Cell division, cell motility and the formation and maintenance of specialized structures in differentiated cells depend directly on the regulated dynamics of the actin cytoskeleton. To understand the mechanisms of these basic cellular processes, the signalling pathways that link external signals to the regulation of the actin cytoskeleton need to be characterized. Here we identify a pathway for the regulation of cofilin, a ubiquitous actin-binding protein that is essential for effective depolymerization of actin filaments. LIM-kinase 1, also known as KIZ, is a protein kinase with two amino-terminal LIM motifs that induces stabilization of F-actin structures in transfected cells. Dominant-negative LIM-kinasel inhibits the accumulation of the F-actin. Phosphorylation experiments in vivo and in vitro provide evidence that cofilin is a physiological substrate of LIM-kinase 1. Phosphorylation by LIM-kinase 1 inactivates cofilin, leading to accumulation of actin filaments. Constitutively active Rac augmented cofilin phosphorylation and LIM-kinase 1 autophosphorylation whereas phorbol ester inhibited these processes. Our results define a mechanism for the regulation of cofilin and hence of actin dynamics in vivo. By modulating the stability of actin cytoskeletal structures, this pathway should play a central role in regulating cell motility and morphogenesis.

The addition or loss of synapses in response to changes in activity, disease, or aging is a major aspect of nervous system plasticity in the adult. The mechanisms that affect the turnover and maintenance of synapses in the adult are poorly understood and are difficult to investigate in the brain. Here, we exploited a unique anatomical arrangement in the neuromuscular system to determine whether subtypes of synapses can differ in anatomical plasticity and vulnerability. In three genetic mouse models of motoneuron disease of diverse origin and severity, we observed a gradual and selective loss of synaptic connections that begun long before the onset of clinical deficits and correlated with the timing of disease progression. A subgroup of fast-type (fast-fatiguable) neuromuscular synapses was highly vulnerable and was lost very early on. In contrast, slow-type synapses resisted up to the terminal phase of the disease. Muscle-specific differences were also evident. Similar selective losses were detected in aged mice. These selective vulnerability properties of synapses coincided with hitherto unrecognized major differences in stimulus-induced anatomical plasticity that could also be revealed in healthy mice. Using paralysis and/or growth-associated protein 43 overexpression to induce synaptic sprouting, we found that slow-type, disease-resistant synapses were particularly plastic. In contrast, fast-type synapses with the highest vulnerability failed to exhibit any stimulus-induced change. The results reveal pronounced subtype specificity in the anatomical plasticity and susceptibility to loss of neuromuscular synapses and suggest that degenerative motoneuron diseases involve a common early pathway of selective and progressive synaptic weakening also associated with aging.

Regulation of neurite outgrowth and structural plasticity may involve the expression of intrinsic determinants controlling growth competence. We have tested this concept by targeting constitutive expression of the growth-associated protein GAP-43 to the neurons of adult transgenic mice. Such mice showed striking spontaneous nerve sprouting at the neuromuscular junction and in the terminal field of hippocampal mossy fibers. In control mice, these nerve fibers did not express GAP-43, and did not sprout spontaneously. Lesion-induced nerve sprouting and terminal arborization during reinnervation were greatly potentiated in GAP-43-overexpressing mice. A mutant GAP-43 that cannot be phosphorylated by PKC had reduced sprout-promoting activity. The results establish GAP-43 as an intrinsic presynaptic determinant for neurite outgrowth and plasticity.

Transgenic mice expressing high levels of familial amyotrophic lateral sclerosis (FALS)-associated mutant superoxide dismutase 1 (SOD1) under the control of a human SOD1 minigene (hMg) accumulate mutant protein ubiquitously and develop motoneuron disease. However, restricted expression of SOD1 mutants in neurons apparently does not cause motor impairments in mice. Here, we investigated the possible pathogenic roles of mutant SOD1 accumulation in motoneurons. First, we used a Thy1 expression cassette to drive high constitutive expression of transgene in postnatal mouse neurons, including upper and lower motoneurons. Second, we expressed human (h) SOD1(G93A) and hSOD1(G85R) as transgenes (i.e., two SOD1 mutants with aggressive pathogenic properties in inducing FALS). Third, in addition to clinical signs of disease, we monitored early signs of disease onset and pathogenesis, including muscle innervation, astrogliosis in the spinal cord, and accumulation of ubiquitinated deposits in motoneurons and astrocytes. We report that high-level expression and accumulation of the mutant proteins in neurons failed to produce any detectable sign of pathology or disease in these transgenic mice. Crossing hMg-SOD1(G93A) mice (Gurney et al., 1994) with Thy1-SOD1(G93A) mice produced double-transgenic mice with spinal cord SOD1(G93A) levels that were approximately twofold higher than in the hMg-SOD1(G93A) single transgenics but did not affect the onset or progression of pathology or motoneuron disease. The accumulation of mutant SOD1 in postnatal motoneurons is thus not sufficient and probably also not critical to induce or accelerate motoneuron disease in FALS mice. The pathogenic process in FALS may involve non-neuronal cells, and selective vulnerability of motoneurons to this process may lead to motoneuron pathology and disease.