Retinal Axon Regeneration in the Lizard Gallotia galloti in the Presence of CNS Myelin and Oligodendrocytes

Retinal ganglion cell (RGC) axons in lizards (reptiles) were found to regenerate after optic nerve injury. To determine whether regeneration occurs because the visual pathway has growth-supporting glia cells or whether RGC axons regrow despite the presence of neurite growth-inhibitory components, the substrate properties of lizard optic nerve myelin and of oligodendrocytes were analyzed in vitro, using rat dorsal root ganglion (DRG) neurons. In addition, the response of lizard RGC axons upon contact with rat and reptilian oligodendrocytes or with myelin proteins from the mammalian central nervous system (CNS) was monitored. Lizard optic nerve myelin inhibited extension of rat DRG neurites, and lizard oligodendrocytes elicited DRG growth cone collapse. Both effects were partially reversed by antibody IN-1 against mammalian 35/250 kD neurite growth inhibitors, and IN-1 stained myelinated fiber tracts in the lizard CNS. However, lizard RGC growth cones grew freely across oligodendrocytes from the rat and the reptilian CNS. Mammalian CNS myelin proteins reconstituted into liposomes and added to elongating lizard RGC axons caused at most a transient collapse reaction. Growth cones always recovered within an hour and regrew. Thus, lizard CNS myelin and oligodendrocytes possess nonpermissive substrate properties for DRG neurons like corresponding structures and cells in the mammalian CNS, including mammalian-like neurite growth inhibitors. Lizard RGC axons, however, appear to be far less sensitive to these inhibitory substrate components and therefore may be able to regenerate through the visual pathway despite the presence of myelin and oligodendrocytes that block growth of DRG neurites

Regeneration of Retinal Axons in the Lizard Gallotia galloti Is Not Linked to Generation of New Retinal Ganglion Cells

Using anterograde tracing with HRP and antibodies (ABs) against neurofilaments, we show that regrowth of retinal ganglion cell (RGC) axons in the lizard Gallotia galloti commences only 2 months after optic nerve transection (ONS) and continues over at least 9 months. This is unusually long when compared to RGC axon regeneration in fish or amphibians. Following ONS, lizard RGCs up-regulate the immediate early gene C-JUN for 9 months or longer, indicating their reactive state. In keeping with the in vivo data, axon outgrowth from lizard retinal explants is increased above control levels from 6 weeks, reaches its maximum as late as 3 months, and remains elevated for at least 1 year after ONS. By means of BrdU incorporation assays and antiproliferating cell nuclear antigen immunohistochemistry, we show that the late axon outgrowth is not derived from new RGCs that might have arisen in reaction to ONS: no labeled cells were detected in lizard retinas at 0.5, 1, 1.5, 3, 6, and 12 months after ONS. Conversely, numbers of RGCs undergoing apoptosis were too low to be detectable in TUNEL assays at any time after ONS. These results demonstrate that retinal axon regeneration in G. galloti is due to axon regrowth from the resident population of RGCs, which remain in a reactive state over an extended time interval. Neurogenesis does not appear to be involved in RGC axon regrowth in G. galloti

G protein-coupled receptor kinase 2 downregulation reduces hepatic steatosis in a mouse model of diet-induced steatohepatitis

Resumen del póster presentado al 1st Joint Meeting of the French-Portuguese-Spanish Biochemical and Molecular Biology Societies y al XL Spanish Society of Biochemistry and Molecular Biology (SEBBM) Congress, celebrado en Barcelona (España) del 23 al 26 de octubre de 2017.Non-alcoholic fatty liver disease (NAFLD) is an extremely prevalent hepatic pathology that affects about one quarter of adults in the developed world. This disease spectrum includes diff erent conditions ranging from simple steatosis, to non-alcoholic steatohepatitis (NASH), fibrosis and cirrhosis. Insulin resistance and obesity are two well-established pathogenic factors associated to NAFLD, and in fact NAFLD is a very common condition among patients with type 2 diabetes. G protein-coupled receptor kinase 2 (GRK2), classically known as a regulator of G protein-coupled receptors, has been described to play a relevant role in the development of IR and obesity in vivo. However, the eff ect of GRK2 in the development of NASH had not been addressed so far. Here we investigate the contribution of GRK2 to NASH in mice subjected to a methionine and choline-deficient diet (MCD), a well-stablished model of NASH that is independent of fat mass accretion. [Results]: We analyzed a variety of metabolic parameters in WT and GRK2+/- mice fed a MCD diet for four weeks. MCD induced similar alterations and a comparable elevation in plasma transaminase activity in WT and GRK2+/- mice. However, steatosis and other negative effects of this diet in the liver were partially alleviated in GRK2 +/- animals. For instance, the increase in hepatic triglyceride accumulation? and in the liver to body weight ratio induced by MCD were significantly lower in GRK2+/- mice. Furthermore, pathophysiological examination revealed signifi cantly lower NAFLD activity score (NAS) in GRK2+/- mice which were protected from overt NASH. GRK2+/- mice also presented protection from some deleterious effects of the MCD in the liver, such as reduced expression of markers of endoplasmic reticulum stress and inflammation. Moreover, GRK2+/- mice retained certain hepatic protective mechanisms after the MCD as revealed using markers of autophagy and mitochondrial fusion processes. We also found that MCD feeding induced an increase in GRK2 protein levels in WT but not in GRK2+/- livers, similar to what is observed in hepatic tissue of mice fed with a high fat diet. Interestingly, a similar increase in GRK2 protein was also observed in human hepatic samples of NAFLD patients by immunohistochemistry. In the same line, we found that Huh7 cells overexpressing GRK2 accumulated more lipids when exposed to palmitic acid compared to controls. [Conclusion]: Our results uncover a role for GRK2 in the regulation of hepatic lipid metabolism, and provide a proof of concept that reducing the levels of this kinase can impair the development of NASH.Peer reviewe

Long-term evolution of local, proximal and remote astrocyte responses after diverse nucleus basalis lesioning (an experimental Alzheimer model): GFAP immunocytochemical study

A study on long-term astrocytic responses (from 1 day to 20 months after lesioning in 4-month-old rats, and from 1 day to 6 months in 20-month-old rats) to diverse unilateral damage of the nucleus basalis (nbM) by injection of 40 nmol of ibotenic acid, or 50 or 100 nmols of quisqualic acid was performed using a histochemical method (immunoreactivity against the glial fibrillary acidic protein GFAP). Glial reactivity (i.e., isolated or clustered hypertrophic and/or hyper-reactive astrocytes) was evaluated in several ipsilateral and contralateral brain regions: the 'local response' within the damaged nbM region; the 'proximal response' (a new concept proposed by us) in the non-damaged structures neighbouring the nbM; and the 'remote response' in the ipsilateral brain cortex and in the contralateral cortex and nbM. In 4-month-old animals, the remote cortical glial responses, independent of the involution of cortical cholinergic activity and randomly located in layers I-V of motor and somatosensory cortical regions, were similar in appearance over a long period (13-20 months), with the highest reactivity 45 days after lesioning. The proximal response lasted from 1 day to 13 months and afterwards tended to disappear. Contralateral reactivity and ipsilateral cortical scars were observed. The local (nbM) glial response was maintained throughout the period studied. Subsets of astrocytes of different reactivities were observed, most of their elements being highly intermeshed. In 20-month-old animals, nbM lesions produced less positive, but similar, glial reactive patterns. This glial reactivity was superposed onto the glial reactivity of old age. All these results are discussed. The maintenance of reactive astrocytes many months after lesioning suggests the existence of cellular factors other than those produced by damaged nbM neurons. Taking into account the role of glial cells under pathological conditions, it is possible that these reactive astrocytes in humans could promote neurodegenerative processes, such as amyloid plaque formation and neurodegeneration (Alzheimer's disease). Along this line, nbM cholinergic involution could then originate cortical involution through induced reactive astrocytosis. Themes: Disorders of the nervous system. Topics: Degenerative diseases: Alzheimer's. Copyright (C) 2000 Elsevier Science B.V.Peer Reviewe

Involvement of G protein-coupled receptor kinase 2 (GRK2) in the development of non-alcoholic steatosis and steatohepatitis in mice and humans

Insulin resistance (IR) and obesity are important risk factors for non-alcoholic fatty liver disease (NAFLD). G protein-coupled receptor kinase 2 (GRK2) is involved in the development of IR and obesity in vivo. However, its possible contribution to NAFLD and/or non-alcoholic steatohepatitis (NASH) independently of its role on IR or fat mass accretion has not been explored. Here, we used wild-type (WT) or GRK2 hemizygous (GRK2±) mice fed a high-fat diet (HFD) or a methionine and choline-deficient diet (MCD) as a model of NASH independent of adiposity and IR. GRK2± mice were protected from HFD-induced NAFLD. Moreover, MCD feeding caused an increased in triglyceride content and liver-to-body weight ratio in WT mice, features that were attenuated in GRK2± mice. According to their NAFLD activity score, MCD-fed GRK2± mice were diagnosed with simple steatosis and not overt NASH. They also showed reduced expression of lipogenic and lipid-uptake markers and less signs of inflammation in the liver. GRK2± mice preserved hepatic protective mechanisms as enhanced autophagy and mitochondrial fusion and biogenesis, together with reduced endoplasmic reticulum stress. GRK2 protein was increased in MCD-fed WT but not in GRK2± mice, and enhanced GRK2 expression potentiated palmitic acid-triggered lipid accumulation in human hepatocytes directly relating GRK2 levels to steatosis. GRK2 protein and mRNA levels were increased in human liver biopsies from simple steatosis or NASH patients in two different human cohorts. Our results describe a functional relationship between GRK2 levels and hepatic lipid accumulation and implicate GRK2 in the establishment and/or development of NASH.Ministerio de Economia y Competitividad (MINECO/FEDER), Spain (grants SAF2014-55511-R and SAF2017-84125-R to F.M and C. M., grant SAF2015-65267-R to A.M.V., grant SAF2016-76028R to L.F. and SAF2017-87301-R to M.L.M.-C); Comunidad de Madrid-B2017/BMD-3671-INFLAMUNE to F.M.; Gobierno Vasco-Departamento de Salud 2013111114 and EITB Maratoia BIO15/CA/014 (to M.L.M.C); European Foundation for the Study of Diabetes (EFSD) Novo Nordisk Partnership for Diabetes Research in Europe Grant (to F.M.); Asociacion Espanola contra el Cancer (M.L.M-C) and Fundacion Ramon Areces (to C.M.). Instituto de Salud Carlos III (with European FEDER contribution) funds CIBERCV (grant CB16/11/00278 to F.M), CIBERDEM and CIBERehdPeer Reviewe