BRAIN ACTIVATION AND CONNECTIVITY IN NON-DISABLED MULTIPLE SCLEROSIS PATIENTS

Multiple sclerosis (MS) is the most common demyelinating disease affecting the central nervous system. There is no cure for MS and current therapies have limited efficacy. While the majority of individuals with MS develop significant clinical disability, a subset experiences a disease course with minimal impairment even in the presence of significant apparent tissue damage on magnetic resonance imaging (MRI). The current studies combined functional MRI and diffusion tensor imaging (DTI) to elucidate brain mechanisms associated with lack of clinical disability in patients with MS. Recent evidence has implicated cortical reorganization as a mechanism to limit the clinical manifestation of the disease. Functional MRI was used to test the hypothesis that non-disabled MS patients (Expanded Disability Status Scale ≤ 1.5) show increased recruitment of cognitive control regions (dorsolateral prefrontal and anterior cingulate cortex) while performing sensory, motor and cognitive tasks. Compared to matched healthy controls, patients increased activation of cognitive control brain regions when performing non-dominant hand movements and the 2-back working memory task. Using dynamic causal modeling, we tested whether increased cognitive control recruitment is associated with alterations in connectivity in the working memory functional network. Patients exhibited similar network connectivity to that of control subjects when performing working memory tasks. We subsequently investigated the integrity of major white matter tracts to assess structural connectivity and its relation to activation and functional integration of the cognitive control system. Patients showed substantial alterations in callosal, inferior and posterior white matter tracts and less pronounced involvement of the corticospinal tracts and superior longitudinal fasciculi (SLF). Decreased structural integrity within the right SLF in patients was associated with decreased performance, and decreased activation and connectivity of the cognitive control system when performing working memory tasks. These studies suggest that patient with MS without clinical disability increase cognitive control system recruitment across functional domains and rely on preserved functional and structural connectivity of brain regions associated with this network. Moreover, the current studies show the usefulness of combining brain activation data from functional MRI and structural connectivity data from DTI to improve our understanding of brain adaptation mechanisms to neurological disease.

Glutamate and nitric oxide as regulatory transmitters in developing rabbit retina

Glutamate is the major excitatory neurotransmitter in the retina and serves as the synaptic messenger for the three classes of neurons which constitute the vertical pathway--the photoreceptors, bipolar cells and ganglion cells. In addition, the glutamate system has been localized morphologically, pharmacologically as well as molecularly during the first postnatal week of development before synaptogenesis occurs. The role which glutamate plays in the maturing visual system is complex but ranges from mediating developmental neurotoxicity to inducing neurite outgrowth.^ Nitric oxide/cGMP is a novel intercellular messenger which is thought to act in concert with the glutamate system in regulating a variety of cellular processes in the brain as well as retina, most notably neurotoxicity. Several developmental activities including programmed cell death, synapse elimination and synaptic reorganization are possible functions of cellular regulation modulated by nitric oxide as well as glutamate.^ The purpose of this thesis is to (1) biochemically characterize the endogenous pools of glutamate and determine what fraction exists extracellularly; (2) examine the morphological expression of NO producing cells in developing retina; (3) test the functional coupling of the NMDA subtype of glutamate receptor to the NO system by examining neurotoxicity which has roles in both the maturing and adult retina.^ Biochemical sampling of perfusates collected from the photoreceptor surface of ex vivo retina demonstrated that although the total pool of glutamate present at birth is relatively modest, a high percentage resides in extracellular pools. As a result, immature neurons without significant synaptic connections survive and develop in a highly glutamatergic environment which has been shown to be toxic in the adult retina.^ The interaction of the glutamate system with the NO system has been postulated to regulate neuronal survival. We therefore examined the developmental expression of the enzyme responsible for producing NO, nitric oxide synthase (NOS), using an antibody to the constitutive form of NOS found in the brain. The neurons thought to produce the majority of NO in the adult retina, a subpopulation of widefield amacrine cells, were not immunoreactive until the end of the second postnatal week. However, a unique developmental expression was observed in the ganglion cell layer and developing outer nuclear layer of the retina during the first postnatal week. We postulate NO producing neurons may not be present in a mature configuration therefore permitting neuronal survival in a highly glutamatergic microenvironment and allowing NO to play a development-specific role at this time.^ The next set of experiments constituted a functional test of the hypothesis that the absence of the prototypic NO producing cells in developing retina protects immature neurons against glutamate toxicity. An explant culture system developed in order to examine cellular responses of immature and adult neurons to glutamate toxicity showed that immature neurons were affected by NMDA but were less responsive to NMDA and NO mediated toxicity. In contrast, adult explants exhibited significant NMDA toxicity which was attenuated by NMDA antagonists, 2-amino-5-phosphonovaleric acid (APV), dextromethorphan (Dex) and N$\rm\sp{G}$-D-methyl arginine (metARG). These results indicated that pan-retinal neurotoxicity via the NMDA receptor and/or NO activation occurred in the adult retina but was not significant in the neonate. (Abstract shortened by UMI.) ^

CYTOLOGICAL, ULTRASTRUCTURAL, AND BIOCHEMICAL STUDIES OF THE STRUCTURE OF PREMATURELY CONDENSED CHROMOSOMES

The phenomenon of premature chromosome condensation, resulting from fusion between mitotic and interphase cells, includes dissolution of the interphase nuclear framework, thus allowing a direct visualization of interphase chromosomes. Light microscope morphology of prematurely condensed chromosomes (PCC) from synchronized HeLa cells supports the model of an interphase "chromosome condensation cycle". PCC are increasingly attenuated as cells progress through G(,1). A maximum degree of decondensation is observed at active sites of DNA replication during S phase, and a condensed morphology is rapidly resumed following completion of replication of a chromosome segment.^ To permit ultrastructural and biochemical studies of PCC, a procedure was developed to induce premature chromosome condensation at high frequency. This was achieved by polyethylene glycol (PEG)-mediated fusion of a dense monolayer of mitotic and interphase cells induced by centrifugation onto lectin-coated culture dishes. Using this method, PCC induction frequencies of 60-90% are routinely obtained.^ Scanning electron microscope analysis of PCC spreads revealed that the extension of PCC during progression through G(,1) is accompanied by a transition of the basic 30 nm chromatin fiber from tightly packed looping fibers to extended longitudinal fibers. Sites of active DNA replication is S-PCC were indicated to be organized a single longitudinal fibers. Following replication of a chromosome segment, a rapid reorganization from the extended longitudinal fiber to packed looping fibers occurs. The postreplication maturation process appears to include the assembly of a chromosome core consisting of multiple longitudinal fibers.^ The role of histone H1 phosphorylation in PCC formation was investigated by acidurea polyacrylamide gel electrophoresis of total histone extracted from metaphase chromosomes and PCC following high frequency fusion. This investigation failed to demonstrate an extensive phosphorylation of H1 associated with PCC formation. However, significant dephosphorylation of superphosphorylated metaphase chromosome H1 was observed, indicating that interphase H1-phosphatase activity is dominant over metaphase H1 kinase activity. These observations provide evidence against models suggesting a role for H1 superphosphorylation in triggering mitotic condensation of chromosomes. ^

Ontogeny and regulation of interleukin-7 expressing thymic epithelial cells

T cell development is a multistage process of differentiation that depends on proper thymocyte-thymic epithelial cell (TEC) interactions. Epithelial cells in the thymus are organized in a three-dimensional network that provides support and signals for thymocyte maturation. Concurrently, proper TEC differentiation in the adult thymus relies on thymocyte-derived signals. TECs produce interleukin-7 (IL-7), a non-redundant cytokine that promotes the survival, differentiation, and proliferation of thymocytes. We have identified IL-7 expressing TECs throughout ontogeny and in the adult thymus by in situ hybridization analysis. IL-7 expression is initiated in the thymic fated domain of the thymic primordium by embryonic day 11.5, in a Foxn1 independent pathway. Marked changes occur in the localization and regulation of IL-7 expressing TECs during development. Whereas IL-7 expressing TECs are present throughout the early thymic rudiment, the majority of IL-7 producing TECs are concentrated in the adult thymic medulla. By analyzing mouse strains that sustain blocks at different stages of thymocyte development, we show that IL-7 expression is initiated independently of hematopoietic-derived signals during thymic organogenesis. However, thymocyte-derived signals play an essential role in regulating IL-7 expression in the adult TEC compartment. Furthermore, distinct thymocyte subsets regulate the expression of IL-7 and keratin 5 in adult cortical epithelium. Intraperitoneal injection of Recombination Activating Gene deficient mice (RAG-2−/−) with anti-CD3ϵ monoclonal antibody (mAb) induces CD4− 8− double negative thymocytes to undergo β-selection and differentiate into CD4+8+ cells. Analysis of the thymic stromal compartment reveals that progression through β-selection renders thymocytes competent to alter the pattern of IL-7 expression in the cortical TEC compartment. RAG-2−/− mice do not generate mature T cells and therefore the RAG-2−/− thymus is devoid of organized medullary regions. Histological examination of RAG-2−/− thymus following anti-CD3ϵ stimulation reveals the emergence of mature thymic medullary regions, as assessed by H & E staining and expression of thymic stromal medullary markers. Stromal medullary reorganization occurs in the absence of T cell receptor αβ expression, suggesting that activation of RAG-2−/− thymocytes by CD3ϵ ligation generates thymocyte-derived signals that induce thymic epithelial reorganization, generating a mature medullary compartment. This model provides a tool to assess the mechanisms underlying thymic medullary development. ^

T cell adhesion regulation from clustering GM1 lipid rafts

Lipid rafts are small laterally mobile cell membrane structures that are highly enriched in lymphocyte signaling molecules. Lipid rafts can form from the assembly of specialized lipids and proteins through hydrophobic associations from saturated acyl chains. GM1 gangliosides are a common lipid raft component and have been shown to be essential in many T cell functions. Current lipid raft theory hypothesizes that certain aspects of T cell signaling can be initiated from the coalescence of these signaling-enriched lipid rafts to sites of receptor engagement. We have described how the specific aggregation of GM1 lipid rafts can cause a reorganization of cell surface molecular associations which include dynamic associations of β1 integrins with GM1 lipid rafts. These associations had pronounced effects on T cell adhesive and migratory states. We show that GM1 lipid raft aggregation can dramatically inhibit T cell migration and chemotaxis on the extracellular matrix constituent fibronectin. This inhibition of migration function was shown to be dependent on the src kinase Lck and PKC-regulated F-actin polymerization to extending pseudopods. Furthermore, GM1 lipid raft clustering could activate T cell adhesion-strengthening mechanisms. These include an increase in cellular rigidity, the creation of polymerized cortical F-actin structures, the induction of high affinity integrin states, an increase in surface area and symmetry of the contact plane, and resistance to shear flow detachment while adherent to fibronectin. This indicates that GM1 lipid raft aggregation defines a novel stimulus to regulate lymphocyte motility and cellular adhesion which could have important implications in T cell homing mechanisms. ^

Regulation of Net1A subcellular localization by the small GTPase Rac1

Activation of Rho family small G proteins is thought to be a critical event in breast cancer development and metastatic progression. Rho protein activation is stimulated by a family of enzymes known as guanine nucleotide exchange factors (Rho GEFs). The neuroepithelioma transforming gene 1 (Net1) is a Rho GEF specific for the RhoA subfamily that is overexpressed in primary breast tumors and breast cancer cell lines. Net1 isoform expression is also required for migration and invasion of breast cancer cells in vitro. These data indicate that Net1 may be a critical regulator of metastatic progression in breast cancer. Net1 activity is negatively regulated by sequestration in the nucleus, and relocalization of Net1 outside the nucleus is required to stimulate RhoA activation, actin cytoskeletal reorganization, and oncogenic transformation. However, regulatory mechanisms controlling the extranuclear localization of Net1 have not been identified. In this study, we have addressed the regulation of Net1A isoform localization by Rac1. Specifically, co-expression of constitutively active Rac1 with Net1A stimulates the relocalization of Net1A from the nucleus to the plasma membrane in breast cancer cells, and results in Net1A activation. Importantly, Net1A localization is also driven by endogenous Rac1 activity. Net1A relocalizes outside the nucleus in cells spreading on collagen, and when endogenous Rac1 expression was silenced by siRNA, Net1A remained nuclear in spreading cells. These data indicate that Rac1 controls the localization of the Net1A isoform and suggests a physiological role for Net1A in breast cancer cell adhesion and motility.

REGULATION OF ALTERNATIVE CARBON METABOLISM IN CANDIDA ALBICANS

Candida albicans is the most important fungal pathogen of humans. Transcript profiling studies show that upon phagocytosis by macrophages, C. albicans undergoes a massive metabolic reorganization activating genes involved in alternative carbon metabolism, including the glyoxylate cycle, β-oxidation and gluconeogenesis. Mutations in key enzymes such as ICL1 (glyoxylate cycle) and FOX2 (fatty acid β-oxidation) revealed that alternative carbon metabolic pathways are required for full virulence in C. albicans. These studies indicate C. albicans uses non-preferred carbon sources allowing its adaptation to microenvironments were nutrients are scarce. It has become apparent that the regulatory networks required for regulation of alternative carbon metabolism in C. albicans are considerably different from the Saccharomyces cerevisiae paradigm and appear more analogous to the Aspergillus nidulans systems. Well-characterized transcription factors in S. cerevisiae have no apparent phenotype or are missing in C. albicans. CTF1 was found to be a single functional homolog of the A. nidulans FarA/FarB proteins, which are transcription factors required for fatty acid utilization. Both FOX2 and ICL1 were found to be part of a large CTF1 regulon. To increase our understanding of how CTF1 regulates its target genes, including whether regulation is direct or indirect, the FOX2 and ICL1 promoter regions were analyzed using a combination of bioinformatics and promoter deletion analysis. To begin characterizing the FOX2 and ICL1 promoters, 5’ rapid amplification of cDNA ends (5’RACE) was used to identify two transcriptional initiation sites in FOX2 and one in ICL1. GFP reporter assays show FOX2 and ICL1 are rapidly expressed in the presence of alternative carbon sources. Both FOX2 and ICL1 harbor the CCTCGG sequence known to be bound by the Far proteins, hence rendering the motif as a putative CTF1 DNA binding element. In this study, the CCTCGG sequence was found to be essential for FOX2 regulation. However, this motif does not appear to be equally important for the regulation of ICL1. This study supports the notion that although C. albicans has diverged from the paradigms of model fungi, C. albicans has made specific adaptations to its transcription-based regulatory network that may contribute to its metabolic flexibility.

Delayed Thrombus Resolution and Fibroproliferative Vascular Wound Healing From Deficiency of Type III Collagen: a Paradoxical Mechanism for Tissue Fragility

Vascular Ehlers-Danlos syndrome is a heritable disease of connective tissue caused by mutations in COL3A1, conferring a tissue deficiency of type III collagen. Cutaneous wounds heal poorly in these patients, and they are susceptible to spontaneous and catastrophic rupture of expansible hollow organs like the gut, uterus, and medium-sized to large arteries, which leads to premature death. Although the predisposition for organ rupture is often attributed to inherent tissue fragility, investigation of arteries from a haploinsufficient Col3a1 mouse model (Col3a1+/-) demonstrates that mutant arteries withstand even supraphysiologic pressures comparably to wild-type vessels. We hypothesize that injury that elicits occlusive thrombi instead unmasks defective thrombus resolution resulting from impaired production of type III collagen, which causes deranged remodeling of matrix, persistent inflammation, and dysregulated behavior by resident myofibroblasts, culminating in the development of penetrating neovascular channels that disrupt the mechanical integrity of the arterial wall. Vascular injury and thrombus formation following ligation of the carotid artery reveals an abnormal persistence and elevated burden of occlusive thrombi at 21 post-operative days in vessels from Col3a1+/- mice, as opposed to near complete resolution and formation of a patent and mature neointima in wild-type mice. At only 14 days, both groups harbor comparable burdens of resolving thrombi, but wild-type mice increase production of type III collagen in actively resolving tissues, while mutant mice do not. Rather, thrombi in mutant mice contain higher burdens of macrophages and proliferative myofibroblasts, which persist through 21 days while wild-type thrombi, inflammatory cells, and proliferation all regress. At the same time that increased macrophage burdens were observed at 14 and 21 days post ligation, the medial layer of mutant arterial walls concurrently harbored a significantly higher incidence of penetrating neovessels compared with those in wild-type mice. To assess whether limited type III collagen production alters myofibroblast behavior, fibroblasts from vEDS patients with COL3A1 missense mutations were seeded into three-dimensional fibrin gel constructs and stimulated with transforming growth factor-β1 to initiate myofibroblast differentiation. Although early signaling events occur similarly in all cell lines, late extracellular matrix- and mechanically-regulated events like transcriptional upregulation of type I and type III collagen secretion are delayed in mutant cultures, while transcription of genes encoding intracellular contractile machinery is increased. Sophisticated imaging of collagen synthesized de novo by resident myofibroblasts visualizes complex matrix reorganization by control cells but only meager remodeling by COL3A1 mutant cells, concordant with their compensatory contraction to maintain tension in the matrix. Finally, administration of immunosuppressive rapamycin to mice following carotid ligation sufficiently halts the initial inflammatory phase of thrombus resolution and fully prevents both myofibroblast migration into the thrombus and the differential development of neovessels between mutant and wild-type mice, suggesting that pathological defects in mutant arteries develop secondarily to myofibroblast dysfunction and chronic inflammatory stimulation, rather than as a manifestation of tissue fragility. Together these data establish evidence that pathological defects in the vessel wall architecture develop in mutant arteries as sequelae to abnormal healing and remodeling responses activated by arterial injury. Thus, these data support the hypothesis that events threatening the integrity of type III collagen-deficient vessels develop not as a result of inherent tissue weakness and fragility at baseline but instead as an episodic byproduct of abnormally persistent granulation tissue and fibroproliferative intravascular remodeling.

Heregulin -beta 1 directly associates with RhoA via the immunoglobulin -like domain thereby modulating breast cancer metastasis-related properties

Heregulins constitute a family of growth factors belonging to the epidermal growth factor (EGF) family. Breast cancers that overexpress specific members of the EGF receptor family (EGFR, ErbB2, ErbB3, ErbB4) have increased metastatic potential, and Heregulin-β1 (HRGβ1), a ligand for ErbB3 and ErbB4, has also been shown to induce metastasis-related properties in breast cancer cells in vitro. The secreted form of the HRGβ1 is composed of five distinct structural domains, including the N-terminal domain, an immunoglobulin-like domain (IgG-like), a glycosylation domain, an EGF-like domain, and a β1-specific domain. Of these, the EGF-like domain is well characterized for its function in metastasis-related properties as well as its structure. However, the contributions of the other HRGβ1 domains in breast cancer metastasis remains unclear. ^ To investigate this, HRGβ1 proteins with targeted domain deletions were purified and subjected to assays for metastasis-related properties, including aggregation, invasion, activation of EGFR family members, and motility of breast cancer cells. These assays showed that retaining the EGF-like domain of HRGβ1 is important for activation of EGFRs. Interestingly, the HRGβ1 protein lacking the IgG-like domain (NGEB) led to a decrease in breast cancer cell motility, indicating the IgG-like domain modulates cell motility, an important step in cancer metastasis. ^ To understand the underlying mechanisms, I performed protein sequence and structural analysis of HRGβ1 and identified that the IgG-like domain of HRGβ1 shares sequence homology and three-dimensional structural similarity with the IgG-like domain of TRIO. TRIO is a cytoplasmic protein that directly associates with RhoA, a GTPase involved in cell reorganization and cell motility. Therefore, I hypothesized that HRGβ1 may translocate inside the breast cancer cells through receptor mediated endocytosis and bind to RhoA via its IgG-like domain. I show wild type HRGβ1 but not NGEB binds RhoA in vitro and in vivo, leading to RhoA activation. Inhibition of HRG-β1 internalization via endocytosis disrupted HRGβ1 binding to RhoA. Additionally, breast cancer cell motility induced by HRG-β1 is reduced after treatment with inhibitors to both endocytosis and RhoA function, similar to levels seen with NGEB treatment. ^ Thus, in addition to the well-known role of HRGβ1 as an extracellular stimulator of the EGFR family members, HRGβ1 also functions within the cell as a binding partner and activator of RhoA to modulate cancer cell motility. ^