Aviva J. Symes

Chondroitin-4-sulfation negatively regulates axonal guidance and growth

Glycosaminoglycan (GAG) side chains endow extracellular matrix proteoglycans with diversity and complexity based upon the length, composition and charge distribution of the polysaccharide chain. Using cultured primary neurons, we show that specific sulfation in the GAG chains of chondroitin sulfate mediates neuronal guidance cues and axonal growth inhibition. Chondroitin-4-sulfate (CS-A), but not chondroitin-6-sulfate (CS-C), exhibits a strong negative guidance cue to mouse cerebellar granule neurons. Enzymatic and gene-based manipulations of 4-sulfation in the GAG side chains alter their ability to direct growing axons. Furthermore, 4-sulfated chondroitin sulfate GAG chains are rapidly and significantly increased in regions that do not support axonal regeneration proximal to spinal cord lesions in mice. Thus, our findings show that specific sulfation along the carbohydrate backbone carries instructions to regulate neuronal function.

The protein tyrosine phosphatase SHP-2 negatively regulates ciliary neurotrophic factor induction of gene expression

Ciliary neurotrophic factor, along with other neuropoietic cytokines, signals through the shared receptor subunit gp130 [1-3], leading to the tyrosine phosphorylation of a number of substrates [4,5], including the transcription factors STAT1 and STAT3 and the protein tyrosine phosphatase SHP-2 [6,7] [8]. SHP-2 (also known as PTP1D, SHPTP2, Syp and PTP2C) is a positive regulatory molecule required for the activation of the mitogen-activated protein kinase pathway and the stimulation of gene expression in response to epidermal growth factor, insulin and platelet-derived growth factor stimulation [9-11]. We have previously shown that cytokines that signal via the gp130 receptor subunit activate transcription of the vasoactive intestinal peptide (VIP) gene through a 180 bp cytokine response element (CyRE) [12,13]. To characterize the role of SHP-2 in the regulation of gp130-stimulated gene expression, we examined the regulation of the VIP CyRE in two systems that prevented ligand-dependent SHP-2 phosphorylation. Inhibition of SHP-2, either by mutating the tyrosine residue in gp130 that mediates the SHP-2 interaction, or by expression of dominant-negative SHP-2, resulted in dramatic increases in gp130-dependent gene expression, through the VIP CyRE and more specifically through multimerized STAT-binding sites. These data suggest that SHP-2 has a negative role in gp130 signaling by modulating STAT-mediated transcriptional activation.

Coordinate Regulation of Choline Acetyltransferase, Tyrosine Hydroxylase, and Neuropeptide mRNAs by Ciliary Neurotrophic Factor and Leukemia Inhibitory Factor in Cultured Sympathetic Neurons

Abstract: The neurotransmitter phenotype switch that occurs in cultures of rat superior cervical ganglion neurons after treatment with leukemia inhibitory factor or ciliary neurotrophic factor is a useful model permitting investigation of the mechanisms of cytokine‐mediated differentiation. Recently the actions of leukemia inhibitory factor and ciliary neurotrophic factor have been linked through their interactions with related receptor complexes. Here we compare the effects of these two cytokines on gene expression in sympathetic neuronal cultures and begin to investigate their mechanisms. We report that, as has been shown for leukemia inhibitory factor, ciliary neurotrophic factor regulates peptides and classical transmitters in these cultures at the mRNA level. In addition, we find that the induction of substance P mRNA by these cytokines is rapid, dependent on protein synthesis, and occurs in 40–50% of superior cervical ganglion neurons in dissociated culture.

Smad3 null mice display more rapid wound closure and reduced scar formation after a stab wound to the cerebral cortex.

Following central nervous system injury, adult mammalian neurons do not regenerate through regions of scar formation. This regenerative failure is due in part to the inhibitory environment of the glial scar at the lesion site. Following injury, transforming growth factor beta (TGF-beta) is strongly induced and is important to many aspects of the response to injury, including deposition of extracellular matrix (ECM) in the glial scar. However, the pathways through which TGF-beta signals to mediate these effects are not known. In order to examine the contribution of the TGF-beta-induced transcription factor, Smad3, to formation of the glial scar after traumatic brain injury, we utilized mice that do not express Smad3. We report that Smad3 null mice heal stab wounds to the cerebral cortex more rapidly than do wild-type mice. In Smad3 null mice many aspects of glial scar formation and the immune response to injury were altered. Fewer neutrophils, macrophages/microglia, NG2-positive cells and GFAP-positive cells were detected immediately around the lesion in Smad3 null mice. Expression of fibronectin and laminin was also reduced. Injury-induced cell proliferation was significantly lower in Smad3 null mice around the lesion. There was no overall difference between wild-type and Smad3 null mice in immunoreactivity for TGF-beta(1) after injury. Thus, our experiments suggest that TGF-beta signaling through Smad3 contributes significantly to the immune response and scar formation after cortical stab wound injury, delaying recovery through multiple mechanisms.

COORDINATE REGULATION OF STAT SIGNALING AND C-FOS EXPRESSION BY THE TYROSINE PHOSPHATASE SHP-2

The src homology 2 (SH2) domain-containing protein-tyrosine phosphatase SHP-2 has been implicated as an important positive regulator of several mitogenic signaling pathways. SHP-2 has more recently been shown to be tyrosine phosphorylated and recruited to the gp130 component of the ciliary neurotrophic factor (CNTF) receptor complex upon stimulation with CNTF. CNTF does not, however, have a proliferative effect on responsive cells, but rather enhances the survival and differentiation of sympathetic, motor, and sensory neurons. In this study, expression of an interfering mutant of SHP-2 in the neuroblastoma cell line NBFL increased CNTF induction of a vasoactive intestinal peptide (VIP) reporter gene, and in cultures of sympathetic neurons, it resulted in an up-regulation of endogenous VIP and substance P (SP) gene expression. Members of the CNTF family of cytokines transmit their signal by activating signaling pathways involving both STAT and Fos-Jun transcription factors. In CNTF-stimulated NBFL cells that constitutively express the SHP-2 interfering mutant, there was increased and prolonged formation of STAT/DNA complexes, but decreased AP-1 binding activity, that mirrored a down-regulation of c-fos expression both at the mRNA and protein level. Taken together, these data indicate that SHP-2 has dual and opposing roles in a signaling cascade triggered by the same ligand, as illustrated by its ability to differentially regulate the levels of activity of both STAT and AP-1 transcription factors.

A Sweat Gland-derived Differentiation Activity Acts through Known Cytokine Signaling Pathways

The sympathetic innervation of sweat glands undergoes a target-induced noradrenergic to cholinergic/peptidergic switch during development. Similar changes are induced in cultured sympathetic neurons by sweat gland cells or by one of the following cytokines: leukemia inhibitory factor (LIF), ciliary neurotrophic factor (CNTF), or cardiotrophin-1 (CT-1). None of these is the sweat gland-derived differentiation activity. LIF, CNTF, and CT-1 act through the known receptors LIF receptor β (LIFRβ) and gp130 and well defined signaling pathways including receptor phosphorylation and STAT3 activation. Therefore, to determine whether the gland-derived differentiation activity was a member of the LIF/CNTF cytokine family, we tested whether it acted via these same receptors and signal cascades. Blockade of LIFRβ inhibited the sweat gland differentiation activity in neuron/gland co-cultures, and extracts of gland-containing footpads stimulated tyrosine phosphorylation of LIFRβ and gp130. An inhibitor (CGX) of molecules that bind the CNTFRα, which is required for CNTF signaling, did not affect the gland-derived differentiation activity. Soluble footpad extracts induced the same changes in NBFL neuroblastoma cells as LIF and CNTF, including increased vasoactive intestinal peptide mRNA, STAT3 dimerization, and DNA binding, and stimulation of transcription from the vasoactive intestinal peptide cytokine-responsive element. Thus, the sweat gland-derived differentiation activity uses the same signaling pathway as the neuropoietic cytokines, and is likely to be a family member.

Oncostatin M regulates VIP expression in a human neuroblastoma cell line.

Oncostatin-M (OM), a recently described glycoprotein cytokine, is structurally and functionally related to cholinergic differentiation factor/leukemia inhibitory factor (CDF/LIF) and ciliary neurotrophic factor (CNTF). To determine whether OM, like CDF/LIF and CNTF, possesses trophic or differentiative functions for neurons we examined the effects of recombinant human OM on ciliary neuron survival and neurotransmitter expression in sympathetic neurons. Like CDF/LIF, but in contrast to CNTF, OM had no effect on ciliary neuronal survival at any concentration tested. OM produced small but reproducible increases in choline acetyl transferase (ChAT) activity and vasoactive intestinal peptide (VIP) levels in rat sympathetic neuron cultures, but this effect was significantly less than that of CNTF or CDF/LIF. To determine if human OM would elicit a more robust response from human cells, we utilized a human neuroblastoma cell line, NBFL, that responds to CNTF and CDF/LIF by altering vasoactive intestinal peptide (VIP) levels. OM specifically elevated VIP and c-fos mRNA levels in NBFL cells and was as potent as CDF/LIF in this assay. Our data provides evidence that OM acts on neurons and identifies a neural cell line responsive to OM, CNTF, CDF/LIF.

Alterations in sulfated chondroitin glycosaminoglycans following controlled cortical impact injury in mice

Chondroitin sulfate proteoglycans (CSPGs) play a pivotal role in many neuronal growth mechanisms including axon guidance and the modulation of repair processes following injury to the spinal cord or brain. Many actions of CSPGs in the central nervous system (CNS) are governed by the specific sulfation pattern on the glycosaminoglycan (GAG) chains attached to CSPG core proteins. To elucidate the role of CSPGs and sulfated GAG chains following traumatic brain injury (TBI), controlled cortical impact injury of mild to moderate severity was performed over the left sensory motor cortex in mice. Using immunoblotting and immunostaining, we found that TBI resulted in an increase in the CSPGs neurocan and NG2 expression in a tight band surrounding the injury core, which overlapped with the presence of 4‐sulfated CS GAGs but not with 6‐sulfated GAGs. This increase was observed as early as 7 days post injury (dpi), and persisted for up to 28 dpi. Labeling with markers against microglia/macrophages, NG2+ cells, fibroblasts, and astrocytes showed that these cells were all localized in the area, suggesting multiple origins of chondroitin‐4‐sulfate increase. TBI also caused a decrease in the expression of aggrecan and phosphacan in the pericontusional cortex with a concomitant reduction in the number of perineuronal nets. In summary, we describe a dual response in CSPGs whereby they may be actively involved in complex repair processes following TBI. J. Comp. Neurol. 520:3295–3313, 2012.

Regulation of Nociceptin/Orphanin FQ Gene Expression by Neuropoietic Cytokines and Neurotrophic Factors in Neurons and Astrocytes

Abstract: We have identified the gene encoding nociceptin/orphanin FQ (N/OFQ), the novel opioid‐like neuropeptide, as responsive to ciliary neurotrophic factor (CNTF). N/OFQ mRNA levels were induced five‐ and ninefold by CNTF in striatal and cortical neurons. In primary astrocytes CNTF also increased N/OFQ mRNA levels. CNTF is a multifunctional cytokine that mediates the development and differentiation of both neurons and astrocytes and supports the survival of various neurons. CNTF is also an injury‐induced factor in the brain playing a crucial role in astrogliosis. The mechanism by which CNTF elicits these effects is not well understood, but it is likely to involve regulation of specific genes. CNTF regulation of N/OFQ expression was sensitive to the kinase inhibitors H‐7 and genistein but not to inhibition of protein synthesis. This pharmacological profile is consistent with CNTF activating the Janus protein tyrosine kinase (JAK)/signal transducers and activators of transcription (STAT) pathway to induce N/OFQ transcription. In nuclear extracts of CNTF‐treated striatal neurons DNA binding of STAT proteins was increased. Radioimmunoassays revealed elevated N/OFQ immunoreactivity in striatal neurons after CNTF treatment. Expression of the related proenkephalin gene was not affected by CNTF in either neuronal or glial cultures. Regulation of N/OFQ expression by CNTF might point to a possible function of N/OFQ during development and after neural injury.

Temporal dynamics of cerebral blood flow, cortical damage, apoptosis, astrocyte-vasculature interaction and astrogliosis in the pericontusional region after traumatic brain injury.

Traumatic brain injury (TBI) results in a loss of brain tissue at the moment of impact in the cerebral cortex. Subsequent secondary injury involves the release of molecular signals with dramatic consequences for the integrity of damaged tissue, leading to the evolution of a pericontusional-damaged area minutes to days after in the initial injury. The mechanisms behind the progression of tissue loss remain under investigation. In this study, we analyzed the spatial–temporal profile of blood flow, apoptotic, and astrocytic–vascular events in the cortical regions around the impact site at time points ranging from 5 h to 2 months after TBI. We performed a mild–moderate controlled cortical impact injury in young adult mice and analyzed the glial and vascular response to injury. We observed a dramatic decrease in perilesional cerebral blood flow (CBF) immediately following the cortical impact that lasted until days later. CBF finally returned to baseline levels by 30 days post-injury (dpi). The initial impact also resulted in an immediate loss of tissue and cavity formation that gradually increased in size until 3 dpi. An increase in dying cells localized in the pericontusional region and a robust astrogliosis were also observed at 3 dpi. A strong vasculature interaction with astrocytes was established at 7 dpi. Glial scar formation began at 7 dpi and seemed to be compact by 60 dpi. Altogether, these results suggest that TBI results in a progression from acute neurodegeneration that precedes astrocytic activation, reformation of the neurovascular unit to glial scar formation. Understanding the multiple processes occurring after TBI is critical to the ability to develop neuroprotective therapeutics to ameliorate the short and long-term consequences of brain injury.