The L-coding region of the DA strain of Theiler's murine encephalomyelitis virus causes dysfunction and death of myelin-synthesizing cells.

The DA strain and other members of the TO subgroup of Theiler's murine encephalomyelitis virus (TMEV) induce an early transient subclinical neuronal disease followed by a chronic progressive inflammatory demyelination, with persistence of the virus in the central nervous system (CNS) for the life of the mouse. Although TMEV-induced demyelinating disease (TMEV-IDD) is thought to be immune mediated, there is also evidence that supports a role for the virus in directly inducing demyelination. In order to clarify the function of DA virus genes, we generated a transgenic mouse that had tamoxifen-inducible expression of the DA L-coding region in oligodendrocytes (and Schwann cells), a cell type in which the virus is known to persist. Tamoxifen-treated young transgenic mice usually developed an acute progressive fatal paralysis, with abnormalities of the oligodendrocytes and Schwann cells and demyelination, but without significant lymphocytic infiltration; later treatment led to transient weakness with demyelination and persistent expression of the recombined transgene. These findings demonstrate that a high level of expression of DA L can cause the death of myelin-synthesizing cells and death of the mouse, while a lower level of L expression (which can persist) can lead to cellular dysfunction with survival. The results suggest that expression of DA L plays an important role in the pathogenesis of TMEV-IDD. Virus-induced infection and death of oligodendrocytes may play a part in the demyelination of other diseases in which an immune-mediated mechanism has been stressed, including multiple sclerosis.

Processing of wild-type and mutant familial Alzheimer's disease-associated presenilin-1 in cultured neurons.

Mutations of presenilin (PS)-1, an endoplasmic reticulum/Golgi transmembrane protein, have been associated with early-onset familial Alzheimer's disease (FAD). In mammalian brain, PS1 exists primarily as its processed fragments; however, the role of this cleavage event in PS1 function remains unclear. Although some investigators have shown that mutant PS1 processing is unaltered (with the exception of PS1-deltaE9, which lacks the cleavage site) in stably transfected cells and PS1-FAD transgenic mice, other investigators have reported altered FAD mutant PS1 and PS2 protein processing in transiently transfected cells and human FAD patients. The present study uses recombinant replication-defective adenoviral vectors to transiently express wild-type (WT) or mutant PS1 in various cells, including primary cultured hippocampal neurons. We show that in contrast to PS1-WT, overexpression of mutant PS1 results in an increased ratio of mutant holoprotein to endoproteolytic products that is dependent on cell type and differentiation state. In addition, mutant PS1 overexpression leads to an increase in caspase-type protease derived fragments above that seen with PS1-WT overexpression. Furthermore, overexpression of at least one mutant significantly alters the processing of coexpressed PS1-WT, suggesting that mutant PS1 may affect PS1-WT function. These findings suggest that a defect in PS1 holoprotein stability may be a general defect seen in cells expressing mutant PS1, especially neuronal cells, and may play a critical role in the pathogenesis of FAD.

Mutant presenilin-1 induces apoptosis and downregulates Akt/PKB.

Most early onset cases of familial Alzheimer's disease (AD) are caused by mutations in presenilin-1 (PS1) and presenilin-2 (PS2). These mutations lead to increased beta-amyloid formation and may induce apoptosis in some model systems. Using primary cultured hippocampal neurons (HNs) and rat pheochromocytoma (PC12) cells transiently transfected with replication-defective recombinant adenoviral vectors expressing wild-type or mutant PS1, we demonstrate that mutant PS1s induce apoptosis, downregulate the survival factor Akt/PKB, and affect several Akt/PKB downstream targets, including glycogen synthase kinase-3beta and beta-catenin. Expression of a constitutively active Akt/PKB rescues HNs from mutant PS1-induced neuronal cell death, suggesting a potential therapeutic target for AD. Downregulation of Akt/PKB may be a mechanism by which mutant PS1 induces apoptosis and may play a role in the pathogenesis of familial AD.

The role of immunophilins in mutant superoxide dismutase-1linked familial amyotrophic lateral sclerosis.

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.

A protein critical for a Theiler's virus-induced immune system-mediated demyelinating disease has a cell type-specific antiapoptotic effect and a key role in virus persistence.

TO subgroup strains of Theiler's murine encephalomyelitis virus (TMEV) induce a persistent central nervous system infection and demyelinating disease in mice. This disease serves as an experimental model of multiple sclerosis (MS) because the two diseases have similar inflammatory white matter pathologies and because the immune system appears to mediate demyelination in both processes. We previously reported (H. H. Chen, W. P. Wong, L. Zhang, P. L. Ward, and R. P. Roos, Nat. Med. 1:927-931, 1995) that TO subgroup strains use an alternative initiation codon (in addition to the AUG used to synthesize the picornavirus polyprotein from one long open reading frame) to translate L*, a novel protein that is out of frame with the polyprotein and which plays a key role in the demyelinating disease. We now demonstrate that L* has antiapoptotic activity in macrophage cells and is critical for virus persistence. The antiapoptotic action of L* as well as the differential translation of L* and virion capsid proteins may foster virus persistence in macrophages and interfere with virus clearance. The regulation of apoptotic activity in inflammatory cells may be important in the pathogenesis of TMEV-induced demyelinating disease as well as MS.

Mutant superoxide dismutase-1-linked familial amyotrophic lateral sclerosis: molecular mechanisms of neuronal death and protection.

Mutations in human Cu/Zn superoxide dismutase-1 (SOD) cause approximately 20% of cases of familial amyotrophic lateral sclerosis (FALS). We investigated the mechanism of mutant SOD-induced neuronal degeneration by expressing wild-type and mutant SODs in neuronal cells by means of infection with replication-deficient recombinant adenoviruses. Expression of two FALS-related mutant SODs (A4V and V148G) caused death of differentiated PC12 cells, superior cervical ganglion neurons, and hippocampal pyramidal neurons. Cell death included many features typical of apoptosis. Death could be prevented by copper (Cu2+) chelators, Bcl-2, glutathione, vitamin E, and inhibitors of caspases. Mutant SOD-expressing PC12 cells had higher rates of superoxide (O2-) production under a variety of conditions. The results support the hypothesis that mutant SOD induced-neurodegeneration is associated with disturbances of neuronal free radical homeostasis.

Ca2+ and reactive oxygen species in staurosporine-induced neuronal apoptosis.

Staurosporine (0.03-0.5 microM) induced a dose-dependent, apoptotic degeneration in cultured rat hippocampal neurons that was sensitive to 24-h pretreatments with the protein synthesis inhibitor cycloheximide (1 microM) or the cell cycle inhibitor mimosine (100 microM). To investigate the role of Ca2+ and reactive oxygen species in staurosporine-induced neuronal apoptosis, we overexpressed calbindin D28K, a Ca2+ binding protein, and Cu/ Zn superoxide dismutase, an antioxidative enzyme, in the hippocampal neurons using adenovirus-mediated gene transfer. Infection of the cultures with the recombinant adenoviruses (100 multiplicity of infection) resulted in a stable expression of the respective proteins assessed 48 h later. Overexpression of both calbindin D28K and Cu/Zn superoxide dismutase significantly reduced staurosporine neurotoxicity compared with control cultures infected with a beta-galactosidase overexpressing adenovirus. Staurosporine-induced neuronal apoptosis was also significantly reduced when the culture medium was supplemented with 10 or 30 mM K+, suggesting that Ca2+ influx via voltage-sensitive Ca2+ channels reduces this apoptotic cell death. In contrast, neither the glutamate receptor agonist NMDA (1-10 microM) nor the NMDA receptor antagonist dizocilpine (MK-801; 1 microM) was able to reduce staurosporine neurotoxicity. Cultures treated with the antioxidants U-74500A (1-10 microM) and N-acetylcysteine (100 microM) also demonstrated reduced staurosporine neurotoxicity. These results suggest a fundamental role for both Ca2+ and reactive oxygen species in staurosprine-induced neuronal apoptosis.

p53 expression induces apoptosis in hippocampal pyramidal neuron cultures.

The tumor suppressor gene p53 has been implicated in the induction of apoptosis in dividing cells. We now show that overexpression of p53 using an adenoviral vector in cultured rat hippocampal pyramidal neurons causes widespread neuronal death with features typical of apoptosis. p53 overexpression did not induce p21, bax, or mdm2 in neurons. X-irradiation of hippocampal neurons induced p53 immunoreactivity and cell death associated with features typical of apoptosis. Overexpression of a constitutively active nonphosphorylatable form of the retinoblastoma gene product blocked x-irradiation-induced neuronal death. However, overexpression of the cyclin-dependent kinase inhibitor p21 did not. Treatment of neurons with transforming growth factor-beta1 protected them from x-irradiation. These results are consistent with a role for p53 in nerve cell death that is distinct from its actions relating to cell cycle arrest.

Protective effect of transforming growth factor-beta 1 on beta-amyloid neurotoxicity in rat hippocampal neurons.

Neurodegeneration associated with Alzheimer's disease is believed to involve toxicity to beta-amyloid (A beta) and related peptides. Treatment of cultured rat hippocampal neurons with A beta 1-40 (1 microM) or the active fragment A beta 25-35 (1 microM) for 5 days led to a approximately 40-50% decrease in neuronal viability. The hydrophilic antioxidant ascorbic acid (300 microM) and the lipophilic antioxidant 2-mercaptoethanol (10 microM) both protected significantly against A beta neurotoxicity. Despite the protective effects of these antioxidants, both acute and chronic treatments with A beta 25-35 did not increase production of superoxide anions, as monitored with the fluorescent probe hydroethidine. Similarly, overexpression of Cu/Zn-superoxide dismutase using adenovirus-mediated gene transfer did not protect against A beta neurotoxicity. A beta neurotoxicity, however, was prevented in cultures infected with a recombinant, replication-defective adenovirus overexpressing the Ca2+ binding protein calbindin D28k. Transforming growth factor-beta 1 (TGF-beta 1) has been shown to protect neurons against both Ca(2+)- and free radical-mediated neuronal degeneration. We found that A beta neurotoxicity was significantly attenuated by single treatments with TGF-beta 1 (0.1-10 ng/ml) and prevented by repetitive treatments (10 ng/ml/day). The protective effects of TGF-beta 1 were associated with a preservation of mitochondrial potential and function, as determined with rhodamine-123-based microfluorimetry. Because both increased oxidative stress and pathophysiological Ca2+ fluxes can impair mitochondrial function, preservation of mitochondrial potential by TGF-beta 1 could be directly associated with its protection against A beta neurotoxicity. The ability of TGF-beta 1 to increase the expression of the anti-apoptotic proteins Bcl-2 and Bcl-XL is discussed in this context.

Expression of human copper/zinc-superoxide dismutase inhibits the death of rat sympathetic neurons caused by withdrawal of nerve growth factor.

Rat superior cervical ganglion neurons require the presence of nerve growth factor (NGF) to develop and survive in culture. If NGF is removed from the culture medium, then the neurons die of programmed cell death. We investigated the potential role of Ca2+ and reactive oxygen species in this process. We found that overexpression of human wild-type copper/zinc-superoxide dismutase in cultured superior cervical ganglion neurons, using an adenovirus-based vector, substantially protected the cells from the effects of NGF withdrawal, although overexpression of the Ca(2+)-binding protein calbindin D28k or the enzyme beta-galactosidase did not. We also observed that treatment of the cells with the cytokine transforming growth factor-beta 1, which has been shown to protect neurons against oxidative injury, delayed cell death produced by NGF withdrawal. These data suggest a role for reactive oxygen species in triggering programmed cell death of rat sympathetic neurons upon growth factor withdrawal.

Regulation of excitatory transmission at hippocampal synapses by calbindin D28k.

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.

CNS gene delivery by retrograde transport of recombinant replication-defective adenoviruses.

The ability to program recombinant gene expression in specific sets of motor and sensory neurons would facilitate the treatment of a number of acquired and inherited central nervous system (CNS) diseases. In this report, we demonstrate that intramuscular injection of replication-defective recombinant adenovirus results in high-level recombinant gene expression, specifically in the CNS motor and sensory neurons that innervate the inoculated muscles. Neural expression of the recombinant genes results from virus transport into the CNS, presumably by retrograde axonal transport. This novel method of neural gene delivery may be of value in studies designed to improve understanding and treatment of inherited and acquired neurological diseases.

Activation of interferon-inducible 2'-5' oligoadenylate synthetase by adenoviral VAI RNA.

2'-5' oligoadenylate (2-5(A)) synthetase and protein kinase, RNA activated (PKR) are the only two known enzymes that bind double-stranded RNA (dsRNA) and get activated by it. We have previously identified their dsRNA binding domains, which do not have any sequence homology. Here, we report a profound difference between the two enzymes with respect to the structural features of the dsRNA that are required for their activation. The adenoviral virus-associated type I (VAI) RNA cannot activate PKR, although it binds to the protein and thereby prevents its activation by authentic dsRNA. In contrast, we observed that VAI RNA can both bind and activate 2-5(A) synthetase. Mutations in VAI RNA, which removed occasional mismatches present in its double-stranded stems, markedly enhanced its 2-5(A) synthetase-activating capacity. These mutants, however, are incapable of activating PKR. Other mutations, which disrupted the structure of the central stem-loop region of the VAI RNA, reduced its ability to activate 2-5(A) synthetase. These debilitated mutants could bind to the synthetase protein, although they fail to bind to PKR.

In vitro analysis of virus-associated RNA I (VAI RNA): inhibition of the double-stranded RNA-activated protein kinase PKR by VAI RNA mutants correlates with the in vivo phenotype and the structural integrity of the central domain.

Adenoviruses use the virus-encoded virus-associated RNA (VAI RNA) as a defense against cellular antiviral response by blocking the activation of the interferon-induced, double-stranded RNA-activated protein kinase PKR. The structure of VAI RNA consists of two long, imperfectly base-paired duplex regions connected by a complex short stem-loop at the center, referred to as the central domain. By using a series of adenovirus mutants with linker-scan mutations in the VAI RNA gene, we recently showed that the critical elements required for function in the VAI RNA molecule are in the central domain and that these same elements of the central domain are also involved in binding to PKR. In virus-infected cells, VAI RNA interacts with latent kinase, which is bound to ribosomes; this interaction takes place in a complex milieu. To more fully understand the relationship between structure and function and to determine whether the in vivo phenotype of these mutants can be reproduced in vitro, we have now analyzed these mutant VAI alleles for their ability to block the activation of a partially purified PKR from HeLa cells. We have also derived the structure of these mutants experimentally and correlated the structure with function. Without exception, when the structure of the short stem-loop of the central domain was perturbed, the mutants failed to inhibit PKR. Structural disruptions elsewhere in the central domain or in the long duplex regions of the molecule were not deleterious for in vitro function. Thus, these results support our previous findings and underscore the importance of the elements present in the central domain of the VAI RNA for its function. Our results also suggest that the interaction between PKR and VAI RNA involves a precise secondary (and tertiary) structure in the central domain. It has been suggested that VAI RNA does not activate PKR in virus-infected cells because of mismatches in the imperfectly base-paired long duplex regions. We constructed mutant VAI genes in which the imperfectly base-paired duplex regions were converted to perfectly base-paired regions and assayed in vitro for the activation of PKR. As with the wild-type VAI RNA, these mutants failed to activate PKR in vitro, while they were able to block the activation of PKR better than did the wild type. These results suggest that the failure of VAI RNA to activate PKR is not the result of mismatches in the long duplex regions.(ABSTRACT TRUNCATED AT 400 WORDS)

Involvement of cardiovirus leader in host cell-restricted virus expression.

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.

Characterization of the mouse monoclonal antibody TJ4C4 directed at human p68 kinase.

This report describes the production and characterization of a mouse monoclonal antibody (mAb) TJ4C4, which is directed against the interferon-induced double-stranded RNA-activated protein kinase p68 (PKR). The mAb TJ4C4 was produced against p68 which was isolated using a partially purified p68 preparation from human cells. The specificity of this mAb is demonstrated in competitive inhibition assays using recombinantly produced p68 protein and by immunoprecipitation. This mAb is of particular value in that it detects an epitope on p68 which is not destroyed in routinely prepared, formalin-fixed paraffin-embedded tissues, or in a variety of other commonly used clinical fixatives. The inhibition of mAb TJ4C4 by recombinantly produced p68, on human tissues sections, validates the specificity of this mAb in histological studies. Therefore, mAb TJ4C4 serves as a specific probe to study p68 expression in clinically derived tissue specimens.

Bacterial allergy in nasal polyposis. A new method for quantifying specific IgE.

OBJECTIVES: To determine (1) if bacteria-specific serum IgE levels can be more effectively measured by first absorbing competing IgG antibodies from serum and (2) if patients with chronic paranasal sinus disease exhibit a high positive prevalence of bacteria-specific serum IgE. DESIGN: A modified radioallergosorbent test method was employed wherein each serum sample was absorbed with recProtein A to remove competing non-IgE antibodies, and purified proteins extracted from 16 individual bacteria were used as potential allergens. PARTICIPANTS: Twenty-four patients with nasal polyposis and 14 with chronic sinusitis, all refractory to conventional medical therapy and requiring endoscopic sinusotomies, were tested. Tested as controls were 10 subjects with chronic allergic rhinitis, without a history of chronic sinus disease, and possessing total serum IgE and inhalant-specific IgE levels equal to or higher than the patient group. RESULTS: (1) Pretreatment of serum samples with recProtein A resulted in an increase of bacteria-specific radioallergosorbent test sensitivity. (2) Seventeen of 24 patients with polyps, eight of 14 with chronic sinusitis, and one of 10 with chronic allergic rhinitis were determined to be IgE positive when tested with this assay. CONCLUSIONS: (1) Bacteria-specific serum IgE can be quantified; (2) most patients with nasal polyposis and/or chronic sinusitis possess bacteria-specific IgE in their serum, while subjects with only allergic rhinitis do not; and (3) multiple bacterial species isolated from chronically infected sinuses are capable of inducing IgE-mediated sensitization.

Correlation of the expression of double-stranded RNA-dependent protein kinase (p68) with differentiation in head and neck squamous cell carcinoma.

p68 is an inducible protein kinase which is believed to be an important factor in the regulation of both viral and cellular protein synthesis. We have produced a monoclonal antibody (TJ4C4) which specifically detects p68, and which can be used to detect this antigen in formalin-fixed, paraffin-embedded tissues. Because p68 plays an important role in cellular protein synthesis, we hypothesized that it may correlate with normal and neoplastic cellular differentiation. One hundred and seventy-seven head and neck squamous cell carcinoma specimens, representing 82 patients, were studied. The relative amount, frequency, and distribution of p68 expression were determined by microscopic evaluation of ABC immunoperoxidase-stained specimens. A spectrum of immunoreactivity was detected in 156 of 177 tumors, as well as within the normal squamous epithelium. Normal, actively proliferating cells, such as the basal layer of squamous epithelium, expressed comparatively little p68. Increased p68 expression was noted to parallel the morphologic features of cellular differentiation. In neoplastic tissue, p68 expression also increased with the degree of cellular differentiation. These data demonstrate that the expression of p68 parallels the degree of cellular differentiation in squamous cell carcinoma of the head and neck region, as well as within normal squamous mucosa. Therefore, p68 may provide an objective biologic measure of cellular differentiation which does not depend on morphologic features.

Immunohistochemical detection of double-stranded-RNA-dependent protein kinase (p68) with a novel monoclonal antibody TJ4C4. A case report of an AIDS-associated Kaposi's sarcoma treated with alpha-interferon.

Double-stranded RNA (dsRNA)-dependent protein kinase (p68) has been shown to be induced by alpha-interferon (IFN-alpha) in mammalian cells. It binds to dsRNA, and is believed to be a factor in the control of both cellular and viral protein synthesis. This report describes the use of a new monoclonal antibody (MAb) TJ4C4, to monitor levels of p68 in a patient with AIDS-associated Kaposi's sarcoma. Using a novel immunoperoxidase/iron staining method, we examined formalin-fixed, paraffin-embedded biopsies prior to, and 4 months after the initiation of IFN therapy. Immunostaining showed low levels (1+ staining) of p68 in the pretreatment tissue, whereas a marked increase (4+ staining) was noted during interferon treatment. This staining suggests an increased level of intracellular p68 expression. This patient has subsequently remained on IFN-alpha therapy and is alive with no evidence of Kaposi's sarcoma, 6 1/2 years after diagnosis. The use of MAb TJ4C4 will greatly facilitate the study of p68 kinase in clinical tissues, and may provide a way to monitor the effects of IFN therapy.