Alzheimer’s disease (AD) is a disabling and progressive illness that causes a high social cost and economic burden. Current diagnosis has been done by clinical appreciation based on theDiagnostic and Statistical Manualof Mental Disorders (DSM–5) and by neuropsychological assessments.

The study ofpostmortembrains derived from AD patients revealed two abnormal aggregates now considered as pathological hallmarks of this illness.

1) The extracellular deposition of neuritic plaques formed by amyloid-β (Aβ) peptides and 2) Intraneuronal neurofibrillary tangles (NFTs) formed by the abnormally phosphorylated microtubule associated protein tau. In addition, NFTs were also described inpostmortemolfactory epithelium biopsies and the abnormal aggregates of hyperphosphorylated tau were observed inside the neurites and in the neuronal soma, resembling those NFTs found in brain tissue.

Fig. 1. Representative scheme of the procedure to obtain olfactory neuronal precursors by exfoliation and the method for detection of neurofibrillary tangles and quantitation of phosphorylated and total tau levels.

In the last years the scientific community made efforts to find biological markers to detect Alzheimer’s disease before the appearance of the first clinical manifestations and to monitor the disease progression. Thus, biomarkers were searched in biological fluids, in brain images and in peripheral tissues. However, the study of these markers in alive neuronal cells of patients were hampered by the inaccessibility of the brain tissue to obtain a biopsy and for ethical reasons for the potential damage that can be caused to the patient.

In recent years the olfactory neuroepithelium has been considered as a subrogate model of the Central Nervous System (CNS) because they have metabolic, biochemical, and physiological characteristics like the CNS. Therefore, the obtaining of neuronal olfactory precursors from the nasal cavity by a non-invasive and low painful method have rendered a suitable alternative for identification and characterization ofantemortembiomarkers in AD patients.

In this paper we describe a highly sensitive method for the detection of NFTs by immunofluorescence labeling and the quantitation of the proteins that constitute them (total tau and phosphorylated tau) levels in extracts of olfactory neuronal precursors obtained by exfoliation of the nasal cavity of AD patients and control participants.

In the article we describe the protocol for stain the NFTs in cultured olfactory neuronal precursors. Also, the preparation of total cellular extracts to simultaneously detect phosphorylated tau isoforms and total tau by ELISA.

Neurofibrillary tangles formation was estimated by the frequency of their appearance in cultured neuronal precursors and we reported absolute values of tau isoforms in AD patients calculated from standard curves.

The procedure to separate tau by two-dimensional gel electrophoresis to distinguish phosphorylated tau variants are also described. We used a specific anti-tau antibody that recognizes the carboxy-terminal region of 17 amino acids conserved in all isoforms of tau. We found 5 isoelectric tau variants in the AD patient that showed heavier molecular weights and more acidic isoelectric points than those of the control subject indicating that tau in the AD patient were more phosphorylated and that incorporated a higher number of phosphate groups.

The amounts of total tau and phosphorylated tau in a specific site (199 and 202 serine amino acids) were determined by subtracting the amount of dephosphorylated tau from the total amount of tau. The results shown here demonstrate that and tau alterations and NFTs are present in cultured neuronal precursors derived from AD patients. Thus, both alterations can be used as biomarkers forantemortemdiagnosis.

Agustin Riquelme-Sandoval¹,Claudia Ivonne Mejía-Mondragón²,Gloria Benítez-King^2
1Present address Laboratory of Cellular Neuroanatomy, Institute of Biomedical Sciences, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil
²Laboratory of Neuropharmacology, Subhead of Clinical Research, National Institute of Psychiatry Ramón de la Fuente Muñíz, Mexico

Publication

Potential Use of Exfoliated and Cultured Olfactory Neuronal Precursors for In Vivo Alzheimer’s Disease Diagnosis: A Pilot Study
Riquelme A, Valdés-Tovar M, Ugalde O, Maya-Ampudia V, Fernández M, Mendoza-Durán L, Rodríguez-Cárdenas L, Benítez-King G
Cell Mol Neurobiol. 2020 Jan

Read offline:

Related Articles:

	Purification and characterization of a better-behaved…Alzheimer’s Disease (AD) is an incurable disease that causes memory, reasoning and thinking to deteriorate over time. AD is marked by brain atrophy and plaque-like deposits of a small peptide…
	Social-economic factors predict state differences in opioid…Media accounts of the opioid overdose epidemic emphasize the role of prescription drugs and, to a lesser extent, declining economic and social conditions. In support of the journalists’ accounts, opioid…
	The suitability of scopolamine as an Alzheimer’s modelAlzheimer’s disease is the most common cause of dementia. About 50 million people worldwide have Alzheimer’s disease or a related form of dementia. Alzheimer’s disease is a terminal illness affecting…
	How can we measure problematic videogame use among…Playing video games is currently one of the most frequent leisure activities in young people and adolescents, but it generates a social alarm because it is associated with negative consequences…
	Global climate change and mental healthIn the past decade, several empirical studies and systematic reviews of the literature have documented the mental health impacts of global climate change. However, the range of impacts and their…
	Someone with higher risk: Seeing through Chinese gamblers…Gambling in various forms has been an important economic activity around the world. Governments are trying to balance the economic benefits of legalized gambling with its social costs. However, measuring,…