Дисертації з теми "Neurotrophic peptide"

Trophic factors have shown great promise in their potential to treat neurological disease. In particular, glial cell line-derived neurotrophic factor (GDNF) has been identified as a potent neurotrophic factor for midbrain dopamine (DA) neurons in the substantia nigra (SN), which lose function in Parkinson’s disease (PD). GDNF progressed to phase II clinical trials, which did not meet proposed endpoints. The large size and binding characteristics of GDNF have been suspected to contribute to some of the shortcomings of GDNF related to delivery to target brain regions. Smaller peptides derived from GDNF (Dopamine-Neuron Stimulating Peptides – DNSPs) have been recently investigated and appear to demonstrate trophic-like effects comparable to GDNF. In the described studies, a time course study was conducted to determine in vivo DA-release characteristics 1-, 2- and 4- weeks after peptide treatment. These studies determined the effects on DA terminals within striatal sub-regions using microelectrodes. A heterogeneous effect on striatal sub-regions was apparent with the maximum effect in the dorsal striatum – corresponding to terminals originating from the SN. Dysregulation of GDNF or GDNF signaling is believed to contribute to motor dysfunction in aging and PD. Thus, it is hypothesized that GDNF is necessary for the maintenance and function of neurons. To extend this line of investigation, in vivo functional measures (DA-release and -uptake) and behavioral and cellular alterations were investigated in a transgenic mouse model (Gdnf+/-) with reduced GDNF protein levels. The described studies determined that both DA-uptake and -release properties were altered in middle-aged Gdnf+/- mice with only modest reductions in DA neurochemical levels. GDNF levels in Gdnf+/- mice were restored to levels comparable to wild-type (WT) counterparts by treatment with GDNF. GDNF protein supplementation led to enhanced motor behavior and increased markers for DA neurons in the SN of Gdnf+/- mice. Gdnf+/- mice appeared to show a heightened sensitivity to GDNF treatment compared to WT counterparts. Overall, this body of work examines novel synthetic peptides with potential to enhance DA-neuron function and expands upon the current understanding of GDNF’s role in the nigrostriatal pathway.

L'objectif principal de cette thèse est d'étudier l'effet neuroprotecteur des plasmalogènes (Pls) et d'explorer le potentiel des nanoparticules lipidiques contre les maladies neurodégénératives. Notre stratégie vise à créer un système auto-assemblé, augmentant l'efficacité des plasmalogènes et d'un neuropeptide, le polypeptide activateur de l'adénylate cyclase hypophysaire (PACAP), pour la neuroprotection. Pls, un groupe distinctif de glycérophospholipides membranaires, contiennent généralement une chaîne d'acyle gras polyinsaturé en position sn-2 et une chaîne alkyle liée par une liaison éther-vinyle en position sn-1 du squelette glycérol. La correction du déclin des niveaux de plasmalogènes chez les personnes âgées offre des perspectives pour les thérapies liées à la maladie de Parkinson, à la maladie d'Alzheimer et à la démence. Nous résumons les progrès des nanoparticules lipidiques (LNPs) dans le ciblage de multiples mécanismes de neurodégénérescence. Notre recherche sur les LNPs chargées en plasmalogène explore leur impact in vitro/in vivo sur des modèles de neurodégénérescence. Notre étude montre la faisabilité d'améliorer l'efficacité du Pls avec les LNPs. Nous utilisons des plasmalogènes naturels pour créer des nanoformulations impliquant un excipient lipidique nonlamellaire (monooléine, divers agents tensioactifs et de petites quantités de vitamine E, curcumine ou coenzyme Q10. En utilisant la méthode SAXS, nous avons identifié des caractéristiques structurelles des LNPs (vésicules, cubosomes et hexosomes). Les évaluations in vitro utilisent des cellules SH-SY5Y, différenciées avec 10 µM d'acide rétinoïque pendant 5 jours. Les tests de viabilité cellulaire indiquent une absente de toxicité à une concentration totale en lipides de 10 µM pour une incubation de 24 heures. Nous avons étudié l'impact des nanoparticules chargées en Pls sur les cellules neuronales en utilisant la neurotoxine 6-OHDA comme modèle in vitro de la maladie de Parkinson. Nous explorons les mécanismes de dommages cellulaires (stress oxydatif et enzymes apoptotiques), identifiant la voie de signalisation ERK-Akt-CREB-BDNF. Cela suggère la nécessité d'adopter plusieurs stratégies dans le traitement des maladies neurodégénératives. Plusieurs composés neuroprotecteurs documentés ont été utilisés pour démontrer la capacité à restaurer les lésions neuronales causées par le 6-OHDA, offrant un modèle de conditions neurodégénératives pour élucider davantage les effets bénéfiques des Pls. Nous nous concentrons ensuite sur la protéine de liaison à l'élément de réponse au cAMP (CREB) et sa phosphorylation conduisant à l'expression des neurotrophines, cruciale pour prévenir les troubles neurologiques. À travers des nano-assemblages lipidiques-peptiques, nous avons étudié l'impact des différentes organisations structurelles des LNPs sur la phosphorylation de CREB dans un modèle in vitro de la maladie de Parkinson. Dans un modèle murin de la maladie de Parkinson, les LNPs de structure vésiculaire et hexosomale ont démontré une efficacité distincte dans la restauration de la fonction motrice. L'intervention intranasale a influencé la régulation génétique liée à la maladie de Parkinson et rééquilibré les profils lipidiques. L'administration nasale de LNPs chargées en Pls a amélioré les symptômes comportementaux de la maladie et a régulé à la baisse des gènes tels que IL33 et Tnfa. Nos résultats indiquent l'impact significatif des nanoformulations hexosomales sur l'atténuation de la maladie, le métabolisme lipidique et les modifications génétiques réactives potentiellement impliquées dans la régénération
The primary aim of this thesis is to investigate the neuroprotective effect of plasmalogens (Pls) and explore the potential of lipid nanoparticles against neurodegenerative diseases. Our strategy aims to create a self-assembled system, enhancing the efficacy of plasmalogens and the neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) for neuroprotection. The Pls, a distinctive group of membrane glycerophospholipids, typically contain a polyunsaturated fatty acyl chain at the sn-2 position and an alkyl chain linked by a vinyl-ether bond at the sn-1 position of the glycerol backbone. Pls, with their unique structure featuring a vinyl ether bond, possess free radical scavenging capabilities and antioxidant properties. Addressing the decline in plasmalogen levels in aging individuals holds promise for therapies related to Parkinson's disease, Alzheimer's disease, and dementia. Recent research has expanded our understanding of their antioxidant effects, anti-inflammation, and their involvement in ferroptosis. However, challenges persist in implementing plasmalogens in treatments of neurodegenerative diseases and in developing suitable drug delivery systems. We summarize the progress in lipid nanoparticles (LNPs) for targeting multiple neurodegeneration mechanisms. Our research on plasmalogen-loaded LNPs explores their fabrication mechanism and in vitro/in vivo impacts on neurodegenerative models. Our study shows the feasibility of enhancing Pls efficacy using LNPs as carriers. We employ natural plasmalogens from scallops to create nanoformulations involving a non-lamellar lipid excipient (MO) for structural stabilization, various surfactants, and small amounts of vitamin E, curcumin, or coenzyme Q10. Using small-angle X-ray scattering (SAXS), we identified the structural features of various LNPs (vesicles, cubosomes, and hexosomes). Our in vitro evaluations utilized human neuroblastoma SH-SY5Y cells, differentiated with 10 µM retinoic acid for 5 days. Cell viability tests indicated non-toxicity of the LNPs at a total lipid concentration of 10 µM for 24-hour incubation. We study the impact of Pls nanoparticles on an in vitro model of Parkinson's disease using neuronal cells induced by the neurotoxin 6-OHDA. Using the SH-SY5Y cell line, we explore cellular damage mechanisms (oxidative stress and apoptotic enzymes) via identifying the impact on the ERK-Akt-CREB-BDNF signaling pathway. Several documented neuroprotective compounds were used to demonstrate the ability to restore neuronal lesions caused by 6-OHDA, offering a model of neurodegenerative conditions to further elucidate the beneficial effects of the Pls-based LNPs. We then focus on the cAMP response element binding protein (CREB) and its phosphorylation leading to neurotrophin expression, crucial in preventing neurological disorders. Through lipid peptide nano-assemblies, we studied the impact of different structural organizations of the LNPs on CREB phosphorylation in an in vitro model of Parkinson's disease. Notably, liquid crystalline lipid nanoparticles loaded with plasmalogens prolonged CREB activation under neurodegenerative conditions, showing potential for enhanced neuroregeneration through sustained CREB activation in response to the neurotrophic nanoassemblies. In a mouse model of Parkinson's disease, vesicle and hexosome LNPs demonstrated distinct effectiveness in restoring motor function. The nanomedicine-mediated intervention influenced Parkinson's disease-related gene regulation and rebalanced lipid profiles. Nasal administration of Pls-loaded LNPs improved disease behavioral symptoms and downregulated genes like IL33 and Tnfa. The obtained results indicated the significant impact of hexosomal LNP nanomedicines on disease attenuation, lipid metabolism, and responsive gene modifications potentially involved in regeneration

Neuropeptides, such as substance P (SP) and bombesin/gastrin-releasing peptide (BN/GRP), and neurotrophins are involved in neuro-immunomodulatory processes and have marked trophic, growth-promoting and inflammation-modulating properties. The impact of these modulators in rheumatoid arthritis (RA) is, however, unclear. An involvement of the innervation, including the peptidergic innervation, is frequently proposed as an important factor for arthritic disease. Many patients with RA, but not all, benefit from treatment with anti-TNF medications. The studies presented here aimed to investigate the roles of neuropeptides, with an emphasis on BN/GRP and SP, and neurotrophins, especially with attention to brain-derived neurotrophic factor (BDNF), in human and murine knee joint tissue. The expression patterns of these substances and their receptors in synovial tissue from patients with either RA or osteoarthritis (OA) were studied in parallel with the levels of these factors in blood and synovial fluid from patients with RA and from healthy controls. Correlation studies were also performed comparing the levels of neuropeptides with those of pro-inflammatory cytokines [tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6)]. Furthermore, the impact of anti-TNF treatment on the levels of BDNF in blood was investigated. In a murine model of RA, the expression of these substances on articular chondrocytes along with their expression in synovial tissue was investigated. The expression of BN/GRP in human synovial tissue was confined to fibroblast-like and mononuclear-like cells whereas SP was detected in nerve-related structures. Receptors for these neuropeptides (GRP-R and NK-1R) were frequently present in blood vessel walls, and on fibroblast-like and mononuclear-like cells. The expression of BDNF and its receptors, p75 neurotrophin receptor and TrkB, was mainly confined to nerve structures. The levels of SP, and particularly those of BN/GRP, in synovial fluid and peripheral blood correlated with the levels of pro-inflammatory cytokines. There were clearly more correlations between SP-BN/GRP and inflammatory parameters than between BDNF and these factors. Plasma levels of BDNF were decreased following anti-TNF-treatment. In the joints of the murine model, there was a marked expression of neurotrophins, neurotrophin receptors and NK-1R/GRP-R in the articular chondrocytes. The expression was down-regulated in the arthritic animals. A neurotrophin system was found to develop in the inflammatory infiltrates of the synovium in the arthritic mice. The results presented suggest that there is a local, and not nerve-related, supply of BN/GRP in the human synovial tissue. Furthermore, BN/GRP and SP have marked effects in the synovial tissue of patients with RA, i.e., there were abundant receptor expressions, and these neuropeptides are, together with cytokines, likely to be involved in the neuro-immunomodulation that occurs in arthritis. The observations do on the whole suggest that the neuropeptides, rather than BDNF, are related to inflammatory processes in the human knee joint. A new effect of anti-TNF treatment; i.e., lowering plasma levels of BDNF, was observed. Severe arthritis, as in the murine model, lead to a decrease in the levels of neurotrophin, and neurotrophin and neuropeptide receptor expressions in the articular cartilage. This fact might be a drawback for the function of the chondrocytes. Certain differences between the expression patterns in the synovial tissue of the murine model and those of human arthritic synovial tissue were noted. It is obvious that local productions in the synovial tissue, nerve-related supply in this tissue and productions in chondrocytes to different extents occur for the investigated substances.

La maladie d'Alzheimer (MA) est une pathologie neurodégénérative caractérisée par la présence de plaques séniles majoritairement composées par la protéine β-amyloïde (Aβ). Afin de caractériser les effets de la toxicité amyloïde, nous avons évalué l'impact au cours du temps d'une injection intracérébroventriculaire (icv) du peptide Aβ25-35 agrégé sur des paramètres comportementaux, physiologiques et biochimiques chez le rat et sur un système neuroprotecteur endogène, le BDNF. Nous avons caractérisé 1, 2, 3 et 6 sem après l'injection icv d'Aβ25-35, les effets sur la mémoire à court- et long-terme, sur les niveaux dans l'amygdale, le cortex frontal, l'hippocampe et l'hypothalamus du stress oxydant, des processus apoptotiques et du BDNF ainsi que ces récepteurs (TrkB et p75). Chez ces animaux, des études immunohistologiques sont également réalisées sur le système BDNF, la neuroinflammation, la neurogénèse et la perte cellulaire hippocampique. Cette étude montre que l'injection d'Aβ25-35 induit des déficits mnésiques, un stress oxydant, de la mitochondrie et du réticulum endoplasmique et des processus apoptotiques. L'Aβ25-35 a un impact sur le système cholinergique, l'intégrité hippocampique, la neurogénèse et la neuroinflammation. Les taux de corticostérone et le système BDNF sont également modifiés. L'injection icv d'Aβ25-35 induit les signes neuropathologiques majeurs de la MA chez le rat et valide ce modèle d'injection comme un bon modèle non-transgénique de la MA. De plus, il semble qu'une partie des effets observés pourraient être le résultat d'une dérégulation du système BDNF dans certaines régions du cerveau
Alzheimer's disease is a neurodegenerative pathology characterized by the presence of senile plaques. The major component of senile plaques is an amyloid-ß protein (Aβ). In this study, we assessed the time-course effects and regional changes observed after a single intracerebroventricular (icv) injection of aggregated Aβ fragment [25-35] (Aβ25-35; 10 µg/rat), on physiological parameters (body weight, general activity and body temperature), behavioral responses (spatial short- and long-term memories), stress parameters (BDNF and CORT levels, oxidative, inflammation, neuroprotection, cellular) and on histological parameters (neuroinflammation, acetylcholine systems, hippocampus integrity, BDNF system). We shown that a single icv injection of Aβ25-35 has a significant impact on short- and long-term memories, HPA axis activity, oxidative stress, brain level of a neuroprotective agent (BDNF) and its receptors (TrkB and p75), ER and mitochondrial stress, apoptotic processes, astrogliosis and microgliosis, cholinergic systems, hippocampus integrity and hippocampal neurogenesis. This study allows to realize the parallel existing between the effects induced by Aβ25-35 icv injection and numerous relevant signs of the pathology observed in patients. It seems that effects observed could be due to differential regulation of BDNF system on cerebral regions

BDNF/TrkB são descritos em diversas neoplasias onde iniciam sinais mitogênicos, facilitam o crescimento tumoral, previnem apoptose e regulam angiogênese e metástase. Outros fatores de crescimento também são importantes para tumorigênese, como GRP/GRPR e EGF/EGFR. O objetivo geral deste trabalho foi investigar o papel de BDNF/TrkB em câncer colorretal avaliando possíveis interações com GRPR e EGFR. Verificamos que BDNF e seu receptor, TrkB, estão presentes em amostras de pacientes com câncer colorretal esporádico, e os níveis de BDNF encontram-se mais elevados no tecido neoplásico que no tecido adjacente ao tumor. O tratamento com RC- 3095, um antagonista de GRPR, na linhagem celular de câncer colorretal humana, HT-29, causa diminuição nos níveis de NGF secretados pelas células e aumento de BDNF em relação ao controle não tratado. RC-3095 inibe a proliferação e viabilidade celular das linhagens HT-29 (EGFR positiva) e SW-620 (EGFR negativa), embora apenas em HT-29 ocorra um aumento significativo na expressão de mRNA de BDNF. Por isso, um anticorpo monoclonal anti-EGFR, cetuximabe, foi combinado a RC-3095, nas células HT-29, sendo capaz de prevenir tal aumento, sugerindo que este efeito seja mediado por EGFR. Os tratamentos com um inibidor de Trks, K252a (1000 nM) ou com cetuximabe (10 nM) também inibem a proliferação celular. Entretanto, a combinação de BDNF a cetuximabe previne este efeito, enquanto que a combinação de doses não efetivas de K252a (10 nM) à cetuximabe (1 nM) inibe a proliferação celular de HT- 29. Além disso, cetuximabe também causa aumento na expressão de mRNA de TrkB e BDNF, após 600 minutos de tratamento. Nossos resultados sugerem que a inibição da proliferação celular in vitro ou do crescimento tumoral in vivo devem acontecer através do bloqueio combinado entre GRPR e TrkB em células de câncer colorretal EGFR positivas, e que BDNF também esteja envolvido em mecanismos de resistência a fármacos. Por isso, o bloqueio de BDNF / TrkB pode emergir como potencial alvo antitumoral.
BDNF / TrkB are described in various cancers where they participate in tumor growth, apoptosis, angiogenesis and metastasis. Furthermore, other growth factors are also important to tumorigenesis as GRP/GRPR and EGF/EGFR. Therefore, the aim of this study was to investigate the role of BDNF/TrkB in colorectal cancer evaluating the interactions with GRPR and EGFR. We found that BDNF and its receptor, TrkB, are present in samples from patients diagnosed with sporadic colorectal cancer, and BDNF levels were higher in tumor tissue compared to adjacent tumor tissue. Treatment with RC-3095, GRPR antagonist, in human colorectal cancer cell line, HT-29 caused a decrease in NGF levels secreted by cells, and generated increase of BDNF when compared to untreated control. RC-3095 inhibited the proliferation and cell viability in HT-29 (EGFR positive) and SW-620 (EGFR negative), but only HT-29 cells showed a significant increase in BDNF mRNA expression. Therefore, a monoclonal anti-EGFR antibody, cetuximab was combined with RC-3095 in HT-29 cells, and was able to prevent such an increase, suggesting that this effect is mediated by EGFR. The treatment with a Trk inhibitor, K252a (1000 nM) or cetuximab (10 nM), inhibited cell proliferation. However, the combination of BDNF with cetuximab prevented this effect, whereas the combination of ineffective doses of K252a (10 nM) with cetuximab (1 nM) still inhibited cell proliferation of HT-29. Furthermore, cetuximab also caused an increase in BDNF and TrkB mRNA expression, 600 minutes after treatment. In summary, our results suggest that inhibition of cell proliferation in vitro or tumor growth in vivo must occur between the combination of GRPR and TrkB in EGFR positive colorectal cancer cells, and that BDNF is also involved in drug resistance mechanisms. Therefore, blockage of BDNF / TrkB may emerge as potential antitumor target.

Le pituitary adenylate cyclase-activating polypeptide (PACAP) est présent en quantités non négligeables dans le cervelet de rat au cours des trois semaines qui suivent la naissance c'est à dire pendant la période de multiplication, migration et différenciation des cellules granulaires, suggérant un rôle neurotrophique du PACAP dans le cervelet des mammifères au cours du développement. L'ontogénèse des sites de liaison du PACAP par autoradiographie quantitative dans le cervelet immature du rat a démontré une expression transitoire des sites de liaison au niveau de la couche granulaire externe (CGE) et de la médulla, depuis la naissance jusqu'au 25ème jour de vie postnatale. La caractérisation pharmacologique des sites de liaison présents dans la CGE a révélé qu'il s'agit de récepteurs PVR1 de haute affinité pour le PACAP et de faible affinité pour le VIP. Sur des cultures de neuroblastes cérébelleux, le PACAP stimule de façon dose-dépendante la production d'AMPc et d'inositols phosphates. En revanche, le VIP est 100 fois moins efficace sur l'activité adenylyl cyclasique et sans effet sur le métabolisme des phosphoïnositides. L'activation des récepteurs PVR1 au niveau des cellules granulaires induit une stimulation de la phospholipase C via une protéine G sensible à la toxine pertussique, indépendemment de son effet stimulateur sur le système adénylyl cyclasique. La présence du PACAP et de ses récepteurs dans le cervelet immature et l'activation de plusieurs systèmes de transduction suggèrent que le PACAP pourrait être impliqué dans l'histogénèse du cortex cérébelleux. L'administration de PACAP pendant 24 ou 48 heures sur des neuroblastes cérébelleux en culture provoque une augmentation de la survie cellulaire alors que le VIP est 1000 fois moins actif. Cet effet du PACAP est spécifiquement bloqué par l'antagoniste PACAP(6-38). De plus, le PACAP induit une augmentation du nombre et de la longueur des neurites. Ces données démontrent que, in vitro, le PACAP exerce un effet neuroprotecteur sur les cellules granulaires immatures, suggérant un rôle physiologique du PACAP dans le développement du cervelet de rat.

Orientador: Francesco Langone
Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia
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Resumo: O fator neurotrófico ciliar (CNTF) despertou grande interesse com a descoberta do seu efeito neuroprotetor sobre motoneurônios após secção de nervos periféricos em ratos neonatos e camundongos adultos. Contudo, seus efeitos colaterais de perda de peso e caquexia impedem seu uso clínico. Uma via alternativa muito promissora, no sentido de eliminar estes efeitos colaterais, parece ser a administração do CNTF conjugado com peptídeos que possuem domínio de translocação de proteínas (PTD-protein transduction domain). Previamente, mostramos que o CNTF conjugado a um PTD derivado da proteína tat do vírus HIV-1 (TAT-CNTF) também tem efeito protetor sobre motoneurônios e não produziu efeitos colaterais no tratamento, por 5 dias, de ratos neonatos (P2-P7) que sofreram secção do nervo isquiático. O presente trabalho teve como objetivo investigar se a administração da TAT-CNTF por tempos mais prolongados seria capaz de manter a sobrevivência de motoneurônios e neurônios sensoriais, bem como de estimular a regeneração axonal, sem a ocorrência dos efeitos colaterais do CNTF. Desta forma, ratos Wistar neonatos (P2) receberam tratamento subcutâneo com CNTF (1,2mg/g/dia) durante 5 dias, TATCNTF (1,2mg/g/dia) ou PBS durante 5 ou 15 dias. Ao final do período de tratamento os animais foram sacrificados por decapitação, sendo coletado para posterior análise o sangue e gorduras marrom interescapular, branca retroperitonial e branca epididimal. Um outro grupo de animais tive o nervo isquiático esquerdo esmagado em P2 e recebeu o mesmo tratamento. No período entre 20 e 60 dias de vida foi realizada a avaliação da recuperação funcional motora por meio de Walking Track Test, e da recuperação funcional sensitiva através da medida do limiar da resposta de retirada da pata a um estímulo elétrico. No 30º ou 60º dia de vida foi realizada marcação retrógrada de neurônios cujos axônios compõem o nervo isquiático utilizando-se Amina Dextrano Biotinilado (BDA). Após 7 dias, os animais foram sacrificados, sendo coletada a intumescência lombar, os gânglios sensoriais L4 e L5 e o nervo isquiático para análise histológica. As análises dos níveis plasmáticos de glicose, triglicérides e ácidos graxos revelaram que o tratamento com TAT-CNTF ou CNTF por igual período (5 dias) produziu modificações no metabolismo energético em relação aos ratos controle (PBS), contudo diferiram quanto à intensidade dos efeitos produzidos. O tratamento prolongado com TAT-CNTF (15 dias) produziu significantes alterações nas concentrações plasmáticas de triglicérides e de colesterol. Entretanto, o tratamento com TAT-CNTF (5 ou 15 dias) produziu efeitos reduzidos sobre os tecidos adiposos quando comparados ao tratamento com CNTF. Os ratos do grupo TAT-CNTF apresentaram peso corporal significativamente menor que aqueles do grupo PBS a partir do 9º dia de tratamento, porém o grupo CNTF apresentou menor peso a partir do 2º dia. Em ambos tratamentos houve reversão desse efeito sobre o peso corporal, porém esta ocorreu mais precocemente nos ratos do grupo TAT-CNTF (P25) quando comparados ao grupo CNTF (P32). A recuperação funcional motora e sensorial dos grupos CNTF e TAT-CNTF foi 50% superior ao grupo controle. Os grupos CNTF e TAT-CNTF também apresentaram maior número de neurônios sensoriais e motores BDA positivos, além de maior número de axônios mielínicos no nervo isquiático quando comparados ao grupo controle. Nossos resultados mostraram que o tratamento com TAT-CNTF, mesmo por períodos mais longos, promoveu a sobrevivência e regeneração axonal de motoneurônios e neurônios sensitivos sem a ocorrência dos efeitos colaterais produzidos pelo tratamento com CNTF. Além disso, tais propriedades da TAT-CNTF contribuíram significativamente para a recuperação funcional motora e sensorial após lesões nervosas periféricas.
Abstract: Ciliary neurotrophic factor (CNTF) is known as a neuroprotective agent on motoneurons after peripheral nerve section in neonatal rats and adult mice. However, side effects like weight loss and caquexia have limited its clinical use. A promising approach for the avoidance of such side effects is the fusion of a protein transduction domain (PTD) with CNTF. Previously we showed that CNTF fused with HIV-1 PTD (TAT-CNTF) also had protective effect on motoneurons and did not produce side effects in a 5 days treatment of sciatic nerve transected neonatal rats (P2-P7). The aim of the present work was investigate if the TAT-CNTF administration for long time was capable to support the motoneurons and sensory neurons survival, as well as to stimulate the axonal regeneration, without CNTF side effects. Thus, neonatal Wistar rats (P2) were subcutaneously treated with CNTF (1.2mg/g/day) for 5 days, TAT-CNTF (1.2mg/g/day) or PBS during 5 or 15 days. By the end of treatment rats were killed by decaptation and blood, intrascapular brown adipose tissue and retroperitonial and epididimal white adipose tissue were collected for further analysis. Another group of animals had the left sciatic nerve crushed (NCE) in P2 and received the same treatment. From 20 to 60 days of age a Walking Track Test was performed in order to evaluate the motor function recovery, and the threshold for paw withdraw was used as a measure of sensitive functional recovery. The retrograde labeling of sciatic nerve neurons using Biotinilated Dextran Amine (BDA) was performed at 30 or 60 days of age. Rats were killed after 7 days and the lumbar enlargement, L4 and L5 dorsal root ganglia and the sciatic nerve were collected for histological analysis. The analysis glucose, triglycerides and fatty acid plasmatic levels demonstrated that 5 days TAT-CNTF or CNTF treatment induced changes in energy metabolism compared to control rats, however the effects of these treatments had different intensities. The long term treatment with TAT-CNTF (15 days) induced important changes in triglycerides and cholesterol plasmatic levels. However TAT-CNTF treatment (5 or 15 days) had reduced effects on adipose tissue when compared to CNTF. After the 9th day of treatment the TAT-CNTF group had a smaller body weight when compared to the PBS group, on the other hand the CNTF group had a smaller body weight after the 2nd day compared to the PBS group. In both treatments (CNTF and TATCNTF) there was a reversion of the body weight effect, however this was earlier in the TAT-CNTF group (P25) than on the CNTF group (P32). The motor and sensorial functional recovery of CNTF and TAT-CNTF groups was 50% greater than the control group. CNTF and TAT-CNTF groups also displayed a greater number of BDA positive motor and sensory neurons, and more myelinic axons in the sciatic nerve compared to the control group. Our results demonstrate that TAT-CNTF long term treatment was able to promote the survival and axonal regeneration of motor and sensory neurons without important CNTF related side effects. Moreover, TAT-CNTF properties had significant contribution for the motor and sensory functional recovery after peripheral nerve lesion.
Doutorado
Biologia Celular
Doutor em Biologia Celular e Estrutural

Le développement neuronal est contrôlé par des facteurs épigénétiques de l'environnement cellulaire. Parmi les facteurs diffusibles, les neurotrophines agissent dans plusieurs régions centrales sur la différenciation morphologique et fonctionnelle des neurones post-mitotiques. Dans ce travail, nous nous sommes intéressés à l'effet du BDNF et de la NT3 sur la différenciation de sous populations neuronales hypothalamiques, et particulièrement celles impliquées dans les régulation neuroendocrin iennes. Nous avons caractérisé la différenciation phénotypique des neurones hypophysiotropes au cours du développement, dans un modèle de culture primaire de neurones provenant des noyaux arqué ou périventriculaire, par RT-PCR sur cellule unique. Dans le noyau arqué, on observe une restriction d'expression des peptides somatolibérine (GHRH), neuropeptide Y et somatostatine (SRIF). Dans le noyau. Arqué, cette restriction est mimée pour le GHRH et le SRIF par la NT3 alors que le BDNF n'a pas d'effet. Au contraire, dans le noyau périventriculaire, seul le BDNF induit une restriction d'expression du SRIF. Dans les deux stmctures, les deux neurotrophines accélèrent de façon identique la synaptogenèse. Des effets très sélectifs des neurotrophines ont été mis en évidence sur deux populations hypophysiotropes présentes dans ces noyaux. La différenciation morphologique et biochimique de neurones dopaminergiques est stimulée par le BDNF dans le noyau périventriculaire et par la NT dans le noyau arqué. Le développement des neurones somatostatinergiques n'est sensible qu’au BDNF, et ce essentiellement dans le noyau péri ventriculaire. Par ailleurs, nous avons montré qu'une protéine récemment découverte, le MCH gène overprinted polypeptide (MGOP), colocalisé dès la période périnatale avec la somatostatine noyau périventriculaire. In vitro, le MGOP module la libération de somatostatine et pourrait donc également influencer le développement des neurones à SRIF.

Tese de doutoramento, Ciências Biomédicas (Neurociências), Universidade de Lisboa, Faculdade de Medicina, 2014
Brain-derived neurotrophic factor (BDNF) and its high-affinity full-length receptor, TrkB-FL, play a central role in the nervous system by providing trophic support to neurons and by regulating synaptic transmission and plasticity. BDNF signalling is impaired in Alzheimer’s disease (AD), a neurodegenerative disorder characterized, among other features, by the accumulation of the amyloid-β (Aβ) peptide. Although the mechanisms implicated in the reduction of BDNF signalling in AD were not clarified, the reestablishment of BDNF actions is considered as a promising strategy for AD treatment. In last decade it became clear that most of synaptic actions of BDNF, including the ones upon synaptic transmission, plasticity or upon neurotransmitter release, are fully dependent on adenosine A2A receptors (A2AR) activation. However, evidences indicate that A2AR antagonists can prevent the deficits in AD animal models. Given the lack of data clarifying the mechanisms behind the changes on BDNF signalling, namely changes on TrkB receptors, and the knowledge that A2AR activation facilitates most of BDNF synaptic actions, the main goal of this project was to study the impact of Aβ peptides and A2AR on BDNF signalling. This work revealed that in rat primary neuronal cultures Aβ selectively increases mRNA levels for the truncated TrkB-T1 and TrkB-T2 isoforms without affecting TrkB fulllength (TrkB-FL) mRNA levels. Moreover, Aβ increases protein levels of total pool of truncated TrkB receptors (TrkB-Tc) and decreases TrkB-FL protein levels. This effect is explained by the Aβ-induced calpain-mediated cleavage on TrkB-FL receptors, downstream of Shc binding site, which results in the formation of a new truncated TrkB receptor (TrkB-T’) and a new intracellular fragment (TrkB-ICD), which is also detected in post-mortem human brain samples. In hippocampal slices it was observed that Aβ impairs BDNF function in a calpaindependent way, upon modulation of GABA and glutamate release from hippocampal nerve terminals, and upon modulation of long-term potentiation (LTP). Finally, the exogenous BDNF strongly reduces the Aβ-induced activation of caspase-3 and calpain in neuronal cultures, an effect not affected by A2AR agonist or antagonist. Moreover, for the first time it was shown that chronic in vivo blockade of A2AR by a selective antagonist, prevents the facilitatory action of BDNF upon ex-vivo CA1 hippocampal LTP and decreases both mRNA and protein levels of the TrkB-FL receptor in rat hippocampus. In conclusion, the present work shows that Aβ induces a TrkB-FL cleavage mediated by calpain and impairs BDNF-mediated effects in synaptic plasticity and neurotransmitter release in a calpain-dependent way. While the BDNF action upon synaptic plasticity is abolished under chronic in vivo A2AR blocking conditions, the protective actions of this neurotrophin against Aβ toxicity were found to be dependent on A2AR activation.
O factor neurotrófico derivado do cérebro (Brain-derived neurotrophic factor- BDNF) e o seu receptor de alta afinidade, TrkB-FL, desempenham um papel central no sistema nervoso, dado que promovem suporte trófico aos neurónios e que regulam a transmissão e plasticidade sinápticas. A sinalização mediada pelo BDNF encontra-se diminuída na doença de Alzheimer (Alzheimer’s disease -AD), uma doença neurodegenerativa na qual ocorre acumulação do péptido beta amilóide (amyloid-beta -Aβ). Apesar dos mecanismos envolvidos na redução da sinalização mediada pelo BDNF na AD não serem totalmente conhecidos, o restabelecimento das acções do BDNF tem sido considerado como uma estratégia promissora para a terapêutica desta doença. Na última década tornou-se claro que a maioria das acções sinápticas do BDNF, incluindo as acções na transmissão e plasticidade sinápticas e também na libertação de neurotransmissores, é dependente da activação dos receptores A2A da adenosina (A2AR). Contudo, o uso de antagonistas dos A2AR tem sido apontado como uma possível estratégia terapêutica para o tratamento da AD. Dada a falta de evidências que clarifiquem os mecanismos envolvidos nas alterações da sinalização mediada pelo BDNF e o conhecimento de que a activação dos A2AR facilita a maioria das acções sinápticas do BDNF, o objectivo principal desta tese foi estudar o impacto dos péptidos Aβ e dos A2AR na sinalização mediada pelo BDNF. Este trabalho revelou que, em culturas primárias de neurónios corticais, o Aβ aumenta os níveis de mRNA dos receptores TrkB truncados, TrkB-T1 e TrkB-T2, sem afectar os níveis de mRNA dos receptores TrkB completos, TrkB-FL. Por outro lado, verificou-se que o Aβ aumenta os níveis proteicos do conjunto de receptores TrkB truncados e que diminui os níveis proteicos dos receptores TrkB-FL, por um mecanismo independente da proliferação glial e da activação de caspases. Foi ainda possível concluir que o Aβ induz a clivagem, mediada por calpaínas, dos receptores TrkB-FL, esta clivagem dá-se após o local de ligação da Shc e antes do início do domínio de cinase de tirosina, pelo que origina um novo receptor TrkB truncado (TrkB-T’), contendo o local de ligação à Shc, e um novo fragmento intracelular (TrkBintracellular domain- ICD), contendo a totalidade do domínio da cinase. No entanto, a presença destes fragmentos, não mostrou afectar a fosforilação do receptor TrkB-FL induzida pela exposição ao BDNF. Interessantemente, foi possível detectar o fragmento TrkB-ICD em uma amostra, post-mortem, de cérebro humano. Mostrou-se também que a inibição das calpaínas previne as alterações dos níveis proteicos das isoformas do TrkB, induzidas pelo Aβ, sem afectar as alterações ao nível do mRNA do TrkB. Por outro lado, este trabalho revelou que o BDNF exógeno reduz a activação da caspase-3 e das calpaínas induzida pelo Aβ, de uma forma independentemente dos A2AR. Em fatias de hipocampo de ratos adultos, este trabalho mostrou que o Aβ diminui as acções do BDNF na plasticidade sináptica, nomeadamente na potenciação de longa duração (Long-term potentiation, LTP) na área CA1 do hipocampo, bem como no seu efeito sobre libertação de neurotransmissores (GABA e glutamato) de sinaptosomas. Notavelmente, o inibidor das calpaínas, MDL28170, mostrou restabelecer os efeitos do BDNF, na presença do péptido Aβ, tanto na plasticidade sináptica como na libertação de neurotransmissores. Este trabalho permitiu ainda concluir que o bloqueio crónico dos A2AR, in-vivo, através da administração de um antagonista selectivo (KW-6002), previne o efeito potenciador do BDNF na LTP, registada ex-vivo na área CA1 do hipocampo, e que diminui os níveis de mRNA e de proteína do receptor TrkB-FL, no hipocampo de rato. Em suma, o presente trabalho revelou que o péptido Aβ induz a clivagem dos receptores TrkB-FL, mediada pelas calpaínas, e que bloqueia as acções mediadas pelo BDNF na plasticidade sináptica e na libertação de GABA e glutamato por um mecanismo dependente da actividade das calpaínas. Se por um lado, o efeito do BDNF na plasticidade sináptica é perdido aquando da inibição crónica dos A2AR, o efeito protector desta neurotrofina contra a toxicidade induzida pelo Aβ mostrou-se independente da activação dos A2AR.
Fundação para a Ciência e a Tecnologia (FCT)

Teneurin C-terminal associated peptide-1 (TCAP-1) is a recently characterized peptide that may act as one potential neuroprotective agent as it has been shown to regulate several stress-associated behaviours in rodents and possesses a number of protective actions on cells, however the mechanism remains unknown. Brain-derived neurotrophic factor (BDNF) is a neurotrophin recognized for mediating survival, differentiation, and proliferation. TCAP-1 may act, in part, via BDNF to provide neuroprotection via modulation of BDNF expression. The aim of this research was to further investigate the mechanism of TCAP’s neuroprotective actions. I show that TCAP-1 is neuroprotective and a potent enhancer of cell numbers under varying levels of oxygen. I also establish that TCAP-1 is able to influence neuronal behaviour by differentially regulating neurite growth. In addition, I indicate that TCAP-1 is able to regulate BDNF expression in immortalized mouse hypothalamic N38 cells, which suggests that TCAP-1’s neuroprotective mechanism may involve BDNF.

Over the years, the Burgess group has been focusing on the preparation and testing of small molecules that mimic protein secondary structures for protein-protein interactions. The most successful compounds made are C10 peptide macrocycles that effectively mimic β-turns and have given promising results from biological testing. These peptide macrocycles have also been dimerized to give even more effective ligands for protein-protein interaction. The successes of the peptide macrocycles have enabled us to look into increasing the chemical diversity of our libraries. This we believe will not only improve our ability to obtain high affinity ligands for the receptors of interest, but will also allow us to investigate other receptors. To achieve this, peptoids were incorporated into the C10 system to replace the peptides in the i+1 and i+2 positions. With the help of Microwave irradiation, semi-peptoid macrocycles were synthesized with a total reaction time of less than 2 h. These compounds were characterized and found to mimic β-turn, and show promising biological activity towards the Insulin-like growth factor 1 receptor (IGF-IR).