Differential gene expression of dorsal root ganglia cells in a rat model of diabetes and diabetic neuropathy

Orientador: Carlos Amilcar ParadaTese (doutorado) - Universidade Estadual de Campinas, Instituto de BiologiaResumo: A Neuropatia Diabética Periférica (NDP) manifesta-se em 50-60% dos pacientes diabéticos tanto do tipo I quanto do tipo II e é a maior causa de amputação não traumática de membros. Muito embora as características eletrofisiológicas e morfológicas da doença já estejam bem descritas, pouco se sabe sobre seu desenvolvimento e progressão, limitando terapias eficazes. A hiperglicemia e deficiências na sinalização de insulina são consideradas como os eventos deflagradores da produção de estresse oxidativo observado nos nervos em processo de degeneração. Diversas hipóteses foram elaboradas na tentativa de explicar o fenômeno, mas até hoje existem muitas lacunas no conhecimento da patogênese e da plasticidade celular associada. Alterações detectadas por técnicas de prospecção em larga escala do transcriptoma e proteoma podem ajudar a compreender os eventos moleculares observados na NDP quando os sintomas começam a aparecer. Com esse intuito, o objetivo deste estudo foi o de analisar, por RNA-Seq e espectrometria de massas quantitativa livre de sonda, o transcriptoma e o proteoma de Gânglios da Raiz Dorsal (DRG) de ratos Wistar modelo para diabetes tipo-I apresentando os primeiros sinais de desenvolvimento de NDP e respectivos controles. Os resultados obtidos demonstraram que as alterações na expressão de genes e proteínas nos DRGs já são detectadas há apenas 2 semanas após a verificação de alterações de sensibilidade mecânica periférica nas patas traseiras de ratos. Apesar da grande similaridade entre os transcriptomas dos animais hiperglicêmicos e controles, a análise comparativa detectou 66 transcritos diferencialmente expressos entre os grupos. Os resultados do estudo de proteoma obtidos também revelaram que existem alterações de expressão nos DRGs dos animais hiperglicêmicos: 3 proteínas foram consideradas diferencialmente expressas entre os dois grupos. De maneira similar, a categorização baseada em função apontou para genes e proteínas diferencialmente expressos envolvidos com estresse oxidativo, inflamação, apoptose/sobrevivência celular, proliferação celular e hiperalgesia/analgesia. Dado os seus papéis fisiológicos, alteração de expressão dessas moléculas sugere uma alteração do programa metabólico celular para um voltado a regeneração/sobrevivência. Nossos resultados mostram que as alterações no proteoma e transcriptoma começam a aparecer precocemente e que essas alterações podem ser um esforço para manter a homeostase celular. Assim, os presentes dados podem indicar como as células do DRG respondem a hiperglicemia neste estágio precoce abrindo a possibilidade futura de se descobrir quais mecanismos ou vias param de responder, levando aos danos e morte celular observados na clínica. Descobrir as primeiras alterações celulares na NDP pode levar a alvos mais robustos para a intervenção farmacêutica, que podem por sua vez prevenir ou retardar mais eficientemente os danos celularesAbstract: Peripheral Diabetic Neuropathy (PDN) manifests in 50-60% of types I and II diabetic patients and is the major cause of non-traumatic limb amputation. Although electrophysiological and morphological aspects are well described, little is known about its development and progression, undermining effective therapies. Hyperglycemia and insulin signaling impairment are considered the triggering events of oxidative stress production observed in the dying nerves. Several hypotheses try to explain the phenomenon, but until now there are still many gaps in the pathogenesis and the plastic changes it generates. Changes in transcriptome and proteome detected by high-throughput techniques, when first symptoms of the disease start to appear, can help understand the molecular events observed in PDN. To achieve this, we proposed a study to analyze, by RNA-Seq and label-free quantitative mass spectrometry, transcriptional and proteomic changes in Dorsal Root Ganglia (DRG) of a rat model for type I diabetes and its respective control. The results presented here show that alterations in genes and proteins expression are already taking place after only two weeks of detection of mechanical sensitivity impairment in hyperglycemic rats¿ hindpaw. Despite the great transcriptome similarity between control and diabetic rats, RNA sequencing comparative analysis already found 66 transcripts differentially expressed between the groups. Proteomic study also revealed alteration in protein expression in hyperglycemic rats DRG¿s: 3 proteins were considered differentially expressed between the two groups. Similarly, function-based categorization pointed to differentially expressed genes and proteins enrolled in oxidative estresse response, inflamation, apoptosis/cell survival, cell proliferation, and hyperalgesia/analgesia. Given their physiological roles, expression alterations in such genes and proteins suggest an alteration in cell metabolic program to switch to a more survival/regenerative one. Our results show that changes in transcriptome and proteomic profile start to appear very early in PDN and that these first alterations might be a strong effort for keeping cell homeostases. Hence, the present data may indicate how DRG cells are responding to hyperglycemia in its early stages and which mechanisms first fail to respond, further leading to cell damage and cell death. Knowing the first cell alterations in PDN might lead to more concrete targets for pharmaceutical interventions, that could more efficiently delay cell damageDoutoradoFisiologiaDoutora em Biologia Funcional e Molecular2011/23764-0FAPES

Genetic and proteomic expression analysis in patients with Alzheimer\'s disease: search for peripheral biomarkers

A Doença de Alzheimer (DA) é uma desordem neurodegenerativa influenciada por elementos genéticos e ambientais. O diagnóstico é baseado em parâmetros clínicos, mas sua confirmação é post-mortem, após avaliação patológica durante a autópsia. A identificação de biomarcadores baseados em tecido periférico como o sangue permitiria um diagnóstico menos invasivo e mais preciso, como também poderia servir como marcador de predisposição, conversão, progressão e resposta à tratamento. Para atingir tal objetivo, métodos de prospecção em larga escala de perfis de expressão gênica e protéica têm sido extensamente aplicados. O presente estudo se propôs a selecionar e validar potenciais candidatos a biomarcadores na DA, e também, de identificar potenciais biomarcadores pelo desenvolvimento de perfis proteômicos de plaquetas de pacientes. Dados publicados de microarray para DA foram comparados e genes com perfil de expressão similar em pelo menos dois estudos independentes foram selecionados. Foram identificados 4 genes de expressão aumentada em pacientes (HLADRB1, HLAB, CIRBP, S100A4) e 4 genes de expressão diminuída (CALM1, ATP5J2, KIF1B, FLOT1), que posteriormente foram submetidos a validação por PCR em tempo-real em amostras de RNA extraído a partir de leucócitos de sangue periférico de 20 pacientes e 20 controles idosos. Ao mesmo tempo, padronizamos e traçamos o perfil proteômico de pools de proteínas de plaquetas de pacientes do sexo feminino e masculino e seus respectivos controles idosos por Eletroforese Bidimensional (2-D). Dentre os genes analisados por meio de PCR em tempo-real, três (ATP5J2, S100A4 e KIF1B) apresentaram expressão aumentada no grupo DA em relação ao grupo controle (p >0,05). Estes genes podem estar envolvidos na resposta inflamatória periférica e indução da apoptose. A padronização do perfil proteômico por 2-DE foi bem sucedida e um conjunto de 5 proteínas diferencialmente expressas para ambos os gêneros foram obtidas, mas não foi possível a sua identificação por Espectrometria de Massas devido a massa limitada dessas proteínas em cada spot. Por ambas as técnicas foi possível identificar perfis diferentes de expressão gênica e protéica entre pacientes e controles idosos, demonstrando que sangue periférico é uma boa matriz de prospecção de biomarcadores de doenças neurodegenerativas.Alzheimer\'s disease (AD) is a neurodegenerative disorder influenced by genetic and environmental elements. The diagnosis is based on clinical parameters, but its confirmation needs post-mortem pathologic evaluation at autopsy. The identification of biomarkers based on peripheral tissue would allow a less invasive and more accurate diagnosis, but could also serve as a marker of predisposition, conversion, progression and response to treatment. To achieve this goal, methods of exploring large-scale gene expression profiles and protein have been extensively applied. The present study proposed to select and validate potential candidate genes for biomarkers in AD, and also identify potential biomarkers from proteomic profiles of platelets of these patients. Published microarray data for AD were compared and genes with similar expression profile in at least two independent studies were selected. We identified four up-regulated genes (HLADRB1, HLAB, CIRBP, S100A4) and four down-regulated genes in patients (CALM1, ATP5J2, KIF1B, FLOT1), which were then submitted to validation by real time PCR in RNA samples extracted from peripheral blood leukocytes of 20 patients and 20 elderly controls. At the same time, standardized, and traced the proteomic profile of platelet proteins pools of female patients and male elders and their respective controls by two-dimensional electrophoresis (2-D). Among the genes analyzed by real time PCR three of them (ATP5J2, S100A4 and KIF1B) showed increased expression in the AD group compared to the control group (p> 0.05). These genes may be involved in peripheral inflammatory response and induction of apoptosis. The standardization of proteomic profile by 2-DE has been successful and a set of five differentially expressed proteins in both genders were obtained, but could not be identified by mass spectrometry due to limited mass of these proteins in each spot. For both techniques were able to identify different patterns of gene and protein expression between patients and elderly controls, demonstrating that peripheral blood is a good prospect source of biomarkers for neurodegenerative diseases

Transcriptome analyses of the cortex and white matter of focal cortical dysplasia type II: Insights into pathophysiology and tissue characterization

IntroductionFocal cortical dysplasia (FCD) is a common cause of pharmacoresistant epilepsy. According to the 2022 International League Against Epilepsy classification, FCD type II is characterized by dysmorphic neurons (IIa and IIb) and may be associated with balloon cells (IIb). We present a multicentric study to evaluate the transcriptomes of the gray and white matters of surgical FCD type II specimens. We aimed to contribute to pathophysiology and tissue characterization.MethodsWe investigated FCD II (a and b) and control samples by performing RNA-sequencing followed by immunohistochemical validation employing digital analyses.ResultsWe found 342 and 399 transcripts differentially expressed in the gray matter of IIa and IIb lesions compared to controls, respectively. Cholesterol biosynthesis was among the main enriched cellular pathways in both IIa and IIb gray matter. Particularly, the genes HMGCS1, HMGCR, and SQLE were upregulated in both type II groups. We also found 12 differentially expressed genes when comparing transcriptomes of IIa and IIb lesions. Only 1 transcript (MTRNR2L12) was significantly upregulated in FCD IIa. The white matter in IIa and IIb lesions showed 2 and 24 transcripts differentially expressed, respectively, compared to controls. No enriched cellular pathways were detected. GPNMB, not previously described in FCD samples, was upregulated in IIb compared to IIa and control groups. Upregulations of cholesterol biosynthesis enzymes and GPNMB genes in FCD groups were immunohistochemically validated. Such enzymes were mainly detected in both dysmorphic and normal neurons, whereas GPNMB was observed only in balloon cells.DiscussionOverall, our study contributed to identifying cortical enrichment of cholesterol biosynthesis in FCD type II, which may correspond to a neuroprotective response to seizures. Moreover, specific analyses in either the gray or the white matter revealed upregulations of MTRNR2L12 and GPNMB, which might be potential neuropathological biomarkers of a cortex chronically exposed to seizures and of balloon cells, respectively

Transcriptome analyses of the cortex and white matter of focal cortical dysplasia type II: Insights into pathophysiology and tissue characterization

Introduction Focal cortical dysplasia (FCD) is a common cause of pharmacoresistant epilepsy. According to the 2022 International League Against Epilepsy classification, FCD type II is characterized by dysmorphic neurons (IIa and IIb) and may be associated with balloon cells (IIb). We present a multicentric study to evaluate the transcriptomes of the gray and white matters of surgical FCD type II specimens. We aimed to contribute to pathophysiology and tissue characterization. Methods We investigated FCD II (a and b) and control samples by performing RNA-sequencing followed by immunohistochemical validation employing digital analyses. Results We found 342 and 399 transcripts differentially expressed in the gray matter of IIa and IIb lesions compared to controls, respectively. Cholesterol biosynthesis was among the main enriched cellular pathways in both IIa and IIb gray matter. Particularly, the genes HMGCS1, HMGCR, and SQLE were upregulated in both type II groups. We also found 12 differentially expressed genes when comparing transcriptomes of IIa and IIb lesions. Only 1 transcript (MTRNR2L12) was significantly upregulated in FCD IIa. The white matter in IIa and IIb lesions showed 2 and 24 transcripts differentially expressed, respectively, compared to controls. No enriched cellular pathways were detected. GPNMB, not previously described in FCD samples, was upregulated in IIb compared to IIa and control groups. Upregulations of cholesterol biosynthesis enzymes and GPNMB genes in FCD groups were immunohistochemically validated. Such enzymes were mainly detected in both dysmorphic and normal neurons, whereas GPNMB was observed only in balloon cells. Discussion Overall, our study contributed to identifying cortical enrichment of cholesterol biosynthesis in FCD type II, which may correspond to a neuroprotective response to seizures. Moreover, specific analyses in either the gray or the white matter revealed upregulations of MTRNR2L12 and GPNMB, which might be potential neuropathological biomarkers of a cortex chronically exposed to seizures and of balloon cells, respectively