Cutaneous inflammation differentially regulates the expression and function of Angiotensin-II types 1 and 2 receptors in rat primary sensory neurons.

Neuropathic and inflammatory pain results from cellular and molecular changes in dorsal root ganglion (DRG) neurons. The type-2 receptor for Angiotensin-II (AT2R) has been involved in this type of pain. However, the underlying mechanisms are poorly understood, including the role of the type-1 receptor for Angiotensin-II (AT1R). Here, we used a combination of immunohistochemistry and immunocytochemistry, RT-PCR and in vitro and in vivo pharmacological manipulation to examine how cutaneous inflammation affected the expression of AT1R and AT2R in subpopulations of rat DRG neurons and studied their impact on inflammation-induced neuritogenesis. We demonstrated that AT2R-neurons express C- or A-neuron markers, primarily IB4, trkA, and substance-P. AT1R expression was highest in small neurons and co-localized significantly with AT2R. In vitro, an inflammatory soup caused significant elevation of AT2R mRNA, whereas AT1R mRNA levels remained unchanged. In vivo, we found a unique pattern of change in the expression of AT1R and AT2R after cutaneous inflammation. AT2R increased in small neurons at 1 day and in medium size neurons at 4 days. Interestingly, cutaneous inflammation increased AT1R levels only in large neurons at 4 days. We found that in vitro and in vivo AT1R and AT2R acted co-operatively to regulate DRG neurite outgrowth. In vivo, AT2R inhibition impacted more on non-peptidergic C-neurons neuritogenesis, whereas AT1R blockade affected primarily peptidergic nerve terminals. Thus, cutaneous-induced inflammation regulated AT1R and AT2R expression and function in different DRG neuronal subpopulations at different times. These findings must be considered when targeting AT1R and AT2R to treat chronic inflammatory pain. Cover Image for this issue: doi: 10.1111/jnc.14737.

Neurite atrophy and apoptosis mediated by PERK signaling after accumulation of GM2-ganglioside.

GM2-gangliosidosis, a subgroup of lysosomal storage disorders, is caused by deficiency of hexosaminidase activity, and comprises the closely related Tay-Sachs and Sandhoff diseases. The enzyme deficiency prevents normal metabolization of ganglioside GM2, usually resulting in progressive neurodegenerative disease. The molecular mechanisms whereby GM2 accumulation in neurons triggers neurodegeneration remain unclear. In vitro experiments, using microsomes from Sandhoff mouse model brain, showed that increase of GM2 content negatively modulates sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) (Pelled et al., 2003). Furthermore, Ca2+ depletion in endoplasmic reticulum (ER) triggers Unfolded Protein Response (UPR), which tends to restore homeostasis in the ER; however, if cellular damage persists, an apoptotic response is initiated. We found that ER GM2 accumulation in cultured neurons induces luminal Ca2+ depletion, which in turn activates PERK (protein kinase RNA [PKR]-like ER kinase), one of three UPR sensors. PERK signaling displayed biphasic activation; i.e., early upregulation of cytoprotective calcineurin (CN) and, under prolonged ER stress, enhanced expression of pro-apoptotic transcription factor C/EBP homologous protein (CHOP). Moreover, GM2 accumulation in neuronal cells induced neurite atrophy and apoptosis. Both processes were effectively modulated by treatment with the selective PERK inhibitor GSK2606414, by CN knockdown, and by CHOP knockdown. Overall, our findings demonstrate the essential role of PERK signaling pathway contributing to neurodegeneration in a model of GM2-gangliosidosis.

Non-cytotoxic Concentration of Cisplatin Decreases Neuroplasticity-Related Proteins and Neurite Outgrowth Without Affecting the Expression of NGF in PC12 Cells.

Cisplatin is the most effective and neurotoxic platinum chemotherapeutic agent. It induces a peripheral neuropathy characterized by distal axonal degeneration that might progress to degeneration of cell bodies and apoptosis. Most symptoms occur nearby distal axonal branches and axonal degeneration might induce peripheral neuropathy regardless neuronal apoptosis. The toxic mechanism of cisplatin has been mainly associated with DNA damage, but cisplatin might also affect neurite outgrowth. Nevertheless, the neurotoxic mechanism of cisplatin remains unclear. We investigated the early effects of cisplatin on axonal plasticity by using non-cytotoxic concentrations of cisplatin and PC12 cells as a model of neurite outgrowth and differentiation. PC12 cells express NGF-receptors (trkA) and respond to NGF by forming neurites, branches and synaptic vesicles. For comparison, we used a neuronal model (SH-SY5Y cells) that does not express trkA nor responds to NGF. Cisplatin did not change NGF expression in PC12 cells and decreased neurite outgrowth in both models, suggesting a NGF/trkA independent mechanism. It also reduced axonal growth (GAP-43) and synaptic (synapsin I and synaptophysin) proteins in PC12 cells, without inducing mitochondrial damage or apoptosis. Therefore, cisplatin might affect axonal plasticity before DNA damage, NGF/trkA down-regulation, mitochondrial damage or neuronal apoptosis. This is the first study to show that neuroplasticity-related proteins might be early targets of the neurotoxic action of cisplatin and their role on cisplatin-induced peripheral neuropathy should be investigated in vivo.

Time-resolved neurite mechanics by thermal fluctuation assessments.

In the absence of simple noninvasive measurements, the knowledge of temporal and spatial variations of axons mechanics remains scarce. By extending thermal fluctuation spectroscopy (TFS) to long protrusions, we determine the transverse amplitude thermal fluctuation spectra that allow direct and simultaneous access to three key mechanics parameters: axial tension, bending flexural rigidity and plasma membrane tension. To test our model, we use PC12 cell protrusions-a well-know biophysical model of axons-in order to simplify the biological system under scope. For instance, axial and plasma membrane tension are found in the range of nano Newton and tens of pico Newtons per micron respectively. Furthermore, our results shows that the TFS technique is capable to distinguish quasi-identical protrusions. Another advantage of our approach is the time resolved nature of the measurements. Indeed, in the case of long term experiments on PC12 protrusions, TFS has revealed large temporal, correlated variations of the protrusion mechanics, displaying extraordinary feedback control over the axial tension in order to maintain a constant tension value.

Biological behavior of mesenchymal stem cells on poly-ε-caprolactone filaments and a strategy for tissue engineering of segments of the peripheral nerves.

INTRODUCTION: Peripheral nerves may fail to regenerate across tube implants because these lack the microarchitecture of native nerves. Bone marrow mesenchymal stem cells (MSC) secrete soluble factors that improve the regeneration of the peripheral nerves. Also, microstructured poly-caprolactone (PCL) filaments are capable of inducing bands of Büngner and promote regeneration in the peripheral nervous system (PNS). We describe here the interaction between PCL filaments and MSC, aiming to optimize PNS tubular implants. METHODS: MSC were plated on PCL filaments for 48 h and the adhesion profile, viability, proliferation and paracrine capacity were evaluated. Also, Schwann cells were plated on PCL filaments covered with MSC for 24 h to analyze the feasibility of the co-culture system. Moreover, E16 dorsal root ganglia were plated in contact with PCL filaments for 4 days to analyze neurite extension. Right sciatic nerves were exposed and a 10 mm nerve segment was removed. Distal and proximal stumps were reconnected inside a 14-mm polyethylene tube, leaving a gap of approximately 13 mm between the two stumps. Animals then received phosphate-buffered saline 1×, PCL filaments or PCL filaments previously incubated with MSC and, after 12 weeks, functional gait performance and histological analyses were made. Statistical analyses were made using Student's unpaired t-test, one-way analysis of variance (ANOVA) or two-way ANOVA followed by Bonferroni post-test. RESULTS: MSC were confined to lateral areas and ridges of PCL filaments, aligning along the longitudinal. MSC showed high viability (90 %), and their proliferation and secretion capabilities were not completely inhibited by the filaments. Schwann cells adhered to filaments plated with MSC, maintaining high viability (90 %). Neurites grew and extended over the surface of PCL filaments, reaching greater distances when over MSC-plated filaments. Axons showed more organized and myelinized fibers and reinnervated significantly more muscle fibers when they were previously implanted with MSC-covered PLC filaments. Moreover, animals with MSC-covered filaments showed increased functional recovery after 12 weeks. CONCLUSIONS: We provide evidence for the interaction among MSC, Schwann cells and PCL filaments, and we also demonstrate that this system can constitute a stable and permissive support for regeneration of segments of the peripheral nerves.

Embryonic neural stem cells in a 3D bioassay for trophic stimulation studies.

Progenitors were discovered in the corpus striatum several years ago, but little is known about their proliferation and differentiation. The aim of this study was to analyze embryonic progenitor cells from the corpus striatum using a bioassay with trophic stimulation. Primary cells obtained from brains of rat embryos at E13-14 were dissected from striatum niches and cultured in stem cell media. These floating dispersed cells clumped together to forming floating bodies like irregular spheres (spheroids), which were placed in type I collagen gel and cultured under basal conditions or with the addition of NGF, NT-3, or NTN. Optimum growth of neurites was obtained, and after 24 and 48 h, they were measured for number and length. The expression of proliferation markers such as PCNA and Ki67, and of neural progenitor markers such as GFAP, nestin, vimentin, O4, A2B5, Pax6, S100, TubIII, and NeuN, was then analyzed. The initial behavior in cell cultures showed distinguishable spheroids that, when placed in 3D gels and with trophic support, generated neurites. A similar effect was observed in glial cell outgrowth from the spheroids. Our assay showed high reproducibility, short culture time, and high resolution for tracing neuron-neurite outgrowth or visualizing glial outgrowth in a few hours.

Insulin-like growth factor (IgF)-I, IgF binding protein-3, and prostate cancer: correlation with gleason score

Introduction Non-androgenic growth factors are involved in the growth regulation of prostate cancer (PCa). Objective This is the first Brazilian study to correlate, in a population of patients operated for PCa, PSA, total testosterone, insulin-like growth factor-I (IGF-I) and insulin-like growth factor-binding protein-3 (IGFBP-3) with Gleason score and to compare with a control group with benign prostate hyperplasia (BPH). Materials and Methods This retrospective single-center study included 49 men with previously diagnosed PCa and 45 with previously diagnosed BPH. PSA, testosterone, IGF-I, IGFBP-3 were determined in both groups. Results PSA and IGFBP-3 levels were significantly higher in the PCa group as compared to the BPH group (p<0.001 and p=0.004, respectively). There was a significant difference when we compared the PSA before surgery (p<0.001) and at the inclusion in the study (p<0.001) and IGFBP3 (0.016) among patients with Gleason <7, ≥7 and BPH. In the PCa group, PSA, testosterone, IGF-I and IGFBP-3 levels were comparable between Gleason <7 and ≥7. Conclusions Our data suggest that in localized PCa, the quantification of PSA and, not of IGF-1, may provide independent significant information in the aggressiveness. IGFBP-3 could be a biochemical marker of disease control in PCa patients. .

Tyrosine 251 at the C-terminus of neuronal glycoprotein M6a is critical for neurite outgrowth.

Neuronal glycoprotein M6a is involved in neuronal plasticity, promoting neurite and filopodia outgrowth and, likely, synaptogenesis. Polymorphisms in the human M6a gene GPM6A have recently been associated with mental illnesses such as schizophrenia, bipolar disorders, and claustrophobia. Nevertheless, the molecular bases underlying these observations remain unknown. We have previously documented that, to induce filopodia formation, M6a depends on the association of membrane lipid microdomains and the activation of Src and mitogen-activated protein kinase kinases. Here, in silico analysis of the phosphorylation of tyrosine 251 (Y251) at the C-terminus of M6a showed that it could be a target of Src kinases. We examined whether phosphorylation of M6a at Y251 affects neurite and filopodia outgrowth and the targets involved in its signal propagation. This work provides evidence that the Src kinase family and the phosphatidylinositide 3-kinase (PI3K), but not Ras, participate in M6a signal cascade leading to neurite/filopodia outgrowth in hippocampal neurons and murine neuroblastoma N2a cells. Phosphorylation of M6a at Y251 is essential only for neurite outgrowth by the PI3K/AKT-mediated pathway and, moreover, rescues the inhibition caused by selective Src inhibitor and external M6a monoclonal antibody treatment. Thus, we suggest that phosphorylation of M6a at Y251 is critical for a specific stage of neuronal development and triggers redundant signaling pathways leading to neurite extension.

Serum-induced neurite retraction in CAD cells--involvement of an ATP-actin retractile system and the lack of microtubule-associated proteins.

Cultured catecholamine-differentiated cells [which lack the microtubule-associated proteins (MAPs): MAP1B, MAP2, Tau, STOP, and Doublecortin] proliferate in the presence of fetal bovine serum, and, in its absence, cease dividing and generate processes similar to the neurites of normal neurons. The reintroduction of serum induces neurite retraction, and proliferation resumes. The neurite retraction process in catecholamine-differentiated cells was partially characterized in this study. Microtubules in the cells were found to be in a highly dynamic state, and tubulin in the microtubules consisted primarily of the tyrosinated and deacetylated isotypes. Increased levels of acetylated or Δ2-tubulin (which are normally absent) did not prevent serum-induced neurite retraction. Treatment of differentiated cells with lysophosphatidic acid or adenosine deaminase induced neurite retraction. Inhibition of Rho-associated protein kinase, ATP depletion and microfilament disruption each (individually) blocked serum-induced neurite retraction, suggesting that an ATP-dependent actomyosin system underlies the mechanism of neurite retraction. Nocodazole treatment induced neurite retraction, but this effect was blocked by pretreatment with the microtubule-stabilizing drug paclitaxel (Taxol). Paclitaxel did not prevent serum-induced or lysophosphatidic acid-induced retraction, suggesting that integrity of microtubules (despite their dynamic state) is necessary to maintain neurite elongation, and that paclitaxel-induced stabilization alone is not sufficient to resist the retraction force induced by serum. Transfection with green fluorescent protein-Tau conferred resistance to retraction caused by serum. We hypothesize that, in normal neurons (cultured or in vivo), MAPs are necessary not only to stabilize microtubules, but also to establish interactions with other cytoskeletal or membrane components to form a stable structure capable of resisting the retraction force.

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.

Rab-mediated trafficking role in neurite formation.

Neuronal cells are characterized by the presence of two confined domains, which are different in their cellular properties, biochemical functions and molecular identity. The generation of asymmetric domains in neurons should logically require specialized membrane trafficking to both promote neurite outgrowth and differential distribution of components. Members of the Rab family of small GTPases are key regulators of membrane trafficking involved in transport, tethering and docking of vesicles through their effectors. RabGTPases activity is coupled to the activity of guanine nucleotide exchange factors or GEFs, and GTPase-activating proteins known as GAPs. Since the overall spatiotemporal distribution of GEFs, GAPs and Rabs governs trafficking through the secretory and endocytic pathways, affecting exocytosis, endocytosis and endosome recycling, it is likely that RabGTPases could have a major role in neurite outgrowth, elongation and polarization. In this review we summarize the evidence linking the functions of several RabGTPases to axonal and dendritic development in primary neurons, as well as neurite formation in neuronal cell lines. We focused on the role of RabGTPases from the trans-Golgi network, early/late and recycling endosomes, as well as the function of some Rab effectors in neuritogenesis. Finally, we also discuss the participation of the ADP-ribosylation factor 6, a member of the ArfGTPase family, in neurite formation since it seems to have an important cross-talk with RabGTPases.

Neuronal polarity: demarcation, growth and commitment.

In a biological sense, polarity refers to the extremity of the main axis of an organelle, cell, or organism. In neurons, morphological polarity begins with the appearance of the first neurite from the cell body. In multipolar neurons, a second phase of polarization occurs when a single neurite initiates a phase of rapid growth to become the neuron's axon, while the others later differentiate as dendrites. Finally, during a third phase, axons and dendrites develop an elaborate architecture, acquiring special morphological and molecular features that commit them to their final identities. Mechanistically, each phase must be preceded by spatial restriction of growth activity. We will review recent work on the mechanisms underlying the polarized growth of neurons.

NeuronGrowth, a software for automatic quantification of neurite and filopodial dynamics from time-lapse sequences of digital images.

We developed NeuronGrowth, a software for the automatic quantification of extension and retraction of neurites and filopodia, from time-lapse sequences of two-dimensional digital micrographs. NeuronGrowth requires a semiautomatic characterization of individual neurites in a reference frame, which is then used for automatic tracking and measurement of every neurite over the whole image sequence. Modules for sequence alignment, background subtraction, flat field correction, light normalization, and cropping have been integrated to improve the quality of the analysis. Moreover, NeuronGrowth incorporates a deconvolution filter that corrects the shadow-cast effect of differential interference contrast (DIC) images. NeuronGrowth was tested by analyzing the formation of outgrowth patterns by individual leech neurons cultured under two different conditions. Phase contrast images were obtained from neurons plated on CNS homogenates and DIC images were obtained from similar neurons plated on ganglion capsules as substrates. Filopodia were measured from fluorescent growth-cones of chick dorsal root ganglion cells. Quantitative data of neurite extension and retraction obtained by three different users applying NeuronGrowth and two other manually operated software packages were similar. However, NeuronGrowth required less user participation and had a better time performance when compared with the other software packages. NeuronGrowth may be used in general to quantify the dynamics of tubular structures such as blood vessels. NeuronGrowth is a free plug-in for the free software ImageJ and can be downloaded along with a user manual, a troubleshooting section and other information required for its use from http://www.ifc.unam.mx or http://www.ifc.unam.mx/ffm/index.html.

Role of phosphatidylcholine during neuronal differentiation.

Neuronal differentiation is characterized by neuritogenesis and neurite outgrowth, processes, which are critically dependent on membrane biosynthesis, and therefore, on the expression and regulation of enzymes involved in phospholipid biosynthesis. During the last decade a great effort was made to clarify where membrane lipids are synthesized, how the newly synthesized membrane components reach the membrane and are inserted during neuritogenesis and to elucidate the mechanism by which the supply of new membrane components is coordinated with the demand for growth. Phosphatidylcholine is the principal and essential component for mammalian membranes. This review updates the mechanism by which phosphatidylcholine biosynthesis takes place and how it is coordinately regulated during neuronal differentiation.

Metabotropic glutamate receptors transduce signals for neurite outgrowth after binding of the prion protein to laminin γ1 chain.

The prion protein (PrP(C)) is highly expressed in the nervous system, and its abnormal conformer is associated with prion diseases. PrP(C) is anchored to cell membranes by glycosylphosphatidylinositol, and transmembrane proteins are likely required for PrP(C)-mediated intracellular signaling. Binding of laminin (Ln) to PrP(C) modulates neuronal plasticity and memory. We addressed signaling pathways triggered by PrP(C)-Ln interaction in order to identify transmembrane proteins involved in the transduction of PrP(C)-Ln signals. The Ln γ1-chain peptide, which contains the Ln binding site for PrP(C), induced neuritogenesis through activation of phospholipase C (PLC), Ca(2+) mobilization from intracellular stores, and protein kinase C and extracellular signal-regulated kinase (ERK1/2) activation in primary cultures of neurons from wild-type, but not PrP(C)-null mice. Phage display, coimmunoprecipitation, and colocalization experiments showed that group I metabotropic glutamate receptors (mGluR1/5) associate with PrP(C). Expression of either mGluR1 or mGluR5 in HEK293 cells reconstituted the signaling pathways mediated by PrP(C)-Ln γ1 peptide interaction. Specific inhibitors of these receptors impaired PrP(C)-Ln γ1 peptide-induced signaling and neuritogenesis. These data show that group I mGluRs are involved in the transduction of cellular signals triggered by PrP(C)-Ln, and they support the notion that PrP(C) participates in the assembly of multiprotein complexes with physiological functions on neurons.

Subcellular rearrangement of hsp90-binding immunophilins accompanies neuronal differentiation and neurite outgrowth.

FKBP51 and FKBP52 (FK506-binding protein 51 and 52) are tetratricopeptide repeat-domain immunophilins belonging to the tetratricopeptide-protein•hsp90•hsp70•p23 heterocomplex bound to steroid receptors. Immunophilins are related to receptor folding, subcellular localization, and hormone-dependent transcription. Also, they bind the immunosuppressant macrolide FK506, which shows neuroregenerative and neuroprotective actions by a still unknown mechanism. In this study, we demonstrate that in both, undifferentiated neuroblastoma cells and embryonic hippocampal neurons, the FKBP52•hsp90•p23 heterocomplex concentrates in a perinuclear structure. Upon cell stimulation with FK506, this structure disassembles and this perinuclear area becomes transcriptionally active. The acquisition of a neuronal phenotype is accompanied by increased expression of ßIII-tubulin, Map-2, Tau-1, but also hsp90, hsp70, p23, and FKBP52. During the early differentiation steps, the perinuclear heterocomplex redistributes along the cytoplasm and nascent neurites, p23 binds to intermediate filaments and microtubules acquired higher filamentary organization. While FKBP52 moves towards neurites and concentrates in arborization bodies and terminal axons, FKBP51, whose expression remains constant, replaces FKBP52 in the perinuclear structure. Importantly, neurite outgrowth is favored by FKBP52 over-expression or FKBP51 knock-down, and is impaired by FKBP52 knock-down or FKBP51 over-expression, indicating that the balance between these FK506-binding proteins plays a key role during the early mechanism of neuronal differentiation.

Evidence for the involvement of Lfc and Tctex-1 in axon formation.

RhoA and Rac play key and opposite roles during neuronal polarization. We now show that Lfc, a guanosine nucleotide exchange factor (GEF), localizes to the Golgi apparatus and growth cones of developing neurons and negatively regulates neurite sprouting and axon formation through a Rho signaling pathway. Tctex-1, a dynein light chain implicated in axon outgrowth by modulating actin dynamics and Rac activity, colocalizes and physically interacts with Lfc, thus inhibiting its GEF activity, decreasing Rho-GTP levels, and functionally antagonizing Lfc during neurite formation.

Assessing the role of endooligopeptidase activity of Ndel1 (nuclear-distribution gene E homolog like-1) in neurite outgrowth.

Ndel1 plays multiple roles in neuronal development but it is unknown whether its reported cysteine protease activity is important for these processes. Ndel1 is known to be critical for neurite outgrowth in PC12 cells where it works co-operatively in a complex with DISC1 to allow normal neuritogenesis. Through an initial interest in understanding the regulation of the expression of Ndel1 during neuronal differentiation, we have been able to show that Ndel1 expression and enzyme activity is up-regulated during neurite outgrowth in PC12 cells induced to neural differentiation. Heterologous expression of wild-type Ndel1 (Ndel1(WT)) in PC12 cells increases the percentage of cells bearing neurites in contrast to the catalytically dead mutant, Ndel1(C273A), which caused a decrease. Furthermore depletion of endogenous Ndel1 by RNAi decreased neurite outgrowth, which was rescued by transfection of the enzymatically active Ndel1(WT), but not by the Ndel1(C273A) mutant. Together these data support the notion that the endooligopeptidase activity of Ndel1 plays a crucial role in the differentiation process of PC12 cells to neurons. Genetic data and protein interaction with DISC1 might suggest a role for Ndel1 in neuropsychiatirc conditions.

Wnt signaling modulates pre- and postsynaptic maturation: therapeutic considerations.

Wnt signaling regulates a wealth of aspects of nervous system development and function in embryonic stages and in adulthood. The expression of Wnt ligands and components of the Wnt signaling machinery in early stages of neural development has been related to its role in neurite patterning and in synaptogenesis. Moreover, its expression in the mature nervous system suggests a role for this pathway in synaptic maintenance and function. Therefore, it is of crucial relevance the understanding of the mechanisms by which Wnt signaling regulates these processes. Herein, we discuss how different Wnt ligands, acting through different Wnt signaling pathways, operate in pre- and postsynaptic regions to modulate synapse structure and function. We also elaborate on the idea that Wnt signaling pathways are a target for the treatment of neurodegenerative diseases that affect synaptic integrity, such as Alzheimer's disease.

Schwann cell chondroitin sulfate proteoglycan inhibits dorsal root ganglion neuron neurite outgrowth and substrate specificity via a soma and not a growth cone mechanism.

Sensory axons do not regenerate into or within the spinal cord because of the presence of the axon regeneration inhibitor chondroitin sulfate proteoglycan (CSPG) on activated astrocytes. In the peripheral nervous system, CSPG associated with denervated Schwann cells retards axon regeneration, but regeneration occurs because the balance of regenerating, inhibiting, and promoting factors favors regeneration. The present experiments were aimed at determining the mechanism by which Schwann cells inhibit adult human dorsal root ganglia (H-DRG) neuron growth cone elongation and substrate specificity, restricting the growth cones to Schwann cell membranes and inhibiting their growth onto a poly-l-lysine/laminin substrate. Neurites of H-DRG neurons free of soma contact with Schwann cells, or after the Schwann cell membranes' CSPG had been digested, were 11.1-fold longer than those of neurons in soma contact with untreated Schwann cells. Growth cones of DRG neuron somas without Schwann cell CSPG showed no outgrowth inhibition or substrate specificity. These results indicate that the Schwann cell CSPG influences act via contact with neuron somas but not growth cones. These results suggest that eliminating CSPG associated with Schwann cells within DRG in vivo will make the neurons' growth cones insensitive to the regeneration inhibitory influences of CSPG, allowing them to regenerate through the dorsal root entry zone and into and within the spinal cord, where they can establish appropriate and functional synaptic connections.