Molecular forms of neurogranin in cerebrospinal fluid.

Faisal Hayat Nazir,Gunnar Brinkmalm,Elena Camporesi,Christina E Toomey,Henrik Zetterberg,Bruno Becker,Hlin Kvartsberg,Kaj Blennow,Tammaryn Lashley

doi:10.1111/jnc.15252

Abstract

Neurogranin (Ng) is a 78 amino acid neuronal protein and a biomarker candidate for Alzheimer's disease (AD). Ng has been suggested to bind to calmodulin and phosphatidic acid via its centrally located IQ domain. Ng is cleaved within this functionally important domain, yielding the majority of fragments identified in cerebrospinal fluid (CSF), suggesting that cleavage of Ng may be a mechanism to regulate its function. Up to now, Ng has been shown to be present in CSF as both C‐terminal fragments as well as full‐length protein. To obtain an overview of the different molecular forms of Ng present in CSF, we show by size exclusion chromatography (SEC), immunoblotting, immunoprecipitation, and MS that Ng is present in CSF as several molecular forms. Besides monomeric full‐length Ng, also higher molecular weight forms of Ng, and C‐terminal‐ and previously not identified N‐terminal fragments were observed. We found by immunodepletion that C‐terminal peptides contribute on average to ~50% of the total‐Ng ELISA signal in CSF samples. There were no differences in the overall C‐terminal fragment/total‐Ng ratios between samples from AD and control groups. In addition, we found that monomeric Ng and its C‐terminal fragments bind to heparin via a heparin‐binding motif, which might be of relevance for their export mechanism from neurons. Taken together, this study highlights the presence of several molecular forms of Ng in CSF, comprising monomeric full‐length Ng, and N‐ and C‐terminal truncations of Ng, as well as larger forms of still unknown composition.

Highlights

Neurogranin (Ng) is a 78 amino acid post-synaptic protein known to be expressed in cerebral cortex, amygdala, caudate, putamen, and the hippocampus in the brain (Alvarez-Castelao & Schuman, 2015; Represa et al, 1990)
Trypsin-digested eluates of NG36 antibody immunoprecipitates from the pooled fractions yielding these higher molecular weight bands revealed the presence of Ng peptide Ng54-68 by mass spectrometry (MS), but at much less abundance than the monomer Ng fractions. This suggests that these molecular forms could either be full-length oligomers of Ng, or Ng complexed with other proteins
We detected different molecular forms of Ng present in cerebrospinal fluid (CSF), which correspond to monomeric and both N-and C-truncated forms of Ng in CSF, as well as higher molecular weight forms of Ng, either representing Ng oligomers or Ng complexed with other proteins present in CSF

Summary

| INTRODUCTION

Neurogranin (Ng) is a 78 amino acid (aa) post-synaptic protein known to be expressed in cerebral cortex, amygdala, caudate, putamen, and the hippocampus in the brain (Alvarez-Castelao & Schuman, 2015; Represa et al, 1990). Research from our group has shown that Ng can be detected at increased concentrations in cerebrospinal fluid (CSF) from Alzheimer's disease (AD) patients, both as full-length protein (Thorsell et al, 2010) and as C-terminal fragments (Kvartsberg et al, 2015). In an effort to estimate the proportion of C-terminal Ng fragments to total-Ng content, we depleted full-length Ng and mainly N-terminal Ng fragments from several individual AD and control CSF samples and measured by sandwich ELISA the concentration of the remaining Ng which represented mainly the C-terminal Ng fragments, allowing estimation of the proportion of C-terminal fragments to total-Ng content On average, this proportion amounted to about 50% and it was the same for the control as well as the AD sample set. We show that full-length Ng and the C-terminal fragments contained a functional heparin-binding motif, which, similar as for tau, may allow their export from neurons via the unconventional transport mechanism (Katsinelos et al, 2018)

| MATERIALS AND METHODS

| DISCUSSION

Findings

| Strengths and limitations

| CONCLUSIONS