To date, there have been few reports of discovery-mode LC-MS/MS analysis of the membrane subproteome in neurodegenerative disease. This study reports changes in the membrane enriched proteome in AD. After statistical analysis of the spectral count data obtained by LC-MS/MS, 13 proteins were found significantly altered in AD. Of these proteins, tau, UCHL1, Munc-18, and A2M were independently validated by immunoblot. Confirming targets that have previously been shown to have an association with AD demonstrates that the membrane enrichment method used in this study indeed does enable sensitive discovery of proteins involved in disease pathogenesis that may be relevant as biomarkers in this or other contexts.
While the membrane enrichment strategy utilized did not produce a pure membrane sample, it did successfully enrich for membrane proteins. The percentage of proteins containing a TMD in this study was somewhat lower than the result reported in a previously published study using a similar method for membrane enrichment from cells in culture. In that study, approximately 50% of the proteins identified by LC-MS/MS had at least one TMD [42
]. The lower percentage observed in brain tissue in this study is likely due in part to the relatively high amount of extracellular proteins such as myelin, versican, tenascin as well as structural proteins, that are associated with tissue rather than cells in culture.
The goal of this study was to examine a comprehensive membrane fraction of human brain tissue. For that reason, a crude membrane enrichment strategy was employed to fractionate samples to ensure that as many membranes as possible (i.e., mitochondrial membranes, plasma membranes, and vesicular membranes) were included in the final membrane enriched fraction. By doing this, it is possible that some of the purity of the sample was sacrificed. Non-transmembrane domain containing proteins, such as tubulin and actin, were still highly abundant in the membrane fraction and there are known points of contact between cytoskeleton and membranes. Nonetheless, a more stringent fractionation procedure would likely reduce the number of cytoskeletal and other non-membrane bound proteins in the membrane sample.
Tau is a microtubule associated protein important for stabilizing the neuronal cytoskeleton. In addition to binding microtubules, it also interacts with the plasma membrane [43
]. This interaction appears to be mediated by phosphorylation; it was recently demonstrated that hyperphosphorylated tau loses its ability to bind the plasma membrane [44
]. This may account for the intracellular accumulation of hyperphospohrylated tau observed in AD. Moreover, mislocalized tau has been shown to mediate Aβ toxicity through increased excitotoxicty [45
]. Despite these findings, this study found that tau was enriched in the membrane fraction of AD cases. However, this study focused on total tau rather than hyperphosphorylated tau which may account for the difference. All of the AD cases in this study were Braak stage V or VI and therefore had high levels of pathological tau. It is possible that neurofibrillary tangles were co-purified with the membrane fraction of our sample, accounting for the increased signal seen with immunoblotting. While tau binds the plasma membrane, it does not contain a transmembrane domain. Therefore it is possible that any membrane-associated tau in the control cases was removed in the high pH wash step. There may be other modifications to tau not contained in neurofibrillary tangles in Alzheimer’s disease that make it more tightly associated with the membrane.
UCHL1 is a deubiquinating enzyme that is found primarily in neurons and neuroendocrine cells. It composes 1-2% of the soluble protein in neurons [46
]. While it plays an important role in ubiquitin recycling, it has also been shown to have ubiquitin ligase activity [47
]. Recently, it was reported that UCHL1 is an important regulator of survival of motor neuron (SMN) expression via ubiquitination in fibroblasts from patients with spinal muscular atrophy [48
]. Increased association of UCHL1 with the membrane fraction due to farnesylation has also been reported in Parkinson’s disease and was shown to mediate alpha-synuclein toxicity in cells [49
]. While certain polymorphisms in UCHL1 are reported to be protective in Parkinson’s disease, this protective effect is not seen in AD [50
]. Given that UCHL1 appears to play a role in many neurodegenerative diseases, it is likely not specific to AD; however, that does not negate the fact that it may also play an important role in AD pathogenesis.
Munc-18 is involved in vesicle docking and exocytosis. It is found exclusively in presynaptic nerve terminals and binds to syntaxin-1, preventing it from interacting with docking fusion proteins [51
]. Phosphorylation of Munc-18 by cyclin dependent kinase (Cdk) 5 causes Munc-18 and syntaxin-1 to dissociate and allows vesicular docking and neurotransmitter release to proceed. Phosphorylation of Munc-18 also enables Munc interacting (Mint) proteins 1 and 2 to bind Munc-18 [51
]. These complexes have been postulated to increase neurotransmission and may play a role in APP processing and Aβ production. Up-regulation of this pathway has been reported in the cortex of patients with Alzheimer’s disease [37
A2M has been implicated in AD pathogenesis due to its role in mediating Aβ clearance [52
]. Several studies have also linked polymorphisms in the A2M gene to an increased risk of developing late-onset AD [38
]. Lower levels of A2M could lead to in an inability to clear Aβ efficiently, thereby contributing to Aβ deposition in plaques. A2M was significantly depleted in AD by spectral counts (p
= 0.0001); however, after immunoblotting individual cases, it became apparent that this finding was being driven by two control cases with high levels of A2M. This result illustrates a pitfall of pooling samples for proteomic experiments. Although pooling decreases inter-individual variability, it becomes difficult to ascertain if differences between pooled samples are the result of a consistent change across cases or whether they are driven by one or two outliers in the pool. This underscores the importance of independently validating findings from these studies across individual cases and that there is a large amount of inherent variability when working with human samples.
While not independently validated by immunoblotting, levels of creatine kinase B (CKB) were also significantly higher in AD cases compared to controls by proteomics analysis. Creatine kinase is a protein found in the intermembrane-space in mitochondria. It utilizes ATP to generate phosphocreatine which can then be used as a source of energy in the cell [55
]. Lynn et al. recently published a study comparing the mitochondrial proteome in AD and control subjects [56
]. Along with other proteins involved in ATP utilization pathways, creatine kinase was found to be enriched in AD. The results from this study support the findings by Lynn et al. and may provide further evidence that ATP utilization is abnormal in patients with AD.
One limitation to this study is that despite improved detection sensitivity, analyzing complex mixtures following LC-MS/MS by spectral counting restricts peptide identification to only the most abundant peptides. The more complex a mixture, the less likely it is that low abundance peptides will be sequenced [57
] at levels high enough to reach significance. However, it is possible that changes in these proteins could have the largest biological impact. Spectral counting is not sensitive enough to detect these changes without using other methods, such as analysis of extracted peptide ion intensity. Spectral counting and G-test analysis do not take peptide signal intensity or protein coverage into consideration when determining which proteins are significantly changing. While this study successfully identified proteins with altered levels in AD, future studies using either a labeled quantitative approach or augmenting spectral count analysis with other label-free quantification methods could be used to identify less abundant proteins changing in disease. Finally, it can be difficult when analyzing post-mortem brain tissue from patients with end stage neurodegenerative diseases to dissect which changes are due to the disease process and which are a non-specific effect of neurodegeneration. In future studies, it would be important to compare these results to those from a disease control to determine whether these findings are specific to AD or represent proteins that are ubiquitously altered in neurodegeneration. Furthermore, the AD cases used in this study were all diagnosed with AD before age 65. While none of the cases had a known familial mutation causing their disease (i.e.
APP (amyloid precursor protein) or presenilin 1 or 2 mutations), it is possible that these cases differ from the more common late-onset AD (diagnosed after age 65). Therefore, it would be important to verify these findings in more representative AD samples over a broader age range.
In conclusion, studying a membrane enriched fraction has several advantages for identifying proteins altered in AD. While the accumulation of Aβ certainly plays a large role in AD pathogenesis, there are other mechanisms, including abnormal vesicular trafficking and synaptic dysfunction that may also contribute to neurodegeneration [10
]. The small sample size in this study limits the generalization of these results and requires that the findings be confirmed with much larger samples. However, it provides support for using a membrane enriched proteome to identify biomarkers in AD. Future analysis of the membrane proteome from post-mortem brain tissue in any number of neurodegenerative diseases may offer mechanistic insights or potential new biomarkers that will aid in the diagnosis of these debilitating diseases.