A loss of FUS/TLS function leads to impaired cellular proliferation.

Fused in sarcoma/translocated in liposarcoma (FUS/TLS or FUS) is a multifunctional RNA/DNA-binding protein that is pathologically associated with cancer and neurodegeneration. To gain insight into the vital functions of FUS and how a loss of FUS function impacts cellular homeostasis, FUS expression was reduced in different cellular models through RNA interference. Our results show that a loss of FUS expression severely impairs cellular proliferation and leads to an increase in phosphorylated histone H3, a marker of mitotic arrest. A quantitative proteomics analysis performed on cells undergoing various degrees of FUS knockdown revealed protein expression changes for known RNA targets of FUS, consistent with a loss of FUS function with respect to RNA processing. Proteins that changed in expression as a function of FUS knockdown were associated with multiple processes, some of which influence cell proliferation including cell cycle regulation, cytoskeletal organization, oxidative stress and energy homeostasis. FUS knockdown also correlated with increased expression of the closely related protein EWS (Ewing's sarcoma). We demonstrate that the maladaptive phenotype resulting from FUS knockdown is reversible and can be rescued by re-expression of FUS or partially rescued by the small-molecule rolipram. These results provide insight into the pathways and processes that are regulated by FUS, as well as the cellular consequences for a loss of FUS function.

Identification of small molecule inhibitors of beta-amyloid cytotoxicity through a cell-based high-throughput screening platform.

Calpain activation is hypothesized to be an early occurrence in the sequence of events resulting in neurodegeneration, as well as in the signaling pathways linking extracellular accumulation of beta-amyloid (Abeta) peptides and intracellular formation of neurofibrillary tangles. In an effort to identify small molecules that prevent neurodegeneration in Alzheimer's disease by early intervention in the cell death cascade, a cell-based assay in differentiated Sh-SY5Y cells was developed using calpain activity as a read-out for the early stages of death in cells exposed to extracellular Abeta. This assay was optimized for high-throughput screening, and a library of approximately 120,000 compounds was tested. It was expected that the compounds identified as calpain inhibitors would include those that act directly on the enzyme and those that prevented calpain activation by blocking an upstream step in the pathway. In fact, of the compounds that inhibited calpain activation by Abeta with IC(50) values of <10 microM and showed little or no toxicity at concentrations up to 30 microM, none inhibit the calpain enzyme directly.

Correlations and fluctuations in phase coarsening.

Three-dimensional phase coarsening at various volume fractions is simulated by employing multiparticle diffusion methods. The dynamic process of phase coarsening is visualized through a three-dimensional movie. The present study also characterizes interparticle spacings in polydispersed particle systems and clarifies the controversial mathematical expressions for interparticle spacings used in the literature for 30 years. Consequently, this study reveals spatial, temporal, and nearest-neighbor correlations in polydispersed particle systems. A new three-dimensional movie of a Voronoi network demonstrating these correlations is provided. Our simulation and experiments show that growth rates of individual particles deviate from those of the mean-field theory, which is caused by their differing local environments. Multiplicative noise provides a good basis to describe the stochastic nature of fluctuations during phase coarsening.

Modeling and simulation for phase coarsening: a comparison with experiment.

The phase coarsening of precipitates is modeled in the framework of Debye-Hückel theory. The interactions observed among a population of precipitates dispersed throughout a matrix can be described by diffusion screening. The relationship between the maximum particle radius and the volume fraction of the phases is established, and the rate of coarsening is related to the volume fraction and the self-similar particle size distribution. We simulated the dynamics of late-stage phase separation using multiparticle diffusion methods. Experimental measurements on the rates of coarsening of delta(') ( Al3 Li) precipitates in binary Al-Li alloys are compared with our results using modeling and simulation. The theoretically predicted particle size distributions and the maximum radius expected for particles in the microstructure agree well with recent experimental results.

Noise of microstructural environments in late-stage phase coarsening.

Multiparticle diffusion equations were modeled to simulate the dynamics of late-stage phase coarsening in the region of lower volume fractions. Local environmental information and particle interactions within each coarsening "locale" are included in our simulations. These studies reveal that locale fluctuations occur in the growth rates of particles due to their differing environments. Multiplicative noise provides a sound basis to describe locale fluctuation in late-stage coarsening. A Fokker-Planck equation for the particle size distribution and its asymptotic solution are obtained.

Evidence for tip velocity oscillations in dendritic solidification.

Dendritic growth experiments were conducted in the reduced-convection environment aboard the space shuttle Columbia on STS-87. Spectral analysis was performed on 30-frame/s video data during growths of isothermal dendrites. Results indicate that pivalic acid dendrites exhibit a subtle oscillatory behavior of the axial growth velocity near the tip, with a frequency component that is associated with the sidebranch formation process.

HAART improves prognosis in HIV-associated progressive multifocal leukoencephalopathy.

Introduction of highly active antiretroviral therapy (HAART) has been associated with many changes in the complications of human immunodeficiency virus (HIV) infection. A cohort of 25 HIV patients with progressive multifocal leukoencephalopathy (PML) treated with HAART experienced a median survival of >46 weeks. This is an improvement in prognosis compared with recent historic experience and correlated with HIV RNA viral load reductions. We conclude that current HIV therapy is important in improving the outlook of PML in the setting of HIV.

Experimental measurements of sidebranching in thermal dendrites under terrestrial-gravity and microgravity conditions.

We perform sidebranch measurements on pure succinonitrile dendrites grown in both microgravity and terrestrial-gravity conditions for a set of supercoolings within the range 0.1-1.0 K. Two distinct types of sidebranch regions, uniform and coarsening, exist, and are characterized by the distance from the tip at which the region began, D(i), and the average spacing of sidebranches within that region, lambda(i). There does not appear to be any significant dependence on either gravity level or supercooling when D(i) or lambda(i) are normalized with respect to the radius of curvature of the tip, R. The apparently constant normalized proportionality factor between D(i), lambda(i), and R, regardless of the relative importance of diffusion and convective heat transport, demonstrates self-similarity between dendrites of different length scales propagating under various heat transfer conditions. However, when the form of the sidebranch envelope is defined by a power law relating the amplitude and relative positions of the sidebranches normalized to the radius of the tip, the form is seen to have significant variations with supercooling between the terrestrial gravity and microgravity growth dendrites. Furthermore, both the amplitude coefficient and exponent from the power-law regressions of the microgravity data are statistically different (95% confidence level) than their terrestrial counterparts.

CEP-1347/KT7515 prevents motor neuronal programmed cell death and injury-induced dedifferentiation in vivo.

CEP-1347, also known as KT7515, a derivative of a natural product indolocarbazole, inhibited motor neuronal death in vitro, inhibited activation of the stress-activated kinase JNK1 (c-jun NH terminal kinase) in cultured spinal motor neurons, but had no effect on the mitogen-activated protein kinase ERK1 in these cells. Results reported here profile the functional activity of CEP-1347/KT7515 in vivo in models of motor neuronal death or dedifferentiation. Application of CEP-1347/KT7515 to the chorioallantoic membrane of embryonic chicks rescued 40% of the lumbar motor neurons that normally die during the developmental period assessed. Peripheral administration of low doses (0.5 and 1 mg/kg daily) of CEP-1347/KT7515 reduced death of motor neurons of the spinal nucleus of the bulbocavernosus in postnatal female rats, with efficacy comparable to testosterone. Strikingly, daily administration of CEP-1347/KT7515 during the 4-day postnatal window of motor neuronal death resulted in persistent long-term motor neuronal survival in adult animals that received no additional CEP-1347/KT7515. In a model of adult motor neuronal dedifferentiation following axotomy, local application of CEP-1347/KT7515 to the transected hypoglossal nerve substantially reduced the loss of choline acetyl transferase immunoreactivity observed 7 days postaxotomy compared to untreated animals. Results from these experiments demonstrate that a small organic molecule that inhibits a signaling pathway associated with stress and injury also reduces neuronal death and degeneration in vivo.

CEP-1347/KT7515, a JNK pathway inhibitor, supports the in vitro survival of chick embryonic neurons.

Developing neurons depend on target-derived trophic factors for survival in vivo and in vitro, which also decrease the activity of c-Jun N-terminal kinase (JNK). We have recently described a survival-promoting effect of inhibitors of cyclin-dependent kinases and JNK on chick peripheral embryonic neurons. Here, we report that the small trophic molecule CEP-1347/KT7515, which has been shown to inhibit the JNK signalling pathway, can promote long term-survival of cultured chick embryonic dorsal root ganglion, sympathetic, ciliary and motor neurons. Because of their pharmacological properties, small trophic molecules such as CEP-1347/KT7515 might be of interest for the treatment of neurodegenerative disorders.

Motoneuron apoptosis is blocked by CEP-1347 (KT 7515), a novel inhibitor of the JNK signaling pathway.

Neurons undergoing apoptosis can be rescued by trophic factors that simultaneously increase the activity of extracellular signal-regulated kinase (ERK) and decrease c-Jun N-terminal kinase (JNK) and p38. We identified a molecule, CEP-1347 (KT7515), that rescues motoneurons undergoing apoptosis and investigated its effect on ERK1 and JNK1 activity. Cultured rat embryonic motoneurons, in the absence of trophic factor, began to die 24-48 hr after plating. During the first 24 hr ERK1 activity was unchanged, whereas JNK1 activity increased fourfold. CEP-1347 completely rescued motoneurons for at least 72 hr with an EC50 of 20 +/- 2 nM. CEP-1347 did not alter ERK1 activity but rapidly inhibited JNK1 activation. The IC50 of CEP-1347 for JNK1 activation was the same as the EC50 for motoneuron survival. Inhibition of JNK1 activation by CEP-1347 was not selective to motoneurons. CEP-1347 also inhibited JNK1 activity in Cos7 cells under conditions of ultraviolet irradiation, osmotic shock, and inhibition of glycosylation. Inhibition by CEP-1347 of the JNK1 signaling pathway appeared to be selective, because CEP-1347 did not inhibit p38-regulated mitogen-activated protein kinase-activated protein kinase-2 (MAPKAP2) activity in Cos7 cells subjected to osmotic shock. The direct molecular target of CEP-1347 was not JNK1, because CEP-1347 did not inhibit JNK1 activity in Cos7 cells cotransfected with MEKK1 and JNK1 cDNA constructs. This is the first demonstration of a small organic molecule that promotes motoneuron survival and that simultaneously inhibits the JNK1 signaling cascade.

Neurotrophic 3,9-bis[(alkylthio)methyl]-and-bis(alkoxymethyl)-K-252a derivatives.

A series of 3,9 disubstituted [(alkylthio)methyl]- and (alkoxymethyl)-K-252a derivatives was synthesized with the aim of enhancing and separating the neurotrophic properties from the undesirable NGF (trk A kinase) and PKC inhibitory activities of K-252a. Data from this series reveal that substitution in the 3- and 9-positions of K-252a with these groups reduces trk A kinase inhibitory properties approximately 100- to > 500-fold while maintaining or in certain cases enhancing the neurotrophic activity. From this research, 3,9-bis[(ethylthio)methyl]-K-252a (8) was identified as a potent and selective neurotrophic agent in vitro as measured by enhancement of choline acetyltransferase activity in embryonic rat spinal cord and basal forebrain cultures. Compound 8 was found to have weak kinase inhibitory activity for trk A, protein kinase C1 protein kinase A, and myosin light chain kinase. On the basis of the in vitro profile, 8 was evaluated in in vivo models suggestive of neurological diseases. Compound 8 was active in preventing degeneration of cholinergic neurons of the nucleus basalis magnocellularis (NBM) and reduced developmentally programmed cell death (PCD) of female rat spinal nucleus of the bulbocavernosus motoneurons and embryonic chick lumbar motoneurons.

Potential utility of rhIGF-1 in neuromuscular and/or degenerative disease.

Neuromuscular/neurodegenerative disorders, such as the death of spinal cord motor neurons in amyotrophic lateral sclerosis (ALS) or the degeneration of spinal cord motor neuron axons in certain peripheral neuropathies, present a unique opportunity for therapeutic intervention with neurotrophic proteins. We have found that in mixed rat embryonic spinal cord cultures or in purified motor neuron preparations, recombinant human insulin-like growth factor 1 (rhIGF-1) enhances the survival of motor neurons at EC50 concentrations of 2 nM, consistent with an interaction at the tyrosine kinase-coupled rhIGF-1 receptor. In a model of programmed cell death in ovo, administration of rhIGF-1 produces a marked survival of motor neurons. In a variety of models of predominantly motor neuron or nerve injury in rodents, administration of rhIGF-1 prevents the death of motor neurons in neonatal facial nerve lesions, attenuates the loss of cholinergic phenotype in adult hypoglossal nerve axotomy and hastens recovery from sciatic nerve crush in mice. In a genetic model of motor neuron compromise, the wobbler mouse, rhIGF-1 (1 mg/kg s.c. daily) delayed the deterioration of grip strength and provided for a more normal distribution of fibre types. In addition, rhIGF-1 (0.3-1.0 mg/kg s.c. daily) prevents the motor and/or sensory neuropathy in rodents caused by vincristine, cisplatinum or Taxol. These combined data indicate that rhIGF-1 has marked effects on the survival of compromised motor neurons and the maintenance of their axons and functional connections. They also suggest the potential utility of rhIGF-1 for the treatment of diseases such as ALS and certain neuropathies.

K-252a promotes survival and choline acetyltransferase activity in striatal and basal forebrain neuronal cultures.

The organic molecule K-252a promoted cell survival, neurite outgrowth, and increased choline acetyltransferase (ChAT) activity in rat embryonic striatal and basal forebrain cultures in a concentration-dependent manner. A two- to threefold increase in survival was observed at 75 nM K-252a in both systems. A single application of K-252a at culture initiation prevented substantial (> 60%) cell death that otherwise occurred after 4 days in striatal or basal forebrain cultures. A 5-h exposure of striatal or basal forebrain cells to K-252a, followed by its removal, resulted in survival equivalent to that observed in cultures continually maintained in its presence. This is in contrast to results found with a 5-h exposure of basal forebrain cultures to nerve growth factor (NGF). Acute exposure of basal forebrain cultures to K-252a, but not to NGF, increased ChAT activity, indicating that NGF was required the entire culture period for maximum activity. Striatal cholinergic and GABAergic neurons were among the neurons rescued by K-252a. Of the protein growth factors tested in striatal cultures (ciliary neurotrophic factor, neurotrophin-3, NGF, brain-derived neurotrophic factor, interleukin-2, basic fibroblast growth factor), only brain-derived neurotrophic factor promoted survival. The enhancement of survival and ChAT activity of basal forebrain and striatal neurons by K-252a defines additional populations of neurons in which survival and/or differentiation is regulated by a K-252a-responsive mechanism. The above results expand the potential therapeutic targets for these molecules for the treatment of neuro-degenerative diseases.

K-252a induces tyrosine phosphorylation of the focal adhesion kinase and neurite outgrowth in human neuroblastoma SH-SY5Y cells.

The protein kinase inhibitor K-252a has been shown to promote cholinergic activity in cultures of rat spinal cord and neuronal survival in chick dorsal root ganglion cultures. To determine the mechanism by which K-252a acts as a neurotrophic factor, we examined the effects of this molecule on a human neuroblastoma cell line, SH-SY5Y. K-252a induced neurite outgrowth in a dose-dependent manner. Coincident with neurite outgrowth was the early tyrosine phosphorylation of 125- and 140-kDa proteins. The phosphorylation events were independent of protein kinase C inhibition because down-regulation of protein kinase C by long-term treatment with phorbol ester did not prevent K252a-induced tyrosine phosphorylation. Similarly, the protein kinase C inhibitors H7, GF-109203X, and calphostin C did not induce the phosphorylation. We have identified one of the phosphosubstrates as the pp125 focal adhesion protein tyrosine kinase (Fak). Induction of phosphorylation coincided with increased Fak activity and appeared to be independent of ligand/integrin interaction. The induction of Fak phosphorylation by K-252a was also observed in LA-N-5 cells and primary cultures of rat embryonic striatal cells but not in PC12 cells. The protein kinase C-independent induction of tyrosine phosphorylation and the identification of Fak as a substrate of K-252a-induced tyrosine kinase activity suggest that this compound mediates neurotrophic effects through a novel signaling pathway.

Insulin-like growth factors: putative muscle-derived trophic agents that promote motoneuron survival.

Treatment of chick embryos in ovo with IGF-I during the period of normal, developmentally regulated neuronal death (embryonic days 5-10) resulted in a dose-dependent rescue of a significant number of lumbar motoneurons from degeneration and death. IGF-II and two variants of IGF-I with reduced affinity for IGF binding proteins, des(1-3) IGF-I and long R3 IGF-I, also elicited enhanced survival of motoneurons equal to that seen in IGF-I-treated embryos. IGF-I did not enhance mitogenic activity in motoneuronal populations when applied to embryos during the period of normal neuronal proliferation (E2-5). Treatment of embryos with IGF-I also reduced two types of injury-induced neuronal death. Following either deafferentation or axotomy, treatment of embryos with IGF-I rescued approximately 75% and 50%, respectively, of the motoneurons that die in control embryos as a result of these procedures. Consistent with the survival-promoting activity on motoneurons in ovo, IGF-I, -II, and des(1-3) IGF-I elevated choline acetyltransferase activity in embryonic rat spinal cord cultures, with des(1-3) IGF-I demonstrating 2.5 times greater potency than did IGF-I. A single addition of IGF-I at culture initiation resulted in the maintenance of 80% of the initial ChAT activity for up to 5 days, during which time ChAT activity in untreated control cultures fell to 9%. In summary, these results demonstrate clear motoneuronal trophic activity for the IGFs. These findings, together with previous reports that IGFs are synthesized in muscle and may participate in motoneuron axonal regeneration and sprouting, indicate that these growth factors may have an important role in motoneuron development, maintenance, and recovery from injury.

K-252a and staurosporine promote choline acetyltransferase activity in rat spinal cord cultures.

The protein kinase inhibitor K-252a increased choline acetyltransferase (ChAT) activity in rat embryonic spinal cord cultures in a dose-dependent manner (EC50 of approximately 100 nM) with maximal stimulatory activity at 300 nM resulting in as much as a fourfold increase. A single application of K-252a completely prevented the marked decline in ChAT activity occurring over a 5-day period following culture initiation. Of 11 kinase inhibitors, only the structurally related inhibitor staurosporine also increased ChAT activity (EC50 of approximately 0.5 nM). Effective concentrations of K-252a were not cytotoxic or mitogenic and did not alter the total protein content of treated cultures. Insulin-like growth factor I, basic fibroblast growth factor, ciliary neurotrophic factor, and leukemia inhibitory factor yielded dose-dependent increases in ChAT activity in spinal cord cultures. The combination of K-252a with insulin-like growth factor-I or basic fibroblast growth factor increased ChAT activity up to eightfold over that of untreated controls, which was greater than that observed with each compound alone. K-252a combined with ciliary neurotrophic factor or leukemia inhibitory factor demonstrated no additive or synergistic effects on ChAT activity. These results suggest that there are multiple mechanisms for the regulation of ChAT activity in spinal cord cultures. The enhancement of spinal cord ChAT activity by K-252a and staurosporine defines a new neurotrophic activity for these small organic molecules and raises the possibility that they may activate some regulatory elements in common with the ciliary neurotrophic factor and leukemia inhibitory factor family of neurotrophic proteins.