Biology subjects

Saraiva, C.

Publications and source records attributed to Saraiva, C..

7 recordsLinked to original sources

bioRxiv2025

Autophagy Dysfunction in iPSCs-Derived Neurons and Midbrain Organoids Carrying a SNCA Triplication

Parkinsons disease (PD), characterized by -Synuclein aggregation and dopaminergic neuronal loss, has no current cure. Autophagy is critical for -Synuclein clearance, yet its real-time dynamics remain challenging to assess in human-relevant systems. Here, we used live-cell imaging to assess autophagy within human neuronal cultures and midbrain organoids (hMOs) derived from induced pluripotent stem cells (iPSCs) of PD patients carrying a triplication of the -Synuclein gene (3xSNCA). Using the LC3-Rosella dual-fluorescent reporter, we quantified autolysosomes dynamics in real time. In 3xSNCA neuronal cultures, we detected early autophagy defects. In 3xSNCA hMOs, reduced autolysosome area, increased total and phosphorylated -Synuclein (pS129), and decreased electrophysiological activity were observed at 50 days of differentiation (DoD). By 70 DoD, autophagy impairment became more pronounced, overlapping with dopaminergic neuron loss. These findings support the use of human iPSCs-derived models to study autophagy dysfunction in PD and demonstrate a temporal correlation between impaired autophagy, -Synuclein pathology and neuronal degeneration.

neuroscience↗

bioRxiv2025

Vascularized midbrain assembloids show neuroinflammation and dopaminergic neuron vulnerability in Parkinsons Disease

The use of micro-physiological systems has rapidly risen in the last years due to their translatability and complex cellular composition. Human midbrain-specific organoids contain neuroectoderm-derived cell types and are suitable for brain region-specific disease modeling. However, the lack of vasculature in these systems reduces oxygenation and nutrient supply. Furthermore, neurovascular interactions cannot be studied, and disease phenotypes affecting vascular and neurovascular structures cannot be assessed. To overcome these limitations, in this work, we successfully incorporated a vascular network into midbrain organoids by fusion with vascular organoids. Midbrain-vascular assembloids are enriched in vascular cells and microglia. We observed a decrease in hypoxia and cell death in these assembloids. Furthermore, microglia and endothelial cells increased their morphological complexity. Assembloids derived from a Parkinsons disease patient carrying a LRRK2-G2019S mutation displayed a pro-inflammatory phenotype and altered electrophysiological properties. Midbrain-vascular assembloids increase the midbrain model complexity and allow for neuroinflammation studies in Parkinsons disease.

neuroscience↗

bioRxiv2024

Deciphering shared molecular dysregulation across Parkinson's disease variants using a multi-modal network-based data integration and analysis

Parkinsons disease (PD) is a progressive neurodegenerative disorder with no effective treatment. Advances in neuroscience and systems biomedicine now enable the use of complex patient-specific in vitro disease models and cutting-edge computational tools for data integration, enhancing our understanding of complex PD mechanisms. To explore common biomedical features across monogenic PD forms, we developed a knowledge graph (KG) by integrating previously published high-content imaging and RNA sequencing data of PD patient-specific midbrain organoids harbouring LRRK2-G2019S, SNCA triplication, GBA-N370S or MIRO1-R272Q mutations with publicly available biological data. Furthermore, we generated a single-cell RNA sequencing dataset of midbrain organoids derived fromidiopathic PD patients (IPD) to stratify IPD patients towards genetic forms of PD. Despite high PD heterogeneity, we found that common transcriptomic dysregulation in monogenic PD forms is reflected in IPD glial cells. In addition, dysregulation in ROBO signalling might be involved in shared pathophysiology between monogenic PD and IPD cases.

systems biology↗

bioRxiv2023

Parkinson's disease-related Miro1 mutation induces mitochondrial dysfunction and loss of dopaminergic neurons in vitro and in vivo

The complex and heterogeneous nature of Parkinsons disease (PD) is still not fully understood, however, increasing evidence supports mitochondrial impairments as a major driver of neurodegeneration in PD. Recently, the regulator of mitochondrial homeostasis Miro1 has been linked genetically and pathophysiologically to PD. Using 2D and 3D patient-based induced pluripotent stem cells models, including an isogenic control, showed that the Miro1 p.R272Q mutation leads to mitochondrial impairments including increased oxidative stress, disrupted mitochondrial bioenergetics and altered metabolism. This was accompanied by increased -synuclein levels in 2D dopaminergic neurons and by a significant reduction of dopaminergic neurons within midbrain organoids. Knock-in mice expressing mutant p.R285Q Miro1 (orthologue of the human p.R272Q mutation) confirmed the PD-specific dopaminergic neuronal loss in the substantia nigra, accumulation of striatal phosphorylated -synuclein accompanied by behavioral alterations. These findings demonstrate that mutant Miro1 is sufficient to comprehensively model PD-relevant phenotypes in vitro and in vivo, reinforcing its pivotal role in PD pathogenesis.

neuroscience↗

bioRxiv2023

Age-induced midbrain-striatum assembloids model early phenotypes of Parkinsons disease

Parkinsons disease (PD), one of the most common aging-associated neurodegenerative disorders, is characterised by nigrostriatal pathway dysfunction, caused by the gradual loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc) of the midbrain and the dopamine depletion in the striatum. State of the art, human in vitro models are enabling the study of the dopaminergic neurons loss, but not the dysregulation of the dopaminergic network in the nigrostriatal pathway. Additionally, these models do not incorporate aging characteristics which potentially contribute to the development of PD. Therefore, it is conceivable that research conducted using these models overlooked numerous processes that contribute to diseases phenotypes. Here we present a nigrostriatal pathway model based on midbrain-striatum assembloids with inducible aging. We show that these assembloids are capable of developing characteristics of the nigrostriatal connectivity, with catecholamine release from the midbrain to striatum and synapse formation between midbrain and striatal neurons. Moreover, Progerin-overexpressing assembloids acquire aging traits that lead to early phenotypes of PD. This new model shall help to reveal the contribution of aging as well as nigrostriatal connectivity to the onset and progression of PD.

neuroscience↗

bioRxiv2022

Microglia integration into human midbrain organoids leads to increased neuronal maturation and functionality.

The human brain is a complex, three-dimensional structure. To better recapitulate brain complexity, recent efforts have focused on the development of human specific midbrain organoids. Human iPSC-derived midbrain organoids consist of differentiated and functional neurons, which contain active synapses, as well as astrocytes and oligodendrocytes. However, the absence of microglia, with their ability to remodel neuronal networks and phagocytose apoptotic cells and debris, represents a major disadvantage for the current midbrain organoid systems. Additionally, neuro-inflammation related disease modeling is not possible in the absence of microglia. So far, no studies about the effects of human iPSC-derived microglia on midbrain organoid neural cells have been published. Here we describe an approach to derive microglia from human iPSCs and integrate them into iPSC-derived midbrain organoids. Using single nuclear RNA Sequencing, we provide a detailed characterization of microglia in midbrain organoids as well as the influence of their presence on the other cells of the organoids. Furthermore, we describe the effects that microglia have on cell death and oxidative stress- related gene expression. Finally, we show that microglia in midbrain organoids affect synaptic remodeling and increase neuronal excitability. Altogether, we show a more suitable system to further investigate brain development, as well as neurodegenerative diseases and neuro- inflammation. Main Points- Macrophage precursors can be efficiently co-cultured with midbrain organoids, they integrate into the tissue and differentiate into microglia in 3D. - Organoids containing microglia have a smaller size and show a down-regulation of oxidative stress-related genes. - Organoids co-cultured with microglia show differences in genes related to synaptic remodeling and action potential, as well as a more spontaneous action potential firing.

neuroscience↗

bioRxiv2021

Synaptic decline precedes dopaminergic neuronal loss in human midbrain organoids harboring a triplication of the SNCA gene

1.Increased levels of the protein alpha-synuclein (-syn) are associated with the development of neurodegenerative diseases like Parkinsons disease (PD). In physiological conditions, -syn modulates synaptic plasticity, neurogenesis and neuronal survival. However, its pathogenic accumulation and aggregation results in toxicity and neurodegeneration. Here, we used a PD patient specific midbrain organoid model derived from induced pluripotent stem cells harboring a triplication in the SNCA gene to study PD-associated phenotypes. The model recapitulates the two main hallmarks of PD, which are -syn aggregation and loss of dopaminergic neurons. Additionally, impairments in astrocyte differentiation were detected. Transcriptomics data indicate that synaptic function is impaired in PD specific midbrain organoids. This is further confirmed by alterations in synapse number and electrophysiological activity. We found that synaptic decline precedes neurodegeneration. Finally, this study substantiates that patient specific midbrain organoids allow a personalized phenotyping, which make them an interesting tool for precision medicine and drug discovery.

neuroscience↗