JNK regulates dendrite and spine architecture in the central nervous system influencing spatial learning and motor tasks

JNK1 is a MAP-kinase that has proven a significant player in the central nervous system. It regulates brain development and the maintenance of dendrites and axons. Several novel phosphorylation targets of JNK1 were identified in a screen performed in the Coffey lab. These proteins were mainly involved in the regulation of neuronal cytoskeleton, influencing the dynamics and stability of microtubules and actin. These structural proteins form the dynamic backbone for the elaborate architecture of the dendritic tree of a neuron. The initiation and branching of the dendrites requires a dynamic interplay between the cytoskeletal building blocks. Both microtubules and actin are decorated by associated proteins which regulate their dynamics. The dendrite-specific, high molecular weight microtubule associated protein 2 (MAP2) is an abundant protein in the brain, the binding of which stabilizes microtubules and influences their bundling. Its expression in non-neuronal cells induces the formation of neurite-like processes from the cell body, and its function is highly regulated by phosphorylation. JNK1 was shown to phosphorylate the proline-rich domain of MAP2 in vivo in a previous study performed in the group. Here we verify three threonine residues (T1619, T1622 and T1625) as JNK1 targets, the phosphorylation of which increases the binding of MAP2 to microtubules. This binding stabilizes the microtubules and increases process formation in non-neuronal cells. Phosphorylation-site mutants were engineered in the lab. The non-phosphorylatable mutant of MAP2 (MAP2- T1619A, T1622A, T1625A) in these residues fails to bind microtubules, while the pseudo-phosphorylated form, MAP2- T1619D, T1622D, Thr1625D, efficiently binds and induces process formation even without the presence of active JNK1. Ectopic expression of the MAP2- T1619D, T1622D, Thr1625D in vivo in mouse brain led to a striking increase in the branching of cortical layer 2/3 (L2/3) pyramidal neurons, compared to MAP2-WT. The dendritic complexity defines the receptive field of a neuron and dictates the output to the postsynaptic cells. Previous studies in the group indicated altered dendrite architecture of the pyramidal neurons in the Jnk1-/- mouse motor cortex. Here, we used Lucifer Yellow loading and Sholl analysis of neurons in order to study the dendritic branching in more detail. We report a striking, opposing effect in the absence of Jnk1 in the cortical layers 2/3 and 5 of the primary motor cortex. The basal dendrites of pyramidal neurons close to the pial surface at L2/3 show a reduced complexity. In contrast, the L5 neurons, which receive massive input from the L2/3 neurons, show greatly increased branching. Another novel substrate identified for JNK1 was MARCKSL1, a protein that regulates actin dynamics. It is highly expressed in neurons, but also in various cancer tissues. Three phosphorylation target residues for JNK1 were identified, and it was demonstrated that their phosphorylation reduces actin turnover and retards migration of these cells. Actin is the main cytoskeletal component in dendritic spines, the site of most excitatory synapses in pyramidal neurons. The density and gross morphology of the Lucifer Yellow filled dendrites were characterized and we show reduced density and altered morphology of spines in the motor cortex and in the hippocampal area CA3. The dynamic dendritic spines are widely considered to function as the cellular correlate during learning. We used a Morris water maze to test spatial memory. Here, the wild-type mice outperformed the knock-out mice during the acquisition phase of the experiment indicating impaired special memory. The L5 pyramidal neurons of the motor cortex project to the spinal cord and regulate the movement of distinct muscle groups. Thus the altered dendrite morphology in the motor cortex was expected to have an effect on the input-output balance in the signaling from the cortex to the lower motor circuits. A battery of behavioral tests were conducted for the wild-type and Jnk1-/- mice, and the knock-outs performed poorly compared to wild-type mice in tests assessing balance and fine motor movements. This study expands our knowledge of JNK1 as an important regulator of the dendritic fields of neurons and their manifestations in behavior.

The central histaminergic neurons in vitro, and their neuroprotective role in excitotoxic cell death in the postnatal rat hippocampus.

Histamine acts as a neurotransmitter in the central nervous system. Brain histamine in synthesized in neurons located to the posterior hypothalamus, from where these neurons send their projections to different parts of the brain. Released histamine participates in the regulation of several physiological functions such as arousal, attention and body homeostasis. Disturbances in the histaminergic system have been detected in diseases such as epilepsy, sleep disorders, anxiety, depression, Alzheimer’s disease, and schizophrenia. The purpose of this thesis was to develop optimal culture conditions for the histaminergic neurons, to study their detailed morphology, and to find out their significance in the kainic acid (KA)-induced neuronal death in the immature rat hippocampus. The morphology of the histaminergic neurons in vitro was comparable with the earlier findings. Histamine-containing vesicles were found in the axon but also in the cell body and dendrites suggesting a possibility for the somatodendritic release. Moreover, histamine was shown to be colocalized with the vesicular monoamine transporter 2 (VMAT2) suggesting that VMAT2 transports histamine to the subcellular storage vesicles. Furthermore, histamine was localized with γ-aminobutyric acid (GABA) in distinct storage vesicles and with neuropeptide galanin partly in the same storage vesicles suggesting different corelease mechanisms for GABA and galanin with histamine. In the organotypic hippocampal slice cultures, KA-induced neuronal death was first detected 12 h after the treatment being restricted mainly to the CA3 subregion. Moreover, cell death was irreversible, since the 48 h recovery period did not save the cells, but instead increased the damage. Finally, neuronal death was suggested to be necrotic, since intracellular apoptotic pathways were not activated, and the morphological changes detected with the electron microscopy were characteristic for necrosis. In the coculture system of the hippocampal and posterior hypothalamic slices, histaminergic neurons significantly decreased epileptiform burst activity and neuronal death in the hippocampal slices, this effect being mediated by histamine 1 (H1) and 3 (H3) receptors. In conclusion, the histaminergic neurons were maintained succesfully in the in vitro conditions exhibiting comparable morphological characteristics as detected earlier in vivo. Moreover, they developed functional innervations within the hippocampal slices in the coculture system. Finally, the KA-induced regionspecific, irreversible and necrotic hippocampal pyramidal cell damage was significantly decreased by the histaminergic neurons through H1 and H3 receptors.

Towards an Integrative Information Society: Studies on Individuality in Speech and Sign

The flow of information within modern information society has increased rapidly over the last decade. The major part of this information flow relies on the individual’s abilities to handle text or speech input. For the majority of us it presents no problems, but there are some individuals who would benefit from other means of conveying information, e.g. signed information flow. During the last decades the new results from various disciplines have all suggested towards the common background and processing for sign and speech and this was one of the key issues that I wanted to investigate further in this thesis. The basis of this thesis is firmly within speech research and that is why I wanted to design analogous test batteries for widely used speech perception tests for signers – to find out whether the results for signers would be the same as in speakers’ perception tests. One of the key findings within biology – and more precisely its effects on speech and communication research – is the mirror neuron system. That finding has enabled us to form new theories about evolution of communication, and it all seems to converge on the hypothesis that all communication has a common core within humans. In this thesis speech and sign are discussed as equal and analogical counterparts of communication and all research methods used in speech are modified for sign. Both speech and sign are thus investigated using similar test batteries. Furthermore, both production and perception of speech and sign are studied separately. An additional framework for studying production is given by gesture research using cry sounds. Results of cry sound research are then compared to results from children acquiring sign language. These results show that individuality manifests itself from very early on in human development. Articulation in adults, both in speech and sign, is studied from two perspectives: normal production and re-learning production when the apparatus has been changed. Normal production is studied both in speech and sign and the effects of changed articulation are studied with regards to speech. Both these studies are done by using carrier sentences. Furthermore, sign production is studied giving the informants possibility for spontaneous speech. The production data from the signing informants is also used as the basis for input in the sign synthesis stimuli used in sign perception test battery. Speech and sign perception were studied using the informants’ answers to questions using forced choice in identification and discrimination tasks. These answers were then compared across language modalities. Three different informant groups participated in the sign perception tests: native signers, sign language interpreters and Finnish adults with no knowledge of any signed language. This gave a chance to investigate which of the characteristics found in the results were due to the language per se and which were due to the changes in modality itself. As the analogous test batteries yielded similar results over different informant groups, some common threads of results could be observed. Starting from very early on in acquiring speech and sign the results were highly individual. However, the results were the same within one individual when the same test was repeated. This individuality of results represented along same patterns across different language modalities and - in some occasions - across language groups. As both modalities yield similar answers to analogous study questions, this has lead us to providing methods for basic input for sign language applications, i.e. signing avatars. This has also given us answers to questions on precision of the animation and intelligibility for the users – what are the parameters that govern intelligibility of synthesised speech or sign and how precise must the animation or synthetic speech be in order for it to be intelligible. The results also give additional support to the well-known fact that intelligibility in fact is not the same as naturalness. In some cases, as shown within the sign perception test battery design, naturalness decreases intelligibility. This also has to be taken into consideration when designing applications. All in all, results from each of the test batteries, be they for signers or speakers, yield strikingly similar patterns, which would indicate yet further support for the common core for all human communication. Thus, we can modify and deepen the phonetic framework models for human communication based on the knowledge obtained from the results of the test batteries within this thesis.

Kainic acid-induced seizures: inflammation and excitotoxic neuronal damage in the developing rat hippocampus

Epileptic seizures are harmful to the developing brain. During epileptic seizures, overactivation of glutamate receptors (GluR) leads to neuronal degeneration, defined as excitotoxicity. The hippocampus is especially vulnerable to excitotoxic neuronal death, but its mechanism has remained incompletely known in the developing brain. Recently, signs of activation of inflammatory processes after epileptic seizures have been detected in the hippocampus. The purpose of this thesis was to study the inflammatory reaction and death mechanisms in excitoxic neurodegeneration induced by the glutamate analogue kainic acid (KA) in the developing hippocampus. Organotypic hippocampal slice cultures (OHCs), prepared from 6-7-day-old rats (P6-7) and treated with KA, served as an in vitro model. KA-induced status epilepticus in P9 and P21 rats was used as an in vivo model. The results showed that the pyramidal cell layers of the hippocampus were the most susceptible to irreversible and age-specific neurodegeneration, which occurred in the juvenile (P21), but not in the immature (P9), rat hippocampus. The primary death mechanism was necrosis as there were no significant changes in the expression of selected apoptosis markers and morphological cellular features of necrosis were found. Inflammatory response was similarly age-dependent after KA treatment as a rapid, fulminant and wide response was detected in the juvenile, but not in the immature, rat brain. An anti-inflammatory drug treatment, given before KA, was not neuroprotective in OHCs, possibly because of the timing of the treatment. In summary, the results suggest that KA induces an age-dependent inflammatory response and necrotic neurodegeneration, which may cause disturbances in hippocampal connectivity and promote epileptogenesis.

Disruptive innovations at the bottom of the pyramid : can they impact the sustainability of today´s top companies?

Due to the different dynamics required for organizations to serve the emerging market which contains billions of people at the bottom of the pyramid (BOP) coupled with the increasing desire for organizations to grow and be more multinational, organizations need to continually innovate. However, the tendency for large and established companies to ignore the BOP market and rather focus on existing markets, gives an indication of the existence of a vulnerability that potentially disruptive innovations from the BOP will not be recognized in good time for a counter measure. This can be deduced from the fact that good management practice advocates that managers should learn and listen to their customers. Therefore majority of the large existing companies continually focus on their main customer/market with sustaining innovations which leaves aspiring new entrants with an underserved BOP market to experiment with. With the aid of research interviews and an agent-based model (ABM) simulation, this thesis examines the attributes of BOP innovations that can qualify them as disruptive and the possibilities of tangible disruptive innovations arising from the bottom of the pyramid and their underlying drivers. The thesis Furthermore, examines the associated impact of such innovations on the future sustainability of established large companies that are operating in the developed world, particularly those with a primary focus which is targeted towards the market at the top of the pyramid (TOP). Additionally, with the use of a scenario planning model, the research provides an evaluation of the possible evolution and potential sustainability impacts that could emerge, from the interplay of innovations at the two pyramidal market levels and the chosen market focus of organizations – TOP or BOP. Using four scenario quadrants, the thesis demonstrates the resulting possibilities from the interaction between the rate of innovations and the segment focused on by organizations with disruptive era characterizing the paradigm shift quadrant. Furthermore, a mathematical model and two theoretical propositions are developed for further research. As recommendations, the thesis also extends the ambidextrous organizational theory, business model innovation and portfolio diversification as plausible recommendations to limit a catastrophic impact, resulting from disruptive innovations.

Motoneuronitautiin liittyvä hengitysvajaus: hengitystoiminnan ja energia-aineenvaihdunnan mittaaminen

Tavoitteet: Tämän tutkimussarjan tavoitteena oli tutkia hengitystoiminnan sekä energia-aineen¬vaihdunnan muutoksia motoneuronitautia (amyotrofinen lateraaliskleroosi, ALS) sairastavilla potilailla. Erityisenä mielenkiinnon kohteena olivat kotihoitoon soveltuvan hengityslaitteen vai¬kutus elinajan ennusteeseen sekä hengitysvajauksen etenemistä kuvaavien keuhkotoimintakokei¬den arviointi ALS-potilailla, epäsuoran kalorimetrian mittaustarkkuus ja perusaineenvaihdunnan (PAV) suuruus kajoavaa hengityslaitetta käyttävillä ALS-potilailla. Aineisto ja menetelmät: Kajoamattoman hengityslaitteen käytön ja iän vaikutusta elinajan en¬nusteeseen arvioitiin 84:llä ja hengitystoiminnan muutoksia 42 ALS-potilaalla. Epäsuoran kalo¬rimetrian mittaustarkkuutta kajoamatonta hengityslaitetta käytettäessä arvioitiin hereillä olevilla 12 vapaaehtoisella mieshenkilöllä. PAV:n suuruutta arvioitiin viidellä kajoavaa hengityslaitetta käyttävällä ALS-potilaalla. Osatöistä kaksi ensimmäistä olivat luonteeltaan havainnoivia (retros¬pektiivisiä) ja kaksi viimeistä seurantatutkimuksia (prospektiivisia). Tulokset: Alle 65-vuotiailla ALS-potilailla ei havaittu eroa elinajan ennusteessa kajoamaton¬ta hengityslaitetta käyttävien ja käyttämättömien potilaiden välillä. Sen sijaan yli 65-vuotiail¬la ALS-potilailla elinajan ennuste piteni merkittävästi kajoamatonta hengityslaitetta käyttävillä potilailla (elinaika diagnoosin jälkeen 22 vs. 8 kk, Hazard Ratio = 0.25, 95 % luottamusväli 0.11 – 0.55, p <0.001). ALS-potilailla, joilla kajoamaton hengityslaite katsottiin tarpeelliseksi kuuden kuukauden kuluessa diagnoosihetkestä, hengitystiheys osoittautui diagnoosihetkellä mer-kittävästi kiihtyneeksi (21/min) ja rintakehän liike merkittävästi alentuneeksi (2.9 cm) verrattuna ALS-potilaisiin, joille kajoamaton hengityslaite katsottiin tarpeelliseksi myöhemmin (16/min ja 4.0 cm). Kajoamattoman hengityslaitehoidon aikana keskimääräinen mitattu PAV vapaaehtoisilla miehillä oli 1858 kcal/vrk kun PAV ilman hengityslaitetta oli 1852 kcal/vrk, p = 0.8. Kajoavaa hengityslaitehoitoa käyttävien viiden ALS-potilaan keskimääräinen PAV vastaavalla mittausase¬telmalla mitattaessa oli 1130 kcal/vrk, kun vastaava PAV laskettuna viidellä eri laskentakaavalla oli 1700 kcal/vrk, p < 0.001. Johtopäätökset: Yli 65-vuotiailla ALS-potilailla, jotka eivät sopeutuneet kajoamattomaan hen¬gityslaitehoitoon, oli nelinkertainen riski menehtyä aiemmin kuin kajoamattomaan hengityslai¬tehoitoon sopeutuneilla ALS-potilailla. Hengitystiheys osoittautui merkittävästi kiihtyneeksi ja rintakehän liike alentuneeksi ALS-potilailla, joille kajoamaton hengityslaitehoito katsottiin ai¬heelliseksi kuuden kuukauden kuluessa diagnoosihetkestä. Kajoamattoman hengityslaitehoidon aikana mitattu PAV ei poikennut mitatusta PAV:sta itsenäisen hengityksen aikana. Näin ollen epäsuoraa kalorimetriamenetelmää voidaan käyttää luotettavasti PAV:n määrittämiseen käytet¬täessä samanaikaisesti kotihoitoon soveltuvaa hengityslaitehoitoa. Elämää ylläpitävää kajoavaa hengityslaitehoitoa käyttävien ALS-potilaiden PAV oli merkittävästi hidastunut laskennallisella menetelmällä arvioituun PAV verrattuna.

Natural born weight gainers: The mechanisms of obesity in transgenic mice overexpressing neuropeptide Y

Neuropeptide Y (NPY) is a neurotransmitter promoting energy storage by activating Y-receptors and thus affecting food intake, thermogenesis and adipose tissue metabolism. NPY is expressed both in the central and sympathetic nervous system. Hypothalamic NPY is known to stimulate feeding, but the effects of noradrenergic neuron NPY are more ambiguous. Chronic stress stimulates fat accumulation via NPY release from noradrenergic neurons. Furthermore, polymorphism in the human Npy gene has been associated with metabolic disturbances and increased NPY secretion after sympathetic stimulation. The main objective of this study was to clarify the mechanisms of noradrenergic neuron NPY in the development of obesity. The metabolic phenotype of a homozygous mouse overexpressing NPY in the brain noradrenergic neurons and sympathetic nervous system (OE-NPYDβH mouse) was characterized. OE-NPYDβH mice had an increased fat mass and body weight, which caused impairments of glucose metabolism and hyperinsulinaemia with age. There were no differences in energy intake or expenditure, but the sympathetic tone was down-regulated and the endocannabinoid system activated. Furthermore, peripheral Y2-receptors in energy-rich conditions played an important role in mediating the fat-accumulating effect of NPY. These results indicate that noradrenergic neuron NPY promotes obesity via direct effects in the periphery and by modulating the sympatho-adrenal and endocannabinoid systems. Additionally, NPY in the central noradrenergic neurons is believed to possess many important roles. The phenotype of the OE-NPYDβH mouse resembles the situations of chronic stress and Npy gene polymorphism and thus these mice may be exploited in testing novel drug candidates for the treatment of obesity.

Late-onset spinal motor neuronopathy- a new neuromuscular disease

The aim of this study was to clarify the clinical phenotype of late-onset spinal motor neuronopathy (LOSMoN), an adult-onset autosomal dominant lower motor neuron disorder identified first in two families in Eastern Finland, in order to clarify its genetic background. Motor neuron disorders (MNDs) are characterized by dysfunction and premature death of motor neurons in the brain and spinal cord. MNDs can manifest at any age of the human lifespan, ranging from pre- or neonatal forms such as spinal muscular atrophy type I (SMA I) to those preferentially affecting the older age groups exemplified by sporadic amyotrophic lateral sclerosis (ALS). With a combination of genetic linkage analysis and genome sequencing using DNA from a total of 55 affected members of 17 families and a whole genome scan, we were able to show that LOSMoN is caused by the c.197G>T p.G66V mutation in the gene CHCHD10. This study showed that LOSMoN has very characteristic features that help to differentiate it from other more malignant forms of motor neuron disease, such as ALS, which was erroneously diagnosed in many patients in our cohort. Lack of fibrillations in the first dorsal interosseus muscle on EMG and extensive grouping of non-atrophic type IIA/2A fibers on muscle biopsy were shown to be common findings in LOSMoN, but rare or absent in ALS patients. The results of this study will help clinicians recognize the characteristic phenotype of LOSMoN disease and thus improve their diagnostic accuracy, and will also allow physicians to provide adequate genetic counseling for patients.