Nerve growth factor (NGF) in the experimental autoimmune Encephalomyelitis

ABSTRACT BREVE(in inglese) Multiple sclerosis (MS) stands out among neurological diseases by virtue of its frequency, chronicity, and tendency to attack young adults. The pathological hallmark of MS is the presenceof multiple lesions (plaques) characterized by various degrees of inflammation, demyelination, and gliosis in the central nervous system (CNS). Experimental allergic encephalomyelitis (EAE) has been extensevely studied and widely used as animal model of MS. The cerebrospinal fluid (CSF) of patients affected by MS and rodents with EAE show elevated levels of Nerve Growth Factor (NGF). This increase correlates with the course of the disease and show a maximum in corrispondence of the pick of the pathology. NGF is the best characterized member of a family of neurotrophic factors and is crucial for development of the CNS, promoting neuronal repair in a number of CNS insults. Moreover, during EAE, an upregulation of the levels of both the protein and the mRNA for NGF has been observed in the CNS tissues that produce NGF under normal conditions. There are several lines of evidence implicating a disorder of the immune system is implicated in the pathogenesis of MS. Since NGF is involved also in the functional regulation of immune cells and in the modulation of the inflammatory response, a role of NGF in EAE and MS autoimmune-inflammation can be hypothesized. Numerous immunosuppressive or immunomodulating agents have been tried even if only few have yielded positive and not long- lasting effects. Stem cells based therapy is of major interest for neurodegenerative disease’s therapy since adult CNS preserve some germinative areas also in the adulthood, in particular in the Subventricular Zone (SVZ) around the lateral ventricles. Stem cells have the potential to differentiate in a number of cell types and could be relevant in order to replace lost or damaged cells in the adult CNS. There is evidence suggesting that NGF could affect stem cells behaviour and improve stem cell repair after EAE induction. In fact, neural stem cells of the SVZ express both p75 and TrkA NGF receptors during EAE. Moreover, it has been shown that the uptake of ventricularly injected radiolabelled-NGF by SVZ cells causes migration of these cells toward the adjacent brain parenchima after EAE is induced. The major aim of my thesis was to investigate the role of neural stem cells of NGF on the SVZ during EAE. The results of my study, support the idea that NGF stimulates neural stem cells in the adulthood and prospect a potential therapeutic relevance of NGF in the autoimmune-demyelination of MS. Key Words (in inglese): Multiple Sclerosis, EAE, NGF, Inflammation, Demyelination, Stem Cells. Tissue Repair ABSTRACT BREVE(in italiano) La Sclerosi Multipla (SM) è una malattia neurologica che si distingue per la sua frequenza, cronicità e tendenza a colpire giovani adulti. La caratteristica patologica della SM è data dalla presenza di lesioni multiple (placche) nel sistema nervoso centrale (SNC) con vario grado di infiammazione, demielinizzazione e gliosi. Il più diffuso modello sperimentale per lo studio della SM è l’Encefalite Allergica Sperimentale (EAS). Il fluido cerebrospinale (FCS) di pazienti affetti da SM e di roditori con EAS presenta elevati livelli di fattore di crescita nervoso (NGF), in corrispondenza del picco massimo di manifestazione della malattia. L’NGF è il capostipite della famiglia delle neurotrofine ed è di cruciale importanza per lo sviluppo del SNC, nonché nei fenomeni di riparo tissutale a seguito di danno neuronale di varia origine e causa. Per di più nei tessuti cerebrali, dove l’NGF è prodotto in condizioni normali, si osserva un incremento sia della proteina che dell’mRNA per NGF durante l’EAS. Numerose lavori sperimentali sembrano indicare come causa della SM una disregolazione del sistema immunitario e l’NGF noto per regolare le cellule immmunitarie come pure le risposte infiammatorie. Tali osservazioni supportano l’idea che l’NGF rivesta un ruolo importante nell’infiammazione autoimmune di EAS e SM. Diversi agenti immunosoppressivi o immunomodulatori sono stati usati nella terapia clinica della SM, ma con pochi effetti positivi, peraltro limitati nel tempo. La terapia basata sull’uso di cellule staminali è di grande interesse al momento per la terapia delle mlattie neurodegenerative. Il SNC conserva alcune aree germinative contenenti cellule staminali anche nell’adulto, come ad esempio la zona subventricolare (SVZ) lungo i ventricoli laterali. Le cellule staminali hanno la potenzialità di differenziare in un gran numero di tipi cellulari diversi e si prospettano rilevanto ai fini del rimpiazzo delle cellue danneggiate o morte nel SNC. Un numero crescente di osservazioni avvalora l’ipotesi che l’NGF possa agire su tali cellule a seguito dell’induzione dell’EAS. Infatti durante l’EAE le cellule della SVZ esprimono entrambi i recettori dell’NGF, p75 e TrkA. Inoltre, se iniettato nel ventricolo, l’NGF viene captato dalle cellule della SVZ che sono recettive all’NGF e che acquisiscono così la capacità di migrare dal ventricolo verso il parenchima adiacente. L’ obiettivo della mia tesi è stato quello di studiare il ruolo dell’NGF sull’SVZ durante l’EAS. I risultati dei miei studi indicano che l’NGF è in grado di stimolare le cellule staminali cerebrali adulte e prospettano un notevole potenziale terapeutico dell’NGF nei processi autoimmunitari – demielinizzanti della SM. Key Words (in italiano): Sclerosi Multipla, EAS, NGF, Infiammazione, Demielinizzazione, Cellule Staminali, Riparo Tissutale ABSTRACT DETTAGLIATO(in inglese) The human disease multiple sclerosis (MS) is an autoimmune-based neuroinflammatory condition of the Central Nervous System (CNS), in which an attack is driven by the immune system against its own myelin. In fact, once blood brain barrier is destructed, self-reactive lymphocytes reach the brain and spinal cord initiating an inflammatory autoimmune response to myelin, the major component of oligodendrocytes (OL). OL are the axon enveloping cells of the CNS and, when damaged, the integrity and the electrical activity of naked axons are lost. Given the complexity of this multitrait disease, the immunomodulatory and anti-inflammatory drugs used in MS therapy have a limited beneficial effect and further information about autoimmune and neurodegenerative events occurring in MS is needed. NGF is an important neurotrophin, known to be involved in autoimmune response, to orchestrate the three major compartments of the body, namely, the nervous, endocrine and immune systems, and, finally, to regulate survival and differentiation of CNS glial cells and neurons under normal and pathological conditions. For all these reasons, NGF is a biological molecule relevant to MS pathology. In order to study MS, we used Experimental Allergic Encephalomyelitis (eae), the animal model resembling the human disease. We induced EAE in the Lewis rodent strain, which is more susceptible than other to develop an autoimmune response. In the first part of this study, EAE rats and eae rats with high circulating levels of anti-Nerve Growth Factor (ngf) antibodies (NGF-deprived EAE rats) were daily monitored for clinical signs of eae paralysis and relapses attacks in the chronic phase. blood, spinal cord and brain stem were used for histological examination and for neurotophin levels measurement, in particular ngf and Brain Derived Neurotrophic Factor (bdnf) evaluation, eighty-five days after eae induction (chronic stage of EAE). the results showed that low cerebral levels of NGF were found in ngf-deprived EAE rats as expected, and that Ngf deprivation caused a severe worsening of the clinical signs of the disease. these effects were not associated to changes in the amount of bdnf in the regions examined, but the spinal cord where BDNF decreased. in this study we have demonstrated that NGF levels parallel acute and chronic inflammation and that its presence in the CNS is relevant to the reduction of inflammatory cells number and to the shutdown of the CNS inflammatory response in ongoing EAE. In the second part of the study, we evaluated whether progenitor cells of the Subventricular Zone (SVZ) of the brain are affected by NGF-deprivation during chronic EAE. Neuroepithelial cells lying in the SVZ of both rodent and human have been demonstrated to be pluripotent cells forming neurospheres and differentiating both in glial and neuronal cells in vitro. These cells are known to express both p75 and TrkA NGF-receptors in Lewis rats after EAe induction and to incorporate NGF following NGF injection in the ventricle. Thus, we studied NGF effect on oligodendrocytes progenitors (OPC) proliferation and SVZ cells migration in EAE and we found that NGF-deprivation lead to an accumulation of migrating cells in the SVZ, and at the same time OPC diminish in number. Since EAE is a neuroinflammatory disorder characterized by cells damage and loss, the possibility that NGF can exert a protective/reparative in EAE, affecting progenitor survival and migration cannot be excluded. In the third part of the study, we studied spinal cord responsiveness to NGF in EAE rats both in the acute and chronic phase EAE. We found that spinal motor neurons of the IX lamina express basal levels of trkA and that TrkA strongly decreased and sometimes almost disappeared in these neurons following EAE induction. Moreover, this region was covered with more numerous astroglial cells with much stronger TrkA immunoreactivity than in controls. In the chronic EAE group it was found that TrkA immunoreactivity in motoneurons returned almost to the basal levels, suggesting the transient nature of the TrkA-immunoreactivity decline seen in acute EAE. No observable shrinkage of motoneurons was seen at the chronic stage of EAE. In the fourth part of the study, we showed that NGF affects both BrdU incorporation and neuronal commitment of progenitors cells in all the adult brain germinal zones examined also in acute EAE. In fact, NGF intracerebroventricular (icv) injected in the brain of rats with ongoing EAE, increases BrdU incorporation in SVZ, dentate gyrus, periventricular white matter and olfactory bulbs, whereas NGF deprivation had an opposite effect on BrdU incorporation in all these regions, compared to control rats. Moreover, we observed that TrkA-expressing migrating cells (NCAM/trkA double immunopositive cells) colonized the SVZ and the adjacent parenchyma. NCAM positivity was also found in some bipolar shaped cells of the adjacent white matter with a typical migratory morphology. Moreover, NGF icv administration in EAE rats stimulates the expression of early neuronal markers on proliferating precursors of the SVZ. The data obtained demonstrated that NGF and its antibody affect TrkA expression, bromodeoxyuridine (BrdU) incorporation and neuronal commitment of SVZ cells in acute EAE. Overall, the results of these studies indicate that NGF is a key factor influencing the course of EAE pathology and improving recovery response, because of the relevance of NGF in the down-regulation of inflammation-autoimmunity and in promoting the CNS reparative events in response to EAE and, probably, also to MS.