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Kyotorphin (KTP, L-tyrosyl-L-arginine) is an endogenous dipeptide, described for the first time in 1979, as a potent analgesic molecule. Its naloxone-reversible opioid-like analgesic effect is indirectly mediated by the inducing the release of Met-enkephalin (Met-enk). It is currently accepted that KTP acts through a specific Gi-coupled receptor (KTPr), inducing Ca2+ influx in a phospholipase C-mediated process. Moreover, this KTPr-mediated action can be antagonized by the dipeptide L-Leucine-L-Arginine (KTPant). Over the last decades, several studies have been revealing KTP role in the modulation of several mechanisms in the central and peripheric nervous systems. KTP has been described as having an antiepileptic, thermoregulatory, anti-hibernation, behavioral and stress modulatory actions, being these non-opioid-mediated effects. However, the vast majority of KTP research has been explored its potential application in pain treatment. More recently, the link between pain and Alzheimer’s Disease (AD), two conditions with high epidemiologic relevance, supported the use of KTP-related drugs as new therapeutic strategies for AD. Evidence shows that chronic pain aggravates AD, and the limited capacity of AD patients to verbally express and perceive pain can worsen disease progression. AD has proven to be a highly complex neurodegenerative disease, of which brain amyloid plaques, mainly constituted by amyloid beta (Aβ) peptide, and neurofibrillary tangles formed by hyperphosphorylated tau (p-Tau) protein, are the two histopathological hallmarks of this disease. Additionally, excitotoxicity, as well as dysregulation of brain-derived neurotrophic factor (BDNF) signaling, are known to be involved in neurodegenerative disorders such as AD. Similarly to what was observed in patients with persistent pain, AD patients have decreased cerebrospinal fluid (CSF) KTP levels, which are inversely correlated to the increase of p-Tau levels in CSF of those patients. Recently, KTP was suggested to be an endogenous neuroprotective agent. In particular, when intracerebroventricular (i.c.v.) injected, KTP ameliorated memory impairments in a rat model of sporadic AD. However, KTP has a limited capacity to cross the blood-brain barrier (BBB). The potential therapeutic value of KTP as a central nervous system (CNS) drug led to the development of synthetic KTP derivatives, which might cross the BBB. Accordingly, the KTP amidated-derivative, the Amidated-Kyotorphin (KTP-NH2), was designed and produced to overcome the BBB. In this work, the main goal was to explore the therapeutic use of KTP-NH2 as a new drug for AD treatment. It started with the characterization and comparison of the impact of KTP and KTP-NH2 in synaptic function under physiological mimetic conditions. Then, given the particular interest of this work in AD treatment, the neuroprotective potential of both peptides was evaluated upon Aβ-induced AD (Chapter 3). Finally, this work correlated the synaptic mechanisms protected by KTP-NH2 action against Aβ-induced toxicity, and the ameliorated memory impairments observed after the systemic administration of KTP-NH2 in a model of sporadic AD in rat (Chapter 4). Electrophysiological recordings obtained in the CA1 area of hippocampal slices prepared from adult male C57BL/6J mice, pre-exposed or superfused with KTP and KTP-NH2, allowed the characterization of the effects of both peptides on synaptic function under non pathological conditions. Results revealed that for concentrations ranging from 5 nM to 50 µM, the peptides affected the basal synaptic transmission in a concentration-dependent manner. While KTP slightly increased synaptic transmission, with a maximal effect measured at 50 nM, KTP-NH2 had a gradual inhibitory effect. At concentrations of 5 mM, largely in excess of what is likely to occur endogenously, peptides’ action rapidly inhibited synaptic transmission, being these effects reversible. In fact, this inhibitory effect was totally or partially eliminated with the respective removal of KTP or KTP-NH2. Thus, under nonpathological conditions, these findings suggested a different effect on synaptic mechanisms. However, neither KTP (50 nM), nor KTP-NH2 (50 nM), significantly affected: 1) synaptic transmission efficiency, evaluated by input/output curves; 2) short-term plasticity, evaluated by post-tetanic potentiation and paired-pulse facilitation; or 3) glutamate release. Together, these findings suggested that the effects of both peptides on synaptic transmission were likely not directly mediated by pre- or post-synaptic mechanisms. Synaptic plasticity is a key mechanism in memory and learning processes. Long-term potentiation (LTP) was evaluated in hippocampal slices, which were either pre-exposed for 3h or acutely superfused with KTP (50 nM) or KTP-NH2 (50 nM). For testing mimetic AD pathophysiological conditions, hippocampal slices were pre-treated with oligomeric Aβ peptide species (200 nM), one of the most soluble toxic Aβ species. Additionally, dendritic spines were evaluated in cultured cortical neurons, after the treatment with KTP (50 nM) or KTP-NH2 (50 nM), and in the presence or absence of Aβ peptide (25 µM) for 24h. Results demonstrated that KTP-NH2, but not KTP, had a neuroprotective effect against Aβ-induced impairments on LTP magnitude. However, these differences contrasted with the similar molecular neuroprotective effect. The action of both peptides restored the density of dendritic spines affected by the action of the Aβ peptide, without inducing toxic effects on neurons. To evaluate whether the neuroprotective effect of KTP-NH2 (50 nM) over LTP could be antagonized by KTPant, slices were pre-treated for 30 min with KTPant (250 nM). The results revealed that KTPant antagonized the neuroprotective effect of KTP-NH2 over Aβ peptide-induced impairments in LTP. However, it is still unclear if KTPant, alone, had a preventive neuroprotective action against Aβ peptide, or which affected mechanism might prompt toxicity when KTPant was added prior to KTP-NH2. Elevation of intracellular Ca2+ levels are a hallmark of KTP-mediated processes. Thus, calcium imaging technique was used to understand whether the increase in Ca2+ levels could be directly caused by KTP activity and/or whether such increases would be necessary for the emergence of KTP-mediated actions. The results revealed that neither the presence of KTP (50 nM) nor KTP-NH2 (50 nM) affected Ca2+ intracellular levels in neurons, so there were no significant changes in neuronal calcium homeostasis. Calpain overactivation happens due to increased intracellular Ca2+ levels, usually as a consequence of Aβ peptide-induced excitotoxicity. As such, the action of both peptides (50 nM) over calpain in vitro activity was assessed using mice cortical tissue homogenates. Results revealed that neither KTP nor KTP-NH2 impacted calpain activation and, consequently, did not prevent calpain-induced cleavage of the BDNF receptor, the tropomyosin receptor kinase B-full length (TrkB-FL), which has a well-known neuroprotective role, and it is a substrate for calpains under Aβ-induced toxicity. Finally, in a rat model of sporadic AD, systemic administration of KTP-NH2 protected against spatial working-memory and episodic memory deficits, without affecting motor activity or inducing an anxiety-like behavior in the animals. Moreover, this KTP-NH2-induced neuroprotective effect was correlated with the prevention of Aβ-induced deficits, in both LTP magnitude of pre-treated hippocampal slices and spine density of cortical neuronal cultures. In conclusion, the present work collected novel evidence of KTP and KTP-NH2 neuromodulatory effects over synaptic function, highlighting the neuroprotective actions of KTP-NH2. Under physiological mimetic conditions, the absence of effects over synaptic function bolsters the therapeutic potential of both peptides, suggesting the absence of sideeffects upon synaptic transmission. At molecular and functional levels, these findings supported the KTP-NH2 neuroprotective effect (Chapter 3), confirming the results obtained through its systemic administration in a rat model of sporadic AD (Chapter 4), which provides important evidence for the use of KTP-NH2 as a drug for treatment of AD and, eventually, other diseases. In light of the complex pathophysiology of AD, in the future, it will be important to determine whether KTP-NH2 neuroprotective effect is potentiated as a standalone treatment, or if its actions should be included as a broader multidrug treatment regimen. Among other aspects, in the treatment of AD, identifying the time-window for therapeutic intervention with KTP-NH2 will be essential to ascertain whether its action relies on the prevention or if this drug is able to recover the molecular and cognitive deficits present in AD pathophysiology. |