Potential neurotoxic activity of diverse molecules released by astrocytes.

Astrocytes are the main support cells of the central nervous system. They also participate in neuroimmune reactions. In response to pathological and immune stimuli, astrocytes transform to reactive states characterized by increased release of inflammatory mediators. Some of these molecules are neuroprotective and inflammation resolving while others, including reactive oxygen species (ROS), nitric oxide (NO), matrix metalloproteinase (MMP)- 9, L-glutamate, and tumor necrosis factor α (TNF), are well-established toxins known to cause damage to surrounding cells and tissues. We hypothesized that similar to microglia, the brain immune cells, reactive astrocytes can release a broader set of diverse molecules that are potentially neurotoxic. A literature search was conducted to identify such molecules using the following two criteria: 1) evidence of their expression and secretion by astrocytes and 2) direct neurotoxic action. This review describes 14 structurally diverse molecules as less-established astrocyte neurotoxins, including C-X-C motif chemokine ligand (CXCL)10, CXCL12/CXCL12(5-67), FS-7-associated surface antigen ligand (FasL), macrophage inflammatory protein (MIP)- 2α, TNF-related apoptosis inducing ligand (TRAIL), pro-nerve growth factor (proNGF), pro-brain-derived neurotrophic factor (proBDNF), chondroitin sulfate proteoglycans (CSPGs), cathepsin (Cat)B, group IIA secretory phospholipase A2 (sPLA2-IIA), amyloid beta peptides (Aß), high mobility group box (HMGB)1, ceramides, and lipocalin (LCN)2. For some of these molecules, further studies are required to establish either their direct neurotoxic effects or the full spectrum of stimuli that induce their release by astrocytes. Only limited studies with human-derived astrocytes and neurons are available for most of these potential neurotoxins, which is a knowledge gap that should be addressed in the future. We also summarize available evidence of the role these molecules play in select neuropathologies where reactive astrocytes are a key feature. A comprehensive understanding of the full spectrum of neurotoxins released by reactive astrocytes is key to understanding neuroinflammatory diseases characterized by the adverse activation of these cells and may guide the development of novel treatment strategies.

Regulation of Macrophage and Dendritic Cell Function by Chondroitin Sulfate in Innate to Antigen-Specific Adaptive Immunity.

Chondroitin sulfate (CS), a type of glycosaminoglycan (GAG), is a linear acidic polysaccharide comprised of repeating disaccharides, modified with sulfate groups at various positions. Except for hyaluronan (HA), GAGs are covalently bound to core proteins, forming proteoglycans (PGs). With highly negative charges, GAGs interact with a variety of physiologically active molecules, including cytokines, chemokines, and growth factors, and control cell behavior during development and in the progression of diseases, including cancer, infections, and inflammation. Heparan sulfate (HS), another type of GAG, and HA are well reported as regulators for leukocyte migration at sites of inflammation. There have been many reports on the regulation of immune cell function by HS and HA; however, regulation of immune cells by CS has not yet been fully understood. This article focuses on the regulatory function of CS in antigen-presenting cells, including macrophages and dendritic cells, and refers to CSPGs, such as versican and biglycan, and the cell surface proteoglycan, syndecan.

The soluble form of LOTUS inhibits Nogo receptor type 1-mediated signaling induced by B lymphocyte stimulator and chondroitin sulfate proteoglycans.

There are global efforts in developing therapeutic strategies for central nervous system (CNS) injuries using multimodal approaches. Nogo receptor type 1 (NgR1) has been known as a primary molecule limiting neuronal regeneration in the adult CNS. We identified lateral olfactory tract usher substance (LOTUS) as an endogenous NgR1 antagonist. Membrane-bound LOTUS interacts with NgR1 and inhibits its function by blocking its ligand binding. Five molecules including Nogo, myelin-associated glycoprotein (MAG), oligodendrocyte myelin glycoprotein (OMgp), B lymphocyte stimulator (BLyS) and chondroitin sulfate proteoglycans (CSPGs) have been identified as NgR1 ligands. These ligands bind to NgR1 and activate NgR1 signaling, leading to axon growth inhibition such as growth cone collapse and neurite outgrowth inhibition. We have recently reported that the soluble form of LOTUS (s-LOTUS) also suppressed NgR1-mediated signaling induced by myelin axonal inhibitors (MAIs) including Nogo, MAG and OMgp by binding with both NgR1 and its co-receptor p75 neurotrophin receptor (p75NTR). Though s-LOTUS has been reported to suppress MAIs, whether s-LOTUS also suppresses NgR1 signaling induced by BLyS and CSPGs remains to be elucidated. Here, we show that s-LOTUS inhibits NgR1-mediated signaling induced by BLyS and CSPGs. Although treatment with s-LOTUS did not suppress BLyS-NgR1 interaction, s-LOTUS inhibited growth cone collapse and neurite outgrowth inhibition induced by BLyS and CSPGs in chick dorsal root ganglion (DRG) neurons. Furthermore, s-LOTUS compensated for the suppressive function of endogenous LOTUS in NgR1-mediated signaling in olfactory bulb neurons of lotus-knockout mice. These findings suggest that s-LOTUS is a potent therapeutic agent for neuronal regeneration in the CNS injuries.

Chondroitin sulfate proteoglycans as novel drivers of leucocyte infiltration in multiple sclerosis.

Multiple sclerosis presents with profound changes in the network of molecules involved in maintaining central nervous system architecture, the extracellular matrix. The extracellular matrix components, particularly the chondroitin sulfate proteoglycans, have functions beyond structural support including their potential interaction with, and regulation of, inflammatory molecules. To investigate the roles of chondroitin sulfate proteoglycans in multiple sclerosis, we used the experimental autoimmune encephalomyelitis model in a time course study. We found that the 4-sulfated glycosaminoglycan side chains of chondroitin sulfate proteoglycans, and the core protein of a particular family member, versican V1, were upregulated in the spinal cord of mice at peak clinical severity, correspondent with areas of inflammation. Versican V1 expression in the spinal cord rose progressively over the course of experimental autoimmune encephalomyelitis. A particular structure in the spinal cord and cerebellum that presented with intense upregulation of chondroitin sulfate proteoglycans is the leucocyte-containing perivascular cuff, an important portal of entry of immune cells into the central nervous system parenchyma. In these inflammatory perivascular cuffs, versican V1 and the glycosaminoglycan side chains of chondroitin sulfate proteoglycans were observed by immunohistochemistry within and in proximity to lymphocytes and macrophages as they migrated across the basement membrane into the central nervous system. Expression of versican V1 transcript was also documented in infiltrating CD45+ leucocytes and F4/80+ macrophages by in situ hybridization. To test the hypothesis that the chondroitin sulfate proteoglycans regulate leucocyte mobility, we used macrophages in tissue culture studies. Chondroitin sulfate proteoglycans significantly upregulated pro-inflammatory cytokines and chemokines in macrophages. Strikingly, and more potently than the toll-like receptor-4 ligand lipopolysaccharide, chondroitin sulfate proteoglycans increased the levels of several members of the matrix metalloproteinase family, which are implicated in the capacity of leucocytes to cross barriers. In support, the migratory capacity of macrophages in vitro in a Boyden chamber transwell assay was enhanced by chondroitin sulfate proteoglycans. Finally, using brain specimens from four subjects with multiple sclerosis with active lesions, we found chondroitin sulfate proteoglycans to be associated with leucocytes in inflammatory perivascular cuffs in all four patients. We conclude that the accumulation of chondroitin sulfate proteoglycans in the perivascular cuff in multiple sclerosis and experimental autoimmune encephalomyelitis boosts the activity and migration of leucocytes across the glia limitans into the central nervous system parenchyma. Thus, chondroitin sulfate proteoglycans represent a new class of molecules to overcome in order to reduce the inflammatory cascades and clinical severity of multiple sclerosis.

Chondroitin sulfate proteoglycans negatively regulate the positioning of mitochondria and endoplasmic reticulum to distal axons.

Chondroitin sulfate proteoglycans (CSPGs) are components of the extracellular matrix that inhibit the extension and regeneration of axons. However, the underlying mechanism of action remains poorly understood. Mitochondria and endoplasmic reticulum (ER) are functionally inter-linked organelles important to axon development and maintenance. We report that CSPGs impair the targeting of mitochondria and ER to the growth cones of chicken embryonic sensory axons. The effect of CSPGs on the targeting of mitochondria is blocked by inhibition of the LAR receptor for CSPGs. The regulation of the targeting of mitochondria and ER to the growth cone by CSPGs is due to attenuation of PI3K signaling, which is known to be downstream of LAR receptor activation. Dynactin is a required component of the dynein motor complex that drives the normally occurring retrograde evacuation of mitochondria from growth cones. CSPGs elevate the levels of p150Glu dynactin found in distal axons, and inhibition of the interaction of dynactin with dynein increased axon lengths on CSPGs. CSPGs decreased the membrane potential of mitochondria, and pharmacological inhibition of mitochondria respiration at the growth cone independent of manipulation of mitochondria positioning impaired axon extension. Combined inhibition of dynactin and potentiation of mitochondria respiration further increased axon lengths on CSPGs relative to inhibition of dynactin alone. These data reveal that the regulation of the localization of mitochondria and ER to growth cones is a previously unappreciated aspect of the effects of CSPGs on embryonic axons. © 2017 Wiley Periodicals, Inc. Develop Neurobiol 77: 1351-1370, 2017.

Thrombin impairs human endometrial endothelial angiogenesis; implications for progestin-only contraceptive-induced abnormal uterine bleeding.

OBJECTIVE: Progestin-only contraceptives induce abnormal uterine bleeding, accompanied by prothrombin leakage from dilated endometrial microvessels and increased thrombin generation by human endometrial stromal cell (HESC)-expressed tissue factor. Initial studies of the thrombin-treated HESC secretome identified elevated levels of cleaved chondroitin sulfate proteoglycan 4 (CSPG4), impairing pericyte-endothelial interactions. Thus, we investigated direct and CSPG4-mediated effects of thrombin in eliciting abnormal uterine bleeding by disrupting endometrial angiogenesis. STUDY DESIGN: Liquid chromatography/tandem mass spectrometry, enzyme-linked immunosorbent assay (ELISA) and quantitative real-time-polymerase chain reaction (PCR) evaluated conditioned medium supernatant and cell lysates from control versus thrombin-treated HESCs. Pre- and post-Depo medroxyprogesterone acetate (DMPA)-administered endometria were immunostained for CSPG4. Proliferation, apoptosis and tube formation were assessed in human endometrial endothelial cells (HEECs) incubated with recombinant human (rh)-CSPG4 or thrombin or both. RESULTS: Thrombin induced CSPG4 protein expression in cultured HESCs as detected by mass spectrometry and ELISA (p<.02, n=3). Compared to pre-DMPA endometria (n=5), stromal cells in post-DMPA endometria (n=5) displayed stronger CSPG4 immunostaining. In HEEC cultures (n=3), total tube-formed mesh area was significantly higher in rh-CSPG4 versus control (p<.05). However, thrombin disrupted HEEC tube formation by a concentration- and time-dependent reduction of angiogenic parameters (p<.05), whereas CSPG4 co-treatment did not reverse these thrombin-mediated effects. CONCLUSION: These results suggest that disruption of HEEC tube formation by thrombin induces aberrant angiogenesis and abnormal uterine bleeding in DMPA users. IMPLICATIONS: Mass spectrometry analysis identified several HESC-secreted proteins regulated by thrombin. Therapeutic agents blocking angiogenic effects of thrombin in HESCs can prevent or minimize progestin-only contraceptive-induced abnormal uterine bleeding.

Dermatopontin augments angiogenesis and modulates the expression of transforming growth factor beta 1 and integrin alpha 3 beta 1 in endothelial cells.

Dermatopontin (DPT) is a matricellular protein with cardinal roles in cutaneous wound healing. The protein is also reported to be altered in various anomalies including cancer. The present study is aimed to unravel the role of DPT in angiogenesis which is imperative in many physiological and pathological processes. DPT's capabilities on promoting angiogenesis were assessed using various in vitro and ex vivo systems. The results indicated that DPT enhances cell motility and induces lamellipodia formation in endothelial cells. Additionally, we noticed that DPT stimulates tube formation in endothelial cells when plated on a matrigel substrate. However, it was observed that DPT had no effect on the proliferation of endothelial cells even at higher concentrations and prolonged treatment periods. Additional experiments on CAM and aortic arch assays apparently depicted that DPT promotes neovascularisation and tube sprouting, thus unraveling its prominent role in angiogenesis. Further, PCR analysis revealed that endothelial cells are devoid of DPT expression, but when exogenously supplied, modulates the expression of transforming growth factor ß1 and integrin α3ß1 which are reported to have crucial roles in endothelial cell behaviour during angiogenesis. In conclusion, DPT possess vital pro-angiogenic properties and thus retains promising therapeutic values in managing chronic wounds and cancer.

Identification of factors promoting ex vivo maintenance of mouse hematopoietic stem cells by long-term single-cell quantification.

The maintenance of hematopoietic stem cells (HSCs) during ex vivo culture is an important prerequisite for their therapeutic manipulation. However, despite intense research, culture conditions for robust maintenance of HSCs are still missing. Cultured HSCs are quickly lost, preventing their improved analysis and manipulation. Identification of novel factors supporting HSC ex vivo maintenance is therefore necessary. Coculture with the AFT024 stroma cell line is capable of maintaining HSCs ex vivo long-term, but the responsible molecular players remain unknown. Here, we use continuous long-term single-cell observation to identify the HSC behavioral signature under supportive or nonsupportive stroma cocultures. We report early HSC survival as a major characteristic of HSC-maintaining conditions. Behavioral screening after manipulation of candidate molecules revealed that the extracellular matrix protein dermatopontin (Dpt) is involved in HSC maintenance. DPT knockdown in supportive stroma impaired HSC survival, whereas ectopic expression of the Dpt gene or protein in nonsupportive conditions restored HSC survival. Supplementing defined stroma- and serum-free culture conditions with recombinant DPT protein improved HSC clonogenicity. These findings illustrate a previously uncharacterized role of Dpt in maintaining HSCs ex vivo.

Guiding migration of transplanted glial progenitor cells in the injured spinal cord.

Transplantation of glial-restricted progenitors (GRPs) is a promising strategy for generating a supportive environment for axon growth in the injured spinal cord. Here we explored the possibility of producing a migratory stream of GRPs via directional cues to create a supportive pathway for axon regeneration. We found that the axon growth inhibitor chondroitin sulfate proteoglycan (CSPG) strongly inhibited the adhesion and migration of GRPs, an effect that could be modulated by the adhesion molecule laminin. Digesting glycosaminoglycan side chains of CSPG with chondroitinase improved GRP migration on stripes of CSPG printed on cover glass, although GRPs were still responsive to the remaining repulsive signals of CSPG. Of all factors tested, the basic fibroblast growth factor (bFGF) had the most significant effect in promoting the migration of cultured GRPs. When GRPs were transplanted into either normal spinal cord of adult rats or the injury site in a dorsal column hemisection model of spinal cord injury, a population of transplanted cells migrated toward the region that was injected with the lentivirus expressing chondroitinase or bFGF. These findings suggest that removing CSPG-mediated inhibition, in combination with guidance by attractive factors, can be a promising strategy to produce a migratory stream of supportive GRPs.

Lumican Inhibits SNAIL-Induced Melanoma Cell Migration Specifically by Blocking MMP-14 Activity.

Lumican, a small leucine rich proteoglycan, inhibits MMP-14 activity and melanoma cell migration in vitro and in vivo. Snail triggers epithelial-mesenchymal transitions endowing epithelial cells with migratory and invasive properties during tumor progression. The aim of this work was to investigate lumican effects on MMP-14 activity and migration of Snail overexpressing B16F1 (Snail-B16F1) melanoma cells and HT-29 colon adenocarcinoma cells. Lumican inhibits the Snail induced MMP-14 activity in B16F1 but not in HT-29 cells. In Snail-B16F1 cells, lumican inhibits migration, growth, and melanoma primary tumor development. A lumican-based strategy targeting Snail-induced MMP-14 activity might be useful for melanoma treatment.

NME2 associates with PTPσ to transduce signals from chondroitin sulfate proteoglycans.

Chondroitin sulfate proteoglycans (CSPGs) are a major component of glial scars, inhibiting axonal growth in the central nervous system. Protein tyrosine phosphatase, receptor type S (PTPσ) has been identified as a receptor for CSPGs, whereas its downstream signaling pathway remains to be fully understood. Here, we report that nucleoside diphosphate kinase 2 (NME2) interacts with PTPσ. We screened proteins associated with PTPσ by mass spectrometry, and obtained NME2. Immunoprecipitation analysis revealed that NME2 associated with the PTPσ intracellular domain in HEK-293T cells. NME2 was expressed in the cytoplasm and nucleus of cortical neurons, and knockdown of NME2 in the cortical neurons completely rescued neurite outgrowth inhibition induced by CSPGs. These results demonstrate that NME2 associates with PTPσ to elicit neurite outgrowth inhibition in response to CSPGs.

Chondroitinase improves midbrain pathway reconstruction by transplanted dopamine progenitors in Parkinsonian mice.

Within the adult central nervous system the lack of guidance cues together with the presence of inhibitory molecules produces an environment that is restrictive to axonal growth following injury. Consequently, while clinical trials in Parkinson's disease (PD) patients have demonstrated the capacity of fetal-derived dopamine neurons to survive, integrate and alleviate symptoms, the non-permissive host environment has contributed to the incomplete re-innervation of the target tissue by ectopic grafts, and even more noticeable, the poor reconstruction of the midbrain dopamine pathways following homotopic midbrain grafting. One such inhibitory molecule is the chondroitin sulfate proteoglycan (CSPG), a protein that has been shown to impede axonal growth during development and after injury. Digestion of CSPGs, by delivery of the bacterial enzyme chondroitinase ABC (ChABC), can improve axonal regrowth following a number of neural injuries. Here we examined whether ChABC could similarly improve axonal growth of transplanted dopamine neurons in an animal model of PD. Acute delivery of ChABC, into the medial forebrain bundle, degraded CSPGs along the nigrostriatal pathway. Simultaneous homotopic transplantation of dopaminergic progenitors, into the ventral midbrain of ChABC treated PD mice, had no effect on graft survival but resulted in enhanced axonal growth along the nigrostriatal pathway and reinnervation of the striatum, compared to control grafted mice. This study demonstrates that removal of axonal growth inhibitory molecules could significantly enhance dopaminergic graft integration, thereby holding implications for future approaches in the development of cell replacement therapies for Parkinsonian patients.

CNS axon regeneration inhibitors stimulate an immediate early gene response via MAP kinase-SRF signaling.

BACKGROUND: CNS axon regeneration inhibitors such as Nogo and CSPGs (Chondroitin Sulfate Proteoglycans) are major extrinsic factors limiting outgrowth of severed nerve fibers. However, knowledge on intracellular signaling cascades and gene expression programs activated by these inhibitors in neurons is sparse. Herein we studied intracellular signaling cascades activated by total myelin, Nogo and CSPGs in primary mouse CNS neurons. RESULTS: Total myelin, Nogo and CSPGs stimulated gene expression activity of the serum response factor (SRF), a central gene regulator of immediate early (IEG) and actin cytoskeletal gene transcription. As demonstrated by pharmacological interference, SRF-mediated IEG activation by myelin, Nogo or CSPGs depended on MAP kinase, to a lesser extent on Rho-GTPase but not on PKA signaling. Stimulation of neurons with all three axon growth inhibitors activated the MAP kinase ERK. In addition to ERK activation, myelin activated the IEG c-Fos, an important checkpoint of neuronal survival vs. apoptosis. Employing Srf deficient neurons revealed that myelin-induced IEG activation requires SRF. This suggests an SRF function in mediating neuronal signaling evoked by axon regeneration associated inhibitors. Besides being a signaling target of axon growth inhibitors, we show that constitutively-active SRF-VP16 can be employed to circumvent neurite growth inhibition imposed by myelin, Nogo and CSPGs. CONCLUSION: In sum, our data demonstrate that axon regeneration inhibitors such as Nogo trigger gene expression programs including an SRF-dependent IEG response via MAP kinases and Rho-GTPases.

Lumican: a new inhibitor of matrix metalloproteinase-14 activity.

We previously showed that lumican regulates MMP-14 expression. The aim of this study was to compare the effect of lumican and decorin on MMP-14 activity. In contrast to decorin, the glycosylated form of lumican was able to significantly decrease MMP-14 activity in B16F1 melanoma cells. Our results suggest that a direct interaction occurs between lumican and MMP-14. Lumican behaves as a competitive inhibitor which leads to a complete blocking of the activity of MMP-14. It binds to the catalytic domain of MMP-14 with moderate affinity (KD∼275 nM). Lumican may protect collagen against MMP-14 proteolysis, thus influencing cell-matrix interaction in tumor progression.

Lumican - derived peptides inhibit melanoma cell growth and migration.

Lumican, a small leucine-rich proteoglycan of the extracellular matrix, presents potent anti-tumor properties. Previous works from our group showed that lumican inhibited melanoma cell migration and tumor growth in vitro and in vivo. Melanoma cells adhered to lumican, resulting in a remodeling of their actin cytoskeleton and preventing their migration. In addition, we identified a sequence of 17 amino acids within the lumican core protein, named lumcorin, which was able to inhibit cell chemotaxis and reproduce anti-migratory effect of lumican in vitro. The aim of the present study was to characterize the anti-tumor mechanism of action of lumcorin. Lumcorin significantly decreased the growth in monolayer and in soft agar of two melanoma cell lines - mice B16F1 and human SK-MEL-28 cells - in comparison to controls. Addition of lumcorin to serum free medium significantly inhibited spontaneous motility of these two melanoma cell lines. To characterize the mechanisms involved in the inhibition of cell migration by lumcorin, the status of the phosphorylation/dephosphorylation of proteins was examined. Inhibition of focal adhesion kinase phosphorylation was observed in presence of lumcorin. Since cancer cells have been shown to migrate and to invade by mechanisms that involve matrix metalloproteinases (MMPs), the expression and activity of MMPs were analyzed. Lumcorin induced an accumulation of an intermediate form of MMP-14 (~59kDa), and inhibited MMP-14 activity. Additionally, we identified a short, 10 amino acids peptide within lumcorin sequence, which was able to reproduce its anti-tumor effect on melanoma cells. This peptide may have potential pharmacological applications.

Effect of lumican on the migration of human mesenchymal stem cells and endothelial progenitor cells: involvement of matrix metalloproteinase-14.

BACKGROUND: Increasing number of evidence shows that soluble factors and extracellular matrix (ECM) components provide an optimal microenvironment controlling human bone marrow mesenchymal stem cell (MSC) functions. Successful in vivo administration of stem cells lies in their ability to migrate through ECM barriers and to differentiate along tissue-specific lineages, including endothelium. Lumican, a protein of the small leucine-rich proteoglycan (SLRP) family, was shown to impede cell migration and angiogenesis. The aim of the present study was to analyze the role of lumican in the control of MSC migration and transition to functional endothelial progenitor cell (EPC). METHODOLOGY/PRINCIPAL FINDINGS: Lumican inhibited tube-like structures formation on Matrigel® by MSC, but not EPC. Since matrix metalloproteinases (MMPs), in particular MMP-14, play an important role in remodelling of ECM and enhancing cell migration, their expression and activity were investigated in the cells grown on different ECM substrata. Lumican down-regulated the MMP-14 expression and activity in MSC, but not in EPC. Lumican inhibited MSC, but not EPC migration and invasion. The inhibition of MSC migration and invasion by lumican was reversed by MMP-14 overexpression. CONCLUSION/SIGNIFICANCE: Altogether, our results suggest that lumican inhibits MSC tube-like structure formation and migration via mechanisms that involve a decrease of MMP-14 expression and activity.

A chemical genetic approach identifies piperazine antipsychotics as promoters of CNS neurite growth on inhibitory substrates.

Injury to the central nervous system (CNS) can result in lifelong loss of function due in part to the regenerative failure of CNS neurons. Inhibitory proteins derived from myelin and the astroglial scar are major barriers for the successful regeneration of injured CNS neurons. Previously, we described the identification of a novel compound, F05, which promotes neurite growth from neurons challenged with inhibitory substrates in vitro, and promotes axonal regeneration in vivo (Usher et al., 2010). To identify additional regeneration-promoting compounds, we used F05-induced gene expression profiles to query the Broad Institute Connectivity Map, a gene expression database of cells treated with >1300 compounds. Despite no shared chemical similarity, F05-induced changes in gene expression were remarkably similar to those seen with a group of piperazine phenothiazine antipsychotics (PhAPs). In contrast to antipsychotics of other structural classes, PhAPs promoted neurite growth of CNS neurons challenged with two different glial derived inhibitory substrates. Our pharmacological studies suggest a mechanism whereby PhAPs promote growth through antagonism of calmodulin signaling, independent of dopamine receptor antagonism. These findings shed light on mechanisms underlying neurite-inhibitory signaling, and suggest that clinically approved antipsychotic compounds may be repurposed for use in CNS injured patients.

Lumican inhibits cell migration through α2ß1 integrin.

Lumican, an extracellular matrix protein of the small leucine-rich proteoglycan family, has been shown to impede melanoma progression by inhibiting cell migration. In the present study, we show that lumican targets α2ß1 integrin thereby inhibiting cell migration. A375 melanoma cells were transfected with siRNA directed against the α2 integrin subunit. Compared to A375 control cells, the anti-migratory effect of lumican was abrogated on transfected A375 cells. Moreover, lumican inhibited the chemotactic migration of Chinese hamster ovary (CHO) cells stably transfected with α2 integrin subunit (CHO-A2) but not that of wild-type CHO cells (CHO-WT) lacking this subunit. In contrast to CHO-WT cells, we observed in time-lapse microscopy a decrease of CHO-A2 cell migration speed in presence of lumican. Focal adhesion kinase phosphorylated at tyrosine-397 (pFAK) and total FAK were analysed in CHO-WT and CHO-A2 cells. A significant decrease of the ratio pFAK/FAK was shown in presence of recombinant human lumican. Using solid phase assays, a direct binding between lumican and the α2ß1 integrin was demonstrated. This interaction did not involve the glycan moiety of lumican and was cation independent. Lumican was also able to bind the activated I domain of the α2 integrin subunit with a K(d)≥200nM. In conclusion, we demonstrated for the first time that the inhibition of cell migration by lumican depends on a direct binding between the core protein of lumican and the α2ß1 integrin.

Amphotericin B, identified from a natural product screen, antagonizes CNS inhibitors to promote axon growth via activation of an Akt pathway in neurons.

One of the major barriers to successful axon regeneration in the adult CNS is the presence of inhibitory molecules that originate from the myelin sheath and glial scar. So far, only a small number of pharmacological compounds have exhibited functional activity against CNS inhibitors in promoting axon regeneration after injury. To search for novel compounds that enhance neurite outgrowth in vitro, we initiated a screen of a collection of natural products. We identified four compounds with the potential to promote growth over a myelin substrate. Of these, Amphotericin B (AmB) was shown to enhance neurite outgrowth and antagonize activities of major myelin associated inhibitors and glial-scar-derived chondroitin sulfate proteoglycans. AmB was found to activate Akt and thereby suppress the activity of glycogen synthase kinase 3 beta. Also, a cell permeable peptide that inhibits Akt activity was shown to block the effect of AmB in promoting axonal growth, while another peptide that increases Akt activity stimulated axonal growth in the presence of the myelin associated inhibitors. Our results suggest that AmB can promote neurite outgrowth over a wide range of inhibitory substrates via a mechanism that involves activation of Akt.

HDAC inhibition promotes neuronal outgrowth and counteracts growth cone collapse through CBP/p300 and P/CAF-dependent p53 acetylation.

Neuronal outgrowth is guided by both extrinsic and intrinsic factors, involving transcriptional regulation. The acetylation of histones and transcription factors, which facilitates promoter accessibility, ultimately promotes transcription, and depends on the balance between histone deacetylases (HDACs) and histone acetyltransferases (HATs) activities. However, a critical function for specific acetylation modifying enzymes in neuronal outgrowth has yet to be investigated. To address this issue, we have used an epigenetic approach to facilitate gene expression in neurons, by using specific HDAC inhibitors. Neurons treated with a combination of HDAC and transcription inhibitors display an acetylation and transcription-dependent increase in outgrowth and a reduction in growth cone collapse on both 'permissive' (poly-D-lysine, PDL) and 'non-permissive' substrates (myelin and chondroitin sulphate proteoglycans (CSPGs)). Next, we specifically show that the expression of the histone acetyltransferases CBP/p300 and P/CAF is repressed in neurons by inhibitory substrates, whereas it is triggered by HDAC inhibition on both permissive and inhibitory conditions. Gene silencing and gain of function experiments show that CBP/p300 and P/CAF are key players in neuronal outgrowth, acetylate histone H3 at K9-14 and the transcription factor p53, thereby initiating a pro-neuronal outgrowth transcriptional program. These findings contribute to the growing understanding of transcriptional regulation in neuronal outgrowth and may lay the molecular groundwork for the promotion of axonal regeneration after injury.