Targeting Synaptic Pathology with a Novel Affinity Mass Spectrometry Approach

Abstract

We report a novel strategy for studying synaptic pathology by concurrently measuring levels of four SNARE complex proteins from individual brain tissue samples. This method combines affinity purification and mass spectrometry and can be applied directly for studies of SNARE complex proteins in multiple species or modified to target other key elements in neuronal function. We use the technique to demonstrate altered levels of presynaptic proteins in Alzheimer disease patients and prion-infected mice. Molecular & Cellular Proteomics 13: 10.1074/mcp. M114.040113, 2584-2592, 2014.One prominent pathological feature of neuropsychiatric disorders such as Alzheimer disease (AD) 1 is severe synaptic loss (1-3). Previous reports of AD patients have shown that presynaptic dysfunction might occur early in the disease process (1, 4). Cortical synapse pathology has also been shown to correlate to the severity of dementia more closely than other pathological hallmarks of AD such as plaques and neurofibrillary tangles (5, 6). The SNARE proteins are essential components for the regulation of neurotransmitter exocytosis at the presynaptic site (7). Animal models suggest that changed expression or modification of SNARE complex proteins (synaptosomal-associated protein 25 (SNAP-25), syntaxin-1, and vesicle-associated membrane protein (VAMP)) alters synaptic function and is an interesting target for the development of therapeutics for neuropsychiatric illness (8, 9). The constituents of the SNARE complex are either localized in synaptic vesicles (VAMPs) or anchored at the presynaptic plasma membrane (SNAP-25 and syntaxin). The SNARE proteins are tightly assembled, and subsequent neurotransmitter release of the complex is quickly dissociated by N-ethylmaleimide-sensitive factor (7, 10 -12). Because they are both strongly associated into complexes and membrane associated, the SNARE proteins are difficult to analyze via mass spectrometry, which is incompatible with most detergents necessary for the solubilization of proteins. Each SNARE complex protein exists in several isoforms that are differently distributed within the central nervous system (13-18). Posttranslational modifications and truncated variants of the SNARE proteins make investigation of the protein expression even more complicated.In this study we developed an approach for the characterization and concurrent quantification of SNARE complex proteins that combines affinity purification by immunoprecipitation and mass spectrometry (IP-MS). We used precipitation with monoclonal antibodies against SNAP-25 to target the SNARE complex proteins and nanoflow LC-tandem mass spectrometry (LC-MS/MS) to characterize the co-immunoprecipitated interaction partners. Selected reaction monitoring (SRM) on a triple quadrupole mass spectrometer coupled to a microflow LC system was used for quantification of the SNARE proteins. To demonstrate the usability of the IP-MS method, we performed a comparison of SNARE complex protein levels in brain tissue from AD patients and agematched controls, a...

Distinctive alteration of presynaptic proteins in the outer molecular layer of the dentate gyrus in Alzheimer’s disease

“…A possible explanation for this finding could be that STX1A levels undergo a compensatory increase in these regions (possibly via increased collateral branching or sprouting) in order to compensate for the reduced input that dentate granule cells receive from the EC layer II, which is evident by the reduced STX1A level in the OML. As opposed to our findings, in a few proteomics study, decreased levels of STX1A were reported in AD brains [7, 32, 58]. However, these studies have used homogenates prepared from bulk tissue of AD brains, and would not be able to tease out cell- and layer-specific discrete changes as the ones observed in sub-hippocampal regions in the current study.…”

“…Although decreased mRNA and/or protein levels of these five presynaptic markers have been previously detected in AD brain [4, 7, 32, 51], these studies have so far been performed on homogenates prepared from cortical or hippocampal bulk tissue, thus not allowing any detailed examination of different hippocampal sub-regions. Here, we investigated the distribution of presynaptic proteins in 10 hippocampal sub-regions of AD and control cases using immunofluorescence.…”

A Parallel Reaction Monitoring Mass Spectrometric Method for Analysis of Potential CSF Biomarkers for Alzheimer's Disease

“…Moreover, differential diagnosis between AD and other forms of dementia is frequently difficult. Early loss of synaptic function is believed to play an important role in AD and recently the CSF levels of peptide products from both presynaptic and postsynaptic proteins have been shown to be altered in AD. Moreover, inflammatory processes and oxidative stress may be involved in AD pathogenesis.…”

Targeting LAMP2 in human cerebrospinal fluid with a combination of immunopurification and high resolution parallel reaction monitoring mass spectrometry

“…In this report a MS and MS/MS resolution setting of 70,000, at 200 m / z , was used. Recently we have developed a similar PRM method targeting synaptic pathology by measuring the relative amount of the SNARE complex protein synaptosomal-associated protein 25 (SNAP-25) in human CSF and brain tissue [ 35 , 36 ]. This highlights the potential of PRM methods in neurobiological biomarker discovery and development.…”