Klotho overexpression protects human cortical neurons from β-amyloid induced neuronal toxicity

Abstract

Klotho, a well-known aging suppressor protein, has been implicated in neuroprotection and the regulation of neuronal senescence. While previous studies have demonstrated its anti-aging properties in human brain organoids, its potential to mitigate neurodegenerative processes triggered by β-amyloid remains underexplored. In this study, we utilised human induced pluripotent stem cells (iPSCs) engineered with a doxycycline-inducible system to overexpress KLOTHO and generated 2D cortical neuron cultures from these cells. These neurons were next exposed to pre-aggregated β-amyloid 1-42 oligomers to model the neurotoxicity associated with Alzheimer's disease. Our data reveal that upregulation of KLOTHO significantly reduced β-amyloid-induced neuronal degeneration and apoptosis, as evidenced by decreased cleaved caspase-3 expression and preservation of axonal integrity. Additionally, KLOTHO overexpression prevented the loss of dendritic branching and mitigated reductions in axonal diameter, hallmark features of neurodegenerative pathology. These results highlight Klotho's protective role against β-amyloid-induced neurotoxicity in human cortical neurons and suggest that its age-related decline may contribute to neurodegenerative diseases such as Alzheimer's disease. Our findings underscore the therapeutic potential of Klotho-based interventions in mitigating age-associated neurodegenerative processes.

Keywords: Alzheimer’s disease; Cortical neurons; Klotho; Neurodegeneration; Neuroprotection; iPSCs; β-amyloid.

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