Analysis of transgenic mice expressing familial amyotrophic lateral sclerosis (ALS)-linked mutations in the enzyme superoxide dismutase (SOD1) have shown that motor neuron death arises from a mutant-mediated toxic property or properties. In testing the disease mechanism, both elimination and elevation of wild-type SOD1 were found to have no effect on mutant-mediated disease, which demonstrates that the use of SOD mimetics is unlikely to be an effective therapy and raises the question of whether toxicity arises from superoxide-mediated oxidative stress. Aggregates containing SOD1 were common to disease caused by different mutants, implying that coaggregation of an unidentified essential component or components or aberrant catalysis by misfolded mutants underlies a portion of mutant-mediated toxicity.

Familial amyotrophic lateral sclerosis-linked mutations in copper-zinc superoxide dismutase cause motor neuron death through one or more acquired toxic properties. An early event in the mechanism of toxicity from such mutants is now demonstrated to be activation of caspase-1. Neuronal death, however, follows only after months of chronic caspase-1 activation concomitantly with activation of the executioner caspase-3 as the final step in the toxic cascade. Thus, a common toxicity of mutant SOD1 is a sequential activation of at least two caspases, caspase-1 that acts slowly as a chronic initiator and caspase-3 acting as the final effector of cell death.

Neurofilaments are essential for establishment and maintenance of axonal diameter of large myelinated axons, a property that determines the velocity of electrical signal conduction. One prominent model for how neurofilaments specify axonal growth is that the 660–amino acid, heavily phosphorylated tail domain of neurofilament heavy subunit (NF-H) is responsible for neurofilament-dependent structuring of axoplasm through intra-axonal crossbridging between adjacent neurofilaments or to other axonal structures. To test such a role, homologous recombination was used to generate NF-H–null mice. In peripheral motor and sensory axons, absence of NF-H does not significantly affect the number of neurofilaments or axonal elongation or targeting, but it does affect the efficiency of survival of motor and sensory axons. Loss of NF-H caused only a slight reduction in nearest neighbor spacing of neurofilaments and did not affect neurofilament distribution in either large- or small-diameter motor axons. Since postnatal growth of motor axon caliber continues largely unabated in the absence of NF-H, neither interactions mediated by NF-H nor the extensive phosphorylation of it within myelinated axonal segments are essential features of this growth.

Medical Sciences. In the article "Activation of protein kinase C correlates with a cardioprotective effect of regular ethanol consumption" by Masami Miyamae, Manuel M. Rodriguez, S. Albert Camacho, Ivan Diamond, Daira Mochly-Rosen, and Vincent M. Figueredo, which appeared in number 14, July 7, 1998, of Proc. Natl. Acad. Sci. USA (95, 8262-8267), the following correction should be noted. On page 8264, an incorrect Fig. 1 was printed. The correct figure and its accompanying legend are reproduced below.Neurobiology. In the article "Caspase-1 is activated in neural cells and tissue with amyotrophic lateral sclerosis-associated mutations in copper-zinc superoxide dismutase" by Piera Pasinelli, David R. Borchelt, Megan K. Houseweart, Don W. Cleveland, and Robert H. Brown, Jr., which appeared in number 26, December 22, 1998, of Proc. Natl. Acad. Sci. USA (95, 15763-15768), the following corrections should be noted. An erroneous version of Fig. 6 was published. The lane indicated as G41D represents lumbo-sacral spinal cord extract from G85R transgenic mice. In Fig. 7a, cell viability is expressed as % of untreated cells and not as % of viability. Physiology FIG. 1. LVDP prior to 45 min of global ischemia and duringreperfusion in four groups of perfused guinea pig hearts (n ϭ 9 for each group): 1, following 8 wk 15% ethanol-derived calories (E); 2, pair-fed controls (‚); 3, following 8 wk of ethanol, before and after 10 mM chelerythrine (F); and 4, pair-fed controls, before and after chelerythrine (s). LVDP recovery is significantly greater in hearts from ethanol-treated animals (P Ͻ 0.05 at each 6-min interval). Chelerythrine abolished ethanol's protective effect on LVDP recovery. Data are presented as mean Ϯ SEM (SEM not included for group 2 but lie well within SEM of groups 3 and 4). 3330Corrections Proc. Natl. Acad. Sci. USA 96 (1999) Edited by Irwin Fridovich, Duke University Medical Center, Durham, NC, and approved October 26, 1998 (received for review August 31, 1998) ABSTRACT 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 (xanthine͞xanthine oxidase), which triggers cleavage and secretion of the interleukin 1␤ converting enzyme substrate, pro-interleukin 1␤, and induces apoptosis. This N2a culture system should be an instructive in vitro model for further investigation of the proapoptotic properties of mutant SOD1.

The inherited epilepsy Unverricht-Lundborg disease (EPM1) is caused by loss-of-function mutations in the cysteine protease inhibitor, cystatin B. Because cystatin B inhibits a class of lysosomal cysteine proteases called cathepsins, we hypothesized that increased proteolysis by one or more of these cathepsins is likely to be responsible for the seizure, ataxia, and neuronal apoptosis phenotypes characteristic of EPM1. To test this hypothesis and to identify which cysteine cathepsins contribute to EPM1, we have genetically removed three candidate cathepsins from cystatin B-deficient mice and tested for rescue of their EPM1 phenotypes. Whereas removal of cathepsins L or S from cystatin B-deficient mice did not ameliorate any aspect of the EPM1 phenotype, removal of cathepsin B resulted in a 36-89% reduction in the amount of cerebellar granule cell apoptosis depending on mouse age. The incidence of an incompletely penetrant eye phenotype was also reduced upon removal of cathepsin B. Because the apoptosis and eye phenotypes were not abolished completely and the ataxia and seizure phenotypes experienced by cystatin B-deficient animals were not diminished, this suggests that another molecule besides cathepsin B is also responsible for the pathogenesis, or that another molecule can partially compensate for cathepsin B function. These findings establish cathepsin B as a contributor to the apoptotic phenotype of cystatin B-deficient mice and humans with EPM1. They also suggest that the identification of cathepsin B substrates may further reveal the molecular basis for EPM1.

Progressive myoclonus epilepsy of the Unverricht-Lundborg type (EPM1) is a recessively inherited neurodegenerative disease caused by loss-of-function mutations in the gene encoding cystatin B, a cysteine protease inhibitor. Mice with disruptions in this gene display myoclonic seizures, progressive ataxia, and cerebellar pathology closely paralleling EPMI in humans. To provide further insight into our understanding of EPM1, we report pathological findings in brains from cystatin B-deficient mice. In addition to confirming the loss of cerebellar granular cell neurons by apoptosis, we identified additional neuronal apoptosis in young mutant mice (3-4 months old) within the hippocampal formation and entorhinal cortex. In older mutant mice (16-18 months old), there was also gliosis most marked in the presubiculum and parasubiculum of the hippocampal formation, as well as the entorhinal cortex, neocortex, and striatum. Furthermore, widespread white matter gliosis was also noted, which may be a secondary phenomenon. Within the cerebral cortex, cellular atrophy was a prominent finding in the superficial neurons of the prosubiculum. Finally, we show that mutant mice in either a "seizure-prone" or "seizure-resistant" genetic background display similar neuropathological changes. These findings indicate that neuronal atrophy is an important consequence of cystatin-B deficiency independent of seizure events, suggesting a physiological role for this protein in the maintenance of normal neuronal structure.

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A new isoform of the actin-neurofilament linker protein BPAG has been found that binds to and stabilizes axonal microtubules. This and other newly identified microtubule-associated proteins are likely to be just the tip of an iceberg of multifunctional proteins that stabilize and crosslink cytoskeletal filament networks.