Toward countering muscle and bone loss with spaceflight: GSK3 as a potential target.

We examined the effects of ∼30 days of spaceflight on glycogen synthase kinase 3 (GSK3) content and inhibitory serine phosphorylation in murine muscle and bone samples from four separate missions (BION-M1, rodent research [RR]1, RR9, and RR18). Spaceflight reduced GSK3ß content across all missions, whereas its serine phosphorylation was elevated with RR18 and BION-M1. The reduction in GSK3ß was linked to the reduction in type IIA fibers commonly observed with spaceflight as these fibers are particularly enriched with GSK3. We then tested the effects of inhibiting GSK3 before this fiber type shift, and we demonstrate that muscle-specific Gsk3 knockdown increased muscle mass, preserved muscle strength, and promoted the oxidative fiber type with Earth-based hindlimb unloading. In bone, GSK3 activation was enhanced after spaceflight; and strikingly, muscle-specific Gsk3 deletion increased bone mineral density in response to hindlimb unloading. Thus, future studies should test the effects of GSK3 inhibition during spaceflight.

Kynurenine metabolism is altered in mdx mice: a potential muscle to brain connection.

NEW FINDINGS: What is the central question in this study? Promoting muscle health with regular aerobic exercise can improve mental health through a kynurenine metabolic pathway: do conditions of muscle disease such as muscular dystrophy negatively influence this pathway? What is the main finding and its importance? The DBA/2J mdx model of Duchenne muscular dystrophy exhibits altered kynurenine metabolism with less kynurenic acid and peroxisome proliferator-activated receptor-γ coactivator 1-α and higher levels of tumour necrosis factor α mRNA - results associated with anxiety-like behaviour. ABSTRACT: Regular exercise can direct muscle kynurenine (KYN) metabolism toward the neuroprotective branch of the kynurenine pathway thereby limiting the accumulation of neurotoxic metabolites in the brain and contributing to mental resilience. However, the effect of muscle disease on KYN metabolism has not yet been investigated. Previous work has highlighted anxiety-like behaviours in approximately 25% of patients with Duchenne muscular dystrophy (DMD), possibly due to altered KYN metabolism. Here, we characterized KYN metabolism in mdx mouse models of DMD. Young (8-10 week old) DBA/2J (D2) mdx mice, but not age-matched C57BL/10 (C57) mdx mice, had lower levels of circulating kynurenic acid (KYNA) and lower KYNA:KYN ratio compared with their respective wild-type (WT) controls. While both C57 and D2 mdx mice displayed signs of anxiety-like behaviour, spending more time in the corners of the arena during a novel object recognition test, this effect was more prominent in D2 mdx mice. Correlational analysis detected a significant negative association between KYNA:KYN levels and time spent in corners in D2 mice, but not C57 mice. In extensor digitorum longus muscles from D2 mdx mice, but not C57 mdx mice, we found lowered protein levels of peroxisome proliferator-activated receptor-γ coactivator 1-α and kynurenine amino transferase-1 enzyme when compared with WT. Furthermore, D2 mdx quadriceps muscles had the highest level of tumour necrosis factor α expression, which is suggestive of enhanced inflammation. Thus, our pilot work shows that KYN metabolism is altered in D2 mdx mice, with a potential contribution from altered muscle health.

Amplified detection of nucleic acids and proteins using an isothermal proximity CRISPR Cas12a assay.

Herein, we describe an isothermal proximity CRISPR Cas12a assay that harnesses the target-induced indiscrimitive single-stranded DNase activity of Cas12a for the quantitative profiling of gene expression at the mRNA level and detection of proteins with high sensitivity and specificity. The target recognition is achieved through proximity binding rather than recognition by CRISPR RNA (crRNA), which allows for flexible assay design. A binding-induced primer extension reaction is used to generate a predesigned CRISPR-targetable sequence as a barcode for further signal amplification. Through this dual amplification protocol, we were able to detect as low as 1 fM target nucleic acid and 100 fM target protein isothermally. The practical applicability of this assay was successfully demonstrated for the temporal profiling of interleukin-6 gene expression during allergen-mediated mast cell activation.

GSK3 inhibition with low dose lithium supplementation augments murine muscle fatigue resistance and specific force production.

Calcineurin is a Ca2+ -dependent serine/threonine phosphatase that dephosphorylates nuclear factor of activated T cells (NFAT), allowing for NFAT entry into the nucleus. In skeletal muscle, calcineurin signaling and NFAT activation increases the expression of proliferator-activated receptor-gamma coactivator 1-alpha (PGC-1α) and slow myosin heavy chain (MHC) I ultimately promoting fatigue resistance. Glycogen synthase kinase 3 (GSK3) is a serine/threonine kinase that antagonizes calcineurin by re-phosphorylating NFAT preventing its entry into the nucleus. Here, we tested whether GSK3 inhibition in vivo with low dose lithium chloride (LiCl) supplementation (10 mg kg-1  day-1 for 6 weeks) in male C57BL/6J mice would enhance muscle fatigue resistance in soleus and extensor digitorum longus (EDL) muscles by activating NFAT and augmenting PGC-1α and MHC I expression. LiCl treatment inhibited GSK3 by elevating Ser9 phosphorylation in soleus (+1.8-fold, p = .007) and EDL (+1.3-fold p = .04) muscles. This was associated with a significant reduction in NFAT phosphorylation (-50%, p = .04) and a significant increase in PGC-1α (+1.5-fold, p = .05) in the soleus but not the EDL. MHC isoform analyses in the soleus also revealed a 1.2-fold increase in MHC I (p = .04) with no change in MHC IIa. In turn, a significant enhancement in soleus muscle fatigue (p = .04), but not EDL (p = .26) was found with LiCl supplementation. Lastly, LiCl enhanced specific force production in both soleus (p < .0001) and EDL (p = .002) muscles. Altogether, our findings show the skleletal muscle contractile benefits of LiCl-mediated GSK3 inhibition in mice.

Colorimetric Polymerase Chain Reaction Enabled by a Fast Light-Activated Substrate Chromogenic Detection Platform.

Miniaturization of nucleic acid tests (NATs) into portable, inexpensive detection platforms may aid disease diagnosis in point-of-care (POC) settings. Colorimetric signals are ideal readouts for portable NATs, and it remains of high demand to develop color readouts that are simple, quantitative, and versatile. Thus motivated, we report a fast light-activated substrate chromogenic polymerase chain reaction (FLASH PCR) that uses DNA intercalating dyes (DIDs) to enable colorimetric nucleic acid detection and quantification. The FLASH system is established on our finding that DID-DNA intercalation can promote the rapid photooxidation of chromogenic substrates through light-induced production of singlet oxygen. Using this principle, we have successfully converted DID-based fluorescent PCR assays into colorimetric FLASH PCR. To demonstrate the practical applicability of FLASH PCR to POC diagnosis, we also fabricated two readout platforms, including a portable electronic FLASH reader and a paper-based FLASH strip. Using the FLASH reader, we were able to detect as low as 60 copies of DNA standards, a limit of detection (LOD) comparable with commercial quantitative PCR. The FLASH strip further enables the reader-free detection of PCR amplicons by converting the colorimetric signal into the visual measurement of distance as a readout. Finally, the practical applicability of the FLASH PCR was demonstrated by the detection and/or quantification of nucleic acid markers in diverse clinical and biological samples.

Attenuation of allergen-mediated mast cell activation by rosemary extract (Rosmarinus officinalis L.).

Mast cells are immune sentinels and a driving force in both normal and pathological contexts of inflammation, with a prominent role in allergic hypersensitivities. Crosslinking of FcεRI by allergen-bound IgE Abs leads to mast cell degranulation, resulting in an early-phase response and release of newly synthesized pro-inflammatory mediators in the late-phase. The MAPK and NF-κB pathways are established as critical intracellular mechanisms directing mast cell-induced inflammation. Rosemary extract (RE) has been shown to modulate the MAPK and NF-κB pathways in other cellular contexts in vitro and in vivo. However, the effect of RE on mast cell activation has not been explored, and thus we aim to evaluate the potential of RE in modulating mast cell activation and FcεRI/c-kit signaling, potentially via these key pathways. Primary murine mast cells were sensitized with anti-TNP IgE and stimulated with cognate allergen (TNP-BSA) under stem cell factor (SCF) potentiation while treated with 0-25 µg/ml RE. RE treatment inhibited phosphorylation of p38 and JNK MAPKs while also impairing NF-кB transcription factor activity. Gene expression and mediator secretion analysis showed that RE treatment decreased IL-6, TNF, IL-13, CCL1, and CCL3, but major component polyphenols do not contribute to these effects. Importantly, RE treatment significantly inhibited early phase mast cell degranulation (down to 15% of control), with carnosic acid and carnosol contributing. These findings indicate that RE is capable of modulating mast cell functional outcomes and that further investigation of the underlying mechanisms and its potential therapeutic properties in allergic inflammatory conditions is warranted.

Low-dose lithium feeding increases the SERCA2a-to-phospholamban ratio, improving SERCA function in murine left ventricles.

NEW FINDINGS: What is the central question of this study? Inhibition of glycogen synthase kinase-3 (GSK3) has been shown to improve cardiac SERCA2a function. Lithium can inhibit GSK3, but therapeutic doses used in treating bipolar disorder can have toxic effects. It has not been determined whether subtherapeutic doses of lithium can improve cardiac SERCA function. What is the main finding and its importance? Using left ventricles from wild-type mice, we found that subtherapeutic lithium feeding for 6 weeks decreased GSK3 activity and increased cardiac SERCA function compared with control-fed mice. These findings warrant the investigation of low-dose lithium feeding in preclinical models of cardiomyopathy and heart failure to determine the therapeutic benefit of GSK3 inhibition. ABSTRACT: The sarco(endo)plasmic reticulum Ca2+ -ATPase (SERCA) pump is responsible for regulating calcium (Ca2+ ) within myocytes, with SERCA2a being the dominant isoform in cardiomyocytes. Its inhibitor, phospholamban (PLN), acts by decreasing the affinity of SERCA for Ca2+ . Changes in the SERCA2a:PLN ratio can cause Ca2+ dysregulation often seen in patients with dilated cardiomyopathy and heart failure. The enzyme glycogen synthase kinase-3 (GSK3) is known to downregulate SERCA function by decreasing the SERCA2a:PLN ratio. In this study, we sought to determine whether feeding mice low-dose lithium, a natural GSK3 inhibitor, would improve left ventricular SERCA function by altering the SERCA2a:PLN ratio. To this end, male wild-type C57BL/6J mice were fed low-dose lithium via drinking water (10 mg kg-1  day-1 LiCl for 6 weeks) and left ventricles were harvested. GSK3 activity was significantly reduced in LiCl-fed versus control-fed mice. The apparent affinity of SERCA for Ca2+ was also increased (pCa50 ; control, 6.09 ± 0.03 versus LiCl, 6.26 ± 0.04, P < 0.0001) along with a 2.0-fold increase in SERCA2a:PLN ratio in LiCl-fed versus control-fed mice. These findings suggest that low-dose lithium supplementation can improve SERCA function by increasing the SERCA2a:PLN ratio. Future studies in murine preclinical models will determine whether GSK3 inhibition via low-dose lithium could be a potential therapeutic strategy for dilated cardiomyopathy and heart failure.

TAK1 signaling activity links the mast cell cytokine response and degranulation in allergic inflammation.

Mast cells drive the inappropriate immune response characteristic of allergic inflammatory disorders via release of pro-inflammatory mediators in response to environmental cues detected by the IgE-FcεRI complex. The role of TGF-ß-activated kinase 1 (TAK1), a participant in related signaling in other contexts, remains unknown in allergy. We detect novel activation of TAK1 at Ser412 in response to IgE-mediated activation under SCF-c-kit potentiation in a mast cell-driven response characteristic of allergic inflammation, which is potently blocked by TAK1 inhibitor 5Z-7-oxozeaenol (OZ). We, therefore, interrogated the role of TAK1 in a series of mast cell-mediated responses using IgE-sensitized murine bone marrow-derived mast cells, stimulated with allergen under several TAK1 inhibition strategies. TAK1 inhibition by OZ resulted in significant impairment in the phosphorylation of MAPKs p38, ERK, and JNK; and mediation of the NF-κB pathway via IκBα. Impaired gene expression and near abrogation in release of pro-inflammatory cytokines TNF, IL-6, IL-13, and chemokines CCL1, and CCL2 was detected. Finally, a significant inhibition of mast cell degranulation, accompanied by an impairment in calcium mobilization, was observed in TAK1-inhibited cells. These results suggest that TAK1 acts as a signaling node, not only linking the MAPK and NF-κB pathways in driving the late-phase response, but also initiation of the degranulation mechanism of the mast cell early-phase response following allergen recognition and may warrant consideration in future therapeutic development.

Neurogranin is expressed in mammalian skeletal muscle and inhibits calcineurin signaling and myoblast fusion.

Calcineurin is a Ca2+/calmodulin (CaM)-dependent phosphatase that plays a critical role in promoting the slow fiber phenotype and myoblast fusion in skeletal muscle, thereby making calcineurin an attractive cellular target for enhancing fatigue resistance, muscle metabolism, and muscle repair. Neurogranin (Ng) is a CaM-binding protein thought to be expressed solely in brain and neurons, where it inhibits calcineurin signaling by sequestering CaM, thus lowering its cellular availability. Here, we demonstrate for the first time the expression of Ng protein and mRNA in mammalian skeletal muscle. Both protein and mRNA levels are greater in slow-oxidative compared with fast-glycolytic muscles. Coimmunoprecipitation of CaM with Ng in homogenates of C2C12 myotubes, mouse soleus, and human vastus lateralis suggests that these proteins physically interact. To determine whether Ng inhibits calcineurin signaling in muscle, we used Ng siRNA with C2C12 myotubes to reduce Ng protein levels by 60%. As a result of reduced Ng expression, C2C12 myotubes had enhanced CaM-calcineurin binding and calcineurin signaling as indicated by reduced phosphorylation of nuclear factor of activated T cells and increased utrophin mRNA. In addition, calcineurin signaling affects the expression of myogenin and stabilin-2, which are involved in myogenic differentiation and myoblast fusion, respectively. Here, we found that both myogenin and stabilin-2 were significantly elevated by Ng siRNA in C2C12 cells, concomitantly with an increased fusion index. Taken together, these results demonstrate the expression of Ng in mammalian skeletal muscle where it appears to be a novel regulator of calcineurin signaling.