WT Levels Research Articles

Inositol 1,4,5-trisphosphate receptor determines intracellular Ca2+ concentration in Trypanosoma cruzi throughout its life cycle.

Regulation of intracellular Ca2+ concentration ([Ca2+]i) is vital for eukaryotic organisms. Recently, we identified a Ca2+ channel (TcIP 3R) associated with intracellular Ca2+ stores in Trypanosoma cruzi, the parasitic protist that causes Chagas disease. In this study, we measured [Ca2+]i during the parasite life cycle and determined whether TcIP 3R is involved in the observed variations. Parasites expressing R‐GECO1, a red fluorescent, genetically encoded Ca2+ indicator for optical imaging that fluoresces when bound to Ca2+, were produced. Using these R‐GECO1‐expressing parasites to measure [Ca2+]i, we found that the [Ca2+]i in epimastigotes was significantly higher than that in trypomastigotes and lower than that in amastigotes, and we observed a positive correlation between TcIP 3 R mRNA expression and [Ca2+]i during the parasite life cycle both in vitro and in vivo. We also generated R‐GECO1‐expressing parasites with TcIP 3R expression levels that were approximately 65% of wild‐type (wt) levels (SKO parasites), and [Ca2+]i in the wt and SKO parasites was compared. The [Ca2+]i in SKO parasites was reduced to approximately 50–65% of that in wt parasites. These results show that TcIP 3R is the determinant of [Ca2+]i in T. cruzi. Since Ca2+ signaling is vital for these parasites, TcIP 3R is a promising drug target for Chagas disease.

Ligand dependent restoration of human TLR3 signaling and death in p53 mutant cells.

Diversity within the p53 transcriptional network can arise from a matrix of changes that include target response element sequences and p53 expression level variations. We previously found that wild type p53 (WT p53) can regulate expression of most innate immune-related Toll-like-receptor genes (TLRs) in human cells, thereby affecting immune responses. Since many tumor-associated p53 mutants exhibit change-of-spectrum transactivation from various p53 targets, we examined the ability of twenty-five p53 mutants to activate endogenous expression of the TLR gene family in p53 null human cancer cell lines following transfection with p53 mutant expression vectors. While many mutants retained the ability to drive TLR expression at WT levels, others exhibited null, limited, or change-of-spectrum transactivation of TLR genes. Using TLR3 signaling as a model, we show that some cancer-associated p53 mutants amplify cytokine, chemokine and apoptotic responses after stimulation by the cognate ligand poly(I:C). Furthermore, restoration of WT p53 activity for loss-of-function p53 mutants by the p53 reactivating drug RITA restored p53 regulation of TLR3 gene expression and enhanced DNA damage-induced apoptosis via TLR3 signaling. Overall, our findings have many implications for understanding the impact of WT and mutant p53 in immunological responses and cancer therapy.

Rhox13 is required for a quantitatively normal first wave of spermatogenesis in mice.

We previously described a novel germ cell-specific X-linked reproductive homeobox gene (Rhox13) that is upregulated at the level of translation in response to retinoic acid (RA) in differentiating spermatogonia and preleptotene spermatocytes. We hypothesize that RHOX13 plays an essential role in male germ cell differentiation, and have tested this by creating a Rhox13 gene knockout (KO) mouse. Rhox13 KO mice are born in expected Mendelian ratios, and adults have slightly reduced testis weights, yet a full complement of spermatogenic cell types. Young KO mice (at ~7-8 weeks of age) have a ≈50% reduction in epididymal sperm counts, but numbers increased to WT levels as the mice reach ~17 weeks of age. Histological analysis of testes from juvenile KO mice reveals a number of defects during the first wave of spermatogenesis. These include increased apoptosis, delayed appearance of round spermatids and disruption of the precise stage-specific association of germ cells within the seminiferous tubules. Breeding studies reveal that both young and aged KO males produce normal-sized litters. Taken together, our results indicate that RHOX13 is not essential for mouse fertility in a controlled laboratory setting, but that it is required for optimal development of differentiating germ cells and progression of the first wave of spermatogenesis.

Protein arginine methylation facilitates KCNQ channel-PIP2 interaction leading to seizure suppression.

KCNQ channels are critical determinants of neuronal excitability, thus emerging as a novel target of anti-epileptic drugs. To date, the mechanisms of KCNQ channel modulation have been mostly characterized to be inhibitory via Gq-coupled receptors, Ca(2+)/CaM, and protein kinase C. Here we demonstrate that methylation of KCNQ by protein arginine methyltransferase 1 (Prmt1) positively regulates KCNQ channel activity, thereby preventing neuronal hyperexcitability. Prmt1+/- mice exhibit epileptic seizures. Methylation of KCNQ2 channels at 4 arginine residues by Prmt1 enhances PIP2 binding, and Prmt1 depletion lowers PIP2 affinity of KCNQ2 channels and thereby the channel activities. Consistently, exogenous PIP2 addition to Prmt1+/- neurons restores KCNQ currents and neuronal excitability to the WT level. Collectively, we propose that Prmt1-dependent facilitation of KCNQ-PIP2 interaction underlies the positive regulation of KCNQ activity by arginine methylation, which may serve as a key target for prevention of neuronal hyperexcitability and seizures.

Protective and detrimental effects of neuroectodermal cell-derived tissue factor in mouse models of stroke.

Within the CNS, a dysregulated hemostatic response contributes to both hemorrhagic and ischemic strokes. Tissue factor (TF), the primary initiator of the extrinsic coagulation cascade, plays an essential role in hemostasis and also contributes to thrombosis. Using both genetic and pharmacologic approaches, we characterized the contribution of neuroectodermal (NE) cell TF to the pathophysiology of stroke. We used mice with various levels of TF expression and found that astrocyte TF activity reduced to ~5% of WT levels was still sufficient to maintain hemostasis after hemorrhagic stroke but was also low enough to attenuate inflammation, reduce damage to the blood-brain barrier, and improve outcomes following ischemic stroke. Pharmacologic inhibition of TF during the reperfusion phase of ischemic stroke attenuated neuronal damage, improved behavioral deficit, and prevented mortality of mice. Our data demonstrate that NE cell TF limits bleeding complications associated with the transition from ischemic to hemorrhagic stroke and also contributes to the reperfusion injury after ischemic stroke. The high level of TF expression in the CNS is likely the result of selective pressure to limit intracerebral hemorrhage (ICH) after traumatic brain injury but, in the modern era, poses the additional risk of increased ischemia-reperfusion injury after ischemic stroke.

Slamf6 negatively regulates autoimmunity

The nine SLAM family (Slamf) receptors are positive or negative regulators of adaptive and innate immune responses, and of several autoimmune diseases. Here we report that the transfer of Slamf6−/− B6 CD4+ T cells into co-isogenic bm12 mice causes SLE-like autoimmunity with elevated levels of autoantibodies. In addition, significantly higher percentages of Tfh cells and IFN-γ-producing CD4+ cells, as well as GC B cells were observed. Interestingly, the expression of the Slamf6-H1 isoform in Slamf6−/− CD4+ T cells did not induce this lupus-like phenotype. By contrast, Slamf1−/− or Slamf5−/− CD4+ T cells caused the same pathology as WT CD4+ T cells. As the transfer of Slamf [1+6]−/− or Slamf [1+5+6]−/− CD4+ T cells induced WT levels of autoantibodies, the presence of Slamf1 was requisite for the induction of increased levels of autoantibodies by Slamf6−/− CD4+ T cells. We conclude that Slamf6 functions as an inhibitory receptor that controls autoimmune responses.

Fitness Trade-Offs in Competence Differentiation of Bacillus subtilis.

In the stationary phase, Bacillus subtilis differentiates stochastically and transiently into the state of competence for transformation (K-state). The latter is associated with growth arrest, and it is unclear how the ability to develop competence is stably maintained, despite its cost. To quantify the effect differentiation has on the competitive fitness of B. subtilis, we characterized the competition dynamics between strains with different probabilities of entering the K-state. The relative fitness decreased with increasing differentiation probability both during the stationary phase and during outgrowth. When exposed to antibiotics inhibiting cell wall synthesis, transcription, and translation, cells that differentiated into the K-state showed a selective advantage compared to differentiation-deficient bacteria; this benefit did not require transformation. Although beneficial, the K-state was not induced by sub-MIC concentrations of antibiotics. Increasing the differentiation probability beyond the wt level did not significantly affect the competition dynamics with transient antibiotic exposure. We conclude that the competition dynamics are very sensitive to the fraction of competent cells under benign conditions but less sensitive during antibiotic exposure, supporting the picture of stochastic differentiation as a fitness trade-off.

TFEB Overexpression in the P301S Model of Tauopathy Mitigates Increased PHF1 Levels and Lipofuscin Puncta and Rescues Memory Deficits.

Transcription factor EB (TFEB) was recently shown to be a master regulator of autophagy lysosome pathway. Here, we successfully generated and characterized transgenic mice overexpressing flag-TFEB. Enhanced autophagy in the flag-TFEB transgenic mice was confirmed by an increase in the cellular autophagy markers, as determined by both immunoblots and transmission electron microscopy. Surprisingly, in the flag-TFEB mice we observed increased activity of senescence-associated β-galactosidase by ∼66% of neurons in the cortex (p < 0.001) and 73% of neurons in the hippocampus (p < 0.001). More importantly, flag-TFEB expression remarkably reduced the levels of paired-helical filament (PHF)-tau from 372% in the P301S model of tauopathy to 171% (p < 0.001) in the cortex, and from 436% to 212% (p < 0.001) in the hippocampus. Significantly, reduced levels of NeuN in the cortex (38%, p < 0.001) and hippocampus (25%, p < 0.05) of P301S mice were almost completely restored to WT levels in the P301S/flag-TFEB double-transgenic mice. Also, levels of spinophilin in both the cortex (p < 0.001) and hippocampus (p < 0.001) were restored almost to WT levels. Most importantly, the age-associated lipofuscin granules, which are generally presumed to be nondegradable, were reduced by 57% (p < 0.001) in the cortex and by 55% (p < 0.001) in the hippocampus in the double-transgenic mice. Finally, TFEB overexpression in the P301S mice markedly reversed learning deficits in terms of spatial memory (Barnes maze), as well as working and reference memories (T maze). These data suggest that the overexpression of TFEB can reliably enhance autophagy in vivo, reduce levels of PHF-tau, and thereby reverse the deposition of lipofuscin granules and memory deficits.

Endothelial Nogo-B regulates sphingolipid biosynthesis to promote pathological cardiac hypertrophy during chronic pressure overload.

We recently discovered that endothelial Nogo-B, a membrane protein of the ER, regulates vascular function by inhibiting the rate-limiting enzyme, serine palmitoyltransferase (SPT), in de novo sphingolipid biosynthesis. Here, we show that endothelium-derived sphingolipids, particularly sphingosine-1-phosphate (S1P), protect the heart from inflammation, fibrosis, and dysfunction following pressure overload and that Nogo-B regulates this paracrine process. SPT activity is upregulated in banded hearts in vivo as well as in TNF-α-activated endothelium in vitro, and loss of Nogo removes the brake on SPT, increasing local S1P production. Hence, mice lacking Nogo-B, systemically or specifically in the endothelium, are resistant to the onset of pathological cardiac hypertrophy. Furthermore, pharmacological inhibition of SPT with myriocin restores permeability, inflammation, and heart dysfunction in Nogo-A/B-deficient mice to WT levels, whereas SEW2871, an S1P1 receptor agonist, prevents myocardial permeability, inflammation, and dysfunction in WT banded mice. Our study identifies a critical role of endothelial sphingolipid biosynthesis and its regulation by Nogo-B in the development of pathological cardiac hypertrophy and proposes a potential therapeutic target for the attenuation or reversal of this clinical condition.

Uptake and Lysosomal Delivery of Recombinant Human Alpha‐N‐Acetylglucosamine‐6‐Sulfatase to Mucopolysaccharidosis IIID Fibroblasts

Sanfilippo syndrome (mucopolysaccharidosis type III; MPS III) is a devastating neurodegenerative lysosomal storage disorder of childhood. There is no cure or effective treatment available. The fundamental cause of MPS III is an inherited mutation in one of the 4 enzymes required to catabolize heparan sulfate (HS), a glycosaminoglycan which plays important structural and functional roles in the brain and elsewhere. We now propose to develop an enzyme replacement treatment (ERT) for MPS IIID that will ameliorate or reverse the catastrophic and fatal neurologic decline caused by this disease. The symptoms of MPS IIID are largely localized to the brain, therefore, our strategy proposes to deliver recombinant human alpha‐N‐acetylglucosamine‐6‐sulfatase (rhGNS) intrathecally to effectively treat the underlying causes of the neurologic symptoms that dominate MPS III pathology. We have purified ~100 ug rhGNS from 1500 mL media of CHO cells (specific activity &gt;10,000 nmol/hr/mg), demonstrated maximal enzymatic activity at pH 5.6 (low activity at neutral pH), which is closer to lysosomal pH, demonstrated good enzymatic activity at 37°C and showed it was stable for over one month at 4°C in artificial cerebrospinal fluid storage buffer. Additionally we have demonstrated intracellular enzymatic activity of rhGNS in MPS IIID fibroblasts when rhGNS is added to the media, shown it colocalizes with lysosomal markers using confocal microscopy, and confirmed radiolabelled HS is diminished (33–65%) to WT levels in MPS IIID fibroblasts treated with rhGNS. These results demonstrate scale‐up is feasible in preparation for proof of concept studies in the MPS IIID knock‐out mouse. Our ultimate goal is bring this ERT to the clinic which will likely have a transformational effect on patients and families.Support or Funding InformationThis work was funded by Grant R41 NS089061‐01 from NIH NINDS

Discovering Palmitoylation Sites on the Dopamine Transporter

The dopamine transporter (DAT) controls synaptic dopamine (DA) levels by reuptake after presynaptic neuronal vesicular release. Parkinson disease, addiction, and ADHD are neurological disorders associated with abnormal DA homeostasis causing DAT dysregulation to be hypothesized as a contributing factor in these disorders. Several binding partner interactions and posttranslational modifications combine to regulate DAT function. One such modification is S‐palmitoylation, the reversible, catalytic addition of palmitate to cysteine via a thioester linkage. S‐palmitoylation can regulate protein‐protein interactions, protein trafficking, transmembrane domain tilting, and membrane stability, and DAT palmitoylation increases DA transport Vmax without altering DAT surface expression and opposes DAT degradation. Because S‐palmitoylation is thought to be an intracellular process, we generated five cytosolic accessible cysteine to alanine mutants at residues Cys6, Cys135, Cys341, Cys522, and Cys580 and analyzed them for palmitoylation by [3H]palmitic acid metabolic labeling. A significant decrease in labeling was seen in only the C580A mutant versus WT control; however, approximately fifty percent of the signal remained, implying the presence of additional S‐palmitoylation site(s). To this end the more sensitive acyl‐biotinyl exchange (ABE) method was used to reanalyze these same mutants, and similar results were obtained confirming Cys580 as a major palmitoylation site with similar palmitoylation reduction in the C580A DAT and approximately 50% remaining residual palmitoylation. To further assess palmitoylation at individual cysteines, all but one of the intracellular cysteines were mutated to alanine, leaving a single cysteine available for palmitoylation. ABE analysis of these mutants showed significantly elevated palmitoylation of DATs containing Cys6 or Cys580 relative to those containing Cys135, Cys341, or Cys522 and mutants containing both Cys6 and Cys580 displayed near WT levels of palmitoylation. To assess if endogenous N‐terminal palmitoylation is present, we performed peptide mapping of rat striatal DAT using endoproteinase Asp‐N digestion prior to ABE and epitope specific immunoblotting. Asp‐N DAT cleavage occurs at Asp174, producing a characteristic 19 kDa peptide fragment containing Cys6 and Cys135 when blotted with a DAT N‐terminal antibody. This analysis revealed palmitoylation of this fragment, indicating one or both of these residues is palmitoylated suggesting further analysis is necessary to distinguish between these sites. Together these results provide evidence supporting DAT palmitoylation at Cys6, implying the potential for tethering of the distal N‐terminus to the plasma membrane. Additionally, Cys6 is adjacent to a known PKC‐mediated phosphorylation site, Ser7, suggesting the potential for interplay between phosphorylation and palmitoylation at these sites and a role for these modifications in DAT regulation.Support or Funding InformationSupported by National Institute on Drug Abuse grants R15 DA031991 (J.D.F.) and R01 DA13147 (R.A.V.) and NIH grants P20GM103442/P20RR016471 (to UND).

Mutations in jasmonoyl-L-isoleucine-12-hydroxylases suppress multiple JA-dependent wound responses in Arabidopsis thaliana

Plants rapidly perceive tissue damage, such as that inflicted by insects, and activate several key defense responses. The importance of the fatty acid-derived hormone jasmonates (JA) in dictating these wound responses has been recognized for many years. However, important features pertaining to the regulation of the JA pathway are still not well understood. One key unknown is the inactivation mechanism of the JA pathway and its relationship with plant response to wounding. Arabidopsis cytochrome P450 enzymes in the CYP94 clade metabolize jasmonoyl-L-isoleucine (JA-Ile), a major metabolite of JA responsible for many biological effects attributed to the JA signaling pathway; thus, CYP94s are expected to contribute to the attenuation of JA-Ile-dependent wound responses. To directly test this, we created the double and triple knock-out mutants of three CYP94 genes, CYP94B1, CYP94B3, and CYP94C1. The mutations blocked the oxidation steps and caused JA-Ile to accumulate 3–4-fold the WT levels in the wounded leaves. Surprisingly, over accumulation of JA-Ile did not lead to a stronger wound response. On the contrary, the mutants displayed a series of symptoms reminiscent of JA-Ile deficiency, including resistance to wound-induced growth inhibition, decreased anthocyanin and trichomes, and increased susceptibility to insects. The mutants, however, responded normally to exogenous JA treatments, indicating that JA perception or signaling pathways were intact. Untargeted metabolite analyses revealed >40% reduction in wound-inducible metabolites in the mutants. These observations raise questions about the current JA signaling model and point toward a more complex model perhaps involving JA derivatives and/or feedback mechanisms. This article is part of a Special Issue entitled: Plant Lipid Biology edited by Kent D. Chapman and Ivo Feussner.

Copper resistance and its regulation in the sulfate-reducing bacterium Desulfosporosinus sp. OT.

Desulfosporosinus sp. OT is a Gram-positive, acidophilic sulfate-reducing firmicute isolated from copper tailings sediment in the Norilsk mining-smelting area in Siberia and represents the first Desulfosporosinus species whose genome has been sequenced. Desulfosporosinus sp. OT is exceptionally copper resistant, which made it of interest to study the resistance mechanism. It possesses a copUAZ operon which is shown here to be involved in copper resistance. The copU gene encodes a CsoR-type homotetrameric repressor. By electrophoretic mobility shift assay, it was shown that CopU binds to the operator/promoter region of the copUAZ operon in the absence of copper and is released from the DNA by Cu+ or Ag+, implying that CopU regulates the operon in a copper/silver-dependent manner. DOT_CopA is a P1B-type ATPase related to other characterized, bacterial copper ATPases. When expressed in a copper-sensitive Escherichia coli ΔcopA mutant, it restores copper resistance to WT levels. His-tagged DOT_CopA was expressed from a plasmid in E. coli and purified by Ni-NTA affinity chromatography. The purified enzyme was most active in the presence of Cu(I) and bacterial phospholipids. These findings indicate that the copUAZ operon confers copper resistance to Desulfosporosinus sp. OT, but do not per se explain the basis of the high copper resistance of this strain.

Fundamental Constants for Activation of Human Endplate Receptors

Receptors have a low probability of being active in the absence of agonists (equilibrium constant E0). Agonists increase this probability because they bind with a higher affinity to active (Jd) vs. resting (Kd) receptors. For a receptor with n equivalent agonist binding sites, En=E0(Kd/Jd)∧n. Some mutations that cause congenital myaesthenic syndromes do nothing more than increase E0 above the WT level. We estimated E0 in adult human endplate AChRs by adding background mutations that increase this constant to known extents, and then extrapolating to the zero-effect intercept (−100 mV). The provisional result is E0(WT)∼8.7E-7 or +8.2 kcal/mol (7.4E-7 or 8.3 kcal/mol in adult mouse AChRs). The effects of mutations in mouse and human AChRs were comparable. For many agonists of mouse endplate AChRs, the resting and active affinities have an exponential relationship, Jd=Kd∧M, where M=1.92 (Jadey and Auerbach 2012, JGP 140:17). Hence, log efficacy and log resting affinity are correlated linearly: log(En)=log (E0)+n(1-M)log(Kd). We are also estimating E0 and M for human endplate AChRs from a linear fit of a log-affinity vs. log-efficacy plot, for different agonists. E0 and M are the fundamental, receptor-specific and agonist-independent parameters that shape the dose-response profile.

The Transcription Factor THO Promotes Transcription Initiation and Elongation by RNA Polymerase I

Although ribosomal RNA represents the majority of cellular RNA, and ribosome synthesis is closely connected to cell growth and proliferation rates, a complete understanding of the factors that influence transcription of ribosomal DNA is lacking. Here, we show that the THO complex positively affects transcription by RNA polymerase I (Pol I). We found that THO physically associates with the rDNA repeat and interacts genetically with Pol I transcription initiation factors. Pol I transcription in hpr1 or tho2 null mutants is dramatically reduced to less than 20% of the WT level. Pol I occupancy of the coding region of the rDNA in THO mutants is decreased to ~50% of WT level. Furthermore, although the percentage of active rDNA repeats remains unaffected in the mutant cells, the overall rDNA copy number increases ~2-fold compared with WT. Together, these data show that perturbation of THO function impairs transcription initiation and elongation by Pol I, identifying a new cellular target for the conserved THO complex.

In vitro cultures of Scutellaria alpina as a source of pharmacologically active metabolites

Callus, cell suspension, and shoot cultures as well as in vitro-derived plants of Scutellaria alpina grown under greenhouse (for 3 months) and field conditions (for 2 years) were established, and their flavonoid (baicalin, wogonoside, luteolin) and phenylethanoid glycoside (verbascoside) contents were determined by ultra performance liquid chromatography. The quantities of individual compounds were influenced by the type of analyzed plant material, its age, and day of culture passage. Undifferentiated callus and cell suspension cultures of S. alpina showed higher amounts of metabolites than the proliferating shoot cultures. The highest baicalin (26.25 mg g−1 dry wt) and wogonoside (15.60 mg g−1 dry wt) levels were found in callus cultured for 1.5–2 years on MS agar medium supplemented with NAA (0.1 mg l−1) and TDZ (0.2 mg l−1), which were similar to these yielded by 3-month-old and 2-year-old plant roots of S. alpina. The maximum level of verbascoside (27.21 mg g−1 dry wt) was achieved in cell suspension culture maintained for 1 year in MS liquid medium with NAA (0.1 mg l−1) and TDZ (0.2 mg l−1). The amount was found to be about 3–4-fold higher than in shoots and roots of S. alpina micropropagated plants grown in field condition (7.2–9.7 mg g−1 dry wt). Our results suggest that in vitro cultures of S. alpina represent effective system for the production of the bioactive compounds with different pharmacological activity during long-term subculturing.

Novel Functional Roles for Ten-Eleven-Translocation 2 (Tet2) in Normal and Leukemic Growth of Mast Cells

KIT receptor signaling plays an important role in mast cell development. Gain-of-function mutations in KIT receptor have been identified in human diseases including gastrointestinal stromal tumors (GIST), systemic mastocytosis (SM) and acute myeloid leukemia (AML). Although KIT mutations found in GIST are sensitive to imatinib, KIT mutation (KITD816V) found in 90% of SM patients is imatinib-resistant and currently no therapies are available to treat the human diseases associated with this mutation. Our recent studies have identified Ten-Eleven-Translocation 2 (TET2) mutations in ~23% of SM patients and are associated with poor prognosis and overall survival. TET2 is a methylcytosine dioxygenase that plays a vital role in active DNA demethylation. Recent studies suggest that patients with mutations in TET2 and KITD816V develop more aggressive form of mastocytosis with worse prognosis. Although it is known that TET2 and KITD816V cooperate in SM patients, it is not clear how they cooperate with each other and what is the physiologic role of TET2 in normal mast cell development. We show that loss of Tet2 results in impaired maturation of mast cells in vivo and in bone marrow-derived mast cells (BMMC) compared to WT controls, which is associated with reduction in 5-hmc levels compared to WT BMMCs. We also observed reduction in the expression of mast cell-specific genesincluding Mast cell proteinase-5 (MCP-5), Mast cell proteinase-6 (MCP-6) and Carboxypeptidase A (CPA). To determine the mechanism behind altered mast cell differentiation in Tet2-/- BMMCs, we performed RNA-seq analysis in WT and Tet2-/- mast cells and observed altered expression of various genes involved in development of mast cells including Kit, FcεR1, Mitf, Notch, and Myc. We further confirmed altered expression of Mitf, Gata-2, and PU.1 in Tet2-/- BMMCs compared to WT BMMCs by western blotting. Since Tet2 regulates DNA demethylation, we tested whether altered BMMC differentiation in Tet2-/- mice is due to enhanced DNA methylation. We treated WT or Tet2-/- BM cells for 3 weeks with vehicle or 5-azacytidine (hypomethylating agent) and analyzed mast cell differentiation. Treatment with 5-azacytidine completely corrected the defective mast cell differentiation in Tet2-/- cells to WT levels. These results suggest that Tet2 plays a significant role in mast cell differentiation by regulating the expression of critical transcription factors including Mitf, Gata-2 and PU.1. We next analyzed the growth of Tet2-/- BMMCs in response to cytokines. Tet2-deficient BMMCs show enhanced cytokine mediated growth compared to WT BMMCs. Hyper-proliferation of Tet2-/- BMMCs is associated with reduced expression of tumor suppressor, PTEN, whose promoter is hypermethylated and a concomitant increase in the activation of the PI3K/AKT pathway. Since loss of function TET2 mutations have been observed in SM patients in addition to KITD816V mutation, we tested whether loss of Tet2 cooperates with KIT mutation in vitro and in vivo. Tet2-deficiency or knockdown in conjunction with the expression of KIT mutation resulted in significantly enhanced growth compared to cells bearing KIT mutation alone or lacking Tet2 expression. Likewise in human mastocytosis xenograft model, significantly enlarged tumors were observed in NSG mice transplanted with human mastocytosis cell line bearing the KITD816V mutation (HMC1.2) and knockdown of TET2 compared to HMC1.2 cells bearing only the KITD816V mutation. The cooperation between loss of Tet2 and KIT mutation was associated with further increase in PI3K/AKT activation and pharmacologic inhibitor treatment with a PI3K inhibitor GDC-0941 (Pan PI3K), but not TGX221 (p110β-specific) or IC87114 (p110δ-specific), significantly reduced the hyper-proliferation of Tet2-/- BMMCs and cell lines as well as primary BM blasts derived from SM patients bearing the KITD816V mutation. Consistently, combined loss of p110α and p110δ subunits of PI3K resulted in the most profound growth repression in oncogenic KIT bearing BM cells, but did not correct altered differentiation in Tet2-/- BMMCs. Taken together our results suggest that combinational therapy involving 5-azacytidine (which corrects the impaired mast cell differentiation) and PI3K inhibitor (which corrects the excessive proliferation) is a better therapeutic option for treating human mastocytosis patients bearing TET2 and KIT mutations. DisclosuresNo relevant conflicts of interest to declare.

Pathological gamma oscillations, impaired dopamine release, synapse loss and reduced dynamic range of unitary glutamatergic synaptic transmission in the striatum of hypokinetic Q175 Huntington mice

Huntington’s disease (HD) is a severe genetically inherited neurodegenerative disorder. Patients present with three principal phenotypes of motor symptoms: choreatic, hypokinetic-rigid and mixed. The Q175 mouse model of disease offers an opportunity to investigate the cellular basis of the hypokinetic-rigid form of HD. At the age of 1year homozygote Q175 mice exhibited the following signs of hypokinesia: Reduced frequency of spontaneous movements on a precision balance at daytime (−55%), increased total time spent without movement in an open field (+42%), failures in the execution of unconditioned avoidance reactions (+32%), reduced ability for conditioned avoidance (−96%) and increased reaction times (+65%) in a shuttle box. Local field potential recordings revealed low-frequency gamma oscillations in the striatum as a characteristic feature of HD mice at rest. There was no significant loss of DARPP-32 immunolabeled striatal projection neurons (SPNs) although the level of DARPP-32 immunoreactivity was lower in HD. As a potential cause of hypokinesia, HD mice revealed a strong reduction in striatal KCl-induced dopamine release, accompanied by a decrease in the number of tyrosine hydroxylase-(TH)- and VMAT2-positive synaptic varicosities. The presynaptic TH fluorescence level was also reduced. Patch-clamp experiments were performed in slices from 1-year-old mice to record unitary EPSCs (uEPSCs) of presumed cortical origin in the absence of G-protein-mediated modulation. In HD mice, the maximal amplitudes of uEPSCs amounted to 69% of the WT level which matches the loss of VGluT1+/SYP+ synaptic terminals in immunostained sections. These results identify impairment of cortico-striatal synaptic transmission and dopamine release as a potential basis of hypokinesia in HD.

Zebrafish lacking functional DNA polymerase gamma survive to juvenile stage, despite rapid and sustained mitochondrial DNA depletion, altered energetics and growth.

DNA polymerase gamma (POLG) is essential for replication and repair of mitochondrial DNA (mtDNA). Mutations in POLG cause mtDNA instability and a diverse range of poorly understood human diseases. Here, we created a unique Polg animal model, by modifying polg within the critical and highly conserved polymerase domain in zebrafish. polg+/− offspring were indistinguishable from WT siblings in multiple phenotypic and biochemical measures. However, polg−/− mutants developed severe mtDNA depletion by one week post-fertilization (wpf), developed slowly and had regenerative defects, yet surprisingly survived up to 4 wpf. An in vivo mtDNA polymerase activity assay utilizing ethidium bromide (EtBr) to deplete mtDNA, showed that polg+/− and WT zebrafish fully recover mtDNA content two weeks post-EtBr removal. EtBr further reduced already low levels of mtDNA in polg−/− animals, but mtDNA content did not recover following release from EtBr. Despite significantly decreased respiration that corresponded with tissue-specific levels of mtDNA, polg−/− animals had WT levels of ATP and no increase in lactate. This zebrafish model of mitochondrial disease now provides unique opportunities for studying mtDNA instability from multiple angles, as polg−/− mutants can survive to juvenile stage, rather than lose viability in embryogenesis as seen in Polg mutant mice.

Characterization of the cardiac succinylome and its role in ischemia–reperfusion injury

Succinylation refers to modification of lysine residues with succinyl groups donated by succinyl-CoA. Sirtuin5 (Sirt5) is a mitochondrial NAD(+)-dependent deacylase that catalyzes the removal of succinyl groups from proteins. Sirt5 and protein succinylation are conserved across species, suggesting functional importance of the modification. Sirt5 loss impacts liver metabolism but the role of succinylation in the heart has not been explored. We combined affinity enrichment with proteomics and mass spectrometry to analyze total succinylated lysine content of mitochondria isolated from WT and Sirt5(-/-) mouse hearts. We identified 887 succinylated lysine residues in 184 proteins. 44 peptides (5 proteins) occurred uniquely in WT samples, 289 (46 proteins) in Sirt5(-/-) samples, and 554 (133 proteins) were common to both groups. The 46 unique proteins in Sirt5(-/-) heart participate in metabolic processes such as fatty acid β-oxidation (Eci2) and branched chain amino acid catabolism, and include respiratory chain proteins (Ndufa7, 12, 13, Dhsa). We performed label-free analysis of the peptides common to WT and Sirt5(-/-) hearts. 16 peptides from 9 proteins were significantly increased in Sirt5(-/-) by at least 30%. The adenine nucleotide transporter 1 showed the highest increase in succinylation in Sirt5(-/-) (108.4 fold). The data indicate that succinylation is widespread in the heart and enriched in metabolic pathways. We examined whether the loss of Sirt5 would impact ischemia-reperfusion (I/R) injury and we found an increase in infarct size in Sirt5(-/-) hearts compared to WT littermates (68.5(+)/-1.1% Sirt5(-/-) vs 39.6(+)/(-) 6.8% WT) following 20min of ischemia and 90-min reperfusion. We further demonstrate that I/R injury in Sirt5(-/-) heart is restored to WT levels by pretreatment with dimethyl malonate, a competitive inhibitor of succinate dehydrogenase (SDH), implicating alteration in SDH activity as causative of the injury.