Identification and investigation of gamma-secretase associated proteins from brain

Abstract

Alzheimer disease (AD) is a progressive neurodegenerative disorder and the most common cause of dementia. The pathological hallmarks in the AD brain are extracellular deposition of amyloid plaques, mainly composed of the amyloid β-peptide (Aβ), and intracellular neurofibrillary tangles made of hyperphosphorylated tau. Several studies have shown that Aβ aggregation provides the initial insult, and the formation of tangles seems to be a secondary effect. Aβ is generated from the amyloid precursor protein (APP) by cleaving, which is sequentially mediated by β-secretase and γ-secretase. γ-Secretase is a transmembrane protease complex responsible for the processing of a multitude of type 1 transmembrane proteins such as APP and Notch. In clinical trials, treatment with γ-secretase inhibitors often results in Notch-related side effects and, thus, more specific inhibition of APP processing is necessary. Four proteins, presenilin, nicastrin, Aph-1 and Pen-2, are necessary and sufficient to produce an active γ-secretase complex. It has been suggested that γ-secretase associated proteins (GSAPs) could be of importance for substrate selection. Here, we have established an affinity purification method using a γ-secretase inhibitor derivative to isolate the native complex from brain material. We have identified several novel GSAPs from brain and studied their effect on Aβ production and Notch processing.

In Paper I, we designed an efficient and selective method for purification and analysis of γ-secretase and GSAPs. Microsomal membranes were incubated with a γ-secretase inhibitor coupled to biotin via a long linker and an S-S bridge (GCB). After pull-down using streptavidin beads, bound proteins were eluted under reducing conditions and digested by trypsin. The tryptic peptides were subjected to liquid chromatography directly coupled to tandem mass spectrometry analysis, and proteins were identified by sequence data from an MS/MS spectra. All of the known γ-secretase components were identified, as well as the previously reported GSAP TMP21 and the PS-associated protein, syntaxin1. Hence, we suggest that the present method can be used to further study the composition of the γ-secretase complex. In Paper II, we investigated novel GSAPs from detergent-resistant membranes (DRMs). Recent studies showed that γ-secretase activity is highly enriched in DRMs. Thus, GSAPs localized to DRMs could be of special interest to study. We employed GCB and identified several novel GSAPs in DRMs from brain. From these identified proteins, silencing of voltage-dependent anion channel 1 (VDAC1) and contactin-associated protein 1 (CNTNAP1) reduced Aβ production. These proteins had a less pronounced effect on Notch processing. We concluded that VDAC1 and CNTNAP1 associate with γ-secretase in DRMs and affect APP processing. In Paper III, we investigated novel GSAPs from synaptic membranes and synaptic vesicles prepared from rat brain. Synaptic degeneration is one of the earliest Indicators of AD and results in loss of cognitive function. We employed GCB and identified several novel GSAPs in synaptic membranes and synaptic vesicles from brain. From these identified proteins, silencing of NADH dehydrogenase [ubiquinone] iron–sulfur protein 7 (NDUFS7) resulted in a decrease in Aβ levels, whereas, silencing of tubulin polymerization promoting protein (TPPP) resulted in an increase in Aβ levels. These proteins had no effect on Notch processing. Association of TPPP and NDUFS7 with γ-secretase was verified using co-immunoprecipitation and Proximity Ligation Assay. In Paper IV, we conduct a large scale affinity purification study of the γ-secretase complex obtained from rat and human brain. Silencing of some of the identified proteins: Probable phospholipid-transporting ATPase IIA (ATP9A), BDNF/NT-3 growth factors receptor precursor (NTRK2), Potassium/sodium hyperpolarization-activated cyclic nucleotide-gated channel 2 (HCN2), DnaJ homolog subfamily A member 2 (DNAJA2) and Proton myo-inositol cotransporter (SLC2A13) reduced Aβ42 secretion in a siRNA dose-dependent manner. Two of these proteins, SLC2A13 and HCN2, had a relatively lower effect on Notch processing. Interestingly, overexpression of SLC2A13 increased Aβ40 generation. The interaction between γ-secretase and ATP9A, NTRK2, HCN2 and SLC2A13 was confirmed by using immunoprecipitation.

In summary, we have established an affinity purification method to isolate the native γ-secretase complex from brain material, and we have identified several novel GSAPs that affect Aβ processing without affecting Notch cleavage. We suggest that these proteins could be the targets in a strategy to lower Aβ to treat AD.