A CRM1-Mediated Nuclear Export Signal Is Essential for Cytoplasmic Localization of Neurogenin 3 in Neurons

Neurogenin 3 (Ngn3), a proneural gene, regulates dendritogenesis and synaptogenesis in mouse hippocampal neurons. Ngn3 is transiently exported from the cell nucleus to the cytoplasm when neuronal polarity is initiated, suggesting that the nucleo-cytoplasmic transport of the protein is important for its action on neuronal development. In this study, we identified for the first time a functional nuclear export sequence (NES2; 131YIWALTQTLRIA142) in Ngn3. The green fluorescent protein (EGFP)-NES2 fusion protein was localized in the cytoplasm and its nucleo-cytoplasmic shuttling was blocked by the CRM1 specific export inhibitor leptomycin B. Mutation of a leucine residue to alanine (L135A) in the NES2 motif resulted in both cytoplasmic and nuclear localization of the EGFP-NES2 fusion protein and in the nuclear accumulation of ectopic full-length myc-Ngn3. In addition, point mutation of the leucine 135 counteracted the effects of Ngn3 on neuronal morphology and synaptic inputs indicating that the cytoplasmic localization of Ngn3 is important for neuronal development. Pharmacological perturbation of the cytoskeleton revealed that cytoplasmic Ngn3 is associated with microtubules. © 2013 Simon-Areces et al.Peer Reviewe

Sex differences and gonadal hormone regulation of brain cardiolipin, a key mitochondrial phospholipid

Cardiolipin (CL) is a phospholipid that is almost exclusively located in the inner mitochondrial membrane of eukaryotic cells. As a result of its unique structure and distribution, CL establishes non-covalent bonds with a long list of proteins involved in ATP production, mitochondria biogenesis, mitophagy and apoptosis. Thus, the amount of CL, as well as its fatty acid composition and location, strongly impacts upon mitochondrial-dependent functions and therefore the metabolic homeostasis of different tissues. The brain is particularly sensitive to mitochondrial dysfunction as a result of its high metabolic demand. Several mitochondrial related-neurodegenerative disorders, as well as physiological ageing, show altered CL metabolism. Furthermore, mice lacking enzymes involved in CL synthesis show cognitive impairments. CL content and metabolism are regulated by gonadal hormones in the developing and adult brain. In neuronal cultures, oestradiol increases CL content, whereas adult ovariectomy decreases CL content and alters CL metabolism in the hippocampal mitochondria. Transient sex differences in brain CL metabolism have been detected during development. At birth, brain CL has a higher proportion of unsaturated fatty acids in the brain of male mice than in the brain of females. In addition, the expression of enzymes involved in CL de novo and recycling synthetic pathways is higher in males. Most of these sex differences are abolished by the neonatal androgenisation of females, suggesting a role for testosterone in the generation of sex differences in brain CL. The regulation of brain CL by gonadal hormones may be linked to their homeostatic and protective actions in neural cells, as well as the manifestation of sex differences in neurodegenerative disorders.This research was funded by the German Research Foundation (DFG), grant number AS547‐1/1 to MA. EAF, AOR and LMGS acknowledge support from Agencia Estatal de Investigación, Spain (BFU2017‐82754‐R), Centro de Investigación Biomédica en Red Fragilidad y Envejecimiento Saludable (CIBERFES), Instituto de Salud Carlos III, Spain and Fondos FEDER

Role of astrocytes in the neuroprotective actions of 17β-estradiol and selective estrogen receptor modulators

Neuroprotective actions of 17β-estradiol (estradiol) are in part mediated by direct actions on neurons. Astrocytes, which play an essential role in the maintenance of the homeostasis of neural tissue, express estrogen receptors and are also involved in the neuroprotective actions of estradiol in the brain. Estradiol controls gliosis and regulates neuroinflammation, edema and glutamate transport acting on astrocytes. In addition, the hormone regulates the release of neurotrophic factors and other neuroprotective molecules by astrocytes. In addition, reactive astrocytes are a local source of neuroprotective estradiol for the injured brain. Since estradiol therapy is not free from peripheral risks, alternatives for the hormone have been explored. Some selective estrogen receptor modulators (SERMs), which are already in use in clinical practice for the treatment of breast cancer, osteoporosis or menopausal symptoms, exert similar actions to estradiol on astrocytes. Therefore, SERMs represent therapeutic alternatives to estradiol for the activation of astroglia-mediated neuroprotective mechanisms. © 2014 Elsevier Ireland Ltd.Peer Reviewe

Lipotoxic Effects of Palmitic Acid on Astrocytes Are Associated with Autophagy Impairment

Obesity is associated with an increase in the brain levels of saturated free fatty acids, such as palmitic acid (PA). Previous studies have shown that PA exerts proinflammatory actions and reduces cell viability in astrocyte cultures. In this study, we have assessed whether an alteration in autophagy is involved in the effects of PA on astrocytes. Primary astrocytes were obtained from the cerebral cortex of male and female CD1 mouse pups and were incubated for 4.5 or 24 h with 250–500 μM PA. PA increased the levels of LC3-II, an autophagosome marker, and reduced LC3-II flux in astrocytes, suggesting a blockade of autophagy. This effect was observed both after 4.5 and 24 h of treatment with PA. PA had additional effects after treatment for 24 h, increasing the expression of proinflammatory cytokines, decreasing cell viability, and increasing the levels of an endoplasmic reticulum stress marker. In addition, PA decreased the expression of estrogen receptors, but only in female astrocytes. However, the treatment with estradiol, estrogen receptor agonists, or inhibitor of estradiol synthesis did not counteract the action of PA on cell viability. Rapamycin, an autophagy inducer, was unable to prevent the effect of PA on cell viability. In addition, hydroxychloroquine, an autophagy blocker, did not cause per se astrocyte death. These findings suggest that the effect of PA on autophagy is not sufficient to induce astrocyte loss, which is only observed when prolonged PA treatment causes other alterations in astrocytes, such as increased inflammation and endoplasmic reticulum stress.This work was supported by grants from Ministerio de Economía, Industria y Competitividad (MINECO), Spain (grant numbers BFU2014–51836-C2-1-R and BFU2017-82754-R); Centro de Investigación Biomédica en Red de Fragilidad y Envejecimiento Saludable (CIBERFES), Instituto de Salud Carlos III, Madrid, Spain; and Fondos FEDER

Changes in cannabinoid receptors, aquaporin 4 and vimentin expression after traumatic brain injury in adolescent male mice. Association with edema and neurological deficit

© 2015 Lopez-Rodriguez et al.Traumatic brain injury (TBI) incidence rises during adolescence because during this critical neurodevelopmental period some risky behaviors increase. The purpose of this study was to assess the contribution of cannabinoid receptors (CB1 and CB2), blood brain barrier proteins (AQP4) and astrogliosis markers (vimentin) to neurological deficit and brain edema formation in a TBI weight drop model in adolescent male mice. These molecules were selected since they are known to change shortly after lesion. Here we extended their study in three different timepoints after TBI, including short (24h), early mid-term (72h) and late mid-term (two weeks). Our results showed that TBI induced an increase in brain edema up to 72 h after lesion that was directly associated with neurological deficit. Neurological deficit appeared 24 h after TBI and was completely recovered two weeks after trauma. CB1 receptor expression decreased after TBI and was negatively correlated with edema formation and behavioral impairments. CB2 receptor increased after injury and was associated with high neurological deficit whereas no correlation with edema was found. AQP4 increased after TBI and was positively correlated with edema and neurological impairments as occurred with vimentin expression in the same manner. The results suggest that CB1 and CB2 differ in the mechanisms to resolve TBI and also that some of their neuroprotective effects related to the control of reactive astrogliosis may be due to the regulation of AQP4 expression on the end-feet of astrocytes.Support was provided by the Ministerio de Economía y Competitividad, Spain (BFU2011-30217-C03-01 and BFU2012-38144), Instituto de Salud Carlos III, Redes temáticas de Investigación Cooperativa en Salud, Red de Trastornos Adictivos (RD2012/0028/0021) and GRUPO UCM (951579)

Notch signaling in astrocytes mediates their morphological response to an inflammatory challenge

In the nervous system, Notch pathway has a prominent role in the control of neuronal morphology and in the determination of the astrocyte fate. However, the role of Notch in morphological astrocyte plasticity is unknown. Here, we have explored the role of Notch activity on the morphological reactivity of primary astrocytes in response to LPS, an inflammatory stimulus. We found that LPS induces reactive astrocyte morphology by the inhibition of Notch signaling via NFκB activation and Jagged upregulation. In contrast, IGF-1, an anti-inflammatory molecule, inhibits LPS-induced reactive astrocyte morphological phenotype by enhancing Notch signaling through the inhibition of NFκB and the activation of MAPK. Therefore, Notch signaling pathway emerges as a mediator of the regulation of astrocyte morphology by inflammatory and anti-inflammatory stimuli.We thank Elisa Baides Rosell for excellent technical assistance. This work was supported by grants from Agencia Estatal de Investigación, Spain (grant number BFU2017-82754-R), Centro de Investigación Biomédica en Red de Fragilidad y Envejecimiento Saludable (CIBERFES), Instituto de Salud Carlos III, Madrid, Spain and Fondos FEDER

Sex differences in glia reactivity after cortical brain injury

© 2015 Wiley Periodicals, Inc. Several brain disorders associated with neuroinflammation show sex differences in their incidence, onset, progression and/or outcome. The different regulation of the neuroinflammatory response in males and females could underlie these sex differences. In this study, we have explored whether reactive gliosis after a penetrating cortical injury exhibits sex differences. Males presented a higher density of Iba1 immunoreactive cells in the proximity of the wound (0-220 μm) than females. This sex difference was due to a higher number of Iba1 immunoreactive cells with nonreactive morphology. In addition microglia/macrophages in that region expressed arginase-1, marker of alternatively activated microglia, and the neuroprotective protein Neuroglobin, in a greater proportion in males than in females. No sex differences were found in the number of astrocytes around the lesion. However, the percentage of astrocytes expressing chemokine (C-C motif) ligand 2 (CCL2), involved in recruitment of immune cells and gliosis regulation, was higher in males. Males also presented a significantly higher density of neurons in the lesion edge than females. These findings indicate that male and female mice have different neuroinflammatory responses after a cortical stab wound injury and suggest that sex differences in reactive gliosis may contribute to sex differences in neuroinflammatory diseases.Peer Reviewe

17β-Oestradiol anti-inflammatory effects in primary astrocytes require oestrogen receptor β-mediated neuroglobin up-regulation.

""Neuroglobin (Ngb), so named after its initial discovery in brain neurones, has received great attention as a result of its neuroprotective effects both in vitro and in vivo. Recently, we demonstrated that, in neurones, Ngb is a 17-oestradiol (E2) inducible protein that is pivotal for hormone-induced anti-apoptotic effects against H2O2 toxicity. The involvement of Ngb in other brain cell populations, as well as in other neuroprotective effects of E2, is completely unknown at present. We demonstrate Ngb immunoreactivity in reactive astrocytes located in the proximity of a penetrating cortical injury in vivo and the involvement of Ngb in the E2-mediated anti-inflammatory effect in primary cortical astrocytes. Upon binding to oestrogen receptor (ER), E2 enhances Ngb levels in a dose-dependent manner. Although with a lesser degree than E2, the pro-inflammatory stimulation with lipopolysaccharide (LPS) also induces the increase of Ngb protein levels via nuclear factor-(NF)B signal(s). Moreover, a negative cross-talk between ER subtypes and NFB signal(s) has been demonstrated. In particular, ER-activated signals prevent the NFB-mediated Ngb increase, whereas LPS impairs the ER-induced up-regulation of Ngb. Therefore, the co-expression of both ER and ER is pivotal for mediating E2-induced Ngb expression in the presence of NFB-activated signals. Interestingly, Ngb silencing prevents the effect of E2 on the expression of inflammatory markers (i.e. interleukin 6 and interferon -inducible protein 10). Ngb can be regarded as a key mediator of the different protective effects of E2 in the brain, including protection against oxidative stress and the control of inflammation, both of which are at the root of several neurodegenerative diseases."