Characterization of GSK'963: a structurally distinct, potent and selective inhibitor of RIP1 kinase.

Necroptosis and signaling regulated by RIP1 kinase activity is emerging as a key driver of inflammation in a variety of disease settings. A significant amount has been learned about how RIP1 regulates necrotic cell death through the use of the RIP1 kinase inhibitor Necrostatin-1 (Nec-1). Nec-1 has been a transformational tool for exploring the function of RIP1 kinase activity; however, its utility is somewhat limited by moderate potency, off-target activity against indoleamine-2,3-dioxygenase (IDO), and poor pharmacokinetic properties. These limitations of Nec-1 have driven an effort to identify next-generation tools to study RIP1 function, and have led to the identification of 7-Cl-O-Nec-1 (Nec-1s), which has improved pharmacokinetic properties and lacks IDO inhibitory activity. Here we describe the characterization of GSK'963, a chiral small-molecule inhibitor of RIP1 kinase that is chemically distinct from both Nec-1 and Nec-1s. GSK'963 is significantly more potent than Nec-1 in both biochemical and cellular assays, inhibiting RIP1-dependent cell death with an IC50 of between 1 and 4 nM in human and murine cells. GSK'963 is >10 000-fold selective for RIP1 over 339 other kinases, lacks measurable activity against IDO and has an inactive enantiomer, GSK'962, which can be used to confirm on-target effects. The increased in vitro potency of GSK'963 also translates in vivo, where GSK'963 provides much greater protection from hypothermia at matched doses to Nec-1, in a model of TNF-induced sterile shock. Together, we believe GSK'963 represents a next-generation tool for examining the function of RIP1 in vitro and in vivo, and should help to clarify our current understanding of the role of RIP1 in contributing to disease pathogenesis.

Ectopic sympathetic preganglionic neurons maintain proper connectivity in the reeler mutant mouse.

The location of sympathetic preganglionic neurons (SPN) in the spinal cord of the reeler mouse mutant is abnormal. Instead of their normal location in the intermediolateral column, the majority of SPN in the reeler cluster around the central canal. To determine whether ectopically located SPN in the reeler form appropriate synaptic connections with their pre- and postsynaptic partners, we examined 1). whether the axons of descending neural pathways that normally terminate on SPN follow them to their ectopic location, and 2). whether the central autonomic neural circuit that controls sympathetic output to the kidney is organized normally in the reeler. Using antibodies against tyrosine hydroxylase, serotonin, neuropeptide Y, substance P and calcitonin gene-related peptide as markers for adrenergic, serotonergic and peptidergic terminals, we found that axons which normally innervate SPN follow these neurons to their ectopic spinal location in the reeler. Injection of pseudorabies virus into the kidney of wild type and reeler mutant mice revealed similar patterns of renal sympathetic and pre-sympathetic control circuits in the spinal cord, brainstem and forebrain. These results indicate that the presynaptic inputs and postsynaptic targets of SPN in the reeler are normal, despite the ectopic spinal location of their cell bodies.

Reelin controls position of autonomic neurons in the spinal cord.

Mutation of the reeler gene (Reln) disrupts neuronal migration in several brain regions and gives rise to functional deficits such as ataxic gait and trembling in the reeler mutant mouse. Thus, the Reln product, reelin, is thought to control cell-cell interactions critical for cell positioning in the brain. Although an abundance of reelin transcript is found in the embryonic spinal cord [Ikeda, Y. & Terashima, T. (1997) Dev. Dyn. 210, 157-172; Schiffmann, S. N., Bernier, B. & Goffinet, A. M. (1997) Eur. J. Neurosci. 9, 1055-1071], it is generally thought that neuronal migration in the spinal cord is not affected by reelin. Here, however, we show that migration of sympathetic preganglionic neurons in the spinal cord is affected by reelin. This study thus indicates that reelin affects neuronal migration outside of the brain. Moreover, the relationship between reelin and migrating preganglionic neurons suggests that reelin acts as a barrier to neuronal migration.

Segmental specificity of chick sympathetic preganglionic projections is influenced by preganglionic neurons from neighboring spinal cord segments.

Sympathetic preganglionic neurons of the chick are located between the brachial and lumbosacral enlargements of the spinal cord. Their axons exit the spinal cord via their adjacent ventral roots and project rostrally or caudally along the sympathetic trunk to innervate sympathetic ganglia. The projections of sympathetic preganglionic neurons are segmentally specific. Neurons from the 16th cervical (C16) and the first thoracic (T1) spinal cord segments project predominantly in the rostral direction, whereas those from the fifth thoracic (T5) to the first lumbar (L1) spinal segments project predominantly in the caudal direction. Neurons from intervening spinal cord segments (T2-T4) project in rostral and caudal directions. In the present study, neural tube manipulations show that the direction of preganglionic projections is altered by both the elimination and addition of preganglionic neurons projecting into the sympathetic trunk from neighboring segments. The present study also compares the projections of preganglionic neurons from transplants of multiple neural tube segments with those from transplants of single neural tube segments reported in a previous study (Yip, 1987). In the previous study when single thoracic neural tube segments were transplanted to the cervical level, preganglionic neurons did not maintain their original projection patterns. The present study found that, when contiguous neighboring segments were transplanted to the cervical level, preganglionic neurons maintained projection patterns characteristic of their original segmental levels. These results indicate that the direction of preganglionic projections can be influenced by neurons from neighboring segments, suggesting that the formation of segmentally specific preganglionic projections during embryogenesis may involve the interactions of preganglionic neurons with those from neighboring spinal cord segments.

Specific projections of sympathetic preganglionic neurons are not intrinsically determined by segmental origins of their cell bodies.

Sympathetic preganglionic projections of the chick are segmentally specific. Neurons from the 16th cervical (C16) and the first thoracic (T1) spinal cord segments project almost exclusively in the rostral direction, while those from the fifth thoracic (T5) to the first lumbar (L1) spinal segments project almost exclusively in the caudal direction. Neurons from the intervening spinal cord segments (T2-4) project in rostral and caudal directions. There is also a tendency for rostrally located neurons in each segment to project rostrally and caudally located neurons to project caudally. To investigate whether specific projections of preganglionic neurons are intrinsically determined by segmental origins of their cell bodies, neural tube segments were transplanted or rotated in embryos at stages 19-26; these stages include times during and after preganglionic cell birth and just prior to axon outgrowth. When the T1 neural tube segment was replaced with the T5 or T7 neural tube segment the transplanted T5 or T7 preganglionic neurons, now in the T1 position, projected rostrally. Conversely, when the T5 or T7 neural tube segment was replaced with the T1 neural tube, the transplanted T1 preganglionic neurons projected caudally. In addition, when individual T3 spinal cord segments were rotated 180 degrees along the A-P axis, neurons which were originally in the caudal part of the segment projected rostrally, whereas neurons originally from the rostral part of the segment projected caudally. These results show that specific projections of preganglionic neurons are not intrinsically determined by segmental origins of their cell bodies.

Effects of MR exposure on axonal outgrowth in the sympathetic nervous system of the chick.

The effects of MR exposure on the rate and specificity of sympathetic preganglionic axonal outgrowth were examined in the chick embryo. Embryos were exposed to a static magnetic field of 1.5 T for 6 hours, 64 MHz RF field pulses, and a switched magnetic field gradient of amplitude 0.6 G/cm for 4 hours. No significant difference in axonal outgrowth was observed between MR-exposed and control embryos. In addition, the distributions of several major extracellular matrix (ECM) molecules, laminin, fibronectin, and collagen IV, were examined. Immunostaining patterns of these ECM molecules during axonal outgrowth showed no difference between MR-exposed and control embryos. Our results suggest that the MR exposure conditions used in this study do not affect axonal outgrowth in the sympathetic nervous system of the chick.

The expression, origin and function of tenascin during peripheral nerve formation in the chick.

During development of the peripheral nervous system, the extracellular matrix molecule tenascin has been found to be closely associated with growing axons. However, its origin and function in peripheral nerve formation are far from clear. In this study, we examined the expression of tenascin during outgrowth of sensory, motor and sympathetic preganglionic axons, and assessed its origin and function in peripheral nerve formation. During outgrowth of sensory and motor axons, a high concentration of tenascin and its mRNA was found to surround sensory and motor axons in the newly formed spinal nerves. The source of this tenascin was examined through a series of surgical manipulations. Neural crest removals did not alter the distribution of tenascin protein or its mRNA surrounding the spinal nerves. Transplantation of quail somites into chick embryos showed that, similar to the distribution of tenascin, there is a high concentration of somitic cells surrounding the spinal nerves. Moreover, somite removals resulted in a reduction of the tenascin and tenascin mRNA surrounding the spinal nerves. Taken together, these results suggest that the majority of the tenascin surrounding the spinal nerves is of somitic origin. Possible functions of tenascin associated with peripheral nerve formation were examined through injections of tenascin or its antiserum into individual somites prior to or during axon outgrowth. Injections of tenascin or its antiserum did not alter the trajectory of peripheral axons in the anterior half of the somite, nor produce gross abnormalities in the morphology of peripheral nerves, suggesting that tenascin does not play a crucial role in the early formation of peripheral nerves.

Effects of MR exposure on cell proliferation and migration of chick motoneurons.

The effect of magnetic resonance (MR) exposure on the proliferation and migration of motoneurons was examined in chick embryos. Embryos were exposed in ovo to a static magnetic field of 1.5 T for 6 hours and to 64-MHz radio-frequency field pulses and a switched magnetic field gradient with an amplitude of 0.6 G/cm for 4 hours. For cell proliferation studies, embryos were exposed to MR fields during the developmental stage at which motoneuron proliferation is most active. For cell migration studies, embryos were exposed to MR fields at the developmental stage just before lateral motoneuron migration. The results show that the birth dates, migration, and proliferation of lateral motoneurons were unaffected by the MR exposure conditions in this study.

Effects of MR exposure at 1.5 T on early embryonic development of the chick.

The potential teratogenicity of magnetic resonance (MR) field exposure on the early development of the chick embryo was examined. Eggs at four developmental stages within the first 42 hours of incubation were exposed to a static magnetic field of 1.5 T for 6 hours and to 64-MHz radio-frequency field pulses and a switched magnetic field gradient with an amplitude of 0.6 G/cm for 4 h. When the embryos were sacrificed shortly thereafter (at approximately 53 hours of incubation), the abnormality and mortality rates of exposed and unexposed (control) embryos in each of the developmental groups did not show a consistent pattern. When embryos were sacrificed on the 6th day of incubation, exposed embryos from all developmental groups showed a trend toward higher abnormality and mortality rates than their controls. More extensive studies are needed to confirm these findings.

A model system and technique to study the effects of ultrasound irradiation on embryonic development.

To study the effects of ultrasound on development it is important to have a system which provides reliable results. We have designed a system which allows for reproducible irradiations of chick embryos in ovo. The irradiation system includes a heated sonation tank with ultrasound absorbers and a PC/AT computer-based data acquisition system for on-line monitoring of irradiations. The ultrasound detection microprobe and irradiation transducers were calibrated against an NBS traceable balance meter. An acoustic spacer was utilized to provide a more uniform profile of the irradiation beam. At the position of the embryo the ultrasound field geometry was determined. To maintain the chick embryo in its natural physiological state while minimizing ultrasonic reflections and standing-wave generation, two diametrically opposed windows were made in the eggshell along the ultrasound pathway and covered with polyethylene membranes. Using this irradiation system at intensity levels as high as 1.1 W/cm2 (spatial average, temporal average) for 10 min, the temperature rise is minimal.

Effect of ultrasound on axonal outgrowth in the sympathetic nervous system of the chick.

The effects of ultrasound exposure on the rate and specificity of sympathetic preganglionic axonal outgrowth were examined in the chick embryo. Using a technique which allows for exact quantitation of exposure for exact quantiation of exposure conditions, embryos were irradiated in ovo for 5 min daily on 3 consecutive days at an intensity of 1 W/cm2 Spatial Average, Temporal Average (SATA), with a frequency of 1.1 MHz pulsed at 1 kHz and a pulse width of 75 microsecond(s). Our results show no significant difference between irradiated and sham-irradiated embryos. In addition, we have examined the distributions of several major extracellular matrix molecules (fibronectin, laminin and collagen IV) in irradiated and sham-irradiated embryos using immunofluorescent staining. No difference in the staining pattern was found. Finally, we found no increase in the incidence of gross abnormalities and no evidence of lesions and malformations in irradiated embryos.

Ultrasound effects on cell proliferation and migration of chick motoneurons.

The effect of ultrasound exposure on proliferation and migration of motoneurons was examined in chick embryos. Using an egg preparation which minimizes ultrasonic reflections and standing wave generation, chick embryos were exposed to ultrasound in ovo for 10 min at an intensity of 2 W/cm2 Spatial Average, Temporal Average (SATA) with a frequency of 1.1 MHz pulsed at 2 kHz, and a pulse width of 75 microseconds. For cell proliferation studies, embryos were irradiated at the developmental stage of the most active lateral motoneuron proliferation. For cell migration studies, embryos were irradiated at the developmental stage just prior to lateral motoneuron migration. Our results show that the birthdates, migration and proliferation of lateral motoneurons are unaffected by the ultrasound exposure parameters used in this study.