Activator Protein-1 and Epidermal Growth Factor Receptor Interplay : In Vivo & In Vitro Studies

Critical cellular decisions such as should the cell proliferate, migrate or differentiate, are regulated by stimulatory signals from the extracellular environment, like growth factors. These signals are transformed to cellular responses through their binding to specific receptors present at the surface of the recipient cell. The epidermal growth factor receptor (EGF-R/ErbB) pathway plays key roles in governing these signals to intracellular events and cell-to-cell communication. The EGF-R forms a signaling network that participates in the specification of cell fate and coordinates cell proliferation. Ligand binding triggers receptor dimerization leading to the recruitment of kinases and adaptor proteins. This step simultaneously initiates multiple signal transduction pathways, which result in activation of transcription factors and other target proteins, leading to cellular alterations. It is known that mutations of EGF-R or in the components of these pathways, such as Ras and Raf, are commonly involved in human cancer. The four best characterized signaling pathways induced by EGF-R are the mitogen-activated protein kinase cascades (MAPKs), the lipid kinase phosphatidylinositol 3 kinase (PI3K), a group of transcription factors called Signal Transducers and Activator of Transcription (STAT), and the phospholipase Cγ; (PLCγ) pathways. The activation of each cascade culminates in kinase translocation to the nucleus to stimulate various transcription factors including activator protein 1 (AP-1). AP-1 family proteins are basic leucine zipper (bZIP) transcription factors that are implicated in the regulation of a variety of cellular processes (proliferation and survival, growth, differentiation, apoptosis, cell migration, transformation). Therefore, the regulation of AP-1 activity is critical for the decision of cell fate and their deregulated expression is widely associated with many types of cancers, such as breast and prostate cancers. The aims of this study were to characterize the roles of EGF-R signaling during normal development and malignant growth in vitro and in vivo using different cell lines and tissue samples. We show here that EGF-R regulates cell proliferation but is also required for regulation of AP-1 target gene expression in fibroblasts in a MAP-kinase mediated manner. Furthermore, EGF-R signaling is essential for enterocyte proliferation and migration during intestinal maturation. EGF-R signaling network, especially PI3-K-Akt pathway mediated AP-1 activity is involved in cellular survival in response to ionizing radiation. Taken together, these results elucidate the connection of EGF-R and AP-1 in various cellular contexts and show their importance in the regulation of cellular behaviour presenting new treatment cues for intestinal perforations and cancer therapy.

Molecular pathogenesis of human parechovirus 1 infection

The Parechoviruses (HPEV) belong to the family Picornaviridae of positive-stranded RNA viruses. Although the parechovirus genome shares the general properties of other picornaviruses, the genus has several unique features when compared to other family members. We found that HPEV1 attaches to αv integrins on the cell surface and is internalized through the clathrin-mediated endocytic pathway. During he course of the infection, the Golgi was found to disintegrate and the ER membranes to swell and loose their ribosomes. The replication of HPEV1 was found to take place on small clusters of vesicles which contained the trans-Golgi marker GalT as well as the viral non-structural 2C protein. 2C was additionally found on stretches of modified ER-membranes, seemingly not involved in RNA replication. The viral non-structural 2A and 2C proteins were studied in further detail and were found to display several interesting features. The 2A protein was found to be a RNA-binding protein that preferably binds to positive sense 3 UTR RNA. It was found to bind also duplex RNA containing 3 UTR(+)-3 UTR(-), but not other dsRNA molecules studied. Mutagenesis revealed that the N-terminal basic-rich region as well as the C-terminus, are important for RNA-binding. The 2C protein on the other hand, was found to have both ATP-diphosphohydrolase and AMP kinase activities. Neither dATP nor other NTP:s were suitable substrates. Furthermore, we found that as a result of theses activities the protein is autophosphorylated. The intracellular changes brought about by the individual HPEV1 non-structural proteins were studied through the expression of fusion proteins. None of the proteins expressed were able to induce membrane changes similar to those seen during HPEV1 infection. However, the 2C protein, which could be found on the surface of lipid droplets but also on diverse intracellular membranes, was partly relocated to viral replication complexes in transfected, superinfected cells. Although Golgi to ER traffic was arrested in HPEV1-infected cells, none of the individually expressed non-structural proteins had any visible effect on the anterograde membrane traffic. Our results suggest that the HPEV1 replication strategy is different from that of many other picornaviruses. Furthermore, this study shows how relatively small differences in genome sequence result in very different intracellular pathology.

Studies of the genetic epidemiology of cardiovascular disease: focus on inflammatory candidate genes

Cardiovascular disease (CVD) is a complex disease with multifactorial aetiology. Both genetic and environmental factors contribute to the disease risk. The lifetime risk for CVD differs markedly between men and women, men being at increased risk. Inflammatory reaction contributes to the development of the disease by promoting atherosclerosis in artery walls. In the first part of this thesis, we identified several inflammatory related CVD risk factors associating with the amount of DNA from whole blood samples, indicating a potential source of bias if a genetic study selects the participants based on the available amount of DNA. In the following studies, this observation was taken into account by applying whole genome amplification to samples otherwise subjected to exclusion due to very low DNA yield. We continued by investigating the contribution of inflammatory genes to the risk for CVD separately in men and women, and looked for sex-genotype interaction. In the second part, we explored a new candidate gene and its role in the risk for CVD. Selenoprotein S (SEPS1) is a membrane protein residing in the endoplasmic reticulum where it participates in retro-translocation of unfolded proteins to cytosolic protein degradation. Previous studies have indicated that SEPS1 protects cells from oxidative stress and that variations in the gene are associated with circulating levels of inflammatory cytokines. In our study, we identified two variants in the SEPS1 gene, which associated with coronary heart disease and ischemic stroke in women. This is, to our knowledge, the first study suggesting a role of SEPS1 in the risk for CVD after extensively examining the variation within the gene region. In the third part of this thesis, we focused on a set of seven genes (angiotensin converting enzyme, angiotensin II receptor type I, C-reactive protein (CRP), and fibrinogen alpha-, beta-, and gamma-chains (FGA, FGB, FGG)) related to inflammatory cytokine interleukin 6 (IL6) and their association with the risk for CVD. We identified one variant in the IL6 gene conferring risk for CVD in men and a variant pair from IL6 and FGA genes associated with decreased risk. Moreover, we identified and confirmed an association between a rare variant in the CRP gene and lower CRP levels, and found two variants in the FGA and FGG genes associating with fibrinogen. The results from this third study suggest a role for the interleukin 6 pathway genes in the pathogenesis of CVD and warrant further studies in other populations. In addition to the IL6 -related genes, we describe in this thesis several sex-specific associations in other genes included in this study. The majority of the findings were evident only in women encouraging other studies of cardiovascular disease to include and analyse women separately from men.

The molecular basis of Marinesco-Sjögren syndrome

Marinesco-Sjögren syndrome (MSS) is a rare autosomal recessive neurodegenerative disorder characterized by cerebellar ataxia due to cerebellar cortical atrophy, infantile- or childhood-onset bilateral cataracts, progressive myopathy, and mild to severe mental retardation. Additional features include hypergonadotropic hypogonadism, various skeletal abnormalities, short stature, and strabismus. The neuroradiologic hallmarks are hypoplasia of both the vermis and cerebellar hemispheres. The histopathologic findings include severe cerebellar atrophy and loss of Purkinje and granule cells. The common pathologic findings in muscle biopsy are variation in muscle fiber size, atrophic fibers, fatty replacement, and rimmed vacuole formation. The presence of marked cerebellar atrophy with myopathy distinguishes MSS from another rare syndrome, the congenital cataracts, facial dysmorphism, and neuropathy syndrome (CCFDN). Previously, work by others had resulted in the identification of an MSS locus on chromosome 5q31. A subtype of MSS with myoglobinuria and neuropathy had been linked to the CCFDN locus on chromosome 18qter, at which mutations in the CTDP1 gene had been identified. We confirmed linkage to the previously identified locus on chromosome 5q31 in two Finnish families with eight affected individuals, reduced the critical region by fine-mapping, and identified SIL1 as a gene underlying MSS. We found a common homozygous founder mutation in all Finnish patients. The same mutation was also present in patient samples from Norway and Sweden. Altogether, we identified eight mutations in SIL1, including nonsense, frameshift, splice site alterations, and one missense mutation. SIL1 encodes a nucleotide exchange factor for the endoplasmic reticulum (ER) resident heat-shock protein 70 chaperone GRP78. GRP78 functions in protein synthesis and quality control of the newly synthesized polypeptides. It senses and responds to stressful cellular conditions. We showed that in mice, SIL1 and GRP78 show highly similar spatial and temporal tissue expression in developing and mature brain, eye, and muscle. Studying endogenous proteins in mouse primary hippocampal neurons, we found that SIL1 and GRP78 colocalize and that SIL1 localizes to the ER. We studied the subcellular localization of two mutant proteins, a missense mutant found in two patients and an artificial mutant lacking the ER retrieval signal, and found that both mutant proteins formed aggregates within the ER. Well in line with our findings and the clinical features of MSS, recent work by Zhao et al. showed that a truncation of SIL1 causes ataxia and cerebellar Purkinje cell loss in the naturally occurring woozy mutant mouse. Prior to Purkinje cell degeneration, the unfolded protein response is initiated and abnormal protein accumulations are present. MSS thus joins the group of protein misfolding and accumulation diseases. These findings highlight the importance of SIL1 and the role of the ER in neuronal function and survival. The results presented in this thesis provide tools for the molecular genetic diagnostics of MSS and give a basis for future studies on the molecular pathogenesis of MSS. Understanding the mechanisms behind this pleiotropic syndrome may provide insights into more common forms of ataxia, myopathy, and neurodegeneration.

The Myopathic Protein Myotilin in Developing Mouse and in Muscle Function

Skeletal muscle cells are highly specialised in order to accomplish their function. During development, the fusion of hundreds of immature myoblasts creates large syncytial myofibres with a highly ordered cytoplasm filled with packed myofibrils. The assembly and organisation of contractile myofibrils must be tightly controlled. Indeed, the number of proteins involved in sarcomere building is impressive, and the role of many of them has only recently begun to be elucidated. Myotilin was originally identified as a high affinity a-actinin binding protein in yeast twohybrid screen. It was then found to interact also with filamin C, actin, ZASP and FATZ-1. Human myotilin is mainly expressed in striated muscle and induces efficient actin bundling in vitro and in cells. Moreover, mutations in myotilin cause different forms of muscle disease, now collectively known as myotilinopathies. In this thesis, consisting of three publications, the work on the mouse orthologue is presented. First, the cloning and molecular characterisation of the mouse myotilin gene showed that human and mouse myotilin share high sequence homology and a similar expression pattern and gene regulation. Functional analysis of the mouse promoter revealed the myogenic factor-binding elements that are required for myotilin gene transcription. Secondly, expression of myotilin was studied during mouse embryogenesis. Surprisingly, myotilin was expressed in a wide array of tissues at some stages of development; its expression pattern became more restricted at perinatal stages and in adult life. Immunostaining of human embryos confirmed broader myotilin expression compared to the sarcomeric marker titin. Finally, in the third article, targeted deletion of myotilin gene in mice revealed that it is not essential for muscle development and function. These data altogether indicate that the mouse can be used as a model for human myotilinopathy and that loss of myotilin does not alter significantly muscle structure and function. Therefore, disease-associated mutant myotilin may act as a dominant myopathic factor.

Animal model and molecular interactions of Cln5

Neuronal ceroid lipofuscinoses (NCLs) are a family of inherited pediatric neurodegenerative disorders, leading to retinal degeneration, death of selective neuronal populations and accumulation of autofluorscent ceroid-lipopigments. The clinical manifestations are generally similar in all forms. The Finnish variant late infantile neuronal ceroid lipofuscinosis (vLINCLFin) is a form of NCL, especially enriched in the Finnish population. The aim of this thesis was to analyse the brain pathology of vLINCLFin utilising the novel Cln5-/- mouse model. Gene expression profiling of the brains of already symptomatic Cln5-/- mice revealed that inflammation, neurodegeneration and defects in myelinization are the major characteristics of the later stages of the disease. Histological characterization of the brain pathology confirmed that the thalamocortical system is affected in Cln5-/- mice, similarly to the other NCL mouse models. However, whereas the brain pathology in all other analyzed NCL mice initiate in the thalamus and spread only months later to the cortex, we observed that the sequence of events is uniquely reversed in Cln5-/- mice; beginning in the cortex and spreading to the thalamus only months later. We could also show that even though neurodegeneration is inititated in the cortex, reactive gliosis and loss of myelin are evident in specific nuclei of the thalamus already in the 1 month old brain. To obtain a deeper insight into the disturbed metabolic pathways, we performed gene expression profiling of presymptomatic mouse brains. We validated these findings with immunohistological analyses, and could show that cytoskeleton and myelin were affected in Cln5-/- mice. Comparison of gene expression profiling results of Cln5-/- and Cln1-/- mice, further highlighted that these two NCL models share a common defective pathway, leading to disturbances in the neuronal growth cone and cytoskeleton. Encouraged by the evidence of this defected pathway, we analyzed the molecular interactions of NCL-proteins and observed that Cln5 and Cln1/Ppt1 proteins interact with each other. Furthermore, we demonstrated that Cln5 and Cln1/Ppt1 share an interaction partner, the F1-ATP synthase, potentially linking both vLINCLFIN and INCL diseases to disturbed lipid metabolism. In addition, Cln5 was shown to interact with other NCL proteins; Cln2, Cln3, Cln6 and Cln8, implicating a central role for Cln5 in the NCL pathophysiology. This study is the first to describe the brain pathology and gene expression changes in the Cln5-/- mouse. Together the findings presented in this thesis represent novel information of the disease processes and the molecular mechanisms behind vLINCLFin and have highlighted that vLINCLFin forms a very important model to analyze the pathophysiology of NCL diseases.

The Role of Genes on Chromosome Locus 4q12 in Human Central Nervous System Tumors

Human central nervous system (CNS) tumors are a heterogeneous group of tumors occurring in brain, brainstem and spinal cord. Malignant gliomas (astrocytic and oligodendroglial tumors), which arise from the neuroepithelial cells are the most common CNS neoplasms in human. Malignant gliomas are highly aggressive and invasive tumors, and have a very poor prognosis. The development and progression of gliomas involve a stepwise accumulation of genetic alterations that generally affect either signal transduction pathways activated by receptor tyrosine kinases (RTKs), or cell cycle arrest pathways. Constitutive activation or deregulated signaling by RTKs is caused by gene amplification, overexpression or mutations. The aberrant RTK signaling results in turn in the activation of several downstream pathways, which ultimately lead to malignant transformation and tumor proliferation. Many genetic abnormalities implicated in nervous system tumors involve the genes located at the chromosomal region 4q12. This locus harbors the receptor tyrosine kinases KIT, PDGFRA and VEGFR2, and other genes (REST, LNX1) with neural function. Gene amplification and protein expression of KIT, PDGFRA, and VEGFR2 was studied using clinical tumor material. REST and LNX1, as well as NUMBL, the interaction partner of LNX1, were studied for their gene mutations and amplifications. In our studies, amplification of LNX1 was associated with KIT and PDGFRA amplification in glioblastomas, and coamplification of KIT, PDGFRA and VEGFR2 was detected in medulloblastomas and CNS primitive neuroectodermal tumors. PDGFRA amplification was also correlated with poor overall survival. Coamplification of KIT, PDGFRA and VEGFR2 was observed in a subset of human astrocytic and oligodendroglial tumors. We suggest that genes at 4q12 could be a part of a larger amplified region, which is deregulated in gliomas, and could be used as a prognostic marker of tumorigenic process. The signaling pathways activated due to gene amplifications, activating gene mutations, and overexpressed proteins may be useful as therapeutic targets for glioma treatment. This study also includes the characterization of KIT overexpressing astrocytes, analyzed by various in vitro functional assays. Our results show that overexpression of KIT in mouse astrocytes promotes cell proliferation and anchorage-independent growth, as well as phenotypic changes in the cells. Furthermore, the increased proliferation is partly inhibited by imatinib, a small molecule inhibitor of KIT. These results suggest that KIT may play a role in astrocyte growth regulation, and might have an oncogenic role in brain tumorigenesis. Elucidation of the altered signaling pathways due to specific gene amplifications, activating gene mutations, and overexpressed proteins may be useful as therapeutic targets for glioma treatment.

Resistance Mechanisms of Non-Hodgkin Lymphomas against Rituximab Treatment

Rituximab, a monoclonal antibody against B-cell specific CD20 antigen, is used for the treatment of non-Hodgkin lymphomas (NHL) and chronic lymphatic leukemia. In combination with chemotherapeutics rituximab has remarkably improved the outcome of NHL patients, but a vast variation in the lengths of remissions remains and the outcome of individual patients is difficult to predict. This thesis has searched for an explanation for this by studying the effector mechanisms of rituximab and by comparing gene expression in lymphoma tissue samples of patients with long- and short-term survival. This work demonstrated that activation of complement (C) system is in vitro more efficient effector mechanism of rituximab than cellular mechanisms or apoptosis. Activation of the C system was also shown in vivo during rituximab treatment. However, intravenously administered rituximab could not enter the cerebrospinal fluid, and neither C activation nor removal of lymphoma cells was observed in central nervous system. In vitro cytotoxicity assays showed that rituximab-induced cell killing could be markedly improved with simultaneous neutralization of the C regulatory proteins CD46 (Membrane cofactor protein), CD55 (Decay-accelerating factor), and CD59 (protectin). In a retrospective study of follicular lymphoma (FL) patients, low lymphoma tissue mRNA expressions of CD59 and CD55 were associated with a good prognosis and in a progressive flow cytometry study high expression of CD20 relative to CD55 was correlated to a longer progression free survival. Gene expression profile analysis revealed that expression of certain often cell cycle, signal transduction or immune response related genes correlate with clinical outcome of FL patients. Emphasizing the role of tumor microenvironment the best differentiating genes Smad1 and EphA1 were demonstrated to be mainly expressed in the non-malignant cells of tumors. In conclusion, this thesis shows that activation of the C system is a clinically important effector mechanism of rituximab and that microenvironment factor in tumors and expression of C regulatory proteins affect markedly the efficacy of immunochemotherapy. This data can be used to identify more accurately the patients for whom immunochemotherapy is given. It may also be beneficial in development of rituximab-containing and other monoclonal antibody therapies against cancer.

Regulation of tumor suppressor protein p53 in cellular stress and tumorigenesis

Functional loss of tumor suppressor protein p53 is a common feature in diverse human cancers. The ability of this protein to sense cellular damage and halt the progression of the cell cycle or direct the cells to apoptosis is essential in preventing tumorigenesis. Tumors having wild-type p53 also respond better to current chemotherapies. The loss of p53 function may arise from TP53 mutations or dysregulation of factors controlling its levels and activity. Probably the most significant inhibitor of p53 function is Mdm2, a protein mediating its degradation and inactivation. Clearly, the maintenance of a strictly controlled p53-Mdm2 route is of great importance in preventing neoplastic transformation. Moreover, impairing Mdm2 function could be a nongenotoxic way to increase p53 levels and activity. Understanding the precise molecular mechanisms behind p53-Mdm2 relationship is thus essential from a therapeutic point of view. The aim of this thesis study was to discover factors affecting the negative regulation of p53 by Mdm2, causing activation of p53 in stressed cells. As a model of cellular damage, we used UVC radiation, inducing a complex cellular stress pathway. Exposure to UVC, as well as to several chemotherapeutic drugs, causes robust transcriptional stress in the cells and leads to activation of p53. By using this model of cellular stress, our goal was to understand how and by which proteins p53 is regulated. Furthermore, we wanted to address whether these pathways affecting p53 function could be altered in human cancers. In the study, two different p53 pathway proteins, nucleophosmin (NPM) and promyelocytic leukemia protein (PML), were found to participate in the p53 stress response following UV stress. Subcellular translocations of these proteins were discovered rapidly after exposure to UV. The alterations in the cellular localizations were connected to transient interactions with p53 and Mdm2, implicating their significance in the regulation of p53 stress response. NPM was shown to control Mdm2-p53 interface and mediate p53 stabilization by blocking the ability of Mdm2 to promote p53 degradation. Furthermore, NPM mediated p53 stabilization upon viral insult. We further detected a connection between cellular pathways of NPM and PML, as PML was found to associate with NPM in UV-radiated cells. The observed temporal UV-induced interactions strongly imply existence of a multiprotein complex participating in the p53 response. In addition, PML controlled the UV response of NPM, its localization and complex formation with chromatin associated factors. The relevance of the UV-promoted interactions was demonstrated in studies in a human leukemia cell line, being under abnormal transcriptional repression due to expression of oncogenic PML-RARa fusion protein. Reversing the leukemic phenotype with a therapeutically significant drug was associated with similar complex formation between p53 and its partners as following UV. In conclusion, this thesis study identifies novel p53 pathway interactions associated with the recovery from UV-promoted as well as oncogenic transcriptional repression.

Human Torque teno virus: epidemiology, cell biology and immunology

Torque teno virus (TTV) was discovered in 1997 in the serum of a Japanese patient who had a post-transfusion hepatitis of unknown etiology. It is a small virus containing a circular single-stranded DNA genome which is unique among human viruses. Within a few years after its discovery, the TTVs were noted to form a large family of viruses with numerous genotypes. TTV is highly prevalent among the general population throughout the world, and persistent infections and co-infections with several genotypes occur frequently. However, the pathogenicity and the mechanism for the sustained occurrence of the virus in blood are at present unclear. To determine the prevalence of TTV in Finland, we set up PCR methods and examined the sera of asymptomatic subjects for the presence of TTV DNA and for genotype-6 DNA. TTV was found to be highly prevalent also in Finland; 85% of adults harbored TTV in their blood, and 4% were infected with genotype-6. In addition, TTV DNA was detected in a number of different tissues, with no tissue-type or symptom specificity. Most cell-biological events during TTV infections are at the moment unknown. Replicating TTV DNA has, however, been detected in liver and the hematopoietic compartment, and three mRNAs are known to be generated. To characterize TTV cell biology in more detail, we cloned in full length the genome of TTV genotype 6. We showed that in human kidney-derived cells TTV produces altogether six proteins with distinct subcellular localizations. TTV mRNA transcription was detected in all cell lines transfected with the full-length clone, and TTV DNA replicated in several of them, including those of erythroid, kidney, and hepatic origin. Furthermore, the viral DNA replication was shown to utilize the cellular DNA polymerases. Diagnoses of TTV infections have been based almost solely on PCR, whereas serological tests, measuring antibody responses, would give more information on many aspects of these infections. To investigate the TTV immunology in more detail, we produced all six TTV proteins for use as antigens in serological tests. We detected in human sera IgM and IgG antibodies to occur simultaneously with TTV DNA, and observed appearance of TTV DNA regardless of pre-existing antibodies, and disappearance of TTV DNA after antibody appearance. The genotype-6 nucleotide sequence remained stable for years within the infected subjects, suggesting that some mechanism other than mutations is used by this minute virus to evade our immune system and to establish chronic infections in immunocompetent subjects.

Molecular and Cellular Mechanisms Behind Juvenile Neuronal Ceroid Lipofuscinosis (JNCL, Batten disease)

Neurodegenerative disorders are chronic, progressive, and often fatal disorders of the nervous system caused by dysfunction, and ultimately, death of neuronal cells. The underlying mechanisms of neurodegeneration are poorly understood, and monogenic disorders can be utilised as disease models to elucidate the pathogenesis. Juvenile neuronal ceroid-lipofuscinosis (JNCL, Batten disease) is a recessively inherited lysosomal storage disorder with progressive neurodegeneration and accumulation of autofluorescent storage material in most tissues. It is caused by mutations in the CLN3 gene; however, the exact function of the corresponding CLN3 protein, as well as the molecular mechanisms of JNCL pathogenesis have remained elusive. JNCL disease exclusively affects the central nervous system leaving other organs unaffected, and therefore it is of a particular importance to conduct studies in brain tissue and neuronal cells. The aim of this thesis project was to elucidate the molecular and cell biological mechanisms underlying JNCL. This was the first study to describe the endogenous Cln3 protein, and it was shown that Cln3 localised to neuronal cells in the mouse brain. At a subcellular level, endogenous Cln3 was localised to the presynaptic terminals and to the synaptosome compartment, but not to the synaptic vesicles. Studies with the CLN3-deficient cells demonstrated an impaired endocytic membrane trafficking, and established an interconnection between CLN3, microtubulus-binding Hook1 and Rab proteins. This novel data was not only important in characterising the roles of CLN3 in cells, but also provided significant information delineating the versatile role of the Rab proteins. To identify affected cellular pathways in JNCL, global gene expression profiling of the knock-out mouse Cln3-/- neurons was performed and systematically analysed; this revealed a slight dysfunction of the mitochondria, cytoskeletal abnormality in the microtubule plus-end, and an impaired recovery from depolarizing stimulus when specific N-type Ca2+ channels were inhibited, thus leading to a prolonged time of higher intracellular calcium. All these defective pathways are interrelated, and may together be sufficient to initiate the neurodegenerative process. Results of this thesis also suggest that in neuronal cells, CLN3 most likely functions at endocytic vesicles at the presynaptic terminal, potentially involved in the regulation of the calcium-mediated synaptic transmission.

Target Cell Topography and Cytoskeletal Reorganization in Natural Killer Cell Activity

The immune system has to recognize and destroy abnormal or infected cells to maintain homeostasis. Natural killer (NK) cells directly recognize and kill transformed or virus-infected cells without prior sensitization. We have studied both virus-infected and tumor cells in order to identify the target structures involved in triggering NK activity. Mouse/human cell hybrids containing various human chromosomes were used as targets. The human chromosome responsible for activating NK cell killing was identified to chromosome number 6. The results suggest that activated NK cells recognize ligands that are encoded on human chromosome 6. We showed that the ligand on the target cell side was intercellular adhesion molecule 2 (ICAM-2). There was no difference in the level of expression of ICAM-2, however, but a drastic difference was seen in the distribution of the molecule: ICAM-2 was evenly distributed on the surface of the NK-resistant cells, but almost totally redistributed to the tip of uropods, bud-like extensions, which were absent from the parental cells. Interestingly, the gene coding for cytoskeletal linker protein ezrin has been localized to human chromosome 6, and there was a colocalization of ezrin and ICAM-2 in the uropods. Furthermore, the transfected human ezrin into NK cell-resistant cells induced uropod formation, ICAM-2 and ezrin redistribution to newly formed uropods, and sensitized target cells to NK cell killing. These data reveal a novel form of NK cell recognition: target structures are already present on normal cells; they become detectable only after abnormal redistribution into hot spots on the target cell membrane. NK cells are central players in the defence against virus infections. They inhibit the spread of infection, allowing time for specific immune responses to develop. The virus-proteins that directly activate human NK cell killing are largely unknown. We studied the sensitivity of virus-specific early proteins of Semliki Forest virus (SFV) to NK killing. The viral non-structural proteins (nsP1-4) translated early in the virus cycle were transfected in NK-resistant cells. Viral early gene nsP1 alone efficiently sensitized target cells to NK activity, and the tight membrane association of nsP1 seems to be critical in the triggering of NK killing. NsP1 protein colocalized with (redistributed) ezrin in filopodia-like structures to which the NK cells were bound. The results suggest that also in viral infections NK cells react to rapid changes in membrane topography. Based on the results of this thesis, a new model of target cell recognition of NK cells can be suggested: reorganization of the cytoskeleton induces alterations in cell surface topography, and this new pattern of surface molecules is recognized as "altered-self".

Molecular Interactions Underlying Neuronal Ceroid Lipofuscinoses CLN1 and CLN5

Disorders resulting from degenerative changes in the nervous system are progressive and incurable. Both environmental and inherited factors affect neuron function, and neurodegenerative diseases are often the sum of both factors. The cellular events leading to neuronal death are still mostly unknown. Monogenic diseases can offer a model for studying the mechanisms of neurodegeneration. Neuronal ceroid lipofuscinoses, or NCLs, are a group of monogenic, recessively inherited diseases affecting mostly children. NCLs cause severe and specific loss of neurons in the central nervous system, resulting in the deterioration of motor and mental skills and leading to premature death. In this thesis, the focus has been on two forms of NCL, the infantile NCL (INCL, CLN1) and the Finnish variant of late infantile NCL (vLINCLFin, CLN5). INCL is caused by mutations in the CLN1 gene encoding for the PPT1 (palmitoyl protein thioesterase 1) enzyme. PPT1 removes a palmitate moiety from proteins in experimental conditions, but its substrates in vivo are not known. In the Finnish variant of late infantile NCL (vLINCLFin), the CLN5 gene is defective, but the function of the encoded CLN5 has remained unknown. The aim of this thesis was to elucidate the disease mechanisms of these two NCL diseases by focusing on the molecular interactions of the defective proteins. In this work, the first interaction partner for PPT1, the mitochondrial F1-ATP synthase, was described. This protein has been linked to HDL metabolism in addition to its well-known role in the mitochondrial energy production. The connection between PPT1 and the F1-ATP synthase was studied utilizing the INCL-disease model, the genetically modified Ppt1-deficient mice. The levels of F1-ATP synthase subunits were increased on the surface of Ppt1-deficient neurons when compared to controls. We also detected several changes in lipid metabolism both at the cellular and systemic levels in Ppt1-deficient mice when compared to controls. The interactions between different NCL proteins were also elucidated. We were able to detect novel interactions between CLN5 and other NCL proteins, and to replicate the previously reported interactions. Some of the novel interactions influenced the intracellular trafficking of the proteins. The multiple interactions between CLN5 and other NCL proteins suggest a connection between the NCL subtypes at the cellular level. The main results of this thesis elicit information about the neuronal function of PPT1. The connection between INCL and neuronal lipid metabolism introduces a new perspective to this rather poorly characterized subject. The evidence of the interactions between NCL proteins provides the basis for future research trying to untangle the NCL disease mechanisms and to develop strategies for therapies.

Antizyme Inhibitor 2 : A Novel Regulator of Cellular Polyamine Homeostasis

Polyamines are organic polycations that participate in various physiological functions, including cell proliferation, differentiation and apoptosis. Cellular polyamines originate from endogenous biosynthesis and exogenous sources. Their subcellular pool is under strict control, achieved by regulating their uptake and metabolism. Polyamine-induced proteins called antizymes (AZ) act as key regulators of intracellular polyamine concentration. They regulate both the transport of polyamines and the activity and degradation of ornithine decarboxylase (ODC), the rate-limiting enzyme in polyamine biosynthesis. AZs themselves are negatively regulated by antizyme inhibitor (AZIN). AZIN functions as a positive regulator of cellular polyamine homeostasis, which by binding to AZs reactivates ODC and induces the uptake of polyamines. In various pathological conditions, including cancer, polyamine levels are misregulated. Polyamine homeostasis has therefore become an attractive target for therapeutic interventions and it is thus crucial to characterize the molecular basis underlying the homeostatic regulation. A novel human AZIN-resembling protein was previously identified in our group. The purpose of this study was to elucidate the function and distribution of this protein, termed as an antizyme inhibitor 2 (AZIN2). According to my results, AZIN2 functions as a novel regulator of polyamine homeostasis. It shows no enzymatic activity, but instead it binds AZs and negates their activity, which subsequently leads to reactivation of ODC and inhibition of its degradation. Expression of AZIN2 is restricted to terminally differentiated cells, such as mast cells (MC) and neurosecretory cells. In these actively secreting cell types, AZIN2 localizes to subcellular vesicles or granules where its function is important for the vesicle-mediated secretion. In MCs, AZIN2 localizes to the serotonin-containing subset of MC granules, and its expression is coupled to MC activation. The functional role of polyamines as potential mediators of MC activity was also investigated, and it was observed that the secretion of serotonin is selectively dependent on activation of ODC. In neurosecretory cells, AZIN2-positive vesicles localize mainly to the trans-Golgi network (TGN). Depletion of AZIN2 or cellular polyamines causes selective fragmentation of the TGN and retards secretion of proteins. Since addition of exogenous polyamines reverses these effects, the data indicate that AZIN2 and its downstream effectors, polyamines, are functionally implicated in the regulation of secretory vesicle transport. My studies therefore reveal a novel function for polyamines as modulators of both constitutive and regulated secretion. Based on the results, I propose that the role of AZIN2 is to act as a local in situ activator of polyamine biosynthesis.

Characterization of the TRIM37 gene and mutations underlying mulibrey nanism

Mulibrey nanism is a hereditary developmental disorder, characterized by prenatal onset growth failure without postnatal catch-up growth, distinctive craniofacial features, progressive cardiopathy and failure of sexual maturation. In addition, the patients develop insulin resistance syndrome and type 2 diabetes and they have an increased risk of developing tumors. The TRIM37 gene that underlies mulibrey nanism encodes for a member of the tripartite motif (TRIM) protein family. The physiological function of TRIM37 and the pathogenetic mechanisms leading from TRIM37 dysfunction to the mulibrey nanism phenotype are unknown. However, TRIM37 localizes at least partially to peroxisomes, and possesses ubiquitin E3-ligase activity. Thus, it may mediate ubiquitin dependent protein degradation, suggesting that accumulation of yet unknown substrate proteins may underlie the disease pathogenesis. In this study, the TRIM37 gene was characterized in detail. A transcription initiation window, with several separate transcription start sites, was identified and the putative promoter region immediately upstream from the transcription initiation window was shown to possess basal promoter activity. Further, several alternative splice variants of the gene were identified, including a highly expressed testis specific variant, encoding for an identical protein product with the main transcript. Expression of TRIM37 mRNA was detected in several different tissues, with highest expression seen in testis and in brain, when the expression patterns of the two major transcripts in different human tissues were studied by quantitative real-time PCR. Several mulibrey nanism patients were studied and thirteen novel mutations in TRIM37 were found, including three mutations (p.Gly322Val, p.Cys109Ser, p.Glu271_Ser287), that are likely to express mutant TRIM37 proteins. These mutations were further shown to alter the subcellular localization of the mutant proteins. Most of the mulibrey nanism associated mutations however, lead to premature termination codons and degradation of mRNA. All the TRIM37 mutations identified to date predict loss-of-function alleles, and thus no phenotype-genotype correlation is seen among the patients. In order to understand the pathogenetic mechanisms underlying mulibrey nanism, an animal model for the disorder is needed. For the development of a Trim37 knock-out mouse, the mouse Trim37 gene was characterized. Alternative splice variants, were identified, including a testis specific variant predicting a longer protein product. Further, a strictly tissue and cell-specific pattern of Trim37 expression was observed in developing and adult mouse tissues, when studied by immunohistochemical methods. This distribution of Trim37 expression in mouse tissues is in agreement with the clinical findings in human mulibrey nanism patients. This thesis work gives new tools for the diagnostics of mulibrey nanism as well as for studying the molecular pathogenesis behind this interesting disorder.