Mingshan Yan

ATM promotes apoptosis and suppresses tumorigenesis in response to Myc

Overexpression of the c-myc oncogene contributes to the development of a significant number of human cancers. In response to deregulated Myc activity, the p53 tumor suppressor is activated to promote apoptosis and inhibit tumor formation. Here we demonstrate that p53 induction in response to Myc overexpression requires the ataxia-telangiectasia mutated (ATM) kinase, a major regulator of the cellular response to DNA double-strand breaks. In a transgenic mouse model overexpressing Myc in squamous epithelial tissues, inactivation of Atm suppresses apoptosis and accelerates tumorigenesis. Deregulated Myc expression induces DNA damage in primary transgenic keratinocytes and the formation of γH2AX and phospho-SMC1 foci in transgenic tissue. These findings suggest that Myc overexpression causes DNA damage in vivo and that the ATM-dependent response to this damage is critical for p53 activation, apoptosis, and the suppression of tumor development.

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

ABSTRACT 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 gPr80env 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, γ-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 gPr80env 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.

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.

Identification and characterization of a novel Nogo‐interacting mitochondrial protein (NIMP)

Nogo is a potent inhibitor of regeneration following spinal cord injury. To develop a better understanding of the mechanisms responsible for regenerative failure we used a yeast two‐ hybrid approach to try and identify proteins that interact with Nogo. We identified a novel mitochondrial protein designated Nogo‐interacting mitochondrial protein (NIMP) in a screen of an adult human brain cDNA library. This interaction was confirmed by co‐immunoprecipitation in both brain tissue (endogenous) and transfected HEK293T cells (overexpressed). In support of these studies we demonstrate that Nogo interacts with the UQCRC1 and UQCRC2 components of complex III, within the mitochondrial respiratory chain. The mitochondrial localization of NIMP was evidenced by confocal image analysis and western blot analysis of isolated mitochondria. NIMP is highly conserved and ubiquitously expressed in mitochondria‐enriched tissues. Within the CNS, NIMP‐like immunoreactivity is present in neurons and astrocytes. These data suggest that NIMP is a novel mitochondrial protein that interacts with Nogo. The interaction of Nogo with mitochondrial proteins may provide insight into the mechanisms for Nogo‐induced inhibition of neurite growth.

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.

Retrovirus-Induced Oxidative Stress with Neuroimmunodegeneration Is Suppressed by Antioxidant Treatment with a Refined Monosodium α-Luminol (Galavit)

ABSTRACT 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 α-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.

The ataxia-telangiectasia gene product may modulate DNA turnover and control cell fate by regulating cellular redox in lymphocytes

The ATM kinase, when activated postnatally, exerts multiple functions to prevent the onset of ataxia‐telangiectasia (AT). Using freshly isolated thymo¬cytes from Atm−/ − mice that were under stress during postnatal differentiation, we noted that thiol redox activity, as indicated by reduction of the tetrazolium MTS, and DNA turnover activity, as indicated by incor¬poration of [3H]thymidine into DNA, were both greatly increased compared with activities in thymocytes from Atm+ / + mice. This increased thymidine incorporation could be suppressed by the thiol N‐acetylcysteine. In primary noncycling splenocytes, mitogens proportion¬ally increased both the rate of [3H]thymidine incorpo¬ration and the rate of reduction of MTS. The mitogeninduced activities in splenocytes were not affected by ATM but were suppressed by the calcineurin‐dependent inhibitor FK‐506, which has no effect on these activities in thymocytes. These findings suggest that increased [3H]thymidine incorporation and reducing power indicate increased cell cycling in mitogenically stimulated splenocytes, whereas these two indicators represent increased FK‐506‐independent DNAturnover activities in thymocytes. Thus, a primary function of ATM is to activate the redox‐sensitive checkpoint re¬quired for down‐regulation of DNA turnover activities in developing lymphocytes. Cell‐cycling checkpoints in undamaged quiescent lymphocytes are not activated by ATM with mitogenic stimulation. ATM may suppress abnormal DNA turnover and the resultant oncogenesis by regulating cellular thiol redox pathways.—Yan, M., Qiang, W., Liu, N., Shen, J., Lynn, W. S., Wong, P. K. Y. The ataxia‐telangiectasia gene product may modulate DNA turnover and control cell fate by regulating cellular redox in lymphocytes. FASEBJ. 15, 1132‐1138 (2001)

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.

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.

Phenylbutyric acid suppresses protein accumulation-mediated ER stress in retrovirus-infected astrocytes and delays onset of paralysis in infected mice

Many neurodegenerative diseases are associated with accumulation of misfolded proteins in cells of the central nervous system (CNS). We have previously reported that accumulation of the precursor envelope protein gPr80(env) of ts1, a mutant of Moloney murine leukemia virus (MoMuLV), in the endoplasmic reticulum (ER) of infected astrocytes, results in ER stress, oxidative stress and cell death, subsequently leading to ts1-mediated neurodegeneration in infected mice. In the present study, we assessed whether treatments that reduce the accumulation of gPr80(env) in the ER of ts1-infected astrocytes provided a protective effect against ER stress and cell death. We show that treatment with phenylbutyric acid (PBA) can prevent the unfolded protein response (UPR), ER stress and cell death in cultured ts1-infected astrocytes. The protective effect of PBA is associated with its ability to reduce gPr80(env) accumulation and to increase the expression of proteins involved in protein folding in the ER, such as protein disulfide isomerase (PDI) and ERp44, rather than by decrease mRNA levels of gPr80(env) or alter the proteasomal degradation process for gPr80(env). In infected mice treated with PBA we also noted a reduction in the severity of the neuropathology in brainstem tissues and a delayed onset of paralysis. These results show that PBA is a potentially effective drug for the treatment of neurodegeneration caused by protein accumulation in cells of the CNS.