Molecular forms of neurogranin in cerebrospinal fluid

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. K E Y W O R D S CSF, heparin-binding motif, neurograninThis is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.

Neurogranin and Neurofilament Light Chain as Preclinical Biomarkers in Scrapie

“…Neurogranin (Ng, formerly named p17, also known as RC3 and BICKS) is a 78 amino acid post-synaptic protein with a molecular weight of ~12 kDa [9,10]. This protein is known to be principally expressed in the telencephalon of adult mammals, especially in the cerebral cortex, hippocampus, and striatum [11], as a neuronal postsynaptic protein, which is specifically located in the neuronal soma and dendrites [12].…”

Bioinformatics tools and data resources for assay development of fluid protein biomarkers

“…One identified cause for concern is the high sequence similarity between neurogranin and neuromodulin within the IQ domain both proteins contain. Interestingly, the expected cross-reactivity with neuromodulin of antibodies binding the IQ domain has been confirmed in a recent study [ 119 ]. Retrospectively, the information collected through bioinformatics resources and tools could have guided researchers towards the development of antibodies with favorable epitopes.…”

“…Similarly, many proteins undergo proteolytic cleavage. Often only the cleaved fragment might serve as a biomarker; examples include the AD biomarkers amyloid- and neurogranin [ 103 , 119 ]. If a fragment is to be detected by an assay, the location of associated cleavage sites is thus essential.…”

“…The assessment of neurogranin by bioinformatics tools agrees with research findings. Indeed, three successful neurogranin immunoassay have been developed with the majority of the antibody epitopes being located in the C-terminal region [ 119 , 159 , 160 ]. All three assays have been compared by Willemse et al establishing high correlation of the assays between each other [ 161 ].…”

Mass spectrometry‐based methods for robust measurement of Alzheimer's disease biomarkers in biological fluids

“…Although Aβ-and tau-related AD biomarkers remain at the center stage of the AT(N) research framework, there are continuous efforts to develop novel diagnostic and prognostic fluid biomarker for AD. For example, several synaptic proteins have been identified in CSF including pre-synaptic synaptosomal-associated protein 25 (SNAP-25) and pre-synaptic proteins synaptotagmin-1 (SYT-1) (Davidsson et al, 1996(Davidsson et al, , 1999, as well as the dendritic (post-synaptic) protein neurogranin (Kvartsberg et al, 2015;Thorsell et al, 2010), which is present in CSF in several molecular forms like monomeric full-length neurogranin, and N-and C-terminal truncations of neurogranin, as well as larger forms of still unknown composition (Nazir et al, 2020). Neuronal pentraxin 2 (NPTX2) and neuronal secretory protein VGF have been found as promising synaptic biomarkers for predicting progression versus no progression from mild cognitive impairment to AD (Hendrickson et al, 2015;Spellman et al, 2015;Swanson et al, 2016).…”