Biomarcadores periféricos e regulação transcricional nos transtornos psiquiátricos : vias moleculares associadas à psicopatologia no transtorno bipolar, transtorno depressivo maior e esquizofrenia

Dados mostram que existe certa sobreposição de características clínicas e de resposta ao tratamento nos transtornos psiquiátricos, como Transtorno Bipolar (TB), Transtorno Depressivo Maior (TDM) e Esquizofrenia (SZ), sugerindo a existência de certos mecanismos fisiopatológicos similares. Os estudos contidos nesta tese tiveram como objetivo investigar biomarcadores periféricos em transtornos mentais, bem como características clínicas comuns entre eles, além de explorar, como foco principal, a regulação transcricional no TB, TDM e SZ. Neste sentido, no primeiro capítulo estudamos a utilização de biomarcadores séricos, além de variáveis clínicas e sociodemográficas, na predição de níveis de ruminação mal adaptativa em uma amostra transdiagnóstica através de análises de machine learning. Observamos que somente algumas variáveis clínicas e demográficas apresentaram associação significativa com os níveis de ruminação, não sendo encontrada relação com os biomarcadores séricos estudados. No segundo capítulo, utilizando algoritmos de machine learning, a proposta foi verificar a utilidade de marcadores inflamatórios, de estresse oxidativo e do BDNF para discriminar entre pacientes com depressão unipolar, depressão bipolar e controles, e constatamos que IL-4, TBARS e IL-10 representam as variáveis mais relevantes na distinção entre depressão bipolar e depressão unipolar; IL-6, IL-4, TBARS, carbonil e IL17-A tiveram destaque para distinguir depressão bipolar e controles; e IL-6, carbonil, BDNF, IL-10, IL17-A, IL-4 e TNF- α demonstraram ser capazes de distinguir entre depressão unipolar e controles. A partir de então, com o intuito de investigar as bases fisiopatológicas do TB, TDM e SZ em nível central, utilizamos uma abordagem transcriptômica valendo-nos de dados de microarranjo e RNA-Seq. No capítulo 3, identificamos potenciais genes que atuam como reguladores mestres (MRs) no córtex pré-frontal para cada um dos três transtornos, bem como o estado de ativação (ativado ou reprimido) de cada um deles, e usamos essas informações para análises de clusterização que pudessem caracterizar os fenótipos das doenças, além de determinar os processos biológicos modulados pelos MRs. Assim, identificamos três grandes grupos: (1) TDM, (2) majoritariamente TB, e (3) TB e SZ. TB e SZ compartilharam diversos processos biológicos relacionados a transporte e homeostase de íons, função sináptica e função imunológica; SZ particularmente mostrou também enriquecimento de processos relativos ao citoesqueleto e estrutura neuronal; e TDM foi relacionado a processos ligados a desenvolvimento glial e metabolismo de ácidos graxos. Ainda, entre os MRs identificados, o early growth response (EGR) 3 estava reprimido e o EGR1 predominantemente reprimido nos três transtornos e, por este motivo, investigamos no capítulo quatro, a expressão gênica de EGR1 e EGR3 perifericamente de maneira mais direcionada, assim como do gene cryptochrome circadian regulator 2 (CRY2) que atua no ritmo circadiano e possui relação com os EGRs. Encontramos uma redução na expressão gênica de EGR1 no TB, TDM e SZ, corroborando com os achados em nível central do capítulo três, o que o coloca em destaque como um alvo molecular transdiagnóstico. Em conjunto, os resultados sugerem que há unidades gênicas regulatórias disfuncionais no córtex pré-frontal de pacientes com TB, TDM e SZ, sendo que o padrão de regulação transcricional e processos biológicos associados aproximam TB e SZ, ou seja, torna mais complexa a distinção entre estes dois transtornos, e os distancia do TDM. Nesta tese, identificamos o EGR1 como unidade regulatória de destaque, com evidências centrais e periféricas de que este regulon está reprimido nesses três transtornos psiquiátricos, o que deve ser ressaltado considerando que o EGR1 responde a estímulos ambientais (como experiências sociais, estresse, tarefas cognitivas) e, por meio dos seus alvos, atua em vias de sinalizações relacionadas à plasticidade sináptica. Em suma, a utilização de biomarcadores séricos para caracterizar transtornos psiquiátricos constitui um campo desafiador, sendo que combinar dados clínicos com dados de genômica e/ou transcriptômica em modelos de análise mais complexos tem o potencial de contribuir para o entendimento da fisiopatologia desses transtornos multifatoriais.Evidence shows some overlapping of clinical symptoms and response to treatment in psychiatric disorders, such as Bipolar Disorder (BD), Major Depressive Disorder (MDD), and Schizophrenia (SZ), also suggesting similar underlying pathophysiological mechanisms. This thesis comprises studies with the aim to investigate peripheral biomarkers in mental disorders and common clinical characteristics among them, as well as exploring, as its main focus, the transcriptional regulation in BD, MDD, and SZ. In this sense, in the first chapter we studied the use of peripheral biomarkers, in addition to clinical and sociodemographic variables, to predict maladaptive rumination levels in a transdiagnostic sample using machine learning analyses. We found that only some clinical and demographic variables, and not the assessed serum biomarkers, were associated with rumination levels. In the second chapter, using machine learning algorithms, we proposed to evaluate whether inflammatory and oxidative stress markers, and BDNF could be used to discriminate patients with unipolar depression, patients with bipolar depression and controls; we found that IL-4, TBARS and IL-10 are the most relevant variables to differentiate bipolar depression and unipolar depression; IL-6, IL-4, TBARS, carbonil and IL17-A were relevant to distinguish between bipolar depression and controls; and IL-6, carbonil, BDNF, IL-10, IL17-A, IL-4 and TNF- α were able to discriminate unipolar depression from controls. Therefore, aiming to investigate the physiopathological basis of BD, MDD and SZ at the central level, we applied a transcriptomic approach using microarray and RNA-Seq data. In the chapter three, we identified potential genes acting as master regulators (MRs) in the prefrontal, as well as their pattern of activation (activate or repressed) in the three disorders, and we used these information in cluster analyses to characterize the disease phenotypes, and to assess the biological processes modulated by the MRs. Then, we identified 3 major clusters: (1) MDD, (2) mostly BD, and (3) TB and SZ. BD and SZ share biological processes related to ionic transport and homeostasis, synaptic function, and immune function; SZ particularly also shows enrichment of processes related to glial development and fatty acids metabolism. Also, among the identified MRs, the early growth response (EGR) 3 was repressed and the EGR1 was mostly repressed in the three disorders. Thus, we investigated, in the chapter four, the gene expression of EGR1 and EGR3 in the periphery, as well as the cryptochrome 2 (CRY2) that is a circadian rhythm gene and associated with the EGRs. We found reduction in EGR1 gene expression in BD, MDD and SZ, supporting the findings at the central level found in the chapter three, which suggests this gene as a transdiagnostic molecular target. Altogether, the results suggest that there are dysfunctional gene regulatory units in the prefrontal cortex of patients with BD, MDD and SZ, with similar transcriptional regulation patterns and their related biological processes in BD and SZ, which makes more complex the discrimination between these two disorders. In this thesis, we identified EGR1 as a key regulatory unit, with central and peripheral evidence that this regulon is repressed in these three psychiatric disorders, which is crucial considering that EGR1 is highly responsive to environmental stimuli (as social experiences, stress, and cognitive tasks) and plays relevant roles, through its targets, in signaling pathways linked to synaptic plasticity. In sum, the use of serum biomarkers to characterize psychiatric disorders represent a great challenge in the field and integrating clinical data with genomic or transcriptomic data using more complexes approaches has a substantial potential to contribute to the understanding of the physiopathology of these multifactorial disorders

Effects of lithium on inflammatory and neurotrophic factors after an immune challenge in a lisdexamfetamine animal model of mania

Objective: To evaluate whether an animal model of mania induced by lisdexamfetamine dimesylate (LDX) has an inflammatory profile and whether immune activation by lipopolysaccharides (LPS) has a cumulative effect on subsequent stimuli in this model. We also evaluated the action of lithium (Li) on inflammatory and neurotrophic factors. Methods: Adult male Wistar rats were subjected to an animal model of mania. After the open-field test, they were given LPS to induce systemic immune activation. Subsequently, the animals’ blood was collected, and their serum levels of brain-derived neurotrophic factor and inflammatory markers (tumor necrosis factor [TNF]-a, interleukin [IL]-6, IL-1b, IL-10, and inducible nitric oxide synthase [iNOS]) were measured. Results: LDX induced hyperactivity in the animals, but no inflammatory marker levels increased except brain-derived neurotrophic factor (BDNF). Li had no effect on serum BDNF levels but prevented iNOS levels from increasing in animals subjected to immune activation. Conclusion: Although Li prevented an LPS-induced increase in serum iNOS levels, its potential antiinflammatory effects in this animal model of mania were conflicting

Brain-derived neurotrophic factor increase during treatment in severe mental illness inpatients

Meta-analytical evidence suggests that brain-derived neurotrophic factor (BDNF) is altered in various psychiatric disorders. However, meta-analyses may be hampered by the heterogeneity of BDNF assays, lack of BDNF standard values and heterogeneity among the populations included in the studies. To address these issues, our study aimed to test, in a ‘true-to-life’ setting, the hypothesis that the serum BDNF level is nonspecifically reduced in acute severe mental illness (SMI) patients and increases during inpatient treatment. Consecutive samples of 236 inpatients with SMI and 100 healthy controls were recruited. SMI includes schizophrenia and severe mood disorders, and is characterized in the sample by the presence of at least 2 years of psychiatric treatment and disability. Generalized estimating equations were used to analyze BDNF serum levels at admission and upon discharge controlled by confounding factors. BDNF levels increased significantly between admission and discharge in SMI patients. BDNF levels showed significant reductions compared with controls both at admission and upon discharge. In addition, BDNF levels showed no difference among SMI patient diagnostic subgroups (unipolar depression, bipolar depression, schizophrenia and manic episode). The increase but non-restoration of BDNF levels, even with the general acute improvement of clinical scores, may reflect the progression of the disorder characteristically seen in these patients. BDNF levels could be considered as a marker for the presence of a nonspecific psychiatric disorder and possibly a transdiagnostic and nonspecific marker of disease activity

Brain-derived neurotrophic factor serum levels and hippocampal volume in mild cognitive impairment and dementia due to Alzheimer disease

Background/Aims: Hippocampal atrophy is a recognized biomarker of Alzheimer disease (AD) pathology. Serum brain-derived neurotrophic factor (BDNF) reduction has been associated with neurodegeneration. We aimed to evaluate BDNF serum levels and hippocampal volume in clinical AD (dementia and mild cognitive impairment [MCI]). Methods: Participants were 10 patients with MCI and 13 with dementia due to AD as well as 10 healthy controls. BDNF serum levels were determined by ELISA and volumetric measures with NeuroQuant ® . Results: MCI and dementia patients presented lower BDNF serum levels than healthy participants; dementia patients presented a smaller hippocampal volume than MCI patients and healthy participants. Discussion: The findings support that the decrease in BDNF might start before the establishment of neuronal injury expressed by the hippocampal reduction

Influência do Estresse Agudo de Restrição sobre Processo Inflamatório Induzido pela Carragenina

O estresse é um fator ambiental que altera respostas fisiológicas envolvendo a liberação de corticóides e catecolaminas. Os glicocorticóides afetam o sistema imune. O objetivo deste trabalho é avaliar o efeito do estresse agudo sobre o processo inflamatório em ratos estressados submetidos à  2h de restrição e controles. Após o estresse, foi realizado o modelo de pleurisia induzida pela carragenina. Os resultados demonstraram diferença nos leucócitos totais antes e depois da pleurisia apenas no grupo controle. Na contagem diferencial de leucócitos circulantes, foram encontradas diferenças no percentual de linfócitos e neutrófilos entre os grupos antes da indução do processo inflamatório. No exsudato pleural não foram encontradas diferenças no total de leucócitos e na análise nas células polimorfo e mononucleares em ambos os grupos. Os resultados sugerem que no estresse agudo, após imediata realização da pleurisia ocorrem alterações imunológicas que parecem não influenciar o processo inflamatório agudo

Increase in serum brain-derived neurotrophic factor levels during early withdrawal in severe alcohol users

Introduction: Changes in brain-derived neurotrophic factor (BDNF) have been linked to the neuroadaptative consequences of chronic alcohol use and associated with disease severity and prognosis. Few studies have evaluated the influence of drug withdrawal and clinical and sociodemographic data on BDNF levels in severe alcohol users. Objectives: Our goals were (1) to evaluate variation in BDNF levels during alcohol withdrawal and, (2) to assess the influence of putative confounding factors on BDNF levels. Methods: Our sample consists of 62 men with alcohol use disorder undergoing a detoxification process. Serum BDNF levels were measured using a commercial sandwich-ELISA kit, at two points: before and after the detoxification period. Results: We found an increase in BDNF levels during alcohol withdrawal (25.4±9.6 at admission vs. 29.8±10.2 ng/ml at discharge; p < 0.001), even after controlling for potential confounders (positive family history, number of days between blood sample collections, and age) (Generalized Estimating Equation: coefficient = -4.37, 95% confidence interval [95%CI] -6.3; -2.4; p < 0.001). Moreover, individuals who had first-degree relative with alcohol dependence had smaller increases in BDNF levels than individuals with no family history (14.8 [95%CI -5.3; 35.6] vs. 35.3 [95%CI 15.4; 74.8]; p = 0.005). Conclusions: In summary, variation in BDNF levels seems to be influenced by withdrawal in severe alcohol users. A positive family history of alcohol dependence could also be a factor that influences variation in this biomarker