Biology subjects

Sierra, A.

Publications and source records attributed to Sierra, A..

27 records · Page 2Linked to original sources

bioRxiv2021

RANK is an independent biomarker of poor prognosis in estrogen receptor-negative breast cancer and a therapeutic target in patient-derived xenografts

Despite strong preclinical data, the therapeutic benefit of the RANKL inhibitor denosumab in BC patients, beyond its bone-related effects, is unclear. Here, we investigated the prognostic value of RANK expression and its functionality in human BC. We analyzed RANK and RANKL expression in more than 1500 BC cases (777 being estrogen receptor-negative (ER-)) from four independent cohorts. We confirmed that RANK is more frequently expressed in ER- tumors, but it is also found in a subset of ER+ tumors. In ER- BC, RANK expression was independently associated with poor outcome, especially in postmenopausal patients and those who received adjuvant chemotherapy. Gene expression analyses unraveled distinct biology associated with RANK in relation to ER expression and menopause, and evidenced enhanced RANK activation in ER- postmenopausal tumors, together with regulation of metabolic pathways. Functional studies and transcriptomic analyses in ER- RANK+ patients-derived orthoxenografts demonstrated that activation of RANK signaling pathway promotes tumor cell proliferation and stemness, and regulates multiple biological processes including tumor immune surveillance and metabolism. Our results demonstrate that RANK expression is an independent poor prognosis biomarker in postmenopausal ER- BC patients and support the rational of using RANK pathway inhibitors in combination with chemotherapy in ER- BC.

cancer biology↗

bioRxiv2021

Microglial phagocytosis dysfunction during stroke is prevented by rapamycin

Microglial phagocytosis is rapidly emerging as a therapeutic target in neurodegenerative and neurological disorders. An efficient removal of cellular debris is necessary to prevent buildup damage of neighbor neurons and the development of an inflammatory response. As the brain professional phagocytes, microglia are equipped with an array of mechanisms that enable them to recognize and degrade several types of cargo, including neurons undergoing apoptotic cell death. While microglia are very competent phagocytes of apoptotic cells under physiological conditions, here we report their dysfunction in mouse and monkey (Macaca fascicularis and Callithrix jacchus) models of stroke by transient occlusion of the medial cerebral artery (tMCAo). The impairment of both engulfment and degradation was related to energy depletion triggered by oxygen and nutrients deprivation (OND), which led to reduced process motility, lysosomal depletion, and the induction of a protective autophagy response in microglia. Basal autophagy, which is in charge of removing and recycling intracellular elements, was critical to maintain microglial physiology, including survival and phagocytosis, as we determined both in vivo and in vitro using knock-out models of autophagy genes and the autophagy inhibitor MRT68921. Notably, the autophagy inducer rapamycin partially prevented the phagocytosis impairment induced by tMCAo in vivo but not by OND in vitro. These results suggest a more complex role of microglia in stroke than previously acknowledged, classically related to the inflammatory response. In contrast, here we demonstrate the impairment of apoptotic cell phagocytosis, a microglial function critical for brain recovery. We propose that phagocytosis is a therapeutic target yet to be explored and provide evidence that it can be modulated in vivo using rapamycin, setting the stage for future therapies for stroke patients.

neuroscience↗

bioRxiv2020

A two-step model of autophagy: autophagosome formation, degradation and net turnover

Autophagy is a complex process that encompasses the enclosure of cytoplasmic debris or dysfunctional organelles in membranous vesicles, the autophagosomes, for their elimination in the lysosomes. A gold-standard method to assess its induction is the analysis of the autophagic flux using as a surrogate the expression of the microtubule-associated light chain protein 3 conjugated to phosphatidylethanolamine (LC3-II) by Western blot, in the presence of lysosomal inhibitors. Therefore, the current definition of autophagy flux actually puts the focus on the degradation stage of autophagy. In contrast, the most important autophagy controlling genes that have been identified in the last few years in fact target early stages of autophagosome formation. From a biological standpoint is therefore conceivable that autophagosome formation and degradation are independently regulated and we argue that both stages need to be systematically analyzed. Here, we propose a simple two-step model to understand changes in autophagosome formation and degradation using data from conventional LC3-II Western blot, and test it using two models of autophagy modulation in cultured microglia, the brain macrophages: rapamycin and the ULK1/2 inhibitor, MRT68921. Our model provides a comprehensive understanding of the autophagy process and will help to unravel the effect of genetic, pharmacological, and environmental manipulations on both the formation and degradation of autophagosomes.

cell biology↗

bioRxiv2020

Microglial phagocytosis dysfunction is related to local neuronal activity in a genetic model of epilepsy

Microglial phagocytosis of apoptotic cells is an essential component of the brain regenerative response in neurodegenerative diseases. Phagocytosis is very efficient in physiological conditions, as well as during apoptotic challenge induced by excitotoxicity or inflammation, but is impaired in mouse and human mesial temporal lobe epilepsy (MTLE). Here we extend our studies to a genetic model of progressive myoclonus epilepsy type 1 (EPM1) in mice lacking cystatin B (CSTB), an inhibitor of cysteine proteases involved in lysosomal proteolysis. We first demonstrated that microglial phagocytosis was impaired in the hippocampus in Cstb knock-out (KO) mice when seizures arise and hippocampal atrophy begins, at 1 month of age. To test if this blockage was related to the lack of Cstb in microglia, we used an in vitro model of phagocytosis and siRNAs to acutely reduce Cstb expression but we found no significant effect in the phagocytosis of apoptotic cells. We then tested whether seizures were involved in the phagocytosis impairment, similar to MTLE, and analyzed Cstb KO mice before seizures begin, at postnatal day 14. Here, phagocytosis impairment was restricted to the granule neuron layer but not to the subgranular zone, where there are no active neurons. Furthermore, we observed apoptotic cells (both phagocytosed and not phagocytosed) in Cstb deficient mice at close proximity to active, cFos+ neurons and used mathematical modeling to demonstrate that the physical relationship between apoptotic cells and cFos+ neurons was specific for Cstb KO mice. These results suggest a complex crosstalk between apoptosis, phagocytosis and neuronal activity, hinting that local neuronal activity could be related to phagocytosis dysfunction in Cstb KO mice. Overall, this data suggest that phagocytosis impairment is an early feature of hippocampal damage in epilepsy and opens novel therapeutic approaches for epileptic patients based on targeting microglial phagocytosis.

neuroscience↗

bioRxiv2020

Automated skull-stripping and segmentation with Multi-Task U-Net in large mouse brain MRI databases

Skull-stripping and region segmentation are fundamental steps in preclinical magnetic resonance imaging (MRI) studies, and these common procedures are usually performed manually. We present Multi-task U-Net (MU-Net), a convolutional neural network designed to accomplish both tasks simultaneously. MU-Net achieved higher segmentation accuracy than state-of-the-art multi-atlas segmentation methods with an inference time of 0.35 seconds and no pre-processing requirements. We evaluated the performance of our network in the presence of skip connections and recently proposed framing connections, finding the simplest network to be the most effective. We tested MU-Net with an unusually large dataset combining several independent studies consisting of 1,782 mouse brain MRI volumes of both healthy and Huntington animals, and measured average Dice scores of 0.906 (striati), 0.937 (cortex), and 0.978 (brain mask). These high evaluation scores demonstrate that MU-Net is a powerful tool for segmentation and skull-stripping, decreasing inter and intra-rater variability of manual segmentation. The MU-Net code and the trained model are publicly available at https://github.com/Hierakonpolis/MU-Net.

bioinformatics↗

bioRxiv2019

DeepACSON: Automated White Matter Segmentation

Automated segmentation techniques are essential to tracing the entirety of ultrastructures in large three-dimensional electron microscopy (3D-EM) images of the brain tissue. Current automated techniques use deep convolutional neural networks (DCNNs) and rely on high-contrast cellular membranes to trace a small number of neuronal processes in very high-resolution EM datasets. We developed DeepACSON to segment large field-of-view, low-resolution 3D-EM datasets of white matter where tens of thousands of myelinated axons traverse the tissue. DeepACSON performs DCNN-based semantic segmentation and shape decomposition-based instance segmentation. With its top-down design, DeepACSON manages to account for severe membrane discontinuities inescapable with the low-resolution imaging. In particular, the instance segmentation of DeepACSON uses the tubularity of myelinated axons, decomposing an under-segmented myelinated axon into its constituent axons. We applied DeepACSON to ten serial block-face scanning electron microscopy datasets of rats after sham-operation or traumatic brain injury, segmenting hundreds of thousands of long-span myelinated axons, thousands of cell nuclei, and millions of mitochondria with excellent evaluation scores. DeepACSON quantified the morphology and spatial aspects of white matter ultrastructures, capturing nanoscopic morphological alterations five months after the injury.

neuroscience↗

bioRxiv2019

Essential omega-3 fatty acids tune microglial phagocytosis of synaptic elements in the developing brain

Omega-3 fatty acids (n-3 polyunsaturated fatty acids; n-3 PUFAs) are essential for the functional maturation of the brain. Westernization of dietary habits in both developed and developing countries is accompanied by a progressive reduction in dietary intake of n-3 PUFAs. Low maternal intake of n-3 PUFAs has been linked to neurodevelopmental diseases in epidemiological studies, but the mechanisms by which a n-3 PUFA dietary imbalance affects CNS development are poorly understood. Active microglial engulfment of synaptic elements is an important process for normal brain development and altered synapse refinement is a hallmark of several neurodevelopmental disorders. Here, we identify a molecular mechanism for detrimental effects of low maternal n-3 PUFA intake on hippocampal development. Our results show that maternal dietary n-3 PUFA deficiency increases microglial phagocytosis of synaptic elements in the developing hippocampus, through the activation of 12/15- lipoxygenase (LOX)/12-HETE signaling, which alters neuronal morphology and affects cognition in the postnatal offspring. While women of child bearing age are at higher risk of dietary n-3 PUFA deficiency, these findings provide new insights into the mechanisms linking maternal nutrition to neurodevelopmental disorders.\n\nOne Sentence SummaryLow maternal omega-3 fatty acids intake impairs microglia-mediated synaptic refinement via 12-HETE pathway in the developing brain.

neuroscience↗

bioRxiv2019

Microglia actively remodels adult hippocampal neurogenesis through the phagocytosis secretome

During adult hippocampal neurogenesis, the majority of newborn cells undergo apoptosis and are rapidly phagocytosed by resident microglia to prevent the spillover of their intracellular contents. Here, we propose that phagocytosis is not merely a passive process of corpse removal but has an active role in maintaining adult hippocampal neurogenesis. First, we found that neurogenesis was disrupted in mice chronically deficient for two microglial phagocytosis pathways (P2Y12 and MerTK/Axl), but was transiently increased in mice in which MerTK expression was conditionally downregulated. Next, we performed a transcriptomic analysis of microglial phagocytosis in vitro and identified genes involved in metabolism, chromatin remodeling, and neurogenesis-related functions. Finally, we discovered that the secretome of phagocytic microglia limits the production of new neurons both in vivo and in vitro. Our data suggest that reprogrammed phagocytic microglia act as a sensor of local cell death, modulating the balance between cell proliferation and cell survival in the neurogenic niche, thereby supporting the long-term maintenance of adult hippocampal neurogenesis.\n\nGRAPHICAL ABSTRACT

cell biology↗

bioRxiv2017

3D Axonal Morphometry of White Matter

Axonal structure underlies white matter functionality and plays a major role in brain connectivity. The current literature on the axonal structure is based on the analysis of two-dimensional (2D) cross-sections, which, as we demonstrate, is precarious. To be able to quantify three-dimensional (3D) axonal morphology, we developed a novel pipeline for automated 3D segmentation and morphometric analysis (ACSON) of the white matter ultrastructure. The automated pipeline eliminates the need for time-consuming manual segmentation of 3D datasets. ACSON segments myelin, myelinated and unmyelinated axons, mitochondria, cells and vacuoles, and analyzes the morphology of axons. We applied the pipeline to serial block-face scanning electron microscopy images of the corpus callosum of sham-operated (n = 2) and brain injured (n = 3) rats 5 months after the injury. The 3D morphometry showed that axonal cross-sections were elliptic rather than spherical, and their diameter varied substantially along axons. It also showed a significant reduction in the axonal diameter of the ipsilateral corpus callosum of rats 5 months after brain injury, indicating ongoing axonal alterations even at this chronic time-point.

neuroscience↗