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Lafora disease (LD) is a fatal rare neurodegenerative disorder that affects young adolescents and has no treatment yet. The hallmark of LD is the presence of polyglucosan inclusions (PGs), called Lafora bodies (LBs), in the brain and peripheral tissues. LD is caused by mutations in either EPM2A or EPM2B genes, which, respectively, encode laforin, a glucan phosphatase, and malin, an E3-ubiquitin ligase, with identical clinical features. LD knockout mouse models (Epm2a - / - and Epm2b - / -) recapitulate PG body accumulation, as in the human pathology, and display alterations in glutamatergic transmission and neuroinflammatory pathways in the brain. In this work, we show the results of four pre-clinical trials based on the modulation of glutamatergic transmission (riluzole and memantine) and anti-neuroinflammatory interventions (resveratrol and minocycline) as therapeutical strategies in an Epm2b - / - mouse model. Drugs were administered in mice from 3 to 5 months of age, corresponding to early stage of the disease, and we evaluated the beneficial effect of the drugs by in vivo behavioral phenotyping and ex vivo histopathological brain analyses. The behavioral assessment was based on a battery of anxiety, cognitive, and neurodegenerative tests and the histopathological analyses included a panel of markers regarding PG accumulation, astrogliosis, and microgliosis. Overall, the outcome of ameliorating the excessive glutamatergic neurotransmission present in Epm2b - / - mice by memantine displayed therapeutic effectiveness at the behavioral levels. Modulation of neuroinflammation by resveratrol and minocycline also showed beneficial effects at the behavioral level. Therefore, our study suggests that both therapeutical strategies could be beneficial for the treatment of LD patients. A mouse model of Lafora disease (Epm2b-/-) was used to check the putative beneficial effect of different drugs aimed to ameliorate the alterations in glutamatergic transmission and/or neuroin

CIBER (Center for Biomedical Network Research; Centro de Investigación Biomédica En Red) is a public national consortium created in 2006 under the umbrella of the Spanish National Institute of Health Carlos III (ISCIII). This innovative research structure comprises 11 different specific areas dedicated to the main public health priorities in the National Health System. CIBERER, the thematic area of CIBER focused on rare diseases (RDs) currently consists of 75 research groups belonging to universities, research centers, and hospitals of the entire country. CIBERER's mission is to be a center prioritizing and favoring collaboration and cooperation between biomedical and clinical research groups, with special emphasis on the aspects of genetic, molecular, biochemical, and cellular research of RDs. This research is the basis for providing new tools for the diagnosis and therapy of low-prevalence diseases, in line with the International Rare Diseases Research Consortium (IRDiRC) objectives, thus favoring translational research between the scientific environment of the laboratory and the clinical setting of health centers. In this article, we intend to review CIBERER's 15-year journey and summarize the main results obtained in terms of internationalization, scientific production, contributions toward the discovery of new therapies and novel genes associated to diseases, cooperation with patients' associations and many other topics related to RD research.

Friedreich's ataxia (FRDA) is a neurodegenerative disorder caused by an unstable GAA repeat expansion within intron 1 of the FXN gene and characterized by peripheral neuropathy. A major feature of FRDA is frataxin deficiency with the loss of large sensory neurons of the dorsal root ganglia (DRG), namely proprioceptive neurons, undergoing dying-back neurodegeneration with progression to posterior columns of the spinal cord and cerebellar ataxia. We used isolated DRGs from a YG8R FRDA mouse model and C57BL/6J control mice for a proteomic study and a primary culture of sensory neurons from DRG to test novel pharmacological strategies. We found a decreased expression of electron transport chain (ETC) proteins, the oxidative phosphorylation (OXPHOS) system and antioxidant enzymes, confirming a clear impairment in mitochondrial function and an oxidative stress-prone phenotype. The proteomic profile also showed a decreased expression in Ca2+ signaling related proteins and G protein-coupled receptors (GPCRs). These receptors modulate intracellular cAMP/cGMP and Ca2+ levels. Treatment of frataxin-deficient sensory neurons with phosphodiesterase (PDE) inhibitors was able to restore improper cytosolic Ca2+ levels and revert the axonal dystrophy found in DRG neurons of YG8R mice. In conclusion, the present study shows the effectiveness of PDE inhibitors against axonal degeneration of sensory neurons in YG8R mice. Our findings indicate that PDE inhibitors may become a future FRDA pharmacological treatment.

Abnormalities in actin cytoskeleton have been linked to Friedreich's ataxia (FRDA), an inherited peripheral neuropathy characterised by an early loss of neurons in dorsal root ganglia (DRG) among other clinical symptoms. Despite all efforts to date, we still do not fully understand the molecular events that contribute to the lack of sensory neurons in FRDA. We studied the adult neuronal growth cone (GC) at the cellular and molecular level to decipher the connection between frataxin and actin cytoskeleton in DRG neurons of the well-characterised YG8R Friedreich's ataxia mouse model. Immunofluorescence studies in primary cultures of DRG from YG8R mice showed neurons with fewer and smaller GCs than controls, associated with an inhibition of neurite growth. In frataxin-deficient neurons, we also observed an increase in the filamentous (F)-actin/monomeric (G)-actin ratio (F/G-actin ratio) in axons and GCs linked to dysregulation of two crucial modulators of filamentous actin turnover, cofilin-1 and the actin-related protein (ARP) 2/3 complex. We show how the activation of cofilin is due to the increase in chronophin (CIN), a cofilin-activating phosphatase. Thus cofilin emerges, for the first time, as a link between frataxin deficiency and actin cytoskeleton alterations.

Friedreich's ataxia (FRDA) is a peripheral neuropathy involving a loss of proprioceptive sensory neurons. Studies of biopsies from patients suggest that axonal dysfunction precedes the death of proprioceptive neurons in a dying-back process. We observed that the deficiency of frataxin in sensory neurons of dorsal root ganglia (DRG) of the YG8R mouse model causes the formation of axonal spheroids which retain dysfunctional mitochondria, shows alterations in the cytoskeleton and it produces impairment of axonal transport and autophagic flux. The homogenous distribution of axonal spheroids along the neurites supports the existence of continues focal damages. This lead us to propose for FRDA a model of distal axonopathy based on axonal focal damages. In addition, we observed the involvement of oxidative stress and dyshomeostasis of calcium in axonal spheroid formation generating axonal injury as a primary cause of pathophysiology. Axonal spheroids may be a consequence of calcium imbalance, thus we propose the quenching or removal extracellular Ca2+ to prevent spheroids formation. In our neuronal model, treatments with BAPTA and o-phenanthroline reverted the axonal dystrophy and the mitochondrial dysmorphic parameters. These results support the hypothesis that axonal pathology is reversible in FRDA by pharmacological manipulation of intracellular Ca2+ with Ca2+ chelators or metalloprotease inhibitors, preventing Ca2+-mediated axonal injury. Thus, the modulation of Ca2+ levels may be a relevant therapeutic target to develop early axonal protection and prevent dying-back neurodegeneration.

La FRDA es una enfermedad caracterizada por la afectación primaria del ganglio dorsal. La falta de frataxina no afecta por igual a todos los tipos neuronales del ganglio dorsal, siendo las neuronas propioceptivas las más dañadas. Esto nos lleva a cuestionarnos porque este tipo neuronal es más sensible a una deficiencia de frataxina. Esta tesis doctoral se ha centrado en la investigación de la biología celular de frataxina en neuronas sensitivas del ganglio dorsal y las consecuencias fisiopatológicas del déficit de frataxina en el modelo murino YG8R. La caracterización molecular y bioquímica de las neuronas sensitivas, especialmente de las propioceptivas, en condiciones fisiológicas y de déficit de frataxina, ha permitido identificar nuevos marcadores selectivos de esta población neuronal e identificar aquellos mecanismos fisiopatológicos que provocan la neurodegeneración primaria del ganglio dorsal en la FRDA. Además, es interesante comprobar cómo estos procesos afectan a la neurona, distinguiendo dos localizaciones: el soma y el axón. La respuesta a estas cuestiones nos ha ayudado a comprender el papel de la frataxina en los mecanismos moleculares relacionados con los procesos de neurodegeneración y ha abierto nuevas vías celulares de estudio y la implementación de nuevos tratamientos farmacológicos en la FRDA.

The effect of COX-2-dependent prostaglandins (PGs) in acute liver injury has been investigated in transgenic mice that express human COX-2 in hepatocytes. We have used three well established models of liver injury: Lipopolysaccharide injury in D-galactosamine preconditioned mice (LPS/D-GalN), the hepatitis induced by concanavalin A (ConA) and the proliferation of hepatocytes in regenerating liver after partial hepatectomy (PH). Our data demonstrate that PGs synthesis decreases in hepatocytes the susceptibility to LPS/D-GalN or ConA-induced liver injury as deduced by significant lower levels of the pro-inflammatory profile and plasmatic aminotransferases in transgenic mice, an effect suppressed by COX-2 selective inhibitors. These transgenic animals express higher levels of anti-apoptotic proteins and exhibit activation of proteins implicated in cell survival, such as Akt and AMP-kinase after injury. The resistance to LPS/D-GalN induced liver apoptosis involves an impairment of procaspase 3 and 8 activation. Protection against ConA-induced injury implies a significant reduction in necrosis. Moreover, hepatocyte commitment to start replication is anticipated in Tg mice after PH, due to the expression of PCNA, cyclin D1 and E. These results show, in a genetic model, that tissue-specific COX-2 dependent PGs exert an efficient protection against acute liver injury by an antiapoptotic/antinecrotic effect and by accelerated early hepatocyte proliferation.

We have investigated the mechanism of COX-2 (cyclo-oxygenase 2)-dependent inhibition of apoptosis in liver, a key pathway underlying proliferative actions of COX-2 in liver cancers, cirrhosis, chronic hepatitis C infection and regeneration after partial hepatectomy. Stable expression of COX-2 in CHL (Chang liver) cells induced proliferation, with an increase in the proportion of cells in S-phase, but no other significant changes in cell-cycle distribution. This was associated with a marked inhibition of the apoptotic response to serum deprivation, an effect mimicked by treating empty-vector-transfected control cells (CHL-V cells) with prostaglandin E2 and prevented in COX-2-expressing cells (CHL-C cells) treated with selective inhibitors of COX-2. Serum-deprived CHL-V cells displayed several indicators of activation of intrinsic apoptosis: caspases 9 and 3 activated within 6 h and caspase 8 within 18 h, Bax expression was induced, cytochrome c was released to the cytosol, and PARP-1 [poly(ADP-ribose) polymerase 1] cleavage was evident in nuclei. COX-2 expression blocked these events, concomitant with reduced expression of p53 and promotion of Akt phosphorylation, the latter indicating activation of survival pathways. CHL cells were resistant to stimulation of the extrinsic pathway with anti-Fas antibody. Moreover, in vivo expression of GFP (green fluorescent protein)-labelled COX-2 in mice by hydrodynamics-based transient transfection conferred resistance to caspase 3 activation and apoptosis induced by stimulation of Fas.