Frataxin knockdown in human astrocytes triggers cell death and the release of factors that cause neuronal toxicity

© 2014 Elsevier Inc. Friedreich's ataxia (FA) is a recessive, predominantly neurodegenerative disorder caused in most cases by mutations in the first intron of the frataxin (FXN) gene. This mutation drives the expansion of a homozygous GAA repeat that results in decreased levels of FXN transcription and frataxin protein. Frataxin (Fxn) is a ubiquitous mitochondrial protein involved in iron-sulfur cluster biogenesis, and a decrease in the levels of this protein is responsible for the symptoms observed in the disease. Although the pathological manifestations of FA are mainly observed in neurons of both the central and peripheral nervous system, it is not clear if changes in non-neuronal cells may also contribute to the pathogenesis of FA, as recently suggested for other neurodegenerative disorders. Therefore, the aims of this study were to generate and characterize a cell model of Fxn deficiency in human astrocytes (HAs) and to evaluate the possible involvement of non-cell autonomous processes in FA. To knockdown frataxin in vitro, we transduced HAs with a specific shRNA lentivirus (shRNA37), which produced a decrease in both frataxin mRNA and protein expression, along with mitochondrial superoxide production, and signs of p53-mediated cell cycle arrest and apoptotic cell death. To test for non-cell autonomous interactions we cultured wild-type mouse neurons in the presence of frataxin-deficient astrocyte conditioned medium, which provoked a delay in the maturation of these neurons, a decrease in neurite length and enhanced cell death. Our findings confirm a detrimental effect of frataxin silencing, not only for astrocytes, but also for neuron-glia interactions, underlining the need to take into account the role of non-cell autonomous processes in FA.“l'Association Française de l'Ataxie de Friedreich” (AFAF), as well as the Spanish National Research Plan (SAF 2012-38042) and the Autonomous Government of Madrid (S20/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

Enhanced production of herpes simplex virus 1 amplicon vectors by gene modification and optimization of packaging cell growth medium

Herpes simplex virus 1 (HSV-1)-derived amplicon vectors are unique in their ability to accommodate large DNA molecules allowing whole genomic loci to be included with all of their regulatory elements. Additional advantages of these amplicons include their minimal toxicity and ability to persist as episomes, with negligible risk of insertional mutagenesis, being particularly well-suited for gene therapy of neurological disorders due to their outstanding ability to deliver genes into neurons and other neural cells. However, extensive gene therapy application has been hindered by difficulties in vector production. This work improved HSV-1 amplicons production by genetic modification of the packaging cell line and optimization of the culture medium. A stably-transfected Vero 2-2 cell line overexpressing the anti-apoptotic Bcl-2 protein was generated, exhibiting an increased resistance to apoptosis, prolonged culture duration, and a significant improvement in viral vector production. Additionally, supplementation of the growth medium with antioxidants, polyamines, amino acids, and reduced glutathione further increased the yield of packaged amplicon vectors. With these modifications, HSV-1 amplicons could be isolated from culture supernatants instead of cell lysates, leading to vector preparations with higher titer and purity and paving the way for generation of stable cell lines that are capable of continuous herpesviral vector productionThis work was supported by grants of the Spanish National Research Plan (SAF 2015–69361-R), Friedreich Ataxia Research Alliance (FARA), FARA Ireland, Spanish FEDAES, GENEFA, and Babel Famil

Future prospects of gene therapy for Friedreich's ataxia

Friedreich’s ataxia is an autosomal recessive neurogenetic disease that is mainly associated with atrophy of the spinal cord and progressive neurodegeneration in the cerebellum. The disease is caused by a GAA-expansion in the first intron of the frataxin gene leading to a decreased level of frataxin protein, which results in mitochondrial dysfunction. Currently, there is no effective treatment to delay neurodegeneration in Friedreich’s ataxia. A plausible therapeutic approach is gene therapy. Indeed, Friedreich’s ataxia mouse models have been treated with viral vectors en-coding for either FXN or neurotrophins, such as brain-derived neurotrophic factor showing promising results. Thus, gene therapy is increasingly consolidating as one of the most promising therapies. However, several hurdles have to be overcome, including immunotoxicity and pheno-toxicity. We review the state of the art of gene therapy in Friedreich’s ataxia, addressing the main challenges and the most feasible solutions for themComunidad Autónoma de Madrid (NEUROMETAB-CM, B2017/BMD-3700) and Spanish Ministerio de Ciencia e Innovación (MICINN, grant PID2019-111338RB-I00), co-financed by Agencia Estatal de Investigación and Fondo Europeo de Desarrollo Regional (AEI/FEDER, EU). We also acknowledge institutional support to the CBMSO from Fundación Ramón Areces. Gabriel Ocaña-Santero receive

Olfactory Ensheathing Glia: Drivers of Axonal Regeneration in the Central Nervous System?

Olfactory ensheathing glia (OEG) accompany olfactory growing axons in their entry to the adult mammalian central nervous system (CNS). Due to this special characteristic, considerable attention has been focused on the possibility of using OEG for CNS regeneration. OEG present a large heterogeneity in culture with respect to their cellular morphology and expressed molecules. The specific characteristics of OEG responsible for their regenerative properties have to be defined. These properties probably result from the combination of several factors: molecular composition of the membrane (expressing adhesion molecules as PSA-NCAM, L1 and/or others) combined with their ability to reduce glial scarring and to accompany new growing axons into the host CNS. Their capacity to produce some neurotrophic factors might also account for their ability to produce CNS regeneration

Phosphorylation of a neuronal-specific β-tubulin isotype

Adult rats were intracraneally injected with [32P] phosphate and brain microtubules isolated. The electrophoretically purified, in vivo phospholabeled, beta-tubulin was digested with the V8-protease and the labeled peptide purified by reversed-phase liquid chromatography. Its amino acid sequence corresponds to the COOH-terminal sequence of a minor neuronal beta 3-tubulin isoform from chicken and human. The phosphorylation site was at serine 444. A synthetic peptide with sequence EMYEDDEEESESQGPK, corresponding to that of the COOH terminus of beta 3-tubulin, was efficiently phosphorylated in vitro by casein kinase II at the same serine 444. The functional meaning of tubulin phosphorylation is still unclear. However, the modification of the protein takes place after microtubule assembly, and phosphorylated tubulin is mainly present in the assembled microtubule protein fraction

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

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