A hipercolesterolemia como um fator de risco para o desenvolvimento de comprometimento cognitivo leve: evidências obtidas em modelos experimentais

Tese (doutorado) - Universidade Federal de Santa Catarina, Centro de Ciências Biológicas, Programa de Pós-Graduação em Neurociências, Florianópolis, 2013.Evidências acumuladas ao longo das duas últimas décadas sugerem umaassociação positiva entre a hipercolesterolemia e o desenvolvimento dadoença de Alzheimer esporádica. Estudos prospectivos demonstraramque esta relação parece ser idade-dependente, sendo ahipercolesterolemia um possível fator de risco para o desenvolvimentoda doença de Alzheimer décadas após. A hipercolesterolemia familiar éuma doença genética do metabolismo das lipoproteínas cujo modo deherança é autossômico codominante e que se caracteriza por níveismuito elevados de colesterol plasmático. O fenótipo clínico dehipercolesterolemia familiar é geralmente decorrente de defeitos no geneLDLR, que codifica o receptor de lipoproteínas de baixa densidade(LDL). Recentemente, um estudo prospectivo demonstrou que pacientescom hipercolesterolemia familiar de meia-idade apresentam umaelevada incidência de comprometimento cognitivo leve em comparaçãoa indivíduos sem hipercolesterolemia familiar. Neste contexto, acaracterização do comprometimento cognitivo leve em modelosexperimentais de hipercolesterolemia familiar torna-se imperativa,visando tanto o desenvolvimento de abordagens terapêuticas epreventivas, quanto o entendimento de mecanismos molecularesresponsáveis pelo declínio cognitivo associado com ahipercolesterolemia. Os resultados desta tese demonstram quecamundongos nocaute para o receptor de LDL (LDLr-/-) de quatorzemeses (?meia-idade?), um modelo experimental de hipercolesterolemiafamiliar, apresentaram prejuízos cognitivos em testes de memóriaoperacional, de referência espacial e de procedimento. Também foiobservado um desequilíbrio no sistema antioxidante dependente daglutationa (GSH) e um aumento na atividade da enzimaacetilcolinesterase (AChE) no córtex pré-frontal dos camundongosLDLR-/- de ?meia-idade?. O fármaco hipolipemiante probucol e oexercício físico voluntário (rodas de correr) diminuíram de modosignificativo os níveis de colesterol plasmático de camundongos LDLr-/-,todavia, apenas o exercício físico voluntário foi capaz de atenuar osprejuízos cognitivos dos camundongos LDLr-/-. Por outro lado, aexposição a uma dieta hipercolesterolêmica (1,25 % de colesterol) pordois meses também induziu déficits cognitivos em camundongos Swiss,bem como aumentou significativamente a atividade da enzima AChE nocórtex pré-frontal e hipocampo. De modo interessante, a exposição decélulas de neuroblastoma humano da linhagem SH-5Y6Y à LDLtambém induziu um aumento na atividade da enzima AChE. Ademais,foi demonstrado que camundongos LDLr-/- parecem apresentar umamaior susceptibilidade a insultos neurotóxicos, como à neurotoxicidadeinduzida pela exposição crônica ao metilmercúrio (MeHg). Emconjunto, os achados do presente estudo caracterizam principalmente apresença de comprometimento cognitivo leve em camundongos LDLr-/-de ?meia-idade?, tal como observado em pacientes comhipercolesterolemia familiar de meia-idade. Os resultados deste estudoencorajam estudos adicionais para investigar o valor terapêutico doexercício físico regular no controle do comprometimento cognitivo leveem pacientes com hipercolesterolemia familiar, bem como a associaçãode fármacos hipolipemiantes com inibidores da enzima AChE notratamento da doença de Alzheimer. Abstract : Accumulating evidence over the past decades compellingly argues thatpeople with high serum cholesterol concentration in midlife have asignificantly higher risk of Alzheimer?s disease (AD) in later life.Familial hypercholesterolemia (FH) is an inherited metabolic disordercharacterized by high levels of plasma low density lipoproteins (LDL).FH remains the most common monogenic disorder of lipoproteinmetabolism, and it is mainly due to mutations in the LDL receptor(LDLr) gene that leads to the plasma accumulation of cholesterol esterladenLDL particles. It was recently showed that middle age patientswith FH show a particularly high incidence of mild cognitiveimpairments (MCI). Thus, the characterization of MCI in experimentalmodels of FH aimed at developing therapeutic and preventiveapproaches for the cognitive decline associated withhypercholesterolemia is imperative. The results presented herein showthat middle-aged low-density lipoprotein receptor-deficient (LDLr-/-)mice, an experimental model of FH, exhibit working, spatial reference,and procedural memory impairments, which were associated with theoccurrence of antioxidant imbalance, oxidative damage, and putativedecrease of cholinergic signaling in the prefrontal cortex. The lipidloweringdrug probucol and physical exercise (voluntary running wheel)similarly decreased total plasma cholesterol levels in LDLr-/- mice,however, only physical exercise mitigated the spatial memory deficits ofLDLr-/- mice. Moreover, mild hypercholesterolemia was modeled inSwiss mice (high fat/cholesterol diet) in order to evaluate primarybiochemical events linked to hypercholesterolemia-induced cognitiveimpairments. An impaired short-term object location memory wasobserved in hypercholesterolemic mice, which was associated with anincreased acetylcholinesterase (AChE) activity within the prefrontalcortex and hippocampus. Notably, LDL cholesterol significantlyincreased in a dose-dependent manner the activity of AChE in SHSY5Yhuman neuroblastoma cells. Finally, hypercholesterolemic LDLr-/- mice were more susceptible to methylmercury (MeHg)-inducedcerebellar glial activation, suggesting that hypercholesterolemia in itselfmay pose a risk factor in MeHg-induced neurotoxicity. Overall, thisstudy corroborated previous clinical data regarding the observation ofMCI in FH patients. The present findings may have importantimplications, since they provide an opportunity for clinical interventionssuch as control of vascular risk factors that minimize, arrest, or evenreverse cognitive deterioration

Glucose Homeostasis Is Not Affected in a Murine Model of Parkinson’s Disease Induced by 6-OHDA

There is a mutual relationship between metabolic and neurodegenerative diseases. However, the causal relationship in this crosstalk is unclear and whether Parkinson’s disease (PD) causes a posterior impact on metabolism remains unknown. Considering that, this study aimed to evaluate the appearance of possible changes in metabolic homeostasis due to 6-hydroxydopamine (6-OHDA) administration, a neurotoxin that damage dopaminergic neurons leading to motor impairments that resemble the ones observed in PD. For this, male Wistar rats received bilateral 6-OHDA administration in the dorsolateral striatum, and the motor and metabolic outcomes were assessed at 7, 21, or 35 days post-surgical procedure. Dexamethasone, a diabetogenic glucocorticoid (GC), was intraperitoneally administered in the last 6 days to challenge the metabolism and reveal possible metabolic vulnerabilities caused by 6-OHDA. Controls received only vehicles. The 6-OHDA-treated rats displayed a significant decrease in locomotor activity, exploratory behavior, and motor coordination 7 and 35 days after neurotoxin administration. These motor impairments paralleled with no significant alteration in body mass, food intake, glucose tolerance, insulin sensitivity, and biochemical parameters (plasma insulin, triacylglycerol, and total cholesterol levels) until the end of the experimental protocol on days 35–38 post-6-OHDA administration. Moreover, hepatic glycogen and fat content, as well as the endocrine pancreas mass, were not altered in rats treated with 6-OHDA at the day of euthanasia (38th day after neurotoxin administration). None of the diabetogenic effects caused by dexamethasone were exacerbated in rats previously treated with 6-OHDA. Thus, we conclude that bilateral 6-OHDA administration in the striatum causes motor deficits in rats with no impact on glucose and lipid homeostasis and does not exacerbate the adverse effects caused by excess GC. These observations indicate that neurodegeneration of dopaminergic circuits in the 6-OHDA rats does not affect the metabolic outcomes

Análise do P&D Brasileiro com Uso da Nanotecnologia em Medicamentos para Uso Humano: pesquisas, tecnologias e produtos

The aim of the present work was to analyze the relation between Science and Technology Institutes and the national pharmaceutical companies with respect to nanotechnology in drug development process. Specifically, it was analyzed the innovative process promoted by nanotechnology in Brazil, by evaluating the scientific research lines, deposits of patents, technologies developed, ongoing and completed clinical trials and, eventually, products in the market. Overall, it was observed a significant increase in the number of ongoing research studies and deposits of patents from national origin; however, the main portion of these deposits is from National Science and Technology Institutes. Noteworthy, there are national industries that developed clinical trials with national academy collaboration, which resulted in the development of pharmaceutical products.Este trabalho teve como objetivo analisar a dinâmica de relações entre Instituições de Ciência e Tecnologia (ICTs) e empresas brasileiras acerca de pesquisas envolvendo nanotecnologia com aplicação ao desenvolvimento de medicamentos para uso em seres humanos. Especificamente, buscou-se apresentar o processo de inovação promovido pela nanotecnologia nas pesquisas de origem brasileira, por meio do levantamento de linhas de pesquisas, depósitos de patentes, tecnologias desenvolvidas, transferências desses conhecimentos, ensaios clínicos em andamento e concluídos, os quais resultaram, em produtos de origem nacional. Observou-se que as empresas brasileiras, em termos de depósitos de patentes, apresentam números incipientes, embora um número expressivo desses depósitos seja oriundo de parceria com ICTs brasileiras. Em especial, algumas empresas brasileiras realizaram ensaios clínicos com parcerias de ICTs, resultando no desenvolvimento de produtos de origem brasileira

Probucol Increases Striatal Glutathione Peroxidase Activity and Protects against 3-Nitropropionic Acid-Induced Pro-Oxidative Damage in Rats

<div><p>Huntington’s disease (HD) is an autosomal dominantly inherited neurodegenerative disease characterized by symptoms attributable to the death of striatal and cortical neurons. The molecular mechanisms mediating neuronal death in HD involve oxidative stress and mitochondrial dysfunction. Administration of 3-nitropropionic acid (3-NP), an irreversible inhibitor of the mitochondrial enzyme succinate dehydrogenase, in rodents has been proposed as a useful experimental model of HD. This study evaluated the effects of probucol, a lipid-lowering agent with anti-inflammatory and antioxidant properties, on the biochemical parameters related to oxidative stress, as well as on the behavioral parameters related to motor function in an <i>in vivo</i> HD model based on 3-NP intoxication in rats. Animals were treated with 3.5 mg/kg of probucol in drinking water daily for 2 months and, subsequently, received 3-NP (25 mg/kg i.p.) once a day for 6 days. At the end of the treatments, 3-NP-treated animals showed a significant decrease in body weight, which corresponded with impairment on motor ability, inhibition of mitochondrial complex II activity and oxidative stress in the striatum. Probucol, which did not rescue complex II inhibition, protected against behavioral and striatal biochemical changes induced by 3-NP, attenuating 3-NP-induced motor impairments and striatal oxidative stress. Importantly, probucol was able to increase activity of glutathione peroxidase (GPx), an enzyme important in mediating the detoxification of peroxides in the central nervous system. The major finding of this study was that probucol protected against 3-NP-induced behavioral and striatal biochemical changes without affecting 3-NP-induced mitochondrial complex II inhibition, indicating that long-term probucol treatment resulted in an increased resistance against neurotoxic events (i.e., increased oxidative damage) secondary to mitochondrial dysfunction. These data appeared to be of great relevance when extrapolated to human neurodegenerative processes involving mitochondrial dysfunction and indicates that GPx is an important molecular target involved in the beneficial effects of probucol.</p> </div

Probucol reduces 3-NP-induced lipid peroxidation in rats.

<p>Treatments were conducted as previously mentioned (see Methods Section). Striatal thiobarbituric acid reactive substance (TBARS) levels are expressed as nmol of MDA/mg protein. The data are presented as the mean ± S.E.M. (n = 6 rats/group). *** p< 0.001 compared with the control group and # # p < 0.01 and # # # p < 0.001 compared with the 3-NP group using two-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison test.</p

3-NP treatment inhibits complex II activity.

<p>Treatments were conducted as previously mentioned (see Methods Section). Complex II activity in striatum is expressed as nmol.min⁻¹.mg protein⁻¹ and presented as the mean ± S.E.M. (n = 6 rats/group). ** p < 0.01 and *** p < 0.001 compared with the control group using two-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison test.</p

Probucol attenuates motor impairment induced by 3-NP in rats.

<p>Treatments were conducted as previously mentioned (see Methods Section). Locomotor (A) and exploratory (B) activities in the open field test as well as the latency for the first fall (C) and the number of falls in the rotarod (D) were evaluated 24 h after the last 3-NP administration. These results are expressed as the total number of crossings (A), total number of rearings (B), the latency for the first fall(s) (C) and the total number of falls. The data are presented as the mean ± S.E.M. (n =10 rats/group). *p < 0.05 and *** p < 0.001 compared with the control group and # p < 0.05, # # p < 0.01 and # # # p < 0.001 compared with the 3-NP group using two-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison test.</p

Figure 7

<p><b>Effects of 3-NP and/or probucol on iNOS, GFAP</b> and <b>caspase 3/cleaved caspase 3 expression.</b></p