Mechanisms of Regulation and Diverse Activities of Tau-Tubulin Kinase (TTBK) Isoforms.

Tau-tubulin kinase 1 (TTBK1) is a CNS-specific, kinase that has been implicated in the pathological phosphorylation of tau in Alzheimer's Disease (AD) and Frontotemporal Dementia (FTD). TTBK1 is a challenging therapeutic target because it shares a highly conserved catalytic domain with its homolog, TTBK2, a ubiquitously expressed kinase genetically linked to the disease spinocerebellar ataxia type 11. The present study attempts to elucidate the functional distinctions between the TTBK isoforms and increase our understanding of them as distinct targets for the treatment of neurodegenerative disease. We demonstrate that in cortical neurons, TTBK1, not TTBK2, is the isoform responsible for tau phosphorylation at epitopes enriched in tauopathies such as Serine 422. In addition, although our elucidation of the crystal structure of the TTBK2 kinase domain indicates almost identical structural similarity with TTBK1, biochemical and cellular assays demonstrate that the enzymatic activity of these two proteins is regulated by a combination of unique extra-catalytic sequences and autophosphorylation events. Finally, we have identified an unbiased list of neuronal interactors and phosphorylation substrates for TTBK1 and TTBK2 that highlight the unique cellular pathways and functional networks that each isoform is involved in. This data address an important gap in knowledge regarding the implications of targeting TTBK kinases and may prove valuable in the development of potential therapies for disease.

Hybridization Liquid Chromatography-Tandem Mass Spectrometry: An Alternative Bioanalytical Method for Antisense Oligonucleotide Quantitation in Plasma and Tissue Samples.

Quantitative bioanalysis in plasma and tissues samples is required to study the pharmacokinetic and pharmacodynamic properties of antisense oligonucleotides (ASOs). To overcome intrinsic drawbacks in specificity, sensitivity, and throughput of traditional ligand-binding assay (LBA) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods, an alternative bioanalytical method was developed by combining oligonucleotide hybridization and LC-MS/MS technologies. Target ASOs were extracted from biological samples by hybridization with biotinylated sense-strand oligonucleotides coupled to streptavidin magnetic beads. Using ion-pairing chromatography and tandem mass spectrometry, this method demonstrated high sensitivity (0.5 ng/mL using 100 µL of plasma), high specificity, wide linear range, complete automation, and generic applications in tests with multiple ASOs. The typical challenge of sensitivity drop in traditional ion-pairing LC-MS/MS was for the first time overcome by the introduction of a ternary pump system. Due to the high specificity, quantitation in various biological matrixes was achieved using calibration standards in plasma, largely improving efficiency and consistency. Another major advantage was the capability of simultaneous quantitation of ASO metabolites. The hybridization LC-MS/MS was considered an improved alternative for quantitation of ASOs and metabolites in plasma and tissue samples, showing a great potential to replace traditional LBA and LC-MS/MS methods.

The Tec Kinase-Regulated Phosphoproteome Reveals a Mechanism for the Regulation of Inhibitory Signals in Murine Macrophages.

Previous work has shown conflicting roles for Tec family kinases in regulation of TLR-dependent signaling in myeloid cells. In the present study, we performed a detailed investigation of the role of the Tec kinases Btk and Tec kinases in regulating TLR signaling in several types of primary murine macrophages. We demonstrate that primary resident peritoneal macrophages deficient for Btk and Tec secrete less proinflammatory cytokines in response to TLR stimulation than do wild-type cells. In contrast, we found that bone marrow-derived and thioglycollate-elicited peritoneal macrophages deficient for Btk and Tec secrete more proinflammatory cytokines than do wild-type cells. We then compared the phosphoproteome regulated by Tec kinases and LPS in primary peritoneal and bone marrow-derived macrophages. From this analysis we determined that Tec kinases regulate different signaling programs in these cell types. In additional studies using bone marrow-derived macrophages, we found that Tec and Btk promote phosphorylation events necessary for immunoreceptor-mediated inhibition of TLR signaling. Taken together, our results are consistent with a model where Tec kinases (Btk, Tec, Bmx) are required for TLR-dependent signaling in many types of myeloid cells. However, our data also support a cell type-specific TLR inhibitory role for Btk and Tec that is mediated by immunoreceptor activation and signaling via PI3K.

B cell-specific lentiviral gene therapy leads to sustained B-cell functional recovery in a murine model of X-linked agammaglobulinemia.

The immunodeficiency disorder, X-linked agammaglobulinemia (XLA), results from mutations in the gene encoding Bruton tyrosine kinase (Btk). Btk is required for pre-B cell clonal expansion and B-cell antigen receptor signaling. XLA patients lack mature B cells and immunoglobulin and experience recurrent bacterial infections only partially mitigated by life-long antibody replacement therapy. In pursuit of definitive therapy for XLA, we tested ex vivo gene therapy using a lentiviral vector (LV) containing the immunoglobulin enhancer (Emu) and Igbeta (B29) minimal promoter to drive B lineage-specific human Btk expression in Btk/Tec(-/-) mice, a strain that reproduces the features of human XLA. After transplantation of EmuB29-Btk-LV-transduced stem cells, treated mice showed significant, albeit incomplete, rescue of mature B cells in the bone marrow, peripheral blood, spleen, and peritoneal cavity, and improved responses to T-independent and T-dependent antigens. LV-treated B cells exhibited enhanced B-cell antigen receptor signaling and an in vivo selective advantage in the peripheral versus central B-cell compartment. Secondary transplantation showed sustained Btk expression, viral integration, and partial functional responses, consistent with long-term stem cell marking; and serial transplantation revealed no evidence for cellular or systemic toxicity. These findings strongly support pursuit of B lineage-targeted LV gene therapy in human XLA.

Development of B-lineage predominant lentiviral vectors for use in genetic therapies for B cell disorders.

Sustained, targeted, high-level transgene expression in primary B lymphocytes may be useful for gene therapy in B cell disorders. We developed several candidate B-lineage predominant self-inactivating lentiviral vectors (LV) containing alternative enhancer/promoter elements including: the immunoglobulin ß (Igß) (B29) promoter combined with the immunoglobulin µ enhancer (EµB29); and the endogenous BTK promoter with or without Eµ (EµBtkp or Btkp). LV-driven enhanced green fluorescent protein (eGFP) reporter expression was evaluated in cell lines and primary cells derived from human or murine hematopoietic stem cells (HSC). In murine primary cells, EµB29 and EµBtkp LV-mediated high-level expression in immature and mature B cells compared with all other lineages. Expression increased with B cell maturation and was maintained in peripheral subsets. Expression in T and myeloid cells was much lower in percentage and intensity. Similarly, both EµB29 and EµBtkp LV exhibited high-level activity in human primary B cells. In contrast to EµB29, Btkp and EµBtkp LV also exhibited modest activity in myeloid cells, consistent with the expression profile of endogenous Bruton's tyrosine kinase (Btk). Notably, EµB29 and EµBtkp activity was superior in all expression models to an alternative, B-lineage targeted vector containing the EµS.CD19 enhancer/promoter. In summary, EµB29 and EµBtkp LV comprise efficient delivery platforms for gene expression in B-lineage cells.

The distinct response of gammadelta T cells to the Nod2 agonist muramyl dipeptide.

Purified gammadelta T cells are primed directly in response to pathogen associated molecular patterns (PAMPs) to better respond to secondary signals and increase expression of chemokine and activation-related genes. Transcripts encoding the innate receptor Nod2 were detected in bovine and human gammadelta T cells. Nod2 is the intracellular receptor for muramyl dipeptide (MDP), functions in regulating innate activities, and was thought to be expressed primarily in APCs. The response of gammadelta T cells to MDP was analyzed by microarray, Q-PCR, proteome array and functional priming assays. MDP had a consistent priming effect on gammadelta T cells, characterized by changes in transcripts and enhanced proliferation response to secondary signaling. Knockdown experiments implicated Nod2 as the receptor for MDP in gammadelta T cell-enriched bovine PBLs. The results indicate priming of gammadelta T cells by MDP, and offer definitive evidence of the expression of functional Nod2 in gammadelta T cells.

Pristane-Accelerated Autoimmune Disease in (SWR X NZB) F1 Mice Leads to Prominent Tubulointerstitial Inflammation and Human Lupus Nephritis-Like Fibrosis.

Mouse models lupus nephritis (LN) have provided important insights into disease pathogenesis, although none have been able to recapitulate all features of the human disease. Using comprehensive longitudinal analyses, we characterized a novel accelerated mouse model of lupus using pristane treatment in SNF1 (SWR X NZB F1) lupus prone mice (pristane-SNF1 mice). Pristane treatment in SNF1 mice accelerated the onset and progression of proteinuria, autoantibody production, immune complex deposition and development of renal lesions. At week 14, the pristane-SNF1 model recapitulated kidney disease parameters and molecular signatures seen in spontaneous disease in 36 week-old SNF1 mice and in a traditional IFNα-accelerated NZB X NZW F1 (BWF1) model. Blood transcriptome analysis revealed interferon, plasma cell, neutrophil, T-cell and protein synthesis signatures in the pristane-SNF1 model, all known to be present in the human disease. The pristane-SNF1 model appears to be particularly useful for preclinical research, robustly exhibiting many characteristics reminiscent of human disease. These include i) a stronger upregulation of the cytosolic nucleic acid sensing pathway, which is thought to be key component of the pathogenesis of the human disease, and ii) more prominent kidney interstitial inflammation and fibrosis, which have been both associated with poor prognosis in human LN. To our knowledge, this is the only accelerated model of LN that exhibits a robust tubulointerstitial inflammatory and fibrosis response. Taken together our data show that the pristane-SNF1 model is a novel accelerated model of LN with key features similar to human disease.

Anti-BDCA2 monoclonal antibody inhibits plasmacytoid dendritic cell activation through Fc-dependent and Fc-independent mechanisms.

Type I interferons (IFN-I) are implicated in the pathogenesis of systemic lupus erythematosus (SLE). In SLE, immune complexes bind to the CD32a (FcγRIIa) receptor on the surface of plasmacytoid dendritic cells (pDCs) and stimulate the secretion of IFN-I from pDCs. BDCA2 is a pDC-specific receptor that, when engaged, inhibits the production of IFN-I in human pDCs. BDCA2 engagement, therefore, represents an attractive therapeutic target for inhibiting pDC-derived IFN-I and may be an effective therapy for the treatment of SLE. In this study, we show that 24F4A, a humanized monoclonal antibody (mAb) against BDCA2, engages BDCA2 and leads to its internalization and the consequent inhibition of TLR-induced IFN-I by pDCs in vitro using blood from both healthy and SLE donors. These effects were confirmed in vivo using a single injection of 24F4A in cynomolgus monkeys. 24F4A also inhibited pDC activation by SLE-associated immune complexes (IC). In addition to the inhibitory effect of 24F4A through engagement of BDCA2, the Fc region of 24F4A was critical for potent inhibition of IC-induced IFN-I production through internalization of CD32a. This study highlights the novel therapeutic potential of an effector-competent anti-BDCA2 mAb that demonstrates a dual mechanism to dampen pDC responses for enhanced clinical efficacy in SLE.

Mucosal lymphatic-derived gammadelta T cells respond early to experimental Salmonella enterocolitis by increasing expression of IL-2R alpha.

To better understand the roles of gammadelta T cells in mucosal infection, we utilized Salmonella enterica serovar Typhimurium (Salmonella serovar Typhimurium) infection in cattle as it closely approximates Salmonella serovar Typhimurium-induced enterocolitis in humans. Protein and gene expression in alphabeta and gammadelta T cells derived from lymphatic ducts draining the gut mucosa in Salmonella serovar Typhimurium-infected calves were analyzed. In calves with enterocolitis, general gene expression trends in gammadelta T cells suggested subtle activation and innate response, whereas alphabeta T cells were relatively quiescent following Salmonella serovar Typhimurium infection. An increase in IL-2R alpha expression on gammadelta T cells from infected calves and results from in vitro assays suggested that gammadelta T cells were primed by Salmonella serovar Typhimurium LPS to better respond to IL-2 and IL-15. Together with gene expression trends in vivo, these data support early priming activation of target tissue gammadelta T cells during Salmonella serovar Typhimurium infection.