Alok Dhar

Adaptive Immune Neuroprotection in G93A-SOD1 Amyotrophic Lateral Sclerosis Mice

Background Innate neuroimmune dysfunction is a pathobiological feature of amyotrophic lateral sclerosis (ALS). However, links, if any, between disease and adaptive immunity are poorly understood. Thus, the role of T cell immunity in disease was investigated in human G93A superoxide dismutase 1 (SOD1) transgenic (Tg) mice and subsequently in ALS patients. Methods and Findings Quantitative and qualitative immune deficits in lymphoid cell and T cell function were seen in G93A-SOD1 Tg mice. Spleens of Tg animals showed reductions in size, weight, lymphocyte numbers, and morphological deficits at terminal stages of disease compared to their wild-type (Wt) littermates. Spleen sizes and weights of pre-symptomatic Tg mice were unchanged, but deficits were readily seen in T cell proliferation coincident with increased annexin-V associated apoptosis and necrosis of lymphocytes. These lymphoid deficits paralleled failure of Copolymer-1 (COP-1) immunization to affect longevity. In addition, among CD4+ T cells in ALS patients, levels of CD45RA+ (naïve) T cells were diminished, while CD45RO+ (memory) T cells were increased compared to age-matched caregivers. In attempts to correct mutant SOD1 associated immune deficits, we reconstituted SOD1 Tg mice with unfractionated naïve lymphocytes or anti-CD3 activated CD4+CD25+ T regulatory cells (Treg) or CD4+CD25− T effector cells (Teff) from Wt donor mice. While naive lymphocytes failed to enhance survival, both polyclonal-activated Treg and Teff subsets delayed loss of motor function and extended survival; however, only Treg delayed neurological symptom onset, whereas Teff increased latency between disease onset and entry into late stage. Conclusions A profound and progressive immunodeficiency is operative in G93A-SOD1 mice and is linked to T cell dysfunction and the failure to elicit COP-1 neuroprotective immune responses. In preliminary studies T cell deficits were also observed in human ALS. These findings, taken together, suggest caution in ascribing vaccination outcomes when these animal models of human ALS are used for study. Nonetheless, the abilities to improve neurological function and life expectancy in G93A-SOD1 Tg mice by reconstitution with activated T cells do provide opportunities for therapeutic intervention.

AID/APOBEC cytosine deaminase induces genome-wide kataegis

Clusters of localized hypermutation in human breast cancer genomes, named “kataegis” (from the Greek for thunderstorm), are hypothesized to result from multiple cytosine deaminations catalyzed by AID/APOBEC proteins. However, a direct link between APOBECs and kataegis is still lacking. We have sequenced the genomes of yeast mutants induced in diploids by expression of the gene for PmCDA1, a hypermutagenic deaminase from sea lamprey. Analysis of the distribution of 5,138 induced mutations revealed localized clusters very similar to those found in tumors. Our data provide evidence that unleashed cytosine deaminase activity is an evolutionary conserved, prominent source of genome-wide kataegis events.ReviewersThis article was reviewed by: Professor Sandor Pongor, Professor Shamil R. Sunyaev, and Dr Vladimir Kuznetsov.

Potential mechanisms for astrocyte-TIMP-1 downregulation in chronic inflammatory diseases.

The pathogenesis of many neurodegenerative disorders, including human immunodeficiency virus (HIV)‐1 associated dementia, is exacerbated by an imbalance between matrix metalloproteinases (MMPs) and their inhibitors, tissue inhibitors of metalloproteinases (TIMPs). In the context of disease, TIMP‐1 has emerged as an important multifunctional protein capable of regulating inflammation. We previously reported differential TIMP‐1 expression in acute versus chronic activation of astrocytes. This study investigates possible mechanisms underlying TIMP‐1 downregulation in chronic neuroinflammation. We used interleukin (IL)‐1β as a model pro‐inflammatory stimulus and measured TIMP‐1 binding to extracellular matrix, cell death, receptor downregulation, TIMP‐1 mRNA stability and transcriptional regulation in activated astrocytes. TIMP‐1 remained localized to the cell body or was secreted into the cell supernatant. DNA fragmentation ELISA and MTT assay showed that prolonged IL‐1β activation of astrocytes induced significant astrocyte death. In acute and chronic IL‐1β‐activated astrocytes, IL‐1 receptor levels were not significantly different. TIMP‐1 mRNA stability was measured in astrocytes and U87 astroglioma cells by real‐time PCR, and TIMP‐1 promoter activation was studied using TIMP‐1‐luciferase reporter constructs in transfected astrocytes. Our results indicated that TIMP‐1 expression is regulated through multiple mechanisms. Transcriptional control and loss of mRNA stabilization are, however, the most likely primary contributors to chronic downregulation of TIMP‐1. These data are important for unraveling the mechanisms underlying astrocyte responses during chronic neuroinflammation and have broader implications in other inflammatory diseases that involve MMP/TIMP imbalance.

Genome-Wide Mutation Avalanches Induced in Diploid Yeast Cells by a Base Analog or an APOBEC Deaminase

Genetic information should be accurately transmitted from cell to cell; conversely, the adaptation in evolution and disease is fueled by mutations. In the case of cancer development, multiple genetic changes happen in somatic diploid cells. Most classic studies of the molecular mechanisms of mutagenesis have been performed in haploids. We demonstrate that the parameters of the mutation process are different in diploid cell populations. The genomes of drug-resistant mutants induced in yeast diploids by base analog 6-hydroxylaminopurine (HAP) or AID/APOBEC cytosine deaminase PmCDA1 from lamprey carried a stunning load of thousands of unselected mutations. Haploid mutants contained almost an order of magnitude fewer mutations. To explain this, we propose that the distribution of induced mutation rates in the cell population is uneven. The mutants in diploids with coincidental mutations in the two copies of the reporter gene arise from a fraction of cells that are transiently hypersensitive to the mutagenic action of a given mutagen. The progeny of such cells were never recovered in haploids due to the lethality caused by the inactivation of single-copy essential genes in cells with too many induced mutations. In diploid cells, the progeny of hypersensitive cells survived, but their genomes were saturated by heterozygous mutations. The reason for the hypermutability of cells could be transient faults of the mutation prevention pathways, like sanitization of nucleotide pools for HAP or an elevated expression of the PmCDA1 gene or the temporary inability of the destruction of the deaminase. The hypothesis on spikes of mutability may explain the sudden acquisition of multiple mutational changes during evolution and carcinogenesis.

Disruption of Transcriptional Coactivator Sub1 Leads to Genome-Wide Re-distribution of Clustered Mutations Induced by APOBEC in Active Yeast Genes.

Mutations in genomes of species are frequently distributed non-randomly, resulting in mutation clusters, including recently discovered kataegis in tumors. DNA editing deaminases play the prominent role in the etiology of these mutations. To gain insight into the enigmatic mechanisms of localized hypermutagenesis that lead to cluster formation, we analyzed the mutational single nucleotide variations (SNV) data obtained by whole-genome sequencing of drug-resistant mutants induced in yeast diploids by AID/APOBEC deaminase and base analog 6-HAP. Deaminase from sea lamprey, PmCDA1, induced robust clusters, while 6-HAP induced a few weak ones. We found that PmCDA1, AID, and APOBEC1 deaminases preferentially mutate the beginning of the actively transcribed genes. Inactivation of transcription initiation factor Sub1 strongly reduced deaminase-induced can1 mutation frequency, but, surprisingly, did not decrease the total SNV load in genomes. However, the SNVs in the genomes of the sub1 clones were re-distributed, and the effect of mutation clustering in the regions of transcription initiation was even more pronounced. At the same time, the mutation density in the protein-coding regions was reduced, resulting in the decrease of phenotypically detected mutants. We propose that the induction of clustered mutations by deaminases involves: a) the exposure of ssDNA strands during transcription and loss of protection of ssDNA due to the depletion of ssDNA-binding proteins, such as Sub1, and b) attainment of conditions favorable for APOBEC action in subpopulation of cells, leading to enzymatic deamination within the currently expressed genes. This model is applicable to both the initial and the later stages of oncogenic transformation and explains variations in the distribution of mutations and kataegis events in different tumor cells.

Novel role of TGF‐β in differential astrocyte‐TIMP‐1 regulation: Implications for HIV‐1‐dementia and neuroinflammation

Astrocyte production of tissue inhibitor of metalloproteinase (TIMP)‐1 is important in central nervous system (CNS) homeostasis and inflammatory diseases such as HIV‐1‐associated dementia (HAD). TIMPs and matrix metalloproteinases (MMPs) regulate the remodeling of the extracellular matrix. An imbalance between TIMPs and MMPs is associated with many pathologic conditions. Our recently published studies uniquely demonstrate that HAD patients have reduced levels of TIMP‐1 in the brain. Astrocyte‐TIMP‐1 expression is differentially regulated in acute and chronic inflammatory conditions. In this and the adjoining report (Gardner et al., 2006 ), we investigate the mechanisms that may be involved in differential TIMP‐1 regulation. One mechanism for TIMP‐1 downregulation is the production of anti‐inflammatory molecules, which can activate signaling pathways during chronic inflammation. We investigated the contribution of transforming growth factor (TGF)‐signaling in astrocyte‐MMP/TIMP‐1‐astrocyte regulation. TGF‐β1 and β2 levels were upregulated in HAD brain tissues. Co‐stimulation of astrocytes with IL‐1β and TGF‐β mimicked the TIMP‐1 downregulation observed with IL‐1β chronic activation. Measurement of astrocyte‐MMP protein levels showed that TGF‐β combined with IL‐1β increased MMP‐2 and decreased proMMP‐1 expression compared to IL‐1β alone. We propose that one of the mechanisms involved in TIMP‐1 downregulation may be through TGF‐signaling in chronic immune activation. These studies show a novel extracellular regulatory loop in astrocyte‐TIMP‐1 regulation.

Bypass of a psoralen DNA interstrand cross-link by DNA polymerases β, ι, and κ in vitro.

Repair of DNA interstrand cross-links in mammalian cells involves several biochemically distinctive processes, including the release of one of the cross-linked strands and translesion DNA synthesis (TLS). In this report, we investigated the in vitro TLS activity of a psoralen DNA interstrand cross-link by three DNA repair polymerases, DNA polymerases β, κ, and ι. DNA polymerase β is capable of bypassing a psoralen cross-link with a low efficiency. Cell extracts prepared from DNA polymerase β knockout mouse embryonic fibroblasts showed a reduced bypass activity of the psoralen cross-link, and purified DNA polymerase β restored the bypass activity. In addition, DNA polymerase ι misincorporated thymine across the psoralen cross-link and DNA polymerase κ extended these mispaired primer ends, suggesting that DNA polymerase ι may serve as an inserter and DNA polymerase κ may play a role as an extender in the repair of psoralen DNA interstrand cross-links. The results demonstrated here indicate that multiple DNA polymerases could participate in TLS steps in mammalian DNA interstrand cross-link repair.

Recombination Is Responsible for the Increased Recovery of Drug-Resistant Mutants with Hypermutated Genomes in Resting Yeast Diploids Expressing APOBEC Deaminases

DNA editing deaminases (APOBECs) are implicated in generation of mutations in somatic cells during tumorigenesis. APOBEC-dependent mutagenesis is thought to occur during transient exposure of unprotected single-stranded DNA. Mutations frequently occur in clusters (kataegis). We investigated mechanisms of mutant generation in growing and resting diploid yeast expressing APOBEC from sea lamprey, PmCDA1, whose kataegistic effect was previously shown to be associated with transcription. We have found that the frequency of canavanine-resistant mutants kept raising after growth cessation, while the profile of transcription remained unchanged. Surprisingly, the overall number of mutations in the genomes did not elevate in resting cells. Thus, mutations were accumulated during vigorous growth stage with both intense replication and transcription. We found that the elevated recovery of can1 mutant clones in non-growing cells is the result of loss of heterozygosity (LOH) leading to clusters of homozygous mutations in the chromosomal regions distal to the reporter gene. We confirmed that recombination frequency in resting cells was elevated by orders of magnitude, suggesting that cells were transiently committed to meiotic levels of recombination, a process referred to in yeast genetics as return-to-growth. In its extreme, on day 6 of starvation, a few mutant clones were haploid, likely resulting from completed meiosis. Distribution of mutations along chromosomes indicated that PmCDA1 was active during ongoing recombination events and sometimes produced characteristic kataegis near initial breakpoints. AID and APOBEC1 behaved similar to PmCDA1. We conclude that replication, transcription, and mitotic recombination contribute to the recovered APOBEC-induced mutations in resting diploids. The mechanism is relevant to the initial stages of oncogenic transformation in terminally differentiated cells, when recombination may lead to the LOH exposing recessive mutations induced by APOBECs in cell’s history and to acquisition of new mutations near original break.

CCR3 Chemokine Receptor

CCR3, a receptor for chemokines such as eotaxin-1,-2,-3, MCP-2,-3,-4, and RANTES, is expressed on the surface of eosinophils, mast cells, basophils, and Th2 lymphocytes and, intracellularly, in mast cells. In addition, CCR3 is also found on dendritic cells, astrocytes, and microglial cells in the brain. CCR3 plays an important role in the pathology of acute and chronic inflammatory conditions such as allergic rhinitis, asthma, and airway hyperresponsiveness (AHR), and seasonal conjunctivitis. CCR3 mediates recruitment of eosinophils, basophils, and Th2 lymphocytes to the site of inflammation. CCR3 in and/or on mast cells also mediates trafficking of progenitors, selective programming of mast cell phenotype and effector function, and differentiation and selective development of mast cells to different subtypes. Therefore, CCR3 constitutes an attractive target for therapeutic intervention.