Referencias científicas Nº 163
Biochemical Analyses of Human Iron–Sulfur Protein Biogenesis and of Related Diseases
Maturation of Fe/S proteins in mammals is an intricate process mediated by two assembly systems located in the mitochondrial and cytosolic–nuclear compartments. Malfunction particularly of the mitochondrial system gives rise to severe neurological, metabolic, or hematological disorders, often with fatal outcome. In this chapter, we describe approaches for the differential biochemical investigation of cellular Fe/S protein maturation in mitochondria, cytosol, and nucleus. The analyses may also facilitate the identification of the affected Fe/S protein assembly step in diseased state. As Fe/S cluster insertion into target apoproteins is a frequent determinant of protein stability, examination of protein steady-state levels in biological samples frequently permits reliable first clues about the maturation process. In some specific cases, this approach allows the assessment of enzymatic or regulatory functions of Fe/S proteins, including the formation of lipoate cofactor by mitochondrial lipoic acid synthase or the posttranscriptional regulation of transferrin receptor and ferritin expression by the cytosolic iron regulatory proteins. More direct Fe/S protein maturation assays like enzymatic analyses may further validate the observed maturation defects. Here, we present a simple protocol for the determination of dihydropyrimidine dehydrogenase enzyme activity by thin-layer chromatography. In order to directly monitor Fe/S cluster insertion into target apoproteins, we have developed a 55Fe radiolabeling technique tracing the in vivo Fe/S cofactor formation in mammalian tissue culture. The combination of the presented techniques represents a comprehensive strategy to assess the multiple facets of Fe/S protein assembly for both mechanistic analyses and for the elucidation of specific defects in Fe/S diseases.
Depressive symptoms in Friedreich ataxia
Síntomas depresivos en la ataxia de Friedreich
Background/Objective: Almost no attention has been paid to depression in Friedreich ataxia (FRDA), a highly disabling cerebellar degenerative disease. Our aim was to study the presence and the profile of depressive symptoms in FRDA and their relationship with demographic-disease variables and cognitive processing speed. Method: The study groups consisted of 57 patients with a diagnosis of FRDA. The Beck Depression Inventory-II was used to assess symptoms of depression. Speed of information processing was measured with a Choice Reaction time task. Results: The mean BDI score for patients was significantly higher than the mean score in the general population. Twenty one percent of participants scored in the moderate/severe range. A Cognitive-Affective score and a Somatic-Motivational score was calculated for each patient. Patients’ scores in both dimensions were significantly higher than the scores in the general population. Demographic and disease variables were not related with symptoms of depression, except for severity of ataxia. Depressive symptoms predict cognitive reaction times. The greater proportion of variance was explained by the Cognitive-Affective dimension. Conclusions: Our data show that both somatic-motivational and cognitive affective symptoms of depression are frequent in individuals with FRDA. In addition, depressive symptoms may influence cognition, especially, the cognitive and affective symptoms.
Mitochondrial pore opening and loss of Ca2 + exchanger NCLX levels occur after frataxin depletion
Frataxin-deficient neonatal rat cardiomyocytes and dorsal root ganglia neurons have been used as cell models of Friedreich ataxia. In previous work we show that frataxin depletion resulted in mitochondrial swelling and lipid droplet accumulation in cardiomyocytes, and compromised DRG neurons survival. Now, we show that these cells display reduced levels of the mitochondrial calcium transporter NCLX that can be restored by calcium-chelating agents and by external addition of frataxin fused to TAT peptide. Also, the transcription factor NFAT3, involved in cardiac hypertrophy and apoptosis, becomes activated by dephosphorylation in both cardiomyocytes and DRG neurons. In cardiomyocytes, frataxin depletion also results in mitochondrial permeability transition pore opening. Since the pore opening can be inhibited by cyclosporin A, we show that this treatment reduces lipid droplets and mitochondrial swelling in cardiomyocytes, restores DRG neuron survival and inhibits NFAT dephosphorylation. These results highlight the importance of calcium homeostasis and that targeting mitochondrial pore by repurposing cyclosporin A, could be envisaged as a new strategy to treat the disease.
Friedreich ataxia: Clinical feature and electrophysiological symptoms
Department of Pathobiological Science and Technology, Faculty of Medicine, Tottori University, Yonago, Japan
Department of Pathobiological Science and Technology, Faculty of Medicine, Tottori University, Nishi-cho 86, Yonago
Friedreich ataxia is inherited as an autosomal recessive disorder involving the spinocerebellar tracts, dorsal columns in the spinal cord, the pyramidal tracts, and the cerebellum and medulla. The majority of patients bear a recessive GAA triplet-repeat expansion on intron 1 of both alleles while a minority carry an expansion on allele and a point mutation or deletion on the other., The disease-causing genotype leads to decreased production of frataxin. Mutations cause oxidative injury associated with excessive iron deposits in mitochondria; frataxin deficiency leads to mitochondrial iron accumulation, deficient production of adenosine triphosphate, and a potential rise in free radical generation.,These events lead to onset of a variety of symptoms, such as gait disturbance, loss of sensation, areflexia, and dyscoordination, beginning usually between ages 5 and 15 years although later onset is not uncommon.
Approximately two-thirds of individuals with Friedreich ataxia have cardiomyopathy and up to 30% have diabetes mellitus. The loss of frataxin function in mitochondria accounts for these pathogenic processes in Friedreich ataxia. Mitochondria are essential for the sensing of nutrients by the β cell and for the generation of signals that trigger and amplify insulin secretion, known as stimulus-secretion coupling. Moreover, in the intrinsic pathway of apoptosis, pro-apoptotic signals converge on mitochondria, resulting in mitochondrial Bax translocation, membrane permeabilization, cytochrome c release and caspase cleavage.
The ataxia is slowly progressive and involves the lower extremities to a greater degree than the upper extremities. In general, results of electrophysiologic studies including visual, auditory brainstem, and somatosensory-evoked potentials are often abnormal.
In the article “Diabetes Mellitus as the Presenting Feature of Friedreich Ataxia,” the authors report a case of an 8-year girl who initially presented with diabetic ketoacidosis and was treated as case of insulin dependent diabetes mellitus. Furthermore, they report an axonal type of generalized sensory neuropathy and lower MCV in tibial nerve. The authors assumed the diagnosis of Friedreich ataxia for the patient. However, neuroimaging and FXN gene analysis were not conducted. The co-morbidity of diabetes mellitus and peripheral neuropathy can also result from mitochondrial disorders, and could represent a complication of hereditary motor and sensory neuropathy in a diabetic patient by chance. In addition, atypical Friedreich ataxia due to compound heterozygosity for FXN GAA expansion and a point mutation may present a greater diagnostic dilemma.
Iron-induced oligomerization of human FXN81-210 and bacterial CyaY frataxin and the effect of iron chelators
- Eva-Christina Ahlgren,
- Mostafa Fekry,
- Mathias Wiemann,
- Christopher A. Söderberg,
- Katja Bernfur,
- Olex Gakh,
- Morten Rasmussen,
- Peter Højrup,
- Cecilia Emanuelsson,
- Grazia Isaya,
- Salam Al-Karadaghi
Patients suffering from the progressive neurodegenerative disease Friedreich’s ataxia have reduced expression levels of the protein frataxin. Three major isoforms of human frataxin have been identified, FXN42-210, FXN56-210 and FXN81-210, of which FXN81-210 is considered to be the mature form. Both long forms, FXN42-210 and FXN56-210, have been shown to spontaneously form oligomeric particles stabilized by the extended N-terminal sequence. The short variant FXN81-210, on other hand, has only been observed in the monomeric state. However, a highly homologous E. coli frataxin CyaY, which also lacks an N-terminal extension, has been shown to oligomerize in the presence of iron. To explore the mechanisms of stabilization of short variant frataxin oligomers we compare here the effect of iron on the oligomerization of CyaY and FXN81-210. Using dynamic light scattering, small-angle X-ray scattering, electron microscopy (EM) and cross linking mass spectrometry (MS), we show that at aerobic conditions in the presence of iron both FXN81-210 and CyaY form oligomers. However, while CyaY oligomers are stable over time, FXN81-210 oligomers are unstable and dissociate into monomers after about 24 h. EM and MS studies suggest that within the oligomers FXN81-210 and CyaY monomers are packed in a head-to-tail fashion in ring-shaped structures with potential iron-binding sites located at the interface between monomers. The higher stability of CyaY oligomers can be explained by a higher number of acidic residues at the interface between monomers, which may result in a more stable iron binding. We also show that CyaY oligomers may be dissociated by ferric iron chelators deferiprone and DFO, as well as by the ferrous iron chelator BIPY. Surprisingly, deferiprone and DFO stimulate FXN81-210oligomerization, while BIPY does not show any effect on oligomerization in this case. The results suggest that FXN81-210 oligomerization is primarily driven by ferric iron, while both ferric and ferrous iron participate in CyaY oligomer stabilization. Analysis of the amino acid sequences of bacterial and eukaryotic frataxins suggests that variations in the position of the acidic residues in helix 1, β-strand 1 and the loop between them may control the mode of frataxin oligomerization.
C-Path And FARA Announce Collaborative Data Aggregation Project For Friedreich’s Ataxia
Tucson, AZ, and Downingtown, PA — Dec. 4, 2017 — Critical Path Institute’s (C-Path) Data Collaboration Center (DCC) and the Friedreich’s Ataxia Research Alliance (FARA) have announced that they will work together to develop an aggregated database of clinical data for Friedreich’s ataxia (FA). Use of this database will promote collaborative research to support the understanding of natural history, potential biomarkers, and potential clinical endpoints for patients with FA, which will help researchers develop more efficient clinical trial protocols to test new therapies more quickly and effectively.
“FA is a rare disease which is progressive, affects multiple organ systems, and is fatal. Treating the disease is an urgent unmet need. FA research has reached a critical juncture, where several therapies have undergone or are in clinical trials, and additional new therapies are expected to start clinical development in the next few years. The purpose of this project is to leverage and share as much information as possible, to more fully understand progression of disease, how that progression can be captured in measurable endpoints, and the effect of placebo treatment. We want to ensure that we are using all the information available to design the most efficient and robust clinical trials, giving potential therapies the best chance of success,” explained Jennifer Farmer, FARA’s Executive Director.
The project will establish an integrated database of clinical data for FA that can be shared and utilized by existing FA researchers. It will enlist companies that have carried out clinical trials in FA to obtain contributions of clinical data, as well as sharing natural history data collected by FARA’s collaborative clinical research network.
Three datasets have been promised for the project, with the first placebo-arm dataset expected to be shared in the next few weeks. Sharing additional datasets is under discussion. The de-identified datasets will be aggregated into a single database in a scientifically rigorous manner by C-Path’s DCC.
Launched in 2014, the DCC was founded to provide large-scale data solutions for scientific research, and it will play a key role in this collaborative project, using its experience in clinical data standards, data aggregation, and data sharing.
“With a long history of expertise in data standards development, curation, and oversight of multiple data sharing initiatives, C-Path has the unique opportunity to help coordinate the collaborative contributions from data owners and integrate that data into a single database for this rare disease. Our approach has been successfully implemented in multiple C-Path consortia, and it is our hope that this proven experience will translate well toward the ongoing success of this important project,” said Richard Liwski, Director of the DCC and C-Path’s Chief Technology Officer.
The role of oxidative stress in Friedreich’s ataxia
- Federica Lupoli1,
- Tommaso Vannocci2,
- Giovanni Longo3,
- Neri Niccolai1and
- Annalisa Pastore2
Oxidative stress and an increase in the levels of free radicals are important markers associated with several pathologies, including Alzheimer’s disease, cancer and diabetes. Friedreich’s ataxia (FRDA) is an excellent paradigmatic example of a disease in which oxidative stress plays an important, albeit incompletely understood, role. FRDA is a rare genetic neurodegenerative disease that involves the partial silencing of frataxin, a small mitochondrial protein that was completely overlooked before being linked to FRDA. More than 20 years later, we now know how important this protein is in terms of being an essential and vital part of the machinery that produces iron-sulfur clusters in the cell. In this review, we revisit the most important steps that have brought us to our current understanding of the function of frataxin and its role in disease. We discuss the current hypotheses on the role of oxidative stress in FRDA and review some of the existing animal and cellular models. We also evaluate new techniques that can assist in the study of the disease mechanisms, as well as in our understanding of the interplay between primary and secondary phenotypes.
Designer molecule points to treatment for diseases caused by DNA repeats
November 30, 2017 By David Tenenbaum For news media
Using a molecule designed to overcome a roadblock formed by a common type of genetic flaw, researchers at the University of Wisconsin–Madison have made progress towards novel molecular treatments for Friedreich’s ataxia — a rare but fatal disorder — in the laboratory dish and in animals.
Friedreich’s, like at least 40 other genetic diseases, is caused by stretches of repetitive DNA that prevent protein from forming correctly.
The repeats can contain hundreds of identical, short sequences of DNA (such as GAAGAAGAAGAA …). In some diseases, including Friedreich’s, the repeats become roadblocks to cellular machines that decode the gene and start making the protein that the cell needs. In other diseases, such as the neurological condition Huntington’s, the repeats can result in excess protein, which itself can become toxic.
In research reported this week in the “first release” section of the journal Science, Aseem Ansari, a professor of biochemistry and genomics at UW–Madison, and colleagues showed that their “molecular prosthesis” can help cellular machinery overcome the blockade posed by the repeats in Friedreich’s ataxia.
Aseem Ansari (center), professor of biochemistry, talks with his research group about their findings in his lab in the DeLuca Biochemistry Laboratories on the UW–Madison campus. PHOTO: BRYCE RICHTER
One component of the prosthesis locates the repeats, then the second helps the cellular machinery soldier past the repeats to properly decode the gene.
Friedreich’s appears in only one American in 50,000, but it’s fatal and untreatable, says Ansari. “These kids accumulate repeats in a gene for a protein called frataxin that mitochondria, the cell’s powerhouse, need to process energy. Without frataxin, tissues that use the most energy get hurt first: the brain, heart and pancreas.”
As early as age 5, movement is impaired “because the brain does not have the energy it needs and it also accumulates DNA damage,” says Ansari, who has a joint appointment with the Genome Center of Wisconsin at UW–Madison. “Most young people with Friedreich’s develop severe heart problems and are wheelchair-bound, but the disease is so rare that few drug companies invest in it.”
In the Ansari group, Graham Erwin, Matthew Grieshop and Asfa Ali formed a team that designed and created the prototype molecule, and also orchestrated collaboration with colleagues at UW–Madison, the pharmaceutical firm Novartis, and a leading medical center in India.
The Science publication rested on two kinds of experiments:
- In studies of cell lines from more than 20 Friedreich’s patients, the molecular prosthesis restored expression of the frataxin protein.
- In mice containing transplanted human cells carrying about 310 GAA repeats, the prosthesis restored expression of a signaling protein to nearly normal.
The molecule being tested is designed to assist the enzyme that reads, or “transcribes,” DNA at the confusing repeats. Once it reaches the other side, the enzyme, called RNA polymerase, reads the gene and makes RNA that in turn codes for frataxin, the protein that is lacking in Friedreich’s ataxia.
One part of the new molecule is “a direction finder that we engineered to locate the problem in the patient’s genome,” Ansari says. Once it finds the troublesome repeats, “the second component brings in the machinery that helps RNA polymerase slog through the repeats and make the correct transcript and subsequently the functional protein.”
“Most young people with Friedreich’s develop severe heart problems and are wheelchair-bound, but the disease is so rare that few drug companies invest in it.”
Understanding the role of frataxin and how repeats block its synthesis started with studies in yeast cells and then fruit flies, Ansari says. “In those simple organisms, scientists teased out exactly what the repeat region was doing, and the same principles were at work in human cells. Without that understanding, we would not have been able to devise our molecules, which highlights the need to study biology at all levels.”
Although the biochemistry is complex, the concept of ignoring repeats is not, Ansari says. “If we compare the enzyme that reads DNA to an engine moving down the ‘track’ of DNA, the repeat may stop it cold, cause it to skip too far ahead, or jam it in the ‘on’ position so it makes a toxic amount of the protein.”
The new results published this week in Science suggest that “we have figured out how to turn on the part of the gene that is being ignored without doing anything anywhere else,” Ansari says.
Even as patients and families yearn for a cure, it’s likely to take several years before drug testing begins, Ansari warns.
Three years ago, Annie Hamilton (center) was diagnosed with Friedreich’s ataxia. Tom and Karen, her parents, have formed a foundation to support research aimed at curing FA and other rare diseases. “We are very hopeful, very optimistic,” says Tom. COURTESY OF CURE FA FOUNDATION
Ansari, who has been trying to unravel repeats for about 15 years at UW–Madison, achieved initial success in 2004 when his group designed “two-headed” molecules with a “DNA reading head” that would deliver the molecule to a specific location in an individual’s genome, and a “docking head” that would dock a cellular machine to force the gene to be read correctly at that site.
Then, Ansari says, researchers found that the molecule got “distracted” while floating in a sea of DNA and failed to distinguish hundreds of “look-alike” sites from its actual target.
By 2013, funding had dried up, and only a grant from the W.M. Keck Foundation enabled Ansari to continue searching to outfox the repeats.
While many efforts to treat Friedreich’s are screening millions of drugs, “we are working from a deeper understanding of the problem,” Ansari says. “Once we understood why the enzyme was getting blocked, we rationally designed seven molecules to help the enzyme pass the obstruction.”
The Wisconsin Alumni Research Foundation has applied for two patents on the discovery, which Ansari thinks could ultimately be applied more broadly. “With recent advances in human genome sequencing, more than 40 diseases have been recognized as resulting from microsatellite repeats,” says Ansari, including fragile X, which causes developmental difficulties, and some types of muscular dystrophy.
“Now that we are starting to understand how to defuse these repeats,” he says, “we see this as a general solution, a molecular engineering principle. We would sequence the genome and figure out the problem, and make a molecule tailored for that individual. It’s a new precision-tailored path to personalized medicine.”
Physical activity in the prevention of human diseases: role of epigenetic modifications
- Elisa Grazioli,
- Ivan Dimauro,
- Neri Mercatelli,
- Guan Wang,
- Yannis Pitsiladis,
Luigi Di Luigi and
- Daniela Caporossi
Epigenetic modification refers to heritable changes in gene function that cannot be explained by alterations in the DNA sequence. The current literature clearly demonstrates that the epigenetic response is highly dynamic and influenced by different biological and environmental factors such as aging, nutrient availability and physical exercise. As such, it is well accepted that physical activity and exercise can modulate gene expression through epigenetic alternations although the type and duration of exercise eliciting specific epigenetic effects that can result in health benefits and prevent chronic diseases remains to be determined. This review highlights the most significant findings from epigenetic studies involving physical activity/exercise interventions known to benefit chronic diseases such as metabolic syndrome, diabetes, cancer, cardiovascular and neurodegenerative diseases.