The role of mast cells in ischemic and hemorrhagic brain injury

Stroke, ischemic or hemorrhagic, belongs among the foremost causes of death and disability worldwide. Massive brain swelling is the leading cause of death in large hemispheric strokes and is only modestly alleviated by available treatment. Thrombolysis with tissue plasminogen activator (TPA) is the only approved therapy in acute ischemic stroke, but fear of TPA-mediated hemorrhage is often a reason for withholding this otherwise beneficial treatment. In addition, recanalization of the occluded artery (spontaneously or with thrombolysis) may cause reperfusion injury by promoting brain edema, hemorrhage, and inflammatory cell infiltration. A dominant event underlying these phenomena seems to be disruption of the blood-brain barrier (BBB). In contrast to ischemic stroke, no widely approved clinical therapy exists for intracerebral hemorrhage (ICH), which is associated with poor outcome mainly due to the mass effect of enlarging hematoma and associated brain swelling. Mast cells (MCs) are perivascularly located resident inflammatory cells which contain potent vasoactive, proteolytic, and fibrinolytic substances in their cytoplasmic granules. Experiments from our laboratory showed MC density and their state of granulation to be altered early following focal transient cerebral ischemia, and degranulating MCs were associated with perivascular edema and hemorrhage. (I) Pharmacological MC stabilization led to significantly reduced ischemic brain swelling (40%) and BBB leakage (50%), whereas pharmacological MC degranulation raised these by 90% and 50%, respectively. Pharmacological MC stabilization also revealed a 40% reduction in neutrophil infiltration. Moreover, genetic MC deficiency was associated with an almost 60% reduction in brain swelling, 50% reduction in BBB leakage, and 50% less neutrophil infiltration, compared with controls. (II) TPA induced MC degranulation in vitro. In vivo experiments with post-ischemic TPA administration demonstrated 70- to 100-fold increases in hemorrhage formation (HF) compared with controls HF. HF was significantly reduced by pharmacological MC stabilization at 3 (95%), 6 (75%), and 24 hours (95%) of follow-up. Genetic MC deficiency again supported the role of MCs, leading to 90% reduction in HF at 6 and 24 hours. Pharmacological MC stabilization and genetic MC deficiency were also associated with significant reduction in brain swelling and in neutrophil infiltration. Importantly, these effects translated into a significantly better neurological outcome and lower mortality after 24 hours. (III) Finally, in ICH experiments, pharmacological MC stabilization resulted in significantly less brain swelling, diminished growth in hematoma volume, better neurological scores, and decreased mortality. Pharmacological MC degranulation produced the opposite effects. Genetic MC deficiency revealed a beneficial effect similar to that found with pharmacological MC stabilization. In sum, the role of MCs in these clinically relevant scenarios is supported by a series of experiments performed both in vitro and in vivo. That not only genetic MC deficiency but also drugs targeting MCs could modulate these parameters (translated into better outcome and decreased mortality), suggests a potential therapeutic approach in a number of highly prevalent cerebral insults in which extensive tissue injury is followed by dangerous brain swelling and inflammatory cell infiltration. Furthermore, these experiments could hint at a novel therapy to improve the safety of thrombolytics, and a potential cellular target for those seeking novel forms of treatment for ICH.

Carotid Stenosis, Endarterectomy, and the Brain : Brain microcirculation, diffusion, and cognitive function before and after carotid endarterectomy in patients with a high-grade carotid stenosis

Carotid atherosclerotic disease is a major cause of stroke, but it may remain clinically asymptomatic. The factors that turn the asymptomatic plaque into a symptomatic one are not fully understood, neither are the subtle effects that a high-grade carotid stenosis may have on the brain. The purpose of this study was to evaluate brain microcirculation, diffusion, and cognitive performance in patients with a high-grade stenosis in carotid artery, clinically either symptomatic or asymptomatic, undergoing carotid endarterectomy (CEA). We wanted to find out whether the stenoses are associated with diffusion or perfusion abnormalities of the brain or variation in the cognitive functioning of the patients, and to what extent the potential findings are affected by CEA, and compare the clinically symptomatic and asymptomatic subjects as well as strictly healthy controls. Coagulation and fibrinolytic parameters were compared with the rate microembolic signals (MES) in transcranial Doppler (TCD) and the macroscopic appearance of stenosing plaques in surgery. Patients (n=92) underwent CEA within the study. Blood samples pertaining to coagulation and fibrinolysis were collected before CEA, and the subjects underwent repeated TCD monitoring for MES. A subpopulation (n= 46) underwent MR imaging and repeated neuropsychological examination (preoperative, as well 4 and 100 days after CEA). In MRI, the average apparent diffusion coefficients were higher in the ipsilateral white matter (WM), and altough the interhemispheric difference was abolished by CEA, the levels remained higher than in controls. Symptomatic stenoses were associated with more sluggish perfusion especially in WM, and lower pulsatility of flow in TCD. All patients had poorer cognitive performance than healthy controls. Cognitive functions improved as expected by learning effect despite transient postoperative worsening in a few subjects. Improvement was greater in patients with deepest hypoperfusion, primarily in executive functions. Symptomatic stenoses were associated with higher hematocrit and tissue plasminogen activator antigen levels, as well as higher rate of MES and ulcerated plaques, and better postoperative improvement of vasoreactivity and pulsatility. In light of the findings, carotid stenosis is associated with differences in brain diffusion, perfusion, and cognition. The effect on diffusion in the ipsilateral WM, partially reversible by CEA, may be associated with WM degeneration. Asymptomatic and symptomatic subpopulations differ from each other in terms of hemodynamic adaptation and in their vascular physiological response to removal of stenosis. Although CEA may be associated with a transient cognitive decline, a true improvement of cognitive performance by CEA is possible in patients with the most pronounced perfusion deficits. Mediators of fibrinolysis and unfavourable hemorheology may contribute to the development of a symptomatic disease in patients with a high-grade stenosis.

Effects of Metabolic Risk Factors and Type 1 Diabetes on Brain Glucose and Brain Metabolites

The metabolic syndrome and type 1 diabetes are associated with brain alterations such as cognitive decline brain infarctions, atrophy, and white matter lesions. Despite the importance of these alterations, their pathomechanism is still poorly understood. This study was conducted to investigate brain glucose and metabolites in healthy individuals with an increased cardiovascular risk and in patients with type 1 diabetes in order to discover more information on the nature of the known brain alterations. We studied 43 20- to 45-year-old men. Study I compared two groups of non-diabetic men, one with an accumulation of cardiovascular risk factors and another without. Studies II to IV compared men with type 1 diabetes (duration of diabetes 6.7 ± 5.2 years, no microvascular complications) with non-diabetic men. Brain glucose, N-acetylaspartate (NAA), total creatine (tCr), choline, and myo-inositol (mI) were quantified with proton magnetic resonance spectroscopy in three cerebral regions: frontal cortex, frontal white matter, thalamus, and in cerebellar white matter. Data collection was performed for all participants during fasting glycemia and in a subgroup (Studies III and IV), also during a hyperglycemic clamp that increased plasma glucose concentration by 12 mmol/l. In non-diabetic men, the brain glucose concentration correlated linearly with plasma glucose concentration. The cardiovascular risk group (Study I) had a 13% higher plasma glucose concentration than the control group, but no difference in thalamic glucose content. The risk group thus had lower thalamic glucose content than expected. They also had 17% increased tCr (marker of oxidative metabolism). In the control group, tCr correlated with thalamic glucose content, but in the risk group, tCr correlated instead with fasting plasma glucose and 2-h plasma glucose concentration in the oral glucose tolerance test. Risk factors of the metabolic syndrome, most importantly insulin resistance, may thus influence brain metabolism. During fasting glycemia (Study II), regional variation in the cerebral glucose levels appeared in the non-diabetic subjects but not in those with diabetes. In diabetic patients, excess glucose had accumulated predominantly in the white matter where the metabolite alterations were also the most pronounced. Compared to the controls values, the white matter NAA (marker of neuronal metabolism) was 6% lower and mI (glia cell marker) 20% higher. Hyperglycemia is therefore a potent risk factor for diabetic brain disease and the metabolic brain alterations may appear even before any peripheral microvascular complications are detectable. During acute hyperglycemia (Study III), the increase in cerebral glucose content in the patients with type 1 diabetes was, dependent on brain region, between 1.1 and 2.0 mmol/l. An every-day hyperglycemic episode in a diabetic patient may therefore as much as double brain glucose concentration. While chronic hyperglycemia had led to accumulation of glucose in the white matter, acute hyperglycemia burdened predominantly the gray matter. Acute hyperglycemia also revealed that chronic fluctuation in blood glucose may be associated with alterations in glucose uptake or in metabolism in the thalamus. The cerebellar white matter appeared very differently from the cerebral (Study IV). In the non-diabetic men it contained twice as much glucose as the cerebrum. Diabetes had altered neither its glucose content nor the brain metabolites. The cerebellum seems therefore more resistant to the effects of hyperglycemia than is the cerebrum.

Brain and Spinal Cavernomas : Helsinki Experience

Cavernomas are rare neurovascular lesions, encountered in up to 10% of patients harboring vascular abnormalities of the CNS. Cavernomas consist of dilated thin-walled sinusoids or caverns covered by a single layer of endothelium. Due to advancements in neuroradiology, the number of cavernoma patients coming to be evaluated in neurosurgical practice is increasing. In the present work, we summarized our results on the treatment of cavernomas. Particular attention was paid to uncommon locations or insufficiently investigated cavernomas, including 1. Intraventricular cavernomas; 2. Multiple cavernomas; 3. Spinal cavernomas; and 4. Temporal lobe cavernomas. After analyzing the patient series with these lesions, we concluded that: 1. IVCs are characterized by a high tendency to cause repetitive hemorrhages in a short period of time after the first event. In most patients, hemorrhages were not life-threatening. Surgery is indicated when re-bleedings are frequent and the mass-effect causes progressive neurological deterioration. Modern microsurgical techniques allow safe removal of the IVC, but surgery on fourth ventricle cavernomas carries increased risk of postoperative cranial nerve deficits. 2. In MC cases, when the cavernoma bleeds or generates drug-resistant epilepsy, microsurgical removal of the symptomatic lesion is beneficial to patients. In our series, surgical removal of the most active cavernoma usually the biggest lesion with signs of recent hemorrhage - was safe and prevented further bleedings. Epilepsy outcome showed the effectiveness of active treatment of MCs. However, due to the remaining cavernomas, epileptogenic activity can persist postoperatively, frequently necessitating long-term use of antiepileptic drugs. 3. Spinal cavernomas can cause severe neurological deterioration due to low tolerance of the spinal cord to mass-effect with progressive myelopathy. When aggravated by extralesional massive hemorrhage, neurological decline is usually acute and requires immediate treatment. Microsurgical removal of a cavernoma is effective and safe, improving neurological deficits. Sensorimotor deficits and pain improved postoperatively at a high rate, whereas bladder dysfunction remained essentially unchanged, causing social discomfort to patients. 4. Microsurgical removal of temporal lobe cavernomas is beneficial for patents suffering from drug-resistant epilepsy. In our series, 69% of patients with this condition became seizure-free postoperatively. Duration of epilepsy did not correlate with seizure prognosis. The most frequent disabling symptom at follow-up was memory disorder, considered to be the result of a complex interplay between chronic epilepsy and possible damage to the temporal lobe during surgery.

Brain imaging of chronic pain

Acute pain has substantial survival value because of its protective function in the everyday environment. Instead, chronic pain lacks survival and adaptive function, causes great amount of individual suffering, and consumes the resources of the society due to the treatment costs and loss of production. The treatment of chronic pain has remained challenging because of inadequate understanding of mechanisms working at different levels of the nervous system in the development, modulation, and maintenance of chronic pain. Especially in unclear chronic pain conditions the treatment may be suboptimal because it can not be targeted to the underlying mechanisms. Noninvasive neuroimaging techniques have greatly contributed to our understanding of brain activity associated with pain in healthy individuals. Many previous studies, focusing on brain activations to acute experimental pain in healthy individuals, have consistently demonstrated a widely-distributed network of brain regions that participate in the processing of acute pain. The aim of the present thesis was to employ non-invasive brain imaging to better understand the brain mechanisms in patients suffering from chronic pain. In Study I, we used magnetoencephalography (MEG) to measure cortical responses to painful laser stimulation in healthy individuals for optimization of the stimulus parameters for patient studies. In Studies II and III, we monitored with MEG the cortical processing of touch and acute pain in patients with complex regional pain syndrome (CRPS). We found persisting plastic changes in the hand representation area of the primary somatosensory (SI) cortex, suggesting that chronic pain causes cortical reorganization. Responses in the posterior parietal cortex to both tactile and painful laser stimulation were attenuated, which could be associated with neglect-like symptoms of the patients. The primary motor cortex reactivity to acute pain was reduced in patients who had stronger spontaneous pain and weaker grip strength in the painful hand. The tight coupling between spontaneous pain and motor dysfunction supports the idea that motor rehabilitation is important in CRPS. In Studies IV and V we used MEG and functional magnetic resonance imaging (fMRI) to investigate the central processing of touch and acute pain in patients who suffered from recurrent herpes simplex virus infections and from chronic widespread pain in one side of the body. With MEG, we found plastic changes in the SI cortex, suggesting that many different types of chronic pain may be associated with similar cortical reorganization. With fMRI, we found functional and morphological changes in the central pain circuitry, as an indication of central contribution for the pain. These results show that chronic pain is associated with morphological and functional changes in the brain, and that such changes can be measured with functional imaging.

Brain oscillatory responses, working memory and cognitive developmental stages in 11- and 14-year-old children: an ERD/ERS study

Aim: So far, most of the cognitive neuroscience studies investigating the development of brain activity in childhood have made comparisons between different age groups and ignored the individual stage of cognitive development. Given the wide variation in the rate of cognitive development, this study argues that chronological age alone cannot explain the developmental changes in brain activity. This study demonstrates how Piaget s theory and information on child s individual stage of development can complement the age-related evaluations of brain oscillatory activity. In addition, the relationship between cognitive development and working memory is investigated. Method: A total of 33 children (17 11-year-olds, 16 14-year-olds) participated in this study. The study consisted of behavioural tests and an EEG experiment. Behavioral tests included two Piagetian tasks (the Volume and Density task, the Pendulum task) and Raven s Standard Progressive Matrices task. During EEG experiment, subjects performed a modified version of the Sternberg s memory search paradigm which consisted of an auditorily presented memory set of 4 words and a probe word following these. The EEG data was analyzed using the event-related desynchronization / synchronization (ERD/ERS) method. The Pendulum task was used to assess the cognitive developmental stage of each subject and to form four groups based on age (11- or 14-year-olds) and cognitive developmental stage (concrete or formal operational stage). Group comparisons between these four groups were performed for the EEG data. Results and conclusions: Both age- and cognitive stage-related differences in brain oscillatory activity were found between the four groups. Importantly, age-related changes similar to those reported by previous studies were found also in this study, but these changes were modified by developmental stage. In addition, the results support a strong link between working memory and cognitive development by demonstrating differences in memory task related brain activity and cognitive developmental stages. Based on these findings it is suggested that in the future, comparisons of development of brain activity should not be based only on age but also on the individual cognitive developmental stage.

Music in the recovering brain

Listening to music involves a widely distributed bilateral network of brain regions that controls many auditory perceptual, cognitive, emotional, and motor functions. Exposure to music can also temporarily improve mood, reduce stress, and enhance cognitive performance as well as promote neural plasticity. However, very little is currently known about the relationship between music perception and auditory and cognitive processes or about the potential therapeutic effects of listening to music after neural damage. This thesis explores the interplay of auditory, cognitive, and emotional factors related to music processing after a middle cerebral artery (MCA) stroke. In the acute recovery phase, 60 MCA stroke patients were randomly assigned to a music listening group, an audio book listening group, or a control group. All patients underwent neuropsychological assessments, magnetoencephalography (MEG) measurements, and magnetic resonance imaging (MRI) scans repeatedly during a six-month post-stroke period. The results revealed that amusia, a deficit of music perception, is a common and persistent deficit after a stroke, especially if the stroke affects the frontal and temporal brain areas in the right hemisphere. Amusia is clearly associated with deficits in both auditory encoding, as indicated by the magnetic mismatch negativity (MMNm) response, and domain-general cognitive processes, such as attention, working memory, and executive functions. Furthermore, both music and audio book listening increased the MMNm, whereas only music listening improved the recovery of verbal memory and focused attention as well as prevented a depressed and confused mood during the first post-stroke months. These findings indicate a close link between musical, auditory, and cognitive processes in the brain. Importantly, they also encourage the use of listening to music as a rehabilitative leisure activity after a stroke and suggest that the auditory environment can induce long-term plastic changes in the recovering brain.

Identification of TGFβ signaling, p53, and actin stress fibers as targets of LKB1 tumor suppressor activity

Tumorigenesis is a consequence of inactivating mutations of tumor suppressor genes and activating mutations of proto-oncogenes. Most of the mutations compromise cell autonomous and non-autonomous restrains on cell proliferation by modulating kinase signal transduction pathways. LKB1 is a tumor suppressor kinase whose sporadic mutations are frequently found in non-small cell lung cancer and cervical cancer. Germ-line mutations in the LKB1 gene lead to Peutz-Jeghers syndrome with an increased risk of cancer and development of benign gastrointestinal hamartomatous polyps consisting of hyperproliferative epithelia and prominent stromal stalk composed of smooth muscle cell lineage cells. The tumor suppressive function of LKB1 is possibly mediated by 14 identified LKB1 substrate kinases, whose activation is dependent on the LKB1 kinase complex. The aim of my thesis was to identify cell signaling pathways crucial for tumor suppression by LKB1. Re-introduction of LKB1 expression in the melanoma cell line G361 induces cell cycle arrest. Here we demonstrated that restoring the cytoplasmic LKB1 was sufficient to induce the cell cycle arrest in a tumor suppressor p53 dependent manner. To address the role of LKB1 in gastrointestinal tumor suppression, Lkb1 was deleted specifically in SMC lineage in vivo, which was sufficient to cause Peutz-Jeghers syndrome type polyposis. Studies on primary myofibroblasts lacking Lkb1 suggest that the regulation of TGFβ signaling, actin stress fibers and smooth muscle cell lineage differentiation are candidate mechanisms for tumor suppression by LKB1 in the gastrointestinal stroma. Further studies with LKB1 substrate kinase NUAK2 in HeLa cells indicate that NUAK2 is part of a positive feedback loop by which NUAK2 expression promotes actin stress fiber formation and, reciprocally the induction of actin stress fibers promote NUAK2 expression. Findings in this thesis suggest that p53 and TGFβ signaling pathways are potential mediators of tumor suppression by LKB1. An indication of NUAK2 in the promotion of actin stress fibers suggests that NUAK2 is one possible mediator of LKB1 dependent TGFβ signaling and smooth muscle cell lineage differentiation.

Tonically Active Kainate Receptors (tKARs) : A Novel Mechanism Regulating Neuronal Function in the Brain

Fast excitatory transmission between neurons in the central nervous system is mainly mediated by L-glutamate acting on ligand gated (ionotropic) receptors. These are further categorized according to their pharmacological properties to AMPA (2-amino-3-(5-methyl-3-oxo-1,2- oxazol-4-yl)propanoic acid), NMDA (N-Methyl-D-aspartic acid) and kainate (KAR) subclasses. In the rat and the mouse hippocampus, development of glutamatergic transmission is most dynamic during the first postnatal weeks. This coincides with the declining developmental expression of the GluK1 subunit-containing KARs. However, the function of KARs during early development of the brain is poorly understood. The present study reveals novel types of tonically active KARs (hereafter referred to as tKARs) which play a central role in functional development of the hippocampal CA3-CA1 network. The study shows for the first time how concomitant pre- and postsynaptic KAR function contributes to development of CA3-CA1 circuitry by regulating transmitter release and interneuron excitability. Moreover, the tKAR-dependent regulation of transmitter release provides a novel mechanism for silencing and unsilencing early synapses and thus shaping the early synaptic connectivity. The role of GluK1-containing KARs was studied in area CA3 of the neonatal hippocampus. The data demonstrate that presynaptic KARs in excitatory synapses to both pyramidal cells and interneurons are tonically activated by ambient glutamate and that they regulate glutamate release differentially, depending on target cell type. At synapses to pyramidal cells these tKARs inhibit glutamate release in a G-protein dependent manner but in contrast, at synapses to interneurons, tKARs facilitate glutamate release. On the network level these mechanisms act together upregulating activity of GABAergic microcircuits and promoting endogenous hippocampal network oscillations. By virtue of this, tKARs are likely to have an instrumental role in the functional development of the hippocampal circuitry. The next step was to investigate the role of GluK1 -containing receptors in the regulation of interneuron excitability. The spontaneous firing of interneurons in the CA3 stratum lucidum is markedly decreased during development. The shift involves tKARs that inhibit medium-duration afterhyperpolarization (mAHP) in these neurons during the first postnatal week. This promotes burst spiking of interneurons and thereby increases GABAergic activity in the network synergistically with the tKAR-mediated facilitation of their excitatory drive. During development the amplitude of evoked medium afterhyperpolarizing current (ImAHP) is dramatically increased due to decoupling tKAR activation and ImAHP modulation. These changes take place at the same time when the endogeneous network oscillations disappear. These tKAR-driven mechanisms in the CA3 area regulate both GABAergic and glutamatergic transmission and thus gate the feedforward excitatory drive to the area CA1. Here presynaptic tKARs to CA1 pyramidal cells suppress glutamate release and enable strong facilitation in response to high-frequency input. Therefore, CA1 synapses are finely tuned to high-frequency transmission; an activity pattern that is common in neonatal CA3-CA1 circuitry both in vivo and in vitro. The tKAR-regulated release probability acts as a novel presynaptic silencing mechanism that can be unsilenced in response to Hebbian activity. The present results shed new light on the mechanisms modulating the early network activity that paves the way for oscillations lying behind cognitive tasks such as learning and memory. Kainate receptor antagonists are already being developed for therapeutic use for instance against pain and migraine. Because of these modulatory actions, tKARs also represent an attractive candidate for therapeutic treatment of developmentally related complications such as learning disabilities.

Functional outcome and health-related quality of life after traumatic brain injury in the framework of the International Classification of Functioning, Disability and Health (ICF)

Traumatic brain injury (TBI) affects people of all ages and is a cause of long-term disability. In recent years, the epidemiological patterns of TBI have been changing. TBI is a heterogeneous disorder with different forms of presentation and highly individual outcome regarding functioning and health-related quality of life (HRQoL). The meaning of disability differs from person to person based on the individual s personality, value system, past experience, and the purpose he or she sees in life. Understanding of all these viewpoints is needed in comprehensive rehabilitation. This study examines the epidemiology of TBI in Finland as well as functioning and HRQoL after TBI, and compares the subjective and objective assessments of outcome. The frame of reference is the International Classification of Functioning, Disability and Health (ICF). The subjects of Study I represent the population of Finnish TBI patients who experienced their first TBI between 1991 and 2005. The 55 Finnish subjects of Studies II and IV participated in the first wave of the international Quality of life after brain injury (QOLIBRI) validation study. The 795 subjects from six language areas of Study III formed the second wave of the QOLIBRI validation study. The average annual incidence of Finnish hospitalised TBI patients during the years 1991-2005 was 101:100 000 in patients who had TBI as the primary diagnosis and did not have a previous TBI in their medical history. Males (59.2%) were at considerably higher risk of getting a TBI than females. The most common external cause of the injury was falls in all age groups. The number of TBI patients ≥ 70 years of age increased by 59.4% while the number of inhabitants older than 70 years increased by 30.3% in the population of Finland during the same time period. The functioning of a sample of 55 persons with TBI was assessed by extracting information from the patients medical documents using the ICF checklist. The most common problems were found in the ICF components of Body Functions (b) and Activities and Participation (d). HRQoL was assessed with the QOLIBRI which showed the highest level of satisfaction on the Emotions, Physical Problems and Daily Life and Autonomy scales. The highest scores were obtained by the youngest participants and participants living independently without the help of other people, and by people who were working. The relationship between the functional outcome and HRQoL was not straightforward. The procedure of linking the QOLIBRI and the GOSE to the ICF showed that these two outcome measures cover the relevant domains of TBI patients functioning. The QOLIBRI provides the patients subjective view, while the GOSE summarises the objective elements of functioning. Our study indicates that there are certain domains of functioning that are not traditionally sufficiently documented but are important for the HRQoL of persons with TBI. This was the finding especially in the domains of interpersonal relationships, social and leisure activities, self, and the environment. Rehabilitation aims to optimize functioning and to minimize the experience of disability among people with health conditions, and it needs to be based on a comprehensive understanding of human functioning. As an integrative model, the ICF may serve as a frame of reference in achieving such an understanding.

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.

Functional regulation of the Neurofibromatosis 2 tumor suppressor merlin

Neurofibromatosis 2 (NF2) is an autosomal dominant disorder manifested by the formation of multiple benign tumors of the nervous system. Affected individuals typically develop bilateral vestibular schwannomas which lead to deafness and balance disorders. The syndrome is caused by inactivation of the NF2 tumor suppressor gene, and mutation or loss of the NF2 product, merlin, is sufficient for tumorigenesis in both hereditary and sporadic NF2-associated tumors. Merlin belongs to the band 4.1 superfamily of cytoskeletal proteins, which also contain the related ezrin, radixin, and moesin (ERM) proteins. The ERM members provide a link between the cell cytoskeleton and membrane by connecting membrane-associated proteins to actin filaments. By stabilizing complexes in the cell cortex, the ERMs modulate morphology, growth, and migration of cells. Despite their structural homology, overlapping subcellular distribution, direct molecular association, and partial overlap of molecular interactions, merlin and ezrin exert opposite effects on cell proliferation. Merlin suppresses cell proliferation, whereas ezrin expression is linked to oncogenic activity. We hypothesized that the regions which differ between the proteins might explain merlin s specificity as a tumor suppressor. We therefore analyzed the regions, which are most diverse between merlin and ezrin; the N-terminal tail and the C-terminus. To determine the properties of the C-terminal region, we studied the two most predominant merlin isoforms together with truncation variants similar to those found in patients. We also focused on the evolutionally conserved C-terminal residues, E545-E547, that harbor disease causing mutations in its corresponding DNA sequence. In addition to inhibiting cell proliferation, merlin regulates cytoskeletal organization. The morphogenic properties of merlin may play a role in tumor suppression, since patient-derived tumor cells demonstrate cytoskeletal abnormalities. We analyzed the mechanisms of merlin-induced extension formation and determined that the C-terminal region of amino acids 538-568 is particularly important for the morphogenic activity. We also characterized the role of C-terminal merlin residues in the regulation of proliferation, phosphorylation, and intramolecular associations. In contrast to previous reports, we demonstrated that both merlin isoforms are able to suppress cell proliferation, whereas C-terminally mutated merlin constructs showed reduced growth inhibition. Phosphorylation serves as a mechanism to regulate the tumor suppressive activity of merlin. The C-terminal serine 518 is phosphorylated in response to both p21-activated kinase (PAK) and protein kinase A (PKA), which inactivates the growth inhibitory function of merlin. However, at least three differentially phosphorylated forms of the protein exist. In this study we demonstrated that also the N-terminus of merlin is phosphorylated by AGC kinases, and that both PKA and Akt phosphorylate merlin at serine 10 (S10). We evaluated the impact of this N-terminal tail phosphorylation, and showed that the phosphorylation state of S10 is an important regulator of merlin s ability to modulate cytoskeletal organization but also regulates the stability of the protein. In summary, this study describes the functional effect of merlin specific regions. We demonstrate that both S10 in the N-terminal tail and residues E545-E547 in the C-terminus are essential for merlin activity and function.

Literature Review: Investigating Drug Transport at the Blood-Brain Barrier and the Effects of P-glycoprotein on the Brain Distribution of Drugs.

The blood-brain barrier (BBB) is a unique barrier that strictly regulates the entry of endogenous substrates and xenobiotics into the brain. This is due to its tight junctions and the array of transporters and metabolic enzymes that are expressed. The determination of brain concentrations in vivo is difficult, laborious and expensive which means that there is interest in developing predictive tools of brain distribution. Predicting brain concentrations is important even in early drug development to ensure efficacy of central nervous system (CNS) targeted drugs and safety of non-CNS drugs. The literature review covers the most common current in vitro, in vivo and in silico methods of studying transport into the brain, concentrating on transporter effects. The consequences of efflux mediated by p-glycoprotein, the most widely characterized transporter expressed at the BBB, is also discussed. The aim of the experimental study was to build a pharmacokinetic (PK) model to describe p-glycoprotein substrate drug concentrations in the brain using commonly measured in vivo parameters of brain distribution. The possibility of replacing in vivo parameter values with their in vitro counterparts was also studied. All data for the study was taken from the literature. A simple 2-compartment PK model was built using the Stella™ software. Brain concentrations of morphine, loperamide and quinidine were simulated and compared with published studies. Correlation of in vitro measured efflux ratio (ER) from different studies was evaluated in addition to studying correlation between in vitro and in vivo measured ER. A Stella™ model was also constructed to simulate an in vitro transcellular monolayer experiment, to study the sensitivity of measured ER to changes in passive permeability and Michaelis-Menten kinetic parameter values. Interspecies differences in rats and mice were investigated with regards to brain permeability and drug binding in brain tissue. Although the PK brain model was able to capture the concentration-time profiles for all 3 compounds in both brain and plasma and performed fairly well for morphine, for quinidine it underestimated and for loperamide it overestimated brain concentrations. Because the ratio of concentrations in brain and blood is dependent on the ER, it is suggested that the variable values cited for this parameter and its inaccuracy could be one explanation for the failure of predictions. Validation of the model with more compounds is needed to draw further conclusions. In vitro ER showed variable correlation between studies, indicating variability due to experimental factors such as test concentration, but overall differences were small. Good correlation between in vitro and in vivo ER at low concentrations supports the possibility of using of in vitro ER in the PK model. The in vitro simulation illustrated that in the simulation setting, efflux is significant only with low passive permeability, which highlights the fact that the cell model used to measure ER must have low enough paracellular permeability to correctly mimic the in vivo situation.