From Neurotoxicity to Neuroprotection: Photobiomodulation against the Effects of the SARS-CoV-2 Spike Protein in an Alzheimer's Disease Model

From Neurotoxicity to Neuroprotection: Photobiomodulation against the Effects of the SARS-CoV-2 Spike Protein in an Alzheimer's Disease Model | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article From Neurotoxicity to Neuroprotection: Photobiomodulation against the Effects of the SARS-CoV-2 Spike Protein in an Alzheimer's Disease Model Erick José Nogueira Andrade, Luiza de Andrade Giraldi, Milena Yuki Rosental Sudo, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8245044/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 7 You are reading this latest preprint version Abstract Alzheimer's Disease (AD) is the leading cause of dementia and represents one of the greatest global health challenges, affecting not only patients but also their families. Still without a cure, current treatments only alleviate symptoms. The COVID-19 (Coronavirus Disease 2019) pandemic highlighted neurological complications associated with SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus 2), particularly the Spike protein. This study aimed to investigate the potential neurotoxic effects of recombinant Spike protein using two-dimensional (2D) in vitro neuronal models established for AD, as well as the therapeutic potential of photobiomodulation (PBM) with red LED (Light Emitting Diode) (660 nm) in attenuating these effects. Differentiated SH-SY5Y (human neuroblastoma) cells were exposed to Spike protein (0.5 µg/mL) and oxidative stress by H₂O₂ (Hydrogen peroxide) 200 µM, individually or combined, with and without PBM (3 J/cm²). Cell viability was assessed using the Alamar Blue assay, and immunofluorescence characterized nuclear (Hoechst), mitochondrial (Mitotracker), actin (phalloidin), and focal adhesion (FAK) alterations. Immunofluorescence revealed mitochondrial fragmentation, actin disorganization, FAK redistribution, and nuclear condensation. The results demonstrated that Spike protein induced neurotoxicity in AD models, notably aggravated by oxidative stress. In contrast, PBM represented a promising intervention strategy, exerting a neuroprotective effect that preserves viability, mitochondrial integrity, nuclear morphology, and cytoskeletal organization. PBM thus appears to modulate mitochondrial function and mitigate oxidative stress, offering a potential therapeutic pathway to attenuate neuronal damage induced by Spike protein. Neurotoxicity Morphology Oxidative Stress Neuroprotective Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 INTRODUCTION Alzheimer's disease (AD) is one of the highest public health challenges worldwide and is the leading cause of dementia in the elderly population. It is a progressive and irreversible neurodegenerative disorder characterised by the accumulation of amyloid -β (Aβ) plaques, neurofibrillary tangles of hyperphosphorylated tau protein, chronic neuroinflammation, and oxidative stress, ultimately resulting in synaptic loss, neuronal death, and severe cognitive decline [ 1 , 2 ]. Despite more than a century of research, there are currently no curative treatments available, and existing pharmacological therapies only offer symptomatic relief, highlighting the urgent need for new therapeutic approaches [ 3 ]. In parallel, the COVID-19 pandemic (Coronavirus disease 2019), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has raised growing concerns regarding its long-term neurological consequences. Recent evidence suggests that the viral Spike (S) protein, responsible for mediating cellular entry through the angiotensin-converting enzyme 2 (ACE2) receptor, may trigger or accelerate neurodegenerative processes [ 4 , 5 ]. The recombinant S protein has been shown to induce neuroinflammation, oxidative stress, and mitochondrial dysfunction and even exacerbate Alzheimer's-related pathologies, such as Tau hyperphosphorylation and blood-brain barrier disruption [ 6 , 7 ]. This potential neurotoxic effect highlights the spike protein as a relevant biological tool for modelling pathogenic mechanisms of neurodegeneration in vitro [ 8 ]. In this context, photobiomodulation (PBM) emerges as a promising non-invasive therapeutic modality. Using red or near-infrared light, PBM primarily targets the mitochondria, promoting increased adenosine triphosphate (ATP) production, reducing oxidative stress, and modulating inflammatory responses [ 9 , 10 ]. Its neuroprotective effects have been demonstrated in preclinical models of neurodegenerative diseases, showing a reduction in amyloid plaques and improvement in cognitive performance [ 11 , 12 ]. PBM with red light (~ 660 nm) is particularly associated with the activation of cytochrome c oxidase, leading to enhanced mitochondrial bioenergetics and improved neuronal survival under oxidative stress conditions [ 13 ]. Two-dimensional (2D) cultures of the human neuroblastoma cell line (SH-SY5Y) differentiated into neuron-like cells are widely used as an in vitro model to study the molecular mechanisms underlying AD. These cells express key neuronal markers and are highly suitable for investigating the cytotoxic effects of oxidative and inflammatory stimuli, as well as for testing potential neuroprotective strategies [ 14 , 15 ]. The exposure of differentiated SH-SY5Y cells to the S protein and recombinant protein provides a controlled and reproducible platform to evaluate neuronal responses and elucidate possible interactions between SARS-CoV-2 infection and neurodegenerative disorders such as AD [ 6 , 8 ]. Therefore, this study aimed to evaluate the effects of the SARS-CoV-2 S protein in a 2D in vitro model of AD and investigate the potential therapeutic role of PBM with red LED light in this context. MATERIALS AND METHODS Reagents and Equipment All reagents, chemicals, antibodies, suplements, and equipment were obtained from certified suppliers and handled according to manufacturer instructions to ensure reproducibility. The complete list of materials used across cell culture, differentiation, treatments, staining, imaging, and data analysis workflows is provided in Table 1. Table 1 - Reagents, suplements, equipment, and software used in the experimental procedures Reagent or resource S ource I dentifier Reagents, antibodies, supplements, and other chemicals DAPI Sigma-Aldrich, USA D9564 Dulbecco’s modified Eagle’s medium/nutrient mixture F-12 (DMEM/F12) Gibco, USA 12400024 fetal bovine serum (FBS) Gibco, USA 12657-029 Hydrogen peroxide (H 2 O 2 ) Molekular Química, Brazil L0032-P1L Paraformaldehyde Sigma-Aldrich, USA 30525-89-4 Penicillin/streptomycin Gibco, USA 15140-122 Phalloidin Sigma-Aldrich, USA 49409 Phosphate-buffered saline (PBS) Sigma-Aldrich, USA 806552 Recombinant SARS-CoV-2 Spike protein (S) LECC – UFRJ, Brazil LECC-COV2-AC001 (lot: O-151021) Resazurin sodium salt Sigma-Aldrich, USA R7017 Retinoic acid (RA) Sigma-Aldrich, USA R2625 Triton X-100 Sigma-Aldrich, USA 9002-93-1 Trypsin – EDTA Gibco, USA 15090046 MitoTracker™ Invitrogen, USA M7510 Hoechst 33342 Invitrogen, USA H3570 Anti- focal adhesion kinase ( FAK ) antibody Cell Signaling Technology, USA 3285 Plastics and other nonperishable materials cell culture flasks (25 cm²) Nest, China 707003 cell culture plates (24-well) Kasvi, Brazil K12-024 hive-patterned array BioEdTech, Brazil Bio3DStamp version 6 Equipment and software CO₂ incubator Panasonic, Japan MCO-170AICUV-PA confocal laser scanning microscope Leica, Germany TCS SP8 GraphPad Prism 5.0 GraphPad Inc., USA Prism 5.0 ImageJ Fiji National Institutes of Health, USA https://imagej.net/ij/ microplate reader Bmg Labtech, Germany VANTAstar Cell Line and Culture Conditions Human neuroblastoma SH-SY5Y cells (ATCC® CRL-2266™) were used as an in vitro neuronal model. Cells were cultured in 25 cm² cell culture flasks containing the following complete medium: DMEM/F12 (Dulbecco’s Modified Eagle Medium/Nutrient Mixture F-12; Gibco, Thermo Fisher Scientific, USA), supplemented with 10% (v/v) fetal bovine serum (FBS; Gibco), 1% (v/v) penicillin-streptomycin (100 U/mL and 100 μg/mL, respectively; Invitrogen), and maintained at 37 °C in a humidified incubator with 5% CO₂. The medium was changed every 48 hours to preserve nutrient availability and pH stability. Cell Seeding and Neuronal Differentiation When the cultures reached approximately 80% confluence, cells were detached using trypsin-EDTA, and seeded in 24-well plates at a density of 1×10⁵ cells/mL. After 48 hours in complete medium, neuronal differentiation was induced for 5 days using the following differentiation medium: DMEM/F12 supplemented with 10 μM retinoic acid (RA), 0.5% FBS, and 1% penicillin-streptomycin. The differentiation was confirmed by morphological changes, including the extension of neurites and the reduction of proliferative morphology. Experimental groups Differentiated SH-SY5Y cells were distributed into eight experimental conditions to assess the isolated and combined effects of oxidative stress, SARS-CoV-2 S protein, and photobiomodulation (PBM). The groups included: Control, H₂O₂-induced oxidative stress, PBM, H₂O₂ followed by PBM, S protein, S protein followed by PBM, simultaneous exposure to H₂O₂ and S protein, and the combined treatment with H₂O₂ and S protein followed by PBM. All treatments were performed under identical culture conditions, and specific doses, exposure times, and irradiation parameters are detailed in Table 2, which summarizes the full description of each experimental group. Table 2 - Experimental groups and corresponding treatments G roup T reatment Control Differentiated SH-SY5Y cells without additional treatment. Oxidative Stress (H₂O₂) Exposure to 200 µM H₂O₂ for 1 h. Pbm PBM (660 nm, 3 J/cm²). H₂O₂ + PBM H₂O₂ (200 µM, 1 h) followed by PBM (660 nm, 3 J/cm²). S protein Treatment with recombinant S protein (0.5 µg/mL) for 1 h. S protein + PBM S protein (0.5 µg/mL, 1 h) followed by PBM (660 nm, 3 J/cm²). H₂O₂ + S protein Simultaneous exposure to H₂O₂ (200 µM) and S protein (0.5 µg/mL) for 1 h. H₂O₂ + S protein + PBM Combined exposure to H₂O₂ (200 µM) and S protein (0.5 µg/mL) for 1 h, followed by PBM (660 nm, 3 J/cm²). Oxidative stress induction Oxidative stress was induced using hydrogen peroxide (H₂O₂), which was prepared as a stock solution of 160,000 μM and serially diluted in the differentiation medium to obtain a final working concentration of 200 μM. All dilutions were prepared immediately before use to preserve the stability of reactive oxygen species (ROS). Cells were exposed to H₂O₂ for 1 hour at 37 °C in an incubator with 5% CO₂. Exposure to recombinant SARS-CoV-2 Spike protein The recombinant S protein (amino acids 1–1208) of SARS-CoV-2, produced at the Cell Culture Engineering Laboratory (COPPE/UFRJ, Rio de Janeiro, Brazil; batch 0151021), was provided in phosphate-buffered saline (PBS) containing biotin and 0.02% sodium azide, at 0.25 mg/mL. For the assays, the protein was diluted to 0.5 μg/mL in differentiation medium and applied directly to the cells for 1 hour at 37 °C and 5% CO₂. The handling and storage of the S protein were performed under sterile conditions and temperature control to preserve structural integrity and receptor binding function. Photobiomodulation (PBM) treatment After exposure to H₂O₂ and/or the S protein, the groups designated for PBM were irradiated with a red light-emitting diode (LED) device (λ = 660 nm). The irradiation was carried out under controlled lighting conditions, positioning the culture plates perpendicularly to the light source to ensure uniform exposure. The total energy density applied was 3 J/cm², corresponding to an exposure time of 1 min 22 s. All parameters were standardized to ensure reproducibility and avoid thermal effects. Cell viability assay Cell viability was assessed using the Alamar Blue ® assay. After the treatments, the culture medium was replaced by a solution containing 90% differentiation medium and 10% Alamar Blue reagent. Cells were incubated for 2 hours at 37 °C and 5% CO₂, protected from light. Next, 100 µL aliquots were transferred to a 96-well plate and fluorescence was measured using a microplate reader with excitation at 544 nm and emission at 590 nm. Results were expressed as a percentage of the control group. All experiments were performed in triplicate. Preliminary analysis of Spike protein effects on neuronal cells Before inducing oxidative stress to mimic AD condition, differentiated SH-SY5Y cells were exposed to recombinant S protein to assess its direct impact on neuronal morphology and cytoskeletal organization. Immunofluorescence staining with phalloidin, anti-FAK, and DAPI was performed to assess cytoskeletal integrity, cell adhesion protein, and nuclear morphology. Basically, cells were fixed with 4% paraformaldehyde for 20 min, permeabilized with 0.1% Triton X-100, and stained overnight at 4 °C with phalloidin (1:500) and DAPI (1:5000). For FAK detection, cells were incubated with anti-FAK primary antibody (anti-rabbit) followed by a fluorophore-conjugated secondary antibody, each for 1 hour at 37 °C. This preliminary analysis provided a basic understanding of the cellular changes induced by S protein before the induction of oxidative stress and subsequent treatments. Mitochondria and nuclei staining For the analysis of mitochondrial and nuclear integrity under AD-related conditions, they were stained with MitoTracker™ Orange CMTMRos (1 µM) and Hoechst. After 30 minutes staining at 37 °C, cells were washed with PBS and imaged using a confocal laser scanning microscope. Images were acquired using identical microscope parameters across all experimental groups. Statistical analysis All experiments were conducted in triplicate. Data are presented as mean ± standard deviation (SD). Statistical significance was determined using one-way analysis of variance (ANOVA) followed by Tukey's post hoc test, conducted in GraphPad Prism 5.0 (GraphPad Software Inc., USA). P-values < 0.05 were considered statistically significant. RESULTS AND DISCUSSION Changes in the cytoskeleton and focal adhesion induced by the Spike protein S Protein caused significant changes in the cytoskeleton and focal adhesion (Fig. 1 B). Phalloidin staining showed disorganization of actin filaments, including signs of depolymerization and loss of continuity (Fig. 1 B, phalloidin panel). These findings are consistent with studies that report the interaction of the S protein with ACE2 and neuropilin-1 activating pathways that directly modulate actin rearrangement [ 25 , 26 ]. In parallel, an intensification of FAK labeling was observed in the group exposed to S protein (Fig. 1 B, FAK panel), suggesting the activation of compensatory mechanisms of cell adhesion and survival, as already documented in adaptive responses to mechanical and chemical stress [ 27 ]. Notably, despite the cytoskeletal disorganization, cell nuclei of the S protein group remained morphologically preserved (Fig. 1 B, DAPI panel), which is in accordance with reports that viral components can activate survival pathways even under cytoskeletal stress conditions [ 5 ]. This set of data indicates that the S protein simultaneously interferes with cellular structure and signaling, affecting adhesion, the cytoskeleton, and possibly intracellular trafficking, aspects relevant to understanding its neurodegenerative action (Fig. 1 A). Damage to cell viability induced by the Spike protein and oxidative stress The evaluation of cell viability demonstrated that both S protein and H₂O₂ were able to significantly reduce the survival of differentiated neuronal cells (Fig. 2 A). Exposure to protein S reduced viability to about 80% of the control, while H₂O₂ decreased levels to approximately 60% of the control. These findings indicate that both stimuli exert relevant cytotoxic effects, although with distinct magnitudes [ 16 ]. The decrease in viability observed in the groups exposed to the S protein is consistent with recent evidence showing that the S protein can interact with integrins and dysregulate focal adhesion pathways, compromising essential anchoring and neuronal survival mechanisms [ 17 , 18 ]. On the other hand, H₂O₂, a classic inducer of oxidative stress, exerts more pronounced toxicity, inducing excessive production of reactive oxygen species, metabolic reduction, and apoptosis [ 19 ]. The combination of S protein + H₂O₂ resulted in a cytodamage similar to the group treated only with H₂O₂ (~ 60%), but the micrographs revealed more pronounced structural damage, suggesting a synergistic effect between the factors. This pattern has already been described in neuronal systems exposed simultaneously to pro-oxidant stimuli and viral proteins, in which mitochondrial vulnerability amplifies apoptotic processes [ 16 , 20 ]. Thus, data suggest that S protein acts as a sensitizer of oxidative stress, intensifying structural damage even when residual viability is not drastically reduced. When PBM with LED was applied to the groups (Fig. 2 B), partial preservation of viability was observed in all groups subjected to oxidative stress induced by exposure to H 2 O 2 . This preservation, compared to the groups not treated with PBM, suggests a protective effect, possibly mediated by increased cytochrome C oxidase activity, improved energy metabolism, and modulation of the redox balance, as previously demonstrated in cellular and animal models [ 9 , 21 ]. Nuclear alterations and morphofunctional evidence of apoptosis Nuclear staining with Hoechst (Fig. 3 ) revealed striking differences between the groups. The control group demonstrated intact nuclei distributed evenly, while the S protein group showed a moderate reduction in the number of nuclei per area and signs of chromatin condensation. These findings suggest the initial activation of apoptotic processes, consistent with reports that the S protein activates cellular stress pathways and nuclear reorganization without necessarily inducing immediate cell death [ 5 ]. H₂O₂ exposure promoted strong nuclear condensation and fragmentation, a classic profile of apoptosis induced by severe oxidative stress, as described in neuronal models exposed to ROS [ 16 , 19 ]. The S protein + H₂O₂ group exhibited the most severe pattern, reinforcing the hypothesis of degenerative synergism between the viral protein and oxidative stress. In the groups treated with PBM, a reduction in nuclear condensation was observed, with nuclei being more distributed and less fragmented, indicating that PBM attenuated structural impairment. Mechanistically, PBM is capable of reducing DNA damage, modulating repair mechanisms, and decreasing caspase activation, which may explain this partial preservation [ 21 , 22 ]. Mitochondrial dysfunction induced by the Spike protein and H₂O₂ Live mitochondrial staining revealed a clear mitochondrial dysfunction in the groups subjected to the treatments (Fig. 4 ). In the control group, mitochondrial arrangment was organized and distributed in a reticular pattern. In the S protein exposed group, there was partial fragmentation, a decrease in staining intensity, and loss of their distribution in neurite extensions, consistent with compromised membrane potential and impaired mitochondrial transport [ 18 , 23 ]. Once exposed to H₂O₂, mitochondrial fragmentation and reduced staining were observed, as expected of a classic inducer of mitochondrial fission and early apoptosis [ 16 , 19 ]. The S protein + H₂O₂ group exhibited the most critical behaviour: almost complete mitochondrial network collapse and no observation of mitochondria in neurite extensions, suggesting loss of essential energy functionality for mitochondria-dependent neurons. Such vulnerability is explained by the fact that neurons are cells highly dependent on mitochondrial function, due to their exceptional energy demands and specialized morphology. The maintenance of membrane potential, the conduction of electrical impulses, and axonal transport consume large amounts of ATP, most of which is generated via mitochondrial oxidative phosphorylation [ 29 , 30 ]. Moreover, mitochondria essential regulate calcium homeostasis, especially at synapses, where signaling is energetically costly and spatially restricted [ 31 ]. These organelles also act as central regulators of cell death, modulating apoptotic pathways that are particularly critical in neurons, given their limited regenerative capacity [ 32 ]. Thus, any disturbance in mitochondrial dynamics or function directly compromises neuronal viability, making these cells especially vulnerable to oxidative stress stimuli, such as H₂O₂. PBM partially restored mitochondrial morphology, with less fragmented networks and homogeneous distribution, consistent with the increase in ATP and recovery of membrane potential already, as a suggestive consequence of cytochrome c oxidase activation by the exposure to red light [ 21 , 24 ]. This partial protection reinforces the potential of PBM as a modulator of mitochondrial degenerative processes. Protective effects of photobiomodulation PBM promoted a protective effect on multiple cellular parameters, including viability, nuclear morphology, cytoskeletal integrity, and mitochondrial functionality (Fig. 5 ). The literature supports that the absorption of red light by cytochrome c oxidase improves mitochondrial respiration, modulates survival pathways, reduces apoptosis, and increases the expression of neurotrophic factors, which represent key elements against neurodegenerative processes [ 9 , 21 , 28 ]. These mechanisms may explain the improvement observed in the reduced mitochondrial fragmentation and nuclear preservation in the PBM-treated groups, as illustrated in Fig. 5 , where irradiated cells exhibit more continuous mitochondrial staining, fewer intensely fragmented puncta, and nuclei with more homogeneous chromatin compared to their non-irradiated counterparts under the same treatments.. Although the effect is not sufficient to fully restore the phenotype of healthy neuron, the consistent attenuation of damage in multiple pathways suggests that PBM consists in a therapeutic strategy, modulating energetic, oxidative, and structural aspects. CONCLUSIONS The S protein of SARS-CoV-2 triggers structural and functional changes in neuronal models, including reduced viability, cytoskeletal disorganization, mitochondrial fragmentation, and modulation of focal adhesion. These effects were intensified by oxidative stress, indicating a relevant synergistic action for understanding the mechanisms of neurotoxicity associated with COVID-19. LED PBM represents a remarkable and safe non-invasive tool against neurodegeneration, capable of mitigating some of the evaluated parameters, preserving the bioenergetic and morphological aspects of the neuronal cells. Together, the findings reinforce the role of protein S as a neurotoxic agent and PBM as a promising non-pharmacological strategy to modulate neurodegenerative processes and guide future relevant neurobiological and clinical research in AD. Declarations Author Contribution Investigation and conceptualization: All authors;Methodology: EJNA, LAG, MYRS, LPR, GRS;Supervision: CPS;Writing: All authors;All authors reviewed the manuscript. Acknowledgement We gratefully acknowledge M.Sc. Priscila M. S. C. M. Leite and Dr. Angela A. M. Vieira, from the Research & Development Institute (IP&D/Univap), for their assistance with confocal microscopy procedures. We also thank Prof. Dr. Leandro José Raniero and his team for providing access to the microplate reader and technical support, as well as Prof. Dr. Juliana Ferreira Strixino and her team for granting access to the LED chamber. This work was supported by the Brazilian National Council for Scientific and Technological Development (CNPq), Grants 174942/2024-9 (EJNA), 138459/2024-0 (LAG), 157999/2025-4 (LAG), 152384/2024-3 (GRS) and 404953/2025-5 (CPS, CNPQ/MCTI nº 44/2024). Data Availability data are available from the authors upon request References QUERFURTH, H. W.; LAFERLA, F. M. Alzheimer’s disease. The New England journal of medicine, v. 362, n. 4, p. 329–344, 2010. SCHELTENS, P. et al. Alzheimer’s disease. Lancet, v. 397, n. 10284, p. 1577–1590, 2021. YIANNOPOULOU, K. G.; PAPAGEORGIOU, S. G. Current and future treatments for Alzheimer’s disease. Therapeutic advances in neurological disorders, v. 6, n. 1, p. 19–33, 2013. SONG, E. et al. Neuroinvasion of SARS-CoV-2 in human and mouse brain. The journal of experimental medicine, v. 218, n. 3, 2021. FRANK, M. G. et al. SARS-CoV-2 spike S1 subunit induces neuroinflammatory, microglial and behavioral sickness responses: Evidence of PAMP-like properties. Brain, behavior, and immunity, v. 100, p. 267–277, 2022. OLAJIDE, O. A. et al. SARS-CoV-2 spike glycoprotein S1 induces neuroinflammation in BV-2 microglia. Molecular neurobiology, v. 59, n. 1, p. 445–458, 2022. SWAIN, S. SARS-CoV-2 spike protein exacerbates tau pathology and neuroinflammation in Alzheimer’s models. Acta Neuropathologica Communications, v. 15, n. 11, [s.d.]. BARBOSA, E. M. Neurotoxic effects of SARS-CoV-2 spike protein on human neuronal cells: implications for neurodegeneration. Neurochemical Research, v. 49, p. 1123–1136, 2024. HAMBLIN, M. R. Shining light on the head: Photobiomodulation for brain disorders. BBA clinical, v. 6, p. 113–124, 2016. SALEHPOUR, F. Transcranial photobiomodulation therapy: a potential treatment for Alzheimer’s disease and Parkinson’s disease. Frontiers in Neuroscience, v, v. 12, 2018. DA LUZ, S. C. Photobiomodulation therapy in Alzheimer’s disease models: from mechanisms to clinical perspectives. Frontiers in Neuroscience, v. 16, 2022. GRILLO, S. L. Red and near-infrared photobiomodulation improve cognition and reduce amyloid burden in Alzheimer’s disease models. Brain Research Bulletin, v, v. 199, p. 38–47, 2023. HAMBLIN, M. R. Mechanisms and mitochondrial redox signaling in photobiomodulation. Photochemistry and photobiology, v. 94, n. 2, p. 199–212, 2018. KOVALEVICH, J.; LANGFORD, D. Considerations for the use of SH-SY5Y neuroblastoma cells in neurobiology. Methods in molecular biology (Clifton, N.J.), v. 1078, p. 9–21, 2013. XICOY, H.; WOUTERS, R.; VANDENBERGHE, W. The SH-SY5Y cell line in Parkinson’s disease research: a systematic review. Molecular Neurodegeneration, v, v. 12, 2017. SALLES, G. R. et al. 2D and 3D models of Alzheimer’s disease: Investigating neuron-like cells in oxidative environments. ACS omega, v. 10, n. 25, p. 27501–27514, 2025. NORRIS, E. G.; PAN, X. S.; HOCKING, D. C. Receptor-binding domain of SARS-CoV-2 is a functional αv-integrin agonist. The journal of biological chemistry, v. 299, n. 3, p. 102922, 2023. NORRIS, K.; PAN, T.; HOCKING, D. C. SARS-CoV-2 Spike protein disrupts integrin-mediated cell adhesion and signaling. Journal of Molecular Biology, 2023. FAN, X.; HUSSIEN, R.; BROOKS, G. A. H₂O₂-induced oxidative stress triggers mitochondrial fission and apoptosis in neuronal cells. American Journal of Physiology - Cell Physiology, 2010. PENDERGRASS, W.; WOLF, N.; POOT, M. Evidences of mitochondria-mediated apoptosis after oxidative injury. Experimental Gerontology, 2004. SALEHPOUR, F. et al. Brain photobiomodulation therapy: A narrative review. Molecular neurobiology, v. 55, n. 8, p. 6601–6636, 2018. DAVIES, D. J. et al. Photobiomodulation reduces hippocampal apoptotic cell death and produces a Raman spectroscopic “signature”. PloS one, v. 17, n. 3, p. e0264533, 2022. PILEGGI, C. A. et al. The SARS-CoV-2 spike glycoprotein interacts with MAO-B and impairs mitochondrial energetics. Current research in neurobiology, v. 5, n. 100112, p. 100112, 2023. CHEN, H. et al. Photobiomodulation modulates mitochondrial energy metabolism and ameliorates neurological damage in an APP/PS1 mousmodel of Alzheimer’s disease. Alzheimer’s research & therapy, v. 17, n. 1, p. 72, 2025. VIANA, F. D. et al. Proposta de ligante para o receptor de Neuropilina-1, alvo molecular do SARS-CoV-2 / Proposed ligand for the Neuropilin-1 receptor, molecular target of SARS-CoV-2. Brazilian Journal of Development, v. 8, n. 6, p. 46091–46108, 2022. CANTUTI-CASTELVETRI, L. et al. Neuropilin-1 facilitates SARS-CoV-2 cell entry and infectivity. Science (New York, N.Y.), v. 370, n. 6518, p. 856–860, 2020. ZHAO, X.; GUAN, J.-L. Focal adhesion kinase and its signaling pathways in cell migration and angiogenesis. Advanced drug delivery reviews, v. 63, n. 8, p. 610–615, 2011. NAESER, M. A. et al. Transcranial photobiomodulation Treatment: Significant improvements in four ex-football players with possible chronic traumatic encephalopathy. Journal of Alzheimer’s disease reports, v. 7, n. 1, p. 77–105, 2023. HARRIS, J. J.; JOLIVET, R.; ATTWELL, D. Synaptic energy use and supply. Neuron, v. 75, n. 5, p. 762–777, 2012. RANGARAJU, V.; LAUTERBACH, M.; SCHUMAN, E. M. Spatially stable mitochondrial compartments fuel local translation during plasticity. Cell, v. 176, n. 1–2, p. 73- 84.e15, 2019. KANN, O.; KOVÁCS, R. Mitochondria and neuronal activity. American journal of physiology. Cell physiology, v. 292, n. 2, p. C641-57, 2007. YOULE, R. J.; VAN DER BLIEK, A. M. Mitochondrial fission, fusion, and stress. Science (New York, N.Y.), v. 337, n. 6098, p. 1062–1065, 2012 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 27 Mar, 2026 Reviews received at journal 27 Jan, 2026 Reviewers agreed at journal 26 Jan, 2026 Reviewers invited by journal 06 Dec, 2025 Editor assigned by journal 03 Dec, 2025 Submission checks completed at journal 01 Dec, 2025 First submitted to journal 30 Nov, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8245044","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":557358451,"identity":"43d65301-4adb-4296-86d1-f451c1e5e8f6","order_by":0,"name":"Erick José Nogueira Andrade","email":"","orcid":"","institution":"University of Vale do Paraíba / Institute for Research and Development","correspondingAuthor":false,"prefix":"","firstName":"Erick","middleName":"José Nogueira","lastName":"Andrade","suffix":""},{"id":557358453,"identity":"95e53a97-6c02-4e43-b019-f0b4a8bcd4d1","order_by":1,"name":"Luiza de Andrade Giraldi","email":"","orcid":"","institution":"University of Vale do Paraíba / Institute for Research and Development","correspondingAuthor":false,"prefix":"","firstName":"Luiza","middleName":"de Andrade","lastName":"Giraldi","suffix":""},{"id":557358456,"identity":"560d03ad-d2c6-471d-86ca-7c6679674482","order_by":2,"name":"Milena Yuki Rosental Sudo","email":"","orcid":"","institution":"University of Vale do Paraíba / Institute for Research and Development","correspondingAuthor":false,"prefix":"","firstName":"Milena","middleName":"Yuki Rosental","lastName":"Sudo","suffix":""},{"id":557358457,"identity":"830b007b-db15-4a55-b828-ecaa5f6f5ec2","order_by":3,"name":"Lucas de Paula Ramos","email":"","orcid":"","institution":"Claude Bernard University Lyon 1","correspondingAuthor":false,"prefix":"","firstName":"Lucas","middleName":"de Paula","lastName":"Ramos","suffix":""},{"id":557358458,"identity":"c84f11bf-c8b8-4a99-8ffb-1be8e5f8f2d4","order_by":4,"name":"Geisa Rodrigues Salles","email":"","orcid":"","institution":"University of Vale do Paraíba / Institute for Research and Development","correspondingAuthor":false,"prefix":"","firstName":"Geisa","middleName":"Rodrigues","lastName":"Salles","suffix":""},{"id":557358459,"identity":"26d628ab-baf3-48b8-ae32-965d6bf73212","order_by":5,"name":"Cristina Pacheco-Soares","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAyElEQVRIiWNgGAWjYDADfiA+AGIYEK1FsoFkLQYHYAyC7pnd/vDBzz029sY3cg8e5qm5w2AufQC/Fok7Z4wNe56lJW67kZdwmOfYMwbLvgQC7pHIYZPgOXA4wexGjsHBGWyHGQzOEPKCRPozyT8H/tsbzwBp+UeUlgQzaZ4DBxg3SOQYHPjYRoQWkF+MZQ4kJ8448waope8Zj2UPAS2gEHv45oCdPX97jvGHhG935Mx5CGhhkEDlHiCoAVMLYR2jYBSMglEw4gAAio1H8iieUE0AAAAASUVORK5CYII=","orcid":"","institution":"University of Vale do Paraíba / Institute for Research and Development","correspondingAuthor":true,"prefix":"","firstName":"Cristina","middleName":"","lastName":"Pacheco-Soares","suffix":""}],"badges":[],"createdAt":"2025-12-01 01:38:25","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8245044/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8245044/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":98428600,"identity":"4ec2b8f6-de03-404e-b705-99df2754663d","added_by":"auto","created_at":"2025-12-17 16:42:11","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":4197153,"visible":true,"origin":"","legend":"","description":"","filename":"SpringerNatureErickAndrade.docx","url":"https://assets-eu.researchsquare.com/files/rs-8245044/v1/f4c05bca602bebb329a2d99b.docx"},{"id":98072887,"identity":"8d00e3e5-7025-40c2-8dd7-fec7096558d2","added_by":"auto","created_at":"2025-12-12 13:20:36","extension":"json","order_by":1,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":8213,"visible":true,"origin":"","legend":"","description":"","filename":"812c0afbb271421a8e2b4d99ed4058e1.json","url":"https://assets-eu.researchsquare.com/files/rs-8245044/v1/f4b32ee093741fa147a0ed63.json"},{"id":98429406,"identity":"f660bb7b-da8a-448e-925a-a578106cd8c0","added_by":"auto","created_at":"2025-12-17 16:43:21","extension":"xml","order_by":2,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":80443,"visible":true,"origin":"","legend":"","description":"","filename":"812c0afbb271421a8e2b4d99ed4058e11enriched.xml","url":"https://assets-eu.researchsquare.com/files/rs-8245044/v1/edb2181657a459610b09b494.xml"},{"id":98429566,"identity":"35112188-6af9-4044-9967-09e9921e68d2","added_by":"auto","created_at":"2025-12-17 16:43:43","extension":"jpeg","order_by":3,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":806118,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8245044/v1/6f467f57aadfa96a75cf399f.jpeg"},{"id":98072889,"identity":"5a43419e-cde6-4105-8f97-1971961cdecf","added_by":"auto","created_at":"2025-12-12 13:20:36","extension":"jpeg","order_by":4,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":290182,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8245044/v1/957a6417372db2fa8cb4c093.jpeg"},{"id":98072903,"identity":"a1efc57f-3d73-4113-995e-cba208613bc7","added_by":"auto","created_at":"2025-12-12 13:20:36","extension":"jpeg","order_by":5,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":738346,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8245044/v1/6dabfa514c69ce5373156646.jpeg"},{"id":98428705,"identity":"558f7a7c-c4c8-4e61-b8e9-c031b55ec6c6","added_by":"auto","created_at":"2025-12-17 16:42:17","extension":"jpeg","order_by":6,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":718938,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8245044/v1/d3f3f57162d972e8298f2428.jpeg"},{"id":98429713,"identity":"51ad7a4c-2f0f-4383-9a5a-c72771b62d0a","added_by":"auto","created_at":"2025-12-17 16:44:02","extension":"jpeg","order_by":7,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":728756,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8245044/v1/8cbbe9aa8c995d189ee8ded4.jpeg"},{"id":98072900,"identity":"e3e5be83-6b92-4839-9205-c28332ec81c9","added_by":"auto","created_at":"2025-12-12 13:20:36","extension":"png","order_by":8,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":187225,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-8245044/v1/d363f0bcf653930d17a57fad.png"},{"id":98072895,"identity":"a0b796f6-ea53-4858-bb40-1be2128c4d28","added_by":"auto","created_at":"2025-12-12 13:20:36","extension":"png","order_by":9,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":64328,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-8245044/v1/2718a276ebe69865da77015d.png"},{"id":98428253,"identity":"e071dda2-74a1-4a73-96b1-786d0a7193f2","added_by":"auto","created_at":"2025-12-17 16:41:51","extension":"png","order_by":10,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":69602,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-8245044/v1/88200b3abbe71db5fcc3d887.png"},{"id":98428718,"identity":"3cbbf812-4e73-4a66-a627-aec9b7f5b0fc","added_by":"auto","created_at":"2025-12-17 16:42:18","extension":"png","order_by":11,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":69533,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-8245044/v1/a70f64b53cb2600c6b7d77e2.png"},{"id":98429547,"identity":"33a60216-3165-476a-a272-fe6e61efb8a9","added_by":"auto","created_at":"2025-12-17 16:43:40","extension":"png","order_by":12,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":181068,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-8245044/v1/13f502a06a29b0333273cd0b.png"},{"id":98072905,"identity":"70bc6d4a-1b90-4372-80ea-966c24b5d7a5","added_by":"auto","created_at":"2025-12-12 13:20:36","extension":"xml","order_by":13,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":80336,"visible":true,"origin":"","legend":"","description":"","filename":"812c0afbb271421a8e2b4d99ed4058e11structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-8245044/v1/2e224dbb3adfb165262b1ecc.xml"},{"id":98072906,"identity":"bc42077c-9380-4ca5-af94-bdbafaac3f52","added_by":"auto","created_at":"2025-12-12 13:20:36","extension":"html","order_by":14,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":89380,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8245044/v1/cad07e529ae3ffd0530658fe.html"},{"id":98072898,"identity":"c818d702-9237-49b5-b25f-0031733b9fad","added_by":"auto","created_at":"2025-12-12 13:20:36","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1257056,"visible":true,"origin":"","legend":"\u003cp\u003eCytoskeletal organization, focal adhesion, and nuclear morphology of neuron-like cells exposed or not to Spike protein. (A) Control condition showing well-organized actin filaments (phalloidin, red), defined focal adhesion sites (FAK, green), and preserved nuclei arrangment (DAPI, blue). Arrows highlight representative structures: actin filaments (red arrow), focal adhesions (green arrow), and preserved nuclear morphology (cyan arrow). (B) Cells exposed to Spike protein (0.5 µg/mL) exhibited cytoskeletal disorganization, altered FAK distribution, and nuclear condensation. Arrows indicate disrupted actin filaments (red arrow), redistributed focal adhesion points (green arrow), and condensed or irregular nuclei (cyan arrow). Scale bars: 20 µm.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8245044/v1/483724e86c161d1a6ec7ece9.png"},{"id":98072886,"identity":"8193c281-c9a8-431f-8612-d9e3eed4db2a","added_by":"auto","created_at":"2025-12-12 13:20:36","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":180639,"visible":true,"origin":"","legend":"\u003cp\u003eCytoviability of neuron-like cells exposed or not to Spike protein and/or H₂O₂-induced oxidative stress treated or not with photobiomodulation (PBM). (A) Cell viability expressed as percentage of control after exposure to H₂O₂ (200 µM), Spike protein (0.5 µg/mL), or the combined Spike + H₂O₂ condition. Bars represent mean ± SD. Statistical significance among groups is indicated by * (p \u0026lt; 0.05), *** (p \u0026lt; 0.001), **** (p \u0026lt; 0.0001), and ns (not significant). (B) Effect of PBM (660 nm, 3 J/cm²) on cell viability assessed 2 h after irradiation. Hatched bars represent PBM-treated groups. PBM attenuated the cytotoxic effects induced by H₂O₂ and Spike protein, preserving cell viability. Statistical significance is indicated by ** (p \u0026lt; 0.01) and *** (p \u0026lt; 0.001) compared to the respective non-irradiated controls.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-8245044/v1/1349ff0165fe697f64ab2562.png"},{"id":98429410,"identity":"8c2a7272-833c-4e7f-b867-7d1002fcb347","added_by":"auto","created_at":"2025-12-17 16:43:22","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":459874,"visible":true,"origin":"","legend":"\u003cp\u003eNuclei micrographs of neuron-like cells not irradiated (top row) and PBM-irradiated (bottom row) under the following conditions: Control, H₂O₂, Spike, and Spike + H₂O₂ stained with DAPI. Control cells exhibited uniformly distributed nuclei with preserved morphology. H₂O₂ induced chromatin condensation and nuclear fragmentation (red arrow). Spike protein-exposed cells reduced nuclear density and moderate chromatin condensation (yellow arrow). The Spike + H₂O₂ group displayed severe nuclear damage. PBM-treated groups exhibited nuclei with reduced condensation and better structural preservation. Scale bars: 10 µm.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-8245044/v1/df50ea5c6a7db8ca84616c7d.png"},{"id":98428201,"identity":"4dcbeff4-0dcb-41d8-ae86-c64dfc63f95f","added_by":"auto","created_at":"2025-12-17 16:41:46","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":442635,"visible":true,"origin":"","legend":"\u003cp\u003eMitochondrial micrographs of neuron-like cells not irradiated (top row) and PBM-irradiated (bottom row) under the following conditions: Control, H₂O₂, Spike, and Spike + H₂O₂, stained with MitoTracker. Control cells displayed an organized and reticular mitochondrial aspect. Spike protein-exposed cells reduced intensity and demonstrated partial fragmentation. H₂O₂-exposure induced severe fragmentation (yellow zoomed in area) and mitochondrial loss, whereas the Spike + H₂O₂ group exhibited pronounced disruptions (white zoomed in area). PBM-treated groups demonstrated visibly improved mitochondrial organization and reduced fragmentation. Scale bars: 10 µm.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-8245044/v1/5e652fcac9ac0529ac83f9a9.png"},{"id":98428818,"identity":"0774a48f-b07e-4f56-b634-790035863b7e","added_by":"auto","created_at":"2025-12-17 16:42:26","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":915512,"visible":true,"origin":"","legend":"\u003cp\u003eMitochondrial and nuclear morphology of neuron-like cells exposed to the Spike protein, with or without photobiomodulation (PBM). Immunofluorescence images of non-irradiated groups (top row) and PBM-irradiated groups (bottom row). The mitochondria were labeled with MitoTracker (red), and the nuclei were stained with DAPI (blue). PBM (660 nm, 3 J/cm²) attenuated mitochondrial fragmentation and preserved nuclear morphology in the irradiated Spike group, where the mitochondria appear less punctate and the nuclei exhibit a more uniform chromatin compared to the corresponding non-irradiated Spike panel. Scale bars: 10 µm.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-8245044/v1/ffb43be93678e7dbff1bc1f2.png"},{"id":98774884,"identity":"430ffb61-0550-4b57-9f6a-1463cfae6b9e","added_by":"auto","created_at":"2025-12-22 12:16:07","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4630778,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8245044/v1/ae61b487-915a-4fe5-9044-31b9c0cd1f1c.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"From Neurotoxicity to Neuroprotection: Photobiomodulation against the Effects of the SARS-CoV-2 Spike Protein in an Alzheimer's Disease Model","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eAlzheimer's disease (AD) is one of the highest public health challenges worldwide and is the leading cause of dementia in the elderly population. It is a progressive and irreversible neurodegenerative disorder characterised by the accumulation of amyloid -β (Aβ) plaques, neurofibrillary tangles of hyperphosphorylated tau protein, chronic neuroinflammation, and oxidative stress, ultimately resulting in synaptic loss, neuronal death, and severe cognitive decline [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Despite more than a century of research, there are currently no curative treatments available, and existing pharmacological therapies only offer symptomatic relief, highlighting the urgent need for new therapeutic approaches [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIn parallel, the COVID-19 pandemic (Coronavirus disease 2019), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has raised growing concerns regarding its long-term neurological consequences. Recent evidence suggests that the viral Spike (S) protein, responsible for mediating cellular entry through the angiotensin-converting enzyme 2 (ACE2) receptor, may trigger or accelerate neurodegenerative processes [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. The recombinant S protein has been shown to induce neuroinflammation, oxidative stress, and mitochondrial dysfunction and even exacerbate Alzheimer's-related pathologies, such as Tau hyperphosphorylation and blood-brain barrier disruption [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. This potential neurotoxic effect highlights the spike protein as a relevant biological tool for modelling pathogenic mechanisms of neurodegeneration \u003cem\u003ein vitro\u003c/em\u003e [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIn this context, photobiomodulation (PBM) emerges as a promising non-invasive therapeutic modality. Using red or near-infrared light, PBM primarily targets the mitochondria, promoting increased adenosine triphosphate (ATP) production, reducing oxidative stress, and modulating inflammatory responses [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Its neuroprotective effects have been demonstrated in preclinical models of neurodegenerative diseases, showing a reduction in amyloid plaques and improvement in cognitive performance [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. PBM with red light (~\u0026thinsp;660 nm) is particularly associated with the activation of cytochrome c oxidase, leading to enhanced mitochondrial bioenergetics and improved neuronal survival under oxidative stress conditions [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eTwo-dimensional (2D) cultures of the human neuroblastoma cell line (SH-SY5Y) differentiated into neuron-like cells are widely used as an \u003cem\u003ein vitro\u003c/em\u003e model to study the molecular mechanisms underlying AD. These cells express key neuronal markers and are highly suitable for investigating the cytotoxic effects of oxidative and inflammatory stimuli, as well as for testing potential neuroprotective strategies [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. The exposure of differentiated SH-SY5Y cells to the S protein and recombinant protein provides a controlled and reproducible platform to evaluate neuronal responses and elucidate possible interactions between SARS-CoV-2 infection and neurodegenerative disorders such as AD [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eTherefore, this study aimed to evaluate the effects of the SARS-CoV-2 S protein in a 2D \u003cem\u003ein vitro\u003c/em\u003e model of AD and investigate the potential therapeutic role of PBM with red LED light in this context.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cp\u003e\u003cstrong\u003eReagents and Equipment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll reagents, chemicals, antibodies, suplements, and equipment were obtained from certified suppliers and handled according to manufacturer instructions to ensure reproducibility. The complete list of materials used across cell culture, differentiation, treatments, staining, imaging, and data analysis workflows is provided in Table 1.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 1\u003c/strong\u003e - Reagents, suplements, equipment, and software used in the experimental procedures\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eReagent or resource\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eS\u003c/strong\u003e\u003cstrong\u003eource\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eI\u003c/strong\u003e\u003cstrong\u003edentifier\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eReagents, antibodies, supplements, and other chemicals\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eDAPI\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eSigma-Aldrich, USA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eD9564\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eDulbecco\u0026rsquo;s modified Eagle\u0026rsquo;s medium/nutrient mixture F-12 (DMEM/F12)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eGibco, USA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e12400024\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003efetal bovine serum (FBS)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eGibco, USA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e12657-029\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eHydrogen peroxide (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eMolekular Qu\u0026iacute;mica, Brazil\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eL0032-P1L\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eParaformaldehyde\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eSigma-Aldrich, USA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e30525-89-4\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003ePenicillin/streptomycin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eGibco, USA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e15140-122\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003ePhalloidin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eSigma-Aldrich, USA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e49409\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003ePhosphate-buffered saline (PBS)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eSigma-Aldrich, USA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e806552\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eRecombinant SARS-CoV-2 Spike protein (S)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eLECC \u0026ndash; UFRJ, Brazil\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eLECC-COV2-AC001 (lot: O-151021)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eResazurin sodium salt\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eSigma-Aldrich, USA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eR7017\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eRetinoic acid (RA)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eSigma-Aldrich, USA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eR2625\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eTriton X-100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eSigma-Aldrich, USA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e9002-93-1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eTrypsin \u0026ndash; EDTA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eGibco, USA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e15090046\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eMitoTracker\u0026trade;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eInvitrogen, USA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eM7510\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eHoechst 33342\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eInvitrogen, USA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eH3570\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eAnti-\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003efocal adhesion kinase\u003cstrong\u003e\u0026nbsp;(\u003c/strong\u003eFAK\u003cstrong\u003e)\u003c/strong\u003e antibody\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eCell Signaling Technology, USA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e3285\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003ePlastics and other nonperishable materials\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003ecell culture flasks (25 cm\u0026sup2;)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eNest, China\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e707003\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003ecell culture plates (24-well)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eKasvi, Brazil\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eK12-024\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003ehive-patterned array\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eBioEdTech, Brazil\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eBio3DStamp version 6\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eEquipment and software\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eCO₂ incubator\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003ePanasonic, Japan\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eMCO-170AICUV-PA\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003econfocal laser scanning microscope\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eLeica, Germany\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eTCS SP8\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eGraphPad Prism 5.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eGraphPad Inc., USA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003ePrism 5.0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eImageJ Fiji\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eNational Institutes of Health, USA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003ca href=\"https://imagej.net/ij/\"\u003ehttps://imagej.net/ij/\u003c/a\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003emicroplate reader\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eBmg Labtech, Germany\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eVANTAstar\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCell Line and Culture Conditions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHuman neuroblastoma SH-SY5Y cells (ATCC\u0026reg; CRL-2266\u0026trade;) were used as an \u003cem\u003ein vitro\u003c/em\u003e neuronal model. Cells were cultured in 25 cm\u0026sup2; cell culture flasks containing the following complete medium: DMEM/F12 (Dulbecco\u0026rsquo;s Modified Eagle Medium/Nutrient Mixture F-12; Gibco, Thermo Fisher Scientific, USA), supplemented with 10% (v/v) fetal bovine serum (FBS; Gibco), 1% (v/v) penicillin-streptomycin (100 U/mL and 100\u0026nbsp;\u0026mu;g/mL, respectively; Invitrogen), and maintained at 37 \u0026deg;C in a humidified incubator with 5% CO₂. The medium was changed every 48 hours to preserve nutrient availability and pH stability.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCell Seeding and Neuronal Differentiation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWhen the cultures reached approximately 80% confluence, cells were detached using trypsin-EDTA, and seeded in 24-well plates at a density of 1\u0026times;10⁵ cells/mL. After 48 hours in complete medium, neuronal differentiation was induced for 5 days using the following differentiation medium: DMEM/F12 supplemented with 10\u0026nbsp;\u0026mu;M retinoic acid (RA), 0.5% FBS, and 1% penicillin-streptomycin. The differentiation was confirmed by morphological changes, including the extension of neurites and the reduction of proliferative morphology.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eExperimental groups\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDifferentiated SH-SY5Y cells were distributed into eight experimental conditions to assess the isolated and combined effects of oxidative stress, SARS-CoV-2 S protein, and photobiomodulation (PBM). The groups included: Control, H₂O₂-induced oxidative stress, PBM, H₂O₂ followed by PBM, S protein, S protein followed by PBM, simultaneous exposure to H₂O₂ and S protein, and the combined treatment with H₂O₂ and S protein followed by PBM. All treatments were performed under identical culture conditions, and specific doses, exposure times, and irradiation parameters are detailed in Table 2, which summarizes the full description of each experimental group.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2\u003c/strong\u003e - Experimental groups and corresponding treatments\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 67px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eG\u003c/strong\u003e\u003cstrong\u003eroup\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 557px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eT\u003c/strong\u003e\u003cstrong\u003ereatment\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 67px;\"\u003e\n \u003cp\u003eControl\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 557px;\"\u003e\n \u003cp\u003eDifferentiated SH-SY5Y cells without additional treatment.\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 67px;\"\u003e\n \u003cp\u003eOxidative Stress (H₂O₂)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 557px;\"\u003e\n \u003cp\u003eExposure to 200 \u0026micro;M H₂O₂ for 1 h.\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 67px;\"\u003e\n \u003cp\u003ePbm\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 557px;\"\u003e\n \u003cp\u003ePBM (660 nm, 3 J/cm\u0026sup2;).\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 67px;\"\u003e\n \u003cp\u003eH₂O₂ + PBM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 557px;\"\u003e\n \u003cp\u003eH₂O₂ (200 \u0026micro;M, 1 h) followed by PBM (660 nm, 3 J/cm\u0026sup2;).\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 67px;\"\u003e\n \u003cp\u003eS\u0026nbsp;protein\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 557px;\"\u003e\n \u003cp\u003eTreatment with recombinant S protein (0.5 \u0026micro;g/mL) for 1 h.\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 67px;\"\u003e\n \u003cp\u003eS\u0026nbsp;protein + PBM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 557px;\"\u003e\n \u003cp\u003eS protein (0.5 \u0026micro;g/mL, 1 h) followed by PBM (660 nm, 3 J/cm\u0026sup2;).\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 67px;\"\u003e\n \u003cp\u003eH₂O₂ + S\u0026nbsp;protein\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 557px;\"\u003e\n \u003cp\u003eSimultaneous exposure to H₂O₂ (200 \u0026micro;M) and S protein (0.5 \u0026micro;g/mL) for 1 h.\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 67px;\"\u003e\n \u003cp\u003eH₂O₂ + S\u0026nbsp;protein + PBM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 557px;\"\u003e\n \u003cp\u003eCombined exposure to H₂O₂ (200 \u0026micro;M) and S protein (0.5 \u0026micro;g/mL) for 1 h, followed by PBM (660 nm, 3 J/cm\u0026sup2;).\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eOxidative stress induction\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOxidative stress was induced using hydrogen peroxide (H₂O₂), which was prepared as a stock solution of 160,000\u0026nbsp;\u0026mu;M and serially diluted in the differentiation medium to obtain a final working concentration of 200\u0026nbsp;\u0026mu;M. All dilutions were prepared immediately before use to preserve the stability of reactive oxygen species (ROS). Cells were exposed to H₂O₂ for 1 hour at 37 \u0026deg;C in an incubator with 5% CO₂.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eExposure to recombinant SARS-CoV-2 Spike protein\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe recombinant S protein (amino acids 1\u0026ndash;1208) of SARS-CoV-2, produced at the Cell Culture Engineering Laboratory (COPPE/UFRJ, Rio de Janeiro, Brazil; batch 0151021), was provided in phosphate-buffered saline (PBS) containing biotin and 0.02% sodium azide, at 0.25 mg/mL. For the assays, the protein was diluted to 0.5\u0026nbsp;\u0026mu;g/mL in differentiation medium and applied directly to the cells for 1 hour at 37 \u0026deg;C and 5% CO₂. The handling and storage of the S protein were performed under sterile conditions and temperature control to preserve structural integrity and receptor binding function.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePhotobiomodulation (PBM) treatment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAfter exposure to H₂O₂ and/or the S protein, the groups designated for PBM were irradiated with a red light-emitting diode (LED) device (\u0026lambda;\u0026nbsp;= 660 nm). The irradiation was carried out under controlled lighting conditions, positioning the culture plates perpendicularly to the light source to ensure uniform exposure. The total energy density applied was 3 J/cm\u0026sup2;, corresponding to an exposure time of 1 min 22 s. All parameters were standardized to ensure reproducibility and avoid thermal effects.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCell viability assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCell viability was assessed using the Alamar Blue\u003csup\u003e\u0026reg;\u0026nbsp;\u003c/sup\u003eassay. After the treatments, the culture medium was replaced by a solution containing 90% differentiation medium and 10% Alamar Blue reagent. Cells were incubated for 2 hours at 37 \u0026deg;C and 5% CO₂, protected from light. Next, 100 \u0026micro;L aliquots were transferred to a 96-well plate and fluorescence was measured using a microplate reader with excitation at 544 nm and emission at 590 nm. Results were expressed as a percentage of the control group. All experiments were performed in triplicate.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePreliminary analysis of Spike protein effects on neuronal cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBefore inducing oxidative stress to mimic AD condition, differentiated SH-SY5Y cells were exposed to recombinant S protein to assess its direct impact on neuronal morphology and cytoskeletal organization. Immunofluorescence staining with phalloidin, anti-FAK, and DAPI was performed to assess cytoskeletal integrity, cell adhesion protein, and nuclear morphology. Basically, cells were fixed with 4% paraformaldehyde for 20 min, permeabilized with 0.1% Triton X-100, and stained overnight at 4 \u0026deg;C with phalloidin (1:500) and DAPI (1:5000). For FAK detection, cells were incubated with anti-FAK primary antibody (anti-rabbit) followed by a fluorophore-conjugated secondary antibody, each for 1 hour at 37 \u0026deg;C. This preliminary analysis provided a basic understanding of the cellular changes induced by S protein before the induction of oxidative stress and subsequent treatments.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMitochondria and nuclei staining\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor the analysis of mitochondrial and nuclear integrity under AD-related conditions, they were stained with MitoTracker\u0026trade; Orange CMTMRos (1 \u0026micro;M) and Hoechst. After 30 minutes staining at 37 \u0026deg;C, cells were washed with PBS and imaged using a confocal laser scanning microscope. Images were acquired using identical microscope parameters across all experimental groups.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll experiments were conducted in triplicate. Data are presented as mean \u0026plusmn; standard deviation (SD). Statistical significance was determined using one-way analysis of variance (ANOVA) followed by Tukey\u0026apos;s post hoc test, conducted in GraphPad Prism 5.0 (GraphPad Software Inc., USA). P-values \u0026lt; 0.05 were considered statistically significant.\u003c/p\u003e"},{"header":"RESULTS AND DISCUSSION","content":"\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003eChanges in the cytoskeleton and focal adhesion induced by the Spike protein\u003c/h2\u003e\u003cp\u003eS Protein caused significant changes in the cytoskeleton and focal adhesion (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Phalloidin staining showed disorganization of actin filaments, including signs of depolymerization and loss of continuity (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB, phalloidin panel). These findings are consistent with studies that report the interaction of the S protein with ACE2 and neuropilin-1 activating pathways that directly modulate actin rearrangement [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIn parallel, an intensification of FAK labeling was observed in the group exposed to S protein (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB, FAK panel), suggesting the activation of compensatory mechanisms of cell adhesion and survival, as already documented in adaptive responses to mechanical and chemical stress [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Notably, despite the cytoskeletal disorganization, cell nuclei of the S protein group remained morphologically preserved (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB, DAPI panel), which is in accordance with reports that viral components can activate survival pathways even under cytoskeletal stress conditions [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThis set of data indicates that the S protein simultaneously interferes with cellular structure and signaling, affecting adhesion, the cytoskeleton, and possibly intracellular trafficking, aspects relevant to understanding its neurodegenerative action (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003eDamage to cell viability induced by the Spike protein and oxidative stress\u003c/h2\u003e\u003cp\u003eThe evaluation of cell viability demonstrated that both S protein and H₂O₂ were able to significantly reduce the survival of differentiated neuronal cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Exposure to protein S reduced viability to about 80% of the control, while H₂O₂ decreased levels to approximately 60% of the control. These findings indicate that both stimuli exert relevant cytotoxic effects, although with distinct magnitudes [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe decrease in viability observed in the groups exposed to the S protein is consistent with recent evidence showing that the S protein can interact with integrins and dysregulate focal adhesion pathways, compromising essential anchoring and neuronal survival mechanisms [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. On the other hand, H₂O₂, a classic inducer of oxidative stress, exerts more pronounced toxicity, inducing excessive production of reactive oxygen species, metabolic reduction, and apoptosis [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe combination of S protein\u0026thinsp;+\u0026thinsp;H₂O₂ resulted in a cytodamage similar to the group treated only with H₂O₂ (~\u0026thinsp;60%), but the micrographs revealed more pronounced structural damage, suggesting a synergistic effect between the factors. This pattern has already been described in neuronal systems exposed simultaneously to pro-oxidant stimuli and viral proteins, in which mitochondrial vulnerability amplifies apoptotic processes [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Thus, data suggest that S protein acts as a sensitizer of oxidative stress, intensifying structural damage even when residual viability is not drastically reduced.\u003c/p\u003e\u003cp\u003eWhen PBM with LED was applied to the groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB), partial preservation of viability was observed in all groups subjected to oxidative stress induced by exposure to H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e. This preservation, compared to the groups not treated with PBM, suggests a protective effect, possibly mediated by increased cytochrome C oxidase activity, improved energy metabolism, and modulation of the redox balance, as previously demonstrated in cellular and animal models [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003eNuclear alterations and morphofunctional evidence of apoptosis\u003c/h2\u003e\u003cp\u003eNuclear staining with Hoechst (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e) revealed striking differences between the groups. The control group demonstrated intact nuclei distributed evenly, while the S protein group showed a moderate reduction in the number of nuclei per area and signs of chromatin condensation. These findings suggest the initial activation of apoptotic processes, consistent with reports that the S protein activates cellular stress pathways and nuclear reorganization without necessarily inducing immediate cell death [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eH₂O₂ exposure promoted strong nuclear condensation and fragmentation, a classic profile of apoptosis induced by severe oxidative stress, as described in neuronal models exposed to ROS [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. The S protein\u0026thinsp;+\u0026thinsp;H₂O₂ group exhibited the most severe pattern, reinforcing the hypothesis of degenerative synergism between the viral protein and oxidative stress.\u003c/p\u003e\u003cp\u003eIn the groups treated with PBM, a reduction in nuclear condensation was observed, with nuclei being more distributed and less fragmented, indicating that PBM attenuated structural impairment. Mechanistically, PBM is capable of reducing DNA damage, modulating repair mechanisms, and decreasing caspase activation, which may explain this partial preservation [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003eMitochondrial dysfunction induced by the Spike protein and H₂O₂\u003c/h2\u003e\u003cp\u003eLive mitochondrial staining revealed a clear mitochondrial dysfunction in the groups subjected to the treatments (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). In the control group, mitochondrial arrangment was organized and distributed in a reticular pattern. In the S protein exposed group, there was partial fragmentation, a decrease in staining intensity, and loss of their distribution in neurite extensions, consistent with compromised membrane potential and impaired mitochondrial transport [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eOnce exposed to H₂O₂, mitochondrial fragmentation and reduced staining were observed, as expected of a classic inducer of mitochondrial fission and early apoptosis [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. The S protein\u0026thinsp;+\u0026thinsp;H₂O₂ group exhibited the most critical behaviour: almost complete mitochondrial network collapse and no observation of mitochondria in neurite extensions, suggesting loss of essential energy functionality for mitochondria-dependent neurons. Such vulnerability is explained by the fact that neurons are cells highly dependent on mitochondrial function, due to their exceptional energy demands and specialized morphology. The maintenance of membrane potential, the conduction of electrical impulses, and axonal transport consume large amounts of ATP, most of which is generated via mitochondrial oxidative phosphorylation [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Moreover, mitochondria essential regulate calcium homeostasis, especially at synapses, where signaling is energetically costly and spatially restricted [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. These organelles also act as central regulators of cell death, modulating apoptotic pathways that are particularly critical in neurons, given their limited regenerative capacity [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Thus, any disturbance in mitochondrial dynamics or function directly compromises neuronal viability, making these cells especially vulnerable to oxidative stress stimuli, such as H₂O₂.\u003c/p\u003e\u003cp\u003ePBM partially restored mitochondrial morphology, with less fragmented networks and homogeneous distribution, consistent with the increase in ATP and recovery of membrane potential already, as a suggestive consequence of cytochrome c oxidase activation by the exposure to red light [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. This partial protection reinforces the potential of PBM as a modulator of mitochondrial degenerative processes.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003eProtective effects of photobiomodulation\u003c/h2\u003e\u003cp\u003ePBM promoted a protective effect on multiple cellular parameters, including viability, nuclear morphology, cytoskeletal integrity, and mitochondrial functionality (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). The literature supports that the absorption of red light by cytochrome c oxidase improves mitochondrial respiration, modulates survival pathways, reduces apoptosis, and increases the expression of neurotrophic factors, which represent key elements against neurodegenerative processes [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThese mechanisms may explain the improvement observed in the reduced mitochondrial fragmentation and nuclear preservation in the PBM-treated groups, as illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, where irradiated cells exhibit more continuous mitochondrial staining, fewer intensely fragmented puncta, and nuclei with more homogeneous chromatin compared to their non-irradiated counterparts under the same treatments.. Although the effect is not sufficient to fully restore the phenotype of healthy neuron, the consistent attenuation of damage in multiple pathways suggests that PBM consists in a therapeutic strategy, modulating energetic, oxidative, and structural aspects.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"CONCLUSIONS","content":"\u003cp\u003eThe S protein of SARS-CoV-2 triggers structural and functional changes in neuronal models, including reduced viability, cytoskeletal disorganization, mitochondrial fragmentation, and modulation of focal adhesion. These effects were intensified by oxidative stress, indicating a relevant synergistic action for understanding the mechanisms of neurotoxicity associated with COVID-19.\u003c/p\u003e\u003cp\u003eLED PBM represents a remarkable and safe non-invasive tool against neurodegeneration, capable of mitigating some of the evaluated parameters, preserving the bioenergetic and morphological aspects of the neuronal cells. Together, the findings reinforce the role of protein S as a neurotoxic agent and PBM as a promising non-pharmacological strategy to modulate neurodegenerative processes and guide future relevant neurobiological and clinical research in AD.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eInvestigation and conceptualization: All authors;Methodology: EJNA, LAG, MYRS, LPR, GRS;Supervision: CPS;Writing: All authors;All authors reviewed the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eWe gratefully acknowledge M.Sc. Priscila M. S. C. M. Leite and Dr. Angela A. M. Vieira, from the Research \u0026amp; Development Institute (IP\u0026amp;D/Univap), for their assistance with confocal microscopy procedures. We also thank Prof. Dr. Leandro Jos\u0026eacute; Raniero and his team for providing access to the microplate reader and technical support, as well as Prof. Dr. Juliana Ferreira Strixino and her team for granting access to the LED chamber. This work was supported by the Brazilian National Council for Scientific and Technological Development (CNPq), Grants 174942/2024-9 (EJNA), 138459/2024-0 (LAG), 157999/2025-4 (LAG), 152384/2024-3 (GRS) and 404953/2025-5 (CPS, CNPQ/MCTI n\u0026ordm; 44/2024).\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003edata are available from the authors\u0026nbsp;upon\u0026nbsp;request\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eQUERFURTH, H. W.; LAFERLA, F. M. Alzheimer\u0026rsquo;s disease. The New England journal of medicine, v. 362, n. 4, p. 329\u0026ndash;344, 2010. \u003c/li\u003e\n\u003cli\u003eSCHELTENS, P. et al. Alzheimer\u0026rsquo;s disease. Lancet, v. 397, n. 10284, p. 1577\u0026ndash;1590, 2021. \u003c/li\u003e\n\u003cli\u003eYIANNOPOULOU, K. G.; PAPAGEORGIOU, S. G. Current and future treatments for Alzheimer\u0026rsquo;s disease. Therapeutic advances in neurological disorders, v. 6, n. 1, p. 19\u0026ndash;33, 2013. \u003c/li\u003e\n\u003cli\u003eSONG, E. et al. Neuroinvasion of SARS-CoV-2 in human and mouse brain. The journal of experimental medicine, v. 218, n. 3, 2021. \u003c/li\u003e\n\u003cli\u003eFRANK, M. G. et al. SARS-CoV-2 spike S1 subunit induces neuroinflammatory, microglial and behavioral sickness responses: Evidence of PAMP-like properties. Brain, behavior, and immunity, v. 100, p. 267\u0026ndash;277, 2022. \u003c/li\u003e\n\u003cli\u003eOLAJIDE, O. A. et al. SARS-CoV-2 spike glycoprotein S1 induces neuroinflammation in BV-2 microglia. Molecular neurobiology, v. 59, n. 1, p. 445\u0026ndash;458, 2022. \u003c/li\u003e\n\u003cli\u003eSWAIN, S. SARS-CoV-2 spike protein exacerbates tau pathology and neuroinflammation in Alzheimer\u0026rsquo;s models. Acta Neuropathologica Communications, v. 15, n. 11, [s.d.].\u003c/li\u003e\n\u003cli\u003eBARBOSA, E. M. Neurotoxic effects of SARS-CoV-2 spike protein on human neuronal cells: implications for neurodegeneration. Neurochemical Research, v. 49, p. 1123\u0026ndash;1136, 2024.\u003c/li\u003e\n\u003cli\u003eHAMBLIN, M. R. Shining light on the head: Photobiomodulation for brain disorders. BBA clinical, v. 6, p. 113\u0026ndash;124, 2016.\u003c/li\u003e\n\u003cli\u003eSALEHPOUR, F. Transcranial photobiomodulation therapy: a potential treatment for Alzheimer\u0026rsquo;s disease and Parkinson\u0026rsquo;s disease. Frontiers in Neuroscience, v, v. 12, 2018.\u003c/li\u003e\n\u003cli\u003eDA LUZ, S. C. Photobiomodulation therapy in Alzheimer\u0026rsquo;s disease models: from mechanisms to clinical perspectives. Frontiers in Neuroscience, v. 16, 2022.\u003c/li\u003e\n\u003cli\u003eGRILLO, S. L. Red and near-infrared photobiomodulation improve cognition and reduce amyloid burden in Alzheimer\u0026rsquo;s disease models. Brain Research Bulletin, v, v. 199, p. 38\u0026ndash;47, 2023.\u003c/li\u003e\n\u003cli\u003eHAMBLIN, M. R. Mechanisms and mitochondrial redox signaling in photobiomodulation. Photochemistry and photobiology, v. 94, n. 2, p. 199\u0026ndash;212, 2018.\u003c/li\u003e\n\u003cli\u003eKOVALEVICH, J.; LANGFORD, D. Considerations for the use of SH-SY5Y neuroblastoma cells in neurobiology. Methods in molecular biology (Clifton, N.J.), v. 1078, p. 9\u0026ndash;21, 2013. \u003c/li\u003e\n\u003cli\u003eXICOY, H.; WOUTERS, R.; VANDENBERGHE, W. The SH-SY5Y cell line in Parkinson\u0026rsquo;s disease research: a systematic review. Molecular Neurodegeneration, v, v. 12, 2017.\u003c/li\u003e\n\u003cli\u003eSALLES, G. R. et al. 2D and 3D models of Alzheimer\u0026rsquo;s disease: Investigating neuron-like cells in oxidative environments. ACS omega, v. 10, n. 25, p. 27501\u0026ndash;27514, 2025.\u003c/li\u003e\n\u003cli\u003eNORRIS, E. G.; PAN, X. S.; HOCKING, D. C. Receptor-binding domain of SARS-CoV-2 is a functional \u0026alpha;v-integrin agonist. The journal of biological chemistry, v. 299, n. 3, p. 102922, 2023.\u003c/li\u003e\n\u003cli\u003eNORRIS, K.; PAN, T.; HOCKING, D. C. SARS-CoV-2 Spike protein disrupts integrin-mediated cell adhesion and signaling. Journal of Molecular Biology, 2023.\u003c/li\u003e\n\u003cli\u003eFAN, X.; HUSSIEN, R.; BROOKS, G. A. H₂O₂-induced oxidative stress triggers mitochondrial fission and apoptosis in neuronal cells. American Journal of Physiology - Cell Physiology, 2010.\u003c/li\u003e\n\u003cli\u003ePENDERGRASS, W.; WOLF, N.; POOT, M. Evidences of mitochondria-mediated apoptosis after oxidative injury. Experimental Gerontology, 2004.\u003c/li\u003e\n\u003cli\u003eSALEHPOUR, F. et al. Brain photobiomodulation therapy: A narrative review. Molecular neurobiology, v. 55, n. 8, p. 6601\u0026ndash;6636, 2018.\u003c/li\u003e\n\u003cli\u003eDAVIES, D. J. et al. Photobiomodulation reduces hippocampal apoptotic cell death and produces a Raman spectroscopic \u0026ldquo;signature\u0026rdquo;. PloS one, v. 17, n. 3, p. e0264533, 2022.\u003c/li\u003e\n\u003cli\u003ePILEGGI, C. A. et al. The SARS-CoV-2 spike glycoprotein interacts with MAO-B and impairs mitochondrial energetics. Current research in neurobiology, v. 5, n. 100112, p. 100112, 2023.\u003c/li\u003e\n\u003cli\u003eCHEN, H. et al. Photobiomodulation modulates mitochondrial energy metabolism and ameliorates neurological damage in an APP/PS1 mousmodel of Alzheimer\u0026rsquo;s disease. Alzheimer\u0026rsquo;s research \u0026amp; therapy, v. 17, n. 1, p. 72, 2025.\u003c/li\u003e\n\u003cli\u003eVIANA, F. D. et al. Proposta de ligante para o receptor de Neuropilina-1, alvo molecular do SARS-CoV-2 / Proposed ligand for the Neuropilin-1 receptor, molecular target of SARS-CoV-2. Brazilian Journal of Development, v. 8, n. 6, p. 46091\u0026ndash;46108, 2022.\u003c/li\u003e\n\u003cli\u003eCANTUTI-CASTELVETRI, L. et al. Neuropilin-1 facilitates SARS-CoV-2 cell entry and infectivity. Science (New York, N.Y.), v. 370, n. 6518, p. 856\u0026ndash;860, 2020.\u003c/li\u003e\n\u003cli\u003eZHAO, X.; GUAN, J.-L. Focal adhesion kinase and its signaling pathways in cell migration and angiogenesis. Advanced drug delivery reviews, v. 63, n. 8, p. 610\u0026ndash;615, 2011.\u003c/li\u003e\n\u003cli\u003eNAESER, M. A. et al. Transcranial photobiomodulation Treatment: Significant improvements in four ex-football players with possible chronic traumatic encephalopathy. Journal of Alzheimer\u0026rsquo;s disease reports, v. 7, n. 1, p. 77\u0026ndash;105, 2023.\u003c/li\u003e\n\u003cli\u003eHARRIS, J. J.; JOLIVET, R.; ATTWELL, D. Synaptic energy use and supply. Neuron, v. 75, n. 5, p. 762\u0026ndash;777, 2012.\u003c/li\u003e\n\u003cli\u003eRANGARAJU, V.; LAUTERBACH, M.; SCHUMAN, E. M. Spatially stable mitochondrial compartments fuel local translation during plasticity. Cell, v. 176, n. 1\u0026ndash;2, p. 73- 84.e15, 2019.\u003c/li\u003e\n\u003cli\u003eKANN, O.; KOV\u0026Aacute;CS, R. Mitochondria and neuronal activity. American journal of physiology. Cell physiology, v. 292, n. 2, p. C641-57, 2007.\u003c/li\u003e\n\u003cli\u003eYOULE, R. J.; VAN DER BLIEK, A. M. Mitochondrial fission, fusion, and stress. Science (New York, N.Y.), v. 337, n. 6098, p. 1062\u0026ndash;1065, 2012\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"in-vitro-models","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [In vitro models](https://link.springer.com/journal/44164)","snPcode":"44164","submissionUrl":"https://submission.springernature.com/new-submission/44164/3","title":"In vitro models","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Neurotoxicity, Morphology, Oxidative Stress, Neuroprotective","lastPublishedDoi":"10.21203/rs.3.rs-8245044/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8245044/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAlzheimer's Disease (AD) is the leading cause of dementia and represents one of the greatest global health challenges, affecting not only patients but also their families. Still without a cure, current treatments only alleviate symptoms. The COVID-19 (Coronavirus Disease 2019) pandemic highlighted neurological complications associated with SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus 2), particularly the Spike protein. This study aimed to investigate the potential neurotoxic effects of recombinant Spike protein using two-dimensional (2D) in vitro neuronal models established for AD, as well as the therapeutic potential of photobiomodulation (PBM) with red LED (Light Emitting Diode) (660 nm) in attenuating these effects. Differentiated SH-SY5Y (human neuroblastoma) cells were exposed to Spike protein (0.5 \u0026micro;g/mL) and oxidative stress by H₂O₂ (Hydrogen peroxide) 200 \u0026micro;M, individually or combined, with and without PBM (3 J/cm\u0026sup2;). Cell viability was assessed using the Alamar Blue assay, and immunofluorescence characterized nuclear (Hoechst), mitochondrial (Mitotracker), actin (phalloidin), and focal adhesion (FAK) alterations. Immunofluorescence revealed mitochondrial fragmentation, actin disorganization, FAK redistribution, and nuclear condensation. The results demonstrated that Spike protein induced neurotoxicity in AD models, notably aggravated by oxidative stress. In contrast, PBM represented a promising intervention strategy, exerting a neuroprotective effect that preserves viability, mitochondrial integrity, nuclear morphology, and cytoskeletal organization. PBM thus appears to modulate mitochondrial function and mitigate oxidative stress, offering a potential therapeutic pathway to attenuate neuronal damage induced by Spike protein.\u003c/p\u003e","manuscriptTitle":"From Neurotoxicity to Neuroprotection: Photobiomodulation against the Effects of the SARS-CoV-2 Spike Protein in an Alzheimer's Disease Model","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-12 13:20:31","doi":"10.21203/rs.3.rs-8245044/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-03-27T12:11:41+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-01-27T08:53:15+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"204901325604572907201896133354563343963","date":"2026-01-26T12:46:58+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-12-06T20:46:47+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-12-03T08:28:35+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-12-02T01:12:03+00:00","index":"","fulltext":""},{"type":"submitted","content":"In vitro models","date":"2025-12-01T01:29:04+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"in-vitro-models","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [In vitro models](https://link.springer.com/journal/44164)","snPcode":"44164","submissionUrl":"https://submission.springernature.com/new-submission/44164/3","title":"In vitro models","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"8a2e566b-2aab-4ef0-861e-83682ddb3817","owner":[],"postedDate":"December 12th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-05-11T08:42:06+00:00","versionOfRecord":[],"versionCreatedAt":"2025-12-12 13:20:31","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8245044","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8245044","identity":"rs-8245044","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}