Label-Free, Real-Time Measurement of Metabolism of Adherent and Suspended Single Cells by In-Cell Fourier Transform Infrared Microspectroscopy.

We used infrared (IR) microscopy to monitor in real-time the metabolic turnover of individual mammalian cells in morphologically different states. By relying on the intrinsic absorption of mid-IR light by molecular components, we could discriminate the metabolism of adherent cells as compared to suspended cells. We identified major biochemical differences between the two cellular states, whereby only adherent cells appeared to rely heavily on glycolytic turnover and lactic fermentation. We also report spectroscopic variations that appear as spectral oscillations in the IR domain, observed only when using synchrotron infrared radiation. We propose that this effect could be used as a reporter of the cellular conditions. Our results are instrumental in establishing IR microscopy as a label-free method for real-time metabolic studies of individual cells in different morphological states, and in more complex cellular ensembles.

The role of interruptions in polyQ in the pathology of SCA1.

At least nine dominant neurodegenerative diseases are caused by expansion of CAG repeats in coding regions of specific genes that result in abnormal elongation of polyglutamine (polyQ) tracts in the corresponding gene products. When above a threshold that is specific for each disease the expanded polyQ repeats promote protein aggregation, misfolding and neuronal cell death. The length of the polyQ tract inversely correlates with the age at disease onset. It has been observed that interruption of the CAG tract by silent (CAA) or missense (CAT) mutations may strongly modulate the effect of the expansion and delay the onset age. We have carried out an extensive study in which we have complemented DNA sequence determination with cellular and biophysical models. By sequencing cloned normal and expanded SCA1 alleles taken from our cohort of ataxia patients we have determined sequence variations not detected by allele sizing and observed for the first time that repeat instability can occur even in the presence of CAG interruptions. We show that histidine interrupted pathogenic alleles occur with relatively high frequency (11%) and that the age at onset inversely correlates linearly with the longer uninterrupted CAG stretch. This could be reproduced in a cellular model to support the hypothesis of a linear behaviour of polyQ. We clarified by in vitro studies the mechanism by which polyQ interruption slows down aggregation. Our study contributes to the understanding of the role of polyQ interruption in the SCA1 phenotype with regards to age at disease onset, prognosis and transmission.

Nuclease-stimulated homologous recombination at the human ß-globin gene.

BACKGROUND: Mutations in the ß-globin gene (HBB) cause haemoglobinopathies where current treatments have serious limitations. Gene correction by homologous recombination (HR) is an attractive approach to gene therapy for such diseases and is stimulated by gene-specific endonucleases, including zinc finger nucleases (ZFNs). Customised nucleases targeting HBB have previously been shown to promote HR-mediated HBB modification in 0.3­60% of drug-selected cells, although frequencies among unselected cells, more relevant to the goal of correcting HBB in primary stem cells, have not been reported. METHODS: ZFNs targeting HBB were tested for HBB binding (two-hybrid assay) or HBB cleavage followed by inaccurate end joining (surveyor assay)in bacteria or human cancer cell lines, respectively. ZFN-stimulated HR was measured in cell lines by a modified fluorescence-based reporter assay or by targeted insertion of a drug-resistance marker into endogenous HBB confirmed by Southern analyses. RESULTS: Although the ZFNs that we assembled in-house showed limited potential, a commercially commissioned nuclease (ZFN4) enhanced HR mediated HBB modification in up to 95% of drug-selected cells. Among unselected cells, however, this frequency was less than 0.2%. Furthermore, ZFN4 cleaved HBB at an efficiency of 1­2% (surveyor assay) and enhanced the HR reporter assay 20-fold less efficiently than a control endonuclease. CONCLUSIONS: With ZFN4, we achieved higher efficiencies of HR-mediated HBB modification than previously reported for drug-selected cells. Our measurements of ZFN4-induced HR in unselected cells, however, suggest that improved nucleases must be developed if therapeutic HBB correction is to be achievable in primary stem cells.

A model for the aggregation of the acylphosphatase from Sulfolobus solfataricus in its native-like state.

Evidence is accumulating that normally folded proteins retain a significant tendency to form amyloid fibrils through a direct assembly of monomers in their native-like conformation. However, the factors promoting such processes are not yet well understood. The acylphosphatase from Sulfolobus solfataricus (Sso AcP) aggregates under conditions in which a native-like state is initially populated and forms, as a first step, aggregates in which the monomers maintain their native-like topology. An unstructured N-terminal segment and an edge beta-strand were previously shown to play a major role in the process. Using kinetic experiments on a set of Sso AcP variants we shall show that the major event of the first step is the establishment of an inter-molecular interaction between the unstructured segment of one Sso AcP molecule and the globular unit of another molecule. This interaction is determined by the primary sequence of the unstructured segment and not by its physico-chemical properties. Moreover, we shall show that the conversion of these initial aggregates into amyloid-like protofibrils is an intra-molecular process in which the Sso AcP molecules undergo conformational modifications. The obtained results allow the formulation of a model for the assembly of Sso AcP into amyloid-like aggregates at a molecular level.

A new tool to determine the cellular metabolic landscape: nanotechnology to the study of Friedreich's ataxia.

Understanding the cell response to oxidative stress in disease is an important but difficult task. Here, we demonstrate the feasibility of using a nanomotion sensor to study the cellular metabolic landscape. This nanosensor permits the non-invasive real-time detection at the single-cell level and offers high sensitivity and time resolution. We optimised the technique to study the effects of frataxin overexpression in a cellular model of Friedreich's ataxia, a neurodegenerative disease caused by partial silencing of the FXN gene. Previous studies had demonstrated that FXN overexpression are as toxic as silencing, thus indicating the importance of a tight regulation of the frataxin levels. We probed the effects of frataxin overexpression in the presence of oxidative stress insults and measured the metabolic response by the nanosensor. We show that the nanosensor provides new detailed information on the metabolic state of the cell as a function of time, that agrees with and complements data obtained by more traditional techniques. We propose that the nanosensor can be used in the future as a new and powerful tool to study directly how drugs modulate the effects of oxidative stress on Friedreich's ataxia patients and, more in general, on other neurodegenerative processes.

The role of oxidative stress in Friedreich's ataxia.

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.

Adding a temporal dimension to the study of Friedreich's ataxia: the effect of frataxin overexpression in a human cell model.

The neurodegenerative disease Friedreich's ataxia is caused by lower than normal levels of frataxin, an important protein involved in iron-sulfur (Fe-S) cluster biogenesis. An important step in designing strategies to treat this disease is to understand whether increasing the frataxin levels by gene therapy would simply be beneficial or detrimental, because previous studies, mostly based on animal models, have reported conflicting results. Here, we have exploited an inducible model, which we developed using the CRISPR/Cas9 methodology, to study the effects of frataxin overexpression in human cells and monitor how the system recovers after overexpression. Using new tools, which range from high-throughput microscopy to in cell infrared, we prove that overexpression of the frataxin gene affects the cellular metabolism. It also leads to a significant increase of oxidative stress and labile iron pool levels. These cellular alterations are similar to those observed when the gene is partly silenced, as occurs in Friedreich's ataxia patients. Our data suggest that the levels of frataxin must be tightly regulated and fine-tuned, with any imbalance leading to oxidative stress and toxicity.

A new cellular model to follow Friedreich's ataxia development in a time-resolved way.

Friedreich's ataxia (FRDA) is a recessive autosomal ataxia caused by reduced levels of frataxin (FXN), an essential mitochondrial protein that is highly conserved from bacteria to primates. The exact role of frataxin and its primary function remain unclear although this information would be very valuable to design a therapeutic approach for FRDA. A main difficulty encountered so far has been that of establishing a clear temporal relationship between the different observations that could allow a distinction between causes and secondary effects, and provide a clear link between aging and disease development. To approach this problem, we developed a cellular model in which we can switch off/on in a time-controlled way the frataxin gene partially mimicking what happens in the disease. We exploited the TALEN and CRISPR methodologies to engineer a cell line where the presence of an exogenous, inducible FXN gene rescues the cells from the knockout of the two endogenous FXN genes. This system allows the possibility of testing the progression of disease and is a valuable tool for following the phenotype with different newly acquired markers.

Analyzing the Effects of a G137V Mutation in the FXN Gene.

Reduced levels of frataxin, an essential mitochondrial protein involved in the regulation of iron-sulfur cluster biogenesis, are responsible for the recessive neurodegenerative Friedreich Ataxia (FRDA). Expansion of a GAA triplet in the first intron of the FRDA is essential for disease development which causes partial silencing of frataxin. In the vast majority of cases, patients are homozygotes for the expansion, but a small number of FRDA patients are heterozygotes for expansion and point mutations in the frataxin coding frame. In this study, we analyze the effects of a point mutation G137V. The patient P94-2, with a history of alcohol and drug abuse, showed a FRDA onset at the border between the classic and late onset phenotype. We applied a combination of biophysical and biochemical methods to characterize its effects on the structure, folding and activity of frataxin. Our study reveals no impairment of the structure or activity of the protein but a reduced folding stability. We suggest that the mutation causes misfolding of the native chain with consequent reduction of the protein concentration in the patient and discuss the possible mechanism of disease.