Chaperone-facilitated copper binding is a property common to several classes of familial amyotrophic lateral sclerosis-linked superoxide dismutase mutants

Mutations in Cu, Zn superoxide dismutase (SOD1) cause the neurodegenerative disease familial amyotrophic lateral sclerosis from an as-yet-unidentified toxic property(ies). Analysis in Saccharomyces cerevisiae of a broad range of human familial amyotrophic lateral sclerosis–linked SOD1 mutants (A4V, G37R, G41D, H46R, H48Q, G85R, G93C, and I113T) reveals one property common to these mutants (including two at residues that coordinate the catalytic copper): Each does indeed bind copper and scavenge oxygen-free radicals in vivo. Neither decreased copper binding nor decreased superoxide scavenging activity is a property shared by all mutants. The demonstration that shows that all mutants tested do bind copper under physiologic conditions supports a mechanism of SOD1 mutant-mediated disease arising from aberrant copper-mediated chemistry catalyzed by less tightly folded (and hence less constrained) mutant enzymes. The mutant enzymes also are shown to acquire the catalytic copper in vivo through the action of CCS, a specific copper chaperone for SOD1, which in turn suggests that a search for inhibitors of this SOD1 copper chaperone may represent a therapeutic avenue.

Caspase-1 is activated in neural cells and tissue with amyotrophic lateral sclerosis-associated mutations in copper-zinc superoxide dismutase

The mechanism by which mutations in the superoxide dismutase (SOD1) gene cause motor neuron degeneration in familial amyotrophic lateral sclerosis (ALS) is unknown. Recent reports that neuronal death in SOD1-familial ALS is apoptotic have not documented activation of cell death genes. We present evidence that the enzyme caspase-1 is activated in neurons expressing mutant SOD1 protein. Proteolytic processing characteristic of caspase-1 activation is seen both in spinal cords of transgenic ALS mice and neurally differentiated neuroblastoma (line N2a) cells with SOD1 mutations. This activation of caspase-1 is enhanced by oxidative challenge (xanthine/xanthine oxidase), which triggers cleavage and secretion of the interleukin 1β converting enzyme substrate, pro-interleukin 1β, and induces apoptosis. This N2a culture system should be an instructive in vitro model for further investigation of the proapoptotic properties of mutant SOD1.

Synaptic sprouting increases the uptake capacities of motoneurons in amyotrophic lateral sclerosis mice

Using adenoviruses encoding reporter genes as retrograde tracers, we assessed the capacity of motoneurons to take up and retrogradely transport adenoviral particles injected into the muscles of transgenic mice expressing the G93A human superoxide dismutase mutation, a model of amyotrophic lateral sclerosis. Surprisingly, transgene expression in the motoneurons was significantly higher in symptomatic mice than in control or presymptomatic mice. Using botulinum toxin to induce nerve sprouting at neuromuscular junctions, we showed that the unexpectedly high level of motoneurons retrograde transduction results, at least in part, from newly acquired uptake properties of the sprouts. These findings demonstrate the remarkable uptake properties of amyotrophic lateral sclerosis motoneurons in response to denervation and the rationale of using intramuscular injections of adenoviruses to overexpress therapeutic proteins in motor neuron diseases.

Meristem-Specific Suppression of Mitosis and a Global Switch in Gene Expression in the Root Cap of Pea by Endogenous Signals1

Two functionally distinct sets of meristematic cells exist within root tips of pea (Pisum sativum): the root apical meristem, which gives rise to the body of the root; and the root cap meristem, which gives rise to cells that differentiate progressively through the cap and separate ultimately from its periphery as border cells. When a specific number of border cells has accumulated on the root cap periphery, mitosis within the root cap meristem, but not the apical meristem, is suppressed. When border cells are removed by immersion of the root tip in water, a transient induction of mitosis in the root cap meristem can be detected starting within 5 min. A corresponding switch in gene expression throughout the root cap occurs in parallel with the increase in mitosis, and new border cells begin to separate from the root cap periphery within 1 h. The induction of renewed border cell production is inhibited by incubating root tips in extracellular material released from border cells. The results are consistent with the hypothesis that operation of the root cap meristem and consequent turnover of the root cap is self-regulated by a signal from border cells.

Inhibition of Auxin Movement from the Shoot into the Root Inhibits Lateral Root Development in Arabidopsis1

In roots two distinct polar movements of auxin have been reported that may control different developmental and growth events. To test the hypothesis that auxin derived from the shoot and transported toward the root controls lateral root development, the two polarities of auxin transport were uncoupled in Arabidopsis. Local application of the auxin-transport inhibitor naphthylphthalamic acid (NPA) at the root-shoot junction decreased the number and density of lateral roots and reduced the free indoleacetic acid (IAA) levels in the root and [3H]IAA transport into the root. Application of NPA to the basal half of or at several positions along the root only reduced lateral root density in regions that were in contact with NPA or in regions apical to the site of application. Lateral root development was restored by application of IAA apical to NPA application. Lateral root development in Arabidopsis roots was also inhibited by excision of the shoot or dark growth and this inhibition was reversible by IAA. Together, these results are consistent with auxin transport from the shoot into the root controlling lateral root development.

Control of Meristem Development by CLAVATA1 Receptor Kinase and Kinase-Associated Protein Phosphatase Interactions1

The CLAVATA1 (CLV1) gene encodes a putative receptor kinase required for the proper balance between cell proliferation and differentiation in Arabidopsis shoot and flower meristems. Impaired CLV1 signaling results in masses of undifferentiated cells at the shoot and floral meristems. Although many putative receptor kinases have been identified in plants, the mechanism of signal transduction mediated by plant receptor-like kinases is largely unknown. One potential effector of receptor kinase signaling is kinase-associated protein phosphatase (KAPP), a protein that binds to multiple plant receptor-like kinases in a phosphorylation-dependent manner. To examine a possible role for KAPP in CLV1-dependent plant development, the interaction of CLV1 and KAPP was investigated in vitro and in vivo. KAPP binds directly to autophosphorylated CLV1 in vitro and co-immunoprecipitates with CLV1 in plant extracts derived from meristematic tissue. Reduction of KAPP transcript accumulation in an intermediate clv1 mutant suppresses the mutant phenotype, and the degree of suppression is inversely correlated with KAPP mRNA levels. These data suggest that KAPP functions as a negative regulator of CLV1 signaling in plant development. This may represent a general model for the interaction of KAPP with receptor kinases.

Mutations in copper-zinc superoxide dismutase that cause amyotrophic lateral sclerosis alter the zinc binding site and the redox behavior of the protein.

A series of mutant human and yeast copper-zinc superoxide dismutases has been prepared, with mutations corresponding to those found in familial amyotrophic lateral sclerosis (ALS; also known as Lou Gehrig's disease). These proteins have been characterized with respect to their metal-binding characteristics and their redox reactivities. Replacement of Zn2+ ion in the zinc sites of several of these proteins with either Cu2+ or Co2+ gave metal-substituted derivatives with spectroscopic properties different from those of the analogous derivative of the wild-type proteins, indicating that the geometries of binding of these metal ions to the zinc site were affected by the mutations. Several of the ALS-associated mutant copper-zinc superoxide dismutases were also found to be reduced by ascorbate at significantly greater rate than the wild-type proteins. We conclude that similar alterations in the properties of the zinc binding site can be caused by mutations scattered throughout the protein structure. This finding may help to explain what is perhaps the most perplexing question in copper-zinc superoxide dismutase-associated familial ALS-i.e., how such a diverse set of mutations can result in the same gain of function that causes the disease.

Expression of calbindin-D28K in motoneuron hybrid cells after retroviral infection with calbindin-D28K cDNA prevents amyotrophic lateral sclerosis IgG-mediated cytotoxicity.

Calbindin-D28K and/or parvalbumin appear to influence the selective vulnerability of motoneurons in amyotrophic lateral sclerosis (ALS). Their immunoreactivity is undetectable in motoneurons readily damaged in human ALS, and in differentiated motoneuron hybrid cells [ventral spinal cord (VSC 4.1 cells)] that undergo calcium-dependent apoptotic cell death in the presence of ALS immunoglobulins. To provide additional evidence for the role of calcium-binding proteins in motoneuron vulnerability, VSC 4.1 cells were infected with a retrovirus carrying calbindin-D28K cDNA under the control of the promoter of the phosphoglycerate kinase gene. Differentiated calbindin-D28K cDNA-infected cells expressed high calbindin-D28K and demonstrated increased resistance to ALS IgG-mediated toxicity. Treatment with calbindin-D28K antisense oligodeoxynucleotides, which significantly decreased calbindin-D28K expression, rendered these cells vulnerable again to ALS IgG toxicity.

A gain-of-function of an amyotrophic lateral sclerosis-associated Cu,Zn-superoxide dismutase mutant: An enhancement of free radical formation due to a decrease in Km for hydrogen peroxide.

Cu,Zn-superoxide dismutase (SOD) is known to be a locus of mutation in familial amyotrophic lateral sclerosis (FALS). Transgenic mice that express a mutant Cu,Zn-SOD, Gly-93--> Ala (G93A), have been shown to develop amyotrophic lateral sclerosis (ALS) symptoms. We cloned the FALS mutant, G93A, and wild-type cDNA of human Cu,Zn-SOD, overexpressed them in Sf9 insect cells, purified the proteins, and studied their enzymic activities for catalyzing the dismutation of superoxide anions and the generation of free radicals with H2O2 as substrate. Our results showed that both enzymes contain one copper ion per subunit and have identical dismutation activity. However, the free radical-generating function of the G93A mutant, as measured by the spin trapping method, is enhanced relative to that of the wild-type enzyme, particularly at lower H2O2 concentrations. This is due to a small, but reproducible, decrease in the value of Km for H2O2 for the G93A mutant, while the kcat is identical for both enzymes. Thus, the ALS symptoms observed in G93A transgenic mice are not caused by the reduction of Cu,Zn-SOD activity with the mutant enzyme; rather, it is induced by a gain-of-function, an enhancement of the free radical-generating function. This is consistent with the x-ray crystallographic studies showing the active channel of the FALS mutant is slightly larger than that of the wild-type enzyme; thus, it is more accessible to H2O2. This gain-of-function, in part, may provide an explanation for the association between ALS and Cu,Zn-SOD mutants.

Transgenic mice carrying a human mutant superoxide dismutase transgene develop neuronal cytoskeletal pathology resembling human amyotrophic lateral sclerosis lesions.

Mutations in the human Cu,Zn superoxide dismutase gene (SOD1) are found in 20% of kindreds with familial amyotrophic lateral sclerosis. Transgenic mice (line G1H) expressing a human SOD1 containing a mutation of Gly-93 --> Ala (G93A) develop a motor neuron disease similar to familial amyotrophic lateral sclerosis, but transgenic mice (line N1029) expressing a wild-type human SOD1 transgene do not. Because neurofilament (NF)-rich inclusions in spinal motor neurons are characteristic of amyotrophic lateral sclerosis, we asked whether mutant G1H and/or N1029 mice develop similar NF lesions. NF inclusions (i.e., spheroids, Lewy body-like inclusions) were first detected in spinal cord motor neurons of the G1H mice at 82 days of age about the time these mice first showed clinical evidence of disease. Other neuronal intermediate filament proteins (alpha-internexin, peripherin) also accumulated in these spheroids. The onset of accumulations of ubiquitin immunoreactivity in the G1H mice paralleled the emergence of vacuoles and NF-rich spheroids in neurons, but they did not colocalize exclusively with spheroids. In contrast, NF inclusions were not seen in the N1029 mice until they were 132 days old, and ubiquitin immunoreactivity was not increased in the N1029 mice even at 199 days of age. Astrocytosis in spinal cord was associated with a marked increase in glial fibrillary acidic protein immunoreactivity in the G1H mice, but not in the N1029 mice. Finally, comparative studies revealed a striking similarity between the cytoskeletal pathology in the G1H transgenic mice and in patients with amyotrophic lateral sclerosis. These findings link a specific SOD1 mutation with alterations in the neuronal cytoskeleton of patients with amyotrophic lateral sclerosis. Thus, neuronal cytoskeletal abnormalities may be implicated in the pathogenesis of human familial amyotrophic lateral sclerosis.

Control of somite patterning by signals from the lateral plate.

The body musculature of higher vertebrates is composed of the epaxial muscles, associated with the vertebral column, and of the hypaxial muscles of the limbs and ventro-lateral body wall. Both sets of muscles arise from different cell populations within the dermomyotomal component of the somite. Myogenesis first occurs in the medial somitic cells that will form the epaxial muscles and starts with a significant delay in cells derived from the lateral somitic moiety that migrate to yield the hypaxial muscles. The newly formed somite is mostly composed of unspecified cells, and the determination of somitic compartments toward specific lineages is controlled by environmental cues. In this report, we show that determinant signals for lateral somite specification are provided by the lateral plate. They result in a blockade of the myogenic program, which maintains the lateral somitic cells as undifferentiated muscle progenitors expressing the Pax-3 gene, and represses the activation of the MyoD family genes. In vivo, this mechanism could account for the delay observed in the onset of myogenesis between muscles of the epaxial and hypaxial domains.

The Lateral Trigger Probability function for the Ultra-High Energy Cosmic Ray showers detected by the Pierre Auger Observatory

In this paper we introduce the concept of Lateral Trigger Probability (LTP) function, i.e., the probability for an Extensive Air Shower (EAS) to trigger an individual detector of a ground based array as a function of distance to the shower axis, taking into account energy, mass and direction of the primary cosmic ray. We apply this concept to the surface array of the Pierre Auger Observatory consisting of a 1.5 km spaced grid of about 1600 water Cherenkov stations. Using Monte Carlo simulations of ultra-high energy showers the LTP functions are derived for energies in the range between 10(17) and 10(19) eV and zenith angles up to 65 degrees. A parametrization combining a step function with an exponential is found to reproduce them very well in the considered range of energies and zenith angles. The LTP functions can also be obtained from data using events simultaneously observed by the fluorescence and the surface detector of the Pierre Auger Observatory (hybrid events). We validate the Monte Carlo results showing how LTP functions from data are in good agreement with simulations.