The evolutionarily conserved splicing regulator neuro-oncological ventral antigen 1 (NOVA1) plays a key role in neural development and function. NOVA1 also includes a protein-coding difference between the modern human genome and Neanderthal and Denisovan genomes. To investigate the functional importance of an amino acid change in humans, we reintroduced the archaic allele into human induced pluripotent cells using genome editing and then followed their neural development through cortical organoids. This modification promoted slower development and higher surface complexity in cortical organoids with the archaic version of NOVA1. Moreover, levels of synaptic markers and synaptic protein coassociations correlated with altered electrophysiological properties in organoids expressing the archaic variant. Our results suggest that the human-specific substitution in NOVA1, which is exclusive to modern humans since divergence from Neanderthals, may have had functional consequences for our species’ evolution.

The cytoskeleton (CSK) is a tensed fiber framework that supports, shapes and stabilizes the cell. The CSK is in a constant state of remodeling, moreover, which is an active non-equilibrium thermodynamic process. We report here that cytoskeletal remodeling involves reconfigurations that are not only sudden but also are transmitted to great distances within the cell in a fashion reminiscent of quakes in the Earth's crust. Remarkably, these events in the cell conform both qualitatively and quantitatively to empirical laws typical of earthquakes, including hierarchical fault structures, cumulative energy distributions following the Gutenberg-Richter law, and rate of after-shocks following Omori's law. While it is well-established that remodeling and stabilization of the cytoskeleton are non-equilibrium process, these new unanticipated observations establish that these processes are also remarkably non-local and strongly cooperative.

Neonatal cardiomyocytes are instrumental for disease modeling, but the effects of different cell extraction methods on basic cell biological processes remain poorly understood. We assessed the influence of two popular methods to extract rat neonatal cardiomyocytes, Pre-plating (PP), and Percoll (PC) on cell structure, metabolism, and function. Cardiomyocytes obtained from PP showed higher gene expression for troponins, titin, and potassium and sodium channels compared to PC. Also, PP cells displayed higher levels of troponin I protein. Cells obtained from PC displayed higher lactate dehydrogenase activity and lactate production than PP cells, indicating higher anaerobic metabolism after 8 days of culture. In contrast, reactive oxygen species levels were higher in PP cells as indicated by ethidium and hydroxyethidium production. Consistent with these data, protein nitration was higher in PP cells, as well as nitrite accumulation in cell medium. Moreover, PP cells showed higher global intracellular calcium under basal and 1 mM isoprenaline conditions. In a calcium-transient assessment under electrical stimulation (0.5 Hz), PP cells displayed higher calcium amplitude than cardiomyocytes obtained from PC and using a traction force microscope technique we observed that PP cardiomyocytes showed the highest relaxation. Collectively, we demonstrated that extraction methods influence parameters related to cell structure, metabolism, and function. Overall, PP derived cells are more active and mature than PC cells, displaying higher contractile function and generating more reactive oxygen species. On the other hand, PC derived cells display higher anaerobic metabolism, despite comparable high yields from both protocols.

Mechanobiologycal and redox processes sinergize to regulate physiological or pathological vascular conditions. More specifically, adaptations to cyclic stretch on vascular smooth muscle cells (VSMC) have been shown to be redox‐regulated. However, mechanisms connecting mechanoadaptation to oxidant generation are still unclear. Previously, we reported that protein disulfide isomerase A1 (PDI), a redox chaperone from endoplasmic reticulum, is targeted to peri/epicellular (pec) space and supports an anti‐constrictive remodeling effect in balloon‐injured arteries by means of cytoskeleton and extracellular matrix architecture organization. We hypothesized that pec PDI acts as a global redox adaptor by connecting oxidant generation with responses to extra‐ or intracellular forces. First, pecPDI inhibition through specific neutralizing antibody (PDI Ab) incubation, prevented stress fiber assembly in response to prolonged exposure to equibiaxial stretch (10–12% at 1 Hz for 24 h). In addition, uniaxial stretch promotes cell repositioning perpendicularly to stretch orientation. Such response was also regulated by pecPDI, as PDI Ab treatment attenuated cell alignment in stretched VSMC at 4h. Through biotinylation experiments followed by western detection, we showed that pecPDI sustains a pro‐oxidant effect on beta1 integrin thiols. To address whether pecPDI organizes intracellular force distribution in such events, we used traction force microscopy, which revealed a significantly decreased net contractile moment in PDGF‐exposed VSMC after pecPDI neutralization (0.70 ±0.08 AU vs control=0.9 ±0.09, p<0.05). Thus, pecPDI involvement in mechanoresponse is not only limited to external forces. The balance between intracellular traction forces and cell adhesion governs cell migration. Indeed, in a model of single VSMC migration, pecPDI impaired migration persistence without affecting total distance or velocity. Since the Rho GTPase RhoA is known to act as a master mechanoregulator, we addressed if downstream pecPDI‐related mechanisms involve RhoA. Neither RhoA expression nor total activity were affected by pecPDI inhibition. However, the polarized distribution of RhoA or caveolin‐3 clusters promoted by cyclic stretch was disrupted by pecPDI inhibition, which promoted a non‐polarized pattern of RhoA/caveolin‐3 cluster colocalization. Moreover, we searched for pecPDI effects on localized RhoA activity using a FRET biosensor. The higher local RhoA activity at cell protrusions compared to perinuclear regions was disrupted by pecPDI neutralization, confirming its regulation of localized RhoA activation. In conclusion, pecPDI acts as a redox organizer able to restrict the noise of cytoskeletal repositioning during mechanoresponses in VSMC. This effect may have several implications, including the role of redox processes and pecPDI on vascular remodeling. Support or Funding Information Supported by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP), CEPID‐Redoxoma grant 2013/07937‐8 and scholarship grant 2013/17115‐5 This abstra...

Agradeço primeiramente ao meu orientador Adriano Mesquita Alencar. Além de inicialmente ter me aceitado como aluna de doutorado, esteve sempre presente, disposto a qualquer ajuda necessária. Um grande exemplo a ser seguido, cujos ensinamentos me acompanharão por toda minha vida acadêmica. Mais do que um orientador, um grande amigo que será levado por toda a vida. Agradeço a todos do grupo e participantes do LabM 2. Especialmente Alexandre, Diana,

Epilepsy is a disorder of the brain characterized by the predisposition to generate recurrent unprovoked seizures, which involves reshaping of neuronal circuitries based on intense neuronal activity. In this review, we first detailed the regulation of plasticity-associated genes, such as ARC, GAP-43, PSD-95, synapsin, and synaptophysin. Indeed, reshaping of neuronal connectivity after the primary, acute epileptogenesis event increases the excitability of the temporal lobe. Herein, we also discussed the heterogeneity of neuronal populations regarding the number of synaptic connections, which in the theoretical field is commonly referred as degree. Employing integrate-and-fire neuronal model, we determined that in addition to increased synaptic strength, degree correlations might play essential and unsuspected roles in the control of network activity. Indeed, assortativity, which can be described as a condition where high-degree correlations are observed, increases the excitability of neural networks. In this review, we summarized recent topics in the field, and data were discussed according to newly developed or unusual tools, as provided by mathematical graph analysis and high-order statistics. With this, we were able to present new foundations for the pathological activity observed in temporal lobe epilepsy.

Critical dynamics have been postulated as an ideal regime for neuronal networks in the brain, considering optimal dynamic range and information processing. Herein, we focused on how information entropy encoded in spatiotemporal activity patterns may vary in critical networks. We employed branching process based models to investigate how entropy can be embedded in spatiotemporal patterns. We determined that the information capacity of critical networks may vary depending on the manipulation of microscopic parameters. Specifically, the mean number of connections governed the number of spatiotemporal patterns in the networks. These findings are compatible with those of the real neuronal networks observed in specific brain circuitries, where critical behavior is necessary for the optimal dynamic range response but the uncertainty provided by high entropy as coded by spatiotemporal patterns is not required. With this, we were able to reveal that information processing can be optimized in neuronal networks beyond critical states.

Anoxia is one of the most prevalent causes of neonatal morbidity and mortality, especially in preterm neonates, constituting an important public health problem due to permanent neurological sequelae observed in patients. Oxygen deprivation triggers a series of simultaneous cascades, culminating in cell death mainly located in more vulnerable metabolic brain regions, such as the hippocampus. In the process of cell death by oxygen deprivation, cytosolic calcium plays crucial roles. Intracellular inositol 1,4,5-trisphosphate receptors (IP3Rs) are important regulators of cytosolic calcium levels, although the role of these receptors in neonatal anoxia is completely unknown. This study focused on the functional role of inositol 1,4,5-trisphosphate receptor type 1 (IP3R1) in rat hippocampus after neonatal anoxia. Quantitative real-time PCR revealed a decrease of IP3R1 gene expression 24 hours after neonatal anoxia. We detected that IP3R1 accumulates specially in CA1, and this spatial pattern did not change after neonatal anoxia. Interestingly, we observed that anoxia triggers translocation of IP3R1 to nucleus in hippocampal cells. We were able to observe that anoxia changes distribution of IP3R1 immunofluorescence signals, as revealed by cluster size analysis. We next examined the role of IP3R1 in the neuronal cell loss triggered by neonatal anoxia. Intrahippocampal injection of non-specific IP3R1 blocker 2-APB clearly reduced the number of Fluoro-Jade C and Tunel positive cells, revealing that activation of IP3R1 increases cell death after neonatal anoxia. Finally, we aimed to disclose mechanistics of IP3R1 in cell death. We were able to determine that blockade of IP3R1 did not reduced the distribution and pixel density of activated caspase 3-positive cells, indicating that the participation of IP3R1 in neuronal cell loss is not related to classical caspase-mediated apoptosis. In summary, this study may contribute to new perspectives in the investigation of neurodegenerative mechanisms triggered by oxygen deprivation.