ProNGF Measurement in Cerebrospinal Fluid Samples of a Large Cohort of Living Patients With Alzheimer's Disease by a New Automated Immunoassay.

Francesca Malerba,Chiara Zecca,Rita Florio,Ivan Arisi,Giancarlo Logroscino,Maria Teresa Dell'Abate,Marialuisa Casella,Serena Camerini,Antonino Cattaneo,Bruno Bruni Ercole

doi:10.3389/fnagi.2021.741414

Abstract

The discovery of new biomarkers for Alzheimer's disease (AD) is essential for an accurate diagnosis, to conceive new strategies of treatments, and for monitoring the efficacy of potential disease-modifying therapies in clinical trials. proNGF levels in the cerebrospinal fluid (CSF) represent a promising diagnostic biomarker for AD, but its validation was hampered by the absence of a reliable immunoassay. In the literature, proNGF is currently measured in postmortem brain tissue by semiquantitative immunoblot. Here we describe the development and validation of a new method to measure proNGF in the CSF of living patients. This method, based on molecular size separation by capillary electrophoresis, is automated and shows a 40-fold increase in sensitivity with respect to the proNGF immunoblot, largely used in literature, and is robust, specific, and scalable to high-throughput. We have measured proNGF in the cerebrospinal fluid of 84 living patients with AD, 13 controls, and 15 subjective memory complaints (SMC) subjects. By comparing the proNGF levels in the three groups, we found a very significant difference between proNGF levels in AD samples compared with both controls and SMC subjects, while no significant difference was found between SMC and controls. Because of the development of this new immunoassay, we are ready to explore the potentiality of proNGF as a new biomarker for AD or subgroups thereof, as well as for other neurodegenerative diseases.

Highlights

Alzheimer’s disease (AD) is characterized by a long preclinical stage and a clinical stage of variable duration that precedes the dementia stage (Jack et al, 2010; Dubois et al, 2016)
Recombinant proNGF from Escherichia coli has a theoretical molecular weight (MW) of 25 kDa, but due to its strong cationic charge, it runs with an apparent MW >30 kDa, in SDS PAGE
ProNGF has been considered a promising biomarker for AD (Counts et al, 2016; Cuello et al, 2019; Mufson et al, 2019; Pentz et al, 2020)

Summary

Introduction

Alzheimer’s disease (AD) is characterized by a long preclinical stage and a clinical stage of variable duration that precedes the dementia stage (Jack et al, 2010; Dubois et al, 2016). The central diagnostic role of imaging and cerebrospinal fluid (CSF) biomarkers have been confirmed in the revised NIA-AA diagnostic criteria (Jack et al, 2018). The discovery of new CSF biomarkers targeting early phases of AD would be essential for an accurate diagnosis, to conceive new strategies of treatments, and for monitoring the efficacy of potential disease-modifying therapies in clinical trials. ProNGF, the nerve growth factor (NGF) precursor, represents a good candidate to become a new CSF biomarker for AD. It was demonstrated that an increase in proNGF level was associated with neurodegeneration in early AD and that proNGF/NGF ratio is an upstream driver for neurodegeneration both in animal models and in humans (Capsoni et al, 2000, 2010; Counts and Mufson, 2005; Capsoni and Cattaneo, 2006; Tiveron et al, 2013; Iulita and Cuello, 2014; Counts et al, 2016; Fasulo et al, 2017)

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