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Live, attenuated vaccines generate humoral and cellular immune memory, increasing the duration of protective immune memory. We previously found that antigens derived from vaccination or viral infection persist within lymphatic endothelial cells (LECs) beyond the clearance of the infection, a process we termed “antigen archiving”. Technical limitations of fluorescent labeling have precluded a complete picture of antigen archiving across cell types in the lymph node. We developed a “molecular tracking device” to follow the distribution, acquisition, and retention of antigen in the lymph node. We immunized mice with an antigen conjugated to a nuclease-resistant DNA tag and used single-cell mRNA sequencing to quantify its abundance in lymph node hematopoietic and non-hematopoietic cell types. At early and late time points after vaccination we found antigen acquisition by dendritic cell populations (DCs), associated expression of genes involved in DC activation and antigen processing, and antigen acquisition and archiving by LECs as well as unexpected stromal cell types. Variable antigen levels in LECs enabled the identification of caveolar endocytosis as a mechanism of antigen acquisition or retention. Molecular tracking devices enable new approaches to study dynamic tissue dissemination of antigens and identify new mechanisms of antigen acquisition and retention at cellular resolution in vivo.

We report the identification by in vitro selection of Zn(2+)/Mn(2+)-dependent deoxyribozymes that glycosylate the 3'-OH of a DNA oligonucleotide. Both β and α anomers of aryl glycosides can be used as the glycosyl donors. Individual deoxyribozymes are each specific for a particular donor anomer.

T4 polynucleotide kinase is widely used for 5'-phosphorylation of DNA and RNA oligonucleotide termini, but no natural protein enzyme is capable of 3'-phosphorylation. Here, we report the in vitro selection of deoxyribozymes (DNA enzymes) capable of DNA oligonucleotide 3'-phosphorylation, using a 5'-triphosphorylated RNA transcript (pppRNA) as the phosphoryl donor. The basis of selection was the capture, during each selection round, of the 3'-phosphorylated DNA substrate terminus by 2-methylimidazole activation of the 3'-phosphate (forming 3'-MeImp) and subsequent splint ligation with a 5'-amino DNA oligonucleotide. Competing and precedented DNA-catalyzed reactions were DNA phosphodiester hydrolysis or deglycosylation, each also leading to a 3'-phosphate but at a different nucleotide position within the DNA substrate. One oligonucleotide 3'-kinase deoxyribozyme, obtained from an N40 random pool and named 3'Kin1, can 3'-phosphorylate nearly any DNA oligonucleotide substrate for which the 3'-terminus has the sequence motif 5'-NKR-3', where N denotes any oligonucleotide sequence, K = T or G, and R = A or G. These results establish the viabilty of in vitro selection for identifying DNA enzymes that 3'-phosphorylate DNA oligonucleotides.

Tau-tubulin kinase-1 (TTBK1) is involved in phosphorylation of tau protein at specific Serine/Threonine residues found in paired helical filaments, suggesting its role in tauopathy pathogenesis. We found that TTBK1 levels were upregulated in brains of human Alzheimer' disease (AD) patients compared with age-matched non-AD controls. To understand the effects of TTBK1 activationin vivo, we developed transgenic mice harboring human full-length TTBK1 genomic DNA (TTBK1-Tg). Transgenic TTBK1 is highly expressed in subiculum and cortical pyramidal layers, and induces phosphorylated neurofilament aggregation. TTBK1-Tg mice show significant age-dependent memory impairment as determined by radial arm water maze test, which is associated with enhancement of tau and neurofilament phosphorylation, increased levels of p25 and p35, both activators of cyclin-dependent protein kinase 5 (CDK5), enhanced calpain I activity, and reduced levels of hippocampal NMDA receptor types 2B (NR2B) and D. Enhanced CDK5/p35 complex formation is strongly correlated with dissociation of F-actin from p35, suggesting the inhibitory mechanism of CDK5/p35 complex formation by F-actin. Expression of recombinant TTBK1 in primary mouse cortical neurons significantly downregulated NR2B in a CDK5- and calpain-dependent manner. These data suggest that TTBK1 in AD brain may be one of the underlying mechanisms inducing CDK5 and calpain activation, NR2B downregulation, and subsequent memory dysfunction.

As part of the innate immune defense against HIV infection, OTK18, a zinc finger protein, is upregulated in human macrophages and reduces viral replication through suppression of viral long-terminal repeat promoter activity. Although we know that the processing products of OTK18 accumulate in the cytoplasm of brain perivascular macrophages in advanced HIV encephalitis cases, the molecular mechanisms behind its post-translational processing are still poorly understood. To characterize OTK18 processing, we assessed a panel of protease inhibitors to identify the candidates involved in the OTK18 processing using human monocyte-derived macrophages (MDM) over-expressing OTK18 by recombinant adenoviral gene transfer. Viral infection of MDM strongly increased the processing of OTK18 into its N-terminal fragment. Treatment of OTK18-expressing MDM with calpain- and proteasome- inhibitors significantly accumulated either full-length or processed OTK18 fragments in time and dose-dependent manners. A series of OTK18 truncation mutants and synthetic peptides were tested to locate the calpain cleavage sites after arginine 359. Finally, we developed an enhanced cyan and yellow fluorescent protein (ECFP and EYFP)-based intramolecular fluorescent resonance energy transfer (intramolecular FRET) system to monitor the OTK18 endoproteolysis in human microglia cell line. Inhibition of proteasome activity significantly increased the intramolecular FRET signal in the nucleus. These data suggest that calpain and proteasome are involved in OTK18 endoproteolysis and degradation. Additionally, intramolecular FRET has proven to be a useful tool for monitoring the processing in live cells.

Deoxyribozymes (DNA enzymes) have been developed for a growing variety of chemical reactions, including with peptide substrates. We recently described the first tyrosine kinase deoxyribozymes, which lacked the ability to discriminate among peptide substrates on the basis of the amino acids surrounding the tyrosine residue. Those deoxyribozymes were identified by in vitro selection using a DNA-anchored peptide substrate in which the residues neighboring tyrosine were all alanine. Here, we performed in vitro selection for tyrosine kinase activity using three peptide substrates in which the neighboring residues included a variety of side chains. For one of these three peptides, we found numerous deoxyribozymes that discriminate strongly in favor of phosphorylating tyrosine when the surrounding residues are specifically those used in the selection process. Three different short peptide sequence motifs of 2-4 amino acids were required for catalysis by three unique deoxyribozymes. For a second peptide substrate, the selection process led to one deoxyribozyme which exhibits partial discrimination among peptide sequences. These findings establish the feasibility of identifying DNA enzymes that catalyze sequence-selective tyrosine phosphorylation, which suggests the downstream practical utility of such deoxyribozymes. More broadly, this outcome reinforces the conclusion that nucleic acid catalysts can discriminate among peptide substrates in the context of biochemically relevant reactions.

We show that DNA catalysts (deoxyribozymes, DNA enzymes) can phosphorylate tyrosine residues of peptides. Using in vitro selection, we identified deoxyribozymes that transfer the γ-phosphoryl group from a 5'-triphosphorylated donor (a pppRNA oligonucleotide or GTP) to the tyrosine hydroxyl acceptor of a tethered hexapeptide. Tyrosine kinase deoxyribozymes that use pppRNA were identified from each of N30, N40, and N50 random-sequence pools. Each deoxyribozyme requires Zn(2+), and most additionally require Mn(2+). The deoxyribozymes have little or no selectivity for the amino acid identities near the tyrosine, but they are highly selective for phosphorylating tyrosine rather than serine. Analogous GTP-dependent DNA catalysts were identified and found to have apparent Km(GTP) as low as ∼20 μM. These findings establish that DNA has the fundamental catalytic ability to phosphorylate the tyrosine side chain of a peptide substrate.

Vaccination therapy of AD animal models and patients strongly suggests an active role of brain mononuclear phagocytes in immune-mediated clearance of amyloid-β peptides (Aβ) in brain. Although Aβ uptake by macrophages can be regulated by pro- and anti-inflammatory cytokines, their effects on macrophage-mediated Aβ degradation are poorly understood. To better understand this mechanism of degradation, we examined whether pro- and anti-inflammatory cytokines affect the degradation of Aβ using primary cultured human monocyte-derived macrophages (MDM) and microglia using pulse-chase analysis of fibrillar and oligomer 125I-Aβ40 and Aβ42. Initial uptake of fibrillar Aβ40 and Aβ42 was 40% and its degradation was saturated by 120 h in both MDM and microglia, compared with an initial uptake of oligomeric Aβ less than 0.5% and saturation of degradation within 24 h. IFN-γ increased the intracellular retention of fibrillar Aβ40 and Aβ42 by inhibiting degradation, whereas IL-4, IL-10, and TGF-β1, but not IL-13 and IL-27, enhanced degradation. Fibrillar Aβ degradation in MDM is sensitive to lysosomal and insulin degrading enzyme inhibitors but insensitive to proteasomal and neprilysin inhibitors. IFN-γ and TNF-α directly reduced the expression of insulin degrading enzyme and chaperone molecules (heat shock protein 70 and heat shock cognate protein 70), which are involved in refolding of aggregated proteins. Coculture of MDM with activated, but not naive T cells, suppressed Aβ degradation in MDM, which was partially blocked by a combination of neutralizing Abs against proinflammatory cytokines. These data suggest that proinflammatory cytokines suppress Aβ degradation in MDM, whereas select anti-inflammatory and regulatory cytokines antagonize these effects.

Correction for ‘DNA-catalyzed glycosylation using aryl glycoside donors’ by Anthony R. Hesser et al., Chem. Commun., 2016, 52, 9259–9262.

beta-Amyloid (Abeta) binding affinities and specificities for six bis-styrylbenzenes with multiple magnetically equivalent fluorine atoms in the form of a tetrafluorophenyl core or symmetrical trifluoromethyl and trifluoromethoxy groups were determined by means of fluorescence titrations with amyloid peptide Abeta1-40 and a novel in vitro fluorescence-based assay using APP/PS1 transgenic mouse brain sections. Bis-styrylbenzenes with a tetrafluorophenyl core had increased Abeta binding affinities compared to their monofluorophenyl or phenyl counterparts. Bis-styrylbenzenes with carboxylic acid functional groups had lower Abeta binding affinities than their neutral counterparts. Selected bis-styrylbenzenes were demonstrated to have good blood-brain barrier penetration capabilities. These data extend the SAR of bis-styrylbenzene Abeta binding and provide direction for the development of a noninvasive probe for early detection of Alzheimer's disease using 19F MRI.

Reactive astrocytes and microglia in Alzheimer's disease surround amyloid plaques and secrete proinflammatory cytokines that affect neuronal function. Relationship between cytokine signaling and amyloid-beta peptide (Abeta) accumulation is poorly understood. Thus, we generated a novel Swedish beta-amyloid precursor protein mutant (APP) transgenic mouse in which the interferon (IFN)-gamma receptor type I was knocked out (APP/GRKO). IFN-gamma signaling loss in the APP/GRKO mice reduced gliosis and amyloid plaques at 14 months of age. Aggregated Abeta induced IFN-gamma production from co-culture of astrocytes and microglia, and IFN-gamma elicited tumor necrosis factor (TNF)-alpha secretion in wild type (WT) but not GRKO microglia co-cultured with astrocytes. Both IFN-gamma and TNF-alpha enhanced Abeta production from APP-expressing astrocytes and cortical neurons. TNF-alpha directly stimulated beta-site APP-cleaving enzyme (BACE1) expression and enhanced beta-processing of APP in astrocytes. The numbers of reactive astrocytes expressing BACE1 were increased in APP compared with APP/GRKO mice in both cortex and hippocampus. IFN-gamma and TNF-alpha activation of WT microglia suppressed Abeta degradation, whereas GRKO microglia had no changes. These results support the idea that glial IFN-gamma and TNF-alpha enhance Abeta deposition through BACE1 expression and suppression of Abeta clearance. Taken together, these observations suggest that proinflammatory cytokines are directly linked to Alzheimer's disease pathogenesis.

Accumulating evidence suggests that bone-marrow (BM)-derived mononuclear phagocytes have an important role in the clearance of soluble and aggregated amyloid-beta peptides (Abeta) in Alzheimer's disease (AD) brains. However, the exact kinetics of Abeta clearance in mononuclear phagocytes derived from transgenic animal models of AD expressing beta-amyloid precursor protein (APP) mutants have been poorly characterized. We have examined whether CCL2 and APP expression affects the clearance of Abeta in conjunction with our control, acetylated low-density lipoprotein (AcLDL), using primary cultured BM-derived macrophages derived from adult APP, CCL2, APP/CCL2, and control littermates. Pulse-chase analysis demonstrated three distinct destinations for Abeta40 and AcLDL: intracellular retention, degradation, and secretion. As predicted, 50% of Abeta remained intracellularly contained even 5 days after pulse, while 40% of degraded and 14% of nondegraded Abeta were secreted. APP/CCL2 macrophages show reduced intracellular Abeta retention, along with enhanced secretion of both degraded and nondegraded Abeta. Abeta accumulation in aggresome is also partially reduced in APP/CCL2 macrophages as compared to other APP, CCL2, or control groups, suggesting impaired sorting of aggregated Abeta in aggresomes. The degradation of intracranially injected (125)I-Abeta40 aggregates was also enhanced in adult APP/CCL2 mice as compared to APP littermates in vivo. These data suggest that APP and CCL2 synergistically enhance BM-derived macrophage-mediated clearance of Abeta. In contrast, the clearance of AcLDL by BM-derived macrophages was not significantly enhanced by the presence of either APP or CCL2.