Of tolerance in mice and men : studies in APECED and Aire

Autoimmune diseases affect 5 % of the population and come in many forms, such as diabetes, rheumatoid arthritis and MS. However, how and why autoimmune diseases arise are not yet fully resolved. In this thesis, the onset of autoimmunity was investigated using both patient samples and a mouse model of autoimmunity. Autoimmune diseases are usually complex, due to a number of different causative genes and environmental factors. However, a few monogenic autoimmune diseases have been described, which are caused by mutations in only one gene per disease. One of such disease is called APECED (autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy) and is enriched in the Finnish population. The causative gene behind APECED is named AIRE from AutoImmune REgulator. How malfunction of just one gene product can cause the multitude of disease components found in APECED is not yet resolved. This thesis sought out to find out more about the functions of AIRE, in order to reveal why APECED and other autoimmune diseases arise and what goes wrong? Usually, immune cells are taught to distinguish between self and non-self during their development. That way, immune cells can fight off bacteria and microbes while leaving the tissues and organs of the host organism itself unharmed. In APECED, the development of immune cells called αβ T cells is incomplete. The cells are not able to fully distinguish between self and non-self. This leads to autodestruction of self tissues and autoimmune disease. One of the achievements of this thesis was the finding that the development of another set of T cells called γδ T cells is not affected by AIRE in mice or in men. Instead, we found that another type of immune cell important in tolerance, called the dendritic cell is defective in APECED patients and is not able to respond to microbial stimulus in a normal fashion. Finally, we studied Aire-deficient mice and found that autoantibodies expressed in the mice were not targeted against the same molecules as those found in APECED patients. This indicates differences in the autoimmune pathology in mice and men. More work is still required before we understand the mechanisms of tolerance and autoimmunity well enough to be able to cure APECED, let alone the more complex autoimmune diseases. Yet altogether, the findings of this thesis work bring us one step closer to finding out why and how APECED and common autoimmune diseases arise.

To reactivate or not to reactivate : Control of KSHV lytic replication is essential for apoptosis in response to p53 restoration

Kaposi's sarcoma herpesvirus (KSHV) is an oncogenic human virus and the causative agent of three human malignancies: Kaposi's sarcoma (KS), Multicentric Castleman's Disease (MCD), and primary effusion lymphoma (PEL). In tumors, KSHV establishes latent infection during which it produces no infectious particles. Latently infected cells can enter the lytic replication cycle, and upon provision of appropriate cellular signals, produce progeny virus. PEL, commonly described in patients with AIDS, represents a diffuse large-cell non-Hodgkin's lymphoma, with median survival time less than six months after diagnosis. As tumor suppressor gene TP53 mutations occur rarely in PEL, the aim of this thesis was to investigate whether non-genotoxic activation of the p53 pathway can eradicate malignant PEL cells. This thesis demonstrates that Nutlin-3, a small-molecule inhibitor of the p53-MDM2 interaction, efficiently restored p53 function in PEL cells, leading to cell cycle arrest and massive apoptosis. Furthermore, we found that KSHV infection activated DNA damage signaling, rendering the cells more sensitive to p53-dependent cell death. We also showed in vivo the therapeutic potential of p53 restoration that led to regression of subcutaneous and intraperitoneal PEL tumor xenografts without adversely affecting normal cells. Importantly, we demonstrated that in a small subset of intraperitoneal PEL tumors, spontaneous induction of viral reactivation dramatically impaired Nutlin-3-induced p53-mediated apoptosis. Accordingly, we found that elevated KSHV lytic transcripts correlated with PEL tumor burden in animals and that inhibition of viral reactivation in vitro restored cytotoxic activity of a small-molecule inhibitor of the p53-MDM2 interaction. Latency provides a unique opportunity for KSHV to escape host immune surveillance and to establish persistent infections. However, to maintain viral reservoirs and spread to other hosts, KSHV must be reactivated from latency and enter into the lytic growth phase. We showed that phosphorylation of nucleolar phosphoprotein nucleophosmin (NPM) by viral cyclin-CDK6 is critical for establishment and maintenance of the KSHV latency. In short, this study provides evidence that the switch between latent phase and lytic replication is a critical step that determines the outcome of viral infection and the pathogenesis of KSHV-induced malignancies. Our data may thus contribute to development of novel targeted therapies for intervention and treatment of KSHV-associated cancers.

Genetic and Epigenetic Characterization of Colorectal and Endometrial Cancer

Colorectal cancer is one of the three most common cancers today, for both men and women. Approximately 90% of the cases are sporadic while the remaining 10% is hereditary. Among this 10% is hereditary nonpolyposis colorectal cancer (HNPCC), an autosomal dominant disease, accounting for up to 13% of these cases. HNPCC is associated with germline mutations in four mismatch repair (MMR) genes, MLH1, MSH2, MSH6, and PMS2, and is characterized by a familial accumulation of endometrial, gastric, urological, and ovarian tumors, in addition to colorectal cancer. An important etiological characteristic of HNPCC is the presence of microsatellite instability (MSI), caused by mutations of the MMR genes. Approximately 15% of sporadic cases share the MSI+ trait. Colon cancer is believed to be a consequence of an accumulation of mutations in tumor suppressor genes and oncogenes, eventually resulting in tumor development. This phenomena is accelerated in HNPCC due the presence of an inherited mutation in the MMR genes, accounting for one of the two hits proposed to be needed by Knudson (1971) in order for the manifestation of the MSI phenotype. MMR alterations alone, however, do not occur in the majority of sporadic colon cancers, prompting searches for other mechanisms. One such mechanism found to play a role in colon cancer development was DNA methylation, which is known to play a role in MLH1 inactivation. Our objective was clarification of mechanisms associated with tumor development in both HNPCC and sporadic colorectal cancer in relation to tumorigenic mechanisms. Of particular interest were underlying mechanisms of MSI in sporadic colorectal cancers, with attention to DNA methylation changes and their correlation to MSI. Of additional interest were the genetic and epigenetic events leading to the HNPCC tumor spectrum, chiefly colon and endometrial cancers, in regards to what extent the somatic changes in target tissue explained this phenomenon. We made a number of important findings pertaining to these questions. First, MSI tumor development differs epigenetically from stable tumor development, possibly underlying developmental pathway differences. Additionally, while epigenetic modification, principally DNA methylation, is a major mechanism in sporadic MSI colorectal cancer MLH1 inactivation it does not play a significant role in HNPCC tumors with germline MLH1 mutations. This is possibly an explanation for tumorigenic pathways and clinicopathological characteristic differences between sporadic and hereditary MSI colorectal cancers. Finally, despite indistinguishable genetic predisposition for endometrial and colorectal cancers, instability profiles highlighting organ-specific differences, may be important HNPCC tumor spectrum determinants.

Cellular Cholesterol Accumulation and Egress from Endosomal Compartments

One of the most important factors determining the development of atherosclerosis is the amount of LDL particles in the circulation. In general, LDL particles are clinically regarded as “bad cholesterol” since these particles get entrapped within the vascular wall, leading to atherosclerosis. Circulating HDL particles are conversely regarded as “good cholesterol” because of their ability to transport cholesterol from peripheral tissues to the liver for secretion as bile salts. Once inside the artery wall LDL particles are engulfed by macrophages, resulting in macrophage foam cells. If the macrophage foam cells are not able to efflux the cholesterol back into the bloodstream, the excessive cholesterol ultimately leads to cell death, and the deposition of cellular debris within the atherosclerotic lesion. The cells ability to secrete cholesterol is mainly dependent on the ABCA1 transporter (ATP-binding cassette transporter A1) which transfers cellular cholesterol to extracellular apoA-I (apolipoprotein A-I) particles, leading to the generation of nascent HDL particles. The process of atherosclerotic plaque development is therefore to a large extent a cellular one, in which the capacity of the macrophages in handling the excessive cholesterol load determines the progression of lesion development. In this work we have studied the cellular mechanisms that regulate the trafficking of LDL-derived cholesterol from endosomal compartments to other parts of the cell. As a basis for the study we have utilized cells from patients with Niemann-Pick type C disease, a genetic disorder resulting from mutations in the NPC1 and NPC2 genes. In these cells, cholesterol is entrapped within the endosomal compartment, and is not available for efflux. By identifying proteins that bypass the cholesterol trafficking defect, we were able to identify the small GTPase Rab8 as an important protein involved in ABCA1 dependent cholesterol efflux. In the study, we show that Rab8 regulates cholesterol efflux in human macrophages by facilitating intracellular cholesterol transport, as well as by regulating the plasma membrane availability of ABCA1. Collectively, these results give new insight in to atherosclerotic lesion development and intracellular cholesterol processing.

Molecular Mechanisms of Androgen Receptor Interactions

The androgen receptor (AR) mediates the effects of the male sex-steroid hormones (androgens), testosterone and 5?-dihydrotestosterone. Androgens are critical in the development and maintenance of male sexual characteristics. AR is a member of the steroid receptor ligand-inducible transcription factor family. The steroid receptor family is a subgroup of the nuclear receptor superfamily that also includes receptors for the active forms of vitamin A, vitamin D3, and thyroid hormones. Like all nuclear receptors, AR has a conserved modular structure consisting of a non-conserved amino-terminal domain (NTD), containing the intrinsic activation function 1, a highly conserved DNA-binding domain, and a conserved ligand-binding domain (LBD) that harbors the activation function 2. Each of these domains plays an important role in receptor function and signaling, either via intra- and inter-receptor interactions, interactions with specific DNA sequences, termed hormone response elements, or via functional interactions with domain-specific proteins, termed coregulators (coactivators and corepressors). Upon binding androgens, AR acquires a new conformational state, translocates to the nucleus, binds to androgen response elements, homodimerizes and recruits sequence-specific coregulatory factors and the basal transcription machinery. This set of events is required to activate gene transcription (expression). Gene transcription is a strictly modulated process that governs cell growth, cell homeostasis, cell function and cell death. Disruptions of AR transcriptional activity caused by receptor mutations and/or altered coregulator interactions are linked to a wide spectrum of androgen insensitivity syndromes, and to the pathogenesis of prostate cancer (CaP). The treatment of CaP usually involves androgen depletion therapy (ADT). ADT achieves significant clinical responses during the early stages of the disease. However, under the selective pressure of androgen withdrawal, androgen-dependent CaP can progress to an androgen-independent CaP. Androgen-independent CaP is invariably a more aggressive and untreatable form of the disease. Advancing our understanding of the molecular mechanisms behind the switch in androgen-dependency would improve our success of treating CaP and other AR related illnesses. This study evaluates how clinically identified AR mutations affect the receptor s transcriptional activity. We reveal that a potential molecular abnormality in androgen insensitivity syndrome and CaP patients is caused by disruptions of the important intra-receptor NTD/LBD interaction. We demonstrate that the same AR LBD mutations can also disrupt the recruitment of the p160 coactivator protein GRIP1. Our investigations reveal that 30% of patients with advanced, untreated local CaP have somatic mutations that may lead to increases in AR activity. We report that somatic mutations that activate AR may lead to early relapse in ADT. Our results demonstrate that the types of ADT a CaP patient receives may cause a clustering of mutations to a particular region of the receptor. Furthermore, the mutations that arise before and during ADT do not always result in a receptor that is more active, indicating that coregulator interactions play a pivotal role in the progression of androgen-independent CaP. To improve CaP therapy, it is necessary to identify critical coregulators of AR. We screened a HeLa cell cDNA library and identified small carboxyl-terminal domain phosphatase 2 (SCP2). SCP2 is a protein phosphatase that directly interacts with the AR NTD and represses AR activity. We demonstrated that reducing the endogenous cellular levels of SCP2 causes more AR to load on to the prostate specific antigen (PSA) gene promoter and enhancer regions. Additionally, under the same conditions, more RNA polymerase II was recruited to the PSA promoter region and overall there was an increase in androgen-dependent transcription of the PSA gene, revealing that SCP2 could play a role in the pathogenesis of CaP.

LKB1 tumor suppression in Peutz-Jeghers Syndrome

Kasvainten, ajatellaan syntyvän yksittäisen solun perimän mutaatioista, jonka seurauksena tuon solun kasvu häiriintyy. Ruoansulatuskanavan polyyppien syntyä käytetään usein mallina siitä, miten nämä epiteelisoluun kerääntyvät mutaatiot aiheuttavat asteittain pahenevan kasvuhäiriön. Peutz–Jeghersin oireyhtymä (PJS) on perinnöllinen polypoosisyndrooma, jossa oireita aiheuttavat erityisesti maha-suolikanavan hamartomatoottiset polyypit. Noin puolella PJS potilaista havaitaan mutaatioita LKB1 kasvunrajoite geenissä. Hiirille joilta toinen Lkb1 alleeli on poistettu (Lkb1+/-) kehittyy PJS-tyypin maha-suolikanavan polyyppeja, joissa on epiteelin liikakasvun lisäksi merkittävä sileälihaskomponentti, aivan kuten PJS polyypeissa. Kuten myös muissa ruoansulatuskanavan polypooseissa, sekä PJS että hiirten polyypeissa Cyclo-oxygenaasi-2:n (COX-2) määrä on usein kohonnut. PJS-polyyppien kehittymisen molekulaarinen mekanismi on kuitenkin selvittämättä. Koska vain osa PJS potilaista kantaa LKB1 mutaatioita, mutaatiot jossakin toisessa lokuksessa saattaisivat selittää osan PJS tapauksista. Jotta PJS:n geneettinen tausta selviäisi, seulottiin kolmen LKB1:n kanssa interaktoivan proteiinin (BRG1, STRADα ja MO25α) geenit PJS potilaista joilla ei ole havaittu LKB1 mutaatioita. Yhdessäkään tutkituista geeneistä ei havaittu tautia aiheuttavia mutaatioita. Näiden kolmen geenin pois sulkeminen, ja uusien menetelmien ansiosta kasvanut havaittujen Lkb1 mutaatioden määrä viittaavat LKB1:n olevan useimpien PJS tapausten taustalla. COX-2:n estäjien käyttö on tehokkaasti vähentänyt polyyppien määrää familiaarisessa adenomatoottisessa polypoosissa. Tästä johtuen COX-2:n eston tehokkuutta tutkittiin PJS polypoosissa. PJS-tyypin polypoosin havaittin pienenevän merkittävästi Lkb1+/- hiirissä, joilta oli lisäksi poistettu toinen tai molemmat COX-2:n alleeleista. Lisäksi farmakologinen COX-2:n esto Celecoxib:lla vähensi polypoosia tehokkaasti. Näin ollen COX-2:n eston tehokkuutta tutkittiin seuraavaksi PJS potilaissa. Kuuden kuukauden Celecoxib hoidon jälkeen polypoosin havaittiin vähentyneen merkittävästi osalla potilaista (2/6). Nämä tulokset osoittavat COX-2:n roolin PJS-polyyppien kehityksessä, ja viittaavat COX-2:n eston vähentävän polypoosia. Kasvunrajoitegeenin klassisen määritelmän mukaan kasvaimen kehitys vaatii perinnöllisen mutaation lisäksi geenin toisenkin alleelin mutaation, mutta PJS-polyyppien häiriintyneestä epiteelistä ei kuitenkaan systemaattisesti löydy toista LKB1:n mutaatiota. Havainto johti tutkimukseen, jossa selvitettiin voisiko LKB1:n kasvun rajoitus välittyäkin epäsuorasti tukikudokseksi ajatelluista sileälihassoluista. Tätä tutkittiin kehittämällä poistogeeninen hiirimalli jossa Lkb1 on mutatoitunut vain sileälihassoluissa. Näille hiirille kehittyi polyyppeja, jotka ovat kaikin tavoin PJS-polyyppien kaltaisia. Lkb1:n menettäneiden solujen havaittiin tuottavan vähemmän transformoivaa kasvutekijä beetaa (TGFß), joka aiheutti solujen välisen viestinnän heikentymisen ja mahdollisesti viereisten epiteelisolujen liikakasvun. Vastaava häiriö havaittiin myös PJS-potilaiden polyypeissa, mikä viittaa siihen, että potilaillakin sileälihassolujen häiriö on polyyppien taustalla. Havainto suuntaa täten hoitokohteiden etsintää ja osoittaa että LKB1 toimii kasvunrajoittajana epätyypillisellä tavalla pitäen naapurisolujen kasvun kurissa.

The function of Bmps and Runx2 in normal tooth development and in the pathogenesis of cleidocranial dysplasia

The development of many embryonic organs is regulated by reciprocal and sequential epithelial-mesenchymal interactions. These interactions are mediated by conserved signaling pathways that are reiteratively used. Cleidocranial dysplasia (CCD) is a congenital syndrome where both bone and tooth development is affected. The syndrome is characterized by short stature, abnormal clavicles, general bone dysplasia, and supernumerary teeth. CCD is caused by mutations in RUNX2, a transcription factor that is a key regulator of osteoblast differentiation and bone formation. The first aim of this study was to analyse the expression of a family of key signal molecules, Bone morphogenetic protein (Bmp) at different stages of tooth development. Bmps have a variety of functions and they were originally discovered as signals inducing ectopic bone formation. We performed a comparative in situ hybridisation analysis of the mRNA expression of Bmp2-7 from initiation of tooth development to differentiation of dental hard tissues. The expression patterns indicated that the Bmps signal between the epithelial and mesenchymal tissues during initiation and morphogenesis of tooth development, as well as during the differentiation of odontoblasts and ameloblasts. Furthermore, they are also part of the signalling networks whereby the enamel knot regulates the patterning of tooth cusps. The second aim was to study the role of Runx2 during tooth development and thereby to gain better understanding of the pathogenesis of the tooth phenotype in CCD. We analysed the tooth phenotype of Runx2 knockout mice and examined the patterns and regulation of Runx2 gene expression.. The teeth of wild-type and Runx2 mutant mice were compared by several methods including in situ hybridisation, tissue culture, bead implantation experiments, and epithelial-mesenchymal recombination studies. Phenotypic analysis of Runx2 -/- mutant tooth development showed that teeth failed to advance beyond the bud stage. Runx2 expression was restricted to dental mesenchyme between the bud and early bell stages of tooth development and it was regulated by epithelial signals, in particular Fgfs. We searched for downstream targets of Runx2 by comparative in situ hybridisation analysis. The expression of Fgf3 was downregulated in the mesenchyme of Runx2 -/- teeth. Shh expression was absent from the enamel knot in the lower molars of Runx2 -/- and reduced in the upper molars. In conclusion, these studies showed that Runx2 regulates key epithelial-mesenchymal interactions that control advancing tooth morphogenesis and histodifferentiation of the epithelial enamel organ. In addition, in the upper molars of Runx2 mutants extra buddings occured at the palatal side of the tooth bud. We suggest that Runx2 acts as an inhibitor of successional tooth formation by preventing advancing development of the buds. Accordingly, we propose that RUNX2 haploinsuffiency in humans causes incomplete inhibition of successional tooth formation and as a result supernumerary teeth.

Palladin, a novel microfilament protein

Palladin is a novel actin microfilament associated protein, which together with myotilin and myopalladin forms a novel cytoskeletal IgC2 domain protein family. Whereas the expression of myotilin and myopalladin is limited mainly to striated muscle, palladin is widely expressed in both epithelial and mesenchymal tissues, including heart and the nervous system. Palladin has a complex genetic structure and it is expressed as several different sized and structured splice variants, which also display differences in their expression pattern and interactions. In muscle cells, all the family members localize to the sarcomeric Z-disc, and in non-muscle cells palladin also localizes to the stress-fiber-dense regions, lamellipodia, podosomes and focal adhesions. A common feature of this protein family is the binding to α-actinin, but other interactions are mostly unique to each member. Palladin has been shown to interact with several proteins, including VASP, profilin, Eps8, LASP-1 and LPP. Its domain structure, lack of enzymatic activity and multiple interactions define it as a molecular scaffolding protein, which links together proteins with different functional modalities into large complexes. Palladin has an important role in cytoskeletal regulation, particularly in stress fiber formation and stabilization. This assumption is supported by several experimental results. First, over-expression of palladin in non-muscle cells results in rapid reorganization of the actin cytoskeleton and formation of thick actin bundles. Second, the knock-down of palladin with anti-sense and siRNA techniques or knock-out by genetic methods leads to defective stress fiber formation. Furthermore, palladin is usually up-regulated in situations requiring a highly organized cytoskeleton, such as differentiation of dendritic cells, trophoblasts and myofibroblasts, and activation of astrocytes during glial scar formation. The protein family members have also direct disease linkages; myotilin missense mutations are the cause of LGMD1A and myofibrillar myopathy. Palladin mutations and polymorphisms, on the other hand, have been linked to hereditary pancreatic cancer and myocardial infarction, respectively. In this study we set out to characterize human palladin. We identified several palladin isoforms, studied their tissue distribution and sub-cellular localization. Four novel interaction partners were identified; ezrin, ArgBP2, SPIN90 and Src-kinase.The previously identified interaction between palladin and α-actinin was also characterized in detail. All the identified new binding partners are actin cytoskeleton associated proteins; ezrin links the plasma membrane to the cytoskeleton, ArgBP2 and SPIN90 localize, among other structures, to the lamellipodia and in cardiomyocytes to the Z-disc. Src is a transforming tyrosine kinase, which besides its role in oncogenesis has also important cytoskeletal associations. We also studied palladin in myofibroblasts, which are specialized cells involved in diverse physiological and pathological processes, such as wound healing and tissue fibrosis. We demonstrated that palladin is up-regulated during the differentiation of myofibroblasts in an isoform specific manner, and that this up-regulation is induced by TGF-β via activation of both the SMAD and MAPK signalling cascades. In summary, the results presented here describe the initial characterization of human palladin and offer a basis for further studies.

Novel conditional mouse models for targeted kidney research : Emphasis on the analysis of the biological function of nephrin

Nephrin is a transmembrane protein belonging to the immunoglobulin superfamily and is expressed primarily in the podocytes, which are highly differentiated epithelial cells needed for primary urine formation in the kidney. Mutations leading to nephrin loss abrogate podocyte morphology, and result in massive protein loss into urine and consequent early death in humans carrying specific mutations in this gene. The disease phenotype is closely replicated in respective mouse models. The purpose of this thesis was to generate novel inducible mouse-lines, which allow targeted gene deletion in a time and tissue-specific manner. A proof of principle model for succesful gene therapy for this disease was generated, which allowed podocyte specific transgene replacement to rescue gene deficient mice from perinatal lethality. Furthermore, the phenotypic consequences of nephrin restoration in the kidney and nephrin deficiency in the testis, brain and pancreas in rescued mice were investigated. A novel podocyte-specific construct was achieved by using standard cloning techniques to provide an inducible tool for in vitro and in vivo gene targeting. Using modified constructs and microinjection procedures two novel transgenic mouse-lines were generated. First, a mouse-line with doxycycline inducible expression of Cre recombinase that allows podocyte-specific gene deletion was generated. Second, a mouse-line with doxycycline inducible expression of rat nephrin, which allows podocyte-specific nephrin over-expression was made. Furthermore, it was possible to rescue nephrin deficient mice from perinatal lethality by cross-breeding them with a mouse-line with inducible rat nephrin expression that restored the missing endogenous nephrin only in the kidney after doxycycline treatment. The rescued mice were smaller, infertile, showed genital malformations and developed distinct histological abnormalities in the kidney with an altered molecular composition of the podocytes. Histological changes were also found in the testis, cerebellum and pancreas. The expression of another molecule with limited tissue expression, densin, was localized to the plasma membranes of Sertoli cells in the testis by immunofluorescence staining. Densin may be an essential adherens junction protein between Sertoli cells and developing germ cells and these junctions share similar protein assembly with kidney podocytes. This single, binary conditional construct serves as a cost- and time-efficient tool to increase the understanding of podocyte-specific key proteins in health and disease. The results verified a tightly controlled inducible podocyte-specific transgene expression in vitro and in vivo as expected. These novel mouse-lines with doxycycline inducible Cre recombinase and with rat nephrin expression will be useful for conditional gene targeting of essential podocyte proteins and to study in detail their functions in the adult mice. This is important for future diagnostic and pharmacologic development platforms.

Identification of two novel human neuronal ceroid lipofuscinosis genes

The neuronal ceroid lipofuscinoses (NCLs) are a group of mostly autosomal recessively inherited neurodegenerative disorders. The aim of this thesis was to characterize the molecular genetic bases of these, previously genetically undetermined, NCL forms. Congenital NCL is the most aggressive form of NCLs. Previously, a mutation in the cathepsin D (CTSD) gene was shown to cause congenital NCL in sheep. Based on the close resemblance of the phenotypes between congenital NCLs in sheep and human, CTSD was considered as a potential candidate gene in humans as well. When screened for mutations by sequencing, a homozygous nucleotide duplication creating a premature stop codon was identified in CTSD in one family with congenital NCL. While in vitro the overexpressed truncated mutant protein was stable although inactive, the absence of CTSD staining in brain tissue samples of patients indicated degradation of the mutant CTSD in vivo. A lack of CTSD staining was detected also in another, unrelated family with congenital NCL. These results imply that CTSD deficiency underlies congenital NCL. While initially Turkish vLINCL was considered a distinct genetic entity (CLN7), mutations in the CLN8 gene were later reported to account for the disease in a subset of Turkish patients with vLINCL. To further dissect the genetic basis of the disease, all known NCL genes were screened for homozygosity by haplotype analysis of microsatellite markers and/or sequenced in 13 mainly consanguineous, Turkish vLINCL families. Two novel, family-specific homozygous mutations were identified in the CLN6 gene. In the remaining families, all known NCL loci were excluded. To identify novel gene(s) underlying vLINCL, a genomewide single nucleotide polymorphism scan, homozygosity mapping, and positional candidate gene sequencing were performed in ten of these families. On chromosome 4q28.1-q28.2, a novel major facilitator superfamily domain containing 8 (MFSD8) gene with six family-specific homozygous mutations in vLINCL patients was identified. MFSD8 transcript was shown to be ubiquitously expressed with a complex pattern of alternative splicing. Our results suggest that MFSD8 is a novel lysosomal integral membrane protein which, as a member of the major facilitator superfamily, is predicted to function as a transporter. Identification of MFSD8 emphasizes the genetic heterogeneity of Turkish vLINCL. In families where no MFSD8 mutations were detected, additional NCL-causing genes remain to be identified. The identification of CTSD and MFSD8 increases the number of known human NCL-causing genes to eight, and is an important step towards the complete understanding of the genetic spectrum underlying NCLs. In addition, it is a starting point for dissecting the molecular mechanisms behind the associated NCLs and contributes to the challenging task of understanding the molecular pathology underlying the group of NCL disorders.

Development of regulatory T cells in the human thymus

The role of the immune system is to protect an organism against pathogens while maintaining tolerance against self. T cells are an essential component of the immune system and they develop in the thymus. The AIRE (autoimmune regulator) gene product plays an important role in T cell development, as it promotes expression of peripheral tissue antigens in the thymus. Developing T cells, thymocytes, which recognize self-antigens with high affinity are deleted. However, this deletion process is not perfect and not all autoreactive T cells are destroyed. When the distinction between self and non-self fails, tolerance breaks and the immune system attacks the host s own tissues. This results in autoimmunity. Regulatory T cells contribute to the maintenance of self-tolerance. They can actively suppress the function of autoreactive cells. Several populations of cells with regulatory properties have been described, but the best characterized population is the natural regulatory T cells (Treg cells), which develop in the thymus and express the transcription factor FOXP3. The thymic development of Treg cells in humans is the subject of this thesis. Thymocytes at different developmental stages were analyzed using flow cytometry. The CD4-CD8- double-negative (DN) thymocytes are the earliest T cell precursors in the T cell lineage. My results show that the Treg cell marker FOXP3 is up-regulated already in a subset of these DN thymocytes. FOXP3+ cells were also found among the more mature CD4+CD8+ double-positive (DP) cells and among the CD4+ and CD8+ single-positive (SP) thymocytes. The different developmental stages of the FOXP3+ thymocytes were isolated and their gene expression examined by quantitative PCR. T cell receptor (TCR) repertoire analysis was used to compare these different thymocyte populations. My data show that in humans commitment to the Treg cell lineage is an early event and suggest that the development of Treg cells follows a linear developmental pathway, FOXP3+ DN precursors evolving through the DP stage to become mature CD4+ Treg cells. Most T cells have only one kind of TCR on their cell surface, but a small fraction of cells expresses two different TCRs. My results show that the expression of two different TCRs is enriched among Treg cells. Furthermore, both receptors were capable of transmitting signals when bound by a ligand. By extrapolating flow cytometric data, it was estimated that the majority of peripheral blood Treg cells are indeed dual-specific. The high frequency of dual-specific cells among human Treg cells suggests that dual-specificity has a role in directing these cells to the Treg cell lineage. It is known that both genetic predisposition and environmental factors influence the development of autoimmunity. It is also known that the dysfunction or absence of Treg cells leads to the development of autoimmune manifestations. APECED (autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy) is a rare monogenic autoimmune disease, caused by mutations in the AIRE gene. In the absence of AIRE gene product, deletion of self-specific T cells is presumably disturbed and autoreactive T cells escape to the periphery. I examined whether Treg cells are also affected in APECED. I found that the frequency of FOXP3+ Treg cells and the level of FOXP3 expression were significantly lower in APECED patients than in controls. Additionally, when studied in cell cultures, the suppressive capacity of the patients' Treg cells was impaired. Additionally, repertoire analysis showed that the TCR repertoire of Treg cells was altered. These results suggest that AIRE contributes to the development of Treg cells in humans and the selection of Treg cells is impaired in APECED patients. In conclusion, my thesis elucidates the developmental pathway of Treg cells in humans. The differentiation of Tregs begins early during thymic development and both the cells dual-specificity and AIRE probably affect the final commitment of Treg cells.

Molecular Genetics of Lactase Deficiencies

Congenital lactase deficiency (CLD) (MIM 223000) is a rare autosomal recessive gastrointestinal disorder characterized by watery diarrhea in infants fed with breast milk or other lactose-containing formulas. The CLD locus was previously assigned by linkage and linkage disequilibrium analyses on 2q21 in 19 Finnish families. In this study, the molecular background of this disorder is reported. The CLD locus was refined in 32 CLD patients in 24 families by using microsatellite and single nucleotide polymorphism (SNP) haplotypes. Mutation analyses were performed by direct sequencing. We identified 5 distinct mutations in the lactase (LCT) gene, encoding the enzyme that hydrolyzes lactose in the intestinal lumen. These findings facilitate genetic testing of CLD in clinical practice and enable genetic counseling. The present data also provide the basis for detailed characterization of the molecular pathogenesis of this disorder. Adult-type hypolactasia (MIM 223100) (lactase non-persistence, lactose intolerance) is an autosomal recessive gastrointestinal condition that is a result of a decline in the activity of lactase in the intestinal lumen after weaning. Adult-type hypolactasia is considered to be a normal phenomenon among mammals and symptoms are remarkably milder than experienced in CLD. Recently, a variant C/T-13910 was shown to associate with the adult-type hypolactasia trait, locating 13.9 kb upstream of the LCT gene. In this study, the functional significance of the C/T-13910 variant was determined by studying the LCT mRNA levels in intestinal biopsy samples in children and adults with different genotypes. RT-PCR followed by solid-phase minisequencing was applied to determine the relative expression levels of the LCT alleles using an informative SNP located in exon 1. In children, the C-13910 allele was observed to be downregulated after five years of age in parallel with lactase enzyme activity. The expression of the LCT mRNA in the intestinal mucosa in individuals with the T-13910 A-22018 alleles was 11.5 times higher than that found in individuals with the C-13910, G-22018 alleles. These findings suggest that the C/T-13910 associated with adult-type hypolactasia is associated with the transcriptional regulation of the LCT gene. The presence of the T-13910 A-22018 allele also showed significant elevation lactase activity. Galactose, the hydrolysing product of the milk sugar lactose, has been hypothesized to be poisonous to ovarian epithelial cells. Hence, consumption of dairy products and lactase persistence has been proposed to be a risk factor for ovarian carcinoma. To investigate whether lactase persistence is related to the risk of ovarian carcinoma the C/T-13910 genotype was determined in a cohort of 782 women with ovarian carcinoma 1331 individuals serving as controls. Lactase persistence did not associate significantly with the risk for ovarian carcinoma in the Finnish, in the Polish or in the Swedish populations. The findings do not support the hypothesis that lactase persistence increases the risk for ovarian carcinoma.

Identification of the Meckel syndrome gene (MKS1) exposes a novel ciliopathy

Meckel syndrome (MKS, MIM 249000) is an autosomal recessive developmental disorder causing death in utero or shortly after birth. The hallmarks of the disease are cystic kidney dysplasia and fibrotic changes of the liver, occipital encephalocele with or without hydrocephalus and polydactyly. Other anomalies frequently seen in the patients are incomplete development of the male genitalia, club feet and cleft lip or palate. The clinical picture has been well characterized in the literature while the molecular pathology underlying the disease has remained unclear until now. In this study we identified the first MKS gene by utilizing the disease haplotypes in Finnish MKS families linked to the MKS1 locus on chromosome 17q23 (MKS1) locus. Subsequently, the genetic heterogeneity of MKS was established in the Finnish families. Mutations in at least four different genes can cause MKS. These genes have been mapped to the chromosomes 17q23 (MKS1), 11q13 (MKS2), 8q22 (MKS3) and 9q33 (MKS4). Two of these genes have been identified so far: The MKS1 gene (this work) and the MKS3 gene. The identified MKS1 gene was initially a novel human gene which is conserved among species. We found three different MKS mutations, one of them being the Finnish founder mutation. The information available from MKS1 orthologs in other species convinced us that the MKS1 gene is required for normal ciliogenesis. Defects of the cilial system in other human diseases and model organisms actually cause phenotypic features similar to those seen in MKS patients. The MKS3 (TMEM67) gene encodes a transmembrane protein and the gene maps to the syntenic Wpk locus in the rat, which is a model with polycystic kidney disease, agenesis of the corpus callosum and hydrocephalus. The available information from these two genes suggest that MKS1 would encode a structural component of the centriole required for normal ciliary functions, and MKS3 would be a transmembrane component most likely required for normal ciliary sensory signaling. The MKS4 locus was localized to chromosme 9q32-33 in this study by using an inbred Finnish family with two affected and two healthy children. This fourth locus contains TRIM32 gene, which is associated to another well characterized human ciliopathy, Bardet Biedl syndrome (BBS). Future studies should identify the MKS4 gene on chromosome 9q and confirm if there are more than two genes causing MKS Finnish families. The research on critical signaling pathways in organogenesis have shown that both Wnt and Hedgehog pathways are dependent on functional cilia. The MKS gene products will serve as excellent model molecules for more detailed studies of the functional role of cilia in organogenesis in more detail.

Molecular mechanisms of cancer predisposition in HNPCC/Lynch syndrome

Hereditary non-polyposis colorectal carcinoma (HNPCC; Lynch syndrome) is among the most common hereditary cancers in man and a model of cancers arising through deficient DNA mismatch repair (MMR). It is inherited in a dominant manner with predisposing germline mutations in the MMR genes, mainly MLH1, MSH2, MSH6 and PMS2. Both copies of the MMR gene need to be inactivated for cancer development. Since Lynch syndrome family members are born with one defective copy of one of the MMR genes in their germline, they only need to acquire a so called second hit to inactivate the MMR gene. Hence, they usually develop cancer at an early age. MMR gene inactivation leads to accumulation of mutations particularly in short repeat tracts, known as microsatellites, causing microsatellite instability (MSI). MSI is the hallmark of Lynch syndrome tumors, but is present in approximately 15% of sporadic tumors as well. There are several possible mechanisms of somatic inactivation (i.e. the second hit ) of MMR genes, for instance deletion of the wild-type copy, leading to loss of heterozygosity (LOH), methylation of promoter regions necessary for gene transcription, or mitotic recombination or gene conversion. In the Lynch syndrome tumors carrying germline mutations in the MMR gene, LOH was found to be the most frequent mechanism of somatic inactivation in the present study. We also studied MLH1/MSH2 deletion carriers and found that somatic mutations identical to the ones in the germline occurred frequently in colorectal cancers and were also present in extracolonic Lynch syndrome-associated tumors. Chromosome-specific marker analysis implied that gene conversion, rather than mitotic recombination or deletion of the respective gene locus accounted for wild-type inactivation. Lynch syndrome patients are predisposed to certain types of cancers, the most common ones being colorectal, endometrial and gastric cancer. Gastric cancer and uroepithelial tumors of bladder and ureter were observed to be true Lynch syndrome tumors with MMR deficiency as the driving force of tumorigenesis. Brain tumors and kidney carcinoma, on the other hand, were mostly MSS, implying the possibility of alternative routes of tumor development. These results present possible implications in clinical cancer surveillance. In about one-third of families suspected of Lynch syndrome, mutations in MMR genes are not found, and we therefore looked for alternative mechanisms of predisposition. According to our results, large genomic deletions, mainly in MSH2, and germline epimutations in MLH1, together explain a significant fraction of point mutation-negative families suspected of Lynch syndrome and are associated with characteristic clinical and family features. Our findings have important implications in the diagnosis and management of Lynch syndrome families.

Rab8 and Rab8-interacting proteins as players in cell polarization

Rab8 and its interacting proteins as regulators of cell polarization During the development of a multi-cellular organism, progenitor cells have to divide and migrate appropriately as well as organize their differentiation with one another, in order to produce a viable embryo. To divide, differentiate and migrate cells have to undergo polarization, a process where internal and external components such as actin, microtubules and adhesion receptors are reorganized to produce a cell that is asymmetric, with functionally different surfaces. Also in the adult organism there is a continuous need for these processes, as cells need to migrate in response to tissue damage and to fight infection. Improper regulation of cell proliferation and migration can conversely lead to disease such as cancer. GTP-binding proteins function as molecular switches by cycling between a GTP-bound (active) conformation and a GDP-bound (inactive) conformation. The Ras super-family of small GTPases are found in all eukaryotic cells. They can be functionally divided into five subfamilies. The Ras family members mainly regulate gene expression, controlling cell proliferation and differentiation. Ras was in fact the first human oncogene to be characterized, and as much as 30% of all human tumors may be directly or indirectly caused by mutations of Ras molecules The Rho family members mainly regulate cytoskeletal reorganization. Arf proteins are known to regulate vesicle budding and Rab proteins regulate vesicular transport. Ran regulates nuclear transport as well as microtubule organization during mitosis. The focus of the thesis of Katarina Hattula, is on Rab8, a small GTPase of the Rab family. Activated Rab8 has previously been shown to induce the formation of new surface extensions, reorganizing both actin and microtubules, and to have a role in directed membrane transport to cell surfaces. However, the exact membrane route it regulates has remained elusive. In the thesis three novel interactors of Rab8 are presented. Rabin8 is a Rab8-specific GEF that localizes to vesicles where it presumably recruits and activates its target Rab8. Its expression in cells leads to remodelling of actin and the formation of polarized cell surface domains. Optineurin, known to be associated with a leading cause of blindness in humans (open-angle glaucoma), is shown to interact specifically with GTP-bound Rab8. Rab8 binds to an amino-terminal region and interestingly, the Huntingtin protein binds a carboxy-terminal region of optineurin. (Aberrant Huntingtin protein is known to be the cause Huntington s disease in humans.) Co-expression of Huntingtin and optineurin enhanced the recruitment of Huntingtin to Rab8-positive vesicular structures. Furthermore, optineurin promoted cell polarization in a similar way to Rab8. A third novel interactor of Rab8 presented in this thesis is JFC1, a member of the synaptogamin-like protein (Slp) family. JFC1 interacts with Rab8 specifically in its GTP-bound form, co-localizes with endogenous Rab8 on tubular and vesicular structures, and is probably involved in controlling Rab8 membrane dynamics. Rab8 is in this thesis work clearly shown to have a strong effect on cell shape. Blocking Rab8 activity by expression of Rab8 RNAi, or by expressing the dominant negative Rab8 (T22N) mutant leads to loss of cell polarity. Conversely, cells expressing the constitutively active Rab8 (Q67L) mutant exhibit a strongly polarized phenotype. Experiments in live cells show that Rab8 is associated with macropinosomes generated at ruffling areas of the membrane. These macropinosomes fuse with or transform into tubules that move toward the cell centre, from where they are recycled back to the leading edge to participate in protrusion formation. The biogenesis of these tubules is shown to be dependent on both actin and microtubule dynamics. The Rab8-specific membrane route studied contained several markers known to be internalized and recycled (1 integrin, transferrin, transferrin receptor, cholera toxin B subunit (CTxB), and major histocompatibility complex class I protein (MHCI)). Co-expression studies revealed that Rab8 localization overlaps with that of Rab11 and Arf6. Rab8 is furthermore clearly functionally linked to Arf6. The data presented in this thesis strongly suggests a role for Rab8 as a regulator for a recycling compartment, which is involved in providing structural and regulatory components to the leading edge to participate in protrusion formation.