Upregulation and Functionality of Neuronal Nicotinic Acetylcholine Receptors

Cigarette smoking is, in developed countries, the leading cause of premature death. In tobacco smoke, the main addictive compound is nicotine, which in the brain binds to neuronal nicotinic acetylcholine receptors (neuronal nAChRs). These have been implicated in addiction, but also in several neurological disorders including Alzheimer's and Parkinson's diseases, Tourette's syndrome, attention-deficit hyperactivity disorder (ADHD), schizophrenia, pain, depression, and autosomal-dominant noctural frontal lobe epilepsy; all of which makes nAChRs an intriguing target of study. Chronic treatment with nicotine leads to an increase in the number of nAChRs (upregulation) in the brain and changes their functionality. Changes in the properties of nAChRs are likely to occur in smokers as well, since they are exposed to nicotine for long periods of time. Several nAChR subtypes likely play a role in the formation of nicotine addiction by participating in the release of dopamine in the striatum. The aim of this study was to clarify at cellular level the changes in nAChR characteristics resulting from chronic nicotine treatment. SH-SY5Y cells, endogenously several nAChR-expressing, and SH-EP1-h-alfa7 cells, transfected with the alfa 7 nAChR subunit gene were treated chronically with nicotine. The localisation of alfa 7 and beta2 subunits was studied with confocal and electron microscopy. Functionality of nAChRs was studied with calcium fluorometry. Effects of long-term treatment with opioid compounds on nAChRs were studied by means of ligand binding. Confocal microscopy showed that in SH-SY5Y cells, alfa7 and beta2 subunits formed clusters, unlike the case in SH-EP1-h alfa7 cells, where alfa7 nAChRs were distributed more diffusely. The majority of nAChR subunits localised on endoplasmic reticulum (ER). The isomers of methadone acted as agonists at alfa7 nAChRs. Acute morphine challenge also stimulated nAChRs. Chronic treatment with methadone or morphine led to an increased number of nAChRs. In animal studies, mice received nicotine for 7 weeks. Electron microscopical analysis of the localisation of nAChRs showed in the striatum that alfa7 and beta2 nAChR subunits localised synaptically, extrasynaptically, and intracellularly, with the majority localising extrasynaptically. Chronic nicotine treatment caused an increase in the number of nAChR subunits at all studied locations. These results suggest that the alfa7 nAChR and beta2 subunit-containing nAChRs respond to chronic nicotine treatment differently. This may indicate that the functional balance of various nAChR subtypes in control of the release of dopamine is altered as a result of chronic nicotine treatment. Compounds binding both to opioid and nACh receptors may be of clinical importance.

The Effects of Nicotine on the Regulation of Neuronal Alpha7 Nicotinic Acetylcholine Receptors and Intracellular Signalling Pathways

Nicotine, the addictive compound of tobacco products, exerts its effects in the brain by binding to neuronal acetylcholine nicotinic receptors (nAChRs). The aim of the present study was to increase the knowledge of nicotine s complex effects, the focus being on homomeric alpha7-nAChRs that are widely expressed in the brain. Nicotinic regulation of differential signalling molecules including transcriptional regulators was also studied. We found that the number of alpha7-nAChRs is increased in specific brain regions in mice, in a time-dependent manner after chronic oral nicotine administration. Our results suggest that in addition to alpha4beta2-nAChRs, the other major nAChR subtype expressed in the brain, the number of alpha7-nAChRs is affected by chronic presence of nicotine. We suggest that when studying the long-term effects of nicotine, the duration on administration is of great importance. Next, we observed that nicotine exposure induces accumulation of cAMP in cell cultures expressing nAChRs. Furthermore, nicotine-induced alpha7-nAChR upregulation was potentiated by treatments enhancing cAMP-signalling, suggesting a role for cAMP in the upregulation process. Protein kinase C (PKC) was found essential for the basal regulation of alpha7-nAChR number. The nicotine-evoked alpha7-nAChR upregulation could be further increased by PKC overexpression. Thirdly, the effects of nicotine on dopamine and cAMP regulated phosphoprotein (DARPP-32) were characterised in rat brain. The results show that DARPP-32 is regulated by both acute and long-term nicotine treatment in the striatal subdivisions. The effect of acute nicotine is dose-dependent and the three striatal regions display differential sensitivities to nicotine. Chronic nicotine is also able to regulate DARPP-32 signalling with prominent effect seen in the nucleus accumbens (NAc), suggesting a role for DARPP-32 in the mediation of long-term effects of nicotine. Finally, the regulation of transcription factors Elk-1 and FosB/deltaFosB by nicotine was investigated. We found that Elk-1 is activated by acute nicotine selectively in the NAc core and hippocampal area CA1, whereas acute nicotine does not affect FosB/deltaFosB. Long-term intermittent or continuous nicotine increases the level of total Elk-1 in the same brain regions as acute nicotine. FosB/deltaFosB is also affected by chronic nicotine. Thus, similarly to other drugs of abuse, nicotine regulates transcriptional regulators Elk-1 and FosB/deltaFosB. These results bring further support for a common mechanism underlying the development of addiction. Nicotine s positive effects on learning and memory might involve the transcription factor Elk-1 based on the changes seen in the hippocampus, the key area in cognitive functions.

Fluorescent labelling of strychnine: A novel approach for recognition of strychnine binding sites on neuronal membrane

trychnine was coupled to fluorescein isothiocyanate to mark strychnine binding sites in spinal cord of rat. Specific binding of strychnine could be demonstrated in synaptosomal fraction. Addition of glycine to the strychninised membrane led to a decrease in fluorescence indicating same receptor loci.

Identification of two novel human neuronal ceroid lipofuscinosis genes

The neuronal ceroid lipofuscinoses (NCLs) are a group of mostly autosomal recessively inherited neurodegenerative disorders. The aim of this thesis was to characterize the molecular genetic bases of these, previously genetically undetermined, NCL forms. Congenital NCL is the most aggressive form of NCLs. Previously, a mutation in the cathepsin D (CTSD) gene was shown to cause congenital NCL in sheep. Based on the close resemblance of the phenotypes between congenital NCLs in sheep and human, CTSD was considered as a potential candidate gene in humans as well. When screened for mutations by sequencing, a homozygous nucleotide duplication creating a premature stop codon was identified in CTSD in one family with congenital NCL. While in vitro the overexpressed truncated mutant protein was stable although inactive, the absence of CTSD staining in brain tissue samples of patients indicated degradation of the mutant CTSD in vivo. A lack of CTSD staining was detected also in another, unrelated family with congenital NCL. These results imply that CTSD deficiency underlies congenital NCL. While initially Turkish vLINCL was considered a distinct genetic entity (CLN7), mutations in the CLN8 gene were later reported to account for the disease in a subset of Turkish patients with vLINCL. To further dissect the genetic basis of the disease, all known NCL genes were screened for homozygosity by haplotype analysis of microsatellite markers and/or sequenced in 13 mainly consanguineous, Turkish vLINCL families. Two novel, family-specific homozygous mutations were identified in the CLN6 gene. In the remaining families, all known NCL loci were excluded. To identify novel gene(s) underlying vLINCL, a genomewide single nucleotide polymorphism scan, homozygosity mapping, and positional candidate gene sequencing were performed in ten of these families. On chromosome 4q28.1-q28.2, a novel major facilitator superfamily domain containing 8 (MFSD8) gene with six family-specific homozygous mutations in vLINCL patients was identified. MFSD8 transcript was shown to be ubiquitously expressed with a complex pattern of alternative splicing. Our results suggest that MFSD8 is a novel lysosomal integral membrane protein which, as a member of the major facilitator superfamily, is predicted to function as a transporter. Identification of MFSD8 emphasizes the genetic heterogeneity of Turkish vLINCL. In families where no MFSD8 mutations were detected, additional NCL-causing genes remain to be identified. The identification of CTSD and MFSD8 increases the number of known human NCL-causing genes to eight, and is an important step towards the complete understanding of the genetic spectrum underlying NCLs. In addition, it is a starting point for dissecting the molecular mechanisms behind the associated NCLs and contributes to the challenging task of understanding the molecular pathology underlying the group of NCL disorders.

Molecular and Cellular Mechanisms Behind Juvenile Neuronal Ceroid Lipofuscinosis (JNCL, Batten disease)

Neurodegenerative disorders are chronic, progressive, and often fatal disorders of the nervous system caused by dysfunction, and ultimately, death of neuronal cells. The underlying mechanisms of neurodegeneration are poorly understood, and monogenic disorders can be utilised as disease models to elucidate the pathogenesis. Juvenile neuronal ceroid-lipofuscinosis (JNCL, Batten disease) is a recessively inherited lysosomal storage disorder with progressive neurodegeneration and accumulation of autofluorescent storage material in most tissues. It is caused by mutations in the CLN3 gene; however, the exact function of the corresponding CLN3 protein, as well as the molecular mechanisms of JNCL pathogenesis have remained elusive. JNCL disease exclusively affects the central nervous system leaving other organs unaffected, and therefore it is of a particular importance to conduct studies in brain tissue and neuronal cells. The aim of this thesis project was to elucidate the molecular and cell biological mechanisms underlying JNCL. This was the first study to describe the endogenous Cln3 protein, and it was shown that Cln3 localised to neuronal cells in the mouse brain. At a subcellular level, endogenous Cln3 was localised to the presynaptic terminals and to the synaptosome compartment, but not to the synaptic vesicles. Studies with the CLN3-deficient cells demonstrated an impaired endocytic membrane trafficking, and established an interconnection between CLN3, microtubulus-binding Hook1 and Rab proteins. This novel data was not only important in characterising the roles of CLN3 in cells, but also provided significant information delineating the versatile role of the Rab proteins. To identify affected cellular pathways in JNCL, global gene expression profiling of the knock-out mouse Cln3-/- neurons was performed and systematically analysed; this revealed a slight dysfunction of the mitochondria, cytoskeletal abnormality in the microtubule plus-end, and an impaired recovery from depolarizing stimulus when specific N-type Ca2+ channels were inhibited, thus leading to a prolonged time of higher intracellular calcium. All these defective pathways are interrelated, and may together be sufficient to initiate the neurodegenerative process. Results of this thesis also suggest that in neuronal cells, CLN3 most likely functions at endocytic vesicles at the presynaptic terminal, potentially involved in the regulation of the calcium-mediated synaptic transmission.

Molecular Interactions Underlying Neuronal Ceroid Lipofuscinoses CLN1 and CLN5

Disorders resulting from degenerative changes in the nervous system are progressive and incurable. Both environmental and inherited factors affect neuron function, and neurodegenerative diseases are often the sum of both factors. The cellular events leading to neuronal death are still mostly unknown. Monogenic diseases can offer a model for studying the mechanisms of neurodegeneration. Neuronal ceroid lipofuscinoses, or NCLs, are a group of monogenic, recessively inherited diseases affecting mostly children. NCLs cause severe and specific loss of neurons in the central nervous system, resulting in the deterioration of motor and mental skills and leading to premature death. In this thesis, the focus has been on two forms of NCL, the infantile NCL (INCL, CLN1) and the Finnish variant of late infantile NCL (vLINCLFin, CLN5). INCL is caused by mutations in the CLN1 gene encoding for the PPT1 (palmitoyl protein thioesterase 1) enzyme. PPT1 removes a palmitate moiety from proteins in experimental conditions, but its substrates in vivo are not known. In the Finnish variant of late infantile NCL (vLINCLFin), the CLN5 gene is defective, but the function of the encoded CLN5 has remained unknown. The aim of this thesis was to elucidate the disease mechanisms of these two NCL diseases by focusing on the molecular interactions of the defective proteins. In this work, the first interaction partner for PPT1, the mitochondrial F1-ATP synthase, was described. This protein has been linked to HDL metabolism in addition to its well-known role in the mitochondrial energy production. The connection between PPT1 and the F1-ATP synthase was studied utilizing the INCL-disease model, the genetically modified Ppt1-deficient mice. The levels of F1-ATP synthase subunits were increased on the surface of Ppt1-deficient neurons when compared to controls. We also detected several changes in lipid metabolism both at the cellular and systemic levels in Ppt1-deficient mice when compared to controls. The interactions between different NCL proteins were also elucidated. We were able to detect novel interactions between CLN5 and other NCL proteins, and to replicate the previously reported interactions. Some of the novel interactions influenced the intracellular trafficking of the proteins. The multiple interactions between CLN5 and other NCL proteins suggest a connection between the NCL subtypes at the cellular level. The main results of this thesis elicit information about the neuronal function of PPT1. The connection between INCL and neuronal lipid metabolism introduces a new perspective to this rather poorly characterized subject. The evidence of the interactions between NCL proteins provides the basis for future research trying to untangle the NCL disease mechanisms and to develop strategies for therapies.

The harmonic torsional oscillations of a thin disk submerged in a fluid with a surfactant surface layer

A formulation in terms of a Fredholm integral equation of the first kind is given for the axisymmetric problem of a disk oscillating harmonically in a viscous fluid whose surface is contaminated with a surfactant film. The equation of the first kind is converted to a pair of coupled integral equations of the second kind, which are solved numerically. The resistive torque on the disk is evaluated and surface velocity profiles are computed for varying values of the ratio of the coefficient of surface shear viscosity to the coefficient of viscosity of the substrate fluid, and the depth of the disk below the surface.

Forced Oscillations of a Contained Rotating Stratified Fluid

The motion generated by forced oscillations in an incompressible inviscid rotating and/or stratified fluid is examined under linear theory taking the density variation on the inertia terms. The solution consists of numerous internal modes in addition to the mode which oscillates with forcing frequency. Resonance occurs when the forcing frequency is equal to one of the frequencies of the internal modes. Some of these modes grow linearly or exponentially with time rendering the motion unstable and eventually may lead to turbulence. Most of the results discussed here will be missed under Boussinesq approximation.

Negative resistance mode of forced oscillations of a string

A new mode of driven nonlinear vibrations of a stretched string is investigated with reference to conditions of existence, properties, and regions of stability. It is shown that this mode exhibits negative resistance properties at all frequencies and driving force amplitudes. Discovery of this mode helps to fill certain gaps in the theory of forced nonlinear vibrations of strings.

Statistical significance of sequential firing patterns in multi-neuronal spike trains

Sequential firings with fixed time delays are frequently observed in simultaneous recordings from multiple neurons. Such temporal patterns are potentially indicative of underlying microcircuits and it is important to know when a repeatedly occurring pattern is statistically significant. These sequences are typically identified through correlation counts. In this paper we present a method for assessing the significance of such correlations. We specify the null hypothesis in terms of a bound on the conditional probabilities that characterize the influence of one neuron on another. This method of testing significance is more general than the currently available methods since under our null hypothesis we do not assume that the spiking processes of different neurons are independent. The structure of our null hypothesis also allows us to rank order the detected patterns. We demonstrate our method on simulated spike trains.

Application of ultraspherical polynomials to forced oscillations of a third-order non-linear system

In this paper the method of ultraspherical polynomial approximation is applied to study the steady-state response in forced oscillations of a third-order non-linear system. The non-linear function is expanded in ultraspherical polynomials and the expansion is restricted to the linear term. The equation for the response curve is obtained by using the linearized equation and the results are presented graphically. The agreement between the approximate solution and the analog computer solution is satisfactory. The problem of stability is not dealt with in this paper.

Studies on bicuculline binding sites on neuronal membrane using fluorescent antibody technique: Comparative binding of gaba and bicuculline

Highly purified fluorescent labelled anti-bicuculline antibodies were used to mark bicuculline binding sites in cerebral cortex of monkey brain. Specific binding of bicuculline could be demonstrated in the synaptosomal fraction, when bicuculline was added both Image and Image . Addition of γ-aminobutyric acid (GABA) to the bicucullinised membrane led to a decrease in fluorescence indicating same receptor loci and establishing GABA-bicuculline antagonism at a molecular level.