Wild-type Littermate Controls Research Articles

A cell type–specific approach to elucidate the role of miR-96 in inner ear hair cells

IntroductionMutations in microRNA-96 (miR-96), a microRNA expressed within the hair cells (HCs) of the inner ear, result in progressive hearing loss in both mouse models and humans. In this study, we present the first HC-specific RNA-sequencing (RNA-seq) dataset from newborn Mir96Dmdo heterozygous, homozygous mutant, and wildtype mice.MethodsBulk RNA-seq was performed on HCs of newborn Mir96Dmdo heterozygous, homozygous mutant, and wildtype mice. Differentially expressed gene analysis was conducted on Mir96Dmdo homozygous mutant HCs compared to wildtype littermate controls, followed by GO term and protein-protein interaction analysis on these differentially expressed genes.ResultsWe identify 215 upregulated and 428 downregulated genes in the HCs of the Mir96Dmdo homozygous mutant mice compared to their wildtype littermate controls. Many of the significantly downregulated genes in Mir96Dmdo homozygous mutant HCs have established roles in HC development and/or known roles in deafness including Myo15a, Myo7a, Ush1c, Gfi1, and Ptprq and have enrichment in gene ontology (GO) terms with biological functions such as sensory perception of sound. Interestingly, upregulated genes in Mir96Dmdo homozygous mutants, including possible miR-96 direct targets, show higher wildtype expression in supporting cells compared to HCs.ConclusionOur data further support a role for miR-96 in HC development, possibly as a repressor of supporting cell transcriptional programs in HCs. The HC-specific Mir96Dmdo RNA-seq data set generated from this manuscript are now publicly available in a dedicated profile in the gene expression analysis resource (gEAR-https://umgear.org/p?l=miR96).

GABA transporter mGat4 is involved in multiple neural functions in mice

γ-Aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the central nervous system. The termination of GABA transmission is through the action of GABA transporters (GATs). mGAT4 (encoded by Slc6a11) is another GAT besides GAT1 (encoded by Slc6a1) that functions in GABA reuptake in CNS. Research on the function of mGAT4 is still in its infancy. We developed an mGat4 knockout mouse model (mGat4−/− mice) and performed a series of behavioral analyses for the first time to study the effect of mGat4 on biological processes in CNS. Our results indicated that homozygous mGat4−/− mice had less depression, anxiety-like behavior and more social activities than their wild-type littermate controls. However, they had weight loss and showed motor incoordination and imbalance. Meanwhile, mGat4−/− mice showed increased pain threshold and hypoalgesia behavior in nociceptive stimulus and learning and memory impairments. The expression of multiple components of the GABAergic system including GAD67, GABAA and KCC2 was altered. There is little or no compensatory change in mGat1. In a word, mGat4 may play a key role in normal motor coordination, sensation, emotion, learning and memory and could be the potential target of neurological disorders.

Deletion of Sigmar1 leads to increased arterial stiffness and altered mitochondrial respiration resulting in vascular dysfunction.

Sigmar1 is a ubiquitously expressed, multifunctional protein known for its cardioprotective roles in cardiovascular diseases. While accumulating evidence indicate a critical role of Sigmar1 in cardiac biology, its physiological function in the vasculature remains unknown. In this study, we characterized the expression of Sigmar1 in the vascular wall and assessed its physiological function in the vascular system using global Sigmar1 knockout (Sigmar1-/-) mice. We determined the expression of Sigmar1 in the vascular tissue using immunostaining and biochemical experiments in both human and mouse blood vessels. Deletion of Sigmar1 globally in mice (Sigmar1-/-) led to blood vessel wall reorganizations characterized by nuclei disarray of vascular smooth muscle cells, altered organizations of elastic lamina, and higher collagen fibers deposition in and around the arteries compared to wildtype littermate controls (Wt). Vascular function was assessed in mice using non-invasive time-transit method of aortic stiffness measurement and flow-mediated dilation (FMD) of the left femoral artery. Sigmar1-/- mice showed a notable increase in arterial stiffness in the abdominal aorta and failed to increase the vessel diameter in response to reactive-hyperemia compared to Wt. This was consistent with reduced plasma and tissue nitric-oxide bioavailability (NOx) and decreased phosphorylation of endothelial nitric oxide synthase (eNOS) in the aorta of Sigmar1-/- mice. Ultrastructural analysis by transmission electron microscopy (TEM) of aorta sections showed accumulation of elongated shaped mitochondria in both vascular smooth muscle and endothelial cells of Sigmar1-/- mice. In accordance, decreased mitochondrial respirometry parameters were found in ex-vivo aortic rings from Sigmar1 deficient mice compared to Wt controls. These data indicate a potential role of Sigmar1 in maintaining vascular homeostasis.

NrCAM-deficient mice exposed to chronic stress exhibit disrupted latent inhibition, a hallmark of schizophrenia.

The neuronal cell adhesion molecule (NrCAM) is widely expressed and has important physiological functions in the nervous system across the lifespan, from axonal growth and guidance to spine and synaptic pruning, to organization of proteins at the nodes of Ranvier. NrCAM lies at the core of a functional protein network where multiple targets (including NrCAM itself) have been associated with schizophrenia. Here we investigated the effects of chronic unpredictable stress on latent inhibition, a measure of selective attention and learning which shows alterations in schizophrenia, in NrCAM knockout (KO) mice and their wild-type littermate controls (WT). Under baseline experimental conditions both NrCAM KO and WT mice expressed robust latent inhibition (p = 0.001). However, following chronic unpredictable stress, WT mice (p = 0.002), but not NrCAM KO mice (F < 1), expressed latent inhibition. Analyses of neuronal activation (c-Fos positive counts) in key brain regions relevant to latent inhibition indicated four types of effects: a single hit by genotype in IL cortex (p = 0.0001), a single hit by stress in Acb-shell (p = 0.031), a dual hit stress x genotype in mOFC (p = 0.008), vOFC (p = 0.020), and Acb-core (p = 0.032), and no effect in PrL cortex (p > 0.141). These results indicating a pattern of differential effects of genotype and stress support a complex stress × genotype interaction model and a role for NrCAM in stress-induced pathological behaviors relevant to schizophrenia and other psychiatric disorders.

Inferring Parameters of Pyramidal Neuron Excitability in Mouse Models of Alzheimer’s Disease Using Biophysical Modeling and Deep Learning

Alzheimer’s disease (AD) is believed to occur when abnormal amounts of the proteins amyloid beta and tau aggregate in the brain, resulting in a progressive loss of neuronal function. Hippocampal neurons in transgenic mice with amyloidopathy or tauopathy exhibit altered intrinsic excitability properties. We used deep hybrid modeling (DeepHM), a recently developed parameter inference technique that combines deep learning with biophysical modeling, to map experimental data recorded from hippocampal CA1 neurons in transgenic AD mice and age-matched wildtype littermate controls to the parameter space of a conductance-based CA1 model. Although mechanistic modeling and machine learning methods are by themselves powerful tools for approximating biological systems and making accurate predictions from data, when used in isolation these approaches suffer from distinct shortcomings: model and parameter uncertainty limit mechanistic modeling, whereas machine learning methods disregard the underlying biophysical mechanisms. DeepHM addresses these shortcomings by using conditional generative adversarial networks to provide an inverse mapping of data to mechanistic models that identifies the distributions of mechanistic modeling parameters coherent to the data. Here, we demonstrated that DeepHM accurately infers parameter distributions of the conductance-based model on several test cases using synthetic data generated with complex underlying parameter structures. We then used DeepHM to estimate parameter distributions corresponding to the experimental data and infer which ion channels are altered in the Alzheimer’s mouse models compared to their wildtype controls at 12 and 24 months. We found that the conductances most disrupted by tauopathy, amyloidopathy, and aging are delayed rectifier potassium, transient sodium, and hyperpolarization-activated potassium, respectively.

Role of human Kallistatin in glucose and energy homeostasis in mice

ObjectiveKallistatin (KST), also known as SERPIN A4, is a circulating, broadly acting human plasma protein with pleiotropic properties. Clinical studies in humans revealed reduced KST levels in obesity. The exact role of KST in glucose and energy homeostasis in the setting of insulin resistance and type 2 diabetes is currently unknown. MethodsKallistatin mRNA expression in human subcutaneous white adipose tissue (sWAT) of 47 people with overweight to obesity of the clinical trial “Comparison of Low Fat and Low Carbohydrate Diets With Respect to Weight Loss and Metabolic Effects (B-SMART)” was measured. Moreover, we studied transgenic mice systemically overexpressing human KST (hKST-TG) and wild type littermate control mice (WT) under normal chow (NCD) and high-fat diet (HFD) conditions. ResultsIn sWAT of people with overweight to obesity, KST mRNA increased after diet-induced weight loss. On NCD, we did not observe differences between hKST-TG and WT mice. Under HFD conditions, body weight, body fat and liver fat content did not differ between genotypes. Yet, during intraperitoneal glucose tolerance tests (ipGTT) insulin excursions and HOMA-IR were lower in hKST-TG (4.42 ± 0.87 AU, WT vs. 2.20 ± 0.27 AU, hKST-TG, p < 0.05). Hyperinsulinemic euglycemic clamp studies with tracer-labeled glucose infusion confirmed improved insulin sensitivity by higher glucose infusion rates in hKST-TG mice (31.5 ± 1.78 mg/kg/min, hKST-TG vs. 18.1 ± 1.67 mg/kg/min, WT, p < 0.05). Improved insulin sensitivity was driven by reduced hepatic insulin resistance (clamp hepatic glucose output: 7.7 ± 1.9 mg/kg/min, hKST-TG vs 12.2 ± 0.8 mg/kg/min, WT, p < 0.05), providing evidence for direct insulin sensitizing effects of KST for the first time. Insulin sensitivity was differentially affected in skeletal muscle and adipose tissue. Mechanistically, we observed reduced Wnt signaling in the liver but not in skeletal muscle, which may explain the effect. ConclusionsKST expression increases after weight loss in sWAT from people with obesity. Furthermore, human KST ameliorates diet-induced hepatic insulin resistance in mice, while differentially affecting skeletal muscle and adipose tissue insulin sensitivity. Thus, KST may be an interesting, yet challenging, therapeutic target for patients with obesity and insulin resistance.

Protein Kinase D Plays a Crucial Role in Maintaining Cardiac Homeostasis by Regulating Post-Translational Modifications of Myofilament Proteins.

Protein kinase D (PKD) enzymes play important roles in regulating myocardial contraction, hypertrophy, and remodeling. One of the proteins phosphorylated by PKD is titin, which is involved in myofilament function. In this study, we aimed to investigate the role of PKD in cardiomyocyte function under conditions of oxidative stress. To do this, we used mice with a cardiomyocyte-specific knock-out of Prkd1, which encodes PKD1 (Prkd1loxP/loxP; αMHC-Cre; PKD1 cKO), as well as wild type littermate controls (Prkd1loxP/loxP; WT). We isolated permeabilized cardiomyocytes from PKD1 cKO mice and found that they exhibited increased passive stiffness (Fpassive), which was associated with increased oxidation of titin, but showed no change in titin ubiquitination. Additionally, the PKD1 cKO mice showed increased myofilament calcium (Ca2+) sensitivity (pCa50) and reduced maximum Ca2+-activated tension. These changes were accompanied by increased oxidation and reduced phosphorylation of the small myofilament protein cardiac myosin binding protein C (cMyBPC), as well as altered phosphorylation levels at different phosphosites in troponin I (TnI). The increased Fpassive and pCa50, and the reduced maximum Ca2+-activated tension were reversed when we treated the isolated permeabilized cardiomyocytes with reduced glutathione (GSH). This indicated that myofilament protein oxidation contributes to cardiomyocyte dysfunction. Furthermore, the PKD1 cKO mice exhibited increased oxidative stress and increased expression of pro-inflammatory markers interleukin (IL)-6, IL-18, and tumor necrosis factor alpha (TNF-α). Both oxidative stress and inflammation contributed to an increase in microtubule-associated protein 1 light chain 3 (LC3)-II levels and heat shock response by inhibiting the mammalian target of rapamycin (mTOR) in the PKD1 cKO mouse myocytes. These findings revealed a previously unknown role for PKD1 in regulating diastolic passive properties, myofilament Ca2+ sensitivity, and maximum Ca2+-activated tension under conditions of oxidative stress. Finally, we emphasized the importance of PKD1 in maintaining the balance of oxidative stress and inflammation in the context of autophagy, as well as cardiomyocyte function.

Influence of the calcium voltage-gated channel auxiliary subunit (CACNA2D1) absence on intraocular pressure in mice

The etiology of elevated intraocular pressure (IOP), a major risk factor for glaucoma (optic nerve atrophy), is poorly understood despite continued efforts. Although the gene variant of CACNA2D1 (encoding α2δ1), a calcium voltage-gated channel auxiliary subunit, has been reported to be associated with primary open-angle glaucoma, and the pharmacological mitigation of α2δ1 activity by pregabalin lowers IOP, the cellular basis for α2δ1 role in the modulation of IOP remains unclear. Our recent findings reveled readily detectable levels of α2δ1 and its ligand thrombospondin in the cytoskeletome fraction of human trabecular meshwork (TM) cells. To understand the direct role of α2δ1 in the modulation of IOP, we evaluated α2δ1 null mice for changes in IOP and found a moderate (∼10%) but significant decrease in IOP compared to littermate wild type control mice. Additionally, to gain cellular insights into α2δ1 antagonist (pregabalin) induced IOP changes, we assessed pregabalin's effects on human TM cell actin cytoskeletal organization and cell adhesive interactions in comparison with a Rho kinase inhibitor (Y27632), a known ocular hypotensive agent. Unlike Y27632, pregabalin did not have overt effects on cell morphology, actin cytoskeletal organization, or cell adhesion in human TM cells. These results reveal a modest but significant decrease in IOP in α2δ1 deficient mice, and this response appears to be not associated with the contractile and cell adhesive characteristics of TM cells based on the findings of pregabalin effects on isolated TM cells. Therefore, the mechanism by which pregabalin lowers IOP remains elusive.

A190 HELICOBACTER PYLORI INDUCES AN AUTO-REACTIVE PHENOTYPE IN PINK1 DEFICIENT MICE THAT CORRELATES WITH MOTOR DYSFUNCTION

Abstract Background Parkinson’s disease (PD) is a chronic neurodegenerative disorder. Several genetic predispositions, including the PTEN-induced kinase 1 (PINK1) mutation, have been implicated in early onset family cases. Besides the loss of dopaminergic neurons in the brain, PD patients have a unique immune phenotype that includes increased inflammation, blood serum, brain levels of proinflammatory cytokines, brain infiltration with cytotoxic CD8 T cells, and loss of regulatory T cells (Treg) and their anti-inflammatory phenotype. The role of the gut microbiota and gastrointestinal infections are increasingly recognized as a cofactor in PD. One of pathogens associated with the risk of PD is Helicobacter pylori. The prevalence of this Gram-negative bacteria in PD patients is higher than in the general population and its eradication in PD patients improves motor function. Loss of the PD-associated PINK1 alters induced Treg function in vitro. We previously have shown that in PINK1 knock-out (KO) mice, gut infection with Gram-negative bacteria, Citrobacter rodentium, induces mitochondrial antigen presentation (MitAP) to the CD8 T cells that later infiltrate the brain. In this model, PINK1 KO mice develop a Parkinson-like L-DOPA-responsive motor phenotype after four C. rodentium infections. Aims Here we aimed to scrutinize potential role of the H. pylori infection in the induction of motor dysfunction and disbalanced immune tolerance in PINK1 KO mice. Methods In addition to standard behavioural testing, at 2- and 6-months post-infection we performed a multiplex cytokine analysis of gastric homogenates and spectral flow cytometry of gastric lamina propria, mesenteric lymph nodes, blood, spleen, and brain infiltrating immunocytes. As well as in vitro immunocytes response and function assays to H.pylori exposure in PINK1 KO and wild-type (WT) littermate controls. Results We show that infection with H. pylori causes a long-lasting inflammation in the stomach; and local and systemic immune phenotype that remains 6 months post-infection. This phenotype, though present in some WT infected mice, is higher in PINK1 KOs and similarly to PD patients includes a decrease in Treg proportion, FoxP3 downregulation in regulatory T cells, Gata3+ CD4 T cell loss, as well as increase of circulating mitochondrial antigen-specific CD8 T cells. Moreover, the immune phenotype in the H. pylori infected PINK1 KO mice correlates with motor dysfunction and CD8 T cell brain infiltration with no such association seen in the WT mice. Conclusions These results provide insight to the gut-immunity-brain axis in the pathogenesis of Parkinson’s disease, and further investigate the role of Gram-negative bacteria in the establishment of immune tolerance-autoreactivity balance. Funding Agencies ASAP

Cxcl17 is a proinflammatory chemokine and promotes neutrophil trafficking.

CXCL17, a novel member of the CXC chemokine class, has been implicated in several human pathologies, but its role in mediating immune response is not well understood. Characteristic features of immune response include resident macrophages orchestrating successive and structured recruitment of neutrophils and monocytes to the insult site. Here, we show that Cxcl17 knockout (KO) mice, compared to the littermate wildtype (WT) control, were significantly impaired in peritoneal neutrophil recruitment post-lipopolysaccharide (LPS) challenge. Further, the KO mice show dysregulated Cxcl1, Cxcr2, and IL6 levels, all of which directly impact neutrophil recruitment. Importantly, the KO mice showed no difference in monocyte recruitment post LPS-challenge or in peritoneal macrophage levels both in unchallenged and LPS-challenged mice. We conclude that Cxcl17 is a proinflammatory chemokine, and that it plays an important role in the early proinflammatory response by promoting neutrophil recruitment to the insult site.

Male Lrp5A214V mice maintain high bone mass during dietary calcium restriction by altering the Vitamin D endocrine system.

Environmental factors and genetic variation individually impact bone. However, it is not clear how these factors interact to influence peak bone mass accrual. Here we tested whether genetically programmed high bone formation driven by missense mutations in the Lrp5 gene (Lrp5A214V) altered the sensitivity of mice to an environment of inadequate dietary calcium (Ca) intake. Weanling male Lrp5A214V mice and wildtype littermates (control) were fed AIN-93G diets with 0.125%, 0.25%, 0.5% (reference, basal), or 1% Ca from weaning until 12 wks of age (i.e. during bone growth). Urinary Ca, serum Ca, and Ca regulatory hormones (PTH, 1,25 dihydroxyvitamin D3 (1,25(OH)2D3), bone parameters (μCT, ash), and renal/intestinal gene expression were analyzed. As expected, low dietary Ca intake negatively impacted bones and Lrp5A214V mice had higher bone mass and ash content. Although bones of Lrp5A214V mice have more matrix to mineralize, their bones were not more susceptible to low dietary Ca intake. In control mice, low dietary Ca intake exerted expected effects on serum Ca (decreased), PTH (increased), and 1,25(OH)2D3 (increased) as well as their downstream actions (i.e. reducing urinary Ca, increasing markers of intestinal Ca absorption). In contrast, Lrp5A214V mice had elevated serum Ca with a normal PTH response but a blunted 1,25(OH)2D3 response to low dietary Ca that was reflected in the renal 1,25(OH)2D3 producing/degrading enzymes, Cyp27b1 and Cyp24a1. Despite elevated serum Ca in Lrp5A214V mice, urinary Ca was not elevated. Despite an abnormal serum 1,25(OH)2D3 response to low dietary Ca, intestinal markers of Ca absorption (Trpv6, S100g mRNA) were elevated in Lrp5A214V mice and responded to low Ca intake. Collectively, our data indicate that the Lrp5A214V mutation induces changes in Ca homeostasis that permit mice to retain more Ca and support their high bone mass phenotype.

P028 A pathogenic role of activated mucosal-associated invariant T cells in an animal model of Inflammatory Bowel Disease

Abstract Background It is known that the mucosal-associated invariant T (MAIT) cells are the innate-like T cells that are restricted by the major histocompatibility complex-related molecule 1 (MR1), and that these cells express a semi-invariant T cell receptor. We have previously reported that the activation status of the circulating MAIT cells in patients with Ulcerative Colitis (UC) is associated with disease activity, and these cells infiltrate the inflamed colonic mucosa. These findings imply that MAIT cells are involved in the pathogenesis of Inflammatory Bowel Disease (IBD). However, the role of MAIT cells in the setting of IBD has not been revealed. Therefore, we investigated the role of MAIT cells in an animal model of UC. Methods To this end, we utilized MR1 deficient mice (MR1-/-), which lack MAIT cells, and isobutyl 6-formyl pterin (i6-FP), which is a synthetic antagonistic MR1 ligand. MR1-/- on the C57BL/6 background, their littermate wild-type controls, and C57BL/6 mice were sensitized by rectal administration of oxazolone to induce colitis. These were then administered an oral i6-FP. Splenocytes (SPL) and colonic lamina propria lymphocytes (LPL) were isolated from mice receiving i6-FP to measure cytokine production. MR1-/-, i6-FP-treated mice and their controls were orally administered FITC-dextran to analyze intestinal permeability. The peripheral blood mononuclear cells (PBMC) from the patients with UC were also isolated to study the effect of i6-FP on cytokine production by MAIT cells. Results MR1 deficiency or i6-FP treatment resulted in reduced severity of oxazolone-induced colitis. Mice treated with i6-FP resulted in reduced MAIT cell production of pro-inflammatory cytokines such as IFN-g and TNF in both SPL and colonic LPL. Similar results were also observed in PBMC isolated from the patients with UC when incubated with i6-FP. Although MR1 deficiency resulted in increased intestinal permeability, i6-FP administration did not affect gut integrity in mice. Conclusion The current studies indicate that MAIT cells have a pathogenic role in colitis and suppressing activation of these cells may reduce the severity of colitis without affecting gut integrity. Thus, MAIT cells may be potential therapeutic targets for IBD including UC.

POMC-specific knockdown of MeCP2 leads to adverse phenotypes in mice chronically exposed to high fat diet

Methyl-CpG binding protein 2 (MeCP2) is an epigenetic factor associated with the neurodevelopmental disorders Rett Syndrome and MECP2 duplication syndrome. Previous studies have demonstrated that knocking out MeCP2 globally in the central nervous system leads to an obese phenotype and hyperphagia, however it is not clear if the hyperphagia is the result of an increased preference for food reward or due to an increase in motivation to obtain food reward. We show that mice deficient in MeCP2 specifically in pro-opiomelanocortin (POMC) neurons have an increased preference for high fat diet as measured by conditioned place preference but do not have a greater motivation to obtain food reward using a progressive ratio task, relative to wildtype littermate controls. We also demonstrate that POMC-Cre MeCP2 knockout (KO) mice have increased body weight after long-term high fat diet exposure as well as elevated plasma leptin and corticosterone levels compared to wildtype mice. Taken together, these results are the first to show that POMC-specific loss-of-function Mecp2 mutations leads to dissociable effects on the rewarding/motivational properties of food as well as changes to hormones associated with body weight homeostasis and stress.

Levodopa Rescues Retinal Function in the Transgenic A53T Alpha-Synuclein Model of Parkinson's Disease.

Loss of substantia nigra dopaminergic cells and alpha-synuclein (α-syn)-rich intraneuronal deposits within the central nervous system are key hallmarks of Parkinson's disease (PD). Levodopa (L-DOPA) is the current gold-standard treatment for PD. This study aimed to evaluate in vivo retinal changes in a transgenic PD model of α-syn overexpression and the effect of acute levodopa (L-DOPA) treatment. Anaesthetised 6-month-old mice expressing human A53T alpha-synuclein (HOM) and wildtype (WT) control littermates were intraperitoneally given 20 mg/kg L-DOPA (50 mg levodopa, 2.5 mg benserazide) or vehicle saline (n = 11-18 per group). In vivo retinal function (dark-adapted full-field ERG) and structure (optical coherence tomography, OCT) were recorded before and after drug treatment for 30 min. Ex vivo immunohistochemistry (IHC) on flat-mounted retina was conducted to assess tyrosine hydroxylase (TH) positive cell counts (n = 7-8 per group). We found that photoreceptor (a-wave) and bipolar cell (b-wave) ERG responses (p < 0.01) in A53T HOM mice treated with L-DOPA grew in amplitude more (47 ± 9%) than WT mice (16 ± 9%) treated with L-DOPA, which was similar to the vehicle group (A53T HOM 25 ± 9%; WT 19 ± 7%). While outer retinal thinning (outer nuclear layer, ONL, and outer plexiform layer, OPL) was confirmed in A53T HOM mice (p < 0.01), L-DOPA did not have an ameliorative effect on retinal layer thickness. These findings were observed in the absence of changes to the number of TH-positive amacrine cells across experiment groups. Acute L-DOPA treatment transiently improves visual dysfunction caused by abnormal alpha-synuclein accumulation. These findings deepen our understanding of dopamine and alpha-synuclein interactions in the retina and provide a high-throughput preclinical framework, primed for translation, through which novel therapeutic compounds can be objectively screened and assessed for fast-tracking PD drug discovery.

Contribution of HSP90 Cleavage to the Cytotoxic Effect of Suberoylanilide Hydroxamic Acid In Vivo and the Involvement of TXNIP in HSP90 Cleavage.

Heat shock protein (HSP) 90 is expressed in most living organisms, and several client proteins of HSP90 are necessary for cancer cell survival and growth. Previously, we found that HSP90 was cleaved by histone deacetylase (HDAC) inhibitors and proteasome inhibitors, and the cleavage of HSP90 contributes to their cytotoxicity in K562 leukemia cells. In this study, we first established mouse xenograft models with K562 cells expressing the wild-type or cleavage-resistant mutant HSP90β and found that the suppression of tumor growth by the HDAC inhibitor suberoylanilide hydroxamic acid (SAHA) was interrupted by the mutation inhibiting the HSP90 cleavage in vivo. Next, we investigated the possible function of thioredoxin interacting protein (TXNIP) in the HSP90 cleavage induced by SAHA. TXNIP is a negative regulator for thioredoxin, an antioxidant protein. SAHA transcriptionally induced the expression of TXNIP in K562 cells. HSP90 cleavage was induced by SAHA also in the thymocytes of normal mice and suppressed by an anti-oxidant and pan-caspase inhibitor. When the thymocytes from the TXNIP knockout mice and their wild-type littermate control mice were treated with SAHA, the HSP90 cleavage was detected in the thymocytes of the littermate controls but suppressed in those of the TXNIP knockout mice suggesting the requirement of TXNIP for HSP90 cleavage. We additionally found that HSP90 cleavage was induced by actinomycin D, β-mercaptoethanol, and p38 MAPK inhibitor PD169316 suggesting its prevalence. Taken together, we suggest that HSP90 cleavage occurs also in vivo and contributes to the anti-cancer activity of various drugs in a TXNIP-dependent manner.

Overexpression of manganese superoxide dismutase mitigates ACL injury-induced muscle atrophy, weakness and oxidative damage

Oxidative stress has been implicated in the etiology of skeletal muscle weakness following joint injury. We investigated longitudinal patient muscle samples following knee injury (anterior cruciate ligament tear). Following injury, transcriptomic analysis revealed downregulation of mitochondrial metabolism-related gene networks, which were supported by reduced mitochondrial respiratory flux rates. Additionally, enrichment of reactive oxygen species (ROS)-related pathways were upregulated in muscle following knee injury, and further investigation unveiled marked oxidative damage in a progressive manner following injury and surgical reconstruction. We then investigated whether antioxidant protection is effective in preventing muscle atrophy and weakness after knee injury in mice that overexpress Mn-superoxide dismutase (MnSOD+/−). MnSOD+/− mice showed attenuated oxidative damage, atrophy, and muscle weakness compared to wild type littermate controls following ACL transection surgery. Taken together, our results indicate that ROS-related damage is a causative mechanism of muscle dysfunction after knee injury, and that mitochondrial antioxidant protection may hold promise as a therapeutic target to prevent weakness and development of disability.

Dietary fibre confers therapeutic effects in a preclinical model of Huntington’s disease

Huntington’s disease (HD) is a neurodegenerative disorder involving psychiatric, cognitive and motor deficits, as well as peripheral symptoms, including gastrointestinal dysfunction. The R6/1 HD mouse model expresses a mutant human huntingtin transgene and has been shown to provide an accurate disease model. Recent evidence of gut microbiome disruption was shown in preclinical and clinical HD. Therefore, we aimed to assess the potential role of gut microbial modulation in the treatment of HD. The R6/1 HD mice and wild-type littermate controls were randomised to receive diets containing different amounts of fibre: high-fibre (10 % fibre), control (5 % fibre), or zero-fibre (0 % fibre), from 6 to 20 weeks of age. We characterized the onset and progression of motor, cognitive and affective deficits, as well as gastrointestinal function and gut morphological changes. Faeces were collected for gut microbiome profiling using 16S rRNA sequencing, at 14 and 20 weeks of age. When compared to the control diet, high-fibre diet improved the performance of HD mice in behavioral tests of cognitive and affective function, as well as the gastrointestinal function of both HD and wild-type mice. While the diets changed the beta diversity of wild-type mice, no statistical significance was observed at 14 or 20 weeks of age within the HD mice. Analysis of Composition of Microbiomes with Bias Correction (ANCOM-BC) models were performed to evaluate microbiota composition, which identified differences, including a decreased relative abundance of the phyla Actinobacteriota, Campylobacterota and Proteobacteria and an increased relative abundance of the families Bacteroidaceae, Oscillospiraceae and Ruminococcaceae in HD mice when compared to wild-type mice after receiving high-fibre diet. PICRUSt2 revealed that high-fibre diet also decreased potentially pathogenic functional pathways in HD. In conclusion, high-fibre intake was effective in enhancing gastrointestinal function, cognition and affective behaviors in HD mice. These findings indicate that dietary fibre interventions may have therapeutic potential in Huntington’s disease to delay clinical onset, and have implications for related disorders exhibiting dysfunction of the gut-brain axis.

The Actin Bundling Protein L-Plastin Mediates Platelet Force Generation and Thrombosis

Within minutes of activation, platelets dramatically change their shape, forming filopodia and lamellipodia, aggregate with other platelets or different cells, and can increase their surface area up to 4 times. This relies on a dynamic actin cytoskeleton, regulated by many actin binding proteins in a time- and spatial-specific manner. Deficiency or inhibition of several actin binding proteins in platelets have been shown with impaired platelet spreading and reduced thrombosis. Recently, we found that an actin bundling protein, L-plastin, is expressed in platelets. Its function in platelets has not been studied. In megakaryocytes, we showed that L-plastin is responsible for maintaining cytoskeletal stiffness; in this study we sought to determine L-plastin's role in platelet functions, including thrombosis. We examined the role of L-plastin in platelets in vivo and ex vivo using L-plastin deficient ( Lcp1 -/-) male and female mice. First, we used the collagen/epinephrine induced pulmonary embolism (PE) model and found that survival times were significantly shorter in Lcp1 -/- mice than in their littermate wildtype (WT) controls (Mean±SEM WT 202±12 sec, Lcp1 -/- 139.5±14 sec, P=0.004), suggesting accelerated thrombosis upon L-plastin deficiency in vivo. Second, we examined L-plastin's role in arterial thrombosis in vivo using the laser-induced cremaster arteriole thrombosis mode. Platelets were labeled with anti-GPIb IgG derivative X488 and the thrombosis formation after laser injury was recorded. Preliminary data indicate that thrombi in Lcp1 -/- mice are larger than those in WT mice. Third, we examined L-plastin's role in platelet adhesion using a microfluidics assay with DiOC6-labelled whole blood. At a shear rate of 200/s, the Lcp1 -/- mice had significantly increased platelet adhesion to both collagen and fibrinogen matrices. Fourth, to evaluate the effect of L-plastin on platelet aggregation in vitro, we stimulated washed platelets with 3 different concentrations of thrombin and measured the platelet aggregation (i.e., 0.1, 0.5 and 1 U/mL). When platelets are stimulated with 0.5U/mL or 1U/mL thrombin, platelets from WT and Lcp1 -/- mice showed comparable aggregation kinetics. At 0.1U/mL thrombin, however, preliminary data showed increased aggregation in the Lcp1 -/- group. The difference between the low and high concentration of thrombin indicates potential regulation of the GPIba mediated actin cytoskeleton rearrangement during platelet aggregation. We next examined under low thrombin concentration, the platelet actin cytoskeleton reorganization and platelet spreading. We seeded washed platelets on a fibrinogen matrix with the presence of thrombin (0.1U/mL). Platelets were then fixed and stained for F-actin and imaged using immunofluorescent confocal microscopy. The spreading area per platelet was calculated using FIJI software. Platelets from Lcp1 -/- mice showed significantly increased average membrane area at 30 min (Median WT 689 pixels, Lcp1 -/- 866 pixels, P&amp;lt;0.0001). This suggests that the actin rearrangement in platelets upon fibrinogen activation and thrombin stimulation is inhibited by L-plastin. Lastly, we examined whether L-plastin is involved in platelet force generation during the actin reorganization. We used a recently developed “black dot” assay. Briefly, a fluorescent pattern (i.e., black dots) was microcontact printed onto a flexible polydimethylsiloxane substrate and coated with fibrinogen. Platelets were seeded onto the matrix and fixed after 30 min. The contractile forces of individual platelets were measured based on the deformation of the fluorescent micropattern as illustrated in Fig 1A. The contractile force in platelets from Lcp1 -/- mice is decreased with altered actin distribution pattern compared to WT mice (Fig 1. Contractile force per platelet, Mean±SEM WT 14.78±0.86nN, Lcp1 -/- 12.03±0.67nN, P=0.01). In summary, we found accelerated thrombosis in Lcp1 -/- mice, associated with accelerated platelet adhesion and spreading ex vivo. Mechanistically, our preliminary data suggest this is due to impaired actin bundle formation and decreased platelet contractile force. L-plastin may be unique among actin binding proteins.

Platelet Mitochondria Calcium Uniporter Regulates ITAM-Dependent Platelet Activation and Signaling

Introduction: The ability of platelets to multitask is critical to arrest bleeding after injury and for the development of arterial thrombosis, which underlies myocardial infarction and stroke. Platelet activation relies heavily on changes in cytoplasmic calcium flux. However, little is known on the role mitochondrial calcium flux directly plays in platelet activation. In other cells, release of calcium from intracellular stores results in calcium entry into channels located in the outer mitochondrial membrane. Once calcium enters the intermembrane space, entry into the mitochondrial matrix is regulated by a multimeric complex, composed of channel-forming subunits and regulatory elements called the mitochondrial calcium uniporter (MCU). MCU has been extensively studied in cardiac tissue where calcium flux through MCU is critical for regulating bioenergetics, ROS formation, and cytoplasmic calcium levels. As these processes are important for platelet activation, these studies would indicate mitochondrial calcium flux may regulate platelet activation. However, the role of MCU in platelet function and activation is poorly understood. Aim: The goals of the current study are 1) to examine the role of MCU and mitochondrial calcium flux in platelet function and 2) to establish if mitochondrial calcium regulates thrombosis. Methods: We generated a platelet-specific MCU-deficient mouse (MCU fl/fl-PF4-cre, KO) and compared them to littermate wild-type controls (MCU fl/fl, WT). Platelet function including activation, aggregation, and mitochondrial calcium flux in response to PAR4 activating peptide (PAR) and ADP (P2Y12), both GPCRs or to collagen (GPVI) and rhodocytin (CLEC-2), both ITAMs, were examined. In addition, we examined ex vivo platelet adhesion under arterial and venous shear to collagen and in vivo thrombosis using a ferric chloride carotid artery model. Results: Mice deficient inplatelet MCU were viable and fertile. In addition, platelet-specific MCU deletion did not alter platelet counts, mean platelet volume, or platelet half-life. MCU KO platelets had significantly reduced (p&amp;lt;0.05) GPVI and CLEC-2-dependent aIIbb3 activation and P-selectin expression while platelet activation in response to P2Y12 and PAR4 stimulation was unchanged. Consistent with our activation results, platelet aggregation was significantly reduced in response to collagen and rhodocytin (p&amp;lt;0.05) in MCU KO platelets, but not thrombin or ADP. MCU KO platelets adhered significantly less (p=0.0012) to collagen compared to MCU WT platelets under arterial and venous shear conditions. In vivo, MCU KO mice had longer occlusion time compared to the WT (p=0.0044). Mechanistically, mitochondrial calcium flux was significantly reduced (p&amp;lt;0.05) in MCU KO platelets compared to WT platelets after GPVI stimulation, but not PAR4 activation. Furthermore, mitochondrial reactive oxygen species (ROS) generation was significantly reduced in MCU KO platelets compared to WT platelets (p=0.0097) after GPVI-dependent activation while PAR4 activation induced no change in mitochondrial ROS. Mitochondrial ROS is known to regulate signaling in other cells. Consistent with this hypothesis, we observed a significant reduction in the ITAM signaling molecules pSyK (p=0.0084) and pPLCγ2 (p=0.012) in MCU KO platelets after GPVI stimulation. Furthermore, inhibiting mitochondrial ROS using MitoTempo, a specific mitochondrial ROS inhibitor, decreased aggregation (p=0.0004) as well as downstream signaling, including pSyk (p=0.0046) and pPLCγ2 (p=0.0493) in WT platelets when treated with a GPVI agonist. In parallel, treating MCU KO platelets with H 2O 2 to induce ROS production increased platelet aggregation (p=0.0036) after GPVI activation. Conclusion(s): Platelet MCU mediates platelet activation and thrombosis in an ITAM-dependent manner by regulating mitochondrial calcium flux and ROS generation as well as downstream ITAM signaling through GPVI and CLEC-2. Our data support a novel role for mitochondria and mitochondria calcium flux in regulating ITAM-dependent platelet activation and demonstrate platelet MCU as a novel anti-platelet target to reduce thrombosis

STAT3 Deficiency Alters the Macrophage Activation Pattern and Enhances Matrix Metalloproteinase 9 Expression during Staphylococcal Pneumonia.

Staphylococcus aureus is a significant cause of morbidity and mortality in pulmonary infections. Patients with autosomal-dominant hyper-IgE syndrome due to STAT3 deficiency are particularly susceptible to acquiring staphylococcal pneumonia associated with lung tissue destruction. Because macrophages are involved in both pathogen defense and inflammation, we investigated the impact of murine myeloid STAT3 deficiency on the macrophage phenotype invitro and on pathogen clearance and inflammation during murine staphylococcal pneumonia. Murine bone marrow-derived macrophages (BMDM) from STAT3 LysMCre+ knockout or Cre- wild-type littermate controls were challenged with S. aureus, LPS, IL-4, or vehicle control invitro. Pro- and anti-inflammatory responses as well as polarization and activation markers were analyzed. Mice were infected intratracheally with S. aureus, bronchoalveolar lavage and lungs were harvested, and immunohistofluorescence was performed on lung sections. S. aureus infection of STAT3-deficient BMDM led to an increased proinflammatory cytokine release and to enhanced upregulation of costimulatory MHC class II and CD86. Murine myeloid STAT3 deficiency did not affect pathogen clearance invitro or invivo. Matrix metalloproteinase 9 was upregulated in Staphylococcus-treated STAT3-deficient BMDM and in lung tissues of STAT3 knockout mice infected with S. aureus. Moreover, the expression of miR-155 was increased. The enhanced inflammatory responses and upregulation of matrix metalloproteinase 9 and miR-155 expression in murine STAT3-deficient as compared with wild-type macrophages during S. aureus infections may contribute to tissue damage as observed in STAT3-deficient patients during staphylococcal pneumonia.