Proteomic Profiling of Cranial (Superior) Cervical Ganglia Reveals Beta-Amyloid and Ubiquitin Proteasome System Perturbations in an Equine Multiple System Neuropathy

Abstract

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. and equine grass Med. Scopus Google Scholar, I.R. The of Clostridium botulinum type C with equine grass A Vet. J. 1999; PubMed Scopus Google Scholar). that the lesion in EGS the glycoprotein biosynthetic pathway of specific neurons the and consistently while including I.R. F. and study of neuronal lesions in autonomic of horses with grass sickness.Equine Vet. J. 25: PubMed Scopus Google Scholar). while the ultrastructural and cellular of affected neurons is the molecular that to The of this study was therefore to identify the molecular processes underpinning neurodegeneration in EGS 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 sympathetic to the and was selected chromatolysis of a of CCG neurons is a of EGS and in equine dysautonomia Pathol. PubMed Scopus (44) Google Scholar)). Here, proteomic analysis was for relative and well in small animal models of human neurodegenerative but not in large animal models or large animal conditions. proteomic analysis was with quantitative fluorescent Western blotting immunohistochemistry and in silico techniques in an to identify the molecular pathways and processes that may be to neurodegeneration in EGS. Here, we in the CCG of EGS including to a of pathways candidates commonly associated with protein misfolding/aggregation responses in human neurodegenerative conditions. study therefore 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 is also the first to and conservation spanning the degenerative molecular cascades from an apparently unrelated condition of large animals to small animal models with altered neuronal and a of human neurological from neurodegenerative as spinal muscular to diseases associated with as this study highlights the feasibility and benefits of applying techniques to the of the neurodegenerative processes in diseases of large animals. In with of and potential be in the is from horses that with the The study was by the and quantitative fluorescent Western CCG from EGS and control and horses of by of CCG selected chromatolysis of a of CCG neurons is a of EGS in equine dysautonomia Pathol. PubMed Scopus (44) Google Scholar). The in and of EGS horses in the study the of horses affected by this neurodegenerative disease the study EGS horses and as by and J. Equine dysautonomia is associated with altered and of Vet. J. 2001; PubMed Scopus Google Scholar). The of and cases for proteomic analysis was as in clinical and of The is the of disease following of the from patients and the and cellular of we that neuronal not and cases to and the that the remains molecular as an to due to as disease and EGS was in cases by including examination of autonomic equine dysautonomia and autonomic Vet. PubMed Scopus Google Scholar). for than neurological CCG by in and CCG from EGS and control and horses as and in and in the by and a with a in with for or and for proteomic by condition and in a in a for with for of for being for The proteins in this was and the was to a to be for as previously and molecular of and and in PubMed Scopus (47) Google Scholar, C.A. J. in a model of spinal muscular PubMed Scopus Google Scholar, C.A. D. of in the nervous PubMed Scopus Google Scholar, C.A. M.M. A. of ubiquitin and signaling spinal muscular 2014; PubMed Scopus Google Scholar). was in and and for The proteomic have to the the with the with and The for with an to The in and of The to for to protein in by in to from the and as previously and molecular of and and in PubMed Scopus (47) Google Scholar, C.A. J. in a model of spinal muscular PubMed Scopus Google Scholar, C.A. D. of in the nervous PubMed Scopus Google Scholar, C.A. M.M. A. of ubiquitin and signaling spinal muscular 2014; PubMed Scopus Google Scholar). to was the to with the a and in of as in the The according to the by the The and and in and a The was a and by a A a The following system was in 20% and in in 20% was to with a from to a from to from the was a and of the in of to and an system to a The a and a from a a of The as was the precursor for by the precursor was and the was to control for and and The for and and The for and for by the and the the of for was as the by for an of the for and protein by and of altered expression for selected proteins was by quantitative fluorescent Western blotting CCG protein and by immunohistochemistry CCG for a of and with equine was as previously protein analysis as a control for quantitative fluorescent Western PubMed Scopus Google Scholar, A to modern quantitative fluorescent blotting with 2014; Google Scholar). of CCG protein by and to an The and with primary according to according to and an of the from to and was with the analysis protein in with for to and the as previously in protein analysis as a control for quantitative fluorescent Western PubMed Scopus Google Scholar, A to modern quantitative fluorescent blotting with 2014; Google Scholar). and was by in for in a The to for A was according to the with and with for in and with in in for primary for controls. and with for and with for in with in and The of of CCG neurons was for and ubiquitin was with in for following in the neuronal was as and was following a and by with the and neuronal in by a with into potential cellular pathways that may be as a of protein identified in the application was as previously and molecular of and and in PubMed Scopus (47) Google Scholar, C.A. J. in a model of spinal muscular PubMed Scopus Google Scholar, C.A. D. of in the nervous PubMed Scopus Google Scholar, C.A. M.M. A. of ubiquitin and signaling spinal muscular 2014; PubMed Scopus Google analysis of proteins potential cellular and protein of by the PubMed Scopus Google Scholar). analysis to and of disease in A approach to clinical in neurodegenerative Med. 2014; PubMed Scopus Google Scholar). of small and disease the of in a than of the in this is and is from the of than of have identified by techniques as In the limited to but be from in the by limited in this study to a of the of according to a a a that into the or proteins of and the of the The the application to the is to the be that this not the or of the remains the of the Further the in be from into and was to be in the or as EGS-affected horses due to disease severity the first of clinical and this of examination commonly that the of CCG neurons neurodegeneration, and neuronal remains and and is to be a of of neurodegeneration from neurons in the of neurodegeneration to with and in equine dysautonomia Pathol. PubMed Scopus (44) Google Scholar)). an quantitative proteomic protein from EGS CCG and control and and for to for and due to the of the analysis of 2,311 proteins and to a of proteins altered in EGS CCG by including proteins identified by with previously to in a of 20% was as previously and molecular of and and in PubMed Scopus (47) Google Scholar, C.A. J. in a model of spinal muscular PubMed Scopus Google Scholar, C.A. D. of in the nervous PubMed Scopus Google Scholar, C.A. M.M. A. of ubiquitin and signaling spinal muscular 2014; PubMed Scopus Google Scholar, analysis of proteins potential cellular and protein of by the PubMed Scopus Google Scholar, The to the increased expression of proteins associated with and of neuronal 2014; PubMed Scopus (17) Google Scholar, C.A. and as protein for spinal muscular Med. 5: PubMed Scopus Google in the of 320 proteins that to have increased in EGS and 186 with decreased expression and therefore this of proteins to be of the molecular by EGS neurodegenerative processes in to the limited molecular investigations into EGS to the of proteins identified with altered expression not previously associated with this the of the proteomic candidates first selected for as of in the proteomic and potential to neurodegenerative disease from the of protein amyloid precursor protein and components of the ubiquitin proteasome system was of as a potential to of EGS. was selected for due with human neurodegenerative conditions, including but not limited to disease A. A. J. A. and in The potential of PubMed Scopus Google diseases A. of amyloid precursor protein for and PubMed Scopus Google neuropathology in and J. Pathol. PubMed Scopus Google neurodegenerative disease and as of an to neuronal A. amyloid protein in the for the of PubMed Scopus Google Scholar). expression of multiple components of the UPS was identified for as specific ubiquitin as ubiquitin and glycoprotein have previously reported in with altered and enteric in EGS D. Mayhew I.G. in in horses with grass sickness.Equine Vet. J. PubMed Scopus Google Scholar, J. of equine grass sickness sympathetic neurons in and Pathol. PubMed Scopus Google and of the UPS have in multiple human neurodegenerative conditions, including but not limited to spinal muscular and diseases C.A. M.M. A. of ubiquitin and signaling spinal muscular 2014; PubMed Scopus Google Scholar, J. A. and in associated with spinal muscular J. PubMed Scopus Google Scholar, R.C. R.C. J.W. of protein 2009; PubMed Scopus Google Scholar, disease the Med. 2004; 10: PubMed Scopus Google Scholar, and proteasome PubMed Scopus Google Scholar). selected candidates for we the of the proteomic by as previously and molecular of and and in PubMed Scopus (47) Google Scholar, C.A. J. in a model of spinal muscular PubMed Scopus Google Scholar, C.A. D. of in the nervous PubMed Scopus Google Scholar, C.A. M.M. A. of ubiquitin and signaling spinal muscular 2014; PubMed Scopus Google Scholar, protein analysis as a control for quantitative fluorescent Western PubMed Scopus Google Scholar, analysis of proteins potential cellular and protein of by the PubMed Scopus Google Scholar). protein we a protein analysis with an protein analysis as a control for quantitative fluorescent Western PubMed Scopus Google Scholar)). protein was than as altered in from neurodegenerative and the EGS CCG proteomic suggested that as a may be by by that and in the and to the by the proteomic Interestingly, it was to identify expression in and that not specific UPS components altered but also that ubiquitin in EGS Here, we an in ubiquitin and a in ubiquitin and therefore that the proteomic analysis is to be an of the molecular in EGS the of proteins associated with protein responses in the of a of neurodegenerative in we to the of proteomic analysis to candidates the cellular While by protein is a for relative protein expression in this it an of protein of or subcellular the cellular of the CCG and the cellular of the proteins selected for by we to expression the cellular Here, we with analysis of and analysis the examination of and from EGS and and control identified by proteomic analysis and by it appears from that the increased expression is predominantly of EGS CCG and is a protein of PubMed Scopus Google to cellular and in neurons A. precursor protein in of in and and with PubMed Google Scholar). we EGS for in cellular Interestingly, the is not but appears as with of the of the The and subcellular of that the in expression increased protein and/or of from the to the with previously reported in the and I.R. F. and study of neuronal lesions in autonomic of horses with grass sickness.Equine Vet. J. 25: PubMed Scopus Google Scholar). is a protein with examination by and an in ubiquitin to and in PubMed Scopus Google and a in was is with increased expression of proteins as of the In neurons, in an in ubiquitin and a in as in EGS ubiquitin CCG relative to have previously reported in to and in PubMed Scopus Google Scholar). of an increased of with and a in the of in EGS horses with controls. therefore to the of the proteomic of the molecular in EGS represent the first demonstration that the neurodegenerative processes in EGS some molecular in neurodegenerative conditions, including and models as the and/or of proteins as and the of the proteomic we to into potential molecular pathways and processes that may be by in silico analysis and application was to identify and molecular pathway and the and the proteins identified as altered in proteomic expressed proteins that to be eligible for in silico the clustered predominantly into the following biofunctions: (1) diseases and disorders, including neurological disease skeletal and muscular disorders and (2) molecular and cellular functions, including cellular assembly and cell-to-cell signaling and interaction including epinephrine, dopamine, and adrenergic signaling and receptor function), and small molecule Interestingly, examination of the diseases and disorders that to 20% of the candidates have previously reported in the as being associated with specific neurodegenerative including but not limited to and motor diseases the that molecular of neurodegenerative processes have we a of candidates identified as being expressed in EGS affected with control with from a model of a neurodegenerative condition C.A. J. in a model of spinal muscular PubMed Scopus Google for and a model of and molecular of and and in PubMed Scopus (47) Google for that candidates identified in EGS also altered in neurodegenerative processes of and processes of While this in is it that may be to the degenerative responses in and that may be responses to a neurodegenerative in and EGS in and that may be in in and of the have as of degenerative processes in disease C.A. M.M. A. of ubiquitin and signaling spinal muscular 2014; PubMed Scopus Google and of the to degenerative processes in disorders A. A.M. of and in models of 2009; PubMed Scopus Google Scholar). with an examination of cellular processes an with neuronal and that of the molecular processes in EGS may be with neurodegenerative and the of applying techniques to the of neurodegeneration in human and animal with of of of EGS protein highlights molecular with from with altered neuronal of the EGS proteomic and that from models of and the human neurodegenerative disease spinal muscular is an neurodegenerative condition and in this have previously in and molecular of and and in PubMed Scopus (47) Google Scholar). a in a The for is following a lesion in tissues signs of neurodegenerative The be in C.A. J. in a model of spinal muscular PubMed Scopus Google Scholar). of candidates and of candidates also in the EGS analysis with neurodegenerative processes the molecular of EGS Interestingly, the candidates that may to the degenerative responses in and that may be responses to a neurodegenerative in and EGS in and that may be in in and with the demonstration that the ubiquitin system and and in EGS and pathway analysis highlighted a that appears to represent a molecular system may be to EGS-induced neurodegenerative processes associated with cell-to-cell signaling and nervous system and and small molecule of candidates from In this is a with and a of the protein components or with than of from or In with the molecular and cellular in this study a of the and cellular of and and with a of human neurodegenerative and proteasome PubMed Scopus Google Scholar, of PubMed Scopus Google Scholar, A. precursor protein in of in and and with PubMed Google for the first as an as a potential for the processes in EGS. the tools to potential of the that may be in the system of A approach to clinical in neurodegenerative Med. 2014; PubMed Scopus Google Scholar). potential or with the expression in a specific of and may in the of the by of the potential highlighted by this analysis for a of have in human neurodegenerative in protein is a J. of neuronal in the of neuronal in the 2014; Scholar, A. diseases and the 2014; PubMed Scopus Google Scholar, disease of of neuronal and in the Pathol. 2014; PubMed Scopus Google Scholar, with to with the with a of 2014; Google Scholar, of protein in 2014; PubMed Scopus Google Scholar). is an potential of reported with a of human neurodegenerative conditions. for of also a potential in that neurodegeneration in and also decreased in EGS Here, we by that is in EGS but in the form is therefore that than protein previously as a potential of the disease in but this and protein was not in of CCG protein from EGS and control horses that is protein the in the analysis of the proteomic supports the and the that may be a protein in degenerative While of neurodegenerative diseases tissues by the represent a molecular of disease processes from horses with a multiple system neuropathy (EGS) in to controls. Importantly, of the cellular from analysis of a of EGS animals that of the identified to be in the of this the of disease CCG examination that CCG neurons a of of neurodegeneration and that the the of EGS CCG reflect the of proteins from neurons of proteins of neuronal as well as of the processes and the neurodegenerative of have identified The altered expression of proteins in the study may not for of the of EGS but also for the of a molecular of the that the from EGS CCG a protein from a or also and While analysis of in the it identified proteins in the of or to be of to the of EGS. is to that this not EGS being by a or remains that the of the may be to a may be or a may not the CCG of EGS in the EGS CCG multiple of the ubiquitin proteasome system Here, we identified ubiquitin expression including a of and and ubiquitin as have previously reported in with altered in D. Mayhew I.G. in in horses with grass sickness.Equine Vet. J. PubMed Scopus Google and autonomic J. of equine grass sickness sympathetic neurons in and Pathol. PubMed Scopus Google in EGS. is a of cellular in the of protein and including the of A. D. amyloid precursor protein and by of PubMed Scopus Google of the UPS have in human neurodegenerative conditions, including; and motor diseases C.A. M.M. A. of ubiquitin and signaling spinal muscular 2014; PubMed Scopus Google Scholar, J. A. and in associated with spinal muscular J. PubMed Scopus Google Scholar, R.C. R.C. J.W. of protein 2009; PubMed Scopus Google Scholar, disease the Med. 2004; 10: PubMed Scopus Google Scholar, and proteasome PubMed Scopus Google Scholar). pathway analysis of the in this we highlighted and molecular processes human neurodegenerative and this equine multiple system neuropathy Further of specific candidates of specific to the neurodegenerative and equine clinical from the of the be in the of the cellular processes identified as being altered in EGS was The expressed proteins associated with and also in presynaptic and proteins have also reported in with and disease J. receptor and by receptor PubMed Scopus Google Scholar, of the of A in the of and 2005; PubMed Scopus Google Scholar, C.A. dysregulation of proteins with PubMed Scopus Google Scholar, de but is not for and 2005; PubMed Scopus Google Scholar, J.W. A. M.J. F. of disease and Med. PubMed Scopus Google Scholar). is therefore that dysregulation of proteins to neurodegeneration in EGS. it appears that the proteins identified by analysis and proteins expression is in neurodegenerative diseases in or with in a analysis of the identified in EGS highlighted a of candidates associated with human neurodegenerative of by protein and/or and F. A. precursor protein as a molecular for amyloid neuronal in PubMed Scopus Google Scholar, J. in human for J. 5: Google Scholar, D. of and as Med. PubMed Scopus Google Scholar)). In with of altered and demonstration of and as a with of the of a therefore appears to be the of the in the is to be a of EGS with potential to and and the for neurodegenerative disease is the first application of modern proteomic tools and in silico techniques to equine neuronal tissues and/or to an inherent neurodegenerative disease of large animals (not a model of human it the of than protein in equine grass sickness cranial (superior) cervical of not previously with EGS. is also the first demonstration of and conservation for neurodegenerative molecular cascades spanning from an apparently unrelated large animal small animal models of altered neuronal vulnerability, and a of human neurological conditions. this study highlights the feasibility and benefits of applying techniques to the of the neurodegenerative processes in diseases of large animals. for and for with