**3. Alzheimer's Disease-Related Oxidative Stress in the Olfactory Epithelium and ONPs**

Oxidative stress is the result of an imbalance between oxidant and antioxidant cellular pathways. One of the most studied oxidant compounds are ROS, which are highly reactive molecules, including peroxide (H2O2), superoxide anion radical (O2 • −), and hydroxyl radical (• OH), among others. These molecules may covalently interact with lipids, proteins, and carbohydrates, generating molecular adducts and cumulative damage that, when sensed by cells, may actively trigger different death programs [55].

It was well established almost three decades ago that oxidative stress damage is linked to AD [14]. Furthermore, it has been proposed that oxidative stress at different brain neuronal and non-neuronal cells might be the earliest event of a pathogenic cascade [13]. Whether oxidative stress is a causative agent or just a consequence in neurodegenerative disorders has been thoroughly debated for several years, but still remains an open question [56–58]. The most parsimonious interpretation of this evidence is that oxidative stress as well as other potential AD causative agents (such as Aβ accumulation) are part of a highly interconnected vicious cycle rather than a linear chain of events with a unique origin. The molecular mechanisms and implications of oxidative stress on the nervous system and, potentially, during AD pathogenesis have been thoroughly reviewed elsewhere [12,59]. Here, we focus on evidence showing AD-associated oxidative stress in the peripheral olfactory system rather than reviewing mechanistic explanations.

Oxidative stress associated with AD is manifested in the olfactory neuroepithelium. Accordingly, increased immunoreactivity of the antioxidant enzyme manganese and Copper-Zinc superoxide dismutases have been detected in ORNs and basal and sustentacular cells of the olfactory neuroepithelium of AD patients compared with age-matched controls [60]. Analogously, AD patients harbor a higher immunoreactivity against the antioxidant protein Metallothionein both in the olfactory neuroepithelium and the Bowman's Glands and the LP [61]. Both results suggest that cells from olfactory neuroepithelium elicit an increased antioxidant defense, due to increased oxidative stress during AD. With respect to the direct measurement of oxidation products, post-mortem staining showed an increase in 3-nitrotyrosine (3-NT) in the brain and olfactory neuroepithelium of AD patients [23]. Figure 3 schematizes the antioxidant response and oxidative damage reported in ONPs and OE from AD patients. It would be of interest to uncover whether some AD genetic factors such as the *ApoE* ε4 allele (*ApoE4*) (the single most important genetic risk factor for AD) also manifests oxidative stress signatures in the olfactory epithelium. It is plausible that this is the case because deficits in odor fluency, identification, recognition memory, and odor threshold sensitivity have been associated with the inheritance of the *ApoE4* genotype in several studies [62–64]. For a more thorough compiling of evidence showing AD-associated oxidative damage across other domains of the nervous system, readers may refer to the following excellent articles [12,59,65].

The relationship between oxidative stress and AD has been extensively studied mainly through cellular and animal models [47,54]. However, these models may not fully capture key features of the disease. This limitation potentially leads to wrong conclusions about the pathogenic mechanisms and ultimately may dampen the development of effective therapies. Alternatively, patient-derived cells of neuronal lineage such as those from the olfactory epithelium may provide a convenient solution to this problem [5,9,42].

**Figure 3.** Oxidative stress associated with AD in the olfactory neuroepithelium. (**a**) ONPs and sustentacular cells in the olfactory epithelium (OE) show an increased antioxidant defense with elevated levels of manganese and copper-zinc superoxide dismutases as well as heme oxygenase-1 due to increased oxidative stress in AD patients compared with age-matched controls. Moreover, there is an increase in 3-nitrotyrosine (3-NT) and 4-hydroxynonenal (lipid peroxidation indicator) levels, suggesting AD-associated oxidative damage. (**b**) The increased generation of superoxide anion activates superoxide dismutases (SOD) as an antioxidant response. The generation of other reactive oxygen species (ROS), such as H2O2, induces the expression of other antioxidant enzymes (heme oxygenase-1). On the other hand, the accumulation of superoxide anion increases the levels of compounds such as 4-hydroxynonenal (4-HNE). Moreover, the increased levels of 3-NT are produced from the interaction of superoxide anion and nitric oxide (NO), whose probable source is located at activated macrophages in the OE of AD patients. Created with BioRender.com.

Interestingly, cultured patient-derived ONPs and other peripheral cells also manifest AD-associated oxidative stress. For example, an increase in the level of hydroxynonenal and N*ε*-(carboxymethyl)lysine) (indicating lipid peroxidation), as well as a higher content of heme oxygenase-1, has been found in ONPs isolated from AD patients compared with age-matched controls (Figure 3) [24]. Furthermore, ONPs from AD patients are also more susceptible to oxidative stress-induced cell death [25]. This is strikingly similar to what has been found by our group in blood-derived lymphocytes from AD patients [66,67]. Indeed, manifestations of oxidative stress associated with AD have been reported in different patient-derived peripheral cells ranging from blood cells to fibroblasts and iPSCs-derived neurons. These changes may include compensatory antioxidant responses and a rise in the concentration of oxidation by-products, as well as increased susceptibility to ROS-induced cell death, which has been demonstrated in different cellular types from AD patients. Many of those findings are summarized in the Table 1. In addition, Table 1 also summarizes similar evidence of other relevant pathogenic mechanisms proposed for AD pathogenesis, including Amyloid/Tau, mitochondria, and ER-stress. Thus, different cells throughout the body show signs of different proposed AD pathogenic mechanisms, including oxidative stress at early stages of the disease continuum. The robustness of this tendency highlights the potential of patient-derived cells, and in particular ONPs, for monitoring oxidative stress associated with AD.

**Table 1.** Signatures of oxidative stress and other AD mechanistic hypotheses are manifested in patient-derived peripheral cells, iPSCs and ONPs.



**Table 1.** *Cont.*
