Quantitative proteomic profiling of the rat substantia nigra places glial fibrillary acidic protein at the hub of proteins dysregulated during aging : implications for idiopathic Parkinson’s disease

This work was made possible by generous funding from the Keele University ACORN scheme and Keele University School of Medicine.There is a strong correlation between aging and onset of idiopathic Parkinson's disease, but little is known about whether cellular changes occur during normal aging that may explain this association. Here, proteomic and bioinformatic analysis was conducted on the substantia nigra (SN) of rats at four stages of life to identify and quantify protein changes throughout aging. This analysis revealed that proteins associated with cell adhesion, protein aggregation and oxidation‐reduction are dysregulated as early as middle age in rats. Glial fibrillary acidic protein (GFAP) was identified as a network hub connecting the greatest number of proteins altered during aging. Furthermore, the isoform of GFAP expressed in the SN varied throughout life. However, the expression levels of the rate‐limiting enzyme for dopamine production, tyrosine hydroxylase (TH), were maintained even in the oldest animals, despite a reduction in the number of dopamine neurons in the SN pars compact(SNc) as aging progressed. This age‐related increase in TH expression per neuron would likely to increase the vulnerability of neurons, since increased dopamine production would be an additional source of oxidative stress. This, in turn, would place a high demand on support systems from local astrocytes, which themselves show protein changes that could affect their functionality. Taken together, this study highlights key processes that are altered with age in the rat SN, each of which converges upon GFAP. These findings offer insight into the relationship between aging and increased challenges to neuronal viability, and indicate an important role for glial cells in the aging process.Publisher PDFPeer reviewe

Anti-epileptic drugs and bone loss: phenytoin reduces pro-collagen I and alters the electrophoretic mobility of osteonectin in cultured bone cells.

Phenytoin is an antiepileptic drug used in the management of partial and tonic-clonic seizures. In previous studies we have shown that valproate, another antiepileptic drug, reduced the amount of two key bone proteins, pro-collagen I and osteonectin (SPARC, BM-40), in both skin fibroblasts and cultured osteoblast-like cells. Here we show that phenytoin also reduces pro-collagen I production in osteoblast-like cells, but does not appear to cause a decrease in osteonectin message or protein production. Instead, a 24h exposure to a clinically relevant concentration of phenytoin resulted in a dose-dependent change in electrophoretic mobility of osteonectin, which was suggestive of a change in post-translational modification status. The perturbation of these important bone proteins could be one of the mechanisms to explain the bone loss that has been reported following long-term treatment with phenytoin

Enhanced expression of the human Survival motor neuron 1 gene from a codon-optimised cDNA transgene in vitro and in vivo

Spinal muscular atrophy (SMA) is a neuromuscular disease particularly characterised by degeneration of ventral motor neurons. Survival motor neuron (SMN) 1 gene mutations cause SMA, and gene addition strategies to replace the faulty SMN1 copy are a therapeutic option. We have developed a novel, codon-optimised hSMN1 transgene and produced integration-proficient and integration-deficient lentiviral vectors with cytomegalovirus (CMV), human synapsin (hSYN) or human phosphoglycerate kinase (hPGK) promoters to determine the optimal expression cassette configuration. Integrating, CMV-driven and codon-optimised hSMN1 lentiviral vectors resulted in the highest production of functional SMN protein in vitro. Integration-deficient lentiviral vectors also led to significant expression of the optimised transgene and are expected to be safer than integrating vectors. Lentiviral delivery in culture led to activation of the DNA damage response, in particular elevating levels of phosphorylated ataxia telangiectasia mutated (pATM) and ?H2AX, but the optimised hSMN1 transgene showed some protective effects. Neonatal delivery of adeno-associated viral vector (AAV9) vector encoding the optimised transgene to the Smn2B/- mouse model of SMA resulted in a significant increase of SMN protein levels in liver and spinal cord. This work shows the potential of a novel codon-optimised hSMN1 transgene as a therapeutic strategy for SMA

Proteomic analysis of age-related changes in ovine cerebrospinal fluid

Cerebrospinal fluid (CSF) circulates through the brain and has a unique composition reflecting the biological processes of the brain. Identifying ageing CSF biomarkers can aid in understanding the ageing process and interpreting CSF protein changes in neurodegenerative diseases. In this study, ovine CSF proteins from young (1-2 year old), middle aged (3-6 year old) and old (7-10 year old) sheep were systemically studied. CSF proteins were labelled with iTRAQ tagging reagents and fractionated by 2-dimensional high performance, liquid chromatography. Tryptic peptides were identified using MS/MS fragmentation ions for sequencing and quantified from iTRAQ reporter ion intensities at m/z 114, 115, 116 and 117. Two hundred thirty one peptides were detected, from which 143 proteins were identified. There were 52 proteins with >25% increase in concentrations in the old sheep compared to the young. 33 of them increased >25% but 50% but 1 fold [i.e. haptoglobin (Hp), haemoglobin, neuroendocrine protein 7B2, IgM, fibrous sheath interacting protein 1, vimentin]. There were 18 proteins with >25% decrease in concentrations in the old sheep compared to the young. 17 of them decreased >25% but <50%, and histone deacetylase 7 (HDAC7) was gradually decreased for over 80%. Glutathione S-transferase was decreased in middle aged CSF compared to both young and old CSF. The differential expressions of 3 proteins (Hp, neuroendocrine protein 7B2, IgM) were confirmed by immunoassays. These data expand our current knowledge regarding ovine CSF proteins, supply the necessary information to understand the ageing process in the brain and provide a basis for diagnosis of neurodegenerative diseases

Contaminants in commercial preparations of ‘purified’ small leucine-rich proteoglycans may distort mechanistic studies

The authors are grateful to Genodisc (EC’s 7th Framework Programme (FP7, 2007-2013) under grant agreement no. HEALTH-F2-2008-201626) and the Orthopaedic Institute Ltd for funding.This paper reports the perplexing results that came about because of seriously impure commercially available reagents. Commercial reagents and chemicals are routinely ordered by scientists and are expected to have been rigorously assessed for their purity. Unfortunately, we found this assumption to be risky. Extensive work was carried out within our laboratory using commercially-sourced preparations of the small leucine-rich proteoglycans, decorin and biglycan, to investigate their influence on nerve cell growth. Unusual results compelled us to analyse the composition and purity of both preparations of these proteoglycans using both mass spectrometry and Western blotting, with and without various enzymatic deglycosylations. Commercial ‘decorin’ and ‘biglycan’ were found to contain a mixture of proteoglycans including not only both decorin and biglycan but also fibromodulin and aggrecan. The unexpected effects of ‘decorin’ and ‘biglycan’ on nerve cell growth could be explained by these impurities. Decorin and biglycan contain either chondroitin or dermatan sulphate glycosaminoglycan chains whilst fibromodulin only contains keratan sulphate and the large (>2,500 kDa), highly glycosylated aggrecan, contains both keratan and chondroitin sulphate. The different structure, molecular weights and composition of these impurities significantly affected our work and any conclusions that could be made. These findings beg the question as to whether scientists need to verify the purity of each commercially obtained reagent used in their experiments. The implications of these findings are vast, since the effects of these impurities may already have led to inaccurate conclusions and reports in the literature with concomitant loss of researchers’ funds and time.Publisher PDFPeer reviewe

Absence of gemin5 from SMN complexes in nuclear Cajal bodies

<p>Abstract</p> <p>Background</p> <p>Spinal muscular atrophy is caused by reduced levels of the survival of motor neurons (SMN) protein. SMN is found in large complexes with Sm proteins and at least eight other proteins, including seven "gemins". These complexes are involved in the assembly of snRNPs in the cytoplasm and their transport into the nucleus, but the precise roles of the individual protein components are largely unknown.</p> <p>Results</p> <p>We have investigated the subcellular distribution of gemins using novel antibodies against gemins 3–7, and existing mAbs against SMN, gemin2, unrip, fibrillarin and profilin II. Most gemins were equally distributed between nuclear and cytoplasmic fractions of HeLa cells, but gemin5 and unrip were more abundant in the cytoplasm. In a cytoplasmic extract obtained by mild disruption of HeLa cells, nearly all the SMN and gemins 2–4 were in large complexes, but most of the gemin5 sedimented separately with a lower S value. Most of the unrip sedimented with gemins 6 and 7 near the top of the sucrose density gradients, separate from both SMN and gemin5. Anti-SMN mAbs pulled down gemin5 from cytoplasmic extracts, but not from nuclear extracts, and gemin5 did not co-sediment with large SMN complexes in nuclear extracts. These data suggest that gemin5 is easily detached from SMN-gemin complexes in the nucleus. By immuno-histochemistry, gemin5 was rarely detectable in nuclear gems/Cajal bodies, although it was accessible to antibody and easily detectable when present. This suggests that gemin5 is normally absent from SMN complexes in these nuclear storage sites.</p> <p>Conclusion</p> <p>We conclude that SMN complexes usually exist without gemin5 in nuclear gems/Cajal bodies. Gemin5 is believed to be involved in capturing snRNA into SMN complexes in the cytoplasm for transport into the nucleus. We hypothesize that gemin5, though present in the nucleus, is no longer needed for SMN complex function during the time these complexes are stored in gems/Cajal bodies.</p

Understanding the molecular consequences of inherited muscular dystrophies:advancements through proteomic experimentation

Introduction: Proteomic techniques offer insights into the molecular perturbations occurring in muscular-dystrophies (MD). Revisiting published datasets can highlight conserved downstream molecular alterations, which may be worth re-assessing to determine whether their experimental manipulation is capable of modulating disease severity. Areas covered: Here, we review the MD literature, highlighting conserved molecular insights warranting mechanistic investigation for therapeutic potential. We also describe a workflow currently proving effective for efficient identification of biomarkers & therapeutic targets in other neurodegenerative conditions, upon which future MD proteomic investigations could be modelled. Expert commentary: Studying disease models can be useful for identifying biomarkers and model specific degenerative cascades, but rarely offer translatable mechanistic insights into disease pathology. Conversely, direct analysis of human samples undergoing degeneration presents challenges derived from complex chronic degenerative molecular processes. This requires a carefully planed & reproducible experimental paradigm accounting for patient selection through to grouping by disease severity and ending with proteomic data filtering and processing

Investigation of the blood proteome in response to spinal cord injury in rodent models

We would like to thank the Institute of Orthopaedics and the Midlands Centre for Spinal Cord Injury (MCSI) for funding this research. This work was also supported by the Wellcome Trust [grant number 094476/Z/10/Z] which funded the purchase of the TripleTOF 5600 mass spectrometer at the BSRC Mass Spectrometry and Proteomics Facility, University of St Andrews.Study Design: Explanatory and mechanistic study. Objective: A better understanding of the 'whole-body' response following spinal cord injury (SCI) is needed to guide future research aimed at developing novel therapeutic interventions and identifying prognostic indicators for SCI. This study aimed to characterise the blood proteome following contusion or complete SCI compared to a sham injury in rat models. Setting: United Kingdom. Methods: Pooled blood samples from one and seven days after a contusion (serum; n = 5) or from 14 days and 112 days post-complete transection SCI (plasma; n = 8) and their sham-injured counterparts were subjected to independent iTRAQ nanoflow liquid chromatography tandem mass-spectrometry proteomic analyses. Pathway analyses of the proteins that were differentially abundant between SCI and their matched sham injured counterparts were completed to indicate biological pathways that may be changed in response to SCI. Results: Eleven and 42 proteins were differentially abundant (≥±2.0 FC; p ≤ 0.05) between the contusion SCI and sham injured animals at 24 h and seven days post-injury, respectively. Seven and tweleve proteins were differentially abundant between complete and sham injured rats at 14 and 112 days post-injury, respectively. Acute-phase response signalling and Liver X Receptor/Retinoic X Receptor activation were identified as differentially regulated pathways in both models of SCI. Conclusions: We have utilised longitudinal preclinical SCI models to provide an insight into the blood proteome changes that result following SCI and to highlight a number of biological pathways of interest for future studies.Publisher PDFPeer reviewe

The rat striatum responds to nigro-striatal degeneration via the increased expression of proteins associated with growth and regeneration of neuronal circuitry

BACKGROUND: Idiopathic Parkinson's disease is marked by degeneration of dopamine neurons projecting from the substantia nigra to the striatum. Although proteins expressed by the target striatum can positively affect the viability and growth of dopaminergic neurons, very little is known about the molecular response of the striatum as nigro-striatal denervation progresses. Here, iTRAQ labelling and MALDI TOF/TOF mass spectrometry have been used to quantitatively compare the striatal proteome of rats before, during, and after 6-OHDA induced dopamine denervation. RESULTS: iTRAQ analysis revealed the differential expression of 50 proteins at 3 days, 26 proteins at 7 days, and 34 proteins at 14 days post-lesioning, compared to the unlesioned striatum. While the denervated striatum showed a reduced expression of proteins associated with the loss of dopaminergic input (e.g., TH and DARPP-32), there was an increased expression of proteins associated with regeneration and growth of neurites (e.g., GFAP). In particular, the expression of guanine deaminase (GDA, cypin) - a protein known to be involved in dendritic branching - was significantly increased in the striatum at 3, 7 and 14 days post-lesioning (a finding verified by immunohistochemistry). CONCLUSIONS: Together, these findings provide evidence to suggest that the response of the normal mammalian striatum to nigro-striatal denervation includes the increased expression of proteins that may have the capacity to facilitate repair and growth of neuronal circuitry

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