Histone deacetylase inhibitors for the epigenetic therapy of proximal spinal muscular atrophy

Die proximal spinale Muskelatrophie (SMA) ist charakterisiert durch eine fortschreitende Degeneration der a-Motoneuronen in the Vorderhörnern des Rückenmarks. Sie wird durch den homozygoten funktionellen Verlust des survival motor neuron Gens 1 (SMN1) verursacht. Allerdings konnte gezeigt werden, dass alle SMA-Patienten zumindest über eine Kopie des SMN2-Genes verfügen. Während SMN1 ausschließlich Volllängen-Transkripte (FL-SMN) produziert, sind 90% aller SMN2 Transkripte alternativ gespleißt. Ihnen fehlt das Exon 7 (SMN2D7), was zu einem instabilen Protein führt. Zwar reicht die Menge and SMN2 Volllängenprotein nicht aus um für den Verlust von SMN1 zu kompensieren, andererseits beeinflusst es jedoch den SMA Phänotyp: Über je mehr SMN2 Kopien ein SMA Patient verfügt, desto milder ist der Krankheitsverlauf. Es konnte gezeigt werden, dass die Verwendung von Histon Deacetylase Inhibitoren (HDACi) einerseits das SMN2 Gen aktivieren und darüberhinaus auch sein Spleißmuster korregieren. Eine Pilotstudie mit SMA-Patienten, die mit dem HDACi VPA behandelt wurden, ergab jedoch ein recht unterschiedliches Bild. In knapp einem Drittel der Patienten ist die Menge an FL-SMN2 im Blut, wie erhofft, angestiegen. In einem weiteren Drittel jedoch konnte keine Änderung festgestellt werden, wohingegen im letzten Drittel die Menge an FL-SMN2 durch VPA-Gabe sogar reduziert war. Die vorliegende Arbeit beschäftigt sich mit der Suche nach den Ursachen, aufgrund derer SMA-Patienten nicht positiv auf VPA reagieren. Von allen SMA-Patienten, die an der Pilot-Studie teilgenommen hatten, wurden Fibroblasten Linien aus Hautstanzen etabliert. Es konnte gezeigt werden, dass in mehr als 60% der Fälle beide Gewebe, Blut wie Fibroblasten, gleichermaßen auf VPA ansprachen. Um zu verstehen, warum SMN2 nicht durch VPA in Non-Respondern aktiviert wird, wurden Chromatin-Immunopräzipitationen (ChIP) durchgeführt. Es zeigte sich, dass das SMN Protein in Non-Respondern unter VPA-Behandlung nicht ansteigt, da VPA nicht zu einer SMN2 Promotor Hyperacetylierung führt. Um die Frage nach den Gründen hierfür zu beantworten, wurde die Transkriptome von Pos- und Non-Responder Fibroblasten mittels Mikro-Array verglichen. Die Auswertung der Daten zeigte, dass in den Non-Respondern kein einziges Transkript differentiell unter VPA-Behandlung exprimiert wurde. Interessanterweise wurden lediglich neun Gene gefunden, die signifikant unterschiedlich zwischen unbehandelten Pos- and Non-Respondern exprimiert wurden. Basierend auf publizierten Daten wurden die Gene Cluster of Differentiation (CD36), IGF-binde Protein 5 (IGFBP5), Retinoic Acid Receptor b (RARb) und Transforming Growth Factor a (TGFa) für weitere Experimente ausgewählt. CD36 und RARb sind beide in Non-Respondern höher exprimiert, wohingegen höherer Menge von IGFBP5 und TGFa in Pos-Respondern gefunden wurden. Da CD36 ein Fettsäuretransporter ist, wurden ebenfalls massenspektroskopische Versuche zur Aufnahme und Metabolisierung von VPA durchgeführt. Eine differentielle Verstoffwechslung von VPA als Ursache für das Auftreten Non-Respondern konnte jedoch ausgeschlossen werden. Ausgehend von diesen Daten wurde die generelle Fettsäureaufnahme von Fibroblasten verglichen. Diese Daten lassen vermuten, dass CD36 zumindest in Fibroblasten eher als Fettsäureexporter denn als -importer fungiert. Ferner konnten wir den HDACi LBH589 und sein strukturell nah verwandtes Derivat JnJ-26481585 als potentielle SMA-Therapeutika identifizieren, die einem enormen Effekt auf die SMN-Protein Menge haben. So stieg die SMN Menge bis zu 10-fach bei LBH589 Konzentration von 400 nM beziehungsweise 1 uM an. Ein höherer Anstieg wurde bis dato noch nicht publiziert. ChIP-Analyse des SMN2 Promoters, sowie die Messung der SMN2 Promotoraktivität in einer Reporterzelllinie, zeigten, dass am SMN2 Promoter HDAC-Inhibition und Promoter-Aktivität in einer 1:1 Stöichiometrie korrelieren. Es wurde eine EC50 von 108 nM LBH589 bestimmt. Auf RNA-Ebene konnten wir zeigen, dass LBH589 einerseits die SMN Expression steigert, aber auch das Spleißmuster durch Hoch-Regulation des Spleißfaktors hTRA2-b1, welcher den Einbezug des SMN2 Exons 7 fördert, revertiert. Mittels Präzipitations-Experimenten konnten wir zeigen, dass die Ubiquitinylierung von SMN stark reduziert ist. Ursächlich hierfür ist vermutlich eine verstärkte SMN-Komplex Bildung, die dann zu einer Anreicherung von SMN im Laufe der Zeit führt. Wir konnten zeigen, dass LBH589 in humanen NSCs wie auch in MEFs von SMA-Mäusen SMN Protein hochreguliert. Abschließend wurden LBH589 subkutan in Mäuse injiziert um zu untersuchen, ob sich eine in vivo Untersuchung von LBH589 anbietet. Die Analyse von Gewebeextrakten aus dem ZNS zeigte, dass die Menge an SMN Protein im Gehirn steigt und gleichermaßen auch die Histonacetylierung zunahm. Daher sollten LBH589, bzw. auch JnJ-26481585, in einer größer angelegten Studie im SMA-Tiermodell untersucht werden

Evaluating human neural tuning curves from a mechanical model of the cochlea by relating them to psychophysical masking data

Thesis (M.S.)--Massachusetts Institute of Technology, Dept. of Electrical Engineering and Computer Science, 1994.Includes bibliographical references (leaves 40-42).by Peter A.Z. Garbes.M.S

Prolonged treatment with pimelic o-aminobenzamide HDAC inhibitors ameliorates the disease phenotype of a Friedreich ataxia mouse model

NOTICE: this is the author’s version of a work that was accepted for publication in Neurobiology of Disease. Changes resulting from the publishing process, such as peer review, editing, corrections, structural formatting, and other quality control mechanisms may not be reflected in this document. Changes may have been made to this work since it was submitted for publication.Friedreich ataxia (FRDA) is an inherited neurodegenerative disorder caused by GAA repeat expansion within the FXN gene, leading to epigenetic changes and heterochromatin-mediated gene silencing that result in a frataxin protein deficit. Histone deacetylase (HDAC) inhibitors, including pimelic o-aminobenzamide compounds 106, 109 and 136, have previously been shown to reverse FXN gene silencing in short-term studies of FRDA patient cells and a knock-in mouse model, but the functional consequences of such therapeutic intervention have thus far not been described. We have now investigated the long-term therapeutic effects of 106, 109 and 136 in our GAA repeat expansion mutation-containing YG8R FRDA mouse model. We show that there is no overt toxicity up to 5 months of treatment and there is amelioration of the FRDA-like disease phenotype. Thus, while the neurological deficits of this model are mild, 109 and 106 both produced an improvement of motor coordination, whereas 109 and 136 produced increased locomotor activity. All three compounds increased global histone H3 and H4 acetylation of brain tissue, but only 109 significantly increased acetylation of specific histone residues at the FXN locus. Effects on FXN mRNA expression in CNS tissues were modest, but 109 significantly increased frataxin protein expression in brain tissue. 109 also produced significant increases in brain aconitase enzyme activity, together with reduction of neuronal pathology of the dorsal root ganglia (DRG). Overall, these results support further assessment of HDAC inhibitors for treatment of Friedreich ataxia.This work was supported by Repligen Corporation; Muscular Dystrophy Association (MDA) USA; Ataxia UK; Friedreich's Ataxia Research Alliance (FARA); GoFAR; and the Wellcome Trust [089757]

CHARACTERISTICS TAKEN AS RELEVANT IN A SUCCESSFUL ENTREPRENEUR: AN EVALUATION CONTRASTING THE PERCEPTION OF POPULATION (COMMON SENSE) AND OF THE OWN ENTREPRENEURS

O objetivo desse estudo foi de realizar uma investigação a cerca das características percebidas como fundamentais em um empreendedor. Para tanto, conduziu-se inicialmente uma revisão da literatura em que se observaram as características consideradas como relevantes em um empreendedor de sucesso. A partir do levantamento destas características, pode-se realizar uma avaliação contrastando (objetivo secundário) a percepção – sobre os fatores condicionantes do sucesso – da população (senso comum) e dos próprios empreendedores. Como resultados, verificou-se como principal característica apontada tanto na pesquisa de opinião pública quanto nas entrevistas com os empreendedores: estabelecer e manter uma rede de relacionamentos sólida como base primordial para o bom andamento de um empreendimento e diferencial dos empreendedores. O estudo de também destacou fatores importantes como a qualidade e aprendizagem contínua na evolução do empreendimento e criação de novas oportunidades.This study objective was to accomplish an investigation about characteristics perceived as fundamental in an entrepreneur. For this, it’s was initially conducted a literature review that it was observed the characteristics taken as relevant in a successful entrepreneur. From these characteristics lifting, it was possible to accomplish an evaluation contrasting (as secondary objective) the perception – about successful conditioning factors – of population (common sense) and of the own entrepreneurs. As results, it was verified as main characteristic pointed in public opinion research as well as in the interviews with entrepreneurs: to establish and maintain a solid relationship network as primordial base to a great entrepreneurship pace and as entrepreneur’s differential. The study also detached important factors as quality and continuous learning for entrepreneurial evolution and new opportunities creation

Molecular spectrum and differential diagnosis in patients referred with sporadic or autosomal recessive osteogenesis imperfecta

Background: Osteogenesis imperfecta (OI) is a heterogeneous bone disorder characterized by recurrent fractures. Although most cases of OI have heterozygous mutations in COL1A1 or COL1A2 and show autosomal dominant inheritance, during the last years there has been an explosion in the number of genes responsible for both recessive and dominant forms of this condition. Herein, we have analyzed a cohort of patients with OI, all offspring of unaffected parents, to determine the spectrum of variants accounting for these cases. Twenty patients had nonrelated parents and were sporadic, and 21 were born to consanguineous relationships. Methods: Mutation analysis was performed using a next-generation sequencing gene panel, homozygosity mapping, and whole exome sequencing (WES). Results: Patients offspring of nonconsanguineous parents were mostly identified with COL1A1 or COL1A2 heterozygous changes, although there were also a few cases with IFITM5 and WNT1 heterozygous mutations. Only one sporadic patient was a compound heterozygote for two recessive mutations. Patients offspring of consanguineous parents showed homozygous changes in a variety of genes including CRTAP, FKBP10, LEPRE1, PLOD2, PPIB, SERPINF1, TMEM38B, and WNT1. In addition, two patients born to consanguineous parents were found to have de novo COL1A1 heterozygous mutations demonstrating that causative variants in the collagen I structural genes cannot be overlooked in affected children from consanguineous couples. Further to this, WES analysis in probands lacking mutations in OI genes revealed deleterious variants in SCN9A, NTRK1, and SLC2A2, which are associated with congenital indifference to pain (CIP) and Fanconi–Bickel syndrome (FBS). Conclusion: This work provides useful information for clinical and genetic diagnosis of OI patients with no positive family history of this disease. Our data also indicate that CIP and FBS are conditions to be considered in the differential diagnosis of OI and suggest a positive role of SCN9A and NTRK1 in bone development

Genetic analysis of osteogenesis imperfecta in the Palestinian population : molecular screening of 49 affected families

Background : Osteogenesis imperfecta (OI) is a heterogeneous hereditary connective tissue disorder clinically hallmarked by increased susceptibility to bone fractures. Methods : We analyzed a cohort of 77 diagnosed OI patients from 49 unrelated Palestinian families. Next-generation sequencing technology was used to screen a panel of known OI genes. Results : In 41 probands, we identified 28 different disease-causing variants of 9 different known OI genes. Eleven of the variants are novel. Ten of the 28 variants are located in COL1A1, five in COL1A2, three in BMP1, three in FKBP10, two in TMEM38B, two in P3H1, and one each in CRTAP, SERPINF1, and SERPINH1. The absence of disease-causing variants in the remaining eight probands suggests further genetic heterogeneity in OI. In general, most OI patients (90%) harbor mainly variants in type I collagen resulting in an autosomal dominant inheritance pattern. However, in our cohort almost 61% (25/41) were affected with autosomal recessive OI. Moreover, we document a 21-kb genomic deletion in the TMEM38B gene identified in 29% (12/41) of the tested probands, making it the most frequent OI-causing variant in the Palestinian population. Conclusion : This is the first genetic screening of an OI cohort from the Palestinian population. Our data are important for genetic counseling of OI patients and families in highly consanguineous populations

Compound heterozygous variants in NBAS as a cause of atypical osteogenesis imperfecta

Background Osteogenesis imperfecta (OI), the commonest inherited bone fragility disorder, affects 1 in 15,000 live births resulting in frequent fractures and reduced mobility, with significant impact on quality of life. Early diagnosis is important, as therapeutic advances can lead to improved clinical outcome and patient benefit. Report Whole exome sequencing in patients with OI identified, in two patients with a multi-system phenotype, compound heterozygous variants in NBAS (neuroblastoma amplified sequence). Patient 1: NBAS c.5741G > A p.(Arg1914His); c.3010C > T p.(Arg1004*) in a 10-year old boy with significant short stature, bone fragility requiring treatment with bisphosphonates, developmental delay and immunodeficiency. Patient 2: NBAS c.5741G > A p.(Arg1914His); c.2032C > T p.(Gln678*) in a 5-year old boy with similar presenting features, bone fragility, mild developmental delay, abnormal liver function tests and immunodeficiency. Discussion Homozygous missense NBAS variants cause SOPH syndrome (short stature; optic atrophy; Pelger-Huet anomaly), the same missense variant was found in our patients on one allele and a nonsense variant in the other allele. Recent literature suggests a multi-system phenotype. In this study, patient fibroblasts have shown reduced collagen expression, compared to control cells and RNAseq studies, in bone cells show that NBAS is expressed in osteoblasts and osteocytes of rodents and primates. These findings provide proof-of-concept that NBAS mutations have mechanistic effects in bone, and that NBAS variants are a novel cause of bone fragility, which is distinguishable from ‘Classical’ OI. Conclusions Here we report on variants in NBAS, as a cause of bone fragility in humans, and expand the phenotypic spectrum associated with NBAS. We explore the mechanism underlying NBAS and the striking skeletal phenotype in our patients

Neurocalcin Delta Suppression Protects against Spinal Muscular Atrophy in Humans and across Species by Restoring Impaired Endocytosis

This document is the Accepted Manuscript version of the following article: Riessland et al., 'Neurocalcin Delta Suppression Protects against Spinal Muscular Atrophy in Humans and across Species by Restoring Impaired Endocytosis', The American Journal of Human Genetics, Vol. 100 (2): 297-315, first published online 26 January 2017. The final, published version is available online at doi: http://dx.doi.org/10.1016/j.ajhg.2017.01.005 © 2017 American Society of Human Genetics.Homozygous SMN1 loss causes spinal muscular atrophy (SMA), the most common lethal genetic childhood motor neuron disease. SMN1 encodes SMN, a ubiquitous housekeeping protein, which makes the primarily motor neuron-specific phenotype rather unexpected. SMA-affected individuals harbor low SMN expression from one to six SMN2 copies, which is insufficient to functionally compensate for SMN1 loss. However, rarely individuals with homozygous absence of SMN1 and only three to four SMN2 copies are fully asymptomatic, suggesting protection through genetic modifier(s). Previously, we identified plastin 3 (PLS3) overexpression as an SMA protective modifier in humans and showed that SMN deficit impairs endocytosis, which is rescued by elevated PLS3 levels. Here, we identify reduction of the neuronal calcium sensor Neurocalcin delta (NCALD) as a protective SMA modifier in five asymptomatic SMN1-deleted individuals carrying only four SMN2 copies. We demonstrate that NCALD is a Ca(2+)-dependent negative regulator of endocytosis, as NCALD knockdown improves endocytosis in SMA models and ameliorates pharmacologically induced endocytosis defects in zebrafish. Importantly, NCALD knockdown effectively ameliorates SMA-associated pathological defects across species, including worm, zebrafish, and mouse. In conclusion, our study identifies a previously unknown protective SMA modifier in humans, demonstrates modifier impact in three different SMA animal models, and suggests a potential combinatorial therapeutic strategy to efficiently treat SMA. Since both protective modifiers restore endocytosis, our results confirm that endocytosis is a major cellular mechanism perturbed in SMA and emphasize the power of protective modifiers for understanding disease mechanism and developing therapies.Peer reviewedFinal Accepted Versio

Utility of Survival Motor Neuron ELISA for Spinal Muscular Atrophy Clinical and Preclinical Analyses

Genetic defects leading to the reduction of the survival motor neuron protein (SMN) are a causal factor for Spinal Muscular Atrophy (SMA). While there are a number of therapies under evaluation as potential treatments for SMA, there is a critical lack of a biomarker method for assessing efficacy of therapeutic interventions, particularly those targeting upregulation of SMN protein levels. Towards this end we have engaged in developing an immunoassay capable of accurately measuring SMN protein levels in blood, specifically in peripheral blood mononuclear cells (PBMCs), as a tool for validating SMN protein as a biomarker in SMA.A sandwich enzyme-linked immunosorbent assay (ELISA) was developed and validated for measuring SMN protein in human PBMCs and other cell lysates. Protocols for detection and extraction of SMN from transgenic SMA mouse tissues were also developed.The assay sensitivity for human SMN is 50 pg/mL. Initial analysis reveals that PBMCs yield enough SMN to analyze from blood volumes of less than 1 mL, and SMA Type I patients' PBMCs show ∼90% reduction of SMN protein compared to normal adults. The ELISA can reliably quantify SMN protein in human and mouse PBMCs and muscle, as well as brain, and spinal cord from a mouse model of severe SMA.This SMN ELISA assay enables the reliable, quantitative and rapid measurement of SMN in healthy human and SMA patient PBMCs, muscle and fibroblasts. SMN was also detected in several tissues in a mouse model of SMA, as well as in wildtype mouse tissues. This SMN ELISA has general translational applicability to both preclinical and clinical research efforts

Overview of Current Drugs and Molecules in Development for Spinal Muscular Atrophy Therapy

The full text of this article can be found here: https://link.springer.com/article/10.1007/s40265-018-0868-