Fig. 1

Klotho upregulation protects human neurons from β-amyloid-induced toxicity. (A) Schematic overview of the protocol for generating mature human neurons from dCas9-VPR iPSCs transduced with 3 gRNAs. Neurons were pretreated with doxycycline (dox) for 1 week to induce KLOTHO expression (KL+), followed by exposure to 5µM β-amyloid for 24–48 h Created with BioRender.com. (B) Immunostaining of iPSC-derived neurons with TUJ1 (Red), NEUN (Green), and KLOTHO (Yellow). Nuclei were counterstained with Hoechst 33,342 (Blue). Zoomed images show distinct KLOTHO expression in neuronal axons. Scale bar = 60 μm; zoomed images scale bar = 10 μm. (C) Liquid-phase transmission electron microscopy images of small and large oligomers of β-amyloid 1–42 pre-aggregated for 1 h at 4 °C. Scale bar = 10 nm. Right graph shows the size distribution of β-amyloid monomers (orange) and oligomers (blue) measure by DLS at 4 °C. (D) Cultured neurons pretreated with and without dox, and exposed to β-amyloid aggregates for 24 h and 48 h. Immunostaining of with TUJ1 (Green) and CLEAVED CASPASE-3 (CC3, Red) after treatment with β-amyloid for 24–48 h. Yellow arrows indicate TUJ1 puncta along neurites. Scale bar = 50 μm; zoomed images scale bar = 10 μm. (E) Quantification of the percentage of neurons expressing CLEAVED CASPASE-3. Data are presented as mean ± standard deviation; p values were measured via One Way ANOVA with Tukey’s multiple comparisons test; n = 4 independent experiments. (F) Violin plots showing the number of primary neurites per neuron in cultures with (KL+) and without (KL-) KLOTHO upregulation exposed to β-amyloid for 0, 24, and 48 h. p values were measured via One Way ANOVA with Tukey’s multiple comparisons test; n = 3 independent experiments; total number of analysed neurons = 565 neurons; d indicates Cohen’s d analysis. (G) Quantification of neurite diameter in neurons exposed to β-amyloid with or without KLOTHO upregulation at 0, 24, and 48 h. Data are presented as mean ± standard deviation; p values were measured via One Way ANOVA with Tukey’s multiple comparisons test; n = 3 independent experiments; total number of analysed neurons = 515 neurons. (H) Distribution of neurite diameters in neurons with (KL+) and without (KL-) KLOTHO upregulation at 0, 24, and 48 h of β-amyloid exposure. (I) Schematic summary illustrating Klotho’s neuroprotective role in mitigating β-amyloid-induced toxicity in human iPSC-derived neurons. KLOTHO upregulation preserves neurite integrity and reduces apoptotic signalling