23 research outputs found
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Specificity Determinants in Neurotrophin-3 and Design of Nerve Growth Factor-Based trkC Agonists by Changing Central Ī²-Strand Bundle Residues to Their Neurotrophin-3 Analogs
Neurotrophic factors mediate their signal by binding to specific cell surface receptors of the trk family. The binding sites of neurotrophin-3 (NT-3) and nerve growth factor (NGF) to their preferred receptors trkC and trkA, respectively, were previously determined by mutational analyses. These and other studies showed that trkA can discriminate between NGF and NT-3 primarily by recognition of their N-terminal residues. The mechanism of trkC discrimination, however, remained unclear, especially since the most important residue in NT-3 involved in binding to trkC, R103, is conserved in all neurotrophins. In this study residues that are part of the central Ī²-strand bundle of NT-3 and are not conserved among the neurotrophins were grafted onto NGF and tested for recruitment of trkC affinity. Exchange of NGF residues at positions 18, 20, 23, 29, 84, and 86 by their NT-3 counterparts resulted in NGF variants that bound to trkC, while maintaining their affinity to trkA, and were able to induce autophosphorylation and differentiation of PC12 cells expressing trkC. These variants show that the amino acid at position 23 (glycine in NGF, threonine in NT-3) is critical for trkC recognition while other residues fine tune the specificity of NT-3 for trkC. The results demonstrate the importance of nonconserved residues of the central Ī²-strand bundle region for the interaction of NT-3 with trkC and emphasize the different mechanism of specificity determination that is employed in the NT-3/trkC and NGF/trkA ligand/receptor pairs
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High Resolution Mapping of the Binding Site of TrkA for Nerve Growth Factor and TrkC for Neurotrophin-3 on the Second Immunoglobulin-like Domain of the Trk Receptors
Phase II trial of the sigma-1 receptor agonist cutamesine (SA4503) for recovery enhancement after acute ischemic stroke
Background and PurposeāThe Ļ-1 receptor (Sig-1R) agonist cutamesine (SA4503) enhanced functional recovery after experimental stroke with a treatment initiation window of 48 hours and chronic treatment for 28 days. We conducted a phase 2 clinical trial exploring the safety, tolerability, dose range, and functional effects of cutamesine in patients with ischemic stroke.<p></p>
MethodsāSubjects were randomized between 48 and 72 hours after stroke to receive cutamesine 1 mg/d, 3 mg/d, or placebo for 28 days. Effects on safety and function were assessed at baseline, at end of treatment (day 28), and at end of follow-up (day 56).<p></p>
ResultsāIn 60 patients, treatment with both cutamesine dosages was safe and well tolerated without significant differences in numbers of treatment emergent or serious adverse events. No significant effect was observed on the primary efficacy measure (change in National Institutes of Health Stroke Scale from baseline to day 56) or modified Rankin Scale and Barthel Index scores. Post hoc analysis of moderately and severely affected patients (baseline National Institutes of Health Stroke Scale, ≥7 and ≥10) showed greater National Institutes of Health Stroke Scale improvements in the 3 mg/d cutamesine group when compared with placebo (P=0.034 and P=0.038, respectively). A trend toward a higher proportion being able to complete a 10m timed walk was observed for cutamesine-treated subjects.<p></p>
ConclusionsāCutamesine was safe and well tolerated at both dosage levels. Although no significant effects on functional end points were seen in the population as a whole, greater improvement in National Institutes of Health Stroke Scale scores among patients with greater pretreatment deficits seen in post hoc analysis warrants further investigation. Additional studies should focus on the patient population with moderate-to-severe stroke
Functional comparison of long and short splice forms of RPTPĪ²: Implications for glioblastoma treatment
The receptor protein tyrosine phosphatase beta (RPTPĪ²/PTPĪ¶) is overexpressed in glioblastoma tumors and plays a functional role in tumor cell migration and adhesion. Glioblastomas express at least three splice variants of RPTPĪ², including long and short receptor forms and a secreted chondroitin sulfate proteoglycan called phosphacan. Here we explore the differences in the expression pattern and function of long RPTPĪ² and short RPTPĪ². The short form of RPTPĪ² lacks exon 12, which encodes 860 amino acids located in the extracellular domain. Until now, functional differences between long and short RPTPĪ² have been difficult to elucidate. In this study, antibodies specific to the splice junction, unique to short RPTPĪ², allowed for the discrimination of the two receptors. A study of normal brain tissue and graded astrocytomas indicates that long and short RPTPĪ² forms have an overlapping expression pattern. In order to study functional differences between long and short RPTPĪ², we created stable U87 glioblastoma cells that expressed these receptors. U87 stable cell lines overexpressing long or short RPTPĪ² migrate faster and adhere more robustly than parental U87 cells. The two forms differ in that long-RPTPĪ² -overexpressing cells migrate and adhere better than short-RPTPĪ² -overexpressing cells. A study of the extracellular domain of short RPTPĪ² indicates that it retains much of the functional capacity of phosphacan. Indeed, the action of recombinant, short-RPTPĪ² extra-cellular domain protein is similar to that of phosphacan as a repulsive substrate for glioblastoma cells. Comparison of the signaling capacity of long RPTPĪ² to that of short RPTPĪ² reveals very similar abilities to activate transcription pathways. Moreover, transient transfection with either long or short RPTPĪ² activates NF-ĪŗB reporter gene transcription. Because of their tumor-restricted and largely overlapping expression patterns in glioblastoma, both RPTPĪ² splice forms are potential therapeutic targets. The involvement of long and short RPTPĪ² in glioma tumor cell biology also contributes to the value of RPTPĪ² as a cancer target
Npas4, a novel helix-loop-helix PAS domain protein, is regulated in response to cerebral ischemia
Basic helix-loop-helix PAS domain proteins form a growing family of transcription factors. These proteins are involved in the process of adaptation to cellular stresses and environmental factors such as a change in oxygen concentration. We describe the identification and characterization of a recently cloned PAS domain protein termed Npas4 in ischemic rat brain. Using gene expression profiling following middle cerebral artery occlusion, we showed that the Npas4 mRNA is differentially expressed in ischemic tissue. The full-length gene was cloned from rat brain and its spatial and temporal expression characterized with in situ hybridization and Northern blotting. The Npas4 mRNA is specifically expressed in the brain and is highly up-regulated in ischemic tissues following both focal and global cerebral ischemic insults. Immunohistochemistry revealed a strong expression in the limbic system and thalamus, as well as in layers 3 and 5 in the cortex of the unchallenged brain. When overexpressed in HEK 293 cells, Npas4 appears as a protein of similar to 100 kDa. In brain samples, however, in addition to the 100 kDa band a specific 200 kDa immunoreactive band was also detected. Ischemic challenge lead to a decrease in the 200 kDa form and a simultaneous increase in the 100 kDa immunoreactivity. This could indicate a novel regulatory mechanism for activation and/or deactivation of this protein in response to ischemic brain injury
The sigma-1 receptor enhances brain plasticity and functional recovery after experimental stroke.
Stroke leads to brain damage with subsequent slow and incomplete recovery of lost brain functions. Enriched housing of stroke-injured rats provides multi-modal sensorimotor stimulation, which improves recovery, although the specific mechanisms involved have not been identified. In rats housed in an enriched environment for two weeks after permanent middle cerebral artery occlusion, we found increased sigma-1 receptor expression in peri-infarct areas. Treatment of rats subjected to permanent or transient middle cerebral artery occlusion with 1-(3,4-dimethoxyphenethyl)-4-(3-phenylpropyl)piperazine dihydrochloride, an agonist of the sigma-1 receptor, starting two days after injury, enhanced the recovery of lost sensorimotor function without decreasing infarct size. The sigma-1 receptor was found in the galactocerebroside enriched membrane microdomains of reactive astrocytes and in neurons. Sigma-1 receptor activation increased the levels of the synaptic protein neurabin and neurexin in membrane rafts in the peri-infarct area, while sigma-1 receptor silencing prevented sigma-1 receptor-mediated neurite outgrowth in primary cortical neuronal cultures. In astrocytic cultures, oxygen and glucose deprivation induced sigma-1 receptor expression and actin dependent membrane raft formation, the latter blocked by sigma-1 receptor small interfering RNA silencing and pharmacological inhibition. We conclude that sigma-1 receptor activation stimulates recovery after stroke by enhancing cellular transport of biomolecules required for brain repair, thereby stimulating brain plasticity. Pharmacological targeting of the sigma-1 receptor provides new opportunities for stroke treatment beyond the therapeutic window of neuroprotection
A Pharmacological Screening Approach for Discovery of Neuroprotective Compounds in Ischemic Stroke
<div><p>With the availability and ease of small molecule production and design continuing to improve, robust, high-throughput methods for screening are increasingly necessary to find pharmacologically relevant compounds amongst the masses of potential candidates. Here, we demonstrate that a primary oxygen glucose deprivation assay in primary cortical neurons followed by secondary assays (i.e. post-treatment protocol in organotypic hippocampal slice cultures and cortical neurons) can be used as a robust screen to identify neuroprotective compounds with potential therapeutic efficacy. In our screen about 50% of the compounds in a library of pharmacologically active compounds displayed some degree of neuroprotective activity if tested in a pre-treatment toxicity assay but just a few of these compounds, including Carbenoxolone, remained active when tested in a post-treatment protocol. When further examined, Carbenoxolone also led to a significant reduction in infarction size and neuronal damage in the ischemic penumbra when administered six hours post middle cerebral artery occlusion in rats. Pharmacological testing of Carbenoxolone-related compounds, acting by inhibition of 11-Ī²-hydroxysteroid dehydrogenase-1 (11Ī²-HSD1), gave rise to similarly potent <i>in vivo</i> neuroprotection. This indicates that the increase of intracellular glucocorticoid levels mediated by 11Ī²-HSD1 may be involved in the mechanism that exacerbates ischemic neuronal cell death, and inhibiting this enzyme could have potential therapeutic value for neuroprotective therapies in ischemic stroke and other neurodegenerative disorders associated with neuronal injury.</p></div
List of compounds displaying post-injury neuroprotective activity in the oxygen glucose deprivation (OGD) assay in cortical neurons.
<p>List of compounds displaying post-injury neuroprotective activity in the oxygen glucose deprivation (OGD) assay in cortical neurons.</p