Trafficking and ethanol-induced inhibition of AMPA receptors

AMPA receptors are an important class of ionotropic glutamate receptors which participate in fast excitatory synaptic transmission in most brain areas. They have a pivotal role in adjustment of cell membrane excitability as their cell membrane expression levels is altered in brain physiology such as in learning and memory formation. AMPA receptor function and trafficking is regulated by several proteins, such as transmembrane AMPA receptor regulatory proteins (TARPs). NMDA-type glutamate receptors are important target molecules of ethanol. The role of AMPA receptors in the actions of ethanol has not been clarified as thoroughly. Furthermore, the regulation of AMPA receptor synthesis and their possible adaptation in neurons with altered inhibitory mechanisms are poorly understood. In this thesis work AMPA receptor pharmacology, trafficking and synaptic localization was studied using patch-clamp electrophysiology. Both native and recombinant AMPA receptors were studied. Hippocampal slices from transgenic Thy1alfa6 mice with altered inhibition were used to study adaptation of AMPA receptors. Ethanol was found to inhibit AMPA receptor function by increasing desensitization of the receptor, as the steady-state current was inhibited more than the peak current. Ethanol inhibition was reduced when cyclothiazide was used to block desensitization and when non-desensitizing mutant receptors were studied. Ethanol also increased the rate of desensitization, which was increased further by the coexpression of TARP-proteins. We found that the agonist binding capability is important for trafficking AMPA receptors from endoplasmic reticulum to the cell membrane. TARP rescues the surface expression of non-binding AMPA receptor mutants in HEK293 cells, but not in native neurons. Studies with Thy1alfa6 mice revealed that decreased inhibition decrease AMPA receptor mediated excitation keeping the neurotransmission in balance. Thy1alfa6 mice also had lower sensitivity to electroshock convulsions, presumably due to the decreased AMPA receptor function. The results suggest that during alcohol intoxication ethanol may inhibit AMPA receptors by increasing the rate and the extent of desensitization. TARPs appear to enhance ethanol inhibition. TARPs also participate in trafficking of AMPA receptors upon their synthesis in the cell. AMPA receptors mediate also long-term adaptation to altered neuronal excitability, which adds to their well-known role in synaptic plasticity.

Heparin-binding growth-associated molecule (HB-GAM) in activity-dependent neuronal plasticity in hippocampus

Cell adhesion and extracellular matrix (ECM) molecules play a significant role in neuronal plasticity both during development and in the adult. Plastic changes in which ECM components are implicated may underlie important nervous system functions, such as memory formation and learning. Heparin-binding growthassociated molecule (HB-GAM, also known as pleiotrophin), is an ECM protein involved in neurite outgrowth, axonal guidance and synaptogenesis during perinatal period. In the adult brain HB-GAM expression is restricted to the regions which display pronounced synaptic plasticity (e.g., hippocampal CA3-CA1 areas, cerebral cortex laminae II-IV, olfactory bulb). Expression of HB-GAM is regulated in an activity-dependent manner and is also induced in response to neuronal injury. In this work mutant mice were used to study the in vivo function of HB-GAM and its receptor syndecan-3 in hippocampal synaptic plasticity and in hippocampus-dependent behavioral tasks. Phenotypic analysis of HBGAM null mutants and mice overexpressing HB-GAM revealed that opposite genetic manipulations result in reverse changes in synaptic plasticity as well as behavior in the mutants. Electrophysiological recordings showed that mice lacking HB-GAM have an increased level of long-term potentiation (LTP) in the area CA1 of hippocampus and impaired spatial learning, whereas animals with enhanced level of HB-GAM expression have attenuated LTP, but outperformed their wild-type controls in spatial learning. It was also found that GABA(A) receptor-mediated synaptic transmission is altered in the transgenic mice overexpressing HB-GAM. The results suggest that these animals have accentuated hippocampal GABAergic inhibition, which may contribute to the altered glutamatergic synaptic plasticity. Structural studies of HB-GAM demonstrated that this protein belongs to the thrombospondin type I repeat (TSR) superfamily and contains two β-sheet domains connected by a flexible linker. It was found that didomain structure is necessary for biological activity of HB-GAM and electrophysiological phenotype displayed by the HB-GAM mutants. The individual domains displayed weaker binding to heparan sulfate and failed to promote neurite outgrowth as well as affect hippocampal LTP. Effects of HB-GAM on hippocampal synaptic plasticity are believed to be mediated by one of its (co-)receptor molecules, namely syndecan-3. In support of that, HB-GAM did not attenuate LTP in mice deficient in syndecan-3 as it did in wild-type controls. In addition, syndecan-3 knockout mice displayed electrophysiological and behavioral phenotype similar to that of HB-GAM knockouts (i.e. enhanced LTP and impaired learning in Morris water-maze). Thus HB-GAM and syndecan-3 are important modulators of synaptic plasticity in hippocampus and play a role in regulation of learning-related behavior.

Neurotrophins and neuronal plasticity in the action of antidepressants and morphine

Neuronal plasticity is a well characterized phenomenon in the developing and adult brain. It refers to capasity of a single neuron to modify morphology, synaptic connections and activity. Neuronal connections and capacity for plastic events are compromised in several pathological disorders, such as major depression. In addition, neuronal atrophy has been reported in depressive patients. Neurotrophins are a group of secretory proteins functionally classified as neuronal survival factors. Neurotrophins, especially brain derived neurotrophic factor (BDNF), have also been associated with promoting neuronal plasticity in dysfunctional neuronal networks. Chronic antidepressant treatment increases plastic events including neurogenesis and arborization and branching of neurites in distinct brain areas, such as the hippocampus. One suggested mode of action is where the antidepressants elevate the synaptic levels of BDNF thus further activating several signaling cascades via trkB-receptor. In our studies we have tried to clarify the mechanisms of action for antidepressants and to resolve the role of BDNF in this process. We found that chronic antidepressant treatment increases amount of markers of neuronal plasticity in both hippocampus and in the medial prefrontal cortex, both of which are closely linked to the etiology of major depression. Secondary actions of antidepressants include rapid activation of the trkB receptor followed by a phosphorylation of transcription factor CREB. In addition, activation of CREB by phosphorylation appears responsible for the regulation of the expression of the BDNF gene. Using transgenic mice we found that BDNF-induced trkB-mediated signaling proved crucial for the behavioral effects of antidepressants in the forced swimming test and for the survival of newly-born neurons in the adult hippocampus. Antidepressants not only increased neurogenesis in the adult hippocampus but also elevated the turnover of hippocampal neurons. During these studies we also discovered that another trkB ligand, NT-4, is involved in morphine-mediated anti-nociception and tolerance. These results present a novel role for trkB-mediated signaling in plastic events present in the opioid system. This thesis evaluates neuronal plasticity and trkB as a target for future antidepressant treatments.

Intraspecific variation in brain size and architecture : population divergence and phenotypic plasticity

Brain size and architecture exhibit great evolutionary and ontogenetic variation. Yet, studies on population variation (within a single species) in brain size and architecture, or in brain plasticity induced by ecologically relevant biotic factors have been largely overlooked. Here, I address the following questions: (i) do locally adapted populations differ in brain size and architecture, (ii) can the biotic environment induce brain plasticity, and (iii) do locally adapted populations differ in levels of brain plasticity? In the first two chapters I report large variation in both absolute and relative brain size, as well as in the relative sizes of brain parts, among divergent nine-spined stickleback (Pungitius pungitius) populations. Some traits show habitat-dependent divergence, implying natural selection being responsible for the observed patterns. Namely, marine sticklebacks have relatively larger bulbi olfactorii (chemosensory centre) and telencephala (involved in learning) than pond sticklebacks. Further, I demonstrate the importance of common garden studies in drawing firm evolutionary conclusions. In the following three chapters I show how the social environment and perceived predation risk shapes brain development. In common frog (Rana temporaria) tadpoles, I demonstrate that under the highest per capita predation risk, tadpoles develop smaller brains than in less risky situations, while high tadpole density results in enlarged tectum opticum (visual brain centre). Visual contact with conspecifics induces enlarged tecta optica in nine-spined sticklebacks, whereas when only olfactory cues from conspecifics are available, bulbus olfactorius become enlarged.Perceived predation risk results in smaller hypothalami (complex function) in sticklebacks. Further, group-living has a negative effect on relative brain size in the competition-adapted pond sticklebacks, but not in the predation-adapted marine sticklebacks. Perceived predation risk induces enlargement of bulbus olfactorius in pond sticklebacks, but not in marine sticklebacks who have larger bulbi olfactorii than pond fish regardless of predation. In sum, my studies demonstrate how applying a microevolutionary approach can help us to understand the enormous variation observed in the brains of wild animals a point-of-view which I high-light in the closing review chapter of my thesis.

Effects of dairy proteins and calcium on diet-induced obesity in mice

Diet high in dairy products is inversely associated with body mass index, risk of metabolic syndrome and prevalence of type 2 diabetes in several populations. Also a number of intervention studies support the role of increased dairy intake in the prevention and treatment of obesity. Dairy calcium has been suggested to account for the effect of dairy on body weight, but it has been repeatedly shown that the effect of dairy is superior to the effect of supplemental calcium. Dairy proteins are postulated to either enhance the effect of calcium or have an independent effect on body weight, but studies in the area are scarce. The aim of this study was to evaluate the potential of dairy proteins and calcium in the prevention and treatment of diet-induced obesity in C57Bl/6J mice. The effect of dairy proteins and calcium on the liver and adipose tissue was also investigated in order to characterise the potential mechanisms explaining the reduction of risk for metabolic syndrome and type 2 diabetes. A high-calcium diet (1.8%) in combination with dietary whey protein inhibited body weight and fat gain and accelerated body weight and fat loss in high-fat-fed C57Bl/6J mice during long-term studies of 14 to 21 weeks. α-lactalbumin, one of the major whey proteins, was the most effective whey protein fraction showing significantly accelerated weight and fat loss during energy restriction and reduced the amount of visceral fat gain during ad libitum feeding after weight loss. The microarray data suggest sensitisation of insulin signalling in the adipose tissue as a result of a calcium-rich whey protein diet. Lipidomic analysis revealed that weight loss on whey protein-based high-calcium diet was characterised by significant decreases in diabetogenic diacylglycerols and lipotoxic ceramide species. The calcium supplementation led to a small, but statistically significant decrease in fat absorption independent of the protein source of the diet. This augments, but does not fully explain the effects of the studied diets on body weight. A whey protein-containing high-calcium diet had a protective effect against a high-fat diet-induced decline of β3 adrenergic receptor expression in adipose tissue. In addition, a high-calcium diet with whey protein increased the adipose tissue leptin expression which is decreased in this obesity-prone mouse strain. These changes are likely to contribute to the inhibition of weight gain. The potential sensitisation of insulin signalling in adipose tissue together with the less lipotoxic and diabetogenic hepatic lipid profile suggest a novel mechanistic link to explain why increased dairy intake is associated with a lower prevalence of metabolic syndrome and type 2 diabetes in epidemiological studies. Taken together, the intake of a high-calcium diet with dairy proteins has a body weight lowering effect in high-fat-fed C57Bl/6J mice. High-calcium diets containing whey protein prevent weight gain and enhance weight loss, α-lactalbumin being the most effective whey protein fraction. Whey proteins and calcium have also beneficial effects on hepatic lipid profile and adipose tissue gene expression, which suggest a novel mechanistic link to explain the epidemiological findings on dairy intake and metabolic syndrome. The clinical relevance of these findings and the precise mechanisms of action remain an intriguing field of future research.

Inflammation-induced atherogenesis, liver alterations, and cardiovascular outcome

Cardiovascular diseases, which presently are considered inflammatory diseases, affect millions of people worldwide. Chronic infections may contribute to the systemic inflammation suggested to increase the risk for cardiovascular diseases. Such chronic infections are periodontitis and Chlamydia pneumoniae infection. They are highly prevalent as approximately 10% of adult population and 30% of people over 50 years old are affected by severe periodontitis and 70-80% of elderly people are seropositive for C. pneumoniae. Our general aim was to investigate the role of infection and inflammation in atherosclerosis both in animal and human studies. We aimed to determine how the two pathogens alter the atherosclerosis-associated parameters, and how they affect the liver inflammation and lipid composition. Furthermore, we evaluated the association between matrix metalloproteinase-8 (MMP-8), a proteinase playing a major role in inflammation, and the future cardiovascular diseases (CVD) events in a population-based cohort. For the animal experiments, we used atherosclerosis-susceptible apolipoprotein E deficient (apoE-/-) mice. They were kept in germ free conditions and fed with a normal chow diet. The bacteria were administered either intravenously (A. actinomycetemcomitans) or intranasally (C. pneumoniae). Several factors were determined from serum as well as from aortic and hepatic tissues. We also determined how cholesterol efflux, a major event in the removal of excess cholesterol from the tissues, and endothelial function were affected by these pathogens. In the human study, serum MMP-8 and its tissue inhibitor (TIMP-1) concentrations were measured and their associations during the follow-up time of 10 years with CVD events were determined. An infection with A. actinomycetemcomitans increased concentrations of inflammatory mediators, MMP production, and cholesterol deposit in macrophages, decreased lipoprotein particle size, and induced liver inflammation. C. pneumoniae infection also elicited an inflammatory response and endothelial dysfunction, as well as induced liver inflammation, microvesicular appearance and altered fatty acid profile. In the population-based cohort, men with increased serum MMP-8 concentration together with subclinical atherosclerosis (carotid artery intima media thickness > 1mm) had a three-fold increased risk for CVD death during the follow-up. The results show that infections with A. actinomycetemcomitans and C. pneumoniae induce proatherogenic changes, as well as affect the liver. These data therefore support the concept that common infections have systemic effects and could be considered as cardiovascular risk factors. Furthermore, our data indicate that, as an independent predictor of fatal CVD event, serum MMP-8 could have a clinical significance in diagnosing cardiovascular diseases.

UV-induced NOy emissions in gas-exchange chambers enclosing Scots pine shoots: an analysis on their origin and significance

It is essential to have a thorough understanding of the sources and sinks of oxidized nitrogen (NOy) in the atmosphere, since it has a strong influence on the tropospheric chemistry and the eutrophication of ecosystems. One unknown component in the balance of gaseous oxidized nitrogen is vegetation. Plants absorb nitrogenous species from the air via the stomata, but it is not clear whether plants can also emit them at low ambient concentrations. The possible emissions are small and difficult to measure. The aim of this thesis was to analyse an observation made in southern Finland at the SMEAR II station: solar ultraviolet radiation (UV) induced NOy emissions in chambers measuring the gas exchange of Scots pine (Pinus sylvestris L.) shoots. Both measuring and modelling approaches were used in the study. The measurements were performed under noncontrolled field conditions at low ambient NOy concentrations. The chamber blank i.e. artefact NOy emissions from the chamber walls, was dependent on the UV irradiance and increased with time after renewing the Teflon film on chamber surfaces. The contribution of each pine shoot to the total NOy emissions in the chambers was determined by testing whether the emissions decrease when the shoots are removed from their chambers. Emissions did decrease, but only when the chamber interior was exposed to UV radiation. It was concluded that also the pine shoots emit NOy. The possible effects of transpiration on the chamber blank are discussed in the summary part of the thesis, based on previously unpublished data. The possible processes underlying the UV-induced NOy emissions were reviewed. Surface reactions were more likely than metabolic processes. Photolysis of nitrate deposited on the needles may have generated the NOy emissions; the measurements supported this hypothesis. In that case, the emissions apparently would consist mainly of nitrogen dioxide (NO2), nitric oxide (NO) and nitrous acid (HONO). Within studies on NOy exchange of plants, the gases most frequently studied are NO2 and NO (=NOx). In the present work, the implications of the emissions for the NOx exchange of pine were analysed with a model including both NOy emissions and NOy absorption. The model suggested that if the emissions exist, pines can act as an NOx source rather than a sink, even under relatively high ambient concentrations.

Expression Analysis of Host-Induced Genes and Proteins of Potato Pathogen Pectobacterium atrosepticum

Pectobacterium atrosepticum on Gram-negatiivinen bakteeri, joka aiheuttaa perunan tyvi- ja märkämätää. P. atrosepticum bakteerin optimilämpötila on melko alhainen ja se on yleinen lauhkeilla alueilla. Tyvimätä leviää pääasiassa siemenperunan välityksellä ja siksi se on ongelma erityisesti siemenperunan tuotannossa. P. atrosepticum kannan SCRI1043 genomi on julkaistu ja sitä tutkitaan malliorganismina märkä- ja tyvimädän taudinaiheuttamisen ymmärtämiseksi. Tämä opportunistinen taudinaiheuttaja voi elää isäntäkasvissa kuukausia piilevänä, aiheuttamatta näkyviä oireita. Suotuisissa olosuhteissa bakteerit alkavat jakautua ja tuottaa kasvin kudoksia hajottavia entsyymejä. Mädäntyvä kasvimassa tarjoaa ravinteita bakteerien kasvuun ja mahdollistaa isäntäkasvin asuttamisen. Soluseiniä hajottavien entsyymien merkitys taudinaiheuttamisessa on hyvin tunnettu, mutta oireettomasta jaksosta ja taudin alkuvaiheista tiedätään vain vähän. Bakteerin genomi sisältää monia toksiineja, adhesiineja, hemolysiineja ja muita proteiineja, joilla saattaa olla merkitys taudinaiheuttamisessa. Tässä työssä käytettiin proteomiikkaa ja mikrosiruanalysiä P. atrosepticum bakteerin erittyvien proteiinien ja geeniekspression tutkimiseen. Proteiinit, jotka eritetään ulos bakteerista, toimivat todennäköisesti taudinaiheuttamisessa, koska ne ovat suorassa kontaktissa isäntäkasvin kanssa. Analyysit suoritettiin olosuhteissa, jotka muistuttavat kasvin soluvälitilaa: matala pH, vähän ravinteita ja matala lämpötila. Isäntäkasvin läsnäolon vaikutusta proteiinien tuottoon ja geeniekspressioon tutkittiin lisäämällä perunauutetta kasvatusalustaan. Tutkimuksessa tunnistettiin P. atrosepticum bakteerin monia jo tunnettuja ja mahdollisesti taudinaiheuttamiseen liittyviä proteiineja. Perunauute lisäsi hiljattain tunnistetun, proteiinien eritysreittiä (tyyppi VI sekreetio, T6SS) koodaavien geenien ilmentymistä. Lisäksi bakteerin havaittiin erittävän useita T6SS:n liittyviä proteiineja kasvualustaan, johon oli lisätty perunauutetta. T6SS:n merkitys bakteereille on vielä epäselvä ja sen vaikutuksesta taudinaiheuttamiseen on julkaistu ristiriitaisia tuloksia. Märkä- ja tyvimädän ymmärtäminen molekulaarisella tasolla luo pohjan tautien kontrollointiin tähtäävään soveltavaan tutkimukseen. Tämä tutkimus lisää tietoa kasvi-patogeeni- interaktiosta ja sitä voidaan tulevaisuudessa käyttää hyväksi esimerkiksi diagnostiikassa, resistenttien perunalajikkeiden jalostuksessa tai viljely- ja varastointiolosuhteiden parantamisessa.