The Proteome of the Dentate Terminal Zone of the Perforant Path Indicates Presynaptic Impairment in Alzheimer Disease.

Synaptic dysfunction is an early pathogenic event in Alzheimer disease (AD) that contributes to network disturbances and cognitive decline. Some synapses are more vulnerable than others, including the synapses of the perforant path, which provides the main excitatory input to the hippocampus. To elucidate the molecular mechanisms underlying the dysfunction of these synapses, we performed an explorative proteomic study of the dentate terminal zone of the perforant path. The outer two-thirds of the molecular layer of the dentate gyrus, where the perforant path synapses are located, was microdissected from five subjects with AD and five controls. The microdissected tissues were dissolved and digested by trypsin. Peptides from each sample were labeled with different isobaric tags, pooled together and pre-fractionated into 72 fractions by high-resolution isoelectric focusing. Each fraction was then analyzed by liquid chromatography-mass spectrometry. We quantified the relative expression levels of 7322 proteins, whereof 724 showed significantly altered levels in AD. Our comprehensive data analysis using enrichment and pathway analyses strongly indicated that presynaptic signaling, such as exocytosis and synaptic vesicle cycle processes, is severely disturbed in this area in AD, whereas postsynaptic proteins remained unchanged. Among the significantly altered proteins, we selected three of the most downregulated synaptic proteins; complexin-1, complexin-2 and synaptogyrin-1, for further validation, using a new cohort consisting of six AD and eight control cases. Semi-quantitative analysis of immunohistochemical staining confirmed decreased levels of complexin-1, complexin-2 and synaptogyrin-1 in the outer two-thirds of the molecular layer of the dentate gyrus in AD. Our in-depth proteomic analysis provides extensive knowledge on the potential molecular mechanism underlying synaptic dysfunction related to AD and supports that presynaptic alterations are more important than postsynaptic changes in early stages of the disease. The specific synaptic proteins identified could potentially be targeted to halt synaptic dysfunction in AD.

Endogenous APP accumulates in synapses after BACE1 inhibition.

BACE1-mediated cleavage of APP is a pivotal step in the production of the Alzheimer related Aß peptide and inhibitors of BACE1 are currently in clinical development for the treatment of Alzheimer disease (AD). While processing and trafficking of APP has been extensively studied in non-neuronal cells, the fate of APP at neuronal synapses and in response to reduced BACE1 activity has not been fully elucidated. Here we examined the consequence of reduced BACE1 activity on endogenous synaptic APP by monitoring N- and C-terminal APP epitopes by immunocytochemistry. In control rodent primary hippocampal neuron cultures, labeling with antibodies directed to N-terminal APP epitopes showed a significant overlap with synaptic vesicle markers (SV2 or synaptotagmin). In contrast, labeling with antibodies directed to C-terminal epitopes of APP showed only a limited overlap with these proteins. In neurons derived from BACE1-deficient mice, and in control neurons treated with a BACE1 inhibitor, both the N-terminal and the C-terminal APP labeling overlapped significantly with synaptic vesicle markers. Moreover, BACE1 inhibition increased the proximity between the APP C-terminus and SV2 as shown by a proximity ligation assay. These results, together with biochemical observations, indicate that BACE1 can regulate the levels of full-length APP at neuronal synapses.