Cholesterol Disturbances in Inherited Neurodegenerative Diseases : Studies on Npc1, Ppt1 and Ctsd deficient mice

The central nervous system (CNS) is the most cholesterol-rich organ in the body. Cholesterol is essential to CNS functions such as synaptogenesis and formation of myelin. Significant differences exist in cholesterol metabolism between the CNS and the peripheral organs. However, the regulation of cholesterol metabolism in the CNS is poorly understood compared to our knowledge of the regulation of cholesterol homeostasis in organs reached by cholesterol-carrying lipoprotein particles in the circulation. Defects in CNS cholesterol homeostasis have been linked to a variety of neurodegenerative diseases, including common diseases with complex pathogenetic mechanisms such as Alzheimer s disease. In spite of intense effort, the mechanisms which link disturbed cholesterol homeostasis to these diseases remain elusive. We used three inherited recessive neurodegenerative disorders as models in the studies included in this thesis: Niemann-Pick type C (NPC), infantile neuronal ceroid lipofuscinosis and cathepsin D deficiency. Of these three, NPC has previously been linked to disturbed intracellular cholesterol metabolism. Elucidating the mechanisms with which disturbances of cholesterol homeostasis link to neurodegeneration in recessive inherited disorders with known genetic lesions should shed light on how cholesterol is handled in the healthy CNS and help to understand how these and more complex diseases develop. In the first study we analyzed the synthesis of sterols and the assembly and secretion of lipoprotein particles in Npc1 deficient primary astrocytes. We found that both wild type and Npc1 deficient astrocytes retain significant amounts of desmosterol and other cholesterol precursor sterols as membrane constituents. No difference was observed in the synthesis of sterols and the secretion of newly synthesized sterols between Npc1 wild type, heterozygote or knockout astrocytes. We found that the incorporation of newly synthesized sterols into secreted lipoprotein particles was not inhibited by Npc1 mutation, and the lipoprotein particles were similar to those excreted by wild type astrocytes in shape and size. The bulk of cholesterol was found to be secreted independently of secreted NPC2. These observations demonstrate the ability of Npc1 deficient astrocytes to handle de novo sterols, and highlight the unique sterol composition in the developing brain. Infantile neuronal ceroid lipofuscinosis is caused by the deficiency of a functional Ppt1 enzyme in the cells. In the second study, global gene expression studies of approximately 14000 mouse genes showed significant changes in the expression of 135 genes in Ppt1 deficient neurons compared to wild type. Several genes encoding for enzymes of the mevalonate pathway of cholesterol biosynthesis showed increased expression. As predicted by the expression data, sterol biosynthesis was found to be upregulated in the knockout neurons. These data link Ppt1 deficiency to disturbed cholesterol metabolism in CNS neurons. In the third study we investigated the effect of cathepsin D deficiency on the structure of myelin and lipid homeostasis in the brain. Our proteomics data, immunohistochemistry and western blotting data showed altered levels of the myelin protein components myelin basic protein, proteolipid protein and 2 , 3 -cyclic nucleotide 3 phosphodiesterase in the brains of cathepsin D deficient mice. Electron microscopy revealed altered myelin structure in cathepsin D deficient brains. Additionally, plasmalogen-derived alkenyl chains and 20- and 24-carbon saturated and monounsaturated fatty acids typical for glycosphingolipids were found to be significantly reduced, but polyunsaturated species were significantly increased in the knockout brains, pointing to a decrease in white matter. The levels of ApoE and ABCA1 proteins linked to cholesterol efflux in the CNS were found to be altered in the brains of cathepsin D deficient mice, along with an accumulation of cholesteryl esters and a decrease in triglycerols. Together these data demonstrate altered myelin architecture in cathepsin D deficient mice and link cathepsin D deficiency to aberrant cholesterol metabolism and trafficking. Basic research into rare monogenic diseases sheds light on the underlying biological processes which are perturbed in these conditions and contributes to our understanding of the physiological function of healthy cells. Eventually, understanding gained from the study of disease models may contribute towards establishing treatment for these disorders and further our understanding of the pathogenesis of other, more complex and common diseases.

Trafficking and ethanol-induced inhibition of AMPA receptors

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

Mitochondrial Malfunction in Tumor Formation and Neuromuscular Diseases : Consequences of defects in fumarate hydratase and in the respiratory chain

The mitochondrion is an organelle of outmost importance, and the mitochondrial network performs an array of functions that go well beyond ATP synthesis. Defects in mitochondrial performance lead to diseases, often affecting nervous system and muscle. Although many of these mitochondrial diseases have been linked to defects in specific genes, the molecular mechanisms underlying the pathologies remain unclear. The work in this thesis aims to determine how defects in mitochondria are communicated within - and interpreted by - the cells, and how this contributes to disease phenotypes. Fumarate hydratase (FH) is an enzyme of the citrate cycle. Recessive defects in FH lead to infantile mitochondrial encephalopathies, while dominant mutations predispose to tumor formation. Defects in succinate dehydrogenase (SDH), the enzyme that precedes FH in the citrate cycle, have also been described. Mutations in SDH subunits SDHB, SDHC and SDHD are associated with tumor predisposition, while mutations in SDHA lead to a characteristic mitochondrial encephalopathy of childhood. Thus, the citrate cycle, via FH and SDH, seems to have essential roles in mitochondrial function, as well as in the regulation of processes such as cell proliferation, differentiation or death. Tumor predisposition is not a typical feature of mitochondrial energy deficiency diseases. However, defects in citrate cycle enzymes also affect mitochondrial energy metabolism. It is therefore necessary to distinguish what is specific for defects in citrate cycle, and thus possibly associated with the tumor phenotype, from the generic consequences of defects in mitochondrial aerobic metabolism. We used primary fibroblasts from patients with recessive FH defects to study the cellular consequences of FH-deficiency (FH-). Similarly to the tumors observed in FH- patients, these fibroblasts have very low FH activity. The use of primary cells has the advantage that they are diploid, in contrast with the aneuploid tumor cells, thereby enabling the study of the early consequences of FH- in diploid background, before tumorigenesis and aneuploidy. To distinguish the specific consequences of FH- from typical consequences of defects in mitochondrial aerobic metabolism, we used primary fibroblasts from patients with MELAS (mitochondrial encephalopathy with lactic acidosis and stroke-like episodes) and from patients with NARP (neuropathy, ataxia and retinitis pigmentosa). These diseases also affect mitochondrial aerobic metabolism but are not known to predispose to tumor formation. To study in vivo the systemic consequences of defects in mitochondrial aerobic metabolism, we used a transgenic mouse model of late-onset mitochondrial myopathy. The mouse contains a transgene with an in-frame duplication of a segment of Twinkle, the mitochondrial replicative helicase, whose defects underlie the human disease progressive external ophthalmoplegia. This mouse model replicates the phenotype in the patients, particularly neuronal degeneration, mitochondrial myopathy, and subtle decrease of respiratory chain activity associated with mtDNA deletions. Due to the accumulation of mtDNA deletions, the mouse was named deletor. We first studied the consequences of FH- and of respiratory chain defects for energy metabolism in primary fibroblasts. To further characterize the effects of FH- and respiratory chain malfunction in primary fibroblasts at transcriptional level, we used expression microarrays. In order to understand the in vivo consequences of respiratory chain defects in vivo, we also studied the transcriptional consequences of Twinkle defects in deletor mice skeletal muscle, cerebellum and hippocampus. Fumarate accumulated in the FH- homozygous cells, but not in the compound heterozygous lines. However, virtually all FH- lines lacked cytoplasmic FH. Induction of glycolysis was common to FH-, MELAS and NARP fibroblasts. In deletor muscle glycolysis seemed to be upregulated. This was in contrast with deletor cerebellum and hippocampus, where mitochondrial biogenesis was in progress. Despite sharing a glycolytic pattern in energy metabolism, FH- and respiratory chain defects led to opposite consequences in redox environment. FH- was associated with reduced redox environment, while MELAS and NARP displayed evidences of oxidative stress. The deletor cerebellum had transcriptional induction of antioxidant defenses, suggesting increased production of reactive oxygen species. Since the fibroblasts do not represent the tissues where the tumors appear in FH- patients, we compared the fibroblast array data with the data from FH- leiomyomas and normal myometrium. This allowed the determination of the pathways and networks affected by FH-deficiency in primary cells that are also relevant for myoma formation. A key pathway regulating smooth muscle differentiation, SRF (serum response factor)-FOS-JUNB, was found to be downregulated in FH- cells and in myomas. While in the deletor mouse many pathways were affected in a tissue-specific basis, like FGF21 induction in the deletor muscle, others were systemic, such as the downregulation of ALAS2-linked heme synthesis in all deletor tissues analyzed. However, interestingly, even a tissue-specific response of FGF21 excretion could elicit a global starvation response. The work presented in this thesis has contributed to a better understanding of mitochondrial stress signalling and of pathways interpreting and transducing it to human pathology.

Susceptibility genes and neurodevelopmental mechanisms in dyslexia

Developmental dyslexia is a specific reading disability, which is characterised by unexpected difficulty in reading, spelling and writing despite adequate intelligence, education and social environment. It is the most common childhood learning disorder affecting 5-10 % of the population and thus constitutes the largest portion of all learning disorders. It is a persistent developmental failure although it can be improved by compensation. According to the most common theory, the deficit is in phonological processing, which is needed in reading when the words have to be divided into phonemes, or distinct sound elements. This occurs in the lowest level of the hierarchy of the language system and disturbs processes in higher levels, such as understanding the meaning of words. Dyslexia is a complex genetic disorder and previous studies have found nine locations in the genome that associate with it. Altogether four susceptibility genes have been found and this study describes the discovery of the first two of them, DYX1C1 and ROBO1. The first clues were obtained from two Finnish dyslexic families that have chromosomal translocations which disrupt these genes. Genetic analyses supported their role in dyslexia: DYX1C1 associates with dyslexia in the Finnish population and ROBO1 was linked to dyslexia in a large Finnish pedigree. In addition a genome-wide scan in Finnish dyslexic families was performed. This supported the previously detected dyslexia locus on chromosome 2 and revealed a new locus on chromosome 7. Dyslexia is a neurological disorder and the neurobiological function of the susceptibility genes DYX1C1 and ROBO1 are consistent with this. ROBO1 is an axon guidance receptor gene, which is involved in axon guidance across the midline in Drosophila and axonal pathfinding between the two hemispheres via the corpus callosum, as well as neuronal migration in the brain of mice. The translocation and decreased ROBO1 expression in dyslexic individuals indicate that two functional copies of ROBO1 gene are required in reading. DYX1C1 was a new gene without a previously known function. Inhibition of Dyx1c1 expression showed that it is needed in normal brain development in rats. Without Dyx1c1 protein, the neurons in the developing brain will not migrate to their final position in the cortex. These two dyslexia susceptibility genes DYX1C1 and ROBO1 revealed two distinct neurodevelopmental mechanisms of dyslexia, axonal pathfinding and neuronal migration. This study describes the discovery of the genes and our research to clarify their role in developmental dyslexia.

The molecular basis of Marinesco-Sjögren syndrome

Marinesco-Sjögren syndrome (MSS) is a rare autosomal recessive neurodegenerative disorder characterized by cerebellar ataxia due to cerebellar cortical atrophy, infantile- or childhood-onset bilateral cataracts, progressive myopathy, and mild to severe mental retardation. Additional features include hypergonadotropic hypogonadism, various skeletal abnormalities, short stature, and strabismus. The neuroradiologic hallmarks are hypoplasia of both the vermis and cerebellar hemispheres. The histopathologic findings include severe cerebellar atrophy and loss of Purkinje and granule cells. The common pathologic findings in muscle biopsy are variation in muscle fiber size, atrophic fibers, fatty replacement, and rimmed vacuole formation. The presence of marked cerebellar atrophy with myopathy distinguishes MSS from another rare syndrome, the congenital cataracts, facial dysmorphism, and neuropathy syndrome (CCFDN). Previously, work by others had resulted in the identification of an MSS locus on chromosome 5q31. A subtype of MSS with myoglobinuria and neuropathy had been linked to the CCFDN locus on chromosome 18qter, at which mutations in the CTDP1 gene had been identified. We confirmed linkage to the previously identified locus on chromosome 5q31 in two Finnish families with eight affected individuals, reduced the critical region by fine-mapping, and identified SIL1 as a gene underlying MSS. We found a common homozygous founder mutation in all Finnish patients. The same mutation was also present in patient samples from Norway and Sweden. Altogether, we identified eight mutations in SIL1, including nonsense, frameshift, splice site alterations, and one missense mutation. SIL1 encodes a nucleotide exchange factor for the endoplasmic reticulum (ER) resident heat-shock protein 70 chaperone GRP78. GRP78 functions in protein synthesis and quality control of the newly synthesized polypeptides. It senses and responds to stressful cellular conditions. We showed that in mice, SIL1 and GRP78 show highly similar spatial and temporal tissue expression in developing and mature brain, eye, and muscle. Studying endogenous proteins in mouse primary hippocampal neurons, we found that SIL1 and GRP78 colocalize and that SIL1 localizes to the ER. We studied the subcellular localization of two mutant proteins, a missense mutant found in two patients and an artificial mutant lacking the ER retrieval signal, and found that both mutant proteins formed aggregates within the ER. Well in line with our findings and the clinical features of MSS, recent work by Zhao et al. showed that a truncation of SIL1 causes ataxia and cerebellar Purkinje cell loss in the naturally occurring woozy mutant mouse. Prior to Purkinje cell degeneration, the unfolded protein response is initiated and abnormal protein accumulations are present. MSS thus joins the group of protein misfolding and accumulation diseases. These findings highlight the importance of SIL1 and the role of the ER in neuronal function and survival. The results presented in this thesis provide tools for the molecular genetic diagnostics of MSS and give a basis for future studies on the molecular pathogenesis of MSS. Understanding the mechanisms behind this pleiotropic syndrome may provide insights into more common forms of ataxia, myopathy, and neurodegeneration.

Animal model and molecular interactions of Cln5

Neuronal ceroid lipofuscinoses (NCLs) are a family of inherited pediatric neurodegenerative disorders, leading to retinal degeneration, death of selective neuronal populations and accumulation of autofluorscent ceroid-lipopigments. The clinical manifestations are generally similar in all forms. The Finnish variant late infantile neuronal ceroid lipofuscinosis (vLINCLFin) is a form of NCL, especially enriched in the Finnish population. The aim of this thesis was to analyse the brain pathology of vLINCLFin utilising the novel Cln5-/- mouse model. Gene expression profiling of the brains of already symptomatic Cln5-/- mice revealed that inflammation, neurodegeneration and defects in myelinization are the major characteristics of the later stages of the disease. Histological characterization of the brain pathology confirmed that the thalamocortical system is affected in Cln5-/- mice, similarly to the other NCL mouse models. However, whereas the brain pathology in all other analyzed NCL mice initiate in the thalamus and spread only months later to the cortex, we observed that the sequence of events is uniquely reversed in Cln5-/- mice; beginning in the cortex and spreading to the thalamus only months later. We could also show that even though neurodegeneration is inititated in the cortex, reactive gliosis and loss of myelin are evident in specific nuclei of the thalamus already in the 1 month old brain. To obtain a deeper insight into the disturbed metabolic pathways, we performed gene expression profiling of presymptomatic mouse brains. We validated these findings with immunohistological analyses, and could show that cytoskeleton and myelin were affected in Cln5-/- mice. Comparison of gene expression profiling results of Cln5-/- and Cln1-/- mice, further highlighted that these two NCL models share a common defective pathway, leading to disturbances in the neuronal growth cone and cytoskeleton. Encouraged by the evidence of this defected pathway, we analyzed the molecular interactions of NCL-proteins and observed that Cln5 and Cln1/Ppt1 proteins interact with each other. Furthermore, we demonstrated that Cln5 and Cln1/Ppt1 share an interaction partner, the F1-ATP synthase, potentially linking both vLINCLFIN and INCL diseases to disturbed lipid metabolism. In addition, Cln5 was shown to interact with other NCL proteins; Cln2, Cln3, Cln6 and Cln8, implicating a central role for Cln5 in the NCL pathophysiology. This study is the first to describe the brain pathology and gene expression changes in the Cln5-/- mouse. Together the findings presented in this thesis represent novel information of the disease processes and the molecular mechanisms behind vLINCLFin and have highlighted that vLINCLFin forms a very important model to analyze the pathophysiology of NCL diseases.

Endoplasmic reticulum stress and cell death mechanisms in the cell model of Huntington’s disease

This thesis clarifies important molecular pathways that are activated during the cell death observed in Huntington’s disease. Huntington’s disease is one of the most common inherited neurodegenerative diseases, which is primarily inherited in an autosomal dominant manner. HD is caused by an expansion of CAG repeats in the first exon of the IT15 gene. IT15 encodes the production of a Huntington’s disease protein huntingtin. Mutation of the IT15 gene results in a long stretch of polyQ residues close to the amino-terminal region of huntingtin. Huntington’s disease is a fatal autosomal neurodegenerative disorder. Despite the current knowledge of HD, the precise mechanism behind the selective neuronal death, and how the disease propagates, still remains an enigma. The studies mainly focused on the control of endoplasmic reticulum (ER) stress triggered by the mutant huntingtin proteins. The ER is a delicate organelle having essential roles in protein folding and calcium regulation. Even the slightest perturbations on ER homeostasis are effective enough to trigger ER stress and its adaptation pathways, called unfolded protein response (UPR). UPR is essential for cellular homeostasis and it adapts ER to the changing environment and decreases ER stress. If adaptation processes fail and stress is excessive and prolonged; irreversible cell death pathways are engaged. The results showed that inhibition of ER stress with chemical agents are able to decrease cell death and formation of toxic cell aggregates caused by mutant huntingtin proteins. The study concentrated also to the NF-κB (nuclear factor-kappaB) pathway, which is activated during ER stress. NF-κB pathway is capable to regulate the levels of important cellular antioxidants. Cellular antioxidants provide a first line of defence against excess reactive oxygen species. Excess accumulation of reactive oxygen species and subsequent activation of oxidative stress damages motley of vital cellular processes and induce cell degeneration. Data showed that mutant huntingtin proteins downregulate the expression levels of NF-κB and vital antioxidants, which was followed by increased oxidative stress and cell death. Treatment with antioxidants and inhibition of oxidative stress were able to counteract these adverse effects. In addition, thesis connects ER stress caused by mutant huntingtin to the cytoprotective autophagy. Autophagy sustains cellular balance by degrading potentially toxic cell proteins and components observed in Huntington’s disease. The results revealed that cytoprotective autophagy is active at the early points (24h) of ER stress after expression of mutant huntingtin proteins. GADD34 (growth arrest and DNA damage-inducible gene 34), which is previously connected to the regulation of translation during cell stress, was shown to control the stimulation of autophagy. However, GADD34 and autophagy were downregulated at later time points (48h) during mutant huntingtin proteins induced ER stress, and subsequently cell survival decreased. Overexpression GADD34 enhanced autophagy and decreased cell death, indicating that GADD34 plays a critical role in cell protection. The thesis reveales new interesting data about the neuronal cell death pathways seen in Huntington’s disease, and how cell degeneration is partly counteracted by various therapeutic agents. Expression of mutant huntingtin proteins is shown to alter signaling events that control ER stress, oxidative stress and autophagy. Despite that Huntington’s disease is mainly an untreatable disorder; these findings offer potential targets and neuroprotective strategies in designing novel therapies for Huntington’s disease.

N-syndecan and HB-GAM in neural migration and differentiation : Modulation of growth factor activity in brain

The juvenile sea squirt wanders through the sea searching for a suitable rock or hunk of coral to cling to and make its home for life. For this task it has a rudimentary nervous system. When it finds its spot and takes root, it doesn't need its brain any more so it eats it. It's rather like getting tenure. Daniel C. Dennett (from Consciousness Explained, 1991) The little sea squirt needs its brain for a task that is very simple and short. When the task is completed, the sea squirt starts a new life in a vegetative state, after having a nourishing meal. The little brain is more tightly structured than our massive primate brains. The number of neurons is exact, no leeway in neural proliferation is tolerated. Each neuroblast migrates exactly to the correct position, and only a certain number of connections with the right companions is allowed. In comparison, growth of a mammalian brain is a merry mess. The reason is obvious: Squirt brain needs to perform only a few, predictable functions, before becoming waste. The more mobile and complex mammals engage their brains in tasks requiring quick adaptation and plasticity in a constantly changing environment. Although the regulation of nervous system development varies between species, many regulatory elements remain the same. For example, all multicellular animals possess a collection of proteoglycans (PG); proteins with attached, complex sugar chains called glycosaminoglycans (GAG). In development, PGs participate in the organization of the animal body, like in the construction of parts of the nervous system. The PGs capture water with their GAG chains, forming a biochemically active gel at the surface of the cell, and in the extracellular matrix (ECM). In the nervous system, this gel traps inside it different molecules: growth factors and ECM-associated proteins. They regulate the proliferation of neural stem cells (NSC), guide the migration of neurons, and coordinate the formation of neuronal connections. In this work I have followed the role of two molecules contributing to the complexity of mammalian brain development. N-syndecan is a transmembrane heparan sulfate proteoglycan (HSPG) with cell signaling functions. Heparin-binding growth-associated molecule (HB-GAM) is an ECM-associated protein with high expression in the perinatal nervous system, and high affinity to HS and heparin. N-syndecan is a receptor for several growth factors and for HB-GAM. HB-GAM induces specific signaling via N-syndecan, activating c-Src, calcium/calmodulin-dependent serine protein kinase (CASK) and cortactin. By studying the gene knockouts of HB-GAM and N-syndecan in mice, I have found that HB-GAM and N-syndecan are involved as a receptor-ligand-pair in neural migration and differentiation. HB-GAM competes with the growth factors fibriblast growth factor (FGF)-2 and heparin-binding epidermal growth factor (HB-EGF) in HS-binding, causing NSCs to stop proliferation and to differentiate, and affects HB-EGF-induced EGF receptor (EGFR) signaling in neural cells during migration. N-syndecan signaling affects the motility of young neurons, by boosting EGFR-mediated cell migration. In addition, these two receptors form a complex at the surface of the neurons, probably creating a motility-regulating structure.

Mechanisms of KCC2 upregulation during development

Gamma-aminobutyric acid (GABA) acting through ionotropic GABAA receptors plays a crucial role in the activity of the central nervous system (CNS). It triggers Ca2+ rise providing trophic support in developing neurons and conducts fast inhibitory function in mature neuronal networks. There is a developmental change in the GABAA reversal potential towards more negative levels during the first two postnatal weeks in rodent hippocampus. This change provides the basis for mature GABAergic activity and is attributable to the developmental expression of the neuron-specific potassium chloride cotransporter 2 (KCC2). In this work we have studied the mechanisms responsible for the control of KCC2 developmental expression. As a model system we used hippocampal dissociated cultures plated from embryonic day (E) 17 mice embryos before the onset of KCC2 expression. We showed that KCC2 was significantly up-regulated during the first two weeks of culture development. Interestingly, the level of KCC2 upregulation was not altered by chronic pharmacological blockage of action potentials as well as GABAergic and glutamatergic synaptic transmission. By in silico analysis of the proximal KCC2 promoter region we identified 10 candidate transcription factor binding sites that are highly conserved in mammalian KCC2 genes. One of these transcription factors, namely early growth response factor 4 (Egr4), had similar developmental profile as KCC2 and considerably increased the activity of mouse KCC2 gene in neuronal cells. Next we investigated the involvement of neurotrophic factors in regulation of Egr4 and KCC2 expression. We found that in immature hippocampal cultures Egr4 and KCC2 levels were strongly up-regulated by brain derived neurotrophic factor (BDNF)and neurturin. The effect of neurotrophic factors was dependent on the activation of a mitogen activated protein kinase (MAPK) signal transduction pathway. Intact Egr4-binding site in proximal KCC2 promoter was required for BDNF-induced KCC2 transcription. In vitro data were confirmed by several in vivo experiments where we detected an upregulation of KCC2 protein levels after intrahippocampal administration of BDNF or neurturin. Importantly, a MAPK-dependent rise in Egr4 and KCC2 expression levels was also observed after a period of kainic acid-induced seizure activity in neonatal rats suggesting that neuronal activity might be involved in Egr4-mediated regulation of KCC2 expression. Finally we demonstrated that the mammalian KCC2 gene (alias Slc12a5) generated two neuron-specific isoforms by using alternative promoters and first exons. A novel isoform of KCC2, termed KCC2a, differed from the previously known KCC2b isoform by 40 unique N-terminal amino acid residues. KCC2a expression was restricted to CNS,remained relatively constant during postnatal development, and contributed 20 50% of total KCC2 mRNA expression in the neonatal mouse brainstem and spinal cord. In summary, our data provide insight into the complex regulation of KCC2 expression during early postnatal development. Although basal KCC2 expression seems to be intrinsically regulated, it can be further augmented by neurotrophic factors or by enhanced activity triggering MAPK phosphorylation and Egr4 induction. Additional KCC2a isoform, regulated by another promoter, provides basal KCC2 level in neonatal brainstem and spinal cord required for survival of KCC2b knockout mice.

GDNF family receptors in peripheral target innervation and hormone production

Glial cell line-derived neurotrophic factor (GDNF) family ligands: GDNF, neurturin, persephin and artemin, signal through a receptor tyrosine kinase Ret by binding first to a co-receptor (GFRα1-4) that is attached to the plasma membrane. The GDNF family factors can support the survival of various peripheral and central neuronal populations and have important functions also outside the nervous system, especially in kidney development. Activating mutations in the RET gene cause tumours in neuroendocrine cells, whereas inactivating mutations in RET are found in patients with Hirschsprung s disease (HSCR) characterized by loss of ganglionic cells along the intestine. The aim of this study was to examine the in vivo functions of neurturin receptor GFRα2 and persephin receptor GFRα4 using knockout (KO) mice. Mice lacking GFRα2 grow poorly after weaning and have deficits in parasympathetic and enteric innervation. This study shows that impaired secretion of the salivary glands and exocrine pancreas contribute to growth retardation in GFRα2-KO mice. These mice have a reduced number of intrapancreatic neurons and decreased cholinergic innervation of the exocrine pancreas as well as reduced excitatory fibres in the myenteric plexus of the small intestine. This study also demonstrates that GFRα2-mediated Ret signalling is required for target innervation and maintenance of soma size of sympathetic cholinergic neurons and sensory nociceptive IB4-binding neurons. Furthermore, lack of GFRα2 in mice results in deficient perception of temperatures above and below thermoneutrality and in attenuated inflammatory pain response. GFRα4 is co-expressed with Ret predominantly in calcitonin-producing thyroid C-cells in the mouse. In this study GFRα4-deficient mice were generated. The mice show no gross developmental deficits and have a normal number of C-cells. However, young but not adult mice lacking GFRα4 have a lower production of calcitonin in thyroid tissue and consequently, an increased bone formation rate. Thus, GFRα4/Ret signalling may regulate calcitonin production. In conclusion, this study reveals that GFRα2/Ret signalling is crucial for the development and function of specific components of the peripheral nervous system and that GFRα4-mediated Ret signalling is required for controlling transmitter synthesis in thyroid C-cells.

Structure-Function Studies of GDNF Family Ligand-RET signalling

Glial cell line-derived neurotrophic factor (GDNF) and its family members neurturin (NRTN), artemin (ARTN) and persephin (PSPN) are growth factors, which are involved in the development, differentiation and maintenance of many neuron types. In addition, they function outside of the nervous system, e.g. in the development of kidney, testis and liver. GDNF family ligand (GFL) signalling happens through a tetrameric receptor complex, which includes two glycosylphosphatidylinositol (GPI)-anchored GDNF family receptor (GFRα) molecules and two RET (rearranged during transfection) receptor tyrosine kinases. Each of the ligands binds preferentially one of the four GFRα receptors: GDNF binds to GFRα1, NRTN to GFRα2, ARTN to GFRα3 and PSPN to GFRα4. The signal is then delivered by RET, which cannot bind the GFLs on its own, but can bind the GFL-GFRα complex. Under normal cellular conditions, RET is only phosphorylated on the cell surface after ligand binding. At least the GDNF-GFRα1 complex is believed to recruit RET to lipid rafts, where downstream signalling occurs. In general, GFRαs consist of three cysteine-rich domains, but all GFRα4s except for chicken GFRα4 lack domain 1 (D1). We characterised the biochemical and cell biological properties of mouse PSPN receptor GFRα4 and showed that it has a significantly weaker capacity than GFRα1 to recruit RET to the lipid rafts. In spite of that, it can phosphorylate RET in the presence of PSPN and contribute to neuronal differentiation and survival. Therefore, the recruitment of RET to the lipid rafts does not seem to be crucial for the biological activity of all GFRα receptors. Secondly, we demonstrated that GFRα1 D1 stabilises the GDNF-GFRα1 complex and thus affects the phosphorylation of RET and contributes to the biological activity. This may be important in physiological conditions, where the concentration of the ligand or the soluble GFRα1 receptor is low. Our results also suggest a role for D1 in heparin binding and, consequently, in the biodistribution of released GFRα1 or in the formation of the GFL-GFRα-RET complex. We also presented the crystallographic structure of GDNF in the complex with GFRα1 domains 2 and 3. The structure differs from the previously published ARTN-GFRα3 structure in three significant ways. The biochemical data verify the structure and reveal residues participating in the interactions between GFRα1 and GDNF, and preliminarily also between GFRα1 and RET and heparin. Finally, we showed that, the precursor of the oncogenic MEN 2B (multiple endocrine neoplasia type 2) form of RET gets phosphorylated already during its synthesis in the endoplasmic reticulum (ER). We also demonstrated that it associates with Src homology 2 domain-containing protein (SHC) and growth factor receptor-bound protein (GRB2) in the ER, and has the capacity to activate several downstream signalling molecules.

Structural and functional roles of KCC2 in developing cortex

Cation chloride cotransporters (CCCs) are critical for controlling intracellular chloride homeostasis. The CCC family is composed of four isoforms of K-Cl cotransporters (KCC1-4), two isoforms of Na-K-2Cl cotransporters (NKCC1-2), one Na-Cl cotransporter (NCC) and two the structurally related proteins with unknown function, CCC8 also known as cation-chloride cotransporter interaction protein, CIP, and CCC9. KCC2 is a neuron-specific isoform, which plays a prominent role in controlling the intracellular Cl- concentration in neurons and is responsible for producing the negative shift of GABAA responses from depolarizing to hyperpolarizing during neuronal maturation. In the present studies we first used in situ hybridization to examine the developmental expression patterns of the cation-chloride cotransporters KCC1-4 and NKCC1. We found that they display complementary expression patterns during embryonic brain development. Most interestingly, KCC2 expression in the embryonic central nervous system strictly follows neuronal maturation. In vitro data obtained from primary and organotypic neuronal cultures support this finding and revealed a temporal correlation between the expression of KCC2 and synaptogenesis. We found that KCC2 is highly expressed in filopodia and mature spines as well as dendritic shaft and investigated the role of KCC2 in spine formation by analyzing KCC2-/- neurons in vitro. Our studies revealed that KCC2 is a key factor in the maturation of dendritic spines. Interestingly, the effect of KCC2 in spine formation is not due to Cl- transport activity, but mediated through the interaction between KCC2 C-terminal and intracellular protein associated with cytoskeleton. The interacting protein we found is protein 4.1N by immunoprecipitation. Our results indicate a structural role for KCC2 in the development of functional glutamatergic synapses and suggest KCC2 as a synchronizer for the functional development of glutamatergic and GABAergic synapses in neuronal network. Studies on the regulatory mechanisms of KCC2 expression during development and plasticity revealed that synaptic activity of both the glutamatergic and GABAergic system is not required for up-regulation of KCC2 during development, whereas in acute mature hippocampal slices which undergo continuous synchronous activity induced by the absence of Mg2+ solution, KCC2 mRNA and protein expression were down-regulated in CA1 pyramidal neurons subsequently leading to a reduced capacity for neuronal Cl- extrusion. This effect is mediated by endogenous BDNF-TrkB down-stream cascades involving both Shc/FRS-2 and PLCγ-CREB signaling. BDNF mediated changes in KCC2 expression indicate that KCC2 is significantly involved in the complex mechanisms of neuronal plasticity during development and pathophysiological conditions.

GABAa Receptor Mediated Signalling in the Brain: Inhibition, Shunting and Excitation

Within central nervous system, the simple division of chemical synaptic transmission to depolarizing excitation mediated by glutamate and hyperpolarizing inhibition mediated by γ-amino butyric acid (GABA), is evidently an oversimplification. The GABAa receptor (GABAaR) mediated responses can be of opposite sign within a single resting cell, due to the compartmentalized distribution of cation chloride cotransporters (CCCs). The K+/Cl- cotransporter 2 (KCC2), member of the CCC family, promotes K+ fuelled Cl- extrusion and sets the reversal potential of GABA evoked anion currents typically slightly below the resting membrane potential. The interesting ionic plasticity property of GABAergic signalling emerges from the short-term and long-term alterations in the intraneuronal concentrations of GABAaR permeable anions (Cl- and HCO3-). The short-term effects arise rapidly (in the time scale of hundreds of milliseconds) and are due to the GABAaR activation dependent shifts in anion gradients, whereas the changes in expression, distribution and kinetic regulation of CCCs are underlying the long-term effects, which may take minutes or even hours to develop. In this Thesis, the differences in the reversal potential of GABAaR mediated responses between dopaminergic and GABAergic cell types, located in the substantia nigra, were shown to be attributable to the differences in the chloride extrusion mechanisms. The stronger inhibitory effect of GABA on GABAergic neurons was due to the cell type specific expression of KCC2 whereas the KCC2 was absent from dopaminergic neurons, leading to a less prominent inhibition brought by GABAaR activation. The levels of KCC2 protein exhibited activity dependent alterations in hippocampal pyramidal neurons. Intense neuronal activity, leading to a massive release of brain derived neurotrophic factor (BDNF) in vivo, or applications of tyrosine receptor kinase B (TrkB) agonists BDNF or neurotrophin-4 in vitro, were shown to down-regulate KCC2 protein levels which led to a reduction in the efficacy of Cl- extrusion. The GABAergic transmission is interestingly involved in an increase of extracellular K+ concentration. A substantial increase in interstitial K+ tends to depolarize the cell membrane. The effects that varying ion gradients had on the generation of biphasic GABAaR mediated responses were addressed, with particular emphasis on the novel idea that the K+/Cl- extrusion via KCC2 is accelerated in response to a rapid accumulation of intracellular Cl-. The KCC2 inhibitor furosemide produced a large reduction in the GABAaR dependent extracellular K+ transients. Thus, paradoxically, both the inefficient KCC2 activity (via increased intracellular Cl-) and efficient KCC2 activity (via increased extracellular K+) may promote excitation.

Mu in vitro Transposition Technology in Functional Genetics and Genomics: Applications on Mouse and Bacteriophages

Transposons are mobile elements of genetic material that are able to move in the genomes of their host organisms using a special form of recombination called transposition. Bacteriophage Mu was the first transposon for which a cell-free in vitro transposition reaction was developed. Subsequently, the reaction has been refined and the minimal Mu in vitro reaction is useful in the generation of comprehensive libraries of mutant DNA molecules that can be used in a variety of applications. To date, the functional genetics applications of Mu in vitro technology have been subjected to either plasmids or genomic regions and entire genomes of viruses cloned on specific vectors. This study expands the use of Mu in vitro transposition in functional genetics and genomics by describing novel methods applicable to the targeted transgenesis of mouse and the whole-genome analysis of bacteriophages. The methods described here are rapid, efficient, and easily applicable to a wide variety of organisms, demonstrating the potential of the Mu transposition technology in the functional analysis of genes and genomes. First, an easy-to-use, rapid strategy to generate construct for the targeted mutagenesis of mouse genes was developed. To test the strategy, a gene encoding a neuronal K+/Cl- cotransporter was mutagenised. After a highly efficient transpositional mutagenesis, the gene fragments mutagenised were cloned into a vector backbone and transferred into bacterial cells. These constructs were screened with PCR using an effective 3D matrix system. In addition to traditional knock-out constructs, the method developed yields hypomorphic alleles that lead into reduced expression of the target gene in transgenic mice and have since been used in a follow-up study. Moreover, a scheme is devised to rapidly produce conditional alleles from the constructs produced. Next, an efficient strategy for the whole-genome analysis of bacteriophages was developed based on the transpositional mutagenesis of uncloned, infective virus genomes and their subsequent transfer into susceptible host cells. Mutant viruses able to produce viable progeny were collected and their transposon integration sites determined to map genomic regions nonessential to the viral life cycle. This method, applied here to three very different bacteriophages, PRD1, ΦYeO3 12, and PM2, does not require the target genome to be cloned and is directly applicable to all DNA and RNA viruses that have infective genomes. The method developed yielded valuable novel information on the three bacteriophages studied and whole-genome data can be complemented with concomitant studies on individual genes. Moreover, end-modified transposons constructed for this study can be used to manipulate genomes devoid of suitable restriction sites.

Novel CDNF/MANF protein family : Molecular structure, expression and neurotrophic activity

Neurotrophic factors (NTFs) are secreted proteins which promote the survival of neurons, formation and maintenance of neuronal contacts and regulate synaptic plasticity. NTFs are also potential drug candidates for the treatment of neurodegenerative diseases. Parkinson’s disease (PD) is mainly caused by the degeneration of midbrain dopaminergic neurons. Current therapies for PD do not stop the neurodegeneration or repair the affected neurons. Thus, search of novel neurotrophic factors for midbrain dopaminergic neurons, which could also be used as therapeutic proteins, is highly warranted. In the present study, we identified and characterized a novel protein named conserved dopamine neurotrophic factor (CDNF), a homologous protein to mesencephalic astrocyte-derived neurotrophic factor (MANF). Others have shown that MANF supports the survival of embryonic midbrain dopaminergic neurons in vitro, and protects cultured cells against endoplasmic reticulum (ER) stress. CDNF and MANF form a novel evolutionary conserved protein family with characteristic eight conserved cysteine residues in their primary structure. The vertebrates have CDNF and MANF encoding genes, whereas the invertebrates, including Drosophila and Caenorhabditis have a single homologous CDNF/MANF gene. In this study we show that CDNF and MANF are secreted proteins. They are widely expressed in the mammalian brain, including the midbrain and striatum, and in several non-neuronal tissues. We expressed and purified recombinant human CDNF and MANF proteins, and tested the neurotrophic activity of CDNF on midbrain dopaminergic neurons using a 6-hydroxydopamine (6-OHDA) rat model of PD. In this model, a single intrastriatal injection of CDNF protected midbrain dopaminergic neurons and striatal dopaminergic fibers from the 6-OHDA toxicity. Importantly, an intrastriatal injection of CDNF also restored the functional activity of the nigrostriatal dopaminergic system when given after the striatal 6-OHDA lesion. Thus, our study shows that CDNF is a potential novel therapeutic protein for the treatment of PD. In order to elucidate the molecular mechanisms of CDNF and MANF activity, we resolved their crystal structure. CDNF and MANF proteins have two domains; an amino (N)-terminal saposin-like domain and a presumably unfolded carboxy (C)-terminal domain. The saposin-like domain, which is formed by five α-helices and stabilized by three intradomain disulphide bridges, may bind to lipids or membranes. The C-terminal domain contains an internal cysteine bridge in a CXXC motif similar to that of thiol/disulphide oxidoreductases and isomerases, and may thus facilitate protein folding in the ER. Our studies suggest that CDNF and MANF are novel potential therapeutic proteins for the treatment of neurodegenerative diseases. Future studies will reveal the neurotrophic and cytoprotective mechanisms of CDNF and MANF in more detail.