Biology subjects

Goto-Silva, L.

Publications and source records attributed to Goto-Silva, L..

11 recordsLinked to original sources

bioRxiv2025

Proteome Complexity Scales with Architecture in Human Neural Models

Mass-spectrometry-based proteomics enables high-throughput identification and quantification of proteins, providing molecular insight into neural development and cellular organization. Applying this approach to in vitro systems of increasing architectural complexity, we compared immortalized monolayer cell lines with two tissue-like neural models. Here, we present a comparative proteomic analysis of three human neural models: SH-SY5Y neuroblastoma cells (2D), neurospheres, and cerebral organoids. Protein profiling revealed a stepwise increase in molecular complexity, with enhanced detection of neural-related proteins linked to axon guidance, synapse formation, and GTPase signaling. This trend was most pronounced in tissue-like models, underscoring their suitability for studying neuronal maturation and circuit assembly. Functional enrichment analyses showed progressive acquisition of neurodevelopmental programs, including synaptic vesicle cycling, presynaptic organization, neurotransmitter regulation, and late-stage gliogenesis, accompanied by increased expression of astrocytic and oligodendrocytic markers. Kinase diversity also increased across models, reaching up to 210 regulatory kinases in organoids, many implicated in neural development and degeneration. Gene set enrichment for neurological pathways mirrored this trend, aligning proteomic complexity with disease relevance. Regional brain mapping further indicated that organoids most closely recapitulate the protein architecture of the human CNS. Together, these findings demonstrate that tissue-like neural models provide richer proteomic landscapes that approximate in vivo brain biology and support the application of integrative proteomics in neuroscience and translational research.

neuroscience↗

bioRxiv2024

LSD Modulates Proteins Involved in Cell Proteostasis, Energy Metabolism and Neuroplasticity in Human Brain Organoids

Proteomic analysis of human cerebral organoids may reveal how psychedelics regulate biological processes, shedding light on drug-induced changes in the brain. This study elucidates the proteomic alterations induced by lysergic acid diethylamide (LSD) in human cerebral organoids. By employing high-resolution mass spectrometry-based proteomics, we quantitatively analyzed the differential abundance of proteins in cerebral organoids exposed to LSD. Our findings indicate changes in proteostasis, energy metabolism, and neuroplasticity-related pathways. Specifically, LSD exposure led to alterations in protein synthesis, folding, autophagy, and proteasomal degradation, suggesting a complex interplay in the regulation of neural cell function. Additionally, we observed modulation in glycolysis and oxidative phosphorylation, crucial for cellular energy management and synaptic function. In support of the proteomic data, complementary experiments demonstrated LSDs potential to enhance neurite outgrowth in vitro, confirming its impact on neuroplasticity. Collectively, our results provide a comprehensive insight into the molecular mechanisms through which LSD may affect neuroplasticity and potentially contribute to therapeutic effects for neuropsychiatric disorders.

neuroscience↗

bioRxiv2021

Proteomic changes induced by harmine in human brain organoids reveal signaling pathways related to neuroprotection

Harmine is a {beta}-carboline found in Banisteriopsis caapi, a constituent of ayahuasca brew. Ayahuasca is consumed as a beverage in native Americans sacred rituals and in religious ceremonies in Brazil. Throughout the years, the beneficial effects of ayahuasca to improve mental health and life quality have been reported, which propelled the investigation of its therapeutic potential to target neurological disorders such as depression and anxiety. Indeed, antidepressant effects of ayahuasca have been described, raising the question of which cellular mechanisms might underlie those effects. Previous animal studies describe potential neuroprotective mechanisms of harmine, including anti-inflammatory and antioxidant activities, and neurotrophin signaling activation. However, the cellular and molecular mechanisms modulated by harmine in human models remain less investigated. Here we analyzed the short-term changes in the proteome of human brain organoids treated with harmine using shotgun mass spectrometry. Harmine upregulates proteins related to synaptic vesicle cycle, cytoskeleton-dependent intracellular transport, cell cycle, glucose transporter-4 translocation, and neurotrophin signaling pathway. In addition, protein expression levels of Akt and phosphorylated CREB were increased after 24 hour-treatment. Our results shed light on the potential mechanisms that may underlie harmine-induced neuroprotective effects.

cell biology↗

bioRxiv2021

DYRK1A role in microtubule-based axonal transport regulates the retrograde dynamics of APP vesicles in human neurons

In Alzheimers Disease (AD) the abnormal intracellular distribution of the amyloid precursor protein (APP) affects its processing and, consequently, the generation of A{beta}. Axonal transport plays key roles in the neuronal distribution of APP. The dual-specificity-tyrosine phosphorylation-regulated-kinase-1A (DYRK1A) has been associated with AD onset since its overexpression was found in Down syndrome and sporadic AD patients. Experimental evidence confirmed that APP and tau phosphorylations are mediated by DYRK1A. Moreover, DYRK1A can regulate the cytoskeletal architecture by phosphorylation of both tubulin subunits and microtubule-associated proteins. Therefore, we tested whether DYRK1A has a role in APP axonal transport regulation. We developed highly-polarized human-derived neurons in 2D cultures. At day 14 after terminal plating we inhibited DYRK1A for 48hs with harmine (7.5 M). DYRK1A overexpression was induced to perform live-cell imaging of APP-loaded vesicles in axons and analyzed transport dynamics. A custom-made MATLAB routine was developed to track and analyze single particle dynamics. Short-term harmine treatment reduced axonal APP vesicles density, due to a reduction in retrograde particles. Contrarily, DYRK1A overexpression enhanced axonal APP density, due to an increase in the retrograde and stationary component. Moreover, both harmine-mediated DYRK1A inhibition and DYRK1A overexpression revealed opposite phenotypes on single particle dynamics, affecting primarily dynein processivity. These results revealed an increased retrieval of distal APP vesicles in axons when DYRK1A is overexpressed and reinforce the suggestion that DYRK1A enhance APP endocytosis.. Taken together our results suggest that DYRK1A has a relevant role in the regulation of axonal transport and sub-cellular positioning of APP vesicles. Therefore, our work shed light on the role of DYRK1A in axonal transport regulation, and the putative use of harmine to restore axonal transport impairments.

neuroscience↗

bioRxiv2021

Quantitative profiling of axonal guidance proteins during the differentiation of human neurospheres

Axon guidance is required for the establishment of brain circuits. Whether much of the molecular basis of axon guidance is known from animal models, the molecular machinery coordinating axon growth and pathfinding in humans remains to be elucidated. The use of induced pluripotent stem cells (iPSC) from human donors has revolutionized in vitro studies of the human brain. iPSC can be differentiated into neuronal stem cells which can be used to generate neural tissue-like cultures, known as neurospheres, that reproduce, in many aspects, the cell types and molecules present in the brain. Here, we analyzed quantitative changes in the proteome of neurospheres during differentiation. Relative quantification was performed at early time points during differentiation using iTRAQ-based labeling and LC-MS/MS analysis. We identified 6,438 proteins, from which 433 were downregulated and 479 were upregulated during differentiation. We show that human neurospheres have a molecular profile that correlates to the fetal brain. During differentiation, upregulated pathways are related to neuronal development and differentiation, cell adhesion, and axonal guidance whereas cell proliferation pathways were downregulated. We developed a functional assay to check for neurite outgrowth in neurospheres and confirmed that neurite outgrowth potential is increased after 10 days of differentiation and is enhanced by increasing cyclic AMP levels. The proteins identified here represent a resource to monitor neurosphere differentiation and coupled to the neurite outgrowth assay can be used to functionally explore neurological disorders using human neurospheres as a model.

neuroscience↗

bioRxiv2021

A cannabinoid receptor agonist shows anti-inflammatory and survival properties in human SARS-CoV-2-infected iPSC-derived cardiomyocytes

Coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which can infect several organs, especially impacting respiratory capacity. Among the extrapulmonary manifestations of COVID-19 is myocardial injury, which is associated with a high risk of mortality. Myocardial injury, caused directly or indirectly by SARS-CoV-2 infection, can be triggered by inflammatory processes that cause damage to the heart tissue. Since one of the hallmarks of severe COVID-19 is the "cytokine storm", strategies to control inflammation caused by SARS-CoV-2 infection have been considered. Cannabinoids are known to have anti-inflammatory properties by negatively modulating the release of pro-inflammatory cytokines. Herein, we investigated the effects of the cannabinoid agonist WIN 55,212-2 (WIN) in human iPSC-derived cardiomyocytes (hiPSC-CMs) infected with SARS-CoV-2. WIN did not modify angiotensin-converting enzyme II protein levels, nor reduced viral infection and replication in hiPSC-CMs. On the other hand, WIN reduced the levels of interleukins 6, 8, 18 and tumor necrosis factor-alpha (TNF-) released by infected cells, and attenuated cytotoxic damage measured by the release of lactate dehydrogenase (LDH). Our findings suggest that cannabinoids should be further explored as a complementary therapeutic tool for reducing inflammation in COVID-19 patients.

cell biology↗

bioRxiv2021

Inhibition of SARS-CoV-2 infection in human cardiomyocytes by targeting the Sigma-1 receptor disrupts cytoskeleton architecture and contractility

Heart dysfunction, represented by conditions such as myocarditis and arrhythmia, has been reported in COVID-19 patients. Therapeutic strategies focused on the cardiovascular system, however, remain scarce. The Sigma-1 receptor (S1R) has been recently proposed as a therapeutic target because its inhibition reduces SARS-CoV-2 replication. To investigate the role of S1R in SARS-CoV-2 infection in the heart, we used human cardiomyocytes derived from induced pluripotent stem cells (hiPSC-CM) as an experimental model. Here we show that the S1R antagonist NE-100 decreases SARS-CoV-2 infection and viral replication in hiPSC-CMs. Also, NE-100 reduces cytokine release and cell death associated with infection. Because S1R is involved in cardiac physiology, we investigated the effects of NE-100 in cardiomyocyte morphology and function. We show that NE-100 compromises cytoskeleton integrity and reduces beating frequency, causing contractile impairment. These results show that targeting S1R to challenge SARS-CoV-2 infection may be a useful therapeutic strategy but its detrimental effects in vivo on cardiac function should not be ignored.

cell biology↗

bioRxiv2020

Non-permissive SARS-CoV-2 infection of neural cells in the developing human brain and neurospheres

Coronavirus disease 2019 (COVID-19) was initially described as a viral infection of the respiratory tract. It is now known, however, that several other organs are affected, including the brain. Neurological manifestations such as stroke, encephalitis, and psychiatric conditions have been reported in COVID-19 patients, but the neurotropic potential of the virus is still debated. Herein, we sought to investigate SARS-CoV-2 infection in human neural cells. We demonstrated that SARS-CoV-2 infection of neural tissue is non-permissive, however, it can elicit inflammatory response and cell damage. These findings add to the hypothesis that most of the neural damage caused by SARS-CoV-2 infection is due to a systemic inflammation leading to indirect harmful effects on the central nervous system despite the absence of local viral replication.

neuroscience↗

bioRxiv2019

d-Lysergic acid diethylamide has major potential as a cognitive enhancer

Psychedelic agonists of serotonin receptors induce neural plasticity and synaptogenesis, but their potential to enhance learning remains uncharted. Here we show that a single dose of d-LSD, a potent serotonergic agonist, increased novel object preference in young and adult rats several days after treatment. d-LSD alone did not increase preference in old animals, but could rescue it to young levels when followed by a 6-day exposure to enriched environment (EE). Mass spectrometry-based proteomics in human brain organoids treated with d-LSD showed upregulation of proteins from the presynaptic active zone. A computational model of synaptic connectivity in the hippocampus and prefrontal cortex suggests that d-LSD enhances novelty preference by combining local synaptic changes in mnemonic and executive regions, with alterations of long-range synapses. Better pattern separation within EE explained its synergy with d-LSD in rescuing novelty preference in old animals. These results advance the use of d-LSD in cognitive enhancement.

neuroscience↗

bioRxiv2018

Retrograde transport of Akt by a neuronal Rab5-APPL1 endosome

Long-distance axonal trafficking plays a critical role in neuronal function, and transport defects have been linked to neurodegenerative disorders. Various lines of evidence suggest that the small GTPase Rab5 plays a role in neuronal signaling via early endosomal transport. Here, we characterized the motility of Rab5 endosomes in primary cultures of mouse hippocampal pyramidal cells by live-cell imaging and showed that they exhibit bi-directional long-range motility in axons, with a strong bias toward retrograde transport. Characterization of key Rab5 effectors revealed that endogenous Rabankyrin-5, Rabenosyn-5 and APPL1 are all present in axons. Further analysis of APPL1-positive endosomes showed that, similar to Rab5-endosomes, they display more frequent long-range retrograde than anterograde movement, with the endosomal levels of APPL1 correlated with faster retrograde movement. Interestingly, APPL1-endosomes transport the neurotrophin receptor TrkB and mediate retrograde axonal transport of the kinase Akt1. FRET analysis revealed that APPL1 and Akt1 interact in an endocytosis-dependent manner. We conclude that Rab5-APPL1 endosomes exhibit the hallmarks of axonal signaling endosomes to transport Akt1 in hippocampal pyramidal cells.

cell biology↗

bioRxiv2018

Computational fluid dynamic analysis reveals the underlying physical forces playing a role in 3D multiplex brain organoid cultures

Organoid cultivation in suspension culture requires agitation at low shear stress to allow for nutrient diffusion, which preserves tissue structure. Multiplex systems for organoid cultivation have been proposed, but whether they meet similar shear stress parameters as the regularly used spinner flask and its correlation with the successful generation of brain organoids, has not been determined. Herein, we used computational fluid dynamics (CFD) analysis to compare two multiplex culture conditions: steering plates on an orbital shaker and the use of a previously described bioreactor. The bioreactor had low speed and high shear stress regions that may affect cell aggregate growth, depending on volume, whereas the CFD parameters of the steering plates were closest to the parameters of the spinning flask. Our protocol improves the initial steps of the standard brain organoid formation, and organoids produced therefrom displayed regionalized brain structures, including retinal pigmented cells. Overall, we conclude that suspension culture on orbital steering plates is a cost-effective practical alternative to previously described platforms for the cultivation of brain organoids for research and multiplex testing.\n\nHighlightsO_LIImprovements to organoid preparation protocol\nC_LIO_LIMultiplex suspension culture protocol successfully generate brain organoids\nC_LIO_LIComputational fluid dynamics (CFD) reveals emerging properties of suspension cultures\nC_LIO_LICFD of steering plates is equivalent to that of spinner flask cultures\nC_LI

bioengineering↗