A monolayer hiPSC culture system for autophagy/mitophagy studies in human dopaminergic neurons.

Macroautophagy/autophagy cytoplasmic quality control pathways are required during neural development and are critical for the maintenance of functional neuronal populations in the adult brain. Robust evidence now exists that declining neuronal autophagy pathways contribute to human neurodegenerative diseases, including Parkinson disease (PD). Reliable and relevant human neuronal model systems are therefore needed to understand the biology of disease-vulnerable neural populations, to decipher the underlying causes of neurodegenerative disease, and to develop assays to test therapeutic interventions in vitro. Human induced pluripotent stem cell (hiPSC) neural model systems can meet this demand: they provide a renewable source of material for differentiation into regional neuronal sub-types for functional assays; they can be expanded to provide a platform for screening, and they can potentially be optimized for transplantation/neurorestorative therapy. So far, however, hiPSC differentiation protocols for the generation of ventral midbrain dopaminergic neurons (mDANs) - the predominant neuronal sub-type afflicted in PD - have been somewhat restricted by poor efficiency and/or suitability for functional and/or imaging-based in vitro assays. Here, we describe a reliable, monolayer differentiation protocol for the rapid and reproducible production of high numbers of mDANs from hiPSC in a format that is amenable for autophagy/mitophagy research. We characterize these cells with respect to neuronal differentiation and macroautophagy capability and describe qualitative and quantitative assays for the study of autophagy and mitophagy in these important cells.Abbreviations: AA: ascorbic acid; ATG: autophagy-related; BDNF: brain derived neurotrophic factor; CCCP: carbonyl cyanide m-chlorophenylhydrazone; dbcAMP: dibutyryl cAMP; DAN: dopaminergic neuron; DAPI: 4',6-diamidino-2-phenylindole; DAPT: N-[N-(3,5-difluorophenacetyl)-L-alanyl]-sphenylglycine; DLG4/PSD95: discs large MAGUK scaffold protein 4; DMEM: Dulbecco's modified eagle's medium; EB: embryoid body; ECAR: extracellular acidification rate; EGF: epidermal growth factor; FACS: fluorescence-activated cell sorting; FCCP: arbonyl cyanide p-triflouromethoxyphenylhydrazone; FGF: fibroblast growth factor; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GDNF: glia cell derived neurotrophic factor; hiPSC: human induced pluripotent stem cell; LAMP2A: lysosomal associated membrane protein 2A; LT-R: LysoTracker Red; MAP1LC3: microtubule associated protein 1 light chain 3; mDAN: midbrain dopaminergic neuron; MEF: mouse embryonic fibroblast; MT-GR: MitoTracker Green; MT-R: MitoTracker Red; NAS2: normal SNCA2; NEM: neuroprogenitor expansion media; NR4A2/NURR1: nuclear receptor subfamily group A member 2; OA: oligomycin and antimycin A; OCR: oxygen consumption rate; PD: Parkinson disease; SHH: sonic hedgehog signaling molecule; SNCA/α-synuclein: synuclein alpha; TH: tyrosine hydroxylase; VTN: vitronectin.

Cofilin dysregulation alters actin turnover in frataxin-deficient neurons.

Abnormalities in actin cytoskeleton have been linked to Friedreich's ataxia (FRDA), an inherited peripheral neuropathy characterised by an early loss of neurons in dorsal root ganglia (DRG) among other clinical symptoms. Despite all efforts to date, we still do not fully understand the molecular events that contribute to the lack of sensory neurons in FRDA. We studied the adult neuronal growth cone (GC) at the cellular and molecular level to decipher the connection between frataxin and actin cytoskeleton in DRG neurons of the well-characterised YG8R Friedreich's ataxia mouse model. Immunofluorescence studies in primary cultures of DRG from YG8R mice showed neurons with fewer and smaller GCs than controls, associated with an inhibition of neurite growth. In frataxin-deficient neurons, we also observed an increase in the filamentous (F)-actin/monomeric (G)-actin ratio (F/G-actin ratio) in axons and GCs linked to dysregulation of two crucial modulators of filamentous actin turnover, cofilin-1 and the actin-related protein (ARP) 2/3 complex. We show how the activation of cofilin is due to the increase in chronophin (CIN), a cofilin-activating phosphatase. Thus cofilin emerges, for the first time, as a link between frataxin deficiency and actin cytoskeleton alterations.

Automated profiling of growth cone heterogeneity defines relations between morphology and motility.

Growth cones are complex, motile structures at the tip of an outgrowing neurite. They often exhibit a high density of filopodia (thin actin bundles), which complicates the unbiased quantification of their morphologies by software. Contemporary image processing methods require extensive tuning of segmentation parameters, require significant manual curation, and are often not sufficiently adaptable to capture morphology changes associated with switches in regulatory signals. To overcome these limitations, we developed Growth Cone Analyzer (GCA). GCA is designed to quantify growth cone morphodynamics from time-lapse sequences imaged both in vitro and in vivo, but is sufficiently generic that it may be applied to nonneuronal cellular structures. We demonstrate the adaptability of GCA through the analysis of growth cone morphological variation and its relation to motility in both an unperturbed system and in the context of modified Rho GTPase signaling. We find that perturbations inducing similar changes in neurite length exhibit underappreciated phenotypic nuance at the scale of the growth cone.

FLIM FRET Visualization of Cdc42 Activation by Netrin-1 in Embryonic Spinal Commissural Neuron Growth Cones.

Netrin-1 is an essential extracellular chemoattractant that signals through its receptor DCC to guide commissural axon extension in the embryonic spinal cord. DCC directs the organization of F-actin in growth cones by activating an intracellular protein complex that includes the Rho GTPase Cdc42, a critical regulator of cell polarity and directional migration. To address the spatial distribution of signaling events downstream of netrin-1, we expressed the FRET biosensor Raichu-Cdc42 in cultured embryonic rat spinal commissural neurons. Using FLIM-FRET imaging we detected rapid activation of Cdc42 in neuronal growth cones following application of netrin-1. Investigating the signaling mechanisms that control Cdc42 activation by netrin-1, we demonstrate that netrin-1 rapidly enriches DCC at the leading edge of commissural neuron growth cones and that netrin-1 induced activation of Cdc42 in the growth cone is blocked by inhibiting src family kinase signaling. These findings reveal the activation of Cdc42 in embryonic spinal commissural axon growth cones and support the conclusion that src family kinase activation downstream of DCC is required for Cdc42 activation by netrin-1.

Integration of shallow gradients of Shh and Netrin-1 guides commissural axons.

During nervous system development, gradients of Sonic Hedgehog (Shh) and Netrin-1 attract growth cones of commissural axons toward the floor plate of the embryonic spinal cord. Mice defective for either Shh or Netrin-1 signaling have commissural axon guidance defects, suggesting that both Shh and Netrin-1 are required for correct axon guidance. However, how Shh and Netrin-1 collaborate to guide axons is not known. We first quantified the steepness of the Shh gradient in the spinal cord and found that it is mostly very shallow. We then developed an in vitro microfluidic guidance assay to simulate these shallow gradients. We found that axons of dissociated commissural neurons respond to steep but not shallow gradients of Shh or Netrin-1. However, when we presented axons with combined Shh and Netrin-1 gradients, they had heightened sensitivity to the guidance cues, turning in response to shallower gradients that were unable to guide axons when only one cue was present. Furthermore, these shallow gradients polarized growth cone Src-family kinase (SFK) activity only when Shh and Netrin-1 were combined, indicating that SFKs can integrate the two guidance cues. Together, our results indicate that Shh and Netrin-1 synergize to enable growth cones to sense shallow gradients in regions of the spinal cord where the steepness of a single guidance cue is insufficient to guide axons, and we identify a novel type of synergy that occurs when the steepness (and not the concentration) of a guidance cue is limiting.

EphrinA/EphA-induced ectodomain shedding of neural cell adhesion molecule regulates growth cone repulsion through ADAM10 metalloprotease.

EphrinA/EphA-dependent axon repulsion is crucial for synaptic targeting in developing neurons but downstream molecular mechanisms remain obscure. Here, it is shown that ephrinA5/EphA3 triggers proteolysis of the neural cell adhesion molecule (NCAM) by the metalloprotease a disintegrin and metalloprotease (ADAM)10 to promote growth cone collapse in neurons from mouse neocortex. EphrinA5 induced ADAM10 activity to promote ectodomain shedding of polysialic acid-NCAM in cortical neuron cultures, releasing a ~ 250 kDa soluble fragment consisting of most of its extracellular region. NCAM shedding was dependent on ADAM10 and EphA3 kinase activity as shown in HEK293T cells transfected with dominant negative ADAM10 and kinase-inactive EphA3 (K653R) mutants. Purified ADAM10 cleaved NCAM at a sequence within the E-F loop of the second fibronectin type III domain (Leu(671) -Lys(672) /Ser(673) -Leu(674) ) identified by mass spectrometry. Mutations of NCAM within the ADAM10 cleavage sequence prevented EphA3-induced shedding of NCAM in HEK293T cells. EphrinA5-induced growth cone collapse was dependent on ADAM10 activity, was inhibited in cortical cultures from NCAM null mice, and was rescued by WT but not ADAM10 cleavage site mutants of NCAM. Regulated proteolysis of NCAM through the ephrin5/EphA3/ADAM10 mechanism likely impacts synapse development, and may lead to excess NCAM shedding when disrupted, as implicated in neurodevelopmental disorders such as schizophrenia. PSA-NCAM and ephrinA/EphA3 coordinately regulate inhibitory synapse development. Here, we have found that ephrinA5 stimulates EphA3 kinase and ADAM10 activity to promote PSA-NCAM cleavage at a site in its second FNIII repeat, which regulates ephrinA5-induced growth cone collapse in GABAergic and non-GABAergic neurons. These findings identify a new regulatory mechanism which may contribute to inhibitory connectivity.

Regulation of axon growth by the JIP1-AKT axis.

The polarisation of developing neurons to form axons and dendrites is required for the establishment of neuronal connections leading to proper brain function. The protein kinase AKT and the MAP kinase scaffold protein JNK-interacting protein-1 (JIP1) are important regulators of axon formation. Here we report that JIP1 and AKT colocalise in axonal growth cones of cortical neurons and collaborate to promote axon growth. The loss of AKT protein from the growth cone results in the degradation of JIP1 by the proteasome, and the loss of JIP1 promotes a similar fate for AKT. Reduced protein levels of both JIP1 and AKT in the growth cone can be induced by glutamate and this coincides with reduced axon growth, which can be rescued by a stabilized mutant of JIP1 that rescues AKT protein levels. Taken together, our data reveal a collaborative relationship between JIP1 and AKT that is required for axon growth and can be regulated by changes in neuronal activity.

Axon targeting of the alpha 7 nicotinic receptor in developing hippocampal neurons by Gprin1 regulates growth.

Cholinergic signaling plays an important role in regulating the growth and regeneration of axons in the nervous system. The α7 nicotinic receptor (α7) can drive synaptic development and plasticity in the hippocampus. Here, we show that activation of α7 significantly reduces axon growth in hippocampal neurons by coupling to G protein-regulated inducer of neurite outgrowth 1 (Gprin1), which targets it to the growth cone. Knockdown of Gprin1 expression using RNAi is found sufficient to abolish the localization and calcium signaling of α7 at the growth cone. In addition, an α7/Gprin1 interaction appears intimately linked to a Gαo, growth-associated protein 43, and CDC42 cytoskeletal regulatory pathway within the developing axon. These findings demonstrate that α7 regulates axon growth in hippocampal neurons, thereby likely contributing to synaptic formation in the developing brain.

Rapid impact of progesterone on the neuronal growth cone.

In the last two decades, sensory neurons and Schwann cells in the dorsal root ganglia (DRG) were shown to express the rate-limiting enzyme of the steroid synthesis, cytochrome P450 side-chain cleavage enzyme (P450scc), as well as the key enzyme of progesterone synthesis, 3ß-hydroxysteroid dehydrogenase (3ß-HSD). Thus, it was well justified to consider that DRG neurons similarly are able to synthesize progesterone de novo from cholesterol. Because direct progesterone effects on axonal outgrowth in peripheral neurons have not been investigated up to now, the present study provides the first insights into the impact of exogenous progesterone on axonal outgrowth in DRG neurons. Our studies including microinjection and laser scanning microscopy demonstrate morphological changes especially in the neuronal growth cones after progesterone treatment. Furthermore, we were able to detect a distinctly enhanced motility only a few minutes after the start of progesterone treatment using time-lapse imaging. Investigation of the cytoskeletal distribution in the neuronal growth cone before, during, and after progesterone incubation revealed a rapid reorganization of actin filaments. To get a closer idea of the underlying receptor mechanisms, we further studied the expression of progesterone receptors in DRG neurons using RT-PCR and immunohistochemistry. Thus, we could demonstrate for the first time that classical progesterone receptor (PR) A and B and the recently described progesterone receptor membrane component 1 (PGRMC1) are expressed in DRG neurons. Antagonism of the classical progesterone receptors by mifepristone revealed that the observed progesterone effects are transmitted through PR-A and PR-B.

ALS-associated mutations in FUS disrupt the axonal distribution and function of SMN.

Mutations in the RNA binding protein fused in sarcoma/translated in liposarcoma (FUS/TLS) cause amyotrophic lateral sclerosis (ALS). Although ALS-linked mutations in FUS often lead to a cytosolic mislocalization of the protein, the pathogenic mechanisms underlying these mutations remain poorly understood. To gain insight into these mechanisms, we examined the biochemical, cell biological and functional properties of mutant FUS in neurons. Expression of different FUS mutants (R521C, R521H, P525L) in neurons caused axonal defects. A protein interaction screen performed to explain these phenotypes identified numerous FUS interactors including the spinal muscular atrophy (SMA) causing protein survival motor neuron (SMN). Biochemical experiments showed that FUS and SMN interact directly and endogenously, and that this interaction can be regulated by FUS mutations. Immunostaining revealed co-localization of mutant FUS aggregates and SMN in primary neurons. This redistribution of SMN to cytosolic FUS accumulations led to a decrease in axonal SMN. Finally, cell biological experiments showed that overexpression of SMN rescued the axonal defects induced by mutant FUS, suggesting that FUS mutations cause axonal defects through SMN. This study shows that neuronal aggregates formed by mutant FUS protein may aberrantly sequester SMN and concomitantly cause a reduction of SMN levels in the axon, leading to axonal defects. These data provide a functional link between ALS-linked FUS mutations, SMN and neuronal connectivity and support the idea that different motor neuron disorders such as SMA and ALS may be caused, in part, by defects in shared molecular pathways.

The sonic hedgehog pathway mediates brain plasticity and subsequent functional recovery after bone marrow stromal cell treatment of stroke in mice.

Bone marrow stromal cells (MSCs) improve neurologic recovery after middle cerebral artery occlusion (MCAo). To examine whether in vivo blockage of the endogenous sonic hedgehog (Shh) pathway affects grafted MSC-induced neurologic benefits, MCAo mice were administered: vehicle (control); cyclopamine (CP)- a specific Shh pathway inhibitor; MSC; and MSC and cyclopamine (MSC-CP). Neurologic function was evaluated after MCAo. Electron microscopy and immunofluorescence staining were employed to measure synapse density, protein expression of tissue plasminogen activator (tPA), and Shh in parenchymal cells in the ischemic boundary zone (IBZ), respectively. Marrow stromal cell treatment significantly enhanced functional recovery after ischemia, concurrent with increases of synaptophysin, synapse density, and myelinated axons along the IBZ, and significantly increased tPA and Shh expression in astrocytes and neurons compared with control. After treatment with MSC-CP or CP, the above effects were reversed. Co-culture of MSCs with cortical neurons confirmed the effect of Shh on MSC-mediated neurite outgrowth. Our data support the hypothesis that the Shh pathway mediates brain plasticity via tPA and thereby functional recovery after treatment of stroke with MSCs.

Stabilization of actin bundles by a dynamin 1/cortactin ring complex is necessary for growth cone filopodia.

Dynamin GTPase, a key molecule in endocytosis, mechanically severs the invaginated membrane upon GTP hydrolysis. Dynamin functions also in regulating actin cytoskeleton, but the mechanisms are yet to be defined. Here we show that dynamin 1, a neuronal isoform of dynamin, and cortactin form ring complexes, which twine around F-actin bundles and stabilize them. By negative-staining EM, dynamin 1-cortactin complexes appeared as "open" or "closed" rings depending on guanine nucleotide conditions. By pyrene actin assembly assay, dynamin 1 stimulated actin assembly in mouse brain cytosol. In vitro incubation of F-actin with both dynamin 1 and cortactin led to the formation of long and thick actin bundles, on which dynamin 1 and cortactin were periodically colocalized in puncta. A depolymerization assay revealed that dynamin 1 and cortactin increased the stability of actin bundles, most prominently in the presence of GTP. In rat cortical neurons and human neuroblastoma cell line, SH-SY5Y, both dynamin 1 and cortactin localized on actin filaments and the bundles at growth cone filopodia as revealed by immunoelectron microscopy. In SH-SY5Y cell, acute inhibition of dynamin 1 by application of dynamin inhibitor led to growth cone collapse. Cortactin knockdown also reduced growth cone filopodia. Together, our results strongly suggest that dynamin 1 and cortactin ring complex mechanically stabilizes F-actin bundles in growth cone filopodia. Thus, the GTPase-dependent mechanochemical enzyme property of dynamin is commonly used both in endocytosis and regulation of F-actin bundles by a dynamin 1-cortactin complex.

Stimulation of trigeminal afferents improves motor recovery after facial nerve injury: functional, electrophysiological and morphological proofs.

Recovery of mimic function after facial nerve transection is poor: the successful regrowth of axotomized motoneurons to their targets is compromised by (1) poor axonal navigation and excessive collateral branching, (2) abnormal exchange of nerve impulses between adjacent regrowing axons, and (3) insufficient synaptic input to facial motoneurons. As a result, axotomized motoneurons get hyperexcitable and unable to discharge. Since improvement of growth cone navigation and reduction of the ephaptic cross talk between axons turn out be very difficult, we concentrated our efforts on the third detrimental component and proposed that an intensification of the trigeminal input to axotomized electrophysiologically silent facial motoneurons might improve specificity of reinnervation. To test our hypothesis we compared behavioral, electrophysiological, and morphological parameters after single reconstructive surgery on the facial nerve (or its buccal branch) with those obtained after identical facial nerve surgery but combined with direct or indirect stimulation of the ipsilateral infraorbital (ION) nerve. We found that in all cases, trigeminal stimulation was beneficial for the outcome by improving the quality of target reinnervation and recovery of vibrissa! motor performance.

The Caenorhabditis elegans Eph receptor activates NCK and N-WASP, and inhibits Ena/VASP to regulate growth cone dynamics during axon guidance.

The Eph receptor tyrosine kinases (RTKs) are regulators of cell migration and axon guidance. However, our understanding of the molecular mechanisms by which Eph RTKs regulate these processes is still incomplete. To understand how Eph receptors regulate axon guidance in Caenorhabditis elegans, we screened for suppressors of axon guidance defects caused by a hyperactive VAB-1/Eph RTK. We identified NCK-1 and WSP-1/N-WASP as downstream effectors of VAB-1. Furthermore, VAB-1, NCK-1, and WSP-1 can form a complex in vitro. We also report that NCK-1 can physically bind UNC-34/Enabled (Ena), and suggest that VAB-1 inhibits the NCK-1/UNC-34 complex and negatively regulates UNC-34. Our results provide a model of the molecular events that allow the VAB-1 RTK to regulate actin dynamics for axon guidance. We suggest that VAB-1/Eph RTK can stop axonal outgrowth by inhibiting filopodia formation at the growth cone by activating Arp2/3 through a VAB-1/NCK-1/WSP-1 complex and by inhibiting UNC-34/Ena activity.

Genetic analysis of DSCAM's role as a Netrin-1 receptor in vertebrates.

Down syndrome cell adhesion molecule (DSCAM) has mainly been characterized for its function as an adhesion molecule in axon growth and in self-recognition between dendrites of the same neuron. Recently, it has been shown that DSCAM can bind to Netrin-1 and that downregulation of DSCAM expression by siRNAs in chick and rodent spinal cords leads to impaired growth and turning response of commissural axons to Netrin-1. To investigate the effect of complete genetic ablation of DSCAM on Netrin-1-induced axon guidance, we analyzed spinal commissural neurons in DSCAM-null mice and found that they extend axons that reach and cross the floor plate and express apparently normal levels of the Netrin receptors DCC (deleted in colorectal carcinoma) and Neogenin. In vitro, commissural neurons in dorsal spinal cord explants of DSCAM-null embryos show normal outgrowth in response to Netrin-1. We therefore conclude that DSCAM is not required for Netrin-induced commissural axon outgrowth and guidance in mice.

Neurofibromatosis-1 heterozygosity impairs CNS neuronal morphology in a cAMP/PKA/ROCK-dependent manner.

Children with the neurofibromatosis-1 (NF1) cancer predisposition syndrome exhibit numerous clinical problems that reflect defective central nervous system (CNS) neuronal function, including learning disabilities, attention deficit disorder, and seizures. These clinical features result from reduced NF1 protein (neurofibromin) expression in NF1+/- (NF1 heterozygosity) brain neurons. Previous studies have shown that mouse CNS neurons are sensitive to the effects of reduced Nf1 expression and exhibit shorter neurite lengths, smaller growth cone areas, and attenuated survival, reflecting attenuated neurofibromin cAMP regulation. In striking contrast, Nf1+/- peripheral nervous system (PNS) neurons are nearly indistinguishable from their wild-type counterparts, and complete neurofibromin loss leads to increased neurite lengths and survival in a RAS/Akt-dependent fashion. To gain insights into the differential responses of CNS and PNS neurons to reduced neurofibromin function, we designed a series of experiments to define the molecular mechanism(s) underlying the unique CNS neuronal sensitivity to Nf1 heterozygosity. First, Nf1 heterozygosity decreases cAMP levels in CNS, but not in PNS, neurons. Second, CNS neurons exhibit Nf1 gene-dependent increases in RAS pathway signaling, but no further decreases in cAMP levels were observed in Nf1-/- CNS neurons relative to their Nf1+/- counterparts. Third, neurofibromin regulates CNS neurite length and growth cone areas in a cAMP/PKA/Rho/ROCK-dependent manner in vitro and in vivo. Collectively, these findings establish cAMP/PKA/Rho/ROCK signaling as the responsible axis underlying abnormal Nf1+/- CNS neuronal morphology with important implications for future preclinical and clinical studies aimed at improving cognitive and behavioral deficits in mice and children with reduced brain neuronal NF1 gene expression.

CRP1, a protein localized in filopodia of growth cones, is involved in dendritic growth.

The cysteine-rich protein (CRP) family is a subgroup of LIM domain proteins. CRP1, which cross-links actin filaments to make actin bundles, is the only CRP family member expressed in the CNS with little known about its function in nerve cells. Here, we report that CRP1 colocalizes with actin in the filopodia of growth cones in cultured rat hippocampal neurons. Knockdown of CRP1 expression by short hairpin RNA interference results in inhibition of filopodia formation and dendritic growth in neurons. Overexpression of CRP1 increases filopodia formation and neurite branching, which require its actin-bundling activity. Expression of CRP1 with a constitutively active form of Cdc42, a GTPase involved in filopodia formation, increases filopodia formation in COS-7 cells, suggesting cooperation between the two proteins. Moreover, we demonstrate that neuronal activity upregulates CRP1 expression in hippocampal neurons via Ca²âº influx after depolarization. Ca²âº/calmodulin-dependent protein kinase IV (CaMKIV) and cAMP response element binding protein mediate the Ca²âº-induced upregulation of CRP1 expression. Furthermore, CRP1 is required for the dendritic growth induced by Ca²âº influx or CaMKIV. Together, these data are the first to demonstrate a role for CRP1 in dendritic growth.

Inductive specification and axonal orientation of spinal neurons mediated by divergent bone morphogenetic protein signaling pathways.

BACKGROUND: Bone morphogenetic protein (BMP)7 evokes both inductive and axon orienting responses in dorsal interneurons (dI neurons) in the developing spinal cord. These events occur sequentially during the development of spinal neurons but in these and other cell types such inductive and acute chemotactic responses occur concurrently, highlighting the requirement for divergent intracellular signaling. Both type I and type II BMP receptor subtypes have been implicated selectively in orienting responses but it remains unclear how, in a given cell, divergence occurs. We have examined the mechanisms by which disparate BMP7 activities are generated in dorsal spinal neurons. RESULTS: We show that widely different threshold concentrations of BMP7 are required to elicit the divergent inductive and axon orienting responses. Type I BMP receptor kinase activity is required for activation of pSmad signaling and induction of dI character by BMP7, a high threshold response. In contrast, neither type I BMP receptor kinase activity nor Smad1/5/8 phosphorylation is involved in the low threshold orienting responses of dI axons to BMP7. Instead, BMP7-evoked axonal repulsion and growth cone collapse are dependent on phosphoinositide-3-kinase (PI3K) activation, plausibly through type II receptor signaling. BMP7 stimulates PI3K-dependent signaling in dI neurons. BMP6, which evokes neural induction but does not have orienting activity, activates Smad signaling but does not stimulate PI3K. CONCLUSIONS: Divergent signaling through pSmad-dependent and PI3K-dependent (Smad-independent) mechanisms mediates the inductive and orienting responses of dI neurons to BMP7. A model is proposed whereby selective engagement of BMP receptor subunits underlies choice of signaling pathway.

Inhibitor of DNA binding 2 promotes sensory axonal growth after SCI.

This study investigated whether neuronal inhibitor of DNA binding 2 (Id2), a regulator of basic helix-loop-helix (bHLH) transcription factors, can activate the intrinsic neuritogenetic mode of dorsal root ganglion (DRG) neurons in adult mice following spinal cord injury (SCI). First, the Id2 developmental expression profile of DRG neurons, along with the correlated activity of Cdh1-anaphase promoting complex (Cdh1-APC), was characterized. Next, a D-box mutant Id2 (Id2DBM) adenoviral vector, resistant to Cdh1-APC degradation, was developed to enhance neuronal Id2 expression. After the vector was introduced into DRG neurons, the effect of Id2 on neurite outgrowth of cultured DRG neurons and sensory axonal regeneration following spinal cord dorsal hemisection was evaluated. The expression of Id2 in DRG neurons was high in the embryonic stage, downregulated after birth, and significantly reduced in the adult. Expression of Cdh1-APC was opposite to Id2, which may be responsible for Id2 degradation during DRG maturation. Overexpression of Id2DBM in DRG neurons enhanced neuritogenesis on both permissive and inhibitory substrates. Following spinal cord dorsal hemisection, overexpression of Id2DBM reduced axon dieback and increased the number and length of regenerative fibers into the lesion gap. Reprogramming the intrinsic growth status of quiescent adult DRG neurons by enhancing Id2 expression results in active neuritogenesis following SCI. Id2 may be a novel target for enhancing sensory axonal regeneration following injuries to the adult spinal cord.