Role of γ1 Laminin and Its KDI Peptide in the Central Nervous System

Since the 1980 s, laminin-1 has been linked to regeneration of the central nervous system (CNS) and promotion of neuronal migration and axon guidance during CNS development. In this thesis, we clarify the role of γ1 laminin and its KDI tripeptide in development of human embryonic spinal cord, in regeneration of adult rat spinal cord injury (SCI), in kainic acid-induced neuronal death, and in the spinal cord tissue of amyotrophic lateral sclerosis (ALS). We demonstrated that γ1 laminin together with α1, β1, and β3 laminins localize at the floor plate region in human embryonic spinal cord. This localization of γ1 laminin is in spatial and temporal correlation with development of the spinal cord and indicates that γ1 laminin may participate in commissural axon guidance during the embryonic development of the human CNS. With in vitro studies using the Matrigel culture system, we demonstrated that the KDI tripeptide of γ1 laminin provides a chemotrophic guidance cue for neurites of the human embryonic dorsal spinal cord, verifying the functional ability of γ1 laminin to guide commissural axons. Results from our experimental SCI model demonstrate that the KDI tripeptide enhanced functional recovery and promoted neurite outgrowth across the mechanically injured area in the adult rat spinal cord. Furthermore, our findings indicate that the KDI tripeptide as a non-competitive inhibitor of the ionotropic glutamate receptors can provide when administered in adequate concentrations an effective method to protect neurons against glutamate-induced excitotoxic cell death. Human postmortem samples were used to study motor neuron disease, ALS (IV), and the study revealed that in human ALS spinal cord, γ1 laminin was selectively over-expressed by reactive astrocytes, and that this over-expression may correlate with disease severity. The multiple ways by which γ1 laminin and its KDI tripeptide provide neurotrophic protection and enhance neuronal viability suggest that the over-expression of γ1 laminin may be a glial attempt to provide protection for neurons against ALS pathology. The KDI tripeptide is effective therapeutically thus far in animal models only. However, because KDI containing γ1 laminin exists naturally in the human CNS, KDI therapies are unlikely to be toxic or allergenic. Results from our animal models are encouraging, with no toxic side-effects detected even at high concentrations, but the ultimate confirmation can be achieved only after clinical trials. More research is still needed until the KDI tripeptide is refined into a clinically applicable method to treat various neurological disorders.

Pathophysiology of Congenital Nephrotic Syndrome of the Finnish type

Congenital nephrotic syndrome of the Finnish type (NPHS1) is an autosomal recessive disease which is highly enriched in the Finnish population. It is caused by mutations in the NPHS1 gene encoding for nephrin, which is a major component of the glomerular filtration barrier in the kidney. Patients with NPHS1 have heavy proteinuria and nephrotic syndrome (NS) from birth and develop renal fibrosis in early childhood. Renal transplantation (TX) is the only curative treatment for NPHS1. These patients form the largest group of pediatric kidney transplant children in our country. The NPHS1 kidneys are removed in infancy and they serve as an excellent human material for studies of the pathophysiology of proteinuric kidney diseases. Sustained proteinuria is a major factor leading to end-stage renal failure and understanding this process is crucial for nephrology. In this study we investigated the glomerular and tubulointerstitial changes that occur in the NPHS1 kidneys during infancy as well as the expression of nephrin in non-renal tissues. We also studied the pathology and management of recurrent proteinuria in kidney grafts transplanted to NPHS1 children. Severe renal lesions evolved in patients with NPHS1 during the first months of life. Glomerular sclerosis developed through progressive mesangial sclerosis, and capillary obliteration was an early consequence of this process. Shrinkage of the glomerular tuft was common, whereas occlusion of tubular opening or protrusion of the glomerular tuft into subepithelial space or through the Bowman's capsule were not detected. Few inflammatory cells were detected in the mesangial area. The glomerular epithelial cells (podocytes) showed severe ultrastructural changes and hypertrophy. Podocyte proliferation and apoptosis were rare, but moderate amounts of podocytes were detached and ended up in the urine. The results showed that endocapillary lesions not extracapillary lesions, as generally believed were important for the sclerotic process in the NPHS1 glomeruli. In the tubulointerstitium, severe lesions developed in NPHS1 kidneys during infancy. Despite heavy proteinuria, tubular epithelial cells (TECs) did not show transition into myofibroblasts. The most abundant chemokines in NPHS1 tissue were neutrophil activating protein-2 (NAP-2), macrophage inhibiting factor (MIF), and monocyte chemoattractant protein-1 (MCP-1). Interstitial inflammation and fibrosis were first detected in the paraglomerular areas and the most abundant inflammatory cells were monocytes/macrophages. Arteries and arterioles showed intimal hypertrophy, but the pericapillary microvasculature remained quite normal. However, excessive oxidative stress was evident in NPHS1 kidneys. The results indicated that TECs were relatively resistant to the heavy tubular protein load. Nephrin was at first thought to be podocyte specific, but some studies especially in experimental animals have suggested that nephrin might also be expressed in non-renal tissues such as pancreas and central nervous system. The knowledge of nephrin biology is important for the evaluation of nephrin related diseases. In our study, no significant amounts of nephrin protein or mRNA were detected in non-renal tissues of man and pig as studied by immunohistochemistry and in situ hybridization. The phenotype analysis of NPHS1 children, who totally lack nephrin, revealed no marked impairment in the neurological, testicular, or pancreatic function speaking against the idea that nephrin would play an important functional role outside the kidney. The NPHS1 kidneys do not express nephrin and antibodies against this major glomerular filter protein have been observed in NPHS1 children after renal TX most likely as an immune reaction against a novel antigen. These antibodies have been associated with the development of recurrent NS in the kidney graft of NPHS1 patients. In our study, a third of the NPHS1 patients homozygous for Fin-Major mutation developed recurrent NS in the transplanted graft. Re-transplantations were performed to patients who lost their graft due to recurrent NS and heavy proteinuria immediately developed in all cases. While 73% of the patients had detectable serum anti-nephrin antibodies, the kidney biopsy findings were minimal. Introduction of plasma exchange (PE) to the treatment of recurrent nephroses increased the remission rate from 54% to 89%. If remission was achieved, recurrent NS did not significantly deteriorate the long term graft function. In conclusion, the results show that the lack of nephrin in podocyte slit diaphragm in NPHS1 kidneys induces progressive mesangial expansion and glomerular capillary obliteration and inflicts interstitial fibrosis, inflammation, and oxidative stress with surprisingly little involvement of the TECs in this process. Nephrin appears to have no clinical significance outside the kidney. Development of antibodies against nephrin seems to be a major cause of recurrent NS in kidney grafts of NPHS1 patients and combined use of PE and cyclophosphamide markedly improved remission rates.

Mitochondrial helicase Twinkle in mtDNA copy number regulation and a late-onset disease model

Mitochondrial diseases are caused by disturbances of the energy metabolism. The disorders range from severe childhood neurological diseases to muscle diseases of adults. Recently, mitochondrial dysfunction has also been found in Parkinson s disease, diabetes, certain types of cancer and premature aging. Mitochondria are the power plants of the cell but they also participate in the regulation of cell growth, signaling and cell death. Mitochondria have their own genetic material, mtDNA, which contains the genetic instructions for cellular respiration. Single cell may host thousands of mitochondria and several mtDNA molecules may reside inside single mitochondrion. All proteins needed for mtDNA maintenance are, however, encoded by the nuclear genome, and therefore, mutations of the corresponding genes can also cause mitochondrial disease. We have here studied the function of mitochondrial helicase Twinkle. Our research group has previously identified nuclear Twinkle gene mutations underlying an inherited adult-onset disorder, progressive external ophthalmoplegia (PEO). Characteristic for the PEO disease is the accumulation of multiple mtDNA deletions in tissues such as the muscle and brain. In this study, we have shown that Twinkle helicase is essential for mtDNA maintenance and that it is capable of regulating mtDNA copy number. Our results support the role of Twinkle as the mtDNA replication helicase. No cure is available for mitochondrial disease. Good disease models are needed for studies of the cause of disease and its progression and for treatment trials. Such disease model, which replicates the key features of the PEO disease, has been generated in this study. The model allows for careful inspection of how Twinkle mutations lead to mtDNA deletions and further causes the PEO disease. This model will be utilized in a range of studies addressing the delay of the disease onset and progression and in subsequent treatment trials. In conclusion, in this thesis fundamental knowledge of the function of the mitochondrial helicase Twinkle was gained. In addition, the first model for adult-onset mitochondrial disease was generated.

Vaikeavammaisen nuoren aikuistuminen : Yksilöllinen ja erilainen elämänkulku

Transition to adulthood of severely disabled adolescents. Diversity in individual life courses. The focus of this study is to examine the transition to adulthood of severely disabled adolescents as part of their life course. The data for this study were gathered through interviews with nine severely disabled adolescents, who were interviewed several times over a period of eight years. At the beginning of the study the adolescents were between 18 and 24 years old. The informants had severe disabilities manifesting themselves as physical incapacity, cerebral palsy, vision or hearing impairment, neurological disease, or developmental disability. One of the adolescents communicated with symbols. All except one used a wheelchair. As severely disabled adolescents, they received benefits from Kela for persons with severe disabilities, such as the higher-rate or special disability allowance or disability pension, the higher-rate or special pensioners' care allowance, or medical rehabilitation services. The interviews focused on a number of selected themes such as relationships, family, education, work, leisure-time activities, dating, decision-making, independence, happiness, and one s self-image and identity. Data were also derived from interviews with five experts. Two of the experts interviewed were severely disabled themselves. The theoritical foundation of the study lies in perviuos research on the severly disabled, the transition to adulthood and the life course. The method of analysis and interpretation is qualitative and based on interviews with the adolescents. In terms of the analytical process, the focus is on recognizing individual events in the transition process to adulthood and identifying the meanings assigned to them by the adolescents. The narratives also provide a method to shed light on the individuality of the transition. The individual situations of severely disabled adolescents vary, and their disability impacts the range of options available to them as they plan their life course. The medical and social models of disability also have an effect on life courses. Although severely disabled adolescents are able to attain some goals, they remain outsiders in many respects. Key words: Disabled person, severely disabled person, adolescent, transition to adulthood, identity, life course.

Identification of genetic susceptibility loci for migraine

Migraine is the common cause of chronic episodic headache, affecting 12%-15% of the Caucasian population (41 million Europeans and some half a million Finns), and causes considerable loss of quality of life to its sufferers, as well as being linked to increased risk for a wide range of conditions, from depression to stroke. Migraine is the 19th most severe disease in terms of disability-adjusted life years, and 9th among women. It is characterized by attacks of headache accompanied by sensitivity to external stimuli lasting 4-72 hours, and in a third of cases by neurological aura symptoms, such as loss of vision, speech or muscle function. The underlying pathophysiology, including what triggers migraine attacks and why they occur in the first place, is largely unknown. The aim of this study was to identify genetic factors associated with the hereditary susceptibility to migraine, in order to gain a better understanding of migraine mechanisms. In this thesis, we report the results of genetic linkage and association analyses on a Finnish migraine patient collection as well as migraineurs from Australia, Denmark, Germany, Iceland and the Netherlands. Altogether we studied genetic information of nearly 7,000 migraine patients and over 50,000 population-matched controls. We also developed a new migraine analysis method called the trait component analysis, which is based on individual patient responses instead of the clinical diagnosis. Using this method, we detected a number of new genetic loci for migraine, including on chromosome 17p13 (HLOD 4.65) and 10q22-q23 (female-specific HLOD 7.68) with significant evidence of linkage, along with five other loci (2p12, 8q12, 4q28-q31, 18q12-q22, and Xp22) detected with suggestive evidence of linkage. The 10q22-q23 locus was the first genetic finding in migraine to show linkage to the same locus and markers in multiple populations, with consistent detection in six different scans. Traditionally, ion channels have been thought to play a role in migraine susceptibility, but we were able to exclude any significant role for common variants in a candidate gene study of 155 ion transport genes. This was followed up by the first genome-wide association study in migraine, conducted on 2,748 migraine patients and 10,747 matched controls followed by a replication in 3,209 patients and 40,062 controls. In this study, we found interesting results with genome-wide significance, providing targets for future genetic and functional studies. Overall, we found several promising genetic loci for migraine providing a promising base for future studies in migraine.

Developmental-like responses in injured mature central neurons

Traumatic insults to the central nervous system are frequently followed by profound and irreversible neuronal loss as well as the inability of the damaged neurons to regenerate. One of the major therapeutic challenges is to increase the amount of surviving neurons after trauma. Thus it is crucial to understand how injury affects neuronal responses and which conditions are optimal for survival to prevent neuronal loss. During development neuronal survival is thought to be dependent on the competition for the availability of survival-promoting molecules called neurotrophic factors. Much less is known on the survival mechanisms of mature neurons under traumatic conditions. Increasing amount of evidence points towards the possibility that after injury neuronal responses might aquire some developmental characteristics. One of the important examples is the change in the responses to the neurotransmitter GABA: it is inhibitory in the intact mature neurons, but can induce excitation during development and after trauma. An important step in the maturation of GABAergic transmission in the CNS is the developmental shift in the action of GABAA receptor from depolarization in immature neurons to hyperpolarization in mature neurons. GABAA-mediated responses are tightly linked to the homeostasis of the chloride anion (Cl-), which in neurons is mainly regulated by Na+-K+-2Cl- cotransporter NKCC1 and K+-Cl- cotransporter KCC2. Trauma-induced functional downregulation of KCC2 promotes a shift from hyperpolarizing GABAA-mediated responses to depolarizing. Other important consequences of neuronal trauma are the emergence of dependency of central neurons on brain-derived neuro¬trophic factor (BDNF) for survival, as well as the upregulation of neurotrophin receptor p75NTR. Our aim was to answer the question whether these post-traumatic events are interrelated, and whether the regulation of BDNF and KCC2 expression is different under traumatic conditions and in intact neurons. To study responses of injured mature central neurons, we used an in vitro and in vivo axotomy models. For in vitro studies, we lesioned organotypic hippocampal slices between CA3 and CA1 regions, which resulted in selective axotomy of the CA3 neurons and denervation of the CA1 neurons. Some experiments were repeated in vivo by lesioning the neurons of the corticospinal tract at the internal capsule level, or by lesioning spinal motoneurons at the ventral root. We show that intact mature neurons do not require BDNF for survival, whereas in axotomized neurons apoptosis is induced upon BDNF deprivation. We further show that post-traumatic dependency on BDNF is mediated by injury-induced upregulation of p75NTR. Post-traumatic increase in p75NTR is induced by GABAA-mediated depolarization, consequent opening of voltage-gated Ca2+ channels, and the activation of Rho kinase ROCK. Thus, post-traumatic KCC2 downregulation leads to the dependency on BDNF through the induction of p75NTR upregulation. Neurons that survive after axotomy over longer period of time lose BDNF dependency and regain normal KCC2 levels. This phenomenon is promoted by BDNF itself, since after axotomy contrary to normal conditions KCC2 is upregulated by BDNF. The developmentally important thyroid hormone thyroxin regulates BDNF expression during development. We show that in mature intact neurons thyroxin downregulates BDNF, whereas after axotomy thyroxin upregulates BDNF. The elevation of BDNF expression by thyroxin promoted survival of injured neurons. In addition, thyroxin also enhanced axonal regeneration and promoted the regaining of normal levels of KCC2. Thus we show that this hormone acts at several levels on the axotomy-initiated chain of events described in the present work, and could be a potential therapeutic agent for the injured neurons. We have also characterized a previously unknown downregulatory interaction between thyroxin and KCC2 in intact neurons. In conclusion, we identified several important interactions at the neurotrophin-protein and hormone-neurotrophin level that acquire immature-like characteristics after axotomy and elucidated an important part of the mechanism by which axotomy leads to the requirement of BDNF trophic support. Based on these findings, we propose a new potential therapeutic strategy where developmentally crucial agents could be used to enhance survival and regeneration of axotomized mature central neurons.

Improving outcome of childhood bacterial meningitis by simplified treatment : Experience from Angola

Background Acute bacterial meningitis (BM) continues to be an important cause of childhood mortality and morbidity, especially in developing countries. Prognostic scales and the identification of risk factors for adverse outcome both aid in assessing disease severity. New antimicrobial agents or adjunctive treatments - except for oral glycerol - have essentially failed to improve BM prognosis. A retrospective observational analysis found paracetamol beneficial in adult bacteraemic patients, and some experts recommend slow β-lactam infusion. We examined these treatments in a prospective, double-blind, placebo-controlled clinical trial. Patients and methods A retrospective analysis included 555 children treated for BM in 2004 in the infectious disease ward of the Paediatric Hospital of Luanda, Angola. Our prospective study randomised 723 children into four groups, to receive a combination of cefotaxime infusion or boluses every 6 hours for the first 24 hours and oral paracetamol or placebo for 48 hours. The primary endpoints were 1) death or severe neurological sequelae (SeNeSe), and 2) deafness. Results In the retrospective study, the mortality of children with blood transfusion was 23% (30 of 128) vs. without blood transfusion 39% (109 of 282; p=0.004). In the prospective study, 272 (38%) of the children died. Of those 451 surviving, 68 (15%) showed SeNeSe, and 12% (45 of 374) were deaf. Whereas no difference between treatment groups was observable in primary endpoints, the early mortality in the infusion-paracetamol group was lower, with the difference (Fisher s exact test) from the other groups at 24, 48, and 72 hours being significant (p=0.041, 0.0005, and 0.005, respectively). Prognostic factors for adverse outcomes were impaired consciousness, dyspnoea, seizures, delayed presentation, and absence of electricity at home (Simple Luanda Scale, SLS); the Bayesian Luanda Scale (BLS) also included abnormally low or high blood glucose. Conclusions New studies concerning the possible beneficial effect of blood transfusion, and concerning longer treatment with cefotaxime infusion and oral paracetamol, and a study to validate our simple prognostic scales are warranted.

Nikotiinireseptoreiden kautta vaikuttavat lääkeaineet nikotiiniriippuvuuden ja neurologisten sairauksien hoidossa

Neuronaaliset nikotiinireseptorit liittyvät tupakkariippuvuuden lisäksi moniin neurologisiin sairauksiin, kuten Alzheimerin tautiin, skitsofreniaan, masennukseen ja tarkkaavaisuus- ja ylivilkkaushäiriöön. Nikotiinireseptorien stimulaation on tutkimuksissa havaittu parantavan kognitiota. Useat lääkeyritykset tutkivat nikotiinireseptoriagonisteja ja -antagonisteja eri neurologisten sairauksien hoidossa. Ongelmana nikotiinireseptori-agonisteja käytettäessä on reseptorissa tapahtuva desensitisaatio. Tällöin reseptori sulkeutuu, eikä aktivoidu vaikka agonistia olisi tarjolla tai sitoutuneena reseptoriin. Varsinkin alfa7-reseptori desensitoituu hyvin nopeasti agonistialtistuksen seurauksena. Reseptorien desensitoituminen voi kliinisessä käytössä aiheuttaa lääkeaineen tehon menetyksen. Perinteisen agonistin sitoutumiskohdan lisäksi nikotiinireseptorissa sijaitsee myös muita sitoutumiskohtia, joita kutsutaan allosteerisiksi sitoutumispaikoiksi. Tutkimuksissa on havaittu, että eräät allosteerisesti sitoutuvat aineet, kuten PNU-120596, voivat vahvistaa agonistin aikaansaamaa vastetta ja/tai estää reseptorin desensitoitumista. Näitä aineita kutsutaan positiivisiksi allosteerisiksi modulaattoreiksi ja niiden ajatellaan olevan vaihtoehto desensitoitumisen aiheuttamaan tehon menetyksen ongelmaan. Nikotiinireseptorien positiivisten allosteeristen modulaattorien tarkkaa vaikutusta ja sitoutumiskohtaa reseptoriin ei vielä tarkkaan tiedetä. Tutkimuksen aiheena oli karakterisoida positiivisten allosteeristen modulaattoreiden vaikutuksia alfa7-nikotiinireseptoriin. Tutkimuksessa tarkoituksena oli käyttää hyväksi laboratoriossa aiemmin tehtyä havaintoa, jonka mukaan alfa7-nikotiinireseptorin transmembraaniosan aminohappoon tehdyn mutaation L247T seurauksena positiiviset allosteeriset modulaattorit muuttuvat agonisteiksi. Haluttiin selvittää, kuinka agonistin sitoutumiskohtaan kohdennettua mutageneesiä käyttäen tehty mutaatio W149M tai W149F vaikuttavat PNU-120596:n kykyyn toimia agonistina alfa7L247T reseptoriin. Asetyylikoliini toimi konventionaalisen agonistin mallina tutkimuksessa. Tutkimuksen toinen tavoite oli tehdä mutaatio M253Lalfa7-reseptorin transmembraaniosaan. Mutaation on todettu estävän allosteeristen potentiaattoreiden kykyä voimistaa agonistin aikaansaamaa vastetta. Tarkoitus oli tutkia millaisia vaikutuksia M253L-mutaatiolla on allosteerisen potentiaattorin kykyyn toimia agonistina L247T-mutaation sisältävään reseptoriin. Mutatoidun reseptorin mRNA mikroinjektoitiin oosyyttiin ja elektrofysiologian avulla tutkittiin ilmennettyjen reseptorien toimintaa käyttäen kahden elektrodin jännitelukitus -menetelmää. Kaikki suunnitellut mutaatiot saatiin tehtyä onnistuneesti alfa7- ja alfa7L247T-reseptoreihin. Ortosteerisen sitoutumiskohdan mutaatio villin tyypin Į7-reseptorissa vaikutti hyvin voimakkaasti joko asetyylikoliinin sitoutumiseen reseptoriin tai reseptorin toimintaan, sillä asetyylikoliinilla ei reseptorista saatu mitattua vasteita. Myöskään PNU-120596 yksinään ei saanut aikaan vasteita alfa7W149M-reseptorissa. Kaksoismutatoidussa alfa7W149M/L247T-reseptorissa puolestaan havaittiin, että asetyylikoliinin annos-vaste -kuvaaja siirtyi huomattavasti enemmän oikealle kuin PNU-120596:n, kun verrattiin annos-vaste –kuvaajia alfa7L247T ja alfa7W149M/L247T–reseptoreiden välillä. Transmembraaniosan mutaatio M253L ei vaikuttanut PNU-120596:n kykyyn toimia agonistina alfa7L247T-reseptoriin, eikä sillä ollut vaikutusta asetyylikoliinin annosvaste-kuvaajiin. Tutkimus tukee aiempia havaintoja siitä, että positiivisten allosteeristen modulaattoreiden sitoutumiskohta nikotiinireseptorissa sijaitsisi transmembraaniosassa. M253L-mutaation osalta tulokset ovat hieman ristiriidassa aiempien tulosten kanssa. L247T-mutaatio vaikuttaa hyvin voimakkaasti nikotiinireseptorin toimintaan sekä sijaitsee aminohapon M253 läheisyydessä. On mahdollista, että se peittää M253L-mutaation vaikutuksen. Toisaalta voi olla, että M253 on aminohappo, joka vaikuttaa vain reseptorivasteiden voimistumiseen eikä allosteeristen potentiaattoreiden sitoutumiseen.

Makrodomeeni-inhibiittorit antiviraalisina yhdisteinä

Alfavirukset ovat positiivissäkeisiä RNA-viruksia, jotka kuuluvat Togaviridea –heimoon. Alfaviruksia levittävät Aedes –suvun hyttyset ja niitä esiintyy Etelämanteretta lukuunottamatta kaikilla mantereilla. Alfaviruksia on tähän mennessä löydetty 29 lajia ja ne voidaan jakaa uuden ja vanhan maailman viruksiin niiden maantieteellisen esiintyvyyden ja taudinaiheuttamiskyvyn mukaan. Chikunkunyavirus (CHIKV) on yksi vanhan maailman alfaviruksista, jota esiintyy muun muassa Afrikassa ja Aasiassa. Ilmaston lämmettyä se on leviämässä myös eteläiseen Eurooppaan. Ihmisessä se aiheuttaa muun muassa kuumetta, päänsärkyä, ihottumaa ja niveltulehdusta, joka voi kestää useita vuosia ja ne voivat olla hyvinkin kivuliaita. Pienillä lapsilla chikungunya on todettu aiheuttavan myös neurologisia oireita kuten aivotulehdusta. Alfaviruksen genomi koodaa neljää rakenneproteiinia ja neljää replikaatioproteiinia. Replikaatioproteiineista nsP3 sisältää makrodomeeniosan. Makrodomeeniproteiinit ovat eliökunnassa konservoituneita, mutta makrodomeeniproteiinien tarkkaa merkitystä ei vielä tunneta. Makrodomeenien on osoitettu sitovan ADP-riboosia ja sen johdannaisia ja alfaviruksen nsP3-proteiinin on osoitettu olevan tärkeä osa viruksen replikaatiossa. Tutkimuksen tavoitteena oli tutkia makrodomeeniproteiiniin sitoutuvien yhdisteiden käyttöä antiviraalisena yhdisteinä. Tietokonemallinnuksella valittiin antiviraalitutkimuksiin 45 yhdistettä, joiden oletettiin sitoutuvan makrodomeeniproteiiniin. Kilpailevassa sitoutumiskokeessa viisi yhdistettä esti yli 50 % poly-ADP-riboosia (PAR) sitoutumasta MDO1-makrodomeeniproteiiniin, jolla tietokonemallinnus oli tehty. SFV-makrodomeeniproteiinilla tehdyssä kokeessa vain yksi yhdiste esti yli 50 % poly-ADP-riboosin sitoutumisen. SFV-antiviraalikokeessa seitsemällä yhdisteellä inhibitioprosentti oli yli 50 %. Näillä yhdisteillä ei kuitenkaan ollut merkittävää vaikutusta poly-ADP-riboosin sitoutumisen estossa. CHIKV-replikonikokeessa yli 50 % inhibitioprosentti oli viidellä yhdisteellä. Muiden mahdollisia vaikutusmekanismeja tutkittiin selvittämällä estävätkö yhdisteet virusta pääsemästä solun sisään. Tässä kokeessa tutkituista yhdisteistä lähes kaikilla oli vaikutusta viruksen soluun pääsyn estossa. Yleisesti ottaen kyky estää PAR:n sitoutuminen makrodomeeniproteiineihin ja antiviraaliset vaikutukset eivät korreloineet keskenään tutkittavilla yhdisteillä. Vaikka antiviraalista vaikutusta omaavat yhdisteet eivät osoittaneetkaan makrodomeeni-inhibiitiota, työssä löydettiin potentiaalisia antiviraalisia yhdisteitä joiden käyttö viruksen soluun pääsyn estäjinä antaa aihetta jatkotutkimuksille.

Studies on Causes of Multiple Sclerosis : From Genes to Transcriptome

Multiple sclerosis (MS) is the most common cause of neurological disability in young adults, affecting more than two million people worldwide. It manifests as a chronic inflammation in the central nervous system (CNS) and causes demyelination and neurodegeneration. Depending on the location of the demyelinated plaques and axonal loss, a variety of symptoms can be observed including deficits in vision, coordination, balance and movement. With a typical age of onset at 20-40 years, the social and economic impacts of MS on lives of the patients and their families are considerable. Unfortunately the current treatments are relatively inefficient and the development of more effective treatments has been impeded by our limited understanding of the causes and pathogenesis of MS. Risk of MS is higher in biological relatives of MS patients than in the general population. Twin and adoption studies have shown that familial clustering of MS is explained by shared genetic factors rather than by shared familial environment. While the involvement of the human leukocyte antigen (HLA) genes was first discovered four decades ago, additional genetic risk factors have only recently been identified through genome-wide association studies (GWAS). Current evidence suggests that MS is a highly polygenic disease with perhaps hundreds of common variants with relatively modest effects contributing to susceptibility. Despite extensive research, the majority of these risk factors still remain to be identified. In this thesis the aim was to identify novel genes and pathways involved in MS. Using genome-wide microarray technology, gene expression levels in peripheral blood mononuclear cells (PBMC) from 12 MS patients and 15 controls were profiled and more than 600 genes with altered expression in MS were identified. Three of five selected findings, DEFA1A3, LILRA4 and TNFRSF25, were successfully replicated in an independent sample. Increased expression of DEFA1A3 in MS is a particularly interesting observation, because its elevated levels have previously been reported also in several other autoimmune diseases. A systematic review of seven microarray studies was then performed leading to identification of 229 genes, in which either decreased or increased expression in MS had been reported in at least two studies. In general there was relatively little overlap across the experiments: 11 of the 229 genes had been reported in three studies and only HSPA1A in four studies. Nevertheless, these 229 genes were associated with several immunological pathways including interleukin pathways related to type 2 and type 17 helper T cells and regulatory T cells. However, whether these pathways are involved in causing MS or related to secondary processes activated after disease onset remains to be investigated. The 229 genes were also compared with loci identified in published MS GWASs. Single nucleotide polymorphisms (SNP) in 17 of the 229 loci had been reported to be associated with MS with P-value less than 0.0001 including variants in CXCR4 and SAPS2, which were the only loci where evidence for correlation between the associated variant and gene expression was found. The CXCR4 variant was further tested for association with MS in a large case-control sample and the previously reported suggestive association was replicated (P-value is 0.0004). Finally, common genetic variants in candidate genes, which had been selected on the basis of showing association with other autoimmune diseases (MYO9B) or showing differential expression in MS in our study (DEFA1A3, LILRA4 and TNFRSF25), were tested for association with MS, but no evidence of association was found. In conclusion, through a systematic review of genome-wide expression studies in MS we have identified several promising candidate genes and pathways for future studies. In addition, we have replicated a previously suggested association of a SNP variant upstream of CXCR4 with MS. Keywords: autoimmune disease, common variant, CXCR4, DEFA1A3, HSPA1A,gene expression, genetic association, GWAS, MS, multiple sclerosis, systematic review

Putative neuroprotective compounds in in vitro models of dopaminergic neurodegeneration

Parkinson´s disease (PD) is a debilitating age-related neurological disorder that affects various motor skills and can lead to a loss of cognitive functions. The motor symptoms are the result of the progressive degeneration of dopaminergic neurons within the substantia nigra. The factors that influence the pathogenesis and the progression of the neurodegeneration remain mostly unclear. This study investigated the role of various programmed cell death (PCD) pathways, oxidative stress, and glial cells both in dopaminergic neurodegeneration and in the protective action of various drugs. To this end, we exposed dopaminergic neuroblastoma cells (SH-SY5Y cells) to 6-OHDA, which produces oxidative stress and activates various PCD modalities that result in neuronal degeneration. Additionally, to explore the role of glia, we prepared rat midbrain primary mixed-cell cultures containing both neurons and glial cell types such as microglia and astroglia and then exposed the cultures to either MPP plus or lipopolysaccharide. Our results revealed that 6-OHDA activated several PCD pathways including apoptosis, autophagic stress, lysosomal membrane permeabilization, and perhaps paraptosis in SH-SY5Y cells. Furthermore, we found that minocycline protected SH-SY5Y cells from 6-OHDA by inhibiting both apoptotic and non-apoptotic PCD modalities. We also observed an inconsistent neuroprotective effect of various dietary anti-oxidant compounds against 6-OHDA toxicity in vitro in SH-SY5Y cells. Specifically, quercetin and curcumin exerted neuroprotection only within a narrow concentration range and a limited time frame, whereas resveratrol and epigallocatechin 3-gallate provided no protection whatsoever. Lastly, we found that molecules such as amantadine may delay or even halt the neurodegeneration in primary cell cultures by inhibiting the release of neurotoxic factors from overactivated microglia and by enhancing the pro-survival actions of astroglia. Together these data suggest that the strategy of dampening oxidative species with anti-oxidants is less effective than preventing the production of toxic factors such as oxidative and pro-inflammatory molecules by pathologically activated microglia. This would subsequently prevent the activation of various PCD modalities that cause neuronal degeneration.

Poststroke survival and ischemic stroke recurrence : the cerebral small-vessel disease perspective

The aim of the present study was to investigate the influence of different manifestations of cerebral SVD on poststroke survival and ischemic stroke recurrence in long-term follow-up. The core imaging features of small-vessel disease (SVD) are confluent and extensive white matter changes (WMC) and lacunar infarcts. These are associated with minor motor deficits but a major negative influence on cognition, mood, and functioning in daily life, resulting from small-vessel lesions in the fronto-subcortical brain network. These sub-studies were conducted as part of the Helsinki Stroke Aging Memory (SAM) study. The SAM cohort consisted of 486 consecutive patients aged 55 to 85 years who were admitted to Helsinki University Central Hospital with acute ischemic stroke. The study included comprehensive clinical, neuropsychological, psychiatric and radiological assessment three months poststroke. The patients were followed up up for 12 years using extensive national registers. The effect of different manifestations of cerebral SVD on poststroke survival and stroke recurrence was analyzed controlling for factors such as age, education, and cardiovascular risk factors. Poststroke dementia and cognitive impairment relate to poor long-term survival. In particular, deficits in executive functions as well as visuospatial and constructional abilities predict poor outcome. The predictive value of cognitive deficits is further underlined by the finding that depression-executive dysfunction syndrome (DES), but not depression in itself, is associated with poor poststroke survival. Delirium is not independently associated with increased risk for long-term poststroke mortality, although it is associated with poststroke dementia. Furthermore, acute index stroke attributable to SVD is associated with poorer long-term survival and a higher risk for cardiac death than other stroke subtypes. Severe WMC, a surrogate of SVD, is independently related to an increased risk of stroke recurrence at five years. In summary, cognitive poststroke outcomes reflecting changes in the executive network brain, and the presence of cerebral SVD are important determinants of poststroke mortality and ischemic stroke recurrence, regardless of whether SVD is the cause of the index stroke or a condition concurrent to some other etiology.

Kielellinen erityisvaikeus : oirekuva ja familiaalisuus potilasasiakirjojen perusteella

Goals. Specific language impairment (SLI) has a negative impact on child s speech and language development and interaction. Disorder may be associated with a wide range of comorbid problems. In clinical speech therapy it is important to see the child as a whole so that the rehabilitation can be targeted properly. The aim of this study was to describe the linguistic-cognitive and comorbid symptoms of children with SLI at the age of five, as well as to provide an overwiew of the developmental disorders in the families. The study is part of a larger research project, which will examine paths of development and quality of life of children with SLI as young adults. Methods. The data consisted of patient documents of 100 5-year old children, who were examined in Lastenlinna mainly at 1998. Majority of the subjects were boys, and children s primary diagnosis was either F80.1 or F80.2, which was most common, or both. The diagnosis and the information about the linguistic-cognitive status and comorbid symptoms were collected from reports of medical doctors and experts of other fields, as well as mentions related to familiality. Linguistic-cognitive symptoms were divided into subclasses of speech motor functions, prosessing of language, comprehension of language and use of language. Comorbid symptoms were divided into subclasses of interaction, activity and attention, emotional and behavior problems and neurologic problems. Statistical analyses were based mainly on Pearson s Chi Square test. Results and conclusions. Problems in language processing and speech motor functions were most common of the linguistic-cognitive symptoms. Most of the children had symptoms from two or three symptom classes, and it seemed that girls had more symptoms than boys. Usually children did not have any comorbid symptoms, or had them from one or three symptom classes. Of the comorbid symptoms the most prevalent ones were problems in activity and attention and neurological symptoms, which consisted mostly of motoric and visuomotoric symptoms. The most common of the comorbid diagnoses was F82, specific developmental disorder of motor function. According to literature children with SLI may have problems in mental health, but the results of this study did not confirm that. Children with diagnosis F80.2 had more linguistic-cognitive and comorbid symptoms than children with diagnosis F80.1. The cluster analyses based on all the symtoms revealed four subgroups of the subjects. Of the subjects 85 percent had a positive family history of developmental disorders, and the most prevalent problem in the families was delayed speech development. This study outlined the symptom profile of children with SLI and laid a foundation for the future longitudinal study. The results suggested that there are differences between linguistic-cognitive symptoms of boys and girls, which is important to notice especially when assessing and diagnosing children with SLI.

FGF signaling in neurogenesis and patterning of the developing midbrain and anterior hindbrain

Embryonic midbrain and hindbrain are structures which will give rise to brain stem and cerebellum in the adult vertebrates. Brain stem contains several nuclei which are essential for the regulation of movements and behavior. They include serotonin-producing neurons, which develop in the hindbrain, and dopamine-producing neurons in the ventral midbrain. Degeneration and malfunction of these neurons leads to various neurological disorders, including schizophrenia, depression, Alzheimer s, and Parkinson s disease. Thus, understanding their development is of high interest. During embryogenesis, a local signaling center called isthmic organizer regulates the development of midbrain and anterior hindbrain. It secretes peptides belonging to fibroblast growth factor (FGF) and Wingless/Int (Wnt) families. These factors bind to their receptors in the surrounding tissues, and activate various downstream signaling pathways which lead to alterations in gene expression. This in turn affects the various developmental processes in this region, such as proliferation, survival, patterning, and neuronal differentiation. In this study we have analyzed the role of FGFs in the development of midbrain and anterior hindbrain, by using mouse as a model organism. We show that FGF receptors cooperate to receive isthmic signals, and cell-autonomously promote cell survival, proliferation, and maintenance of neuronal progenitors. FGF signaling is required for the maintenance of Sox3 and Hes1 expression in progenitors, and Hes1 in turn suppresses the activity of proneural genes. Loss of Hes1 is correlated with increased cell cycle exit and premature neuronal differentiation. We further demonstrate that FGF8 protein forms an antero-posterior gradient in the basal lamina, and might enter the neuronal progenitors via their basal processes. We also analyze the impact of FGF signaling on the various neuronal nuclei in midbrain and hindbrain. Rostral serotonergic neurons appear to require high levels of FGF signaling in order to develop. In the absence of FGF signaling, these neurons are absent. We also show that embryonic meso-diencephalic dopaminergic domain consists of two populations in the anterior-posterior direction, and that these populations display different molecular profiles. The anterior diencephalic domain appears less dependent on isthmic FGFs, and lack several genes typical of midbrain dopaminergic neurons, such as Pitx3 and DAT. In Fgfr compound mutants, midbrain dopaminergic neurons begin to develop but soon adopt characteristics which highly resemble those of diencephalic dopaminergic precursors. Our results indicate that FGF signaling regulates patterning of these two domains cell-autonomously.