**1. Introduction**

Alzheimer's disease (AD) is the most common cause of dementia and the sixth cause of death in the world, constituting a major health problem for aging societies [1]. This disease is a neurodegenerative continuum with well-established pathology hallmarks, namely the deposition of amyloid-β (Aβ) peptides in extracellular plaques and intracellular hyperphosphorylated forms of the microtubule associated protein tau forming neurofibrillary tangles (NFTs), accompanied by neuronal and synaptic loss [2]. Interestingly, patients who will eventually develop AD manifest brain pathology decades before clinical symptoms appear [3,4]. Nevertheless, AD is still frequently diagnosed when symptoms are highly disabling and yet there is no satisfactory treatment.

**Citation:** Gómez-Virgilio, L.; Luarte, A.; Ponce, D.P.; Bruna, B.A.; Behrens, M.I. Analyzing Olfactory Neuron Precursors Non-Invasively Isolated through NADH FLIM as a Potential Tool to Study Oxidative Stress in Alzheimer's Disease. *Int. J. Mol. Sci.* **2021**, *22*, 6311. https://doi.org/ 10.3390/ijms22126311

Academic Editor: Anne Vejux

Received: 22 March 2021 Accepted: 29 April 2021 Published: 12 June 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

Although the manifestations of AD are preponderantly cerebral, cumulative evidence shows that AD is a systemic disorder [5]. Accordingly, molecular changes associated with AD are not exclusively manifested in the brain but include cells from different parts of the body, ranging from the blood and skin to peripheral olfactory cells. More recently, neurons derived from induced pluripotent stem cells (iPSCs) from AD patients have contributed to glean a more realistic insight of brain pathogenic mechanisms [6]. Alternatively, the culture of olfactory neuronal precursors (ONPs) has emerged as a relatively simpler tool to study different brain disorders, taking advantage of their neuronal lineage and their readily non-invasive isolation [7,8]. For instance, patient-derived ONPs manifest abnormal amyloid components together with tau hyperphosphorylation, which have recently led to the proposal of these cells as a novel diagnostic tool for AD [9–11].

Different hypotheses have attempted to explain AD pathogenesis. Some of them include Aβ cascade, tau hyperphosphorylation, mitochondrial damage, endoplasmic reticulum (ER) stress, and oxidative stress. Interestingly, although it has been difficult to establish a prevailing causative mechanism, increased levels of oxidative stress seem to be a common feature for many of these models. Furthermore, oxidative stress due to increased levels of reactive oxygen species (ROS) has been broadly recognized as a very early signature during the course of AD [12–14]. Interestingly, AD-related oxidative stress is by no means restricted to neuronal cells but is also related to astrocytes' oxidative damage and antioxidant capacity [15]. Indeed, since the acknowledgment of the tripartite synapse, it has become increasingly clear that different antioxidant mechanisms of astrocytes can be harnessed by synaptically active neurons and surrounding cells [16–18]. In the tripartite synapse, the astrocyte's endfeet are close to synapses and can be activated by the spillover of synaptic glutamate to provide a timely antioxidant response [19,20]. Moreover, it is not entirely understood how other glial cells such as pericytes may contribute to the damage induced by AD-related oxidative stress. For instance, oxidative damage may compromise the integrity of pericytes, which in turn could alter the blood-brain barrier's integrity, favoring the infiltration of cytotoxic cells and the emergence of brain edema [21,22]. In coherence with a broader systemic manifestation of this disease, the peripheral olfactory system shows AD-associated oxidative stress, which has been measured both in the olfactory neuroepithelium and in cultured ONPs [23–25]. However, while the intriguing relationship between oxidative stress and AD has been long known, their translational impact has remained limited.

Interestingly, the oxidative status of cells is highly correlated with the content of autofluorescent metabolic co-factors such as NADH and its phosphorylated version NADPH [26–29]. In addition, NADH is required to synthesize NADPH, which is at the core of the antioxidant response of different cells by sustaining the synthesis of antioxidants such as glutathione (GSH) and thioredoxin [30]. Furthermore, it has been shown in AD animal models that the provision of NADH is upstream the levels of GSH in order to counterbalance increased ROS levels and neuronal death [27]. Interestingly, external manipulation of oxidative or reducing conditions of cultured neurons are directly manifested as changes in mitochondrial and cytosolic NADH content [28]. As such, by imaging NADH autofluorescence, it might be possible to obtain a real-time monitoring of redox imbalance without the need to use exogenous staining or recombinant sensors. Complementary to methodologies purely based on fluorescence intensity, Fluorescence Lifetime Imaging Microscopy (FLIM) has received increasing attention [31,32]. Fluorescence lifetime is the average time in which a fluorophore remains excited to emit photons before descending to the ground state, providing unique information about its biochemical environment. Importantly, NADH FLIM can be harnessed to increase the sensitivity to its autofluorescence and to discriminate its binding to enzymes from different signaling pathways. In this review, we explore the idea of using ONPs non-invasively isolated coupled to NADH FLIM to reveal AD-associated oxidative stress. This approach may have a broad impact for early AD diagnosis and treatment.
