Electrophysiological characterization of activation state-dependent Cav2 channel antagonist TROX-1 in spinal nerve injured rats

AbstractPrialt, a synthetic version of Cav2.2 antagonist ω-conotoxin MVIIA derived from Conus magus, is the first clinically approved voltage-gated calcium channel blocker for refractory chronic pain. However, due to the narrow therapeutic window and considerable side effects associated with systemic dosing, Prialt is only administered intrathecally. N-triazole oxindole (TROX-1) is a novel use-dependent and activation state-selective small-molecule inhibitor of Cav2.1, 2.2 and 2.3 calcium channels designed to overcome the limitations of Prialt. We have examined the neurophysiological and behavioral effects of blocking calcium channels with TROX-1. In vitro, TROX-1, in contrast to state-independent antagonist Prialt, preferentially inhibits Cav2.2 currents in rat dorsal root ganglia (DRG) neurons under depolarized conditions. In vivo electrophysiology was performed to record from deep dorsal horn lamina V/VI wide dynamic range neurons in non-sentient spinal nerve-ligated (SNL) and sham-operated rats. In SNL rats, spinal neurons exhibited reduced responses to innocuous and noxious punctate mechanical stimulation of the receptive field following subcutaneous administration of TROX-1, an effect that was absent in sham-operated animals. No effect was observed on neuronal responses evoked by dynamic brushing, heat or cold stimulation in SNL or sham rats. The wind-up response of spinal neurons following repeated electrical stimulation of the receptive field was also unaffected. Spinally applied TROX-1 dose dependently inhibited mechanically evoked neuronal responses in SNL but not sham-operated rats, consistent with behavioral observations. This study confirms the pathological state-dependent actions of TROX-1 through a likely spinal mechanism and reveals a modality selective change in calcium channel function following nerve injury

Axonal remodeling for motor recovery after traumatic brain injury requires downregulation of γ-aminobutyric acid signaling

Remodeling of the remnant neuronal network after brain injury possibly mediates spontaneous functional recovery; however, the mechanisms inducing axonal remodeling during spontaneous recovery remain unclear. Here, we show that altered γ-aminobutyric acid (GABA) signaling is crucial for axonal remodeling of the contralesional cortex after traumatic brain injury. After injury to the sensorimotor cortex in mice, we found a significant decrease in the expression of GABAAR-α1 subunits in the intact sensorimotor cortex for 2 weeks. Motor functions, assessed by grid walk and cylinder tests, spontaneously improved in 4 weeks after the injury to the sensorimotor cortex. With motor recovery, corticospinal tract (CST) axons from the contralesional cortex sprouted into the denervated side of the cervical spinal cord at 2 and 4 weeks after the injury. To determine the functional implications of the changes in the expression of GABAAR-α1 subunits, we infused muscimol, a GABA R agonist, into the contralesional cortex for a week after the injury. Compared with the vehicle-treated mice, we noted significantly inhibited recovery in the muscimol-treated mice. Further, muscimol infusion greatly suppressed the axonal sprouting into the denervated side of the cervical spinal cord. In conclusion, recovery of motor function and axonal remodeling of the CST following cortical injury requires suppressed GABAAR subunit expression and decreased GABAergic signaling

The Leucine Zipper Domains of the Transcription Factors GCN4 and c-Jun Have Ribonuclease Activity

Basic-region leucine zipper (bZIP) proteins are one of the largest transcription factor families that regulate a wide range of cellular functions. Owing to the stability of their coiled coil structure leucine zipper (LZ) domains of bZIP factors are widely employed as dimerization motifs in protein engineering studies. In the course of one such study, the X-ray structure of the retro-version of the LZ moiety of yeast transcriptional activator GCN4 suggested that this retro-LZ may have ribonuclease activity. Here we show that not only the retro-LZ but also the authentic LZ of GCN4 has weak but distinct ribonuclease activity. The observed cleavage of RNA is unspecific, it is not suppressed by the ribonuclease A inhibitor RNasin and involves the breakage of 3′,5′-phosphodiester bonds with formation of 2′,3′-cyclic phosphates as the final products as demonstrated by HPLC/electrospray ionization mass spectrometry. Several mutants of the GCN4 leucine zipper are catalytically inactive, providing important negative controls and unequivocally associating the enzymatic activity with the peptide under study. The leucine zipper moiety of the human factor c-Jun as well as the entire c-Jun protein are also shown to catalyze degradation of RNA. The presented data, which was obtained in the test-tube experiments, adds GCN4 and c-Jun to the pool of proteins with multiple functions (also known as moonlighting proteins). If expressed in vivo, the endoribonuclease activity of these bZIP-containing factors may represent a direct coupling between transcription activation and controlled RNA turnover. As an additional result of this work, the retro-leucine zipper of GCN4 can be added to the list of functional retro-peptides

Human Umbilical Cord Blood Cells Restore Brain Damage Induced Changes in Rat Somatosensory Cortex

Intraperitoneal transplantation of human umbilical cord blood (hUCB) cells has been shown to reduce sensorimotor deficits after hypoxic ischemic brain injury in neonatal rats. However, the neuronal correlate of the functional recovery and how such a treatment enforces plastic remodelling at the level of neural processing remains elusive. Here we show by in-vivo recordings that hUCB cells have the capability of ameliorating the injury-related impairment of neural processing in primary somatosensory cortex. Intact cortical processing depends on a delicate balance of inhibitory and excitatory transmission, which is disturbed after injury. We found that the dimensions of cortical maps and receptive fields, which are significantly altered after injury, were largely restored. Additionally, the lesion induced hyperexcitability was no longer observed in hUCB treated animals as indicated by a paired-pulse behaviour resembling that observed in control animals. The beneficial effects on cortical processing were reflected in an almost complete recovery of sensorimotor behaviour. Our results demonstrate that hUCB cells reinstall the way central neurons process information by normalizing inhibitory and excitatory processes. We propose that the intermediate level of cortical processing will become relevant as a new stage to investigate efficacy and mechanisms of cell therapy in the treatment of brain injury

Restoring brain function after stroke - bridging the gap between animals and humans

Stroke is the leading cause of complex adult disability in the world. Recovery from stroke is often incomplete, which leaves many people dependent on others for their care. The improvement of long-term outcomes should, therefore, be a clinical and research priority. As a result of advances in our understanding of the biological mechanisms involved in recovery and repair after stroke, therapeutic opportunities to promote recovery through manipulation of poststroke plasticity have never been greater. This work has almost exclusively been carried out in preclinical animal models of stroke with little translation into human studies. The challenge ahead is to develop a mechanistic understanding of recovery from stroke in humans. Advances in neuroimaging techniques now enable us to reconcile behavioural accounts of recovery with molecular and cellular changes. Consequently, clinical trials can be designed in a stratified manner that takes into account when an intervention should be delivered and who is most likely to benefit. This approach is expected to lead to a substantial change in how restorative therapeutic strategies are delivered in patients after stroke

Transgenic tobacco plants that express an antisense construct derived from a Medicago sativa cDNA encoding a rac-related small GTP-binding protein fail to develop necrotic lesions upon elicitor infiltration

Schiene K, Pühler A, Niehaus K. Transgenic tobacco plants that express an antisense construct derived from a Medicago sativa cDNA encoding a rac-related small GTP-binding protein fail to develop necrotic lesions upon elicitor infiltration. MOLECULAR AND GENERAL GENETICS. 2000;263(5):761-770.Using an RT-PCR approach a vac-related cDNA clone, designated Ms-rac1, was isolated from Medicago sativa (alfalfa). Ms-rac1 encodes a putative protein of 197 amino acids, which is closely related to known Rac-related GTP-binding proteins from Pisum sativum and Arabidopsis thaliana. RT-PCR analysis demonstrated that Ms-rac1 is ubiquitously expressed in various tissues in alfalfa. Expression of Ms-rac1 in suspension cultures occurred independently of treatment with elicitor, indicating that it is constitutively expressed. Heterologous expression of an antisense Ms-rac1 cDNA construct in transgenic tobacco plants was associated with poor growth and retarded flowering. Following infiltration with yeast elicitor, transgenic tobacco plants transformed with either the empty vector or Ms-rac1 in sense orientation developed brown necrotic lesions and subsequently cell death was observed within the infiltrated tissues. In contrast, the majority of the antisense transformants neither formed necrotic lesions nor showed any other visible defence reactions, demonstrating that Rac-related GTPases play an important role in the establishment of plant defence reactions

Characterization of a small GTP-binding protein of the rab 5 family in Mesembryanthemum crystallinum with increased level of expression during early salt stress

Bolte S, Schiene K, Dietz K-J. Characterization of a small GTP-binding protein of the rab 5 family in Mesembryanthemum crystallinum with increased level of expression during early salt stress. PLANT MOLECULAR BIOLOGY. 2000;42(6):923-935.A cDNA encoding a member of the Ypt/Rab family of small GTP-binding proteins was cloned from the facultative CAM plant Mesembryanthemum crystallinum. Mcrab5b includes an open reading frame of 201 amino acids. The deduced amino acid sequence shows 91% similarity to LjRAB5b isolated from Lotus japonicus. The amino acid sequence of McRAB5b provides interesting features suggesting that McRAB5b and its homologue from Lotus japonicus represent a new subclass of Ypt/Rab proteins. The fact that proteins like McRAB5b and LjRAB5b were only found in plants and not in yeast or vertebrates suggests that they have plant-specific functions. The expression of Mcrab5b as investigated by northern blot hybridization and RT-PCR was stimulated under salt stress. After heterologous expression in Escherichia coli an antibody was raised against recombinant McRAB5b protein. Western blot analysis revealed that McRAB5b was bound to membranes. It is present in a monomeric and a dimeric form in vitro and in vivo. In vitro only the monomeric protein exhibits a binding capacity for radiolabelled GTP, while the dimer is unable to do so, indicating that the activity may be regulated by monomer/dimer transition