Synapses are a primary pathological target in neurodegenerative diseases. Identifying therapeutic targets at the synapse could delay progression of numerous conditions. The mitochondrial protein SFXN3 is a neuronally enriched protein expressed in synaptic terminals and regulated by key synaptic proteins, including α‐synuclein. We first show that SFXN3 uses the carrier import pathway to insert into the inner mitochondrial membrane. Using high‐resolution proteomics on Sfxn3 ‐KO mice synapses, we then demonstrate that SFXN3 influences proteins and pathways associated with neurodegeneration and cell death (including CSPα and Caspase‐3), as well as neurological conditions (including Parkinson's disease and Alzheimer’s disease). Overexpression of SFXN3 orthologues in Drosophila models of Parkinson's disease significantly reduced dopaminergic neuron loss. In contrast, the loss of SFXN3 was insufficient to trigger neurodegeneration in mice, indicating an anti‐ rather than pro‐neurodegeneration role for SFXN3. Taken together, these results suggest a potential role for SFXN3 in the regulation of neurodegeneration pathways.
Abstract There is growing evidence suggesting that the lysosome or lysosome dysfunction is associated with Alzheimer’s disease (AD). Pathway analysis of post mortem brain-derived proteomic data from AD patients shows that the lysosomal system is perturbed relative to similarly aged unaffected controls. However, it is unclear if these changes contributed to the pathogenesis or are a response to the disease. Consistent with the hypothesis that lysosome dysfunction contributes to AD pathogenesis, whole genome sequencing data indicate that heterozygous pathogenic mutations and predicted protein-damaging variants in multiple lysosomal enzyme genes are enriched in AD patients compared to matched controls. Heterozygous loss-of-function mutations in the palmitoyl protein thioesterase-1 ( PPT1 ), α-L-iduronidase ( IDUA ), β-glucuronidase ( GUSB ), N-acetylglucosaminidase ( NAGLU ), and galactocerebrosidase ( GALC ) genes have a gene-dosage effect on Aβ 40 levels in brain interstitial fluid in C57BL/6 mice and significantly increase Aβ plaque formation in the 5xFAD mouse model of AD, thus providing in vivo validation of the human genetic data. A more detailed analysis of PPT1 heterozygosity in 18-month-old mice revealed changes in α-, β-, and γ-secretases that favor an amyloidogenic pathway. Proteomic changes in brain tissue from aged PPT1 heterozygous sheep are consistent with both the mouse data and the potential activation of AD pathways. Finally, CNS-directed, AAV-mediated gene therapy significantly decreased Aβ plaques, increased life span, and improved behavioral performance in 5xFAD/PPT1+/- mice. Collectively, these data strongly suggest that heterozygosity of multiple lysosomal enzyme genes represent risk factors for AD and may identify precise therapeutic targets for a subset of genetically-defined AD patients. Significance Statement Lysosomes play a role in the degradation of aggregation-prone proteins such as amyloid β (Aβ). Homozygous lysosomal enzyme gene defects result in fatal pediatric lysosomal storage diseases and, historically, carriers were considered normal. However, a human genetic analysis identified deleterious heterozygous variants in multiple lysosomal enzyme genes that are enriched in Alzheimer’s disease (AD) patients. Those findings were validated in vivo by demonstrating that heterozygous loss-of-function (LoF) mutations in five different lysosomal enzyme genes affect Aβ processing and exacerbate Aβ plaque formation. CNS-directed gene therapy ameliorated the effects of a heterozygous LoF mutation in one of those genes in a mouse model of AD. These findings provide insights into the role of lysosomes in AD and have important therapeutic implications.
Western blotting has been a key technique for determining the relative expression of proteins within complex biological samples since the first publications in 1979. Recent developments in sensitive fluorescent labels, with truly quantifiable linear ranges and greater limits of detection, have allowed biologists to probe tissue specific pathways and processes with higher resolution than ever before. However, the application of quantitative Western blotting (QWB) to a range of healthy tissues and those from degenerative models has highlighted a problem with significant consequences for quantitative protein analysis: how can researchers conduct comparative expression analyses when many of the commonly used reference proteins (e.g. loading controls) are differentially expressed? Here we demonstrate that common controls, including actin and tubulin, are differentially expressed in tissues from a wide range of animal models of neurodegeneration. We highlight the prevalence of such alterations through examination of published “–omics” data, and demonstrate similar responses in sensitive QWB experiments. For example, QWB analysis of spinal cord from a murine model of Spinal Muscular Atrophy using an Odyssey scanner revealed that beta-actin expression was decreased by 19.3±2% compared to healthy littermate controls. Thus, normalising QWB data to β-actin in these circumstances could result in ‘skewing’ of all data by ∼20%. We further demonstrate that differential expression of commonly used loading controls was not restricted to the nervous system, but was also detectable across multiple tissues, including bone, fat and internal organs. Moreover, expression of these “control” proteins was not consistent between different portions of the same tissue, highlighting the importance of careful and consistent tissue sampling for QWB experiments. Finally, having illustrated the problem of selecting appropriate single protein loading controls, we demonstrate that normalisation using total protein analysis on samples run in parallel with stains such as Coomassie blue provides a more robust approach.
Equine grass sickness (EGS) is an acute, predominantly fatal, multiple system neuropathy of grazing horses with reported incidence rates of ∼2%. An apparently identical disease occurs in multiple species, including but not limited to cats, dogs, and rabbits. Although the precise etiology remains unclear, ultrastructural findings have suggested that the primary lesion lies in the glycoprotein biosynthetic pathway of specific neuronal populations. The goal of this study was therefore to identify the molecular processes underpinning neurodegeneration in EGS. Here, we use a bottom-up approach beginning with the application of modern proteomic tools to the analysis of cranial (superior) cervical ganglion (CCG, a consistently affected tissue) from EGS-affected patients and appropriate control cases postmortem. In what appears to be the proteomic application of modern proteomic tools to equine neuronal tissues and/or to an inherent neurodegenerative disease of large animals (not a model of human disease), we identified 2,311 proteins in CCG extracts, with 320 proteins increased and 186 decreased by greater than 20% relative to controls. Further examination of selected proteomic candidates by quantitative fluorescent Western blotting (QFWB) and subcellular expression profiling by immunohistochemistry highlighted a previously unreported dysregulation in proteins commonly associated with protein misfolding/aggregation responses seen in a myriad of human neurodegenerative conditions, including but not limited to amyloid precursor protein (APP), microtubule associated protein (Tau), and multiple components of the ubiquitin proteasome system (UPS). Differentially expressed proteins eligible for in silico pathway analysis clustered predominantly into the following biofunctions: (1) diseases and disorders, including; neurological disease and skeletal and muscular disorders and (2) molecular and cellular functions, including cellular assembly and organization, cell-to-cell signaling and interaction (including epinephrine, dopamine, and adrenergic signaling and receptor function), and small molecule biochemistry. Interestingly, while the biofunctions identified in this study may represent pathways underpinning EGS-induced neurodegeneration, this is also the first demonstration of potential molecular conservation (including previously unreported dysregulation of the UPS and APP) spanning the degenerative cascades from an apparently unrelated condition of large animals, to small animal models with altered neuronal vulnerability, and human neurological conditions. Importantly, this study highlights the feasibility and benefits of applying modern proteomic techniques to veterinary investigations of neurodegenerative processes in diseases of large animals. Equine grass sickness (EGS) is an acute, predominantly fatal, multiple system neuropathy of grazing horses with reported incidence rates of ∼2%. An apparently identical disease occurs in multiple species, including but not limited to cats, dogs, and rabbits. Although the precise etiology remains unclear, ultrastructural findings have suggested that the primary lesion lies in the glycoprotein biosynthetic pathway of specific neuronal populations. The goal of this study was therefore to identify the molecular processes underpinning neurodegeneration in EGS. Here, we use a bottom-up approach beginning with the application of modern proteomic tools to the analysis of cranial (superior) cervical ganglion (CCG, a consistently affected tissue) from EGS-affected patients and appropriate control cases postmortem. In what appears to be the proteomic application of modern proteomic tools to equine neuronal tissues and/or to an inherent neurodegenerative disease of large animals (not a model of human disease), we identified 2,311 proteins in CCG extracts, with 320 proteins increased and 186 decreased by greater than 20% relative to controls. Further examination of selected proteomic candidates by quantitative fluorescent Western blotting (QFWB) and subcellular expression profiling by immunohistochemistry highlighted a previously unreported dysregulation in proteins commonly associated with protein misfolding/aggregation responses seen in a myriad of human neurodegenerative conditions, including but not limited to amyloid precursor protein (APP), microtubule associated protein (Tau), and multiple components of the ubiquitin proteasome system (UPS). Differentially expressed proteins eligible for in silico pathway analysis clustered predominantly into the following biofunctions: (1) diseases and disorders, including; neurological disease and skeletal and muscular disorders and (2) molecular and cellular functions, including cellular assembly and organization, cell-to-cell signaling and interaction (including epinephrine, dopamine, and adrenergic signaling and receptor function), and small molecule biochemistry. Interestingly, while the biofunctions identified in this study may represent pathways underpinning EGS-induced neurodegeneration, this is also the first demonstration of potential molecular conservation (including previously unreported dysregulation of the UPS and APP) spanning the degenerative cascades from an apparently unrelated condition of large animals, to small animal models with altered neuronal vulnerability, and human neurological conditions. Importantly, this study highlights the feasibility and benefits of applying modern proteomic techniques to veterinary investigations of neurodegenerative processes in diseases of large animals. Equine grass sickness (EGS, or equine dysautonomia) is a predominantly fatal, rapid multiple system neuropathy of grazing horses with reported incidence rates of 2.1–2.3% (reviewed by (1.Pirie R.S. Grass sickness.Clin. Tech. Equine Practice. 2006; 5: 30-36Crossref Scopus (17) Google Scholar, 2.Pirie R.S. Jago R.C. Hudson N.P. Equine grass sickness.Equine Vet. J. 2014; 46: 545-553Crossref PubMed Scopus (34) Google Scholar)). An apparently identical disease occurs in cats, dogs, hares, rabbits, llamas, and possibly sheep (3.Sharp N.J.H. Nash A.S. Griffiths I.R. Feline dysautonomia (the Key-Gaskell syndrome): A clinical and pathological study of forty cases.J. Small Animal Practice. 1984; 25: 599-615Crossref Scopus (44) Google Scholar, 4.Whitwell K.E. Do hares suffer from grass sickness?.Vet. Rec. 1991; 128: 395-396Crossref PubMed Scopus (27) Google Scholar, 5.Longshore R.C. O'Brien D.P. Johnson G.C. Grooters A.M. Kroll R.A. Dysautonomia in dogs: A retrospective study.J. Vet. Intern. Med. 1996; 10: 103-109Crossref PubMed Scopus (34) Google Scholar, 6.Kik M.J. van der Hage M.H. Cecal impaction due to dysautonomia in a llama (Lama glama).J. Zoo Wildl. Med. 1999; 30: 435-438PubMed Google Scholar, 7.Pruden S.J. McAllister M.M. Schultheiss P.C. O'Toole D. Christensen D.E. Abomasal emptying defect of sheep may be an acquired form of dysautonomia.Vet. Pathol. 2004; 41: 164-169Crossref PubMed Scopus (22) Google Scholar, 8.Hahn C.N. Whitwell K.E. Mayhew I.G. Neuropathological lesions resembling equine grass sickness in rabbits.Vet. Rec. 2005; 156: 778-779Crossref PubMed Scopus (15) Google Scholar, 9.Lewis C.A. Bozynski C.C. Johnson G.C. Harral C.M. Williams 3rd, F. Tyler J.W. Colonic impaction due to dysautonomia in an alpaca.J. Vet. Intern. Med. 2009; 23: 1117-1122Crossref PubMed Scopus (9) Google Scholar). EGS is associated with chromatolysis of sympathetic and parasympathetic postsynaptic neurons, particularly in the enteric nervous system, as well as autonomic presynaptic and somatic lower motor neurons in the brainstem and spinal cord (10.Hahn C.N. Mayhew I.G. de Lahunta A. Central neuropathology of equine grass sickness.Acta Neuropathol. 2001; 102: 153-159Crossref PubMed Scopus (47) Google Scholar). EGS is subdivided into acute, subacute, and chronic forms according to the severity of clinical signs that largely reflect enteric and autonomic neurodegeneration, including dysphagia, generalized ileus, sweating, salivation, ptosis, rhinitis sicca, and tachycardia. While the etiology of EGS remains unknown, some evidence supports it being a toxic infection with Clostridium botulinum type C or D (11.Poxton I.R. Hunter L.C. Brown R. Lough H.G. Miller J.K. Clostridia and equine grass sickness.Rev. Med. Microbiol. 1997; 8: S52Crossref Scopus (14) Google Scholar, 12.Hunter L.C. Miller J.K. Poxton I.R. The association of Clostridium botulinum type C with equine grass sickness: A toxicoinfection?.Equine Vet. J. 1999; 31: 492-499Crossref PubMed Scopus (104) Google Scholar). Ultrastructural studies suggest that the lesion in EGS primarily involves the glycoprotein biosynthetic pathway of specific neurons since the rough endoplasmic reticulum and Golgi complexes are consistently affected, while other organelles, including mitochondria, appear relatively normal (13.Griffiths I.R. Kyriakides E. Smith S. Howie F. Deary A.W. Immunocytochemical and lectin histochemical study of neuronal lesions in autonomic ganglia of horses with grass sickness.Equine Vet. J. 1993; 25: 446-452Crossref PubMed Scopus (23) Google Scholar). However, while the ultrastructural and cellular appearance of affected neurons has been studied extensively, little is known about the molecular mechanisms that contribute to neurodegeneration. The overarching aim of this study was therefore to identify the molecular processes underpinning neurodegeneration in EGS using a bottom-up approach beginning with the application of modern proteomic tools to the analysis of cranial (superior) cervical ganglion (CCG, a consistently affected tissue) from EGS-affected patients and appropriate control cases postmortem. The cranial (superior) cervical ganglion (CCG), which supplies sympathetic innervation to the head and neck, was selected because chromatolysis of a high proportion of CCG neurons is a consistent feature of EGS (Fig. 1 and Supplemental Fig. 1 (14.Pogson D.M. Doxey D.L. Gilmour J.S. Milne E.M. Chisholm H.K. Autonomic neurone degeneration in equine dysautonomia (grass sickness).J. Comp. Pathol. 1992; 107: 271-283Crossref PubMed Scopus (44) Google Scholar)). Here, proteomic analysis was carried out using isobaric tag for relative and absolute quantitation (iTRAQ) tools, which are now well established in small animal models of human neurodegenerative conditions but which are not routinely utilized in large animal models or large animal intrinsic conditions. This proteomic analysis was coupled with quantitative fluorescent Western blotting (QFWB), immunohistochemistry (IHC), and in silico based techniques in an attempt to identify the molecular pathways and processes that may be contributing to neurodegeneration in EGS. Here, we report widespread changes in the CCG of EGS horses, including significant disruption to a broad range of functional pathways clustering around candidates commonly associated with protein misfolding/aggregation responses in human neurodegenerative conditions. This study therefore represents the first application of modern proteomic tools to equine neuronal tissues and/or to an inherent neurodegenerative disease of large animals (not a model of human disease). It is also the first to demonstrate correlation and conservation spanning the degenerative molecular cascades from an apparently unrelated condition of large animals to small animal models with altered neuronal vulnerability and a range of human neurological conditions from childhood neurodegenerative conditions such as spinal muscular atrophy through to diseases associated with advancing age such as Alzheimer's. Finally, this study highlights the feasibility and benefits of applying differential proteomics techniques to the investigation of the neurodegenerative processes in diseases of large animals. In accordance with MCP guidelines, more detailed discussion of experimental design and potential limitations can be found in the accompanying Supplementary Discussion file. General methodology is provided below. Tissue samples were collected at necropsy from horses that were euthanized on humane grounds, with the horse owners' consent. The study was approved by the local ethics committee. For proteomics and quantitative fluorescent Western blotting, CCG were collected from six EGS (median age 6 years, range 3–17) and six control (14, 6–30 years) mixed-breed and mixed-gender horses within 60 min of euthanasia by administration of barbiturates (Table I). CCG were selected because chromatolysis of a high proportion of CCG neurons is a consistent feature of EGS (see Fig. 1) (14.Pogson D.M. Doxey D.L. Gilmour J.S. Milne E.M. Chisholm H.K. Autonomic neurone degeneration in equine dysautonomia (grass sickness).J. Comp. Pathol. 1992; 107: 271-283Crossref PubMed Scopus (44) Google Scholar). The heterogeneity in breed, sex, and age of EGS horses used in the study reflects the spectrum of horses affected by this spontaneous neurodegenerative disease during the study period. EGS horses comprised three acute and three subacute cases, as categorized by McGorum and Kirk (15.McGorum B.C. Kirk J. Equine dysautonomia (grass sickness) is associated with altered plasma amino acid levels and depletion of plasma sulphur amino acids.Equine Vet. J. 2001; 33: 473-477Crossref PubMed Scopus (30) Google Scholar). The grouping of acute and subacute cases for proteomic analysis was carried out as there are only minor differences in clinical features and pathology of these phenotypes. The main difference is the length of disease process following diagnosis. All of the samples are from post mortem terminal patients and the anatomical and cellular hallmarks are consistent at end stage regardless of case type. Moreover, we can demonstrate that neuronal density does not differ between acute and subacute cases (Supplemental Fig. 1). Due to these considerations and the fact that the initiating insult remains unproven, molecular analyses are currently performed on pooled samples as an attempt to reduce "noise" due to interanimal variability through factors such as individual disease response, age, and breed, among other considerations. EGS was confirmed in all cases by necropsy, including histopathological examination of autonomic ganglia (16.Doxey D.L. Pogson D.M. Milne E.M. Gilmour J.S. Chisholm H.K. Clinical equine dysautonomia and autonomic neuron damage.Res. Vet. Sci. 1992; 53: 106-109Crossref PubMed Scopus (39) Google Scholar). Controls were euthanized on humane grounds for reasons other than neurological disease. Immediately after collection, CCG were rapidly frozen by immersion in dry ice pellets and stored at −80 °C. For immunohistochemistry, CCG were collected from six EGS (median age 8 years, range 2–20) and six control (median age 11, 6–15 years) mixed-breed and mixed-gender horses as described above (Table I) and fixed in 10% neutral buffered formalin and embedded in paraffin wax.Table ISubject information Ganglia were partially thawed, the outer fascia removed by dissection, and a portion macerated with a scalpel before partial homogenization in either radioimmune precipitation assay buffer with protease inhibitor mixture (Roche) for QFWB (see below) or iTRAQ extraction buffer containing 6 m Urea, 2 m thiourea, 2% CHAPS, 0.5% SDS, and protease inhibitor mixture (Roche) for proteomic processing (see below). Samples were pooled by condition and manually homogenized in a dounce glass homogenizer. Homogenized samples were sonicated in a cup style sonicator six times for 15 s at power level 7.5 with vortexing for 30 s between each round of sonication. Samples were left on ice for 10 min before being revortexed then centrifuged at 20,000 g for 30 min at 4 °C. The resulting pellet containing proteins insoluble when processed in this manner was stored at −80 °C, and the supernatant was transferred to a fresh 1.5 ml tube to be processed for iTRAQ labeling as previously described (17.Wishart T.M. Rooney T.M. Lamont D.J. Wright A.K. Morton A.J. Jackson M. Freeman M.R. Gillingwater T.H. Combining comparative proteomics and molecular genetics uncovers regulators of synaptic and axonal stability and degeneration in vivo.PLoS Genet. 2012; 8: e1002936Crossref PubMed Scopus (47) Google Scholar, 18.Wishart T.M. Huang J.P. Murray L.M. Lamont D.J. Mutsaers C.A. Ross J. Geldsetzer P. Ansorge O. Talbot K. Parson S.H. Gillingwater T.H. SMN deficiency disrupts brain development in a mouse model of severe spinal muscular atrophy.Hum. Mol. Genet. 2010; 19: 4216-4228Crossref PubMed Scopus (86) Google Scholar, 19.Comley L.H. Fuller H.R. Wishart T.M. Mutsaers C.A. Thomson D. Wright A.K. Ribchester R.R. Morris G.E. Parson S.H. Horsburgh K. Gillingwater T.H. ApoE isoform-specific regulation of regeneration in the peripheral nervous system.Hum. Mol. Genet. 2011; 20: 2406-2421Crossref PubMed Scopus (26) Google Scholar, 20.Wishart T.M. Mutsaers C.A. Riessland M. Reimer M.M. Hunter G. Hannam M.L. Eaton S.L. Fuller H.R. Roche S.L. Somers E. Morse R. Young P.J. Lamont D.J. Hammerschmidt M. Joshi A. Hohenstein P. Morris G.E. Parson S.H. Skehel P.A. Becker T. Robinson I.M. Becker C.G. Wirth B. Gillingwater T.H. Dysregulation of ubiquitin homeostasis and beta-catenin signaling promote spinal muscular atrophy.J. Clin. Invest. 2014; 124: 1821-1834Crossref PubMed Scopus (134) Google Scholar). Protein was extracted in iTRAQ extraction buffer (6 m Urea, 2 m thiourea, 2% CHAPS, 0.5% SDS, and protease inhibitor mixture (Roche, Burgess Hill, UK), (pH 7.4)) before acetone precipitation and labeling for iTRAQ analysis. The Mass spectrometry proteomic data have been deposited to the ProteomeXchange consortium via the PRIDE partner repository with the dataset identifier PXD002956. Protein extracts (n = 6 ganglia per group; see Fig. 2) were precipitated with −20 °C chilled acetone (1:4, v/v) and stored at −20 °C overnight. The precipitates were spun at 4 °C for 10 min then washed with an acetone:water mixture (4:1, v/v) twice prior to air drying. The pellets were then resuspended in iTRAQ sample buffer (25 μl 500 mm tetraethylammonium bromide, 1 μl denaturant (2% SDS), and 2 μl of reducing agent Tris(2-carboxyethyl)phophine (TCEP)). The samples were allowed to incubate for 1 h at 60 °C prior to protein estimation in triplicate (3 × 1 μl) by microBCA assay (Pierce, Paisley, UK). Samples were run in duplicate to utilize all four tags from the 4plex kit and increase peptide identification yield as previously described (17.Wishart T.M. Rooney T.M. Lamont D.J. Wright A.K. Morton A.J. Jackson M. Freeman M.R. Gillingwater T.H. Combining comparative proteomics and molecular genetics uncovers regulators of synaptic and axonal stability and degeneration in vivo.PLoS Genet. 2012; 8: e1002936Crossref PubMed Scopus (47) Google Scholar, 18.Wishart T.M. Huang J.P. Murray L.M. Lamont D.J. Mutsaers C.A. Ross J. Geldsetzer P. Ansorge O. Talbot K. Parson S.H. Gillingwater T.H. SMN deficiency disrupts brain development in a mouse model of severe spinal muscular atrophy.Hum. Mol. Genet. 2010; 19: 4216-4228Crossref PubMed Scopus (86) Google Scholar, 19.Comley L.H. Fuller H.R. Wishart T.M. Mutsaers C.A. Thomson D. Wright A.K. Ribchester R.R. Morris G.E. Parson S.H. Horsburgh K. Gillingwater T.H. ApoE isoform-specific regulation of regeneration in the peripheral nervous system.Hum. Mol. Genet. 2011; 20: 2406-2421Crossref PubMed Scopus (26) Google Scholar, 20.Wishart T.M. Mutsaers C.A. Riessland M. Reimer M.M. Hunter G. Hannam M.L. Eaton S.L. Fuller H.R. Roche S.L. Somers E. Morse R. Young P.J. Lamont D.J. Hammerschmidt M. Joshi A. Hohenstein P. Morris G.E. Parson S.H. Skehel P.A. Becker T. Robinson I.M. Becker C.G. Wirth B. Gillingwater T.H. Dysregulation of ubiquitin homeostasis and beta-catenin signaling promote spinal muscular atrophy.J. Clin. Invest. 2014; 124: 1821-1834Crossref PubMed Scopus (134) Google Scholar). Each sample equivalent to 100ug was processed separately using the filter aided sample preparation (FASP) method prior to digestion with trypsin (sequencing grade, Roche). After digestion, the samples were dried using a SpeedVac concentrator and then resuspended in 25 μl of dissolution buffer as provided in the iTRAQ Reagents Multiplex kit (AB Sciex, Warrington, UK). The samples were then labeled, respectively, according to the protocol provided by the manufacturer (AB Sciex). The four labeled samples (Control-115 and 117, EGS-114 and 116) were then pooled together in equal proportions and subsequently dried using a SpeedVac concentrator. The pooled iTRAQ 4plex sample was then desalted using a homemade porous R2 ZipTip column and then fractionated by Strong Cation eXchange (SCX) using a Polysulfoethyl A column (2.1 × 200 mm, 5 μm, PolyLC) on a Ultimate U3000 (Dionex, Loughborough, UK) hplc system. The following buffer system was used Buffer A: 5 mm KH2PO4 in 20% CH3CN (pH 2.7) and buffer B: 500 mm NaCl in 5 mm KH2PO4 in 20% CH3CN (pH 2.7).The flow rate was set to 0.2 ml per minute with a linear gradient from 0 to 50% B over 25 min then a linear gradient from 50 to 100% B over 9 min. Each fraction from the SCX fractionation was then dried using a SpeedVac concentrator and stored at −80 °C. Stored SCX fractions of the pooled iTRAQ 4plex sample were then resuspended in 10 μl of 5% formic acid, diluted to 1% formic acid, and then 15 μl aliquots injected onto an Ultimate RSLC nano UHPLC system coupled to a LTQ Orbitrap Velos Pro (Thermo Scientific, Loughborough, UK). The iTRAQ-labeled peptides were injected onto a trapping column (Acclaim PepMap 100, 100 μm × 2 cm, C18, 5 μm, nanoViper) and then separated using a 2 h linear gradient from 2–40% B (80% acetonitrile, 0.1% formic acid) on a separation column (Acclaim PepMap RSLC, 75 μm × 15 cm, C18, 2 μm, nanoViper) at a flow rate of 300 nl/min. The mass spectrometry parameters were set as follows: Fourier transform mass spectrometry (FT-MS) (survey scan) resolution was set at 60,000; the 15 most-intense precursor ions were chosen for fragmentation by high energy collisional-induced dissociation (HCD); the precursor isolation window was set at 1.2 Da; and the ms/ms scan resolution was set to 7,500. Automatic gain control (AGC) values for FT-MS and FT-MS/MS were set at 1e6 and 5e4 ions, respectively. The maximum fill times for FT-MS and FT-MS/MS were set at 500 and 200 ms, respectively. The raw data were extracted using Proteome Discoverer (Version 1.4.1, Thermo Scientific) for both quantitation and for identification by searching against the NCBI mammalia (database: NCBInr 20,121,028–21,171,493 sequences; 7,255,144,311 residues; taxonomy: mammalia (mammals) 1,173,629 sequences) and NCBI bacteria (database: NCBInr 20,130,811–31,351,517 sequences; 10,835,265,410 residues; taxonomy: bacteria (eubacteria) 20,989,102 sequences) databases using the Mascot Search Engine (Version 2.4.1, Matrix Science, London, UK). Search parameters included the following: enzyme: trypsin/P; fixed modifications (carbamidomethyl (C)), iTRAQ4plex (N-term); variable modifications: oxidation (M), dioxidation (M), acetyl (N-term), Gln->pyro-Glu (N-term Q), iTRAQ4plex (K), iTRAQ4plex (Y); peptide mass tolerance ± 10 ppm; fragment mass tolerance ± 0.06 Da; maximum of two miss-cleavages. Threshold score/expectation value for accepting individual spectra was based on Mascot ion score threshold (0.05) as the standard ion score threshold specifically calculated by Mascot for each database search. As an indication of identification certainty, the false discovery rate for peptide and protein matches above identity threshold were calculated by Peptide Validator at 1.0% (strict) and 5.0% (relaxed), respectively. Validation of altered expression levels for selected candidate proteins was carried out by quantitative fluorescent Western blotting using CCG protein extracts and by immunohistochemistry on CCG sections. See Table II for a list of antibodies and their compatibility with equine neural tissues.Table IIAntibody information QFWB was carried out as previously described (21.Eaton S.L. Roche S.L. Llavero Hurtado M. Oldknow K.J. Farquharson C. Gillingwater T.H. Wishart T.M. Total protein analysis as a reliable loading control for quantitative fluorescent Western blotting.PLoS ONE. 2013; 8: e72457Crossref PubMed Scopus (261) Google Scholar, 22.Eaton S.L. Hurtado M.L. Oldknow K.J. Graham L.C. Marchant T.W. Gillingwater T.H. Farquharson C. Wishart T.M. A guide to modern quantitative fluorescent western blotting with troubleshooting strategies.J. Vis. Exp. 2014; : e52099PubMed Google Scholar). Briefly, 15 μg of CCG protein were separated by SDS-polyacrylamide gel electrophoresis on 4–12% precast NuPage BisTris gradient gels (Invitrogen, Paisley, UK) and then transferred to PVDF membrane using an iBLOT fast transfer device (Invitrogen). The membranes were then blocked using Odyssey blocking buffer (LICOR Biosciences, Cambridge, MA) and incubated with primary antibodies according to manufacturers' instructions (see Table II). Odyssey secondary antibodies were added according to manufacturers' instructions (goat anti-rabbit IRDye 680 and goat anti-mouse IRDye 800). Blots were imaged using an Odyssey Infrared Imaging System (LI-COR Biosciences). Scan resolution of the instrument ranges from 21 to 339 μm, and blots were imaged at 169 μm. Quantification was performed on single channels with the analysis software provided. Total protein stain gels, loaded in parallel with those used for membrane transfer, were used to ensure equivocal sample loading and were analyzed using the Odyssey Infrared Imaging System as previously described in (21.Eaton S.L. Roche S.L. Llavero Hurtado M. Oldknow K.J. Farquharson C. Gillingwater T.H. Wishart T.M. Total protein analysis as a reliable loading control for quantitative fluorescent Western blotting.PLoS ONE. 2013; 8: e72457Crossref PubMed Scopus (261) Google Scholar, 22.Eaton S.L. Hurtado M.L. Oldknow K.J. Graham L.C. Marchant T.W. Gillingwater T.H. Farquharson C. Wishart T.M. A guide to modern quantitative fluorescent western blotting with troubleshooting strategies.J. Vis. Exp. 2014; : e52099PubMed Google Scholar). Formalin-fixed, paraffin-wax-embedded CCGs were dewaxed and rehydrated. Antigen retrieval was performed by heating sections in 0.1 m citrate buffer (pH 6.0) for 15 min in a pressure cooker. The slides were then left to cool for 20 min. A commercial immunolabeling kit (DakoCytomation EnVision+ System-HRP; DAB K4001; DAKO, Ely, UK) was used according to the manufacturer's instructions. Slides were rinsed with Tris buffered saline containing 0.5% Tween (pH 7.5; TBST) and incubated with peroxidase blocking agent (Dako Real Peroxidase Blocker S2023) for 10 min. Slides were rinsed in TBST and incubated with murine monoclonal anti-human synaptophysin (Dako M0776) diluted 1 in 20 in TBST for 60 min at 25 °C. TBST replaced primary antibody for negative controls. Slides were rinsed and incubated with horseradish peroxidase-labeled polymer for 30 min then rinsed and incubated with substrate chromogen solution (Liquid DAB; ImmPact Dab SK4105; Vector Laboratories, Peterborough, UK) for 10 min. Slides were rinsed once in distilled water, counterstained with Harris's hematoxylin (1 min), dipped in Scott's tap water substitute, dehydrated, cleared using ethanol then xylene, and mounted under DPX. The intensity of labeling of CCG neurons was assessed blindly. IHC for B-APP, total Tau, and ubiquitin was carried out with antibodies detailed in Table II, for 2 h at room temperature, following microwave pretreatment in citrate buffer (pH 6), then labeled using the EnVision tracer system. For neuronal cell number assessment BIII-tubulin (Table II) IHC was carried out as described above and Neurotrace (Life-Technologies, Paisley, UK, Table II) was employed following manufacturer's instructions. Sections were visualized using a Nikon Eclipse E800 microscope, and whole sections were montaged by aligning approximately 80 10X images per ganglia section using Adobe Photoshop CS5.1 (V12.4) with the integrated Bridge module. BIII-tubulin and neurotrace-based neuronal counts were carried out in image j by overlaying a 10 × 10 square grid with each individual square measuring 100 μm2 (see Supplemental Fig. 1). To obtain further insight into potential cellular pathways that may be modified as a result of protein changes identified in our experiments, the Ingenuity Pathways Analysis (IPA) application (Ingenuity Systems, Silicon Valley, CA) was used as previously described (17.Wishart T.M. Rooney T.M. Lamont D.J. Wright A.K. Morton A.J. Jackson M. Freeman M.R. Gillingwater T.H. 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European red deer (Cervus elaphus elaphus) are susceptible to the agent of bovine spongiform encephalopathy, one of the transmissible spongiform encephalopathies, when challenged intracerebrally but their susceptibility to alimentary challenge, the presumed natural route of transmission, is unknown. To determine this, eighteen deer were challenged via stomach tube with a large dose of the bovine spongiform encephalopathy agent and clinical signs, gross and histological lesions, presence and distribution of abnormal prion protein and the attack rate recorded. Only a single animal developed clinical disease, and this was acute with both neurological and respiratory signs, at 1726 days post challenge although there was significant (27.6%) weight loss in the preceding 141 days. The clinically affected animal had histological lesions of vacuolation in the neuronal perikaryon and neuropil, typical of transmissible spongiform encephalopathies. Abnormal prion protein, the diagnostic marker of transmissible encephalopathies, was primarily restricted to the central and peripheral nervous systems although a very small amount was present in tingible body macrophages in the lymphoid patches of the caecum and colon. Serial protein misfolding cyclical amplification, an in vitro ultra-sensitive diagnostic technique, was positive for neurological tissue from the single clinically diseased deer. All other alimentary challenged deer failed to develop clinical disease and were negative for all other investigations. These findings show that transmission of bovine spongiform encephalopathy to European red deer via the alimentary route is possible but the transmission rate is low. Additionally, when deer carcases are subjected to the same regulations that ruminants in Europe with respect to the removal of specified offal from the human food chain, the zoonotic risk of bovine spongiform encephalopathy, the cause of variant Creutzfeldt-Jakob disease, from consumption of venison is probably very low.
CLN1 disease, also called infantile neuronal ceroid lipofuscinosis (NCL) or infantile Batten disease, is a fatal neurodegenerative lysosomal storage disorder resulting from mutations in the CLN1 gene encoding the soluble lysosomal enzyme palmitoyl-protein thioesterase 1 (PPT1). Therapies for CLN1 disease have proven challenging because of the aggressive disease course and the need to treat widespread areas of the brain and spinal cord. Indeed, gene therapy has proven less effective for CLN1 disease than for other similar lysosomal enzyme deficiencies. We therefore tested the efficacy of enzyme replacement therapy (ERT) by administering monthly infusions of recombinant human PPT1 (rhPPT1) to PPT1-deficient mice (Cln1–/–) and CLN1R151X sheep to assess how to potentially scale up for translation. In Cln1–/– mice, intracerebrovascular (i.c.v.) rhPPT1 delivery was the most effective route of administration, resulting in therapeutically relevant CNS levels of PPT1 activity. rhPPT1-treated mice had improved motor function, reduced disease-associated pathology, and diminished neuronal loss. In CLN1R151X sheep, i.c.v. infusions resulted in widespread rhPPT1 distribution and positive treatment effects measured by quantitative structural MRI and neuropathology. This study demonstrates the feasibility and therapeutic efficacy of i.c.v. rhPPT1 ERT. These findings represent a key step toward clinical testing of ERT in children with CLN1 disease and highlight the importance of a cross-species approach to developing a successful treatment strategy.
Abstract Airway inflammation is highly prevalent in horses, with the majority of non-infectious cases being defined as equine asthma. Currently, cytological analysis of airway derived samples is the principal method of assessing lower airway inflammation. Samples can be obtained by tracheal wash (TW) or by lavage of the lower respiratory tract (bronchoalveolar lavage (BAL) fluid; BALF). Although BALF cytology carries significant diagnostic advantages over TW cytology for the diagnosis of equine asthma, sample acquisition is invasive, making it prohibitive for routine and sequential screening of airway health. However, recent technological advances in sample collection and processing have made it possible to determine whether a wider range of analyses might be applied to TW samples. Considering that TW samples are relatively simple to collect, minimally invasive and readily available in the horse, it was considered appropriate to investigate whether, equine tracheal secretions represent a rich source of cells and both transcriptomic and proteomic data. Similar approaches have already been applied to a comparable sample set in humans; namely, induced sputum. Sputum represents a readily available source of airway biofluids enriched in proteins, changes in the expression of which may reveal novel mechanisms in the pathogenesis of respiratory diseases, such as asthma and chronic obstructive pulmonary disease. The aim of this study was to establish a robust protocol to isolate macrophages, protein and RNA for molecular characterization of TW samples and demonstrate the applicability of sample handling to rodent and human pediatric bronchoalveolar lavage fluid isolates. TW samples provided a good quality and yield of both RNA and protein for downstream transcriptomic/proteomic analyses. The sample handling methodologies were successfully applicable to BALF for rodent and human research. TW samples represent a rich source of airway cells, and molecular analysis to facilitate and study airway inflammation, based on both transcriptomic and proteomic analysis. This study provides a necessary methodological platform for future transcriptomic and/or proteomic studies on equine lower respiratory tract secretions and BALF samples from humans and mice.
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