Abstract
Synaptic degeneration and neuronal loss are early events in Alzheimer’s disease (AD), occurring long before symptom onset, thus making synaptic biomarkers relevant for enabling early diagnosis. The postsynaptic protein neurogranin (Ng) is a cerebrospinal fluid (CSF) biomarker for AD, also in the prodromal phase. Here we tested the hypothesis that during AD neurodegeneration, processing of full-length Ng into endogenous peptides in the brain is increased. We characterized Ng in post-mortem brain tissue and investigated the levels of endogenous Ng peptides in relation to full-length protein in brain tissue of patients with sporadic (sAD) and familial Alzheimer’s disease (fAD), healthy controls and individuals who were cognitively unaffected but amyloid-positive (CU-AP) in two different brain regions. Brain tissue from parietal cortex [sAD (n = 10) and age-matched controls (n = 10)] and temporal cortex [sAD (n = 9), fAD (n = 10), CU-AP (n = 13) and controls (n = 9)] were included and all the samples were analyzed by three different methods. Using high-resolution mass spectrometry, 39 endogenous Ng peptides were identified while full-length Ng was found to be modified including disulfide bridges or glutathione. In sAD parietal cortex, the ratio of peptide-to-total full-length Ng was significantly increased for eight endogenous Ng peptides compared to controls. In the temporal cortex, several of the peptide-to-total full-length Ng ratios were increased in both sAD and fAD cases compared to controls and CU-AP. This finding was confirmed by western blot, which mainly detects full-length Ng, and enzyme-linked immunosorbent assay, most likely detecting a mix of peptides and full-length Ng. In addition, Ng was significantly associated with the degree of amyloid and tau pathology. These results suggest that processing of Ng into peptides is increased in AD brain tissue, which may reflect the ongoing synaptic degeneration, and which is also mirrored as increased levels of Ng peptides in CSF.
Highlights
Alzheimer’s disease (AD) is the most common form of dementia, affecting tens of millions of people worldwide and with numbers increasing each year [1]
By using a combination of hybrid immunoaffinity–mass spectrometry (HI–MS) and high-resolution MS/MS we found that several post-translational modifications (PTMs) were present on full-length Ng
In 2015 we used HI–MS to show that several endogenous Ng peptides are present in both cerebrospinal fluid (CSF) and brain tissue and Ng48–76 was increased in sAD CSF [32] we aimed to investigate Ng in brain tissue in more detail by combining HI–MS, ELISA and WB analysis to find an explanation to why Ng seems to be decreased in sAD brains, as shown previously [15, 49], and increased in CSF from sAD patients
Summary
Alzheimer’s disease (AD) is the most common form of dementia, affecting tens of millions of people worldwide and with numbers increasing each year [1]. Acta Neuropathologica (2019) 137:89–102 hallmarks in the brain including extracellular plaques consisting of amyloid β (Aβ) peptides and intracellular neurofibrillary tangles composed of hyperphosphorylated tau (p-tau) protein [9]. The degree of dementia has been found to be more associated with synaptic loss compared to amyloid plaques and tangles [8, 16, 37, 52, 58]. Studies using immunohistochemistry and immunoelectronmicroscopy suggest that synaptic loss occur without clear relation to plaque pathology [8, 36]. Synaptic loss is especially pronounced in certain areas of the brain such as the hippocampus [52, 53]. Synaptic proteins have the potential to be highly suited as biomarkers for early AD diagnosis in addition to monitoring disease progression and evaluating novel disease-modifying therapeutics
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