No description available.

This research was conducted to investigate the expression of porcine intestinal nutrient transporters along the neonatal crypt-villus axis and during the postnatal development. First, we examined the transport kinetics of Na+-glucose co-tranporter 1 (SGLT1) and Na+-dependent neutral amino acid (AA) transporter B0AT1 and then the protein and mRNA abundances of SGLT1, B0AT1 and Na+-dependent neutral AA exchanger ASCT2 along the jejunal crypt-villus axis in the neonatal pig and the potential mechanisms associated with their regulations. Our results suggested that: 1) high levels of apical maximal SGLT1 and B0AT1 uptake activities were shown to exist along the entire jejunal crypt-villus axis in the neonatal pig; 2) there were no significant differences in the SGLT1, B0AT1 and ASCT2 protein abundances in spite of their different mRNA abundances among the crypt-villus axis, suggesting unique posttranscriptional regulatory mechanisms; and 3) global protein translational efficiency, as assessed by examining some of the key protein translational initiation and elongation factors, was higher in the crypt cells than in the upper villus cells, likely playing a regulatory role for maintaining apical nutrient transporter abundances in crypt cells of the neonate. Second, we further examined the protein and mRNA abundances of jejunal neutral AA transporters B0AT1 and ASCT2 and acidic AA transporter EAAC1 during the postnatal development in pigs at the ages of d 1, 4, 6, 12, 20, 28 (1-wk post-weaning), and 70 (mature gut at grower phase), respectively. Our results showed that the jejunal apical B0AT1, ASCT2 and EAAC1 protein abundances were dramatically decreased during the postnatal development and were likely regulated at both the transcriptional and post-transcriptional levels. These substantial decreases in the small intestinal apical Na+-dependent AA transporter abundances may contribute to increased intestinal microbial catabolism of AA, which may be partially responsible for the reduced whole body efficiency of nitrogen utilization during the postnatal growth in pigs. Collectively, our results suggest that apical nutrient transporters SGLT1, B0AT1 and ASCT2 are abundantly expressed along the entire jejunal crypt-villus axis in the neonatal pig, whereas abundances of jejunal apical AA transporters EAAC1, B0AT1 and ASCT2 declined substantially during the postnatal growth in pigs.

No description available.

Drug active transport into milk is a major concern for breastfeeding mothers and healthcare providers. Studies from the literature indicated that breast cancer resistance protein (ABCG2) plays an important role in drug transfer into milk. There has been limited study on stereoselective interactions with ABCG2. A mechanistic analysis of flux across cell monolayer model is a critical first step toward extrapolating in vitro results for predicting in vivo disposition (including distribution into milk), drug disposition or drug-drug interactions. The objectives of this thesis were (1) to establish a “Chemical knockout model” in rat for studying drug accumulation into milk, (2) to investigate the impact of stereoselective interaction between ABCG2/Abcg2 and pantoprazole on drug transport in milk, (3) to understand in vitro monolayer flux model using experimental data and a mechanistic mathematical model. Quantitive PCR, Western blotting and immunohistochemistry results indicated that Abcg2 was up-regulated during lactation and localized on apical side of epithelial cells in mammary gland. In vitro and in vivo experiments confirmed that Abcg2 is responsible for nitrofurantoin active transport in rat milk and GF120918 was established as a chemical knockout model. Abcg2 interacts stereoselectively with pantoprazole isomers. A significant different apical flux between two pantoprazole isomers was observed in Abcg2-MDCKII cell line. The milk to serum (M/S) ratio of (-)-pantoprazole was almost 3 times as that of (+)-pantoprazole in lactating rats. Administration GF120918 decreased M/S of (-)-pantoprazole (p<0.001) but not (+)-pantoprazole (p>0.05). A stably transfected ABCG2/Abcg2 overexpressing MDCKII cell line was successfully created and used to explore the theoretical relationships in a monolayer flux model. Based on the profiles of pantoprazole isomer transport, a simple three compartment model for drug transfer into breast milk incorporating the permeability-surface area products for passive diffusion (PSD), paracellular flux (PSPC) and apically efflux ABCG2 (PSA,E) transfection was developed. The mathematical model was developed to more fully understand the interplay of paracellular, passive diffusion, active transport, and flux kinetic parameters (Km, Vmax, IC50 and Ki). This model provided useful insights into the meaning and limitation of parameters obtained from monolayer flux.

Recent literature has established that breast cancer resistance protein (ABCG2) is upregulated during lactation and is responsible for the greater than predicted accumulation of many drugs in breast milk. The objectives of this project were (1) to investigate the role of this transporter in the reported apically-directed nitrofurantoin flux in the CIT3 cell culture model of lactation, (2) to develop a mathematical model for drug transfer into breast milk to relate initial flux rates, steady-state concentrations, efflux ratios, and in vivo milk to serum ratios (M/S) and (3) to identify xenobiotic transporters that are highly expressed, and therefore potentially important for drug accumulation during lactation in mice and humans. Expression, localization, and functional assays confirmed that Abcg2 is the molecular mechanism for the apically-directed nitrofurantoin flux in CIT3 cells despite an unchanged expression level following lactogenic hormone stimulation in this model. A simple three compartment model for drug transfer into breast milk incorporating the permeability-surface area products for passive diffusion (PSD), paracellular flux (PSPC), endogenous transporters (PSB,U, PSA,E, PSB,E, and PSA,U), and ABCG2 (PSA,E(ABCG2)) transfection was developed. A stably transfected ABCG2 overexpressing MDCKII cell line was successfully created and used to explore the theoretical relationships of this new model. Derivations and correlations presented herein show the relationships between the calculated efflux ratios, PSA,E(ABCG2), and M/S attributed to ABCG2. Six xenobiotic transporters (Abcg2, Slc22a1, Slc15a2, Slc29a1, Slc16a1, and Abcc5) were identified as upregulated during lactation in murine developmental datasets analyzed by microarray expression profiling. As existing methods were inadequate to obtain pure populations of luminal epithelial cells in sufficient numbers from human breast milk or reduction mammoplasty samples for microarray analysis, a new fluorescence activated cell sorting method was developed and validated. ABCG2, SLC15A2, SLC22A12, SLC6A14, and SLCO4C1 were significantly upregulated 164-, 70-, 41-, 8-, and 2-fold during lactation, respectively. ABCC10, SLC10A1, SLC16A1, SLC22A4, SLC22A5, SLC22A9, SLC28A3, SLC29A1, SLC29A2, and SLCO4A1 had an expression level similar to, or greater than, levels in the kidney or liver. The significant upregulation of SLCO4C1 with ABCG2 is a novel finding that suggests a coordinated vectorial pathway for substrate movement into breast milk.

Multidrug resistance-associated protein 1 (MRP1) is a member of the adenosine 5’-triphosphate (ATP)-binding cassette (ABC) transporters. MRP1 actively effluxes a variety of endogenous and exogenous substrates from cells, ultimately, working to remove these compounds from the body. MRP1 was initially discovered based on its ability to confer resistance against a variety of chemotherapeutics when overexpressed in cancer cells lines. MRP1 function is important for a number of physiological processes, including regulating cellular and extracellular levels of the anti-inflammatory leukotriene C4 (LTC4) and the antioxidant glutathione (GSH). Our studies have focused on the role of MRP1 in regulating hematopoietic stem cell (HSC) self-renewal and differentiation and the role of CK2 as a regulator of MRP1 function. Reactive Oxygen Species (ROS) cellular levels are tightly regulated and fluctuations in ROS levels affect many cellular processes, including the self-renewal and differentiation of hematopoietic stem cells and kinase signaling pathways. MRP1 regulates ROS through the transport of reduced and oxidized GSH. MRP1 is highly expressed in HSCs, therefore we hypothesized that MRP1 regulates ROS levels in HSCs via efflux of GSH. We have shown that MRP1 regulates HSC self-renewal by modulating cellular ROS via the efflux of GSH. The decrease in ROS results in downregulation of p38 activity and altered expression of a number of redox response genes. CK2 is a master regulator of the cell and controls cell growth, proliferation, death and survival. Yeast studies from our lab using Ycf1p (a homologue of MRP1) and Cka1p (a homologue of CK2) have found that Cka1p regulates Ycf1p function. This result suggests that CK2 regulates MRP1 function via phosphorylation. We have found that CK2 does regulate MRP1 function via phosphorylation of the N-terminal extension at Thr249. Using A549, H460, and HeLa cancer cell lines, we found that inhibition of CK2 with tetrabromobenzimidazole (TBBz) reduces MRP1 function and increases cellular toxicity to known MRP1 substrates.

ABCD2 (D2) has been proposed as a peroxisomal long-chain acyl-CoA transporter that is essential for very long chain fatty acid metabolism. In the livers of mice, D2 is highly induced by fenofibrate, a PPARα ligand that has been widely used as a lipid lowering agent in the treatment of hypertriglyceridemia. To determine if D2 is a modifier of fibrate responses, wild-type and D2 deficient mice were treated with fenofibrate for 14 days. The absence of D2 altered expression of gene clusters associated with lipid metabolism, including PPARα signaling. Using 3T3-L1 adipocytes, which express high levels of D2, we confirmed that knock-down of D2 modified genomic responses to fibrate treatment. We next evaluated the impact of D2 on effects of fibrates in a mouse model of dietinduced obesity. Fenofibrate treatment opposed the development of obesity, hypertriglyceridemia, and insulin resistance. However, these effects were unaffected by D2 genotype. We concluded that D2 can modulate genomic responses to fibrates, but that these effects are not sufficiently robust to alter the effects of fibrates on diet-induced obesity phenotypes. Although proposed as a peroxisomal transporter, the intracellular localization of D2, especially in adipose tissue, has not been validated with direct experimental evidence. Sequential centrifugation of mouse adipose homogenates generated a fraction enriched with D2, but lacked well-known peroxisome markers including catalase, PEX19, and ABCD3 (D3). Electron microscopic imaging of this fraction confirmed the presence of D2 protein on an organelle with evidence of a dense matrix and a diameter of ~200 nm, the typical structure and size of a microperoxisome. D2 and PEX19 antibodies recognized distinct structures in mouse adipose. Immunoisolation of the D2-containing compartment from adipose tissue confirmed the scarcity of PEX19. Proteomic profiling of the D2 compartment revealed the presence of proteins associated peroxisome, endoplasmic reticulum (ER), and mitochondria. We conclude that D2 is localized to a distinct subclass of peroxisomes that lack many peroxisome proteins and may physically associate with mitochondria and the ER.

Parkinson’s disease (PD) is a common neurodegenerative disorder that is characterized by the degeneration of dopaminergic (DA) neurons in the substantia nigra and motor deficits. Although the majority of PD cases are sporadic, several genetic defects in rare familial cases have been identified. Animal models of these genetic defects have been created and have provided unique insights into the molecular mechanisms of the pathogenesis of PD. However, the etiology of PD is still not well understood. Here, taking advantage of the unique features offered by zebrafish, I characterized the functions of PINK1 (PTEN-induced kinase 1) gene, which is associated with recessive familial PD, in the development and survival of DA neurons. In zebrafish, antisense morpholino knockdown of pink1 did not cause a large loss of DA neurons in the ventral diencephalon (vDC), but the patterning of these neurons and their projections were perturbed. The pink1 morphants also showed impaired response to touch stimuli and reduced swimming behaviour. Moreover, the pink1 knockdown caused a significant reduction in the number of mitochondria, as well as mitochondrial morphological defects such as smaller size or loss of cristae, thus affecting mitochondrial function. These results suggest that zebrafish pink1 plays conserved important roles in the development of DA neurons and in the mitochondrial morphology and function. To better follow DA neurons after injury or administration of toxins, I generated a transgenic zebrafish line, Tg(dat:EGFP), in which the green fluorescent protein (GFP) is expressed under the control of cis-regulatory elements of dopamine transporter (dat). In Tg(dat:EGFP) fish, all major groups of DA neurons are correctly labeled with GFP, especially the ones in the vDC, which are analogous to the ascending midbrain DA neurons in mammals. In addition, we observed that the DA neurons in the vDC could partially be replaced after severe laser cell ablation. This suggests that zebrafish may have the unique capacity of regenerating DA neurons after injury. Taken together, my studies suggested that zebrafish could be a useful alternative animal model for the study of the molecular mechanisms underlying PD and for the screening of potential therapeutic compounds for PD.

The aim of this thesis was the functional characterization of the zebrafish parl (Presenilin-Associated Rhomboid-Like) genes which code for mitochondrial proteins involved in cell survival. A mutation in PARL has been described in Parkinson’s disease patients. I investigated the role of mitochondrial PD-related proteins using a zebrafish parla and parlb deficiency model. I found that the knockdown of both parl genes is lethal. Parla plays a larger role in patterning of the DA neurons in the ventral diencephalon than Parlb. The human PARL rescued the double morphant phenotype, suggesting function conservation between zebrafish and humans. I was able to rescue the mortality and DA neuron mispatterning observed in double morphants with synthetic pink1 mRNA. This suggests that parl genes are epistatic to pink1 in zebrafish. To visualize mitochondria specifically in dopaminergic neurons of live zebrafish, I established a transgenic line Tg(dat:tom20 MLS-mCherry) where regulatory elements of the dopamine transporter (dat) were used to drive expression of a Tom20-mCherry fusion protein that is targeted to the mitochondria. I characterised the expression of Tom20-mCherry to the mitochondria of the majority of DA neuron groups. In addition, I observed a decrease in mCherry fluorescence following MPTP exposure of live fish. The PD-related mutation in PARL is located in a cleavage site of the mammalian protein, which is necessary for the production of the beta peptide; however, this site is predicted to be absent in the zebrafish Parls. To establish the cleavage patterns of the zebrafish Parls and compare them to those of human PARL, I examined the cleavage of Parl-Flag constructs in cultured cells. I detected one band for Parla-Flag and two bands representing Parlb-Flag. The parla and parlb deficiency model along with the characterization of the cleavage patterns of Parl and the Tg(dat:tom20 MLS-mCherry) transgenic line are tools which will help elucidate the role of mitochondrial proteins in PD research.

Nerve cells are organized into complex networks that comprise the building blocks of our nervous system. Neurons communicate by transmitting messenger molecules released from synaptic vesicles. Alterations in neuronal circuitry and synaptic signaling contribute to a wide range of neurological conditions, often with consequences for movement. Intrinsic neuronal networks in the spinal cord serve to coordinate vital rhythmic motor functions. In spite of extensive efforts to address the organization of these neural circuits, much remains to be revealed regarding the identity and function of specific interneuron cell types and how neuromodulation tune network activity. In this thesis, two novel genes initially identified as markers for spinal neuronal populations were investigated: Slc10a4 and Dmrt3. The orphan transporter SLC10A4 was found to be expressed on synaptic vesicles of the cholinergic system, including motor neurons, as well as in the monoaminergic system, including dopaminergic, serotonergic and noradrenergic nuclei. Thus, it constitutes a novel molecular denominator shared by these classic neuromodulatory systems. SLC10A4 was found to influence vesicular transport of dopamine and affect neuronal release and reuptake efficiency in the striatum. Mice lacking Slc10a4 displayed impaired monoamine homeostasis and were hypersensitive to the drugs amphetamine and tranylcypromine. These findings demonstrate that SLC10A4 is capable of modulating the modulatory systems of the brain with potential clinical relevance for neurological and mental disorders. The transcription factor encoded by Dmrt3 was found to be expressed in a population of inhibitory commissural interneurons originating from the dorsal interneuron 6 (dI6) domain in the spinal cord. In parallel, a genome-wide association study revealed that a non-sense mutation in horse DMRT3 is permissive for the ability to perform pace among other alternate gaits. Further analysis of Dmrt3 null mutant mice showed that Dmrt3 has a central role for spinal neuronal network development with consequences for locomotor behavior. The dI6 class has been suggested to take part in motor circuits but remains one of the least studied classes due to lack of molecular markers. To further investigate the Dmrt3-derived neurons, and the dI6 population in general, a Dmrt3Cre mouse line was generated which allowed for characterization on the molecular, cellular and  behavioral level. It was found that Dmrt3 neurons synapse onto motor neurons, receive extensive synaptic inputs from various neuronal sources and are rhythmically active during fictive locomotion. Furthermore, silencing of Dmrt3 neurons in Dmrt3Cre;Viaatlx/lx mice led to impaired motor coordination and alterations in gait, together demonstrating the importance of this neuronal population in the control of movement.