Teses / dissertações sobre o tema "Central nervous system-associated macrophages"

L’HIC est une pathologie grave pour laquelle il n’existe actuellement aucune stratégie thérapeutique efficace. Une meilleure compréhension des mécanismes sous-jacents à l’HIC et de ses conséquences est donc essentielle pour identifier de nouvelles cibles thérapeutiques potentielles. Parmi les axes de recherche émergents, le rôle des microglies dans la clairance de l’hématome suscite un intérêt croissant. Toutefois, les microglies ne représentent pas le seul type de macrophages résidents du SNC. En effet, les CAMs comprenant les macrophages résidents des plexus choroïdes, des méninges et les macrophages périvasculaire constituent les macrophages résidents des bordures du SNC. Des études réalisées sur l’HSA ont mis en évidence le rôle majeur des CAMs dans les processus inflammatoires et l’élimination des érythrocytes. Par ailleurs, les CAMs influencent certains facteurs de risque associés à l’HIC, tels que le vieillissement, l’hypertension, l’angiopathie amyloïde cérébrale, et pourraient ainsi moduler directement ou indirectement l’HIC. Sur la base de ces observations, nous avons émis l’hypothèse que les CAMs pourraient donc jouer un rôle central dans l’HIC. Pour vérifier cette hypothèse, nous avons mis en place un modèle expérimental d’HIC par injection intracorticale de collagénase chez des souris mâles et femelles, avec ou sans déplétions des CAMs. Nos résultats mettent en évidence le rôle essentiel des CAMs dans l’évolution des HIC, notamment en influençant l’homéostasie de l’unité neurovasculaire et en participant à la résorption endogène de l’hématome. À notre connaissance, cette étude est la première à explorer le rôle des CAMs dans l’HIC tout en incluant une analyse comparative entre les mâles et les femelles
ICH is a severe pathology for which there is currently no effective therapeutic strategy. A deeper understanding of the mechanisms underlying ICH and its consequences is therefore crucial for identifying new potential therapeutic targets. While the role of microglia in hematoma clearance has gained increasing attention, it is important to note that microglia are not the only resident macrophages in the CNS. CAMs, which include choroid plexus macrophages, meningeal macrophages, and perivascular macrophages, are the resident macrophages located at the borders of the CNS.Studies in the context of SAH have demonstrated that CAMs play a pivotal role in inflammatory processes and in clearance of erythrocytes. Additionally, CAMs influence key risk factors associated with ICH, including aging, hypertension, and cerebral amyloid angiopathy, thereby modulating ICH outcomes both directly and indirectly. Based on these findings, we hypothesized that CAMs could have a central role in the progression of ICH.To test this hypothesis, we conducted loss-of-function studies by establishing an experimental model of ICH through intracortical collagenase injection in both male and female mice, with or without CAM depletion. Our results highlight that CAMs play a critical in the progression of ICH, by maintaining the homeostasis of the neurovascular unit and contributing to the endogenous clearance of the hematoma. the best of our knowledge, this study is the first to investigate the role of CAMs in ICH while including a comparative analysis between males and females

Thesis advisor: Kenneth C. Williams
Thesis advisor: Welkin Johnson
Neuropathogenesis of HIV-associated neurocognitive disorders (HAND) is likely instigated by chronic immune activation in response to residual infection in the central nervous system (CNS), where combined antiretroviral therapy (cART) has limited access. Monocyte/macrophages (Mo/Mϕ) constitute the predominant population of infected cells in the CNS and play a major role in HIV-induced neuropathogenesis. Emergence of compartmentalized HIV subpopulations in the brain corresponds with accumulation of HIV-infected Mo/Mϕ and is consistent with acquired immune deficiency syndrome (AIDS)-related neuropathology. We used a rhesus macaque model of neuroAIDS to elucidate the role of Mo/Mϕ in establishing CNS infection and the emergence of compartmentalized virus in the brain. To do this, we: 1) performed phylogenetic analysis of viral sequences from peripheral and CNS compartments and determined the incidence of Mo/Mϕ infection in CNS tissues to identify sources of CNS viral subpopulations that emerge with AIDS-related neuropathology; 2) optimized a method for obtaining single genome viral sequences from Mo/Mϕ populations extracted from tissues and 3) performed phylogenetic analysis of viral sequences from bone marrow (BM) and CNS Mo/Mϕ and determined the incidence of Mo/Mϕ infection in the BM to assess whether BM Mo/Mϕ are sources of infected Mo/Mϕ that accumulate in the CNS with AIDS-related neuropathology. We found that animals with AIDS-related neuropathology had a higher incidence of Mo/Mϕ infection and compartmentalized SIV subpopulations in CNS tissues compared to animals without neuropathology. Additionally, CSF virus, which is used to assess the presence of CNS virus compartmentalization in living patients, was not compartmentalized even with significant compartmentalization in the brain and severe AIDS-related neuropathology (Chapter 2). Relative to animals without CNS pathology, animals with AIDS-related neuropathology had a higher incidence of Mo/Mϕ infection in the BM and viral sequences from BM and CNS perivascular Mo/Mϕ clustered with sequences from trafficking monocytes and CNS tissues (Chapter 4). The results suggest that infected Mo/MΦ in CNS tissues are sources of compartmentalized virus and that infected Mo/Mϕ in the BM are sources of infected Mo/Mϕ that accumulate in the CNS with AIDS-related neuropathology. In summary, the data in this dissertation suggest that targeting Mo/Mϕ may prevent CNS infection and inflammation associated with HIV-induced neuropathogenesis
Thesis (PhD) — Boston College, 2018
Submitted to: Boston College. Graduate School of Arts and Sciences
Discipline: Biology

Thesis (Ph. D.)--University of California, Riverside, 2010.
Includes abstract. Available via ProQuest Digital Dissertations. Title from first page of PDF file (viewed May 17, 2010). Includes bibliographical references. Also issued in print.

Mental disorders, such as Attention deficit hyperactivity disorder (ADHD), and 22q11 deletion syndrome (22q11DS) are both related to a dopaminergic system dysfunction. Several studies have shown that dopamine (DA) not only mediates interactions into the nervous system, but can also contribute to the modulation of immunity via receptors expressed in the immune cells (Nagakome et al., 2011; Nakano et al., 2011). Autoimmunity in the central nervous system has been suggested to play a pivotal role in the pathogenesis of neurodevelopmental disorders (ND) (Schwartz. 2013), but how an immune system alteration might lead to ADHD and participate to the psychiatric features of the 22q11DS is still unclear. The importance of immune system for life-long maintenance of the brain is demonstrated by its role in the formation of new neurons, support to normal cognitive performance and neurogenesis regulation. These evidences have broken the theory of the central nervous system (CNS) as immune privileged organ, indicating that peripheral immune cells can contribute to brain homeostasis and repair. In particular, it was demonstrated that circulating blood macrophages recruitment in brain parenchyma is needed for CNS health and homeostasis. The immune cells access to the CNS is regulated by choroid plexus (CP) activation, promoted by pro-inflammatory signals from both brain parenchyma or peripheral T cells accumulated in CP (Baruch et al., 2015). Importantly, a possible role of the neuropeptide adrenomedullin (ADM) in ND has been suggested, since several studies reported a role of this peptide as a biomarker of different psychiatric disorders, such as Alzheimer (Fernandez et al., 2016), autism (Zoroglu et al., 2003), bipolar disorder (Akpinar et al., 2013), schizophrenia (Chia-Hsing Huang et al., 2004) and ADHD (Fernandez et al., 2008). ADM is a regulatory peptide involved in thymic immune tolerance mechanisms (Rulle et al., 2012; Castellani et al., 2016), which are suggested to be altered in ND. On the basis of these considerations, in this study we investigated the involvement of immunological mechanisms and their dependence to ADM both in the periphery and in CNS in mouse models of ADHD and 22q11DS, in order to define whether an immunomodulatory approach can be useful in the management of these diseases.
I disturbi mentali, come il disturbo da deficit di attenzione e iperattività (ADHD) e la sindrome da delezione 22q11 (22q11DS) sono entrambi correlati a una disfunzione del sistema dopaminergico. Diversi studi hanno dimostrato che la dopamina (DA) non solo media diverse interazioni nel sistema nervoso, ma può anche contribuire alla modulazione dell'immunità attraverso dei recettori espressi nelle cellule immunitarie (Nagakome et al., 2011; Nakano et al., 2011). È stato suggerito che l'autoimmunità nel sistema nervoso centrale svolga un ruolo fondamentale nella patogenesi di disturbi del neurosviluppo (ND) (Schwartz, 2013), ma come una alterazione del sistema immunitario possa portare all'ADHD e indurre alterazioni psichiatriche caratteristiche del 22q11DS rimane tuttora poco chiaro. L'importanza del sistema immunitario per il supporto del sistema nervoso centrale (SNC) a lungo termine è dimostrata dal suo ruolo nella formazione di nuovi neuroni, nel supporto alle normali prestazioni cognitive e nella regolazione della neurogenesi. Queste evidenze hanno rivelato i limiti della precedente teoria del SNC come organo immuno-privilegiato, indicando che le cellule immunitarie periferiche possano contribuire all'omeostasi e alla riparazione del cervello. In particolare, è stato dimostrato che il reclutamento dei macrofagi circolanti, nel parenchima cerebrale, è necessario per la salute e l'omeostasi del SNC. L'accesso delle cellule immunitarie al SNC è regolato dall'attivazione del plesso coroideo (CP), promossa da segnali pro-infiammatori provenienti sia dal parenchima cerebrale che dalle cellule T periferiche accumulate in CP (Baruch et al., 2015). Inoltre, è stato suggerito un possibile ruolo del neuropeptide adrenomedullina (ADM) in ND. Diversi studi hanno infatti riportato un ruolo di questo peptide come biomarcatore in diversi disturbi psichiatrici, come Alzheimer (Fernandez et al., 2016), autismo (Zoroglu et al., 2003), disturbo bipolare (Akpinar et al., 2013), schizofrenia (Chia-Hsing Huang et al., 2004) e ADHD (Fernandez et al., 2008). ADM è un peptide regolatorio coinvolto in meccanismi di tolleranza immunitaria che hanno luogo nel timo (Rulle et al., 2012; Castellani et al., 2016), che sembra essere modificato in ND. Sulla base di queste considerazioni, in questo studio abbiamo studiato il coinvolgimento di meccanismi immunologici e la loro dipendenza dall'ADM, sia in periferia che nel SNC di modelli murini di ADHD e 22q11DS, al fine di definire se un approccio immunomodulatorio possa essere utile nel trattamento di queste malattie.

The concept of antibody–associated central nervous system (CNS) disorders is relatively recent. The classical CNS paraneoplastic neurological syndromes are thought to be T-cell mediated, and the onconeural antibodies merely biomarkers for the presence of the tumour. Accordingly it was thought that antibodies rarely, if ever, caused CNS disease. Recently, however, it has become increasingly clear that there are CNS syndromes associated with antibodies that bind to cell surface determinants on neuronal cells and are likely to be pathogenic. Over the last ten years identification of autoimmune forms of encephalitis with antibodies against neuronal surface (NS) antigens, particularly the VGKC-complex proteins or the glutamate NMDA receptor, have shown that CNS disorders, often without associated tumours, can be antibody-mediated and benefit from immunomodulatory therapies. The clinical spectrum of these diseases is not yet fully explored, there may be others yet to be discovered, and some types of more common disorders, as epilepsy or psychosis, may prove to have an autoimmune basis. Here, we call these antibodies neuronal surface antibodies (NSAbs), and the diseases associated with them NSAb syndromes or NSAS. The aim of the research performed in the three years of PhD study has been to characterize NSAS from both a laboratory and a clinical point of view. The first year of the PhD was spent at the Oxford laboratory under the supervision of Prof. Angela Vincent. During this period I contributed to the laboratory characterization of the two most important groups of NSAS: encephalitis associated with VGKC-complex-Abs and NMDAR-Abs. The results of these studies have been published (Irani et al., Brain 2010a; Irani et al., 2010b). My personal contribution in these studies included the development and optimization of immunofluorescence techniques for the detection of autoantibodies against neuronal surface antigens (NSAbs) using both primary cultures of rat hippocampal neurons and mammalian cells transfected with the antigen of interest. In addition, during my time at Oxford I investigated a possible novel proteomics approach to identify new NSAb by means of immunoprecipitation and mass spectrometry. These experiments have subsequently been applied to screen samples of patients with autoimmune cerebellar syndromes. The results of this study are described in the third part of the thesis. The doctoral thesis is divided into three parts. In the first part I have reviewed the state of the art of syndromes associated with neuronal surface antibodies, starting with a comparison of the conditions associated with antibodies to intracellular antigens and those associated with antibodies to cell surface antigens. I have then summarised the main clinical and paraclinical features of the syndromes that have already been identified. On the basis of these observations, I have designed guidelines for recognizing these and other immune-mediated conditions in the future. The guidelines work has been supervised by Prof. Angela Vincent (Oxford, UK) and critically reviewed by Prof. Francesc Graus (Barcelona, Spain), Prof. Christian Bien (Bielefeld, Germany) and Dr. Bruno Giometto (Treviso, Italy). The second part of the thesis describes the clinical and immunological characterization of a cohort of patients with suspected autoimmune encephalitis and VGKC-complex-Abs that I collected and reviewed in Padua during the 3-year period of the PhD. Recently the Oxford group showed that most antibodies previously attributed to VGKC (by radioimmunoprecipitation) target two proteins complexed with the channels Lgi1 and Caspr2, which also tend to associate with LE and Morvan’s syndrome phenotypes, respectively (see (Irani et al., Brain 2010b)). I therefore decided to focus my research on patients with autoimmune encephalitis associated with ‘VGKC’-complex-Abs in order to report their clinical and immunological profile, with a view to showing a possible antigen-phenotype correlation. During the 3-year period of my PhD, I screened 503 samples for NSAbs from 366 patients with suspected autoimmune encephalitis sent to our laboratory between January 2005 and December 2011: 279 serum (and cerebrospinal fluid) samples from 232 patients were tested for VGKC-complex-Abs; in addition 226 samples were tested for NMDAR-Abs, 59 for AMPAR1,2- and GABAbR-Abs, 91 for GAD-Abs; 21 samples were also tested on live hippocampal neurons. Screening methods included: radioimmunoprecipitation (RIA) assay for VGKC-Abs and GAD65-Abs; cell-based assay for antibodies binding human NMDAR, AMPAR1, AMPAR2, GABAbR, Lgi1 and Caspr2; indirect immunofluorescence on live hippocampal cell cultures. Thirty-two of the 232 patients tested for VGKC-complex-Abs were positive. Clinical data were available for 18 patients: 10 patients harboured Lgi1-Abs in their sera, 3 Caspr2-Abs, 5 patients were positive on RIA but not on IIF. Two patients had Morvan’s syndrome, one associated with Lgi1-ab and the other with Caspr2-Ab and a thymoma. Response to immune modulation was reported in all patients with the exception of a patient who died from lung cancer. A consistently high VGKC-complex-Abs titre despite clinical remission prompted the discovery and subsequent removal of an enlarged hyperplastic thymus in a patient with Lgi1-Abs associated LE; one Caspr2-Abs positive patient had mild encephalitis overlapping with an anti-phospholipid syndrome; another patient with an immunotherapy-responsive encephalomyelitis tested negative on IIF but had high-titre VGKC-complex-Abs on RIA. Striatal hypermetabolism was found in 4 patients with Lgi1-Abs in one case associated with faciobrachial dystonic seizures. In conclusion, these results have expanded the clinical spectrum of autoimmune encephalopathy associated with VGKC-complex antibody. It has been shown that VGKC-LE can associate not only with thymomas, but also with a hyperplastic thymus, similarly to the well-known finding in myasthenia gravis and therefore that the persistence of high VGKC-Abs titres should prompt the search for an enlarged thymus. The third part of the thesis describes a study whose aim was to identify new autoantibodies in cerebellar syndromes of possible autoimmune origin. Dr. Esther Becker from Oxford University contributed equally to me in this work. Relatively few studies have searched for potentially pathogenic antibodies in non-paraneoplastic patients with cerebellar ataxia. We first screened sera from more 52 idiopathic ataxia patients for the binding of serum IgG antibodies to cerebellar neurons. One strong-binding serum was selected for immunoprecipitation and mass spectrometry, which resulted in the identification of contactin-associated protein 2 (Caspr2) as a major antigen. Caspr2 antibodies were then found by a cell-based assay in 9/88 (10%) ataxia patients, compared to 3/144 (2%) multiple sclerosis or dementia controls. Caspr2 is strongly expressed in the cerebellum, only partly in association with voltage-gated potassium channels. Prospective studies are needed to see whether identification of Caspr2-antibodies has relevance for the diagnosis and treatment of idiopathic cerebellar ataxia.
Il concetto di sindromi del sistema nervoso centrale (SNC) associate ad anticorpi è relativamente recente. Si ritiene che le classiche sindromi paraneoplastiche neurologiche abbiano una patogenesi immunitaria prevalentemente cellulo-mediata e che gli anticorpi onconeurali costituiscano semplicemente dei biomarker. Sulla base di questo assunto si è ritenuto a lungo improbabile che autoanticorpi potessero determinare malattie del SNC. Recentemente si è resa sempre più evidente l’esistenza di sindromi del SNC associate ad anticorpi diretti contro antigeni presenti sulla superficie neuronale e pertanto potenzialmente patogenetici. L’identificazione negli ultimi dieci anni di forme di encefaliti autoimmuni con anticorpi diretti contro antigeni della superficie neuronale, ed in particolare le proteine del complesso dei canali del potassio voltaggio-dipendenti (VGKC-complex) e i recettori del glutammato di tipo NMDA, ha dimostrato l’esistenza di sindromi del SNC, generalmente non associate alla presenza di neoplasie, che possono essere anticorpo-mediate e pertanto potenzialmente trattabili. Lo spettro clinico di queste malattie non è stato ancora pienamente esplorato, vi potrebbero essere altre malattie che devono ancora essere scoperte, e vi sono forme di epilessia o sindromi psichiatriche che potrebbero avere una base autoimmune. Definiamo questi anticorpi neuronal surface antibodies (NSAbs) e le malattie ad essi associate NSAS (Neuronal Surface Antibody Syndrome). Lo scopo della ricerca effettuata in questo percorso di tre anni di dottorato è stato la caratterizzazione clinica e laboratoristica di alcune NSAS. Il primo anno di dottorato è stato effettuato presso l’Università di Oxford sotto la supervisione della Prof. Angela Vincent. Durante questo periodo ho contribuito alla caratterizzazione delle due più importanti forme di NSAS, le encefaliti associate ad anticorpi anti-VGKC-complex e ad anti-NMDAR. I risultati di questi studi sono stati pubblicati (Irani et al., Brain 2010 a e b). Il mio personale contributo è consistito nello sviluppo e ottimizzazione di tecniche di immunofluorescenza per l’identificazione di NSAbs, utilizzando sia colture primarie di neuroni ippocampali che cellule di mammifero transfettate con l’antigene di interesse. Inoltre durante il soggiorno presso il laboratorio di Oxford ho cercato di mettere a punto un approccio di proteomica per l’identificazione di nuovi NSAbs mediante immunoprecipitazione e spettrometria di massa. Tale approccio è stato successivamente applicato nello screening di pazienti con sindromi cerebellari autoimmuni. I risultati di questo studio sono descritti nella terza parte della tesi. La tesi di dottorato è divisa in tre parti. Nella prima parte ho esaminato lo stato dell’arte delle sindromi associate ad anticorpi contro antigeni di superficie iniziando con un confronto tra disordini associati ad anticorpi contro antigeni intracellulari e antigeni di superficie. Ho quindi riassunto i maggiori aspetti clinici e paraclinici delle sindromi che sono state già identificate, e da queste osservazioni ho disegnato delle linee guida per il futuro riconoscimento di queste e altre condizioni potenzialmente mediate da NSAbs. Il lavoro di stesura di queste linee guida è stato supervisionato dalla Prof. Angela Vincent e riesaminato criticamente dai Prof. Francesc Graus (Barcelona, Spain) e Prof. Christian Bien (Bielefeld, Germany) e dal Dr. Bruno Giometto (Treviso, Italy). La seconda parte della tesi descrive la caratterizzazione clinica e immunologica di una coorte di pazienti con sospetta encefalite autoimmune e anticorpi anti-VGKC-complex che ho raccolto ed esaminato a Padova nei 3 anni di dottorato. Recentemente è stato dimostrato dal gruppo di lavoro di Oxford che la maggior parte degli anticorpi precedentemente attribuiti ai canali del potassio VGKC (mediante immunoprecipitazione) in realtà hanno come bersaglio due proteine canale complessate con i VGKC, Lgi1 e Caspr2; anticorpi diretti contro queste due proteine tendono ad associarsi rispettivamente con encefalite limbica (EL) e sindrome di Morvan (vedi Irani et al. Brain 2010b). Ho quindi deciso di focalizzare la mia indagine su pazienti con encefaliti autoimmuni associate ad anticorpi anti-VGKC-complex con l’obiettivo di riportare il profilo clinico e immunologico di questi pazienti e possibilmente dimostrare una correlazione tra antigene e fenotipo. Durante i tre anni di dottorato ho analizzato 503 campioni da 366 pazienti con sospetta encefalite autoimmune inviati al nostro laboratorio tra gennaio 2005 e dicembre 2011: 279 campioni di siero (e/o di liquido cerebrospinale) da 232 pazienti sono stati testati per anti-VGKC-complex; inoltre 226 campioni sono stati testati per anticorpi anti-NMDAR, 59 per anti-AMPAR1 e 2 e GABAbR, 91 per anti-GAD; 21 campioni inoltre sono stati testati su neuroni ippocampali. Le metodiche di screening hanno incluso: radioimmunoprecipitazione (RIA) per anti-VGKC e -GAD65; immunofluorescenza indiretta (IFI) su cellule transfettate con NMDAR, AMPAR1, AMPAR2, GABAbR, Lgi1 and Caspr2; IFI su neuroni ippocampali. I dati raccolti dai pazienti risultati positivi per anti-VGKC-complex sono quindi stati analizzati. Trentadue pazienti su 232 testati sono risultati positivi per anti-VGKC-complex. Informazioni cliniche sono risultate disponibili per 18 pazienti: 10 positivi per anti-Lgi1, 3 per anti-Caspr2 mentre 5 sono risultati positivi su RIA ma non su IFI. Due pazienti hanno presentato un quadro sindromico compatibile con la sindrome di Morvan, in un caso in associazione ad anti-Lgi1 e nell’altro ad anti-Caspr2 e timoma. In tutti i pazienti in cui è stato somministrato un trattamento immunomodulante si è assistito ad un miglioramento clinico con l’eccezione di una paziente deceduta a causa di un microcitoma polmonare. Un titolo anticorpale persistentemente elevato nonostante la remissione clinica in una paziente con EL e anticorpi anti-Lgi1 ha condotto all’individuazione e alla successiva rimozione di un timo iperplastico; un paziente con anti-Caspr2 ha manifestato un’encefalite di grado lieve in concomitanza di una sindrome da anticorpi anti-fosfolipidi; un altro paziente con una forma di encefalomielite responsiva al trattamento è risultato negativo in IFI ma ha presentato un alto titolo anticorpale per anti-VGKC su RIA. Un ipermetabolismo dei gangli della base è stato individuato in 4 pazienti con anti-Lgi1, in un caso associato alla presenza di crisi epilettiche distoniche facio-brachiali. In conclusione i risultati di questo studio hanno esteso lo spettro delle manifestazioni cliniche note associate ad anticorpi anti-VGKC-complex. E’ stato dimostrato che le EL con anti-VGKC possono associare non solo a timomi ma anche ad iperplasia del timo, in maniera analoga a quanto avviene nella miastenia gravis. La persistenza di un alto titolo anticorpale dovrebbe pertanto indurre un accurato imaging del timo. La terza parte della tesi descrive uno studio sperimentale che ha avuto lo scopo di identificare nuovi autoanticorpi nelle sindromi cerebellari di possibile origine autoimmune. La Dr.ssa Esther Becker dell’Università di Oxford ha contribuito in egual misura al sottoscritto in questo studio. Pochi studi in precedenza avevano ricercato anticorpi potenzialmente patogenetici in pazienti non paraneoplastici con atassia cerebellare. Inizialmente 52 pazienti con atassia cerebellare idiopatica sono stati sottoposti a screening mediante immunofluorescenza su neuroni cerebellari. Un siero che ha dimostrato un intensa reattività sui neuroni è stato selezionato per gli esperimenti di immunoprecipitazione e spettrometria di massa. Tale approccio ha portato all’identificazione di Caspr2 (contactin-associated protein 2) come maggiore antigene. Anticorpi anti-Caspr2 sono stati poi identificati mediante immunofluorescenza su cellule transfettate in 9 pazienti su 88 con atassia (10%), in confronto a 3 pazienti su 144 di controllo affetti da sclerosi multipla o demenza. Caspr2 è altamente espresso nel cervelletto, e solo in parte in associazione ai canali del potassio voltaggio-dipendenti. Studi prospettici saranno necessari per valutare se l’identificazione di anticorpi anti-Caspr2 abbia valore nella diagnosi e trattamento delle sindromi cerebellari.

Adult mammalian central nervous system (CNS) neurons are unable to extend axons after injury, partially owing to the inhibitory myelin and chondroitin sulfate proteoglycans (CSPGs) present in the environment. A neuron's intrinsic state is also important for determining its regenerative potential. Peripheral nervous system (PNS) neurons, unlike their CNS counterparts, have increased ability to regrow their axons after injury, even in the presence of inhibitory molecules. With the goal of discovering novel regeneration associated genes, we have isolated the genes differentially expressed by PNS neurons. We then developed a high throughput neuronal transfection method to test whether these genes were sufficient to modify neurite growth in vitro. Using high content screening, we measured the ability of cerebellar neurons to initiate neurite outgrowth on inhibitory and permissive substrates. This combination of technologies (subtractive hybridization, microarray, high throughput electroporation and high content screening) allowed phenotypic examination of neurons after the overexpression of over a thousand genes. Additionally, kinases and phosphatases were assayed for their ability to modify neurite outgrowth in hippocampal neurons. Results from both of these large unbiased screens confirmed many of the existing candidates for neurite growth during development and regeneration. We also discovered many novel genes which promoted neurite outgrowth such as GPX3, EIF2B5, RBMX, CHKA, IRF6, and PKN2. To accurately interpret the large volume of data, new methods of analysis were performed. Finally, we developed novel techniques that took advantage of public databases to cluster genes and determine whether those clusters produced robust changes in neurite growth. In summary, we have provided a vast repository of functional data to study axon development and regeneration after injury as well as developing the tools needed to interpret that data.

Thesis (Honors)--Ohio State University, 2005.
Title from first page of PDF file. Document formattted into pages: contains 51 p.; also includes graphics. Includes bibliographical references (p. 29-33). Available online via Ohio State University's Knowledge Bank.

The clinical profile of Gulf War Illness (GWI) is characterized by the presence of the central nervous systems (CNS) symptoms, which include memory impairment, anxiety, widespread pain and motor problems. Now, even twenty years later, veterans with GWI continue to suffer from these persistent symptoms. Currently, there is no treatment available for treating GWI, which is largely due to the complexity of the clinical presentation of this illness and the heterogeneity of OW exposures. The main goals of this thesis were to develop and characterize GW agent exposure mouse models that recapitulate the CNS symptoms of GWI and to identify the underlying aberrant biological pathways. Three major objectives were undertaken to accomplish these goals: (1) Two mouse models of OW-agent exposure were developed using combination of the anti-nerve gas treatment pyridostigmine bromide (PB), pesticide (permethrin), an insect repellent (N,N-Diethyl-meta-toluamide) and stress. Neurobehavioral studies show that combined exposure to GW agents produced anxiety and sensorimotor deficits in one mouse model and anxiety and cognitive impairment in the other. Neuropathological studies showed a presence of astrogliosis in both models. (2) Exploratory proteomic studies suggested that lipid-metabolism and immune/inflammation were associated with GW-agent exposure. (3) As lipid dysfunction is upstream of the inflammatory pathways, a lipidomics approach was used to identify the OW-agent exposure dependent lipid profiles. Lipid profiles of mouse models of OW-agent exposure were compared with those of other neurological conditions to identify profiles that were unique to GW-agent exposure. Lipid profiles were interrogated to identify lipid-metabolism pathways that may be amenable to therapeutic targeting. Studies described in this thesis provide novel insight into the pathobiology of GWI and suggest that pathways involved in phosphatidylcholine metabolism might be of therapeutic value in treating the CNS symptoms of GWI.

3-iodothyronamine (T1AM) is an endogenous high-affinity ligand for the trace amine-associated receptor 1 (TAAR1) found circulating in mammals and accumulating in many tissues including the brain. Its functional effects are now known to include decrease in heart rate, cardiac output, body temperature, and metabolic rate, as well as stimulation of lipid vs carbohydrate catabolism and neurological effects, including prolearning and antiamnestic action, reduced pain threshold, and reduction of non-REM sleep. The close structural similarity with thyroid hormone (TH) induced to speculate that T1AM might be synthetized from TH through decarboxylation and deiodination, but this hypothesis still requires validation. Notably, in addition to the structural similarities between T1AM and TH, T1AM contains the same arylethylamine scaffold also found in monoamine neurotransmitters, implicating an intriguing role for T1AM as both a neuromodulator and a hormone-like molecule constituting a part of thyroid hormone action. Recent evidence indicates that targeting TAAR1 may provide a new therapeutic approach for the treatment of a range of neuropsychiatric and metabolic disorders. Consistently, T1AM and recently developed halogen free biaryl-methane thyronamine-like TAAR1 agonists, SG-1 and SG-2, have emerged as rapid modulators of behavior and metabolism, with SG-2 showing a potency almost comparable to that of T1AM. To assess the therapeutic potential of TAAR1 agonists for neuroprotection, in the present thesis we investigated whether T1AM and its corresponding halogen free synthetic analogue SG-2 improve learning and memory when systemically administered to mice at submicromolar doses. In addition, since autophagy is key for neuronal plasticity, T1AM and newly developed thyronamine-like TAAR1 agonists SG-1 and SG-2 were evaluated as autophagy inducers in cell lines. T1AM is known to induce pro-learning and anti-amnestic responses when administered icv at very low doses (1.32 to 4 μg.kg-1) to mice, but its effects on memory after systemic administration are currently unknown. To address this point, we evaluated the behavior of CD-1 mice injected i.p. with T1AM or SG-2 (1.32, 4 and 11 μg.kg-1), in the passive avoidance test, while measuring in specific brain areas the activation of typical signalling proteins involved in memory acquisition, including pERK and transcription factor c-fos. The passive avoidance test showed that when administered i.p. at 11 μg.kg-1 either T1AM or SG-2 induced significant memory enhancement. T1AM also significantly increased retention when administered at a lower dosage (4 μg.kg-1). Memory acquisition and storage are typically associated with increased ERK1/2 phosphorylation. After treatment with T1AM and SG-2 at doses of 1.32 μg·kg-1, pERK could be detected, but its levels were not significantly different from those found in vehicle injected mice. However, after treatment with T1AM and SG-2 at doses that proved to be effective on memory acquisition and retention (i.e. 4 and 11 μg·kg-1) pERK turned out to be significantly higher (p<0.01) than that observed in the vehicle group. Similar to pERK, c-fos was not significantly changed in any brain area by treatment with 1.32 μg·kg-1 of T1AM or SG-2. For both compounds, only when injected at the highest dose (i.e. 11μg·kg-1) moderate changes of c-fos expression were observed. At the dosages active on memory, T1AM and SG-2 also proved to have an hyperalgesic effect. In addition, our pain threshold experiments revealed that TA1 and SG-6, the oxidative metabolites of T1AM and SG-2, respectively, seem to play a critical role for the action of the corresponding amine. In fact, at our settings, pretreatment with clorgyline abolished almost completely the effect on pain of T1AM or SG-2 administered i.p. at the highest dosage (i.e., 11 μg/kg). The same effect was also shown to involve the histaminergic system, as it disappeared after pretreatment with the histamine H1-receptor antagonist, pyrilamine. Preliminary results from a LC/MS-MS pharmacokinetic study of TA1 bio-distribution after sistemic administration, indicated that there is a difference in the capacity of clearing the acid by different tissues, with the brain conserving TA1 levels for longer than plasma or liver. Therefore, the brain might represent one important target for TA1 action. Autophagy (ATG) is one of the most important mechanisms of neuroprotection. Inducing autophagy may represent a reasonable strategy to develop therapeutics for the treatment of neurodegenerative diseases, including Alzheimer's disease. Therefore, to explore the possible link between TAAR1 activation and neuroprotection, we investigated the ability of T1AM, SG-1 and SG-2 to induce autophagy in human glioblastoma cell lines (U-87MG) by examining the formation of autophagosomes and autophagic flux. During the process of autophagy, LC3I is converted to its lipidated form LC3II, which is one of the hallmarks of autophagy and essential for the formation of autophagosome. P62 is used as a marker of autophagic flux and inversely correlates with autophagic activity. It binds directly to LC3 and degrades during autophagy. In cultured U-87MG cells exposed to 1μM T1AM, SG-1, SG-2 (for 0.5, 4, 8 and 24h) transmission electron microscopy (TEM) and immunofluorescence (IF) showed a significant (p<0.01) time-dependent increase of autophagy vacuoles and microtubule-associated protein 1 light chain 3 (LC3), with T1AM and SG-1 being the most effective agents. Consistently, western blotting results showed that test compounds increased the conversion of LC3-I to LC3-II while decreasing the levels of P62 in an obvious time-dependent manner. We then proceeded to elucidate which pathway was involved in ATG induction by assessing the effect of our test compounds on the PI3K–AKT–mTOR pathway. We found that 1μM T1AM, SG-1 and SG-2 decreased pAKT/AKT ratio at 0.5 and 4h after treatment (p<0.01), suggesting that these compounds induce autophagy through a reduction of pAkt level. Taking into account the key role played by ATG and/or Ubiquitine-proteasome (UP) in all clearing pathways modulating cell survival and disease mechanisms, and considering their involvement in a wide range of disorders, including neurodegenerative diseases (NDDs), we were also able to prove the ability of T1AM, SG-1 and SG2 to modulate the UP system. Indeed, our TEM analysis showed increased 20S proteasome recruitment to autophagosome in U-87MG cells after treatment with 1μM T1AM, SG-1 and SG-2, suggesting that these compounds might modulate both ATG and UP protein clearing pathways within the autophagoproteasomes. A growing body of evidence has suggested the presence of a strong correlation between obesity and neurodegeneration. NDDs, such as Alzheimer Disease (AD) and Parkinson Disease (PD), are characterized by a progressive loss of memory and cognition, which can ultimately lead to death. This deterioration is majorly a result of inflammation due to aberrant protein deposition, oxidative stress and modification in lipid pathways. Recent studies demonstrated acute and chronic T1AM treatment to have potent effects on shifting whole body macro-nutrient metabolism in mammals. On the basis of these findings, we decided to explore whether T1AM, SG-2 and recently designed synthetic thyronamine/thyroid hormone-hybrid analogs IS25 and TG46 could promote lipolysis in HepG2 cells. Oil Red O staining of HepG2 revealed that all test compounds reduced total lipid accumulation into lipid droplets compared to the control. Moreover, the analysis of free glycerol release confirmed that decreased accumulation of lipids in HepG2 cells observed after Oil Red O staining was caused by increased lipolysis. The involvement of AMPK/ACC modulation was also confirmed by western blot analysis that showed an enhancement of both pAMPK/AMPK and pACC/ACC ratios. AMPK stimulation by test compounds led indeed to the phosphorylation and consequent inactivation of acetyl coenzyme A carboxylase (ACC), the major regulator of fatty acids synthesis. In conclusion, taken together our data displayed that T1AM and thyronamine-like compounds SG-1 and SG-2 have neuroprotective properties, which also involve the induction of autophagy and the regulation of Ubiquitine-Proteasome System. Indirectly, together with newly designed thyronamine/thyroid hormone- hybrid analogs, they could exert a protective action through lipolytic effects. This novel aspect requires further investigation.

Neurological disorders – disorders of the brain, spine and associated nerves – are a leading contributor to global disease burden with a sizable economic cost. Adeno-associated viral (AAV) vectors have emerged as an effective platform for CNS gene therapy and have shown early promise in clinical trials. These trials involve direct infusion into brain parenchyma, an approach that may be suboptimal for treatment of neurodegenerative disorders, which often involve more than a single structure in the CNS. However, overall neuronal transduction efficiency of vectors derived from naturally occurring AAV capsids after systemic administration is relatively low. We have developed novel capsids AAV-AS and AAV-B1 that lead to widespread gene delivery throughout the brain and spinal cord, particularly to neuronal populations. Both transduce the adult mouse brain >10-fold more efficiently than the clinical gold standard AAV9 upon intravascular infusion, with gene transfer to multiple neuronal sub-populations. These vectors are also capable of neuronal transduction in a normal cat. We have demonstrated the efficacy of AAV-AS in the context of Huntington's disease by knocking down huntingtin mRNA 33-50% after a single intravenous injection, which is better than what can be achieved by AAV9 at the particular dose. AAVB1 additionally transduces muscle, beta cells, pulmonary alveoli and retinal vasculature at high efficiency, and has reduced sensitivity to neutralizing antibodies in human sera. Generation of this vector toolbox represents a major step towards gaining genetic access to the entire CNS, and provides a platform to develop new gene therapies for neurodegenerative disorders.

Neurological disorders – disorders of the brain, spine and associated nerves – are a leading contributor to global disease burden with a sizable economic cost. Adeno-associated viral (AAV) vectors have emerged as an effective platform for CNS gene therapy and have shown early promise in clinical trials. These trials involve direct infusion into brain parenchyma, an approach that may be suboptimal for treatment of neurodegenerative disorders, which often involve more than a single structure in the CNS. However, overall neuronal transduction efficiency of vectors derived from naturally occurring AAV capsids after systemic administration is relatively low. We have developed novel capsids AAV-AS and AAV-B1 that lead to widespread gene delivery throughout the brain and spinal cord, particularly to neuronal populations. Both transduce the adult mouse brain >10-fold more efficiently than the clinical gold standard AAV9 upon intravascular infusion, with gene transfer to multiple neuronal sub-populations. These vectors are also capable of neuronal transduction in a normal cat. We have demonstrated the efficacy of AAV-AS in the context of Huntington's disease by knocking down huntingtin mRNA 33-50% after a single intravenous injection, which is better than what can be achieved by AAV9 at the particular dose. AAVB1 additionally transduces muscle, beta cells, pulmonary alveoli and retinal vasculature at high efficiency, and has reduced sensitivity to neutralizing antibodies in human sera. Generation of this vector toolbox represents a major step towards gaining genetic access to the entire CNS, and provides a platform to develop new gene therapies for neurodegenerative disorders.

Blood-brain barrier breakdown is a common feature of neuroinflammatory diseases, such as multiple sclerosis (MS). Indeed, blood proteins may play a role in the pathology of the disease. Here we investigate the link between the presence of blood-borne components in the CNS parenchyma and the expression of neurological deficits. Male Sprague-Dawley rats were injected intraspinally with vascular endothelial growth factor (VEGF), which causes breakdown of the blood-brain barrier, or saline as control. The injection was made unilaterally at the T13 – L1 junction, namely the spinal level that controls hind limb movement. In VEGF-injected animals alone, hind limb motor and sensory deficits consistently appeared at day two after surgery, but not at days 1 or 3 (onwards), as assessed by behavioural and locomotor tests (horizontal ladder test, walking treadmill, von Frey hair test, inclined plane, burrowing). Histological examination revealed the presence of a blood-brain barrier leakage within the spinal cord as assessed by the presence of extravascular IgG and fibrinogen. Activated microglia/macrophages (ED1 + , MHC class II + and OX-42 + cells) were present at the injection site, peaking at day two, along with activated astrocytes. The mechanism responsible for the neurological deficit was investigated by attempting to antagonize specific VEGF signalling pathways, assessing a potential hypoxic state using immunohistochemical techniques, and characterizing neuronal excitability by electrophysiological methods. VEGF injection provokes opening of the blood-brain barrier which is temporally and spatially associated with neuroinflammation and loss of function. The findings indicate a potential mechanism underlying loss of function in inflammatory neurological diseases such as MS.

Recent studies examining cell-cell interactions during CNS development and following disease or trauma have highlighted our limited understanding of the in vivo functions of the myelinating cell of the CNS, the oligodendrocyte. With this in mind, our laboratory has developed techniques by which a profile of proteins derived from or regulated by oligodendrocytes can be elucidated. Specifically, we have demonstrated that oligodendrocytes can be selectively eliminated from one optic nerve of a rat by treating the animal with a unilateral exposure of X-irradiation at the time of birth. Consequently, this approach allowed us to experimentally create, within the same animal, one optic nerve devoid of oligodendrocytes and their progenitors (the X- irradiated side) and one optic nerve containing the normal oligodendrocyte population (the untreated side). Using this experimental animal model we hypothesized that uncharacterized proteins, derived from and regulated by oligodendrocytes, which are crucial for CNS development can be identified. Specifically, by comparing protein profiles found within the normal myelinating optic nerve versus the X-irradiated optic nerve, where oligodendrocytes are absent, potential oligodendrocyte-derived proteins can be quickly identified. Further verification that these proteins are indeed related to oligodendrocytes and/or the processes of myelination can be obtained by their reappearance in the 2-D gel protein profile of P28 X-irradiated nerves that, as we have shown previously, undergo a delayed myelination. We then employed mass spectrometric analysis to determine the identities of oligodendrocyte derivedregulated proteins. In this thesis, I will begin by describing our current knowledge of the proteins expressed by oligodendrocytes and their role(s) in oligodendrocyte function. This will be followed by a detailed description of the experimental model system we utilized in an attempt to elucidate the complete repertoire of oligodendrocyte-regulated proteins. We will then describe the results generated fiom our methodology and discuss the implications of our findings in relation to the functional cooperation between oligodendrocytes and other cells of the developing central nervous system. The results generated fiom this project should lead to a clearer understanding of the role of oligodendrocytes and'of the array of proteins whose expression patterns are associated with these cells during CNS development.

Abstract Lymphomas are a heterogeneous group of malignancies that arise from lymphatic tissues. Diffuse large B-cell lymphoma (DLBCL) is the most common lymphoma sub-type. It is an aggressive malignancy with an increasing incidence. The prognosis of DLBCL has improved significantly, but problems also remain. The clinical significance of central nervous system (CNS) relapses has become increasingly important. As secondary CNSL (sCNSL) and primary CNS lymphoma (PCNSL) are known to have poor prognoses; the prevention of sCNSL is of crucial importance. Peripheral T-cell lymphomas (PTCL) are rare neoplasms and include several lymphoma subtypes that possess complex and also overlapping morphological and immunophenotypic characteristics. The identification of different entities has improved, but the biological knowledge remains scarce when compared to DLBCL. The optimal treatment schemas for PTCLs are still lacking and they have long been treated with the same therapies as B-cell lymphomas, mainly with suboptimal treatment results. The aim of this study was to identify poor prognostic markers in DLBCL and PTCLs and potential biological markers for the prediction of DLBCL CNS relapse. The study material included patients with systemic DLBCL without CNS affision (sDLBCL), sCNSL, PCNSL and PTCLs. The expression of epithelial-mesenchymal transition (EMT) transcription factors (TFs), chemokines and their receptors and adhesion-, migration- and inflammatory responses-associated molecules were studied by means of immunohistochemistry. IEM was used to verify the specific subcellular location of the studied molecules. GEP was performed on 12 PTCL samples in order to compare the poor prognosis group with the good prognosis group and on one sDLBCL and one sCNSL sample from the time of primary diagnosis. The EMT TFs were found to be expressed in both DLBCL and PTCLs, where they ultimately proved to have prognostic relevance as well. In PTCLs, these TFs were able to delineate a disease group with a specific gene-expression profile. CXCR4, CXCR5, ITGA10, PTEN and CD44 were found to be differently expressed between DLBCL cases with CNS affision when compared to those without CNS disease. These molecules seem to play a role in the development of CNS relapse and hopefully, if further verified, will lead towards the identification of biological markers for CNS relapse prediction
Tiivistelmä Lymfoomat ovat heterogeeninen ryhmä imukudossyöpiä, joista diffuusi suurisoluinen B-solulymfooma (DLBCL) on yleisin alatyyppi. Se on aggressiivinen maligniteetti, jonka insidenssi on noussut viime vuosina. DLBCL potilaiden ennuste on parantunut merkittävästi, mutta yhä osa potilaista menehtyy tautiinsa. DLBCL:n keskushermostorelapsin kliininen merkitys on tänä päivänä aiempaa suurempi. Sekundaarisen keskushermostolymfooman (sCNSL) ja primaarin aivolymfooman (PCNSL) ennusteet ovat nykyhoidoilla huonoja, joten keskushermostorelapsin ennaltaehkäiseminen on tärkeää. Perifeeriset T-solulymfoomat (PTCLs) ovat ryhmä harvinaisia neoplasioita, joka sisältää useita eri alatyyppejä, joiden morfologiset ja immunofenotyyppiset ominaisuudet ovat monimuotoisia ja osin päällekkäisiä. Eri entiteettien indentifiointi on parantunut, mutta PTCL:ien biologinen tietämys on yhä DLBCL:aa heikompaa. PTCL:ien optimaalinen hoito ei ole selvillä ja tätä tautiryhmää on pitkään hoidettu samoilla hoidoilla kuin DLBCL:aa, mutta huonommilla hoitotuloksilla. Tutkimuksen tavoitteena oli löytää huonon ennusteen markkereita, joilla myös pystyttäisiin ennustamaan DLBCL:n keskushermostorelapsia. Aineisto koostui DLBCL, sCNSL, PCNSL ja PTCL näytteistä. Immunohistokemiallisilla värjäyksillä tutkittiin epiteliaalis-mesenkymaalisen transition (EMT) transkriptiotekijöitä (TF), kemokiinireseptoreita sekä adheesioon-, migraatioon ja inflammaatioon assosioituja molekyylejä. Immunoelektronimikroskopialla varmennettiin molekyylien lokalisaatio soluissa. Geeniekspressioprofiloinnilla (GEP) verrattiin kahdentoista hyvän ja huonon ennusteen ryhmään kuuluvan PTCL näytteen välisiä geeniekspressioeroja sekä kahden DLBCL potilaan näytteitä, joista toiselle kehittyi keskushermostorelapsi. EMT TF:ien ekspressiota nähtiin DLBCL ja PTCL näytteissä, joissa niillä myös todettiin olevan ennusteellista merkitystä. PTCL:ssa TF:t pystyivät erottelemaan tautiryhmän, jolla oli oma spesifinen geeniekspressioprofiilinsa. CXCR4, CXCR5, ITGA10, PTEN ja CD44 ekspressio oli erilaista systeemisissä DLBCL tapauksissa verrattuna sCNSL tapauksiin. Edellä mainituilla molekyyleillä näyttää olevan oma roolinsa keskushermostotaudin kehittymisessä ja jos nämä tulokset pystytään vahvistamaan tulevissa tutkimuksissa, johtavat ne toivottavasti kohti keskushermostorelapsiriskin tarkempaa tunnistamista

Investigations of neurological disease in neonatal calves were conducted over a four and a half year period. The studies commenced with so called Hereditary Neuraxial Oedema of Poll Hereford calves. It was determined that two distinct disease entities in this breed had led to confusion regarding the diagnosis of this disorder.

Alcohol is a commonly consumed beverage, a drug of abuse and an important molecule affecting nearly every organ-system in the body. This project seeks to investigate the interplay between alcohol’s effects on critical organ-systems making up gut-liver-brain axis. Alcohol initially interacts with the gastrointestinal tract. Our research describes the alterations seen in intestinal microbiota following alcohol consumption in an acute-on-chronic model of alcoholic hepatitis and indicates that reducing intestinal bacteria using antibiotics protects from alcohol-induced intestinal cytokine expression, alcoholic liver disease and from inflammation in the brain. Alcohol-induced liver injury can occur due to direct hepatocyte metabolic dysregulation and from leakage of bacterial products from the intestine that initiates an immune response. Here, we will highlight the importance of this immune response, focusing on the role of infiltrating immune cells in human patients with alcoholic hepatitis and alcoholic cirrhosis. Using a small molecule inhibitor of CCR2/CCR5 chemokine receptor signaling in mice, we can protect the liver from damage and alcohol-induced inflammation. In the brain, we observe that chronic alcohol leads to the infiltration of macrophages in a region-specific manner. CCR2/CCR5 inhibition reduced macrophage infiltration, alcohol-induced inflammation and microglial changes. We also report that chronic alcohol shifts excitatory/inhibitory synapses in the hippocampus, possibly through complement-mediated remodeling. Finally, we show that anti-inflammasome inhibitors altered behavior by reducing alcohol consumption in female mice. Together, these data advance our understanding of the gut-liver-brain axis in alcoholism and suggest novel avenues of therapeutic intervention to inhibit organ pathology associated with alcohol consumption and reduce drinking.

Axonal degeneration is a major pathological feature of the central nervous system (CNS) inflammatory demyelinating disease multiple sclerosis (MS). This axonal degeneration has major consequences, as functional axonal regeneration in the CNS is largely absent. Cumulative axonal degeneration is the likely cause of the majority of progressive MS-related disability, and therefore, the need for novel neuroprotective therapies for MS exists. Experimental autoimmune encephalomyelitis (EAE), an animal model of MS pathology, also produces axonal injury. In particular, the optic nerve and spinal cord are key sites of neuroinflammation in mouse EAE. By utilizing this model, the short term and long term effects of the putative neuroprotective cytokine, leukaemia inhibitory factor (LIF), were investigated in the optic nerve and spinal cord utilising a number of outcome measures of axonal dysfunction. These included MRI measures of water diffusivity along (ADC ||) and across (ADC┴) the optic nerves, serum levels of phosphorylated neurofilament heavy chain subunit (pNF-H) and histological morphometric measures. LIF treatment reduced EAE grade and pNF-H plasma levels, decreased ADC┴, but had no effect on ADC ||, axon counts or inflammatory infiltration.
In contrast, genetic deletion of LIF and its sister cytokine ciliary neurotrophic factor (CNTF), not only increased EAE grade and pNF-H levels, but also decreased optic nerve ADC|| and optic nerve and spinal cord axon densities. After reviewing current literature, we hypothesize that the target cell for endogenously upregulated LIF in EAE may be the neuron or axon, whereas the target cell for exogenously administered therapeutic LIF may be another cell type, possibly infiltrating macrophages and activated microglial cells. LIF antagonist treatment did not have any affect on EAE grade, pNF-H levels or MRI parameters. This lack of effect may be due to the inability of the LIF antagonist to enter the CNS, supporting the hypothesis that endogenous LIF has a centrally acting mechanism.

The receptor tyrosine kinase, RON, is expressed on tissue-resident macrophages. RON functions by activating genes that promote wound repair and resolve inflammation, while repressing genes that perpetuate tissue damage and cell death. Chronic HIV infection is associated with dysregulated inflammation, and we hypothesize that diminished macrophage RON expression contributes to the development of end organ diseases including HIV-associated central nervous system (CNS) inflammation. We utilized CNS tissue from a SIV macaque model to examine the temporal regulation of RON in the brain during infection. Following prolonged SIV infection, RON expression was inversely correlated with the development of CNS disease: RON was highly expressed in animals that did not develop CNS lesions and lower in SIV infected macaques that demonstrated moderate to severe inflammatory lesions. Arginase-1 expression was low during late infection whereas expression of the inflammatory genes, IL-12 p40 and TNF &alpha, was elevated compared to uninfected animals. To validate a role for RON in regulating HIV, we infected human tonsillar tissue-resident macrophages. RON inhibited HIV replication in tissue-resident macrophages. Furthermore, HIV infection diminished RON in tonsil macrophages. We propose a model in which RON expression is decreased, genes that quell inflammation are repressed, and inflammatory mediators are induced to promote tissue inflammation following chronic HIV infection in the brain. The cyclin dependent kinase inhibitor p21 is a factor that, like RON, negatively regulates HIV transcription. Elevated expression of p21 in HIV+ elite controllers, or by ectopic expression in primary CD4+ T cells, resulted in reduced HIV expression. Furthermore, these elite controllers had increased binding of factors that negatively regulate transcription elongation at the HIV long terminal repeat. RON and p21 are examples of cellular factors that limit HIV transcription and contribute to HIV latency. Latently infected cells are not targeted by anti-retroviral therapy and permit rapid rebound of viremia following treatment interruption. Understanding intrinsic mechanisms that establish latency may provide targets for purging these HIV reservoirs or maintaining their transcriptionally silent state.

Genetically based Central Nervous System (CNS) disorders remain a largely unresolved issue in the world today. Our genome is the source of our greatest strengths and weaknesses. For this reason, intelligent modification of the genome's DNA is a profoundly beneficial goal in the maximization of the overall health of the human race. Potential benefit in this field is currently limited in both effectiveness and safety in regards to the delivery of therapeutic genes into the nucleus, which is protected by many evolution-based barriers. Evolution has also favored the development of highly specialized and infectiously effective viruses capable of overcoming such boundaries. By neutering the naturally occurring and pathologically benign Adeno-Associated Virus (AAV) we have transformed what was once a virus, into a "pure" vector, taking full advantage of evolution's diligent enhancement of these genetic hijackers without introducing unacceptable danger to patients. We utilized the logically engineered, castrated form of AAV serotype 9 (recombinant AAV9/rAAV9) to act as a vehicle for two reporter genes, Enhanced Green Fluorescent Protein (EGFP) and Firefly Luciferase (Fluc) with the goal of assessing and improving the efficiency of vector transduction in murine CNS. We found that rAAV9, when infused into the intrathecal space of mice is capable of extensive and intensive transduction of both neurons and astrocytes throughout the entire length of the SC as well as the hind regions of the brain (brainstem and cerebellum). We also found that efficiency of transduction was best in our highest dose groups, 1E+12 genome copies (GC) in Experiment 1 and 2E+12 GC in Experiment 2, both of which received rAAV9 particles via the two commercially available (100μL and the 200μL) ALZET® Osmotic Pump designs. Administering dosage higher than this directly into the intrathecal space was limited by the size of the pump reservoir and rAAV9 production titer. We are currently attempting to achieve a more complete CNS transduction by performing another experiment in which we place the pump cannula directly into the intracerebroventricular (ICV) space of the lateral ventricles. Our findings reveal that infusion of rAAV9 by intrathecal placed pump cannula is more effective than any other method tested in this study int the transduction of neurons and glial cells of adult&ndashmouse CNS. By elucidating a mode of delivery that maximizes the robustness of transduction efficiency, our results are a critical building block in designing a cure for the array of genetic-based diseases of the CNS, which are currently untreatable

HIV-associated sensory neuropathy (HIV-SN) is a common complication associated with human immunodeficiency virus (HIV)-infection. A common symptom of HIV-SN is pain. Variation at specific loci within certain candidate genes has been suggested to alter susceptibility to developing HIV-SN as well as the susceptibility to developing pain and the intensity of the pain experienced. Few studies, however, have been conducted in individuals of black African ancestry. The aim of the current research was to conduct an in-depth study, in a black Southern African population, of genes previously associated with susceptibility to developing HIV-SN (TNFA and surrounding genes and IL12B) and variations in pain susceptibility and intensity (GCH1, KCNS1, IL1B, IL6, CCL2 and CCR2) in other neuropathic pain states. Single nucleotide polymorphisms (SNPs) identified in the literature were supplemented with population appropriate tagSNPs to improve assessment of the genes in an African population. Genotyping of previously collected deoxyribonucleic acid (DNA) samples was carried out using a GoldenGate assay with VeraCode microbeads and data were read on an Illumina BeadXpress Reader. Data were statistically analysed to assess the association of the genetic variants with susceptibility to developing HIV-SN and pain and variability of pain intensity in those patients with painful HIV-SN. Although some SNPs and haplotypes in the genes investigated associated with HIV-SN susceptibility (TNFA region), pain susceptibility (TNFA region, IL12B, KCNS1, IL6 and CCR2) or pain intensity (TNFA region, KCNS1, IL1B and IL6), none of the results were consistent with that which has been found in previous studies in non-African populations. Reasons for this may be that associations are population-specific or model-specific. Limitations of the study included the use of a relatively small sample, the method of sampling (convenience sampling), genotyping failure and tagging inefficiency in some instances, and the fact that there is no Southern African population dataset to use for tagSNP selection. My findings emphasise the importance of conducting genetic association studies in separate ethnic groups.

Recent experiments examining the development and plasticity of the chick embryonic spinal cord have described its inherent ability to recover from injury suffered prior to embryonic day (E) 13. Severing the spinal cord on or before E12 resulted in complete anatomical and functional recovery, which defined the permissive period for repair. Transections performed on E13 or later resulted in paraplegia characteristic of the restrictive period for repair. Previous studies have described the expression of growth-associated proteins (GAPs) such as GAP 43 and a-tubulin which were expressed at high levels during axon extension, then down-regulated at the time of target contact. These proteins were also expressed during abortive attempts at regeneration, and characterized a genetic growth program which was recruited after injury. This study examined the neuronal response to injury as reflected by changes in growth-associated gene expression in the hindbrain measured using Northern blotting. Levels of mRNA for GAP 43 and total a-tubulin during normal development were found to peak at E10-12, the period of maximal outgrowth of brainstem-spinal projections. Complete spinal cord transections performed on Ell (successful repair) or E14 (unsuccessful repair) did not detectably alter total a-tubulin mRNA levels. GAP 43 mRNA levels were not detectably altered after Eli transection. In contrast, transection on E14 (unsuccessful repair) resulted in a maintained increase in GAP 43 mRNA levels at least until 7 days post-transection, the longest survival period studied. Northern blotting was likely not sensitive enough to detect the full complement of changes which occurred after injury. However, transaction during the restrictive period for repair resulted in a maintained increase in GAP43 mRNA expression. These data suggested that some brainstem-spinal projection neurons injured on E14 retained the inherent ability to re-express at least part of the axonal growth program, indicated by the appropriate re-expression of GAP 43 mRNA. This suggests that the presence of inhibitory influences (or the absence of facilitatory influences) after E13 may have prevented the re-growth of axons and the re-formation of appropriate synapses.

Dissertação de Mestrado em Biotecnologia Farmacêutica apresentada à Faculdade de Farmácia
Nas últimas décadas, os vetores Virais Adeno-Associados (VAAs) têm-se destacado como um sistema de entrega promissor na área da terapia génica. De facto, vários estudos pré-clínicos demonstraram um conjunto de propriedades únicas deste tipo de vírus, incluindo um perfil robusto de segurança, uma transdução eficiente e uma baixa imunogenicidade. Foram ainda reportadas outras características relevantes, como a expressão prolongada dos seus transgenes em diversos órgãos, assim como um tropismo seletivo dos diferentes serótipos. Tendo tudo isto em consideração, a comunidade científica sentiu-se confiante em explorar o uso dos VAAs recombinantes num contexto clínico; daí que, actualmente, mais de 100 estudos clínicos usando VAAs estão em curso. No entanto, apesar dos resultados promissores obtidos em vários ensaios clínicos, há uma necessidade emergente para o desenvolvimento e otimização de protocolos de produção e purificação que possibilitem o fabrico de vetores VAAs altamente puros e em grande escala. Assim, o principal objetivo deste estudo foi simplificar e melhorar a purificação de vetores VAAs, através do desenvolvimento de protocolos simples e eficientes, baseados em cromatografia, que tirem partido das propriedades bioquímicas naturais dos diferentes serótipos de VAAs. Deste modo, vetores virais do tipo mosaico (rVAA1/2) foram purificados utilizando três colunas de cromatografia de afinidade distintas e os respetivos protocolos experimentais otimizados. A eficiência das três estratégias de purificação foi avaliada através da caracterização dos rVAAs purificados, em termos de pureza, propriedades físicas e atividade biológica. Os resultados obtidos demonstraram que os três protocolos de purificação foram eficientes na obtenção de vetores virais com elevado título e grau de pureza, num sistema passível de ser usado em larga escala. Adicionalmente, os stocks purificados de vetores rVAA1/2 evidenciaram resultados promissores em experiências de transfeção in vitro, relevando-se capazes de transfetar com sucesso linhas primárias, bem como de expressar o gene de interesse. Em resumo, este estudo fornece evidências claras da rapidez, eficiência e potencialidade para o seu uso em grande escala dos métodos baseados em colunas de cromatografia de afinidade para a purificação de rVAAs, diretamente dos lisados de células produtoras, com uma relação custo-benefício atrativa.
Over the past decades, Adeno-Associated Viruses (AAVs) have arisen as a promising delivery system for human gene therapy. Indeed, several in vitro and in vivo preclinical studies have already shown a handful of properties which are unique to this kind of viruses, including a strong safety profile, high gene transfer efficiency and low immunogenicity. Moreover, a prolonged gene expression in several tissues as well as a selective tissue tropism has also been reported. Having all the above in mind, the scientific community felt compelled to resource to the use of recombinant AAVs (rAAVs) for therapeutic gene transfer into patients in the clinical setting, with more than one hundred studies using AAVs currently taking place. However, despite the promising results obtained from several clinical trials, it has also become clear the emerging need for the development of production and purification protocols for the manufacturing of large amounts of highly pure rAAV vectors.Therefore, the main goal of this project was to simplify and improve rAAV vector purification by the establishment of simple, but efficient chromatography-based protocols, taking advantage of the natural biochemical properties of AAV serotypes.In order to do this, mosaic rAAV1/2 vectors were purified using three affinity chromatography columns and the efficiency of the different strategies was evaluated through the characterization of the purified rAAVs in terms of purity, physical properties and biological activity.Overall, the obtained results show that our scalable purification protocols were efficient in obtaining AAV vectors with high titer and purity. Moreover, the purified rAAV1/2 stocks have been successfully used in in vitro transfection experiments.In summary, this study provides compelling evidence of fast, efficient, cost effective, and scalable column-based methods for large-scale rAAV purification of various recombinant adeno-associated viruses directly from the lysates of producer cells.
Outro - This work was funded by the ERDF through the Regional Operational Program Center 2020, Competitiveness Factors Operational Program (COMPETE 2020, POCI) and National Funds through FCT (Foundation for Science and Technology) - BrainHealth2020 projects (CENTRO-01-0145-FEDER-000008), UID/NEU/04539/2019, ViraVector (CENTRO-01-0145-FEDER-022095), CortaCAGs (PTDC/NEU-NMC/0084/2014|POCI-01-0145-FEDER-016719), SpreadSilencing POCI-01-0145-FEDER-029716, Imagene POCI-01-0145-FEDER-016807, CancelStem POCI-01-0145-FEDER-016390, POCI-01-0145-FEDER-030737, POCI-01-0145-FEDER-032309, as well as SynSpread, ESMI and ModelPolyQ under the EU Joint Program - Neurodegenerative Disease Research (JPND), the last two co-funded by the European Union H2020 program, GA No.643417; by National Ataxia Foundation (USA), the American Portuguese Biomedical Research Fund (APBRF) and the Richard Chin and Lily Lock Machado-Joseph Disease Research Fund.

Relatório de Estágio do Mestrado Integrado em Ciências Farmacêuticas apresentado à Faculdade de Farmácia
O presente documento contém o Relatório de Estágio em Farmácia Comunitária, o Relatório de Estágio em Indústria Farmacêutica e a Monografia intitulada "The CSF route of delivery to the brain for molecular-based therapies".O Relatório de Estágio em Farmácia Comunitária é referente ao estágio curricular que realizei na Farmácia Moço, em Coimbra, e foi elaborado segundo a metodologia SWOT (do inglês strengths, weaknesses, opportunities and threats).O Relatório de Estágio em Indústria Farmacêutica diz respeito ao estágio curricular que realizei na Bluepharma Indústria Farmacêutica e, à semelhança do anterior, foi igualmente elaborado segundo a metodologia SWOT (do inglês strengths, weaknesses, opportunities and threats).A Monografia, tal como o título sugere, foca-se na administração de terapias de base molecular, tal como os oligonucleótidos anti-sense, no líquido cefalorraquidiano. Os oligonucleótidos anti-sense e os vetores virais adeno-associados têm sido recentemente considerados promissores para o tratamento de doenças que afetam o sistema nervoso central, doenças essas amplamente conhecidas como das mais desafiantes de tratar. Contudo, estes sistemas terapêuticos, devido à sua elevada dimensão, apresentam uma penetração limitada da Barreira Hematoencefálica, o que dificulta a administração dos mesmos por vias comuns, tais como a via intravenosa. É desse desafio que surge o presente trabalho, que se foca, portanto, na viabilidade da administração destes fármacos diretamente no líquido cefalorraquidiano, dado que, para além da função protetora que desempenha, é também um componente ativo e dinâmico do Sistema Nervoso Central, permitindo o acesso direto destes fármacos, deste modo contornando a barreira hematoencefálica.
This document contains the Community Pharmacy Internship Report, the Pharmaceutical Industry Internship Report and the Monograph entitled "The CSF route of delivery to the brain for molecular-based therapies".The Community Pharmacy Internship Report refers to the curriculum internship that I carried out at Farmácia Moço, in Coimbra, and was elaborated according to the SWOT methodology (strengths, weaknesses, opportunities and threats).The Internship Report in the Pharmaceutical Industry concerns the curricular internship I carried out at Bluepharma Pharmaceutical Industry and, similarly to the previous one, was also prepared according to the SWOT methodology (strengths, weaknesses, opportunities and threats).The Monograph, as the title suggests, focuses on the administration of molecular-based therapies, such as antisense oligonucleotides, into the cerebrospinal fluid. Anti-sense oligonucleotide and adeno-associated viral vectors have been considered promising therapeutical options for the treatment of neurological diseases, broadly known as the most challenging to treat. However, these compounds are large therapeutic systems with limited capacity to transpose the Blood Brain Barrier, which derails the common routes of administration, such as the intravenous route. It is from that hurdle that arouses the subject of the present paper, which focuses on the CSF route of delivery to the brain for molecular-based therapies, once this fluid, in addition to its protective role, is also an active and dynamic component of the Central Nervous System, allowing for these promising drugs to circumvent the Blood Brain Barrier, and consequently, directly access the brain.

Dissertação de Mestrado em Investigação Biomédica apresentada à Faculdade de Medicina
A doença de Machado-Joseph (DMJ), também conhecida como ataxia espinocerebelosa do tipo 3 é uma doença neurodegenerativa caracterizada por uma expansão anormal do tripleto CAG na região codificante do gene MJD1/ATXN3, o que se traduz numa expansão de uma cadeia de poliglutaminas na proteína ataxina-3. Esta expansão de CAGs anormalmente longa confere toxicidade à proteína ataxina-3 produzida que através de múltiplos mecanismos patogénicos culminando em neurodegenerescência em diversas regiões do cérebro.As abordagens terapêuticas atuais consistem principalmente em sessões de fisioterapia e no alívio farmacológico sintomático de sintomas específicos, constituindo a força motriz para o desenvolvimento de novas abordagens terapêuticas.Os diversos modelos existentes de DMJ têm sido ferramentas indispensáveis para a identificação dos mecanismos intrínsecos da doença, bem como para a validação de novas terapias. No entanto, modelos transgénicos de murganho são altamente dispendiosos, necessitam de longos períodos para o desenvolvimento de fenótipo, não recapitulando algumas das características desta doença. O nosso grupo foi pioneiro no desenvolvimento de um modelo roedor da DMJ com base em vetores lentivirais. Apesar das inúmeras vantagens deste modelo, este envolve intervenção cirúrgica (craniotomia), injeção localizada no parênquima cerebral, estando a patologia confinada ao local de injeção. Estas evidências demonstram a urgência no desenvolvimento de novos modelos que expressem ataxina-3 mutante de forma ubíqua no Sistema Nervoso Central (SNC), providenciando novas perspetivas relacionadas com os mecanismos da doença, permitindo ainda a avaliação do potencial de novas terapias.O rápido desenvolvimento de ferramentas baseadas em Vírus Adeno-Associados (AAV), tornou-se um dos mais promissores sistemas de entrega de genes a uma grande variedade de tipos celulares, através de diferentes vias de administração.O objetivo do presente trabalho foi o desenvolvimento de novas estratégias baseadas no uso de vetores mosaico de AAV para gerar modelos in vitro e in vivo de DMJ. Para tal, foram desenvolvidos vetores mosaico AAV1/2 e AAV2/9 codificando para a ataxina-3 humana mutada direcionados para o SNC. Esta abordagem providenciou o desenvolvimento de modelos de DMJ com elevada relevância fisiológica em tempo-útil e com uma boa relação custo-benefício, ultrapassando algumas das limitações mencionadas anteriormente.Resumidamente, este estudo fornece forte evidências que os vetores AAVs gerados são capazes de transduzir o SNC após injeção intracraniana (AAV1/2) ou intravenosa (AAV2/9), sobre expressando a ataxina-3 mutante completa não só em modelos in vitro como também in vivo, recapitulando algumas das principais características da DMJ.
Machado-Joseph Disease (MJD) or Spinocerebellar Ataxia type 3 (SCA3) is a neurodegenerative disorder characterized by an abnormal expansion of the CAG triplet in the coding region of MJD-1/ATXN3 gene, translating into an expanded polyglutamine tract within the ataxin-3 protein. This abnormally long CAG expansion, confers a toxic gain of function to the ataxin-3 protein that through multiple pathogenic mechanisms leads to neurodegeneration in several brain regions. Current therapeutic approaches consist mainly in the use of physiotherapy and in the pharmacological alleviation of specific symptoms, thus encouraging further investigation towards possible therapeutic approaches.The several existing models of MJD have been useful tools that largely contributed to the identification of intrinsic pathways affecting the disease as well as the validation of new therapies. However, transgenic mouse models are expensive, take long periods of time to develop a phenotype, or do not recapitulate some of the hallmarks of this disease. Our group was pioneer in developing cost-effective lentiviral-based rodent models of MJD. Despite the numerous advantages of this model, it involves craniotomy, in situ injection in the brain parenchyma and the pathology is only confined to the local of injection. In light of these evidences, there is an urgent need for new mouse models that widely express mutant ataxin-3 throughout the Central Nervous System (CNS), potentially providing new insights into the disease mechanisms and allowing screening of novel therapies.The rapidly expanding Adeno-Associated Virus (AAV) vector toolkit has become one of the most promising viral vectors delivering genetic cargo to a wide range of cell types through different routes of administration. In the present work, we aimed to develop new strategies based on the use of mosaic rAAV vectors, to generate in vitro and in vivo models of MJD. For that purpose, mosaic vectors AAV1/2, and AAV2/9 encoding the mutant human ataxin-3 have been developed to efficiently target and transduce the CNS. This approach provided physiologically relevant, time-effective, and cost-effective models for MJD, circumventing some of the limitations of above-mentioned models.In summary, this study provides compelling evidence that the generated mosaic rAAVs are able to efficiently transduce the CNS upon intracranial (AAV1/2) or intravenous injection (AAV2/9), and overexpress full-length mutant ataxin-3 both in vitro and in vivo, recapitulating some of the hallmarks of MJD.
FCT
Outro - American Portuguese Biomedical Research Fund (APBRF)
Outro - BrainHealth2020 (CENTRO-01-0145-FEDER-000008)
Outro - CortaCAGs (POCI-01-0145-FEDER-016719)
Outro - H2020 da União Europeia, GA No. 643417
Outro - “National Ataxia Foundation” (USA)
Outro - POCI-01-0145-FEDER-007440
Outro - Richard Chin and Lily Lock Machado Joseph Disease Research Fund
Outro - ViraVector (CENTRO-01-0145-FEDER- 022095)

Progressive degeneration in the central nervous system (CNS) of Huntington’s disease (HD) patients is a relentless debilitating process, resulting from the inheritance of a single gene mutation. With limited knowledge of the underlying pathological molecular mechanisms, pharmaceutical intervention has to-date not provided any effective clinical treatment strategies to attenuate or compensate the neuronal cell death. Attention has therefore turned to biotherapeutic molecules and novel treatment approaches to promote restoration and protection of selectively vulnerable populations of neurons in the HD brain. Rapid advances in vectorology and gene-based medicine over the past decade have opened the way for safe and efficient delivery of biotherapeutics to the CNS. With numerous factors known to regulate the development, plasticity and maintenance of the mammalian nervous system many proteins have emerged as potential therapeutic agents to alleviate HD progression. This investigative study utilised gene delivery vectors derived from the non-pathogenic adeno-associated virus (AAV) to direct high-level expression of brain-derived neurotrophic factor (BDNF), glial cell-line derived neurotrophic factor (GDNF), Bcl-xL or X-linked inhibitor of apoptosis protein (XIAP) within the rodent striatum. Maintenance of the basal ganglia and functional behaviour deficits were assessed following excitotoxic insult of the striatum by quinolinic acid (QA), a neurotoxic model of HD pathology. Enhanced striatal expression of BDNF prior to QA-induced lesioning provided maintenance of the striosome-matrix organisation of the striatum, attenuating impairments of sensorimotor behaviour with a 36-38% increase in the maintenance of DARPP-32 / krox-24 expressing striatal neurons, reduced striatal atrophy and increased maintenance of striatonigral projections. Higher levels of BDNF however induced seizures and weight-loss highlighting the need to provide regulatable control over biotherapeutic protein expression. Continuous high-expression of BDNF or GDNF resulted in a downregulation of intracellular signal mediating proteins including DARPP-32, with AAV-GDNF not found to enhance the overall maintenance of striatal neurons. Neither of the anti-apoptotic factors provided significant protection of transduced striatal neurons but tended towards ameliorating QA-induced behavioural deficits, displaying behaviour – pathology correlations with the survival of parvalbumin-expressing neurons in the globus pallidus. The results of this thesis suggest BDNF as a promising putative biotherapeutic for HD, but emphasises the requirement to control expression following gene delivery, and for further elucidation of the physiological impact that enhanced expression of endogenous factors has on the host cells. Additionally the maintenance of neural networks beyond the caudate-putamen will be vital to ensuring efficient clinical outcomes for HD.
Auckland Medical Research Foundation. Foundation for Research, Science and Technology. The University of Auckland.