OGG1 Mouse

What is OGG1 and what is its function in mice?

OGG1 in mice is a DNA repair enzyme that specifically recognizes and excises 8-oxoguanine (8-oxoG), a common oxidative DNA lesion induced by reactive oxygen species (ROS). The mouse Ogg1 gene is located on Chromosome 6 and consists of 7 exons spanning approximately 6 kb . The encoded protein contains DNA-binding motifs including helix-hairpin-helix and C2H2 zinc finger-like domains that are encoded in exons 4 through 5 .

Functionally, mouse OGG1 possesses both DNA glycosylase activity (removing 8-oxoG from DNA) and AP lyase activity (cleaving the DNA backbone at the resulting abasic site) . These enzymatic activities are essential for initiating the base excision repair pathway to prevent G:C to T:A transversion mutations that would occur if 8-oxoG lesions remained unrepaired during DNA replication . The properties of mouse OGG1 are similar to human and yeast OGG1 proteins in terms of glycosylase/lyase activities and substrate specificities .

What are the phenotypic characteristics of OGG1 knockout mice?

When OGG1 deficiency is combined with other genetic modifications in disease models, more severe phenotypes emerge. For example, in Alzheimer's disease models, combined MTH1/OGG1 deficiency leads to accelerated neurodegeneration at 4-5 months of age . These mice exhibit increased accumulation of 8-oxoG in nuclear DNA of activated microglia in the cortex and hippocampal dentate gyrus, regions where severe neurodegeneration occurs .

Cells derived from Ogg1-/- mice show altered responses to oxidative DNA damage. Cortical neurons isolated from adult MTH1/OGG1-deficient mice accumulate high levels of 8-oxoG in mitochondrial DNA under oxidative conditions, exhibiting mitochondrial dysfunction and impaired neuritogenesis, though not necessarily neuronal death .

What methodological approaches are used for studying OGG1 activity in mouse models?

Researchers employ various techniques to study OGG1 activity in mouse models:

• Genetic manipulation techniques:

  • OGG1 knockout mice (Ogg1-/-) created through targeted gene disruption

  • CRISPR-Cas9 approaches for OGG1 gene editing

  • shRNA-mediated knockdown of OGG1 in specific tissues or cells

• Enzymatic activity assays:

  • In vitro assays using double-stranded oligonucleotides harboring either an 8-oxoG residue or a tetrahydrofuran to measure OGG1 and AP-lyase activities in tissue extracts

  • Measurement of repair activities using purified proteins or tissue lysates

• Detection of 8-oxoG in genomic DNA:

  • Modified comet assay with OGG1 treatment to detect genomic OGG1 substrates

  • Measurement of 8-oxoG levels in purified genomic DNA

• Expression analysis:

  • RT-PCR and quantitative PCR to measure OGG1 mRNA levels using reference genes like cyclophilin A

  • In situ hybridization to localize OGG1 mRNA in tissue sections, particularly useful for understanding tissue-specific expression patterns

  • Immunohistochemistry to detect OGG1 protein and co-localization with other repair enzymes in tissues

• Functional studies:

  • DNA damage assessment using γH2AX staining to detect DNA double-strand breaks

  • Replication stress evaluation through DNA fiber assays measuring replication fork rates

  • Cell cycle analysis and proliferation assays to determine cellular responses to OGG1 perturbation

How can researchers assess the impact of OGG1 deficiency on DNA replication and genome stability?

OGG1 deficiency impacts DNA replication and genome stability in ways that can be measured through several experimental approaches:

These methodologies reveal that OGG1 deficiency leads to replication stress characterized by reduced replication fork progression and S-phase-specific DNA damage, ultimately affecting cellular proliferation and viability.

How does OGG1 deficiency affect cancer development in mouse models?

OGG1 deficiency exhibits complex effects on cancer development that vary depending on the context:

• Increased cancer susceptibility:

  • Ogg1-/- mice show an elevated incidence of lung cancer at 18 months of age, suggesting a tumor suppressor role for OGG1

  • The relatively late onset indicates that additional genetic alterations are required for tumor development

• Paradoxical effects on established cancer cells:

  • OGG1 depletion obstructs growth of A3 T-cell lymphoblastic acute leukemia cells in vitro and in vivo

  • In H460 lung cancer cells, OGG1 depletion using inducible shRNA reduces clonogenic ability

  • A3 cells transduced with OGG1-targeting shRNA divide normally for 48 hours post-induction but then show slower proliferation and loss of viability

• In vivo tumor regression:

  • In xenograft models where A3 cells expressing luciferase and inducible OGG1-targeting shRNA were injected into nude mice, induction of OGG1 knockdown caused tumor regression

  • After seven weeks, only mice injected with OGG1-knockdown cells remained alive, demonstrating that OGG1 protects cancer cells from oncogenic stress in vivo

• Mechanisms of growth inhibition:

  • OGG1 inhibition or depletion causes S-phase DNA damage, replication stress, and proliferation arrest or cell death

  • These effects represent a novel mechanistic approach to target cancer cells that rely on efficient DNA repair due to their elevated ROS levels

These findings suggest a dual role for OGG1 in cancer: while its absence increases long-term cancer risk, targeting OGG1 in established cancers may be therapeutically beneficial due to cancer cells' heightened dependence on efficient oxidative DNA damage repair mechanisms.

What is the role of OGG1 in neurodegenerative disease models?

OGG1 plays a crucial protective role in neurodegenerative disease models, particularly in Alzheimer's disease (AD):

• Prevention of accelerated neurodegeneration:

  • MTH1/OGG1-deficient AD mice (3xTg-AD/Mth1-/-/Ogg1-/-) develop neurodegeneration at 4-5 months of age, much earlier than AD mice with functional MTH1/OGG1

  • Standard 3xTg-AD mice with wild-type Mth1/Ogg1 alleles exhibit neuronal mitochondrial dysfunction but not neuronal loss or neurodegeneration at this age

• Protection against microglial 8-oxoG accumulation:

  • In MTH1/OGG1-deficient AD mice, significantly increased accumulation of 8-oxoG occurs in nuclear DNA of activated microglia in both the cortex and hippocampal dentate gyrus

  • This accumulation correlates with regions where severe neurodegeneration is observed

• Prevention of harmful microglial activation:

  • OGG1 deficiency leads to increased 8-oxoG accumulation in microglial nuclear DNA, triggering harmful microglial activation

  • This creates a vicious cycle where activated microglia produce more ROS during phagocytosis, further enhancing 8-oxoG accumulation and leading to chronic microglial activation (microgliosis)

• Maintenance of low 8-oxoG levels:

  • MTH1 and OGG1 together maintain low levels of 8-oxoG during AD progression, especially in microglial nuclear DNA

  • This prevents harmful activation of microglia and subsequent neurodegeneration

These findings suggest that efficient suppression of 8-oxoG accumulation in brain genomes represents a potential approach for prevention and treatment of AD . The protective roles of OGG1 in neurodegenerative disease models highlight the importance of oxidative DNA damage repair in maintaining neuronal health and preventing pathological microglial activation.

How do OGG1 and MTH1 cooperate to maintain genomic integrity?

OGG1 and MTH1 work through complementary mechanisms to protect genomic DNA from oxidative damage:

• Complementary protection strategies:

  • MTH1 hydrolyzes oxidized purine nucleoside triphosphates (like 8-oxo-dGTP) in the nucleotide pool, preventing their incorporation during DNA synthesis

  • OGG1 removes 8-oxoG lesions already present in DNA through its glycosylase/AP lyase activity

  • Together, they provide two-tiered protection against 8-oxoG-related mutagenesis and genomic instability

• Synergistic protection in disease models:

  • In AD mouse models, combined MTH1/OGG1 deficiency leads to accelerated neurodegeneration compared to single knockout models

  • This synergistic effect suggests that both mechanisms are required for comprehensive protection against oxidative DNA damage

• Compartment-specific cooperation:

  • In cortical neurons, MTH1/OGG1 deficiency leads to preferential accumulation of 8-oxoG in mitochondrial DNA under oxidative conditions

  • In microglia, combined deficiency results in harmful nuclear 8-oxoG accumulation and pathological activation

• Prevention of vicious cycles:

  • Without both MTH1 and OGG1, increased 8-oxoG accumulation in microglial nuclear DNA leads to microglial activation, which produces more ROS

  • This creates a feed-forward loop of ROS production, DNA damage, and chronic inflammation that ultimately contributes to neurodegeneration

The cooperative relationship between MTH1 and OGG1 demonstrates the importance of integrated DNA damage prevention and repair systems in maintaining genomic integrity under oxidative stress conditions . This understanding provides potential targets for therapeutic intervention in diseases associated with oxidative stress and DNA damage.

What are the cellular consequences of OGG1 inhibition versus genetic deletion?

The cellular consequences of OGG1 inhibition and genetic deletion show both similarities and differences:

These observations indicate that both pharmacological inhibition and genetic depletion of OGG1 lead to similar cellular consequences, primarily through inducing replication stress and S-phase-specific DNA damage. The comparable effects validate OGG1 as a potential therapeutic target and suggest that small molecule inhibitors can effectively mimic genetic deletion of OGG1 function.

What challenges exist in studying OGG1 function in mice?

Studying OGG1 function in mice presents several methodological and conceptual challenges:

• Mild phenotype under normal conditions:

  • Ogg1-/- mice are viable and develop normally, with cancer predisposition only appearing late in life (18 months)

  • This mild phenotype necessitates additional stressors or disease models to fully reveal OGG1's protective functions

• Detection of oxidative DNA damage:

  • Measuring 8-oxoG levels in genomic DNA is technically challenging

  • In some experiments, 8-oxoG levels did not rise above assay background levels even after OGG1 inhibition

  • More sensitive methods like modified comet assays are needed to detect subtle changes in OGG1 substrate levels

• Functional redundancy:

  • Other DNA repair enzymes may partially compensate for OGG1 deficiency

  • Combined knockout models (e.g., MTH1/OGG1 double knockout) are often needed to observe clear phenotypes

• Tissue and cell-type specificity:

  • OGG1 expression and function vary across different tissues and cell types

  • Cell-type-specific analyses are required to fully understand OGG1's role in complex tissues like the brain and retina

• Context-dependent effects:

  • OGG1 deficiency has paradoxical effects in different contexts (cancer prevention vs. cancer cell growth inhibition)

  • Understanding these context-dependent effects requires careful experimental design and interpretation

• Dual roles in DNA repair and gene regulation:

  • Beyond its DNA repair function, OGG1 is involved in transcriptional regulation through non-catalytic binding to oxidized DNA in promoter regions

  • Separating these distinct functions experimentally remains challenging

These challenges highlight the need for integrated approaches combining genetic models with biochemical assays and careful phenotypic characterization under various stress conditions to fully understand OGG1's functions in mice.

What are the implications of mouse OGG1 research for human disease treatment?

Research on OGG1 in mouse models has significant implications for understanding and potentially treating various human diseases:

• Cancer therapy:

  • OGG1 inhibition arrests cancer cell proliferation by inducing replication stress and DNA damage in S-phase

  • OGG1 inhibitors target a wide range of cancer cells with a favorable therapeutic index compared to non-transformed cells

  • This adds OGG1 to the list of BER factors (like PARP1) as potential cancer treatment targets

• Neurodegenerative disease prevention:

  • Efficient suppression of 8-oxoG accumulation in brain genomes represents a potential approach for preventing and treating Alzheimer's disease

  • Understanding the mechanisms of microglial activation due to 8-oxoG accumulation provides new targets for intervention in neuroinflammatory processes

• Retinal disease insights:

  • The retina is highly exposed to oxidative stress due to high oxygen consumption and light exposure

  • OGG1's role in protecting retinal cells from oxidative DNA damage suggests potential applications in retinal degenerative diseases

• Biomarker development:

  • Understanding how 8-oxoG levels correlate with disease progression may enable the development of biomarkers for oxidative stress-related conditions

  • Mouse models provide platforms for validating such biomarkers before human application

• Therapeutic targeting considerations:

  • While OGG1 inhibition may be beneficial in cancer treatment, the increased cancer susceptibility in aged Ogg1-/- mice suggests potential long-term risks

  • Context-specific targeting strategies may be needed to maximize therapeutic benefits while minimizing adverse effects

  • Combination approaches targeting both MTH1 and OGG1 might provide more comprehensive protection against oxidative DNA damage in neurodegenerative diseases

These findings from mouse models provide a foundation for developing novel diagnostic and therapeutic approaches for human diseases associated with oxidative stress and DNA damage. The translation of these insights to human applications represents a promising frontier in medicine.