Page 524 - IJB-10-5

International Journal of Bioprinting 3D model of neurogenesis in Alzheimer’s disease

without Aβ oligomers and cultured for the same periods) 3.3. Exposing cells to Aβ oligomers induces
(Figure 4C). These results corroborate a recent study by morphological changes and neurogenesis
Esteve et al., which demonstrated that an Aβ-induced Three days after bioprinting, an evident morphological
increase in reactive oxygen species (ROS) levels led to DNA change was observed on the surface of constructs with and
damage and subsequently induced cell cycle arrest with an without Aβ oligomers (Figure 5D). SEM images revealed
increase in cadherin-1 (Cdh1) and a decrease in cyclin B1 that constructs without neurotoxic aggregates presented a
and cyclin-dependent kinase 5 (Cdk5)/p35 expression. homogeneous and smooth surface, whereas adding 1 µM
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Moreover, it was demonstrated that exposing neurospheres Aβ oligomers made the constructs’ surface rougher.
from prenatal rat cerebral cortical regions to Aβ decreases A reduction of NSCs (Figure 6; Nestin cells, stained
+
cell viability and induces degeneration of neurites and in red) and an increase of mature neurons (Figure 6;
apoptosis. Bernabeu-Zornoza et al. reported that 1 µM MAP-2 cells, stained in green) within the neurospheres
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101
+
Aβ , the same concentration we used, induced cell death in constructs with Aβ oligomers is presented in Figure
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in the human NSC cell line hNS1. 6C (p < 0.0001, compared with constructs without Aβ
As the neurosphere area and proliferative assay oligomers). The 3D reconstitution of a stained neurosphere
demonstrated small and lowly proliferative cells on day displays more mature neurons in the constructs with Aβ
8 in constructs with Aβ oligomers, the following assays oligomers than NSCs, compared to constructs without
+
were performed 2–3 days after bioprinting. On day 2 after Aβ oligomers (Figure 6A and B). Nestin cells represent
bioprinting, the live/dead assay demonstrated that cells 42.4 ± 9.9% of the total stained area of neurospheres in
in the neurospheres within the constructs are viable, and constructs without Aβ oligomers, while in the presence
phalloidin staining revealed that the neurospheres have a of the aggregates, the percentage decreases to 26.8 ± 9.6%
well-structured spheroid shape and a complex and dense (Figure 6C). Conversely, mature neurons correspond to
cytoskeleton arrangement (Figure 5A). 14.8 ± 8.6% of the stained area of constructs without Aβ
aggregates, while in constructs with Aβ oligomers, mature
Oxidative stress is one of the main pathological neurons represent 29.6 ± 9.3% (Figure 6C).
events in AD, causing damage to biomolecules, such It is important to emphasize that NSCs in the
as neuronal membrane proteins and lipids, affecting neurospheres were not stimulated to differentiate by
their integrity, contributing to neuronal death, and adding a differentiation medium, which can also modulate
directly impairing NSC proliferation and survival. Aβ neurogenesis and neurosphere morphology (Figures S2
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oligomers may trigger the generation of ROS, which and S3, Supporting Information). Neurogenesis observed
impairs neurogenesis. There is a vicious cycle whereby in Figure 6 is mainly induced by the 3D bioink environment
Aβ oligomers induce increased ROS levels and oxidative or the presence of Aβ. In vivo, as AD progresses, the
stress, consequently raising Aβ production and leading neurotoxicity of Aβ increases, and when coupled with
to AD. 104,105 ROS can cause DNA damage, leading to other elements, such as glucose uptake imbalance and
9
genomic instability in NSCs. As displayed in Figure dysregulated insulin signaling, adult neurogenesis is
5B and C, oxidative stress significantly increased in increasingly inhibited. Notably, bioprinting Aβ oligomers
constructs containing Aβ oligomers in the bioink as early instead of incorporating them into the neurospheres
as 2 days after bioprinting. The same increase in oxidative allowed our model to mimic the extracellular deposit of
stress in NSCs exposed to Aβ oligomers was observed by Aβ aggregates that surround the neurogenic niches in the
105
Chiang et al. , and the oxidative stress also increased the brain of patients with AD.
expression of pro-inflammatory cytokines TNF-α and
IL-1β. Consequently, the ability of NSCs to proliferate Neurospheres used in this study are derived from six-
and generate functional neurons may be compromised, week-old mice, representing adult but not aged conditions.
contributing to cognitive decline. In adults, Aβ oligomers distributed in the 3D environment
may stimulate neuronal differentiation as a mechanism
Interestingly, oxidative stress can be transiently to overcome future impairment in AD progression. By
generated by neurogenesis, 106,107 which could explain the increasing oxidative stress, autophagy is induced to meet
increased ROS production and enhanced neurogenesis in high energy demands. 109,110 Consequently, neurogenesis is
constructs containing Aβ oligomers (Figures 5 and 6). Some increased as a response to NSC impairment caused by the
evidence indicates that NSCs are well-adapted to protect disease. Another hypothesis is that in earlier stages of AD,
their host environment from oxidative stress, leading to a Aβ oligomers can activate a compensatory mechanism to
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synergistic effect between ROS and neurogenesis when the replace lost or damaged cells by increasing the differentiation
brain is trying to protect or compensate for neuronal loss. of neuronal progenitors into new neurons. However, as

Volume 10 Issue 5 (2024) 516 doi: 10.36922/ijb.3751