Increased mitochondrial activity upon CatSper channel activation is required for mouse sperm capacitation

To fertilize an oocyte, sperm must undergo several biochemical and functional changes known as capacitation. A key event in capacitation is calcium influx through the cation channel of sperm (CatSper). However, the molecular mechanisms of capacitation downstream of this calcium influx are not completely understood. Capacitation is also associated with an increase in mitochondrial oxygen consumption, and several lines of evidence indicate that regulated calcium entry into mitochondria increases the efficiency of oxidative respiration. Thus, we hypothesized that calcium influx through CatSper during capacitation increases mitochondrial calcium concentration and mitochondrial efficiency and thereby contributes to sperm hyperactivation and fertilization capacity. To test this hypothesis, we used high-resolution respirometry to measure mouse sperm mitochondrial activity. We also measured mitochondrial membrane potential, ATP/ADP exchange during capacitation, and mitochondrial calcium concentration in sperm from wild-type and CatSper knockout mice. We show that the increase in mitochondrial activity in capacitated wild-type sperm parallels the increase in mitochondrial calcium concentration. This effect is blunted in sperm from CatSper knockout mice. Importantly, these mechanisms are needed for optimal hyperactivation and fertilization in wild-type mice, as confirmed by using mitochondrial inhibitors. Thus, we describe a novel mechanism of sperm capacitation. This work contributes to our understanding of the role of mitochondria in sperm physiology and opens the possibility of new molecular targets for fertility treatments and male contraception

The Nanomechanical Properties of CLL Cells Are Linked to the Actin Cytoskeleton and Are a Potential Target of BTK Inhibitors.

Chronic lymphocytic leukemia (CLL) is an incurable disease characterized by an intense trafficking of the leukemic cells between the peripheral blood and lymphoid tissues. It is known that the ability of lymphocytes to recirculate strongly depends on their capability to rapidly rearrange their cytoskeleton and adapt to external cues; however, little is known about the differences occurring between CLL and healthy B cells during these processes. To investigate this point, we applied a single-cell optical (super resolution microscopy) and nanomechanical approaches (atomic force microscopy, real-time deformability cytometry) to both CLL and healthy B lymphocytes and compared their behavior. We demonstrated that CLL cells have a specific actomyosin complex organization and altered mechanical properties in comparison to their healthy counterpart. To evaluate the clinical relevance of our findings, we treated the cells in vitro with the Bruton's tyrosine kinase inhibitors and we found for the first time that the drug restores the CLL cells mechanical properties to a healthy phenotype and activates the actomyosin complex. We further validated these results in vivo on CLL cells isolated from patients undergoing ibrutinib treatment. Our results suggest that CLL cells' mechanical properties are linked to their actin cytoskeleton organization and might be involved in novel mechanisms of drug resistance, thus becoming a new potential therapeutic target aiming at the normalization of the mechanical fingerprints of the leukemic cells.CS project is supported by Associazione Italiana per la Ricerca sul Cancro AIRC under IG 2018 - ID 21332 project. OO gratefully acknowledges financial support from the German Federal Ministry of Education and Research (ZIK grant to OO under grant agreement no. 03Z22CN11) as well as from the German Center for Cardiovascular Research (Postdoc start-up grant to OO under grant agreement no. 81X3400107). CAM acknowledges financial support from the Italian Ministry of University and Research (MIUR) Department of Excellence project PREMIA (PREcision MedIcine Approach: bringing biomarker research to clinics). STED microscopy was conducted at the Microscopy & Dynamic Imaging Unit, CNIC, ICTS-ReDib, co-funded by MCIN/AEI/10.13039/501100011033, and FEDER “Una manera de hacer Europa” (#ICTS-2018-04-CNIC-16). The CNIC is supported by the Ministerio de Ciencia e Innovación and the Pro CNIC Foundation and is a Severo Ochoa Center of Excellence (CEX2020-001041-S). Schemes in figures 1, 2, 3 and 4 have been generated with BioRender.com. Funding for the project was provided by the European Union’s Seventh Framework Programme (FP7/2007-2013) under grant agreement no 282510 – BLUEPRINT.S

Modulation of Astrocytic Mitochondrial Function by Dichloroacetate Improves Survival and Motor Performance in Inherited Amyotrophic Lateral Sclerosis

Mitochondrial dysfunction is one of the pathogenic mechanisms that lead to neurodegeneration in Amyotrophic Lateral Sclerosis (ALS). Astrocytes expressing the ALS-linked SOD1G93A mutation display a decreased mitochondrial respiratory capacity associated to phenotypic changes that cause them to induce motor neuron death. Astrocyte-mediated toxicity can be prevented by mitochondria-targeted antioxidants, indicating a critical role of mitochondria in the neurotoxic phenotype. However, it is presently unknown whether drugs currently used to stimulate mitochondrial metabolism can also modulate ALS progression. Here, we tested the disease-modifying effect of dichloroacetate (DCA), an orphan drug that improves the functional status of mitochondria through the stimulation of the pyruvate dehydrogenase complex activity (PDH). Applied to astrocyte cultures isolated from rats expressing the SOD1G93A mutation, DCA reduced phosphorylation of PDH and improved mitochondrial coupling as expressed by the respiratory control ratio (RCR). Notably, DCA completely prevented the toxicity of SOD1G93A astrocytes to motor neurons in coculture conditions. Chronic administration of DCA (500 mg/L) in the drinking water of mice expressing the SOD1G93A mutation increased survival by 2 weeks compared to untreated mice. Systemic DCA also normalized the reduced RCR value measured in lumbar spinal cord tissue of diseased SOD1G93A mice. A remarkable effect of DCA was the improvement of grip strength performance at the end stage of the disease, which correlated with a recovery of the neuromuscular junction area in extensor digitorum longus muscles. Systemic DCA also decreased astrocyte reactivity and prevented motor neuron loss in SOD1G93A mice. Taken together, our results indicate that improvement of the mitochondrial redox status by DCA leads to a disease-modifying effect, further supporting the therapeutic potential of mitochondria-targeted drugs in ALS

Dissecting the Shared Genetic Architecture of Suicide Attempt, Psychiatric Disorders, and Known Risk Factors

Background Suicide is a leading cause of death worldwide, and nonfatal suicide attempts, which occur far more frequently, are a major source of disability and social and economic burden. Both have substantial genetic etiology, which is partially shared and partially distinct from that of related psychiatric disorders. Methods We conducted a genome-wide association study (GWAS) of 29,782 suicide attempt (SA) cases and 519,961 controls in the International Suicide Genetics Consortium (ISGC). The GWAS of SA was conditioned on psychiatric disorders using GWAS summary statistics via multitrait-based conditional and joint analysis, to remove genetic effects on SA mediated by psychiatric disorders. We investigated the shared and divergent genetic architectures of SA, psychiatric disorders, and other known risk factors. Results Two loci reached genome-wide significance for SA: the major histocompatibility complex and an intergenic locus on chromosome 7, the latter of which remained associated with SA after conditioning on psychiatric disorders and replicated in an independent cohort from the Million Veteran Program. This locus has been implicated in risk-taking behavior, smoking, and insomnia. SA showed strong genetic correlation with psychiatric disorders, particularly major depression, and also with smoking, pain, risk-taking behavior, sleep disturbances, lower educational attainment, reproductive traits, lower socioeconomic status, and poorer general health. After conditioning on psychiatric disorders, the genetic correlations between SA and psychiatric disorders decreased, whereas those with nonpsychiatric traits remained largely unchanged. Conclusions Our results identify a risk locus that contributes more strongly to SA than other phenotypes and suggest a shared underlying biology between SA and known risk factors that is not mediated by psychiatric disorders.Peer reviewe

Respiratory analysis of coupled mitochondria in cryopreserved liver biopsies

The aim of this work was to develop a cryopreservation method of small liver biopsies for in situ mitochondrial function assessment. Herein we describe a detailed protocol for tissue collection, cryopreservation, high-resolution respirometry using complex I and II substrates, calculation and interpretation of respiratory parameters. Liver biopsies from cow and rat were sequentially frozen in a medium containing dimethylsulfoxide as cryoprotectant and stored for up to 3 months at −80 °C. Oxygen consumption rate studies of fresh and cryopreserved samples revealed that most respiratory parameters remained unchanged. Additionally, outer mitochondrial membrane integrity was assessed adding cytochrome c, proving that our cryopreservation method does not harm mitochondrial structure. In sum, we present a reliable way to cryopreserve small liver biopsies without affecting mitochondrial function. Our protocol will enable the transport and storage of samples, extending and facilitating mitochondrial function analysis of liver biopsies. Keywords: Cryopreservation, Mitochondria, Biopsy, Oxygen consumption rate, High-resolution respirometry, Mitochondrial functio

Informe final del proyecto: Modulación metabólica de la glía como estrategia para reducir la muerte neuronal en la Esclerosis Lateral Amiotrófica

La esclerosis lateral amiotrófica (ELA) se caracteriza por una degeneración progresiva de las neuronas motoras (MN, por sus siglas en inglés). Diversos estudios utilizando modelos celulares y animales de ELA que expresan la mutación SOD1G93A, indican que existe una compleja interacción entre las MN y las células no neuronales vecinas, como los astrocitos. Los astrocitos en la ELA muestran menor capacidad para mantener la supervivencia de las MN en comparación con aquellos no asociados a la enfermedad, lo cual está fuertemente correlacionado con una baja actividad respiratoria mitocondrial. La inhibición farmacológica de la piruvato deshidrogenasa quinasa (PDK) por dicloroacetato en los astrocitos SOD1G93A condujo a un aumento en la respiración mitocondrial y restauró la supervivencia de las MN. En este proyecto nos propusimos analizar la habilidad de los astrocitos SOD1G93A para mantener la supervivencia de MN cuando se silencia la PDK utilizando ARN de interferencia específico para PDK2 y en segundo lugar, la expresión génica de los astrocitos con inhibición mitocondrial con el fin de identificar nuevos genes con potencial de ser silenciados y restablecer la capacidad trófica de los astrocitos. La supresión de PDK2 aumentó la respiración mitocondrial en los astrocitos SOD1G93A asociado a una mejora en el mantenimiento de la supervivencia de las MN, respaldando aún más el papel principal de la actividad respiratoria mitocondrial en las interacciones astrocito-MN. Los astrocitos con inhibición mitocondrial mostraron diferencias en la expresión de algunos genes que coinciden con las modificaciones encontradas con astrocitos SOD1G93A. Estos estudios arrojaron nuevos blancos moleculares potenciales para enlentecer la progresión de la muerte de MN inducida por astrocitos en la ELA. Además, se concretaron una tesis de doctorado y una de maestría próxima a finalizar y se generaron un manuscrito y otro en preparación.Agencia Nacional de Investigación e Innovació

Nitro-Arachidonic Acid Prevents Angiotensin II-Induced Mitochondrial Dysfunction in a Cell Line of Kidney Proximal Tubular Cells.

Nitro-arachidonic acid (NO2-AA) is a cell signaling nitroalkene that exerts anti-inflammatory activities during macrophage activation. While angiotensin II (ANG II) produces an increase in reactive oxygen species (ROS) production and mitochondrial dysfunction in renal tubular cells, little is known regarding the potential protective effects of NO2-AA in ANG II-mediated kidney injury. As such, this study examines the impact of NO2-AA on ANG II-induced mitochondrial dysfunction in an immortalized renal proximal tubule cell line (HK-2 cells). Treatment of HK-2 cells with ANG II increases the production of superoxide (O2●-), nitric oxide (●NO), inducible nitric oxide synthase (NOS2) expression, peroxynitrite (ONOO-) and mitochondrial dysfunction. Using high-resolution respirometry, it was observed that the presence of NO2-AA prevented ANG II-mediated mitochondrial dysfunction. Attempting to address mechanism, we treated isolated rat kidney mitochondria with ONOO-, a key mediator of ANG II-induced mitochondrial damage, in the presence or absence of NO2-AA. Whereas the activity of succinate dehydrogenase (SDH) and ATP synthase (ATPase) were diminished upon exposure to ONOO-, they were restored by pre-incubating the mitochondria with NO2-AA. Moreover, NO2-AA prevents oxidation and nitration of mitochondrial proteins. Combined, these data demonstrate that ANG II-mediated oxidative damage and mitochondrial dysfunction is abrogated by NO2-AA, identifying this compound as a promising pharmacological tool to prevent ANG II-induced renal disease