The smoothened agonist SAG reduces mitochondrial dysfunction and neurotoxicity of frataxin-deficient astrocytes

Background: Friedreich’s ataxia is a rare hereditary neurodegenerative disease caused by decreased levels of the mitochondrial protein frataxin. Similar to other neurodegenerative pathologies, previous studies suggested that astrocytes might contribute to the progression of the disease. To fully understand the mechanisms underlying neurodegeneration in Friedreich’s ataxia, we investigated the reactivity status and functioning of cultured human astrocytes after frataxin depletion using an RNA interference-based approach and tested the effect of pharmacologically modulating the SHH pathway as a novel neuroprotective strategy. Results: We observed loss of cell viability, mitochondrial alterations, increased autophagy and lipid accumulation in cultured astrocytes upon frataxin depletion. Besides, frataxin-deficient cells show higher expression of several A1-reactivity markers and release of pro-inflammatory cytokines. Interestingly, most of these defects were prevented by chronically treating the cells with the smoothened agonist SAG. Furthermore, in vitro culture of neurons with conditioned medium from frataxin-deficient astrocytes results in a reduction of neuronal survival, neurite length and synapse formation. However, when frataxin-deficient astrocytes were chronically treated with SAG, we did not observe these alterations in neurons. Conclusions: Our results demonstrate that the pharmacological activation of the SHH pathway could be used as a target to modulate astrocyte reactivity and neuron–glia interactions to prevent neurodegeneration in Friedreich’s ataxiaThis study was supported by research grants from Comunidad Autónoma de Madrid (NEUROMETAB-CM, B2017/BMD-3700) to J.D-N., Spanish Ministerio de Ciencia e Innovación (MICINN, grant PID2019-111338RB-I00) to J.D-N. and A.G-C., Instituto de Salud Carlos III (PI20/00934, co-funded by Fondo Europeo de Desarrollo Regional, FEDER) and Association Française de l’Ataxie de Friedreich (AFAF) to F.L. A.V-A. is supported by a contract from Comunidad Autónoma de Madrid (NEUROMETAB-CM, B2017/BMD-3700

Thioredoxin reductase 1 suppresses adipocyte differentiation and insulin responsiveness

Recently thioredoxin reductase 1 (TrxR1), encoded by Txnrd1, was suggested to modulate glucose and lipid metabolism in mice. Here we discovered that TrxR1 suppresses insulin responsiveness, anabolic metabolism and adipocyte differentiation. Immortalized mouse embryonic fibroblasts (MEFs) lacking Txnrd1 (Txnrd1−/−) displayed increased metabolic flux, glycogen storage, lipogenesis and adipogenesis. This phenotype coincided with upregulated PPARγ expression, promotion of mitotic clonal expansion and downregulation of p27 and p53. Enhanced Akt activation also contributed to augmented adipogenesis and insulin sensitivity. Knockdown of TXNRD1 transcripts accelerated adipocyte differentiation also in human primary preadipocytes. Furthermore, TXNRD1 transcript levels in subcutaneous adipose tissue from 56 women were inversely associated with insulin sensitivity in vivo and lipogenesis in their isolated adipocytes. These results suggest that TrxR1 suppresses anabolic metabolism and adipogenesis by inhibition of intracellular signaling pathways downstream of insulin stimulationThis study was supported by funding to ESJA from Karolinska Institutet, The Swedish Research Council, The Swedish Cancer Society, to MR from the Strategic Research Program in Diabetes and to ACG from Diabetesfonden and a “Ramón y Cajal” fellowship (RYC-2014-15792) from Spanish Ministerio de Economía y Competitivida

Delivery of the 135kb human frataxin genomic DNA locus gives rise to different frataxin isoforms

© 2015 Elsevier Inc. Friedreich's ataxia (FRDA) is the most common form of hereditary ataxia caused by recessive mutations in the FXN gene. Recent results have indicated the presence of different frataxin isoforms due to alternative gene expression mechanisms. Our previous studies demonstrated the advantages of using high-capacity herpes simplex virus type 1 (HSV-1) amplicon vectors containing the entire FXN genomic locus (iBAC-FXN) as a gene-delivery vehicle capable of ensuring physiologically-regulated and long-term persistence. Here we describe how expression from the 135. kb human FXN genomic locus produces the three frataxin isoforms both in cultured neuronal cells and also in vivo. Moreover, we also observed the correct expression of these frataxin isoforms in patient-derived cells after delivery of the iBAC-. FXN. These results lend further support to the potential use of HSV-1 vectors containing entire genomic loci whose expression is mediated by complex transcriptional and posttranscriptional mechanisms for gene therapy applications.Spanish National Research Plan (SAF 2012-38042) and the Autonomous Government of Madrid (S2010/BMD-2331). The Center for Biomedical Research on Rare Diseases (“Centro de Investigación Biomédica en Red sobre Enfermedades Raras”, CIBERER) is an initiative supported by the “Instituto de Salud Carlos III”.Peer Reviewe

Disrupted neuroglial metabolic coupling after peripheral surgery

Immune-related events in the periphery can remotely affect brain function, contributing to neurodegenerative processes and cognitive decline. In mice, peripheral surgery induces a systemic inflammatory response associated with changes in hippocampal synaptic plasticity and transient cognitive decline, however, the underlying mechanisms remain unknown. Here we investigated the effect of peripheral surgery on neuronal-glial function within hippocampal neuronal circuits of relevance to cognitive processing in male mice at 6, 24, and72hpostsurgery. At 6hwedetect theproinflammatorycytokineIL-6inthehippocampus, followedupbyalterations in them RNA and protein expression of astrocyticandneuronal proteinsnecessaryfor optimal energysupplytothebrainandfor thereuptakeandrecycling of glutamate in the synapse. Similarly, at 24 h postsurgery the mRNA expression of structural proteins (GFAP and AQP4) was compromised. At this time point, functional analysis in astrocytes revealed a decrease in resting calcium signaling. Examination of neuronal activity by whole-cell patch-clamp shows elevated levels of glutamatergic transmission and changes in AMPA receptor subunit composition at 72 h postsurgery. Finally, lactate, an essential energy substrate produced by astrocytes and critical for memory formation, decreases at 6 and 72 h after surgery. Based on temporal parallels with our previous studies, we propose that the previously reported cognitive decline observed at 72 h postsurgery in mice might be the consequence of temporal hippocampal metabolic, structural, and functional changes in astrocytes that lead to a disruption of the neuroglial metabolic coupling and consequently to a neuronal dysfunction.This work was supported by a “Ramón y Cajal” fellowship (RYC-2014-15792 to A.G.-C.) from Spanish “Ministerio de Economía y Competitividad”, the Swedish Research Council, the confocal microscope used in the study by Knut and Alice Wallenberg Foundation (Grant KAW2008.0149), and NIH/NIA R01AG057525 to N.T

Absence of R-Ras1 and R-Ras2 causes mitochondrial alterations that trigger axonal degeneration in a hypomyelinating disease model

Fast synaptic transmission in vertebrates is critically dependent on myelin for insulation and metabolic support. Myelin is produced by oligodendrocytes (OLs) that maintain multilayered membrane compartments that wrap around axonal fibers. Alterations in myelination can therefore lead to severe pathologies such as multiple sclerosis. Given that hypomyelination disorders have complex etiologies, reproducing clinical symptoms of myelin diseases from a neurological perspective in animal models has been difficult. We recently reported that R-Ras1 and/or R-Ras2 mice, which lack GTPases essential for OL survival and differentiation processes, present different degrees of hypomyelination in the central nervous system with a compounded hypomyelination in double knockout (DKO) mice. Here, we discovered that the loss of R-Ras1 and/or R-Ras2 function is associated with aberrant myelinated axons with increased numbers of mitochondria, and a disrupted mitochondrial respiration that leads to increased reactive oxygen species levels. Consequently, aberrant myelinated axons are thinner with cytoskeletal phosphorylation patterns typical of axonal degeneration processes, characteristic of myelin diseases. Although we observed different levels of hypomyelination in a single mutant mouse, the combined loss of function in DKO mice lead to a compromised axonal integrity, triggering the loss of visual function. Our findings demonstrate that the loss of R-Ras function reproduces several characteristics of hypomyelinating diseases, and we therefore propose that R-Ras1 and R-Ras2 neurological models are valuable approaches for the study of these myelin pathologies.Spanish Ministry of Economy and Competitiveness (RTI2018-096303B-C33) to B. C., (RTI2018-096303B-C31) to F. W., and RTI2018-095166B-I00 to C. G. R. and P. L. and Instituto de Salud Carlos III and co-funded by the European Regional Development Fund (ERDF) within the “Plan Estatal de Investigación Científica y Técnica y de Innovación 2017–2020” (RD16/0008/0020; FIS/PI 18-00754

BACE1 activity impairs neuronal glucose oxidation:rescue by beta-hydroxybutyrate and lipoic acid

Glucose hypometabolism and impaired mitochondrial function in neurons have been suggested to play early and perhaps causative roles in Alzheimer's disease (AD) pathogenesis. Activity of the aspartic acid protease, beta-site amyloid precursor protein (APP) cleaving enzyme 1 (BACE1), responsible for beta amyloid peptide generation, has recently been demonstrated to modify glucose metabolism. We therefore examined, using a human neuroblastoma (SH-SY5Y) cell line, whether increased BACE1 activity is responsible for a reduction in cellular glucose metabolism. Overexpression of active BACE1, but not a protease-dead mutant BACE1, protein in SH-SY5Y cells reduced glucose oxidation and the basal oxygen consumption rate, which was associated with a compensatory increase in glycolysis. Increased BACE1 activity had no effect on the mitochondrial electron transfer process but was found to diminish substrate delivery to the mitochondria by inhibition of key mitochondrial decarboxylation reaction enzymes. This BACE1 activity-dependent deficit in glucose oxidation was alleviated by the presence of beta hydroxybutyrate or α-lipoic acid. Consequently our data indicate that raised cellular BACE1 activity drives reduced glucose oxidation in a human neuronal cell line through impairments in the activity of specific tricarboxylic acid cycle enzymes. Because this bioenergetic deficit is recoverable by neutraceutical compounds we suggest that such agents, perhaps in conjunction with BACE1 inhibitors, may be an effective therapeutic strategy in the early-stage management or treatment of AD

Structural insights and biological effects of glycogen synthase kinase 3-specific inhibitor AR-A014418

Glycogen synthase kinase 3 (GSK3) is a serine/threonine kinase that has been implicated in pathological conditions such as diabetes and Alzheimer's disease. We report the characterization of a GSK3 inhibitor, AR-A014418, which inhibits GSK3 (IC50 = 104 ± 27 nM), in an ATP-competitive manner (K i = 38 nM). AR-A014418 does not significantly inhibit cdk2 or cdk5 (IC50 > 100 μM) or 26 other kinases demonstrating high specificity for GSK3. We report the co-crystallization of AR-A014418 with the GSK3β protein and provide a description of the interactions within the ATP pocket, as well as an understanding of the structural basis for the selectivity of AR-A014418. AR-A014418 inhibits tau phosphorylation at a GSK3-specific site (Ser-396) in cells stably expressing human four-repeat tau protein. AR-A014418 protects N2A neuroblastoma cells against cell death mediated by inhibition of the phosphatidylinositol 3-kinase/protein kinase B survival pathway. Furthermore, AR-A014418 inhibits neurodegeneration mediated by β-amyloid peptide in hippocampal slices. AR-A014418 may thus have important applications as a tool to elucidate the role of GSK3 in cellular signaling and possibly in Alzheimer's disease. AR-A014418 is the first compound of a family of specific inhibitors of GSK3 that does not significantly inhibit closely related kinases such as cdk2 or cdk5.Supported by grants from the Comunidad de Madrid, the Fundacion “La Caixa,” the Lilly Foundation, the Spanish Comision Interministerial de Ciencia y Tecnologia, and an institutional grant from the Fundacion Ramon Areces.Peer reviewe