Unravelling Molecular and Cellular Disease Mechanisms in Infantile Neuronal Ceroid Lipofuscinosis (INCL)

Studying neurodegeneration provides an opportunity to gain insights into normal cell physiology, and not just pathophysiology. In this thesis work the focus is on Infantile Neuronal Ceroid Lipofuscinosis (INCL). It is a recessively inherited lysosomal storage disorder. The disease belongs to the neuronal ceroid lipofuscinoses (NCLs), a group of common progressive neurodegenerative diseases of the childhood. Characteristic accumulation of autofluorescent storage material is seen in most tissues but only neurons of the central nervous system are damaged and eventually lost during the course of the disease leaving most other cell types unaffected. The disease is caused by mutations in the CLN1 gene, but the physiological function of the corresponding protein the palmitoyl protein thioesterase (PPT1) has remained elusive. The aim of this thesis work was to shed light on the molecular and cell biological mechanisms behind INCL. This study pinpointed the localization of PPT1 in axonal presynapses of neurons. It also established the role of PPT1 in early neuronal maturation as well as importance in mature neuronal synapses. This study revealed an endocytic defect in INCL patient cells manifesting itself as delayed trafficking of receptor and non-receptor mediated endocytic markers. Furthermore, this study was the first to connect the INCL storage proteins the sphingolipid activator proteins (SAPs) A and D to pathological events on the cellular level. Abnormal endocytic processing and intracellular re-localization was demonstrated in patient cells and disease model knock-out mouse neurons. To identify early affected cellular and metabolic pathways in INCL, knock-out mouse neurons were studied by global transcript profiling and functional analysis. The gene expression analysis revealed changes in neuronal maturation and cell communication strongly associated with the regulated secretory system. Furthermore, cholesterol metabolic pathways were found to be affected. Functional studies with the knock-out mouse model revealed abnormalities in neuronal maturation as well as key neuronal functions including abnormalities in intracellular calcium homeostasis and cholesterol metabolism. Together the findings, introduced in this thesis work, support the essential role of PPT1 in developing neurons as well as synaptic sites of mature neurons. Results of this thesis also elucidate early events in INCL pathogenesis revealing defective pathways ultimately leading to the neurodegenerative process. These results contribute to the understanding of the vital physiological function of PPT1 and broader knowledge of common cellular mechanisms behind neurodegeneration. These results add to the knowledge of these severe diseases offering basis for new approaches in treatment strategies.

Neuronal K-Cl Cotransporter : Transcriptional Mechanisms of KCC2 Gene Regulation

K-Cl cotransporter 2 (KCC2) maintains a low intracellular Cl concentration required for fast hyperpolarizing responses of neurons to classical inhibitory neurotransmitters γ-aminobutyric acid (GABA) and glycine. Decreased Cl extrusion observed in genetically modified KCC2-deficient mice leads to depolarizing GABA responses, impaired brain inhibition, and as a consequence to epileptic seizures. Identification of mechanisms regulating activity of the SLC12A5 gene, which encodes the KCC2 cotransporter, in normal and pathological conditions is, thus, of extreme importance. Multiple reports have previously elucidated in details a spatio-temporal pattern of KCC2 expression. Among the characteristic features are an exclusive neuronal specificity, a dramatic upregulation during embryonic and early postnatal development, and a significant downregulation by neuronal trauma. Numerous studies confirmed these expressional features, however transcriptional mechanisms predetermining the SLC12A5 gene behaviour are still unknown. The aim of the presented thesis is to recognize such transcriptional mechanisms and, on their basis, to create a transcriptional model that would explain the established SLC12A5 gene behaviour. Up to recently, only one KCC2 transcript has been thought to exist. A particular novelty of the presented work is the identification of two SLC12A5 gene promoters (SLC12A5-1a and SLC12A5-1b) that produce at least two KCC2 isoforms (KCC2a and KCC2b) differing by their N-terminal parts. Even though a functional 86Rb+ assay reveals no significant difference between transport activities of the isoforms, consensus sites for several protein kinases, found in KCC2a but not in KCC2b, imply a distinct kinetic regulation. As a logical continuation, the current work presents a detailed analysis of the KCC2a and KCC2b expression patterns. This analysis shows an exclusively neuron-specific pattern and similar expression levels for both isoforms during embryonic and neonatal development in rodents. During subsequent postnatal development, the KCC2b expression dramatically increases, while KCC2a expression, depending on central nervous system (CNS) area, either remains at the same level or moderately decreases. In an attempt to explain both the neuronal specificity and the distinct expressional kinetics of the KCC2a and KCC2b isoforms during postnatal development, the corresponding SLC12A5-1a and SLC12A5-1b promoters have been subjected to a comprehensive bioinformatical analysis. Binding sites of several transcription factors (TFs), conserved in the mammalian SLC12A5 gene orthologs, have been identified that might shed light on the observed behaviour of the SLC12A5 gene. Possible roles of these TFs in the regulating of the SLC12A5 gene expression have been elucidated in subsequent experiments and are discussed in the current thesis.

Herpes Simplex Virus Infection, Pathological Pain and Recurrent Lymphocytic Meningitis

Background: Aims of the study were: (i) to characterise the clinical picture, immunological features and changes in brain morphology and function in patients with widespread unilateral pain and HSV-infections, and (ii) to analyse the prevalence, clinical symptoms and immunological predisposing factors of HSV-2 induced recurrent lymphocytic meningitis (RLM) in Southern Finland. Patients and methods: Patients for the studies were recruited from the Pain Clinic, and from the Department of Neurology, at Helsinki University Central Hospital. Plasma concentrations of IgM, IgA, IgG, and IgG1-4, and serum concentrations of C3, C4 were measured. Serological anti-HSV-1 and -2 antibody status was tested. C4 genotyping, HLA-A, HLA-B and HLA-DRB1 typing, MBL2 genotyping, and IgG1 and IgG3 allotyping (Gm) were performed. Clinical neurological examination, quantitative sensory testing, skin biopsy, and functional magnetic resonance imaging were also performed. Results: HSV probably has a role in the generation of a pathological pain state. Low serum IgG1 and IgG3 levels, made the patients vulnerable for recurring HSV infections. Both functional and structural changes were observed in the brain pain-processing areas in the patients: they had less pain-related activity in the insular cortices bilaterally, in the anterior cingular cortex (ACC), and in the thalamus, and the gray matter density was lower in the ACC, in the frontal and prefrontal cortices. In the meningitis studies it was shown that RLM is more common and less benign than previously reported, and that neuropathic pain is frequently present both during and after meningitis episodes. HLA-DRB1*01, HLA-B*27, and low IgG1 levels are predisposing factors for RLM. Conclusions: Patients are vulnerable to recurrent HSV infections because of subtle immunological abnormalities. HSV causes diverse clinical manifestations. First, the herpes simplex virus, or the inflammatory process triggered by it, may cause pathological widespread pain probably by activating glial cells in the CNS. In these patients, signs of alterations in the brain pain-processing areas can be demonstrated by functional brain imaging methods. Secondly, HSV-2 induced RLM is a rare complication of HSV-2 virus. The predisposing factors include low IgG1 subclass levels, HLA-DRB1*01 and HLA –B*27 genotypes. Neuropathic pain is frequently associated with RLM.

Pathological changes at the target tissue level in Sjögren's syndrome and their effect on the exocrine function of the salivary glands

Sjögren s syndrome (SS) is a common autoimmune disease affecting the lacrimal and salivary glands. SS is characterized by a considerable female predominance and a late age of onset, commonly at the time of adreno- and menopause. The levels of the androgen prohormone dehydroepiandrosterone-sulphate (DHEA-S) in the serum are lower in patients with SS than in age- and sex-matched healthy control subjects. The eventual systemic effects of low androgen levels in SS are not currently well understood. Basement membranes (BM) are specialized layers of extracellular matrix and are composed of laminin (LM) and type IV collagen matrix networks. BMs deliver messages to epithelial cells via cellular LM-receptors including integrins (Int) and Lutheran blood group antigen (Lu). The composition of BMs and distribution of LM-receptors in labial salivary glands (LSGs) of normal healthy controls and patients with SS was assessed. LMs have complex and highly regulated distribution in LSGs. LMs seem to have specific tasks in the dynamic regulation of acinar cell function. LM-111 is important for the normal acinar cell differentiation and its expression is diminished in SS. Also LM-211 and -411 seem to have some acinar specific functional tasks in LSGs. LM-311, -332 and -511 seem to have more general structure maintaining and supporting roles in LSGs and are relatively intact also in SS. Ints α3β1, α6β1, α6β4 and Lu seem to supply structural basis for the firm attachment of epithelial cells to the BM in LSGs. The expression of Ints α1β1 and α2β1 differed clearly from other LM-receptors in that they were found almost exclusively around the acini and intercalated duct cells in salivons suggesting some type of acinar cell compartment-specific or dominant function. Expression of these integrins was lower in SS compared to healthy controls suggesting that the LM-111 and -211-to-Int α1β1 and α2β1 interactions are defective in SS and are crucial to the maintenance of the acini in LSGs. DHEA/DHEA-S concentration in serum and locally in saliva of patients with SS seems to have effects on the salivary glands. These effects were first detected using the androgen-dependent CRISP-3 protein, the production and secretion of which were clearly diminished in SS. This might be due to the impaired function of the intracrine DHEA prohormone metabolizing machinery, which fails to successfully convert DHEA into its active metabolites in LSGs. The progenitor epithelial cells from the intercalated ductal area of LSGs migrate to the acinar compartment and then undergo a phenotype change into secretory acinar cells. This migration and phenotype change seem to be regulated by the LM-111-to-Int α1β1/Int α2β1 interactions. Lack of these interactions could be one factor limiting the normal remodelling process. Androgens are effective stimulators of Int α1β1 and α2β1 expression in physiologic concentrations. Addition of DHEA to the culture medium had effective stimulating effect on the Int α1β1 and α2β1 expression and its effect may be deficient in the LSGs of patients with SS.

Selective attention in a synchronising bushcricket: physiology

Synchronising bushcricket males achieve synchrony by delaying their chirps in response to calling neighbours. In multi-male choruses, males that delay chirps in response to all their neighbours would remain silent most of the time and be unable to attract mates. This problem could be overcome if the afferent auditory system exhibited selective attention, and thus a male interacted only with a subset of neighbours. We investigated whether individuals of the bushcricket genus Mecopoda restricted their attention to louder chirps neurophysiologically, behaviourally and through spacing. We found that louder leading chirps were preferentially represented in the omega neuron but the representation of softer following chirps was not completely abolished. Following chirps that were 20 dB louder than leading chirps were better represented than leading chirps. During acoustic interactions, males synchronised with leading chirps even when the following chirps were 20 dB louder. Males did not restrict their attention to louder chirps during interactions but were affected by all chirps above a particular threshold. In the field, we found that males on average had only one or two neighbours whose calls were above this threshold. Selective attention is thus achieved in this bushcricket through spacing rather than neurophysiological filtering of softer signals.

Selective attention in a synchronising bushcricket: physiology, behaviour and ecology

Synchronising bushcricket males achieve synchrony by delaying their chirps in response to calling neighbours. In multi-male choruses, males that delay chirps in response to all their neighbours would remain silent most of the time and be unable to attract mates. This problem could be overcome if the afferent auditory system exhibited selective attention, and thus a male interacted only with a subset of neighbours. We investigated whether individuals of the bushcricket genus Mecopoda restricted their attention to louder chirps neurophysiologically, behaviourally and through spacing. We found that louder leading chirps were preferentially represented in the omega neuron but the representation of softer following chirps was not completely abolished. Following chirps that were 20 dB louder than leading chirps were better represented than leading chirps. During acoustic interactions, males synchronised with leading chirps even when the following chirps were 20 dB louder. Males did not restrict their attention to louder chirps during interactions but were affected by all chirps above a particular threshold. In the field, we found that males on average had only one or two neighbours whose calls were above this threshold. Selective attention is thus achieved in this bushcricket through spacing rather than neurophysiological filtering of softer signals.

Selective attention in a synchronising bushcricket: physiology, behaviour and ecology

Synchronising bushcricket males achieve synchrony by delaying their chirps in response to calling neighbours. In multi-male choruses, males that delay chirps in response to all their neighbours would remain silent most of the time and be unable to attract mates. This problem could be overcome if the afferent auditory system exhibited selective attention, and thus a male interacted only with a subset of neighbours. We investigated whether individuals of the bushcricket genus Mecopoda restricted their attention to louder chirps neurophysiologically, behaviourally and through spacing. We found that louder leading chirps were preferentially represented in the omega neuron but the representation of softer following chirps was not completely abolished. Following chirps that were 20 dB louder than leading chirps were better represented than leading chirps. During acoustic interactions, males synchronised with leading chirps even when the following chirps were 20 dB louder. Males did not restrict their attention to louder chirps during interactions but were affected by all chirps above a particular threshold. In the field, we found that males on average had only one or two neighbours whose calls were above this threshold. Selective attention is thus achieved in this bushcricket through spacing rather than neurophysiological filtering of softer signals.

POP-inhibiittorin KYP-2047 vaikutukset angiogeneesiin in vitro

Angiogeneesi on tärkeä ilmiö elimistön fysiologiassa, mutta myös lukuisissa patologisissa tiloissa. Angiogeneesi on monivaiheinen prosessi, joka sisältää angiogeneesiä indusoivia ja sitä inhiboivia tekijöitä tasapainossa keskenään. Useat tutkimukset puoltavat sitä, että tymosiini ȕ4 (Tȕ4) ja tetrapeptidi Ac-SDKP (N-asetyyliseryyli- aspartyyli-lysyyli-proliini) indusoivat angiogeneesiä in vitro ja in vivo. Tutkimukset viittaavat myös siihen, että prolyylioligopeptidaasi (POP) hydrolysoi peptidifragmentin Ac- SDKP Tȕ4:n (43 ah) proliinin jälkeen. POP on laajalti esiintyvä seriiniproteaasi, joka pystyy pilkkomaan vain alle 30 aminohapon oligopeptidejä. Tȕ4:n tulee siksi pilkkoutua ensin jonkin, vielä tuntemattoman peptidaasin johdosta. POP:ia on löydetty eniten aivoista, minkä vuoksi sitä on tutkittu varsinkin muistin ja oppimisen häiriötiloissa sekä neurodegeneratiivisten sairausten yhteydessä. POP:in todellinen fysiologinen merkitys on kuitenkin vielä selvittämättä. Tämän pro gradun kirjallisuusosiossa selvitetään angiogeneesiin liittyvien tekijöiden yhteyksiä sekä kuvataan angiogeenisten Tȕ4:n, Ac-SDKP:n ja POP:in ominaisuuksia, esiintymistä ja toimintaa. Kokeellisen osion tarkoituksena oli osoittaa, osallistuvatko POP ja Tȕ4 tetrapeptidin Ac-SDKP muodostumiseen ja kapillaarimuodostumiseen ja edelleen, voidaanko POPaktiivisuutta, tetrapeptidi- ja kapillaarimuodostumista estää spesifisellä POP-inhibiittorilla, KYP-2047:llä. Kokeellinen osa oli kaksiosainen. Ensimmäisessä osassa tutkittiin POPaktiivisuutta ja suoritettiin Ac-SDKP –pitoisuusmittauksia ajanjaksolla 0-180 min Wistarkannan rotista tehdyillä homogenaateilla. Tutkimusryhminä olivat 0,1 ja 0,5 μM KYP-2047 (+2 μM Tȕ4), 1:20 (0,625 μM) humaaniperäinen rekombinantti-POP (+ 2 μM Tȕ4), 2 μM Tȕ4 (pos. kontrolli) ja raakahomogenaatti (neg. kontrolli). Toisessa osassa tutkittiin kapillaarimuodostumista ajanjaksolla 0-180 min humaaniperäisillä napanuoralaskimon primaariendoteelisoluilla MatrigelTM Matrix -päällystetyllä 48- kuoppalevyllä, jolle oli siirrostettu 50 000 solua/kuoppa. Naudan seerumilla ja antibiooteilla käsitellyt tutkimusryhmät olivat 5 ja 10 μM KYP-2047 (+4 μM Tȕ4), 1:20 (0,625 μM) humaaniperäinen rekombinantti-POP (+4 μM Tȕ4), 4 μM Tȕ4 (pos. kontrolli) ja DMEM (neg. kontrolli). Kuoppia inkuboitiin ja kapillaarimuodostuminen kuvattiin valomikroskoopilla digitaalikameralla. Kutakin tutkimusryhmää pipetoitiin kolmeen rinnakkaiseen kuoppaan ja kokeet toistettiin neljästi. Sulkeutuneiden kapillaarien lukumäärä laskettiin manuaalisesti ja tuloksista tehtiin tilastollinen analyysi. 7ȕ4:n ja POP:in havaittiin molempien osallistuvan tetrapeptidin AC-SDKP muodostumiseen munuaishomogenaateissa. Primaariendoteelisolut muodostivat selkeitä kapillaareja Matrigelilla, erityisesti POP- ja Tȕ4–ryhmissä. KYP-2047 inhiboi tehokkaasti POP:ia kaikissa kokeissa osoittautuen hyväksi antiangiogeeniseksi yhdisteeksi. Angiogeneesin mekanismien ja POP:in, Tȕ4:n ja Ac-SDKP:n yhteyksien selvittäminen vaatii luonnollisesti vielä lisätutkimuksia.

Enhanced non-homologous end joining contributes toward synthetic lethality of pathological RAD51C mutants with poly (ADP-ribose) polymerase

Here, we show that PARP inhibitor-mediated cell death of RAD51C-deficient cells occur by NHEJ-driven illegitimate repair of one-ended double-strand breaks, and the hypomorphic RAD51C pathological mutant cells can be targeted by `synergistic toxicity' induced by low-dose PARP inhibitor and IR.Poly (ADP-ribose) polymerase 1 (PARP1) inhibitors are actively under clinical trials for the treatment of breast and ovarian cancers that arise due to mutations in BRCA1 and BRCA2. The RAD51 paralog RAD51C has been identified as a breast and ovarian cancer susceptibility gene. The pathological RAD51C mutants that were identified in cancer patients are hypomorphic with partial repair function. However, targeting cancer cells that express hypomorphic mutants of RAD51C is highly challenging. Here, we report that RAD51C-deficient cells can be targeted by a `synthetic lethal' approach using PARP inhibitor and this sensitivity was attributed to accumulation of cells in the G(2)/M and chromosomal aberrations. In addition, spontaneous hyperactivation of PARP1 was evident in RAD51C-deficient cells. Interestingly, RAD51C-negative cells exhibited enhanced recruitment of non-homologous end joining (NHEJ) proteins onto chromatin and this accumulation correlated with increased activity of error-prone NHEJ as well as genome instability leading to cell death. Notably, inhibition of DNA-PKcs or depletion of KU70 or Ligase IV rescued this phenotype. Strikingly, stimulation of NHEJ by low dose of ionizing radiation (IR) in the PARP inhibitor-treated RAD51C-deficient cells and cells expressing pathological RAD51C mutants induced enhanced toxicity `synergistically'. These results demonstrate that cancer cells arising due to hypomorphic mutations in RAD51C can be specifically targeted by a `synergistic approach' and imply that this strategy can be potentially applied to cancers with hypomorphic mutations in other homologous recombination pathway genes.

A Kunitz-type serine protease inhibitor from Butea monosperma seed and its influence on developmental physiology of Helicoverpa armigera

A protease inhibitor from the seeds of Butea monosperma (BmPI) was purified, characterized and studied for its influence on developmental physiology of Helicover-pa armigera. BmPI on two-dimensional separations indicated the presence of a 14 kDa protein with an isoelectric point in the acidic region (pl 5.6). Multiple Sequence Analysis data suggested that the BmPI contains a sequence motif which is conserved in various trypsin and chymotrypsin inhibitors of Kunitz-type. The inhibitor exhibited trypsin inhibitory activity in a broad range of pH (4-10) and temperature (10-80 degrees C). The enzyme kinetic studies revealed BmPI as a competitive inhibitor with a K-i value of 1.2 x 10(-9) M. In vitro studies with BmPI indicated measurable inhibitory activity on total gut proteolytic enzymes of H. armigera (IC(50)2.0 mu g/ml) and bovine trypsin. BmPI supplemented artificial diet caused dose dependent mortality and reduction in growth and weight. The fertility and fecundity of H. armigera, declined whereas the larval-pupal duration of the insect life cycle extended. These detrimental effects on H. armigera suggest the usefulness of BmPl in insect pest management of food crops. (C) 2014 Elsevier Ltd. All rights reserved.

Novel Functions of (p)ppGpp and Cyclic di-GMP in Mycobacterial Physiology Revealed by Phenotype Microarray Analysis of Wild-Type and Isogenic Strains of Mycobacterium smegmatis

The bacterial second messengers (p)ppGpp and bis-(3'-5')-cyclic dimeric GMP (c-di-GMP) regulate important functions, such as transcription, virulence, biofilm formation, and quorum sensing. In mycobacteria, they regulate long-term survival during starvation, pathogenicity, and dormancy. Recently, a Pseudomonas aeruginosa strain lacking (p) ppGpp was shown to be sensitive to multiple classes of antibiotics and defective in biofilm formation. We were interested to find out whether Mycobacterium smegmatis strains lacking the gene for either (p)ppGpp synthesis (Delta rel(Msm)) or c-di-GMP synthesis (Delta dcpA) would display similar phenotypes. We used phenotype microarray technology to compare the growth of the wild-type and the knockout strains in the presence of several antibiotics. Surprisingly, the Delta rel(Msm) and Delta dcpA strains showed enhanced survival in the presence of many antibiotics, but they were defective in biofilm formation. These strains also displayed altered surface properties, like impaired sliding motility, rough colony morphology, and increased aggregation in liquid cultures. Biofilm formation and surface properties are associated with the presence of glycopeptidolipids (GPLs) in the cell walls of M. smegmatis. Thin-layer chromatography analysis of various cell wall fractions revealed that the levels of GPLs and polar lipids were reduced in the knockout strains. As a result, the cell walls of the knockout strains were significantly more hydrophobic than those of the wild type and the complemented strains. We hypothesize that reduced levels of GPLs and polar lipids may contribute to the antibiotic resistance shown by the knockout strains. Altogether, our data suggest that (p)ppGpp and c-di-GMP may be involved in the metabolism of glycopeptidolipids and polar lipids in M. smegmatis.

First human hNT neurons patterned on parylene-C/silicon dioxide substrates: Combining an accessible cell line and robust patterning technology for the study of the pathological adult human brain

In this communication, we describe a new method which has enabled the first patterning of human neurons (derived from the human teratocarcinoma cell line (hNT)) on parylene-C/silicon dioxide substrates. We reveal the details of the nanofabrication processes, cell differentiation and culturing protocols necessary to successfully pattern hNT neurons which are each key aspects of this new method. The benefits in patterning human neurons on silicon chip using an accessible cell line and robust patterning technology are of widespread value. Thus, using a combined technology such as this will facilitate the detailed study of the pathological human brain at both the single cell and network level. © 2010 Elsevier B.V.

Ca2+ Influx and Tyrosine Kinases Trigger Bordetella Adenylate Cyclase Toxin (ACT) Endocytosis. Cell Physiology and Expression of the CD11b/CD18 Integrin Major Determinants of the Entry Route

Humans infected with Bordetella pertussis, the whooping cough bacterium, show evidences of impaired host defenses. This pathogenic bacterium produces a unique adenylate cyclase toxin (ACT) which enters human phagocytes and catalyzes the unregulated formation of cAMP, hampering important bactericidal functions of these immune cells that eventually cause cell death by apoptosis and/or necrosis. Additionally, ACT permeabilizes cells through pore formation in the target cell membrane. Recently, we demonstrated that ACT is internalised into macrophages together with other membrane components, such as the integrin CD11b/CD18 (CR3), its receptor in these immune cells, and GM1. The goal of this study was to determine whether ACT uptake is restricted to receptor-bearing macrophages or on the contrary may also take place into cells devoid of receptor and gain more insights on the signalling involved. Here, we show that ACT is rapidly eliminated from the cell membrane of either CR3-positive as negative cells, though through different entry routes, which depends in part, on the target cell physiology and characteristics. ACT-induced Ca2+ influx and activation of non-receptor Tyr kinases into the target cell appear to be common master denominators in the different endocytic strategies activated by this toxin. Very importantly, we show that, upon incubation with ACT, target cells are capable of repairing the cell membrane, which suggests the mounting of an anti-toxin cell repair-response, very likely involving the toxin elimination from the cell surface.

Role of G protein coupled inwardly rectifier K+ channels (GIRK) on the neurobiology depression

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