Guanosina previne alterações mitocondriais, o efeito tipo-depressivo e o déficit olfatório induzido por modelos experimentais da doença de parkinson

Tese (doutorado) - Universidade Federal de Santa Catarina, Centro de Ciências Biológicas, Programa de Pós-Graduação em Bioquímica, Florianópolis, 2019A doença de Parkinson (DP) é a segunda doença neurodegenerativa mais prevalente no mundo e é classicamente descrita como uma doença de sintomas motores. Apesar dos tratamentos atuais serem efetivos para os sintomas motores, os mesmos apresentam efeitos colaterais severos e não impedem a progressão da doença. Diante disso, o nucleosídeo guanosina (GUO) demonstrou eficácia como um agente antiparkinsoniano em modelos clássicos de sintomas motores, além de apresentar efeitos neuroprotetores em modelos celulares in vitro da DP. O objetivo desse trabalho foi demonstrar o efeito da GUO em um protocolo in vitro que mimetiza eventos celulares iniciais da DP e em modelos animais que mimetizam os sintomas não-motores. In vitro, avaliamos os efeitos protetores da GUO contra danos na função mitocondrial de fatias de córtex, estriado e hipocampo do cérebro de ratos. Observamos que a 6-hidroxidopamina (6-OHDA, 100 µM) apresenta toxicidade seletiva em fatias estriatais em alterações bioquímicas relacionados a mitocôndria. Nas fatias estriatais, a 6-OHDA diminuiu a viabilidade celular, induziu um aumento da geração de espécies reativas ao oxigênio (EROs), causou alteração do potencial de membrana mitocondrial, a depleção dos níveis intracelulares de ATP e o aumento da permeabilidade de membrana celular. A co-incubação com GUO (100 µM) protegeu as fatias estriatais contra o aumento de EROs, alteração do potencial de membrana mitocondrial e a depleção dos níveis de ATP intracelular. Em um protocolo in vivo, destacamos a avaliação de sintomas não-motores, como a depressão e o prejuízo olfatório. O comportamento tipo-depressivo foi avaliado utilizando protocolo de infusão bilateral de 6-OHDA (10 µg/hemisfério) no estriado dorsolateral de ratos. A infusão de 6-OHDA induziu comportamento tipo-anedônico no teste do ?splash? e aumentou o tempo de imobilidade no teste do nado forçado. As análises bioquímicas demonstraram alteração do potencial de membrana mitocondrial nas fatias de hipocampo, enquanto que a geração de EROs não foi alterada nas fatias corticais, estriatais e hipocampais dos animais que receberam a infusão de 6-OHDA. O tratamento com GUO 7,5 mg/kg i.p. durante 21 dias após a infusão de 6-OHDA apresentou um efeito parcialmente protetor no teste do?splash?, preveniu comportamento tipo-depressivo no teste do nado forçado e protegeu as fatias hipocampais da alteração do potencial de membrana mitocondrial induzido pela 6-OHDA. Ainda, o efeito do tratamento com GUO 7,5 mg/kg i.p. durante 20 dias em ratos submetidos à infusão intranasal de MPTP (1 mg/narina) foi avaliado. A infusão de MPTP induziu prejuízo nos testes de discriminação olfatória e de reconhecimento social. Adicionalmente, foram observadas alterações no potencial de membrana mitocondrial e aumento da permeabilidade de membrana nas fatias de bulbo olfatório. No hipocampo também foi observado aumento da permeabilidade de membrana. Nenhuma alteração foi observada na expressão total ou na fosforilação dos sítios Ser19, Ser31 e Ser40 da enzima tirosina hidroxilase (TH). A GUO preveniu o prejuízo à habilidade olfatória e à memória de reconhecimento social, a alteração do potencial de membrana mitocondrial e a permeabilidade de membrana no bulbo olfatório. Em síntese, concluímos que a GUO apresenta um potencial efeito neuroprotetor em parâmetros relacionados à função mitocondrial e a sintomas não-motores da DP, mimetizados em modelos in vitro e in vivo pelas toxinas 6-OHDA e MPTP.Abstract: Parkinson's disease (PD) is the second most prevalent neurodegenerative disease worldwide and classically described as a motor symptoms disorder. Although current treatments presented effectiveness against motor symptoms, it is not possible to stop the progression of the disease or avoid the side effects. The guanine nucleoside (GUO) has been shown as an efficient antiparkinsonian agent against motor symptoms in classical animal models of PD, besides showing neuroprotective effects in in vitro models of PD. Here, the aim was to demonstrate the effectiveness of GUO in in vitro model, which reproduces early cellular events related to PD and in in vivo, using animal models which mimic non-motor symptoms of PD. In vitro, we evaluated the protective effects of GUO against 6-OHDA-induced toxicity in slices from the cortex, striatum and hippocampal of rats. 6-OHDA (100 µM) presented selective toxicity on striatal slices in parameters related to mitochondrial function. In striatal slices, 6-OHDA decreases cell viability, increased reactive oxygen species (ROS) generation, disrupted mitochondrial membrane potential, induce intracellular ATP levels depletion and increased cell membrane permeability. Co-incubation with GUO protected striatal slices against increased ROS, mitochondrial membrane potential disruption and intracellular ATP depletion. In in vivo protocol, we highlight non-motor symptoms as depression and olfactory impairment. Depressive-like symptoms were mimicked using a 6-OHDA (10 µg/hemisphere) bilateral infusion model into the dorsolateral striatum of rats. The 6-OHDA infusion induced anhedonic-like behavior in the splash test and increased the immobility time in the forced swimming test. Biochemical analyses demonstrated the disruption of the mitochondrial membrane potential in the hippocampal slices, whereas the ROS generation was not altered in the cortical, striatal and hippocampal slices in animals which received 6-OHDA infusion. Meanwhile, treatment with GUO (7.5 mg/kg, i.p.) during the 21 days after 6-OHDA infusion revealed a partial protective effect in the splash test, prevented increased immobility time in the forced swimming test, and finally protected the hippocampal slices 6-OHDA-induced disruption on mitochondrial membrane potential. Finally, the treatment with GUO 7.5 mg/kg i.p. for 20 days in rats submitted to an intranasal infusion of MPTP was evaluated. MPTP infusion induced behavioral impairments in the olfactory discrimination and the social recognition test. Additionally, MPTP-induced disruption on the mitochondrial membrane potential in olfactory bulb slices and increased cell membrane permeability in the olfactory bulb and hippocampal slices were observed. Regarding tyrosine hydroxylase (TH) regulation, no changes in the expression or phosphorylation of the Ser19, Ser31, and Ser40 sites were observed 21 days after MPTP, although we demonstrated a different profile of phosphorylation of TH in the brain regions analyzed. Finally, we demonstrated the protective effects of GUO on the olfactory discrimination deficits, social recognition memory impairment and also on disruption of mitochondrial membrane potential in the olfactory bulb induced by MPTP. Thus, we conclude that GUO has a potential neuroprotective effect on parameters related to mitochondrial function and non-motor symptoms of PD mimicked in vitro and in vivo models based in the toxins 6-OHDA and MPTP

Role of Prefrontal Cortex on Recognition Memory Deficits in Rats following 6-OHDA-Induced Locus Coeruleus Lesion

Degeneration of the locus coeruleus (LC), the main source of cerebral noradrenaline (NA), has been reported in diverse neurodegenerative diseases, including Parkinson’s diseases (PD). There is increasing evidence indicating the role of NA deficiency in the prefrontal cortex (PFC) and the development of early cognitive impairments in PD. Here, we evaluated whether a selective noradrenergic lesion of LC caused by 6-hydroxydopamine (6-OHDA) may induce memory deficits and neurochemical alterations in the PFC. Adult male Wistar rats received stereotaxic bilateral injections of 6-OHDA (5 μg/2 μl) into the LC, and two stainless-steel guide cannulas were implanted in the PFC. The SHAM group received just vehicle. To induce a selective noradrenergic lesion, animals received nomifensine (10 mg/kg), a dopamine transporter blocker, one hour before surgery. 6-OHDA-lesioned rats displayed impairments of the short- and long-term object recognition memory associated to reduced content of tyrosine hydroxylase in the LC. Neurochemical analysis revealed an altered mitochondrial membrane potential in LC. Regarding the PFC, an increased ROS production, cell membrane damage, and mitochondrial membrane potential disruption were observed. Remarkably, bilateral NA (1 μg/0.2 μl) infusion into the PFC restored the recognition memory deficits in LC-lesioned rats. These findings indicate that a selective noradrenergic LC lesion induced by 6-OHDA deregulates a noradrenergic network in the PFC, which could be involved in the early memory impairments observed in nondemented PD patients

Guanosine Prevents Spatial Memory Impairment and Hippocampal Damage Following Amyloid-β1–42 Administration in Mice

Alzheimer’s disease (AD) is a progressive neurodegenerative illness responsible for cognitive impairment and dementia. Accumulation of amyloid-beta (Aβ) peptides in neurons and synapses causes cell metabolism to unbalance, and the production of reactive oxygen species (ROS), leading to neuronal death and cognitive damage. Guanosine is an endogenous nucleoside recognized as a neuroprotective agent since it prevents glutamate-induced neurotoxicity by a mechanism not yet completely elucidated. In this study, we evaluated behavioral and biochemical effects in the hippocampus caused by the intracerebroventricular (i.c.v.) infusion of Aβ1–42 peptide (400 pmol/site) in mice, and the neuroprotective effect of guanosine (8 mg/kg, i.p.). An initial evaluation on the eighth day after Aβ1–42 infusion showed no changes in the tail suspension test, although ex vivo analyses in hippocampal slices showed increased ROS production. In the second protocol, on the tenth day following Aβ1–42 infusion, no effect was observed in the sucrose splash test, but a reduction in the recognition index in the object location test showed impaired spatial memory. Analysis of hippocampal slices showed no ROS production and mitochondrial membrane potential alteration, but a tendency to increase glutamate release and a significant lactate release, pointing to a metabolic alteration. Those effects were accompanied by decreased cell viability and increased membrane damage. Guanosine treatment prevented behavioral and biochemical alterations evoked by Aβ1–42, suggesting a potential role against behavioral and biochemical damage evoked by Aβ in the hippocampus

Preclinical development of kinetin as a safe error-prone SARS-CoV-2 antiviral able to attenuate virus-induced inflammation

The search for antivirals against SARS-CoV-2 continue due to the emergence of variants of concerns, able to escape the vaccinal humoral response. In this work, authors pre-clinically explore the potential of kinetin against SARS-CoV-2, which could be used alone or in combination with other antivirals