Gi/o-coupled G-protein coupled receptors (GPCRs) can exert an inhibitory effect on vesicle release through several G-protein driven mechanisms, more than one of which may be concurrently present in individual presynaptic terminals. G protein betagamma subunits inhibit exocytosis via directly binding to the synaptosomal-associated protein of 25 kDa (SNAP25), competing with the fusogenic calcium sensor synaptotagmin 1 (Syt1) in a calcium-dependent manner for binding sites on SNAP25. Here, we generate several SNAP25 C-terminal mutants that are deficient in G protein betagamma binding while retaining normal vesicle release. The SNAP25-8A mutant features 8 G protein betagamma-binding residues mutated to Ala, and the SNAP25delta3 mutant, in which residue G204 is replaced by a stop codon, both feature a partial reduction in G protein beta1gamma2 binding in vitro. SNAP25-8A exhibits a reduction in the ability of the lamprey serotonin receptor to reduce excitatory postsynaptic current (EPSC) amplitudes, an effect previously shown to be mediated through the G protein betagamma-SNARE interaction. Syt1 binding to these mutants is largely intact. We conclude that the extreme C-terminus of SNAP25 is a critical region for the G protein betagamma-SNARE interaction. To further investigate the physiological relevance of the G protein betagamma-SNARE interaction, we have developed small molecule modulators of the G protein betagamma-SNARE interaction with micromolar potency. A transgenic mouse has been made containing the SNAP25delta3 mutation using the CRISPR-Cas9 reaction. Characterization of the phenotype of this animal is ongoing. In summary, we have identified key residues for the G protein betagamma-SNARE interaction and generated new experimental tools to investigate the importance of this interaction in tissues and disease states where its relevance is not currently known.

Dysregulation of the mesolimbic dopamine (DA) system is a hallmark of the pathophysiology of drug addiction and many other prevalent diseases. The nucleus accumbens (NAc), a region essential for the incentive and hedonic properties of drugs of abuse, is a key biological substrate. At least 90% of the neurons in the NAc are medium spiny neurons (MSNs), which provide the sole projections from the region. MSNs can divided into two classes by biochemistry and anatomy: D1(+) MSNs project primarily to midbrain DA areas, while A2A/D2(+) MSNs send afferents to the ventral pallidum. MSNs generally rest at relatively hyperpolarized membrane potentials, so excitatory drive is essential to governing the output of the NAc and subsequent complex behavioral outcomes. The prefrontal cortex (PFC), ventral hippocampus, and basolateral amygdala provide major excitatory inputs to the NAc and have been examined recently in the context of cocaine exposure. However, despite its comparable anatomical denseness, little is known about how afferents from the midline nuclei of the thalamus (mThal) to the NAc modulate reward-related behaviors and learning and memory processes. While much remains to be understood, drug-induced modifications of excitatory signaling (i.e. synaptic plasticity) in the NAc have been suggested to underlie the maladaptive behaviors observed in addiction. N-methyl-D-aspartate receptors (NMDARs) are of paramount importance in regulating excitatory synaptic strength and learning and memory. Therefore we aimed to assess the function of NMDARs in NAc core D1(+) MSNs, with a particular emphasis on mThal inputs. We determined that cocaine sensitization and abstinence enhances NMDAR function at mThal-D1(+), mThal-D1(-), and PFC-D1(+) synapses. At mThal-D1(+) synapses specifically, we demonstrated that cocaine enhances GluN2C/D function and NMDAR-dependent synaptic plasticity. The role for these NMDARs cocaine-conditioned behaviors is evidenced by the finding that mice with a D1-specific GluN1 genetic deletion did not reinstate a place preference to cocaine. Collectively these data emphasize the emerging role for D1-NMDARs in reward learning, and highlight mThal inputs and GluN2C/D subunits as novel targets for the treatment of psychostimulant use disorders.

<p>Normal development and homeostasis require dynamic and tight regulation of epithelial architecture. Abnormal epithelial physiology is associated with various pathological conditions including cancers, and may be induced by changes in epithelial polarity, morphology and/or movement. Among the signaling pathways modulating epithelial physiology are those downstream of integrins and receptor tyrosine kinases (RTKs). Although roles of multiple integrins and RTKs in epithelium homeostasis have been established, the identity of signals regulating the functions of these surface receptors and the pathways connecting them to the regulation of epithelial architecture remain largely unknown. In this dissertation, I have identified the Abl family of non-receptor tyrosine kinases (Abl and Arg) as regulators of beta1 integrin and Met receptor tyrosine kinase signaling.</p><p> </p><p>Abl family kinases are hyper-activated in multiple solid tumors and implicated in epithelial polarity regulation. Dysfunction of beta1 integrin is also associated with carcinoma development. To study the role of the Abl family member Arg in epithelial cell polarity, I have taken advantage of a three-dimensional (3D) cell culture system, where Madin Darby canine kidney type II (MDCKII) cells grown in collagen gels develop into polarized cyst structures. I have found that expression of active Arg kinase results in the formation of cysts with inverted apical polarity and that active Arg modulates epithelial polarity by regulating beta1 integrin and small GTPases pathways. In addition, I have shown that Arg regulates the Rap1-beta1 integrin pathway independently of the Rac1 pathway which promotes basal laminin assembly. I have also found that Abl family kinases function downstream of Met and that Abl kinase hyperactivity correlates with Met activation in a mouse mammary tumor model. Abl kinases are activated by HGF which is the ligand for Met, and active Abl kinases are recruited to the Met receptor and promote its tyrosine phosphorylation. Using fluorescence resonance energy transfer (FRET), I have found that Abl kinases regulate RhoA GTPase activity which contributes to actomyosin contractility induced by Met receptor activation. Further, Abl kinases positively regulate Met-dependent migration and invasion induced by HGF in several breast cancer cell lines. </p><p>In conclusion, I have identified novel functions of the Abl kinases in epithelial architecture regulation: modulation of epithelial polarity by targeting beta1 integrin function and promotion of Met signaling required for migration and invasion. This has important implications as it suggests potential roles for Abl kinases in carcinoma initiation mediated by beta1 integrin dysfunction, and development of Abl kinases inhibitors for treatment of cancers driven by hyper-activation of HGF-Met signaling.</p> / Dissertation

Obsessive-compulsive disorder (OCD) is a chronic, disabling condition with inadequate treatment options that leave most patients with substantial residual symptoms. Structural, neurochemical, and behavioral findings point to a significant role for basal ganglia circuits and for the glutamate system in OCD. Genetic linkage and association studies in OCD point to SLC1A1, which encodes the neuronal glutamate/aspartate/cysteine transporter EAAT3/EAAC1. Despite this, no previous studies have investigated EAAT3 in basal ganglia circuits or in relation to OCD-related behavior. Here, we report a new model of Slc1a1 loss based on an excisable STOP cassette that yields successful ablation of EAAT3 expression and function. Using amphetamine as a probe, we found that EAAT3 loss prevents expected increases in 1) locomotor activity, 2) stereotypy, and 3) immediate early gene induction in the dorsal striatum following amphetamine administration. Further, Slc1a1-STOP mice showed diminished grooming in an SKF-38393 challenge experiment, a pharmacologic model of OCD-like grooming behavior. This is accompanied by reduced D1 receptor binding in the dorsal striatum of Slc1a1-STOP mice. Slc1a1-STOP mice also exhibit reduced extracellular dopamine concentrations both at baseline and following amphetamine challenge in the dorsal striatum. Viral-mediated restoration of Slc1a1/EAAT3 expression in the midbrain but not in the striatum results in partial rescue of amphetamine induced locomotion and stereotypy in Slc1a1-STOP mice, consistent with an impact of EAAT3 loss on pre-synaptic dopaminergic function. Collectively, these findings represent the first indication that the most consistently associated OCD candidate gene impacts basal ganglia-dependent repetitive behaviors.

Unacceptable pharmacokinetics (PK) relating to aldehyde oxidase (AO) metabolism have resulted in clinical failure of several promising drug candidates, yet reliable and standardized methods to predict the human PK and drug-drug interaction (DDI) liability of AO-metabolized drugs remains to be established. Investigations into the DDI liability associated with AO substrates also metabolized by cytochrome P450 and the potential utility of allometric scaling methods to predict human clearance (CL) of AO substrates were conducted. These investigations indicate a susceptibility of mixed AO/P450-metabolized drugs to DDI with P450 inhibitors, resulting in elevated AO metabolite exposure. In addition, data indicate allometric scaling with multiple or single species may be useful to predict human CL when appropriate species are utilized. Evaluations of biotransformation, fraction metabolized by AO (Fm,AO), hepatic extraction ratio (E), and allometric scaling of in vivo and in vitro CL indicate guinea pig and monkey may be most useful for single-species scaling, while minipig, rat, and mouse may also be of use in multispecies allometry.

Highly reactive acyclic levuglandin-like gamma-ketoaldehydes (gamma-KA, isoketals, or IsoKs) are formed as products of the isoprostane pathway of lipid peroxidation. IsoKs are known to covalently adduct ε-amino groups in lysyl residues of proteins, forming stable adducts and intramolecular cross-links. Increased IsoK-lysyl-lactam adducts are found in a number of disease conditions, including atherosclerosis, end-stage renal disease, and Alzheimerâs disease. A selective IsoK scavenger, salicylamine (SA), was developed and tested in Caenorhabditis elegans to probe the pathophysiological processes in IsoK-mediated oxidative injury, and consequently provide the groundwork for the development of new rational therapeutic interventions to limit oxidative damage. Administration of SA extends adult nematode longevity by nearly 56% and prevents multiple deleterious age-related biochemical and functional changes. Testing of a variety of molecular targets for SAâs action revealed the sirtuin SIR-2.1 as the leading candidate. When SA was administered to a SIR-2.1 knockout strain, the effects on lifespan and healthspan extension were abolished. The SIR-2.1-dependent effects of SA were not mediated by large changes in gene expression programs or by significant changes in mitochondrial function. However, expression array analysis did show SA-dependent regulation of the transcription factor ets-7 and associated genes. In ets-7 knockout worms, SAâs longevity effects were abolished, similar to sir-2.1 knockouts. However, SA dose-dependently increases ets-7 mRNA levels in non-functional SIR-2.1 mutant, suggesting that both are necessary for SAâs complete lifespan and healthspan extension.

Heart development depends on coordinated proliferation and differentiation of cardiac progenitor cells, but how the two processes are synchronized is not well understood. The data herein show that the secreted BMP antagonist GREMLIN 2 (GREM2) is induced in cardiac progenitor cells shortly after cardiac mesoderm specification during differentiation of human pluripotent stem cells. GREM2 expression follows cardiac lineage differentiation independently of the differentiation method used, or the origin of the pluripotent stem cells, suggesting that GREM2 is linked to cardiogenesis. Addition of GREM2 protein strongly increases cardiomyocyte output compared to established pro-cardiogenic differentiation methods. These data show that inhibition of canonical BMP signaling by GREM2 is necessary to promote proliferation of cardiac progenitor cells. However, canonical BMP signaling inhibition alone is not sufficient to induce cardiac differentiation, which depends on subsequent JNK pathway activation specifically by GREM2. These findings may have broader implications in the design of approaches to orchestrate growth and differentiation of pluripotent stem cell-derived lineages that depend on precise regulation of BMP signaling.

No description available.

Reduced platelet aggregation and a mild bleeding phenotype has been observed in patients chronically taking selective serotonin reuptake inhibitors (SSRIs) that block the serotonin transporter (SERT). Here, I explore this relationship between platelet activation, specifically αIIbβ3 activation, and the loss of SERT function. First, using a pharmacological approach, we investigate the role of acute loss of SERT function on platelet αIIbβ3 activation. We find that acute treatment with SSRIs does not alter αIIbβ3 activation but reduced αIIbβ3-mediated platelet spreading. Next, two models of sustained loss of SERT function were used to investigate platelet αIIbβ3 activation; the SERT knockout mouse (SERT-/-) mice and mice treated with citalopram for 6-days. Both models replicate the mild bleeding phenotypes noted in human patients taking SSRIs. Following transfusion of wild-type platelets into SERT-/- mice, SERT-/- bleeding times were reduced to WT levels suggesting defects in platelet function. We examined αIIbβ3 activation (JON/A binding) and granule exocytosis with P-selectin surface expression (CD62p binding) following ADP stimulation and found reduced ADP-mediated αIIbβ3 activation in SERT-/- platelets. Acute treatment of platelets with SSRIs (paroxetine and citalopram) to prevent serotonin uptake during activation did not alter ADP-mediated αIIbβ3 activation. However, 5HT2AR antagonists significantly reduced ADP-mediated αIIbβ3 activation. These findings suggest that serotonin synergizes with ADP through 5HT2AR activation, but not acute SERT uptake. Furthermore, SERT-/- platelets displayed reduced serotonin enhanced ADP-mediated αIIbβ3 activation, likely due to reduced 5HT2AR cell surface levels. This again suggests acute function of SERT does not alter ADP-mediated αIIbβ3 activation but that sustained loss of SERT function alters surface expression of 5HT2AR and ultimately reduces ADP-mediated αIIbβ3 activation. Lastly, I discuss the hypercoaguability phenotype of the β3 integrin mutation, which models the human PIA2 polymorphism. This mutation leads to a primed αIIbβ3 integrin characterized by increased baseline Src-signaling. The hypercoaguability phenotype can be rescued with the Src inhibitor SKI606 but, interestingly, is also hyperserotonergic. We find that blockage of the SERT transporter with 6-day citalopram treatment is also able to rescue the KI thrombin clotting time. These findings further establish a connection between serotonergic regulation and αIIbβ3 activation in platelet function. This body of work expands the current knowledge of serotonin in platelet biology and supports a more physiologically based understanding of the effects of SSRIs on platelet function.

BACKGROUND: Obesity has become increasingly prevalent due to over-nutrition and sedentary lifestyle. While many past studies have identified a link between metabolic dysfunction and tissue inflammation, molecular mechanisms that initiate and propagate inflammation remain unclear. In this study, we investigated the role of Toll-like receptor-2 (TLR2) in modulating inflammatory response in mice and examined the effect of high-fat diet (HFD)-induced myelopoiesis and systemic inflammation. OBJECTIVE: To investigate the role of TLR2 in regulating the HFD-induced myelopoiesis and systemic inflammation. METHODS: After HFD-feeding for 12 weeks, mouse bone marrow (BM) cells and peripheral blood leukocytes were isolated. Mouse BM hematopoietic progenitor cells (HSPCs) were enriched using a HSPC enrichment kit. Isolated HSPCs were used for RNA purification, FACS analysis, and RT-qPCR. Statistical analysis was performed on FACS and RT-qPCR data. RESULTS: Compared to WT mice, TLR2 KO mice demonstrated significant reduction in the mRNA expression in HSPCs of C/EBPα, C/EBPε, GFI-1, PU.1, and Runx1, which are all transcription factors involved in myeloid cell differentiation. FACS analysis showed a substantial percentage reduction in BM neutrophils and increase in BM lymphocytes in TLR2 KO mice in comparison to WT mice. Interestingly, the percentage of blood B-lymphocyte of TLR2 KO mice was also markedly decreased. CONCLUSIONS: HFD feeding activates TLR-dependent C/EBPα-GFI-1 pathway required for myelopoiesis and systemic inflammation. Given that the deletion of TLR2 is sufficient to reverse the long-term HFD-induced molecular changes and neutrophil production, TLR2 may be involved in obesity-related systemic tissue inflammation. / 2019-07-11T00:00:00Z