Mammalian Scribble is a target for proteins encoded by human papilloma virus, retro- and flaviviruses. Tick-borne encephalitis virus (TBEV) is a flavivirus that have evolved distinct strategies to escape antiviral responses. Information of how flaviviruses intrude on cell integrity comes from understanding of the roles that host-factors play when they interfere with viruses. The first part of this thesis describes a novel interaction between the TBEVNS5 protein and Scribble. The importance of the interaction was demonstrated by RNAi-mediated depletion of Scribble, which prevented suppression of JAK-STAT signaling by NS5. Together, these results define Scribble as a novel target for NS5. TBEV is known to cause central nervous system disease TBE in humans that can lead to cognitive dysfunction. A unifying theme in CNS related diseases are defects in neuronal extensions. We therefore addressed the effects of TBEV expression in PC12 cell differentiation, which is characterized by extensive neurite growth. Our data show that TBEVNS5 suppresses neurite outgrowth through the Rho GTPase Rac1. These findings provide evidence that Rac1 is an indirect target of NS5 in neurite inhibition. Scribble was recently implicated in spine morphogenesis. Thus, we tested the role of Scribble in neurite elongation. Depletion of Scribble in PC12 cells, reduced neurite density but increased length of those remaining. Moreover, Scribble bound components in the Ras/ERK cascade in a growth factor dependent manner. Together, these results demonstrate that Scribble controls neurite elongation by scaffolding MAPK components. Moreover, as loss of dendritic spines, actin-rich protrusions on neurons, is a feature in cognitive dysfunction we speculate that cognitive dysfunction in TBE might involve disturbed Scribble expression by NS5. We also investigated the binding between NS1 of Influenza A virus and Scribble. The PDZ domains of Scribble are usually selective for specific C-terminal motifs in proteins. Because NS1 has a canonical PDZ motif we tested if binding to Scribble depends on this motif. We found that Scribble binds NS1; the association is dependent on the NS1 C-terminus that is recognized by PDZ3-4 of Scribble. Together, these results suggest that Scribble is a target for the H5N1 NS1 protein / At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 2: In press. Paper 3: Manuscript. Paper 4: Manuscript.

Glycogen synthase kinase-3 (GSK-3) is a serine/threonine protein kinase which plays a key role in several signaling pathways and its homologues have been identified in most eukaryotes. Since GSK3£]is an essential protein kinase that regulates numerous functions within the cell, an effort to survey possible GSK3£]- interacting proteins from a human testis cDNA library using the yeast two-hybrid system is made. Two interesting candidates are chosen to characterize their functions in this study. One is a centrosomal protein, hNinein, and the other is a novel inhibitor of GSK3£], designated as GSKIP (GSK3£] interaction protein). In the first part of the present thesis we describe the identification of four diverse CCII-termini of human hNinein isoforms, including a novel isoform 6, by differential expression in a tissue-specific manner. In a kinase assay, the CCII region of hNinein isoforms provides a differential phosphorylation site by GSK3£]. In addition, either N-terminal or CCIIZ domain disruption may cause hNinein conformational change which recruits £^-tubulin to centrosomal or non-centrosomal hNinein-containing sites. Further, depletion of all hNinein isoforms caused a significant decrease in the £^-tubulin signal in the centrosome. In domain swapping, it clearly shows that the CCIIX-CCIIY region provides docking sites for £^-tubulin. Moreover, nucleation of microtubules from the centrosome is significantly affected by the overexpression of either the full-length hNinein or CCIIX-CCIIY region. Taken together, these results show that the centrosomal targeting signals of hNinein have a role not only in regulating hNinein conformation, resulting in localization change, but also provide docking sites to recruit £^-tubulin at centrosomal and non-centrosomal sites. In the second part of the thesis we describe another candidate, GSK3£]interaction protein (GSKIP), to characterize its functions in neuron differentiation. We use human neuroblastoma SH-SY5Y cells as a model of neuronal cell differentiation. When overexpression of GSKIP prevents neurite outgrowth from RA-mediated differentiation, this result is similar to the presence of LiCl or SB415286, an inhibitor of GSK3£]. Further, GSKIP regulates the activity of GSK3£] through protein-protein interactions rather than post-modulation and GSKIP may affect GSK3£] on neurite outgrowth via inhibiting the specific phosphorylation site of tau. In addition to inhibition of neurite outgrowth, GSKIP overexpressed in SH-SY5Y cells also promotes cell cycle progression by analyzing cell proliferation with cell growth and MTT assay. Furthermore, GSKIP raises the level of £]-catenin and cyclin D1 through inhibition of GSK3£] activity in RA-mediated differentiation SH-SY5Y cells. Taken together, the data suggest that GSKIP, a dual functional molecule, is able to inhibit neurite outgrowth and promote cell proliferation via negative regulation of GSK3£] activity in RA-mediated differentiation of SH-SY5Y cells.

Aggregating proteoglycans (PG) bearing chondroitin sulfate (CS) side chains are well-known inhibitors of neural plasticity and associate with hyaluronan and tenascin-R to form a complex of extracellular matrix (ECM) in the central nervous system (CNS). Little is known about whether proteolytic cleavage of the core protein affects neural plasticity. Several members of the ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) family of metalloproteinases are glutamyl-endopeptidases that cleave aggregating PGs. Our initial studies determined that neural cultures secrete a brevican-containing matrix, and that these neural cultures also produced ADAMTS4, a protease that cleaves brevican. Furthermore, this brevican-containing matrix in astrocytes could be modulated by treatment with transforming growth factor beta (TGFbeta) through the inhibition of the activity of the ADAMTSs.Once it was established that neural cultures produce a brevican-rich matrix, we s ought to utilize this matrix to determine whether cleavage of aggregating PGs, especially brevican, by the ADAMTSs influences neurite outgrowth in cultured neurons. Transfection of rat neurons with ADAMTS4 cDNA induced longer neurites, and interestingly, this effect proved to be independent of the proteolytic action of the ADAMTSs. Addition of recombinant ADAMTS4 or ADAMTS5 protein to immature neuronal cultures similarly enhanced neurite extension, an action dependent on the activation of extracellular signal-related kinase (ERK)1/2 (MAP kinase 42/44), resulting in the first evidence that ADAMTSs may induce intracellular signaling events. Studies of dendritic spine morphology and levels of synaptic proteins in response to ADAMTS4 treatment were also undertaken. Neuronal cultures treated with ADAMTS4 showed increased length of dendritic spines and increased percent of immature spines detected. A concurrent decrease in post-synaptic protein staining was detected on the neurites of yo ung neurons overexpressing ADAMTS4 or expressing proteolytically-inactive mutant ADAMTS4 protein. Thus, ADAMTS4 may promote plasticity in neurons in vitro by preventing the formation, maturation, and/or stabilization of synapses. Overall, these experiments provide evidence that implicate the ADAMTSs as mediators of neural plasticity, and while primarily known only as proteases, these studies demonstrate that the ADAMTSs exert actions distinct from these proteolytic properties that require the induction of intracellular signaling events.

The growth of a neuronal dendritic tree depends on the neuron’s internal state and the environment within which it is situated. Different types of neuron develop dendritic trees with specific characteristics, such as the average number of terminal branches and the average length of terminal and intermediate segments. A key aspect of the growth process is the construction of the microtubule cytoskeleton within the dendritic tree. Neurite elongation requires assembly of microtubules from free tubulin at the growth cone. The stability of microtubule bundles is an important factor in determining how likely it is for a growth cone to split to form new daughter branches. Microtubule assembly rates and bundle stability are controlled by microtubule-associated proteins, principally MAP2 in dendrites. Extending previous work (Hely et al, J. Theor. Biol. 210:375-384, 2001) I have developed a mathematical model of neurite outgrowth in which elongation and branching rates are determined by the phosphorylation state of MAP2 at the tips of each terminal branch. Tubulin and MAP2 are produced in the cell body and transported along the neurite by a combination of diffusion and active transport. Microtubule (dis)assembly at neurite tips is a function of tubulin concentration. The rate of assembly depends on the amount of unphosphorylated MAP2 bound to the microtubules and linking them together. Phosphorylation of MAP2 destroys its linking capability and destabilises the microtubule bundles. Each terminal has a probability of branching that depends on the phosphorylation of MAP2 which, in turn, is a function of calcium concentration. Results from this model show that changes in the (de)phosphorylation rates of MAP2 affect the topology of the final dendritic tree. Higher phosphorylation promotes branching and results in trees with many short terminal branches and relatively long intermediate segments. Reducing phosphorylation promotes elongation and inhibits branching.

Neovibsanin A and B are natural products which induced neurite outgrowth in PC12 cells. They belong in the neovibsane class under the rare vibsane natural product family, whose structures are characterized by polycyclic, polyoxygenated cores. Based on a proposed biosynthesis, the synthetic strategy towards neovibsanin A and B involved synthesizing a key enone intermediate. Initial investigation using this intermediate lead to the total synthesis of 2-O-methylneovibsanin H. Crucial to this concise synthesis was an acid-catalyzed, one-pot, four-step cascade reaction. Modifying the reaction condition leads to a different five-step cascade pathway, resulting in the total synthesis of 4,5-bis-epi-neovibsanin A and B. The synthetic trials and tribulations encountered on the road to these final compounds are explored. It is envisaged that other related neovibsane natural products may arise based on this synthetic sequence. 4,5-Bis-epi-neovibsanin A and B, as well as several other structural analogues collected during the synthesis, were biologically assayed using NGF-stimulated PC12 cells. All compounds induced a significant proportion of neurons to extend neurite processes compared to control cultures. The structure-activity relationship studies indicated that the tricyclic core, as well as the 3,3-dimethylacroyl enol ester side chain, may be responsible for promoting a biological response.

Neovibsanin A and B are natural products which induced neurite outgrowth in PC12 cells. They belong in the neovibsane class under the rare vibsane natural product family, whose structures are characterized by polycyclic, polyoxygenated cores. Based on a proposed biosynthesis, the synthetic strategy towards neovibsanin A and B involved synthesizing a key enone intermediate. Initial investigation using this intermediate lead to the total synthesis of 2-O-methylneovibsanin H. Crucial to this concise synthesis was an acid-catalyzed, one-pot, four-step cascade reaction. Modifying the reaction condition leads to a different five-step cascade pathway, resulting in the total synthesis of 4,5-bis-epi-neovibsanin A and B. The synthetic trials and tribulations encountered on the road to these final compounds are explored. It is envisaged that other related neovibsane natural products may arise based on this synthetic sequence. 4,5-Bis-epi-neovibsanin A and B, as well as several other structural analogues collected during the synthesis, were biologically assayed using NGF-stimulated PC12 cells. All compounds induced a significant proportion of neurons to extend neurite processes compared to control cultures. The structure-activity relationship studies indicated that the tricyclic core, as well as the 3,3-dimethylacroyl enol ester side chain, may be responsible for promoting a biological response.

Neovibsanin A and B are natural products which induced neurite outgrowth in PC12 cells. They belong in the neovibsane class under the rare vibsane natural product family, whose structures are characterized by polycyclic, polyoxygenated cores. Based on a proposed biosynthesis, the synthetic strategy towards neovibsanin A and B involved synthesizing a key enone intermediate. Initial investigation using this intermediate lead to the total synthesis of 2-O-methylneovibsanin H. Crucial to this concise synthesis was an acid-catalyzed, one-pot, four-step cascade reaction. Modifying the reaction condition leads to a different five-step cascade pathway, resulting in the total synthesis of 4,5-bis-epi-neovibsanin A and B. The synthetic trials and tribulations encountered on the road to these final compounds are explored. It is envisaged that other related neovibsane natural products may arise based on this synthetic sequence. 4,5-Bis-epi-neovibsanin A and B, as well as several other structural analogues collected during the synthesis, were biologically assayed using NGF-stimulated PC12 cells. All compounds induced a significant proportion of neurons to extend neurite processes compared to control cultures. The structure-activity relationship studies indicated that the tricyclic core, as well as the 3,3-dimethylacroyl enol ester side chain, may be responsible for promoting a biological response.

Neovibsanin A and B are natural products which induced neurite outgrowth in PC12 cells. They belong in the neovibsane class under the rare vibsane natural product family, whose structures are characterized by polycyclic, polyoxygenated cores. Based on a proposed biosynthesis, the synthetic strategy towards neovibsanin A and B involved synthesizing a key enone intermediate. Initial investigation using this intermediate lead to the total synthesis of 2-O-methylneovibsanin H. Crucial to this concise synthesis was an acid-catalyzed, one-pot, four-step cascade reaction. Modifying the reaction condition leads to a different five-step cascade pathway, resulting in the total synthesis of 4,5-bis-epi-neovibsanin A and B. The synthetic trials and tribulations encountered on the road to these final compounds are explored. It is envisaged that other related neovibsane natural products may arise based on this synthetic sequence. 4,5-Bis-epi-neovibsanin A and B, as well as several other structural analogues collected during the synthesis, were biologically assayed using NGF-stimulated PC12 cells. All compounds induced a significant proportion of neurons to extend neurite processes compared to control cultures. The structure-activity relationship studies indicated that the tricyclic core, as well as the 3,3-dimethylacroyl enol ester side chain, may be responsible for promoting a biological response.

Neovibsanin A and B are natural products which induced neurite outgrowth in PC12 cells. They belong in the neovibsane class under the rare vibsane natural product family, whose structures are characterized by polycyclic, polyoxygenated cores. Based on a proposed biosynthesis, the synthetic strategy towards neovibsanin A and B involved synthesizing a key enone intermediate. Initial investigation using this intermediate lead to the total synthesis of 2-O-methylneovibsanin H. Crucial to this concise synthesis was an acid-catalyzed, one-pot, four-step cascade reaction. Modifying the reaction condition leads to a different five-step cascade pathway, resulting in the total synthesis of 4,5-bis-epi-neovibsanin A and B. The synthetic trials and tribulations encountered on the road to these final compounds are explored. It is envisaged that other related neovibsane natural products may arise based on this synthetic sequence. 4,5-Bis-epi-neovibsanin A and B, as well as several other structural analogues collected during the synthesis, were biologically assayed using NGF-stimulated PC12 cells. All compounds induced a significant proportion of neurons to extend neurite processes compared to control cultures. The structure-activity relationship studies indicated that the tricyclic core, as well as the 3,3-dimethylacroyl enol ester side chain, may be responsible for promoting a biological response.

Neovibsanin A and B are natural products which induced neurite outgrowth in PC12 cells. They belong in the neovibsane class under the rare vibsane natural product family, whose structures are characterized by polycyclic, polyoxygenated cores. Based on a proposed biosynthesis, the synthetic strategy towards neovibsanin A and B involved synthesizing a key enone intermediate. Initial investigation using this intermediate lead to the total synthesis of 2-O-methylneovibsanin H. Crucial to this concise synthesis was an acid-catalyzed, one-pot, four-step cascade reaction. Modifying the reaction condition leads to a different five-step cascade pathway, resulting in the total synthesis of 4,5-bis-epi-neovibsanin A and B. The synthetic trials and tribulations encountered on the road to these final compounds are explored. It is envisaged that other related neovibsane natural products may arise based on this synthetic sequence. 4,5-Bis-epi-neovibsanin A and B, as well as several other structural analogues collected during the synthesis, were biologically assayed using NGF-stimulated PC12 cells. All compounds induced a significant proportion of neurons to extend neurite processes compared to control cultures. The structure-activity relationship studies indicated that the tricyclic core, as well as the 3,3-dimethylacroyl enol ester side chain, may be responsible for promoting a biological response.