White Light Stimulation at Gamma Frequency to Modify the Aβ42 and tau Proteins in SH-SY5Y Cells

Abstract

Background

Non-invasive 40 Hz gamma frequency stimulation has shown improved cognition and memory recall in Alzheimer’s disease (AD).

Purpose

The present study investigates the effects of white light stimulation at gamma frequency on SH-SY5Y cells using Delta M + BrainCare Light without causing discomfort.

Methods

SH-SY5Y cells were exposed to The Delta M + BrainCare Light for 15, 30, 45, and 60 minutes. The secretion of amyloid-β42 (Aβ42), p-AKT (Ser473), AKT, p-mTOR (Ser2448), mTOR, p-4E-BP1 (Thr37/46), 4E-BP1, p-tau (Thr181), and tau were analyzed. Aβ42 fibril aggregation and phagocytosis of FITC-Aβ42 in BV-2 cells were also observed.

Results

Delta M + BrainCare Light reduced tau, AKT, and p-mTOR and increased p-4E-BP1 in SH-SY5Y cells. Further, it inhibited secretion and aggregation of Aβ42, alongside increased phagocytosis in microglial cells.

Conclusion

These findings underscore the potential of the Delta M + BrainCare Light in mitigating the associated proteins and pathways implicated in AD.

Significance Statements

(1) Exposure to white light at gamma frequency decreases the phosphorylation of AD-related proteins.

(2) The secretion and aggregation of Aβ42 may be inhibited by white light stimulation at gamma frequency, while Aβ40 may not.

(3) White light stimulation at gamma frequency may help eliminate Aβ42 fibril because the microglial cells can engulf Aβ42 fibril.

Introduction

Alzheimer’s disease (AD), the leading cause of dementia in the elderly population, presents a significant healthcare challenge. Presently, acetyl-cholinesterase inhibitors or N-methyl-D-aspartic acid antagonists provide symptomatic relief for approximately 40%-60% of AD patients. However, the disease has few disease-modifying pharmacological treatment options, highlighting the urgency for early diagnosis and intervention to improve patient outcomes. ¹ The etiology of AD is multifactorial, involving a complex interplay of neuropathological events.^2,3 Initially, the aberrant cleavage and aggregation of amyloid β (Aβ) protein precipitate intracellular amyloid plaque formation. Subsequently, neurofibrillary tangles accumulate extracellularly, owing to excessive phosphorylation of tau protein and the subsequent microtubular degradation.^4,5 Further, dysregulated phagocytosis in microglial cells exacerbates the aggregation of toxic Aβ protein.^6,7 Consequently, neuroinflammation triggered by Aβ toxicity and neurofibrillary tangles leads to neuronal and synaptic loss, disrupting network oscillations. Notably, many AD animal models exhibit a reduction in the power of gamma frequency oscillations.^8,9 A decrease in gamma frequency oscillations might be linked to the diminished expression of parvalbumin (PV) fast-spiking interneurons possessing GABA transmission in AD patients’ hippocampal neurons. Interestingly, optogenetic stimulation of PV-positive interneurons, which generate robust gamma oscillations upon receiving patterned 40 Hz sensory stimuli, decreased amyloid load in 5XFAD mice.^10,11 Remarkably, when 40 Hz gamma entrainment using sensory stimuli (GENUS) was implemented, dose-dependent and time-dependent neuroprotection was observed in 5XFAD mice. ¹² This beneficial impact of GENUS could be attributed to its ability to decrease amyloid levels, including Aβ40 and Aβ42, plaque formation, and modulate microglial phagocytic activity and inflammation.^12,13 Furthermore, multisensory stimuli were found to reduce hyper-phosphorylated tau levels in the hippocampus of AD mice. ¹⁴ However, in humans, light flickering has induced adverse effects. ¹⁵

In this study, we propose a novelty lighting, the Delta M + BrainCare Light, developed by Delta Electronics. This device comprises an inner lamp flickering at gamma frequency, masked by an un-flickering outer lamp, which may cause fewer adverse effects. Our research aims to examine the molecular changes induced by exposure to the Delta M + BrainCare Light in cells, primarily assessing its neuroprotective effects. We also aim to investigate its potential to modulate amyloid load, phosphorylated tau, and other relevant phagocytic markers in SH-SY5Y neuroblastoma cells and BV-2 microglia cells.

Methods

For each of the 25 ms periods of the flicker signal, with a duty cycle of 20%, the first 5 milliseconds (ms) was a 40 Hz signal, and the remaining 20 ms was a silent signal. A white light LED(light-emitting diode) luminaire was used to provide the 40 Hz visual flicker, and the 40 Hz visual flicker was generated with a 5 ms light-on and 20 ms light-off period, which means multi-frequency for mixed lighting. Meanwhile, the 40 Hz visual flicker was accompanied by a non-flickering general lighting in the same luminaire. The luminaire was controlled by a programmable LED driver provided by Delta Electronics Corporation, and the lighting was provided by a type of recessed light. Measured by a lux-meter, the intensity of the light signal received by the cells is approximately 1222-1242 lumens, with an intensity per area of .4 mW/mm². The mixed lighting used 2 types of light sources, both placed in the same luminaire for the inner (adjustable visual flicker lighting)and outer circles (general lighting). The light source with adjustable visual flicker lighting was set to a zero Hz frequency (non-flickering) for the control groups. The light source with adjustable visual flicker lighting was set to 40 Hz for the experimental groups. Cells were exposed to the luminaire for 0, 15, 30, 45, and 60 minutes respectively. The stimulation occurred at 22°C in a laminar flow hood and ensured that the temperature stimulation was maintained at 22 to 24°C during the stimulation. To explore the effects of light on the formation of the proteins, we measured intracellular total tau, p-tau (Thr181), Aβ40, and Aβ42 with Western blots, and ELISA assays in the human neuroblastoma cell line (SH-SY5Y). Transmission electron microscope (TEM) imaging was utilized to ensure the aggregation of Aβ42 peptide into fibrils. A stock solution of FITC-Aβ42 peptide was prepared by dissolving the peptide in dimethyl sulfoxide (DMSO), followed by a one-minute vortex and five-minute sonication. Then, we used confocal microscopy to observe and analyze the FITC-Aβ42 uptake in BV-2 cells. The details of chemicals and reagents used in cell cultures and the uptake of FITC-Aβ42 in BV-2 cells were shown. (Supplementary Materials)

Statistical analyses were conducted using SigmaPlot software. Significant differences between groups were determined by applying the student’s t test. A P-value of <.05 was deemed statistically significant. All presented figures originated from 1 representative experiment out of 3 independent trials and are expressed as the mean ± standard deviation (SD).

Results

The Delta M + BrainCare Light, capable of emitting white light at gamma frequency, was utilized to expose SH-SY5Y cells. This was conducted with the presence or absence of gamma frequency at 15, 30, 45, and 60-minute intervals. As demonstrated in Figure 1(A), the mixed lighting significantly inhibited tau (Thr181) phosphorylation at the 45 and 60-minute exposures in the SH-SY5Y cells. Even after the correction for GAPDH (Figure 1(B)), the significance remained. Conversely, total tau showed no significant inhibition in SHSY5Y cells under identical experimental conditions (Figure 1(A) and (C)).

Figure 1.

Inhibition of tau phosphorylation by the Delta M + BrainCare Light at gamma frequency. SH-SY5Y cells were exposed to the Delta M + BrainCare Light with or without gamma frequency for 15, 30, 45, and 60 minutes. (A) The expression levels of total tau, p-tau (Thr181), and GAPDH proteins were assessed via western blotting. Three independent experiments yielded similar results. (B and C) The expression of total tau and p-tau (Thr181) proteins were quantified using Image J software and normalized against the corresponding GAPDH levels. Data are expressed as mean ± SD. *P < .05, **P < .01, and ***P < .001 (t test).

For this experiment, the SH-SY5Y cells were exposed to the Delta M + BrainCare Light, both with and without gamma frequency, across 15, 30, 45, and 60 minutes. It was observed that p-mTOR (Ser2448) protein expression was notably inhibited at the 15, 45, and 60 minute exposures by the white light stimulation at gamma frequency (Figure 2(A) and (B)). However, mTOR (Ser2448) protein did not demonstrate a similar significant inhibition (Figure 2(CC). The expression of p-AKT (Ser473) protein was significantly inhibited at 15 and 30 minutes under exposure to the white light stimulation at gamma frequency (Figure 2(A) and (D)). Furthermore, the Delta M + BrainCare Light notably induced the expression of p-4E-BP1 (Thr37/46) protein in the SH-SY5Y cells for only 15 minutes (Figure 2(A) and (F)). Remarkably, in line with our previous study, the light stimulation at gamma frequency notably inhibited the expression of p-mTOR (Ser2448) and p-AKT (Ser473) proteins while inducing the expression of p-4E-BP1 (Thr37/46) in the SH-SY5Y cells, when compared to cells unexposed to the gamma frequency.

Figure 2.

Inhibition of AKT and mTOR phosphorylation and induction of 4E-BP1 phosphorylation by the Delta M + BrainCare Light. SH-SY5Y cells were exposed to the Delta M + BrainCare Light with or without gamma frequency for 15, 30, 45, and 60 minutes. (A) The expression levels of p-mTOR (Ser2448), mTOR, p-AKT (Ser473), AKT, p-4E-BP1 (Thr37/46), 4E-BP1, and GAPDH proteins were assessed by western blotting. Three independent experiments yielded similar results. (B-G) The expression levels of p mTOR (Ser2448), mTOR, p-AKT (Ser473), AKT, p-4E-BP1 (Thr37/46), and 4E-BP1 proteins were quantified using Image J software and normalized against the corresponding GAPDH levels. Data are expressed as mean ± SD. *P < .05, **P < .01, and ***P < .001 (t test).

We determined the secretion of Aβ40, Aβ42, total tau, and p-tau (Thr181) from SH-SY5Y cells via ELISA following a 24-hour incubation. Remarkably, we discovered that the Delta M + BrainCare Light with gamma frequency selectively inhibited the secretion of Aβ42 while leaving the levels of Aβ40, total tau, and p-tau (Thr181) unaffected (Figure 3(A) to (D)). To ascertain if the white light stimulation at gamma frequency could inhibit Aβ42, we incubated the Aβ42 peptide for 7 days to pre-form Aβ42 fibrils, confirmed via transmission electron microscopy (Figure 4(B)). Subsequently, the Aβ42 fibrils were subjected to the Delta M + BrainCare Light either with or without the gamma frequency for durations of 15, 30, 45, and 60 minutes. The ThT assay revealed a time-dependent significant reduction in Aβ42 fibrils after exposure to the white light stimulation at gamma frequency (Figure 4(A)).

Figure 3.

Differential effect of Delta M + BrainCare Light with gamma frequency on protein secretion in SH-SY5Y cells. The white light stimulation at gamma frequency selectively inhibited the secretion of Aβ42 but had no significant impact on Aβ40, total tau, and p-tau (Thr181). SH-SY5Y cells were exposed to white light stimulation with or without gamma frequency for 15, 30, 45, and 60 minutes, followed by a 24 hour incubation. The secreted levels of (A) total tau, (B) p-tau (Thr181), (C) Aβ40, and (D) Aβ42 were assessed by ELISA. These data, expressed as the mean ± SD, were obtained from 3 independent experiments. *P < .05, **P < .01, and ***P < .001 (t test).

Figure 4.

Inhibition of Aβ42 aggregation in vitro by the Delta M + BrainCare Light. Aβ42 fibrils were exposed to the Delta M + BrainCare Light with or without gamma frequency for 0, 15, 30, 45, and 60 minutes. (A) The aggregation rate of Aβ42 was measured by ThT assay. Similar results were obtained from 3 independent experiments. Data are expressed as mean ± SD. *P < .05, **P < .01, and ***P < .001 (t test). (B) Aβ42 fibrils were pre-formed from the Aβ42 peptide over 7 days. These fibrils were observed by transmission electron microscopy (TEM). The scale bar is 200 nm.

BV-2 cells were administered with FITC-Aβ42 and then subjected to the Delta M + BrainCare Light with or without gamma frequency for 15, 30, 45, and 60 minutes, followed by a 2-hour incubation. The fluorescent intensity demonstrated a significant difference across all time points when comparing BV-2 cells exposed to the white light stimulation with or without gamma frequency (Figure 5(C)). This indicated that the Delta M + BrainCare Light emitting gamma frequency prompted phagocytosis of FITC-Aβ42 by BV-2 cells Figure 6. The co-localization of FITC-Aβ 42 peptide (green) in BV-2 cells stained with GAPDH (red) and DAPI (blue) suggested the uptake of fluorescently labeled Aβ42 (FITC‐Aβ42) by BV-2 cells (Figure 5(A) and (B)).

Figure 5.

Induction of phagocytosis of FITC-Aβ42 by BV-2 microglial cells via the Delta M + BrainCare Light. BV-2 cells were exposed to the Delta M + BrainCare Light with or without gamma frequency for 0, 15, 30, 45, and 60 minutes. (A) The phagocytic activity of BV-2 cells was measured after 2 hour incubation with FITC-Aβ42 (green) peptide. (B) BV-2 cells were stained with anti-GAPDH (red) and DAPI (blue) for immunofluorescent analysis via confocal fluorescence microscopy. Images depict the co-localization of FITC-Aβ42 peptide within BV-2 cells, indicating phagocytosis. Orthogonal views were captured from different planes (XY, XZ, or YZ), and the images were magnified 1000 times. (C) The difference in FITC-Aβ42 intensity was demonstrated by comparing cells exposed to white light stimulation with and without 40 Hz gamma frequency.

Figure 6.

Summary of the study findings: We first demonstrated that the AKT/mTOR/4EBP-1/tau signaling pathway was activated in SH-SY5Y cells, then showed that Aβ42 aggregation was inhibited, and finally, we illustrated that the Delta M + BrainCare Light induced cellular phagocytosis in BV-2 cells.

Discussion

In this present study, we elucidated the influence of a novelty white light stimulation, Delta M + BrainCare Light, exhibiting gamma frequency, on the phosphorylation of p-tau (Thr181), p-mTOR (Ser2448), p AKT (Ser473), and p-4E-BP1 proteins—crucial components of the PI3-K/AKT/mTOR pathway—in SH-SY5Y cells. The white light stimulation, operating at gamma frequency, demonstrated a significant reduction in the secretion of Aβ42 but not Aβ40 when contrasted with a white light stimulation without gamma frequency. Literature evidence documenting the effect of gamma frequency on tau protein expression in animal models is increasingly becoming robust.^14,16 This is corroborated by recent studies demonstrating tau inhibition in the motor and somatosensory cortex of tau P301S and CK-p25 mice following vibrotactile stimulation at 40 Hz over several weeks. ¹⁷ The data collected from our work on SH-SY5Y cells aligns with previous human and animal studies. The suppression of the AKT/mTOR pathway by gamma frequency light could account for the observed results.^14,16 Furthermore, hyperactivation of the PI3K/AKT/mTOR pathway is implicated in the accumulation of Aβ and hyperphosphorylation of tau protein, symptomatic of Alzheimer’s disease. Suppression of mTOR might consequently mitigate Aβ accumulation and development of phosphorylated tau, ¹⁸ thereby curtailing neurofibrillary tangle formation and Aβ plaques characteristic of Alzheimer’s disease. ¹⁹ Our findings underscore the potential role of white light stimulation at gamma frequency light in attenuating tau phosphorylation by modulating the PI3K/AKT/mTOR pathway, a significant factor in AD-related neurodegeneration. The mTOR pathway exists in mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2). The phosphorylation of S6K, 4-EBP, and AKT proteins might be integral to signal transmission in the mTOR pathway, suggesting that the 40 Hz gamma frequency light could perturb the mTOR pathway at the cellular level. ²⁰

Extracellular Aβ deposition, a hallmark of AD pathogenesis, leads to network dysfunction through synaptic loss and neuronal degeneration, instigating the AD pathological cascade.^21-23 This is primarily facilitated by sequential proteolytic processing of the amyloid precursor protein (APP), a ubiquitous transmembrane protein known to be cleaved during subcellular trafficking when co-compartmentalized with active APP secretases. ²⁴ Notably, Aβ production predominantly occurs at the Golgi apparatus and endosomes via active β- and γ-secretases, ²⁵ where the rate-limiting step is BACE-1 cleavage.^26,27 A recent study highlighted the potential of a non-invasive 40 Hz light-flickering regime to reduce APP cleavage intermediates and Aβ40 and Aβ42 levels,^12,14 suggesting the possibility of modulating APP trafficking with 40 Hz gamma frequency light. Our previous study corroborates this finding. ¹⁶ Our current study demonstrates that a white light generator operating at gamma frequency can significantly decrease Aβ42 levels without affecting Aβ40 levels, though the precise molecular and cellular mechanisms remain elusive. There is emerging evidence that APP can modulate the expression of KCC2 and hippocampal GABAergic inhibition in APP knock-out models.^10,22 Chronic exposure to Aβ42 has been shown to affect the expression of cation–chloride cotransporters (CCC), namely K-Cl-2 (KCC2) and N-K-Cl-1 (NKCC1), in the hippocampus of AD mice, potentially disrupting the balance of excitatory and inhibitory signals. ²³ Additionally, 40 Hz gamma frequency light flickering may enhance KCC2 expression and PKC-dependent phosphorylation of APP on a serine residue. ²⁸ This could enhance APP trafficking to the plasma membrane, thereby reducing the Aβ load in AD. In brief, the white light stimulation at gamma frequency may exert a more pronounced effect on Aβ42 levels, possibly via modulating KCC2 expression and APP trafficking to the plasma membrane.

Emerging evidence posits a significant role for the neuroimmune system in neuropsychiatric disorders, particularly in the context of Alzheimer’s disease. ²⁹ In AD pathology, activated microglia present a common yet fluctuating feature across various stages. Notably, enhanced microglial inflammatory activity coupled with impaired microglial-mediated clearance has been identified in AD. However, the mechanisms governing non-pharmacological modulation of the brain’s immune system remain elusive. Recent investigations have demonstrated that exogenous stimulation of neural electrical activity at the gamma frequency (30-50 Hz), facilitated by sensory stimulation, can enhance microglial activity, a vital component of the neuroimmune system, in AD.^12,30 Moreover, several studies have reported that 40 Hz gamma frequency flicker induces temporary modifications in the phosphorylation of proteins within the NF-κB and MAPK pathways and modulates cytokine expression, thereby recruiting microglia within the brain. ³¹ Aligning with these findings, our study uniquely illustrates that white light stimulation at a gamma frequency could potentially stimulate microglia to engulf Aβ42, consequently mitigating the disease burden in AD.

Another strength of our study is the device’s accessibility in clinical practice. Previous methodological studies have reported few side effects (asthenopia, fatigue, and dazzling) in normal individuals; ¹⁵ however, the potential health risk of inducing photosensitive seizure cannot be excluded. ³² With the mixed lighting of gamma frequency and masked non-flickering general light, Delta M + BrainCare Light has been proven that patients with AD may have good compliance and tolerability in a small validation study (data not shown).

The synergy between SH-SY5Y cells and microglia provides a unique platform to explore the interactions between phosphorylated tau, other associated microglia-mediated endocytosis phenomena, and gamma frequency. This model also has the potential to evaluate time-dependent and dose-induced effects of gamma frequency using white light stimulation. A future research trajectory informed by our study may involve probing the mechanistic regulation by specific illumination, wavelength, or in conjunction with other frequencies. Given the integral role of these brain regions in the initiation of AD-like pathology, modulating the activity of this pathway may either mitigate or exacerbate the disease state. Subsequent investigations may also seek to elucidate the impact of gamma frequency on the Alzheimer ’s-linked neural proteome or appraise additional mouse models of the disease. Collectively, the use of white light stimulation at gamma frequency presents a novel, promising intervention to assess gamma frequency’s influence on cellular neurodegeneration models. This approach harbors the potential to identify a non-invasive method that may confer beneficial health outcomes for older people.

Conclusions

Our findings suggest that white light stimulation operating at gamma frequency could potentially influence mechanisms related to tau phosphorylation, the AKT/mTOR pathway, and Aβ42 secretion via microglial phagocytosis. The implications of our research suggest that gamma frequency may serve as a non-invasive modality to mitigate AD progression. Given the robustness of these potential associations, additional exploration in both clinical and preclinical populations to verify this hypothesis is warranted. Collectively, our data provide compelling evidence that gamma frequency may modulate specific molecular mechanisms associated with tau and related pathways, potentially expanding its application in treating AD and preventive care in vulnerable populations.

Supplemental Material

Supplemental Material - White Light Stimulation at Gamma Frequency to Modify the Aβ42 and tau Proteins in SH-SY5Y Cells

Supplemental Material for White Light Stimulation at Gamma Frequency to Modify the Aβ42 and tau Proteins in SH-SY5Y Cells by Yang-Pei Chang, Ching-Fang Chien, Ling-Chun Huang, Chih-Pin Chuu, Hsi-Wen Chang, Tzyh-Chyuan Hour, and Yuan-Han Yang in American Journal of Alzheimer's Disease & Other Dementias®