ACTIN Anchors the Highly Oligomeric DRP1 at Mitochondria-Sarcoplasmic Reticulum Contact Sites in Adult Murine Heart: Its Functional Implication

Rationale: Mitochondrial fission and fusion are relatively infrequent in adult cardiomyocytes compared to other cell types. This is surprising considering that proteins involved in mitochondrial dynamics are highly expressed in the heart. It has been previously reported that dynamin related protein 1 (DRP1) has a critical role in mitochondrial fitness and cardiac protection. Cardiac DRP1 ablation in the adult heart evokes a progressive dilated cardiac myopathy and lethal heart failure. Nevertheless, the conditional cardiacspecific DRP1 knock out animals present a significantly longer survival rate compared with global DRP1 KO models. We have described before the great importance for cardiac physiology of the strategic positioning of mitochondrial proteins in the cardiac tissue. Therefore, we hypothesize that DRP1 plays a regulatory role in cardiac physiology and mitochondrial fitness by preferentially accumulating at mitochondria and junctional sarcoplasmic reticulum (jSR) contact sites, where the high Ca2+ microdomain is formed during excitation-contraction (EC) coupling. Objective: This study aims to determine whether mitochondria-associated DRP1 is preferentially accumulated in the mitochondria and jSR contact sites and if indeed this is the case, what is the mechanism responsible for such a biased distribution and what is the functional implication. Methods and Results: Using high-resolution imaging approaches, we found that mitochondria-associated DRP1 in cardiomyocytes was localized in the discrete regions where T-tubule, jSR, and mitochondria are adjacent to each other. Western blot results showed that mitochondria-bound DRP1 was restricted to the mitochondria-associated membranes (MAM), with undetectable levels in purified mitochondria. Furthermore, in comparison to the cytosolic DRP1, the membrane-bound DRP1 in SR and MAM fractions formed high molecular weight oligomers. In both electrically paced adult cardiomyocytes and Langendorff-perfused beating hearts, the oscillatory Ca2+ pulses preserved MAM-associated DRP1 accumulation. Interestingly, similar to DRP1, all mitochondria-bound βACTIN only exists in MAM and not in the purified mitochondria. Additionally, co-immunoprecipitation pulls down both DRP1 and βACTIN together. Inhibition of βACTIN polymerization with Cytochalasin D disrupts the tight association between DRP1 and βACTIN. In cardiac specific DRP1 knockout mouse after 6 weeks of tamoxifen induction the cardiomyocytes show disarray of sarcomere, a decrease of cardiac contraction, loss of mitochondrial membrane potential significantly decreased spare respiratory capacity, and frequent occurrence of earl after contraction, suggesting the heart is susceptible for failure and arrhythmias. Despite of this phenotype, DRP1icKo animal have a longer life spam than other DRP1 KO models. We also observed that DRP1icKO. Strikingly, DRP1 levels are is only modestly decreased in the MAM when compared with the rest of the cellular fractions. These preserved levels were accompanied with preservation of the mitochondrial pool in the MAM fraction obtained from the DRP1icKO hearts. Conclusions: The results show that in adult cardiomyocytes, mitochondria bound DRP1 clusters in high molecular weight protein complexes at MAM. This clustering is fortified by EC coupling mediated Ca2+ transients and requires its interaction with βACTIN. Together with the better preserved dRP1 levels in the DRP1icKO model in the MAM, we conclude that DRP1 is anchored in mitochondria-SR interface through βACTIN and position itself to play a fundamental role in regulating mitochondrial quality control in the working heart.

RCAN1 Reduces FBXW7β Transcription by Inhibiting Calcineurin/NFAT Signaling Pathway

Regulator of calcineurin 1 (RCAN1), a crucial endogenous regulator of calcineurin, is implicated in multiple important physiological and pathological processes. Aberrant expression of RCAN1 is commonly found in brains of patients with Down syndrome (DS) or Alzheimer’s disease (AD), accounting for impaired neurodevelopment in DS and neuronal degeneration in AD, respectively. However, the mechanism of RCAN1 in brain development and neurodegeneration remains unclear. FBXW7 functions as vital factor in neurodevelopment and neurodegeneration via mediating proteasomal degradation of its substrates. Deficiency of FBXW7 contributes to impaired neurodevelopment and accelerating neurodegeneration. Here, we show that increased RCAN1 reduces the level of β isoform of FBXW7 (FBXW7β). RCAN1 inhibits FBXW7β transcription in a calcineurin dependent manner. Potential NFAT binding sites are identified within the promoter of FBXW7β, and NFAT is also demonstrated to activate the promoter activity of FBXW7β. In summary, our work implies that RCAN1 can regulate FBXW7β expression by inhibiting FBXW7β transcription via calcineurin/NFAT signaling pathway. It could provide more understanding on the mechanism of FBXW7 regulation and suggest a potential mechanism on functional implication of RCAN1 with impaired brain function in some neurodevelopmental and neurodegenerative diseases.

High Endothelial Venules: A Vascular Perspective on Tertiary Lymphoid Structures in Cancer

High endothelial venules (HEVs) are specialized postcapillary venules composed of cuboidal blood endothelial cells that express high levels of sulfated sialomucins to bind L-Selectin/CD62L on lymphocytes, thereby facilitating their transmigration from the blood into the lymph nodes (LN) and other secondary lymphoid organs (SLO). HEVs have also been identified in human and murine tumors in predominantly CD3+T cell-enriched areas with fewer CD20+B-cell aggregates that are reminiscent of tertiary lymphoid-like structures (TLS). While HEV/TLS areas in human tumors are predominantly associated with increased survival, tumoral HEVs (TU-HEV) in mice have shown to foster lymphocyte-enriched immune centers and boost an immune response combined with different immunotherapies. Here, we discuss the current insight into TU-HEV formation, function, and regulation in tumors and elaborate on the functional implication, opportunities, and challenges of TU-HEV formation for cancer immunotherapy.

Crystal structure and functional implication of a bacterial cyclic AMP–AMP–GMP synthetase

Mammalian cyclic GMP-AMP synthase (cGAS) and its homologue dinucleotide cyclase in Vibrio cholerae (VcDncV) produce cyclic dinucleotides (CDNs) that participate in the defense against viral infection. Recently, scores of new cGAS/DncV-like nucleotidyltransferases (CD-NTases) were discovered, which produce various CDNs and cyclic trinucleotides (CTNs) as second messengers. Here, we present the crystal structures of EcCdnD, a CD-NTase from Enterobacter cloacae that produces cyclic AMP-AMP-GMP, in its apo-form and in complex with ATP, ADP and AMPcPP, an ATP analogue. Despite the similar overall architecture, the protein shows significant structural variations from other CD-NTases. Adjacent to the donor substrate, another nucleotide is bound to the acceptor binding site by a non-productive mode. Isothermal titration calorimetry results also suggest the presence of two ATP binding sites. GTP alone does not bind to EcCdnD, which however binds to pppApG, a possible intermediate. The enzyme is active on ATP or a mixture of ATP and GTP, and the best metal cofactor is Mg2+. The conserved residues Asp69 and Asp71 are essential for catalysis, as indicated by the loss of activity in the mutants. Based on structural analysis and comparison with VcDncV and RNA polymerase, a tentative catalytic pathway for the CTN-producing EcCdnD is proposed.

Carbon dioxide and bicarbonate accumulation in caiman erythrocytes during diving

The ability of crocodilian haemoglobins to bind HCO3− has been appreciated for more than half a century, but the functional implication of this is exceptional mechanism has not previously been assessed in vivo. Therefore, the goal of the present study was to address the hypothesis that CO2 primarily binds to Hb, rather than being accumulated in plasma as in other vertebrates, during diving in caimans. Here, we demonstrate that CO2 primarily accumulates within the erythrocyte during diving and that most of the accumulated CO2 is bound to haemoglobin. Furthermore, we show that this HCO3−-binding is tightly associated with the progressive blood deoxygenation during diving, therefore, crocodilians differ from the classic vertebrate pattern, where HCO3− accumulates in the plasma upon excretion from the erythrocytes by the Cl−-HCO3−-exchanger.

Fear response-based prediction for stress susceptibility to PTSD-like phenotypes

Most individuals undergo traumatic stresses at some points in their life, but only a small proportion develop stress-related disorders such as anxiety diseases and posttraumatic stress disorder (PTSD). Although stress susceptibility is one determinant of mental disorders, the underlying mechanisms and functional implication remain unclear yet. We found that an increased amount of freezing that animals exhibited in the intertrial interval (ITI) of a stress-enhanced fear learning paradigm, predicts ensuing PTSD-like symptoms whereas resilient mice show ITI freezing comparable to that of unstressed mice. To examine the behavioral features, we developed a systematic analytical approach for ITI freezing and stress susceptibility. Thus, we provide a behavioral parameter for prognosis to stress susceptibility of individuals in the development of PTSD-like symptoms as well as a new mathematical means to scrutinize freezing behavior.