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It has been reported that expression of familial amyotrophic lateral sclerosis (FALS)-associated mutant Cu/Zn superoxide dismutase-1 (SOD) induces apoptosis of neuronal cells in culture associated with an increase in reactive oxygen species. SOD recently has been shown to prevent calcineurin inactivation, initiating the present investigations examining the role of calcineurin in mutant SOD-induced cell death. Wild-type or mutant SOD was expressed in neuronal cells by infection with replication-deficient adenoviruses. PC12 cells overexpressing human wild-type SOD exhibited higher calcineurin activity than cells expressing FALS-related mutant SOD (SODV148G); however, cells expressing SODV148G had calcineurin activity equal to mock-infected cells, suggesting that cell death induced by mutant SOD was not related to a decrease in calcineurin activity. Calcineurin antagonists such as cyclosporin A and FK506, as well as nonimmunosuppressant analogs of cyclosporin A, significantly enhanced SODV148G- and SODA4V-induced cell death. Because both groups of drugs inhibit the rotamase activity of cyclophilins (CyP), but only the immunosuppressant analogs inhibit calcineurin activity, these data suggest that rotamase inhibition underlies the enhanced cell death after SODV148G expression. The importance of rotamase activity in mutant SOD-mediated apoptosis was supported by experiments showing that overexpressed wild-type cyclophilin A (CyPA), but not CyPA with a rotamase active site point mutation, protected cells from death after SODV148G expression. These data suggest that mutant SOD produces a greater need for rotamase and, also, highlights possible new therapeutic strategies in FALS.

Distinct subpopulations of neurons in the brain contain one or more of the Ca(2+)-binding proteins calbindin D28k, calretinin, and parvalbumin. Although it has been shown that these high-affinity Ca(2+)-binding proteins can increase neuronal Ca2+ buffering capacity, it is not clear which aspects of neuronal physiology they normally regulate. To investigate this problem, we used a recently developed method for expressing calbindin D28k in the somatic and synaptic regions of cultured hippocampal pyramidal neurons. Ninety-six hours after infection with a replication-defective adenovirus containing the calbindin D28k gene, essentially all cultured hippocampal pyramidal neurons robustly expressed calbindin D28k. Our results demonstrate that while calbindin D28k does not alter evoked neurotransmitter release at excitatory pyramidal cell synapses, this protein has a profound effect on synaptic plasticity. In particular, we show that calbindin D28k expression suppresses posttetanic potentiation.

Theiler murine encephalomyelitis virus designates a number of picornavirus strains that are classified into two subgroups on the basis of their different biological activities. DA strain and other members of the TO subgroup produce a chronic demyelinating disease in which the virus persists and manifests a restricted expression. Mutagenesis studies of the DA strain leader (L) coding region, which is located at the 5' end of the polyprotein coding region, demonstrate that L is completely dispensable for infection of some cells; in addition, nucleotides can be inserted into the L coding region with no loss in infectivity, indicating that Theiler murine encephalomyelitis virus may be used as a vector for delivering foreign sequences. In other cells, L is critical for plaque formation and efficient viral multiplication. These findings raise the possibility that L may play a role in the DA-induced demyelinating restricted infection. The functions of L, and even its presence within the genome, vary among picornaviruses, reflecting the various requirements for viral growth among different host cells.

In human cells infected with adenovirus, the virus-associated RNA VAI blocks the activation of the interferon-induced double-stranded-RNA-dependent 68-kDa protein kinase (p68) and maintains normal levels of protein synthesis at late times after infection. VAI antagonizes the kinase activity by binding to p68. The structure of VAI consists of two long, base-paired stems connected by a complex short stem-loop structure. Previous work using a series of adenovirus mutants showed that the structural determinants of the VAI RNA that are essential for function reside in the central complex short stem-loop structure and adjacent base-paired regions (functional domain); the long duplex regions were found to be dispensable for function. To determine whether binding of VAI to p68 correlates with function and whether the structural determinants that are essential for function are also essential for binding, we studied the interaction of wild-type and several mutant VAI RNAs with p68 in whole cells. The p68-VAI complexes from mutant- and wild-type-infected cells were immunoprecipitated by an anti-p68 monoclonal antibody. The mutant RNAs that functioned efficiently in the cells bound to p68 efficiently in the cells, whereas functionally impaired mutants failed to bind to p68, indicating that the binding of the VAI RNA to p68 correlates well with function. In vitro binding assays with immunopurified p68 confirmed these observations. Secondary-structure analysis of several mutant VAI RNAs suggests that the binding does not depend on the long duplex regions but requires all the elements of the functional domain. We propose that the functional domain and the p68-binding domain of the VAI RNA are identical.