amount of DCX that bound to tubulin decreased in Cdk5?/? brain homogenates, which indicates that phosphorylation of DCX by JNK is critical for the regulation of DCX binding to tubulin. These results suggest the physiological importance of phosphorylation of DCX for its function. © 2010 Wiley Periodicals, Inc. Develop Neurobiol 70: 929–942, 2010     

Doublecortin association with actin filaments is regulated by neurabin II

Mutations in the human Doublecortin (DCX) gene cause X-linked lissencephaly, a neuronal migration disorder affecting the neocortex and characterized by mental retardation and epilepsy. Because dynamic cellular asymmetries such as those seen in cell migration critically depend on a cooperation between the microtubule and actin cytoskeletal filament systems, we investigated whether Dcx, a microtubule-associated protein, is engaged in cytoskeletal cross-talk. We now demonstrate that Dcx co-sediments with actin filaments (F-actin), and using light and electron microscopy and spin down assays, we show that Dcx induces bundling and cross-linking of microtubules and F-actin in vitro. It has recently been shown that binding of Dcx to microtubules is negatively regulated by phosphorylation of the Dcx at Ser-47 or Ser-297. Although the phosphomimetic green fluorescent protein (GFP)-Dcx(S47E) transfected into COS-7 cells had a reduced affinity for microtubules, we found that pseudophosphorylation was not sufficient to cause Dcx to bind to F-actin. When cells were co-transfected with neurabin II, a protein that binds F-actin as well as Dcx, GFP-Dcx and to an even greater extent GFP-Dcx(S47E) became predominantly associated with filamentous actin. Thus Dcx phosphorylation and neurabin II combinatorially enhance Dcx binding to F-actin. Our findings raise the possibility that Dcx acts as a molecular link between microtubule and actin cytoskeletal filaments that is regulated by phosphorylation and neurabin II.     

Colocalization of doublecortin with the microtubules: an ex vivo colocalization study of mutant doublecortin

Doublecortin (DCX) plays an important role in neuronal migration and development, and the participation of DCX in neuronal migration has been demonstrated by intensive mutational analysis for patients with X-linked or sporadic lissencephaly, and/or subcortical laminar heterotopia. Although a previous search for protein similarity showed that DCX has a region homologous to the putative Ca(2+)/calmodulin-dependent protein kinase, the function of the DCX gene (DCX) has remained unknown. We show here that mouse DCX colocalizes with the microtubules and provide evidence that its conformational structure is important for its subcellular localization by means of mutant doublecortin expression study. The results of our study may suggest that the cytoskeleton involving DCX mediates the neuronal migration during brain development.     

Doublecortin is a microtubule-associated protein and is expressed widely by migrating neurons.

Doublecortin (DCX) is required for normal migration of neurons into the cerebral cortex, since mutations in the human gene cause a disruption of cortical neuronal migration. To date, little is known about the distribution of DCX protein or its function. Here, we demonstrate that DCX is expressed in migrating neurons throughout the central and peripheral nervous system during embryonic and postnatal development. DCX protein localization overlaps with microtubules in cultured primary cortical neurons, and this overlapping expression is disrupted by microtubule depolymerization. DCX coassembles with brain microtubules, and recombinant DCX stimulates the polymerization of purified tubulin. Finally, overexpression of DCX in heterologous cells leads to a dramatic microtubule phenotype that is resistant to depolymerization. Therefore, DCX likely directs neuronal migration by regulating the organization and stability of microtubules.     

Topology determination and functional analysis of the Escherichia coli TatC protein

Misfolding of the prion protein yields amyloidogenic isoforms, and it shows exacerbating neuronal damage in neurodegenerative disorders including prion diseases. Pituitary adenylate cyclase-activating polypeptide (PACAP) and vasoactive intestinal peptide (VIP) potently stimulate neuritogenesis and survival of neuronal cells in the central nervous system. Here, we tested these neuropeptides on neurotoxicity in PC12 cells induced by the prion protein fragment 106-126 [PrP (106-126)]. Concomitant application of neuropeptide with PrP(106-126) (5x10(-5) M) inhibited the delayed death of neuron-like PC12 cells. In particular, PACAP27 inhibited the neurotoxicity of PrP(106-126) at low concentrations (>10(-15) M), characterized by the deactivation of PrP(106-126)-stimulated caspase-3. The neuroprotective effect of PACAP27 was antagonized by the selective PKA inhibitor, H89, or the MAP kinase inhibitor, U0126. These results suggest that PACAP27 attenuates PrP(106-126)-induced delayed neurotoxicity in PC12 cells by activating both PKA and MAP kinases mediated by PAC1 receptor.     

Stimulation of the ERK pathway by GTP-loaded Rap1 requires the concomitant activation of Ras,protein kinase C,and protein kinase A in neuronal cells

The small GTPases Ras or Rap1 were suggested to mediate the stimulatory effect of some G protein-coupled receptors on ERK activity in neuronal cells. Accordingly, we reported here that pituitary adenylate cyclase-activating polypeptide (PACAP), whose G protein-coupled receptor triggers neuronal differentiation of the PC12 cell line via ERK1/2 activation, transiently activated Ras and induced the sustained GTP loading of Rap1. Ras mediated peak stimulation of ERK by PACAP, whereas Rap1 was necessary for the sustained activation phase. However, PACAP-induced GTP-loading of Rap1 was not sufficient to account for ERK activation by PACAP because 1) PACAP-elicited Rap1 GTP-loading depended only on phospholipase C, whereas maximal stimulation of ERK by PACAP also required the activity of protein kinase A (PKA), protein kinase C (PKC), and calcium-dependent signaling; and 2) constitutively active mutants of Rap1, Rap1A-V12, and Rap1B-V12 only minimally stimulated the ERK pathway compared with Ras-V12. The effect of Rap1A-V12 was dramatically potentiated by the concurrent activation of PKC, the cAMP pathway, and Ras, and this potentiation was blocked by dominant-negative mutants of Ras and Raf. Thus, this set of data indicated that GPCR-elicited GTP loading of Rap1 was not sufficient to stimulate efficiently ERK in PC12 cells and required the permissive co-stimulation of PKA, PKC, or Ras.     

Phagocytic activity of neuronal progenitors regulates adult neurogenesis

Whereas thousands of new neurons are generated daily during adult life, only a fraction of them survive and become part of neural circuits; the rest die, and their corpses are presumably cleared by resident phagocytes. How the dying neurons are removed and how such clearance influences neurogenesis are not well understood. Here, we identify an unexpected phagocytic role for the doublecortin (DCX)-positive neuronal progenitor cells during adult neurogenesis. Our in vivo and ex vivo studies demonstrate that DCX(+) cells comprise a significant phagocytic population within the neurogenic zones. Intracellular engulfment protein ELMO1, which promotes Rac activation downstream of phagocytic receptors, was required for phagocytosis by DCX(+) cells. Disruption of engulfment in vivo genetically (in Elmo1-null mice) or pharmacologically (in wild-type mice) led to reduced uptake by DCX(+) cells, accumulation of apoptotic nuclei in the neurogenic niches and impaired neurogenesis. Collectively, these findings indicate a paradigm wherein DCX(+) neuronal precursors also serve as phagocytes, and that their phagocytic activity critically contributes to neurogenesis in the adult brain.     

DCX, a new mediator of the JNK pathway

Mutations in the X-linked gene DCX result in lissencephaly in males, and abnormal neuronal positioning in females, suggesting a role for this gene product during neuronal migration. In spite of several known protein interactions, the involvement of DCX in a signaling pathway is still elusive. Here we demonstrate that DCX is a substrate of JNK and interacts with both c-Jun N-terminal kinase (JNK) and JNK interacting protein (JIP). The localization of this signaling module in the developing brain suggests its functionality in migrating neurons. The localization of DCX at neurite tips is determined by its interaction with JIP and by the interaction of the latter with kinesin. DCX is phosphorylated by JNK in growth cones. DCX mutated in sites phosphorylated by JNK affected neurite outgrowth, and the velocity and relative pause time of migrating neurons. We hypothesize that during neuronal migration, there is a need to regulate molecular motors that are working in the cell in opposite directions: kinesin (a plus-end directed molecular motor) versus dynein (a minus-end directed molecular motor).     

Global developmental gene expression and pathway analysis of normal brain development and mouse models of human neuronal migration defects

Heterozygous LIS1 mutations are the most common cause of human lissencephaly, a human neuronal migration defect, and DCX mutations are the most common cause of X-linked lissencephaly. LIS1 is part of a protein complex including NDEL1 and 14-3-3ε that regulates dynein motor function and microtubule dynamics, while DCX stabilizes microtubules and cooperates with LIS1 during neuronal migration and neurogenesis. Targeted gene mutations of Lis1, Dcx, Ywhae (coding for 14-3-3ε), and Ndel1 lead to neuronal migration defects in mouse and provide models of human lissencephaly, as well as aid the study of related neuro-developmental diseases. Here we investigated the developing brain of these four mutants and wild-type mice using expression microarrays, bioinformatic analyses, and in vivo/in vitro experiments to address whether mutations in different members of the LIS1 neuronal migration complex lead to similar and/or distinct global gene expression alterations. Consistent with the overall successful development of the mutant brains, unsupervised clustering and co-expression analysis suggested that cell cycle and synaptogenesis genes are similarly expressed and co-regulated in WT and mutant brains in a time-dependent fashion. By contrast, focused co-expression analysis in the Lis1 and Ndel1 mutants uncovered substantial differences in the correlation among pathways. Differential expression analysis revealed that cell cycle, cell adhesion, and cytoskeleton organization pathways are commonly altered in all mutants, while synaptogenesis, cell morphology, and inflammation/immune response are specifically altered in one or more mutants. We found several commonly dysregulated genes located within pathogenic deletion/duplication regions, which represent novel candidates of human mental retardation and neurocognitive disabilities. Our analysis suggests that gene expression and pathway analysis in mouse models of a similar disorder or within a common pathway can be used to define novel candidates for related human diseases.     

PlexinD1 signaling controls morphological changes and migration termination in newborn neurons

Newborn neurons maintain a very simple, bipolar shape, while they migrate from their birthplace toward their destinations in the brain, where they differentiate into mature neurons with complex dendritic morphologies. Here, we report a mechanism by which the termination of neuronal migration is maintained in the postnatal olfactory bulb (OB). During neuronal deceleration in the OB, newborn neurons transiently extend a protrusion from the proximal part of their leading process in the resting phase, which we refer to as a filopodium‐like lateral protrusion (FLP). The FLP formation is induced by PlexinD1 downregulation and local Rac1 activation, which coincide with microtubule reorganization and the pausing of somal translocation. The somal translocation of resting neurons is suppressed by microtubule polymerization within the FLP. The timing of neuronal migration termination, controlled by Sema3E‐PlexinD1‐Rac1 signaling, influences the final positioning, dendritic patterns, and functions of the neurons in the OB. These results suggest that PlexinD1 signaling controls FLP formation and the termination of neuronal migration through a precise control of microtubule dynamics.     

Ndel1 operates in a common pathway with LIS1 and cytoplasmic dynein to regulate cortical neuronal positioning

Correct neuronal migration and positioning during cortical development are essential for proper brain function. Mutations of the LIS1 gene result in human lissencephaly (smooth brain), which features misplaced cortical neurons and disarrayed cerebral lamination. However, the mechanism by which LIS1 regulates neuronal migration remains unknown. Using RNA interference (RNAi), we found that the binding partner of LIS1, NudE-like protein (Ndel1, formerly known as NUDEL), positively regulates dynein activity by facilitating the interaction between LIS1 and dynein. Loss of function of Ndel1, LIS1, or dynein in developing neocortex impairs neuronal positioning and causes the uncoupling of the centrosome and nucleus. Overexpression of LIS1 partially rescues the positioning defect caused by Ndel1 RNAi but not dynein RNAi, whereas overexpression of Ndel1 does not rescue the phenotype induced by LIS1 RNAi. These results provide strong evidence that Ndel1 interacts with LIS1 to sustain the function of dynein, which in turn impacts microtubule organization, nuclear translocation, and neuronal positioning.     

Pituitary adenylate cyclase activating polypeptide regulates the basal production of nitric oxide in PC12 cells

Vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP), two members of the VIP/secretin/glucagon family, modulate neurotransmission via stimulation of protein kinases including cAMP-dependent protein kinase (PKA) and protein kinase C (PKC) in the central and peripheral nervous systems. They are reported to co-exist with nitric oxide synthases (NOSs) and other neuropeptides within the nervous system and peripheral tissues. In the present study, we investigated the neuronal role of these peptides in NO production in PC12 cells. We showed that PACAP decreased NO production in a dose-dependent manner, and the activators of protein kinase A and C also inhibited the NO production in PC12 cells. RT-PCR experiments demonstrated that PC12 cells constitutively express the mRNAs for neuronal NOS and the PACAP-specific (PAC1) receptor, and we concluded that PACAP plays an important role in the regulation of nNOS activity through PAC1 receptor in PC12 cells.     

Smooth,rough and upside-down neocortical development

Lissencephaly, which means 'smooth cortex', is caused by defective neuronal migration during development of the cerebral cortex and has devastating clinical consequences. 'Classical' lissencephaly seems to reflect mutations in regulators of the microtubule cytoskeleton, whereas 'cobblestone' lissencephaly is caused by mutations in genes needed for the integrity of the basal lamina of the central nervous system. Reelin, which is mutated in a third type of lissencephaly, may represent a unifying link because it encodes an extracellular protein that regulates neuronal migration and may also regulate the microtubule cytoskeleton.     

Doublecortin microtubule affinity is regulated by a balance of kinase and phosphatase activity at the leading edge of migrating neurons

Doublecortin (Dcx) is a microtubule-associated protein that is mutated in X-linked lissencephaly (X-LIS), a neuronal migration disorder associated with epilepsy and mental retardation. Although Dcx can bind ubiquitously to microtubules in nonneuronal cells, Dcx is highly enriched in the leading processes of migrating neurons and the growth cone region of differentiating neurons. We present evidence that Dcx/microtubule interactions are negatively controlled by Protein Kinase A (PKA) and the MARK/PAR-1 family of protein kinases. In addition to a consensus MARK site, we identified a serine within a novel sequence that is crucial for the PKA- and MARK-dependent regulation of Dcx's microtubule binding activity in vitro. This serine is mutated in two families affected by X-LIS. Immunostaining neurons with an antibody that recognizes phosphorylated substrates of MARK supports the conclusion that Dcx localization and function are regulated at the leading edge of migrating cells by a balance of kinase and phosphatase activity.     

Neuronal migration.

Like other motile cells, neurons migrate in three schematic steps, namely leading edge extension, nuclear translocation or nucleokinesis, and retraction of the trailing process. In addition, neurons are ordered into architectonic patterns at the end of migration. Leading edge extension can proceed at the extremity of the axon, by growth cone formation, or from the dendrites, by formation of dendritic tips. Among both categories of leading edges, variation seems to be related to the rate of extension of the leading process. Leading edge extension is directed by microfilament polymerization following integration of extracellular cues and is regulated by Rho-type small GTPases. In humans, mutations of filamin, an actin-associated protein, result in heterotopic neurons, probably due to defective leading edge extension. The second event in neuron migration is nucleokinesis, a process which is critically dependent on the microtubule network, as shown in many cell types, from slime molds to vertebrates. In humans, mutations in the PAFAH1B1 gene (more commonly called LIS1) or in the doublecortin (DCX) gene result in type 1 lissencephalies that are most probably due to defective nucleokinesis. Both the Lis1 and doublecortin proteins interact with microtubules, and two Lis1-interacting proteins, Nudel and mammalian NudE, are components of the dynein motor complex and of microtubule organizing centers. In mice, mutations of Cdk5 or of its activators p35 and p39 result in a migration phenotype compatible with defective nucleokinesis, although an effect on leading edge formation is also likely. The formation of architectonic patterns at the end of migration requires the integrity of the Reelin signalling pathway. Other known components of the pathway include members of the lipoprotein receptor family, the intracellular adaptor Dab1, and possibly integrin alpha 3 beta 1. Defective Reelin leads to poor lamination and, in humans, to a lissencephaly phenotype different from type 1 lissencephaly. Although the action of Reelin is unknown, it may trigger some recognition-adhesion among target neurons. Finally, pattern formation requires the integrity of the external limiting membrane, defects of which lead to overmigration of neurons in meninges and to human type 2 lissencephaly.     

Doublecortin kinase-2, a novel doublecortin-related protein kinase associated with terminal segments of axons and dendrites

The microtubule (MT)-associated DCX protein plays an essential role in the development of the mammalian cerebral cortex. We report on the identification of a protein kinase, doublecortin kinase-2 (DCK2), with a domain (DC) highly homologous to DCX. DCK2 has MT binding activity associated with its DC domain and protein kinase activity mediated by a kinase domain, organized in a structure in which the two domains are functionally independent. Overexpression of DCK2 stabilizes the MT cytoskeleton against cold-induced depolymerization. Autophosphorylation of DCK2 strongly reduces its affinity for MTs. DCK2 and DCX mRNAs are nervous system-specific and are expressed during the period of cerebrocortical lamination. DCX is down-regulated postnatally, whereas DCK2 persists in abundance into adulthood, suggesting that the DC sequence has previously unrecognized functions in the mature nervous system. In sympathetic neurons, DCK2 is localized to the cell body and to the terminal segments of axons and dendrites. DCK2 may represent a phosphorylation-dependent switch for the reversible control of MT dynamics in the vicinity of neuronal growth cones.     

Genetic mechanisms underlying abnormal neuronal migration in classical lissencephaly

Classical lissencephaly is a human developmental brain disorder characterized by a paucity of cortical gyration and thickening of the cortical gray matter, leading to severe epilepsy and mental retardation. Loss-of-function mutations in the microtubule-associated protein encoding genes, PAFAH1B1 (encoding the protein LIS1), DCX and TUBA1A have been implicated in the pathogenesis of the condition. Animal models are required to understand the basis of this disease, which is a challenge, given that mice normally have a smooth cortex. Recent advances toward this goal have come from stepwise reduction in gene function, deletion of redundant genes and acute gene inactivation using short hairpin RNA (shRNA). These approaches have implicated genes that regulate the microtubule cytoskeleton during neuronal division, migration and maturation.     

MEGAP impedes cell migration via regulating actin and microtubule dynamics and focal complex formation

Over the past several years, it has become clear that the Rho family of GTPases plays an important role in various aspects of neuronal development including cytoskeleton dynamics and cell adhesion processes. We have analysed the role of MEGAP, a GTPase-activating protein that acts towards Rac1 and Cdc42 in vitro and in vivo, with respect to its putative regulation of cytoskeleton dynamics and cell migration. To investigate the effects of MEGAP on these cellular processes, we have established an inducible cell culture model consisting of a stably transfected neuroblastoma SHSY-5Y cell line that endogenously expresses MEGAP albeit at low levels. We can show that the induced expression of MEGAP leads to the loss of filopodia and lamellipodia protrusions, whereas constitutively activated Rac1 and Cdc42 can rescue the formation of these structures. We have also established quantitative assays for evaluating actin dynamics and cellular migration. By time-lapse microscopy, we show that induced MEGAP expression reduces cell migration by 3.8-fold and protrusion formation by 9-fold. MEGAP expressing cells also showed impeded microtubule dynamics as demonstrated in the TC-7 3x-GFP epithelial kidney cells. In contrast to the wild type, overexpression of MEGAP harbouring an artificially introduced missense mutation R542I within the functionally important GAP domain did not exert a visible effect on actin and microtubule cytoskeleton remodelling. These data suggest that MEGAP negatively regulates cell migration by perturbing the actin and microtubule cytoskeleton and by hindering the formation of focal complexes.     

Differential involvement of PKA and PKC in regulation of catecholamine enzyme genes by PACAP.

Roles of protein kinase A (PKA) and protein kinase C (PKC) in regulation of tyrosine hydroxylase, dopamine beta-hydroxylase, and phenylethanolamine N-methyltransferase expression by pituitary adenylate cyclase-activating polypeptide (PACAP) were determined in primary cultured bovine chromaffin cells. DBH up-regulation by PACAP was reduced by H-89 and not further increased by forskolin showing involvement of cAMP/PKA. It was not mediated by PKC, as 12-O-tetradecanoylphorbol-13-acetate and sphingosine exerted no effect. Tyrosine hydroxylase induction by PACAP was mediated by both kinases. The PACAP-activated PKA up-regulated phenylethanolamine N-methyltransferase expression whereas PKC caused down-regulation. PACAP increased tyrosine hydroxylase and dopamine beta-hydroxylase activities, but slightly lowered phenylethanolamine N-methyltransferase activity, resulting in a preferential rise in norepinephrine over epinephrine.