Annika Öhrfelt1, Julien Dumurgier2, Henrik Zetterberg3,4,5,6, Agathe Vrillon2, Nicholas J Ashton3,7,8,9, Hlin Kvartsberg3,4, Elodie Bouaziz-Amar10, Jacques Hugon2, Claire Paquet2, Kaj Blennow3,4. 1. Department of Psychiatry and Neurochemistry, Institute of Neuroscience and Physiology, The Sahlgrenska Academy at the University of Gothenburg, Sahlgrenska University Hospital/Mölndal, SE-431 80, Mölndal, Sweden. annika.ohrfelt@neuro.gu.se. 2. Université de Paris, INSERM U1144, Center of Cognitive Neurology, Lariboisière - Fernand-Widal Hospital, APHP, Paris, France. 3. Department of Psychiatry and Neurochemistry, Institute of Neuroscience and Physiology, The Sahlgrenska Academy at the University of Gothenburg, Sahlgrenska University Hospital/Mölndal, SE-431 80, Mölndal, Sweden. 4. Clinical Neurochemistry Laboratory, Sahlgrenska University Hospital, Mölndal, Sweden. 5. Department of Neurodegenerative Disease, UCL Institute of Neurology, London, UK. 6. UK Dementia Research Institute, London, UK. 7. Wallenberg Centre for Molecular and Translational Medicine, University of Gothenburg, Gothenburg, Sweden. 8. Department of Old Age Psychiatry, Institute of Psychiatry, Psychology and Neuroscience, King's College London, London, UK. 9. NIHR Biomedical Research Centre for Mental Health and Biomedical Research Unit for Dementia at South London and Maudsley NHS Foundation, London, UK. 10. Department of Biochemistry, Lariboisière - Fernand-Widal Hospital, Université de Paris, INSERMU1144, APHP, Paris, France.
Abstract
BACKGROUND: Neurogranin (Ng) is a neuron-specific and postsynaptic protein that is abundantly expressed in the brain, particularly in the dendritic spine of the hippocampus and cerebral cortex. The enzymatic cleavage of Ng produces fragments that are released into cerebrospinal (CSF), which have been shown to be elevated in Alzheimer's disease (AD) patients and predict cognitive decline. Thus, quantification of distinctive cleavage products of Ng could elucidate different features of the disease. METHODS: In this study, we developed novel ultrasensitive single molecule array (Simoa) assays for measurement of full-length neurogranin (FL-Ng) and C-terminal neurogranin (CT-Ng) fragments in CSF. The Ng Simoa assays were evaluated in CSF samples from AD patients (N = 23), mild cognitive impairment due to AD (MCI-AD) (N = 18), and from neurological controls (N = 26). RESULTS: The intra-assay repeatability and inter-assay precision of the novel methods had coefficients of variation below 7% and 14%, respectively. CSF FL-Ng and CSF CT-Ng median concentrations were increased in AD patients (6.02 ng/L, P < 0.00001 and 452 ng/L, P = 0.00001, respectively) and in patients with MCI-AD (5.69 ng/L, P < 0.00001 and 566 ng/L, P < 0.00001) compared to neurological controls (0.644 ng/L and 145 ng/L). The median CSF ratio of CT-Ng/FL-Ng were decreased in AD patients (ratio = 101, P = 0.008) and in patients with MCI-AD (ratio = 115, P = 0.016) compared to neurological controls (ratio = 180). CSF of FL-Ng, CT-Ng, and ratio of CT-Ng/FL-Ng could each significantly differentiate AD patients from controls (FL-Ng, AUC = 0.907; CT-Ng, AUC = 0.913; CT-Ng/FL-Ng, AUC = 0.775) and patients with MCI-AD from controls (FL-Ng, AUC = 0.937; CT-Ng, AUC = 0.963; CT-Ng/FL-Ng, AUC = 0.785). CONCLUSIONS: Assessments of the FL-Ng and CT-Ng levels in CSF with the novel sensitive immunoassays provide a high separation of AD from controls, even in early phase of the disease. The novel Ng assays are robust and highly sensitive and may be valuable tools to study synaptic alteration in AD, as well as to monitor the effect on synaptic integrity of novel drug candidates in clinical trials.
BACKGROUND:Neurogranin (Ng) is a neuron-specific and postsynaptic protein that is abundantly expressed in the brain, particularly in the dendritic spine of the hippocampus and cerebral cortex. The enzymatic cleavage of Ng produces fragments that are released into cerebrospinal (CSF), which have been shown to be elevated in Alzheimer's disease (AD) patients and predict cognitive decline. Thus, quantification of distinctive cleavage products of Ng could elucidate different features of the disease. METHODS: In this study, we developed novel ultrasensitive single molecule array (Simoa) assays for measurement of full-length neurogranin (FL-Ng) and C-terminal neurogranin (CT-Ng) fragments in CSF. The Ng Simoa assays were evaluated in CSF samples from ADpatients (N = 23), mild cognitive impairment due to AD (MCI-AD) (N = 18), and from neurological controls (N = 26). RESULTS: The intra-assay repeatability and inter-assay precision of the novel methods had coefficients of variation below 7% and 14%, respectively. CSF FL-Ng and CSF CT-Ng median concentrations were increased in ADpatients (6.02 ng/L, P < 0.00001 and 452 ng/L, P = 0.00001, respectively) and in patients with MCI-AD (5.69 ng/L, P < 0.00001 and 566 ng/L, P < 0.00001) compared to neurological controls (0.644 ng/L and 145 ng/L). The median CSF ratio of CT-Ng/FL-Ng were decreased in ADpatients (ratio = 101, P = 0.008) and in patients with MCI-AD (ratio = 115, P = 0.016) compared to neurological controls (ratio = 180). CSF of FL-Ng, CT-Ng, and ratio of CT-Ng/FL-Ng could each significantly differentiate ADpatients from controls (FL-Ng, AUC = 0.907; CT-Ng, AUC = 0.913; CT-Ng/FL-Ng, AUC = 0.775) and patients with MCI-AD from controls (FL-Ng, AUC = 0.937; CT-Ng, AUC = 0.963; CT-Ng/FL-Ng, AUC = 0.785). CONCLUSIONS: Assessments of the FL-Ng and CT-Ng levels in CSF with the novel sensitive immunoassays provide a high separation of AD from controls, even in early phase of the disease. The novel Ng assays are robust and highly sensitive and may be valuable tools to study synaptic alteration in AD, as well as to monitor the effect on synaptic integrity of novel drug candidates in clinical trials.
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