Brain organoids, self-organized three-dimensional aggregates derived from human induced pluripotent stem cells (iPSC), have emerged as a novel and translational model for studying human brain development and developmental diseases. Brain organoids recapitulate key aspects of human brain structure, including representative cell populations and a complex 3D architecture (Figure 1). Utilizing this complex tool to also model neurodegenerative diseases is an emerging field and has opened new dimensions for understanding genetic disease components. However, the use of organoids as a model system in this field also contains certain challenges and limitations, as cells are juvenile and may not be fully mature for a certain time span.
Figure 1: Representative images of cerebral organoids on (A) DIV35 and (B) DIV48. (A) 3D imaging of whole cerebral organoid fixed at DIV35. The organoid was stained as whole for neuronal marker MAP2 and astrocytic marker GFAP. (B) Organoid fixed at DIV48. Organoid was sectioned and a 10 µM section was subject to immunohistochemistry for the neuronal marker MAP2 and the inflammasome marker ASC. Scalebar (A) 200 µM (B) 100 µM.
Age-related accumulation of senescent cells in the nervous system is relevant for the onset and progression of neurodegenerative diseases and has recently even been addressed in clinical Alzheimer’s disease trials. Cerebral organoids were thus treated with D-galactose (D-Gal) for up to 2 weeks to induce senescence. D-Gal treatment was started at DIV35.
The significant reduction in size across the organoids treated with D-Gal suggests a general trend of cellular atrophy or diminished proliferation. This effect is associated with a reduction in NeuN levels, indicating a decrease in mature neuronal density or health. The concomitant increase in NfL secretion after 1 week of treatment supports this finding, as elevated NfL is indicative of neurodegeneration (Figure 2).
Figure 2: D-Gal-induced changes in organoid size, neurofilament light chain (NfL) secretion and mature neurons. Measurement of organoid size (A), given as the diameter of the organoids in µm and (B) NfL secretion 1 and 2 weeks after start of D-Gal treatment. NfL was analyzed with Uman Diagnostics ELISA. Protein levels of NeuN (C), a marker for mature neurons, was measured 1 week after D-Gal treatment. NeuN was quantified via automated western blot WES. A-B: Two-way ANOVA followed by Šídák’s multiple comparisons test comparing vehicle control (VC) versus D-Gal-treated organoids. C: Unpaired T-test. Mean + SEM (n=6 per group). *p<0.05, ***p<0.001.
p21 is a cyclin-dependent kinase inhibitor that is tightly regulated by the tumor suppressor protein p53. It plays a key role in cell cycle arrest, particularly in response to DNA damage or other stress signals. Both markers were significantly upregulated in D-Gal-treated cerebral organoids, as shown in the automated western blot analysis, indicating that D-Gal induced senescence. Additionally, the autophagy marker p62 was significantly increased after 1 week of D-Gal treatment (Figure 3).
Figure 3: Quantification of senescence and autophagy markers in D-Gal-treated cerebral organoids. Organoids were treated with D-Gal for 1 week and analyzed for the senescence marker p21 (A), tumor suppressor protein p53 (B), and autophagy marker p62 (C) via automated western blot WES. Mean + SEM (n=4-6 per group). Unpaired t-test. *p<0.05, **p<0.01; ***p<0.001.
D-Gal treatment of cerebral organoids leads to increased senescence and negatively impacts neuronal maturation. Accelerated aging of cerebral organoids can serve as a valuable tool in drug development by providing a fast and efficient means of testing potential therapeutics for age-related neurological conditions such as Alzheimer’s disease.
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