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Las cardiolipinas (CL) son fosfolípidos localizados exclusivamente en las mitocondrias, imprescindibles para el correcto funcionamiento mitocondrial. En condiciones de homeostasis, las CL se localizan en la membrana mitocondrial interna (MMI), pero ante condiciones de estrés son externalizadas hacia la membrana mitocondrial externa (MME), donde tienen un papel esencial en el ensamblaje del inflamasoma y, por tanto, en la generación de una respuesta inflamatoria. La lipina-2 es una enzima del metabolismo lipídico, mutaciones en el gen que la codifica en humanos, produce un síndrome autoinflamatorio conocido como Síndrome de Majeed. Resultados previos del grupo demuestran que los macrófagos Lpin2-/- presentan niveles elevados de CL, junto con un fenotipo proinflamatorio exacerbado y una mayor actividad de la vía glucolítica. Utilizando macrófagos inmortalizados derivados de médula ósea de ratones wt y Lpin2-/-, nos propusimos estudiar la expresión de las enzimas de síntesis, remodelación y degradación de CL, el efecto del silenciamiento de siCrls1 y las posibles diferencias en la externalización de CL entre ambos genotipos. Los resultados indican que los iBMdM Lpin2-/- presentan una mayor expresión de las enzimas de síntesis de CL, junto con una mayor externalización de CL. El silenciamiento de Crls1 reduce la inflamación en células carentes de lipina-2, aunque no se aprecian indicios de reversión de la sobrexpresión de genes relacionados con el metabolismo glucídico en estas condiciones. Estos resultados, permiten plantear nuevos estudios donde la CL sea una diana molecular que modular para paliar los síntomas del síndrome de Majeed., Departamento de Biología Celular, Histología y Farmacología, Máster en Investigación Biomédica y Terapias Avanzadas

Ischemic stroke is the leading cause of neurologic death and long-term adult disability worldwide. Ischemia-induced oxidative stress compromises genome integrity, resulting in DNA damage. Failure of neurons to efficiently repair DNA double-strands breaks (DSB) contributes to cerebral damage and impairs functional recovery after stroke. However, the molecular machinery that regulates DNA repair in this neurological disorder remains unknown. Here we describe that WRAP53 (WD40 encoding RNA Antisense to p53), a scaffold protein implicated in telomere elongation and DNA repair in tumor cells, plays an essential role in neuronal survival after ischemia. We found that experimental ischemia, performed by oxygen and glucose deprivation (OGD), promoted oxidative stress-induced DSB in neurons. These events spatiotemporally correlated with WRAP53 upregulation and nuclear translocation to activate DSBs repair response. Mechanistically, OGD triggered a burst in reactive oxygen species that induced both DSB and translocation of WRAP53 to the nucleus to promote DNA repair, a pathway that was confirmed in an in vivo mouse model of stroke. Noticeably, nuclear translocation of WRAP53 occurred faster in OGD neurons expressing the Wrap53 human nonsynonymous single-nucleotide polymorphism (SNP) rs2287499 (c.202C>G). Patients carrying this SNP showed less infarct volume and better functional outcome after stroke. These results provide a new signaling role for reactive oxygen species in the mechanism of DSB repair in neurons and unravel the role of WRAP53 to maintaining genome integrity and survival after stroke.

Stroke is a major cause of death and long-term disability in the adult. Neuronal apoptosis plays an essential role in the pathophysiology of ischemic brain damage and impaired functional recovery after stroke. The tumor suppressor protein p53 regulates key cellular processes, including cell cycle arrest, DNA repair, senescence, and apoptosis. Under cellular stress conditions, p53 undergoes post-translational modifications, which control protein localization, stability, and proapoptotic activity. After stroke, p53 rapidly accumulates in the ischemic brain, where it activates neuronal apoptosis through both transcriptional-dependent and -independent programs. Over the last years, subcellular localization of p53 has emerged as an important regulator of ischemia-induced neuronal apoptosis. Upon an ischemic insult, p53 rapidly translocates to the mitochondria and interacts with B-cell lymphoma-2 family proteins, which activate the mitochondrial apoptotic program, with higher efficacy than through its activity as a transcription factor. Moreover, the identification of a human single nucleotide polymorphism at codon 72 of the Tp53 gene that controls p53 mitochondrial localization and cell susceptibility to apoptosis supports the important role of the p53 mitochondrial program in neuronal survival and functional recovery after stroke. In this article, we review the relevance of mitochondrial and nuclear localization of p53 on neuronal susceptibility to cerebral ischemia and its impact on functional outcome of stroke patients.

One of the most important mechanisms of preconditioning-mediated neuroprotection is the attenuation of cell apoptosis, inducing brain tolerance after a subsequent injurious ischemia. In this context, the antiapoptotic PI3K/AKT signaling pathway plays a key role by regulating cell differentiation and survival. Active AKT is known to increase the expression of murine double minute-2 (MDM2), an E3-ubiquitin ligase that destabilizes p53 to promote the survival of cancer cells. In neurons, we recently showed that the MDM2–p53 interaction is potentiated by pharmacological preconditioning, based on subtoxic stimulation of NMDA glutamate receptor, which prevents ischemia-induced neuronal apoptosis. However, whether this mechanism contributes to the neuronal tolerance during ischemic preconditioning (IPC) is unknown. Here, we show that IPC induced PI3K-mediated phosphorylation of AKT at Ser473, which in turn phosphorylated MDM2 at Ser166. This phosphorylation triggered the nuclear stabilization of MDM2, leading to p53 destabilization, thus preventing neuronal apoptosis upon an ischemic insult. Inhibition of the PI3K/AKT pathway with wortmannin or by AKT silencing induced the accumulation of cytosolic MDM2, abrogating IPC-induced neuroprotection. Thus, IPC enhances the activation of PI3K/AKT signaling pathway and promotes neuronal tolerance by controlling the MDM2–p53 interaction. Our findings provide a new mechanistic pathway involved in IPC-induced neuroprotection via modulation of AKT signaling, suggesting that AKT is a potential therapeutic target against ischemic injury.

Ischemia-induced oxidative stress compromises genome integrity, which results in DNA damage and neuronal loss after stroke. We described that reactive oxygen species (ROS) generated during ischemia upregulate WRAP53 (WD40 encoding RNA antisense to p53) and trigger its translocation to the nucleus, where it promotes DNA repair [1]. However, the molecular mechanism remains unknown. Transcription factor Sp1 acts as a pleiotropic oxidative stress response protein in neurons. Particularly, ROS-induced Sp1 expression promotes neuroprotection against ischemia [2].i nterestingly, Wrap53 promoter contains putative consensus sequences (GC boxes) for Sp1. We analyze the role of Sp1 as a modulating factor of WRAP53-mediated neuronal survival and its impact on brain repair after stroke.

Failure of neurons to efficiently repair DNA double-strand breaks (DSBs) contributes to cerebral damage after stroke. However, the molecular machinery that regulates DNA repair in this neurological disorder is unknown. Here, we found that DSBs in oxygen/glucose-deprived (OGD) neurons spatiotemporally correlated with the up-regulation of WRAP53 (WD40-encoding p53-antisense RNA), which translocated to the nucleus to activate the DSB repair response. Mechanistically, OGD triggered a burst in reactive oxygen species that induced both DSBs and translocation of WRAP53 to the nucleus to promote DNA repair, a pathway that was confirmed in an in vivo mouse model of stroke. Noticeably, nuclear translocation of WRAP53 occurred faster in OGD neurons expressing the Wrap53 human nonsynonymous single-nucleotide polymorphism (SNP) rs2287499 (c.202C>G). Patients carrying this SNP showed less infarct volume and better functional outcome after stroke. These results indicate that WRAP53 fosters DNA repair and neuronal survival to promote functional recovery after stroke.

Attenuation of cell apoptosis has been involved in endogenous neuroprotection induced during brain tolerance related to ischemic preconditioning (IPC). The PI3K/Akt pathway regulates cell development, growth, and survival. Several studies have reported that activated Akt, a serine-threonine specific protein kinase B enhances MDM2-mediated p53 destabilization triggering survival in cancer cells. Recently, we demonstrated that IPC prevents the activation of p53/PUMA/Caspase-3 apoptotic pathway by increasing MDM2/p53 interaction in cortical neurons. Here, we aimed to clarify the role of Akt in the IPC-induced neuronal tolerance against lethal ischemia. To do that, primary cortical neurons were exposed to a validated in vitro model of IPC (oxygen glucose deprivation; OGD; 20 min) prior to prolonged ischemia (OGD, 90min). Akt levels were modulated by specific siRNA and PI3K/Akt activity was inhibited by wortmannin. Protein levels were determined by Western blotting. Neuronal apoptosis (Annexin-V-staining and caspase-3 activation) was analyzed by flow cytometry and fluorimetry. For ectopic human MDM2 expression, a plasmid construction expressing YFP-tagged Mdm2 was used. Our results show that IPC promoted early phosphorylation of Akt, followed by an increase in Akt-MDM2 interaction. Indeed, Akt activation promoted stabilization of phosphorylated MDM2, which enhanced IPC-promoted nuclear MDM2-p53 complex at 4 hours of reoxygenation after ischemia. Furthermore, PI3K/Akt specific inhibition by wortmannin and siRNA against Akt, completely induced a cytosolic MDM2 translocation, leading to the prevention of IPC-mediated neuroprotection. In conclusion, the PI3K/Akt signaling pathway is involved in ischemic tolerance, through controlling the MDM2/p53 interaction.

Ischemic stroke causes DNA damage in neurons, thereby contributing to cell death unless promptly repaired. An adequate DNA damage response is essential to survive after cerebral ischemia, therefore is indispensable to preserve the integrity of the transcribed genome in post-mitotic neurons; however, the molecular mechanism underlying neuronal survival remains unknown. Here we describe that WRAP53 (WD40 encoding RNA Antisense to p53), a scaffold protein implicated in telomere elongation and DNA repair in tumor cells, also plays an essential role in neuronal survival after ischemia. We observed that ischemia induces oxidative stress and promotes DNA damage, as revealed by the accumulation of yH2AX in neurons. The ischemic insult also promoted a time-dependent increase in Wrap53 gene expression and protein abundance. Remarkably, the ischemic-induced oxidative stress triggers WRAP53 traffic to the nucleus, where WRAP53 is involved in DNA repair processes. By siRNA analysis we confi rm that WRAP53 depletion prevents 53BP1 foci formation, therefore increasing neuronal susceptibility to ischemia-induced apoptosis, whereas WRAP53 expression in ischemic neurons promotes DNA repair events and cell survival. Our results demonstrate the relevance of nuclear accumulation of WRAP53 to maintain genome integrity in neurons and preserve neuronal survival after ischemia.

The Fizzy-related protein 1 (Fzr1) gene encodes Cdh1 protein, a coactivator of the E3 ubiquitin ligase anaphase-promoting complex/cyclosome (APC/C). Previously, we found that genetic ablation of Fzr1 promotes the death of neural progenitor cells leading to neurogenesis impairment and microcephaly in mouse. To ascertain the possible translation of these findings in humans, we searched for mutations in the Fzr1 gene in 390 whole exomes sequenced in trio in individuals showing neurodevelopmental disorders compatible with a genetic origin. We found a novel missense (p.Asp187Gly) Fzr1 gene mutation (c.560A>G) in a heterozygous state in a 4-year-old boy, born from non-consanguineous Spanish parents, who presents with severe antenatal microcephaly, psychomotor retardation, and refractory epilepsy. Cdh1 protein levels in leucocytes isolated from the patient were significantly lower than those found in his parents. Expression of the Asp187Gly mutant form of Cdh1 in human embryonic kidney 293T cells produced less Cdh1 protein and APC/C activity, resulting in altered cell cycle distribution when compared with cells expressing wild-type Cdh1. Furthermore, ectopic expression of the Asp187Gly mutant form of Cdh1 in cortical progenitor cells in primary culture failed to abolish the enlargement of the replicative phase caused by knockout of endogenous Cdh1. These results indicate that the loss of function of APC/C-Cdh1 caused by Cdh1 Asp187Gly mutation is a new cause of prenatal microcephaly, psychomotor retardation, and severe epilepsy. Open science badges: This article has received a badge for *Open Materials* because it provided all relevant information to reproduce the study in the manuscript. The complete Open Science Disclosure form for this article can be found at the end of the article. More information about the Open Practices badges can be found at https://cos.io/our-services/open-science-badges/. (Figure presented.). Open Science: This manuscript was awarded with the Ope

[Background and Purpose] The E3 ubiquitin ligase MDM2 (murine double minute 2) is the main negative regulator of the p53 protein—a key player in neuronal apoptosis after ischemia. A functional single-nucleotide polymorphism in the human MDM2 gene promoter (rs2279744) regulates MDM2 protein expression. We investigated whether the MDM2 SNP309, by controlling p53-mediated apoptosis, determines functional outcome after stroke., [Methods] Primary cortical neurons were subjected to oxygen and glucose deprivation. Mice were subjected to ischemic (transient middle cerebral artery occlusion) or hemorrhagic (collagenase injection) stroke models. Protein and mRNA levels of MDM2 and p53 were measured in both neuronal and brain extracts. The interaction of MDM2 with p53 was disrupted by neuronal treatment with nutlin-3a. siRNA was used to knockdown MDM2 expression. We analyzed the link between the MDM2 SNP309 and functional outcome, measured by the modified Rankin Scale scores, in 2 independent hospital-based stroke cohorts: ischemic stroke cohort (408 patients) and intracerebral hemorrhage cohort (128 patients)., [Results] Experimental stroke and oxygen and glucose deprivation induced the expression of MDM2 in the brain and neurons, respectively. Moreover, oxygen and glucose deprivation promoted MDM2 binding with p53 in neurons. Disruption of the MDM2-p53 interaction with nutlin-3a, or MDM2 knockdown by siRNA, triggered p53 accumulation, which increased neuronal susceptibility to oxygen and glucose deprivation-induced apoptosis. Finally, we showed that patients harboring the G allele in the MDM2 promoter had higher MDM2 protein levels and showed better functional outcome after stroke than those harboring the T/T genotype. The T/T genotype was also associated with large infarct volume in ischemic stroke and increased lesion volume in patients with intracerebral hemorrhage., [Conclusions] Our results reveal a novel role for the MDM2-p53 interaction in neuronal apoptosis after ischemia and show that the MDM2 SNP309 determines the functional outcome of patients after stroke.

[Background] The correct formation and long-term maintenance of the dendritic network are essential for the normal functioning of the brain. In the adult brain, dendrite stability confers mature neurons the ability to maintain long-term dendritic arbor integrity and integration within networks. Dendritic disruption and loss of dendritic spines and synapses have been reported in neurodegenerative conditions, including Alzheimer’s disease (AD). [Material and methods] We first generated CamKIIalpha-Cre-mediated Cdh1 conditional knockout mice (Cdh1 cKO) for the depletion of Cdh1 in glutamatergic neurons of the cortex and hippocampus from the adult brain. Double-transgenic Cdh1 cKO mice were crossed with Thy1-YFP-H animals, which express yellow fluorescent protein in the pyramidal neurons of the previously mentioned areas. To inhibit Rock activity, fasudil (20 mg/kg body weight; 60 days) was injected intraperitoneally every other day into 30-day-old mice. [Results] Here we show that postnatal deletion of Cdh1, a cofactor of the anaphase promoting complex/cyclosome (APC/C) ubiquitin ligase in neurons (Cdh1 cKO), disrupts dendrite arborization and causes dendritic spine and synapse loss in the cortex and hippocampus, concomitant with memory impairment and neurodegeneration in adult mice. We found that the dendrite destabilizer Rho protein kinase 2 (Rock2), which accumulates in the brain of AD patients, is an APC/CCdh1 substrate in vivo and that Rock2 protein and activity increased in the cortex and hippocampus of Cdh1 cKO mice. In these animals, inhibition of Rock activity, using the clinical approved drug fasudil, prevented dendritic network disorganization, memory loss, and neurodegeneration. [Conclusions] APC/CCdh1-mediated degradation of Rock2 maintains the dendritic network, memory formation, and neuronal survival, suggesting that pharmacological inhibition of aberrantly accumulated Rock2 may be a suitable therapeutic strategy against neurodegeneration.

Disruption of neuronal morphology contributes to the pathology of neurodegenerative disorders such as Alzheimer's disease (AD). However, the underlying molecular mechanisms are unknown. Here, we show that postnatal deletion of Cdh1, a cofactor of the anaphasepromoting complex/cyclosome (APC/C) ubiquitin ligase in neurons [Cdh1 conditional knockout (cKO)], disrupts dendrite arborization and causes dendritic spine and synapse loss in the cortex and hippocampus, concomitant with memory impairment and neurodegeneration, in adult mice. We found that the dendrite destabilizer Rho protein kinase 2 (Rock2), which accumulates in the brain of AD patients, is an APC/C substrate in vivo and that Rock2 protein and activity increased in the cortex and hippocampus of Cdh1 cKO mice. In these animals, inhibition of Rock activity, using the clinically approved drug fasudil, prevented dendritic network disorganization, memory loss, and neurodegeneration. Thus, APC/C-mediated degradation of Rock2 maintains the dendritic network, memory formation, and neuronal survival, suggesting that pharmacological inhibition of aberrantly accumulated Rock2 may be a suitable therapeutic strategy against neurodegeneration.