Research areas at ECB Javeriana

There is currently a gap in the understanding of the mechanisms that regulate neuroprotection in human neuronal and non-neuronal cells which must promptly be addressed by consolidating strategies that result in the development of therapies and treatments in traumatic brain injury and neurodegenerative diseases with common pathophysiologies. This calls for cooperative efforts to generate new data sets and new methodologies in the analysis and evaluation of data obtained during research. From the products generated during the research carried out by the Cellular and Molecular Therapy group of the Pontificia Universidad Javeriana, some collaborators, as well as from the advances and trends at national and international level. 

The neurosteroid Tibolone is a promising synthetic compound for its use as neuroprotector in astrocytes as a strategy for the treatment of brain damage and diseases such as Parkinson, Alzheimer, and processes such as neuroinflammation, astrogliosis, among others. However, little progress has been made in this field and even less using cells of human origin, in order to improve the possibilities and satisfactory results in human trials and, for example, to facilitate their evaluation during clinical trials. Thus, there is a need to improve the quality of life of such patients, alleviate the costs associated with both the different health care providers and the families who assume a large part of the care and economic support.

Neurosteroids such as tibolone are envisioned as a therapeutic target that could prevent, delay the onset, extend survival and improve the quality of life of patients with Neurological Disease, recent research has determined that tibolone acts on estrogen receptors found in different nerve cells, without generating the carcinogenic side effects that have been associated with estrogenic activity . Evidence indicates that estrogen receptors (ER) can regulate processes of apoptosis, regeneration and neuronal survival in areas affected by Neurological Disease, such as the amygdala, cortex, basal ganglia, hippocampus, hypothalamus and septum. However, the mechanisms of action and metabolic effects of this substance at the nervous system level remain unknown, due to the poor understanding of the biochemical basis of its action.

It is therefore necessary to explore this issue with new high-throughput technologies that allow simultaneous analysis of biomolecules at various cellular levels. In this regard, metabolomics is an approach that allows the analysis of metabolites from an integral perspective of cellular physiology. This approach discriminates cellular metabolic profiles in different biological contexts such as those associated with Neurological Diseases , specific metabolic lesions and exposure to protective therapeutic substances. However, the separate study of the metabolome does not provide a deep understanding, because the behavior of biological systems is determined by the complex interactions built between its components; therefore, an integrated approach is needed to study biological systems. 

In recent years, there have been innovative developments in computational and analytical technologies that have enabled the integration of large-scale data into computational systemic models, an approach known as systems biology. The integration of these technologies allows bidirectional benefits to be obtained. By virtue of this, 1) systems biology provides what is arguably the best context for interpreting metabolomics data , predicting different pathophysiological states of a system, through the analysis of modeling the structure of the biological network under study and 2) metabolomics data reduce and refine the solution space, generating predictions of metabolic phenotypes that are more accurate and much closer to the actual physiological state. In this context, the use of these tools enables the identification of metabolic mechanisms involved in physiological and pathological responses, the active search for therapeutic targets and the determination of biomarkers in different astrocyte biological scenarios. 

Therefore, the central problem of this project is the need to provide experimental and computational evidence to predict the mechanisms, enzymes, metabolic pathways involved, direct or indirect metabolic effects of tibolone and its role in protective processes in the astrocyte, in order to design more specific therapeutic strategies against metabolic insults such as lipotoxicity in glial cells.

Recently, it was shown that systemic injection of synthetic miRNAs, which had been suggested to act as tumor suppressors, successfully prevented the growth of metastases in animal models. This evidence suggests that therapies based on miRNAs could be used to treat cancer in metastatic stages or to prevent metastasis formation in the early stage of the disease, as well as shed new light on the path to new cancer therapies. One of the challenges for the establishment of miRNA-based therapies for cancer treatment is the selection of appropriate target molecules, which are the key to efficient therapy.

Among the miRNAs identified in humans, the C19MC miRNA cluster (Chromosome 19 miRNA Cluster) is mapped to chromosomal sub-band 19q13. This miRNA cluster plays a crucial role in reproduction, development and differentiation. This important role related to reproduction is reflected in its restricted expression to placental tissue, as previously demonstrated by different authors . Although its expression has also been demonstrated in pluripotent embryonic stem cells and its expression in the brain of fetuses, denoting its importance in the fine regulation of biological processes essential for the proper development of the central nervous system, without being found expressed in tumor cells. Notably, this placental tissue, has the ability to proliferate and invade the myometrium, using molecular mechanisms controlled by diverse genes, which are also involved in the initial stages of migration and invasion of the metastatic process in tumor cells, but are finely regulated in the placenta, C19MC cluster miRNAs attenuate trophoblast migration, regulating the invasive phenotype. Along the same lines, different bioinformatics analyses showed that some cluster members negatively regulate some genes closely associated with the promotion of cell proliferation and invasion, as well as genes involved in cell survival signaling through the PIK3/ATK, TNFs/NF-B and TRAIL pathway

The relevance of using the molecular mechanisms that control biological processes in the placenta as possible targets for study in cancer models is based on the similarities observed between germ cells, placental trophoblasts and cancer cells. John Beard proposed in 1906 the trophoblastic theory of cancer. This theory assumes that tumor cells show similar characteristics to trophoblasts, such as cell migration, invasion of the extracellular matrix, higher proliferation rates, and induction of angiogenesis. These biological processes are tightly controlled in trophoblasts, but their regulation is imbalanced in tumor cells, leading to cancer. In the placenta, a possible mechanism of control of these processes may be coordinated by miRNAs that are expressed exclusively in this tissue. These findings highlight the hypothesis that the C19MC cluster is expressed in placental tissue, for proper placental development, and its lack of expression in other tissues, including tumor, may imply that these miRNAs may act as important regulators of proliferation and invasion, making them tumor suppressors, but so far, the biological function and expression profiles of the C19MC cluster in cancer cells has not been systematically examined, nor is it known in a comprehensive manner.

In this context, the C19MC miRNA cluster becomes an important candidate to be studied within the framework of tumor development and progression. Some of the target genes of the C19MC miRNA cluster have been characterized in several types of cancer, such as breast and cervical cancer, in relation to cell proliferation processes by inhibition of apoptosis and promotion of cell migration and invasion. However, the role of members of the C19MC miRNA cluster of miRNAs in the tumor process is still unclear. Taking all of the above into consideration, we set out to evaluate the role of the C19MC microRNAs cluster on the regulation of biological functions related to tumor progression.

This project aims to identify the role of aromatase (CYP19A1) enzyme regulation in human astrocytes subjected to metabolic stress with fatty acids (GA), from the characterization of the sex-dependent mitochondrial response, through the differential analysis of the protein expression profile. Important contributions have been made on the protective role of steroids in the nervous system, through pharmacological treatment with estrogenic compounds or some similar molecules that modulate this response. However, very few studies take into account sexual dimorphism, a condition that has been described that could alter the response to this type of treatment.  One of the main approaches in the characterization of the sexual response is the study of the hormonal component. The brain is exposed to high concentrations of endogenous estrogenic compounds, where aromatase regulates the production and transformation of several of these compounds and can be used in a model that contributes both to the study of sexual differences and to the understanding of the protective mechanisms mediated by the endogenous hormonal component in the brain. This could be of great interest in order to propose more effective, specific and selective therapeutic mechanisms in the prevention or treatment of pathologies affecting the central nervous system (CNS).

We intend to develop an experimental model based on aromatase silencing in human male and female astrocytes through the Crispr/Cas9 system, ensuring the inhibition of endogenous estrogen production. Subsequently, they will be stimulated with GA characterizing parameters such as cell viability, astrocyte activation and mitochondrial function among others. For the proteomic analysis, we intend to approach the problem from the extraction, quantification and expression of proteins using biochemical techniques (Western Blot), but we also intend to complement this research with bioinformatics tools that are invaluable, since 1) they successfully simulate changes at the systemic level, 2) they are ideal to integrate omic information to improve its biological representation and 3) they allow us to identify a considerable amount of proteins that are being regulated in the model. Within this, we intend to find not only the differential expression of mitochondrial proteins involved in protective pathways and mechanisms, but also the influence that sex may have. This suggests that, depending on sex, there could be differences at the mitochondrial level, which could promote a deterioration in cellular integrity, generating the appearance of pathologies that affect the CNS.

Obesity as well as other related metabolic disorders can lead to neurodegeneration phenomena. In this sense, many studies have shown that subjects suffering from obesity show a higher risk of developing different neurodegenerative diseases . Obesity is considered a syndrome of multifactorial etiology characterized by an excessive accumulation and release of fatty acids (FA) in adipose and non-adipose tissue. In this way, the excess of FA generates a metabolic condition known as lipotoxicity; which triggers pathological cellular and molecular responses that can produce dysregulation of homeostasis and decrease in cell viability. This condition is a hallmark for these diseases, that represent a heterogeneous group of disorders that are characterized by progressive dysfunction of neurons and astrocytes. 

Astrocytes are particularly sensitive to lipotoxicity since the effects of this condition are more impactful in these cells given their crucial role in energy production and oxidative stress management in the brain. Recent findings suggest that astrocytes play a critical role in the function and protection of the central nervous system (CNS). For this reason, loss of normal astrocytic function may be a primary contributor to neurodegeneration. However, analyzing cellular mechanisms associated to these conditions represents a challenge. In this regard, metabolomics is an approach that allows biochemical analysis from a comprehensive perspective of cell physiology. This technique allows determining cellular metabolic profiles in different biological contexts such as those associated with NDs and specific metabolic insults such as lipotoxicity. Nonetheless, the data provided by metabolomics can be complex and hence hard to interpretate. 

For this reason, alternative data analysis techniques such as Machine Learning have been growing exponentially in areas related to omics data. Here, we created a ML model that with results of 93% of area under the ROC curve, sensibility value of 80% and specificity value of 93%. In this work the goal is to analyze the metabolomic profiles of astrocytes in lipotoxic conditions in order to provide powerful insights such as potential biomarkers for scenarios of lipotoxicity induced by palmitic acid, that to our knowledge haven´t been identified in human astrocytes and are proposed as candidates for further research and validation.