Affinity maturation of TCR-like antibodies using phage display guided by structural modeling.

TCR-like antibodies represent a unique type of engineered antibodies with specificity toward pHLA, a ligand normally restricted to the sensitive recognition by T cells. Here, we report a phage display-based sequential development path of such antibodies. The strategy goes from initial lead identification through in silico informed CDR engineering in combination with framework engineering for affinity and thermostability optimization, respectively. The strategy allowed the identification of HLA-DQ2.5 gluten peptide-specific TCR-like antibodies with low picomolar affinity. Our method outlines an efficient and general method for development of this promising class of antibodies, which should facilitate their utility including translation to human therapy.

AKAP18δ Anchors and Regulates CaMKII Activity at Phospholamban-SERCA2 and RYR.

BACKGROUND: The sarcoplasmic reticulum (SR) Ca2+-ATPase 2 (SERCA2) mediates Ca2+ reuptake into SR and thereby promotes cardiomyocyte relaxation, whereas the ryanodine receptor (RYR) mediates Ca2+ release from SR and triggers contraction. Ca2+/CaMKII (CaM [calmodulin]-dependent protein kinase II) regulates activities of SERCA2 through phosphorylation of PLN (phospholamban) and RYR through direct phosphorylation. However, the mechanisms for CaMKIIδ anchoring to SERCA2-PLN and RYR and its regulation by local Ca2+ signals remain elusive. The objective of this study was to investigate CaMKIIδ anchoring and regulation at SERCA2-PLN and RYR. METHODS: A role for AKAP18δ (A-kinase anchoring protein 18δ) in CaMKIIδ anchoring and regulation was analyzed by bioinformatics, peptide arrays, cell-permeant peptide technology, immunoprecipitations, pull downs, transfections, immunoblotting, proximity ligation, FRET-based CaMKII activity and ELISA-based assays, whole cell and SR vesicle fluorescence imaging, high-resolution microscopy, adenovirus transduction, adenoassociated virus injection, structural modeling, surface plasmon resonance, and alpha screen technology. RESULTS: Our results show that AKAP18δ anchors and directly regulates CaMKIIδ activity at SERCA2-PLN and RYR, via 2 distinct AKAP18δ regions. An N-terminal region (AKAP18δ-N) inhibited CaMKIIδ through binding of a region homologous to the natural CaMKII inhibitor peptide and the Thr17-PLN region. AKAP18δ-N also bound CaM, introducing a second level of control. Conversely, AKAP18δ-C, which shares homology to neuronal CaMKIIα activator peptide (N2B-s), activated CaMKIIδ by lowering the apparent Ca2+ threshold for kinase activation and inducing CaM trapping. While AKAP18δ-C facilitated faster Ca2+ reuptake by SERCA2 and Ca2+ release through RYR, AKAP18δ-N had opposite effects. We propose a model where the 2 unique AKAP18δ regions fine-tune Ca2+-frequency-dependent activation of CaMKIIδ at SERCA2-PLN and RYR. CONCLUSIONS: AKAP18δ anchors and functionally regulates CaMKII activity at PLN-SERCA2 and RYR, indicating a crucial role of AKAP18δ in regulation of the heartbeat. To our knowledge, this is the first protein shown to enhance CaMKII activity in heart and also the first AKAP (A-kinase anchoring protein) reported to anchor a CaMKII isoform, defining AKAP18δ also as a CaM-KAP.

Peptides containing the PCNA interacting motif APIM bind to the ß-clamp and inhibit bacterial growth and mutagenesis.

In the fight against antimicrobial resistance, the bacterial DNA sliding clamp, ß-clamp, is a promising drug target for inhibition of DNA replication and translesion synthesis. The ß-clamp and its eukaryotic homolog, PCNA, share a C-terminal hydrophobic pocket where all the DNA polymerases bind. Here we report that cell penetrating peptides containing the PCNA-interacting motif APIM (APIM-peptides) inhibit bacterial growth at low concentrations in vitro, and in vivo in a bacterial skin infection model in mice. Surface plasmon resonance analysis and computer modeling suggest that APIM bind to the hydrophobic pocket on the ß-clamp, and accordingly, we find that APIM-peptides inhibit bacterial DNA replication. Interestingly, at sub-lethal concentrations, APIM-peptides have anti-mutagenic activities, and this activity is increased after SOS induction. Our results show that although the sequence homology between the ß-clamp and PCNA are modest, the presence of similar polymerase binding pockets in the DNA clamps allows for binding of the eukaryotic binding motif APIM to the bacterial ß-clamp. Importantly, because APIM-peptides display both anti-mutagenic and growth inhibitory properties, they may have clinical potential both in combination with other antibiotics and as single agents.

N6-methyladenosine regulates the stability of RNA:DNA hybrids in human cells.

R-loops are nucleic acid structures formed by an RNA:DNA hybrid and unpaired single-stranded DNA that represent a source of genomic instability in mammalian cells1-4. Here we show that N6-methyladenosine (m6A) modification, contributing to different aspects of messenger RNA metabolism5,6, is detectable on the majority of RNA:DNA hybrids in human pluripotent stem cells. We demonstrate that m6A-containing R-loops accumulate during G2/M and are depleted at G0/G1 phases of the cell cycle, and that the m6A reader promoting mRNA degradation, YTHDF2 (ref. 7), interacts with R-loop-enriched loci in dividing cells. Consequently, YTHDF2 knockout leads to increased R-loop levels, cell growth retardation and accumulation of γH2AX, a marker for DNA double-strand breaks, in mammalian cells. Our results suggest that m6A regulates accumulation of R-loops, implying a role for this modification in safeguarding genomic stability.

Secretoneurin Is an Endogenous Calcium/Calmodulin-Dependent Protein Kinase II Inhibitor That Attenuates Ca2+-Dependent Arrhythmia.

BACKGROUND: Circulating SN (secretoneurin) concentrations are increased in patients with myocardial dysfunction and predict poor outcome. Because SN inhibits CaMKIIδ (Ca2+/calmodulin-dependent protein kinase IIδ) activity, we hypothesized that upregulation of SN in patients protects against cardiomyocyte mechanisms of arrhythmia. METHODS: Circulating levels of SN and other biomarkers were assessed in patients with catecholaminergic polymorphic ventricular tachycardia (CPVT; n=8) and in resuscitated patients after ventricular arrhythmia-induced cardiac arrest (n=155). In vivo effects of SN were investigated in CPVT mice (RyR2 [ryanodine receptor 2]-R2474S) using adeno-associated virus-9-induced overexpression. Interactions between SN and CaMKIIδ were mapped using pull-down experiments, mutagenesis, ELISA, and structural homology modeling. Ex vivo actions were tested in Langendorff hearts and effects on Ca2+ homeostasis examined by fluorescence (fluo-4) and patch-clamp recordings in isolated cardiomyocytes. RESULTS: SN levels were elevated in patients with CPVT and following ventricular arrhythmia-induced cardiac arrest. In contrast to NT-proBNP (N-terminal pro-B-type natriuretic peptide) and hs-TnT (high-sensitivity troponin T), circulating SN levels declined after resuscitation, as the risk of a new arrhythmia waned. Myocardial pro-SN expression was also increased in CPVT mice, and further adeno-associated virus-9-induced overexpression of SN attenuated arrhythmic induction during stress testing with isoproterenol. Mechanistic studies mapped SN binding to the substrate binding site in the catalytic region of CaMKIIδ. Accordingly, SN attenuated isoproterenol induced autophosphorylation of Thr287-CaMKIIδ in Langendorff hearts and inhibited CaMKIIδ-dependent RyR phosphorylation. In line with CaMKIIδ and RyR inhibition, SN treatment decreased Ca2+ spark frequency and dimensions in cardiomyocytes during isoproterenol challenge, and reduced the incidence of Ca2+ waves, delayed afterdepolarizations, and spontaneous action potentials. SN treatment also lowered the incidence of early afterdepolarizations during isoproterenol; an effect paralleled by reduced magnitude of L-type Ca2+ current. CONCLUSIONS: SN production is upregulated in conditions with cardiomyocyte Ca2+ dysregulation and offers compensatory protection against cardiomyocyte mechanisms of arrhythmia, which may underlie its putative use as a biomarker in at-risk patients.

Visualization of PML nuclear import complexes reveals FG-repeat nucleoporins at cargo retrieval sites.

Selective nuclear import in eukaryotic cells involves sequential interactions between nuclear import receptors and phenylalanine-glycine (FG)-repeat nucleoporins. Traditionally, binding of cargoes to import receptors is perceived as a nuclear pore complex independent event, while interactions between import complexes and nucleoporins are thought to take place at the nuclear pores. However, studies have shown that nucleoporins are mobile and not static within the nuclear pores, suggesting that they may become engaged in nuclear import before nuclear pore entry. Here we have studied post-mitotic nuclear import of the tumor suppressor protein PML. Since this protein forms nuclear compartments called PML bodies that persist during mitosis, the assembly of putative PML import complexes can be visualized on the surface of these protein aggregates as the cell progress from an import inactive state in mitosis to an import active state in G1. We show that these post-mitotic cytoplasmic PML bodies incorporate a multitude of peripheral nucleoporins, but not scaffold or nuclear basket nucleoporins, in a manner that depends on FG-repeats, the KPNB1 import receptor, and the PML nuclear localization signal. The study suggests that nucleoporins have the ability to target certain nuclear cargo proteins in a nuclear pore-uncoupled state, before nuclear pore entry.

Axitinib blocks Wnt/ß-catenin signaling and directs asymmetric cell division in cancer.

Oncogenic mutations of the Wnt (wingless)/ß-catenin pathway are frequently observed in major cancer types. Thus far, however, no therapeutic agent targeting Wnt/ß-catenin signaling is available for clinical use. Here we demonstrate that axitinib, a clinically approved drug, strikingly blocks Wnt/ß-catenin signaling in cancer cells, zebrafish, and Apc(min/+) mice. Notably, axitinib dramatically induces Wnt asymmetry and nonrandom DNA segregation in cancer cells by promoting nuclear ß-catenin degradation independent of the GSK3ß (glycogen synthase kinase3ß)/APC (adenomatous polyposis coli) complex. Using a DARTS (drug affinity-responsive target stability) assay coupled to 2D-DIGE (2D difference in gel electrophoresis) and mass spectrometry, we have identified the E3 ubiquitin ligase SHPRH (SNF2, histone-linker, PHD and RING finger domain-containing helicase) as the direct target of axitinib in blocking Wnt/ß-catenin signaling. Treatment with axitinib stabilizes SHPRH and thereby increases the ubiquitination and degradation of ß-catenin. Our findings suggest a previously unreported mechanism of nuclear ß-catenin regulation and indicate that axitinib, a clinically approved drug, would provide therapeutic benefits for cancer patients with aberrant nuclear ß-catenin activation.

Regulation of Human Endonuclease V Activity and Relocalization to Cytoplasmic Stress Granules.

Endonuclease V (EndoV) is an enzyme with specificity for inosines in nucleic acids. Whereas the bacterial homologs are active on both DNA and RNA, the mammalian variants only cleave RNA, at least when assayed with recombinant proteins. Here we show that ectopically expressed, as well as endogenously expressed human (h)EndoV, share the same enzymatic properties as the recombinant protein and cleaves RNA with inosine but not DNA. In search for proteins interacting with hEndoV, polyadenylate-binding protein C1 (PABPC1) was identified. The association between PABPC1 and hEndoV is RNA dependent and furthermore, PABPC1 stimulates hEndoV activity and affinity for inosine-containing RNA. Upon cellular stress, PABPC1 relocates to cytoplasmic stress granules that are multimolecular aggregates of stalled translation initiation complexes formed to aid cell recovery. Arsenite, as well as other agents, triggered relocalization also of hEndoV to cytoplasmic stress granules. As inosines in RNA are highly abundant, hEndoV activity is likely regulated in cells to avoid aberrant cleavage of inosine-containing transcripts. Indeed, we find that hEndoV cleavage is inhibited by normal intracellular ATP concentrations. The ATP stores inside a cell do not overlay stress granules and we suggest that hEndoV is redistributed to stress granules as a strategy to create a local environment low in ATP to permit hEndoV activity.

Crystal structure and MD simulation of mouse EndoV reveal wedge motif plasticity in this inosine-specific endonuclease.

Endonuclease V (EndoV) is an enzyme with specificity for deaminated adenosine (inosine) in nucleic acids. EndoV from Escherichia coli (EcEndoV) acts both on inosines in DNA and RNA, whereas the human homolog cleaves only at inosines in RNA. Inosines in DNA are mutagenic and the role of EndoV in DNA repair is well established. In contrast, the biological function of EndoV in RNA processing is largely unexplored. Here we have characterized a second mammalian EndoV homolog, mouse EndoV (mEndoV), and show that mEndoV shares the same RNA selectivity as human EndoV (hEndoV). Mouse EndoV cleaves the same inosine-containing substrates as hEndoV, but with reduced efficiencies. The crystal structure of mEndoV reveals a conformation different from the hEndoV and prokaryotic EndoV structures, particularly for the conserved tyrosine in the wedge motif, suggesting that this strand separating element has some flexibility. Molecular dynamics simulations of mouse and human EndoV reveal alternative conformations for the invariant tyrosine. The configuration of the active site, on the other hand, is very similar between the prokaryotic and mammalian versions of EndoV.

TRIM21 Immune Signaling Is More Sensitive to Antibody Affinity Than Its Neutralization Activity.

Ab-coated viruses can be detected in the cytosol by the FcR tripartite motif-containing 21 (TRIM21), which rapidly recruits the proteasomal machinery and triggers induction of immune signaling. As such, TRIM21 plays a key role in intracellular protection by targeting invading viruses for destruction and alerting the immune system. A hallmark of immunity is elicitation of a balanced response that is proportionate to the threat, to avoid unnecessary inflammation. In this article, we show how Ab affinity modulates TRIM21 immune function. We constructed a humanized monoclonal IgG1 against human adenovirus type 5 (AdV5) and a panel of Fc-engineered variants with a wide range of affinities for TRIM21. We found that IgG1-coated viral particles were neutralized via TRIM21, even when affinity was reduced by as much as 100-fold. In contrast, induction of NF-κB signaling was more sensitive to reduced affinity between TRIM21 and the Ab variants. Thus, TRIM21 mediates neutralization under suboptimal conditions, whereas induction of immune signaling is balanced according to the functional affinity for the incoming immune stimuli. Our findings have implications for engineering of antiviral IgG therapeutics with tailored effector functions.

Synthetic Routes to N-9 Alkylated 8-Oxoguanines; Weak Inhibitors of the Human DNA Glycosylase OGG1.

The human 8-oxoguanine DNA glycosylase OGG1 is involved in base excision repair (BER), one of several DNA repair mechanisms that may counteract the effects of chemo- and radiation therapy for the treatment of cancer. We envisage that potent inhibitors of OGG1 may be found among the 9-alkyl-8-oxoguanines. Thus we explored synthetic routes to 8-oxoguanines and examined these as OGG1 inhibitors. The best reaction sequence started from 6-chloroguanine and involved N-9 alkylation, C-8 bromination, and finally simultaneous hydrolysis of both halides. Bromination before N-alkylation should only be considered when the N-substituent is not compatible with bromination conditions. The 8-oxoguanines were found to be weak inhibitors of OGG1. 6-Chloro-8-oxopurines, byproducts in the hydrolysis of 2,6-halopurines, turned out to be slightly better inhibitors than the corresponding 8-oxoguanines.

Structural basis for incision at deaminated adenines in DNA and RNA by endonuclease V.

Deamination of the exocyclic amines in adenine, guanine and cytosine forms base lesions that may lead to mutations if not removed by DNA repair proteins. Prokaryotic endonuclease V (EndoV/Nfi) has long been known to incise DNA 3' to a variety of base lesions, including deaminated adenine, guanine and cytosine. Biochemical and genetic data implicate that EndoV is involved in repair of these deaminated bases. In contrast to DNA glycosylases that remove a series of modified/damaged bases in DNA by direct excision of the nucleobase, EndoV cleaves the DNA sugar phosphate backbone at the second phosphodiester 3' to the lesion without removing the deaminated base. Structural investigation of this unusual incision by EndoV has unravelled an enzyme with separate base lesion and active site pockets. A novel wedge motif was identified as a DNA strand-separation feature important for damage detection. Human EndoV appears inactive on DNA, but has been shown to incise various RNA substrates containing inosine. Inosine is the deamination product of adenosine and is frequently found in RNA. The structural basis for discrimination between DNA and RNA by human EndoV remains elusive.

Molecular basis of calpain cleavage and inactivation of the sodium-calcium exchanger 1 in heart failure.

Cardiac sodium (Na(+))-calcium (Ca(2+)) exchanger 1 (NCX1) is central to the maintenance of normal Ca(2+) homeostasis and contraction. Studies indicate that the Ca(2+)-activated protease calpain cleaves NCX1. We hypothesized that calpain is an important regulator of NCX1 in response to pressure overload and aimed to identify molecular mechanisms and functional consequences of calpain binding and cleavage of NCX1 in the heart. NCX1 full-length protein and a 75-kDa NCX1 fragment along with calpain were up-regulated in aortic stenosis patients and rats with heart failure. Patients with coronary artery disease and sham-operated rats were used as controls. Calpain co-localized, co-fractionated, and co-immunoprecipitated with NCX1 in rat cardiomyocytes and left ventricle lysate. Immunoprecipitations, pull-down experiments, and extensive use of peptide arrays indicated that calpain domain III anchored to the first Ca(2+) binding domain in NCX1, whereas the calpain catalytic region bound to the catenin-like domain in NCX1. The use of bioinformatics, mutational analyses, a substrate competitor peptide, and a specific NCX1-Met(369) antibody identified a novel calpain cleavage site at Met(369). Engineering NCX1-Met(369) into a tobacco etch virus protease cleavage site revealed that specific cleavage at Met(369) inhibited NCX1 activity (both forward and reverse mode). Finally, a short peptide fragment containing the NCX1-Met(369) cleavage site was modeled into the narrow active cleft of human calpain. Inhibition of NCX1 activity, such as we have observed here following calpain-induced NCX1 cleavage, might be beneficial in pathophysiological conditions where increased NCX1 activity contributes to cardiac dysfunction.

Inosine in DNA and RNA.

Deamination of the nucleobases in DNA and RNA is a result of spontaneous hydrolysis, endogenous or environmental factors as well as deaminase enzymes. Adenosine is deaminated to inosine which is miscoding and preferentially base pairs with cytosine. In the case of DNA, this is a premutagenic event that is counteracted by DNA repair enzymes specifically engaged in recognition and removal of inosine. However, in RNA, inosine is an essential modification introduced by specialized enzymes in a highly regulated manner to generate transcriptome diversity. Defect editing is seen in various human disease including cancer, viral infections and neurological and psychiatric disorders. Enzymes catalyzing the deaminase reaction are well characterized and recently an unexpected function of Endonuclease V in RNA processing was revealed. Whereas bacterial Endonuclease V enzymes are classified as DNA repair enzymes, it appears that the mammalian enzymes are involved in processing of inosine in RNA. This yields an interesting yet unexplored, link between DNA and RNA processing. Further work is needed to gain understanding of the impact of inosine in DNA and RNA under normal physiology and disease progression.

A dominant STIM1 mutation causes Stormorken syndrome.

Stormorken syndrome is a rare autosomal-dominant disease with mild bleeding tendency, thrombocytopathy, thrombocytopenia, mild anemia, asplenia, tubular aggregate myopathy, miosis, headache, and ichthyosis. A heterozygous missense mutation in STIM1 exon 7 (c.910C>T; p.Arg304Trp) (NM_003156.3) was found to segregate with the disease in six Stormorken syndrome patients in four families. Upon sensing Ca(2+) depletion in the endoplasmic reticulum lumen, STIM1 undergoes a conformational change enabling it to interact with and open ORAI1, a Ca(2+) release-activated Ca(2+) channel located in the plasma membrane. The STIM1 mutation found in Stormorken syndrome patients is located in the coiled-coil 1 domain, which might play a role in keeping STIM1 inactive. In agreement with a possible gain-of-function mutation in STIM1, blood platelets from patients were in a preactivated state with high exposure of aminophospholipids on the outer surface of the plasma membrane. Resting Ca(2+) levels were elevated in platelets from the patients compared with controls, and store-operated Ca(2+) entry was markedly attenuated, further supporting constitutive activity of STIM1 and ORAI1. Thus, our data are compatible with a near-maximal activation of STIM1 in Stormorken syndrome patients. We conclude that the heterozygous mutation c.910C>T causes the complex phenotype that defines this syndrome.

Endonuclease V cleaves at inosines in RNA.

Endonuclease V orthologues are highly conserved proteins found in all kingdoms of life. While the prokaryotic enzymes are DNA repair proteins for removal of deaminated adenosine (inosine) from the genome, no clear role for the eukaryotic counterparts has hitherto been described. Here we report that human endonuclease V (ENDOV) and also Escherichia coli endonuclease V are highly active ribonucleases specific for inosine in RNA. Inosines are normal residues in certain RNAs introduced by specific deaminases. Adenosine-to-inosine editing is essential for proper function of these transcripts and defects are linked to various human disease. Here we show that human ENDOV cleaves an RNA substrate containing inosine in a position corresponding to a biologically important site for deamination in the Gabra-3 transcript of the GABA(A) neurotransmitter. Further, human ENDOV specifically incises transfer RNAs with inosine in the wobble position. This previously unknown RNA incision activity may suggest a role for endonuclease V in normal RNA metabolism.

Genotype-phenotype analysis of S326C OGG1 polymorphism: a risk factor for oxidative pathologies.

8-Oxoguanine DNA glycosylase (OGG) activity was measured by an in vitro assay in lymphocytes of healthy volunteers genotyped for various OGG1 polymorphisms. Only homozygous carriers of the polymorphic C326 allele showed a significantly lower OGG activity compared to the homozygous S326 genotype. The purified S326C OGG1 showed a decreased ability to complete the repair synthesis step in a base excision repair reaction reconstituted in vitro. The propensity of this variant to dimerize as well as its catalytic impairment were shown to be enhanced under oxidizing conditions. Mass spectrometry revealed that the extra cysteine of the variant protein is involved in disulfide bonds compatible with significant conformational changes and/or dimerization. We propose that the S326C OGG1 catalytic impairment and its susceptibility to dimerization and disulfide bond formation in an oxidizing environment all concur to decrease repair capacity. Consequently, the C326 homozygous carriers may be at increased risk of oxidative pathologies.

Single transmembrane peptide DinQ modulates membrane-dependent activities.

The functions of several SOS regulated genes in Escherichia coli are still unknown, including dinQ. In this work we characterize dinQ and two small RNAs, agrA and agrB, with antisense complementarity to dinQ. Northern analysis revealed five dinQ transcripts, but only one transcript (+44) is actively translated. The +44 dinQ transcript translates into a toxic single transmembrane peptide localized in the inner membrane. AgrB regulates dinQ RNA by RNA interference to counteract DinQ toxicity. Thus the dinQ-agr locus shows the classical features of a type I TA system and has many similarities to the tisB-istR locus. DinQ overexpression depolarizes the cell membrane and decreases the intracellular ATP concentration, demonstrating that DinQ can modulate membrane-dependent processes. Augmented DinQ strongly inhibits marker transfer by Hfr conjugation, indicating a role in recombination. Furthermore, DinQ affects transformation of nucleoid morphology in response to UV damage. We hypothesize that DinQ is a transmembrane peptide that modulates membrane-dependent activities such as nucleoid compaction and recombination.

The human homolog of Escherichia coli endonuclease V is a nucleolar protein with affinity for branched DNA structures.

Loss of amino groups from adenines in DNA results in the formation of hypoxanthine (Hx) bases with miscoding properties. The primary enzyme in Escherichia coli for DNA repair initiation at deaminated adenine is endonuclease V (endoV), encoded by the nfi gene, which cleaves the second phosphodiester bond 3' of an Hx lesion. Endonuclease V orthologs are widespread in nature and belong to a family of highly conserved proteins. Whereas prokaryotic endoV enzymes are well characterized, the function of the eukaryotic homologs remains obscure. Here we describe the human endoV ortholog and show with bioinformatics and experimental analysis that a large number of transcript variants exist for the human endonuclease V gene (ENDOV), many of which are unlikely to be translated into functional protein. Full-length ENDOV is encoded by 8 evolutionary conserved exons covering the core region of the enzyme, in addition to one or more 3'-exons encoding an unstructured and poorly conserved C-terminus. In contrast to the E. coli enzyme, we find recombinant ENDOV neither to incise nor bind Hx-containing DNA. While both enzymes have strong affinity for several branched DNA substrates, cleavage is observed only with E. coli endoV. We find that ENDOV is localized in the cytoplasm and nucleoli of human cells. As nucleoli harbor the rRNA genes, this may suggest a role for the protein in rRNA gene transactions such as DNA replication or RNA transcription.

Modeling the impact of mitochondrial DNA damage in forebrain neurons and beyond.

We have generated an inducible transgenic mouse model, which expresses a mutated version of UNG1 (mutUNG1) that removes thymine, in addition to uracil from mitochondrial DNA. The abasic-sites (AP-sites) generated by removal of thymine or uracil are a threat to genomic integrity, and are particularly harmful in mitochondria due to inhibition of mitochondrial DNA polymerase. MutUNG1, accompanied by a luciferase reporter-gene, is controlled by the Tet-on system. Transgene expression is spatially regulated by the forebrain specific CaMKIIα-promoter, and temporally by the addition of doxycycline. Mice harboring this transgene develop compromised mitochondrial dynamics, neurodegeneration and impaired behavior.