Characterization of basal pseudopod-like processes in ileal and colonic PYY cells.

The peptide tyrosine tyrosine (PYY) is produced and secreted from L cells of the gastrointestinal mucosa. To study the anatomy and function of PYY-secreting L cells, we developed a transgenic PYY-green fluorescent protein mouse model. PYY-containing cells exhibited green fluorescence under UV light and were immunoreactive to antibodies against PYY and GLP-1 (glucagon-like peptide-1, an incretin hormone also secreted by L cells). PYY-GFP cells from 15 μm thick sections were imaged using confocal laser scanning microscopy and three-dimensionally (3D) reconstructed. Results revealed unique details of the anatomical differences between ileal and colonic PYY-GFP cells. In ileal villi, the apical portion of PYY cells makes minimal contact with the lumen of the gut. Long pseudopod-like basal processes extend from these cells and form an interface between the mucosal epithelium and the lamina propria. Some basal processes are up to 50 μm in length. Multiple processes can be seen protruding from one cell and these often have a terminus resembling a synapse that appears to interact with neighboring cells. In colonic crypts, PYY-GFP cells adopt a spindle-like shape and weave in between epithelial cells, while maintaining contact with the lumen and lamina propria. In both tissues, cytoplasmic granules containing the hormones PYY and GLP-1 are confined to the base of the cell, often filling the basal process. The anatomical arrangement of these structures suggests a dual function as a dock for receptors to survey absorbed nutrients and as a launching platform for hormone secretion in a paracrine fashion.

The role of B cells in solid organ transplantation.

The role of antibodies in chronic injury to organ transplants has been suggested for many years, but recently emphasized by new data. We have observed that when immunosuppressive potency decreases either by intentional weaning of maintenance agents or due to homeostatic repopulation after immune cell depletion, the threshold of B cell activation may be lowered. In human transplant recipients the result may be donor-specific antibody, C4d+ injury, and chronic rejection. This scenario has precise parallels in a rhesus monkey renal allograft model in which T cells are depleted with CD3 immunotoxin, or in a CD52-T cell transgenic mouse model using alemtuzumab to deplete T cells. Such animal models may be useful for the testing of therapeutic strategies to prevent DSA. We agree with others who suggest that weaning of immunosuppression may place transplant recipients at risk of chronic antibody-mediated rejection, and that strategies to prevent this scenario are needed if we are to improve long-term graft and patient outcomes in transplantation. We believe that animal models will play a crucial role in defining the pathophysiology of antibody-mediated rejection and in developing effective therapies to prevent graft injury. Two such animal models are described herein.

Huntingtin is required for normal excitatory synapse development in cortical and striatal circuits.

Huntington's disease (HD) is a neurodegenerative disease caused by the expansion of a poly-glutamine (poly-Q) stretch in the huntingtin (Htt) protein. Gain-of-function effects of mutant Htt have been extensively investigated as the major driver of neurodegeneration in HD. However, loss-of-function effects of poly-Q mutations recently emerged as potential drivers of disease pathophysiology. Early synaptic problems in the excitatory cortical and striatal connections have been reported in HD, but the role of Htt protein in synaptic connectivity was unknown. Therefore, we investigated the role of Htt in synaptic connectivity in vivo by conditionally silencing Htt in the developing mouse cortex. When cortical Htt function was silenced, cortical and striatal excitatory synapses formed and matured at an accelerated pace through postnatal day 21 (P21). This exuberant synaptic connectivity was lost over time in the cortex, resulting in the deterioration of synapses by 5 weeks. Synaptic decline in the cortex was accompanied with layer- and region-specific reactive gliosis without cell loss. To determine whether the disease-causing poly-Q mutation in Htt affects synapse development, we next investigated the synaptic connectivity in a full-length knock-in mouse model of HD, the zQ175 mouse. Similar to the cortical conditional knock-outs, we found excessive excitatory synapse formation and maturation in the cortices of P21 zQ175, which was lost by 5 weeks. Together, our findings reveal that cortical Htt is required for the correct establishment of cortical and striatal excitatory circuits, and this function of Htt is lost when the mutant Htt is present.

Menthol attenuates respiratory irritation and elevates blood cotinine in cigarette smoke exposed mice.

Addition of menthol to cigarettes may be associated with increased initiation of smoking. The potential mechanisms underlying this association are not known. Menthol, likely due to its effects on cold-sensing peripheral sensory neurons, is known to inhibit the sensation of irritation elicited by respiratory irritants. However, it remains unclear whether menthol modulates cigarette smoke irritancy and nicotine absorption during initial exposures to cigarettes, thereby facilitating smoking initiation. Using plethysmography in a C57Bl/6J mouse model, we examined the effects of L-menthol, the menthol isomer added to cigarettes, on the respiratory sensory irritation response to primary smoke irritants (acrolein and cyclohexanone) and smoke of Kentucky reference 2R4 cigarettes. We also studied L-menthol's effect on blood levels of the nicotine metabolite, cotinine, immediately after exposure to cigarette smoke. L-menthol suppressed the irritation response to acrolein with an apparent IC₅₀ of 4 ppm. Suppression was observed even at acrolein levels well above those necessary to produce a maximal response. Cigarette smoke, at exposure levels of 10 mg/m³ or higher, caused an immediate and marked sensory irritation response in mice. This response was significantly suppressed by L-menthol even at smoke concentrations as high as 300 mg/m³. Counterirritation by L-menthol was abolished by treatment with a selective inhibitor of Transient Receptor Potential Melastatin 8 (TRPM8), the neuronal cold/menthol receptor. Inclusion of menthol in the cigarette smoke resulted in roughly a 1.5-fold increase in plasma cotinine levels over those observed in mice exposed to smoke without added menthol. These findings document that, L-menthol, through TRPM8, is a strong suppressor of respiratory irritation responses, even during highly noxious exposures to cigarette smoke or smoke irritants, and increases blood cotinine. Therefore, L-menthol, as a cigarette additive, may promote smoking initiation and nicotine addiction.

Spectrotemporal CT data acquisition and reconstruction at low dose.

PURPOSE: X-ray computed tomography (CT) is widely used, both clinically and preclinically, for fast, high-resolution anatomic imaging; however, compelling opportunities exist to expand its use in functional imaging applications. For instance, spectral information combined with nanoparticle contrast agents enables quantification of tissue perfusion levels, while temporal information details cardiac and respiratory dynamics. The authors propose and demonstrate a projection acquisition and reconstruction strategy for 5D CT (3D+dual energy+time) which recovers spectral and temporal information without substantially increasing radiation dose or sampling time relative to anatomic imaging protocols. METHODS: The authors approach the 5D reconstruction problem within the framework of low-rank and sparse matrix decomposition. Unlike previous work on rank-sparsity constrained CT reconstruction, the authors establish an explicit rank-sparse signal model to describe the spectral and temporal dimensions. The spectral dimension is represented as a well-sampled time and energy averaged image plus regularly undersampled principal components describing the spectral contrast. The temporal dimension is represented as the same time and energy averaged reconstruction plus contiguous, spatially sparse, and irregularly sampled temporal contrast images. Using a nonlinear, image domain filtration approach, the authors refer to as rank-sparse kernel regression, the authors transfer image structure from the well-sampled time and energy averaged reconstruction to the spectral and temporal contrast images. This regularization strategy strictly constrains the reconstruction problem while approximately separating the temporal and spectral dimensions. Separability results in a highly compressed representation for the 5D data in which projections are shared between the temporal and spectral reconstruction subproblems, enabling substantial undersampling. The authors solved the 5D reconstruction problem using the split Bregman method and GPU-based implementations of backprojection, reprojection, and kernel regression. Using a preclinical mouse model, the authors apply the proposed algorithm to study myocardial injury following radiation treatment of breast cancer. RESULTS: Quantitative 5D simulations are performed using the MOBY mouse phantom. Twenty data sets (ten cardiac phases, two energies) are reconstructed with 88 μm, isotropic voxels from 450 total projections acquired over a single 360° rotation. In vivo 5D myocardial injury data sets acquired in two mice injected with gold and iodine nanoparticles are also reconstructed with 20 data sets per mouse using the same acquisition parameters (dose: ∼60 mGy). For both the simulations and the in vivo data, the reconstruction quality is sufficient to perform material decomposition into gold and iodine maps to localize the extent of myocardial injury (gold accumulation) and to measure cardiac functional metrics (vascular iodine). Their 5D CT imaging protocol represents a 95% reduction in radiation dose per cardiac phase and energy and a 40-fold decrease in projection sampling time relative to their standard imaging protocol. CONCLUSIONS: Their 5D CT data acquisition and reconstruction protocol efficiently exploits the rank-sparse nature of spectral and temporal CT data to provide high-fidelity reconstruction results without increased radiation dose or sampling time.

A Peptide Uncoupling BDNF Receptor TrkB from Phospholipase Cγ1 Prevents Epilepsy Induced by Status Epilepticus.

The BDNF receptor tyrosine kinase, TrkB, underlies nervous system function in both health and disease. Excessive activation of TrkB caused by status epilepticus promotes development of temporal lobe epilepsy (TLE), revealing TrkB as a therapeutic target for prevention of TLE. To circumvent undesirable consequences of global inhibition of TrkB signaling, we implemented a novel strategy aimed at selective inhibition of the TrkB-activated signaling pathway responsible for TLE. Our studies of a mouse model reveal that phospholipase Cγ1 (PLCγ1) is the dominant signaling effector by which excessive activation of TrkB promotes epilepsy. We designed a novel peptide (pY816) that uncouples TrkB from PLCγ1. Treatment with pY816 following status epilepticus inhibited TLE and prevented anxiety-like disorder yet preserved neuroprotective effects of endogenous TrkB signaling. We provide proof-of-concept evidence for a novel strategy targeting receptor tyrosine signaling and identify a therapeutic with promise for prevention of TLE caused by status epilepticus in humans.

Immunity and Arginine Deprivation in Alzheimer's Disease

The pathogenesis of Alzheimer’s disease (AD) is a critical unsolved question, and while recent studies have demonstrated a strong association between altered brain immune responses and disease progression, the mechanistic cause of neuronal dysfunction and death is unknown. We have previously described the unique CVN-AD mouse model of AD, in which immune-mediated nitric oxide is lowered to mimic human levels, resulting in a mouse model that demonstrates the cardinal features of AD, including amyloid deposition, hyperphosphorylated and aggregated tau, behavioral changes and age-dependent hippocampal neuronal loss. Using this mouse model, we studied longitudinal changes in brain immunity in relation to neuronal loss and, contrary to the predominant view that AD pathology is driven by pro-inflammatory factors, we find that the pathology in CVN-AD mice is driven by local immune suppression. Areas of hippocampal neuronal death are associated with the presence of immunosuppressive CD11c+ microglia and extracellular arginase, resulting in arginine catabolism and reduced levels of total brain arginine. Pharmacologic disruption of the arginine utilization pathway by an inhibitor of arginase and ornithine decarboxylase protected the mice from AD-like pathology and significantly decreased CD11c expression. Our findings strongly implicate local immune-mediated amino acid catabolism as a novel and potentially critical mechanism mediating the age-dependent and regional loss of neurons in humans with AD.

There is a large interest in identifying, lineage tracing, and determining the physiologic roles of monophagocytes in Alzheimer’s disease. While Cx3cr1 knock-in fluorescent reporting and Cre expressing mice have been critical for studying neuroimmunology, mice that are homozygous null or hemizygous for CX3CR1 have perturbed neural development and immune responses. There is, therefore, a need for similar tools in which mice are CX3CR1+/+. Here, we describe a mouse where Cre is driven by the Cx3cr1 promoter on a bacterial artificial chromosome (BAC) transgene (Cx3cr1-CreBT) and the Cx3cr1 locus is unperturbed. Similarly to Cx3cr1-Cre knock-in mice, these mice express Cre in Ly6C-, but not Ly6C+, monocytes and tissue macrophages, including microglia. These mice represent a novel tool that maintains the Cx3cr1 locus while allowing for selective gene targeting in monocytes and tissue macrophages.

The study of immunity in Alzheimer’s requires the ability to identify and quantify specific immune cell subsets by flow cytometry. While it is possible to identify lymphocyte subsets based on cell lineage-specific markers, the lack of such markers in brain myeloid cell subsets has prevented the study of monocytes, macrophages and dendritic cells. By improving on tissue homogenization, we present a comprehensive protocol for flow cytometric analysis, that allows for the identification of several cell types that have not been previously identified by flow cytometry. These cell types include F4/80hi macrophages, which may be meningeal macrophages, IA/IE+ macrophages, which may represent perivascular macrophages, and dendritic cells. The identification of these cell types now allows for their study by flow cytometry in homeostasis and disease.

Functional Evaluation of Causal Mutations Identified in Human Genetic Studies

Human genetics has been experiencing a wave of genetic discoveries thanks to the development of several technologies, such as genome-wide association studies (GWAS), whole-exome sequencing, and whole genome sequencing. Despite the massive genetic discoveries of new variants associated with human diseases, several key challenges emerge following the genetic discovery. GWAS is known to be good at identifying the locus associated with the patient phenotype. However, the actually causal variants responsible for the phenotype are often elusive. Another challenge in human genetics is that even the causal mutations are already known, the underlying biological effect might remain largely ambiguous. Functional evaluation plays a key role to solve these key challenges in human genetics both to identify causal variants responsible for the phenotype, and to further develop the biological insights from the disease-causing mutations.

We adopted various methods to characterize the effects of variants identified in human genetic studies, including patient genetic and phenotypic data, RNA chemistry, molecular biology, virology, and multi-electrode array and primary neuronal culture systems. Chapter 1 is a broader introduction for the motivation and challenges for functional evaluation in human genetic studies, and the background of several genetics discoveries, such as hepatitis C treatment response, in which we performed functional characterization.

Chapter 2 focuses on the characterization of causal variants following the GWAS study for hepatitis C treatment response. We characterized a non-coding SNP (rs4803217) of IL28B (IFNL3) in high linkage disequilibrium (LD) with the discovery SNP identified in the GWAS. In this chapter, we used inter-disciplinary approaches to characterize rs4803217 on RNA structure, disease association, and protein translation.

Chapter 3 describes another avenue of functional characterization following GWAS focusing on the novel transcripts and proteins identified near the IL28B (IFNL3) locus. It has been recently speculated that this novel protein, which was named IFNL4, may affect the HCV treatment response and clearance. In this chapter, we used molecular biology, virology, and patient genetic and phenotypic data to further characterize and understand the biology of IFNL4. The efforts in chapter 2 and 3 provided new insights to the candidate causal variant(s) responsible for the GWAS for HCV treatment response, however, more evidence is still required to make claims for the exact causal roles of these variants for the GWAS association.

Chapter 4 aims to characterize a mutation already known to cause a disease (seizure) in a mouse model. We demonstrate the potential use of multi-electrode array (MEA) system for the functional characterization and drug testing on mutations found in neurological diseases, such as seizure. Functional characterization in neurological diseases is relatively challenging and available systematic tools are relatively limited. This chapter shows an exploratory research and example to establish a system for the broader use for functional characterization and translational opportunities for mutations found in neurological diseases.

Overall, this dissertation spans a range of challenges of functional evaluations in human genetics. It is expected that the functional characterization to understand human mutations will become more central in human genetics, because there are still many biological questions remaining to be answered after the explosion of human genetic discoveries. The recent advance in several technologies, including genome editing and pluripotent stem cells, is also expected to make new tools available for functional studies in human diseases.

Targeted Gene Repression Technologies for Regenerative Medicine, Genomics, and Gene Therapy

Gene regulation is a complex and tightly controlled process that defines cell function in physiological and abnormal states. Programmable gene repression technologies enable loss-of-function studies for dissecting gene regulation mechanisms and represent an exciting avenue for gene therapy. Established and recently developed methods now exist to modulate gene sequence, epigenetic marks, transcriptional activity, and post-transcriptional processes, providing unprecedented genetic control over cell phenotype. Our objective was to apply and develop targeted repression technologies for regenerative medicine, genomics, and gene therapy applications. We used RNA interference to control cell cycle regulation in myogenic differentiation and enhance the proliferative capacity of tissue engineered cartilage constructs. These studies demonstrate how modulation of a single gene can be used to guide cell differentiation for regenerative medicine strategies. RNA-guided gene regulation with the CRISPR/Cas9 system has rapidly expanded the targeted repression repertoire from silencing single protein-coding genes to modulation of genes, promoters, and other distal regulatory elements. In order to facilitate its adaptation for basic research and translational applications, we demonstrated the high degree of specificity for gene targeting, gene silencing, and chromatin modification possible with Cas9 repressors. The specificity and effectiveness of RNA-guided transcriptional repressors for silencing endogenous genes are promising characteristics for mechanistic studies of gene regulation and cell phenotype. Furthermore, our results support the use of Cas9-based repressors as a platform for novel gene therapy strategies. We developed an in vivo AAV-based gene repression system for silencing endogenous genes in a mouse model. Together, these studies demonstrate the utility of gene repression tools for guiding cell phenotype and the potential of the RNA-guided CRISPR/Cas9 platform for applications such as causal studies of gene regulatory mechanisms and gene therapy.

Discovery and Characterization of a Novel Fatty Acid Synthase Inhibitor with Antineoplastic Activity against Breast Cancer

During oncogenesis, cancer cells go through metabolic reprogramming to maintain their high growth rates and adapt to changes in the microenvironment and the lack of essential nutrients. Several types of cancer are dependent on de novo fatty acid synthesis to sustain their growth rates by providing precursors to construct membranes and produce vital signaling lipids. Fatty acid synthase (FASN) catalyze the terminal step of de novo fatty acid synthesis and it is highly expressed in many types of cancers where it’s up-regulation is correlated with cancer aggressiveness and low therapeutic outcome. Many FASN inhibitors were developed and showed potent anticancer activity however, only one inhibitor advanced to early stage clinical trials with some dose limiting toxicities. Using a modified fluorescence-linked enzyme chemoproteomic strategy (FLECS) screen, we identified HS-106, a thiophenopyrimiden FASN inhibitor that has anti-neoplastic activity against breast cancer in vitro and in vivo. HS-106 was able to inhibit both; purified human FASN activity and cellular fatty acid synthesis activity as evaluated by radioactive tracers incorporation into lipids experiments. In proliferation and apoptosis assays, HS-106 was able to block proliferation and induce apoptosis in several breast cancer cell lines. Several rescue experiment and global lipidome analysis were performed to probe the mechanism by which HS-106 induces apoptosis. HS-106 was found to induce several changes in lipids metabolism: (i) inhibit fatty acids synthesis. (ii) Inhibit fatty acids oxidation as indicated by the ability of inhibiting Malonyl CoA accumulation to block HS-106 induced apoptosis and the increase in the abundance of ceramides. (iii) Increase fatty acids uptake and neutral lipids formation as confirmed 14C Palmitate uptake assay and neutral lipids staining. (iv)Inhibit the formation of phospholipids by inhibiting de novo fatty acid synthesis and diverting exogenous fatty acids to neutral lipids. All of these events would lead to disruption in membranes structure and function. HS-106 was also tested in Lapatinib resistant cell lines and it was able to induce apoptosis and synergizes Lapatinib activity in these cell lines. This may be due the disruption of lipid rafts based on the observation that HS-106 reduces the expression of both HER2 and HER3. HS-106 was found to be well tolerated and bioavailable in mice with high elimination rate. HS-106 efficacy was tested in MMTV neu mouse model. Although did not significantly reduced tumor size (alone), HS-106 was able to double the median survival of the mice and showed potent antitumor activity when combined with Carboplatin. Similar results were obtained when same combinations and dosing schedule was used in C3Tag mouse model except for the inability of HS-106 affect mice survival.

From the above, HS-106 represent a novel FASN inhibitor that has anticancer activity both in vivo and in vitro. Being a chemically tractable molecule, the synthetic route to HS-106 is readily adaptable for the preparation of analogs that are similar in structure, suggesting that, the pharmacological properties of HS-106 can be improved.

Posttranscriptional Regulation of Embryonic Neurogenesis by the Exon Junction Complex

The six-layered neuron structure in the cerebral cortex is the foundation for human mental abilities. In the developing cerebral cortex, neural stem cells undergo proliferation and differentiate into intermediate progenitors and neurons, a process known as embryonic neurogenesis. Disrupted embryonic neurogenesis is the root cause of a wide range of neurodevelopmental disorders, including microcephaly and intellectual disabilities. Multiple layers of regulatory networks have been identified and extensively studied over the past decades to understand this complex but extremely crucial process of brain development. In recent years, post-transcriptional RNA regulation through RNA binding proteins has emerged as a critical regulatory nexus in embryonic neurogenesis. The exon junction complex (EJC) is a highly conserved RNA binding complex composed of four core proteins, Magoh, Rbm8a, Eif4a3, and Casc3. The EJC plays a major role in regulating RNA splicing, nuclear export, subcellular localization, translation, and nonsense mediated RNA decay. Human genetic studies have associated individual EJC components with various developmental disorders. We showed previously that haploinsufficiency of Magoh causes microcephaly and disrupted neural stem cell differentiation in mouse. However, it is unclear if other EJC core components are also required for embryonic neurogenesis. More importantly, the molecular mechanism through which the EJC regulates embryonic neurogenesis remains largely unknown. Here, we demonstrated with genetically modified mouse models that both Rbm8a and Eif4a3 are required for proper embryonic neurogenesis and the formation of a normal brain. Using transcriptome and proteomic analysis, we showed that the EJC posttranscriptionally regulates genes involved in the p53 pathway, splicing and translation regulation, as well as ribosomal biogenesis. This is the first in vivo evidence suggesting that the etiology of EJC associated neurodevelopmental diseases can be ribosomopathies. We also showed that, different from other EJC core components, depletion of Casc3 only led to mild neurogenesis defects in the mouse model. However, our data suggested that Casc3 is required for embryo viability, development progression, and is potentially a regulator of cardiac development. Together, data presented in this thesis suggests that the EJC is crucial for embryonic neurogenesis and that the EJC and its peripheral factors may regulate development in a tissue-specific manner.

Genetic Abrogation of Adenosine A3 Receptor Prevents Uninephrectomy and High Salt-Induced Hypertension.

BACKGROUND: Early-life reduction in nephron number (uninephrectomy [UNX]) and chronic high salt (HS) intake increase the risk of hypertension and chronic kidney disease. Adenosine signaling via its different receptors has been implicated in modulating renal, cardiovascular, and metabolic functions as well as inflammatory processes; however, the specific role of the A3 receptor in cardiovascular diseases is not clear. In this study, gene-modified mice were used to investigate the hypothesis that lack of A3 signaling prevents the development of hypertension and attenuates renal and cardiovascular injuries following UNX in combination with HS (UNX-HS) in mice. METHODS AND RESULTS: Wild-type (A3 (+/+)) mice subjected to UNX-HS developed hypertension compared with controls (mean arterial pressure 106±3 versus 82±3 mm Hg; P<0.05) and displayed an impaired metabolic phenotype (eg, increased adiposity, reduced glucose tolerance, hyperinsulinemia). These changes were associated with both cardiac hypertrophy and fibrosis together with renal injuries and proteinuria. All of these pathological hallmarks were significantly attenuated in the A3 (-/-) mice. Mechanistically, absence of A3 receptors protected from UNX-HS-associated increase in renal NADPH oxidase activity and Nox2 expression. In addition, circulating cytokines including interleukins 1β, 6, 12, and 10 were increased in A3 (+/+) following UNX-HS, but these cytokines were already elevated in naïve A3 (-/-) mice and did not change following UNX-HS. CONCLUSIONS: Reduction in nephron number combined with chronic HS intake is associated with oxidative stress, chronic inflammation, and development of hypertension in mice. Absence of adenosine A3 receptor signaling was strongly protective in this novel mouse model of renal and cardiovascular disease.