Physiological Aβ Concentrations Produce a More Biomimetic Representation of the Alzheimer's Disease Phenotype in iPSC Derived Human Neurons

Abstract

Alzheimer's disease (AD) is characterized by slow, progressive neurodegeneration leading to severe neurological impairment, but current drug development efforts are limited by the lack of robust, human-based disease models. Amyloid-β (Aβ) is known to play an integral role in AD progression as it has been shown to interfere with neurological function. However, studies into AD pathology commonly apply Aβ to neurons for short durations at nonphysiological concentrations to induce an exaggerated dysfunctional phenotype. Such methods are unlikely to elucidate early stage disease dysfunction, when treatment is still possible, since damage to neurons by these high concentrations is extensive. In this study, we investigated chronic, pathologically relevant Aβ oligomer concentrations to induce an electrophysiological phenotype that is more representative of early AD progression compared to an acute high-dose application in human cortical neurons. The high, acute oligomer dose resulted in severe neuronal toxicity as well as upregulation of tau and phosphorylated tau. Chronic, low-dose treatment produced significant functional impairment without increased cell death or accumulation of tau protein. This in vitro phenotype more closely mirrors the status of early stage neural decline in AD pathology and could provide a valuable tool to further understanding of early stage AD pathophysiology and for screening potential therapeutic compounds.

Keywords: Alzheimer’s disease; Aβ oligomers; chronic; electrophysiology; human.

Figure 1. Treatment of hiPSC derived neurons with physiological doses of Aβ does not induce changes in gross cell morphology or viability

A) Representative phase contrast images of hiPSC-derived neurons comparing untreated, chronic, and acute Aβ treated cells at 30 DIV. Scale = 50 μm. B) Viability of hiPSC-derived neurons determined by MTT assay at 30 DIV following treatment with Aβ. Acute 5 μM treatment significantly reduced cell viability (P < 0.05) whereas acute and chronic 10 nM treatments had no significant effect on cell survival.

Figure 2. Electrophysiological evaluation of untreated, acute and chronic Aβ-treated hiPSC-derived neurons

Patch-clamp recordings were taken between 28–30 DIV in control (column A), 10 nM chronic (column B), and 5 μM acute (column C) treated cortical neurons. Recordings from cells acutely treated with 10 nM Aβ were also taken but there were no significant difference from the control (p > 0.05). (Top row) Voltage-clamp recording of inward sodium and outward potassium currents (−70 mV holding potential, 10 mV steps). (Second row) Neurons fired repeatedly in response to depolarizing current injections at a −70 mV holding potential. (Third row). A single AP was generated using a 3 ms depolarizing current pulse of 1–2 nA. (Bottom row) Gap free recordings were performed for up to 5 m to measure spontaneous activity of patched cells. (Inset) Phase contrast image of patched neuron. Scale = 20 μM.

Figure 3. Changes in electrophysiological characteristics of hiPSC-derived neurons treated with either acute or chronic Aβ

A) Resting membrane potential, B) peak inward current, C) peak outward current, D) maximum APs fired in response to depolarizing current injections at −70 mV, E) and peak action AP are represented at 28 DIV. Whereas the resting membrane potential, peak inward and outward currents, and maximum number of APs were maintained across control and 10 nM conditions, these parameters were significantly reduced in acute, 5 μM treated cultures (p < 0.05). Treatment with 5 μM of Aβ resulted in an even more significant drop in spontaneous neural firing activity (p < 0.01). Time to peak AP and AP duration, and represented by rise time (F) and FWHM (G) respectively was significantly increased in 5 μM treatment of Aβ (p < 0.05). Inversely, the maximum rate of rise (Vmax; H) was significantly reduced in 5 μM treatment of Aβ but was unaffected in other conditions (p < 0.03). 10 nM Aβ acute treatment did not have a significant effect on spontaneous APs fired during gap-free recordings (I) but chronic 10 nM Aβ resulted in a significant drop in the spontaneous activity of these cultures (p = 0.02). Error bars represent SEM.

Figure 4. Tau protein is upregulated in hiPSC-derived neurons treated with higher-than-physiologic concentrations of Aβ

(A) Cells were fixed at culture endpoints (28–30 DIV) and stained for beta-ill tubulin (red), tau (green), and DAPI (blue). Scale bar = 20 μm. (B) Image analysis of tau protein intensity was analyzed relative to control tau positive staining while accounting for differences in cell viability (p = 0.057).