Charles E. Schmidt College of Medicine

Oxidative stress (OS) is strongly implicated in age-related neurodegeneration and
other diseases. Under OS, the production of excessive oxidants leads to increased
damages to cellular components. Recently, RNA has been discovered as a major target of
oxidative damage, including the creation of abasic sites. In this work, we developed a
method for quantifying abasic RNA in cell. Using this method, we have examined the
potential role of the RNA-processing cellular foci, stress granule (SG) and processing
bodies (PB) in eliminating abasic RNA in situ. We demonstrated that RNA is a major
target of oxidative damage, constituting the majority of OS-induced abasic nucleic acids
in HeLa cell. Importantly, the level of abasic RNA is strongly correlated with SG
abundance. Furthermore, inhibition of SG/PB formation causes accumulation of abasic
RNA, suggesting that SG/PB participates in removing oxidized RNA and protects cells
under OS, which offers novel targets for therapeutic intervention in age-related diseases.

It is of interest to understand how new neurons incorporate themselves into the
existing circuitry of certain neuronal populations. One such population of neurons is that
which are born in the subventricular zone (SVZ) and migrate to the olfactory bulb where
they differentiate into granule cells. Another area of interest is the role of brain-derived
neurotrophic factor (BDNF) on the survival and overall health of these neurons. This
study aimed to test whether or not BDNF is a survival factor for adult-born granule cells.
Here were utilized a transgenic mouse model over-expressing BDNF under the α-
calcium/calmodulin-dependent protein kinase II (CAMKIIα) promoter, and tested its
effect on olfactory granule cells under sensory deprived conditions. Results from this
experiment indicated that there was no significant difference in cell death or cell survival when comparing transgenic and wild type animals. We concluded that BDNF is not a
survival factor for adult-born granule cells.

The aim of this study was to investigate the current level of awareness and use of the Great American Smokeout (GASO) and the extent to which that may vary by lifetime smoking, socioeconomic status, age and gender. We hypothesized and found that the current levels of awareness and use were significantly less than the most recent reports available (over 13 years ago), and that awareness of the GASO was more likely in lifetime smokers, those with more education, and older age groups, but found no significant effect related to gender. The drop in awareness and use of the GASO was more than half and almost five times, respectively, compared to that of most recent previous reports. These findings highlight a critical need for more attention and resources to reinvigorate the GASO as a useful event for smokers.

Matrix metalloproteinases (MMPs) constitute the major class of enzymes capable
of degrading all protein components of extracellular matrix (ECM) and have important
roles in normal physiologic processes of maintaining tissue integrity and remodeling.
However, excess MMP activities are associated with many diseases including rheumatoid
arthritis and osteoarthritis, cardiomyopathy, and macular degeneration. The activity of
MMPs is regulated by their endogenous protein inhibitors, the tissue inhibitors of
metalloproteinases (TIMPs) which are avid broad-spectrum inhibitors of numerous
human matrixins (MMPs and ADAMs). Uncontrolled matrix degradation occurs when
the balance between TIMPs and MMPs is disrupted, resulting in serious diseases such as
cancer, arthritis and chronic tissue ulcers. Thus, the engineering of TIMPs to produce
highly selective and efficacious inhibitors of individual MMPs may be utilized for future
treatment of diseases. Such engineering requires detailed analysis for the structural and
biophysical information of MMP-TIMP interaction. Changes in the dynamics of proteins and solvent that accompany their
associations with different binding partners, influence the specificity of binding through
entropic effects. From the current studies it appears that the interactions of the inhibitory
domains of TIMPs-1 and -2 (N-TIMPs) with MT1-MMP are driven by entropy increases
that are partitioned between solvent and conformational entropy (ΔSsolv and ΔSconf), and a
large conformational entropy penalty is responsible for the weak inhibition of MT1-MMP
by NT1.We investigated how mutations that modify N-TIMP selectivity affect the
thermodynamics of interactions with MMP1, MMP3 and MT1-MMP. The weak
inhibition of MT1-MMP by N-TIMP-1 is enhanced by mutation of threonine 98, on the
edge of the binding ridge, to leucine. This mutation increases the large ΔSconf cost for
binding to MT1-MMP but this is offset by a greater increase in ΔSsolv. In contrast, this
mutation enhances binding to MMP3 by increasing ΔSconf for the interaction. ΔSsolv and
ΔSconf show mutual compensation for all interactions, with characteristic ranges for each
MMP. Distinct electrostatic and dynamic features of MMPs are key factors in their
selective inhibition.

The lens is a crystallin tissue of the anterior part of the eye that focuses light onto the
retina. Aged-related cataract, which is the result of loss of lens transparency, is the most
common cause of blindness in the world. Being constantly exposed to UV-light, lens is
significantly affected by its UVA spectrum. UV-light exposure has been shown to result
in apoptosis of lens cells which can lead to cataract formation. This suggests the need for
molecular mechanisms to remove apoptotic debris from the lens. In the set of
experiments it was proven that integrin αvβ5-mediated pathway is involved in
phagocytosis of apoptotic cell debris in the ocular lens, thus contributing to its
homeostasis. Additionally, it was shown that exposure to UV-light plays role in cataract
formation by influencing integrin αvβ5-mediated phagocytosis function.

This study aimed to identify changes in the expression of serotonin (5-hydroxytryptamine; 5-HT) receptor 5-HT2a mRNA in paraformaldehyde prefixed rat brain tissue after exposure to 3, 4 methylenedioxymethamphetamine (MDMA). Rather than use traditional, time-consuming methods that were highly prone to error, such as the use of radioactive riboprobes, we used recent advancements in the development of rapid in-situ hybridization to our advantage. The use of rapid in-situ hybridization (such as the RNAscope assay) has allowed us to use paraformaldehyde prefixed brain tissue sections, which are more widely available and easier to use, instead of fresh frozen tissue. Here we describe the results from the use of the RNAscope assay to measure the amount of dapB (a bacterial gene), ppiB (a eukaryotic housekeeping gene), and htr2a (a eukaryotic gene coding for 5-HT2A receptors) mRNA.

Metastasis is the primary cause of mortality in women with breast cancer. Recently, elevated serum levels of a glycoprotein known as chitinase-3 likeprotein- 1 (CHI3L1) has been correlated with poor prognosis and shorter survival of patients with cancer and inflammatory diseases. The biological and physiological functions of CHI3L1 in tumor progression have not yet been elucidated. In this document, we describe the role of CHI3L1 in tumor growth and metastasis and its relationship with inflammation.
Using well-established models of breast cancer, we show that CHI3L1 is increased in the serum of tumor bearing mice. We found that CHI3L1 levels are increased at both the “pre-metastatic” and “metastatic stage” and that tumor cells, splenic, alveolar and interstitial macrophages; and myeloid derived population produce CHI3L1. Furthermore, we demonstrated that CHI3L1 has an inhibitory role on the expression of interferon-gamma (IFN γ) by T cells, while enhancing the production of pro-inflammatory mediators by macrophages such as Cchemokine ligand 2 (CCL2/MCP-1), Chemokine CX motif ligand 2 (CXCL2/IL-8) and matrix metalloproteinase-9 (MMP-9), all of which promote tumor growth and metastasis. We demonstrated that in vivo treatment of tumor-bearing mice with chitin microparticles, a TH1 adjuvant and a substrate for CHI3L1, promoted immune effector functions with increased production of IFN-γ but decreased CCL2/MCP-1, CXCL2/IL-8 and MMP-9 expression by splenic and pulmonary macrophages. Significantly, in vivo administration of chitin microparticles decreased tumor growth and pulmonary metastasis in mammary tumor bearing mice. These results suggest that CHI3L1 may play a role in tumor progression. Inflammation plays a pivotal role during tumor progression and metastasis by promoting the production of pro-inflammatory molecules such as CHI3L1. However, little is known about how CHI3L1 expression can affect secondary sites to enhance metastasis. In these studies, we demonstrated that CHI3L1 alters the cellular composition and inflammatory mediators that aid in the establishment of a metastatic niche for the support of infiltrating tumor cells leading to accelerated tumor progression. Since previous studies showed that CHI3L1 modulates inflammation, we determined the role of CHI3L1 in the context of pre-existing inflammation and metastasis. We found that CHI3L1 deficient mice with preexisting inflammation had decreased pro-inflammatory mediators, and significant reduction in tumor volume and metastasis compared to wild type controls. Preexisting inflammation and CHI3L1 may be driving the establishment of a premetastatic milieu in the lungs and aiding in the establishment of metastasis. Understanding the role of CHI3L1 in inflammation during tumor progression could result in the design of targeted therapies for breast cancer patients.

The molecular mechanisms by which pregnancy malaria affects the outcome of fetal development are unknown. Megalin, which has been well studied in kidney, has high expression in the placenta from early stages to term, and is proposed to be an important factor in extensive maternofetal exchange during development of the fetus. Pregnancy malaria (PM) is characterized by inflammation in placenta and is associated with low birthweight (LBW), stillborn birth, and other pathologies. It is hypothesized that PM disturbs megalin function/expression/distribution in the brush boarder of syncytiotrophoblast which, in turn, may contribute significantly to pathology of LBW. Our studies show that the presence of infected erythrocytes in placenta at the time of delivery negatively affects protein abundance for megalin and Dab2. This is the first report associating the abundance of placental megalin system proteins with the birth weight of newborn babies, and associating PM with changes in megalin system protein abundance.

The mitochondrion is the powerhouse of the cell. Therefore, it is critical to the homeostasis of the cell that populations of mitochondria that are damaged or in excess are degraded. The process of targeted elimination of damaged or excess mitochondria by autophagy is called mitophagy. In this report, analysis of the mitophagy regulators PINK1/PARKIN and BNIP3L and their roles are assessed in the lens. PARKIN, an E3 ubiquitin ligase, has been shown to play a role in directing damaged mitochondria for degradation. While BNIP3L, an outer mitochondrial membrane protein, increases in expression in response to excess mitochondria and organelle degradation during cellular differentiation. We have shown that PARKIN is both induced and translocates from the cytoplasm to the mitochondria in human epithelial lens cells upon oxidative stress exposure. In addition, our findings also show that overexpression of BNIP3L causes premature clearance of mitochondria and other organelles, while loss of BNIP3L results in lack of clearance. Prior to this work, PARKIN mediated mitophagy had not been shown to act as a protective cellular response to oxidative stress in the lens. This project also resulted in the novel finding that BNIP3L-mediated mitophagy mechanisms are required for targeted organelle degradation in the lens.

There is compelling evidence that smokers are less responsive to vaccination. We reported that both therapeutic and prophylactic vaccines fail to protect and cure animals from disease due to negative effects of nicotine on DCs’ ability to generate effector T cells. We have been investigating whether vaccine formulated with TLR agonist(s) could potentially overcome the immunosuppressive effects of nicotine on human DC-NK cross-talk essential for effector T cell generation. Monocyte-derived DCs and nicDCs were stimulated with individual and combined TLR agonists prior to co-culture with purified T cells. The phenotypes and cytokine profiles of T cell were assessed using Flow Cytometry and ELISA, respectively. We found nicDCs cultured with TLR-8/7 alone or in combination with TLR-3 produce quantitatively and qualitatively similar IFN-γ producing effector T cells when compared to control DCs. Our data suggest that the addition of appropriate TLR agonist to vaccine formulation could potentially overcome the immunosuppression seen in smokers, thereby containing the spread of infectious disease to vulnerable population