Abstract
The purpose of this study was to develop ELISAs for key neural proteins, three synaptic and one glial, that exist in different intracellular compartments, which would be used as a measure of synaptic phenotype. These assays would be valuable to neurologically phenotype transgenic mouse models of human disease and also human disease itself using minimal amounts of post-mortem tissue. We showed that supernatant from crude brain tissue homogenates extracted in RIPA buffer containing 0.1% SDS bind to synaptophysin, synaptosome-associated protein of 25 kDa (SNAP-25), post-synaptic density-95 (PSD-95), and glial fibrillary acidic protein (GFAP) antibody pairs with high affinity and selectivity. Overall, RIPA + 0.1% SDS were more efficient than RIPA + 2% SDS or a buffer containing only 1% Triton-X-100. Diluting the brain extracts resulted in dose-dependent binding to the antibody pairs for each neural protein, with EC50s that varied from 8.6 µg protein for PSD-95 to 0.23 µg for GFAP. The assays were used to measure synaptic marker protein levels at various times during mouse development and GFAP in a model of disease accompanied by neuroinflammation. Comparison of ELISAs with Western blots by measuring marker levels in brain extract from developing mice showed a greater relative difference in values derived from ELISA. These ELISAs should be valuable to phenotype the synapse in neurological disease and their rodent models.
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References
Ansari MA, Roberts KN, Scheff SW (2008) Oxidative stress and modification of synaptic proteins in hippocampus after traumatic brain injury. Free Radic Biol Med
Aya-ay J, Mayer J, Eakin AK, Muffly BG, Anello M, Sandy JD, Gottschall PE (2005) The effect of hypoxic-ischemic brain injury in perinatal rats on the abundance and proteolysis of brevican and NG2. Exp Neurol 193:149–162
Calhoun ME, Jucker M, Martin LJ, Thinakaran G, Price DL, Mouton PR (1996) Comparative evaluation of synaptophysin-based methods for quantification of synapses. J Neurocytol 25:821–828
Counts SE, Nadeem M, Lad SP, Wuu J, Mufson EJ (2006) Differential expression of synaptic proteins in the frontal and temporal cortex of elderly subjects with mild cognitive impairment. J Neuropathol Exp Neurol 65:592–601
DeKosky ST, Scheff SW (1990) Synapse loss in frontal cortex biopsies in Alzheimer’s disease: correlation with cognitive severity. Ann Neurol 27:457–464
Dong H, Martin MV, Chambers S, Csernansky JG (2007) Spatial relationship between synapse loss and beta-amyloid deposition in Tg2576 mice. J Comp Neurol 500:311–321
Fonseca MI, Zhou J, Botto M, Tenner AJ (2004) Absence of C1q leads to less neuropathology in transgenic mouse models of Alzheimer’s disease. J Neurosci 24:6457–6465
Glantz LA, Gilmore JH, Hamer RM, Lieberman JA, Jarskog LF (2007) Synaptophysin and postsynaptic density protein 95 in the human prefrontal cortex from mid-gestation into early adulthood. Neuroscience 149:582–591
Holcomb L, Gordon MN, McGowan E, Yu X, Benkovic S, Jantzen P, Wright K, Saad I, Mueller R, Morgan D, Sanders S, Zehr C, O’Campo K, Hardy J, Prada CM, Eckman C, Younkin S, Hsiao K, Duff K (1998) Accelerated Alzheimer-type phenotype in transgenic mice carrying both mutant amyloid precursor protein and presenilin 1 transgenes. Nat Med 4:97–100
Honer WG, Falkai P, Bayer TA, Xie J, Hu L, Li HY, Arango V, Mann JJ, Dwork AJ, Trimble WS (2002) Abnormalities of SNARE mechanism proteins in anterior frontal cortex in severe mental illness. Cereb Cortex 12:349–356
Hsiao K, Chapman P, Nilsen S, Eckman C, Harigaya Y, Younkin S, Yang F, Cole G (1996) Correlative memory deficits, Abeta elevation, and amyloid plaques in transgenic mice. Science 274:99–102
Jacobsen JS, Wu CC, Redwine JM, Comery TA, Arias R, Bowlby M, Martone R, Morrison JH, Pangalos MN, Reinhart PH, Bloom FE (2006) Early-onset behavioral and synaptic deficits in a mouse model of Alzheimer’s disease. Proc Natl Acad Sci USA 103:5161–5166
Kim E, Sheng M (2004) PDZ domain proteins of synapses. Nat Rev Neurosci 5:771–781
King DL, Arendash GW (2002) Maintained synaptophysin immunoreactivity in Tg2576 transgenic mice during aging: correlations with cognitive impairment. Brain Res 926:58–68
Knott GW, Holtmaat A, Wilbrecht L, Welker E, Svoboda K (2006) Spine growth precedes synapse formation in the adult neocortex in vivo. Nat Neurosci 9:1117–1124
Mayer J, Hamel MG, Gottschall PE (2005) Evidence for proteolytic cleavage of brevican by the ADAMTSs in the dentate gyrus after excitotoxic lesion of the mouse entorhinal cortex. BMC Neurosci 6:52
Mokin M, Keifer J (2006) Quantitative analysis of immunofluorescent punctate staining of synaptically localized proteins using confocal microscopy and stereology. J Neurosci Methods 157:218–224
Nithianantharajah J, Levis H, Murphy M (2004) Environmental enrichment results in cortical and subcortical changes in levels of synaptophysin and PSD-95 proteins. Neurobiol Learn Mem 81:200–210
O’Callaghan JP, Imai H, Miller DB, Minter A (1999) Quantitative immunoblots of proteins resolved from brain homogenates: underestimation of specific protein concentration and of treatment effects. Anal Biochem 274:18–26
Pfeiffer BE, Huber KM (2007) Fragile X mental retardation protein induces synapse loss through acute postsynaptic translational regulation. J Neurosci 27:3120–3130
Porchet R, Probst A, Bouras C, Draberova E, Draber P, Riederer BM (2003) Analysis of glial acidic fibrillary protein in the human entorhinal cortex during aging and in Alzheimer’s disease. Proteomics 3:1476–1485
Ross GW, O’Callaghan JP, Sharp DS, Petrovitch H, Miller DB, Abbott RD, Nelson J, Launer LJ, Foley DJ, Burchfiel CM, Hardman J, White LR (2003) Quantification of regional glial fibrillary acidic protein levels in Alzheimer’s disease. Acta Neurol Scand 107:318–323
Sans N, Petralia RS, Wang YX, Blahos J 2nd, Hell JW, Wenthold RJ (2000) A developmental change in NMDA receptor-associated proteins at hippocampal synapses. J Neurosci 20:1260–1271
Scheff SW, Price DA, Hicks RR, Baldwin SA, Robinson S, Brackney C (2005) Synaptogenesis in the hippocampal CA1 field following traumatic brain injury. J Neurotrauma 22:719–732
Scheff SW, Price DA, Schmitt FA, Mufson EJ (2006) Hippocampal synaptic loss in early Alzheimer’s disease and mild cognitive impairment. Neurobiol Aging 27:1372–1384
Scheff SW, Price DA, Schmitt FA, DeKosky ST, Mufson EJ (2007) Synaptic alterations in CA1 in mild Alzheimer disease and mild cognitive impairment. Neurology 68:1501–1508
Schlaf G, Salje C, Poethke R, Felgenhauer K, Mader M (1996) A novel enzyme-linked immunosorbent assay for determination of synaptophysin as compared with other quantification procedures. J Neuroimmunol 67:59–65
Schmidt GR, Hossner KL, Yemm RS, Gould DH, O’Callaghan JP (1999) An enzyme-linked immunosorbent assay for glial fibrillary acidic protein as an indicator of the presence of brain or spinal cord in meat. J Food Prot 62:394–397
Siew LK, Love S, Dawbarn D, Wilcock GK, Allen SJ (2004) Measurement of pre- and post-synaptic proteins in cerebral cortex: effects of post-mortem delay. J Neurosci Methods 139:153–159
Smith R, Klein P, Koc-Schmitz Y, Waldvogel HJ, Faull RL, Brundin P, Plomann M, Li JY (2007) Loss of SNAP-25 and rabphilin 3a in sensory-motor cortex in Huntington’s disease. J Neurochem 103:115–123
Sorensen JB (2005) SNARE complexes prepare for membrane fusion. Trends Neurosci 28:453–455
Sudhof TC, Lottspeich F, Greengard P, Mehl E, Jahn R (1987) A synaptic vesicle protein with a novel cytoplasmic domain and four transmembrane regions. Science 238:1142–1144
Tabuchi K, Blundell J, Etherton MR, Hammer RE, Liu X, Powell CM, Sudhof TC (2007) A neuroligin-3 mutation implicated in autism increases inhibitory synaptic transmission in mice. Science 318:71–76
Tanzi RE (2005) The synaptic Abeta hypothesis of Alzheimer disease. Nat Neurosci 8:977–979
Terry RD (2000) Cell death or synaptic loss in Alzheimer disease. J Neuropathol Exp Neurol 59:1118–1119
Tsai J, Grutzendler J, Duff K, Gan WB (2004) Fibrillar amyloid deposition leads to local synaptic abnormalities and breakage of neuronal branches. Nat Neurosci 7:1181–1183
Acknowledgments
This work was supported in part by the National Institutes of Health (AG022101) and Alzheimer’s Association (Grant no. IIRG-02-3758). The authors would like to thank Christopher C. Leonardo and Yun Bai for their assistance.
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Gottschall, P.E., Ajmo, J.M., Eakin, A.K. et al. Panel of synaptic protein ELISAs for evaluating neurological phenotype. Exp Brain Res 201, 885–893 (2010). https://doi.org/10.1007/s00221-010-2182-x
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DOI: https://doi.org/10.1007/s00221-010-2182-x