Postulated Vasoactive Neuropeptide Autoimmunity in Fatigue-Related Conditions: A Brief Review and Hypothesis

Disorders such as chronic fatigue syndrome (CFS) and gulf war syndrome (GWS) are characterised by prolonged fatigue and a range of debilitating symptoms of pain, intellectual and emotional impairment, chemical sensitivities and immunological dysfunction. Sudden infant death syndrome (SIDS) surprisingly may have certain features in common with these conditions. Post-infection sequelae may be possible contributing factors although ongoing infection is unproven. Immunological aberration may prove to be associated with certain vasoactive neuropeptides (VN) in the context of molecular mimicry, inappropriate immunological memory and autoimmunity

Targeting VIP and PACAP receptor signalling: new therapeutic strategies in multiple sclerosis

MS (multiple sclerosis) is a chronic autoimmune and neurodegenerative pathology of the CNS (central nervous system) affecting approx. 2.5 million people worldwide. Current and emerging DMDs (disease-modifying drugs) predominantly target the immune system. These therapeutic agents slow progression and reduce severity at early stages of MS, but show little activity on the neurodegenerative component of the disease. As the latter determines permanent disability, there is a critical need to pursue alternative modalities. VIP (vasoactive intestinal peptide) and PACAP (pituitary adenylate cyclase-activating peptide) have potent anti-inflammatory and neuroprotective actions, and have shown significant activity in animal inflammatory disease models including the EAE (experimental autoimmune encephalomyelitis) MS model. Thus, their receptors have become candidate targets for inflammatory diseases. Here, we will discuss the immunomodulatory and neuroprotective actions of VIP and PACAP and their signalling pathways, and then extensively review the structure–activity relationship data and biophysical interaction studies of these peptides with their cognate receptors

Regulation of neural progenitor cell proliferation and fate by proteolytic pathways and inflammatory signals in the brain

Neural progenitor cells (NPCs) are present in the developing and adult neuroepithelium of the brain and are regulated by internal and external signals that influence neurogenesis and tissue homeostasis. NPCs are multipotent tissue stem cells that can arouse all neural cell types, including neurons and glial cells. In culture, NPCs grow preferentially as cell aggregates called neurospheres. This suggests that interactions between cells are essential to regulate NPC behavior and development. Interactions between cells may be facilitated by cell surface-attached proteases and their inhibitors that play an important role in development and during tissue remodeling after injury. Neuroinflammation, an innate immune response of the nervous system, is part of many neurodegenerative diseases. Neuroinflammation involves activation of microglia and production of proinflammatory cytokines. Inflammation may have negative effects on NPCs and thus, agents that protect NPCs could serve as a therapeutic potential for neuronal injuries and neurodegenerative diseases by enabling local tissue repair in the brain. The aim of this thesis was to study the regulation of NPC development by membrane-associated proteins and the effects of inflammation on NPCs. Glucocorticoid hormone (GH) levels increase in inflammation and after stress. GHs have previously been shown to decrease NPC proliferation and neurogenesis. We have studied the effects of a synthetic GH dexamethasone on the cytosolic membrane-associated and anti-apoptotic protein BRUCE, and how BRUCE affects NPC behaviour. In addition, we have studied the secretion of cytokine interferon-gamma (IFN-gamma) after microglial activation and further the influence of IFN-gamma on NPCs. To address the role of cell surface-associated protease inhibitors during NPC development, we have studied the expression and function of Kunitz type serine protease inhibitors HAI-1 and HAI-2 in NPCs. The results show that dexamethasone enhances degradation of BRUCE by the ubiquitin-proteasome system (UPS), which leads to decreased NPC proliferation. NPC division was negatively affected also by IFN-gamma produced by microglial cells as well as protease inhibitors HAI-1 and HAI-2. Moreover, IFN-gamma induced NPC cell death that was rescued by a neuropeptide PACAP. In the developing NPCs, HAI-1 and HAI-2 expression was increased by bone morphogenetic protein-2 (BMP-2) and BMP-4, which inhibited NPC proliferation and increased glial cell differentiation partly in a HAI-dependent manner. This thesis provides knowledge about interplay between immune cells and NPCs as well as developmental signaling systems, including proteolytic pathways, that affect NPC behaviour. In NPCs, proteolytic pathways may be regulated by external signals, like cytokines, from the neighboring cells. Proteolysis is involved also in the UPS that regulates the cell cycle machinery and thus, cell division. This thesis also deals with NPC survival, which is of importance for stem cell therapies. Knowledge of reciprocal effects of IFN-gamma and PACAP on NPCs is relevant when designing treatment for brain inflammation and disease.Aivojen kehityksen aikana lukuisat viestintäreitit solujen välillä sekä solun sisällä säätelevät hermoston kantasoluja. Hermoston kantasolut ovat niin kutsuttuja kudoskantasoluja, joilla on kyky uusiutua. Ne voivat myös erilaistua hermoston eri solutyypeiksi, kuten hermosoluiksi ja hermotukisoluiksi. Hermoston kantasolujen sijainti aivoissa vaikuttaa niiden kehittymiseen ja ne jakautuvat usein vain tietyssä mikroympäristössä, joka säätelee niiden erilaistumista. Viljelmässä hermoston kantasolut kasvavat aggregaatteina, mikä tukee niiden kasvua ja jakautumista viitaten solujen välisten vuorovaikutusten olevan tärkeitä niiden kehitykselle. Solujen välisiä vuorovaikutuksia voivat välittää esimerkiksi solun pinnalla sijaitsevat proteiinit. Tulehdus on läsnä monissa hermoston tautitiloissa. Aivojen tulehdusreaktioissa mikroglia-solut aktivoituvat ja erittävät tulehdusta edistäviä tulehdusvälittäjäaineita, kuten sytokiinejä, jotka välittävät elimistön immuunijärjestelmän vuorovaikutuksia. Hermoston kantasolut voivat olla vuorovaikutuksessa immuunijärjestelmän kanssa ja saattavat vähentää aivojen tulehdusvastetta. Aivojen tulehdusreaktioissa ja aivovaurioissa molekyylejä, jotka suojelevat hermoston kantasoluja, voitaisiin käyttää vaurion hoidossa lisäämään tuhoutuneen kudoksen uusiutumispotentiaalia. Väitöskirja tutkii hermoston kantasolujen jakautumista ja erilaistumista sekä sitä, miten eri tekijät, kuten sytokiinit ja solukalvoilla sijaitsevat proteiinit vaikuttavat näiden solujen kehittymiseen sekä niiden säätelyyn aivojen tulehdusreaktioissa. Väitöskirjatyö osoittaa, että hermoston kantasolujen pinnalla esiintyvät proteiinien pilkkoutumista säätelevät proteaasiestäjät ohjaavat sytokiinien säätelemänä näiden solujen jakautumista ja erilaistumista aivojen kehityksen aikana. Lisäksi proteasomihajotuksella on rooli hermoston kantasolujen säätelyssä vaikuttamalla jakautumisessa tarvittavien proteiinien ilmentymiseen. Väitöskirja myös osoittaa aivojen mikroglia-solujen vaikuttavan sytokiinien välityksellä hermoston kantasoluihin. Hermoston kantasolut ovat herkkiä tulehdusvälittäjäaineille, ja niiden jakautuminen saattaa häiriintyä hermoston tautitiloissa ja aivovaurion seurauksena. Siksi niiden kasvun ja jakautumisen säätely on tärkeää etenkin stressi- ja tulehdusreaktioissa ja myös suunniteltaessa kantasoluterapiaa

Role of functionalized liposomes in drug delivery through the blood-brain barrier

The central nervous system (CNS), one of the most delicate microenvironments of the body, is protected by the blood-brain barrier (BBB). The protective properties of the BBB are conferred by the intricate architecture of its endothelium coupled with multiple specific transport systems expressed on the surface of endothelial cells in the brain vasculature. Although the BBB has a key role in regulation of neural biochemical environment, essential for maintaining neuronal integrity, it limits drug delivery to the CNS. In fact, less than 2% of all US Food and Drug Agency (FDA)-approved small-molecule drugs cross the intact BBB to varying degrees. Due to their flexible physicochemical and biophysical properties, the liposomes represent an attractive tool to deliver therapeutic molecules across the BBB. Moreover, their surface can be easily modified with ligands to improve their target and delivery. The peptide gH625, identified as a membrane-perturbing domain in glycoprotein H (gH) of Herpes Simplex virus 1, has been used extensively for vector-mediated strategies that enable passage of a large variety of small molecules as well as proteins across cell membranes in vitro. The goal of this Ph.D. project was to develop a new carrier system to deliver therapeutic molecules through the BBB with high efficacy and minimal toxicity. The project was divided in two parts. In the first part, in vitro and in vivo experiments were performed to investigate the capacity of gH625 peptide to enter and accumulate in neuron and astrocyte cell lines, and its ability to cross the blood-brain barrier in rats. In the second part of the project, the efficiency of liposomes functionalized with gH625 was evaluated on both in vitro model of rat BBB by using a neuroprotective peptide like PACAP (pituitary adenylate cyclase-activating polypeptide) and on in vivo mouse brain by using a hypothermic neuropeptide. The results show that gH625 peptide has a significant ability to penetrate brain cells. In fact, gH625 can be efficiently incorporated by human neuroblastoma (SH-SY5Y) and glioblastoma-astrocytoma cell lines (U-87 MG) without alteration of their cell viability. Furthermore, the in vivo experiments demonstrate that, despite the blood filtration action of the liver, carried out through the gH625 uptake of Kupffer cells, gH625 substantially reaches the brain BBB vessels. In particular, gH625 can be highly accumulated in endothelial cells of the BBB and taken up in some neurons. The results of second part of project show that the functionalization of liposomes with gH625 improves their passage through the endothelium of in vitro BBB model, thus resulting in an increased transport of PACAP and its less accumulation in the endothelial cells. Moreover, the toxicity studies reveal that the gH625-liposomes are nontoxic and do not affect tight junction organization in the BBB endothelium. Finally, further in vivo results demonstrated that the gH625 peptide may improve the efficiency of liposomes in mice. The results of this study suggest that gH625 peptide is a valuable tool to develop functionalized nanosystems for drug delivery to the brain. In particular, gH625-mediated liposomes represent a promising strategy to deliver therapeutic agents to CNS. Taken together, these data may have importance for the treatment of brain diseases and tracking of nanosystems in vivo

Die Rolle der Neuropeptide Calcitonin gene-related Peptide und Pituitary adenylate cyclase-activating Peptide im Superoxiddismutase 1 Mausmodell der amyotrophen Lateralsklerose

Bei der amyotrophen Lateralsklerose (ALS) handelt es sich um eine tödlich verlaufende, neurodegenerative Erkrankung, für die bisher keine effektiven Therapiemöglichkeiten zur Verfügung stehen. Gekennzeichnet ist die ALS durch den Verlust der Motoneurone, der zur Denervierung der Muskulatur und letztendlich dem Tod durch Atemlähmung führt. Die molekularen Ursachen der ALS sind vielfältig und bis heute nur unzureichend aufgeklärt, aktuell stehen die Rolle der begleitenden Neuroinflammation und die molekularen Unterschiede zwischen ALS-vulnerablen und ALS-resistenen Motoneuronen im Zentrum der Forschung. Obwohl der neuroprotektive Einfluss verschiedener Neuropeptide in anderen neurodegenerativen Erkrankungen, Ischämie oder Axotomie lange bekannt ist, wurde deren Rolle bei der ALS bisher kaum untersucht. In der vorliegenden Arbeit sollte die Beteiligung der aussichtsreichsten Kandidaten Calcitonin gene-related Peptide (CGRP) und Pituitary adenylate cyclaseactivating Polypeptide (PACAP) am Krankheitsverlauf des SOD1G93A-Mausmodells der ALS aufgeklärt werden. Im Gegensatz zu Axotomie oder peripherer Inflammation war in somatomotorischen Neuronen keine Hochregulation der CGRP-Expression in ALS-kranken Mäusen zu beobachten. Doch traten beginnend am postnatalen Tag (P) 40 CGRP-immunoreaktive Vakuolen in den Neuriten auf, die im Zeitverlauf deutlich an Volumen zunahmen und nach der Degeneration der Motoneurone frei im Neuropil vorlagen. Diese Veränderung der CGRPImmunoreaktivität ging dem Einsetzen der klinischen Symptomatik an P90-100 deutlich voraus und war räumlich und zeitlich eng mit einer Aktivierung der Astrozyten assoziiert. Zudem traten die Vakuolen, ebenfalls beginnend an P40, in extramotorischen Arealen wie dem Hypothalamus und der Substantia nigra auf. Da auch bei ALS-Patienten Störungen nigraler und extramotorischer Funktionen beobachtet werden, ist diese Beobachtung im Mausmodell ein wichtiger Hinweis darauf, dass es sich bei der ALS nicht ausschließlich um eine Motoneuronen-, sondern eine Multisystemerkrankung mit unterschiedlichen Subtypen handeln könnte. Desweiteren wurde mit CGRP erstmalig ein Biomarker für vulnerable Motoneurone identifiziert. So konnte gezeigt werden, dass es einerseits Motoneurone ohne CGRP-Expression (nonCGRP) gibt und diese gegen die ALS-Pathologie resistent zu sein scheinen, während andererseits Motoneurone mit starker oder schwacher CGRP-Expression (high und lowCGRP) im Zeitverlauf der Erkrankung selektiv degenerieren, und zwar umso früher und umso stärker, je mehr CGRP sie exprimieren. Die bisher vorgenommene Abgrenzung der resistenten, oculomotorischen Nuclei von den vulnerablen motorischen Nuclei im Hirnstamm resultiert ebenfalls aus der CGRP-Expression, da nachgewiesen werden konnte, dass sich die oculomotorischen Kerne in der Mehrheit aus nonCGRP-Neuronen zusammensetzen, während stark vulnerable Kerne wie der Nucleus ambiguus hauptsächlich aus highCGRP-Motoneuronen bestehen. Während eine Deletion von αCGRP keinen Effekt auf den Krankheitsverlauf des SOD1G93A-Mausmodell hatte, resultierte die Deletion der CGRP-spezifischen Rezeptorkomponente Receptor activity modifying protein 1 (RAMP1) in einem früheren motorischen Krankheitsbeginn mit einem anschließend verlangsamten Verlauf der Symptomatik. Histopathologisch waren diese Veränderungen mit einer reduzierten Neuroinflammation, insbesondere einer geringeren Aktivierung der Astrozyten und einem Ausbleiben der Lymphozyteninfiltration assoziiert. Im Umkehrschluss bedeutet dies, dass CGRP in der ALS-Pathologie einen stimulierenden Einfluss auf die Neuroinflammation hat und diese zu Beginn der Erkrankung einen protektiven, später einen toxischen Effekt auf die Motoneurone ausübt. Im bereits erkrankten Patienten könnte der therapeutische Einsatz von CGRP-Antagonisten ebenfalls einen hemmenden Einfluss auf die neurotoxischen Komponenten der Neuroinflammation ausüben und somit die Krankheitsprogression verlangsamen. Während Motoneurone auf Axotomie mit einer starken Induktion der PACAP-Expression reagieren, war im Zuge der hier untersuchten ALS-Pathologie nur in wenigen Einzelneuronen eine Induktion des Neuropeptids zu beobachten. In früheren Studien an anderen neuronalen Erkrankungen bzw. Schädigungen wie Morbus Parkinson und Alzheimer, Ischämie oder Axotomie hatte PACAP grundsätzliche eine neuroprotektive Wirkung, ein Unterbinden der Signalkaskade sollte also in einem progressiveren Krankheitsverlauf resultieren. Entgegen dieser Erwartung verlängerte sich bei der Einkreuzung einer PACAP-Defizienz in das SOD1G93A-Mausmodell die durchschnittliche Lebenserwartung der Tiere jedoch um ca. 5,5%. Im Gegensatz zu anderen neurdegenerativen Erkrankungen scheint PACAP in der ALS also keinen protektiven Effekt auszuüben, sondern die Pathogenese zu beschleunigen. Histopathologisch war im Endstadium SOD1G94A-transgener Tiere unter der PACAP-Deletion eine amöbiode Morphologie der Mikroglia zu beobachten, während PACAP-kompetente Tiere bereits hypertrophe und degenerierende Mikroglia aufwiesen. Dies deutete auf eine geringere oder verzögerte Aktivierung der Mikroglia in PACAP-defizienten Tieren hin. Die damit einhergehende Reduktion der neurotoxischen Wirkung der Neuroinflammation könnte die Verzögerung des Krankheitsverlaufs erklären. Entsprechend besitzt auch im Falle von PACAP der Einsatz von Antagonisten ein therapeutisches Potential bezüglich einer Hemmung der Krankheitsprogression in der ALS-Pathogenese

Systematic review and meta-analysis of experimental multiple sclerosis studies

Background: Multiple sclerosis (MS) is the most common cause of disability in young people and yet there are no interventions available which reliably alter disease progression. This is despite several decades of research using the most common animal model of multiple sclerosis, experimental autoimmune encephalomyelitis (EAE). There is now emerging evidence across the neurosciences to suggest that limited internal validity (measures to reduce bias) and external validity (e.g. using a clinically relevant animal model) may influence the translational success. Aim and objectives: To provide an unbiased summary of the scope of the literature on candidate drugs for MS tested in EAE to identify potential reasons for the failures to translate efficacy to clinical trials. My objectives were, across all of the identified publications, to: (1) describe the reporting of measures to reduce bias and to assess their impact on measures of drug efficacy; (2) assess the relationship between treatment related effects measured using different outcome measures; (3) assess the prevalence and impact of any publication bias; (4) compare findings from the above with another disease with limited translational success (Parkinson’s disease; PD). Methods: I used systematic searches of three online databases to identify relevant publications. Estimates of efficacy were extracted for neurobehavioural scores, inflammation, demyelination and axon loss. For PD experiments, we searched for dopamine agonists tested in animal models of PD with outcome assessed as change in neurobehavioural scores. I calculated normalised mean difference or standardised mean difference effect sizes and combined these in a meta-analysis using a random effects model. I used stratified meta-analysis or meta-regression to assess the extent to which different study design characteristics explained differences in reported efficacies. These characteristics included: measures to reduce bias (random allocation to group and blinded assessment of outcome), the animal species, sex, time of drug administration, route of drug administration and the number of animals per group. Publication bias was assessed using funnel plotting, Egger regression and “trim and fill”. Results: I identified 1464 publications reporting drugs tested in EAE. Reported study quality was poor: 11% reported random allocation to group, 17% reported blinded assessment of neurobehavioural outcomes, 28% reported blinded assessment of histological outcomes, and <1% reported a sample size calculation. Estimates of efficacy measured as the reduction in inflammation were substantially higher in unblinded studies (47.1% reduction (95% CI 41.8-52.4)) versus blinded studies (33.1% (25.8-40.4). Moreover, the same finding was identified for 121 publications on dopamine agonists tested in experimental PD models where efficacy was measured as change in neurobehavioural outcomes. For EAE studies we were unable to include data from 631 publications describing original research. Usually this was because the publication did not include basic details such as the number of animals in each group (115 publications), the observed variance (592) or suitable control data (49). For each category of outcome I found evidence of a substantial publication bias. Interventions were most commonly administered on or before the induction of EAE with shorter times to treatment associated with higher estimates of efficacy for the reduction in mean severity scores (a neurobehavioural outcome). Treatment related effects were found to vary across different outcome measures with the largest effect being for the reduction in axon loss. Where neurobehavioural scores and axon loss were measured in the same cohort of animals, the concordance between efficacies in these increased with later times to treatment. Conclusions: In this, the largest systematic review and meta-analysis of animal studies in any domain, I have found that a large number of publications present incomplete data. In addition, measures to reduce bias are seldom reported, the lack of which is associated with overstatements of efficacy for both a measure of drug efficacy in EAE and experimental PD studies. Translational success may have also been affected by the majority of studies administering drugs on or before EAE induction which is of limited relevance in the clinical setting where patients do not present at that stage of disease. Moreover, my analysis of the relationship between outcome measures provides empirical evidence from systematically identified studies to suggest that targeting axon loss as later time points is most strongly associated with improvements in neurobehavioural scores. Therefore drugs which are successfully able to target axon loss at these time points may offer substantial hope for clinical success. Overall, improvements in the conduct and reporting of preclinical studies are likely to improve their utility, and the prospects for translational success. While my findings relate predominately to the animal modelling of MS and PD it is likely that they also hold for other animal research