Proteomics-based identification and characterisation of spinal muscular atrophy disease pathways

Spinal muscular atrophy (SMA) is a debilitating genetic disorder, characterised by homozygous loss of the survival of motor neuron 1 (SMN1) gene, resulting in insufficient levels of ubiquitously expressed survival of motor neuron (SMN) protein. The traditional picture of SMA as a neuromuscular disease is slowly being changed by the findings of systemic pathology in SMA patients. This is also one of the reasons why therapeutic strategies aimed at increasing the levels of functional SMN protein are not completely effective, since they would often target neuromuscular pathology only. Here, the research was focused on identifying and characterising new disease pathways in SMA that could potentially be used as therapeutic targets in SMA. In the first results chapter, six proteins, lamin A/C, UBA1, ANXA2, GAPDH, NCAM and GAP43, identified previously in a multi-study comparison as having consistent direction of differential expression across three proteomic studies of SMA, were investigated in SMA tissues and cells using western blotting. Of these, lamin A/C, UBA1, ANXA2 and GAPDH showed widespread dysregulation across a range of tissues from severe Taiwanese mouse model of SMA and in fibroblast cells from SMA patients, suggesting that each one of these proteins might have a role in SMA disease pathways. Selection of drugs that can target these proteins proved to be very challenging, and further efforts are needed to identify appropriate therapeutic strategies. In the second results chapter, the potential role for lamin A/C in SMA disease pathways was further highlighted by findings of mechanistic link between lamin A/C and UBA1, a known SMA modifier. Lamin A/C dysregulation might be especially relevant in the context of heart pathology, where increased levels of lamin A/C would likely stiffen the cardiomyocytes and impair normal heart function. In the third results chapter, multi-study comparison of published proteomic studies of amyotrophic lateral sclerosis (ALS) identified core protein changes in ALS tissues and cells, and biochemical investigation of two proteins, ALDOA and GAPDH, highlighted defects in cell metabolism as an important disease mechanism in ALS. Of these, fifteen proteins were also differentially expressed across at least two proteomic studies of SMA, however, when investigated biochemically in SMA and ALS mouse spinal cords, these proteins showed very little evidence that two diseases converge on the same molecular mechanisms. When taken together, results presented here show that published proteomic studies contain a wealth of information that often get overlooked when examined in isolation. These datasets were exploited here to identify core proteins and molecular mechanisms that drive disease pathogenesis in SMA and ALS. Knowledge of general disease mechanisms may allow development of therapies that can systemically target peripheral pathology in SMA. For example, systemic restoration of lamin A/C levels might prove useful for correcting defects in a range of tissues, with an emphasis on stiff tissues like the heart and muscles, and when combined with an SMN-targeted approach, it might bring greater therapeutic benefit to SMA patients compared to SMN-targeted approach alone. In conclusion, several proteins identified in a multi-study comparison, including lamin A/C, GAPDH and ALDOA, were linked to different SMA and ALS disease pathways, providing evidence that multi-study comparison has the power to identify core disease-related protein changes. This thesis also opened a range of new questions that need to be addressed in the future work, including the selection of drugs that can restore defects in the expression or activity or target proteins. Many other proteins, identified in multi-study comparisons, could not be investigated here in more detail. These proteins may help to further expand the knowledge of SMA and ALS disease pathways and therefore demand further experimental attention

The effect of perinatal exposure to increased serotonin concentrations on histological structure of somatosensory cortex of adult rats

Serotonin (5HT) je biološki aktivan amin prisutan u mozgu i perifernim tkivima sisavaca gdje izvršava različite funkcije, ovisno o odjeljku u kojem se nalazi. U mozgu regulira razvoj serotonergičnih neurona i ciljanih tkiva tijekom neurogeneze, dok u razvijenom mozgu preuzima ulogu neurotransmitera. Cilj ovog diplomskog rada bio je ispitati utjecaj perinatalno inducirane povišene razine serotonina, primjenom neposrednog prekursora sinteze serotonina, 5-hidroksitriptofana (5HTP, 25 mg/kg), ili inhibitora razgradnje serotonina, tranilcipromina (TCP, 2mg/kg) na razvoj bačvastog polja u somatosenzoričkoj kori mozga štakora. Mozgovi su prethodno izdvojeni neposredno po završetku tretmana (PND 21) ili u odrasloj dobi (PND 70). Tangencijalne rezove somatosenzoričke kore podvrgnula sam histološkoj metodi bojanja po Nisslu, te histokemijskoj metodi bojanja citokrom oksidazom (CO). Kvalitativna analiza bačvastog polja vršena je pregledavanjem rezova Nissla pod mikroskopom. Bačvasta polja na rezovima bojanim CO pronašli smo pod mikroskopom i fotografirali. Programom ImageJ ocrtali smo bačve unutar bačvastog polja, te izmjerili površinu koja je služila za kvantitativnu usporedbu eksperimentalnih skupina s kontrolnom, tretiranom fiziološkom otopinom. Povišene razine serotonina imale su značajan učinak na bačvasto polje mladunaca, pri čemu je tretman 5HTP-om rezultirao manjom površinom bačvastog polja i pojedinačnih bačvi, dok je je TCP narušio strukturu bačvi. Učinci tretmana 5HTP-om, vidljivi i u mozgu odraslih životinja, ukazuju na smanjeno grananje talamokortikalnih neurona tijekom prenatalnog dijela tretmana. Učinci TCP-a nisu zadržani u odraslih životinja, pa pretpostavljamo da je došlo do odgođenog sazrijevanja bačvastog polja uslijed kronične izloženosti povišenim koncentracijama serotonina u postnatalnom dijelu tretmana.Serotonin (5HT) is biologically active amine present in the brain and peripheral tissues of mammals where it has various functions, depending on the compartment. In the brain, during neurogenesis, it regulates the development of serotonergic neurons and target tissues, while later it assumes the function of a neurotransmitter. The aim of this graduation thesis was to examine the effect of perinatally induced high serotonin levels on the development of barrel field in the rat somatosensory cortex, induced by treatment with the immediate serotonin precursor ,5-hydroxytryptophan (5HTP, 25 mg/kg), and inhibitor of serotonin degradation, tranylcypromine (TCP, 2 mg/kg). Brains were collected on PND 21 or PND 70. Tangential sections of somatosensory cortex where used for histological processing with Nissl staining method, and histochemical method for cytochrome oxidase staining (CO). Qualitative analysis of barrel field were conducted by examination of Nissl sections under the microscope. We found barrel field on CO sections under the microscope and photographed them. In software ImageJ we draw barrels in barrel field, measured their area surface and conducted analysis of quantitative differences between treated and control group. High serotonin levels had significant effect on barrel field of the pups, with 5HTP treatment resulting in smaller barrel field and barrel area, and TCP treatment disrupting their structure. Treatment effects of 5HTP, still visible in adult rats, point to the decreased branching of thalamo-cortical axons during the prenatal phase of the treatment. Since the effects of TCP were not present in the brains of adult animals, we assume that the cronic exposure to high serotonin levels in the postnatal phase of the treatmen only delayed maturation of the barrel field

Multi-Study Proteomic and Bioinformatic Identification of Molecular Overlap between Amyotrophic Lateral Sclerosis (ALS) and Spinal Muscular Atrophy (SMA)

Unravelling the complex molecular pathways responsible for motor neuron degeneration in amyotrophic lateral sclerosis (ALS) and spinal muscular atrophy (SMA) remains a persistent challenge. Interest is growing in the potential molecular similarities between these two diseases, with the hope of better understanding disease pathology for the guidance of therapeutic development. The aim of this study was to conduct a comparative analysis of published proteomic studies of ALS and SMA, seeking commonly dysregulated molecules to be prioritised as future therapeutic targets. Fifteen proteins were found to be differentially expressed in two or more proteomic studies of both ALS and SMA, and bioinformatics analysis identified over-representation of proteins known to associate in vesicles, and molecular pathways, including metabolism of proteins and vesicle-mediated transport; both of which converge on ER-Golgi trafficking processes. Calreticulin, a calcium-binding chaperone found in the ER, was associated with both pathways and we independently confirm that its expression was decreased in spinal cords from SMA and increased in spinal cords from ALS mice. Together, these findings offer significant insights into potential common targets that may help to guide the development of new therapies for both diseases

AAV9-mediated SMN gene therapy rescues cardiac desmin but not lamin A/C and elastin dysregulation in Smn2B/- spinal muscular atrophy mice

This work was supported by Great Ormond Street Hospital Charity (GOSH) and SPARKS Children’s Medical Research Charity (Grant No. V5018 to H.R.F.). M.B. acknowledges general financial support from SMA Angels Charity, SMA UK, Muscular Dystrophy UK, Action Medical Research, Academy of Medical Sciences and Association Française contre les Myopathies for SMA research in her laboratory. H.K.S. and T.H.G. acknowledge support from the Euan MacDonald Centre for Motor Neuron Disease Research and SMA Europe. E.M.C. was partially funded by a scholarship from Royal Holloway University of London. R.J.Y.-M. acknowledges general financial support from SMA UK (formerly The SMA Trust), through the UK SMA Research Consortium, for SMA research in his laboratory.Structural, functional and molecular cardiac defects have been reported in spinal muscular atrophy (SMA) patients and mouse models. Previous quantitative proteomics analyses demonstrated widespread molecular defects in the severe Taiwanese SMA mouse model. Whether such changes are conserved across different mouse models, including less severe forms of the disease, has yet to be established. Here, using the same high-resolution proteomics approach in the less-severe Smn2B/− SMA mouse model, 277 proteins were found to be differentially abundant at a symptomatic timepoint (post-natal day (P) 18), 50 of which were similarly dysregulated in severe Taiwanese SMA mice. Bioinformatics analysis linked many of the differentially abundant proteins to cardiovascular development and function, with intermediate filaments highlighted as an enriched cellular compartment in both datasets. Lamin A/C was increased in the cardiac tissue, whereas another intermediate filament protein, desmin, was reduced. The extracellular matrix (ECM) protein, elastin, was also robustly decreased in the heart of Smn2B/− mice. AAV9-SMN1-mediated gene therapy rectified low levels of survival motor neuron protein and restored desmin levels in heart tissues of Smn2B/− mice. In contrast, AAV9-SMN1 therapy failed to correct lamin A/C or elastin levels. Intermediate filament proteins and the ECM have key roles in cardiac function and their dysregulation may explain cardiac impairment in SMA, especially since mutations in genes encoding these proteins cause other diseases with cardiac aberration. Cardiac pathology may need to be considered in the long-term care of SMA patients, as it is unclear whether currently available treatments can fully rescue peripheral pathology in SMA.Publisher PDFPeer reviewe

The effect of perinatal exposure to increased serotonin concentrations on histological structure of somatosensory cortex of adult rats

Serotonin (5HT) je biološki aktivan amin prisutan u mozgu i perifernim tkivima sisavaca gdje izvršava različite funkcije, ovisno o odjeljku u kojem se nalazi. U mozgu regulira razvoj serotonergičnih neurona i ciljanih tkiva tijekom neurogeneze, dok u razvijenom mozgu preuzima ulogu neurotransmitera. Cilj ovog diplomskog rada bio je ispitati utjecaj perinatalno inducirane povišene razine serotonina, primjenom neposrednog prekursora sinteze serotonina, 5-hidroksitriptofana (5HTP, 25 mg/kg), ili inhibitora razgradnje serotonina, tranilcipromina (TCP, 2mg/kg) na razvoj bačvastog polja u somatosenzoričkoj kori mozga štakora. Mozgovi su prethodno izdvojeni neposredno po završetku tretmana (PND 21) ili u odrasloj dobi (PND 70). Tangencijalne rezove somatosenzoričke kore podvrgnula sam histološkoj metodi bojanja po Nisslu, te histokemijskoj metodi bojanja citokrom oksidazom (CO). Kvalitativna analiza bačvastog polja vršena je pregledavanjem rezova Nissla pod mikroskopom. Bačvasta polja na rezovima bojanim CO pronašli smo pod mikroskopom i fotografirali. Programom ImageJ ocrtali smo bačve unutar bačvastog polja, te izmjerili površinu koja je služila za kvantitativnu usporedbu eksperimentalnih skupina s kontrolnom, tretiranom fiziološkom otopinom. Povišene razine serotonina imale su značajan učinak na bačvasto polje mladunaca, pri čemu je tretman 5HTP-om rezultirao manjom površinom bačvastog polja i pojedinačnih bačvi, dok je je TCP narušio strukturu bačvi. Učinci tretmana 5HTP-om, vidljivi i u mozgu odraslih životinja, ukazuju na smanjeno grananje talamokortikalnih neurona tijekom prenatalnog dijela tretmana. Učinci TCP-a nisu zadržani u odraslih životinja, pa pretpostavljamo da je došlo do odgođenog sazrijevanja bačvastog polja uslijed kronične izloženosti povišenim koncentracijama serotonina u postnatalnom dijelu tretmana.Serotonin (5HT) is biologically active amine present in the brain and peripheral tissues of mammals where it has various functions, depending on the compartment. In the brain, during neurogenesis, it regulates the development of serotonergic neurons and target tissues, while later it assumes the function of a neurotransmitter. The aim of this graduation thesis was to examine the effect of perinatally induced high serotonin levels on the development of barrel field in the rat somatosensory cortex, induced by treatment with the immediate serotonin precursor ,5-hydroxytryptophan (5HTP, 25 mg/kg), and inhibitor of serotonin degradation, tranylcypromine (TCP, 2 mg/kg). Brains were collected on PND 21 or PND 70. Tangential sections of somatosensory cortex where used for histological processing with Nissl staining method, and histochemical method for cytochrome oxidase staining (CO). Qualitative analysis of barrel field were conducted by examination of Nissl sections under the microscope. We found barrel field on CO sections under the microscope and photographed them. In software ImageJ we draw barrels in barrel field, measured their area surface and conducted analysis of quantitative differences between treated and control group. High serotonin levels had significant effect on barrel field of the pups, with 5HTP treatment resulting in smaller barrel field and barrel area, and TCP treatment disrupting their structure. Treatment effects of 5HTP, still visible in adult rats, point to the decreased branching of thalamo-cortical axons during the prenatal phase of the treatment. Since the effects of TCP were not present in the brains of adult animals, we assume that the cronic exposure to high serotonin levels in the postnatal phase of the treatmen only delayed maturation of the barrel field