Generation and Characterization of a Human Neuronal In Vitro Model for Rett Syndrome Using a Direct Reprogramming Method.

Rett Syndrome (RTT) is a severe neurodevelopmental disorder, afflicting 1 in 10,000 female births. It is caused by mutations in the X-linked methyl-CpG-binding protein gene (MECP2), which encodes for the global transcriptional regulator methyl CpG binding protein 2 (MeCP2). As human brain samples of RTT patients are scarce and cannot be used for downstream studies, there is a pressing need for in vitro modeling of pathological neuronal changes. In this study, we use a direct reprogramming method for the generation of neuronal cells from MeCP2-deficient and wild-type human dermal fibroblasts using two episomal plasmids encoding the transcription factors SOX2 and PAX6. We demonstrated that the obtained neurons exhibit a typical neuronal morphology and express the appropriate marker proteins. RNA-sequencing confirmed neuronal identity of the obtained MeCP2-deficient and wild-type neurons. Furthermore, these MeCP2-deficient neurons reflect the pathophysiology of RTT in vitro, with diminished dendritic arborization and hyperacetylation of histone H3 and H4. Treatment with MeCP2, tethered to the cell penetrating peptide TAT, ameliorated hyperacetylation of H4K16 in MeCP2-deficient neurons, which strengthens the RTT relevance of this cell model. We generated a neuronal model based on direct reprogramming derived from patient fibroblasts, providing a powerful tool to study disease mechanisms and investigating novel treatment options for RTT.

Expression, Purification, Characterization and Cellular Uptake of MeCP2 Variants.

The transcriptional regulator Methyl-CpG-binding protein 2 (MeCP2) is an intrinsically disordered protein, mutations in which, are implicated in the onset of Rett Syndrome, a severe and debilitating neurodevelopmental disorder. Delivery of this protein fused to the cell-penetrating peptide TAT could allow for the intracellular replenishment of functional MeCP2 and hence potentially serve as a prospective Rett Syndrome therapy. This work outlines the expression, purification and characterization of various TAT-MeCP2 constructs as well as their full-length and shortened eGFP fusion variants. The latter two constructs were used for intracellular uptake studies with subsequent analysis via western blotting and live-cell imaging. All purified MeCP2 samples exhibited high degree of stability and very little aggregation propensity. Full length and minimal TAT-MeCP2-eGFP were found to efficiently transduce into human dermal and murine fibroblasts and localize to cell nuclei. These findings clearly support the utility of MeCP2-based protein replacement therapy as a potential Rett Syndrome treatment option.

TAT-MeCP2 protein variants rescue disease phenotypes in human and mouse models of Rett syndrome.

Rett syndrome (RTT) is a neurodevelopmental disorder caused by pathogenic variants leading to functional impairment of the MeCP2 protein. Here, we used purified recombinant MeCP2e1 and MeCP2e2 protein variants fused to a TAT protein transduction domain (PTD) to evaluate their transduction ability into RTT patient-derived fibroblasts and the ability to carry out their cellular function. We then assessed their transduction ability and therapeutic effects in a RTT mouse model. In vitro, TAT-MeCP2e2-eGFP reversed the pathological hyperacetylation of histones H3K9 and H4K16, a hallmark of abolition of MeCP2 function. In vivo, intraperitoneal administration of TAT-MeCP2e1 and TAT-MeCP2e2 extended the lifespan of Mecp2-/y mice by >50%. This was accompanied by rescue of hippocampal CA2 neuron size in animals treated with TAT-MeCP2e1. Taken together, these findings provide a strong indication that recombinant TAT-MeCP2 can reach mouse brains following peripheral injection and can ameliorate the phenotype of RTT mouse models. Thus, our study serves as a first step in the development of a potentially novel RTT therapy.

An Electrochemiluminescence-Based Assay for MeCP2 Protein Variants.

The ECLIA is a versatile method which is able to quantify endogenous and recombinant protein amounts in a 96-well format. To demonstrate ECLIA efficiency, this assay was used to analyze intrinsic levels of MeCP2 in mouse brain tissue and the uptake of TAT-MeCP2 in human dermal fibroblasts. The MeCP2-ECLIA produces highly accurate and reproducible measurements with low intra- and inter-assay error. In summary, we developed a quantitative method for the evaluation of MeCP2 protein variants that can be utilized in high-throughput screens.

An electrochemiluminescence based assay for quantitative detection of endogenous and exogenously applied MeCP2 protein variants.

Methyl-CpG-binding protein 2 (MeCP2) is a multifunctional chromosomal protein that plays a key role in the central nervous system. Its levels need to be tightly regulated, as both deficiency and excess of the protein can lead to severe neuronal dysfunction. Loss-of-function mutations affecting MeCP2 are the primary cause of Rett syndrome (RTT), a severe neurological disorder that is thought to result from absence of functional protein in the brain. Several therapeutic strategies for the treatment of RTT are currently being developed. One of them is the use of stable and native TAT-MeCP2 fusion proteins to replenish its levels in neurons after permeation across the blood-brain barrier (BBB). Here we describe the expression and purification of various transactivator of transcription (TAT)-MeCP2 variants and the development of an electrochemiluminescence based assay (ECLIA) that is able to measure endogenous MeCP2 and recombinant TAT-MeCP2 fusion protein levels in a 96-well plate format. The MeCP2 ECLIA produces highly quantitative, accurate and reproducible measurements with low intra- and inter-assay error throughout a wide working range. To underline its broad applicability, this assay was used to analyze brain tissue and study the transport of TAT-MeCP2 variants across an in vitro model of the blood-brain barrier.

No changes in heme synthesis in human Friedreich´s ataxia erythroid progenitor cells.

Friedreich's ataxia (FRDA) is a neurodegenerative disease caused by reduced expression of the protein frataxin. Frataxin is thought to play a role in iron-sulfur cluster biogenesis and heme synthesis. In this study, we used erythroid progenitor stem cells obtained from FRDA patients and healthy donors to investigate the putative role, if any, of frataxin deficiency in heme synthesis. We used electrochemiluminescence and qRT-PCR for frataxin protein and mRNA quantification. We used atomic absorption spectrophotometry for iron levels and a photometric assay for hemoglobin levels. Protoporphyrin IX and Ferrochelatase were analyzed using auto-fluorescence. An "IronChip" microarray analysis followed by a protein-protein interaction analysis was performed. FRDA patient cells showed no significant changes in iron levels, hemoglobin synthesis, protoporphyrin IX levels, and ferrochelatase activity. Microarray analysis presented 11 genes that were significantly changed in all patients compared to controls. The genes are especially involved in oxidative stress, iron homeostasis and angiogenesis. The mystery about the involvement of frataxin on iron metabolism raises the question why frataxin deficiency in primary FRDA cells did not lead to changes in biochemical parameters of heme synthesis. It seems that alternative pathways can circumvent the impact of frataxin deficiency on heme synthesis. We show for the first time in primary FRDA patient cells that reduced frataxin levels are still sufficient for heme synthesis and possibly other mechanisms can overcome reduced frataxin levels in this process. Our data strongly support the fact that so far no anemia in FRDA patients was reported.

Detection of survival motor neuron protein in buccal cells through electrochemiluminescence-based assay.

Spinal muscular atrophy (SMA) is a severe autosomal recessive disorder affecting one in every 10,000 live births. The disease is characterized by loss of alpha-motor neurons in the spinal cord that leads to progressive atrophy and weakness of limb and trunk muscles. This neuromuscular disorder results from deletions and/or mutations within the survival motor neuron 1 (SMN1) gene, leading to a pathologically decreased expression of functional full-length SMN protein. Here we report on the investigation to measure SMN protein levels through electrochemiluminescence immunoassay (ECLIA). This simple assay is a highly quantitative method able to measure SMN protein levels in human, mouse, and rat samples throughout a wide working range with low intra- and interassay error. The sensitivity for human SMN is 30 pg/mL and provides a new tool for the set up of high-throughput screening for basic research. Moreover, we describe a novel tool for a noninvasive assessment of SMN in buccal cells derived from healthy donors, SMA carriers, and SMA patients. The availability of a validated quantitative ECLIA should improve the investigation of novel compounds for the treatment of SMA.

Identification and molecular characterisation of a homozygous missense mutation in the ADAMTS10 gene in a patient with Weill-Marchesani syndrome.

Weill-Marchesani syndrome is a rare disorder of the connective tissue. Functional variants in ADAMTS10 are associated with Weill-Marchesani syndrome-1. We identified a homozygous missense mutation, c.41T>A, of the ADAMTS10 gene in a 19-year-old female with typical symptoms of WMS1: proportionate short stature, brachydactyly, joint stiffness, and microspherophakia. The ADAMTS10 missense mutation was analysed in silico, with conflicting results as to its effects on protein function, but it was predicted to affect the leader sequence. Molecular characterisation in HEK293 Ebna cells revealed an intracellular mis-targeting of the ADAMTS10 protein with a reduced concentration of the polypeptide in the endoplasmic reticulum. A large reduction in glycosylation of the cytoplasmic fraction of the mutant ADAMTS10 protein versus the wild-type protein and a lack of secretion of the mutant protein are also evident in our results.In conclusion, we identified a novel missense mutation of the ADAMTS10 gene and confirmed the functional consequences suggested by the in silico analysis by conducting molecular studies.

Correlation of frataxin content in blood and skeletal muscle endorses frataxin as a biomarker in Friedreich ataxia.

BACKGROUND: Friedreich ataxia is an autosomal recessive disorder caused by mutations in the frataxin gene, leading to reduced levels of the mitochondrial protein frataxin. Assays to quantitatively measure frataxin in peripheral blood have been established. To determine the validity of frataxin as a biomarker for clinical trials, we assessed frataxin in clinically affected tissue. METHODS: In 7 patients with Friedreich ataxia, frataxin content was measured in blood and skeletal muscle before and after treatment with recombinant human erythropoietin, applying the electrochemiluminescence immunoassay. RESULTS: We found frataxin content to be correlated in peripheral blood mononuclear cells and skeletal muscle in drug-naive patients with Friedreich ataxia. The correlation of frataxin content in both compartments remained significant after 8 weeks of treatment. Skeletal-muscle frataxin values correlated with ataxia using the Scale for the Assessment and Rating of Ataxia score. CONCLUSIONS: Our results endorse frataxin measurements in peripheral blood cells as a valid biomarker in Friedreich ataxia.

Effects of erythropoietin on frataxin levels and mitochondrial function in Friedreich ataxia--a dose-response trial.

Friedreich ataxia (FRDA) is an autosomal recessive inherited neurodegenerative disorder leading to reduced expression of the mitochondrial protein frataxin. Previous studies showed frataxin upregulation in FRDA following treatment with recombinant human erythropoietin (rhuEPO). Dose-response interactions between frataxin and rhuEPO have not been studied until to date. We administered escalating rhuEPO single doses (5,000, 10,000 and 30,000 IU) in monthly intervals to five adult FRDA patients. Measurements of frataxin, serum erythropoietin levels, iron metabolism and mitochondrial function were carried out. Clinical outcome was assessed using the "Scale for the assessment and rating of ataxia". We found maximal erythropoietin serum concentrations 24 h after rhuEPO application which is comparable to healthy subjects. Frataxin levels increased significantly over 3 months, while ataxia rating did not reveal clinical improvement. All FRDA patients had considerable ferritin decrease. NADH/NAD ratio, an indicator of mitochondrial function, increased following rhuEPO treatment. In addition to frataxin upregulation in response to continuous low-dose rhuEPO application shown in previous studies, our results indicate for a long-lasting frataxin increase after single high-dose rhuEPO administration. To detect frataxin-derived neuroprotective effects resulting in clinically relevant improvement, well-designed studies with extended time frame are required.

S-carbamoylation impairs the oxidant scavenging activity of cysteine: its possible impact on increased LDL modification in uraemia.

Carbamoylation is the non-enzymatic reaction of cyanate with amino-, hydroxy- or thiol groups. In vivo, amino group modification (N-carbamoylation) resulting in altered function of proteins/amino acids has been observed in patients suffering from uraemia due to urea-derived cyanate. Uraemia has been linked to impaired antioxidant defense. As thiol-compounds like cysteine, N-acetyl cysteine and GSH have oxidant scavenging properties one may speculate that thiol-group carbamoylation (S-carbamoylation) may impair their protective activity. Here we report on the effect of S-carbamoylation on the ABTS free radical and HOCl scavenging property of cysteine as well on its ability to protect LDL from atherogenic modification induced by AAPH generated peroxylradicals or HOCl. The results show that S-carbamoylation impaired the ABTS free radical and HOCl scavenging property of the thiol-compounds tested. The ability of the thiols to protect LDL from lipid oxidation and apolipoprotein modification was strongly diminished by S-carbamoylation. The data indicate that S-carbamoylation could impair the free radical and HOCl scavenging of thiol-amino acids reducing their protective property against LDL atherogenic modification by these oxidant species. As S-carbamoylation is most effective at pH 7 to 5 in vivo thiol-carbamoylation may especially occur at sites of acidic extracellular pH as in hypoxic/inflammatory macrophage rich areas like the atherosclerotic plaque where increased LDL oxidation has been found and may contribute to the higher oxidative stress in uraemia.

In vitro study on the effects of iron sucrose, ferric gluconate and iron dextran on redox-active iron and oxidative stress.

Concerns exist that administration of intravenous (i.v.) iron preparations is associated with oxidative stress. Therefore iron sucrose (CAS 8047-67-4), ferric gluconate (CAS 34098-81-1) and iron dextran (CAS 9004-66-4) were assessed for redox-active iron by a dichlorofluorescein assay and for intracellular reactive oxygen species (ROS) generation and cytotoxicity in HepG2 cells. Examining each i.v. iron preparation at its maximum concentration achieved following clinically frequently used doses in a 70 kg individual in in vitro experiments, redox-active iron was highest with ferric gluconate, followed by iron dextran and iron sucrose. Interestingly, when the i.v. iron preparations were diluted in human serum instead of buffer, redox-active iron was highest with iron dextran, followed by iron sucrose, and practically disappeared with ferric gluconate. ROS production in HepG2 cells was increased by all i.v. iron preparations. However, in the neutral red cytotoxicity assay all i.v. iron preparations were non-toxic. In conclusion, ferric gluconate showed the highest increase in intracellular ROS-production in HepG2 cells and the highest amount of redox-active iron in buffer in the in vitro assays. In contrast to the other i.v. iron preparations, redox-active iron from ferric gluconate was rendered completely redox-inactive by serum, indicating that redox-active iron in the various preparations has different chemical properties.

Carbamylated erythropoietin increases frataxin independent from the erythropoietin receptor.

BACKGROUND: Friedreich's ataxia (FRDA) is a neurodegenerative disorder caused by decreased expression of the mitochondrial protein frataxin. Recently we showed in a clinical pilot study in Friedreich's ataxia patients that recombinant human erythropoietin (rhuEPO) significantly increases frataxin-expression. In this in vitro study, we investigated the role of the erythropoietin receptor (EPO-R) in the frataxin increasing effect of rhuEPO and if nonerythropoietic carbamylated erythropoietin (CEPO), which cannot bind to the classical EPO-R increases frataxin expression. MATERIALS AND METHODS: In our experiments human erythroleukaemic K562 cells (+ EPO-R), human monocytic leukemia THP-1 cells (- EPO-R) and isolated primary lymphocytes from healthy control and FRDA patients were incubated with different concentrations of rhuEPO or CEPO. Frataxin-expression was detected by an electrochemical luminescence immunoassay (based on the principle of an ELISA). RESULTS: We show that rhuEPO increases frataxin-expression in K562 cells (expressing EPO-R) as well as in THP-1 cells (without EPO-R expression). These results were confirmed by the finding that CEPO, which cannot bind to the classical EPO-R increased frataxin expression in the same concentration range as rhuEPO. In addition, we show that both EPO derivatives significantly increase frataxin-expression in vitro in control and Friedreich's ataxia patients primary lymphocytes. CONCLUSION: Our results provide a scientific basis for further studies examining the effectiveness of nonerythropoietic derivatives of erythropoietin for the treatment of Friedreich's ataxia patients.

A high throughput electrochemiluminescence assay for the quantification of frataxin protein levels.

Friedreich's ataxia (FRDA) is an autosomal recessive neurodegenerative disease affecting 1 in 50,000 people and is caused by a GAA-trinucleotide expansion in the frataxin gene located on chromosome locus 9q13 which results in a markedly reduced expression of frataxin, a small mitochondrial protein. The exact function of frataxin is still unknown and currently there is no approved treatment available. In the near future there will be a high demand for measuring frataxin protein levels due to the development of therapeutic strategies for FRDA based on manipulating frataxin expression levels in vivo. In this paper we describe the development of an electrochemiluminescence assay (ECLIA) to measure frataxin protein levels in a 96-well plate format. The ECLIA for frataxin is able to measure human and mouse samples and is highly quantitative, accurate and reproducible, with low intra- and inter-assay error throughout a wide working range. The assay has an excellent precision and provides a new tool for the set up of high-throughput screening for basic research and for clinical studies with FRDA patients.

Hydrogen sulfide scavenges the cytotoxic lipid oxidation product 4-HNE.

Highly reactive alpha,beta-unsaturated aldehydes like 4-hydroxy-2-nonenal (4-HNE), generated from oxidation of polyunsaturated fatty acids, can bind to proteins, polynucleotides and exert cytotoxicity. 4-HNE is known to react readily with thiol and amino groups on free or bound amino acids. Recently, hydrogen sulfide (H(2)S) has been identified as an endogenous vascular gasotransmitter and neuromodulator which can reach up to 160 micromol/l in the brain. Markedly higher 4-HNE concentrations were reported in the brain of patients suffering from Alzheimer's disease. Assuming that the low molecular thiol H(2)S may react with 4-HNE, we have tested the ability of H(2)S to counteract the cytotoxic and protein-modifying activity of 4-HNE. The results show that H(2)S at physiologically relevant concentrations could effectively protect neuronal cells (SH-SY5Y) from the cytotoxic action of 4-HNE. The HNE-modification of cellular proteins was also inhibited in presence of H(2)S. These data suggest that H(2)S may be an important protective factor against carbonyl stress by inactivating/modulating the action of highly reactive alpha,beta-unsaturated aldehydes like 4-HNE in the brain.

Neurological effects of recombinant human erythropoietin in Friedreich's ataxia: a clinical pilot trial.

In a "proof-of-concept" study, we demonstrated that recombinant human erythropoietin (rhuEPO) increases frataxin levels in Friedreich's ataxia (FRDA) patients. We now report a 6-month open-label clinical pilot study of safety and efficacy of rhuEPO treatment in FRDA. Eight adult FRDA patients received 2.000 IU rhuEPO thrice a week subcutaneously. Clinical outcome measures included Ataxia Rating Scales. Frataxin levels and indicators for oxidative stress were assessed. Hematological parameters were monitored biweekly. Scores in Ataxia Rating Scales such as FARS (P = 0.0063) and SARA (P = 0.0045) improved significantly. Frataxin levels increased (P = 0.017) while indicators of oxidative stress such as urine 8-OHdG (P = 0.012) and peroxide levels decreased (P = 0.028). Increases in hematocrit requiring phlebotomies occurred in 4 of 8 patients. In this explorative open-label clinical pilot study, we found an evidence for clinical improvement together with a persistent increase of frataxin levels and a reduction of oxidative stress parameters in patients with FRDA receiving chronic treatment with rhuEPO. Safety monitoring with regular blood cell counts and parameters of iron metabolism is a potential limitation of this approach.