Paul T. Massa - US grants

DESCRIPTION (provided by applicant): We have shown that the neurotropic virus, Theiler's encephalomyelitis virus (TMEV), replicates to a much higher degree and causes more extensive demyelination in the CNS of mice genetically lacking the protein tyrosine phosphatase SHP-1 compared to wild type mice expressing SHP-1. Further, oligodendrocytes isolated from these mice replicate TMEV to higher levels in vitro than oligodendrocytes of normal littermates containing SHP-1. This indicates that SHP-1, which we have shown is expressed in CNS glia including oligodendrocytes, plays a role in resisting replication of TMEV in these cells and may relate to increased demyelination seen in infected SHP-1-deficient mice. SHP-1 has been shown to control mitogen-activated protein kinase (MAPK) activities arising from growth factor or neurotrophic factor receptors. Preliminary data show that SHP-1 controls constitutive Erk1/2 activation in CNS oligodendrocytes. Activation of multiple MAPK pathways has been shown to promote numerous classes of viruses. We propose that a main role for SHP-1 under the control of a virus-inducible promoter in CNS oligodendrocytes is to modulate MAPK pathways during TMEV infections thus effectively limiting virus replication. In the absence of SHP-1, we hypothesize that TMEV fully utilizes MAPK to promote virus replication. As such, SHP-1 may be considered an important antiviral state gene in the CNS. Since induction of SHP-1 is believed to be primarily epithelial cell-specific, we believe that these studies will be particularly relevant to the microglial populations of the CNS including oligodendrocytes and astrocytes, which are epithelial in origin. However, the role of constitutive expression of SHP-1 through the constitutive hematopoietic promoter in resisting TMEV infection will also be analyzed because these cells are also important targets for TMEV in the CNS. Finally, we show that expression of a virus-inducible anti-viral state gene, inducible nitric oxide synthase (iNOS), and its product nitric oxide is profoundly and specifically deficient in oligodendrocytes and astrocytes of SHP-1-deficient mice compared to those of normal littermates indicating a severe defect in this important anti-viral system. To elucidate the role of SHP-1 in TMEV infections in the CNS we propose the following specific aims. In specific aim 1, we will study the mechanism by which SHP-1 promotes iNOS expression in oligodendrocytes, astrocytes, and microglia with attention on possible tissue-specific differences in regulation of iNOS between these cells that relate to observed differences in virus replication in these populations. Second, we show that SHP-1 specifically inhibits Erk1/2 activation in oligodendrocytes and will study related mechanisms by which SHP-1 controls replication of TMEV especially at the translational level in specific aim 2. Third, we have shown that SHP-1 expression is induced during virus infections. The mechanism for virus-inducible SHP-1 at the level of the epithelial promoter 1 will be studied in specific aim 3. We believe that information gained under these specific aims will elucidate mechanisms whereby SHP-1 controls virus infections in oligodendrocytes and perhaps other cells of the CNS.

Numerous studies have shown that the protein tyrosine phosphatase SHP-1 modulates the expression of proinflammatory genes in leukocytes and glia that have been shown to cause inflammatory demyelination in the CNS. Accordingly, genetic deficiency in SHP-1 causes increased inflammatory demyelination in multiple animal models of multiple sclerosis (MS). Most recently, we demonstrated that SHP-1 deficiency in blood-borne monocytes is responsible for monocyte infiltration and macrophage- mediated inflammatory demyelination in the CNS of mice. With regards to MS, we published a report describing a deficiency in SHP-1 expression and function in peripheral blood leukocytes of MS patients compared to normal human subjects. We showed that SHP-1 deficiency plays a direct role in the hyper- activation of signal transducers and activators of transcription (STAT6 and STAT1)) and NF-?B. Increased activation of these transcription factors correlated with corresponding heightened expression of multiple pro-inflammatory genes in MS leukocytes compared to normal subject cells. Therefore, we propose that SHP-1 deficiency is a biologically important abnormality in leukocytes of MS patients that promotes inflammatory demyelination. This hypothesis is supported by in vivo and in vitro experiments in which induction of SHP-1 to normal levels by IFN-? reduces the expression of pro-inflammatory genes in lymphocytes and macrophages of MS patients to normal levels. Thus, specific aims 1 and 2 of this research proposal describe experimental approaches to further characterize deficiency in expression and function of SHP-1 in various leukocyte subsets from MS patients and control subjects. Adding to our preliminary report, increased numbers of MS patients with various disease sub-classifications will be included in future studies. Additionally, an expanded group of control subjects with other neurological diseases and non-MS autoimmune diseases will be included. Lastly, we have identified a specific deficiency in the transcriptional activity of one of two known promoters (promoter II) of the human SHP-1 gene in MS leukocytes compared to those of normal subjects. Interestingly, IFN-2 corrects promoter II deficiency in MS leukocytes by a currently unknown mechanism. Therefore, specific aim 3 is focused on determining recently described epigenetic alterations and transcription factor activity on promoter II that may be responsible for deficiency and cytokine-mediated effects in SHP-1 promoter II transcript expression in MS leukocytes. In summary, these studies are directed at further characterizing the abnormal expression of SHP-1 in MS and multiple downstream inflammatory pathways regulated by SHP-1 in lymphocytes and macrophages that are known to play a role in MS.

DESCRIPTION (provided by applicant): Cells of the innate immune system such as macrophages express specific genes that control both virus replication and virus-induced inflammation. Dysregulation of these genes has been shown to result in increased susceptibility to either viral infections or inflammatory diseases. Our laboratory is focused on genes that control specific virus-cell interactions in monocyte-lineage cells including macrophages that cause virus- induced inflammatory demyelination in the central nervous system (CNS). These studies are particularly relevant to understanding the genetic mechanisms for initiation and sustained inflammatory demyelination mediated by monocyte-lineage cells in the human demyelinating disease multiple sclerosis (MS). Of particular importance is that we have discovered a deficiency in the key anti-inflammatory gene SHP-1 in macrophages of MS patients that results in high levels of inflammatory activity in these cells similar to that seen in MS lesions. To further define the role of SHP-1 deficiency in demyelinating disease, we are studying both anti- inflammatory and anti-viral activities in monocytes of mice that are genetically deficient in SHP-1. Recently, we have shown that SHP-1-deficient mice are profoundly susceptible to virus-induced demyelinating disease compared to wild type mice following a peripheral inoculation with relatively low amounts of Theiler's murine encephalomyelitis virus (TMEV). TMEV infection appears to predominant initially in the spleen and in inflammatory monocytes in the blood prior to entry of these cells and TMEV into the CNS. These new observations have posed important questions on how peripheral virus infection of monocytes may elicit a relatively specific targeting of monocyte-mediated inflammation in the CNS white matter. Thus, the specific aims are designed to address how TMEV infects and then stimulates monocytes in the periphery to enter the CNS in large numbers and subsequently mediate inflammatory demyelination. Specific aim 1 will characterize the initial infection and spreading of TMEV within monocyte populations following peripheral inoculation. Specific aim 2 will focus on the role for the chemokine MCP-1 and its receptor CCR2 in the spreading of TMEV to monocytes in peripheral tissues and eventual movement of these infected monocytes to the CNS. Specific aim 3 will determine whether infected inflammatory monocytes that enter the white matter following peripheral TMEV inoculation differentiate into either mature macrophages or dendritic cells within demyelinating lesions and function in demyelination. Finally, we will characterize the role for SHP-1 deficiency in allowing heightened infection and persistence of TMEV in monocyte-lineage cells that may be an essential condition for development of CNS disease. Together, the research plan presents novel approaches to further elucidate the importance of SHP-1 in monocyte-lineage cells in controlling multiple key events critical for virus-induced inflammatory diseases in the CNS. PUBLIC HEALTH RELEVANCE: An essential effector cell in the human demyelinating disease, multiple sclerosis (MS), is the macrophage. Macrophages in MS white matter lesions express multiple inflammatory cytokines, toxic molecules, and phagocytic activity that cause destruction of both myelin-forming oligodendrocytes and myelin sheathes. Yet, our knowledge of macrophage biology in demyelinating disease is incomplete. We have found that a key molecule SHP-1 stringently modulates inflammatory activities of macrophages in demyelinating disease. The present project is thus focused on defining the essential role of SHP-1 in macrophage-mediated demyelinating disease.

DESCRIPTION (provided by applicant): Oligodendrocytes and their connected myelin sheathes are extensively damaged in CNS inflammatory demyelinating lesions in multiple sclerosis (MS). Targeting of oligodendrocytes and myelin in MS is thought to be initiated by either autoimmunity to myelin antigens and/or to inflammatory immune responses to viruses that specifically infect oligodendrocytes. Irrespective of the mode of initiation, common features of white matter damage are oxidative stress of the oligodendrocytes and oxidation of myelin lipids and proteins. The source of oxidative changes in MS white matter is typically assigned to production of reactive oxygen species (ROS) by inflammatory macrophages, microglia, and astrocytes within or near to inflammatory demyelinating lesions. However, we have evidence that major cells responsible for ROS production are oligodendrocytes. Further, we have identified that the protein tyrosine phosphatase SHP-1 is a critical regulator of ROS production in oligodendrocytes and oxidative damage to myelin. As such, we believe that SHP-1 is a key factor in susceptibility to demyelinating disease by pathways that commonly involve ROS generation in the active phase of disease when inflammatory lesions are produced. The latter proposition stems from our related observations of increased susceptibility to demyelinating disease in SHP-1-deficient mice and SHP-1- deficiency in oligodendrocytes in MS brain (Gruber et al, 2011, submitted). The present project is focused on elucidating the mechanisms of ROS production in oligodendrocytes exposed to proinflammatory cytokines that exist in MS lesions and how SHP-1 controls production of ROS in these cells. To do this, we propose 3 specific aims. The first is to determine how SHP-1 regulates constitutive and TNF-¿-inducible ROS production in oligodendrocytes. These studies will identify the sources of superoxide production in oligodendrocytes including ROS-producing complexes localized within early endosomes and mitochondria that are controlled by SHP-1. The second aim is to determine the consequences of increased ROS production in SHP-1-deficient oligodendrocytes. Analysis of lipid peroxidation, protein oxidation, and oxidation of anti-inflammatory PTP in SHP-1-deficient and wild type oligodendrocytes and myelin will be performed using both in vivo and in vitro approaches. Finally, the third specific aim involves a novel approach to ascertain the function of SHP-1 in vivo using targeted conditional deletion of the SHP-1 gene in either mature oligodendrocytes or in macrophages/microglia. These studies will be critical to defining the autonomous activity of SHP-1 in controlling ROS production in oligodendrocytes and resulting demyelinating processes in vivo.

Ant1 is the muscle/heart/central nervous system (CNS) isoform of adenine nucleotide translocase that is primarily involved in ATP/ADP exchange across the inner mitochondrial membrane (IMM). An increasing number of missense mutations in Ant1 are found to cause dominant diseases that affect skeletal muscle and the central nervous system. These diseases are commonly manifested by fractional mtDNA deletions and mild bioenergetic defects. The mechanism of neuromuscular damage in the diseases is poorly understood. Interestingly, our recent studies in yeast and cultured human cells suggested that the mutant Ant1 is misfolded. This leads to cell death by a novel mechanism that we named mitochondrial Precursor Overaccumulation Stress (mPOS). mPOS is characterized by the toxic accumulation and aggregation of un-imported mitochondrial preproteins in the cytosol. These findings led to the central hypothesis that the mutant Ant1 primarily affects mitochondrial protein import. This results in mPOS in the cytosol, which plays an important role in inducing neural and muscular degeneration. Fractional mtDNA deletions occur independent of nucleotide transport activity, likely as a secondary damage collateral to reduced mitochondrial protein import. In this application, we propose to directly test this hypothesis in mouse models. We successfully generated knock-in (KI) mouse lines expressing misfolded variants of Ant1. Preliminary studies indicated that these mice develop phenotypes consistent with neural and muscular degeneration. In Specific Aim 1, we will use these unique experimental models to test the hypothesis that misfolded Ant1 induces neural and muscular degeneration and mtDNA instability independent of nucleotide transport. In Specific Aim 2, we will use various experimental tools that we developed in yeast, cultured human cells and the Ant1-KI mice to test the hypothesis that the misfolded Ant1 (or Aac2 in yeast) causes structural and functional damage to the mitochondrial protein import machinery and induces mPOS in the cytosol. In Specific Aim 3, we will determine the mechanisms that protect cells against Ant1-induced protein import stress and mPOS. Success of the project will establish a mouse model of protein import stress associated with mPOS. Particularly, validation of the mPOS model would help reconciling the mitochondrial and proteostatic pathways in many neural and muscular degenerative diseases. Finally, the results could have important implications for the understanding and therapy of Ant1-induced diseases, as well as many other clinical conditions that directly or indirectly affect mitochondrial protein import.