N-Acetylcysteine in Combination with IGF-1 Enhances Neuroprotection against Proteasome Dysfunction-Induced Neurotoxicity in SH-SY5Y Cells.

Ubiquitin proteasome system (UPS) dysfunction has been implicated in the development of many neuronal disorders, including Parkinson's disease (PD). Previous studies focused on individual neuroprotective agents and their respective abilities to prevent neurotoxicity following a variety of toxic insults. However, the effects of the antioxidant N-acetylcysteine (NAC) on proteasome impairment-induced apoptosis have not been well characterized in human neuronal cells. The aim of this study was to determine whether cotreatment of NAC and insulin-like growth factor-1 (IGF-1) efficiently protected against proteasome inhibitor-induced cytotoxicity in SH-SY5Y cells. Our results demonstrate that the proteasome inhibitor, MG132, initiates poly(ADP-ribose) polymerase (PARP) cleavage, caspase 3 activation, and nuclear condensation and fragmentation. In addition, MG132 treatment leads to endoplasmic reticulum (ER) stress and autophagy-mediated cell death. All of these events can be attenuated without obvious reduction of MG132 induced protein ubiquitination by first treating the cells with NAC and IGF-1 separately or simultaneously prior to exposure to MG132. Moreover, our data demonstrated that the combination of the two proved to be significantly more effective for neuronal protection. Therefore, we conclude that the simultaneous use of growth/neurotrophic factors and a free radical scavenger may increase overall protection against UPS dysfunction-mediated cytotoxicity and neurodegeneration.

Furanodiene alters mitochondrial function in doxorubicin-resistant MCF-7 human breast cancer cells in an AMPK-dependent manner.

Furanodiene is a bioactive sesquiterpene isolated from the spice-producing Curcuma wenyujin plant (Y. H. Chen and C. Ling) (C. wenyujin), which is a commonly prescribed herb used in clinical cancer therapy by modern practitioners of traditional Chinese medicine. Previously, we have shown that furanodiene inhibits breast cancer cell growth both in vitro and in vivo, however, the mechanism for this effect is not yet known. In this study, therefore, we asked (1) whether cultured breast cancer cells made resistant to the chemotherapeutic agent doxorubicin (DOX) via serial selection protocols are susceptible to furanodiene's anticancer effect, and (2) whether AMP-activated protein kinase (AMPK), which is a regulator of cellular energy homeostasis in eukaryotic cells, participates in this effect. We show here (1) that doxorubicin-resistant MCF-7 (MCF-7/DOX(R)) cells treated with furanodiene exhibit altered mitochondrial function and reduced levels of ATP, resulting in apoptotic cell death, and (2) that AMPK is central to this effect. In these cells, furanodiene (as opposed to doxorubicin) noticeably affects the phosphorylation of AMPK and AMPK pathway intermediates, ACLY and GSK-3ß, suggesting that furanodiene reduces mitochondrial function and cellular ATP levels by way of AMPK activation. Finally, we find that the cell permeable agent and AMPK inhibitor compound C (CC), abolishes furanodiene-induced anticancer activity in these MCF-7/DOX(R) cells, with regard to cell growth inhibition and AMPK activation; in contrast, AICAR (5-aminoimidazole-4-carboxamide-1-ß-4-ribofuranoside, acadesine), an AMPK activator, augments furanodiene-induced anticancer activity. Furthermore, specific knockdown of AMPK in MCF-7/DOX(R) cells protects these cells from furanodiene-induced cell death. Taken together, these findings suggest that AMPK and its pathway intermediates are promising therapeutic targets for treating chemoresistant breast cancer, and that furanodiene may be an important chemical agent incorporated in next-generation chemotherapy protocols.

Mycoplasma genitalium promotes epithelial crossing and peripheral blood mononuclear cell infection by HIV-1.

BACKGROUND: Mycoplasma genitalium co-infection in HIV-infected individuals has been reported to increase the shedding of HIV in the urogenital region of females. To better understand this relationship, we investigated the influence of M. genitalium on the transmission and replication of HIV using an in vitro model. METHODS: The Transwell co-culture system was employed to assess the crossing of an endocervical cell barrier by HIV-1. Immunocytochemistry and confocal microscopy were used to assess the distribution of the nectin-1 molecule on M. genitalium-infected epithelial cells of the End1/E6E7 endocervical cell line, grown as monolayers in the insert wells. Peripheral blood mononuclear cells (PBMC) were cultured in the bottom wells to assess the effects of M. genitalium, passing through the semipermeable culturing membrane, on subsequent HIV infection of susceptible target cells. RESULTS: Infection of the endocervical cells with the adhesion-positive M. genitalium G37 strain (wild-type) significantly elevated the passage of HIV across the epithelial cell barrier relative to HIV transfer across endocervical cells infected with the adhesion-negative M. genitalium JB1 strain. Immunostaining of the M. genitalium-G37-infected epithelial cells disclosed capping and internalization of the junctional regulatory protein nectin-1, in association with reduced transepithelial resistance (TER) in the cell monolayer. When PBMC were cultured beneath insert wells containing M. genitalium-G37-infected epithelial cell monolayers, we observed significantly enhanced infectivity and replication of HIV added afterward to the cultures. CONCLUSIONS: M. genitalium influences events on both sides of a cultured mucosal epithelial monolayer: (1) by infecting the epithelial cells and reducing the integrity of the barrier itself, and (2) by activating HIV target cells below it, thereby promoting HIV infection and progeny virus production.

Activation of AMP-activated protein kinase in cerebella of Atm-/- mice is attributable to accumulation of reactive oxygen species.

Ataxia telangiectasia (A-T) is an inherited disease, the most prominent feature of which is ataxia caused by degeneration of cerebellar neurons and synapses. The mechanisms underlying A-T neurodegeneration are still unclear, and many factors are likely to be involved. AMP-activated protein kinase (AMPK) is a sensor of energy balance, and research on its function in neural cells has gained momentum in the last decade. The dual roles of AMPK in neuroprotection and neurodegeneration are complex, and they need to be identified and characterized. Using an Atm (ataxia telangiectasia mutated) gene deficient mouse model, we showed here that: (a) upregulation of AMPK phosphorylation and elevation of reactive oxygen species (ROS) coordinately occur in the cerebella of Atm-/- mice; (b) hydrogen peroxide induces AMPK phosphorylation in primary mouse cerebellar astrocytes in an Atm-independent manner; (c) administration of the novel antioxidant monosodium luminol (MSL) to Atm-/- mice attenuates the upregulation of both phosphorylated-AMPK (p-AMPK) and ROS, and corrects the neuromotor deficits in these animals. Together, our results suggest that oxidative activation of AMPK in the cerebellum may contribute to the neurodegeneration in Atm-/- mice, and that ROS and AMPK signaling pathways are promising therapeutic targets for treatment of A-T and other neurodegenerative diseases.

Biochemical and physiological characterization of the GTP-binding protein Obg of Mycobacterium tuberculosis.

BACKGROUND: Obg is a highly conserved GTP-binding protein that has homologues in bacteria, archaea and eukaryotes. In bacteria, Obg proteins are essential for growth, and they participate in spore formation, stress adaptation, ribosome assembly and chromosomal partitioning. This study was undertaken to investigate the biochemical and physiological characteristics of Obg in Mycobacterium tuberculosis, which causes tuberculosis in humans. RESULTS: We overexpressed M. tuberculosis Obg in Escherichia coli and then purified the protein. This protein binds to, hydrolyzes and is phosphorylated with GTP. An anti-Obg antiserum, raised against the purified Obg, detects a 55 kDa protein in immunoblots of M. tuberculosis extracts. Immunoblotting also discloses that cultured M. tuberculosis cells contain increased amounts of Obg in the late log phase and in the stationary phase. Obg is also associated with ribosomes in M. tuberculosis, and it is distributed to all three ribosomal fractions (30 S, 50 S and 70 S). Finally, yeast two-hybrid analysis reveals that Obg interacts with the stress protein UsfX, indicating that M. tuberculosis Obg, like other bacterial Obgs, is a stress related protein. CONCLUSIONS: Although its GTP-hydrolyzing and phosphorylating activities resemble those of other bacterial Obg homologues, M. tuberculosis Obg differs from them in these respects: (a) preferential association with the bacterial membrane; (b) association with all three ribosomal subunits, and (c) binding to the stress protein UsfX, rather than to RelA. Generation of mutant alleles of Obg of M. tuberculosis, and their characterization in vivo, may provide additional insights regarding its role in this important human pathogen.

alpha-Luminol prevents decreases in glutamate, glutathione, and glutamine synthetase in the retinas of glaucomatous DBA/2J mice.

OBJECTIVE: To test the hypothesis that in DBA/2J mice, oxidative stress decreases glutamine synthetase (GS) levels resulting in a loss of neuronal glutamate and that the antioxidant alpha-luminol (GVT) decreases this stress and glutamate loss in some types of glaucoma. ANIMALS: DBA/2J mice were separated into two groups, of which one was not treated, and the other treated with GVT in the drinking water. At 7 months of age, retinas were examined from five untreated DBA/2J mice, seven GVT-treated mice, and five C57BL/6 mice (negative controls). METHODS: Serial 0.5 microm plastic sections were immunogold stained for glutamate, GS, and total glutathione, followed by image analysis for staining patterns and density. RESULTS: Focal decreases in glutamate immunostaining were common in the inner nuclear layer (INL) of DBA/2J retinas, but not in C57BL/6 or GVT-treated DBA/2J retinas. Decreases in glutathione and GS immunostaining were found in DBA/2J retinal regions where neuronal glutamate immunostaining was reduced. Retinas from GVT-treated DBA/2J had no significant decreases in INL levels of glutamate, glutathione, or GS. CONCLUSIONS: Retinas of dogs with primary glaucoma are reported to have focal depletion of neuronal glutamate. In DBA/2J mice, similar changes occur prior to the development of clinical disease. In these focal glutamate-depleted regions, levels of glutathione and GS are also reduced, consistent with the hypothesis that oxidative stress contributes to retinal changes in glaucoma. The ability of GVT, an antioxidant, to inhibit retinal abnormalities in DBA/2J mice provides further support for this hypothesis.

Attenuation of oxidative stress, inflammation and apoptosis by minocycline prevents retrovirus-induced neurodegeneration in mice.

The ts1 mutant of the Moloney murine leukemia virus (MoMuLV) causes neurodegeneration in infected mice that resembles HIV-associated dementia. We have shown previously that ts1 infects glial cells in the brain, but not neurons. The most likely mechanism for ts1-mediated neurodegeneration is loss of glial redox support and glial cell toxicity to neurons. Minocycline has been shown to have neuroprotective effects in various models of neurodegeneration. This study was designed to determine whether and how minocycline prevents paralysis and death in ts1-infected mice. We show here that minocycline delays neurodegeneration in ts1-infected mice, and that it prevents death of cultured astrocytes infected by ts1 through attenuating oxidative stress, inflammation and apoptosis. Although minocycline reduces virus titers in the CNS of infected mice, it does not affect virus titers in infected mice thymi, spleens or infected C1 astrocytes. In addition, minocycline prevents death of primary neurons when they are cocultured with ts1-infected astrocytes, through mechanisms involving both inhibition of oxidative stress and upregulation of the transcription factor NF-E2-related factor 2 (Nrf2), which controls cellular antioxidant defenses. We conclude that minocycline delays retrovirus ts1-induced neurodegeneration involving antioxidant, anti-inflammation and anti-apoptotic mechanisms.

The drug monosodium luminol (GVT) preserves thymic epithelial cell cytoarchitecture and allows thymocyte survival in mice infected with the T cell-tropic, cytopathic retrovirus ts1.

A mutant of MoMuLV, called ts1, causes an AIDS-like syndrome in susceptible strains of mice. In mice infected at birth, thymic atrophy, CD4+ T cell loss, body wasting, and death occur by approximately 30-40 days postinfection (dpi). We have shown previously that the death of ts1-infected cells is not caused by viral replication per se, but by oxidative stress and apoptosis following their accumulation the ts1 viral envelope precursor protein, gPr80(env). In infected mice treated with the antioxidant monosodium alpha-luminol (GVT), T cell loss and thymic atrophy are delayed for many weeks, and body wasting and death do not occur until long after infected, untreated control mice have died. We show here that GVT treatment of ts1-infected mice maintains the thymic epithelial cell (TEC) cytoarchitecture and cytokeratin gradients required for thymocyte differentiation. It also suppresses thymocyte reactive oxygen species (ROS) levels, upregulates and stabilizes levels of the antioxidant-regulating transcription factor Nrf2, and prevents accumulation of gPr80(env) in thymocytes. We conclude that GVT treatment can make ts1 a non-cytopathic virus for thymocytes, although it cannot prevent thymocyte infection. Since oxidative stress also contributes to the loss of T cells in HIV-AIDS, the antioxidant effects of GVT may make it a useful therapeutic adjunct to HAART treatment.

The drug monosodium luminol (GVT) preserves crypt-villus epithelial organization and allows survival of intestinal T cells in mice infected with the ts1 retrovirus.

Of the cytopathic retroviruses that affect mammals, including HIV-1, many selectively infect CD4+ T cells and cause immunosuppressive syndromes. These diseases destroy both the thymus and the small and large intestines, after infecting and killing T-lineage cells in both tissues. A mutant of the murine leukemia retrovirus MoMuLV-TB, called ts1, causes this syndrome in susceptible strains of mice. In FVB/N strain mice that are infected at birth, thymic atrophy, CD4+ T cell loss, intestinal collapse, body wasting, and death occur by approximately 30-40 days postinfection (dpi). Apoptosis of ts1-infected T-lineage cells, in the thymus, peripheral lymphoid system and intestines is caused by accumulation of the ts1 mutant viral envelope preprotein gPr80(env), which is inefficiently cleaved into the mature viral proteins gp70 and PrP15E. We show here that ts1 infection in the small intestine is followed by loss of intestinal epithelial cell (IEC) thyroid-stimulating hormone (TSH) and cell cycling gradients (along the crypt-villus axes), accumulation of gPr80(env) in intestinal cells, apoptosis of developing T cells in the lamina propria (LP), and intestinal collapse by approximately 30 dpi. In infected mice treated with the antioxidant drug monosodium luminol (GVT), however, normal intestinal epithelial cell gradients are still in place at 30 dpi, and IECs covering both the crypts and villi contain large amounts of the antioxidant transcription factor Nrf2. In addition, no apoptotic cells are present, and accumulated gpr80(env) is absent from the tissue at this time. We conclude that GVT treatment can make ts1 a noncytopathic virus for intestinal lymphoid cells, as it does for thymocytes [25]. As in the thymus, GVT may protect the intestine by reducing oxidant stress in infected intestinal T cells, perhaps by prevention of gPr80(env) accumulation via Nrf2 upregulation in the IECs. These results identify GVT as a potential therapy for intestinal diseases or inflammatory conditions, including HIV-AIDS, in which oxidative stress is a triggering or exacerbating factor.

The glucocorticoid receptor is increased in Atm-/- thymocytes and in Atm-/- thymic lymphoma cells, and its nuclear translocation counteracts c-myc expression.

We have previously demonstrated that spontaneous DNA synthesis in immature thymocytes of Atm-/- mice is elevated, and that treatment with the glucocorticoid dexamethasone (Dex) attenuates this increased DNA synthesis and prevents the development of thymic lymphomas. Deregulation of c-myc may drive the uncontrolled proliferation of Atm-/- thymocytes, since upregulation of c-myc parallels the elevated DNA synthesis in the cells. In this study, we show that the glucocorticoid receptor (GR) is expressed at high levels in Atm-/- thymocytes and in Atm-/- thymic lymphoma cells, although serum glucocorticoid (GC) levels in Atm-/- mice are similar to those in Atm+/+ mice. In cultured Atm-/- thymic lymphoma cells treated with Dex, GR nuclear translocation occurs, resulting in suppression of DNA synthesis and c-myc expression at both the mRNA and protein levels. Interestingly, the GR antagonist RU486 also causes GR nuclear translocation, but does not affect DNA synthesis and c-myc expression in Atm-/- thymic lymphoma cells. As expected, RU486 reverses the suppressive effects of Dex on DNA synthesis and c-myc expression. Administration of Dex to Atm-/- mice decreases the elevated c-Myc protein levels in their thymocytes. These findings suggest that GC/GR signaling plays an important role in regulating c-myc expression in Atm-/- thymocytes and thymic lymphoma cells.

ATM controls c-Myc and DNA synthesis during postnatal thymocyte development through regulation of redox state.

The oncoprotein c-Myc is essential for thymocyte development, and its dysregulation causes lymphoid malignancies. We have demonstrated previously that spontaneous DNA synthesis in Atm(-/-) thymocytes is markedly increased over that of Atm(+/+) thymocytes and that glucocorticoid dexamethasone suppresses thymocyte DNA synthesis and prevents the ultimate development of thymic lymphoma in Atm(-/-) mice. Recently, we reported that in Atm(-/-) thymic lymphoma cells c-Myc is overexpressed compared with the levels of c-Myc in primary thymocytes from wild-type or Atm(-/-) mice. In this study, we show that c-Myc expression progressively increases with age in primary thymocytes from Atm(-/-) mice and that the upregulation of c-Myc parallels the elevated DNA synthesis in the cells, suggesting that deregulation of c-Myc may drive the uncontrolled proliferation of thymocytes in Atm(-/-) mice. Here we also demonstrate that Atm(-/-) thymocytes exhibit increased levels of hydrogen peroxide, NF-E2-related factor (Nrf-2), peroxiredoxin-1, and intracellular glutathione relative to thymocytes from Atm(+/+) mice. Importantly, reduction of hydrogen peroxide by administration of the antioxidant N-acetylcysteine to Atm(-/-) mice attenuates the elevation of Nrf-2, c-Myc, and DNA synthesis in their thymocytes, suggesting that ATM may control c-Myc and DNA synthesis during postnatal thymocyte development by preventing accumulation of reactive oxygen species.

Retrovirus-induced oxidative stress with neuroimmunodegeneration is suppressed by antioxidant treatment with a refined monosodium alpha-luminol (Galavit).

Oxidative stress is involved in many human neuroimmunodegenerative diseases, including human immunodeficiency virus disease/AIDS. The retrovirus ts1, a mutant of Moloney murine leukemia virus, causes oxidative stress and progressive neuro- and immunopathology in mice infected soon after birth. These pathological changes include spongiform neurodegeneration, astrogliosis, thymic atrophy, and T-cell depletion. Astrocytes and thymocytes are directly infected and killed by ts1. Neurons are not infected, but they also die, most likely as an indirect result of local glial infection. Cytopathic effects of ts1 infection in cultured astrocytes are associated with accumulation of the viral envelope precursor protein gPr80env in the endoplasmic reticulum (ER), which triggers ER stress and oxidative stress. We have reported (i) that activation of the Nrf2 transcription factor and upregulation of antioxidative defenses occurs in astrocytes infected with ts1 in vitro and (ii) that some ts1-infected astrocytes survive infection by mobilization of these pathways. Here, we show that treatment with a refined monosodium alpha-luminol (Galavit; GVT) suppresses oxidative stress and Nrf2 activation in cultured ts1-infected astrocytes. GVT treatment also inhibits the development of spongiform encephalopathy and gliosis in the central nervous system (CNS) in ts1-infected mice, preserves normal cytoarchitecture in the thymus, and delays paralysis, thymic atrophy, wasting, and death. GVT treatment of infected mice reduces ts1-induced oxidative stress, cell death, and pathogenesis in both the CNS and thymus of treated animals. These studies suggest that oxidative stress mediates ts1-induced neurodegeneration and T-cell loss.

Astrocytes survive chronic infection and cytopathic effects of the ts1 mutant of the retrovirus Moloney murine leukemia virus by upregulation of antioxidant defenses.

The ts1 mutant of Moloney murine leukemia virus (MoMuLV) induces a neurodegenerative disease in mice, in which glial cells are infected by the retrovirus but neurons are not. ts1 infection of primary astrocytes, or of the immortalized astrocytic cell line C1, results in accumulation of the ts1 gPr80(env) envelope protein in the endoplasmic reticulum (ER), with ER and oxidative stress. Notably, only about half of the infected astrocytes die in these cultures, while the other half survive, continue to proliferate, and continue to produce virus. To determine how these astrocytes survive ts1 infection in culture, we established a chronically infected subline of the living cells remaining after the death of all acutely infected cells in an infected C1 cell culture (C1-ts1-S). We report here that C1-ts1-S cells proliferate more slowly, produce less virus, show reduced H2O2 levels, increase their uptake of cystine, and maintain higher levels of intracellular GSH and cysteine compared to acutely infected or uninfected C1 cells. C1-ts1-S cells also upregulate their thiol antioxidant defenses by activation of the transcription factor NF-E2-related factor 2 (Nrf2) and its target genes. Interestingly, despite maintenance of higher levels of intracellular reduced thiols, C1-ts1-S cells are more sensitive to cystine deprivation than uninfected C1 cells. We conclude that some ts1-infected astrocytes survive and adapt to virus-induced oxidative stress by successfully mobilizing their thiol redox defenses.

Interaction between endoplasmic reticulum stress and caspase 8 activation in retrovirus MoMuLV-ts1-infected astrocytes.

The murine retrovirus, MoMuLV-ts1, induces progressive paralysis and immune deficiency in FVB/N mice. We have reported previously that ts1 infection causes apoptosis in astrocytes via endoplasmic reticulum (ER) and mitochondrial stress (Liu, N., Kuang, X., Kim, H.T., Stoica, G., Qiang, W., Scofield, V.L., Wong, P.K.Y. Wong. 2004. Possible involvement of both endoplasmic reticulum- and mitochondria-dependent pathways in MoMuLV-ts1-induced apoptosis in astrocytes. J. NeuroVirol. 10, 189-198). In the present study, we show that caspase 8 activation in these cells is mediated through ER stress-associated elevation of death receptor DR5 and the C/EBP homologous protein (GADD153/CHOP), an ER stress-initiated transcription factor, rather than through TNFalpha and TNF-R1 interactions on the cell surface. Treatment with Z-IETD-FMK, a specific inhibitor of caspase 8 enzymatic activity, reduced ER stress by two mechanisms: by inhibiting caspase 8 activation, and by preventing cleavage of the ER-associated membrane protein BAP31 into BAP20, which exacerbates the ER stress response. These findings suggest that caspase 8- and ER stress-associated apoptotic pathways are linked in ts1-infected astrocytes.

ATM deficiency induces oxidative stress and endoplasmic reticulum stress in astrocytes.

ATM kinase, the product of the ataxia telangiectasia mutated (Atm) gene, is activated by genomic damage. ATM plays a crucial role in cell growth and development. Here we report that primary astrocytes isolated from ATM-deficient mice grow slowly, become senescent, and die in culture. However, before reaching senescence, these primary Atm(-/-) astrocytes, like Atm(-/-) lymphocytes, show increased spontaneous DNA synthesis. These astrocytes also show markers of oxidative stress and endoplasmic reticulum (ER) stress, including increased levels of heat shock proteins (HSP70 and GRP78), malondialdehyde adducts, Cu/Zn superoxide dismutase, procaspase 12 cleavage, and redox-sensitive phosphorylation of extracellular signal-regulated protein kinase 1 and 2 (ERK1/2). In addition, HSP70 and ERK1/2 phosphorylation are upregulated in the cerebella of ATM-deficient mice. This increase in ERK1/2 phosphorylation is seen primarily in cerebellar astrocytes, or Bergmann glia, near degenerating Purkinje cells. ERK1/2 activation and astrogliosis are also found in other parts of the brain, for example, the cortex. We conclude that ATM deficiency induces intrinsic growth defects, oxidative stress, ER stress, and ERKs activation in astrocytes.

Up-regulation of astrocyte cyclooxygenase-2, CCAAT/enhancer-binding protein-homology protein, glucose-related protein 78, eukaryotic initiation factor 2 alpha, and c-Jun N-terminal kinase by a neurovirulent murine retrovirus.

In susceptible strains of mice, infection with the mutant retrovirus MoMuLV-ts1 causes a neurodegeneration and immunodeficiency syndrome that resembles human human immunodeficiency virus-acquired immunodeficiency syndrome (HIV-AIDS). In this study the authors show increased expression of cyclooxygenase-2 (COX-2) in the brainstem tissues of ts1-infected mice. Up-regulated central nervous system (CNS) levels of this enzyme are associated with HIV-associated dementia and other inflammatory and neurodegenerative diseases such as amyotrophic lateral sclerosis, Alzheimer's disease, and Parkinson's disease. In brainstem sections, the authors find that astrocytes surrounding spongiform lesions contain increased amounts of immunoreactive COX-2. COX-2 is also up-regulated in cultured ts1-infected cells from the C1 astrocytic cell line, and activation of c-Jun N-terminal kinase, or JNK, pathway. Markers of endoplasmic reticulum (ER) stress, specifically the CCAAT/enhancer-binding protein (CHOP), the glucose-related protein 78 (GRP78), and phosphorylated eukaryotic initiation factor 2 alpha (eIF2 alpha), were also up-regulated in ts1-infected C1 astrocytes. Up-regulation of COX-2 and the above ER signaling factors was reversed by treatment of the infected cells with curcumin which specifically inhibits the JNK/c-Jun pathway. These findings indicate that the JNK/c-Jun pathway is most likely responsible for COX-2 expression induced by ts1 in astrocytes, and that ts1 infection in astrocytes may lead to up-regulation of both inflammatory and ER stress pathways in the central nervous system. Because COX-2 inhibitors are now widely used to treat inflammatory conditions in animals and humans, this finding suggests that these drugs may be useful for therapeutic intervention in neurodegenerative syndromes as well.

Intestinal TSH production is localized in crypt enterocytes and in villus 'hotblocks' and is coupled to IL-7 production: evidence for involvement of TSH during acute enteric virus infection.

The immune and neuroendocrine systems have been shown to work conjointly in a number of ways. One aspect of this has to do with a potential role for thyroid stimulating hormone (TSH) in the regulation of the mucosal immune system, although the mechanisms by which this occurs remain vague. To more thoroughly understand how TSH participates in intestinal intraepithelial lymphocyte (IEL) development and immunity, experiments have been conducted to define local sites of intestinal TSH production, and to characterize changes that occur in the synthesis of TSH during acute enteric virus infection. Here, we demonstrate that TSH in the small intestine is specifically localized to regions below villus crypts as seen by immunocytochemical staining, which revealed high-level TSH staining in lower crypts in the absence of IL-7 staining, and TSH and IL-7 co-staining in upper crypt regions. Additionally, prominent TSH staining was evident in TSH 'hotblocks' sparsely dispersed throughout the epithelial layer. In rotavirus-infected mice, the TSH staining pattern differed significantly from that of non-infected animals. Notably, at 2 and 3 days post-infection, TSH expression was high in and near apical villi where virus infection was greatest. These findings lend credence to the notion that TSH plays a role both in the development of intestinal T cells, and in the process of local immunity during enteric virus infection.

Control of Atm-/- thymic lymphoma cell proliferation in vitro and in vivo by dexamethasone.

AIM: Ataxia telangiectasia (A-T) is an autosomal recessive disease in humans caused by mutations in the Atm (A-T mutated) gene. The disease involves multiple organ systems, and is associated with a high incidence of leukemias and lymphomas that develop in childhood. We have reported previously that thymic lymphoma development in Atm knockout (Atm-/-) mice is associated with elevated spontaneous DNA synthesis in thymocytes, and that dexamethasone (Dex) attenuates the elevated DNA synthesis and prevents thymic lymphoma development. The primary objectives of the present study were (1) to investigate possible mechanisms underlying the tumor-suppressing effect of Dex on Atm-/- thymic lymphoma cells, and (2) to determine whether Dex is an effective tumor-suppressing treatment in mice bearing transplanted Atm-/- thymic tumors. METHODS: Establishment of a number of Atm-/- thymic lymphoma (ATL) cell lines from Atm-/- mice, cell proliferation assays, cell cycle analyses, Western blotting and Hoechst nuclear staining were used to analyze the effects of Dex on Atm-/- thymic lymphoma cells. Atm-/- tumor cells were transplanted into the right flanks of Atm+/+ mice prior to the initiation of Dex treatment. RESULTS: Atm-/- tumor cells were highly sensitive to Dex, both in culture and in vivo as ectopic tumors in mice. In cultured ATL-1 cells, Dex induced apoptosis, arrested the cell cycle at the G1 phase and downregulated NF-kappaB and multiple cell cycle regulators, while upregulating the NF-kappaB inhibitor IkappaBalpha. In Atm+/+ mice transplanted subcutaneously with ATL-1 cells, tumor growth was either prevented completely or significantly suppressed by Dex treatment. CONCLUSIONS: Our findings identify potential mechanisms by which Dex affects the proliferation and survival of ATL-1 cells in culture, and provide evidence that Dex can suppress the proliferation of Atm-/- thymic lymphoma cells growing in the body. Together these results add to our earlier published data suggesting that the cellular pathways regulated by Dex may be promising therapeutic targets for prevention and treatment of thymic lymphomas in A-T individuals.

Fluorescent proteins expressed in mouse transgenic lines mark subsets of glia, neurons, macrophages, and dendritic cells for vital examination.

To enable vital observation of glia at the neuromuscular junction, transgenic mice were generated that express proteins of the green fluorescent protein family under control of transcriptional regulatory sequences of the human S100B gene. Terminal Schwann cells were imaged repetitively in living animals of one of the transgenic lines to show that, except for extension and retraction of short processes, the glial coverings of the adult neuromuscular synapse are stable. In other lines, subsets of Schwann cells were labeled. The distribution of label suggests that Schwann cells at individual synapses are clonally related, a finding with implications for how these cells might be sorted during postnatal development. Other labeling patterns, some present in unique lines, included astrocytes, microglia, and subsets of cerebellar Bergmann glia, spinal motor neurons, macrophages, and dendritic cells. We show that lines with labeled macrophages can be used to follow the accumulation of these cells at sites of injury.

Activation of transcription factor Nrf-2 and its downstream targets in response to moloney murine leukemia virus ts1-induced thiol depletion and oxidative stress in astrocytes.

The neuroimmunodegenerative syndrome that develops in mice infected with ts1, a mutant of Moloney murine leukemia virus, resembles human AIDS. Both ts1 and human immunodeficiency virus type 1 infect astrocytes, microglia, and oligodendrocytes but do not infect neurons. Oxidative stress has been implicated in the neuropathology of AIDS dementia and other neurodegenerative diseases. We report here that ts1 infection of astrocytes (both transformed C1 cells and primary cultures) also induces thiol (i.e., glutathione and cysteine) depletion and reactive oxygen species (ROS) accumulation, events occurring in parallel with viral envelope precursor gPr80(env) accumulation and upregulated expression of endoplasmic reticulum chaperones GRP78 and GRP94. Furthermore, ts1-infected astrocytes mobilize their thiol redox defenses by upregulating levels of the Nrf-2 transcription factor, as well its targets, the xCT cystine/glutamate antiporter, gamma-glutamylcysteine ligase, and glutathione peroxidase. Depleting intracellular thiols by treating uninfected astrocytes with buthionine sulfoximine (BSO), a glutathione synthesis inhibitor, or by culturing in cystine-deficient medium, also induces ROS accumulation, activates Nrf-2, and upregulates Nrf-2 target gene expression in these astrocytes. Overexpression of Nrf-2 in astrocytes specifically increases expression of the above thiol synthesis-related proteins. Further treatment with BSO or N-acetylcysteine in transfected cells modulates this expression. Thiol depletion also accelerates cell death, while thiol supplementation promotes survival of ts1-infected cells. Together, our results indicate that ts1 infection of astrocytes, along with ts1-induced gPr80(env) accumulation, endoplasmic reticulum stress, thiol depletion, and oxidative stress, accelerates cell death; in response to the thiol depletion and oxidative stress, astrocytes activate their Nrf-2-mediated thiol antioxidant defenses, promoting cell survival.