Browsing by Subject "neuroscience"

Sort by: Order: Results:

Now showing items 1-20 of 23
  • Saure, Emma (Helsingin yliopisto, 2018)
    Background and objectives: Autism spectrum disorders (ASD) are developmental neuropsychiatric disorders in which core symptoms are problems in communication and interaction as well as restrictive and repetitive behaviour and interests. ASD is 2-5 times more common in males than in females. In recent years, researchers have found, that there are differences between females and males in ASD symptoms, neuropsychological characteristics, comorbid problems, neurobiology and etiology. The purpose of this systematic review is to give a comprehensive picture about the role of female sex/gender in ASD. To establish this, the review covers symptoms of autism, neuropsychology, neurobiology, comorbidity, neurogenetics and neuroendocrinology. Research questions were the following: 1) Is there evidence of sex/gender differences in ASD symptoms and comorbidity disorders? 2) Are there sex/gender differences to be found in ASD etiology? 3) What kind of support different explanations about sex/gender bias have gotten in various research areas? The purpose of the study is also to integrate the existing theories into one model that takes account to different aspects of sex/gender differences in ASD. Methods: The protocol of this systematic review follows "The Preferred Reporting Items for Systematic Reviews and Meta-Analyses" (PRISMA) when applicable. Eligibly criteria and search terms were selected in a way that would offer the widest range of articles covering the subjects of this study. Literature search was conducted using the Medline and PsychINFO as search engines. The final sample consisted of a total of 129 articles. Data was extracted on all relevant variables of the study, that were the number of participants, age of participants, specific diagnoses, methods and results. Results: Sex/gender differences in ASD were found in all areas that were included in this systematic review. Females with high function ASD (HFASD) were found to have less problems in social communication and interaction and less repetitive and restricted behavior and interests than males with HFASD. In addition, HFASD were found to have better language skills than males with HFASD. However, females with ASD were found to have more sensory processing problems, mental health problems and epilepsy than males with ASD. Females with ASD were also found to have lower full-scale intelligence quotient than males with ASD. In the context of etiology, it has been found that there are sex/gender differences in neuroanatomy, susceptibility genes and hormone levels. Conclusions: Results from this systematic review suggest that females with HFASD are underdiagnosed. This results from etiological sex/gender differences that cause partially different clinical presentation of ASD between females and males. ASD research has also concentrated mostly on males with ASD while ignoring females with ASD. Underdiagnosing can have many unfavorable consequences for females with HFASD since if they do not have a diagnosis, they do not get support. In the future, it is crucial to pay attention to females with ASD in the clinical work and scientific research.
  • Tokariev, Maksym (Helsingin yliopisto, 2020)
    Structural and functional development of the brain during the maturation results in an improvement of higher cognitive abilities such as attention, working memory (WM), and executive functions. The most pronounced changes take place in the frontal, parietal and temporal association cortices which orchestrate the goal-directed behaviour in response to the incoming information. During development, a set of brain regions establishes a processing system of functionally coupled large-scale networks which maintains higher cognition. As an example, the frontal and parietal areas constitute the fronto-parietal network which supports WM and selective attention. The protracted maturation of the association brain regions such as the prefrontal cortex (PFC), which also regulates activity in posterior brain regions, is reflected in the age-related gradual improvement of cognitive abilities in children. The neuroanatomical basis of higher cognition starts to shape during the third trimester of pregnancy when the brain undergoes significant structural changes and forms the precursors of neuronal networks that will support a wide range of cognitive functions. This critical stage of brain development is extremely sensitive to endogenous and environmental factors. Extreme preterm birth (< 28 gestational week) poses a high risk of developing lifelong neurological and cognitive impairments. This thesis investigated the functional organization of brain regions underlying attention and WM, and the network functional connectivity in young, school-aged children. Study I investigated task-related activation and top-down regulation induced by PFC in the category-specific visual areas in healthy children and young adults. In three studies of the thesis the consequences of extremely preterm birth were investigated in 7.5-year-old children. Study II investigated the distribution of brain activation during visuospatial n-back tasks and Study IV the dynamics of functional connectivity between two brain states, resting state and WM task performance. Moreover, the effects of extremely preterm birth on the white matter microstructure were investigated in Studies II and III. The data for thesis were obtained using functional magnetic resonance imaging and diffusion tensor imaging (DTI) techniques. Study I found that both adults and children activated the retrosplenial complex (RSC) and parahippocampal place area (PPA) during scene processing, although the RSC responded less than the PPA. Adults demonstrated weaker task-related modulation of activity in the RSC compared with the PPA, while this modulation in children was comparable between the two regions. Together, these results suggest that cognitive control over category specific regions is still under development in 7–11-year-old children. Study II investigated brain responsivity during WM n-back tasks and the white mater microstructure in extremely preterm-born (EPB) and term-born (TB) children. The EPB, compared with TB children, showed weaker WM-related brain activation in the PFC and weaker deactivation of the right temporal lobe. Moreover, they failed to recruit additional neuronal resources when the cognitive load was increased. In addition, the EPB children performed the n-back tasks poorer than the TB children during the scanning. The EPB children also showed alteration of the white matter microstructure in multiple white matter tracts. Unlike the EPB children, the TB children showed significant associations between the microstructure of the anatomical connections and performance of the n-back tasks. Overall, these results suggest that even in EPB children without neurological or neurosensory impairments, the microstructure of the white matter neural tracts and recruitment of brain areas related to WM are compromised. Study III presents the protocol that was used in Study II for the preprocessing and analysis of DTI data acquired from young school-aged children. The protocol pointed out several problems related to the DTI from child populations, and presented solutions to the collecting of the data, preprocessing, template generation, data registration and statistical analysis. Study IV explored differences in the functional architecture of the brain networks between EPB and TB children during resting state and task performance. Tasks performance showed stronger functional connectivity (FC) within and between the regions of the default mode and fronto-parietal networks that are involved in WM processing, whereas the resting state connectivity was largely characterized by stronger between-network FC. The EPB, compared with TB controls, exhibited weaker dynamic modulation of FC between resting state and task performance. In both groups, larger modulation of FC between brain states associated with better performance of the tasks. These results underline the importance of flexible network connectivity for cognitive performance and demonstrate that this ability may be compromised in preterm-born children with no obvious cognitive impairments. The thesis demonstrates a protracted functional specialization for the visual category-selective brain regions and their top-down regulation in school-aged children. The thesis also shows that extreme prematurity may be reflected on the white matter microstructure, brain responsiveness and functional connectivity even in school-aged children with normal global cognitive abilities.  
  • Merezhko, Maria (Helsingin yliopisto, 2020)
    Neurodegenerative disorders are progressive, age-dependent, devastating conditions with only symptomatic treatment available. The progressive accumulation and spread of misfolded proteins in the nervous system is the common attribute of multiple neurodegenerative diseases. In Alzheimer’s disease (AD), two types of aggregates accumulate and spread through the brain: extracellular amyloid plaques composed of β-amyloid peptide and intracellular neurofibrillary tangles composed of hyperphosphorylated tau protein. β-amyloid peptide originates from the pathological processing of amyloid precursor protein (APP). APP processing is susceptible to various stimuli and controlled by multiple proteins and interactions with lipids. The regulation of APP processing, however, is not fully understood. Tau is one of the major microtubule-associated proteins in neurons. In AD and other tauopathies, however, tau becomes hyperphosphorylated, detaches from microtubules, and first forms small soluble oligomers and then larger, insoluble aggregates. Like several other neurodegeneration-related proteins, pathological tau spreads through the brain via cell-to-cell transmission, which involves secretion and internalization stages, and can initiate templated misfolding of normal tau in recipient cells. Unfortunately, the mechanisms of cell-to-cell transfer and templated misfolding of tau are rather elusive. The aim of this thesis is to (1) develop novel assays to advance the understanding of the regulation of processing and trafficking of neurodegenerative-related proteins and (2) investigate the molecular mechanisms of tau secretion. In this thesis, two novel in vitro live-cell assays were developed based on protein-fragment complementation to study APP, tau, and other neurodegeneration-associated proteins. The first assay can generate multiple readouts, reflecting cellular fates of APP: total cellular APP level, total secreted sAPP level in the media, APP-BACE1 interaction in cells, and in culture media. The second assay can monitor protein localization to dynamic nanoscale cholesterol/sphingomyelin-rich microdomains at the plasma membrane, usually called lipid rafts. This assay may be beneficial for neurodegenerative disease research, as many misfolded proteins associate with lipid rafts, including APP and tau. Additionally, this thesis addressed the molecular mechanisms of tau secretion. In N2A cells overexpressing human tau as well as in primary neurons, tau secretion to the extracellular space was shown to occur via an unconventional, vesicle-free mechanism. Imaging studies have revealed that tau clusters at the plasma membrane in the discrete microdomains and does not localize to membranous intracellular organelles. Instead, tau secretion depended on the lipid composition of the plasma membrane, particularly on lipid-organizing lipid rafts, such as cholesterol and sphingolipids. Tau secretion was also shown to depend on its oligomerization state and heparan sulfate proteoglycans at the cell surface. The data collectively suggest that tau secretion happens via translocation through the plasma membrane, which likely occurs in lipid rafts. In summary, the studies included in this thesis provide both methodological and conceptual insights in the field of neurodegeneration.
  • Bourbia, Nora (Helsingin yliopisto, 2015)
    The central nucleus of amygdala (CeA) is known to be involved in pain and nociception, but the mechanisms or its role in descending control of pain-related behavior is poorly understood. The aim of this study was to investigate the involvement of the neuropeptide corticotropin-releasing factor (CRF) and the glutamatergic system of the CeA in pain and nociception in healthy control animals and in an animal model of chronic neuropathic pain induced by spared-nerve injury (SNI). Two aspects of pain were studied: emotional-like pain behavior was assessed by using the aversive place-avoidance paradigm and sensory-discriminative was assessed by determining the mechanical limb-withdrawal threshold and the thermal (heat) limb-withdrawal latency. Moreover, the aims were to determine whether medullospinal serotoninergic pathways and the midbrain periaqueductal grey (PAG), respectively, were involved in relaying pain-modulation induced by the CeA in SNI and healthy control animals. Additionally, hemisphere of the CeA and submodality of pain stimulus were among studied parameters. Surgical procedures and electrophysiological recordings were performed under general anesthesia. The studies on the role of the CeA in the emotional-like aspect of pain in SNI rats revealed that activation and blocking of the group I metabotropic glutamate receptors (mGluRs) facilitates and inhibits, respectively, the aversive aspect of pain. Furthermore, increase of endogenous CRF as well as blocking glutamatergic N-methyl-D-aspartate (NMDA) receptors in the CeA reduced the aversive aspect of neuropathic pain. The studies on the sensory-discriminative aspect of pain revealed that an increase of endogenous CRF in the CeA is pronociceptive in both control and SNI rats. CeA injection of a high dose of glutamate had a mechanical antinociceptive effect that was mediated by NMDA receptors in healthy but not SNI rats. A low dose of glutamate had a pronociceptive effect mediated by NMDA receptors in SNI rats. Furthermore, tonic descending pronociception induced by NMDA receptors and the mGluR1 in the CeA contributes to the maintenance of neuropathic hypersensitivity. The investigation on the role of serotonergic neurons of the rostroventromedial medulla (RVM) in modulation of spinal nociception by amygdaloid glutamate in SNI rats indicated that the RVM is a relay for both descending pro- and antinociceptive effects from the CeA. The investigation on the role of the PAG in the descending control of nociception induced by glutamate in the CeA of healthy rats indicated that the PAG is a relay in the descending control of nociception induced by amygdaloid glutamate. Furthermore, the right-hemispheric lateralization of the pronociceptive effect by amygdaloid CRF in controls was lost in SNI rats. However, descending antinociception induced by the glutamatergic system of the CeA showed no hemispheric lateralization in healthy controls; a high dose of glutamate in both the left and right CeA induced equal attenuations of mechanical and thermal nociception, which effects were, respectively, NMDA-dependent and NDMA-independent.
  • Visala, Aku (2021)
    Some philosophers and scientists have argued that we humans cannot be held morally responsible for anything. Invoking results of the neurosciences and the cognitive sciences, they argue that humans lack the kind of conscious control and awareness required for moral responsibility. For theological ethics and Christian theology as a whole, moral responsibility is indispensable. I will begin by outlining some empirical results that are invoked in support of moral responsibility skepticism. I will, then, examine the subsequent discussion and the question why conscious awareness is central to moral responsibility. Consciousness contributes to morally relevant control over action in multiple ways. I will briefly examine some accounts of conscious control that are resistant to the skeptical challenge. Although the empirical results might lead us to revise the degree and range of conscious control, there seems to be enough of it to ground many everyday practices of responsibility. I will conclude the article with some theological reflections.
  • Luchkina, Natalia V. (Helsingin yliopisto, 2015)
    Activity-dependent synaptic plasticity, and long-term potentiation in particular, represents the predominant model of memory and learning at the cellular level. In addition, synaptic plasticity plays a critical role in the activity-dependent refinement and fine-tuning of neuronal circuits during development by maintaining and stabilising certain synaptic connections and eliminating others. The main goal of this project was to increase our understanding of the molecular mechanisms underlying activity-dependent synaptic plasticity in the developing brain, with particular emphasis on the mechanisms that are specific to early postnatal development. First, we characterise in detail the properties of developmentally restricted neonatal presynaptic long-term potentiation (LTP) in CA1 area of the hippocampus and demonstrate its susceptibility to regulation via protein kinase C (PKC) signalling. Next, we explore the physiological functions of GluA4 subunit-containing AMPA type glutamate receptors, predominantly expressed at developing CA3 CA1 synapses. We show that GluA4 expression is necessary for protein kinase A (PKA)-dependent LTP at immature synapses. Further, the loss of GluA4 expression in parallel with circuit maturation explains the developmental switch in LTP signalling requirements from PKA- to Ca2+/calmodulin-dependent protein kinase II (CaMKII)-dependent. Further, we also explore the role of GluA4 C-terminal interaction partners in synaptic trafficking of GluA4-containing AMPA receptors and its importance for synapse maturation. We confirm a critical role for the membrane proximal region of GluA4 C-terminal domain in trafficking and identify a novel mechanism for activity-dependent synaptic delivery of GluA4 by the extreme C-terminal region. Finally, we show an important role of the GluA4 subunit in strengthening of AMPA receptor-mediated transmission, observed during early postnatal development. In summary, we provide novel information on the pre- and postsynaptic plasticity mechanisms operating at hippocampal CA3 CA1 synapses during the critical period of activity-dependent maturation of glutamatergic neuronal circuitry in rodents. This expands our knowledge on the cellular mechanisms guiding development of synaptic connectivity in the brain. Dysfunction of such mechanisms may play fundamental roles in the underlying pathophysiological causes of various neurodevelopmental disorders.
  • Demontis, Gian Carlo; Pezzini, Francesco; Margari, Elisa; Bianchi, Marzia; Longoni, Biancamaria; Doccini, Stefano; Lalowski, Maciej Maurycy; Santorelli, Filippo Maria; Simonati, Alessandro (2020)
    CLN1 disease (OMIM #256730) is an inherited neurological disorder of early childhood with epileptic seizures and premature death. It is associated with mutations in CLN1 coding for Palmitoyl-Protein Thioesterase 1 (PPT1), a lysosomal enzyme which affects the recycling and degradation of lipid-modified (S-acylated) proteins by removing palmitate residues. Transcriptomic evidence from a neuronal-like cellular model derived from differentiated SH-SY5Y cells disclosed the potential negative roles of CLN1 overexpression, affecting the elongation of neuronal processes and the expression of selected proteins of the synaptic region. Bioinformatic inquiries of transcriptomic data pinpointed a dysregulated expression of several genes coding for proteins related to voltage-gated ion channels, including subunits of calcium and potassium channels (VGCC and VGKC). In SH-SY5Y cells overexpressing CLN1 (SH-CLN1 cells), the resting potential and the membrane conductance in the range of voltages close to the resting potential were not affected. However, patch-clamp recordings indicated a reduction of Ba2+ currents through VGCC of SH-CLN1 cells; Ca2+ imaging revealed reduced Ca2+ influx in the same cellular setting. The results of the biochemical and morphological investigations of CACNA2D2/α2δ-2, an accessory subunit of VGCC, were in accordance with the downregulation of the corresponding gene and consistent with the hypothesis that a lower number of functional channels may reach the plasma membrane. The combined use of 4-AP and NS-1643, two drugs with opposing effects on Kv11 and Kv12 subfamilies of VGKC coded by the KCNH gene family, provides evidence for reduced functional Kv12 channels in SH-CLN1 cells, consistent with transcriptomic data indicating the downregulation of KCNH4. The lack of compelling evidence supporting the palmitoylation of many ion channels subunits investigated in this study stimulates inquiries about the role of PPT1 in the trafficking of channels to the plasma membrane. Altogether, these results indicate a reduction of functional voltage-gated ion channels in response to CLN1/PPT1 overexpression in differentiated SH-SY5Y cells and provide new insights into the altered neuronal excitability which may underlie the severe epileptic phenotype of CLN1 disease. It remains to be shown if remodeling of such functional channels on plasma membrane can occur as a downstream effect of CLN1 disease.
  • Kislin, Mikhail (Helsingin yliopisto, 2015)
    Acute brain trauma and ischemia are severe injuries that have no adequate treatment to date. In vivo two-photon microscopy allows studying longitudinally the process of injury development and brain recovery. This thesis summarizes the work on: i) animal model of acute brain injury and role of extracellular matrix in neuronal recovery and plasticity, ii) investigation of mitochondria during physiological/pathological calcium elevations in vitro and further implementation of quantitative microscopic analysis of neuronal mitochondrial morphology in vivo, iii) mitochondrial damage and recovery in animal models of acute neurodegenerative disorders in the neocortex of anesthetized mice, iv) a novel approach for awake head-fixed recordings. The results described in this work provide novel approaches for intravital morphological analysis of neurons and of their mitochondria, increase our understanding of pathogenesis after traumatic and ischemic injury in neocortex of rodents, and enable the development of novel therapies for CNS injuries.
  • Pavlov, Ivan (Helsingin yliopisto, 2006)
    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.
  • Abdurakhmanova, Shamsiiat (Helsingin yliopisto, 2020)
    The hypothalamus is one of the oldest brain structures and it is responsible for the regulation of vital homeostatic functions. A small population of the neurons containing neurotransmitters histamine and GABA, reside in the posterior hypothalamus. Activation of these histamine/GABA neurons promotes attentive wakefulness and vigilance state. In addition to its main role in supporting wake state, histamine released from histamine/GABA neurons is involved in controlling appetite, water intake and energy expenditure. Although the role of GABA released from the histamine/GABA neurons has been much less studied, it is suggested to provide tonic inhibition in the cortical and striatal regions. There are three major topics addressed in the current work. First, we aimed to further probe the hypothesis of the dual histamine/GABA neurotransmitter phenotype of the hypothalamic histaminergic neurons. We were especially interested in whether histamine/GABA neurons are able to release GABA into the synapse via the vesicular transport mechanism. Our second aim was to investigate the consequences of global histamine deficiency with a focus on the cortico-striatal system and brain dopamine-histamine interactions. Lastly, we studied the possible role of GABA released from histamine/GABA neurons in tonic extrasynaptic inhibition. It is well established that histaminergic neurons are also GABAergic, based on the presence of GABA producing enzyme - glutamic acid decarboxylase (GAD) and GABA itself in histaminergic neurons, as demonstrated by immunohistochemical methods. Contradictory findings have been reported on the presence of the vesicular GABA transporter (Vgat) in these neurons. We used double fluorescence in situ hybridization (dFISH) to simultaneously detect GABAergic markers (GAD67 or Vgat mRNA) with a marker for histaminergic neurons - histidine decarboxylase (Hdc) mRNA. We confirmed that histamine/GABA neurons express Vgat mRNA and are able to release GABA via a classical Vgat-dependent mechanism. Previous research has shown the interaction of histamine and dopamine systems at the level of the striatum and proposed involvement of these two systems in neuropsychiatric disorders such as Gilles de la Tourette syndrome. Using a mouse line lacking the histamine-synthesizing enzyme (Hdc KO mice), we investigated the role of histamine in the regulation of the striatal system and its interaction with dopamine at the striatal level. We measured striatal dopamine and its metabolites levels with high performance liquid chromatography (HPLC) at the baseline and after treatment with dopamine precursor l-3,4-dihydroxyphenylalanine (L-Dopa). By quantitative polymerase chain reaction (qPCR), we compared the levels of striatal prodynorphin and proenkephalin transcripts in Hdc WT and KO mice. The transcript level of prodynorphin and proenkephalin is tightly regulated by the activity of principal striatal neurons, medium spiny neurons (MSN) and various neurotransmitters such as dopamine and acetylcholine. Furthermore, we performed detailed analyses of exploratory open field behavior of Hdc KO mice and used a stereological approach to assess the morphology and cytoarchitecture of the corticostriatal system in these mice. We found that Hdc KO mice had increased dopamine turnover in the striatum and impaired expression of striatal prodynorphin and proenkephalin transcripts. We hypothesized that global deficiency of histamine leads to upregulation of the dopaminergic system in the striatum, which in turn leads to altered behavioral structure observed in the novel open field test. Impaired dynorphin/κ-opioid receptor inhibitory feedback on the dopaminergic terminals might be responsible for the increased striatal dopamine release. Finally, we provided new evidence suggesting that GABA released from the histamine/GABA neurons acts on the extrasynaptic δ subunit containing GABAA receptors and provides tonic inhibition. Pharmacological activation of the histamine/GABA neurons in the mice lacking GABAA δ subunit (Gabrd KO) led to a hypervigilant phenotype in these mice as was shown by EEG recordings. In conclusion, we showed that histamine together with dopamine regulates striatal circuits and that GABA released from the histamine/GABA neurons regulates brain arousal state at least partially through the extrasynaptic δ subunit containing GABAA receptors.
  • Tigistu-Sahle, Feven (Helsingin yliopisto, 2017)
    The application of human bone marrow derived mesenchymal stromal cells (hBMSCs) for regenerative or immunomodulatory therapies, e.g. treatment of the graft-versus-host disease, requires in vitro expansion of the cells. The hBMSCs undergo subtle changes during expansion which may compromise their functionality. In order to evaluate these changes lipidomics techniques were applied and the fatty acid (FA) and glycerophospholipid (GPL) profiles of hBMSCs were determined. During the cell passaging, arachidonic acid (20:4n-6) -containing species of phosphatidylcholine (PC) and phosphatidyl-ethanolamine (PE) accumulated while the species containing monounsaturated fatty acids (MUFA) or n-3 polyunsaturated fatty acids (n-3 PUFAs) decreased. The accumulation of 20:4n-6 and deficiency of n-3 PUFAs correlated with the decreased immunosuppressive capacity of the hBMSCs, which suggests that extensive expansion of hBMSCs harmfully modulates membrane GPLs profiles, affects lipid signaling and eventually impairs the functionality of the cells. Experiments, in which hBMSCs were cultured with different PUFA supplements revealed that the cells may limit the proinflammatory 20:4n-6 signaling by elongating this precursor with high biological activity to the less active precursor, 22:4n-6. It was also found that the ability of hBMSCs to produce long chain highly unsaturated fatty acids from C18 PUFA precursors was limited apparently due to the low desaturase activity of the cells. Thus, when the n-3 PUFA precursor, 18:3n-3, had little potency to reduce the GPL 20:4n-6 content, the eicosapentaenoic (20:5n-3) and docosahexaenoic (22:6n-3) acid supplements efficiently displaced the 20:4n-6 acyls, allowing attenuation of inflammatory signaling. These findings call for specifically designed optimal PUFA supplements for the cultures with sufficiently 20:5n-3 and 22:6n-3 but moderately 20:4n-6. Studies on the dynamics of PUFA incorporation into the major GPL classes revealed that the PUFAs in PC are remodeled at first, then those of the PEs and phosphatidylserines (PS). These results demonstrate that not only the type of PUFA administered but also the treatment time largely determines the resulting composition of the membrane GPL species, which serve as PUFA donors for the synthesis of lipid mediators. This thesis work highlights the importance of using lipidomics data to complement genomics or proteomics approaches when aiming at understanding of the therapeutic mechanisms of stem/stromal cells. The work provides tools to develop the protocols of hBMSCs culture and manipulate the functionality of the cells.
  • Körber, Inken (2017)
    The autosomal recessively inherited progressive myoclonus epilepsy of Unverricht- Lundborg type (EPM1, OMIM 254800) is a neurodegenerative disease severely affecting patients motor coordination. Its onset lies around 6 to 16 years of age and the presenting symptoms are severely incapacitating, stimulus-sensitive myoclonus and/or tonic-clonic seizures. Later during disease course, the patients develop ataxia. EPM1 is caused by mutations in the cystatin B (CSTB, OMIM 601145) gene, encoding the cysteine protease inhibitor CSTB. The CSTB-deficient mouse (Cstb-/- mice) is characterized by neuronal hyperexcitability and brain atrophy. In addition, histological studies revealed that the neuronal pathology is accompanied by early microglial activation. Aberrant activation of microglia and neuroinflammation has previously been linked to neuropathology and neurodegenerative diseases. Therefore, we aimed to characterize microglia of Cstb-/- mice in more detail. Our results identified impaired interferon signaling as a potential molecular pathway underlying the phenotype of Cstb-/- mice. In addition, functional properties of activated primary Cstb-/- microglia are altered in vitro. For example, their chemokine release is enhanced, but their phagocytic activity is reduced. In pre-symptomatic Cstb-/- mouse brains at postnatal day 14, the activation of Cstb-/- microglia is shifted towards an anti-inflammatory activation, and it switches towards a pro-inflammatory activation in early symptomatic Cstb-/- mice at postnatal day 30. In line with this, inflammatory markers are upregulated, and T lymphocytes and granulocytes exist in the brain of Cstb-/- mice, which suggests an infiltration of peripheral immune cells. Pro-inflammatory activated macrophages in the spleen imply a widespread immune system activation in Cstb-/- mice. In conclusion, we show that microglia in Cstb-/- mice are dysfunctional and that their activation is aberrant. We link CSTB deficiency in young mice to inflammatory processes and suggest that microglial dysfunction might contribute to the pathology of EPM1.
  • Albert, Katrina (Helsingin yliopisto, 2019)
    The neurodegenerative disorder Parkinson’s disease is diagnosed when motor symptoms appear, which is caused by death of the substantia nigra dopamine neurons. Most disease cases are idiopathic, and there are currently no disease-modifying therapies. Since the mechanism underlying Parkinson’s disease is still unknown, bringing treatments to the clinic has been difficult. Alpha-synuclein (α-syn) is a protein found abundantly in the central nervous system of vertebrates. Its importance for Parkinson’s disease was confirmed when it was discovered that mutations in the gene led to an autosomal dominant disease form and that it is the majority protein in what is considered a pathological marker of the disease, Lewy bodies. Cerebral dopamine neurotrophic factor (CDNF) is a conserved protein with neurotrophic-like properties. It has been shown to protect dopamine neurons in toxin models of Parkinson’s disease and is currently in Phase I/II clinical trials. It has not been tested in α-syn animal models and therefore the aim was to model α-syn-based Parkinson’s disease and to test whether CDNF can intervene with α-syn aggregation and has a therapeutic effect. We generated two models that used α-syn to model sporadic Parkinson’s disease and test CDNF on: adeno-associated virus (AAV) and preformed fibrils. We used an AAV to overexpress human wild-type α-syn and were able to model nigrostriatal dopamine loss accompanied by behavioural deficits. However, the variation in the success of the model was too high to consider it feasible to test CDNF on. This, combined with concerns about controls, led us to conclude that it may not be an ideal model of sporadic Parkinson’s disease. Using a preformed α-syn fibrils model to seed endogenous α-syn, we observed modest behavioural deficits that were ameliorated by CDNF, however the model did not result in dopamine neuron loss with the measures used. Although, we were able to model the spreading of Lewy body- and neurite-like inclusions that were positive for phosphorylated α-syn. From parallel in vitro studies we can conclude that CDNF is affecting the preformed α-syn fibrils model, but further studies are needed to clarify this. Since CDNF has been successful in the 6-hydroxydopamine (6-OHDA) model after striatal injection, we tested injection to the substantia nigra and characterized the injection in naïve rats to further study CDNF. We expected similar effects of CDNF on dopamine neurons and behaviour using nigral injection, however issues with the injection paradigm and that CDNF was given as a single injection meant only minor behavioural effects and no restoration of dopamine neurons. Though when CDNF was injected to the substantia nigra of naïve rats, it was not transported to the striatum, but rather diffused around the midbrain. Lastly, we used a proteasomal inhibitor, the lactacystin toxin. When lactacystin was injected we observed a buildup of α-syn, nigrostriatal dopamine loss, neuroinflammation, and mild behavioural deficits. In general, this was repeated successfully and could be used for therapeutic studies. In conclusion, we used four different methods to model Parkinson’s disease to varying degrees of success in order to test CDNF. Our results indicate the importance of having proper controls and outcome measures. Additionally, we had success in modeling the progressive spreading of Lewy-like pathology, a phenomenon that is occurring in Parkinson’s disease. Notably, CDNF had some success and future studies will explore this further.
  • Priyadarshini, Madhusmita (Helsingin yliopisto, 2013)
    Parkinson's disease (PD) is the second most common neurodegenerative disorder after Alzheimer's disease (AD). The quest for better therapies to modify the progression of PD is still ongoing. During the last two decades, the concept of the etiological basis of PD has changed, which has been driven by genetics, the recognition of familial forms, knowledge of the effects of the environment and toxins, and genome-wide association studies. Although most cases are sporadic, approximately 5-10% of PD cases are due to genetic mutations that give rise to the familial forms. Studies using neurotoxins and also genetic mutations that underlie familial PD have implicated mitochondrial dysfunction in the pathogenesis of PD. Among the different genes associated with familial PD, PTEN-induced putative kinase1 (Pink1), responsible for the autosomal recessive type, is strongly linked to the mitochondria. To investigate in depth the underlying mechanisms of Pink1, we inhibited the function of pink1 in zebrafish using morpholino oligonucleotides (MOs). The MO was first thoroughly characterized with all necessary control experiments to avoid unspecific effects. Since the dopaminergic system is affected in PD, a marker for dopamine, tyrosine hydroxylase (TH), was used to assess damage to the system. Due to a genome duplication event that occurred early in the evolution of teleosts after the divergence from the mammals, two TH non-allelic isoforms were identified in zebrafish: th1 and th2. In the pink1 morphants, both the TH gene isoforms were altered. With in situ hybridization, the loss of th1 was found in the ventral diencephalon (dopaminergic cell groups 5, 6, 11) and th2 was reduced in the caudal hypothalamus (cell group 10b). Similar results were obtained with the cell counting method for TH1 immunoreactive cells. TH-ir indicated the loss of cells in the pretectum (group 7) and the ventral diencephalic cluster represented by cell groups 5,6,11. These pink1 morphants were exposed to subeffective doses of the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). This amalgamation of the toxin and genetic manipulation caused a locomotor deficit and also facilitated the loss of TH-ir in the same cell populations in the larval brains as was instigated by pink1 knockdown alone. To investigate the involvement of pink1 in cell damage, we used a two-color gene expression-based microarray and identified a number of genes that were potentially involved in the pathogenic mechanism of the disease. After successful data analysis, the changes in critical genes were successfully validated by quantitative real-time PCR. The gene expression changes in the morphants, identified by the microarray, were rescued by pink1 mRNA injections, suggesting the specific involvement of pink1 in the differentially expressed gene regulation. One of the significant findings was HIF signaling, an important pathway affected by pink1 knockdown. Individual factors and genes in the same pathway were validated by independent methods in the pink1 morphants to reveal whether pink1 affected hif1α or the cascade of events in the signaling pathway. Changes in the VEGF transcripts, erythropoiesis, and reactive oxygen species were observed, as were other antioxidant system genes, including cat and sod2. These pathways may provide new targets for drug development in PD. To study the mechanisms underlying the involvement of pink1 in oxidative stress-mediated PD pathology using zebrafish as a tool, we generated a transgenic line, Tg(pink1:EGFP). The Tol2 transgenic approach was used to generate Tg(pink1:EGFP) by using the zebrafish pink1 promoter. Expression of the pink1 transgene was detected in the telencephalon, midbrain, and rhombencephalon in the CNS, and in the muscle, heart, and liver among the peripheral organs. The transgenic fish line was used to study the effect of oxidative stress. When subjected to a low concentration of hydrogen peroxide (H2O2), which had no effect on the mortality or phenotype of the fish, the transgenic fish showed an increase in pink1 transgene activity in the brain of the larval zebrafish. Oxidative stress-mediated changes in TH expression are valuable for PD study. H2O2 administration did not affect the th1 transcript levels, but it significantly increased pink1 expression and reduced the th2 transcript levels. This transgenic model will be highly useful for drug development and the screening of new potential therapeutic approaches as an in vivo model.
  • Ning, Lin (Helsingin yliopisto, 2013)
    In the central nervous system, neurons are connected through local contacting structures called synapses. The initial contacts between axons and dendrites undergo changes in morphology and protein composition, and differentiate into fully functional synapses. Precise regulation on synapse formation is essential for normal brain functioning. Cell adhesion molecules are the key regulators during this process, by connecting the membranes of synapses and initiating signaling cascades that mediate synapse formation. A neuron-specific cell adhesion molecule ICAM-5 (telencephalin) controls immune responses by suppressing T-cell activation or by mediating neuron-microglia interaction. On the other hand, ICAM-5 is so far the only identified CAM that slows synaptic development. Deletion of ICAM-5 from mice leads to accelerated synapse formation, enhanced synaptic capacity, and improved memory and learning. Clinically, changes in ICAM-5 levels are associated with various diseases, such as acute encephalitis, epilepsy, and Alzheimer s disease. The major goal of my thesis is to address the molecular mechanisms by which ICAM-5 regulates synapse formation as well as maturation of dendritic spines, the post-synaptic components of excitatory synapses. In my study, ICAM-5 was observed as a substrate for MMP-2 and -9. Activation NMDA receptors in neurons elevated the level of MMP activity, and subsequently induced ICAM-5 ectodomain cleavage, which in turn promoted spine maturation. In addition, I identified the pre-synaptic β1-integrins as counter-receptors for ICAM-5. This trans-synaptic interaction inhibited the MMP-induced ICAM-5 cleavage, and thereby prevented spine maturation. At the intracellular side, α-actinin, an actin cross-linking protein, was found as a binding partner for ICAM-5. This binding linked ICAM-5 to the actin cytoskeleton and was important for the membrane distribution of ICAM-5 and neurite outgrowth. The GluN1 subunit of the NMDAR is also known to bind to α-actinin. We found here that GluN1 and ICAM-5 competed for the same binding region in α-actinin. Activation of NMDAR changed α-actinin binding property to ICAM-5, resulting in α-actinin accumulation and actin reorganization. In conclusion, my thesis defines a novel, ICAM-5-dependent mechanism, which regulates synapse formation, spine maturation and remodeling.
  • Popova, Dina (Helsingin yliopisto, 2015)
    Dynamic modifications of synaptic connectivity enables the brain to adequately respond to environmental challenges. This ability, known as synaptic plasticity, peaks during the early postnatal period, yet it is maintained throughout life. Interestingly, antidepressants (ADs) and AD-like drugs can promote neuronal plasticity in the adult brain, a phenomenon recently suggested to contribute to the mood-improving effects of ADs. However, the mechanisms underlying AD-induced neuronal network refinement are still poorly understood. The main goal of this thesis was to advance our understanding of the mechanisms associated with pharmacologically-enhanced plasticity in the adult brain. Two pharmacologically distinct compounds with AD-like actions, namely the selective serotonin reuptake inhibitor fluoxetine (Flx) and the volatile anesthetic isoflurane were used to enhance synaptic plasticity in the rodent cortex and hippocampus. After drug exposure, behavioral, molecular, histological and in vitro electrophysiological approaches were utilized to investigate the effects of Flx and ISO on synaptic function and plasticity. Using electrophysiological recordings in brain slices, we show that chronic Flx treatment results in increased short- and long-term plasticity as well as enhanced basal transmission in excitatory CA3-CA1 synapses in the hippocampus. These changes were paralleled by an activity-dependent enhancement in the expression of proteins related to vesicular trafficking and release, such as synaptophysin, synaptotagmin 1, mammalian uncoordinated protein 18 (Munc 18) and syntaxin 1. Moreover, Flx treatment reduced the percentage of parvalbumin-expressing GABAergic neurons, increased the expression of polysialylated-neural cell adhesion molecule (PSA-NCAM) and decreased the expression of the potassium-chloride co-transporter 2 (KCC2) in the basolateral amygdala and in the medial prefrontal cortex (mPFC). All the above findings are likely to be attributed to increased dynamic range of synaptic plasticity induced by Flx. Our behavioral findings demonstrate that long term Flx administration in combination with extinction training results in long-term loss of fearful memories while the Flx treatment alone failed to influence fear behavior. These data suggest that behavioral training is indispensable for the guidance of Flx-induced network plasticity. Exposure to isoflurane promotes long-term synaptic plasticity and enhances basal synaptic transmission in excitatory CA3-CA1 synapses in the mouse hippocampus. These changes were correlated with increased tropomyosin receptor kinase B (TrkB) signaling through the mammalian target of rapamycin (mTOR) pathway in the prefrontal cortex and hippocampus and led to rapid antidepressant-like behavioral effects in the forced swim test. Taken together, our findings highlight that Flx and isoflurane enhance synaptic plasticity in hippocampal and cortical excitatory synapses, however, the underlying molecular mechanisms as well as behavior improvements were different. In conclusion, the results described in this work provide a mechanistic background for adult brain plasticity and network tuning, with high practical significance to the design of clinical therapy.
  • Yan, Xu (Helsingin yliopisto, 2017)
    Progressive development of pathology in neuroanatomically connected brain regions is a common feature of many neurodegenerative diseases. The spread of disease pathology is suggested to be dependent on the transmissibility of disease-associated proteins, particularly soluble aggregates of misfolded proteins. Emerging evidence suggests that many disease-associated proteins such as α-synuclein (aSyn) and tau, in certain misfolded and aggregated states convert from physiologically normal proteins into forms that lead to progression of disease pathology in a template-dependent manner, which is also known as seeding . The propagation and the proteinopathy have been suggested to occur via cell-to-cell transmission. The exact mechanisms involved in the seeding and spreading process are incompletely understood. In this thesis work, three critical steps of the seeding pathway (a process involves multiple steps), the intracellular aggregation, cellular release and uptake of aSyn and tau, were carefully studied primarily via a newly developed platform based on protein-fragment complementation assay. The main findings of this thesis are: a)Prolyl oligopeptidase (PREP) is a serine peptidase that was previously known to accelerate the process of aSyn aggregation and suppress autophagy clearance in cells and transgenic aSyn mice. The results of this thesis show that PREP directly interacts with aSyn in neuro2A cells and cell-free environment, and enhances aSyn dimerization, which is an early event in aSyn aggregation pathway. In addition, the PREP-mediated aSyn dimerization can be antagonized by KYP-2047, a small-molecule PREP inhibitor. b) Late-onset Alzheimer s disease (LOAD) susceptibility genes affect the individual risk of developing Alzheimer s disease, which is one of the common tauopathies. In this work, the functional connection between selected LOAD susceptibility genes and cell-to-cell transmission of tau was studied in vitro. We observed that RNAi knockdown of CD2AP and FRMD4A reduced tau secretion, and knockdown of APOE reduced tau uptake in HEK293T cells. Further mechanistic studies revealed that FRMD4A modulates tau secretion via the FRMD4A-cytohesin-Arf6 signalling pathway and the Par6/aPKC polarity signalling complex. This data, for the first time, demonstrates a functional connection between LOAD risk genes and cell-to-cell propagation of tau. c) Following internalization, extracellular, hyperphosphorylated tau was found to be recruited to stress granules, transient non-membraneous cytosolic structures composed of RNA and self-aggregating RNA-binding proteins. Tau recruitment was dependent on TIA-1, an RNA-binding stress granule protein. Importantly, the stress granules induced by and containing internalized tau were resistant to normal clearance and associated with increased sensitivity of cells to other stresses. This data describe a previously unrecognized mechanism and pathological consequence of cell-to-cell propagation of tau-mediated by stress granules, which have previously been associated with the pathophysiology of various neurodegenerative diseases. Overall, the work described in this thesis provides several novel findings that improve our understanding of cellular mechanisms underlying the development and spreading of aSyn and tau-related neurodegenerative pathologies. These pieces of knowledge may be potential avenues towards the development of crucial therapeutics against aSyn and tau-related neurodegenerative diseases.
  • Magalhães, Ana Cathia Dias (Helsingin yliopisto, 2016)
    The time of arrival of interneurons and oligodendrocytes to the neocortex is critical for proper functional brain development. Aberrances in this sequence can be detrimental, and involved in different developmental diseases. Thus, understanding the mechanisms for temporal control of the genesis and migration of neural cells is crucial. The aim of this study was to focus on the ventral telencephalon, a major source of interneurons and oligodendrocytes, in more detail. A more sensitive method was developed for detecting and quantifying oligodendrocyte precursor cells, e.g. Olig2. The device decloaking chamber was compared to the microwave oven-based heat-induced epitope retrieval (HIER) method by studying the labeling of Olig2 marker in paraffin-embedded sections from embryonic mouse brain. The results demonstrated that the decloaking chamber-based HIER method is the most suitable technique for the detection of single Olig2-labeled cells in the ventral telencephalon. This qualitative result was reflected in the quantitative analyses: more Olig2-labeled cells were quantifiable with the decloaking chamber- than with the microwave oven-approach. Thus, the decloaking chamber-based HIER method constitutes a sensitive technique for the detection of oligodendrocyte precursor cells, and therefore for its quantification in the developing ventral telencephalon. The development of telencephalon depends on fundamental processes, which include proliferation and migration of neural cells. The Na-K-Cl cotransporter isoform 1 (NKCC1) is an important protein for the process of volume regulation, and has been implicated in cell division. Within the developing brain, the ventral telencephalon showed the highest expression of NKCC1. This expression corresponded to neural progenitor cells in the lateral ganglionic eminence (LGE). Using NKCC1 knockout mice, it was demonstrated that NKCC1 influenced cell cycle reentry. Consequently, mice lacking NKCC1 have impaired Sp8-expressing interneurons and Olig2-labeled cells. Thus, NKCC1 is crucial in vivo for cell cycle decision, thereby altering the production of oligodendrocyte and interneuron progenitor cells in the LGE. Once interneurons are born, they migrate to the neocortex. The implication of syndecan-3 was assessed in their tangential migration. The results showed that the Glial cell line-derived neurotrophic factor GDNF interacts with syndecan-3 to promote the tangential migration of calbindin-expressing interneurons within the telencephalon. Consistently, mice lacking syndecan-3 have an accumulation of migrating interneurons in the LGE. In summary, two important mechanisms were found for temporal and spatial control of cortical oligodendrocytes and interneurons.
  • Ahmad, Faraz (Helsingin yliopisto, 2012)
    KCC2 is an important K+-Cl- co-transporter that along with Na+-K+-2Cl- co-transporter, NKCC1 is largely responsible for the regulation of intracellular chloride concentration in neurons which determines whether the ionotropic GABAergic/glycinergic responses are depolarizing or hyperpolarizing. There are spatiotemporal differences in the intracellular chloride concentration in individual neurons which are attributable to a differential temporal and spatial activation of cation chloride co-transport mediated by KCC2 and NKCC1. Post-translational modulation of proteins is a fundamental cellular mechanism for such spatiotemporal regulation of protein activity. This thesis deals with the work that has been ongoing in our laboratory to understand the mechanisms of post-translational regulation of KCC2 function. In Study I, we have demonstrated a fast post-translational increase in KCC2 co-transport function in neonatal rat hippocampus after a single seizure episode. This TrkB-dependent effect was caused by an increased surface expression of KCC2. Study II deals with the establishment of a modified protease cleavage method for quantitative analysis of surface expression of proteins using a cold-adapted trypsin. This can serve as a fast and reliable procedure and can be easily applied to brain slice preparations as well as cell culture systems. In study II, we have also shown that KCC2 has a low surface expression in the rat hippocampus but a very fast turn-over rate of the plasmalemmal pool. Not surprisingly, modifications in the turn-over rate of the surface pool can be employed as a mechanism to regulate the surface expression of KCC2 and consequently its function. Study III deals with another post-translational cellular strategy to regulate KCC2 function in the rat hippocampus under patho-physiological conditions. While the KCC2 protein is quite stable in the rat hippocampus and has a slow turn-over rate under basal conditions, epileptiform activity and excitotoxicity can induce a rapid calpain-mediated cleavage of KCC2 with a consequent loss of its co-transport function.
  • Kumar, Anmol (Helsingin yliopisto, 2014)
    Neurotrophic factors such as brain¬-derived neurotrophic factor (BDNF) and glial cell line¬¬¬-derived neurotrophic factor (GDNF) are a family of proteins which play an important role inside and outside central nervous system. While precise regulation of BDNF and GDNF levels in time and space in an organism is crucial in determining the biological outcome, mechanisms involved in controlling their levels are not fully understood. Messenger RNAs (mRNAs) play a critical role in gene expression by conveying genetic information from DNA to protein synthesis. 3ʹ untranslated region (3ʹUTR) is a part of mRNA sequence which regulates gene expression by binding to microRNAs (miRs), RNA-binding proteins (RBPs) and other trans-acting factors. In this thesis, we investigated the 3ʹUTR mediated regulation of BDNF and GDNF. We demonstrate the presence of regulatory elements in the 3ʹUTR of BDNF and GDNF and, show that BDNF is regulated by four different miRs, namely miR-1, miR-10b, miR-155 and miR-191 and, RBP tristetraprolin (TTP) in different cell lines. Further, we show that GDNF is regulated by multiple miRs in cell lines and identify binding sites for miR-146a and miR-96 in the GDNF 3ʹUTR. Finally, we demonstrate that replacement of GDNF 3ʹUTR in mice with a 3ʹUTR with reduced responsiveness to negative regulators including miRs leads to elevated level of endogenous GDNF mRNA and protein in various organs with profound biological effects including in the brain dopamine system function in mice. We conclude that 3ʹUTR mediated regulation of BDNF and GDNF is biologically important and propose that 3ʹUTR replacement is an informative way to study gene function in vivo.