Small-molecule induction of Aβ-42 peptide production in human cerebral organoids to model Alzheimer's disease associated phenotypes

Abstract

Human mini-brains (MB) are cerebral organoids that recapitulate in part the complexity of the human brain in a unique three-dimensional in vitro model, yielding discrete brain regions reminiscent of the cerebral cortex. Specific proteins linked to neurodegenerative disorders are physiologically expressed in MBs, such as APP-derived amyloids (Aβ), whose physiological and pathological roles and interactions with other proteins are not well established in humans. Here, we demonstrate that neuroectodermal organoids can be used to study the Aβ accumulation implicated in Alzheimer's disease (AD). To enhance the process of protein secretion and accumulation, we adopted a chemical strategy of induction to modulate post-translational pathways of APP using an Amyloid-β Forty-Two Inducer named Aftin-5. Secreted, soluble Aβ fragment concentrations were analyzed in MB-conditioned media. An increase in the Aβ42 fragment secretion was observed as was an increased Aβ42/Aβ40 ratio after drug treatment, which is consistent with the pathological-like phenotypes described in vivo in transgenic animal models and in vitro in induced pluripotent stem cell-derived neural cultures obtained from AD patients. Notably in this context we observe time-dependent Aβ accumulation, which differs from protein accumulation occurring after treatment. We show that mini-brains obtained from a non-AD control cell line are responsive to chemical compound induction, producing a shift of physiological Aβ concentrations, suggesting that this model can be used to identify environmental agents that may initiate the cascade of events ultimately leading to sporadic AD. Increases in both Aβ oligomers and their target, the cellular prion protein (PrPC), support the possibility of using MBs to further understand the pathophysiological role that underlies their interaction in a human model. Finally, the potential application of MBs for modeling age-associated phenotypes and the study of neurological disorders is confirmed.

Fig 1. Mini-Brain maturation through neural induction and differentiation.

(A) Neural induction matures the neuroectodermic layers at the outer surface of the EB, the edge of which appears optically translucent (arrow). (B) Neural differentiation in Matrigel droplets expands neuroepithelial buds (arrowheads) and increases their size (primitive organoid). (C) Organoids develop in size and morphology for 2 months, reaching their maximal size of 3–4 mm in diameter at the end of this period. (D) Hematoxylin and eosin staining shows the presence of discrete brain regions such as ventricle like-cavities (upper panel, 1-month old MBs) and choroid plexus-like structures (lower panel, 4-month old MBs). Scale bars 250 μm (A, B, C) and 100 μm (D).

Fig 2. Chemical induction of APP cleavage in vitro upregulates both Aβ₄₂ and Aβ₄₂/Aβ₄₀ ratio without altering Aβ₄₀ levels.

(A) Schematic representation of treatments with Aftin-5; 1- and 2-month-old MBs (upper and lower representations respectively) were treated once with Aftin-5 for 4 days before collecting conditioned media. (B) Aβ₄₀ (left) and Aβ₄₂ (right) mean concentration levels in conditioned media measured by ELISA (MSD); concentrations of soluble Aβ₄₀ and Aβ₄₂ peptides from supernatant of vehicle-treated with DMSO (Ctrl) were compared with concentrations obtained from MBs treated with Aftin-5 (concentration of 150μM (A150)). (C) Aβ₄₂/Aβ₄₀ ratios corresponding to 1 month- and 2-month old organoids measured by ELISA (MSD) in conditioned media of MBs treated with control vehicle or with Aftin-5. Statistical analysis: unpaired nonparametric Mann-Whitney test. On charts *: p = 0.02; ***: p = 0.009; ns: not significant.

Fig 3. APP and PrP^C expression in MBs during culture.

(A) Real-time PCR analysis shows APP and PRNP gene expression at different times during culture. Expression during MB generation and differentiation was normalized with iPSCs expression, which was used as the baseline. (1) iPSCs; (2) embryoid bodies, (EBs); (3) embryoid bodies after neural induction (NI); (4) MBs after 19 days of culture (d19); (5) 2-month old MBs (2m); (6) 4-month old MBs (4m). (B) Western Blot analysis of APP and PrP^C using specific antibodies: PrP^C antibody SAF32 is specific for the octapeptide repeat region at the N-terminal site which contains the octarepeat region; full-length diglycosylated PrPC appears at ∼ 35 kDa (arrow) whereas unglycosylated and truncated forms appear at a lower molecular weight. 22C11 antibody recognizes amino acid sequence 66–81 and allows identification of a full-length APP695 isoform at ∼ 100 kDa (arrow) as well as an N-truncated form of APP protein. The Bradford method was used to measure the protein load (5 μg of total protein per well); α-tubulin (∼ 55 kDa) was used as loading control. (4) MBs after 19 days of culture (d19); (5) 2-month old MBs (2m); (6) 4-month old MBs (4m); (>6) 7-month old MBs (7m). (C) Tissue sections of 1 month- (left panel) and 3-month old MBs (right panel), immunostained with specific antibodies recognizing PrP^C (SAF32 antibody, specific for an N-terminal epitope of protein, upper panel) and Aβ peptides (BAM10 antibody, lower panel).